Construction and identification of eukaryotic expression vector of human angiopoietin-related protein-1 angioarrestin / 第二军医大学学报

ABSTRACT

Objective: To clone human angio poietin- related protein-1 angioarrestin genes and construct their recombinant eukaryotic expression vector. Methods: Full length sequence of angioarrestin gene and its C-FD domain were amplified from human liver cDNA library by PCR and were subsequently inserted into the eukaryotic expression vector pcDNA3. 1/ His-Myc (-)B. Then angioarrestin and FD recombinant plasmids were stablely transfected in NCI-H460 cell line. The positive clones were identified by RT-PCR and Western blot. Results: Full length fragment of angioarrestin gene (1 473 bp) and C-FD domain (560 bp) were successfully amplified from human liver cDNA library by PCR. The pcDNA3. 1-ARP and pcDNA3. 1-FD recombinant plasmids were also constructed successfully as identified by PCR and enzyme digestion. RT-PCR and Western blot showed the expression of target mRNA and protein in NCI-1-1460 cells. Conclusion: The eukaryotic expression vectors of angioarrestin and C-FD gene have been successfully constructed and expressed in NCI-H460 cells, which pave a way for further study on the anti-angiogenesis function of angioarrestin in cancer.