Preparation Of Anti-ndv Monoclonal Antibodies And Identification Of Partial Antigenic Epitopes In Np Protein

Newcastle Disease Virus (NDV) is an important pathogen which causes great losses to poultry industry in the world. Currently much research has been done on NDV; however the structure and function of the proteins encoded by NDV and the mechanism on pathogencity of the virus are not fully elucidated. Using monoclonal antibodies (MAbs) is still a basic tool to do research on the structure and function analyses of the proteins encoded by NDV, epitope identification and differentiate diagnosis of NDV.The prokaryotic expression vector containing the whole F gene and its partial segment of FA(1-357bp), FB(336-1047bp)and FC(948-1662bp)was constructed, after induction by IPTG, the fragment of the fused protein was expressed with a molecular weight of 30.5ku, 43.6ku and 43.7ku, respectively. Among them FA corresponding to F2, FB and FC corresponding to partial overlapped two fragment of F1. The result of Western-blot showed that expressed protein FB and FC have good reactivity. Two MAbs were obtained by immunization with the prokaryotic expressed fragment FB and FC and named as FB-B9 and FC-B5, respectively.6-week-old female BALB/c mice were immunized with purified NDV QY97 strain for the preparation of MAbs by cell-fusion technology. After screening by indirect ELISA and IFA, 15 MAbs were obtained. Among them, MAb 5A12 belongs to IgG2a subgroup; MAb 18C6 belongs to IgG2b subgroup; MAbs 31B4 and 33F12 belong to IgM subgroup; and the others belong to IgG1 subgroup. All these MAbs have no neutralization and HI activities. Cross-reactivity of MAbs with 10 NDV strains of different genotype were detected by sandwich ELISA and different reactivity was found.The purified NDV QY97 strain was used as antigen to do Western-blot with MAbs obtained in the study. The results of western-blot showed that 18C6, 24F9 and 25C12 reacted with 55ku protein fragment of NDV, so these three MAbs may react with linear antigenic epitope on structural proteins of NDV; the protein recognized by MAbs is NP or F protein. While other MAbs showed no obvious reactions with NDV proteins, these MAbs may react with conformational antigenic epitope on structural proteins of NDV.Ten NDV strains from genotype I, II, VI, IX and VII were used as typical strain and cross-reactivity of three MAbs which recognize linear epitope with different NDV strains were detected by indirect ELISA and Western-blot. The results are the same with the results of sandwich ELISA; MAb 18C6 could react only with NDV CK/CH/HLJ/1/06(ZY), CK/CH/HN/1/07(LHN), QY97 and Mallard/CH/HLJ01/06(JL01). MAb 24F9 did not react with NDV strain in genotypeâ…¥, but could react with strains in other genotype; while MAb 25C12 could react with all the strains used.Serials of truncated NP protein were expressed by prokaryotic expression system and used as antigen to map the linear antigenic epitope recognized by MAbs 18C6 and 24F9 by Western-blot, respectively. The results showed that these two MAbs could react with C-terminal of NP protein. Antigenic epitope recognized by 18C6 lines in amino acid sequence of 473-481 and its sequence is (473)^PPPTPGASQ⁴⁸¹; while the antigenic epitope recognized by 24F9 lines in amino acid sequence of 470-478 and its sequence is (470)^HPEPPPTPG⁴⁷⁸. Two antigenic epitopes are overlapped and they have the same core sequence (473)^PPPTPG⁴⁷⁸.The results of NP gene analysis showed that NP gene is relatively conservative, but the differences are obvious in the C-terminal 400-489aa of NP protein. The homology of amino acid sequence in C-terminal 400-489aa of avirulent strain is high and is 83.5% to the lowest level. The homology of amino acid sequence in this segment in the virulent strains is related to genotype, it is above 84.4% in the same genotype; while the homology is 67% between some virulent and avirulent strain. The difference in 400-489aa in the C-terminal of NP protein showed the variation of NP has its pattern in the virus evolution process.There are some differences in amino acid sequence of the antigenic epitope corresponding to the position of 470-478 recognized by MAb 24F9 in NDV strains from different genotypes. It is conservative in the NDV strains from genotype VII and the amino acid sequence is (470)^HPEPPPTPG⁴⁷⁸; while the amino acid sequence is (470)^H(P/L)EP(L/P)(S/P)HE⁴⁷⁸in NDV strains belong to genotype VI, His in position 477 was not seen in the virus from other genotypes; the amino acid sequence of avirulent virus strains are(470)^Q(S/L)GPPPTPG⁴⁷⁸, there are at least three amino acid difference with virulent strains.15 MAbs were obtained by NDV immunization and 2 MAbs were obtained by immunization with prokaryotic expressed F protein peptide segment in this study, respectively. Different reactivity was found among MAbs with NDV of different genotype. Sequence analysis of NP gene showed that there are some differences in amino acid sequence in the C-terminal of NP protein in the virulent and avirulent NDV strains from different genotypes. Two overlapped epitopes were identified by two MAbs and they have same core sequence. This study laid foundation for the further study on the structure and function analyses of the proteins encoded by NDV, epitope identification and differentiate diagnosis of NDV.