婴儿利什曼原虫<i>Pepck</i>和<i>Gp</i>63优势表位的真核与原核重组载体的构建与表达

张建辉; 王玉洁; 何金蕾; 李焕卿; 陈劲宇; 陈达丽; 陈建平

doi:10.12182/20210160104

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

摘要

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 63×10³, 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74×10³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。

Abstract:
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 63×10³(GP63) epitopes identified by the same method in our laboratory, the dominant epitope genes of Pepck and Gp63 were used to construct pET32a-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 NIH3T3 cells by liposome transfection.
Results There were multiple dominant epitopes in Pepck and there were Pepck-Gp63 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 NIH3T3 cells transfected by pVAX1-Pepck-Gp63 were positive.
Conclusion The recombinant prokaryotic expression plasmids pET32a-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 NIH3T3 cells, respectively, providing a preliminary experimental basis for the subsequent study of immunization strategies.

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