China Animal Husbandry and Veterinary Medicine ›› 2021, Vol. 48 ›› Issue (9): 3423-3431.doi: 10.16431/j.cnki.1671-7236.2021.09.034

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Preparation and Identification of Monoclonal Antibody Against Newcastle Disease Virus M Protein

ZENG Jianyu, DONG Zhenyuan, JIA Wenfeng, ZHANG Guozhong, XUE Jia   

  1. College of Veterinary Medicine, China Agricultural University, Beijing 100193, China
  • Received:2021-04-02 Online:2021-09-20 Published:2021-09-17

Abstract: To obtain the monoclonal antibodies against Newcastle disease virus (NDV) M protein, the gene encoding the protein was cloned, the recombinant protein was expressed and purified. Mice were immunized with recombinant M protein, and an appropriate ELISA screening method was established. After the serum titer of mice was determined by ELISA, the mouse spleen cells with the highest serum antibody titer were screened and fused with SP2/0 cells. Hybridoma cell lines that could stably produce monoclonal antibodies against NDV M protein were obtained. Immunofluorescence, Western blotting, subclass identification, chromosome count of hybridoma cells, preparation of mouse ascites, and titer determination of monoclonal antibody in ascites were carried out. The results of PCR, recombinant plasmid sequencing and double enzyme digestion showed that this study successfully amplified the NDV M gene, which was about 1 095 bp in size. SDS-PAGE and Western blotting analysis showed that the recombinant M protein was successfully expressed with about 60 ku molecular weight, and it could react with positive serum of NDV. In the established ELISA screening method, the optimal working concentration or dilution of recombinant M protein, His tagged protein and antibody were 0.5 μg/mL, 0.5 μg/mL and 1∶256 000, respectively. Immunofluorescence, Western blotting and subclass identification of the antibody showed that the hybridoma cells produced antibody could specifically bind to NDV SG10 strain and recombinant M protein, and its light chain was κ and heavy chain was IgG2A. Chromosome counts of C9-G2 and D3-F2 hybridioma cells were 97 and 101, respectively. The ELISA titers of the supernatant of the hybridioma cell lines were both 1∶6 400. And the ELISA titers of ascites were 1∶409 600 and 1∶102 400, respectively. In this study, monoclonal antibodies against NDV M protein were successfully prepared, which could provide tools for further study of the function of M protein.

Key words: Newcastle disease virus (NDV); M protein; monoclonal antibody

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