摘要 为了实现微小隐孢子虫(Cryptosporidium parvum)E2F样结构域包含转录因子编码基因cpeld的原核和真核表达,设计2对特异性引物,以C.parvum卵囊cDNA为模板,PCR扩增得到cpeld基因,将其分别插入pET32a和p3XFLAG-CMV14载体。同时设计引物,扩增微小隐孢子虫类钙调蛋白(C.parvum calmoduin-like proteins)的编码基因CpCML,插入pcmv-HA载体。将鉴定正确的重组质粒分别转入BL21(DE3)和293T细胞进行表达,Western blot观察重组蛋白的反应原性。结果显示,成功构建了重组原核表达质粒pET32a-cpeld和真核表达质粒p3XFLAG-CMV14-cpeld以及pcmv-HA-CML;SDS-PAGE显示,重组质粒转化菌经IPTG诱导后表达出了分子质量约为55 ku的重组蛋白;Western blot分析显示,原核表达的重组蛋白能与鼠抗His单克隆抗体,真核表达重组CpELD和CpCML蛋白能分别与抗FLAG和HA单克隆抗体反应。结果表明,成功实现了cpeld基因的原核和真核表达以及CpCML基因的真核表达,表达产物具有良好的反应原性,为后续开展隐孢子虫CpELD和CpCML的功能和作用机制研究、新型的防控技术研发奠定了基础。 In order to achieve the prokaryotic and eukaryotic expressions of the Cryptosporidium parvum E2F-like domain containing transcription factor encoding gene cpeld,the C.parvum oocyst cDNA was used as a template,and the cpeld genes were amplified by PCR with 2 pairs of specific primers.The PCR products were inserted into pET32a and p3XFLAG-CMV14 vector,respectively.At the same time,a pair of primers were designed to amplify the C.parvum calmoduin-like protein encoding gene CpCML and inserted into pcmv-HA vector.The identified recombinant plasmids were transferred into BL21(DE3)and 293T cells respectively,and expressed.The reactionogenicities of the recombinant proteins were observed by Western blot.The results showed that the recombinant prokaryotic expression plasmid pET32a-cpeld and the eukaryotic expression plasmid p3XFLAG-CMV14-cpeld and pcmv-HA-CML were successfully constructed.SDS-PAGE showed that the recombinant plasmid transforming bacteria successfully expressed a 55 ku recombinant protein after IPTG induction.Western blot analysis showed that the recombinant protein expressed in prokaryotic cells could react with mouse anti-His monoclonal antibodies,eukaryotic expressed recombinant CpELD and CpCML proteins could react with anti-FLAG and HA monoclonal antibodies,respectively.These results indicated that the cpeld gene was prokaryotically and eukaryotically expressed,the CpCML gene was eukaryotically expressed successfully,and the expression products had good reactogenicity.It laid a foundation for the follow-up research on the function and mechanism of CpELD and CpCML and the development of new prevention and control methods.
机构地区 中国农业科学院上海兽医研究所
出处 《动物医学进展》 北大核心 2021年第5期50-57,共8页 Progress In Veterinary Medicine
基金 国家自然科学基金项目(No.31702025) 上海市科技兴农项目(No.2019-02-08-00-08-F01151) 国家农产品质量安全风险评估计划项目(No.GJFP2019027) 中央级公益性科研院所基本科研业务费项目(No.2019JB13)。
关键词 微小隐孢子虫 cpeld基因 CpCML基因 原核表达 真核表达 Cryptosporidium parvum cpeld gene CpCML gene prokaryotic expression eukaryotic expression
