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

摘要

试验旨在筛选坏死梭杆菌 (Fusobacterium necrophorum, Fn) 外膜蛋白 (outer membrane protein, Omp) 中的候选抗原蛋白, 为Fn的亚单位疫苗研究奠定基础.采用Uniprot对Fn Omp序列进行预测, 基于预测结果筛选拟表达蛋白, 并根据GenBank中登录的Fn Omp相应基因序列设计特异性引物, 以QL03株为模板进行PCR扩增、原核表达, 以及SDS-PAGE和Western blotting分析, 并通过血清杀菌试验和小鼠免疫攻毒保护试验, 鉴定重组蛋白的免疫原性, 筛选最佳候选蛋白.结果显示, 预测得到121个蛋白, 顺次命名为1-Fn～121-Fn, 根据预测结果筛选出8-Fn、11-Fn、41-Fn、95-Fn和102-Fn 5个候选蛋白, 其PCR扩增产物大小分别为672、1 164、570、1 059、729bp.SDS-PAGE和Western blotting分析显示, 除8-Fn未表达外, 其余4个蛋白均成功表达, 大小分别为60、39、59、44ku, 命名为P11-Fn、P41-Fn、P95-Fn和P102-Fn, 均能与Fn阳性血清反应.血清杀菌试验结果显示, 4个重组蛋白均有一定杀菌效果, 其中抗P102-Fn血清的杀菌效果最佳, 杀菌率可达40.49％.小鼠免疫保护试验结果显示, P11-Fn、P41-Fn、P95-Fn和P102-Fn对小鼠均具有一定的保护效果, 其中P102-Fn保护效率最高, 达60％.结果表明, P11-Fn、P41-Fn、P95-Fn和P102-Fn均具有良好的免疫原性, 其中P102-Fn的免疫原性及诱导机体产生保护免疫反应能力最佳, 最有望成为Fn亚单位疫苗的候选蛋白.%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.