The Construction And Preliminary Identification Of The Eukaryotic Expression Vector Of Pcsk9 Mutants With Gain Of Function And Loss Of Function

Objective To construct the eukaryotic expression vector of pcsk9 mutants with gain of function and loss of function and to complete the preliminary identification. Consequently, a vector for the further study of the relationship between pcsk9 and the endothelial cell(EC) injury as well as the formation of foam cell(FC),and a new idea for investigating the mechanism and prevention of atherosclerosis(As) are expected.Methods Human liver cell line (BLE7402)were clutured, then collected to extract total RNA.After DNA was removed from the RNA,the cDNA got by reverse transcription .The full coding region of pcsk9 gene was amplified with the template of cDNA , and amplified product was identificated.According to the instruction of the Target clone-plus kit , pcsk9 was combined with pMD18-T vector. The pMD18-T-pcsk9 vector was transformed into E. coli DH-5α, which was clutured in solid plate with AmpLB . Enough plasmid was extracted and sequenced after a monoclonal was selected to culture. Suitable primers were designed to add a Flag tag to pMD18-T-pcsk9 'end ,then the pMD18-T-pcsk9 and pcDNA4.0 were recombined together.Finally, endotoxin was removed.from the wild-type expression vector.Suitable primers were designed according to gain of function S127R and loss of function L253F,and then site-directed mutagenesis was conducted with the template of wild-type expression vector to get the expression vector of the two kinds of mutants.Finally,two expression vectors of mutants were identified as above.THP-1 cells were cultured in 24-well plates. According to the instruction of Lipofectamine LTX Transfection, pcsk9 wild-type plasmid, pcsk9-S127R plasmid, pcsk9-L253F plasmid were transfected into THP-1 cells,respectively.24h.later, those transfected cells were incubated with phorbol ester for 24h.Finally,those cells were observed under a fluorescence microscope. mRNA and protein of pcsk9 and rotein of LDLR were detected in those transfected cells by RT-PCR and Western blot .analysis.Results The OD value of extracted RNA was 1.9 when it was diluted to 1/100 and the electropherogram showed that there were three bands ,28S rRNA, 18S rRNA and 5S rRNA .The former two bands were clear and 28S rRNA band was 1.5-2.0 times brighter than 18S rRNA band;The band 5S rRNA was fuzzy. The coded region of pcsk9 gene was amplified and the electropherogram showed there was a band in the position of 2.1kb which matched with the size of pcsk9 gene (2079bp). The sequencing of pMD18-T- pcsk9 showed that identity rate of the measured gene was 100%,and base deletion rate was 0%, compared with pcsk9 in GeneBank.The wild-type expression vector was digested by Hind III and XbaI and the electropherogram showed there were a band in the position of 2.1 kb and a band in the position of 5.1 kb which were matched with the size of pcsk9 (2079bp) and pcDNA4 .0 (5078bp) ,respectively. The sequencing of two expression vectors of mutants showed the 381 base in the DNA of pcDNA4.0-pcsk9-S127R was changed from T into A ,and the 757 base in the DNA of pcDNA4.0-pcsk9-L253F was changed from C into T.The view of transfected cells under a fluorescence microscope showed there were no green fluorescent cells in the normal control group ,but some in groups of wild-type, pcsk9-S127R and pcsk9-L253F.Detecting mRNA and protein of pcsk9 showed there was no band in the normal control group, but some in groups of wild-type, pcsk9-S127R and pcsk9-L253F in the right position of electropherogram.Western blot analysis showed there was no band in the normal control group, but some in groups of wild-type, pcsk9-S127R and pcsk9-L253F in the position of 60KD (pcsk9).It also showed the gray value of bands in the position of 93KD(LDLR) were significantly different in four groups (p <0.05).Conclusion We successfully constructed the eukaryotic expression vector of pcsk9's mutants with gain of function (S127R) and loss of function (L253F) which supplied a vector for the further study of the relationship between pcsk9 and the EC damage as well as the formation of FC ,and gave some new idea to investigate the mechanism and prevention of As.