中国畜牧兽医2019，Vol.46Issue(1)：215-222,8.DOI:10.16431/j.cnki.1671-7236.2019.01.025
坏死梭杆菌亚单位疫苗候选抗原蛋白的筛选、表达及免疫原性分析
Screening, Expression and Immunogenicity Analysis of Sub-unit Vaccine Candidate Antigen Protein of Fusobacterium necrophorum
摘要
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 tothe 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 059and729 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 44ku, 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 4recombinant 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 4recombinant 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.关键词
坏死梭杆菌/原核表达/外膜蛋白/免疫原性/杀菌试验Key words
Fusobacterium necrophorum (Fn) /prokaryotic expression/outer membrane protein (Omp) /immunogenicity/bactericidal assay分类
农业科技引用本文复制引用
肖璐,廖党金,潘梦,叶健强,戴卓建,谢晶,康润敏,叶勇刚,于吉锋,曹冶,李兴玉,魏勇,林毅..坏死梭杆菌亚单位疫苗候选抗原蛋白的筛选、表达及免疫原性分析[J].中国畜牧兽医,2019,46(1):215-222,8.基金项目
四川省财政运行专项(SASA2014CZYX009) （SASA2014CZYX009）
四川省科技成果转化(2017CC0053) （2017CC0053）
四川省重点研发计划(2018NZ0003) （2018NZ0003）
