婴儿利什曼原虫Pepck和Gp63优势表位的真核与原核重组载体的构建与表达  

Construction and Expression of Eukaryotic and Prokaryotic Recombinant Vectors of Pepck and Gp63 Dominant Epitopes of Leishmania infantum

作　　者：张建辉 王玉洁 何金蕾 李焕卿 陈劲宇 陈达丽[1] 陈建平[1] ZHANG Jian-hui;WANG Yu-jie;HE Jin-lei;LI Huan-qing;CHEN Jin-yu;CHEN Da-li;CHEN Jian-ping(Parasitology Teaching and Research Center,Department of Pathogenic Biology,West China School of Basic Medical Sciences and Forensic Medicine,Sichuan University,Chengdu 610041,China;Department of Preventive Medicine,West China School of Public Health and West China Fourth Hospital,Sichuan University,Chengdu 610041,China)

机构地区：[1]四川大学华西基础医学与法医学院病原生物学系寄生虫教研室,成都610041 [2]四川大学华西公共卫生学院/四川大学华西第四医院预防医学系,成都610041

出　　处：《四川大学学报（医学版）》2021年第2期194-201,共8页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金面上项目(No.81672048、No.31572240和No.31872959);大学生创新训练计划项目(No.C2020108116)资助。

摘　　要：目的选取婴儿利什曼原虫的Pepck和Gp63的优势表位基因,构建Pepck-Gp63二联优势表位的原核与真核重组表达载体并表达蛋白。方法用计算机分析预测磷酸烯醇丙酮酸羧基酶(phosphoenolpyruvate carboxylase,PEPCK)的二级结构及HLA表位,筛选出优势表位。根据本实验室之前对表面蛋白酶63(glycoprotein of 63×10^(3),GP63)用相同的方法分析而筛选的表位结果,把Pepck和Gp63的优势表位基因通过重叠PCR和酶切连接反应构建出重组原核表达质粒pET32a-Pepck-Gp63,并诱导其在大肠杆菌中表达;构建出重组真核表达质粒pVAX1-Pepck-Gp63,用脂质体转染法把真核质粒转染到NIH3T3细胞中表达。结果Pepck存在多个优势表位;测序结果表明二联优势表位基因Pepck-Gp63连接成功;PEPCK-GP63蛋白在大肠杆菌中以包涵体形式表达并在SDS-PAGE电泳-考马斯亮蓝染色的结果中呈现相对分子质量为74×10^(3)大小的条带;细胞免疫荧光中被pVAX1-Pepck-Gp63转染的NIH3T3细胞呈阳性。结论成功构建重组原核表达质粒pET32a-Pepck-Gp63和重组真核表达质粒pVAX1-Pepck-Gp63,并分别在大肠杆菌和NIH3T3细胞中验证重组质粒能表达相应目的蛋白,为后续的免疫策略研究提供初步实验基础。Objective To construct eukaryotic and prokaryotic recombinant vectors containing Pepck-Gp63 and to achieve protein expression by selecting the dominant epitope genes of Pepck and Gp63 of Leishmania infantum.Methods The secondary structure and HLA epitopes of phosphoenolpyruvate carboxylase(PEPCK)were predicted by in silico analysis,and the dominant epitopes were picked out.According to the analysis results of glycoprotein of 63×10^(3)(GP63)epitopes identified by the same method in our laboratory,the dominant epitope genes of Pepck and Gp63 were used to construct pET32 a-Pepck-Gp63 and pVAX1-Pepck-Gp63 by overlapping PCR and enzyme reaction.Then,for protein expression,the prokaryotic vectors were transfected into E.coil while the eukaryotic vectors were transfected into NIH3 T3 cells by liposome transfection.Results There were multiple dominant epitopes in Pepck and there were PepckGp63 sequences in the polyclonal site of expression vector.The expression of Pepck-Gp63 in E.coil appeared in inclusion form and led to 74 kDa band in SDS-PAGE.The immunofluorescence results of NIH3 T3 cells transfected by pVAX1-Pepck-Gp63 were positive.Conclusion The recombinant prokaryotic expression plasmids pET32 a-Pepck-Gp63 and eukaryotic expression plasmids pVAX1-Pepck-Gp63 were successfully constructed,and it was shown that the recombinant plasmids were able to express the corresponding target proteins in E.coli and NIH3 T3 cells,respectively,providing a preliminary experimental basis for the subsequent study of immunization strategies.

关 键 词：婴儿利什曼原虫 表位疫苗 Pepck基因 Gp63基因 

分 类 号：R392[医药卫生—免疫学] Q78[医药卫生—基础医学]