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

作　　者：呼高伟[1,2] 程天印[1] 米荣升[2] 秦培兰[2] 黄燕[2] 周鹏[2] 曹薇[2] 陈兆国[2] 

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128 [2]中国农业科学院上海兽医研究所,农业部动物寄生虫学重点开放实验室,中国农业科学院动物源性食品安全研究中心,上海200241

出　　处：《安徽农业科学》2012年第13期7739-7741,7803,共4页Journal of Anhui Agricultural Sciences

基　　金：国家科技重大专项项目(2012ZX10004220-008);中央级公益性科研院所基本科研业务费专项资金项目(2010JB12;2012JB16);上海市科技兴农重点攻关项目[沪农科攻字(2005)第3-4号]

摘　　要：[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.

关 键 词：cp-miR-2980 前体 真核表达载体 RT-PCR 

分 类 号：S852.723[农业科学—基础兽医学]