Construction of an angiopoietin-like protein 2 expression vector and its expression in human endothelial cells / 医学研究生学报

ABSTRACT

Objective: To construct an angiopoietin-like protein 2(ANGPTL2) expression vector and obtain ANGPTL2 over-expression endothelial cell strains.Methods: Plasmid phrGFP-C was used to amplify hrGFP protein coding sequence by polymerase chain reaction.The amplified sequence was cloned into the A multiple cloning sites of pIRES to construct plasmid pIRES-hrGFP.Complementary oligonucleotides containing the recognition sequence of BamH I,Sal I,Xba I and SSe8387 I were synthesized,annealed and cloned into a BamH I site on the backbone of plasmid pIRES-hrGFP to obtain vector pIRES-hrGFP-MS.ANGPTL2 cDNA was cloned by RT-PCR while human renal RNA was used as the templet and then inserted into the B multiple cloning sites of the vector pIRES-hrGFP-MS.The newly constructed ANGPTL2 expression vector pIRES-hrGFP-MS-ANGPTL2 was linearized by Xba I and introduced into human umbilical vein endothelial cells.ANGPTL2 over-expressed endothelial clones were screened out by G418 selection and identified by the expression levels of both hrGFP and ANGPTL2 genes in these cell clones.Results: The ANGPTL2 expression vector pIRES-hrGFP-MS-ANGPTL2 was constructed successfully and two ANGPTL2 over-expression endothelial strains were obtained.The cells displayed a significantly extended appearance quite different from that of the control cells.Conclusion: The successful construction of the ANGPTL2 expression vector pIRES-hrGFP-MS-ANGPTL2 and the obtainment of two ANGPTL2 over-expression HUVEC strains have paved the way for further investigation into the function of ANGPTL2 and its possible role in diabetic nephropathy.