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目的 构建隐孢子虫CP15/60真核表达载体pcDNA3.1-15/60,观察其在Hela细胞中的表达。方法用Xho I和EcoR I从pMD18-T-15/60中酶切得到CP 15/60基因,将其插入真核表达载体pCR3.1（+）的 XhoI和EcoRI位点,构建CP15/60真核表达载体pcDNA3.1-15/60,用脂质体介导法将其转染Hela细胞,并用G418加压筛选,用RT-PCR方法检测外源CP15/60基因的转录,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pcDNA3.1-15/60,外源CP15/60基因能在转染细胞中有效转录,经检测表达产物具有良好的生物活性。结论 已成功地构建了pcDNA3.1-15/60真核表达载体,并在Hela细胞中具有良好的表达。
Objective To construct the eukaryotic expression vector pcDNA3.1-15 / 60 of Cryptosporidium parvum CP15 / 60 and observe its expression in Hela cells. Methods The CP 15/60 gene was digested with Xho I and EcoR I from pMD18-T-15/60 and inserted into the XhoI and EcoRI sites of the eukaryotic expression vector pCR3.1 (+) to construct CP15 / 60 The recombinant plasmid pcDNA3.1-15 / 60 was transfected into Hela cells by liposome-mediated method and was screened by G418. The transcription of exogenous CP15 / 60 gene was detected by RT-PCR, and the indirect ELISA Immunofluorescence was used to detect the activity. Results Restriction endonuclease digestion showed that the recombinant eukaryotic expression vector pcDNA3.1-15 / 60 was successfully constructed. The exogenous CP15 / 60 gene can be transcribed efficiently in the transfected cells. The expressed product has good bioactivity. Conclusion The pcDNA3.1-15 / 60 eukaryotic expression vector has been successfully constructed and expressed well in Hela cells.