Construction Of Angiopoietin-1Gene Eukaryotic Expression Vector And Expression In HEK293Cells

Objective:Purpose of this study was to construct the recombinant eukaryotic expressionvector pSecTag2B-Ang-1, then the recombinant plasmid was transiently transfectedinto human embryonic kidney293cells via lipofectamine2000and its expressionlevel of the mRNA and protein was investigated. It was aimed at providingexperimental basis for gene therapy on coronary heart disease.Methods:Primers were designed and synthesized according to the human angiopoietin-1gene. The angiopoietin-1gene fragments were amplified with DNA template byhuman heart and verified by the agarose gel electrophoresis. Then the angiopoietin-1gene fragments were purified by DNA Gel Extraction kit and cloned into theeukaryote expression vector pSecTag2B. Firstly, the recombinant vector ofpSecTag-Ang-1was digested and identified by electrophoresis. Secondly, it wastransformed into Escherichia coli DH5α and amplificated. Lastly, the positive clonewere sequenced by Shanghai sangon corporation. Then the recombinant plasmid wastransfected into human embryonic kidney293cells as the experimental group, and theempty plasmid was transfected into human embryonic kidney293cells as the controlgroup. The angiopoietin-1mRNA and protein expression was investigated by themethod of Quantitative real time polymerase chain reaction and Western blot.Results:The recombinant eukaryotic expression vector pSecTag2B-Ang-1wasconstructed successfully. Then the recombinant vector pSecTag2B-Ang-1wastransfected into HEK293cells via lipofectamine2000. QPCR result showed that theexpression of angiopoietin-1mRNA in experimental group was higher than those inthe control group (p<0.05). Results detected by western blot showed that the expression of angiopoietin-1protein in experimental group was higher than those inthe control group (p<0.05). These results showed that the human embryonickidney293cells transfected with recombinant plasmid could express angiopoietin-1mRNA and protein efficiently and stably.Conclusion:1. The recombinant plasmid of pSecTag2B-Ang-1was successfully constructed.2. The angiopoietin-1mRNA and protein can be expressed by the HEK293cellstransfected with recombinant plasmid efficiently and stably.