微小隐孢子虫CP15基因高效真核表达载体的构建及其在Hela细胞中的表达

Construction of a eukaryotic expressing vector containing CP15 gene of C.parvum and its expression in Hela cells

目的 :构建隐孢子虫CP15真核表达载体pCR3 1 15 ,观察其在Hela细胞中的表达。方法 :用BglⅡ从pMD18 T 15中酶切得到CP15基因 ,将其插入真核表达载体pCR3 1(+)的BamHⅠ位点 ,构建CP15真核表达载体pCR3 1 15 ,脂质体介导法将其转染Hela细胞 ,并用G4 18加压筛选 ,用RT PCR方法检测外源CP15基因的转录 ,用ELISA法和间接免疫荧光法检测其活性。结果 :酶切鉴定表明已成功构建了重组真核表达载体pCR3 1 15 ;外源CP15基因能在转染细胞中有效转录 ;ELISA法和间接免疫荧光法实验结果表明表达产物具有良好的生物活性。结论 :构建的pCR3 1 15真核表达载体在Hela细胞中具有良好的表达活性。

Objective:To construct an eukaryotic expressing vector pCR3 1 15 containing CP15 gene of Crypstosporidium parvum(C.parvum) and express it in Hela cells Methods:CP15 gene of C parvum was obtained from pMD18 T 15 disgested by BglⅡ and was inserted into eukaryotic expressing vector pCR3 1(+) in BamHⅠ site,and then Hela cells were transfected with recombinant by liposomes The transcription and expressed products of CP15 in the transfected Hela cells were assayed by RT PCR,ELISA and indiret immunofluorescence assay after screening with G418 Results:It showed that pCR3 1 15 was constructed successfully CP15 gene was transcripted in transfectants and CP15 protein with obvious biological activity was highly expressed in Hela cells Conclusion:CP15 gene in recombinant vector is proved to be expressed in Hela cells and obvious biological activity of expression production in transfected cells was detected