摘要
为构建牛瑟氏泰勒虫双拷贝p23表面蛋白基因真核表达质粒,根据GenBank牛瑟氏泰勒虫p23表面蛋白基因序列(D84447),分别设计2对特异性引物,利用全血基因组DNA提取试剂盒提取牛瑟氏泰勒虫基因组DNA,采用SOE-PCR技术构建双拷贝p23基因,克隆到pMD-18一T载体上,经过PCR、酶切鉴定及测序后,亚克隆到pVAX-I真核表达载体上,经过鉴定后采用脂质体法将重组质粒pVA XI-2p23转染到BHK-21细胞,用IFA和RT-PCR来鉴定目的基因的表达情况.结果表明,成功构建了牛瑟氏泰勒虫双拷贝p23表面蛋白基因真核表达质粒,并在BHK-21细胞中获得表达.
To construct double-copy p23 major suface protein gene eukaryotic expression plasmid of Theileria sergenti, two pairs of specific oligonucleotide primers designed according to the gene encoding p23 surface protein of Theileria sergenti （D84447）, genomic DNA which Theileria sergenti in cattle was used for template,according SOE-PCR construct identical p23 gene, and cloned into pMD18-T-Simple vector. The right plasmid was determined by PCR and Digestion and Sequencing was subcloned into the eukaryotic expression vector pVAX1 as pVAX1-2p23. The plasmid was detected,pVAX1-2p23 was transfected with Lipofectamine to BHK-21 cells,the 2p23 genes expression were identified by IFA and RT-PCR. Double-copy p23 major suface protein gene eukaryotic expression plasmid of Theileria sergenti has been constructed. A base for further study of Theileria. sergenti as basis of immune.
出处
《延边大学农学学报》
2011年第4期242-247,共6页
Agricultural Science Journal of Yanbian University
基金
延边大学"211工程"三期重点建设项目
