微小隐孢子虫miR-2980真核表达载体的构建及初步鉴定

Construction and Preliminary Identification of Eukaryotic Expression Vector of Cryptosporidium parvum miR-2980

[目的]构建微小隐孢子虫的miR-2980真核表达载体,并进行鉴定。[方法]从微小隐孢子虫(Cryptosporidium parvum)基因组DNA扩增cp-miR-2980前体,克隆到pMD18-T载体上,经测序鉴定正确后亚克隆到pVAXⅠ载体上,转染HCT-8细胞,提取细胞总RNA,RT-PCR检测cp-miR-2980的表达。[结果]成功构建了cp-miR-2980真核表达载体pVAX-miR2980,RT-PCR检测证实其能在真核细胞HCT-8中表达。[结论]构建的真核表达载体pVAX-miR2980能在HCT-8细胞中成功表达cp-miR-2980,为后续研究cp-miR-2980的功能奠定了基础。

[ Objective ] This study aimed to construct and preliminarily identify the eukaryotic expression vector of C. parvum miR-2980. [ Method] The cp-miR-2980 precursor was amplified from C. parvum genomic DNA and cloned into pMD18-T vector. The amplified precursor was then subcloned into pVAX I vector and identified with restriction endonuclease digestion and sequencing. The recombinant plasmid pVAX- miR2980 was transfected into HCT-8 cells. Total RNA was extracted and the expression of cp-miR-2980 was evaluated by RT-PCR detection. [ Result] The results showed that the recombinant eukaryotic expression vector pVAX-miR2980 was successfully constructed, which can ex- oress co-miR-2980 in HCT-8 cell [ Conclusion] This study laid the foundation for further exDlorin~ the biolozical function of cD-miR-2980.