人DNMT3B7原核表达载体的构建和表达

Construction and Expression of Prokaryotic Expression Plasmid of Human DNMT3B7

目的:构建人类DNA甲基转移酶(DNMT)3B7原核表达载体,并进行初步表达。方法:根据Genebank中人DNMT3B7 cDNA序列设计并合成特异性引物;提取HCT116细胞总RNA并进行反转录,利用PCR技术扩增出DNMT3B7;将扩增产物克隆到原核表达载体PGEX-4T.3中,并对重组质粒进行鉴定;然后将重组质粒转化E.coli BL21,IPTG诱导表达目的蛋白。结果:PCR扩增得到人DNMT3B7的cDNA片段大小为1.1 kb,重组子利用限制性内切酶进行酶切、PCR鉴定和DNA序列分析得到原核表达载体PGEX-4T.3-DNMT3B7;IPTG诱导表达后,SDS-PAGE分析显示目的蛋白在E.coli BL21中高效表达,分子量约为40 kD。结论:成功构建了人DNMT3B7原核表达载体,完成了其在E.coli BL21中的诱导表达,为进一步在体外研究DNMT3B异构体提供了条件。

Objective： To construct the prokaryotic expression plasmid of human PGEX-4T·3-DNMT3B7 and express DNMT3B7 in E.coli BL21（DE3）.Methods： According to the published human cDNA sequence in Genebank,a pair of primers were respectively designed and synthesized.The total RNA was isolated from HCT116 cells.After amplification with reverse transcription polymerase chain reaction（RT-PCR）,the product was cloned into PGEX-4T·3 vector.The recombinants were finally sequenced and identified by restrictive endonuclease digestion.Then the recombinants were transferred into E.coli BL21 for expression.Induced by 1 mmol/L IPTG,the expression product of DNMT3B7 gene was analyzed by SDS-PAGE.Results： PGEX-4T·3-DNMT3B7 prokaryotic expression vectors was successfully constructed,and it can express DNMT3B7 in E.coli BL21.Conclusion： The DNMT3B7 prokaryotic expression vectors is successfully constructed and DNMT3B7 protein has been expressed successfully.