ACE2-mCherry融合基因表达载体的构建及功能验证  

Construction and Functional Verification of ACE2-mCher⁃ry Fusion Gene Expression Vector

作　　者：蒋晓祎 侯欣妤 张波 任禾 牛祖彪 郑幽 王雨琪 孙强 黄红艳 JIANG Xiao-Yi;HOU Xin-Yu;ZHANG Bo;REN He;NIU Zu-Biao;ZHENG You;WANG Yu-Qi;SUN Qiang;HUANG Hong-Yan(Department of Medical Oncology,Beijing Shijitan Hospital,Capital Medical University,Beijing 100038;Beijing Institute of Biotechnology,Beijing 100071,Chin)

机构地区：[1]首都医科大学附属北京世纪坛医院肿瘤内科,北京100038 [2]军事医学研究院生物工程研究所,北京100071

出　　处：《生物技术通讯》2020年第6期675-680,共6页Letters in Biotechnology

基　　金：北京市教委-市自然基金联合项目/北京市教委科技计划重点项目(KZ202110025029)。

摘　　要：目的:构建带有荧光标记的血管紧张素转化酶2(ACE2)表达载体,并初步验证该表达载体的功能。方法:采用PCR方法,从载体pcDNA3.1-ACE2-3xFlag中扩增得到目的基因片段ACE2,用限制性内切酶XhoⅠ和AgeⅠ对载体质粒pQCXIP-mCherry-Lamp进行双酶切,回收得到线性化载体后与扩增的ACE2片段同源重组,得到目的载体pQCXIP-ACE2-mCherry。测序成功后,将构建的pQCXIP-ACE2-mCherry重组质粒及表达SARS-CoV-2的S蛋白的质粒pSec-SARS-CoV-2-S同时瞬时转染293T细胞,48 h后通过活细胞拍摄观察胞膜融合现象,Western印迹检测蛋白表达。结果:构建了表达人ACE2蛋白以及荧光标记蛋白mCherry的真核表达载体pQCXIP-ACE2-mCherry,与pSec-SARS-CoV-2-S共转染293T细胞后胞膜发生融合。结论:通过同源重组技术构建了载体pQCXIP-ACE2-mCherry,其研究思路可供SARS-CoV-2病毒研究借鉴。Objective:To construct a expression vector containing fluorescently labeled angiotensin-converting en⁃zyme 2(ACE2),and then examine the vecctor function.Methods:DNA fragment containing ACE2 was amplified by PCR from pcDNA3.1-ACE2-3xFlag as template,meanwhile,the vector pQCXIP-mCherry-Lamp was cut by re⁃striction enzymes XhoⅠand AgeⅠto provide the linearized vector.And then the linearized vector and ACE2 frag⁃ment were linked by homologous recombination to obtain a new vector pQCXIP-ACE2-mCherry.After this plasmid was sequenced,it was co-transfected into 293T cells with the plasmid pSec-SARS-CoV-2-S expressing the S pro⁃tein of SARS-CoV-2.After 48 hours,cell fusion was observed by the live cell photography and protein expres⁃sion was detected by Western blotting.Results:A large number of syncytia by cell fusion in 293T cells was ob⁃served when co-transfected with pQCXIP-ACE2-mCherry and pSec-SARS-CoV-2-S.Conclusion:The vector pQCXIP-ACE2-mCherry was successfully constructed by homologous recombination and which would be helpful for further investigation of SARS-CoV-2.

关 键 词：血管紧张素转化酶2 同源重组 SARS-CoV-2 

分 类 号：Q78[生物学—分子生物学]