# Patent

## Title
Nucleotide sequences of the nucleocapsid (NP) gene of a Malaysian velogenic Newcastle disease virus strain AF2240

## Abstract

The present invention relates to nucleotide sequences encoding the nucleocapsid (NP) protein and phosphoprotein (P) of Newcastle disease virus (NDV) and the production of the corresponding proteins with recombinant plasmids bearing the nucleotide sequences in Escherichia coli.

## Background
FIELD OF THE INVENTION
The present invention relates to nucleotide sequences encoding the nucleocapsid (NP) protein and phosphoprotein (P) of Newcastle disease virus (NDV) strain AF2240, and the production of the corresponding proteins with recombinant plasmids bearing the nucleotide sequences in Escherichia coli. 
DESCRIPTION OF THE PRIOR ART
Newcastle disease virus (NDV) is the prototype of avian paramyxovirus, which causes a highly contagious disease known as Newcastle disease (ND) in many avian species. This disease is of great economic importance requiring control by vaccination or quarantine with slaughter of all birds in confirmed outbreaks, resulting in substantial losses in the poultry industry worldwide. Therefore, development of an improved vaccine and also a rapid and sensitive diagnostic test are greatly desired by the poultry industry.
A Malaysian heat resistant NDV strain AF2240, which causes 100% mortality in susceptible chicken flocks has been reported by Abdul Rahman et al. (1976) and Lai, C. M. (1985). Further studies by Idris et al. (1993) revealed that the thermostabilities of haemagglutination and neuraminidase activities of this AF2240 strain were found to be higher than those of other strains. The basis giving rise to these unique features is still unknown. However a comprehensive understanding of the viral proteins would provide some solutions and useful information for the development of heat stable recombinant vaccines and diagnostic tests.
The genome of NDV is a linear, non-segmented, single-stranded negative sense RNA with a molecular weight of 5.2-5.7×106 Daltons, or approximately 15,000 bases which encodes six main structural proteins. The genomic RNA is associated with the nucleocapsid (NP), phosphoprotein (P) and large (L) proteins. These macromolecules form the transcriptive complex of the virus, which in turn is surrounded by a lipid bilayer membrane derived from the host cell. Embedded in the membrane are the haemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins. Beneath the lipid bilayer is a shell of protein known as the matrix (M) protein, which is believed to interact with the transcriptive complex. The HN and F glycoproteins are associated with the host cell receptor during infection. The NP encapsidates the viral RNA together with the L protein which is thought to be the transcriptase, and a P protein with an unknown reason.
The genes encoding for the HN (EMBL/Gen Bank/DDBJ accession No.X79092), F (EMBL/Gen Bank/DDBJ accession No.AFO48763) and M (EMBL/Gen Bank/DDBJ accession No. AF060563) proteins of the NDV strain AF2240 have been completely sequenced by Tan et al. (1995), Salih et al. (2000) and Jemain, S. F. P. (1999) respectively. From the HN gene sequence of strain AF2240, it was quite clear that this strain is different from he other published NDV strains. The HN protein lacked the Arg (403) residue and contained 581 amino acids. At the time when the project was initiated, there was no information available on the coding sequences for the NP and P proteins of NDV strain AF2240. Therefore it remained a problem to prepare cDNA for the cloning of the NP and P genes of NDV.
The inventors have now successfully determined the nucleotide sequences encoding the NP and P proteins of NDV strain AF2240. The accession numbers for the genes encoding the NP and P proteins are EMBL/Gen Bank/DDBJ No. AF284646 and AF284647 respectively. The inventors had discovered that the proteins, in either non-fusion or fusion forms bearing the myc epitope and six residues of His at their carboxyl terminal end could be successfully produced in E. coli by means of recombinant DNA technologies. The NP and P proteins were expressed to a substantial level in the bacteria and can be recognised by chicken anti-NDV serum.
SUMMARY OF THE INVENTION
The present invention provides nucleotides encoding the full length NP and P polypeptides of Newcastle disease virus strain AF2240. Whereas the genome of NDV is of length approximately 15,000 nucleotides, it has been determined, by this invention, that the portion coding for the NP polypeptide is approximately 1470 nucleotides long and the portion that codes for the P polypeptide is approximately 1188 nucleotides long. Accordingly, one aspect of the present invention provides for the coding regions of the nucleocapsid (NP) and phosphoprotein (P) genes of Newcastle disease virus strain AF2240. Both the nucleotide sequences are as listed below:












NP coding region (SEQ ID NO: 1)











        10         20         30         40         50         60



ATGTCTTCCG TATTCGATGA ATACGAGCAG CTCCTCGCTG CTCAGACTCG CCCCAATGGA


        70         80         90        100        110        120


GCTCACGGAG GGGGAGAGAG AGGGAGCACT TTAAGAGTTG AGGTCCCAGT ATTCACTCTT


       130        140        150        160        170        180


AACAGTGACG ATCCAGAAGA TAGATGGAAT TTTGCGGTAT TCTGTCTTCG GATTGCTGTT


       190        200        210        220        230        240


AGCGAGGACG CCAACAAACC GCTCAGGCAA GGTGCTCTCA TATCCCTCCT GTGCTCCCAT


       250        260        270        280        290        300


TCTCAAGTGA TGAGGAACCA TGTTGCCCTT GCAGGAAAAC AGAATGAGGC TACACTGACT


       310        320        330        340        350        360


GTTCTTGAGA TCGATGGTTT TACCAGCAGC GTGCCTCAGT TCAACAACAG GAGTGGGGTG


       370        380        390        400        410        420


TCTGAGGAGA GAGCACAGAG ATTCATGGTG ATAGCAGGGT CTCTCCCTCG GGCGTCCACT


       430        440        450        460        470        480


AACGGTACTC CGTTCGTCAC GGCTGGGGTT GAAGATGATG CACCAGAAGA TATCACTGAT


       490        500        510        520        530        540


ACTCTGGAAA GAATCCTGTC TATCCAGGCT CAGGTATGGG TCACAGTAGC GAAGGCCATG


       550        560        570        580        590        600


ACTGCATATG AGACAGCAGA TGAGTCGGAA ACAAGAAGAA TCAATAAGTA CATGCAGCAA


       610        620        630        640        650        660


GGCAGAGTCC AGAAGAAGTA CATCCTCCAC CCTGTATGCA GGAGTGCAAT TCAACTCACA


       670        680        690        700        710        720


ATCAGACATT CTCTGGCAGT CCGCATTTTC TTAGTTAGCG AGCTTAAGAG AGGCCGCAAT


       730        740        750        760        770        780


ACGGCAGGTG GGAGCTCCAC GTATTACAAC TTAGTAGGGG ATGTAGACTC ATACATCAGG


       790        800        810        820        830        840


AACACCGGAC TTACTGCATT CTTCCTTACA CTCAAATATG GAATTAATAC CAAGACATCA


       850        860        870        880        890        900


GCCCTAGCAC TCAGCAGCCT CACAGGCGAT ATCCAAAAGA TGAAGCAGCT CATGCGTTTA


       910        920        930        940        950        960


TATCGGATGA AGGGAGAAAA TGCGCCGTAC ATGACATTGC TAGGTGACAG TGATCAGATG


       970        980        990       1000       1010       1020


AGCTTTGCAC CGGCTGAGTA TGCACAGCTT TATTCTTTTG CCATGGGCAT GGCATCAGTC


      1030       1040       1050       1060       1070       1080


TTAGATAAAG GAACTGGCAA ATACCAATTC GCCAGAGACT TCATGAGCAC ATCATTCTGG


      1090       1100       1110       1120       1130       1140


AGACTCGGGG TGGAGTATGC TCAGGCTCAG GGGAGTAGCA TCAACGAACA CATGGCTGCT


      1150       1160       1170       1180       1190       1200


GAGCTAAAC TAACCCCGGC AGCAAGAAGG GGCCTGGCAG CTGCTGCCCA ACGAGTGTCT


      1210       1220       1230       1240       1250       1260


GAGGAAACTG GCAGCGTGGA TATTCCTACT CAACAAGCCG GGGTCCTCAC TGGGCTCAGC


      1270       1280       1290       1300       1310       1320


GATGGAGGCC CCCGAGCCTC TCAGGGTGGA TCGAACAAGT CGCAAGGGCA ACCAGATGCC


      1330       1340       1350       1360       1370       1380


GGAGATGGGG AGACCCAATT CTTGGATTTG ATGAGAGCAG TGGCGAACAG CATGCGAGAA


      1390       1400       1410       1420       1430       1440


GCGCCAAACT CCGCACAGAG CACCACCCAC CCGGAACCCC CCCCGACTCC CGGGCCATCA


      1450       1460       1470       1480       1490       1500


CAAGATAACG ACACCGACTG GGGGTATTGA .......... .......... ..........










P gene coding region (SEQ ID NO: 2)











        10         20         30         40         50         60



ATGGCCACCT TTACAGATGC GGAGATAGAT GATATATTTG AGACCAGTGG AACTGTCATT


        70         80         90        100        110        120


GACAGCATAA TTACGGCCCA GGGTAAATCA GCAGAGACTG TCGGAAGGAG CGCAATCCCA


       130        140        150        160        170        180


CAAGGCAAGA CCAAAGCGCT GAGCATAGCA TGGGAGAAGC ATGGGAGCAT CCAACCATCC


       190        200        210        220        230        240


ACCAGCCAGG ACAACCCCGA CCAACAGGAT AGACCAGACA AACAGCTATC CACACCTGAG


       250        260        270        280        290        300


CAGGCGACCC CACACAACAG CTCGCCAGCC ACATCCGCCG AACCGCTCCC CACTCAGGCC


       310        320        330        340        350        360


GCAGGTGAGG CCGGCGACAC ACAGCTCAAG ACCGGAGCAA GCAACTCTCT TCTGTCTATG


       370        380        390        400        410        420


CTCGACAAGC TGAGCAATAA ACCATCTAAT GCTAAAAAGG GCCCATGGTC GAGTCCCCAG


       430        440        450        460        470        480


GAAGGATATC ATCAACCTCC GACCCAACAA CATGGGGATC AGCCGAACCG CGGAAACAGC


       490        500        510        520        530        540


CAGGAGAGGC TGCGGCACCA AGCCAAGGCC GCCCCTGGAA GCCGGGGCAC AGACGCGAGC


       550        560        570        580        590        600


ACAGCATATC ATGGACAATG GAAGGAGTCA CAACTATCAG CTGGTGCAAC CCCTCATGTG


       610        620        630        640        650        660


CTCCAATCAG GGCAGAGCCA AGACAGTACT CCTGTACCTG TGGATCATGT CCAGCCACCT


       670        680        690        700        710        720


GTCGACTTTG TGCAGGCGAT GATGACTATG ATGGAGGCGT TATCACAGAA GGTAAGTAAA


       730        740        750        760        770        780


GTCGACTATC AGCTAGACCT AGTCTTAAAG CAGACATCCT CCATCCCTAT GATGCGGTCT


       790        800        810        820        830        840


GAAATCCAAC AGCTAAAAAC ATCTGTTGCG GTCATGGAAG CTAATTTAGG CATGATGAAA


       850        860        870        880        890        900


ATTCTGGACC CTGGTTGTGC TAACATTTCA TCCTTAAGTG ATCTGCGGGC AGTCGCCCGG


       910        920        930        940        950        960


TCCCACCCAG TTTTAATTTC AGGCCCCGGA GATCCGTCCC CCTACGTGAC ACAAGGGGGT


       970        980        990       1000       1010       1020


GAGATGACAC TCAATAAACT CTCACAACCA GTACAACACC CTTCCGAGTT AATTAAATCT


      1030       1040       1050       1060       1070       1080


GCCACAGCGG GCGGACCTGA TATGGGAGTG GAAAAGGACA CTGTCCGTGC ATTGATCACC


      1090       1100       1110       1120       1130       1140


TCGCGCCCGA TGCATCCAAG CTCCTCAGCT AAGCTCCTGA GTAAGCTGGA TGCAGCCGGG


      1150       1160       1170       1180       1190       1200


TCGATTGAAG AGATCAGAAA GATCAAGCGC CTTGCACTAA ATGGCTAA.. ..........









Further, the present invention provides the amino acid sequences of both the NP and P proteins as listed below:
NP gene (SEQ ID NO: 1): amino acid sequence (SEQ ID NO: 3)












NP gene (SEQ ID NO: 1): amino acid sequence (SEQ ID NO: 3)












  1
 M   S   S   V   F   D   E   Y   E   Q   L   L   A   A   Q   T
 16



ATG TCT TCC GTA TTC GAT GAA TAC GAG CAG CTC CTC GCT GCT CAG ACT



1          10           20           30            40


 17
 R   P   N   G   A   H   G   G   G   E   R   G   S   T   L   R
 32



CGC CCC AAT GGA GCT CAC GGA GGG GGA GAG AGA GGG AGC ACT TTA AGA



50           60            70           80           90


 33
 V   E   V   P   V   F   T   L   N   S   D   D   P   E   D   R
 48



GTT GAG GTC CCA GTA TTC ACT CTT AAC AGT  GAC GAT CCA GAA GAT AGA



  100          110          120           130          140


 49
 W   N   F   A   V   F   C   L   R   I   A   V   S   E   D   A
 64



TGG AAT TTT GCG GTA TTC TGT CTT CGG ATT GCT GTT AGC GAG GAC GCC



    150           160          170          180           190


 65
 N   K   P   L   R   Q   G   A   L   I   S   L   L   C   S   H
 80



AAC AAA CCG CTC AGG CAA GGT GCT CTC ATA TCC CTC CTG TGC TCC CAT



      200           210          220           230          240


 81
 S   Q   V   M   R   N   H   V   A   L   A   G   K   Q   N   E
 96



TCT CAA GTG ATG AGG AAC CAT GTT GCC CTT GCA GGA AAA CAG AAT GAG



          250          260          270           280


 97
 A   T   L   T   V   L   E   I   D   G   F   T   S   S   V   P
112



GCT ACA CTG ACT GTT CTT GAG ATC GAT GGT TTT ACC AGC AGC GTG CCT



290         300          310          320          330


113
 G   F   N   N   R   S   G   V   S   E   E   R   A   Q   R   F
128



CAG TTC AAC AAC AGG AGT GGG GTG TCT GAG GAG AGA GCA CAG AGA TTC



  340          350          360           370          380


129
 M   V   I   A   G   S   L   P   R   A   C   S   N   G   T   P
144



ATG GTG ATA GCA GGG TCT CTC CCT CGG GCG TGC AGT AAC GGT ACT CCG



    390           400          410          420           430


145
 F   V   T   A   G   V   E   D   D   A   P   E   D   I   T   D
160



TTC GTC ACG GCT GGG GTT GAA GAT GAT GCA CCA GAA GAT ATC ACT GAT



       440          450           460          470          480


161
 T   L   E   R   I   L   S   I   Q   A   Q   V   W   V   T   V
176



ACT CTG GAA AGA ATC CTG TCT ATC CAG GCT CAG GTA TGG GTC ACA GTA



          490          500          510           520


177
 A   K   A   M   T   A   Y   E   T   A   D   E   S   E   T   R
192



GCG AAG GCC ATG ACT GCA TAT GAG ACA GCA GAT GAG TCG GAA ACA AGA



530         540           550          560          570


193
 R   I   N   K   Y   M   Q   Q   G   R   V   Q   K   K   Y   I
208



AGA ATC AAT AAG TAC ATG CAG CAA GGC AGA GTC CAG AAG AAG TAC ATC



  580          590          600           610          620


209
 L   H   P   V   C   R   S   A   I   Q   L   T   I   R   H   S
224



CTC CAC CCT GTA TGC AGG AGT GCA ATT CAA CTC ACA ATC AGA CAT TCT



    630           640          650          660           670


225
 L   A   V   R   I   F   L   V   S   E   L   K   R   G   R   N
240



CTG GCA GTC CGC ATT TTC TTA GTT AGC GAG CTT AAG AGA GGC CGC AAT



       680          690           700          710          720


241
 T   A   G   G   S   S   T   Y   Y   N   L   V   G   D   V   D
256



ACG GCA GGT GGG AGC TCC ACG TAT TAC AAC TTA GTA GGG GAT GTA GAC



          730          740          750           760


257
 S   Y   I   R   N   T   G   L   T   A   F   F   L   T   L   K
272



TCA TAC ATC AGG AAC ACC GGA CTT ACT GCA TTC TTC CTT ACA CTC AAA



770         780          790          800          810


273
 Y   G   I   N   T   K   T   S   A   L   A   L   S   S   L   T
288



TAT GGA ATT AAT ACC AAG ACA TCA GCC CTA GCA CTC AGC AGC CTC ACA



  820          830          840           850           860


289
 G   D   I   Q   K   M   K   Q   L   M   R   L   Y   R   M   K
304



GGC GAT ATC CAA AAG ATG AAG CAG CTC ATG CGT TTA TAT CGG ATG AAG



    870           880          890          900           910


305
 G   E   N   A   P   Y   M   T   L   L   G   D   S   D   Q   M
320



GGA GAA ATT GCG CCG TAC ATG ACA TTG CTA GGT GAC AGT GAT CAG ATG



       920          930           940          950          960


321
 S   F   A   P   A   E   Y   A   Q   L   Y   S   F   A   M   G
336



AGC TTT GCA CCG GCT GAG TAT GCA CAG CTT TAT TCT TTT GCC ATG GGC



          970          980       990          1000


337
 M   A   S   V   L   D   K   G   T   G   K   Y   Q   F   A   R
352



ATG GCA TCA GTC TTA GAT AAA GGA ACT GGC AAA TAC CAA TTC GCC AGA



1010       1020          1030         1040         1050


353
 D   F   M   S   T   S   F   W   R   L   G   V   E   Y   A   Q
368



GAC TTC ATG AGC ACA TCA TTC TGG AGA CTC GGG GTG GAG TAT GCT CAG



 1060         1070         1080          1090         1100


369
 S   Q   G   S   S   I   N   E   D   M   A   A   E   L   K   L
384



GCT CAG GGG AGT AGC ATC AAC GAA CAG ATG GCT GCT GAG CTA AAA CTA



   1110          1120         1130         1140           1150


385
 T   P   A   A   R   R   G   L   A   A   A   A   G   R   V   S
400



ACC CCG GCA GCA AGA AGG GGC CTG GCA GCT GCT GCC CAA CGA GTG TCT



      1160         1170          1180         1190         1200


401
 E   E   T   G   S   V   D   I   P   T   Q   Q   A   G   V   L
416



GAG GAA ACT GGC AGC GTG GAT ATT CCT ACT CAA CAA GCC GGG GTC CTC



         1210         1220         1230          1240


417
 T   G   L   S   D   G   G   P   R   A   S   Q   G   G   S   N
432



 ACT GGG CTC AGC GAT GGA GGC CCC CGA GCC TCT CAG GGT GGA TCG AAC



1250       1260          1270         1280         1290


433
 K   S   Q   G   Q   P   D   A   G   D   G   E   T   Q   F   L
448



AAG TCG CAA GGG CAA CCA GAT GCC GGA GAT GGG GAG ACC CAA TTC TTG



 1300         1310         1320          1330         1340


449
 D   L   M   R   A   V   A   N   S   M   R   E   A   P   N   S
464



GAT TTG ATG AGA GCA GTG GCG AAC AGC ATG CGA GAA GCG CCA AAC TCC



   1350          1360         1370         1380          1390


465
 A   Q   S   T   T   H   P   E   P   P   P   T   P   G   P   S
480



GCA CAG AGC ACC ACC CAC CCG GAA CCC CCC CCG ACT CCC GGG CCA TCC



      1400         1410          1420         1430         1440


481
 Q   D   N   D   T   D   W   G   Y   *
490



CAA GAT AAC GAC ACC GAC TGG GGG TAT TGA



         1450         1460         1470










P gene: (SEQ ID NO: 2): amino acid sequence (SEQ ID NO: 4)












  1
 M   A   T   F   T   D   A   E   I   D   D   I   F   E   T   S
 16



ATG GCC ACC TTT ACA GAT GCG GAG ATA GAT GAT ATA TTT GAG ACC AGT



1          10           20           30            40


 17
 G   T   V   I   D   S   I   I   T   A   Q   G   K   S   A   E
 32



GGA ACT GTC ATT GAC AGC ATA ATT ACG GCC CAG GGT AAA TCA GCA GAG



50           60            70           80           90


 33
 T   V   G   R   S   A   I   P   Q   G   K   T   K   A   L   S
 48



ACT GTC GGA AGG AGC GCA ATC CCA CAA GGC AAG ACC AAA GCG CTG AGC



  100          110          120           130          140


 49
 I   A   W   E   K   H   G   S   I   Q   P   S   T   S   Q   D
 64



ATA GCA TGG GAG AAG CAT GGG AGC ATC CAA CCA TCC ACC AGC CAG GAC



    150           160          170          180           190


 65
 N   P   D   Q   Q   D   R   P   D   K   Q   L   S   T   P   E
 80



AAC CCC GAC CAA CAG GAT AGA CCA GAC AAA CAG CTA TCC ACA CCT GAG



       200          210           220          230          240


 81
 Q   A   T   P   H   N   S   S   P   A   T   S   A   E   P   L
 96



CAG GCG ACC CCA CAC AAC AGC TCG CCA GCC ACA TCC GCC GAA CCG CTC



          250          260          270           280


 97
 P   T   Q   A   A   G   E   A   G   D   T   Q   L   K   T   G
112



CCC ACT CAG GCC GCA GGT GAG GCC GGC GAC ACA CAG CTC AAG ACC GGA



290         300           310          320          330


113
 A   S   N   S   L   L   S   M   L   D   K   L   S   N   K   P
128



GCA AGC AAC TCT CTT CTG TCT ATG CTC GAC AAG CTG AGC AAT AAA CCA



  340          350          360           370          380


129
 S   N   A   K   K   G   P   W   S   S   P   Q   E   G   Y   H
144



TCT AAT GCT AAA AAG GGC CCA TGG TCG AGT CCC CAG GAA GGA TAT CAT



    390           400          410          420           430


145
 Q   P   P   T   Q   Q   H   C   D   Q   P   N   R   G   N   S
160



CAA CCT CCG ACC CAA CAA CAT GGG GAT CAG CCG AAC CGC GGA AAC AGC



       440          450           460          470          480


161
 Q   E   R   L   R   H   Q   A   K   A   A   P   G   S   R   G
176



CAG GAG AGG CTG CGG CAC CAA GCC AAG GCC GCC CCT GGA AGC CGG GGC



          490          500          510           520


177
 T   D   A   S   T   A   Y   H   G   Q   W   K   E   S   Q   L
192



ACA GAC GCG AGC ACA GCA TAT CAT GGA CAA TGG AAG GAG TCA CAA CTA



530         540           550          560          570


193
 S   A   G   A   T   P   H   V   L   Q   S   G   Q   S   G   D
208



TCA GCT GGT GCA ACC CCT CAT GTG CTC CAA TCA GGG CAG AGC CAA GAC



  580          590           600           610          620


209
 S   T   P   V   P   V   D   H   V   Q   P   P   V   D   F   V
224



AGT ACT CCT GTA CCT GTG GAT CAT GTC CAG CCA CCT GTC GAC TTT GTG



    630           640          650          660           670


225
 Q   A   M   M   T   M   M   E   A   L   S   Q   K   V   S   K
240



CAG GCG ATG ATG ACT ATG ATG GAG GCG TTA TCA CAG AAG GTA AGT AAA



       680          690           700          710          720


241
 V   D   Y   Q   L   D   L   V   L   K   Q   T   S   S   I   P
256



GTC GAC TAT CAG CTA GAC CTA GTC TTA AAG CAG ACA TCC TCC ATC CCT



          730          740          750           760


257
 M   M   R   S   E   I   Q   Q   L   K   T   S   V   A   V   M
272



ATG ATG CGG TCT GAA ATC CAA CAG CTA AAA ACA TCT GTT GCG GTC ATG



770         780           790          800          810


273
 E   A   N   L   G   M   M   K   I   L   D   P   G   C   A   N
288



GAA GCT AAT TTA GGC ATG ATG AAA ATT CTG GAC CCT GGT TGT GCT AAC



  820          830          840           850          860


289
 I   S   S   L   S   D   L   R   A   V   A   R   S   H   P   V
304



ATT TCA TCC TTA AGT GAT CTG CGG GCA GTC GCC CGG TCC CAC CCA GTT



    870           880          890          900           910


305
 L   I   S   G   P   G   D   P   S   P   Y   V   T   Q   G   G
320



TTA ATT TCA GGC CCC GGA GAT CCG TCC CCC TAC GTG ACA CAA GGG GGT



       920          930           940          950          960


321
 E   M   T   L   N   K   L   S   Q   P   V   Q   H   P   S   E
336



GAG ATG ACA CTC AAT AAA CTC TCA CAA CCA GTA CAA CAC CCT TCC GAG



          970          980          990          1000


337
 L   I   K   S   A   T   A   G   G   P   D   M   G   V   E   K
352



TTA ATT AAA TCT GCC ACA GCG GGC GGA CCT GAT ATG GGA GTG GAA AAG



1010       1020          1030         1040         1050


353
 D   T   V   R   A   L   I   T   S   R   P   M   H   P   S   S
368



GAC ACT GTC CGT GCA TTG ATC ACC TCG CGC CCG ATG CAT CCA AGC TCC



 1060         1070         1080          1090         1100


369
 S   A   K   L   L   S   K   L   D   A   A   G   S   I   E   E
384



TCA GCT AAG CTC CTG AGT AAG CTG GAT GCA GCC GGG TCG ATT GAA GAG



   1110          1120         1130         1140          1150


385
 I   R   K   I   K   R   L   A   L   N   G   *
396



ATC AGA AAG ATC AAG CGC CTT GCA CTA AAT GGC TAA



      1160         1170          1180









A primary use of the nucleotides as defined above is for the creation of plasmids using recombinant DNA technologies. The resulting recombinant molecule can then be introduced into an appropriate host. The plasmids thus created can be used to encode NP and P proteins. For expression of the NP and P proteins, any of the common expression vectors, especially the bacterial vectors can be used. The usable bacterial hosts for the vectors include any of the conventional prokaryotic cells. In this invention, the bacterial host used was Escherichia coli. Accordingly, a further aspect of the present invention provides for a prokaryotic cell, such as for example a bacterial cell and in particular an E. coli cell containing the nucleotides as defined above for the production of NP and P proteins.
The NP and P proteins, produced using recombinant plasmids in accordance with the present invention, can be in the fusion or non-fusion forms. In accordance with the embodiment of the present invention, it provides a method for producing the fusion and non-fusion forms of both the NP and P proteins of NDV virus strain AF2240 in an E. coli system. The preferred method for producing the fusion and non-fusion forms of both the NP and P proteins of NDV virus strain AF2240 comprises culturing the transformed E. coli of the present invention on an appropriate medium to express the said nucleocapsid protein and phosphoprotein, and isolating and purifying the expressed fusion proteins from the cultures.
While the invention will now be described in connection with certain preferred embodiments in the following experiments so that aspects thereof may be more fully understood and appreciated, it is not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the scope of the invention as defined by the appended claims.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a western blot of NDV nucleocapsid protein (NP) expressed by transformed E. coli TOP10 containing plasmid pTrcHis2-NP
FIG. 2 is a western blot of NDV phosphoprotein (P) expressed by transformed E. coli TOP10 containing plasmid pTrcHis2-P



DETAILED DESCRIPTION OF THE INVENTION
The present invention was accomplished through the employment of the recombinant DNA techniques which comprises the amplification of the NP and P coding regions of NDV strain AF2240, the cloning of the genes into the expression vector, the production of the transformed E. coli, the cultivation of the transformant, the expression of the NP and P proteins and the purification of the expressed fusion proteins.
The NP and P coding regions of NDV strain AF2240 which had been cloned into the expression vector were prepared through reverse transcription-polymerase chain reaction (RT-PCR). Three primers were used for each gene, which consisted of one forward and two reverse primers as listed below:
For the Amplification of the NP Gene








NPf1 (20 mer):
5′-cct tct gcc aac atg tct tc -3′



(Forward primer; SEQ ID NO: 5)


NPr1 (20 mer):
5′-tca ata ccc cca gtc ggt gt -3′



(Reverse primer SEQ ID NO: 6)


NPr2 (18 mer):
5′-ata ccc cca gtc ggt gtc -3′



(Reverse primer; SEQ ID NO: 7)






For the Amplification of the P Gene









Pf1 (20 mer):
5′-atg gcc acc ttt aca gat gc -3′



(Forward primer; SEQ ID NO: 8)


Pr1 (23 mer):
5′-taa tta gcc att tag tgc aag gc -3′



(Reverse primer; SEQ ID NO: 9)


Pr2 (21 mer):
5′-gcc att tag tgc aag gcg ctt -3′



(Reverse primer; SEQ ID NO: 10)






Incorporation of primers designated as NPf1 and NPr1 (for the NP gene), or Pf1 and Pr1 (for the P gene) during PCR had amplified gene products containing a stop codon at their 3′ ends, while the presence of primers NPf1 and NPr2 (for the NP gene) or Pf1 and Pr2 (for the P gene) gave rise to genes without any no stop codon. For cloning and expression purposes, a commercially available expression vector, pTrcHis2 (Invitrogen, USA) containing the coding regions for the myc epitope and 6 His residues downstream of the multiple cloning site was used. After cloning of the respective coding regions of NP and P genes into the pTrcHis2 vector, they were subsequently introduced into a bacterial host E. coli TOP10. The resulting plasmid harbouring the NP gene was designated as pTrcHis2-NP while the other one with the P gene as an insert was denoted as pTrcHis2-P. Both the NP and P proteins were expressed in E. coli TOP10 cells as non-fusion and fusion proteins. The latter forms contain the myc epitope and 6 His residues at their C termini. For protein identification, protein samples were analysed with SDS-PAGE and then followed by immunoblotting with the anti-NDV chicken serum and the anti-myc monoclonal antibody. The western blots for NP and P proteins are as shown in FIG. 1 and FIG. 2, respectively.
The expressed NP fusion protein was purified with affinity chromatography (nickel column), and was judged to be more than 90% pure by SDS-PAGE.
The nucleotide sequences of the NP and P genes were determined by the ABI PRISM® automated sequencer, model 377. The recombinant plasmids, pTrcHis2-N and pTrcHis2-P, were used as templates and the synthetic primers used in the sequencing reactions of the NP and P genes are as follows:
For the Sequencing of the NP Gene Coding Region








pTrcHis2F
5′-gag gta tat att aat gta tcg -3′


(21 mer):
(SEQ ID NO: 11)


sNPf1 (21 mer):
5′-gac tca tac atc agg aac acc -3′



(SEQ ID NO: 12)


sNPf2 (21 mer):
5′-gat gag agc agt ggc gaa cag -3′



(SEQ ID NO: 13)


pTrcHis2R
5′-gat tta atc tgt atc agg -3′


(18 mer):
(SEQ ID NO: 14)


sNPr1 (20 mer):
5′-tca ata ccc cca gtc ggt gt -3′



(SEQ ID NO: 15)


sNPr2 (21 mer):
5′-cta agt tgt aat acg tgg agc -3′



(SEQ ID NO: 16)


sNPr3 (21 mer):
5′-cca tcg atc tca aga aca tgc -3′



(SEQ ID NO: 17)






For the Sequencing of the P Gene Coding Region










pTrcHis2F
5′-gag gta tat att aat gta tcg -3′



(21 mer):
(SEQ ID NO: 18)


sPf (21 mer):
5′-gtc gac ttt gtg cag gcg atg -3′



(SEQ ID NO: 19)


sPf2 (21 mer):
5′-gga cac tgt ccg tgc att gat -3′



(SEQ ID NO: 20)


pTrcHis2.R
5′-gat tta atc tgt atc agg -3′


(18 mer):
(SEQ ID NO: 21)


sPr1 (21 mer):
5′-cca ggg tcc aga att ttc atc -3′



(SEQ ID NO: 22)


sPr2 (22 mer):
5′-ggt gtg gat agc tgt ttg tct g -3′



(SEQ ID NO: 23)






Both the NP and P coding regions were sequenced from 5′ to 3′ direction and reversely from 3′ to 5′ direction.
Example I illustrates the recombinant DNA techniques employed in obtaining bacterial clones harbouring a plasmid containing inserts of NP and P coding cDNA for NDV genomic RNA, the nucleotide sequences of the NP and P genes, and also the expressed NP and P proteins.
EXAMPLE I
Virus Propagation
The stock of NDV strain AF2240 was originally obtained from the Veterinary Research Institute (VRI), Ipoh. The virus was grown in the allantoic cavity of 8 to 9 day-old chicken embryonated eggs according to the procedures of Blaskovic and Styk (1967). After 3-4 days of incubation at 37° C., the eggs were chilled overnight at 4° C. The allantoic fluid was then harvested and the presence of the viruses was determined by haemagglutination (HA) test. The allantoic fluid which showed positive reaction of HA test was then clarified by centrifugation at 6000×g for 20 min at 4° C. (Beckman, JA14 rotor, USA) to remove debris.
Genomic RNA Extraction
Total RNA was extracted using the TRIZOL® LS reagent (Gibco BRL, USA). Briefly, 250 μl of the virus infected allantoic fluid was mixed with 750 μl Trizol LS reagent and incubated for 5 min at room temperature. After incubation, 100 μl of 1-bromo-3-chloropropane (BCP) (MRC, UK) was added and the mixtures were mixed vigorously for about 15 s and again incubated at room temperature for 10 min. The mixtures were base separated by microcentrifuging at 13,000×g for 15 min at 4° C. (Jouan MR 1812, France). The RNA was then precipitated by adding 500 μl of isopropanol (Merck) to the aqueous phase and left at room temperature for 10 min. The precipitated RNA was microcentrifuged at 13,000×g for 10 min and the pellet obtained was washed once with 75% (v/v) diethyl pyrocarbonate (DEPC) (Sigma, USA) treated ethanol (Hamburg). The pellet was dissolve in 20 μl of DEPC treated dH2O.
cDNA Synthesis and Amplification of Nucleocapsid (NP) and Phosprotein (P) Genes by RT-PCR
The amplification reactions were carried out in a programmed thermal cycler (MJ Research Inc. USA). Synthesis of the first strand cDNA was performed in a final volume of 30 μl. The reaction mixture contained 0.4 μM of each the forward and reverse primers, 0.2 mM deoxynucleoside triphosphate (MBI Fermentas, Inc. USA), 5 U of AMV reverse transcriptase (Promega, USA), 8 U of RNase inhibitor (Gibco BRL, USA), 1.5 mM of MgCl2 and 1× of reaction buffer (50 mM Tris-HCl, 15 mM (NH4)2SO4, 0.1% Triton X-100). The mixture was incubated at 42° C. for 30 min to synthesise the first strand of cDNA, and then 94° C. for 3 min to inactivate the reverse transcriptase.
For the amplification of the respective NP and P genes, another 20 μl of reaction mixture containing 1 U of DYNAZYMETM EXT DNA polymerase (FINNZYMES), 1.5 mM of MgCl2 and 1× of reaction buffer was added to the top of the above cDNA mixture which was held at 94° C. in the thermal cycler. The PCR profile for the amplification of NP gene comprising denaturation at 94° C. for 30 s, annealing at 55° C. for 50 s and extension at 72° C. for 1 min for a total of 30 cycles. To ensure a complete synthesis of the PCR product, the extension step at 72° C. was prolonged for 7 min after the last cycle. The PCR profile for the amplification of P gene was basically similar to that of NP gene, except the annealing step was carried out at 55° C. for 30 s.
Purification of the Amplified PCR Products
A total of 40 μl of the amplified PCR product was analysed on 1% TAE agarose gel. After the staining with ethidium bromide, the band with the correct size was excised from the gel and purified with the WIZARD® PCR Preps DNA Purification System (Promega, USA) according to the manufacturer's procedures. After purification, 5 μl of the PCR product was again analysed with agarose gel electrophoresis to determine the recovery of the PCR product, which would be used in TA CLONING®.
TOP® TA Cloning of NP and P genes
Four μl of the purified NP or P DNA fragments carrying an A overhang at their 3′ ends was mixed with 1 μl of the pTrcHis2 TOPO® expression vector (Invitrogen, USA) and the ligation reaction was carried out at room temperature (25° C.) for 5 min to form the desired recombinant plasmid.
Transformation
For transformation, 5 μl of the ligation mixture was added to 50 μl of TOP10 E. coli competent cells (Invitrogen, USA). The transformation mixture was incubated on ice for 30 min and the cells were heated at 42° C. for 30 to 60 s. This was followed by the adding of 250 μl SOC medium (2% trypton, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) and the incubation of the reaction mixture at 37° C. for 30 to 60 min with shaking at 250 rpm. Thirty-50 μl of the transformation mixture was spread on a LB plate containing 50 μg/ml ampicillin and 0.5% of glucose, and the plates were then incubated overnight at 37° C.
Screening for Positive Clones
Ten single colonies were randomly chosen and cultured overnight in 3 to 5 ml of LB medium containing 50 μg/ml ampicillin and 0.5% glucose. Plasmid DNA was isolated by using the alkaline lysis method and the orientation of the insert in the positive clones was confirmed by PCR.
Protein Expression
The identified positive clones were cultured overnight in LB medium containing 50 μg/ml ampicillin. The next day, 10 ml of LB medium containing 50 μg/ml ampicillin was inoculated with 0.2 ml of the overnight culture and incubated at 37° C. with shaking at 250 rpm. Once the cells reached the optical density of 0.6 to 0.8 at A600, 1 mM IPTG was added into the culture and continued shaking for 3 to 5 hours. The cells were harvested from the culture by centrifugation and then subjected to polyacrylamide gel electrophoresis (SDS-PAGE).
SDS-PAGE and Western Blotting
The cell pellets (from 1 ml culture solution) were resuspended in 50 to 100 μl of 1X SDS-PAGE sample buffer and boiled for 10 min. Five to 10 μl of the sample was loaded onto 12% SDS-PAGE gel and eletrophoresesed for 70 to 80 min at 32 volt. The proteins on SDS-PAGE gel were then electro-transferred onto a nitrocellulose membrane for 1 h. Western blotting was carried out by blocking the membrane first with skim milk for 1 h to saturate unoccupied regions on the membrane. This was followed by adding the anti-NDV chicken serum or anti-myc monoclonal antibody (for fusion protein) onto the membrane and this was shaken for 1 h at room temperature. The membrane was then washed four times with TTBS washing solution (TBS containing 0.5% Tween 20), 5 to 10 min for each wash to remove the unbound antibodies. After washing, peroxidase-labelled antibody was added to react with the primary antibody and left shaking for another 1 h. The membrane was further washed four times with TTBS solution, each for 5 to 10 min, and lastly BCIP/NBT solution was added as substrate for the peroxidase. The molecular weight of NP and P proteins was about 55 kDa while the fusion form for both the NP and P proteins gave rise to an apparent molecular weight of about 60 kDa.
Purification of NP Fusion Protein Using PROBONDTM Column Two hundred μl of LB medium containing 50 μg/ml ampicillin was cultured with 2 ml of overnight culture of transformant harbouring plasmid pTrcHis2-NP (carrying the NP insert without a stop codon), and the cells were grown to an OD600 of 0.6 to 0.8. Protein expression was then induced by adding 1 mM IPTG and the cells were grown for another 5 h. The cells were harvested by centrifugation at 2000×g for 15 min at 4° C. The cell pellet was first resuspended in 10 ml of binding buffer (500 mM NaCl, 20 mM NaH2PO4, pH 7.8), then 100 μg/ml of lysozyme was added and incubated for 15 min on ice. The cells were lysed by sonication until the cell lysate is no longer viscous. The cell lysate was then treated with RNase and DNase I, both at a concentration of 5 μg/ml for 15 min at 30° C. The cell lysate was then centrifuged at 10,000×g for 20 min to remove all the cell debris. The supernatant was collected and p sed through a 0.45 μm filter. This cell lysate was incubated with the PROBONDTM resin (Invitrogen, USA) for 30 min and then allowed to drip through the resin. The column was washed with 10 ml of washing buffer (50 mM Imidazole, 500 mM NaCl, 20 mM NaH2PO4, pH 6.0), and the proteins were then eluted with 5 ml of elution buffer (500 mM Imidazole, 500 mM NaCl, 20 mM NaH2PO4, pH 6.0). The elute was collected as 1 ml fractions. Samples from each fractions were analysed on 12% SDS-PAGE to check the purity of the protein.
References
Abdul Rahman, M. S., Chee, Y. S. and Lim, S. S. (1976) Observation on the response of breeder flocks to ranikhet standard vaccination. Kajian Vet. 8: 48-53.
Blaskovic, D. and Stvk, B. (1967) Laboratory methods of virus transmission in multicellular organisms. In: Maramorasch, K. and Koprocvski, H. (Eds.), Virology, Vol. 1. Academic Press, New York, pp. 194-197.
Idris, Z., Yusoff, K. Shamaan, N. A. and Ibrahim, A. L. (1993) The Effect of temperature on different strains of Newcastle disease virus. 2nd. UNESCO National Workshop on Promotion of Microbiology in Malaysia, 38.
Jemain S. F. P., (1999) Sequence determination of the Matrix gene in Newcastle disease virus strain AF2240. MS thesis, Universiti Putra Malaysia.
Lai C. M., (1985) A Study on a velogenic viscerotropic Newcastle disease virus in-vitro and in-vivo. PhD thesis, Universiti Pertanian Malaysia.
Salih, O., Omar, A. R., Ali, A. M. and Yusoff, K. (2000) Nucleotide sequence analysis of the F protein gene of a Malaysian velogenic NDV strain AF2240._Journal of Biochemistry, Molecular Biology and Biophysics 4: 51-57.
Tan, W. S., Lau, C. H., Ng, B. K., Ibrahim, A. L. and Yusoff, K. (1995) Nucleotide sequence of the haemagglutinin-neuraminidase (HN) gene of a Malaysian heat resistant viscerotropic-velogenic Newcastle disease virus (NDV) strain AF2240. DNA Sequence 6: 47-50.

## Claims

1. An isolated nucleic acid molecule encoding the full-length nucleocapsid (NP) protein of Newcastle disease virus (NDV), wherein the full length nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 1.

2. A recombinant expression plasmid containing the isolated nucleic acid molecule as claimed in claim 1.

3. The recombinant expression plasmid according claim 2, wherein the expression plasmid comprises coding regions for a myc epitope and 6 His residues downstream of a multiple cloning site into which the isolated nucleic acid is inserted.

4. An Escherichia coli cell transformed with the recombinant expression plasmid of claim 2.

5. The Escherichia coli cell according to claim 4, which has a genotype of F31  mcrA Δ(mrr-hsdRMS-mcrBC)φ80lacZΔM15ΔlacX74 recA1 araAΔ139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG.

6. An Escherichia coli cell transformed with the recombinant expression plasmid of claim 3.

7. The Escherichia coli cell according to claim 6, which has a genotype of F31  mcrA Δ(mrr-hsdRMS-mcrBC)φ80lacZΔM15 ΔlacX74 recA1 araΔ139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG.