Sited-Directed Mutagenesis Of Human Apolipoprotein M And Assay Of Its Biological Function

Objective Sited-directed mutagenesis of human apolipoprotein M genes to cnstructwild-type and mutant human apolipoprotein M expression plasmid and assay of itsbiological function. Methods Trizol extract liver cell total RNA L02, RT-PCRamplification ApoM full-length gene, gene fragment will be recombined into PMD18-Tplasmid construct PMD18-T-ApoM recombinant plasmid and transformed into E coliJM109.The positive clones, plasmids extracted digestion and sequencing. Sited-directedmutagenesis using site-directed mutagenesis kit ApoM genes in vitro site-directedmutagenesis, DNA sequencing success of site-directed mutagenesis. Respectively, bydouble enzyme digestion of wild type and mutant ApoM directed to connect to the geneexpression vector pGEX-KG and pEGFP-N3to construct wild-type and mutantpGEX-KG-ApoM and pEGFP-N3-ApoM recombinants were transformed into into E coliDH5α, the extraction of plasmid restriction enzyme digestion and sequencing. Will buildgood prokaryotic expression plasmid transformed into E. coli DE3(BL21),SDS-polyacrylamide gel electrophoresis (sds-page) showed that wild-type prokaryoticexpression plasmid of fusion protein could be highly expressed ApoM. The constructedvectors were transiently transfected into7402cells, using western blot techniquetransfected7402cells Which were detected ApoM protein expression.Using in vitroexperiments,using cholesterol, cholesterol efflux and antioxidant assay experimentstesting wild-type and mutant protein biological functions ApoM. Results Full-lengthgene was successfully cloned and built ApoM wild type and mutant expression vectors ofApoM. sds-page electrophoresis showed that wild-type prokaryotic expression plasmid offusion protein could be highly expressed ApoM. Western blot showed that transfectedwild-type recombinant plasmid ApoM7402cells, ApoM protein levels were increased,while the empty vector empty liposome group and ApoM group of mutant recombinant protein were lower ApoM expression. THP-1cells are involved in cholesterol effluxexperiments. With the empty vector, empty liposome group, and ApoM mutation carriergroup, the wild-type carrier group has significantly promoted in the outflow of cholesterolfrom the role of foam cells, the difference was significant (P <0.05); ApoM mutationcarrier group, vector group and empty liposome groups, the difference was notstatistically significant (P>0.05). Using the CuSO4oxidation experiments showed that,ApoM expression of wild-type recombinant protein and recombinant ApoM expression ofmutant protein compared to the antioxidant capacity of the former is significantly strongerthan the latter and the mutant group, the difference was significant (P <0.05); ApoMmutant recombinant plasmid, empty vector and empty liposomes was not statisticallysignificant between groups (P>0.05). Conclusion RT-PCR, site-directed mutagenesis andrecombinant DNA technology was successfully constructed recombinant wild-type andmutant expression plasmids. Transformation experiments efficiently convert the originalwild-type expression plasmid ApoM fusion protein. Transient transfection of wild-typerecombinant plasmid in7402cells can effectively, ApoM protein highly expressed, andApoM group of mutant recombinant protein were lower exprssion. In cholesterol effluxand antioxidant experiment, the wild-type recombinant group has significantly promotecholesterol efflux from foam cells while in the antioxidant the role of mutant recombinantApoM group significantly reduced or loss of these functions. All this results maycontribuilt to further research the role of ApoM in the lipid metabolism and the role of thedevelopment of CHD basis.