人tPA信号肽和Yersinia Pestis保护性抗原F1-V融合基因的构建及其免疫效果观察

为获得含有鼠疫F1和V抗原编码基因以及人tPA信号肽基因的重组质粒tPA-pVAX1/F1-V，并测定其诱导特异性免疫应答的能力, 用PCR扩增鼠疫菌F1和V编码基因，分别与pGEM-T连接测序，构建pVAX1/F1-V融合重组质粒.PCR扩增tPA信号肽片段并将其插入到F1-V的上游，构建tPA-pVAX1/F1-V融合重组质粒；转染COS-7细胞，Western blot法鉴定目的蛋白的表达.重组质粒tPA-pVAX1/F1-V加GM-CSF佐剂免疫BALB/c小鼠，观察免疫效果.400个LD50强毒鼠疫菌皮下攻毒观察保护率.结果表明,tPA-pVAX1/F1-V在COS-7细胞中表达；免疫鼠体内产生特异性抗体；抗体亚型分析、细胞因子等指标的测定表明,所构建DNA疫苗以诱发Th1型免疫为主；攻毒保护率达90%.结果提示，已成功构建F1-V融合蛋白真核表达载体tPA-pVAX1/F1-V，且具有诱导特异性细胞免疫和体液免疫应答的能力, 对强毒鼠疫菌皮下攻毒有一定的保护效力，为鼠疫菌新型疫苗研制奠定了基础.

Construction and Immunogenicity of Fused Gene Containing Protective Antigen F1-V of Yersinia Pestis and Signal Peptide of Human tPA

To construct recombinant eukaryotic expression vector tPA-pVAX1/F1-V based on the coding-gene of F1-V antigen and signal of tPA and then study its immunogenicity,Yersinia pestis F1-V gene was amplified from EV76 line by PCR.They were inserted into the eukaryotic expression vector pVAX1,tPA signal encoding sequence was amplified by PCR and then inserted to the 5'-terminus of F1-V to construct secretion eukaryotic expression vector tPA-pVAX1/F1-V.Then the recombinant plasmid was injected into BALB/c mice via an intramuscular route together with plasmid DNA carrying GM-CSF gene.Mice were challenged via subcutaneous injection of virulent Yersinia pestis 10 days after the fourth immunization and observed twice daily for morbidity and mortality.tPA-pVAX1/F1-V recombinant plasmid was successfully constructed.Western blot analysis indicated that F1-V fusion protein was expressed in COS-7 cells.Animals immunized with the recombinant plasmid induced antigen specific antibody.Furthmore,the recombinant plasmid induced typical T-helper 1-dominated immune response as determined by immunoglobulin G isotype and cytokine analysis.It was able to induce a effective immuno-protection against subcutaneous challenge of virulent Yersinia pestis with 400 LD 50,the survival rate was 90%.tPA-pVAX1/F1-V can induce both humoral and cellular immune response,it could be used as potential candidate vaccine for the control of Yersinia Pestis.