SJIR-2基因真核表达载体的构建及在真核细胞内表达的研究

Construction of eukaryotic expression vector carrying SJIR-2 gene and investigation of its expression in eukaryotic cells

目的构建SJIR-2基因的真核表达载体,检测该基因在真核细胞内的表达,为进一步研究该基因作为血吸虫免疫疫苗打下基础。方法 PCR反应扩增出目的基因SJIR-2后插入到真核表达载体pEGFP中,双酶切鉴定并进行序列测定,以脂质体介导的方法转染293T细胞,再以Western blot方法检测该基因的编码蛋白在293T细胞内的表达情况。结果 PCR反应扩增出目的基因SJIR-2,构建了p EGFP-SJIR-2真核表达载体,双酶切鉴定插入片段大小正确,DNA测序结果与GeneBank中的SJIR-2基因序列同源性为100%,重组基因成功转染进293T细胞中,Western blot检测出该蛋白在293T细胞内的表达。结论成功构建了pEGFP-SJIR-2真核表达载体,该基因编码的蛋白可以在真核细胞内表达,为我们进一步探索SJIR-2基因的重组疫苗在血吸虫病防治中的作用打下基础。

Objective To construct eukaryotic expression vector of SJIR-2 and detect its expression in eukaryotic cells for further research this gene as the role of schistosomiasis vaccine in prevention and control of schistosomiasis. Methods Targeted SJIR-2 gene was amplified with PCR,and then inserted into eukaryotic expression vector p EGFP that was undergone double enzyme digestion and sequencing. The recombinant gene was transfected into 293 T cells with liposome mediated technique,and Western blot was performed to detect the expression of the protein encoding in eukaryotic cells. Results Targeted SJIR-2 gene was successfully amplified by PCR reactions,and pEGFP-SJIR-2 eukaryotic expression vector was finally constructed. Double enzyme digestion indicated errorlessly inserted fragment size,and DNA sequencing demonstrated 100% homology with SJIR-2 by Genebank reference. The recombinant gene was successfully transfected into 293 T cells,which were expressed in the eukaryotic cells by Western blot confirmation. Conclusion pEGFP-SJIR-2 vector was successfully constructed,and expressed in the eukaryotic cells,which may lay a foundation for further study of the vaccine on the recombinant SJIR-2 gene basis for prevention and control of schistosomiasis.