中国动物传染病学报Issue(6)：33-38,6.
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
CONSTRUCTION OF RECOMBINANT PLASMIDS OF EIMERIATENELLA CALCIUM-DEPENDENT PROTEIN KINASES 3 GENE AND ITS EXPRESSION IN 293T CELLS
摘要
Abstract
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.关键词
柔嫩艾美耳球虫/钙依赖蛋白激酶3基因/293T细胞/真核表达Key words
Eimeriatenella/calcium-dependent protein kinases 3/293T cell/erkaryotic expression分类
农业科技引用本文复制引用
王晔,韩红玉,董辉,姜连连,赵其平,朱顺海,薛璞,舒凡帆,黄兵..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[J].中国动物传染病学报,2013,(6):33-38,6.基金项目
中央级公益性科研院所基本科研业务费项目 （）
