tPA信号肽和鼠疫杆菌保护性抗原V融合基因的构建及免疫效果鉴定

获得含有鼠疫杆菌V抗原编码基因以及tPA信号肽编码序列的重组质粒,并测定其诱导特异性免疫应答的能力。采用PCR扩增鼠疫菌杆菌V基因构建到pVAX1质粒中产生pVAX1/V重组质粒,PCR扩增tPA信号肽编码序列片段并将其插入到pVAX1/V中V基因的上游,构建tPA-pVAX1/V重组质粒;转染COS-7细胞,免疫细胞化学方法鉴定V蛋白的表达;二重组质粒分别加mGM-CSF质粒免疫BALB/c小鼠,观察免疫应答反应;以400个LD50强毒鼠疫杆菌皮下攻击免疫小鼠观察保护效率。结果显示,tPA-pVAX1/V在COS-7细胞中表达了V蛋白;免疫小鼠血清产生了特异性抗体和细胞免疫应答;攻毒保护率达80%。成功构建了分泌型V蛋白的真核表达质粒载体,具有诱导特异性细胞免疫和体液免疫应答的能力,对强毒鼠疫杆菌攻毒有一定的保护效力,为鼠疫杆菌新型疫苗研制奠定了基础。

Construction of fused plasmid containing protective antigen V of Yersinia pestis and signal peptide of tPA and its immuno genicity

To construct recombination secretory eukaryotic expression vector tPA-pVAX1/V based on the coding-gene of V antigen and signal of tPA and then investigate its immunogenicity.Yersinia pestis V gene was amplified from EV76 line by PCR,and it was inserted into the eukaryotic expression vector pVAX1,tPA signal encoding sequence was amplified by PCR method and then was inserted to the 5' terminus of V to construct secretory eukaryotic expression plasmid tPA-pVAX1/V.Then the recombinant plasmid was injected into BALB/c mice via an intramuscular route together with plasmid DNA carrying GM-CSF gene as an adjuvant.Mice were challenged via subcutaneous injection of virulent Yersinia pestis 10 days after the fourth immunization and observed twice daily for morbidity and mortality.The recombinant plasmid tPA-pVAX1/V was successfully constructed;Immunocytohist ochemistry method analysis indicated that V protein was expressed in COS-7 cells;Animals immunized with the recombinant plasmid induced antigen specific antibody;Furthmore,the recombinant plasmid enhanced typical T-helper 1-dominated immune response as determined by immunoglobulin G isotype and cytokine analysis.It was able to induce an effective immuno-protection against subcutaneous challenge of virulent Yersinia pestis of 400 LD_(50),and the survival rate was 80%.It is concluded that tPA-pVAX1/V can induce both humoral and cellular immune responses,it could be used as potential candidate vaccine for the control of Yersinia pestis infections.