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.

Description:
FIELD OF THE INVENTION  
         [0001]    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  
         [0002]    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.  
           [0003]    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.  
           [0004]    The genome of NDV is a linear, non-segmented, single-stranded negative sense RNA with a molecular weight of 5.2-5.7×10 6  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.  
           [0005]    The genes encoding for the HN (EMBL/Gen Bank/DDBJ accession No.X70092), 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 the 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.  
           [0006]    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  
         [0007]    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                            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                            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.. ..........                  
 
           [0008]    Further, the present invention provides the amino acid sequences of both the NP and P proteins as listed below:  
                                                                       NP gene: amino acid sequence                 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 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           A  AC 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           250          260          270           280   10  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             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             TCA TAC ATC AGG ACC ACC GGA CTT ACT GCA 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   T   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      A   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: amino acid sequence                      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           560           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 GAC 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                  
 
           [0009]    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.  
           [0010]    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.  
           [0011]    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  
       [0012]    [0012]FIG. 1 is a western blot of NDV nucleocapsid protein (NP) expressed by transformed  E. coli  TOP10 containing plasmid pTrcHis2-NP  
         [0013]    [0013]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  
       [0014]    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.  
         [0015]    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:  
         [0016]    For the Amplification of the NP Gene  
                                   NPf1 (20 mer):   5′-cct tct gcc aac atg tct tc -3′   (Forward primer)                   NPr1 (20 mer):   5′-tca ata ccc cca gtc ggt gt -3′   (Reverse primer)               NPr2 (18 mer):   5′-ata ccc cca gtc ggt gtc -3′   (Reverse primer)          
 
         [0017]    For the Amplification of the P Gene  
                                   Pf1 (20 mer):   5′-atg gcc acc ttt aca gat gc -3′   (Forward primer)                   Pr1 (23 mer):   5′-taa tta gcc att tag tgc aag gc -3′   (Reverse primer)               Pr2 (21 mer):   5′-gcc att tag tgc aag gcg ctt -3′   (Reverse primer)          
 
         [0018]    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.  
         [0019]    The expressed NP fusion protein was purified with affinity chromatography (nickel column), and was judged to be more than 90% pure by SDS-PAGE.  
         [0020]    The nucleotide sequences of the NP and P genes were determined by the ABI PRISM automated sequencer, model 377. The recombinant plasmids, pTrcHis2-NP 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:  
         [0021]    For the Sequencing of the NP Gene Coding Region  
                               pTrcHis2F   5′-gag gta tat att aat gta tcg -3′           (21 mer):               sNPf1 (21 mer):   5′-gac tca tac atc agg aac acc -3′               sNPf2 (21 mer):   5′-gat gag agc agt ggc gaa cag -3′               pTrcHis2R   5′-gat tta atc tgt atc agg -3′       (18 mer):               sNPr1 (20 mer):   5′-tca ata ccc cca gtc ggt gt -3′               sNPr2 (21 mer):   5′-cta agt tgt aat acg tgg agc -3′               sNPr3 (21 mer):   5′-cca tcg atc tca aga aca tgc -3′          
 
         [0022]    For the Sequencing of the P Gene Coding Region  
                               pTrcHis2F   5′-gag gta tat att aat gta tcg -3′           (21 mer):               sPf (21 mer):   5′-gtc gac ttt gtg cag gcg atg -3′               sPf2 (21 mer):   5′-gga cac tgt ccg tgc att gat -3′               pTrcHis2.R   5′-gat tta atc tgt atc agg -3′       (18 mer):               sPr1 (21 mer):   5′-cca ggg tcc aga att ttc atc -3′               sPr2 (22 mer):   5′-ggt gtg gat agc tgt ttg tct g -3′          
 
         [0023]    Both the NP and P coding regions were sequenced from 5′ to 3′ direction and reversely from 3′ to 5′ direction.  
         [0024]    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  
       [0025]    Virus Propagation  
         [0026]    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.  
         [0027]    Genomic RNA Extraction  
         [0028]    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 phase separated by microcentrifugating 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 dissolved in 20 μl of DEPC treated dH 2 O.  
         [0029]    cDNA Synthesis and Amplification of Nucleocapsid (NP) and Phosprotein (P) Genes by RT-PCR  
         [0030]    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 MgCl 2  and 1× of reaction buffer (50 mM Tris-HCl, 15 mM (NH 4 ) 2 SO 4 , 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.  
         [0031]    For the amplification of the respective NP and P genes, another 20 μl of reaction mixture containing 1 U of DyNAzyme EXT DNA polymerase (FINNZYMES), 1.5 mM of MgCl 2  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.  
         [0032]    Purification of the Amplified PCR Products  
         [0033]    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&#39;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.  
         [0034]    TOPO TA Cloning of NP and P Genes  
         [0035]    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 (Invitogen, USA) and the ligation reaction was carried out at room temperature (25° C.) for 5 min to form the desired recombinant plasmid.  
         [0036]    Transformation  
         [0037]    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 MgCl 2 , 10 mM MgSO 4 , 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.  
         [0038]    Screening for Positive Clones  
         [0039]    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.  
         [0040]    Protein Expression  
         [0041]    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 A 600 , 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).  
         [0042]    SDS-PAGE and Western Blotting  
         [0043]    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.  
         [0044]    Purification of NP Fusion Protein Using ProBond Column  
         [0045]    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 OD 600  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 NaH 2 PO 4 , 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 passed through a 0.45 μm filter. This cell lysate was incubated with the ProBond resin (Invirogen, 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 NaH 2 PO 4 , pH 6.0), and the proteins were then eluted with 5 ml of elution buffer (500 mM Imidazole, 500 mM NaCl, 20 mM NaH 2 PO 4 , 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  
       [0046]    Abdul Rahman, M. S., Cheer Y. S. and Lim, S. S. (1976) Observation on the response of breeder flocks to ranikhet standard vaccination. Kajian Vet. 8: 48-53.  
         [0047]    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.  
         [0048]    Idris, Z., Yusoff, K. Shamaan, N. A. and Ibrahim, A. L. (1993) The Effect of temperature on different strains of Newcastle disease virus. 2 nd . UNESCO National Workshop on Promotion of Microbiology in Malaysia, 38.  
         [0049]    Jemain S. F. P., (1999) Sequence determination of the Matrix gene in Newcastle disease virus strain AF2240. MS thesis, Universiti Putra Malaysia.  
         [0050]    Lai C. M., (1985) A Study on a velogenic viscerotropic Newcastle disease virus in-vitro and in-vivo. PhD thesis, Universiti Pertanian Malaysia.  
         [0051]    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.  
         [0052]    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.  
     
       
       
         1 
         
           
             23  
           
           
             1  
             1470  
             DNA  
             Newcastle disease virus  
           
            1 

atgtcttccg tattcgatga atacgagcag ctcctcgctg ctcagactcg ccccaatgga     60 

gctcacggag ggggagagag agggagcact ttaagagttg aggtcccagt attcactctt    120 

aacagtgacg atccagaaga tagatggaat tttgcggtat tctgtcttcg gattgctgtt    180 

agcgaggacg ccaacaaacc gctcaggcaa ggtgctctca tatccctcct gtgctcccat    240 

tctcaagtga tgaggaacca tgttgccctt gcaggaaaac agaatgaggc tacactgact    300 

gttcttgaga tcgatggttt taccagcagc gtgcctcagt tcaacaacag gagtggggtg    360 

tctgaggaga gagcacagag attcatggtg atagcagggt ctctccctcg ggcgtgcagt    420 

aacggtactc cgttcgtcac ggctggggtt gaagatgatg caccagaaga tatcactgat    480 

actctggaaa gaatcctgtc tatccaggct caggtatggg tcacagtagc gaaggccatg    540 

actgcatatg agacagcaga tgagtcggaa acaagaagaa tcaataagta catgcagcaa    600 

ggcagagtcc agaagaagta catcctccac cctgtatgca ggagtgcaat tcaactcaca    660 

atcagacatt ctctggcagt ccgcattttc ttagttagcg agcttaagag aggccgcaat    720 

acggcaggtg ggagctccac gtattacaac ttagtagggg atgtagactc atacatcagg    780 

aacaccggac ttactgcatt cttccttaca ctcaaatatg gaattaatac caagacatca    840 

gccctagcac tcagcagcct cacaggcgat atccaaaaga tgaagcagct catgcgttta    900 

tatcggatga agggagaaaa tgcgccgtac atgacattgc taggtgacag tgatcagatg    960 

agctttgcac cggctgagta tgcacagctt tattcttttg ccatgggcat ggcatcagtc   1020 

ttagataaag gaactggcaa ataccaattc gccagagact tcatgagcac atcattctgg   1080 

agactcgggg tggagtatgc tcaggctcag gggagtagca tcaacgaaga catggctgct   1140 

gagctaaaac taaccccggc agcaagaagg ggcctggcag ctgctgccca acgagtgtct   1200 

gaggaaactg gcagcgtgga tattcctact caacaagccg gggtcctcac tgggctcagc   1260 

gatggaggcc cccgagcctc tcagggtgga tcgaacaagt cgcaagggca accagatgcc   1320 

ggagatgggg agacccaatt cttggatttg atgagagcag tggcgaacag catgcgagaa   1380 

gcgccaaact ccgcacagag caccacccac ccggaacccc ccccgactcc cgggccatca   1440 

caagataacg acaccgactg ggggtattga                                    1470 

 
           
             2  
             1188  
             DNA  
             Newcastle disease virus  
           
            2 

atggccacct ttacagatgc ggagatagat gatatatttg agaccagtgg aactgtcatt     60 

gacagcataa ttacggccca gggtaaatca gcagagactg tcggaaggag cgcaatccca    120 

caaggcaaga ccaaagcgct gagcatagca tgggagaagc atgggagcat ccaaccatcc    180 

accagccagg acaaccccga ccaacaggat agaccagaca aacagctatc cacacctgag    240 

caggcgaccc cacacaacag ctcgccagcc acatccgccg aaccgctccc cactcaggcc    300 

gcaggtgagg ccggcgacac acagctcaag accggagcaa gcaactctct tctgtctatg    360 

ctcgacaagc tgagcaataa accatctaat gctaaaaagg gcccatggtc gagtccccag    420 

gaaggatatc atcaacctcc gacccaacaa catggggatc agccgaaccg cggaaacagc    480 

caggagaggc tgcggcacca agccaaggcc gcccctggaa gccggggcac agacgcgagc    540 

acagcatatc atggacaatg gaaggagtca caactatcag ctggtgcaac ccctcatgtg    600 

ctccaatcag ggcagagcca agacagtact cctgtacctg tggatcatgt ccagccacct    660 

gtcgactttg tgcaggcgat gatgactatg atggaggcgt tatcacagaa ggtaagtaaa    720 

gtcgactatc agctagacct agtcttaaag cagacatcct ccatccctat gatgcggtct    780 

gaaatccaac agctaaaaac atctgttgcg gtcatggaag ctaatttagg catgatgaaa    840 

attctggacc ctggttgtgc taacatttca tccttaagtg atctgcgggc agtcgcccgg    900 

tcccacccag ttttaatttc aggccccgga gatccgtccc cctacgtgac acaagggggt    960 

gagatgacac tcaataaact ctcacaacca gtacaacacc cttccgagtt aattaaatct   1020 

gccacagcgg gcggacctga tatgggagtg gaaaaggaca ctgtccgtgc attgatcacc   1080 

tcgcgcccga tgcatccaag ctcctcagct aagctcctga gtaagctgga tgcagccggg   1140 

tcgattgaag agatcagaaa gatcaagcgc cttgcactaa atggctaa                1188 

 
           
             3  
             489  
             PRT  
             Newcastle disease virus  
           
            3 

Met Ser Ser Val Phe Asp Glu Tyr Glu Gln Leu Leu Ala Ala Gln Thr 
1               5                   10                  15 

Arg Pro Asn Gly Ala His Gly Gly Gly Glu Arg Gly Ser Thr Leu Arg 
            20                  25                  30 

Val Glu Val Pro Val Phe Thr Leu Asn Ser Asp Asp Pro Glu Asp Arg 
        35                  40                  45 

Trp Asn Phe Ala Val Phe Cys Leu Arg Ile Ala Val Ser Glu Asp Ala 
    50                  55                  60 

Asn Lys Pro Leu Arg Gln Gly Ala Leu Ile Ser Leu Leu Cys Ser His 
65                  70                  75                  80 

Ser Gln Val Met Arg Asn His Val Ala Leu Ala Gly Lys Gln Asn Glu 
                85                  90                  95 

Ala Thr Leu Thr Val Leu Glu Ile Asp Gly Phe Thr Ser Ser Val Pro 
            100                 105                 110 

Gln Phe Asn Asn Arg Ser Gly Val Ser Glu Glu Arg Ala Gln Arg Phe 
        115                 120                 125 

Met Val Ile Ala Gly Ser Leu Pro Arg Ala Cys Ser Asn Gly Thr Pro 
    130                 135                 140 

Phe Val Thr Ala Gly Val Glu Asp Asp Ala Pro Glu Asp Ile Thr Asp 
145                 150                 155                 160 

Thr Leu Glu Arg Ile Leu Ser Ile Gln Ala Gln Val Trp Val Thr Val 
                165                 170                 175 

Ala Lys Ala Met Thr Ala Tyr Glu Thr Ala Asp Glu Ser Glu Thr Arg 
            180                 185                 190 

Arg Ile Asn Lys Tyr Met Gln Gln Gly Arg Val Gln Lys Lys Tyr Ile 
        195                 200                 205 

Leu His Pro Val Cys Arg Ser Ala Ile Gln Leu Thr Ile Arg His Ser 
    210                 215                 220 

Leu Ala Val Arg Ile Phe Leu Val Ser Glu Leu Lys Arg Gly Arg Asn 
225                 230                 235                 240 

Thr Ala Gly Gly Ser Ser Thr Tyr Tyr Asn Leu Val Gly Asp Val Asp 
                245                 250                 255 

Ser Tyr Ile Arg Asn Thr Gly Leu Thr Ala Phe Phe Leu Thr Leu Lys 
            260                 265                 270 

Tyr Gly Ile Asn Thr Lys Thr Ser Ala Leu Ala Leu Ser Ser Leu Thr 
        275                 280                 285 

Gly Asp Ile Gln Lys Met Lys Gln Leu Met Arg Leu Tyr Arg Met Lys 
    290                 295                 300 

Gly Glu Asn Ala Pro Tyr Met Thr Leu Leu Gly Asp Ser Asp Gln Met 
305                 310                 315                 320 

Ser Phe Ala Pro Ala Glu Tyr Ala Gln Leu Tyr Ser Phe Ala Met Gly 
                325                 330                 335 

Met Ala Ser Val Leu Asp Lys Gly Thr Gly Lys Tyr Gln Phe Ala Arg 
            340                 345                 350 

Asp Phe Met Ser Thr Ser Phe Trp Arg Leu Gly Val Glu Tyr Ala Gln 
        355                 360                 365 

Ala Gln Gly Ser Ser Ile Asn Glu Asp Met Ala Ala Glu Leu Lys Leu 
    370                 375                 380 

Thr Pro Ala Ala Arg Arg Gly Leu Ala Ala Ala Ala Gln Arg Val Ser 
385                 390                 395                 400 

Glu Glu Thr Gly Ser Val Asp Ile Pro Thr Gln Gln Ala Gly Val Leu 
                405                 410                 415 

Thr Gly Leu Ser Asp Gly Gly Pro Arg Ala Ser Gln Gly Gly Ser Asn 
            420                 425                 430 

Lys Ser Gln Gly Gln Pro Asp Ala Gly Asp Gly Glu Thr Gln Phe Leu 
        435                 440                 445 

Asp Leu Met Arg Ala Val Ala Asn Ser Met Arg Glu Ala Pro Asn Ser 
    450                 455                 460 

Ala Gln Ser Thr Thr His Pro Glu Pro Pro Pro Thr Pro Gly Pro Ser 
465                 470                 475                 480 

Gln Asp Asn Asp Thr Asp Trp Gly Tyr 
                485 

 
           
             4  
             395  
             PRT  
             Newcastle disease virus  
           
            4 

Met Ala Thr Phe Thr Asp Ala Glu Ile Asp Asp Ile Phe Glu Thr Ser 
1               5                   10                  15 

Gly Thr Val Ile Asp Ser Ile Ile Thr Ala Gln Gly Lys Ser Ala Glu 
            20                  25                  30 

Thr Val Gly Arg Ser Ala Ile Pro Gln Gly Lys Thr Lys Ala Leu Ser 
        35                  40                  45 

Ile Ala Trp Glu Lys His Gly Ser Ile Gln Pro Ser Thr Ser Gln Asp 
    50                  55                  60 

Asn Pro Asp Gln Gln Asp Arg Pro Asp Lys Gln Leu Ser Thr Pro Glu 
65                  70                  75                  80 

Gln Ala Thr Pro His Asn Ser Ser Pro Ala Thr Ser Ala Glu Pro Leu 
                85                  90                  95 

Pro Thr Gln Ala Ala Gly Glu Ala Gly Asp Thr Gln Leu Lys Thr Gly 
            100                 105                 110 

Ala Ser Asn Ser Leu Leu Ser Met Leu Asp Lys Leu Ser Asn Lys Pro 
        115                 120                 125 

Ser Asn Ala Lys Lys Gly Pro Trp Ser Ser Pro Gln Glu Gly Tyr His 
    130                 135                 140 

Gln Pro Pro Thr Gln Gln His Gly Asp Gln Pro Asn Arg Gly Asn Ser 
145                 150                 155                 160 

Gln Glu Arg Leu Arg His Gln Ala Lys Ala Ala Pro Gly Ser Arg Gly 
                165                 170                 175 

Thr Asp Ala Ser Thr Ala Tyr His Gly Gln Trp Lys Glu Ser Gln Leu 
            180                 185                 190 

Ser Ala Gly Ala Thr Pro His Val Leu Gln Ser Gly Gln Ser Gln Asp 
        195                 200                 205 

Ser Thr Pro Val Pro Val Asp His Val Gln Pro Pro Val Asp Phe Val 
    210                 215                 220 

Gln Ala Met Met Thr Met Met Glu Ala Leu Ser Gln Lys Val Ser Lys 
225                 230                 235                 240 

Val Asp Tyr Gln Leu Asp Leu Val Leu Lys Gln Thr Ser Ser Ile Pro 
                245                 250                 255 

Met Met Arg Ser Glu Ile Gln Gln Leu Lys Thr Ser Val Ala Val Met 
            260                 265                 270 

Glu Ala Asn Leu Gly Met Met Lys Ile Leu Asp Pro Gly Cys Ala Asn 
        275                 280                 285 

Ile Ser Ser Leu Ser Asp Leu Arg Ala Val Ala Arg Ser His Pro Val 
    290                 295                 300 

Leu Ile Ser Gly Pro Gly Asp Pro Ser Pro Tyr Val Thr Gln Gly Gly 
305                 310                 315                 320 

Glu Met Thr Leu Asn Lys Leu Ser Gln Pro Val Gln His Pro Ser Glu 
                325                 330                 335 

Leu Ile Lys Ser Ala Thr Ala Gly Gly Pro Asp Met Gly Val Glu Lys 
            340                 345                 350 

Asp Thr Val Arg Ala Leu Ile Thr Ser Arg Pro Met His Pro Ser Ser 
        355                 360                 365 

Ser Ala Lys Leu Leu Ser Lys Leu Asp Ala Ala Gly Ser Ile Glu Glu 
    370                 375                 380 

Ile Arg Lys Ile Lys Arg Leu Ala Leu Asn Gly 
385                 390                 395 

 
           
             5  
             20  
             DNA  
             Newcastle disease virus  
           
            5 

ccttctgcca acatgtcttc                                                 20 

 
           
             6  
             20  
             DNA  
             Newcastle disease virus  
           
            6 

tcaatacccc cagtcggtgt                                                 20 

 
           
             7  
             18  
             DNA  
             Newcastle disease virus  
           
            7 

atacccccag tcggtgtc                                                   18 

 
           
             8  
             20  
             DNA  
             Newcastle disease virus  
           
            8 

atggccacct ttacagatgc                                                 20 

 
           
             9  
             23  
             DNA  
             Newcastle disease virus  
           
            9 

taattagcca tttagtgcaa ggc                                             23 

 
           
             10  
             21  
             DNA  
             Newcastle disease virus  
           
            10 

gccatttagt gcaaggcgct t                                               21 

 
           
             11  
             21  
             DNA  
             Newcastle disease virus  
           
            11 

gaggtatata ttaatgtatc g                                               21 

 
           
             12  
             21  
             DNA  
             Newcastle disease virus  
           
            12 

gactcataca tcaggaacac c                                               21 

 
           
             13  
             21  
             DNA  
             Newcastle disease virus  
           
            13 

gatgagagca gtggcgaaca g                                               21 

 
           
             14  
             18  
             DNA  
             Newcastle disease virus  
           
            14 

gatttaatct gtatcagg                                                   18 

 
           
             15  
             20  
             DNA  
             Newcastle disease virus  
           
            15 

tcaatacccc cagtcggtgt                                                 20 

 
           
             16  
             21  
             DNA  
             Newcastle disease virus  
           
            16 

ctaagttgta atacgtggag c                                               21 

 
           
             17  
             21  
             DNA  
             Newcastle disease virus  
           
            17 

ccatcgatct caagaacatg c                                               21 

 
           
             18  
             21  
             DNA  
             Newcastle disease virus  
           
            18 

gaggtatata ttaatgtatc g                                               21 

 
           
             19  
             21  
             DNA  
             Newcastle disease virus  
           
            19 

gtcgactttg tgcaggcgat g                                               21 

 
           
             20  
             21  
             DNA  
             Newcastle disease virus  
           
            20 

ggacactgtc cgtgcattga t                                               21 

 
           
             21  
             18  
             DNA  
             Newcastle disease virus  
           
            21 

gatttaatct gtatcagg                                                   18 

 
           
             22  
             21  
             DNA  
             Newcastle disease virus  
           
            22 

ccagggtcca gaattttcat c                                               21 

 
           
             23  
             22  
             DNA  
             Newcastle disease virus  
           
            23 

ggtgtggata gctgtttgtc tg                                              22