减毒沙门菌介导牛结核杆菌多表位疫苗的构建及免疫反应性研究

Construction and immune response of attenuated Salmonella-mediated bovine tuberculosis multi-epitope vaccine

目的构建以减毒沙门菌为载体的牛结核杆菌多表位疫苗,免疫小鼠后观察其刺激产生的免疫反应。方法运用BepiPred和IEBD对牛结核杆菌Ag85B、MPT63和HSP65的B、T细胞表位进行预测分析,设计新的表位基因肽段AMH,通过ProtParam、VaxiJen、ProtScale、TMHMM、SOPMA和SWISS-MODEL在线软件对AMH的理化性质、免疫原性、亲水性、跨膜区和空间结构进行分析,利用C-IMMSIM进行免疫模拟。将AMH、Ag85B、MPT63和HSP65基因序列用T2A和P2A连接后构建入真核表达载体pEGFP-N1。将重组质粒导入减毒沙门菌LH430,对重组减毒沙门菌进行PCR检测。取重组减毒沙门菌以每只2×10^(7)cfu/200μL的剂量灌胃免疫小鼠2次,检测末次免疫后小鼠血清抗体和细胞因子水平及脾淋巴细胞中的T细胞亚群百分率。结果共筛选出17条表位肽段,构建的表位蛋白序列AMH分子质量为30.21339 ku。生物信息学预测AMH结构稳定,亲水性高,无跨膜区,二级结构松散易与抗体结合。免疫模拟显示AMH可引起B细胞和TH细胞免疫反应,刺激抗体IgM、IgG和细胞因子IFN-γ、IL-2水平升高。重组质粒经双酶切得到4713 bp和3133 bp两条目的片段,PCR扩增的目的基因片段大小与预期相符,且重组减毒沙门菌经PCR扩增获得3133 bp的片段。与PBS组和空载减毒沙门菌组相比,小鼠经重组减毒沙门菌加强免疫后血清IgG水平显著升高(均P<0.01),细胞因子IFN-γ、IL-4和IL-10水平显著升高(均P<0.05),脾脏CD4^(+)T淋巴细胞占比和CD4^(+)/CD8^(+)值均显著升高(均P<0.05)。结论重组减毒沙门菌能激发小鼠的体液免疫和细胞免疫反应,为牛结核病新型疫苗的研制奠定了基础。

Objective To construct a multi-epitope vaccine of bovine tuberculosis with attenuated Salmonella as the carrier,and to observe the immune response in mice after immunization.MethodsBepiPred and IEBD were used to predict and analyze the B and T cell epitopes of bovine tuberculosis Ag85B,MPT63 and HSP65,and a new epitope gene peptide AMH was designed.The physical and chemical properties,immunogenicity,hydrophilicity,transmembrane region and spatial structure of AMH were analyzed by ProtParam,VaxiJen,ProtScale,TMHMM,SOPMA and SWISS-MODEL online software.C-IMMSIM was used for immune simulation.The AMH,Ag85B,MPT63 and HSP65 gene sequences were ligated with T2A and P2A to construct the eukaryotic expression vector pEGFP-N1.The recombinant plasmid was introduced into attenuated Salmonella LH43O,and the recombinant attenuated Salmonella was detected by PCR.The mice were immunized twice with recombinant attenuated Salmonella at a dose of 21o'cfu/200μL per mouse.The levels of serum antibodies and cytokines and the percentage of T cell subsets in spleen lymphocytes were detected after the last immunization.Results A total of 17 epitope peptides were screened,and the molecular weight of the constructed epitope protein sequence AMH was 30.21339 ku.Bioinformatics predicts that AMH has stable structure,high hydrophilicity,no transmembrane region,loose secondary structure and easy binding to antibodies.Immunological simulation showed that AMH could induce immune responses of B cells and TH cells,and increase the levels of IgM,IgG and cytokines IFN-and IL-2.The recombinant plasmid was double-digested to obtain two target fragments of 4713 bp and 3133 bp.The size of the target gene fragment amplified by PCR was consistent with the expectation,and the recombinant attenuated Salmonella was amplified by PCR to obtain a 3133 bp fragment.Compared with PBS group and empty attenuated Salmonella group,the serum IgG level of mice after booster immunization with recombinant attenuated Salmonella was significantly increased(all P<0.01),the levels of cytokines IFN-,IL-4 and IL-10 were significantly increased(all P<0.05),and the proportion of spleen CD4^(+)T lymphocytes and CD4^(+)/CD8^(+)values were significantly increased(all P<0.05).Conclusion The recombinant attenuated Salmonella can stimulate the humoral and cellular immune responses in mice,which lays a foundation for the development of a new vaccine for bovine tuberculosis.