大鼠ACE2腺病毒载体的构建及其在INS-1细胞中的表达

目的构建携带大鼠血管紧张素转化酶2(angiotensin-converting enzyme 2,ACE2)基因的重组腺病毒表达载体并确定其对INS-1细胞的感染效率。方法采用RT-PCR方法,从大鼠肾脏组织中扩增出ACE2基因的全长cDNA序列,将ACE2基因定向克隆到穿梭质粒载体pShuttle-GFP-CMV,经与腺病毒骨架质粒pAdxsi载体同源重组后得到携带大鼠ACE2基因的重组腺病毒(pAdxsi-GFP-CMV-ACE2),采用PCR的方法对重组腺病毒进行鉴定,转染HEK293细胞进行包装和扩增,氯化铯密度梯度离心法纯化,半数组织培养感染剂量(50%tissue culture infective dose,TCID50)方法测定重组腺病毒的滴度。体外转染INS-1细胞,绿色荧光观察绿色荧光蛋白(GFP)和Western blotting检测ACE2蛋白的表达。结果克隆得到大鼠ACE2基因,经PCR鉴定和测序证实结果正确,成功构建得到高滴度的重组腺病毒,并能高效感染INS-1细胞。结论成功构建了表达ACE2的复制缺陷型重组腺病毒,为深入研究ACE2基因在胰岛细胞中的生物学功能提供了一定的工作基础。

Construction of Rat ACE2 Adenovirus Vector and Its Expression in INS-1 Cells

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.