人载脂蛋白M野生型和突变型表达载体的构建及鉴定

Construction and identification of wild-type and mutant human apolipoprotein M expression plasmid

目的:构建人载脂蛋白M(ApoM)野生型和突变型原核表达载体pGEX-KG-ApoM及真核表达载体pcDNA3.1(+)-ApoM。方法:Trizol法抽提HepG2细胞总RNA,RT-PCR扩增ApoM全长基因,将基因片断重组到PMD18-T质粒中构建PMD18-T-ApoM重组质粒载体,转化到大肠埃希菌JM109后筛选阳性克隆,提取质粒酶切和测序鉴定。采用Sited-directedMutagenesis定点诱变试剂盒对ApoM基因进行体外定点诱变,DNA测序鉴定定点诱变成功与否。用双酶切方法分别将ApoM野生型和突变型基因定向连接到原核表达载体pGEX-KG和真核表达载体pcDNA3.1(+)中,构建野生型和突变型pGEX-KG-ApoM重组体和pcDNA3.1(+)-ApoM重组体,分别转化到大肠埃希菌DH5α内,提取质粒酶切鉴定和测序鉴定。结果:成功克隆了ApoM并构建了野生型和突变型原核表达载体和真核表达载体。结论:通过RT-PCR,定点诱变及基因重组技术成功构建野生型和突变型重组表达质粒pGEX-KG-ApoM和pcDNA3.1(+)-ApoM,为进一步研究ApoM的功能奠定了基础。

Objective：To construct the wild-type and mutant human apolipoprotein M（ApoM） expression plasmid.Methods：The total RNA was extracted from HepG2 cells with Trizol.ApoM gene was amplified by RT-PCR,and inserted into PMD18-T vectors.After ApoM vector was transformed into E.coli JM109,the positive clones were obtained.Then the recombinant plasmids were identified by restriction enzyme digestion and DNA sequencing.In vitro site-directed mutagenesis was carried out with site-directed mutagenesis kit.Successful site-directed mutagenesis was conformed by DNA sequencing.The wild-type and mutant coding genes were subcloned into prokaryotic expression vector pGEX-KG and eukaryotic expression vector pcDNA3.1（＋）.The recombinant plasmids were transformed into E.coli DH5α and identified by restriction endonuclease digestion and DNA sequencing.Results：The human ApoM gene was successfully cloned.The wild-type and mutant pGEX-KG-ApoM and pcDNA3.1（＋）-ApoM recombinant plasmids were constructed successfully.Conclusions：With the techniques of RT-PCR,sited-directed mutagenesis and gene recombination,the wild-type and mutant pGEX-KG-ApoM and pcDNA3.1（＋）-ApoM were successfully constructed,which lays the foundation for further investigating the role of ApoM.