Recombinant Expression And Purification Of Secretory Phospholipase A2 And Its Effect On Cholesterol Content In Foam Cells

Objective:To investigate the expression and purification of Human secreted phospholipase A2GIIE(sPLA2-hGIIE),and the effect of exogenous addition of sPLA2-hGIIE on cholesterol content in foam cells.The recombinant expression system of Pichia pastoris was used to express human secreted phospholipase A2 GIB(sPLA2-hGIB)and its proenzyme(sPLA2-Pro hGIB),and the protein was purified using chromatography.Methods:sPLA2-hGIIE and sPLA2-hGIB were expressed and purified by Pichia pastoris expression system,and purified by ion exchange and molecular rejection.The macrophage-derived foam cell model was constructed.The experimental group and the control group were designed to detect the effect of exogenically added sPLA2-hGIIE protein on the cholesterol content of foam cells with a cholesterol assay kit.The m RNA expression of genes related to foam cell formation was analyzed by fluorescence quantitative PCR.This study includes the expression and purification of sPLA2-hGIIE,the construction of foam cell model,the effect of sPLA2-hGIIE on cholesterol content of foam cells,the construction of sPLA2-hGIB expression vector,and the expression and purification of sPLA2-hGIB protein.(1)Expression and purification of sPLA2-hGIIEFirst,the frozen strain X33-sPLA2-hGIIE was resuscitated and separated on a solid plate by strebing.The BSM medium was used for amplification culture.The supernatant of fermentation was collected by high speed centrifugation and filtered,concentrated and diluted.The samples were preliminarily purified by cationic exchange method and fine purified by molecular sieve.The concentration of target protein was determined by BSA method,and the enzyme activity of target protein was determined by enzyme activity detection kit.(2)Construction of foam cell modelOxidized low density lipoprotein(ox-LDL)with different concentration gradient was used to treat macrophages,and its effect on macrophage survival rate was determined by CCK-8 kit.Macrophages were treated with oxidized low density lipoprotein at different time periods(24h,36 h,48h),and the content and distribution of lipids in the cells were analyzed by oil red O staining.(3)Effect of sPLA2-hGIIE on cholesterol content in foam cellsMacrophage-derived foam cells were treated with sPLA2-hGIIE of different concentration gradients,and intracellular cholesterol levels were measured with total cholesterol(TC)and free cholesterol(FC)kits.The inhibition of foam cell formation by sPLA2-hGIIE was evaluated by oil red O staining.Finally,real-time quantitative PCR was used to detect m RNA expression of genes related to cholesterol synthesis,esterification,hydrolysis,and lipid inflow and outflow from macrophages.(4)Construction of sPLA2-hGIB expression vectorPrimers were designed according to sPLA2-hGIB nucleic acid protein sequence,target genes were amplified by polymerase chain reaction,the plasmid pGAPZαA was extracted by plasmid extraction kit,and the target genes and plasmids were linked to construct recombinant vectors after double enzyme digestion.The recombinant plasmid was extracted from the top 10 receptor cells of Escherichia coli.Linearized endonucliase was used to linearize the recombinant plasmid and electrotransfer it into yeast cell X33.The transferred bacterial solution was coated on YPD solid medium.(5)Expression and purification of sPLA2-hGIB proteinThe positive monoclonal bacteria were screened and selected,and cultured by YPD and BSM medium,respectively.The supernatant was collected by high-speed centrifugation,filtered,concentrated and diluted.The supernatant was preliminally purified by ion exchange chromatography,and then purified by molecular exclusion layer.Results:1.The results of CCK-8 showed that 80mg/L ox-LDL had no significant toxic effect on the survival rate of macrophages,and oil red O staining showed that macrophages treated with 80mg/L ox-LDL could stably establish foam cell model after 24 h.2.Compared with the modeling group,the total cholesterol and free cholesterol in macrophage derived foam cells were decreased by exogenous addition of sPLA2-hGIIE.The TC and FC in macrophages were decreased to 40.9% and 41.1% respectively by adding 75mg/ml sPLA2-hGIIE protein.3.The real-time quantitative PCR detection results showed that the expression levels of genes related to cholesterol synthesis HMGCS,cholesterol esterification(ACAT-1)and hydrolysis(n CEH)were decreased in the experimental group.the expression levels of macrophage surface lipid inflow related genes(CD36,SR-A)and the expression levels of macrophage surface lipid efflux related genes of ABCG1 were decreased.4.The recombinant vectors pGAPZαA-hGIB and pGAPZαA-Pro-hGIB were constructed successfully.5.The expression strains of pichia pastoris X33-pGAPZαA-hGIB and X33-pGAPZαA-Pro-hGIB were successfully constructed by electrotransfer method.The hierarchical culture method was used to expand the culture.6.The concentration of sPLA2-Pro-hGIB protein was 0.233mg/m L,and approximately 0.2mg of target protein was obtained by using cationic column and molecular sieve.The preliminary results of the cation column purification of sPLA2-hGIB showed that the protein concentration was too low,so the expression scheme of sPLA2-hGIB should be further optimized.Conclusion:The sPLA2-hGIIE protein was successfully expressed and purified,and macrophage derived foam cells were constructed.The exogenous addition of sPLA2-hGIIE could reduce the cholesterol content in cells.Build pGAPZαA-hGIB,pGAPZαA-Pro-hGIB recombinant vector and protein expression using the Pichia pastoris expression system.