# Patent

## Title
Hybrid expression of neisserial proteins

## Abstract

Two or more Neisserial proteins (e.g. A and B) are expressed as a single hybrid protein which can be represented simply by the formula NH2-A-B—COOH.

## Background
This application is a Continuation application of prior application Ser. No. 10/220,480, filed Aug. 28, 2002, which was the National Stage of International Application No. PCT/IB01/00420, filed Feb. 28, 2001, which claims the benefit of GB 0004695.3, filed Feb. 28, 2000 and GB 0027675.8, filed Nov. 13, 2000, from which applications priority is claimed pursuant to the provision of 35 U.S.C. §§119/120 and which applications are incorporated by reference in their entireties.


SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 23002099501SUBSEQLIST.txt, date recorded: Mar. 29, 2010, size: 5,840 KB).
All documents cited herein are incorporated by reference in their entirety.
TECHNICAL FIELD
This invention is in the field of protein expression. In particular, it relates to the heterologous expression of proteins from Neisseria (e.g. N. gonorrhoeae or, preferably, N. meningitidis).
BACKGROUND ART
International patent applications WO99/24578, WO99/36544, WO99/57280 and WO00/22430 disclose proteins from Neisseria meningitidis and Neisseria gonorrhoeae. These proteins are typically described as being expressed in E. coli (i.e. heterologous expression) as either N-terminal GST-fusions or C-terminal His-tag fusions, although other expression systems, including expression in native Neisseria, are also disclosed.
It is an object of the present invention to provide alternative and improved approaches for the heterologous expression of these proteins. These approaches will typically affect the level of expression, the ease of purification, the cellular localisation of expression, and/or the immunological properties of the expressed protein.
DISCLOSURE OF THE INVENTION
In accordance with the invention, two or more (e.g. 3, 4, 5, 6 or more) proteins of the invention are expressed as a single hybrid protein. It is preferred that no non-Neisserial fusion partner (e.g. GST or poly-His) is used.
This offers two advantages. Firstly, a protein that may be unstable or poorly expressed on its own can be assisted by adding a suitable hybrid partner that overcomes the problem. Secondly, commercial manufacture is simplified—only one expression and purification need be employed in order to produce two separately-useful proteins.
Thus the invention provides a method for the simultaneous heterologous expression of two or more proteins of the invention, in which said two or more proteins of the invention are fused (i.e. they are translated as a single polypeptide chain).
The method will typically involve the steps of: obtaining a first nucleic acid encoding a first protein of the invention; obtaining a second nucleic acid encoding a second protein of the invention; ligating the first and second nucleic acids. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector.
Where just two proteins are joined, the hybrid protein can be represented simply by the formula NH2-A-B—COOH. A and B can each be selected from any Neisserial proteins, and in particular those represented by SEQ#s 122-4447. The method is well suited to the expression of proteins orf1, orf4, orf25, orf40, Orf46/46.1, orf83, 233, 287, 292L, 564, 687, 741, 907, 919, 953, 961 and 983.
The 42 hybrids indicated by ‘X’ in the following table of form NH2-A-B—COOH are preferred:













B














A
ORF46.1
287
741
919
953
961
983





ORF46.1

X
X
X
X
X
X


287
X

X
X
X
X
X


741
X
X

X
X
X
X


919
X
X
X

X
X
X


953
X
X
X
X

X
X


961
X
X
X
X
X

X


983
X
X
X
X
X
X









Preferred proteins to be expressed as hybrids are thus ORF46.1, 287, 741, 919, 953, 961 and 983. These may be used in their essentially full-length form, or poly-glycine deletions (ΔG) forms may be used (e.g. ΔG-287, ΔGTbp2, ΔG741, ΔG983 etc.), or truncated forms may be used (e.g. Δ1-287, Δ2-287 etc.), or domain-deleted versions may be used (e.g. 287B, 287C, 287BC, ORF461-433, ORF46433-608, ORF46, 961c etc.) and so on.
Particularly preferred are: (a) a hybrid protein comprising 919 and 287; (b) a hybrid protein comprising 953 and 287; (c) a hybrid protein comprising 287 and ORF46.1; (d) a hybrid protein comprising ORF1 and ORF46.1; (e) a hybrid protein comprising 919 and ORF46.1; (f) a hybrid protein comprising ORF46.1 and 919; (g) a hybrid protein comprising ORF46.1, 287 and 919; (h) a hybrid protein comprising 919 and 519; and (i) a hybrid protein comprising ORF97 and 225.
Further embodiments are shown in the drawings and include ΔG287-919, ΔG287-953, ΔG287-961, ΔG983-ORF46.1, ΔG983-741, ΔG983-961, ΔG983-961C, ΔG741-961, ΔG741-961C, ΔG741-983, ΔG741-ORF46.1, ORF46.1-741, ORF46.1-961, ORF46.1-961C, 961-ORF46.1, 961-741, 961-983, 961C-ORF46.1, 961C-741, 961C-983, 961CL-ORF46.1, 961CL-741, and 961CL-983.
Where 287 is used, it is preferably at the C-terminal end of a hybrid; if it is to be used at the N-terminus, if is preferred to use a ΔG form of 287 is used (e.g. as the N-terminus of a hybrid with ORF46.1, 919, 953 or 961).
Where 287 is used, this is preferably from strain 2996 or from strain 394/98.
Where 961 is used, this is preferably at the N-terminus. Domain forms of 961 may be used.
Alignments of polymorphic forms of ORF46, 287, 919 and 953 are disclosed in WO00/66741. Any of these polymorphs can be used according to the present invention.
Preferably, the constituent proteins (A and B) in a hybrid protein according to the invention will be from the same strain.
The fused proteins in the hybrid may be joined directly, or may be joined via a linker peptide e.g. via a poly-glycine linker (i.e. Gn where n=3, 4, 5, 6, 7, 8, 9, 10 or more) or via a short peptide sequence which facilitates cloning. It is evidently preferred not to join a ΔG protein to the C-terminus of a poly-glycine linker.
The fused proteins may lack native leader peptides or may include the leader peptide sequence of the N-terminal fusion partner.
Host
It is preferred to utilise a heterologous host. The heterologous host may be prokaryotic or eukaryotic. It is preferably E. coli, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonenna typhimurium, Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea, Mycobateria (e.g. M. tuberculosis), yeast etc.
Vectors, Hosts etc.
As well as the methods described above, the invention provides (a) nucleic acid and vectors useful in these methods (b) host cells containing said vectors (c) proteins expressed or expressable by the methods (d) compositions comprising these proteins, which may be suitable as vaccines, for instance, or as diagnostic reagents, or as immunogenic compositions (e) these compositions for use as medicaments (e.g. as vaccines) or as diagnostic reagents (f) the use of these compositions in the manufacture of (1) a medicament for treating or preventing infection due to Neisserial bacteria (2) a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria, and/or (3) a reagent which can raise antibodies against Neisserial bacteria and (g) a method of treating a patient, comprising administering to the patient a therapeutically effective amount of these compositions.
Sequences
The invention also provides a protein or a nucleic acid having any of the sequences set out in the following examples. It also provides proteins and nucleic acid having sequence identity to these. As described above, the degree of ‘sequence identity’ is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more).
Nomenclature Herein
The 2166 protein sequences disclosed in WO99/24578, WO99/36544 and WO99/57280 are referred to herein by the following SEQ# numbers:












Application
Protein sequences
SEQ# herein







WO99/24578
Even SEQ IDs 2-892
SEQ#s 122-567


WO99/36544
Even SEQ IDs 2-90
SEQ#s 568-612


WO99/57280
Even SEQ IDs 2-3020
SEQ#s 613-2122



Even SEQ IDs 3040-3114
SEQ#s 2123-2160



SEQ IDs 3115-3241
SEQ#s 2161-2287









In addition to this SEQ# numbering, the naming conventions used in WO99/24578, WO99/36544 and WO99/57280 are also used (e.g. ‘ORF4’, ‘ORF40’, ‘ORF40-1’ etc. as used in WO99/24578 and WO99/36544; ‘m919’, ‘g919’ and ‘a919’ etc. as used in WO99/57280).
The 2160 proteins NMB0001 to NMB2160 from Tettelin et al. [Science (2000) 287:1809-1815] are referred to herein as SEQ#s 2288-4447 [see also WO00/66791].
The term ‘protein of the invention’ as used herein refers to a protein comprising:

    
    
        (a) one of sequences SEQ#s 122-4447; or
        (b) a sequence having sequence identity to one of SEQ#s 122-4447; or
        (c) a fragment of one of SEQ#s 122-4447.
    
    


The degree of ‘sequence identity’ referred to in (b) is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more). This includes mutants and allelic variants [e.g. see WO00/66741]. Identity is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence.
The ‘fragment’ referred to in (c) should comprise at least n consecutive amino acids from one of SEQ#s 122-4447 and, depending on the particular sequence, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more). Preferably the fragment comprises an epitope from one of SEQ#s 122-4447. Preferred fragments are those disclosed in WO00/71574 and WO01/04316.
Preferred proteins of the invention are found in N. meningitidis serogroup B.
Preferred proteins for use according to the invention are those of serogroup B N. meningitidis strain 2996 or strain 394/98 (a New Zealand strain). Unless otherwise stated, proteins mentioned herein are from N. meningitidis strain 2996. It will be appreciated, however, that the invention is not in general limited by strain. References to a particular protein (e.g. ‘287’, ‘919’ etc.) may be taken to include that protein from any strain.
It will be appreciated that references to “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.



BRIEF DESCRIPTION OF DRAWINGS
FIGS. 1 to 26 show hybrid proteins according to the invention.
FIG. 1: Gene construct used to express the ΔG287-919 hybrid protein
FIG. 2: Gene construct used to express the ΔG287-953 hybrid protein
FIG. 3: Gene construct used to express the ΔG287-961 hybrid protein
FIG. 4: Gene construct used to express the ΔG287NZ-919 hybrid protein
FIG. 5: Gene construct used to express the ΔG287NZ-953 hybrid protein
FIG. 6: Gene construct used to express the ΔG287NZ-961 hybrid protein
FIG. 7: Gene construct used to express the ΔG983-ORF46.1 hybrid protein
FIG. 8: Gene construct used to express the ΔG983-741 hybrid protein
FIG. 9: Gene construct used to express the ΔG983-961 hybrid protein
FIG. 10: Gene construct used to express the ΔG983-961c hybrid protein
FIG. 11: Gene construct used to express the ΔG741-961 hybrid protein
FIG. 12: Gene construct used to express the ΔG741-961c hybrid protein
FIG. 13: Gene construct used to express the ΔG741-983 hybrid protein
FIG. 14: Gene construct used to express the ΔG741-ORF46.1 hybrid protein
FIG. 15: Gene construct used to express the ORF46.1-741 hybrid protein
FIG. 16: Gene construct used to express the ORF46.1-961 hybrid protein
FIG. 17: Gene construct used to express the ORF46.1-961c hybrid protein
FIG. 18: Gene construct used to express the 961-ORF46.1 hybrid protein
FIG. 19: Gene construct used to express the 961-741 hybrid protein
FIG. 20: Gene construct used to express the 961-983 hybrid protein
FIG. 21: Gene construct used to express the 961c-ORF46.1 hybrid protein
FIG. 22: Gene construct used to express the 961c-741 hybrid protein
FIG. 23: Gene construct used to express the 961c-983 hybrid protein
FIG. 24: Gene construct used to express the 961cL-ORF46.1 hybrid protein
FIG. 25: Gene construct used to express the 961cL-741 hybrid protein
FIG. 26: Gene construct used to express the 961cL-983 hybrid protein



MODES FOR CARRYING OUT THE INVENTION
Example 1
Hybrids of ORF46
The complete ORF46 protein from N. meningitidis (serogroup B, strain 2996) has the following sequence (SEQ ID NO: 1):









1
LGISRKISLI LSILAVCLPM HAHASDLAND SFIRQVLDRQ HFEPDGKYHL



 


51
FGSRGELAER SGHIGLGKIQ SHQLGNLKIQ QAAIKGNIGY IVRFSDHGHE


 


101
VHSPFDNHAS HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD GYDGPQGGGY


 


151
PAPKGARDIY SYDIKGVAQN IRLNLTDNRS TGQRLADRFH NAGSMLTQGV


 


201
GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE IVGAGDAVQG


 


251
ISEGSNIAVM HGLGLLSTEN KMARINDLAD MAQLKDYAAA AIRDWAVQNP


 


301
NAAQGIEAVS NIFMAAIPIK GIGAVRGKYG LGGITAHPIK RSQMGAIALP


 


351
KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LBQRYGKENI TSSTVPPSNG


 


401
KNVKLADQRH PKTGVPFDGK GFPNFEKHVK YDTKLDIQEL SGGGIPKAKP


 


451
VSDAKPRWEV DRKLNKLTTR EQVEKNVQEI RNGNKNSNFS QHAQLEREIN


 


501
KLKSADEINF ADGMGKFTDS MNDKAFSRLV KSVKENGFTN PVVEYVEING


 


551
KAYIVRGNNR VFAARYLGRI HELKFKKVDF PVPNTSWKNP TDVLNESGNV


 


601
KRPRYRSK*






The leader peptide is underlined.
The sequences of ORF46 from other strains can be found in WO00/66741.
ORF46 has been fused at its C-terminus and N-terminus with 287, 919, and ORF1. The hybrid proteins were generally insoluble, but gave some good ELISA and bactericidal results (against the homologous 2996 strain):














Protein
ELISA
Bactericidal Ab



















Orf1-Orf46.1-His
850
256



919-Orf46.1-His
12900
512



919-287-Orf46-His
n.d.
n.d.



Orf46.1-287His
150
8192



Orf46.1-919His
2800
2048



Orf46.1-287-919His
3200
16384









For comparison, ‘triple’ hybrids of ORF46.1, 287 (either as a GST fusion, or in ΔG287 form) and 919 were constructed and tested against various strains (including the homologous 2996 strain) versus a simple mixture of the three antigens. FCA was used as adjuvant:

















2996
BZ232
MC58
NGH38
F6124
BZ133





















Mixture
8192
256
512
1024
>2048
>2048


ORF46.1-
16384
256
4096
8192
8192
8192


287-919his


ΔG287-
8192
64
4096
8192
8192
16384


919-ORF46.


1his


ΔG287-
4096
128
256
8192
512
1024


ORF46.1-


919his









Again, the hybrids show equivalent or superior immunological activity.
Hybrids of two proteins (strain 2996) were compared to the individual proteins against various heterologous strains:














1000
MC58
F6124 (MenA)




















ORF46.1-His
<4
4096
<4



ORF1-His
8
256
128



ORF1-ORF46.1-His
1024
512
1024









Again, the hybrid shows equivalent or superior immunological activity.
Example 2
Hybrids of ΔG287
The deletion of the (Gly)6 sequence in 287 was found to have a dramatic effect on protein expression. The protein lacking the N-terminal amino acids up to GGGGGG is called ‘ΔG287’. In strain MC58, its basic sequence (leader peptide underlined) is (SEQ ID NO: 2):








    SPDVKS ADTLSKPAAP VVSEKETEAK EDAPQAGSQG QGAPSAQGSQ DMAAVSEENT 



 


GNGGAVTADN PKNEDEVAQN DMPQNAAGTD SSTPNHTPDP NMLAGNMENQ ATDAGESSQP


 


ANQPDMANAA DGMQGDDPSA GGQNAGNTAA QGANQAGNNQ AAGSSDPIPA SNPAPANGGS


 


NFGRVDLANG VLIDGPSQNI TLTHCKGDSC SGNNFLDEEV QLKSEFEKLS DADKISNYKK


 


DGKNDKFVGL VADSVQMKGI NQYIIFYKPK PTSFARFRRS ARSRRSLPAE MPLIPVNQAD


 


TLIVDGEAVS LTGHSGNIFA PEGNYRYLTY GAEKLPGGSY ALRVQGEPAK GEMLAGAAVY


 


NGEVLHPHTE NGRPYPTRGR FAAKVDFGSK SVDGIIDSGD DLHMGTQKFK AAIDGNGFKG


 


TWTENGSGDV SGKFYGPAGE EVAGKYSYRP TDAEKGGFGV FAGKKEQD*






ΔG287, with or without His-tag (‘ΔG287-His’ and ‘ΔG287K’, respectively), are expressed at very good levels in comparison with the ‘287-His’ or ‘287untagged’,
On the basis of gene variability data, variants of ΔG287-His were expressed in E. coli from a number of MenB strains, in particular from strains 2996, MC58, 1000, and BZ232. The results were also good—each of these gave high ELISA titres and also serum bactericidal titres of >8192. ΔG287K, expressed from pET-24b, gave excellent titres in ELISA and the serum bactericidal assay.
Deletion of poly-Gly sequences is also applicable to Tbp2 (NMB0460), 741 (NMB 1870) and 983 (NMB1969). When cloned in pET vector and expressed in E. coli without the sequence coding for their leader peptides and without poly-Gly (i.e. as “ΔG forms”), the same effect was seen—expression was good in the clones carrying the deletion of the poly-glycine stretch, and poor or absent if the glycines were present in the expressed protein.
ΔG287 was fused directly in-frame upstream of 919, 953, 961 (sequences shown below) and ORF46.1:








ΔG287-919



(SEQ ID NO: 3)



ATGGCTAGCCCCGATGTTAAATCGGCGGACACGCTGTCAAAACCGGCCGCTCCTGTTGTTGC



 


TGAAAAAGAGACAGAGGTAAAAGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGC


 


CATCCACACAAGGCAGCCAAGATATGGCGGCAGTTTCGGCAGAAAATACAGGCAATGGCGGT


 


GCGGCAACAACGGACAAACCCAAAAATGAAGACGAGGGACCGCAAAATGATATGCCGCAAAA


 


TTCCGCCGAATCCGCAAATCAAACAGGGAACAACCAACCCGCCGATTCTTCAGATTCCGCCC


 


CCGCGTCAAACCCTGCACCTGCGAATGGCGGTAGCAATTTTGGAAGGGTTGATTTGGCTAAT


 


GGCGTTTTGATTGATGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTG


 


TAATGGTGATAATTTATTGGATGAAGAAGCACCGTCAAAATCAGAATTTGAAAATTTAAATG


 


AGTCTGAACGAATTGAGAAATATAAGAAAGATGGGAAAAGCGATAAATTTACTAATTTGGTT


 


GCGACAGCAGTTCAAGCTAATGGAACTAACAAATATGTCATCATTTATAAAGACAAGTCCGC


 


TTCATCTTCATCTGCGCGATTCAGGCGTTCTGCACGGTCGAGGAGGTCGCTTCCTGCCGAGA


 


TGCCGCTAATCCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTG


 


ACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGC


 


GGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTGCAAGGCGAACCGGCAAAAGGCGAAA


 


TGCTTGCTGGCACGGCCGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGT


 


CCGTACCCGACTAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGG


 


CATTATCGACAGCGGCGATGATTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATG


 


GAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAGGTTTTAC


 


GGCCCGGCCGGCGCGGAAGTGGCGGGAAAATACAGCTATCGCCCGACAGATGCGGAAAAGGG


 


CGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGATGCCAAA


 


GCAAGAGCATCCAAACCTTTCCGCAACCCGACACATCCGTCATCAACGGCCCGGACCGGCCG


 


GCCGGTCGGCATCCCCGACCCCGCCGGAACGACGGTCGGCGGCGGCGGGGCCGTCTATACCG


 


TTGTACCGCACCTGTCCCTGCCCCACTGGGCGGCGCAGGATTTCGCCAAAAGCCTGCAATCC


 


TTCCGCCTCGGCTGCGCCAATTTGAAAAACCGCCAAGGCTGGCAGGATGTGTGCGCCCAAGC


 


CTTTCAAACCCCCGTCCATTCCTTTCAGGCAAAACAGTTTTTTGAACGCTATTTCACGCCGT


 


GGCAGGTTGCAGGCAACGGAAGCCTTGCCGGTACGGTTACCGGCTATTACGAGCCGGTGCTG


 


AAGGGCGACGACAGGCGGACGGCACAAGCCVGTTCCCGATTTACGGTATTCCCGACGATTTT


 


ATCTCCGTCCCCCTGCCTGCCGGTTTGCGGAGCGGAAAAGCCCTTGTCCGCATCAGGCAGAC


 


GGGAAAAAACAGCGGCACAATCGACAATACCGGCGGCACACATACCGCCGACCTCTCCCGAT


 


TCCCCATCACCGCGCGCACAACGGCAATCAAAGGCAGGTTTGAAGGAAGCCGCTTCCTCCCC


 


TACCACACGCGCAACCAAATCAACGGCGGCGCGCTTGACGGCAAAGCCCCGATACTCGGTTA


 


CGCCGAAGACCCCGTCGAACTTTTTTTTATGCACATCCAAGGCTCGGGCCGTCTGAAACCCC


 


GTCCGGCAAATACATCGGCTATGCCGACAAAAACGAACATCCCTACGTTTCCATCGGACGCT


 


ATATGGCGGACAAAGGCTACCTCAAGCTCGGGCAGACCTCGATGCAGGGCATCAAAGCCTAT


 


ATGCGGCAAAATCCGCAACGCCTCGCCGAAGTTTTGGGTCAAAACCCCAGCTATATCTTTTT


 


CCGCGAGCTTGCCGGAAGCAGCAATGACGGTCCCGTCGGCGCACTGGGCACGCCGTTGATGG


 


GGGAATATGCCGGCGCAGTCGACCGGCACTACATTACCTTGGGCGCGCCCTTATTTGTCGCC


 


ACCGCCCATCCGGTTACCCGCAAAGCCCTCAACCGCCTGATTATGGCGCAGGATACCGGCAG


 


CGCGATTAAAGGCGCGGTGCGCGTGGATTATTTTTGGGGATACGGCGACGAAGCCGGCGAAC


 


TTGCCGGCAAACAGAAAACCACGGGTTACGTCTGGCAGCTCCTACCCAACGGTATGAAGCCC


 


GAATACCGCCCGTAACTCGAG


 


(SEQ ID NO: 4)










1
MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM



 


51
AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS


 


101
DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL


 


151
LDEEAPSKSE FENLNESERI EKYKKDGKSD KPTNLVATAV QANGTNKYVI


 


201
IYKDKSASSS SASFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG


 


251
HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE


 


301
VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI


 


351
DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GRYSYRPTDA EKGGFGVFAG


 


401
KKEQDGSGGG GCQSKSIQTF PQPDTSVING PDRPVGIPDP AGTTVGGGGA


 


451
VYTVVPHLSL PHWAAQDFAK SLQSFRLGCA NLKNRQGWQD VCAQAFQTPV


 


501
HSFQAKQFFE RYFTPWQVAG NGSLAGTVTG YYEPVLKGDD RRTAQARFPI


 


551
YGIPDDFISV PLPAGLRSGK ALVRIRQTGK NSGTIDNTGG THTADLSRFP


 


601
ITARTTAIKG RFEGSRFLPY HTRNQINGGA LDGKAPILGY AEDPVELFFM


 


651
HIQGSGRLKT PSGKYIRIGY ADKNEHPYVS IGRYMADKGY LKLGQTSMQG


 


701
IKAYMRQNPQ RLAEVLGQNP SYIFFRELAG SSNDGPVGAL GTPLMGEYAG


 


751
AVDRHYITLG APLFVATAHP VTRKALNRLI MAQDTGSAIK GAVRVDYFWG


 


801
YGDEAGELAG KQKTTGYVWQ LLPNGMKPEY RP*


 








ΔG287-953



(SEQ ID NO: 5)



ATGGCTAGCCCCGATGTTAAATCGGCGGACACGCTGTCAAAACCGGCCGCTCCTGTTGTTGC



 


TGAAAAAGAGACAGAGGTAAAAGAAGATGCGCCACAGGCAGGACAGGGCGCGCCATCCACAC


 


AAGGCAGCCAAGATATGGCGGCAGTTTCGGCAGAAAATACAGGCAATGGCGGTGCGGCAACA


 


ACGGACAAACCCAAAAATGAAGACGAGGGACCGCAAAATGATATGCCGCAAAATTCCGCCGA


 


ATCCGCAAATCAAACAGGGAACAACCAACCCGCCGATTCTTCAGATTCCGCCCCCGCGTCAA


 


ACCTGCACCTGCGAATGGCGGTAGCAATTTTGGAAGGGTTGATTTGGCTAATGGCGTTTTGA


 


TTGATGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAATGGTGAT


 


AATTTATTGGATGAAGAAGCACCGTCAAAATCAGAATTTGAAAATTTAAATGAGTCTGAACG


 


AATTGAGAAATATAAGAAAGATGGGAAAAGCGATAAATTTACTAATTTGGTTGCGACAGCAG


 


TTCAAGCTAATGGAACTAACAAATATGTCATCATTTATAAAGACAAGTCCGCTTCATCTTCA


 


TCTGCGCGATTCAGGCGTTCTGCACGGTCGAGGAGGTCGCTTCCTGCCGAGATGCCGCTAAT


 


CCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATT


 


CCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTG


 


CCCGGCGGATCGTATGCCCTCCGTGTGCAAGGCGAACCGGCAAAAGGCGAAATGCTTGCTGG


 


CACGGCCGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTACCCGA


 


CTAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACA


 


GCGGCGATGATTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGTTTA


 


AGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAGGTTTTACGCCCGGCCGGCG


 


AGGAAGTGGCGGGAAAATACAGCTATCGCCCGACAGATGCGGAAAAGGGCGGATTGGCGTGT


 


TTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGAGCCACCTACAAAGTGGACGAA


 


TATCACGCCAACGCCCGTTTCGCCATCGACCATTTCAACACCAGCACCAACGTCGGCGGTTT


 


TTACGGTCTGACCGGTTCCGTCGAGTTCGACCAAGCAAAACGCGACGGTAAAATCGACATCA


 


CCATCCCCGTTGCCAACCTGCAAAGCGGTTCGCAACACTTTACCGACCACCTGAAATCAGCC


 


GACATCTTCGATGCCGCCCAATATCCGGACATCCGCTTTGTTTCCACCAAATTCAACTTCAA


 


CGGCAAAAAACTGGTTTCCGTTGACGGCAACCTGACCATGCACGGCAAAACCGCCCCCGTCA


 


AACTCAAAGCCGAAAAATTCAACTGCTACCAAAGCCCGATGGCGAAAACCGAAGTTTGCGGC


 


GGCGACTTCAGCACCACCATCGACCGCACCAAATGGGGCGTGGACTACCTCGTTAACGTTGG


 


TATGACCAAAAGCGTCCGCATGACATCCAAATCGAGGCAGCCAAACAATAACTCGAG


 


(SEQ ID NO: 6)










1
MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM



 


51
AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS


 


101
DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL


 


151
LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI


 


201
IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG


 


251
HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE


 


301
VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI


 


351
DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG


 


401
KKEQDGSGGG GATYKVDEYH ANARFAIDHF NTSTNVGGFY GLTGSVEFDQ


 


451
AKRDGKIDIT IPVANLQSGS QHFTDHLKSA DIFDAAQYPD IRFVSTKFNF


 


501
NGKKLVSVDG NLTMHGKTAP VKLKAEKFNC YQSPMAKTEV CGGDFSTTID


 


551
RTKWGVDYLV NVGMTKSVRI DIQIEAAKQ*


 








ΔG287-961



(SEQ ID NO: 7)



ATGGCTAGCCCCGATGTTAAATCGGCGGACACGCTGTCAAAACCGGCCGCTCCTGTTGTTGC



 


TGAAAAAGAGACAGAGGTAAAAGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGC


 


CATCCACACAAGGCAGCCAAGATGGCGGCAGTTTCGGCAGAAAATACAGGCAATGGCGGTGC


 


GGCAACAACGGACAAACCCAAAAATGAAGACGAGGGACCGCAAAATGATATGCCGCAAAATT


 


CCGAATCCGCAAATCAAACAGGGAACAACCAACCCGCCGATTCTTCAGATTCCGCCCCCGCG


 


TCAAACCCTGCACCTGCGAATGGCGGTAGCAATTTTGGAAGGGTTGATTTGGCTAATGGCGT


 


TTTGATTGATGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAATG


 


GTGATAATTTATTGGATGAAGAAGCACCGTCAAAATCAGAATTTGAAAATTTAAATGAGTCT


 


GAACGAATTGAGAAATATAAGAAAGATGGGAAAAGCGATAAATTTACTAATTTGGTTGCGAC


 


AGCAGTTCAAGCTAATGGAACTAACAAATATGTCATCATTTATAAAGACAAGTCCGCTTCAT


 


CTTCATCTGCGCGATTCAGGCGTTCTGCACGGTCGAGGAGGTCGCTTCCTGCCGAGATGCCG


 


CTAATCCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGG


 


GCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAA


 


AATTGCCCGGCGGATCGTATGCCCTCCGTGTGCAAGGCGAACCGGCAAAAGGCGAAATGCTT


 


GCTGGCACGGCCGTGTACAACGGCGAAGTGCTGCATTTCATACGGAAAACGGCCGTCCGTAC


 


CCGACTAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTAT


 


CGACAGCGGCGATGATTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACG


 


GCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAGGTTTTACGGCCCG


 


GCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCGACAGATGCGGAAAAGGGCGGATT


 


CGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGAGCCACAAACGACG


 


ACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATC


 


AACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAA


 


AGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCG


 


TGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCT


 


GCAGAATCTGAAATAGAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGAT


 


ACTGATCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACA


 


TTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGA


 


TACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACA


 


CTAAGGCAGACGAAGCCGTCAAACGCCAATGAAGCCAAACAGACGGCCGAAGAACCAACAAA


 


ACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGC


 


ACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGC


 


TGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCA


 


GAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTG


 


GACACAGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTG


 


GATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCT


 


CTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCT


 


ACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAACTTTGCCGCCA


 


AAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAAT


 


TACGAGTGGTAACTCGAG


 


(SEQ ID NO: 8)










1
MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM



 


51
AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS


 


101
DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL


 


151
LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI


 


201
IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG


 


251
HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE


 


301
VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI


 


351
DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG


 


401
KKEQDGSGGG GATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE


 


451
DGTITKKDAT AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE


 


501
SEIEKLTTKL ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV


 


551
KIDEKLEAVA DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE


 


601
TKQNVDAKVK AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN


 


651
KDNIAKKANS ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH


 


701
DTRLNGLDKT VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK


 


751
SESAVAIGTG FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEW*



















ELISA
Bactericidal



















ΔG287-953-His
3834
65536



ΔG287-961-His
108627
65536









The bactericidal efficacy (homologous strain) of antibodies raised against the hybrid proteins was compared with antibodies raised against simple mixtures of the component antigens (using 287-GST) for 919 and ORF46.1:













Mixture with 287
Hybrid with ΔG287



















919
32000
128000



ORF46.1
128
16000









Data for bactericidal activity against heterologous MenB strains and against serotypes A and C were also obtained:















919

ORF46.1














Strain
Mixture
Hybrid
Mixture
Hybrid
















NGH38
1024
32000
—
16384



MC58
512
8192
—
512



BZ232
512
512
—
—



MenA (F6124)
512
32000
—
8192



MenC (C11)
>2048
>2048
—
—



MenC (BZ133)
>4096
64000
—
8192









The hybrid proteins with ΔG287 at the N-terminus are therefore immunologically superior to simple mixtures, with ΔG287-ORF46.1 being particularly effective, even against heterologous strains. ΔG287-ORF46.1K may be expressed in pET-24b.
The same hybrid proteins were made using New Zealand strain 394/98 rather than 2996:








ΔG287NZ-919



 (SEQ ID NO: 9)



ATGGCTAGCCCCGATGTCAAGTCGGCGGACACGCTGTCAAAACCTGCCGC



 


CCCTGTTGTTTCTGAAAAAGACAGAGGCAAAGGAAGATGCGCCACAGGCA


 


GGTTCTCAAGGACAGGGCGCGCCATCCGCACAAGGCGGTCAAGATATGGC


 


GGCGGTTTCGGAAGAAAATACAGGCAATGGCGGTGCGGCAGCAACGGACA


 


AACCCAAAAATGAAGACGAGGGGGCGCAAAATGATATGCCGCAAAATGCC


 


GCCGATACAGATAGTTTGACACCGAATCACACCCCGGCTTCGAATATGCC


 


GGCCGGAAATATGGAAAACCAAGCACCGGATGCCGGGAATCGGAGCAGCC


 


GGCAAACCAACCGGATATGGCAAATACGGCGGACGGAATGCAGGGTGACG


 


ATCCGTCGGCAGGCGGGGAAAATGCCGGCAATACGGCTGCCCAAGGTACA


 


AATCAAGCCGAAAACAATCAAACCGCCGGTTCTCAAAATCCTGCCTCTTC


 


AACCAATCCTAGCGCCACGAATAGCGGTGGTGATTTTGGAAGGACGAACG


 


TGGGCAATTCTGTTGTGATTGACGGGCCGTCGCAAAATATAACGTTGACC


 


CACTGTAAAGGCGATTCTTGTAGTGGCAATAATTTCTTGGATGAAGAAGT


 


ACAGCTAAAATCAGAATTTGAAAAATTAAGTGATGCAGACAAAATAAGTA


 


ATTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGTCGGT


 


TTGGTTGCCGATAGTGTGCAGATGAAGGGAATCAATCAATATATTATCTT


 


TTATAAACCTAAACCCACTTCATTTGCGCGATTTAGGCGTTCTGCACGGT


 


CGAGGCGGTCGCTTCCGGCCGAGATGCCGCTGATTCCCGTCAATCAGGCG


 


GATACGCTGATTGTCGATGGGAAGCGGTCAGCCTGACGGGCATTCCGGCA


 


ATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAA


 


AAATTGCCCGGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACCTTCAAA


 


AGGCGAAATGCTCGCGGGCACGGCAGTGTACAACGGCGAAGTGCTGCATT


 


TTCATACGGAAAACGGCCGTCCGTCCCCGTCCAGAGGCAGGTTTGCCGCA


 


AAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGA


 


TGGTTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACG


 


GCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAAG


 


TTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCA


 


ACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCA


 


GGATGGATCCGGAGGAGGAGGATGCCAAAGCAAGAGCATCCAAACCTTTC


 


CGCAACCCGACACATCCGTCATCAACGGCCCGGACCGGCCGGTCGGCATC


 


CCCGCCGGAACGACGGTCGGCGGCGGCGGGGCCGTCATATACCGTTGTAC


 


CGCACCTGTCCCTGCCCCACTGGGCGGCGCAGGATTTCGCCAAAAGCCTG


 


CAATCCTTCCGCCTCGGCTGCGCCAATTTGAAAAACCGCCAAGGCTGGCT


 


GGCAGGATGTGTGCGCCCAAGCCTTTCAAACCCCCGTCCATTCCCTTTCA


 


GGCAAAACAGTTTTTTGAACGCTATTTCACGCCGTGGCAGGTTGCAGGCA


 


ACGGAAGCCTTGCCGGTACGGTTACCGGCTATTACGAGCCGGTGCTGAAG


 


GGCGACGACAGGCGGACGGCACAAGCCCGCTTCCCGATTTACGTATTCCC


 


GACGATTTTATCTCCGTCCCCCTGCCTGCCGGTTTGCGGAGCGGAAAAGC


 


CCTTGTCCGCATCAGGCAGACGGGAAAAAACAGCGGCACAATCGACAATA


 


CCGGCGGCACACATACCGCCGACCTCTCCCGATTCCCCATCACCGCGCGC


 


ACAACGGCAATCAAAGGCAGGTTTGAAGGAAGCCGCTTCCTCCCCTACCA


 


CACGCGCAACCAAATCAACGGCGGCGCGCTTGACGGCAAAGCCCCGATAC


 


TCGGTTACGCCGAAGACCCCGTCGAACTTTTTTTTATGCACATCCAAGGC


 


TCGGGCCGTCTGAAAACCCCGTCCGGCAAATACATCCGCATCGGCTATGC


 


CGACAAAAACGAACATCCCTACGTTTCCATCGGACGCTATATGGCGGACA


 


AGGCTACCTCAAGCTCGGGCAGACCTCGATGCAGGGCATCAAAGCCTATA


 


TGCGGCAAAATCCGCAACGCCTCGCCGAAGTTTTGGGTCAAAACCCCAGC


 


TATATCTTTTTCCGCGAGCTTGCCGGAAGCAGCAATGACGGTCCCGTCGG


 


CGCACTGGGCACGCCGTTGATGGGGGAATATGCCGGCGCAGTCGACCGGC


 


AGTCGACCGGCACTACATTACCTTGGGCGCGCCCTTATTTGTCGCCACCG


 


CCCATCCGGTTACCCGCAAAGCCCTCAACCGCCTGATTATGGCGCAGGAT


 


ACCGGCAGCGCGATTAAAGGCGCGGTGCGCGTGGATTATTTTTGGGGATA


 


CGGCGACGAAGCCGGCGAACTTGCCGGCAAACAGAAAACCACGGGTTACG


 


TCTGGCAGCTCCTACCCAACGGTATGAAGCCCGAATACCGCCCGTAAAAG


 


CTT


 


(SEQ ID NO: 10)










1
MASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGQQDM



 


51
AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM


 


101
PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG


 


151
TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL


 


201
THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV


 


251
GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ


 


301
ADTLIVDGEA VSLTGHSGNI PAPEGNYRYL TYGAEKLPGG SYALRVQGEP


 


351
SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS


 


401
GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY


 


451
RPTDAEKGGF GVFAGKKEQD GSGGGGCQSK SIQTFPQPDT SVINGPDRPV


 


501
GIPDPAGTTV GGGGAVYTVV PHLSLPHWAA QDFAKSLQSF RLGCANLKNR


 


551
QGWQDVCAQA FQTPVHSFQA KQFFERYFTP WQVAGNGSLA GTVTGYYEPV


 


601
LKGDDRRTAQ ARFPIYGIPD DFISVPLPAG LRSGKALVRI RQTGKNSGTI


 


651
DNTGGTHTAD LSRFPITART TAIKGEFEGS RFLPYHTRNQ INGGALDGKA


 


701
PILGYAEDPV ELFFMHIQGS GRLKTPSGKY IRIGYADKNE HPYVSIGRYM


 


751
ADKGYLKLGQ TSMQGIKAYM RQNPQRLAEV LGQNPSYIFF RELAGSSNDG


 


801
PVGALGTPLM GEYAGAVDRH YITLGAPLFV ATAHPVTRKA LNRLIMAQDT


 


851
GSAIKGAVRV DYFWGYGDEA GELAGKQKTT GYVWQLLPNG MKPEYRP*


 








ΔG287NZ-953



 (SEQ ID NO: 11)



ATGGCTAGCCCCGATGTCAAGTCGGCGGACACGCTGTCAAAACCTGCCGC



 


CCCTGTTGTTTGAAAAAGAGACAGAGGCAAAGGAAGATGCGCCACAGGCA


 


GGTTCTCAAGGACAGGGCGCGCCATCCGCACAAGGCGGTCAAGATATGGC


 


GGCGGTTTCGGAAGAAAATACAGGCAATGGCGGTGCGGCAGCAACGGACA


 


AACCCAAAAATGAAGACGAGGGGGCAAAATGATATGCCGCAAAATGCCGC


 


CGATACAGATAGTTTGACACCGAATCACACCCCGGCTTCGAATATGCCGG


 


CCGGAAATATGGAAAACCAAGCACCGGATGCCGGGGAATCGGAGCAGCCG


 


GCAAACCAACCGGATATGGCAAATACGGCGGACGGAATGCAGGGTGACGA


 


TCCGTCGGCAGGCGGGGAAAATGCCGGCAATACGGCTGCCCAAGGTACAA


 


ATCAAGCCGAAAACAATCAAACCGCCGGTTCTCAAAATCCTGCCTCTTCA


 


ACCAATCCTAGCGCCACGAATAGCGGTGGTGATTTTGGAAGGACGAACGT


 


GGGCAATTCTGTTGTGATTGACGGGCCGTCGCAAAATATAACGTTGACCC


 


ACTGTAAAGGCGATTCTTGTAGTGGCAATAATTTCTTGGATGAAGAAGTA


 


CAGCTAAAATCAGAATTTGAAAAATTAAGTGATGCAGACAAAATAAGTAA


 


TTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGTCGGTT


 


TGGTTGCCGATAGTGTGCAGATGAAGGGAATCAATCAATATATTATCTTT


 


TATAAACCTAAACCCACTTCATTTGCGCGATTTAGGCGTTCTGCACGGTC


 


GAGGCGGTCGCTTCCGGCCGAGATGCCGCTGATTCCCGTCAATCAGGCGG


 


ATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGC


 


AATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGA


 


AAAATTGCCCGGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACCTTCAA


 


AAGGCGAAATGCTCGCGGGCACGGCAGTGTACAACGGCGAAGTGCTGCAT


 


TTTCATACGGAAAACGGCCGTCCGTCCCCGTCCAGAGGCAGGTTTGCCGC


 


AAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCG


 


ATGGTTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAAC


 


GGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAA


 


GTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCC


 


CAACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAG


 


CAGGATGGATCCGGAGGAGGAGGAGCCACCTACAAAGTGGACGAATATCA


 


CGCCAACGCCCGTTTCGCCATCGACCATTTCAACACCAGCACCAACGTCG


 


GCGGTTTTTACGGTCTGACCGGTTCCGTCGAGTTCGACCAAGCAAAACGC


 


GACGGTAAAATCGACATCACCATCCCCGTTGCCAACCTGCAAAGCGGTTC


 


GCAACACTTTACCGACCACCTGAAATCAGCCGACATCTTCGATGCCGCCC


 


AATATCCGGACATCCGCTTTGTTTCCACCAAATTCAACTTCAACGGCAAA


 


AAACTGGTTTCCGTTGACGGCAACCTGACCATGCACGGCAAAACCGCCCC


 


CGTCAAACTCAAAGCCGAAAAATTCAACTGCTACCAAAGCCCGATGGCGA


 


AAACCGAAGTTTGCGGCGGCGACTTCAGCACCACCATCGACCGCACCAAA


 


TGGGGCGTGGACTACCTCGTTAACGTTGGTATGACCAAAAGCGTCCGCAT


 


CGACATCCAAATCGAGGCAGCCAAACAATAAAAGCTT


 


(SEQ ID NO: 12)










1
MASPDVKSAD TLSKPAAPVV SEKETEAKED APQASQGQG APSAQGGQDM



 


51
AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM


 


101
PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG


 


151
TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL


 


201
THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV


 


251
GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ


 


301
ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP


 


351
SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS


 


401
GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY


 


451
RPTDAEKGGF GVFAGKKEQD GSGGGGATYK VDEYHANARF AIDHFNTSTN


 


501
VGGFYGLTGS VEFDQAKRDG KIDITIPVAN LQSGSQHFTD HLKSADIFDA


 


551
AQYPDIRFVS TKFNFNGKKL VSVDGNLTMH GKTAPVKLKA EKFNCYQSPM


 


601
AKTEVCGGDF STTIDRTKWG VDYLVNVGMT KSVRIDIQIE AAKQ*


 








ΔG287NZ-961



 (SEQ ID NO: 13)



ATGGCTAGCCCCGATGTCAAGTCGGCGGACACGCTGTCAAAACCTGCCGC



 


CCCTGTTGTTTCTGAAAAAGAGACAGAGGCAAAGGAAGATGCGCCACAGG


 


CAGGTTCTCAAGGACAGGGCGCGCCATCCGCACAAGGCGGTCAAGATATG


 


GCCGGCGGTTTCGGAAGAAAATACAGGCAATGGCGGTGCGGCAGCAACGG


 


ACAAACCCAAAAATGAAGACGAGGGGGCGCAAAATGATATGCCGCAAAAT


 


GCCGCCGATACAGATAGTTTGACACCGAATCACACCCCGGCTTCGAATAT


 


GCCGGCCGGAAATATGGAAAACCAAGCACCGGATGCCGGGGAATCGGAGC


 


AGCCGGCAAACCAACCGGATATGGCAAATACGGCGGACGGAATGCAGGGT


 


GACGATCCGTCGGCAGGCGGGGAAAATGCCGGCAATACGGCTGCCCAAGG


 


TACAAATCAAGCCGAAAACAATCAAACCGCCGGTTCTCAAAATCCTGCCT


 


CTTCAACCAATCCTAGCGCCACGAATAGCGGTGGTGATTTTGGAAGGACG


 


AACGTGGGCAATTCTGTTGTGATTGACGGGCCGTCGCAAAATATAACGTT


 


GACCCACTGTAAAGGCGATTCTTGTAGTGGCAATAATTTCTTGGATGAAG


 


AAGTACAGCTAAAATCAGAATTTGAAAAATTAAGTGATGCAGACAAAATA


 


AGTAATTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGT


 


CGGTTTGGTTGCCGATAGTGTGCAGATGAAGGGAATCAATCAATATATTA


 


TCTTTTATAAACCTAAACCCACTTCATTTGCGCGATTTAGGCGTTCTGCA


 


CGGTCGAGGCGGTCGCTTCCGGCCGAGATGCCGCTGATTCCCGTCAATCA


 


GGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATT


 


CCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGG


 


GCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACC


 


TTCAAAAGGCGAAATGCTCGCGGGCACGGCAGTGTACAACGGCGAAGTGC


 


TGCATTTTCATACGGAAAACGGCCGTCCGTCCCCGTCCAGAGGCAAGGTT


 


TGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACA


 


GCGGCGATGGTTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGAT


 


GGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTC


 


CGGAAAGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCT


 


ATCGCCCAACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAA


 


AAAGAGCAGGATGGATCCGGAGGAGGAGGAGCCACAAACGACGACGATGT


 


TAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAG


 


AAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGAC


 


GGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGA


 


CTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCG


 


TCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCT


 


GAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGC


 


AGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGG


 


GAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAA


 


ATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGA


 


AGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAG


 


ACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACC


 


AAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAA


 


AGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAG


 


CTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAA


 


GATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGA


 


GTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAA


 


AATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGAT


 


ACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAAC


 


CCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTT


 


ACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCC


 


GAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTACCGAAAACTTTGCC


 


GCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTA


 


CCATGTCGGCGTCAATTACGAGTGGTAAAAGCTT


 


(SEQ ID NO: 14)










1
MASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM



 


51
AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM


 


101
PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG


 


151
TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL


 


201
THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV


 


251
GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ


 


301
ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP


 


351
SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS


 


401
GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY


 


451
RPTDAEKGGF GVFAGKKEQD GSGGGGATND DDVKKAATVA IAAAYNNGQE


 


501
INGFKAGETI YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV


 


551
NENKQNVDAK VKAAESEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG


 


601
ENITTFAEET KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD


 


651
EAVKTANEAK QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA


 


701
VAAKVTDIKA DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK


 


751
LDTRLASAEK SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY


 


801
NVGRFNVTAA VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY


 


851
HVGVNYEW*






Example 3
Hybrids of ΔG983
Protein 983 has the following sequence:








983 (SEQ ID NO: 15)                           ΔG983 










1
MRTTPTFPTK TFKPTAMALA VATTLSACLG GGGGGTSAPD FNAGGTGIGS



 


51
NSRATTAKSA AVSYAGIKNE MCKDRSMLCA GRDDVAVTDR DAKINAPPPN


 


101
LHTGDFPNPN DAYKNLINLK PAIEAGYTGR GVEVGIVDTG ESVGSISFPE


 


151
LYGRKEHGYN ENYKNYTAYM RKEAPEDGGG KDIEASFDDE AVIETEAKPT


 


201
DIRHVKEIGH IDLVSHIIGG RSVDGRPAGG IAPDATLHIM NTNDETKNEM


 


251
MVAAIRNAWV KLGERGVRIV NNSFGTTSRA GTADLFQIAN SEEQYRQALL


 


301
DYSGGDKTDE GIRLMQQSDY GNLSYHIRNK NMLFIPSTGN DAQAQPNTYA


 


351
LLPFYEKDAQ KGIITVAGVD RSGEKFKREM YGEPGTEPLE YGSNHCGITA


 


401
MWCLSAPYEA SVRFTRTNPI QIAGTSFSAP IVTGTAALLL QKYPWMSNDN


 


451
LRTTLLTTAQ DIGAVGVDSK FGWGLLDAGK AMNGPASFPF GDFTADTKGT


 


501
SDIAYSFRND ISGTGGLIKK GGSQLQLHGN NTYTGKTIIE GGSLVLYGNN


 


551
KSDMRVETKG ALIYNGAASG GSLNSDGIVY LADTDQSGAN ETVHIKGSLQ


 


601
LDGKGTLYTR LGKLLKVDGT AIIGGKLYMS ARGKGAGYLN STGRRVPFLS


 


651
AAKIGQDYSF FTNIETDGGL LASLDSVEKT AGSEGDTLSY YVRRGNAART


 


701
ASAAAHSAPA GLKHAVEQGG SNLENLMVEL DASESSATPE TVETAAADRT


 


751
DMPGIRPYGA TFRAAAAVQH ANAADGVRIF NSLAATVYAD STAAHADMQG


 


801
RRLKAVSDGL DHNGTGLRVI AQTQQDGGTW EQGGVEGKMR GSTQTVGIAA


 


851
KTGENTTAAA TLGMGRSTWS ENSANAKTDS ISLFAGIRHD AGDIGYLKGL


 


901
FSYGRYKNSI SRSTGADEHA EGSVNGTLMQ LGALGGVNVP FAATGDLTVE


 


951
GGLRYDLLKQ DAFAEKGSAL GWSGNSLTEG TLVGLAGLKL SQPLSDKAVL


 


1001
FATAGVERDL NGRDYTVTGG FTGATAATGK TGARNMPHTR LVAGLGADVE


 


1051
FGNGWNGLAR YSYAGSKQYG NHSGRVGVGY RF*


 








ΔG983 thus has the following basic sequence (SEQ ID NO: 16):



 


                                      TSAPD FNAGGTGIGS


 


NSRATTAKSA AVSYAGIKNE MCKDRSMLCA GRDDVAVTDR DAKINAPPPN


 


LHTGDFPNPN DAYKNLINLK PAIEAGYTGR GVEVGIVDTG ESVGSISFPE


 


LYGRKEHGYN ENYKNYTAYM RKEAPEDGGG KDIEASFDDE AVIETEAKPT


 


DIRHVKEIGH IDLVSHIIGG RSVDGRPAGG IAPDATLHIM NTNDETKNEM


 


MVAAIRNAWV KLGERGVRIV NNSFGTTSRA GTADLFQIAN SEEQYRQALL


 


DYSGGDKTDE GIRLMQQSDY GNLSYHIRNK NMLFIPSTGN DAQAQPNTYA


 


LLPFYEKDAQ KGIITVAGVD RSGEKFKREM YGEPGTEPLE YGSNHCGITA


 


MWCLSAPYEA SVRFTRTNPI QIAGTSFSAP IVTGTAALLL QKYPWMSNDN


 


LRTTLLTTAQ DIGAVGVDSK FGWGLLDAGK AMNGPASFPF GDFTADTKGT


 


SDIAYSFRND ISGTGGLIKK GGSQLQLHGN NTYTGKTIIE GGSLVLYGNN


 


KSDMRVETKG ALIYNGAASG GSLNSDGIVY LADTDQSGAN ETVHIKGSLQ


 


LDGKGTLYTR LGKLLKVDGT AIIGGKLYMS ARGKGAGYLN STGRRVPFLS


 


AAKIGQDYSF FTNIETDGGL LASLDSVEKT AGSEGDTLSY YVRRGNAART


 


ASAAAHSAPA GLKHAVEQGG SNLENLMVEL DASESSATPE TVETAAADRT


 


DMPGIRPYGA TFRAAAAVQH ANAADGVRIF NSLAATVYAD STAAHADMQG


 


RRLKAVSDGL DHNGTGLRVI AQTQQDGGTW EQGGVEGKMR GSTQTVGIAA


 


KTGENTTAAA TLGMGRSTWS ENSANAKTDS ISLFAGIRHD AGDIGYLKGL


 


FSYGRYKNSI SRSTGADEHA EGSVNGTLMQ LGALGGVNVP FAATGDLTVE


 


GGLRYDLLKQ DAFAEKGSAL GWSGNSLTEG TLVGLAGLKL SQPLSDKAVL


 


FATAGVERDL NGRDYTVTGG FTGATAATGK TGARNMPHTR LVAGLGADVE


 


FGNGWNGLAR YSYAGSKQYG NHSGRVGVGY RF*


 


ΔG983 was expressed as a hybrid, with ORF46.1, 


741,961 or 961c at its C-terminus:


 


ΔG983-ORF46.1


(SEQ ID NO: 17)



ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAA



 


CAGAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAA


 


GAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACG


 


TTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTG


 


CATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAA


 


CCTAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGG


 


TATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGT


 


ATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCG


 


TATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGC


 


TTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATA


 


TCCGCCACGTAAAAGAAATCGAACACATCGATTTGGTCTCCCATATTATT


 


GGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGC


 


GACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGG


 


TTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGC


 


ATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCT


 


TTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACT


 


ATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGC


 


GATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCAT


 


CTTTTCGACAGGCAATGACGCACAAGCTAGCCCAACACATATGCCCTATT


 


GCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCG


 


TAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGT


 


ACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTG


 


GTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACC


 


CGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACG


 


GGGCGGCTCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGT


 


ACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAG


 


CAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCG


 


CGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGAT


 


ATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAA


 


AAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCA


 


AAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCG


 


GATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATC


 


CGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACC


 


AATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGAC


 


GGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGG


 


TACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGG


 


CAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCC


 


AAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGG


 


CCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCG


 


ACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCG


 


GCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGG


 


CGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCAT


 


CCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATG


 


CCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACA


 


GCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTA


 


CCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGC


 


CTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGT


 


CATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTG


 


AAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACC


 


GGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATG


 


GAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAG


 


GCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCC


 


TACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACA


 


TGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCG


 


GTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGT


 


CTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGC


 


TTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCG


 


CGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCA


 


ACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGG


 


CGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATA


 


TGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGC


 


AACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTA


 


CGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGACGGTG


 


GTGGCGGAGGCACTGGATCCTCAGATTTGGCAAACGATTCTTTTATCCGG


 


CAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATT


 


CGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAA


 


AAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATT


 


AAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGT


 


CCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCG


 


GTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATAC


 


GAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCC


 


CGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTG


 


CCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCCGACAACGG


 


CTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGG


 


CGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGG


 


GCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATC


 


ATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCAT


 


AAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCA


 


CCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTC


 


AAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAA


 


TGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCC


 


CCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATC


 


ACGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAA


 


AGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAAT


 


ACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGT


 


TACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAA


 


AAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTG


 


ACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTC


 


GAGCACCACCACCACCACCACTGA


 


(SEQ ID NO: 18)










1
MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD



 


51
VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV


 


101
GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE


 


151
ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD


 


201
ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD


 


251
LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF


 


301
IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP


 


351
GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG


 


401
TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG


 


451
PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT


 


501
GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT


 


551
DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK


 


601
GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE


 


651
GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE


 


701
SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA


 


751
ATVYADSTAA HADMQGRRLK AVSDGLDHNQ TGLRVIAQTQ QDGGTWEQGG


 


801
VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NARTDSISLF


 


851
AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL


 


901
GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG


 


951
LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR


 


1001
NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLD


 


1051
GGGGTGSSDL ANDSFIRQVL DRQHFEPDGK YHLFGSRGEL AERSGHIGLG


 


1101
KIQSHQLGNL MIQQAAIKGN IGYIVRFSDH GHEVHSPFDN HASHSDSDEA


 


1151
GSPVDGFSLY RIHWDGYEHH PADGYDGPQG GGYPAPKGAR DIYSYDIKGV


 


1201
AQNIRLNLTD NRSTGQRLAD RFHNAGSMLT QGVGDGFKRA TRYSPELDRS


 


1251
GNAAEAFNGT ADIVKNIIGA AGEIVGAGDA VQGISEGSNI AVMHGLGLLS


 


1301
TENKMARIND LADMAQLKDY AAAAIRDWAV QNPNAAQGIE AVSNIFMAAI


 


1351
PIKGIGAVRG KYGLGGITAH PIKRSQMGAI ALPKGKSAVS DNFADAAYAK


 


1401
YPSPYHSRNI RSNLEQRYGK ENITSSTVPP SNGKNVKLAD QRHPKTGVPF


 


1451
DGKGFPNFEK HVKYDTLEHH HHHH*


 








ΔQ983-741



(SEQ ID NO: 19)



ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAA



 


CAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCA


 


AGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGAC


 


GTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCT


 


GCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCA


 


ACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTA


 


GGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACT


 


GTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGG


 


CGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAA


 


GCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGA


 


TATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTA


 


TTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGAT


 


GCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGAT


 


GGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGC


 


GCATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGAC


 


CTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGA


 


CTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGA


 


GCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTC


 


ATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCT


 


ATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAG


 


GCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCG


 


GGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCAT


 


GTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAA


 


ACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGC


 


ACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCT


 


GCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGG


 


ACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGA


 


CCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATC


 


CGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGA


 


TCAAAAAAGGCGGCAGCAACTGCAACTGCACGGCAACAACACCTATACGG


 


GCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAA


 


TCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGC


 


ATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCG


 


ACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTG


 


GACGGCAAAGGTACGCTGTACACACGTITGGGCAAACTGCTGAAAGTGGA


 


CGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGG


 


GGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCC


 


GCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGG


 


CGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAG


 


GCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCT


 


TCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACA


 


GGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAAT


 


CATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGAT


 


ATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGT


 


ACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCG


 


CTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGC


 


CGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCG


 


CGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTG


 


TTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAA


 


ACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCAC


 


ATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTG


 


CAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTC


 


TCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGA


 


ACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGG


 


GCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGC


 


GGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAG


 


TGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGAC


 


TCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTT


 


GCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAAC


 


GGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCA


 


ATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTC


 


GGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACA


 


GTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAGG


 


GATCCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGAT


 


GCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGAC


 


GCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAG


 


GTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTG


 


AAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGA


 


CGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAG


 


CCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGC


 


ATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCG


 


GGCGAACATACATCTTGACAAGCTTCCCGAGGCGGCAGGGCGACATATCG


 


CGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCA


 


TAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCG


 


CCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAA


 


ACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAAAGGC


 


AGTTACTCCCTCGGTATCTTTGGCGGAAAGCCCAGGAAGTTGCCGGCAGC


 


GCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCC


 


AAGCAACTCGAGCACCACCACCACCACCACCACTGA


 


(SEQ ID NO: 20)










1
MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD



 


51
VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV


 


101
GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE


 


151
ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD


 


201
ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVPIVNNSF GTTSRAGTAD


 


251
LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF


 


301
IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP


 


351
GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG


 


401
TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG


 


451
PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT


 


501
GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT


 


551
DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK


 


601
GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE


 


651
GDTLSYYVER GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE


 


701
SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA


 


751
ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG


 


801
VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF


 


851
AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL


 


901
GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG


 


951
LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR


 


1001
NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE


 


1051
GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR KNEKLKLAAQ


 


1101
GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL ESGEFQVYKQ


 


1151
SHSALTAFQT EQIQDSEHSG KMVAKRQPRI GDIAGEHTSF DKLPEGGRAT


 


1201
YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD


 


1251
GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK TVNGIRHIGL


 


1301
AAKQLEHHHH HH*


 








ΔG983-961



(SEQ ID NO: 21)



ATGCATTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAA



 


CAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCA


 


AGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGAC


 


GTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCT


 


GCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCA


 


ACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGGGGTAGAGGTAGG


 


TATCGTCGACACACGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGT


 


ATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCG


 


TATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGC


 


TTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATA


 


TCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATT


 


GGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTSTTGCGCCCGATGC


 


GACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATGG


 


TTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGC


 


ATCGTCAATAACAGTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTT


 


TTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAGCGTTGCTCGACTAT


 


TCCGGCGGTGATAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATT


 


ACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTT


 


TCGACAGGCAATGACCACAAGCTCAGCCCAACACATATGCCCTATTGCCA


 


TTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGA


 


CCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAG


 


AACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGC


 


CTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGAT


 


TCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGG


 


CTCTGCTGCAGAAATACCGTGGATGAGCAACGACAACCTGCGTACCACGT


 


TGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTC


 


GGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCGCGTCCTT


 


TCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATAATGCCT


 


ACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGC


 


GGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACCAT


 


TATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGC


 


GCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGCGGCATCCGGCGGCA


 


GCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGC


 


GCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGG


 


TACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGA


 


TTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTAT


 


CTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGG


 


GCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGG


 


CTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTG


 


TCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGC


 


ACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCA


 


ATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCCATGCG


 


GAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGT


 


CAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGC


 


GCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTG


 


GGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGG


 


TCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGG


 


CGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGC


 


TTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCC


 


GCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACG


 


GCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGC


 


AACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAGGGTGGCGG


 


AGGCACTGGATCCGCCACAAACGACGACGATGTTAAAAAAGCCACTGTGG


 


CCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCT


 


GGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGA


 


CGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGA


 


AAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAAC


 


GTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAAC


 


CAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGG


 


ATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTT


 


GCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGC


 


CGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCG


 


ATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCC


 


AATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAA


 


AGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCA


 


CAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACC


 


GACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGC


 


AAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCA


 


GAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCT


 


TCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGA


 


TAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAAC


 


AAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAAT


 


GTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGG


 


TACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAG


 


TCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTAC


 


GATGGCTCGAGCACCACCACCACCACCACTGA


 


(SEQ ID NO: 22)










1
MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD



 


51
VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV


 


101
GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE


 


151
ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD


 


201
ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVPIVNNSF GTTSRAGTAD


 


251
LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF


 


301
IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP


 


351
GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG


 


401
TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG


 


451
PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT


 


501
GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT


 


551
DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK


 


601
GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE


 


651
GDTLSYYVER GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE


 


701
SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA


 


751
ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG


 


801
VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF


 


851
AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL


 


901
GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG


 


951
LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR


 


1001
NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE


 


1051
GGGGTGSATN DDDVKKAATV AIAAAYNNGQ EINGFKAGET IYDIDEDGTI


 


1101
TKKDATAADV EADDFKGLGL KKVVTNLTKT VNENKQNVDA KVKAAESEIE


 


1151
KLTTKLADTD AALADTDAAL DATTNALNKL GENITTFAEE TKTNIVKIDE


 


1201
KLEAVADTVD KHAEAFNDIA DSLDETNTKA DEAVKTANEA KQTAEETKQN


 


1251
VDAKVKAAET AAGKAEAAAG TANTAADKAE AVAAKVTDIK ADIATNKDNI


 


1301
AKKANSADVY TREESDSKFV RIDGLNATTE KLDTRLASAE KSIADHDTRL


 


1351
NGLDKTVSDL RKETRQGLAE QAALSGLFQP YNVGRFNVTA AVGGYKSESA


 


1401
VAIGTGFRFT ENFAAKAGVA VGTSSGSSAA YHVGVNYEWL EHHHHHH*


 








ΔG983-961c



(SEQ ID NO: 23)



ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAA



 


CAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTACGCCGGTATCAA


 


GAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACG


 


TTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTG


 


CATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAA


 


CCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAG


 


GTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTG


 


TATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGC


 


GTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAG


 


CTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCGACGGAT


 


ATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTAT


 


TGGCGGGCGTTCCGTGGACAGCAGACCTGCAGGCGGTATTGCGCCCGATG


 


CGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATG


 


GTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCG


 


CATCGTCAATAACAGTTTTGGACAACATCGAGGGCAGGCACTGCCGACCT


 


TTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACT


 


ATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGC


 


GATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCAT


 


CTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTAT


 


TGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGC


 


GTAGACCGCAGTGGAGAAAGTTCAAACGGGAAATGTATGGAGAACCGGGT


 


ACAGAACCGCTTGAGTATGGCTCCAACCATTGCGAATTACTGCCATGTGG


 


TGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCC


 


GATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGGCACGG


 


CGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGT


 


ACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAG


 


CAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCCG


 


CGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGAT


 


ATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAA


 


AAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAA


 


AACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAAATCGGATA


 


TGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGC


 


GGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATC


 


CGGCGCAAACGAAACCGTACACATCAAAGCAGTCTGCAGCTGGACGGCAA


 


AGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGG


 


CGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGC


 


TATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAAT


 


CGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGC


 


TGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACG


 


CTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGC


 


GGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCA


 


GCAATCTGGAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAA


 


CACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGC


 


ATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGC


 


GAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCT


 


ATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAA


 


GCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGC


 


GCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCA


 


AAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAA


 


AATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGA


 


AAACATGTCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATAC


 


GGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGA


 


CGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGA


 


AGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCA


 


ACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGC


 


TACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGG


 


CTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTC


 


TAGCTGTCGAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGC


 


GTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTAC


 


CGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACA


 


CCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGG


 


AACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCA


 


CAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAGGGTGGCACTGGAT


 


CCGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCT


 


GCTGCCTACAACAATGGCCACAAGAAATCAACGGTTTCAAAGCTGGAGAG


 


ACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAAC


 


TGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAG


 


TCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGAT


 


GCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTT


 


AGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAA


 


CCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAA


 


GAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAAGCCGTGG


 


CTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCA


 


TTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGA


 


AGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAA


 


AAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCT


 


AATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACAT


 


CAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACA


 


GTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATT


 


GATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTCTGCTGAAA


 


AATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTG


 


TCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCT


 


CTCCGGTCTGTTCCAACCTTACAACGTGGGTCTCGAGCACCACCACCACC


 


ACCACTGA


 


(SEQ ID NO: 24)










1
MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD



 


51
VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV


 


101
GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE


 


151
ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD


 


201
ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVPIVNNSF GTTSRAGTAD


 


251
LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF


 


301
IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP


 


351
GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG


 


401
TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG


 


451
PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT


 


501
GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT


 


551
DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK


 


601
GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE


 


651
GDTLSYYVER GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE


 


701
SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA


 


751
ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG


 


801
VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF


 


851
AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL


 


901
GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG


 


951
LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR


 


1001
NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE


 


1051
GGGGTGSATN DDDVKKAATV AIAAAYNNGQ EINGFKAGET IYDIDEDGTI


 


1101
TKKDATAADV EADDFKGLGL KKVVTNLTKT VNENKQNVDA KVKAAESEIE


 


1151
KLTTKLADTD AALADTDAAL DATTNALNKL GENITTFAEE TKTNIVKIDE


 


1201
KLEAVADTVD KHAEAFNDIA DSLDETNTKA DEAVKTANEA KQTAEETKQN


 


1251
VDAKVKAAET AAGKAEAAAG TANTAADKAE AVAAKVTDIK ADIATNKDNI


 


1301
AKKANSADVY TREESDSKFV RIDGLNATTE KLDTRLASAE KSIADHDTRL


 


1351
NGLDKTVSDL RKETRQGLAE QAALSGLFQP YNVGLEHHHH HH*






Example 4
Hybrids of ΔG741
Protein 741 has the following sequence (SEQ ID NO: 25):









1
VNRTAFCCLS LTTALILTAC SSGGGGVAAD IGAGLADALT APLDHKDKGL



 


51
QSLTLDQSVR KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSEFDFIRQ


 


101
IEVDGQLITL ESGEFQVYKQ SHSALTAFQT EQIQDSEHSG KMVAKRQFRI


 


151
GDIAGEHTSF DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI


 


201
EHLKSPELNV DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA


 


251
QEVAGSAEVK TVNGIRHIGL AAKQ*


 








ΔG741 thus has the following basic sequence (SEQ ID NO: 26):



 


                            VAAD IGAGLADALT APLDHKDKGL


 


QSLTLDQSVR KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSEFDFIRQ


 


IEVDGQLITL ESGEFQVYKQ SHSALTAFQT EQIQDSEHSG KMVAKRQFRI


 


GDIAGEHTSF DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI


 


EHLKSPELNV DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA


 


QEVAGSAEVK TVNGIRHIGL AAKQ*


 


ΔG741 was fused directly in-frame upstream of


proteins 961, 961c, 983 and ORF46.1:


 


ΔG741-961


(SEQ ID NO: 27)



ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACC



 


GCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCG


 


TCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACT


 


TATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGT


 


CAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTA


 


CCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTA


 


ACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGAT


 


GGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACAT


 


CTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCG


 


TTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGC


 


CGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCA


 


ATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCC


 


GTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTC


 


CCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAG


 


TGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTC


 


GAGGGTGGCGGAGGCACTGGATCCGCCACAAACGACGACGATGTTAAAAA


 


AGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCA


 


ACGGTTTCAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAT


 


TACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAG


 


GTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAA


 


AACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGA


 


AAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTG


 


ATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAAT


 


ATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGA


 


AAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCA


 


ACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCC


 


GTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAA


 


CGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAG


 


CTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCT


 


GCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATAT


 


TGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACA


 


GCAAATTTGTCAGAATTATGGTCTGAACGCTACTACCGAAAAATTGGACA


 


CACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTG


 


AACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGG


 


CCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGG


 


GTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCG


 


GCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAA


 


AGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATG


 


TCGGCGTCAATTACGAGTGGCTCGAGCACCACCACCACCACCACTGA


 


(SEQ ID NO: 28)










1
MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT



 


51
YGNGDSLNTG KLKNDKVSPF DFIRQIEVDG QLITLESGEF QVYKQSHSAL


 


101
TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA


 


151
FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA


 


201
VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL


 


251
EGGGGTGSAT NDDDVKKAAT VAIAAAYNNG QEINGFKAGE TIYDIDEDGT


 


301
ITKKDATAAD VEADDFKGLG LKKVVTNLTK TVNENKQNVD AKVKAAESEI


 


351
EKLTTKLADT DAALADTDAA LDATTNALNK LGENITTFAE ETKTNIVKID


 


401
EKLEAVADTV DKHAEAFNDI ADSLDETNTK ADEAVKTANE AKQTAEETKQ


 


451
NVDAKVKAAE TAAGKAEAAA GTANTAADKA EAVAAKVTDI KADIATNKDN


 


501
IAKKANSADV YTREESDSKF VRIDGLNATT EKLDTRLASA EKSIADHDTR


 


551
LNGLDKTVSD LRKETRQGLA EQAALSGLFQ PYNVGRFNVT AAVGGYKSES


 


601
AVAIGTGFRF TENFAAKAGV AVGTSSGSSA AYHVGVNYEW LEHHHHHH*


 








ΔG741-961c



(SEQ ID NO: 29)



ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACC



 


GCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCG


 


TCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACT


 


TATGGAAACGGTGACAGCCTCAATACGGGCAATTGAGAACGACAAGGTCA


 


GCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACC


 


TTGGAGAGTGGAGAGTTCCAGTATACAAACAAAGCCATTCCGCCTTAACC


 


GCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGT


 


TGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTT


 


TTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTC


 


GGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGC


 


CAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATG


 


TCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTC


 


ATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCT


 


CGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGA


 


AAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAG


 


GGTGGCGGAGGCACTGGATCCGCCACAAACGACGACGATGTTAAAAAAGC


 


TGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACG


 


GTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATT


 


ACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGG


 


TCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAA


 


ACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAA


 


AAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGA


 


TGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATA


 


TAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAA


 


AAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAA


 


CGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCG


 


TCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAAC


 


GTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGC


 


TGCCGCTGGCACAGCTAATACTCAGCCGACAAGGCCGAAGCTGTCGCTGC


 


AAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTG


 


CTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGC


 


AAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACAC


 


ACGCTTGGCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACG


 


GTTTGGATAAAACAGTGTCAGACCTCCGCGCAAAGAAACCCGCCAAGGCC


 


TTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGT


 


CTCGAGCACCACCACCACCACCACTGA


 


(SEQ ID NO: 30)










1
MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT



 


51
YGNGDSLNTG KLKNDKVSPF DFIRQIEVDG QLITLESGEF QVYKQSHSAL


 


101
TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA


 


151
FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA


 


201
VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL


 


251
EGGGGTGSAT NDDDVKKAAT VAIAAAYNNG QEINGFKAGE TIYDIDEDGT


 


301
ITKKDATAAD VEADDFKGLG LKKVVTNLTK TVNENKQNVD AKVKAAESEI


 


351
EKLTTKLADT DAALADTDAA LDATTNALNK LGENITTFAE ETKTNIVKID


 


401
EKLEAVADTV DKHAEAFNDI ADSLDETNTK ADEAVKTANE AKQTAEETKQ


 


451
NVDAKVKAAE TAAGKAEAAA GTANTAADKA EAVAAKVTDI KADIATNKDN


 


501
IAKKANSADV YTREESDSKF VRIDGLNATT EKLDTRLASA EKSIADHDTR


 


551
LNGLDKTVSD LRKETRQGLA EQAALSGLFQ PYNVGLEHHH HHH*


 








ΔG741-983



(SEQ ID NO: 31)



ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACC



 


GCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCG


 


TCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACT


 


TATGGAAACGGTGACAGCCTCAATACGGGCAATTGAGAACGACAAGGTCA


 


GCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACC


 


TTGGAGAGTGGAGAGTTCCAGTATACAAACAAAGCCATTCCGCCTTAACC


 


GCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGT


 


TGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTT


 


TTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTC


 


GGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGC


 


CAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATG


 


TCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTC


 


ATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCT


 


CGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGA


 


AAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAG


 


GGATCCGCCGGAGGCGGCACTTCTGCGCCCGACTTCATGCAGGCGGTACC


 


GGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATC


 


TTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTG


 


CCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCATGCCC


 


CCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGAVGCATAC


 


AAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACG


 


CGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATAT


 


CCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTAC


 


AAAAACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGG


 


TAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAG


 


CAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTG


 


GTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGG


 


TATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCA


 


AGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGC


 


GAACGTGGCGTGCGCGCATCGTCAATAACAGTTTTGGAACAACATCGAGG


 


GCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCG


 


CCAAGCGTTGCTCGACTATCCGGCGGTGATAAAACAGACGAGGGTATCCG


 


CCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATA


 


AAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCC


 


AACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCAT


 


TATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAA


 


TGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGC


 


GGAATTACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCG


 


TTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCAC


 


CCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATG


 


AGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGG


 


TGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTA


 


AGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGAT


 


ACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGG


 


CACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCA


 


ACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTG


 


TACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGAT


 


TTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCT


 


ATCTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAA


 


GGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAA


 


ACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGT


 


CGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTT


 


CCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAA


 


CATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAA


 


CAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAAT


 


GCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAA


 


ACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAAC


 


TGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCGGCA


 


GCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCG


 


CGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCT


 


CAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCG


 


ATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAAC


 


GGCACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTG


 


GGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCG


 


GCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGC


 


ATGGGACGCAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAG


 


CATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATC


 


TCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGC


 


ACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCA


 


GCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATT


 


TGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTC


 


GCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGG


 


CACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATA


 


AAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGC


 


GACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAA


 


GACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCG


 


CGGATGTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTAC


 


GCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTA


 


CCGGTTCCTCGAGCACCACCACCACCACCACTGA


 


(SEQ ID NO: 32)










1
MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT



 


51
YGNGDSLNTG KLKNDKVSPF DFIRQIEVDG QLITLESGEF QVYKQSHSAL


 


101
TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA


 


151
FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA


 


201
VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL


 


251
EGSGGGGTSA PDFNAGGTGI GSNSRATTAK SAAVSYAGIK NEMCKDRSML


 


301
CAGRDDVAVT DRDAKINAPP PNLHTGDFPN PNDAYKNLIN LKPAIEAGYT


 


351
GRGVEVGIVD TGESVGSISF PELYGRKEHG YNENYKNYTA YMRKEAPEDG


 


401
GGKDIEASFD DEAVIETEAK PTDIRHVKEI GHIDLVSHII GGRSVDGRPA


 


451
GGIAPDATLH IMNTNDETKN EMMVAAIRNA WVKLGERGVR IVNNSFGTTS


 


501
RAGTADLFQI ANSEEQYRQA LLDYSGGDKT DEGIRLMQQS DYGNLSYHIR


 


551
NKNMLFIFST GNDAQAQPNT YALLPFYEKD AQKGIITVAG VDRSGEKFKR


 


601
EMYGEPGTEP LEYGSNHCGI TAMWCLSAPY EASVRFTRTN PIQIAGTSFS


 


651
APIVTGTAAL LLQKYPWMSN DNLRTTLLTT AQDIGAVGVD SKFGWGLLDA


 


701
GKAMNGPASF PFGDFTADTK GTSDIAYSFR NDISGTGGLI KKGGSQLQLH


 


751
GNNTYTGKTI IEGGSLVLYG NNKSDMRVET KGALIYNGAA SGGSLNSDGI


 


801
VYLADTDQSG ANETVHIKGS LQLDGKGTLY TRLGKLLKVD GTAIIGGKLY


 


851
MSARGKGAGY LNSTGRRVPF LSAAKIGQDY SFFTNIETDG GLLASLDSVE


 


901
KTAGSEGDTL SYYVERGNAA RTASAAAHSA PAGLKHAVEQ GGSNLENLMV


 


951
ELDASESSAT PETVETAAAD RTDMPGIRPY GATFRAAAAV QHANAADGVR


 


1001
IFNSLAATVY ADSTAAHADM QGRRLKAVSD GLDHNGTGLR VIAQTQQDGG


 


1051
TWEQGGVEGK MRGSTQTVGI AAKTGENTTA AATLGMGRST WSENSANAKT


 


1101
DSISLFAGIR HDAGDIGYLK GLFSYGRYKN SISRSTGADE HAEGSVNGTL


 


1151
MQLGALGGVN VPFAATGDLT VEGGLRYDLL KQDAFAEKGS ALGWSGNSLT


 


1201
EGTLVQLAGL KLSQPLSDKA VLFATAGVER DLNGRDYTVT GGFTGATAAT


 


1251
GKTGARNMPH TRLVAGLGAD VEFGNGWNGL ARYSYAGSKQ YGNHSGRVGV


 


1301
GYRFLEHHHH HH*


 








ΔG741-ORF46.1



(SEQ ID NO: 33)



ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACC



 


GCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCG


 


TCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACT


 


TATGGAAACGGTGACAGCCTCAATACGGGCAATTGAGAACGACAAGGTCA


 


GCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACC


 


TTGGAGAGTGGAGAGTTCCAGTATACAAACAAAGCCATTCCGCCTTAACC


 


GCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGT


 


TGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTT


 


TTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTC


 


GGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGC


 


CAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATG


 


TCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTC


 


ATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCT


 


CGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGA


 


AAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAC


 


GGTGGCGGAGGCACTGGATCCTCAGATTTGGCAAACGATTCTTTTATCCG


 


GCAGGTTCTCGACCGTCCAGCATTTCGAACCCGACGGGAAATACCACCTA


 


TTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGA


 


AAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCAT


 


TAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAG


 


TCCATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCC


 


GGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATA


 


CGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATC


 


CCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTT


 


GCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACG


 


GCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAG


 


GCGACGGATTCAAACGCGCCACCCGATACAGCCCGAGCTGGACAGATCGG


 


GCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATC


 


ATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCAT


 


AAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCA


 


CCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTC


 


AAAGACTATGCCGCAGCAGCCATCCGCGGGGCAGTCCAAAACCCCAATGC


 


CGCACAATGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCA


 


TCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACG


 


GCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGG


 


GAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAATACCC


 


GTCCCCTTACCATCCCGAAATATCCGTTCATTGGAGCAGCGTTACGGCAA


 


AGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCA


 


AACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTACGGTAAAG


 


GGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCAC


 


CACCACCACCACTGA


 


(SEQ ID NO: 34)










1
MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT



 


51
YGNGDSLNTG KLKNDKVSPF DFIRQIEVDG QLITLESGEF QVYKQSHSAL


 


101
TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA


 


151
FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA


 


201
VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL


 


251
DGGGGTGSSD LANDSFIRQV LDRQHFEPDG KYHLFGSRGE LAERSGHIGL


 


301
GKIQSHQLGN LMIQQAAIKG NIGYIVRFSD HGHEVESPFD NHASESDSDE


 


351
AGSPVDGFSL YRIHWDGYEH HPADGYDGPQ GGGYPAPKGA RDIYSYDIKG


 


401
VAQNIRLNLT DNRSTGQRLA DRFHNAGSML TQGVGDGFKR ATRYSPELDR


 


451
SGNAAEAFNG TADIVKNIIG AAGEIVGAGD AVQGISEGSN IAVMHGLGLL


 


501
STENKHAPIN DLADMAQLKD YAAAAIRDWA VQNPNAAQGI EAVSNIFMAA


 


551
IPIKGIGAVR GKYGLGGITA HPIKRSQMGA IALPKGKSAV SDNFADAAYA


 


601
KYPSPYHSRN IRSNLEQRYG KENITSSTVP PSNGKNVKLA DQRHPKTGVP


 


651
FDGKGFPNFE KHVKYDTLEH HHHHH*






Example 5
Hybrids of 287
Expression of 287 as full-length with a C-terminal His-tag, or without its leader peptide but with a C-terminal His-tag, gives fairly low expression levels. Better expression is achieved using a N-terminal GST-fusion. As an alternative to using GST as an N-terminal fusion partner, 287 was placed at the C-terminus of protein 919 (‘919-287’), of protein 953 (‘953-287’), and of proteins ORF46.1 (‘ORF46.1-287’). In both cases, the leader peptides were deleted, and the hybrids were direct in-frame fusions.
To generate the 953-287 hybrid, the leader peptides of the two proteins were omitted by designing the forward primer downstream from the leader of each sequence; the stop codon sequence was omitted in the 953 reverse primer but included in the 287 reverse primer. For the 953 gene, the 5′ and the 3′ primers used for amplification included a NdeI and a BamHI-restriction sites respectively, whereas for the amplification of the 287 gene the 5′ and the 3′ primers included a BamHI and a XhoI restriction sites respectively. In this way a sequential directional cloning of the two genes in pET21b+, using NdeI-BamHI (to clone the first gene) and subsequently BamHI-XhoI (to clone the second gene) could be achieved.
The 919-287 hybrid was obtained by cloning the sequence coding for the mature portion of 287 into the XhoI site at the 3′-end of the 919-His clone in pET21b+. The primers used for amplification of the 287 gene were designed for introducing a SalI restriction site at the 5′- and a XhoI site at the 3′- of the PCR fragment. Since the cohesive ends produced by the SalI and XhoI restriction enzymes are compatible, the 287 PCR product digested with SalI-XhoI could be inserted in the pET21b-919 clone cleaved with XhoI.
The ORF46.1-287 hybrid was obtained similarly.
The bactericidal efficacy (homologous strain) of antibodies raised against the hybrid proteins was compared with antibodies raised against simple mixtures of the component antigens:













Mixture with 287
Hybrid with 287



















919
32000
16000



953
8192
8192



ORF46.1
128
8192









Data for bactericidal activity against heterologous MenB strains and against serotypes A and C were also obtained for 919-287 and 953-287:















919
953
ORF46.1













Strain
Mixture
Hybrid
Mixture
Hybrid
Mixture
Hybrid
















MC58
512
1024
512
1024
—
1024


NGH38
1024
2048
2048
4096
—
4096


BZ232
512
128
1024
16
—
—


MenA (F6124)
512
2048
2048
32
—
1024


MenC (C11)
>2048
n.d.
>2048
n.d.
—
n.d.


MenC (BZ133)
>4096
>8192
>4096
<16
—
2048









Hybrids of ORF46.1 and 919 were also constructed. Best results (four-fold higher titre) were achieved with 919 at the N-terminus.
Hybrids 919-519His, ORF97-225His and 225-ORF97His were also tested. These gave moderate ELISA titres and bactericidal antibody responses.
As hybrids of two proteins A & B may be either NH2A-B—COOH or NH2B-A-COOH, the “reverse” hybrids with 287 at the N-terminus were also made, but using ΔG287. A panel of strains was used, including homologous strain 2996. FCA was used as adjuvant:















287 & 919
287 & 953
287 & ORF46.1














ΔG287-

ΔG287-

ΔG287-
46.1-


Strain
919
919-287
953
953-287
46.1
287
















2996
128000
16000
65536
8192
16384
8192


BZ232
256
128
128
<4
<4
<4


1000
2048
<4
<4
<4
<4
<4


MC58
8192
1024
16384
1024
512
128


NGH38
32000
2048
>2048
4096
16384
4096


394/98
4096
32
256
128
128
16


MenA (F6124)
32000
2048
>2048
32
8192
1024


MenC (BZ133)
64000
>8192
>8192
<16
8192
2048









Better bactericidal titres are generally seen with 287 at the N-terminus.
When fused to protein 961 [NH2-ΔG287-961-COOH— sequence shown above], the resulting protein is insoluble and must be denatured and renatured for purification. Following renaturation, around 50% of the protein was found to remain insoluble. The soluble and insoluble proteins were compared, and much better bactericidal titres were obtained with the soluble protein (FCA as adjuvant):

















2996
BZ232
MC58
NGH38
F6124
BZ133





















Soluble
65536
128
4096
>2048
>2048
4096


Insoluble
8192
<4
<4
16
n.d.
n.d.









Titres with the insoluble form were, however, improved by using alum adjuvant instead:


















Insoluble
32768
128
4096
>2048
>2048
2048









961c was also used in hybrid proteins (see above). As 961 and its domain variants direct efficient expression, they are ideally suited as the N-terminal portion of a hybrid protein.
Example 6
Further Hybrids
Further hybrid proteins of the invention are shown in the drawings and have the sequences set out below. These are advantageous when compared to the individual proteins:








ORF46.1-741



(SEQ ID NO: 35)



ATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCA



 


GCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAAC


 


TTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAG


 


TTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTA


 


CATTGTCCGCTTGTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACA


 


ACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGA


 


TTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGA


 


CGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGA


 


GGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTC


 


AACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCA


 


CAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCG


 


CCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCC


 


TTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGA


 


AATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACA


 


TTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCG


 


CGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGC


 


AGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCA


 


TAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATT


 


GGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTAT


 


CAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCG


 


TCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTAC


 


CATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAA


 


CATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGG


 


CAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTT


 


CCGAATTTTGAGAGCACGTGAAATATGATACGGGATCCGGAGGGGGTGGT


 


GTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCT


 


CGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCA


 


GGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCAGAAAAACTTATG


 


GAAACGGTACAGCCTCAATACGGGCAAATTGAGAACGACAAGGTCAGCCG


 


TTTCGACTTTATCCGCCAAATCGAAGTGGGACGGGCAGCTCATTACCTTG


 


GAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGC


 


CTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTG


 


CGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTT


 


GACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGG


 


TTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCA


 


AGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTC


 


GACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCAT


 


CAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCG


 


GTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAA


 


ACCGTAAACGCATACGCCATATCGGCCTGCCGCAAGCAACTCGAGCACCA


 


CCACCACCACCACTGA


 


(SEQ ID NO: 36)










1
MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ



 


51
LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG


 


101
FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL


 


151
NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA


 


201
FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA


 


251
RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG


 


301
AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH


 


351
SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP


 


401
NFEKHVKYDT GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR


 


451
KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL


 


501
ESGEFQVYKQ SHSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF


 


551
DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV


 


601
DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK


 


651
TVNGIRHIGL AAKQLEHHHH HH*


 








ORF46.1-961



(SEQ ID NO: 37)



ATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCA



 


GCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAAC


 


TTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAG


 


TTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTA


 


CATTGTCCGCTTGTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACA


 


ACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGA


 


TTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGA


 


CGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGA


 


GGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTC


 


AACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCA


 


CAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCG


 


CCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCC


 


TTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGA


 


AATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACA


 


TTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCG


 


CGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGC


 


AGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCA


 


TAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATT


 


GGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTAT


 


CAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCG


 


TCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTAC


 


CATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAA


 


CATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGG


 


CAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTT


 


CCGAATTTTGAGAGCACGTGAAATATGATACGGGATCCGGAGGAGGAGGA


 


GCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGC


 


TGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCA


 


TCTACGACATTGATGAAQACGGCACAATTACCAAAAAAGACGCAACTGCA


 


GCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGT


 


GACTAACCTGACCAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAA


 


GTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGA


 


CACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCA


 


ACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACT


 


AAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATAC


 


CGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATG


 


AAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAA


 


CAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGC


 


AGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTG


 


CAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCT


 


GATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGA


 


CGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTAGAATTGATGGTCT


 


GAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAAT


 


CCATTGCCGATCACGATACTCGCCTGACGGTTTGGATAAAACAGTGTCAG


 


ACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCC


 


GGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGT


 


CGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCT


 


TTACCGAAAACTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCG


 


GTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGCTCGAGCAC


 


CACCACCACCACCACTGA


 


(SEQ ID NO: 38)










1
MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ



 


51
LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG


 


101
FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL


 


151
NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA


 


201
FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA


 


251
RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG


 


301
AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH


 


351
SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP


 


401
NFEKHVKYDT GSGGGGATND DDVKKAATVA IAAAYNNGQE INGFKAGETI


 


451
YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV NENKQNVDAK


 


501
VKAAESEIEK LTTKLADTDA ALDATDAALD ATTNALNKLG ENITTFAEET


 


551
KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD EAVKTANEAK


 


601
QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA VAAKVTDIKA


 


651
DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK LDTRLASAEK


 


701
SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY NVGRFNVTAA


 


751
VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY HVGVNYEWLE


 


801
HHHHHH*


 








ORF46.1-961c



(SEQ ID NO: 39)



ATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCA



 


GCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAAC


 


TTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAG


 


TTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTA


 


CATTGTCCGCTTGTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACA


 


ACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGA


 


TTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGA


 


CGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGA


 


GGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTC


 


AACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCA


 


CAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCG


 


CCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCC


 


TTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGA


 


AATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACA


 


TTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCG


 


CGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGC


 


AGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCA


 


TAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATT


 


GGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTAT


 


CAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCG


 


TCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTAC


 


CATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAA


 


CATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGG


 


CAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTT


 


CCGAATTTTGAGAGCACGTGAAATATGATACGGGATCCGGAGGAGGAGGA


 


GCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGC


 


TGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCA


 


TCTACGACATTGATGAAQACGGCACAATTACCAAAAAAGACGCAACTGCA


 


GCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGT


 


GACTAACCTGACCAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAA


 


GTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGA


 


CACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCA


 


ACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACT


 


AAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATAC


 


CGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATG


 


AAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAA


 


CAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGC


 


AGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTG


 


CAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCT


 


GATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGA


 


CGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTAGAATTGATGGTCT


 


GAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAAT


 


CCATTGCCGATCACGATACTCGCCTGACGGTTTGGATAAAACAGTGTCAG


 


ACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCC


 


GGTCTGTTCCAACCTTACAACGTGGGTCTCGAGCACCACCACCACCACCA


 


CTGA


 


(SEQ ID NO: 40)










1
MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ



 


51
LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG


 


101
FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL


 


151
NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA


 


201
FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA


 


251
RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG


 


301
AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH


 


351
SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP


 


401
NFEKHVKYDT GSGGGGATND DDVKKAATVA IAAAYNNGQE INGFKAGETI


 


451
YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV NENKQNVDAK


 


501
VKAAESEIEK LTTKLADTDA ALDATDAALD ATTNALNKLG ENITTFAEET


 


551
KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD EAVKTANEAK


 


601
QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA VAAKVTDIKA


 


651
DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK LDTRLASAEK


 


701
SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY NVGLEHHHHH


 


751
H*


 








961-ORF46.1



(SEQ ID NO: 41)



ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGC



 


TGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGA


 


CCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACT


 


GCAGCCGATGTTGAAGCCGACGACTAAAGGTCTGGGTCTGAAAAAAGTCG


 


TGACTAACCTGACCAAAACCGTCTGAAAACAAACAAACGTCGATGCCAAA


 


GTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGA


 


CACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCA


 


ACGCCTGAATAAATTGGGAAAAATATAACGACATTTGCTGAAGAGACTAA


 


GACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCG


 


TCGACAAGCATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACC


 


ACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGA


 


GACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTG


 


ATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTG


 


GATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGCTGA


 


TATCGCTACGAACAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGT


 


GTACACCAGAGAAGAGTCTGACAGCAAATGTCAGAATTGATGGTCTGAAC


 


GCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCAT


 


TGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAAACAGTGTCAGAC


 


CTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGG


 


TCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCG


 


GCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTT


 


ACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGG


 


TTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGGGATCCGGAG


 


GAGGAGGATCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGAC


 


CGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGG


 


GGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCC


 


ATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATC


 


GGCTACATTGTCCGCTTTTCCGATCACGGGCACGAATCCATTCCCCCTTC


 


GACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGA


 


CGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCATCCCG


 


CCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGC


 


GCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCG


 


CCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTT


 


TCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAA


 


CGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGA


 


AGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAG


 


GAGAAATGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAA


 


ACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAAOAGATGG


 


CGCGCATCAACGATTTGATATGGCAACTCAAAGACTATGCCGCAGCAGCC


 


ATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGCATAGAAGCC


 


GCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGG


 


GGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCA


 


GATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATT


 


TTGCCGATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAAT


 


ATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTC


 


AACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCC


 


ACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAG


 


AAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCACTGA


 


(SEQ ID NO: 42)










1
MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT



 


51
AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL


 


101
ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA


 


151
DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK


 


201
AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS


 


251
ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT


 


301
VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG


 


351
FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGS DLANDSFIRQ


 


401
VLDRQHFEPD GKYHLFGSRG ELAERSGHIG LGKIQSHQLG NLMIQQAAIK


 


451
GNIGYIVRFS DHGHEVHSPF DNHASHSDSD EAGSPVDGFS LYRIHWDGYE


 


501
HHPADGYDGP QGGGYPAPKG ARDIYSYDIK GVAQNIRLNL TDNRSTGQRL


 


551
ADRFHNAGSM LTQGVGDGFK RATRYSPELD RSGNAAEAFN GTADIVKNII


 


601
GAAGEIVGAG DAVQGISEGS NIAVMHGLGL LSTENKMARI NDLADMAQLK


 


651
DYAAAAIRDW AVQNPNAAQG IEAVSNIFMA AIPIKGIGAV RGKYGLGGIT


 


701
AHPIKRSQMG AIALPKGKSA VSDNPADAAY AKYPSPYHSR NIRSNLEQRY


 


751
GKENITSSTV PPSNGKNVKL ADQRHPKTGV PFDGKGFPNF EKHVKYDTLE


 


801
HHHHHH*


 








961-741



(SEQ ID NO: 43)



ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGC



 


TGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGA


 


CCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACT


 


GCAGCCGATGTTGAAGCCGACGACTAAAGGTCTGGGTCTGAAAAAAGTCG


 


TGACTAACCTGACCAAAACCGTCTGAAAACAAACAAACGTCGATGCCAAA


 


GTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGA


 


CACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCA


 


ACGCCTGAATAAATTGGGAAAAATATAACGACATTTGCTGAAGAGACTAA


 


GACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCG


 


TCGACAAGCATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACC


 


ACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGA


 


GACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTG


 


ATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTG


 


GATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGCTGA


 


TATCGCTACGAACAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGT


 


GTACACCAGAGAAGAGTCTGACAGCAAATGTCAGAATTGATGGTCTGAAC


 


GCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCAT


 


TGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAAACAGTGTCAGAC


 


CTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGG


 


TCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCG


 


GCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTT


 


ACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGG


 


TTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGGGATCCGGAG


 


GGGGTGGTGTCGCCGCCGACATCGGTGCGGGGTTGCCGATGCACTAACCG


 


CACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTGACGCTGGATCAGT


 


CAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGA


 


AAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAATTGAAGAACGA


 


CAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGC


 


TCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCC


 


GCCTTAACCGCCTTTCAGACCGAGCAAATACATCGGAGCATTCCGGGAAT


 


GGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCCGAACATACA


 


TCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGC


 


GTTCGGTTCAGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCG


 


CCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAAT


 


GTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGT


 


CATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCC


 


TCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTG


 


AAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGA


 


GCACCACCACCACCACCACTGA


 


(SEQ ID NO: 44)










1
MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT



 


51
AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL


 


101
ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA


 


151
DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK


 


201
AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS


 


251
ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT


 


301
VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG


 


351
FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGV AADIGAGLAD


 


401
ALTAPLDHKD KGLQSLTLDQ SVRKNEKLKL AAQGAEKTYG NGDSLNTGRL


 


451
KNDKVSRFDF IRQIEVDGQL ITLESGEFQV YKQSHSALTA FQTEQIQDSE


 


501
HSGKMVAKRQ FRIGDIAGEH TSFDKLPEGG RATYRGTAFG SDDAGGKLTY


 


551
TIDFAAKQGN GKIEHLKSPE LNVDLAAADI KPDGKRHAVI SGSVLYNQAE


 


601
KGSYSLGIFG GKAQEVAGSA EVKTVNGIRH IGLAAKQLEH HHHHH*


 








961-983



(SEQ ID NO: 45)



ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGC



 


TGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGA


 


CCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACT


 


GCAGCCGATGTTGAAGCCGACGACTAAAGGTCTGGGTCTGAAAAAAGTCG


 


TGACTAACCTGACCAAAACCGTCTGAAAACAAACAAACGTCGATGCCAAA


 


GTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGA


 


CACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCA


 


ACGCCTGAATAAATTGGGAAAAATATAACGACATTTGCTGAAGAGACTAA


 


GACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCG


 


TCGACAAGCATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACC


 


ACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGA


 


GACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTG


 


ATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTG


 


GATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGCTGA


 


TATCGCTACGAACAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGT


 


GTACACCAGAGAAGAGTCTGACAGCAAATGTCAGAATTGATGGTCTGAAC


 


GCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCAT


 


TGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAAACAGTGTCAGAC


 


CTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGG


 


TCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCG


 


GCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTT


 


ACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGG


 


TTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGGGATCCGGCG


 


GAGGCGGCACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGC


 


AGCAACAGCAGCAACAACAACAGCGAAATCAGCAGCAGTATCTTACGCCG


 


GTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGG


 


GATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCC


 


GAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATT


 


TGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGQACGCGGGGTA


 


GAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTTTCC


 


CGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACT


 


ATACGGCGTATATGCGGAAGGAAGCGCCTGAGACGGAGGCGGTAAAGACA


 


TTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCG


 


ACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCA


 


TATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGC


 


CCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAA


 


ATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGG


 


CGTGCGCATCGTCAATAACAGTTTTGGACAACATCGAGGGCAGGCACTGC


 


CGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGC


 


TCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCOGCCTGATGCAA


 


CAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATGCT


 


TTTCATTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGC


 


CCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCG


 


CAGGCGTAGACCGCAGTGGAGAAAAGTTAACGGGAAATGTATGGAGAACC


 


GGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCA


 


TGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACA


 


AACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCGG


 


CACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACG


 


AACCTGCGTACCACGTTGOTGACGACGGCTCAQGACATCGQTGCAGTCGG


 


CGTGGACAGCAAGTTCGGCTGGGGACTGGATGCGGGTAAGGCCATGAACG


 


GACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACA


 


TCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCT


 


GATCAAAAAAGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATAC


 


GGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACA


 


AATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCG


 


GCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATAC


 


CGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGC


 


TGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGTG


 


GACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAA


 


GGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTG


 


CCGCCAAAATCGGGCAGGATTATTCTTTCTCACAAACATCGAAACCGACG


 


GCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAA


 


GGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGC


 


TTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAAC


 


AGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAA


 


TCATCCGCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGA


 


TATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCG


 


TACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCC


 


GCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACG


 


CCGCCTGAAAGCCGTATCGGACGGGGTTGGACCACAACGGCACGGGTCTG


 


CGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGG


 


TGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCCGCGA


 


AAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGC


 


ACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTT


 


TGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGT


 


TCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGAC


 


GAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACT


 


GGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAG


 


GCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGC


 


AGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGG


 


ACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGT


 


TGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAA


 


CGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGC


 


AATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATT


 


CGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAAC


 


AGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCCTCGAG


 


CACCACCACCACCACCACTGA


 


(SEQ ID NO: 46)










1
MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT



 


51
AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL


 


101
ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA


 


151
DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK


 


201
AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS


 


251
ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT


 


301
VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG


 


351
FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGT SAPDFNAGGT


 


401
GIGSNSRATT AKSAAVSYAG IKNEMCKDRS MLCAGRDDVA VTDRDAKINA


 


451
PPPNLHTGDF PNPNDAYKNL INLKPAIEAG YTGRGVEVGI VDTGESVGSI


 


501
SFPELYGRKE HGYNENYKNY TAYMRKEAPE DGGGKDIEAS PDDEAVIETE


 


551
AKPTDIRHVK EIGHIDLVSH IIGGRSVDGR PAGGIAPDAT LHIMNTNDET


 


601
KNEMMVAAIR NAWVKLGERG VRIVNNSFGT TSRAGTADLF QIANSEEQYR


 


651
QALLDYSGGD KTDEGIRLMQ QSDYGNLSYH IRNKNMLFIF STGNDAQAQP


 


701
NTYALLPFYE KDAQKGIITV AGVDRSGEKF KREMYGEPGT EPLEYGSNHC


 


751
GITAMWCLSA PYEASVRFTR TNPIQIAGTS FSAPIVTGTA ALLLQKYPWM


 


801
SNDNLRTTLL TTAQDIGAVG VDSKFGWGLL DAGKAMNGPA SFPFGDFTAD


 


851
TKGTSDIAYS FRNDISGTGG LIKKGGSQLQ LHGNNTYTGK TIIEGGSLVL


 


901
YGNNKSDMRV ETKGALIYNG AASGGSLNSD GIVYLADTDQ SGANETVHIK


 


951
GSLQLDGKGT LYTRLGKLLK VDGTAIIGGK LYMSARGRGA GYLNSTGRRV


 


1001
PFLSAAKIGQ DYSFFTNIET DGGLLASLDS VEKTAGSEGD TLSYYVRRGN


 


1051
AARTASAAAH SAPAGLKHAV EQGGSNLENL MVELDASESS ATPETVETAA


 


1101
ADRTDMPGIR PYGATFRAAA AVQHANAADG VRIFNSLAAT VYADSTAAHA


 


1151
DMQGRRLKAV SDGLDHNGTG LRVIAQTQQD GGTWEQGGVE GKMRGSTQTV


 


1201
GIAAKTGENT TAAATLGMGR STWSENSANA KTDSISLFAG IRHDAGDIGY


 


1251
LKGLPSYGRY KNSISRSTGA DEHAEGSVNG TLMQLGALGG VNVPFAATGD


 


1301
LTVEGGLRYD LLKQDAFAEK GSALGWSGNS LTEGTLVGLA GLKLSQPLSD


 


1351
KAVLFATAGV ERDLNGRDYT VTGGFTGATA ATGKTGAPNM PHTRLVAGLG


 


1401
ADVEFGNGWN GLARYSYAGS KQYGNHSGRV GVGYRFLEHH HHHH*


 








961c-ORF46.1



(SEQ ID NO: 47)



ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGC



 


TGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGA


 


CCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACT


 


GCAGCCGATGTTGAAGCCGACGACTAAAGGTCTGGGTCTGAAAAAAGTCG


 


TGACTAACCTGACCAAAACCGTCTGAAAACAAACAAACGTCGATGCCAAA


 


GTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGA


 


CACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCA


 


ACGCCTGAATAAATTGGGAAAAATATAACGACATTTGCTGAAGAGACTAA


 


GACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCG


 


TCGACAAGCATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACC


 


ACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGA


 


GACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTG


 


ATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTG


 


GATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGCTGA


 


TATCGCTACGAACAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGT


 


GTACACCAGAGAAGAGTCTGACAGCAAATGTCAGAATTGATGGTCTGAAC


 


GCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCAT


 


TGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAAACAGTGTCAGAC


 


CTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGG


 


TCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGAGGAGGATCAGATT


 


TGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAA


 


CCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGACCG


 


CAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACC


 


TGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGC


 


CGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGC


 


CTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCC


 


TTTACCGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTAT


 


GACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATAT


 


ATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGA


 


CCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCC


 


GGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCG


 


ATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACG


 


GAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGAC


 


AGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAA


 


AAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCCGATGCCGTG


 


CAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCT


 


GCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGG


 


CGCAACTCAAAGACTATGCCGCAGCCAGCCATCCGCGATTGGGCAGTCCA


 


AAACCCCAATGCCGCACAAGGCATAGAACCGTCAGCAATATCTTTCAAGC


 


GGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGC


 


CAGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACG


 


GCAAAAATGTCAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGT


 


TTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACG


 


CTCAGCACCACCACCACCACCACTGA


 


(SEQ ID NO: 48)










1
MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT



 


51
AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL


 


101
ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA


 


151
DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK


 


201
AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS


 


251
ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT


 


301
VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGSDLAND SFIRQVLDRQ


 


351
HFEPDGKYHL FGSRGELAER SGHIGLGKIQ SHQLGNLMIQ QAAIKGNIGY


 


401
IVRFSDHGHE VHSPFDNHAS HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD


 


451
GYDGPQGGGY PAPKGARDIY SYDIKGVAQN IRLNLTDNRS TGQRLADRFH


 


501
NAGSMLTQGV GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE


 


551
IVGAGDAVQG ISEGSNIAVM HGLGLLSTEN KMARINDLAD MAQLKDYAAA


 


601
AIRDWAVQNP NAAQGIEAVS NIFMAAIPIK GIGAVRGKYG LGGITAHPIK


 


651
RSQMGAIALP KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LEQRYGKENI


 


701
TSSTVPPSNG KNVKLADQRH PKTGVPFDGK GFPNFEKHVK YDTLEHHHHH


 


751
H*


 








961c-741



(SEQ ID NO: 49)



ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGC



 


TGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGA


 


CCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACT


 


GCAGCCGATGTTGAAGCCGACGACTAAAGGTCTGGGTCTGAAAAAAGTCG


 


TGACTAACCTGACCAAAACCGTCTGAAAACAAACAAACGTCGATGCCAAA


 


GTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGA


 


CACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCA


 


ACGCCTGAATAAATTGGGAAAAATATAACGACATTTGCTGAAGAGACTAA


 


GACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCG


 


TCGACAAGCATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACC


 


ACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGA


 


GACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTG


 


ATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTG


 


GATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGCTGA


 


TATCGCTACGAACAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGT


 


GTACACCAGAGAAGAGTCTGACAGCAAATGTCAGAATTGATGGTCTGAAC


 


GCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCAT


 


TGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAAACAGTGTCAGAC


 


CTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGG


 


TCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGGGGTGGTGTCGCCG


 


CCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCAT


 


AAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAA


 


CGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACG


 


GTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTC


 


GACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAG


 


TGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTC


 


AGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAA


 


CGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTGACAAG


 


CTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGA


 


CGATGCCGGCGGAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGG


 


AAACGGCAAAATCGAACATTTGAAATCGCCAGAATCAATGTCGACCTGGC


 


CGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTT


 


CCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTT


 


GGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAA


 


CGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACC


 


ACCACCACTGA


 


(SEQ ID NO: 50)










1
MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT



 


51
AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL


 


101
ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA


 


151
DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK


 


201
AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS


 


251
ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT


 


301
VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGVAADIG AGLADALTAP


 


351
LDHKDKGLQS LTLDQSVRKN EKLKLAAQGA EKTYGNGDSL NTGKLKNDKV


 


401
SRFDFIRQIE VDGQLITLES GEFQVYKQSH SALTAFQTEQ IQDSEHSGKM


 


451
VAKRQFRIGD IAGEHTSFDK LPEGGRATYR GTAFGSDDAG GKLTYTIDFA


 


501
AKQGNGKIEH LKSPELNVDL AAADIKPDGK RHAVISGSVL YNQAEKGSYS


 


551
LGIFGGKKQE VAGSAEVKTV NGIRHIGLAA KQLEHHHHHH *


 








961c-983



(SEQ ID NO: 51)



ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGC



 


TGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGA


 


CCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACT


 


GCAGCCGATGTTGAAGCCGACGACTAAAGGTCTGGGTCTGAAAAAAGTCG


 


TGACTAACCTGACCAAAACCGTCTGAAAACAAACAAACGTCGATGCCAAA


 


GTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGA


 


CACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCA


 


ACGCCTGAATAAATTGGGAAAAATATAACGACATTTGCTGAAGAGACTAA


 


GACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCG


 


TCGACAAGCATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACC


 


ACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGA


 


GACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTG


 


ATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTG


 


GATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGCTGA


 


TATCGCTACGAACAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGT


 


GTACACCAGAGAAGAGTCTGACAGCAAATGTCAGAATTGATGGTCTGAAC


 


GCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCAT


 


TGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAAACAGTGTCAGAC


 


CTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGG


 


TCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGCGGCACTTCTGCGC


 


CCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAcACAAC


 


AGCGAATCAGCAGCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAA


 


AGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACA


 


GGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTT


 


CCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAACCTGCAATT


 


GAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGG


 


CGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAAC


 


ACGGCGGATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGA


 


AGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATG


 


AGGCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAA


 


GAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGT


 


GGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAA


 


TGAATACGAATGATGAAACCAAGAACGAATGATGGTTGCAGCCATCCGCA


 


ATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGT


 


TTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAA


 


TTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATA


 


AACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGT


 


CCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAAT


 


GACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTTATGAAA


 


AAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGGAGTGGA


 


GAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGA


 


GTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCAC


 


CCTATGGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGG


 


AACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGC


 


AGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACG


 


ACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGG


 


ACTGCTGGATGCGGGTAAGGCCATGACGGACCCGCGTCCTTTCCGTTCGG


 


CGACTTTACCGCCGATACGAAAGTACATCCGATATTGCCTACTCCTTCCG


 


TAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAAC


 


TGCAACTGCACGGCAACAACACCTATACGGGCAAAACCATTATCGAAGGC


 


GGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAAC


 


CAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACA


 


GCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCAAACGAA


 


ACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTA


 


CACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCG


 


GCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGT


 


ACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTA


 


TTCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCG


 


AAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATG


 


TCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCG


 


CCCGCCGGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAA


 


CCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGG


 


TTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTAC


 


GGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGA


 


CGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGCCAGTA


 


CCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGAC


 


GGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACCCAACA


 


GGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCA


 


GTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCA


 


GCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAA


 


TGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGG


 


GCGATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAAC


 


AGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAA


 


CGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTG


 


CCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTC


 


AAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAA


 


CAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGC


 


AACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGC


 


GACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGAC


 


TGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGG


 


TTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGCTTG


 


GCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACG


 


AGTCGGCGTAGGCTACCGGTTCCTCGAGCACCACCACCACCACCACTGA


 


(SEQ ID NO: 52)










1
MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT



 


51
AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL


 


101
ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA


 


151
DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK


 


201
AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS


 


251
ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT


 


301
VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGTSAPDF NAGGTGIGSN


 


351
SEATTAKSAA VSYAGIKNEM CKDRSMLCAG RDDVAVTDRD AKINAPPPNL


 


401
HTGDFPNPND AYKNLINLKP AIEAGYTGRG VEVGIVDTGE SVGSISFPEL


 


451
YGRKEHGYNE NYKNYTAYMR KEAPEDGGGK DIEASFDDEA VIETEAKPTD


 


501
IRHVKEIGHI DLVSHIIGGR SVDGRPAGGI APDATLHIMN TNDETKNEMM


 


551
VAAIRNAWVK LGERGVRIVN NSFGTTSRAG TADLFQIANS EEQYRQALLD


 


601
YSGGDKTDEG IRLMQQSDYG NLSYHIRNKN MLFIFSTGND AQAQPNTYAL


 


651
LPFYEKDAQK GIITVAGVDR SGEKFKREMY GEPGTEPLEY GSNHCGITAM


 


701
WCLSAPYEAS VRFTRTNPIQ IAGTSFSAPI VTGTAALLLQ KYPWMSNDNL


 


751
RTTLLTTAQD IGAVGVDSKF GWGLLDAGKA MNGPASFPFG DFTADTKGTS


 


801
DIAYSFRNDI SGTGGLIKKG GSQLQLHGNN TYTGKTIIEG GSLVLYGNNK


 


851
SDMRVETKGA LIYNGAASGG SLNSDGIVYL ADTDQSQANE TVHIKGSLQL


 


901
DGKGTLYTRL GKLLKVDGTA IIGGKLYMSA RGKGAGYLNS TGRRVPFLSA


 


951
AKIGQDYSFF TNIETDGGLL ASLDSVEKTA GSEGDTLSYY VRRGNAARTA


 


1001
SAAAHSAPAG LKHAVEQGGS NLENLMVELD ASESSATPET VETAAADRTD


 


1051
MPGIRPYGAT FEAAAAVQHA NAADGVRIFN SLAATVYADS TAAHADMQGR


 


1101
RLKAVSDGLD HNGTGLRVIA QTQQDGGTWE QGGVEGKMRG STQTVGIAAK


 


1151
TGENTTAAAT LGMGRSTWSE NSANAKTDSI SLFAGIRHDA GDIGYLKGLF


 


1201
SYGRYKNSIS RSTGADEHAE GSVNGTLMQL GALGGVNVPF AATGDLTVEG


 


1251
GLRYDLLKQD AFAEKGSALG WSGNSLTEGT LVGLAGLKLS QPLSDKAVLF


 


1301
ATAGVEEDLN GRDYTVTGGF TGATAATGKT GARNMPHTRL VAGLGADVEF


 


1351
GNGWNGLARY SYAGSKQYGN HSGRVGVGYR FLEHHHHHH*


 








961cL-ORF46.1



(SEQ ID NO: 53)



ATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTT



 


CTGTAGCGGCGCACTGGCAGCCACAAACGACGACGATGTTAAAAAAGCTG


 


CCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGT


 


TTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTAC


 


CAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGTCT


 


GGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACA


 


AACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAG


 


TTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTATGCC


 


GCTCTGGATGCAACCACCAACGCCTTGAATAAAATTGGGAGAAAATATAA


 


CGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAA


 


TTAGAAGCCGATAGGCTGATACCGTCGkCAAGCATGCCGAAGCATTCAAC


 


GATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGT


 


CAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACG


 


TCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCT


 


GCCGCTGGCACAGCTAATACTGCAGCCGACAGGCCGAAGCTGTCGCTGCA


 


AAAGTTACCGACATCAAAGCTGATATCGCTACGACAAAGATAATATTGCT


 


AAAAAGCAAACAGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATT


 


TGTCAGAATTGATGGTCTGAGCGCTACTACCGAAAAATTGGACACACGCT


 


TGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGT


 


TTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGC


 


AGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGAT


 


CCGGAGGAGGAGGATCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTT


 


CTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAG


 


CAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATAC


 


AAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAAGGA


 


AATATCGGCTACACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCAT


 


TCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAG


 


TCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAAC


 


ACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCT


 


CCCAAAGGCGCGAAAGGATATATACAGCTACGACATAAAAGGCGTTGCCC


 


AAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTT


 


GCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGA


 


CGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCA


 


ATGCCGCCGCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCA


 


TCGGCGCGGCAGGAGAAATTGTCGCCGCAGGCGATGCCGTGCAGGGCATA


 


AGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCCAC


 


CGAAAACAAGATGGCGCGCATCAGCGATTTGGCAGATATGGCGCAACTCA


 


AAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAAT


 


GCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCC


 


CATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCA


 


CGGCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAA


 


GGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATA


 


CCCGTCCCCTTACCATTCCCGAATATCCGTTCAACTTACGGAGCAGCGTT


 


ACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAA


 


AATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGA


 


CGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGTAAC


 


TCGAG


 


(SEQ ID NO: 54)










1
MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING



 


51
FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN


 


101
KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI


 


151
TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV


 


201
KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA


 


251
KVTDIKADIA TNKDNIAKKA NSHDVYTREE SDSKFVRIDG LNATTEKLDT


 


301
RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLPQPYNVG


 


351
GSGGGGSDLA NDSFIRQVLD RQHFEPDGKY HLFQSRGELA ERSGHIGLGK


 


401
IQSHQLGNLM IQQAAIKGNI GYIVRFSDHG HEVESPFDNH ASHSDSDEAG


 


451
SPVDGFSLYR IHWDGYEHHP ADGYDGPQGG GYPAPKGARD IYSYDIRGVA


 


501
QNIRLNLTDN RSTGQRLADR FHNAGSMLTQ GVGDGFKRAT RYSPELDRSG


 


551
NAAEAFNGTA DIVKNIIGAA GEIVGAGDAV QGISEGSNIA VMHGLGLLST


 


601
ENKMARINDL ADMAQLKDYA AAAIRDWAVQ NPNAAQGIEA VSNIFMAAIP


 


651
IKGIGAVRGK YGLGGITAHP IKRSQMGAIA LPKGKSAVSD NFADAAYAKY


 


701
PSPYHSRNIR SNLEQRYGKE NITSSTVPPS NGKNVKLADQ RHPKTGVPFD


 


751
GKGFPNFEKH VKYDT*


 








961cL-741



(SEQ ID NO: 55)



ATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTT



 


CTGTAGCGGCGCACTGGCAGCCACAAACGACGACGATGTTAAAAAAGCTG


 


CCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGT


 


TTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTAC


 


CAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGTCT


 


GGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACA


 


AACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAG


 


TTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTATGCC


 


GCTCTGGATGCAACCACCAACGCCTTGAATAAAATTGGGAGAAAATATAA


 


CGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAA


 


TTAGAAGCCGATAGGCTGATACCGTCGkCAAGCATGCCGAAGCATTCAAC


 


GATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGT


 


CAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACG


 


TCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCT


 


GCCGCTGGCACAGCTAATACTGCAGCCGACAGGCCGAAGCTGTCGCTGCA


 


AAAGTTACCGACATCAAAGCTGATATCGCTACGACAAAGATAATATTGCT


 


AAAAAGCAAACAGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATT


 


TGTCAGAATTGATGGTCTGAGCGCTACTACCGAAAAATTGGACACACGCT


 


TGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGT


 


TTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGC


 


AGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGAT


 


CCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCA


 


CTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCT


 


GGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTG


 


CGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAG


 


AACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGG


 


GCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCC


 


ATTCCGCCTTAACCGCCTTTCADACCGAGCAAATACAAATTCGGAGCATT


 


CCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGC


 


GAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCG


 


CGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCA


 


TAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCG


 


CCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAA


 


ACGCCATGCCGTCATCAGCGGTTCCGTCCTTPACAACCAAGCCGAGAAAG


 


GCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGC


 


AGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGC


 


CAAGCAACTCGAGCACCACCACCACCACCACTGA


 


(SEQ ID NO: 56)










1
MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING



 


51
FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN


 


101
KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI


 


151
TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV


 


201
KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA


 


251
KVTDIKADIA TNKDNIAKKA NSHDVYTREE SDSKFVRIDG LNATTEKLDT


 


301
RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLPQPYNVG


 


351
GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR KNEKLKLAAQ


 


401
GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL ESGEFQVYKQ


 


451
SHSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF DKLPEGGRAT


 


501
YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD


 


551
GKRHAVISGS VLYNQAEKGS YSLGIPGGKA QEVAGSAEVK TVNGIRHIGL


 


601
AAKQLEHHHH HH*


 








961cL-983



(SEQ ID NO: 57)



ATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTT



 


CTGTAGCGGCGCACTGGCAGCCACAAACGACGACGATGTTAAAAAAGCTG


 


CCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGT


 


TTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTAC


 


CAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGTCT


 


GGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACA


 


AACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAG


 


TTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTATGCC


 


GCTCTGGATGCAACCACCAACGCCTTGAATAAAATTGGGAGAAAATATAA


 


CGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAA


 


TTAGAAGCCGATAGGCTGATACCGTCGkCAAGCATGCCGAAGCATTCAAC


 


GATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGT


 


CAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACG


 


TCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCT


 


GCCGCTGGCACAGCTAATACTGCAGCCGACAGGCCGAAGCTGTCGCTGCA


 


AAAGTTACCGACATCAAAGCTGATATCGCTACGACAAAGATAATATTGCT


 


AAAAAGCAAACAGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATT


 


TGTCAGAATTGATGGTCTGAGCGCTACTACCGAAAAATTGGACACACGCT


 


TGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGT


 


TTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGC


 


AGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGAT


 


CCGGCGGAGGCGGCACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGT


 


CGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACG


 


CCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGT


 


CGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAATGCCCCCCC


 


CCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGA


 


ATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGG


 


GTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCCTT


 


TCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAA


 


ACTATACGGCGTATATGCGGAAGGAAGCGCCTGACGAAGGAGGCGGTAAG


 


ACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAG


 


CCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTC


 


CCATATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTG


 


CGCCCGATGCGACGCTACACATAATGATACGAATGATGAAACCAAGAACG


 


AAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGT


 


GGCGTGCGCATCGTCATAACAGTTTTGGAACAACATCGAGGGCAGGCACT


 


GCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTT


 


GCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGC


 


AACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAATAAAAACATG


 


CTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATA


 


TGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATATCACAGT


 


CGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTATGGAG


 


AACCGGGTACAGACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTG


 


CCATGTGGTGCCTGTCGGCACCCTATGAGCAAGCGTCCGTTCACCCGTAC


 


AAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACCG


 


GCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAAC


 


CTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGT


 


GGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACG


 


GACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACA


 


TCCGATATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCT


 


GATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATA


 


CGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAAC


 


AAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGC


 


GGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATA


 


CCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAG


 


CTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTGAAAGT


 


GGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCA


 


AGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGT


 


GCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGA


 


CGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTG


 


AAGGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACT


 


GCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGA


 


ACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCG


 


AATCATCCGCAACACCCQAGACGGTTGAAACTGCGGCAGCCGACCGCACA


 


GATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGC


 


CGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTATCAACAGTCTC


 


GCCGCAGTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGATATGCAG


 


GGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGG


 


TCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGG


 


GCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCC


 


GCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACG


 


CAGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTC


 


TGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGC


 


CTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGC


 


GGACGAACATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCG


 


CACTGGGCGGTGTCACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCG


 


AAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAA


 


GGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGT


 


CGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCC


 


TGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACG


 


GTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACGGGGGC


 


ACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCG


 


AATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCC


 


AAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCTG


 


ACTCGAG


 


(SED ID NO: 58)










1
MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING



 


51
FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN


 


101
KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI


 


151
TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV


 


201
KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA


 


251
KVTDIKADIA TNKDNIAKKA NSHDVYTREE SDSKFVRIDG LNATTEKLDT


 


301
RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLPQPYNVG


 


351
GSGGGGTSAP DFNAGGTGIG SNSRATTAKS AAVSYAGIKN EMCKDRSMLC


 


401
AGRDDVAVTD RDAKINAPPP NLHTGDFPNP NDAYKNLINL KPAIEAGYTG


 


451
RGVEVGIVDT GESVGSISFP ELYGRKEHGY NENYKNYTAY MRKEAPEDGG


 


501
GKDIEASFDD EAVIETEAKP TDIRHVKEIG HIDLVSHIIG GRSVDGRPAG


 


551
GIAPDATLHI MNTNDETKNE MMVAAIRNAW VKLGERGVRI VNNSFGTTSR


 


601
AGTADLFQIA NSEEQYRQAL LDYSGGDKTD EGIRLHQQSD YGNLSYHIRN


 


651
KNMLFIFSTG NDAQAQPNTY ALLPFYEKDA QKGIITVAGV DRSGEKFKRE


 


701
MYGEPGTEPL EYGSNHCGIT AMWCLSAPYE ASVEPTRTNP IQIAGTSFSA


 


751
PIVTGTAALL LQKYPWMSND NLRTTLLTTA QDIGAVGVDS KFGWGLLDAG


 


801
KAMNGPASFP FGDFTADTKG TEDIAYSFRN DISGTGGLIK KGGSQLQLHG


 


851
NNTYTGKTII EGGSLVLYGN NKSDMRVETK GALIYNGAAS GGSLNSDGIV


 


901
YLADTDQSGA NETVHIKGSL QLDGKGTLYT RLGKLLKVDG TAIIGGKLYM


 


951
SARGKGAGYL NSTGRRVPFL SAAKIGQDYS FFTNIETDGG LLASLDSVEK


 


1001
TAGSEGDTLS YYVRRGNAAR TASAAAHSAP AGLKHAVEQG GSNLENLMVE


 


1051
LDASESSATP ETVETAAADR TDMPGIRPYG ATFRAAAAVQ HANAADGVRI


 


1101
FNSLAATVYA DSTAAHADMQ GRRLKAVSDG LDHNGTGLRV IAQTQQDGGT


 


1151
WEQGGVEGKM RGSTQTVGIA AKTGENTTAA ATLGMGRSTW SENSANAKTD


 


1201
SISLFAGIRH DAGDIGYLKG LFSYGRYKNS ISRSTGADEH AEGSVNGTLM


 


1251
QLGALGGVNV PFAATGDLTV EGGLRYDLLK QDAFAEKGSA LGWSGNSLTE


 


1301
GTLVGLAGLK LSQPLSDKAV LFATAGVERD LNGRDYTVTG GFTGATAATG


 


1351
KTGARNMPHT RLVAQLQADV EFGNGWNGLA RYSYAGSKQY QNHSGRVGVG


 


1401
YRF*






It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. For instance, the use of proteins from other strains is envisaged [e.g. see WO00/66741 for polymorphic sequences for ORF4, ORF40, ORF46, 225, 235, 287, 519, 726, 919 and 953].
EXPERIMENTAL DETAILS
Cloning Strategy and Oligonucleotide Design
Genes coding for antigens of interest were amplified by PCR, using oligonucleotides designed on the basis of the genomic sequence of N. meningitidis B MC58. Genomic DNA from strain 2996 was always used as a template in PCR reactions, unless otherwise specified, and the amplified fragments were cloned in the expression vector pET21b+ (Novagen) to express the protein as C-terminal His-tagged product, or in pET-24b+(Novagen) to express the protein in ‘untagged’ form (e.g. ΔG 287K).
Where a protein was expressed without a fusion partner and with its own leader peptide (if present), amplification of the open reading frame (ATG to STOP codons) was performed.
Where a protein was expressed in ‘untagged’ form, the leader peptide was omitted by designing the 5′-end amplification primer downstream from the predicted leader sequence.
The melting temperature of the primers used in PCR depended on the number and type of hybridising nucleotides in the whole primer, and was determined using the formulae:

Tm1=4(G+C)+2(A+T)  (tail excluded)

Tm2=64.9+0.41(% GC)−600/N  (whole primer)

The melting temperatures of the selected oligonucleotides were usually 65-70° C. for the whole oligo and 50-60° C. for the hybridising region alone.
Oligonucleotides were synthesised using a Perkin Elmer 394 DNA/RNA Synthesizer, eluted from the columns in 2.0 ml NH4OH, and deprotected by 5 hours incubation at 56° C. The oligos were precipitated by addition of 0.3M Na-Acetate and 2 volumes ethanol. The samples were centrifuged and the pellets resuspended in water.















SEQ
Restriction



Sequences
ID NO
site



















fu (961)-
Fwd
CGCGGATCC-GGAGGGGGTGGTGTCG
59
BamHI


741(MC58)-His
Rev
CCCGCTCGAG-TTGCTTGGCGGCAAGGC
60
XhoI


 


fu (961)-983-His
Fwd
CGCGGATCC-GGCGGAGGCGGCACTT
61
BamHI



Rev
CCCGCTCGAGG-GAACCGGTAGCCTACG
62
XhoI


 


fu (961)- Orf46.1-
Fwd
CGCGGATCCGGTGGTGGTGGT-
63
BamHI


His

TCAGATTTGGCAAACGATTC





Rev
CCCGCTCGAG-CGTATCATATTTCACGTGC
64
XhoI


 


fu (961 c-L)-
Fwd
CGCGGATCCC-GGAGGGGGTGGTGTCG
65
BamHI


741(MC58)







Rev
CCCGCTCGAG-TTATTGCTTGGCGGCAAG
66
XhoI


 


fu (961c-L)-983
Fwd
CGCGGATCC-GGCGGAGGCGGCACTT
67
BamHI



Rev
CCCGCTCGAG-TCAGAACCGGTAGCCTAC
68
XhoI


 


fu (961c.L)-
Fwd
CGCGGATCCGGTGGTGGTGGT-
69
BamHl


Orf46.1

TCAGATTTGGCAAACGATTC





Rev
CCCGCTCGAG-TTACGTATCATATTTCACGTGC
70
XhoI


 


fu-(AG287)-919-
Fwd
CGCGGATCCGGTGGTGGTGGT-
71
BamHI


His

CAAAGCAAGAGCATCCAAACC





Rev
CCCAAGCTT-TTCGGGCGGTATTCGGGCTTC
72
HindIII


 


fu(ΔG287)-953-
Fwd
CGCGGATCCGGTGGTGGTGGT-
73
BamHI


His

GCCACCTACAAAGTGGAC





Rev
GCCCAAGCTT-TTGTTGGCTGCCCTCGAT
74
HindIII


 


fu-(ΔG287)-961-
Fwd
CGCGGATCCGGTGGTGGTGGT-ACAAGCGACGACG
75
BamHI



Rev
GCCCAAGCTT-CCACTCGTAATTGACGCC
76
HindIII


 


fu-(ΔG287)-
Fwd
CGCGGATCCGGTGGTGGTGGT-
77
BamHI


Orf46.1-His

TCAGATTTGGCAAACGATTC





Rev
CCCAAGCTT-CGTATCATATTTCACGTGC
78
HindIII


 


fu-(ΔG287-919)-
Fwd
CCCAAGCTTGGTGGTGGTGGTGGT-
79
HindIII


Orf46.1-His

TCAGATTTGGCAAACGATTC





Rev
CCCGCTCGAG-CGTATCATATTTCACGTGC
80
XhoI


 


fu-(ΔG287-
Fwd
CCCAAGCTTGGTGGTGGTGGTGGT-
81
HindIII


Orf46.1)-919-His

CAAAGCAAGAGCATCCAAACC





Rev
CCCGCTCGAG-CGGGCGGTATTCGGGCTT
82
XhoI


 


fu ΔG287(394.98)-
Fwd
CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC
83
NbeI


. . .
Rev
CGGGGATCC-ATCCTGCTCTTTTTTGCCGG
84
BamHI


 


fu Orf1-(Orf46.1)-
Fwd
CGCGGATCCGCTAGC-GGACACACTTATTTCGGCATC
85
NheI


His
Rev
CGCGGATCC-CCAGCGGTAGCCTAATTTTGAT
86



 


fu (Orf1)-Orf46.1-
Fwd
CGCGGATCCGGTGGTGGTGGT-
87
BamHI


His

TCAGATTTGGCAAACGATTC





Rev
CCCAAGCTT-CGTATCATATTTCACGTGC
88
HindIII


 


fu (919)-Orf46.1-
Fwd1
GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAG
89
SalI


His
Fwd2
GGAGGCACTGGATCCTCAGATTTGGCAAACGATTC
90




Rev
CCCGCTCGAG-CGTATCATATTTCACGTGC
91
XhoI


 


fu (orf46)-287-His
Fwd
CGGGGATCCGGGGGCGGCGGTGGCG
92
BamHI



Rev
CCCAAGCTTATCCTGCTCTTTTTTGCCGGC
93
HindIII


 


Fu (orf46)-919-His
Fwd
CGCGGATCCGGTGGTGGTGGTCAAAGCAAGAGCATCCA
94
BamHI




AACC





Rev
CCCAAGCTTCGGGCGGTATTCGGGCTTC
95
HindIII


 


Fu (orf46-919)-
Fwd
CCCCAAGCTTGGGGGCGGCGGTGGCG
96
HindIII


287-His
Rev
CCCGCTCGAGATCCTGCTCTTTTTTGCCGGC
97
XhoI


 


Fu (orf46-287)-
Fwd
CCCAAGCTTGGTGGTGGTGGTGGTCAAAGCAAGAGCAT
98
HindIII


919-His

CCAAACC





Rev
CCCGCTCGAGCGGGCGGTATTCGGGCTT
99
XhoI


 


(ΔG741)-961c-His
Fwd1
GGAGGCACTGGATCCGCAGCCACAAACGACGACGA
100
XhoI



Fwd2
GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG
101




Rev
CCCGCTCGAG-ACCCAGCTTGTAAGGTTG
102
XhoI


 


(ΔG741)-961-His
Fwd1
GGAGGCACTGGATCCGCAGCCACAAACGACGACGA
103
XhoI



Fwd2
GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG
104




Rev
CCCGCTCGAG-CCACTCGTAATTGACGC
105
XhoI


 


(ΔG741)-983-His
Fwd
GCGGCCTCGAG-
106
XhoI




GGATCCGGCGGAGGCGGCACTTCTGCG





Rev
CCCGCTCGAG-GAACCGGTAGCCTACG
107
XhoI


 


(ΔG741)-orf46.1-
Fwd1
GGAGGCACTGGATCCTCAGATTTGGCAAACGATTC
108
SalI


His
Fwd2
GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAGA
109




Rev
CCCGCTCGAG-CGTATCATATTTCACGTGC
110
XhoI


 


(ΔG983)-
Fwd
GCGGCCTCGAG-GGATCCGGAGGGGGTGGTGTCGCC
111
XhoI


741(MC58) -His
Rev
CCCGCTCGAG-TTGCTTGGCGGCAAG
112
XhoI


 


(ΔG983)-961c-His
Fwd1
GGAGGCACTGGATCCGCAGCCACAAACGACGACGA
113
XhoI



Fwd2
GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG
114




Rev
CCCGCTCGAG-ACCCAGCTTGTAAGGTTG
115
XhoI


 


(ΔG983)-961-His
Fwd1
GGAGGCACTGGATCCGCAGCCACAAACGACGACGA
116
XhoI



Fwd2
GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG
117




Rev
CCCGCTCGAG-CCACTCGTAATTGACGCC
118
XhoI


 


(ΔG983)-Orf46.1-
Fwd1
GGAGGCACTGGATCCTCAGATTTGGCAAACGATC
119
SalI


His
Fwd2
GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAGA
120




Rev
CCCGCTCGAG-CGTATCATATTTCACGTGC
121
XhoI





* This primer was used as a Reverse primer for all the C terminal fusions of 287 to the His-tag.


§Forward primers used in combination with the 287-His Reverse primer.


NB - All PCR reactions use strain 2996 unless otherwise specified (e.g. strain MC58)






In all constructs starting with an ATG not followed by a unique NheI site, the ATG codon is part of the NdeI site used for cloning. The constructs made using NheI as a cloning site at the 5′ end (e.g. all those containing 287 at the N-terminus) have two additional codons (GCT AGC) fused to the coding sequence of the antigen.
Preparation of Chromosomal DNA Templates
N. meningitidis strains 2996, MC58, 394.98, 1000 and BZ232 (and others) were grown to exponential phase in 100 ml of GC medium, harvested by centrifugation, and resuspended in 5 ml buffer (20% w/v sucrose, 50 mM Tris-HCl, 50 mM EDTA, pH 8). After 10 minutes incubation on ice, the bacteria were lysed by adding 10 ml of lysis solution (50 mM NaCl, 1% Na-Sarkosyl, 50 μg/ml Proteinase K), and the suspension incubated at 37° C. for 2 hours. Two phenol extractions (equilibrated to pH 8) and one CHCl3/isoamylalcohol (24:1) extraction were performed. DNA was precipitated by addition of 0.3M sodium acetate and 2 volumes of ethanol, and collected by centrifugation. The pellet was washed once with 70% (v/v) ethanol and redissolved in 4.0 ml TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The DNA concentration was measured by reading OD260.
PCR Amplification
The standard PCR protocol was as follows: 200 ng of genomic DNA from 2996, MC581000, or BZ232 strains or 10 ng of plasmid DNA preparation of recombinant clones were used as template in the presence of 40 μM of each oligonucletide primer, 400-800 μM dNTPs solution, 1×PCR buffer (including 1.5 mM MgCl2), 2.5 units TaqI DNA polymerase (using Perkin-Elmer AmpliTaQ, Boerhingher Mannheim ExpandTM Long Template).
After a preliminary 3 minute incubation of the whole mix at 95° C., each sample underwent a two-step amplification: the first 5 cycles were performed using the hybridisation temperature that excluded the restriction enzyme tail of the primer (Tm1). This was followed by 30 cycles according to the hybridisation temperature calculated for the whole length oligos (Tm2). Elongation times, performed at 68° C. or 72° C., varied according to the length of the Orf to be amplified. In the case of Orf1 the elongation time, starting from 3 minutes, was increased by 15 seconds each cycle. The cycles were completed with a 10 minute extension step at 72° C.
The amplified DNA was either loaded directly on a 1% agarose gel. The DNA fragment corresponding to the band of correct size was purified from the gel using the Qiagen Gel Extraction Kit, following the manufacturer's protocol.
Digestion of PCR Fragments and of the Cloning Vectors
The purified DNA corresponding to the amplified fragment was digested with the appropriate restriction enzymes for cloning into pET-21b+, pET22b+ or pET-24b+. Digested fragments were purified using the QIAquick PCR purification kit (following the manufacturer's instructions) and eluted with either H2O or 10 mM Tris, pH 8.5. Plasmid vectors were digested with the appropriate restriction enzymes, loaded onto a 1.0% agarose gel and the band corresponding to the digested vector purified using the Qiagen QIAquick Gel Extraction Kit.
Cloning
The fragments corresponding to each gene, previously digested and purified, were ligated into pET21b+, pET22b+ or pET-24b+. A molar ratio of 3:1 fragment/vector was used with T4 DNA ligase in the ligation buffer supplied by the manufacturer.
Recombinant plasmid was transformed into competent E. coli DH5 or HB101 by incubating the ligase reaction solution and bacteria for 40 minutes on ice, then at 37° C. for 3 minutes. This was followed by the addition of 800 μl LB broth and incubation at 37° C. for 20 minutes. The cells were centrifuged at maximum speed in an Eppendorf microfuge, resuspended in approximately 200 μl of the supernatant and plated onto LB ampiclllin (100 mg/ml) agar.
Screening for recombinant clones was performed by growing randomly selected colonies overnight at 37° C. in 4.0 ml of LB broth+100 μg/ml ampicillin. Cells were pelleted and plasmid DNA extracted using the Qiagen QIAprep Spin Miniprep Kit, following the manufacturer's instructions. Approximately 1 μg of each individual miniprep was digested with the appropriate restriction enzymes and the digest loaded onto a 1-1.5% agarose gel (depending on the expected insert size), in parallel with the molecular weight marker (1 kb DNA Ladder, GIBCO). Positive clones were selected on the basis of the size of insert.
Expression
After cloning each gene into the expression vector, recombinant plasmids were transformed into E. coli strains suitable for expression of the recombinant protein. 1 μl of each construct was used to transform E. coli BL21-DE3 as described above. Single recombinant colonies were inoculated into 2 ml LB+Amp (100 μg/ml), incubated at 37° C. overnight, then diluted 1:30 in 20 ml of LB+Amp (100 μg/ml) in 100 ml flasks, to give an OD600 between 0.1 and 0.2. The flasks were incubated at 30° C. or at 37° C. in a gyratory water bath shaker until OD600 indicated exponential growth suitable for induction of expression (0.4-0.8 OD). Protein expression was induced by addition of 1.0 mM IPTG. After 3 hours incubation at 30° C. or 37° C. the OD600 was measured and expression examined. 1.0 ml of each sample was centrifuged in a microfuge, the pellet resuspended in PBS and analysed by SDS-PAGE and Coomassie Blue staining.
Purification of His-Tagged Proteins
Various forms of 287 were cloned from strains 2996 and MC58. They were constructed with a C-terminus His-tagged fusion and included a mature form (aa 18-427), constructs with deletions (Δ1, Δ2, Δ3 and Δ4) and clones composed of either B or C domains. For each clone purified as a His-fusion, a single colony was streaked and grown overnight at 37° C. on a LB/Amp (100 μg/ml) agar plate. An isolated colony from this plate was inoculated into 20 ml of LB/Amp (100 μg/ml) liquid medium and grown overnight at 37° C. with shaking. The overnight culture was diluted 1:30 into 1.0 L LB/Amp (100 μg/ml) liquid medium and allowed to grow at the optimal temperature (30 or 37° C.) until the OD550 reached 0.6-0.8. Expression of recombinant protein was induced by addition of IPTG (final concentration 1.0 mM) and the culture incubated for a further 3 hours. Bacteria were harvested by centrifugation at 8000 g for 15 min at 4° C. The bacterial pellet was resuspended in 7.5 ml of either (i) cold buffer A (300 mM NaCl, 50 mM phosphate buffer, 10 mM imidazole, pH 8.0) for soluble proteins or (ii) buffer B (10 mM Tris-HCl, 100 mM phosphate buffer, pH 8.8 and, optionally, 8M urea) for insoluble proteins. Proteins purified in a soluble form included 287-His, Δ1, Δ2, Δ3 and Δ4287-His, Δ4287MC58-His, 287c-His and 287cMC58-His. Protein 287bMC58-His was insoluble and purified accordingly. Cells were disrupted by sonication on ice four times for 30 sec at 40 W using a Branson sonifier 450 and centrifuged at 13000×g for 30 min at 4° C. For insoluble proteins, pellets were resuspended in 2.0 ml buffer C (6 M guanidine hydrochloride, 100 mM phosphate buffer, 10 mM Tris-HCl, pH 7.5 and treated with 10 passes of a Dounce homogenizer. The homogenate was centrifuged at 13000 g for 30 min and the supernatant retained. Supernatants for both soluble and insoluble preparations were mixed with 150 μl Ni2+-resin (previously equilibrated with either buffer A or buffer B, as appropriate) and incubated at room temperature with gentle agitation for 30 min. The resin was Chelating Sepharose Fast Flow (Pharmacia), prepared according to the manufacturer's protocol. The batch-wise preparation was centrifuged at 700 g for 5 min at 4° C. and the supernatant discarded. The resin was washed twice (batch-wise) with 10 ml buffer A or B for 10 min, resuspended in 1.0 ml buffer A or B and loaded onto a disposable column. The resin continued to be washed with either (i) buffer A at 4° C. or (ii) buffer B at room temperature, until the OD280 of the flow-through reached 0.02-0.01. The resin was further washed with either (i) cold buffer C (300 mM NaCl, 50 mM phosphate buffer, 20 mM imidazole, pH 8.0) or (ii) buffer D (10 mM Tris-HCl, 100 mM phosphate buffer, pH 6.3 and, optionally, 8M urea) until OD280 of the flow-through reached 0.02-0.01. The His-fusion protein was eluted by addition of 700 μl of either (i) cold elution buffer A (300 mM NaCl, 50 mM phosphate buffer, 250 mM imidazole, pH 8.0) or (ii) elution buffer B (10 mM Tris-HCl, 100 mM phosphate buffer, pH 4.5 and, optionally, 8M urea) and fractions collected until the OD280 indicated all the recombinant protein was obtained 20 μl aliquots of each elution fraction were analysed by SDS-PAGE. Protein concentrations were estimated using the Bradford assay.
Renaturation of Denatured His-Fusion Proteins.
Denaturation was required to solubilize 287bMC8, so a renaturation step was employed prior to immunisation. Glycerol was added to the denatured fractions obtained above to give a final concentration of 10% v/v. The proteins were diluted to 200 μg/ml using dialysis buffer I (10% v/v glycerol, 0.5M arginine, 50 mM phosphate buffer, 5.0 mM reduced glutathione, 0.5 mM oxidised glutathione, 2.0M urea, pH 8.8) and dialysed against the same buffer for 12-14 hours at 4° C. Further dialysis was performed with buffer II (10% v/v glycerol, 0.5M arginine, 50 mM phosphate buffer, 5.0 mM reduced glutathione, 0.5 mM oxidised glutathione, pH 8.8) for 12-14 hours at 4° C. Protein concentration was estimated using the formula:

Protein (mg/ml)=(1.55×OD280)−(0.76×OD260)

Immunization

Balb/C mice were immunized with antigens on days 0, 21 and 35 and sera analyzed at day 49.
Sera Analysis—ELISA
The acapsulated MenB M7 and the capsulated strains were plated on chocolate agar plates and incubated overnight at 37° C. with 5% CO2. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following OD620. The bacteria were let to grow until the OD reached the value of 0.4-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and bacteria were washed twice with PBS, resuspended in PBS containing 0.025% formaldehyde, and incubated for 1 hour at 37° C. and then overnight at 4° C. with stirring. 100 μl bacterial cells were added to each well of a 96 well Greiner plate and incubated overnight at 4° C. The wells were then washed three times with PBT washing buffer (0.1% Tween-20 in PBS). 200 μl of saturation buffer (2.7% polyvinylpyrrolidone 10 in water) was added to each well and the plates incubated for 2 hours at 37° C. Wells were washed three times with PBT. 200 μl of diluted sera (Dilution buffer. 1% BSA, 0.1% Tween-20, 0.1% NaN3 in PBS) were added to each well and the plates incubated for 2 hours at 37° C. Wells were washed three times with PBT. 100 μl of HRP-conjugated rabbit anti-mouse (Dako) serum diluted 1:2000 in dilution buffer were added to each well and the plates were incubated for 90 minutes at 37° C. Wells were washed three times with PBT buffer. 100 μl of substrate buffer for HRP (25 ml of citrate buffer pH 5, 10 mg of O-phenildiamine and 10 μl of H2O2) were added to each well and the plates were left at room temperature for 20 minutes. 100 μl 12.5% H2SO4 was added to each well and OD490 was followed. The ELISA titers were calculated abitrarely as the dilution of sera which gave an OD490 value of 0.4 above the level of preimmune sera. The ELISA was considered positive when the dilution of sera with OD490 of 0.4 was higher than 1:400.
Sera Analysis—FACS Scan Bacteria Binding Assay
The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37° C. with 5% CO2. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 4 tubes containing 8 ml each Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following OD620. The bacteria were let to grow until the OD reached the value of 0.35-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and the pellet was resuspended in blocking buffer (1% BSA in PBS, 0.4% NaN3) and centrifuged for 5 minutes at 4000 rpm. Cells were resuspended in blocking buffer to reach OD620 of 0.05. 100 μl bacterial cells were added to each well of a Costar 96 well plate. 100 μl of diluted (1:100, 1:200, 1:400) sera (in blocking buffer) were added to each well and plates incubated for 2 hours at 4° C. Cells were centrifuged for 5 minutes at 4000 rpm, the supernatant aspirated and cells washed by addition of 200 μl/well of blocking buffer in each well. 100 μl of R-Phicoerytrin conjugated F(ab)2 goat anti-mouse, diluted 1:100, was added to each well and plates incubated for 1 hour at 4° C. Cells were spun down by centrifugation at 4000 rpm for 5 minutes and washed by addition of 200 μl/well of blocking buffer. The supernatant was aspirated and cells resuspended in 200 μl/well of PBS, 0.25% formaldehyde. Samples were transferred to FACScan tubes and read. The condition for FACScan (Laser Power 15 mW) setting were: FL2 on; FSC-H threshold: 92; FSC PMT Voltage: E 01; SSC PMT: 474; Amp. Gains 6.1; FL-2 PMT: 586; compensation values: 0.
Sera Analysis—Bactericidal Assay
N. meningitidis strain 2996 was grown overnight at 37° C. on chocolate agar plates (starting from a frozen stock) with 5% CO2. Colonies were collected and used to inoculate 7 ml Mueller-Hinton broth, containing 0.25% glucose to reach an OD620 of 0.05-0.08. The culture was incubated for approximately 1.5 hours at 37 degrees with shacking until the OD620 reached the value of 0.23-0.24. Bacteria were diluted in 50 mM Phosphate buffer pH 7.2 containing 10 mM MgCl2, 10 mM CaCl2 and 0.5% (w/v) BSA (assay buffer) at the working dilution of 105 CFU/ml. The total volume of the final reaction mixture was 50 μl with 25 μl of serial two fold dilution of test serum, 12.5 μl of bacteria at the working dilution, 12.5 μl of baby rabbit complement (final concentration 25%).
Controls included bacteria incubated with complement serum, immune sera incubated with bacteria and with complement inactivated by heating at 56° C. for 30′. Immediately after the addition of the baby rabbit complement, 10 μl of the controls were plated on Mueller-Hinton agar plates using the tilt method (time 0). The 96-wells plate was incubated for 1 hour at 37° C. with rotation. 7 μl of each sample were plated on Mueller-Hinton agar plates as spots, whereas 10 μl of the controls were plated on Mueller-Hinton agar plates using the tilt method (time 1). Agar plates were incubated for 18 hours at 37 degrees and the colonies corresponding to time 0 and time 1 were counted.
Sera Analysis—Western Blots
Purified proteins (500 ng/lane), outer membrane vesicles (5 μg) and total cell extracts (25 μg) derived from MenB strain 2996 were loaded onto a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The transfer was performed for 2 hours at 150 mA at 4° C., using transfer buffer (0.3% Tris base, 1.44% glycine, 20% (v/v) methanol). The membrane was saturated by overnight incubation at 4° C. in saturation buffer (10% skimmed milk, 0.1% Triton X100 in PBS). The membrane was washed twice with washing buffer (3% skimmed milk, 0.1% Triton X100 in PBS) and incubated for 2 hours at 37° C. with mice sera diluted 1:200 in washing buffer. The membrane was washed twice and incubated for 90 minutes with a 1:2000 dilution of horseradish peroxidase labelled anti-mouse Ig. The membrane was washed twice with 0.1% Triton X100 in PBS and developed with the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.
The OMVs were prepared as follows: N. meningitidis strain 2996 was grown overnight at 37 degrees with 5% CO2 on 5 GC plates, harvested with a loop and resuspended in 10 ml of 20 mM Tris-HCl pH 7.5, 2 mM EDTA. Heat inactivation was performed at 56° C. for 45 minutes and the bacteria disrupted by sonication for 5 minutes on ice (50% duty cycle, 50% output, Branson sonifier 3 mm microtip). Unbroken cells were removed by centrifugation at 5000 g for 10 minutes, the supernatant containing the total cell envelope fraction recovered and further centrifuged overnight at 50000 g at the temperature of 4° C. The pellet containing the membranes was resuspended in 2% sarkosyl, 20 mM Tris-HCl pH 7.5, 2 mM EDTA and incubated at room temperature for 20 minutes to solubilise the inner membranes. The suspension was centrifuged at 1000 g for 10 minutes to remove aggregates, the supernatant was further centrifuged at 50000 g for 3 hours. The pellet, containing the outer membranes was washed in PBS and resuspended in the same buffer. Protein concentration was measured by the D.C. Bio-Rad Protein assay (Modified Lowry method), using BSA as a standard.
Total cell extracts were prepared as follows: N. meningitidis strain 2996 was grown overnight on a GC plate, harvested with a loop and resuspended in 1 ml of 20 mM Tris-HCl. Heat inactivation was performed at 56° C. for 30 minutes.

## Claims

1. A hybrid protein of formula NH2-A-B—COOH, wherein A and B are different Neisserial proteins and are each selected from orf1, orf4, orf25, orf40, orf46, orf83, 233, 287, 2921, 564, 687, 741, 907, 919, 953, 961 and 983, wherein when the different Neisserial proteins are 953 and 287, A is 953 and B is 287.

2. The protein of claim 1, wherein A and B are each selected from ORF46, 287, 741, 919, 953, 961 and 983.

3. The protein of claim 2, wherein at least one of said ORF46, 287, 741, 919, 953, 961 and 983 consists essentially of the full-length form of the protein.

4. The protein of claim 2, wherein at least one of said ORF46, 287, 741, 919, 953, 961 and 983 has a deletion.

5. The protein of claim 4, wherein A and/or B has a poly-glycine deletion (‘ΔG’).

6. The protein of claim 5, wherein A and/or B is ΔG741 or ΔG983 or B is ΔG-287.

7. The protein of claim 4, wherein A and/or B is a truncated protein.

8. The protein of claim 7, wherein B is Δ1-287, Δ2-287, Δ3-287 or Δ4-287.

9. The protein of claim 1, wherein A and B are: (a) 919 and 287; (b) 953 and 287; (c) 287 and ORF46; (d) ORF1 and ORF46; (e) 919 and ORF46; (f) ORF46 and 919; (g) ORF46 and 741; (h) ORF46 and 961; (i) 961 and ORF46; (j) 961 and 741; or (k) 961 and 983.

10. The protein of claim 4, wherein the protein is ΔG287-919, ΔG287-961, ΔG983-ORF46, ΔG983-741, ΔG983-961, ΔG983-961C, ΔG741-961, ΔG741-961C, ΔG741-983, ΔG741-ORF46, ORF46-961C, 961C-ORF46, 961C-741, 961C-983, 961CL-ORF46, 961CL-741 or 961CL-983.

11. The protein of claim 2, wherein B is 287.

12. The protein of claim 1, wherein B is ΔG-287.

13. The protein of claim 12, wherein A is ORF46, 919, 953 or 961.

14. The protein of claim 11, wherein 287 is from strain 2996 or 394/98.

15. The protein of claim 2, wherein A is 961.

16. The protein of claim 1, wherein A and B are from the same strain.

17. The protein of claim 1, wherein A and B are joined directly.

18. The protein of claim 1, wherein A and B are joined via a linker peptide.

19. The protein of claim 18, wherein the linker peptide is a poly-glycine linker, with the proviso that B is not a AG protein.

20. The protein of claim 11, wherein 287 is from strain 2996 or 394/98.

21. The protein of claim 12, wherein 287 is from strain 2996 or 394/98.

22. The protein of claim 13, wherein 287 is from strain 2996 or 394/98.

23. The protein of claim 9, wherein A and B are from the same strain.

24. The protein of claim 10, wherein A and B are from the same strain.

25. The protein of claim 9, wherein A and B are joined directly.

26. The protein of claim 10, wherein A and B are joined directly.

27. The protein of claim 9, wherein A and B are joined via a linker peptide.

28. The protein of claim 10, wherein A and B are joined via a linker peptide.

29. A method for producing the hybrid protein of claim 1 comprising expressing in a cell a nucleic acid encoding the hybrid protein of claim 1.

30. The method of claim 29, wherein A and B are: (a) 919 and 287; (b) 953 and 287; (c) 287 and ORF46; (d) ORF1 and ORF46; (e) 919 and ORF46; (f) ORF46 and 919; (g) ORF46 and 741; (h) ORF46 and 961; (i) 961 and ORF46; (j) 961 and 741; or (k) 961 and 983.

31. The method of claim 29, in which the cell is an E. coli cell.

32. The method of claim 30, in which the cell is an E. coli cell.

33. A composition comprising the hybrid protein of claim 1 and an aluminum salt.

34. The composition of claim 33, wherein A and B are: (a) 919 and 287; (b) 953 and 287; (c) 287 and ORF46; (d) ORF1 and ORF46; (e) 919 and ORF46; (f) ORF46 and 919; (g) ORF46 and 741; (h) ORF46 and 961; (i) 961 and ORF46; (j) 961 and 741; or (k) 961 and 983.