›› 2019, Vol. 46 ›› Issue (1): 215-222.doi: 10.16431/j.cnki.1671-7236.2019.01.025

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Screening,Expression and Immunogenicity Analysis of Sub-unit Vaccine Candidate Antigen Protein of Fusobacterium necrophorum

XIAO Lu1,2, XIE Jing1,2, KANG Runmin1,2, YE Yonggang1,2, YU Jifeng1,2, CAO Ye1,2, LI Xingyu1,2, WEI Yong1,2, LIN Yi1,2, LIAO Dangjin1,2, PAN Meng1,2, YE Jianqiang1,2, DAI Zhuojian1,2   

  1. 1. Sichuan Animal Science Academy, Chengdu 610066, China;
    2. Key Laboratory of Animal Genetics and Breeding of Sichuan Province, Chengdu 610066, China
  • Received:2018-06-11 Online:2019-01-20 Published:2019-01-19

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Abstract:

This study was aimed to screen the sub-unit vaccine candidate antigen protein of outer membrane protein (Omp) of Fusobacterium necrophorum (Fn),and lay the foundation for the development sub-unit vaccine of Fn.Fn Omp sequence was predicted by Uniprot,and the expression protein was screened based on the predicted results.Specific primers were designed according to the corresponding gene sequence of Fn Omp in GenBank,the QL03 strain was used as template for PCR amplification,prokaryotic expression,SDS-PAGE and Western blotting analysis.The proteins immunogenicity were analyzed by bactericidal assays and vaccination efficacy assessment in mice to screen the best candidate protein.The results showed that 121 proteins were obtained through the predicting outcomes,named 1-Fn to 121-Fn in turn,and 8-Fn,11-Fn,41-Fn,95-Fn and 102-Fn were screened to be candidate proteins,the fragments of 672,1 164,570,1 059 and 729 bp were amplified by PCR.SDS-PAGE and Western blotting results showed that 8-Fn expressed unsuccessfully,and the sizes of the others expressed proteins were 60,39,59 and 44 ku,which named P11-Fn,P41-Fn,P95-Fn and P102-Fn,and all proteins could react with Fn positive serum.The bactericidal assays results showed the 4 recombinant proteins had certain bactericidal effects,and the anti-P102-Fn serum was the best,the sterilizing rate reached 40.49%.The vaccination efficacy assessment results in mice showed the 4 recombinant proteins could protect mice from the challenge of QL03,and the P102-Fn was the best,the protection rate reached 60%.These results indicated that P11-Fn,P41-Fn,P95-Fn and P102-Fn were all of effective immunogenicity,P102-Fn could induce an immune response and it might be useful as an antigen for vaccine.

Key words: Fusobacterium necrophorum (Fn); prokaryotic expression; outer membrane protein (Omp); immunogenicity; bactericidal assay

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