摘要
为获取具有生物学活性的弓形虫 P30蛋白 ,采用定向克隆的方法 ,自行设计引物通过 PCR扩增得到 P30基因片段 ,用 Eco R 和 Sal 双酶切后 ,连接到同样双酶切的原核表达载体 (p BV2 2 0和 p MAL P2 )上 ,转化大肠杆菌 DH5 α,分别得到含重组质粒 p BV2 2 0 - P30和 p MAL P2 - P30的工程菌。扩菌提取质粒经过酶切分析和 PCR扩增鉴定后 ,证实 P30基因的两个原核表达载体构建成功 。
To obtain biological activity of P30 protein of Toxoplasma gondii , we constructed two prokaryotic expression vector contained a gene encoding P30 gene T. gondii . A pair of primes were designed and synthesized according to the published gene sequence of the P30 gene. At the 5′ end of sens and antisens strand of primers EcoRⅠ and SalⅠ enzyme sites were added respectively. Using PCR, the P30 gene which has 976 bp was amplified from genomic of RH strain T. gondii . After digested by EcoRⅠ and SalⅠ, the amplified gene fragments and plasmid vector(one is non fusion type vector pBV220,the other is fusion type vector pMALP 2) were ligated to construct recombinant plasmids. Then the ligation were transferred into E. coli DH5α. The positive clones were primarily confirmed by anti ampicillin selecting, restriction enzymes digestion and PCR. The successful construction of prokaryotic expression vector would be a good basis for the expression of P30 protein.
出处
《中国寄生虫病防治杂志》
CSCD
2000年第3期172-174,共3页
Chinese Journal of Parasitic Disease Control
基金
山东省卫生厅资助项目
关键词
弓形体
P30基因
克隆
Toxoplasma gondii
P30 gene
cloning
