摘要
根据已发表的弓形虫主要表面抗原Ｐ３０基因序列，自行设计合成了一对引物，分别在其正义链和反义链的５′末端加上了ＢａｍＨⅠ和ＥｃｏＲⅠ的酶切位点，利用ＰＣＲ技术获取Ｐ３０抗原基因整个开放阅读框架的碱基序列。ＰＣＲ产物经ＢａｍＨⅠ和ＥｃｏＲⅠ双酶切后，与经同样两种内切酶切割的质粒载体ｐＢｌｕｅｓｃｒｉｐｔＳＫ连接构成重组质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ－Ｐ３０，转化大肠杆菌ＤＨ５α。通过遗传标记筛选、ＰＣＲ扩增及酶切分析对重组子进行了鉴定，为进一步选择真核系统或原核系统体外表达该抗原打下了必要的基础。
A pair of primers were designed and synthesized according to the published gene sequence of the major surface antigen(P30) of Toxoplasma gondii . BamHⅠ and EcoRⅠ sites were added, respectively, at the 5′ end of sens and anti sens strand of the primers. Using PCR, the whole coding sequence of P30 gene were amplified. The amplified gene fragments and plasmid pBluescript SK were digested with BamHⅠ and EcoRⅠ, and then ligated to construct recombinant plasmids pBluescript SK P30. The recombinant DNA were primarily confirmed by genetic marker PCR and digestion with restriction enzymes. The research laid a foundation for expression of P30 gene in procaryotic or eucaryotic system.
出处
《中国寄生虫病防治杂志》
CSCD
1998年第4期284-287,共4页
Chinese Journal of Parasitic Disease Control
关键词
弓形虫
抗原
PCR
基因克隆
Toxoplasma gondii , antigen, PCR, gene cloning
