柔嫩艾美耳球虫棒状体颈部蛋白2基因真核表达载体的构建及在293T细胞中表达

为构建柔嫩艾美耳球虫棒状体颈部蛋白2(Eimeria tenella rhoptry neck protein 2,EtRON2)基因的重组质粒pCAGGS-EtRON2,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扩增其核心编码区的一部分,将其克隆至pGEM-Teasy载体,构建pGEM-Teasy-EtRON2质粒,双酶切出目的片段后与相应酶切的真核表达载体pCAGGS连接,构建真核表达质粒pCAGGS-EtRON2。该重组质粒经酶切和测序鉴定后转染293T细胞进行表达,分别用免疫印迹和间接免疫荧光鉴定EtRON2基因的表达情况。所构建的真核表达质粒pCAGGS-EtRON2经过双酶切鉴定,可见一条大小约为1172 bp的目的条带,测序结果与GenBank所登录序列完全一致;免疫印迹实验可见大小约为43 kDa的目的蛋白条带,间接免疫荧光实验可以检测到特异性红色荧光。研究结果表明已成功构建了EtRON2的真核表达质粒pCAGGS-EtRON2,并在真核细胞中获得表达,为深入研究EtRON2的生物学功能奠定了基础。

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR OF EIMERIA TENELLA RHOPTRY NECK PROTEIN 2 GENE AND ITS EXPRESSION IN 293T CELLS

To construct an eukaryotic expression vector for Eimeria tenella rhoptry neck protein 2 （EtRON2） gene and study the expresstion of EtRON2 gene in transfected 293T cells, a part of EtRON2 gene was amplified by PCR from the cDNA of Eimeria tenella sporulated oocyst and cloned into PGEM-Teasy vector to construct a vector named pGEM-Teasy-EtRON2. The pGEM-Teasy-EtRON2 and the pCAGGS vector were digested by the same restriction enzymes and the recombinant eukaryotic expression vector pCAGGS- EtRON2 was subsequently constructed. The recombinant plasmid pCAI3GS-EtRON2 was identified and transfectcd into 293T cells. The expression of EtRON2 protein was verified by indirect immunofluorescence assay and Western blot. Restriction enzyme digestion obtained correct length ofEtRON2 gene of about 1172 bp. Sequence analysis confirmed EtRON2 gene sequence that was exactly the same as that deposited in GenBank. The results of Western blot showed that the molecular weight of expressed protein was around 43 kDain accordance with the target protein. Red fluorescence was observed in indirect immunofluorescence assay. Taken together, the eukaryotic expression vector of pCAGGS-EtRON2 was constructed and expressed in 293T cells, which would establish the foundation for the future research on its biological functions.