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.

Description: HYBRID EXPRESSION OF NEISSERIAL PROTEINS 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 NH 2 -A — B-COOH. A and B can each be selected from any Neisserial proteins, and in particular those represented by SEQ#s 1-4326. The method is well suited to the expression of proteins orfl, 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 NH 2 -A— B-COOH are preferred: 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. Δl-287, Δ2-287 etc.), or domain-deleted versions may be used (e.g. 287B, 287C, 287BC, ORF46ι- 33, ORF46 433 . 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. G„ 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: 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 2167-4326 [see also WO00/66791]. The term 'protein of the invention' as used herein refers to a protein comprising: (a) one of sequences SEQ#s 1-4326; or (b) a sequence having sequence identity to one of SEQ#s 1-4326; or (c) a fragment of one of SEQ#s 1-4326. 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=l. 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 1-4326 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 1-4326. 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 Figures 1 to 26 show hybrid proteins according to the invention. 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: 1 LGISRKIS I LSILAVCLPM HAHASDLAND SFIRQVLDRQ HFEPDGKYHL 51 FGSRGELAER SGHIGLGKIQ SHQLGNLMIQ QAAIKGNIGY IVRFSDHGHE 101 VHSPFDNHAS HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD GYDGPQGGGY 151 PAPKGARDIY SYDIKGVAQN IR NLTDNRS TGQRLADRFH NAGSMLTQGV 201 GDGFKRATRY SPE DRSGNA AEAFNGTADI VK IIGAAGE IVGAGDAVQG 251 ISEGSNIAVM HGLGLLSTEN K ARIND AD MAQLKDYAAA AIRDWAVQNP 301 NAAQGIEAVS NIFMAAIPIK GIGAVRGKYG LGGITAHPIK RSQMGAIALP 351 KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LEQRYG ENI TSSTVPPSNG 401 KNVKLADQRH PKTGVPFDGK GFPNFEKHVK YDTK DIQE SGGGIPKAKP 451 VSDA PR EV DRK N L.TTR EQVEK VQEI RNGNKNSNFS QHAQ EREIN 501 KLKSADEINF ADGMGKFTDS MNDKAFSRLV KSVKENGFTN PWEYVEING 551 KAYIVRG NR VFAAEY GRI HELKFKKVDF PVPNTSWKNP TDV NESG V 601 RPRYRSK* 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): 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: 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: Again, the hybrid shows equivalent or superior immunological activity. Example 2 - hybrids ofAG287 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: SPDVKS ADT SKPAAP WSEKETEA EDAPQAGSQG QGAPSAQGSQ DMAAVSEENT GNGGAVTADN PKNEDEVAQN DMPQNAAGTD SSTPNHTPDP NMLAGNMENQ ATDAGESSQP A QPD ANAA DG QGDDPSA GGQNAGNTAA QGANQAGN Q AAGSSDPIPA SNPAPANGGS NFGRVDLANG VLIDGPSQNI TLTHCKGDSC SG FLDEEV QLKSEFEKLS DADKIS Y K DGKNDKFVGL VADSVQMKGI NQYIIFYKPK PTSFARFRRS ARSRRSLPAE MPLIPVNQAD TLIVDGEAVS TGHSGNIFA PEGNYRYLTY GAEKLPGGSY ALRVQGEPAK GEMLAGAAVY NGEV HFHTE NGRPYPTRGR FAAKVDFGSK SVDGIIDSGD DLHMGTQKFK AAIDGNGFKG TWTENGSGDV SG FYGPAGE EVAGKYSYRP TDAE GGFGV 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 '287 unta s §ed \ 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 (NMB 1969). 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 ATGGCTAGCCCCGATGTTAAATCGGCGGACACGCTGTCAAAACCGGCCGCTCCTGTTGTTGCTGAAAAAGAGACAGAG GTAAAAGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCACACAAGGCAGCCAAGATATGGCGGCA GTTTCGGCAGAAAATACAGGCAATGGCGGTGCGGCAACAACGGACAAACCCAAAAATGAAGACGAGGGACCGCAAAAT GATATGCCGCAAAATTCCGCCGAATCCGCAAATCAAACAGGGAACAACCAACCCGCCGATTCTTCAGATTCCGCCCCC GCGTCAAACCCTGCACCTGCGAATGGCGGTAGCAATTTTGGAAGGGTTGATTTGGCTAATGGCGTTTTGATTGATGGG CCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAATGGTGATAATTTATTGGATGAAGAAGCACCG TCAAAATCAGAATTTGAAAATTTAAATGAGTCTGAACGAATTGAGAAATATAAGAAAGATGGGAAAAGCGATAAATTT ACTAATTTGGTTGCGACAGCAGTTCAAGCTAATGGAACTAACAAATATGTCATCATTTATAAAGACAAGTCCGCTTCA TCTTCATCTGCGCGATTCAGGCGTTCTGCACGGTCGAGGAGGTCGCTTCCTGCCGAGATGCCGCTAATCCCCGTCAAT CAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGG AATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTGCAAGGCGAACCGGCA AAAGGCGAAATGCTTGCTGGCACGGCCGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTAC CCGACTAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGAT TTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGC GGGGATGTTTCCGGAAGGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCGACAGATGCG GAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGATGCCAAAGCAAGAGC ATCCAAACCTTTCCGCAACCCGACACATCCGTCATCAACGGCCCGGACCGGCCGGTCGGCATCCCCGACCCCGCCGGA ACGACGGTCGGCGGCGGCGGGGCCGTCTATACCGTTGTACCGCACCTGTCCCTGCCCCACTGGGCGGCGCAGGATTTC GCCAAAAGCCTGCAATCCTTCCGCCTCGGCTGCGCCAATTTGAAAAACCGCCAAGGCTGGCAGGATGTGTGCGCCCAA GCCTTTCAAACCCCCGTCCATTCCTTTCAGGCAAAACAGTTTTTTGAACGCTATTTCACGCCGTGGCAGGTTGCAGGC AACGGAAGCCTTGCCGGTACGGTTACCGGCTATTACGAGCCGGTGCTGAAGGGCGACGACAGGCGGACGGCACAAGCC CGCTTCCCGATTTACGGTATTCCCGACGATTTTATCTCCGTCCCCCTGCCTGCCGGTTTGCGGAGCGGAAAAGCCCTT GTCCGCATCAGGCAGACGGGAAAAAACAGCGGCACAATCGACAATACCGGCGGCACACATACCGCCGACCTCTCCCGA TTCCCCATCACCGCGCGCACAACGGCAATCAAAGGCAGGTTTGAAGGAAGCCGCTTCCTCCCCTACCACACGCGCAAC CAAATCAACGGCGGCGCGCTTGACGGCAAAGCCCCGATACTCGGTTACGCCGAAGACCCCGTCGAACTTTTTTTTATG CACATCCAAGGCTCGGGCCGTCTGAAAACCCCGTCCGGCAAATACATCCGCATCGGCTATGCCGACAAAAACGAACAT CCCTACGTTTCCATCGGACGCTATATGGCGGACAAAGGCTACCTCAAGCTCGGGCAGACCTCGATGCAGGGCATCAAA GCCTATATGCGGCAAAATCCGCAACGCCTCGCCGAAGTTTTGGGTCAAAACCCCAGCTATATCTTTTTCCGCGAGCTT GCCGGAAGCAGCAATGACGGTCCCGTCGGCGCACTGGGCACGCCGTTGATGGGGGAATATGCCGGCGCAGTCGACCGG CACTACATTACCTTGGGCGCGCCCTTATTTGTCGCCACCGCCCATCCGGTTACCCGCAAAGCCCTCAACCGCCTGATT ATGGCGCAGGATACCGGCAGCGCGATTAAAGGCGCGGTGCGCGTGGATTATTTTTGGGGATACGGCGACGAAGCCGGC GAACTTGCCGGCAAACAGAAAACCACGGGTTACGTCTGGCAGCTCCTACCCAACGGTATGAAGCCCGAATACCGCCCG TAACTCGAG 1 MASPDVKSAD TLSKPAAPW AEKETEVKED APQAGSQGQG APSTQGSQDM 51 AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESA Q TGNNQPADSS 101 DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNIT THC KGDSCNGDNL 151 DEEAPSKSE FEN NESERI EKYKKDGKSD KFTNLVATAV QA GT KYVI 201 IYKDKSASSS SARFRRSARS RRS PAE PL IPVNQADTLI VDGEAVSLTG 251 HSGNIFAPEG NYRYLTYGAE K PGGSYALR VQGEPAKGE AGTAVYNGE 301 VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDD H MGTQKFKAAI 351 DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG 401 KEQDGSGGG GCQSKSIQTF PQPDTSVING PDRPVGIPDP AGTTVGGGGA 451 VYTWPHLSL PH AAQDFAK S QSFRLGCA N KNRQGWQD VCAQAFQTPV 501 HSFQAKQFFE RYFTPWQVAG NGSLAGTVTG YYEPV KGDD RRTAQARFPI 551 YGIPDDFISV PLPAGLRSGK A VRIRQTGK NSGTIDNTGG THTADLSRFP 601 ITARTTAIKG RFEGSRFLPY HTRNQINGGA LDGKAPILGY AEDPVELFFM 651 HIQGSGRLKT PSGKYIRIGY ADKNEHPYVS IGRYMADKGY LK GQTSMQG 701 IKAYMRQNPQ RLAEVLGQNP SYIFFRELAG SSNDGPVGAL GTPLMGEYAG 751 AVDRHYIT G AP FVATAHP VTRKA NR I MAQDTGSAIK GAVRVDYFWG 801 YGDEAGELAG KQKTTGYVWQ L PNGMKPEY RP* ΔG287-953 ATGGCTAGCCCCGATGTTAAATCGGCGGACACGCTGTCAAAACCGGCCGCTCCTGTTGTTGCTGAAAAAGAGACAGAG GTAAAAGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCACACAAGGCAGCCAAGATATGGCGGCA GTTTCGGCAGAAAATACAGGCAATGGCGGTGCGGCAACAACGGACAAACCCAAAAATGAAGACGAGGGACCGCAAAAT GATATGCCGCAAAATTCCGCCGAATCCGCAAATCAAACAGGGAACAACCAACCCGCCGATTCTTCAGATTCCGCCCCC GCGTCAAACCCTGCACCTGCGAATGGCGGTAGCAATTTTGGAAGGGTTGATTTGGCTAATGGCGTTTTGATTGATGGG CCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAATGGTGATAATTTATTGGATGAAGAAGCACCG TCAAAATCAGAATTTGAAAATTTAAATGAGTCTGAACGAATTGAGAAATATAAGAAAGATGGGAAAAGCGATAAATTT ACTAATTTGGTTGCGACAGCAGTTCAAGCTAATGGAACTAACAAATATGTCATCATTTATAAAGACAAGTCCGCTTCA TCTTCATCTGCGCGATTCAGGCGTTCTGCACGGTCGAGGAGGTCGCTTCCTGCCGAGATGCCGCTAATCCCCGTCAAT CAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGG AATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTGCAAGGCGAACCGGCA AAAGGCGAAATGCTTGCTGGCACGGCCGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTAC CCGACTAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGAT TTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGC GGGGATGTTTCCGGAAGGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCGACAGATGCG GAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGAGCCACCTACAAAGTG GACGAATATCACGCCAACGCCCGTTTCGCCATCGACCATTTCAACACCAGCACCAACGTCGGCGGTTTTTACGGTCTG ACCGGTTCCGTCGAGTTCGACCAAGCAAAACGCGACGGTAAAATCGACATCACCATCCCCGTTGCCAACCTGCAAAGC GGTTCGCAACACTTTACCGACCACCTGAAATCAGCCGACATCTTCGATGCCGCCCAATATCCGGACATCCGCTTTGTT TCCACCAAATTCAACTTCAACGGCAAAAAACTGGTTTCCGTTGACGGCAACCTGACCATGCACGGCAAAACCGCCCCC GTCAAACTCAAAGCCGAAAAATTCAACTGCTACCAAAGCCCGATGGCGAAAACCGAAGTTTGCGGCGGCGACTTCAGC ACCACCATCGACCGCACCAAATGGGGCGTGGACTACCTCGTTAACGTTGGTATGACCAAAAGCGTCCGCATCGACATC CAAATCGAGGCAGCCAAACAATAACTCGAG 1 MASPDVKSAD TLSKPAAPW 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 DGNGFKGT T 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 ATGGCTAGCCCCGATGTTAAATCGGCGGACACGCTGTCAAAACCGGCCGCTCCTGTTGTTGCTGAAAAAGAGACAGAG GTAAAAGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCACACAAGGCAGCCAAGATATGGCGGCA GTTTCGGCAGAAAATACAGGCAATGGCGGTGCGGCAACAACGGACAAACCCAAAAATGAAGACGAGGGACCGCAAAAT GATATGCCGCAAAATTCCGCCGAATCCGCAAATCAAACAGGGAACAACCAACCCGCCGATTCTTCAGATTCCGCCCCC GCGTCAAACCCTGCACCTGCGAATGGCGGTAGCAATTTTGGAAGGGTTGATTTGGCTAATGGCGTTTTGATTGATGGG CCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGTAATGGTGATAATTTATTGGATGAAGAAGCACCG TCAAAATCAGAATTTGAAAATTTAAATGAGTCTGAACGAATTGAGAAATATAAGAAAGATGGGAAAAGCGATAAATTT ACTAATTTGGTTGCGACAGCAGTTCAAGCTAATGGAACTAACAAATATGTCATCATTTATAAAGACAAGTCCGCTTCA TCTTCATCTGCGCGATTCAGGCGTTCTGCACGGTCGAGGAGGTCGCTTCCTGCCGAGATGCCGCTAATCCCCGTCAAT CAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGCCTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGG AATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCCGGCGGATCGTATGCCCTCCGTGTGCAAGGCGAACCGGCA AAAGGCGAAATGCTTGCTGGCACGGCCGTGTACAACGGCGAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTAC CCGACTAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGCAGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGAT TTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGATGGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGC GGGGATGTTTCCGGAAGGTTTTACGGCCCGGCCGGCGAGGAAGTGGCGGGAAAATACAGCTATCGCCCGACAGATGCG GAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAGCAGGATGGATCCGGAGGAGGAGGAGCCACAAACGACGAC GATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGA GAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGAC TTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCC AAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACT GATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAG ACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGAT ATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCC GAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGC ACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAAC AAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATT GATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACT CGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTC TCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCA GTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGT TCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGTAACTCGAG 1 MASPDVKSAD TLSKPAAPW 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 GLGLKKWTN 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* 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: Data for bactericidal activity against heterologous MenB strains and against serotypes A and C were also obtained: 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 ATGGCTAGCCCCGATGTCAAGTCGGCGGACACGCTGTCAAAACCTGCCGCCCCTGTTGTTTCTGAAAAAGAGACAGAG GCAAAGGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCGCACAAGGCGGTCAAGATATGGCGGCG GTTTCGGAAGAAAATACAGGCAATGGCGGTGCGGCAGCAACGGACAAACCCAAAAATGAAGACGAGGGGGCGCAAAAT GATATGCCGCAAAATGCCGCCGATACAGATAGTTTGACACCGAATCACACCCCGGCTTCGAATATGCCGGCCGGAAAT ATGGAAAACCAAGCACCGGATGCCGGGGAATCGGAGCAGCCGGCAAACCAACCGGATATGGCAAATACGGCGGACGGA ATGCAGGGTGACGATCCGTCGGCAGGCGGGGAAAATGCCGGCAATACGGCTGCCCAAGGTACAAATCAAGCCGAAAAC AATCAAACCGCCGGTTCTCAAAATCCTGCCTCTTCAACCAATCCTAGCGCCACGAATAGCGGTGGTGATTTTGGAAGG ACGAACGTGGGCAATTCTGTTGTGATTGACGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGT AGTGGCAATAATTTCTTGGATGAAGAAGTACAGCTAAAATCAGAATTTGAAAAATTAAGTGATGCAGACAAAATAAGT AATTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGTCGGTTTGGTTGCCGATAGTGTGCAGATGAAG GGAATCAATCAATATATTATCTTTTATAAACCTAAACCCACTTCATTTGCGCGATTTAGGCGTTCTGCACGGTCGAGG CGGTCGCTTCCGGCCGAGATGCCGCTGATTCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGC CTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCC GGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACCTTCAAAAGGCGAAATGCTCGCGGGCACGGCAGTGTACAACGGC GAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTCCCCGTCCAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGC AGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGGTTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGAT GGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAAGTTTTACGGCCCGGCCGGCGAG GAAGTGGCGGGAAAATACAGCTATCGCCCAACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAG CAGGATGGATCCGGAGGAGGAGGATGCCAAAGCAAGAGCATCCAAACCTTTCCGCAACCCGACACATCCGTCATCAAC GGCCCGGACCGGCCGGTCGGCATCCCCGACCCCGCCGGAACGACGGTCGGCGGCGGCGGGGCCGTCTATACCGTTGTA CCGCACCTGTCCCTGCCCCACTGGGCGGCGCAGGATTTCGCCAAAAGCCTGCAATCCTTCCGCCTCGGCTGCGCCAAT TTGAAAAACCGCCAAGGCTGGCAGGATGTGTGCGCCCAAGCCTTTCAAACCCCCGTCCATTCCTTTCAGGCAAAACAG TTTTTTGAACGCTATTTCACGCCGTGGCAGGTTGCAGGCAACGGAAGCCTTGCCGGTACGGTTACCGGCTATTACGAG CCGGTGCTGAAGGGCGACGACAGGCGGACGGCACAAGCCCGCTTCCCGATTTACGGTATTCCCGACGATTTTATCTCC GTCCCCCTGCCTGCCGGTTTGCGGAGCGGAAAAGCCCTTGTCCGCATCAGGCAGACGGGAAAAAACAGCGGCACAATC GACAATACCGGCGGCACACATACCGCCGACCTCTCCCGATTCCCCATCACCGCGCGCACAACGGCAATCAAAGGCAGG TTTGAAGGAAGCCGCTTCCTCCCCTACCACACGCGCAACCAAATCAACGGCGGCGCGCTTGACGGCAAAGCCCCGATA CTCGGTTACGCCGAAGACCCCGTCGAACTTTTTTTTATGCACATCCAAGGCTCGGGCCGTCTGAAAACCCCGTCCGGC AAATACATCCGCATCGGCTATGCCGACAAAAACGAACATCCCTACGTTTCCATCGGACGCTATATGGCGGACAAAGGC TACCTCAAGCTCGGGCAGACCTCGATGCAGGGCATCAAAGCCTATATGCGGCAAAATCCGCAACGCCTCGCCGAAGTT TTGGGTCAAAACCCCAGCTATATCTTTTTCCGCGAGCTTGCCGGAAGCAGCAATGACGGTCCCGTCGGCGCACTGGGC ACGCCGTTGATGGGGGAATATGCCGGCGCAGTCGACCGGCACTACATTACCTTGGGCGCGCCCTTATTTGTCGCCACC GCCCATCCGGTTACCCGCAAAGCCCTCAACCGCCTGATTATGGCGCAGGATACCGGCAGCGCGATTAAAGGCGCGGTG CGCGTGGATTATTTTTGGGGATACGGCGACGAAGCCGGCGAACTTGCCGGCAAACAGAAAACCACGGGTTACGTCTGG CAGCTCCTACCCAACGGTATGAAGCCCGAATACCGCCCGTAAAAGCTT 1 MASPDVKSAD TLSKPAAPW SEKETEAKED APQAGSQGQG APSAQGGQDM 51 AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM 101 PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG 151 TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSW 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 GSGGGGCQSK SIQTFPQPDT SVINGPDRPV 501 GIPDPAGTTV GGGGAVYTW PHLSLPHWAA QDFAKSLQSF RLGCANLKNR 551 QGWQDVCAQA FQTPVHSFQA KQFFERYFTP WQVAGNGSLA GTVTGYYEPV 601 LKGDDRRTAQ ARFPIYGIPD DFISVPLPAG LRSGKALVRI RQTGKNSGTI 651 DNTGGTHTAD LSRFPITART TAIKGRFEGS 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 ATGGCTAGCCCCGATGTCAAGTCGGCGGACACGCTGTCAAAACCTGCCGCCCCTGTTGTTTCTGAAAAAGAGACAGAG GCAAAGGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCGCACAAGGCGGTCAAGATATGGCGGCG GTTTCGGAAGAAAATACAGGCAATGGCGGTGCGGCAGCAACGGACAAACCCAAAAATGAAGACGAGGGGGCGCAAAAT GATATGCCGCAAAATGCCGCCGATACAGATAGTTTGACACCGAATCACACCCCGGCTTCGAATATGCCGGCCGGAAAT ATGGAAAACCAAGCACCGGATGCCGGGGAATCGGAGCAGCCGGCAAACCAACCGGATATGGCAAATACGGCGGACGGA ATGCAGGGTGACGATCCGTCGGCAGGCGGGGAAAATGCCGGCAATACGGCTGCCCAAGGTACAAATCAAGCCGAAAAC AATCAAACCGCCGGTTCTCAAAATCCTGCCTCTTCAACCAATCCTAGCGCCACGAATAGCGGTGGTGATTTTGGAAGG ACGAACGTGGGCAATTCTGTTGTGATTGACGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGT AGTGGCAATAATTTCTTGGATGAAGAAGTACAGCTAAAATCAGAATTTGAAAAATTAAGTGATGCAGACAAAATAAGT AATTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGTCGGTTTGGTTGCCGATAGTGTGCAGATGAAG GGAATCAATCAATATATTATCTTTTATAAACCTAAACCCACTTCATTTGCGCGATTTAGGCGTTCTGCACGGTCGAGG CGGTCGCTTCCGGCCGAGATGCCGCTGATTCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGC CTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCC GGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACCTTCAAAAGGCGAAATGCTCGCGGGCACGGCAGTGTACAACGGC GAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTCCCCGTCCAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGC AGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGGTTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGAT GGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAAGTTTTACGGCCCGGCCGGCGAG GAAGTGGCGGGAAAATACAGCTATCGCCCAACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAG CAGGATGGATCCGGAGGAGGAGGAGCCACCTACAAAGTGGACGAATATCACGCCAACGCCCGTTTCGCCATCGACCAT TTCAACACCAGCACCAACGTCGGCGGTTTTTACGGTCTGACCGGTTCCGTCGAGTTCGACCAAGCAAAACGCGACGGT AAAATCGACATCACCATCCCCGTTGCCAACCTGCAAAGCGGTTCGCAACACTTTACCGACCACCTGAAATCAGCCGAC ATCTTCGATGCCGCCCAATATCCGGACATCCGCTTTGTTTCCACCAAATTCAACTTCAACGGCAAAAAACTGGTTTCC GTTGACGGCAACCTGACCATGCACGGCAAAACCGCCCCCGTCAAACTCAAAGCCGAAAAATTCAACTGCTACCAAAGC CCGATGGCGAAAACCGAAGTTTGCGGCGGCGACTTCAGCACCACCATCGACCGCACCAAATGGGGCGTGGACTACCTC GTTAACGTTGGTATGACCAAAAGCGTCCGCATCGACATCCAAATCGAGGCAGCCAAACAATAAAAGCTT 1 MASPDVKSAD TLSKPAAPW SEKETEAKED APQAGSQGQG APSAQGGQDM 51 AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM 101 PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG 151 TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSW 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 ATGGCTAGCCCCGATGTCAAGTCGGCGGACACGCTGTCAAAACCTGCCGCCCCTGTTGTTTCTGAAAAAGAGACAGAG GCAAAGGAAGATGCGCCACAGGCAGGTTCTCAAGGACAGGGCGCGCCATCCGCACAAGGCGGTCAAGATATGGCGGCG GTTTCGGAAGAAAATACAGGCAATGGCGGTGCGGCAGCAACGGACAAACCCAAAAATGAAGACGAGGGGGCGCAAAAT GATATGCCGCAAAATGCCGCCGATACAGATAGTTTGACACCGAATCACACCCCGGCTTCGAATATGCCGGCCGGAAAT ATGGAAAACCAAGCACCGGATGCCGGGGAATCGGAGCAGCCGGCAAACCAACCGGATATGGCAAATACGGCGGACGGA ATGCAGGGTGACGATCCGTCGGCAGGCGGGGAAAATGCCGGCAATACGGCTGCCCAAGGTACAAATCAAGCCGAAAAC AATCAAACCGCCGGTTCTCAAAATCCTGCCTCTTCAACCAATCCTAGCGCCACGAATAGCGGTGGTGATTTTGGAAGG ACGAACGTGGGCAATTCTGTTGTGATTGACGGGCCGTCGCAAAATATAACGTTGACCCACTGTAAAGGCGATTCTTGT AGTGGCAATAATTTCTTGGATGAAGAAGTACAGCTAAAATCAGAATTTGAAAAATTAAGTGATGCAGACAAAATAAGT AATTACAAGAAAGATGGGAAGAATGACGGGAAGAATGATAAATTTGTCGGTTTGGTTGCCGATAGTGTGCAGATGAAG GGAATCAATCAATATATTATCTTTTATAAACCTAAACCCACTTCATTTGCGCGATTTAGGCGTTCTGCACGGTCGAGG CGGTCGCTTCCGGCCGAGATGCCGCTGATTCCCGTCAATCAGGCGGATACGCTGATTGTCGATGGGGAAGCGGTCAGC CTGACGGGGCATTCCGGCAATATCTTCGCGCCCGAAGGGAATTACCGGTATCTGACTTACGGGGCGGAAAAATTGCCC GGCGGATCGTATGCCCTCCGTGTTCAAGGCGAACCTTCAAAAGGCGAAATGCTCGCGGGCACGGCAGTGTACAACGGC GAAGTGCTGCATTTTCATACGGAAAACGGCCGTCCGTCCCCGTCCAGAGGCAGGTTTGCCGCAAAAGTCGATTTCGGC AGCAAATCTGTGGACGGCATTATCGACAGCGGCGATGGTTTGCATATGGGTACGCAAAAATTCAAAGCCGCCATCGAT GGAAACGGCTTTAAGGGGACTTGGACGGAAAATGGCGGCGGGGATGTTTCCGGAAAGTTTTACGGCCCGGCCGGCGAG GAAGTGGCGGGAAAATACAGCTATCGCCCAACAGATGCGGAAAAGGGCGGATTCGGCGTGTTTGCCGGCAAAAAAGAG CAGGATGGATCCGGAGGAGGAGGAGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCC TACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACC AAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTG ACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACA ACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAA TTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTG GCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGAC GAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCT GCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCT GCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTG TACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGC TTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGC AAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAAT GTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTT GCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGG TAAAAGCTT 1 MASPDVKSAD TLSKPAAPW SEKETEAKED APQAGSQGQG APSAQGGQDM 51 AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM 101 PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG 151 TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSW 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 KWTNLTKTV 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 LDTRLASAΞK SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY 801 NVGRFNVTAA VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY 851 HVGVNYEW* Example 3 - hybrids ofΔG983 Protein 983 has the following sequence: 983 + ■ 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 NMLFIFSTGN 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 9.51 GGLRYDLLKQ DAFAEKGSAL GWSGNSLTEG TLVGLAGLKL SQPLSDKAVL 1001 FATAGVERDL NGRDYTVTGG FTGATAATGK TGARNMPHTR LVAGLGADVE 1051 FGNGWNGLAR YSYAGSKQYG NHSGRVGVGY RF* ΔG983 thus has the following basic sequence: 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 NMLFIFSTGN 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-QRF46.1 ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCA GCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCG GTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCA TACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGAC ACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAA AACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAG GCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCAT ATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACG AATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGC ATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAG TACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTAC GGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCC AACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGT GGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATT ACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGA ACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAAC CTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTG GATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGAT ATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCAC GGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATG CGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTAT CTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACG CTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGC GGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTAT TCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAA GGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCC GGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCC GCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTC CGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTAT GCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGC ACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGC GGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGC AGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGC GATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAA CATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACG GGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTG GGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGAT AAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACC GGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGAT GTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGA CGAGTCGGCGTAGGCTACCGGTTCCTCGACGGTGGCGGAGGCACTGGATCCTCAGATTTGGCAAACGATTCTTTTATC CGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCCGAG CGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATTAAA GGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCACAT TCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACACCAT CCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTACGAC ATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGTTTC CACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTGGAC AGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAAATT GTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTTTCC ACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATCCGC GATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCCATC AAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATGGGC GCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCCCCT TACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCGCCG TCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTTCCG AATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCACTGA 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 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 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* ΔG983-741 ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCA GCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCG GTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCA TACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGAC ACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAA AACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAG GCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCAT ATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACG AATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGC ATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAG TACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTAC GGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCC AACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGT GGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATT ACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGA ACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAAC CTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTG GATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGAT ATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCAC GGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATG CGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTAT CTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACG CTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGC GGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTAT TCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAA GGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCC GGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCC GCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTC CGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTAT GCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGC ACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGC GGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGC AGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGC GATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAA CATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACG GGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTG GGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGAT AAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACC GGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGAT GTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGA CGAGTCGGCGTAGGCTACCGGTTCCTCGAGGGATCCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCC GATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAAC GAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAG AACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAG TTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGG AAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGC GGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCC GCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAG CCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGT ATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATCGGCCTT GCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA 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 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 KMVAKRQFRI GDIAGEHTSF DKLPEGGRAT 1201 YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD 1251 GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK TVNGIRHIGL 1301 AAKQLEHHHH HH* ΔG983-961 ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCA GCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCG GTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCA TACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGAC ACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAA AACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAG GCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCAT ATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACG AATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGC ATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAG TACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTAC GGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCC AACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGT GGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATT ACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGA ACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAAC CTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTG GATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGAT ATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCAC GGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATG CGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTAT CTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACG CTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGC GGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTAT TCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAA GGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCC CATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACG GGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTG GGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGAT AAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACC GGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGAT GTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGA CGAGTCGGCGTAGGCTACCGGTTCCTCGAGGGTGGCGGAGGCACTGGATCCGCCACAAACGACGACGATGTTAAAAAA GCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTAC GACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTG GGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCT GCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTG GATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTA AAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCA TTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAA CAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACT GCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATT GCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAAC GCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGT TTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTC CAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGAATCGGCAGTCGCCATCGGT ACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTTCGTCCGGTTCTTCCGCAGCC TACCATGTCGGCGTCAATTACGAGTGGCTCGAGCACCACCACCACCACCACTGA 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 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 KKWTNLTKT 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 ATGACTTCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCA GCAGTATCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCG GTTACAGACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCA TACAAGAATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGAC ACAGGCGAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAA AACTATACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAG GCCGTTATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCAT ATTATTGGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACG AATGATGAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGC ATCGTCAATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAG TACCGCCAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTAC GGCAACCTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCC AACACATATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGT GGAGAAAAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATT ACTGCCATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGA ACATCCTTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAAC CTGCGTACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTG GATGCGGGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGAT ATTGCCTACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCAC GGCAACAACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATG CGCGTCGAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTAT CTGGCAGATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACG CTGTACACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGC GGCAAGGGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTAT TCTTTCTTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAA GGCGACACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCC GGTCTGAAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCC GCAACACCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTC CGCGCAGCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTAT GCCGACAGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGC ACGGGTCTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGC GGCAGTACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGC AGCACATGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGC GATATCGGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAA CATGCGGAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACG GGAGATTTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTG GGCTGGAGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGAT AAAGCCGTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACC GGCGCGACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGAT GTCGAATTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGA CGAGTCGGCGTAGGCTACCGGTTCCTCGAGGGTGGCGGAGGCACTGGATCCGCCACAAACGACGACGATGTTAAAAAA GCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTAC GACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTG GGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCT GCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTG GATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTA AAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCA TTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAA CAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACT GCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGATAATATT GCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTCAGAATTGATGGTCTGAAC GCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCACGATACTCGCCTGAACGGT TTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCCGCGCTCTCCGGTCTGTTC CAACCTTACAACGTGGGTCTCGAGCACCACCACCACCACCACTGA 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 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 KKWTNLTKT 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 ofΔG741 Protein 741 has the following sequence: 1 VNRTAFCCLS LTTALILTAC SSGGGGVAAD IGAGLADALT APLDHKDKGL 51 QSLTLDQSVR KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ 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: VAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ 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 ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAG TCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGA AACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTG GACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAG ACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGC GAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCC GGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAA CTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTAC AACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTG AAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGGGTGGCGGAGGCACTGGATCCGCCACA AACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTC AAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAA GCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAAC GTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTA GCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAA GAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCA TTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAA CAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCT GCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATC GCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTT GTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGAT CACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAA GCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCC GAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACT TCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGCTCGAGCACCACCACCACCACCACTGA 1 MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT 51 YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL 101 TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA 151 FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA 201 VISGSVLYNQ AEKGSYSLGI FGGKAQΞVAG SAEVKTVNGI RHIGLAAKQL 251 EGGGGTGSAT NDDDVKKAAT VAIAAAYNNG QEINGFKAGE TIYDIDEDGT 301 ITKKDATAAD VEADDFKGLG LKKWTNLTK 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* ΔG7 1-961C ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAG TCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGA AACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTG GACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAG ACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGC GAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCC GGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAA CTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTAC AACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTG AAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGGGTGGCGGAGGCACTGGATCCGCCACA AACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTC AAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAA GCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAAC GTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTA GCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAA GAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCA TTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAA CAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCT GCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATC GCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTT GTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGAT CACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAA GCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCTCGAGCACCACCACCACCACCACTGA 1 MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT 51 YGNGDSLNTG KLKNDKVSRF 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 LKKWTNLTK 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 ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAG TCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGA AACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTG GACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAG ACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGC GAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCC GGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAA CTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTAC AACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTG AAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGGGATCCGGCGGAGGCGGCACTTCTGCG CCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTAC GCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGG GATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTG ATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCC GTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCG TATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAG ACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGG CGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACC AAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAAC AGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCG TTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCC TACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCC CTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTC AAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGG TGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCC GCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACG TTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAG GCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCC TTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACC TATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACC AAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACC GACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGT TTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCA GGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACA AACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTG TCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACAC GCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAG ACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCA GCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACC GCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGC GTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAA ACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGC GAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTAT CTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGC AGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACG GTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGC AACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTG TTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCA GCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGC AACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTA GGCTACCGGTTCCTCGAGCACCACCACCACCACCACTGA 1 MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT 51 YGNGDSLNTG KLKNDKVSRF 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 SYYVRRGNAA 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 EGTLVGLAGL KLSQPLSDKA VLFATAGVER DLNGRDYTVT GGFTGATAAT 1251 GKTGARNMPH TRLVAGLGAD VEFGNGWNGL ARYSYAGSKQ YGNHSGRVGV 1301 GYRFLEHHHH HH* ΔG741-ORF46.1 ATGGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAG TCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGA AACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTG GACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAG ACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGC GAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCC GGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAA CTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTAC AACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTG AAAACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGACGGTGGCGGAGGCACTGGATCCTCAGAT TTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGC AGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATG ATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCC TTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCAT TGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCG AGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGA CAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACC CGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATC ATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATG CACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGAC TATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATC TTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCT ATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCA TACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATC ACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCG TTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCACTGA 1 MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT 51 YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL 101 TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA 151 FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PΞLNVDLAAA DIKPDGKRHA 201 VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL 251 DGGGGTGSSD LANDSFIRQV LDRQHFEPDG KYHLFGSRGE LAERSGHIGL 301 GKIQSHQLGN LMIQQAAIKG NIGYIVRFSD HGHEVHSPFD NHASHSDSDE 351 AGSPVDGFSL YRIHWDGYEH HPADGYDGPQ GGGYPAPKGA RDIYSYDIKG 401 VAQNIRLNLT DNRSTGQRLA DRFHNAGSML TQGVGDGFKR ATRYSPELDR 451 SGNAAEAFNG TADIVKNIIG AAGEIVGAGD AVQGISEGSN I VMHGLGLL 501 STENKMARIN 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 Ndel and a BamBI restriction sites respectively, whereas for the amplification of the 287 gene the 5' and the 3' primers included a BamHL and a Xhol restriction sites respectively. In this way a sequential directional cloning of the two genes in pET21b+, using Ndel-BamHl (to clone the first gene) and subsequently Bamϋl-Xhoϊ (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 Xhol 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 Sail restriction site at the 5'- and a Xhol site at the 3'- of the PCR fragment. Since the cohesive ends produced by the Sαfl and Xhol restriction enzymes are compatible, the 287 PCR product digested with Sall-Xhol could be inserted in the pET21b-919 clone cleaved with Xhol. 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: Data for bactericidal activity against heterologous MenB strains and against serotypes A and C were also obtained for 919-287 and 953-287: 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 fitres and bactericidal antibody responses. As hybrids of two proteins A & B may be either NH 2 -A-B-COOH or NH 2 -B-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: Better bactericidal titres are generally seen with 287 at the N-terminus. When fused to protein 961 [NH 2 -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): Titres with the insoluble form were, however, improved by using alum adjuvant instead: 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 23 -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: QRF46.1-741 ATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCAC CTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGC AACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTC CATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTAC CGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCC AAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGC AGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAA CGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTT AAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATT GCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAA CTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTC AGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACG GCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCC GATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAA GAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACA GGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGGGATCCGGAGGGGGTGGT GTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCT TTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAAC GGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGAC GGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACC GAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAA CATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGC GGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTC AATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAAC CAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAA ACCGTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA 1 MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ 51 LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDΞAGSPVDG 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* QRF46.1-961 ATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCAC CTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGC AACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTC CATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTAC CGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCC AAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGC AGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAA CGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTT AAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATT GCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAA CTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTC AGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACG GCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCC GATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAA GAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACA GGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGGGATCCGGAGGAGGAGGA GCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAAC GGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGAT GTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAA CAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCC GCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTT GCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCC GAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAA GCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCC GAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCT GATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGC AAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATT GCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCA GAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTAC AAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTC GGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGCTCGAGCACCACCACCACCACCAC TGA 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 KWTNLTKTV NENKQNVDAK 501 VKAAESEIEK LTTKLADTDA ALADTDAALD 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* 0RF46.1-961C ATGTCAGATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCAC CTATTCGGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGC AACCTGATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTC CATTCCCCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTAC CGCATCCATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCC AAAGGCGCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGC AGCACCGGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAA CGCGCCACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTT AAAAACATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATT GCTGTCATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAA CTCAAAGACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTC AGCAATATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACG GCACATCCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCC GATGCGGCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAA GAAAACATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACA GGCGTACCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGGGATCCGGAGGAGGAGGA GCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAAC GGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGAT GTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAA CAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCC GCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTT GCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCC GAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAA GCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCC GAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCT GATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGC AAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATT GCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCA GAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCTCGAGCACCACCACCACCACCACTGA 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 KWTNLTKTV NENKQNVDAK 501 VKAAESEIEK LTTKLADTDA ALADTDAALD 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-QRF46.1 ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATC AACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCC GATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAAC AAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGAT GCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACA TTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCAT GCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAAT GAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAA GCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAA GCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGAC AGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCC ATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTT GCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGC TACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCA GTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGGGATCCGGAGGAGGAGGATCA GATTTGGCAAACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTC GGCAGCAGGGGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTG ATGATTCAACAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCC CCCTTCGACAACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATC CATTGGGACGGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGC GCGAGGGATATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACC GGACAACGGCTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCC ACCCGATACAGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAAC ATCATCGGCGCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTC ATGCACGGCTTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAA GACTATGCCGCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAAT ATCTTTATGGCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACAT CCTATCAAGCGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCG GCATACGCCAAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAAC ATCACCTCCTCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTA CCGTTTGACGGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCAC TGA 1 MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT 51 AADVEADDFK GLGLKKWTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL 101 ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA 1 15511 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 4 40011 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 6 65511 DYAAAAIRDW AVQNPNAAQG IEAVSNIFMA AIPIKGIGAV RGKYGLGGIT 701 AHPIKRSQMG AIALPKGKSA VSDNFADAAY AKYPSPYHSR NIRSNLEQRY 751 GKΞNITSSTV PPSNGKNVKL ADQRHPKTGV PFDGKGFPNF EKHVKYDTLE 801 HHHHHH* 961-741 ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATC AACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCC GATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAAC AAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGAT GCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACA TTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCAT GCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAAT GAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAA GCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAA GCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGAC AGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCC ATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTT GCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGC TACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCA GTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGGGATCCGGAGGGGGTGGTGTC GCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTG ACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGT GACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGG CAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAG CAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACAT ACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGA AAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAAT GTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAA GCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACC GTAAACGGCATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA 1 MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT 51 AADVEADDFK GLGLKKWTN 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 NGDSLNTGKL 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 ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATC AACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCC GATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAAC AAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGAT GCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACA TTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCAT GCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAAT GAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAA GCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAA GCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGAC AGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCC ATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTT GCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGC TACAAATCCGAATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCA GTCGGCACTTCGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGGGATCCGGCGGAGGCGGCACT TCTGCGCCCGACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTA TCTTACGCCGGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACA GACAGGGATGCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAG AATTTGATCAACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGC GAATCCGTCGGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTAT ACGGCGTATATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTT ATAGAGACTGAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATT GGCGGGCGTTCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGAT GAAACCAAGAACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTC AATAACAGTTTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGC CAAGCGTTGCTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAAC CTGTCCTACCACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACA TATGCCCTATTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAA AAGTTCAAACGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCC ATGTGGTGCCTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCC TTTTCCGCACCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGT ACCACGTTGCTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCG GGTAAGGCCATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCC TACTCCTTCCGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAAC AACACCTATACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTC GAAACCAAAGGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCA GATACCGACCAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTAC ACACGTTTGGGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAG GGGGCAGGCTATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTC TTCACAAACATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGAC ACGCTGTCCTATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTG AAACACGCCGTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACA CCCGAGACGGTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCA GCGGCAGCCGTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGAC AGTACCGCCGCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGT CTGCGCGTCATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGT ACCCAAACCGTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACA TGGAGCGAAAACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATC GGCTATCTCAAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCG GAAGGCAGCGTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGAT TTGACGGTCGAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGG AGCGGCAACAGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCC GTCCTGTTTGCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCG ACTGCAGCAACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAA TTCGGCAACGGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTC GGCGTAGGCTACCGGTTCCTCGAGCACCACCACCACCACCACTGA 1 MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT 51 AADVEADDFK GLGLKKWTN 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 FDDEAVIETE 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 ETKGA IYNG AASGGSLNSD GIVYLADTDQ SGANETVHIK 951 GSLQLDGKGT LYTRLGKLLK VDGTAIIGGK LYMSARGKGA 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 LKGLFSYGRY KNSISRSTGA DEHAEGSVNG TLMQLGALGG VNVPFAATGD 1301 LTVEGGLRYD LLKQDAFAEK GSALGWSGNS LTEGTLVGLA GLKLSQPLSD 1351 KAVLFATAGV ERDLNGRDYT VTGGFTGATA ATGKTGARNM PHTRLVAGLG 1401 ADVEFGNGWN GLARYSYAGS KQYGNHSGRV GVGYRFLEHH HHHH* 961C-ORF46.1 ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATC AACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCC GATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAAC AAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGAT GCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACA TTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCAT GCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAAT GAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAA GCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAA GCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGAC AGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCC ATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTT GCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGAGGAGGATCAGATTTGGCA AACGATTCTTTTATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGG GGGGAACTTGCCGAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAA CAGGCGGCCATTAAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGAC AACCATGCCTCACATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGAC GGATACGAACACCATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGAT ATATACAGCTACGACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGG CTTGCCGACCGTTTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATAC AGCCCCGAGCTGGACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGC GCGGCAGGAGAAATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGC TTGGGTCTGCTTTCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCC GCAGCAGCCATCCGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATG GCAGCCATCCCCATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAG CGGTCGCAGATGGGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCC AAATACCCGTCCCCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCC TCAACCGTGCCGCCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGAC GGTAAAGGGTTTCCGAATTTTGAGAAGCACGTGAAATATGATACGCTCGAGCACCACCACCACCACCACTGA 1 MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT 51 AADVEADDFK GLGLKKWTN 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 ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATC AACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCC GATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAAC AAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGAT GCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACA TTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCAT GCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAAT GAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAA GCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAA GCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGAC AGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCC ATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTT GCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGGGGTGGTGTCGCCGCCGAC ATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGAT CAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTC AATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATT ACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAA GATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTT GACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACC TACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTG GCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAA GGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGC ATACGCCATATCGGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA 1 MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT 51 AADVEADDFK GLGLKKWTN 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 LGIFGGKAQE VAGSAEVKTV NGIRHIGLAA KQLEHHHHHH * 961C-983 ATGGCCACAAACGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATC AACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCC GATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAAC AAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGAT GCCGCTTTAGCAGATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACA TTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCAT GCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAAT GAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAA GCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAA GCTGATATCGCTACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGAC AGCAAATTTGTCAGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCC ATTGCCGATCACGATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTT GCAGAACAAGCCGCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGCGGAGGCGGCACTTCTGCGCCC GACTTCAATGCAGGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCC GGTATCAAGAACGAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGAT GCCAAAATCAATGCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATC AACCTCAAACCTGCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTC GGCAGCATATCCTTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTAT ATGCGGAAGGAAGCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACT GAAGCAAAGCCGACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGT TCCGTGGACGGCAGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAG AACGAAATGATGGTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGT TTTGGAACAACATCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTG CTCGACTATTCCGGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTAC CACATCCGTAATAAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTA TTGCCATTTTATGAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAA CGGGAAATGTATGGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGC CTGTCGGCACCCTATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCA CCCATCGTAACCGGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTG CTGACGACGGCTCAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCC ATGAACGGACCCGCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTC CGTAACGACATTTCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTAT ACGGGCAAAACCATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAA GGTGCGCTGATTTATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGAC CAATCCGGCGCAAACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTG GGCAAACTGCTGAAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGC TATCTCAACAGTACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAAC ATCGAAACCGACGGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCC TATTATGTCCGTCGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCC GTAGAACAGGGCGGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACG GTTGAAACTGCGGCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCC GTACAGCATGCGAATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCC GCCCATGCCGATATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTC ATCGCGCAAACCCAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACC GTCGGCATTGCCGCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAA AACAGTGCAAATGCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTC AAAGGCCTGTTCTCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGC GTCAACGGCACGCTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTC GAAGGCGGTCTGCGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAAC AGCCTCACTGAAGGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTT GCAACGGCGGGCGTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCA ACCGGCAAGACGGGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAAC GGCTGGAACGGCTTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGC TACCGGTTCCTCGAGCACCACCACCACCACCACTGA 1 MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT 51 AADVEADDFK GLGLKKWTN 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 SRATTAKSAA VSYAGIKNEM CKDRSMLCAG RDDVAVTDRD AKINAPPPNL 401 HTGDFPNPND AYKNLINLKP AIEAGYTGRG VEVGIVDTGE SVGSISFPEL 451 YGRKEHGYNE NYKNYTAYMR KEAPEDGGGK DIEASFDDEA VIETEAKPTD 501 IRHVKEIGHI DLVSHIIGGR SVDGRPAGGI APDATLHIMN TNDΞTKNEMM 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 ADTDQSGANE TVHIKGSLQL 901 DGKGTLYTRL GKLLKVDGTA IIGGKLYMSA RGKGAGYLNS TGRRVPFLSA 951 AKIGQDYSFF TNIETDGGLL ASLDSVEKTA GSEGDTLSYY VRRGNAARTA 1001 SAAAHSAPAG LKHAVEQGGS NLENLMVELD ASESSATPET VETAAADRTD 1051 MPGIRPYGAT FRAAAAVQHA 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 ATAGVERDLN GRDYTVTGGF TGATAATGKT GARNMPHTRL VAGLGADVEF 1351 GNGWNGLARY SYAGSKQYGN HSGRVGVGYR FLEHHHHHH* 961cL-ORF46.1 ATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAAC GACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAA GCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCC GACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTC GATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCA GATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAG ACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTC AACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAG ACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCC GCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCT ACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTC AGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCAC GATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCC GCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGAGGAGGATCAGATTTGGCAAACGATTCTTTT ATCCGGCAGGTTCTCGACCGTCAGCATTTCGAACCCGACGGGAAATACCACCTATTCGGCAGCAGGGGGGAACTTGCC GAGCGCAGCGGCCATATCGGATTGGGAAAAATACAAAGCCATCAGTTGGGCAACCTGATGATTCAACAGGCGGCCATT AAAGGAAATATCGGCTACATTGTCCGCTTTTCCGATCACGGGCACGAAGTCCATTCCCCCTTCGACAACCATGCCTCA CATTCCGATTCTGATGAAGCCGGTAGTCCCGTTGACGGATTTAGCCTTTACCGCATCCATTGGGACGGATACGAACAC CATCCCGCCGACGGCTATGACGGGCCACAGGGCGGCGGCTATCCCGCTCCCAAAGGCGCGAGGGATATATACAGCTAC GACATAAAAGGCGTTGCCCAAAATATCCGCCTCAACCTGACCGACAACCGCAGCACCGGACAACGGCTTGCCGACCGT TTCCACAATGCCGGTAGTATGCTGACGCAAGGAGTAGGCGACGGATTCAAACGCGCCACCCGATACAGCCCCGAGCTG GACAGATCGGGCAATGCCGCCGAAGCCTTCAACGGCACTGCAGATATCGTTAAAAACATCATCGGCGCGGCAGGAGAA ATTGTCGGCGCAGGCGATGCCGTGCAGGGCATAAGCGAAGGCTCAAACATTGCTGTCATGCACGGCTTGGGTCTGCTT TCCACCGAAAACAAGATGGCGCGCATCAACGATTTGGCAGATATGGCGCAACTCAAAGACTATGCCGCAGCAGCCATC CGCGATTGGGCAGTCCAAAACCCCAATGCCGCACAAGGCATAGAAGCCGTCAGCAATATCTTTATGGCAGCCATCCCC ATCAAAGGGATTGGAGCTGTTCGGGGAAAATACGGCTTGGGCGGCATCACGGCACATCCTATCAAGCGGTCGCAGATG GGCGCGATCGCATTGCCGAAAGGGAAATCCGCCGTCAGCGACAATTTTGCCGATGCGGCATACGCCAAATACCCGTCC CCTTACCATTCCCGAAATATCCGTTCAAACTTGGAGCAGCGTTACGGCAAAGAAAACATCACCTCCTCAACCGTGCCG CCGTCAAACGGCAAAAATGTCAAACTGGCAGACCAACGCCACCCGAAGACAGGCGTACCGTTTGACGGTAAAGGGTTT CCGAATTTTGAGAAGCACGTGAAATATGATACGTAACTCGAG 1 MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING 51 FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKW TNLTKTVNEN 101 KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI 151 TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV 201 KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA 251 KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT 301 RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG 351 GSGGGGSDLA NDSFIRQVLD RQHFEPDGKY HLFGSRGELA ERSGHIGLGK 401 IQSHQLGNLM IQQAAIKGNI GYIVRFSDHG HEVHSPFDNH ASHSDSDEAG 451 SPVDGFSLYR IHWDGYEHHP ADGYDGPQGG GYPAPKGARD IYSYDIKGVA 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 NITΞSTVPPS NGKNVKLADQ RHPKTGVPFD 751 GKGFPNFEKH VKYDT* 961CI.-741 ATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAAC GACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAA GCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCC GACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTC GATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCA GATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAG ACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTC AACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAG ACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCC GCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCT ACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTC AGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCAC GATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCC GCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGG CTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGG AAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAA TTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGT GGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCAT TCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCC GAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGAT TTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGAT ATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCC CTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCGGCAGCGCGGAAGTGAAAACCGTAAACGGCATACGCCATATC GGCCTTGCCGCCAAGCAACTCGAGCACCACCACCACCACCACTGA 1 MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING 51 FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKW TNLTKTVNEN 101 KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI 151 TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV 201 KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA 251 KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT 301 RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG 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 YSLGIFGGKA QEVAGSAEVK TVNGIRHIGL 601 AAKQLEHHHH HH* 961cL-983 ATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAAC GACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTGCTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAA GCTGGAGAGACCATCTACGACATTGATGAAGACGGCACAATTACCAAAAAAGACGCAACTGCAGCCGATGTTGAAGCC GACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGTGACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTC GATGCCAAAGTAAAAGCTGCAGAATCTGAAATAGAAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCA GATACTGATGCCGCTCTGGATGCAACCACCAACGCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAG ACTAAGACAAATATCGTAAAAATTGATGAAAAATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTC AACGATATCGCCGATTCATTGGATGAAACCAACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAG ACGGCCGAAGAAACCAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCC GCTGGCACAGCTAATACTGCAGCCGACAAGGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCT ACGAACAAAGATAATATTGCTAAAAAAGCAAACAGTGCCGACGTGTACACCAGAGAAGAGTCTGACAGCAAATTTGTC AGAATTGATGGTCTGAACGCTACTACCGAAAAATTGGACACACGCTTGGCTTCTGCTGAAAAATCCATTGCCGATCAC GATACTCGCCTGAACGGTTTGGATAAAACAGTGTCAGACCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCC GCGCTCTCCGGTCTGTTCCAACCTTACAACGTGGGTGGATCCGGCGGAGGCGGCACTTCTGCGCCCGACTTCAATGCA GGCGGTACCGGTATCGGCAGCAACAGCAGAGCAACAACAGCGAAATCAGCAGCAGTATCTTACGCCGGTATCAAGAAC GAAATGTGCAAAGACAGAAGCATGCTCTGTGCCGGTCGGGATGACGTTGCGGTTACAGACAGGGATGCCAAAATCAAT GCCCCCCCCCCGAATCTGCATACCGGAGACTTTCCAAACCCAAATGACGCATACAAGAATTTGATCAACCTCAAACCT GCAATTGAAGCAGGCTATACAGGACGCGGGGTAGAGGTAGGTATCGTCGACACAGGCGAATCCGTCGGCAGCATATCC TTTCCCGAACTGTATGGCAGAAAAGAACACGGCTATAACGAAAATTACAAAAACTATACGGCGTATATGCGGAAGGAA GCGCCTGAAGACGGAGGCGGTAAAGACATTGAAGCTTCTTTCGACGATGAGGCCGTTATAGAGACTGAAGCAAAGCCG ACGGATATCCGCCACGTAAAAGAAATCGGACACATCGATTTGGTCTCCCATATTATTGGCGGGCGTTCCGTGGACGGC AGACCTGCAGGCGGTATTGCGCCCGATGCGACGCTACACATAATGAATACGAATGATGAAACCAAGAACGAAATGATG GTTGCAGCCATCCGCAATGCATGGGTCAAGCTGGGCGAACGTGGCGTGCGCATCGTCAATAACAGTTTTGGAACAACA TCGAGGGCAGGCACTGCCGACCTTTTCCAAATAGCCAATTCGGAGGAGCAGTACCGCCAAGCGTTGCTCGACTATTCC GGCGGTGATAAAACAGACGAGGGTATCCGCCTGATGCAACAGAGCGATTACGGCAACCTGTCCTACCACATCCGTAAT AAAAACATGCTTTTCATCTTTTCGACAGGCAATGACGCACAAGCTCAGCCCAACACATATGCCCTATTGCCATTTTAT GAAAAAGACGCTCAAAAAGGCATTATCACAGTCGCAGGCGTAGACCGCAGTGGAGAAAAGTTCAAACGGGAAATGTAT GGAGAACCGGGTACAGAACCGCTTGAGTATGGCTCCAACCATTGCGGAATTACTGCCATGTGGTGCCTGTCGGCACCC TATGAAGCAAGCGTCCGTTTCACCCGTACAAACCCGATTCAAATTGCCGGAACATCCTTTTCCGCACCCATCGTAACC GGCACGGCGGCTCTGCTGCTGCAGAAATACCCGTGGATGAGCAACGACAACCTGCGTACCACGTTGCTGACGACGGCT CAGGACATCGGTGCAGTCGGCGTGGACAGCAAGTTCGGCTGGGGACTGCTGGATGCGGGTAAGGCCATGAACGGACCC GCGTCCTTTCCGTTCGGCGACTTTACCGCCGATACGAAAGGTACATCCGATATTGCCTACTCCTTCCGTAACGACATT TCAGGCACGGGCGGCCTGATCAAAAAAGGCGGCAGCCAACTGCAACTGCACGGCAACAACACCTATACGGGCAAAACC ATTATCGAAGGCGGTTCGCTGGTGTTGTACGGCAACAACAAATCGGATATGCGCGTCGAAACCAAAGGTGCGCTGATT TATAACGGGGCGGCATCCGGCGGCAGCCTGAACAGCGACGGCATTGTCTATCTGGCAGATACCGACCAATCCGGCGCA AACGAAACCGTACACATCAAAGGCAGTCTGCAGCTGGACGGCAAAGGTACGCTGTACACACGTTTGGGCAAACTGCTG AAAGTGGACGGTACGGCGATTATCGGCGGCAAGCTGTACATGTCGGCACGCGGCAAGGGGGCAGGCTATCTCAACAGT ACCGGACGACGTGTTCCCTTCCTGAGTGCCGCCAAAATCGGGCAGGATTATTCTTTCTTCACAAACATCGAAACCGAC GGCGGCCTGCTGGCTTCCCTCGACAGCGTCGAAAAAACAGCGGGCAGTGAAGGCGACACGCTGTCCTATTATGTCCGT CGCGGCAATGCGGCACGGACTGCTTCGGCAGCGGCACATTCCGCGCCCGCCGGTCTGAAACACGCCGTAGAACAGGGC GGCAGCAATCTGGAAAACCTGATGGTCGAACTGGATGCCTCCGAATCATCCGCAACACCCGAGACGGTTGAAACTGCG GCAGCCGACCGCACAGATATGCCGGGCATCCGCCCCTACGGCGCAACTTTCCGCGCAGCGGCAGCCGTACAGCATGCG AATGCCGCCGACGGTGTACGCATCTTCAACAGTCTCGCCGCTACCGTCTATGCCGACAGTACCGCCGCCCATGCCGAT ATGCAGGGACGCCGCCTGAAAGCCGTATCGGACGGGTTGGACCACAACGGCACGGGTCTGCGCGTCATCGCGCAAACC CAACAGGACGGTGGAACGTGGGAACAGGGCGGTGTTGAAGGCAAAATGCGCGGCAGTACCCAAACCGTCGGCATTGCC GCGAAAACCGGCGAAAATACGACAGCAGCCGCCACACTGGGCATGGGACGCAGCACATGGAGCGAAAACAGTGCAAAT GCAAAAACCGACAGCATTAGTCTGTTTGCAGGCATACGGCACGATGCGGGCGATATCGGCTATCTCAAAGGCCTGTTC TCCTACGGACGCTACAAAAACAGCATCAGCCGCAGCACCGGTGCGGACGAACATGCGGAAGGCAGCGTCAACGGCACG CTGATGCAGCTGGGCGCACTGGGCGGTGTCAACGTTCCGTTTGCCGCAACGGGAGATTTGACGGTCGAAGGCGGTCTG CGCTACGACCTGCTCAAACAGGATGCATTCGCCGAAAAAGGCAGTGCTTTGGGCTGGAGCGGCAACAGCCTCACTGAA GGCACGCTGGTCGGACTCGCGGGTCTGAAGCTGTCGCAACCCTTGAGCGATAAAGCCGTCCTGTTTGCAACGGCGGGC GTGGAACGCGACCTGAACGGACGCGACTACACGGTAACGGGCGGCTTTACCGGCGCGACTGCAGCAACCGGCAAGACG GGGGCACGCAATATGCCGCACACCCGTCTGGTTGCCGGCCTGGGCGCGGATGTCGAATTCGGCAACGGCTGGAACGGC TTGGCACGTTACAGCTACGCCGGTTCCAAACAGTACGGCAACCACAGCGGACGAGTCGGCGTAGGCTACCGGTTCTGA CTCGAG 1 MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING 51 FKAGETIYDI DΞDGTITKKD ATAADVEADD FKGLGLKKW TNLTKTVNEN 101 KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI 151 TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV 201 KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA 251 KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT 301 RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG 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 EGIRLMQQSD YGNLSYHIRN 651 KNMLFIFSTG NDAQAQPNTY ALLPFYEKDA QKGIITVAGV DRSGEKFKRE 701 MYGEPGTEPL EYGSNHCGIT AMWCLSAPYE ASVRFTRTNP IQIAGTSFSA 751 PIVTGTAALL LQKYPWMSND NLRTTLLTTA QDIGAVGVDS KFGWGLLDAG 801 KAMNGPASFP FGDFTADTKG TSDIAYSFRN 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 RLVAGLGADV EFGNGWNGLA RYSYAGSKQY GNHSGRVGVG 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: Tmi = 4 (G+C)+ 2 (A+T) (tail excluded) T m2 = 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 2.0ml NH 4 OH, 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. * 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 Nhel site, the ATG codon is part of the Ndel site used for cloning. The constructs made using Nhel 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 100ml of GC medium, harvested by centrifugation, and resuspended in 5ml buffer (20% w/v sucrose, 50mM Tris-HCl, 50mM EDTA, pH8). After 10 minutes incubation on ice, the bacteria were lysed by adding 10ml of lysis solution (50mM NaCl, 1% Na-Sarkosyl, 50μg/ml Proteinase K), and the suspension incubated at 37°C for 2 hours. Two phenol extractions (equilibrated to pH 8) and one CHCls/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 4.0ml TE buffer (lOmM Tris-HCl, ImM EDTA, pH 8.0). The DNA concentration was measured by reading OD 6( PCR Amplification The standard PCR protocol was as follows: 200ng of genomic DNA from 2996, MC581000, or BZ232 strains or lOng of plasmid DNA preparation of recombinant clones were used as template in the presence of 40μM of each oligonucletide primer, 400-800 μM dNTPs solution, lx PCR buffer (including 1.5mM MgCl 2 ), 2.5 units Taql DNA polymerase (using Perkin-Elmer AmpliTaQ, Boerhingher Mannheim Expand™ 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 (T ml ). This was followed by 30 cycles according to the hybridisation temperature calculated for the whole length oligos (T m ). Elongation times, performed at 68°C or 72°C, varied according to the length of the Orf to be amplified. In the case of Orf 1 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 H 2 O or lOmM 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 800μl LB broth and incubation at 37°C for 20 minutes. The cells were centrifuged at maximum speed in an Eppendorf microfuge, resuspended in approximately 200μl of the supernatant and plated onto LB ampicillin (lOOmg/ml ) agar. Screening for recombinant clones was performed by growing randomly selected colonies overnight at 37°C in 4.0ml of LB broth + lOOμg/ml ampicillin. Cells were pelleted and plasmid DNA extracted using the Qiagen QIAprep Spin Miniprep Kit, following the manufacturer's instructions. Approximately lμg 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 (lkb 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, lμl of each construct was used to transform E.coli BL21-DE3 as described above. Single recombinant colonies were inoculated into 2ml LB+Amp (lOOμg/ml), incubated at 37°C overnight, then diluted 1:30 in 20ml of LB+Amp (lOOμg/ml) in 100ml flasks, to give an OD 600 between 0.1 and 0.2. The flasks were incubated at 30°C or at 37°C in a gyratory water bath shaker until OD 6 oo indicated exponential growth suitable for induction of expression (0.4-0.8 OD). Protein expression was induced by addition of l.OmM IPTG. After 3 hours incubation at 30°C or 37°C the OD 6 oo was measured and expression examined. 1.0ml 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 Narious 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 (Δl, Δ 2, 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 20ml 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 OD 550 reached 0.6-0.8. Expression of recombinant protein was induced by addition of IPTG (final concentration l.OmM) and the culture incubated for a further 3 hours. Bacteria were harvested by centrifugation at 8000g 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 M imidazole, pH 8.0) for soluble proteins or (ii) buffer B (lOmM 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, Δl, 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 40W using a Branson sonifier 450 and centrifuged at 13000xg 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- HC1, pH 7.5 and treated with 10 passes of a Dounce homogenizer. The homogenate was centrifuged at 13000g for 30 min and the supernatant retained. Supernatants for both soluble and insoluble preparations were mixed with 150μl Ni 2+ -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 lOOg for 5 min at 4°C and the supernatant discarded. The resin was washed twice (batch-wise) with 10ml 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 OD 280 of the flow-through reached 0.02-0.01. The resin was further washed with either (i) cold buffer C (300mM NaCl, 50mM phosphate buffer, 20mM imidazole, pH 8.0) or (ii) buffer D (lOmM Tris-HCl, lOOmM phosphate buffer, pH 6.3 and, optionally, 8M urea) until OD 280 of the flow-through reached 0.02-0.01. The His-fusion protein was eluted by addition of 700μl of either (i) cold elution buffer A (300 mM NaCl, 50mM 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 OD 280 indicated all the recombinant protein was obtained. 20μl 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, 50mM phosphate buffer, 5.0mM reduced glutathione, 0.5mM oxidised glutathione, pH 8.8) for 12-14 hours at 4°C. Protein concentration was estimated using the formula: Protein (mg/ml) = (1.55 x OD 2 so) - (0.76 x OD260) Immunization B alb/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% CO . 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 OD 620 . 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 4000rpm. 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. lOOμl 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). 200μl 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. 200μl of diluted sera (Dilution buffer: 1% BSA, 0.1% Tween-20, 0.1% NaN 3 in PBS) were added to each well and the plates incubated for 2 hours at 37°C. Wells were washed three times with PBT. lOOμl 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. lOOμl of substrate buffer for HRP (25ml of citrate buffer pH5, lOmg of O-phenildiamine and lOμl of H O ) were added to each well and the plates were left at room temperature for 20 minutes. lOOμl 12.5% H 2 SO 4 was added to each well and OD 49 o was followed. The ELISA titers were calculated abitrarely as the dilution of sera which gave an OD 49 o value of 0.4 above the level of preimmune sera. The ELISA was considered positive when the dilution of sera with OD o 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% CO 2 . Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 4 tubes containing 8ml each Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following OD 620 . 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 4000rpm. The supernatant was discarded and the pellet was resuspended in blocking buffer (1% BSA in PBS, 0.4% NaN 3 ) and centrifuged for 5 minutes at 4000rpm. Cells were resuspended in blocking buffer to reach OD 620 of 0.05. lOOμl bacterial cells were added to each well of a Costar 96 well plate. lOOμl 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 4000rpm, the supernatant aspirated and cells washed by addition of 200μl/well of blocking buffer in each well. lOOμl 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 4000rpm for 5 minutes and washed by addition of 200μl/well of blocking buffer. The supernatant was aspirated and cells resuspended in 200μl/well of PBS, 0.25% formaldehyde. Samples were transferred to FACScan tubes and read. The condition for FACScan (Laser Power 15mW) 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% CO 2 . Colonies were collected and used to inoculate 7ml Mueller-Hinton broth, containing 0.25% glucose to reach an OD 620 of 0.05-0.08. The culture was incubated for approximately 1.5 hours at 37 degrees with shacking until the OD 6 o reached the value of 0.23-0.24. Bacteria were diluted in 50mM Phosphate buffer pH 7.2 containing lOmM MgCl 2 , lOmM CaCl 2 and 0.5% (w/v) BSA (assay buffer) at the working dilution of 10 5 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, lOμl 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. 7μl of each sample were plated on Mueller-Hinton agar plates as spots, whereas lOμl 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 (500ng/lane), outer membrane vesicles (5μg) and total cell extracts (25μg) 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 150mA 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 XI 00 in PBS and developed with the Opti- 4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water. The OMNs were prepared as follows: N. meningitidis strain 2996 was grown overnight at 37 degrees with 5% CO on 5 GC plates, harvested with a loop and resuspended in 10 ml of 20mM 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 5000g for 10 minutes, the supernatant containing the total cell envelope fraction recovered and further centrifuged overnight at 50000g at the temperature of 4°C . The pellet containing the membranes was resuspended in 2% sarkosyl, 20mM 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 lOOOOg for 10 minutes to remove aggregates, the supernatant was further centrifuged at 50000g 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 1ml of 20mM Tris-HCl. Heat inactivation was performed at 56°C for 30 minutes.

Claims: CLAIMS 1. A method for the simultaneous heterologous expression of two or more proteins of the invention, in which (a) two or more proteins of the invention are fused. 2. The method of claim 23, in which the two or more proteins are: (a) 919 and 287; (b) 953 and 287; (c) 287 and ORF46.1; (d) ORF1 and ORF46.1; (e) 919 and ORF46.1; (f) ORF46.1, 287 and 919; (g) 919 and 519; and (h) ORF97 and 225. 3. The method of claim 24, in which 287 is at the C-terminal end of protein (a), (b) or (c). 4. The method of any preceding claim, in which the expression is in an E.coli host. 5. A protein expressed by the method of any preceding claim. 6. A hybrid protein of formula NH -A — B-COOH, wherein A and B are different Neisserial proteins. 7. The protein of claim 6, wherein A and B are each selected from orfl, orf4, orf25, orf 40, orf46, orf83, 233, 287, 2921, 564, 687, 741, 907, 919, 953, 961 and 983. 8. The protein of claim 7, wherein A and B are each selected from ORF46.1, 287, 741, 919, 953, 961 and 983. 9. The protein of claim 8, wherein at least one of said ORF46.1, 287, 741, 919, 953, 961 and 983 is used in essentially full-length form 10. The protein of claim 8 or claim 9, wherein at least one of said ORF46.1, 287, 741, 919, 953, 961 and 983 has a deletion. 11. The protein of claim 10, wherein A and/or B has a poly-glycine deletion ('ΔG'). 12. The protein of claim 11, wherein A and/or B is ΔG-287, ΔGTbp2, ΔG741, or ΔG983. 13. The protein of claim 10, wherein A and/or B is a truncated protein. 14. The protein of claim 13, wherein A and/or B is Δl-287, Δ2-287, Δ3-287 or Δ4-287. 15. The protein of claim 10, wherein a domain of A and/or B is deleted. 16. The protein of claim 15, wherein A and/or B is 287B, 287C, 287BC, ORF461-433, ORF46433-608, ORF46, or 961c. 17. The protein of claim 6, wherein A and B are: (a) 919 and 287; (b) 953 and 287; (c) 287 and ORF46.1; (d) ORF1 and ORF46.1; (e) 919 and ORF46.1; (f) ORF46.1 and 919; (g) 919 and 519; or (h) ORF97 and 225. 18. The protein of claim 17, wherein the protein is 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, or 961CL-983. 19. The protein of claim 8, wherein A or B is 287. 20. The protein of claim 19, wherein B is 287 21. The protein of claim 19, wherein A is ΔG-287 22. The protein of claim 21, wherein B is ORF46, 919, 953 or 961. 23. The protein of any one of claims 19 to 22, wherein 287 is from strain 2996 or 394/98. 24. The protein of claim 8, wherein A is 961. 25. The protein of any one of claims 6 to 24, wherein A and B are from the same strain. 26. The protein of any one of claims 6 to 24, wherein A and B are joined directly 27. The protein of any one of claims 6 to 24, wherein A and B are joined via a linker peptide. 28. The protein of claim 27, wherein the linker peptide is a poly-glycine linker, with the proviso that B is not a ΔG protein.

Inventors: ARICO MARIA BEATRICE, COMANDUCCI MAURIZIO, GALEOTTI CESIRA, GIULIANI MARZIA MONICA, MASIGNANI VEGA, PIZZA MARIAGRAZIA, ARICO, MARIA, BEATRICE, COMANDUCCI, MAURIZIO, GALEOTTI, CESIRA, GIULIANI, MARZIA, MONICA, MASIGNANI, VEGA, PIZZA, MARIAGRAZIA
Applicants: ARICO MARIA BEATRICE, CHIRON SPA, COMANDUCCI MAURIZIO, GALEOTTI CESIRA, GIULIANI MARZIA MONICA, MASIGNANI VEGA, PIZZA MARIAGRAZIA, ARICO, MARIA, BEATRICE, COMANDUCCI, MAURIZIO, GALEOTTI, CESIRA, GIULIANI, MARZIA, MONICA, MASIGNANI, VEGA, PIZZA, MARIAGRAZIA
Application year: 2001
Main classification: C12N15
IPC codes: A61K38, C07K14, C07K19, C12N15, C12P21, C12Q1