血管紧张素Ⅱ2型受体基因重组腺病毒简便快速的构建方法

Construction of AT2R gene recombinant adenovirus by an efficent method

目的:利用细菌内同源重组法构建含血管紧张素Ⅱ2型受体(AT2R)基因的重组腺病毒。方法:应用PCR技术从质粒PUHD AT2R中克隆出AT2R基因片段,定向克隆到腺病毒穿梭质粒pAdTrackCMV中,PmeⅠ酶切线性化后转化Adeasier1细胞,抽提经鉴定含有目的基因的重组腺病毒质粒,PacⅠ酶切后用脂质体转染293细胞,包装成重组腺病毒Ad AT2R。用PCR方法对重组腺病毒进行鉴定,利用穿梭质粒中带有GFP报告基因,对病毒滴度进行监测。结果:PCR检测表明重组腺病毒已含有目的基因AT2R,滴度为1.5×1012pfu/ml。结论:细菌内同源重组法构建腺病毒比传统的细胞内同源重组法具有成功率高、方法简便、快捷和实验周期短的优点。AT2R基因重组腺病毒的成功构建为进一步实验研究奠定了基础。

Objective:To construct the recombinant adenovirus of AT2R by using the method of homogenous recombination in bacteria. Methods:AT2R gene was get from the vector of PUHD-AT2R by PCR ,and subcloned into shuttle vector of pAdTrack-CMV, forming transfer vector of pAdTrack-CMV/AT2R.Then it was linearized with PmeⅠ and transformed into Adeasier-1 cell. The DNA of identified recombinant plasmid was digested with PacⅠ and transfected to 293cells to package adenovirus. The PCR technique was used to detect target gene . The titre of the Ad-AT2R was measured with the aid of GFP expression. Results:PCR test indicated each the recombinant adenovirus contained the insert of AT2R. The titre of purified recombinant adenovirus was 1.5×10 12pfu/ml. Conclusion:The method of homologous recombination in bacteria is more convenient and efficient compared with that in cell .The prepared Ad-AT2R paves a sound foundation for further study.