柔嫩艾美耳球虫钙依赖蛋白激酶3在293T细胞中的表达研究

摘要

为构建柔嫩艾美耳球虫钙依赖蛋白激酶3（Eimeria tenella calcium-dependent protein kinases 3, EtCDPK3）基因的重组质粒pCAGGS-EtCDPK3，并转染293T细胞进行表达，以柔嫩艾美耳球虫孢子化卵囊cDNA为模板，经PCR扩增其含完整开放阅读框的序列，将其克隆至pGEM-T easy载体，构建pGEM-Teasy-EtCDPK3重组质粒，双酶切回收目的片段后与相应酶切的真核表达载体pCAGGS连接，构建真核重组表达质粒pCAGGS-EtCDPK3。该重组质粒经酶切和测序鉴定正确后转染293T细胞进行表达，分别用免疫印迹和间接免疫荧光鉴定EtCDPK3基因的表达情况。结果显示所构建的真核重组表达质粒pCAGGS-EtCDPK3经过双酶切鉴定，可见1条大小约为1302 bp的目的条带，测序结果与实验室已获得的EtCDPK3序列完全一致；Western blot结果可见大小约为49 kDa的目的蛋白条带，间接免疫荧光实验可以检测到红色荧光。这些研究结果表明已成功构建了EtCDPK3的真核重组表达质粒pCAGGS-EtCDPK3，并在真核细胞中获得表达，为深入研究EtCDPK3的功能和制备DNA疫苗奠定了基础。%The objective of the present study was to construct a recombinant vector of Eimeria tenella calcium-dependent protein kinases 3 (EtCDPK3) gene and study expression of EtCDPK3 gene in transfected 293T cells. The full-length cDNA of EtCDPK3 gene was amplified in PCR from cDNA of E. tenella sporulated oocysts and cloned into pGEM-Teasy vector to construct a recombinant plasmid pGEM-Teasy-EtCDPK3. The pGEM-Teasy-EtCDPK3 and pCAGGS vector were digested with the same restriction enzymes and recombinant plasmid pCAGGS-EtCDPK3 was subsequently constructed, which was then transfected into 293T cells. The results suggested that the target strip of app. 1302 bp was visualized after digestion of pCAGGS-EtCDPK3 with restriction enzymes. The DNA sequencing confirmed that EtCDPK3 gene sequence was exactly the same as that reported previously. The expression of EtCDPK3 protein was verified in indirect immunofluorescence assay and Western blotting. The molecular weight of expressed EtCDPK3 protein was app.49 kDa. Furthermore, red fluorescence was observed in indirect immunofluorescence assay. The construction of eukaryotic recombinant plasmid pCAGGS-EtCDPK3 and successful expression of EtCDPK3 in 293T cells have established the foundation for future research on its biological functions and development of DNA vaccine.