HAX1和EGFP共表达重组腺病毒载体的构建、鉴定  

Construction and Identification of Recombinant Adenovirus Vector Co-expressing HAX1 and Enhanced Green Fluorescent Protein

作　　者：李小兰[1] 徐向上[1] 李兆明[1] 王桂华[1] 魏欣[1] 罗学来[1] 刘慎沛[1] 肖徽[1] 陶德定[1] 胡俊波[1] 龚建平[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院胃肠外科/肿瘤研究所,武汉市430030

出　　处：《医学分子生物学杂志》2010年第1期16-22,共7页Journal of Medical Molecular Biology

基　　金：国家重点基础研究发展规划项目（973计划）（No.2004CB518705）

摘　　要：目的构建和鉴定HAX1和EGFP双基因共表达重组腺病毒载体。方法采用DNA重组技术，将目的基因HAX1克隆至含有报告基因EGFP的穿梭质粒pAdTrack—CMV中，并转化于大肠埃希菌DH5a；筛选出重组质粒pAdTrack—CMV—HAX1，并在BJ5183细菌中与pAdEasy-1质粒进行同源重组，产生重组腺病毒载体；用lipofectamine将其转染HEK293细胞，包装携带全长HAX1的重组复制缺陷型腺病毒pad—HAX1-EGFP，酶切和序列测定鉴定；用制备好的Ad—HAX1-EGFP感染HEK293细胞，流式细胞术检测其感染效率，RT—PCR、Western印迹鉴定外源基因HAX1的表达。BrdU检测感染了Ad—HAX1-EGFP的HEK293细胞增殖情况。结果pAdTrack—CMV—HAX1重组质粒构建成功。pAdTrack—CMV—HAX1质粒与pAdEasy-1质粒同源重组后与预期结果相符。构建好的Ad—HAX1-EGFP能有效感染HEK293细胞；外源基因能在239细胞中有效表达。HAX1高表达的HEK293细胞其增殖率得以提高。结论成功构建了表达HAX1和EGFP共表达的重组腺病毒载体，HAX1能够促进结肠癌细胞HEK293细胞的增殖。Objective To construct and identify a recombinant adenovirus vector of co-expression HAX1 and enhanced green fluorescent protein. Methods Used DNA recombination tecniques, intested gene HAX1 into a shuttle plasmid pAdTrack：CMV, and transformed the plasmid into E. coli DH5α, screened the recombinant plasmid pAdTrack-CMV-HAX1 and transformed it into BJ5183 to perform homologous recombination with pAdEasy-1 and then to acquire a recombinant adenovirus vector, Then transfected it into HEK293 cells to prepare the replication deficient adenovirus Ad- HAX1. Then infected HEK293 cells by Ad-HAX1-EGFP and detected the efficiency of infection by flow cytometry and detected the expression of HAXI by RT-PCR and western blot and detected the influence of HAX1 on the proliferation of HEK293 cells by BrdU. Results （1）7 500 bp and 850 bp fragments were detected by gel electrophoresis, which matched the prospective straps, and they were further confirmed by sequencing. These data indicated that we have successfully constructed recombinant plasmid pAdTrack-CMV-HAX1, （2）after recombination of pAdTrack-CMV- HAX1 and pAdEasy-1, the product was digested by restriction endonuclease Pac I and then performed in gel electrophoresis as the 3.0 kb segment which is the same segment as prospection. （3）Ad-HAX1-EGFP could infect HEK293 cells efficiently and the extrinsic genes could express in cells. （4）High expression of HAX1 could promote the proliferation of HEK293 cells. Conclusion The recombinant adenoviral vector Ad-HAX1-EGFP of HEK293 cells.

关 键 词：重组腺病毒 HAX1 载体构建 增殖 

分 类 号：R373[医药卫生—病原生物学]