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

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.