Construction Of PEGFP-sPLA2-ⅡA Recombinant Plasmid And Expression In Human Umbilical Vein Endothelial Cells

Background:Atherosclerosis disease is one of the principal diseases that threaten human health. It's the primar cause of death in developed countries. Even in developing countries,the incidence of atherosclerosis disease increased year by year. At present, the pathogenesy of atherosclerosis disease is not fully studied. Current studies have shown that inflammation plays an essential role in the development and procedure of atherosclerosis disease. The extent of pathological changes and vulnerability of plaque can be determined indirectly by serum inflammatory factor level.Phospholipases are classifed as esterases and ubiquitous in mammal organisms. Among the subtypes, secretory phospholipase A₂ of group IIA is very popular at present. As one of the inflammation factors, secretory phospholipase A₂ of group IIA, a subtype of lipoprotein-related phosphatidolipase A₂, is mainly produced by smooth muscle cells and macrophages and belongs to calcium-depended phosphatidolipase family, which locates mainly in macrophages gathering place, lipid core area and extracellular matrix of pathological intima. Currently, it's thought to be related with the development of atherosclerosis by possibly hydrolyzing cellular membrane and phosphatidylcholine in lipoprotein to produce proinflammatory reaction and atherosclerotic lysophosphatidylcholine and oxidized fatty acid .Objective:Human sPLA₂-â…¡A was synthesized by using the gene sythesis technology in vitro. To construct eukaryotic expressing recombinant plasmid of pEGFP-sPLA₂-â…¡A gene. The reconstructed plasmid was transfected into Human umbilical vein endothelial cells, and research the expression level of sPLA₂-â…¡A in Human umbilical vein endothelial cellsMethods: Human sPLA₂-â…¡A was synthesized by whole-genome synthesis technology according to the gene sequences from gene bank. The gene fragment of sPLA₂-â…¡A was connected with the pGEMT vector, and then inserted into the eukaryotic expression plasmid pEGFP-N1 after amplification and digestion. Cultured the human umbilical vein endothelial cells in vitro. The reconstructed plasmid was transfected into human umbilical vein cells. Observe the expression of green fluorescent protein(GFP) by inverted microscope. The human umbilical vein endothelial cells were cellected in the control groups and the experimental groups after 24 hours. The mRNA was extracted, and then reversed transcription into cDNA. Detect the mRNA level by Real-time PCR and the fusion protein by Western blot . All results were statistically analyzed with SPSS 13.0. P<0.05 was considered statistic difference,P<0.01 was considered significantly statistic difference.Results:1. The human sPLA₂-â…¡A was synthesized, and the eukaryotic expression plasmid pEGFP-sPLA₂-â…¡A was acquired. The reconstructed plasmid of pEGFP-sPLA₂-â…¡A was confirmed by the sequence analysis and electrophoresis.2. Cultured the human umbilical vein endothelial cells in vitro, and the cells growed in good condition. The reconstructed plasmid of pEGFP-sPLA₂-â…¡A was transfected into human umbilical vein cells. The green fluorescent protein(GFP) was observed by inverted microscope which indicated the success of plasmid transfection.3. The mRNA level of sPLA₂-â…¡A in transfected group increased compared with the control group(P <0.05) .4. The fusion protein of pEGFP-sPLA₂-â…¡A can be detected by Western blot.Conclusions:The pEGFP-sPLA₂-â…¡A plasmid is constructed successfully and it can be transfected into human umbilical vein endothelial cells. The mRNA level of sPLA₂-â…¡A in transfected group increased. The fusion protein of sPLA₂-â…¡A can be expression. This provides the necessary condition for research between sPLA₂-â…¡A and atherosclerosis disease.