关键词: 血管紧张素转化酶2, 腺病毒载体, INS-1细胞

Abstract: Objective To construct the recombinant adenovirus vector carrying rat angiotensin-converting enzyme 2(ACE2), and infect the INS-1 cells. Methods The full cDNA sequence was obtained from rat kidney tissue using RT-PCR. The ACE2 gene was cloned into pShuttle-GFP-CMV vector which was subsequently homologously recombined with pAdxsi vector in the HEK293 cells to package the recombinant adenovirus vector carrying rat ACE2(pAdxsi-GFP-CMV-ACE2). After verified by PCR, we amplified pAdxsi-GFP-CMV-ACE2 in HEK293 cells and purified it by CsCl gradient purification, titrated it using 50% tissue culture infective dose(TCID50) assay. INS-1 cells were infected with adenoviruses and ACE2 expression were detected by the intensity of green fluorescence under fluorescence microscope and western blot. Results The ACE2 gene was cloned and verified by sequencing and high tittered virus was produced by a construct carrying ACE2 gene, and ACE2 was expressed efficiently in the INS-1 cells after infection. Conclusion The newly constructed adenovirus vector containing rat ACE2 provides a potent tool to investigate its biological function in islet cells.

Key words: angiotensin-converting enzyme 2(ACE2), adenvirus vector, INS-1 cells