动物医学进展2021，Vol.42Issue(5)：50-57,8.
微小隐孢子虫E2F样结构域包含转录因子CpELD基因的原核和真核表达
Prokaryotic and Eukaryotic Expressions of Cryptosporidium parvum E2F-like Domain Containing Transcription Factor CpELD Gene
摘要
Abstract
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-CMI 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.关键词
微小隐孢子虫/cpeld基因/CpCML基因/原核表达/真核表达Key words
Cryptosporidium parvum/cpeld gene/CpCML gene/prokaryotic expression/eukaryotic expression分类
农业科技引用本文复制引用
陈宇,米荣升,龚海燕,程龙,王旭,韩先干,黄燕,陈兆国..微小隐孢子虫E2F样结构域包含转录因子CpELD基因的原核和真核表达[J].动物医学进展,2021,42(5):50-57,8.基金项目
国家自然科学基金项目(No.31702025) （No.31702025）
上海市科技兴农项 目(No.2019-02-08-00-08-F01151) （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) （No.GJFP2019027）
中央级公益性科研院所基本科研业务费项目(No.2019JB13). （No.2019JB13）
