人过氧化氢酶基因重组腺病毒的构建及其酶活性的测定  

Construction and expression of recombinant adenovirus containing human catalase gene in vitro

作　　者：欧阳晓玲 李爱玲[1,3] 宁启兰[1] 杨旭东[1] 许楠[1] 王慧莲[1] 

机构地区：[1]西安交通大学医学院遗传与分子生物学系,陕西西安710061 [2]郴州市第一人民医院,湖南郴州423000 [3]陕西教育学院生物科学与技术系,陕西西安710061

出　　处：《浙江大学学报（医学版）》2011年第3期291-296,共6页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金(No:30971220)

摘　　要：目的:构建含人过氧化氢酶(catalase,CAT)基因的重组腺病毒载体,为基因治疗提供一种新的方法。方法:从人血中分离白细胞,提取总RNA,利用RT-PCR法获得人CAT的全长cDNA,克隆到pcDNA3.1+中,构建pcDNA3.1+-CAT载体,酶切和基因测序鉴定阳性克隆;利用酶切法把CAT基因克隆至入门载体pENTR1A中,构建pENTR1A-CAT载体;利用LR反应,体外重组pENTR1A-CAT和pAd/CMV/V5-DEST,得重组腺病毒表达载体pAd/CMV/V5-DEST-CAT。经测序鉴定,并用Pac I线性化pAd/CMV/V5-DEST-CAT后,与脂质体2 000共转染293A细胞,重组病毒颗粒的包装、扩增和滴度测定。利用免疫印迹和240 nm紫外光吸收法测定重组病毒中CAT蛋白的表达量和CAT的活性。结果:Ad-CAT、(A组)Ad-LacZ(B组)及PBS(C组)感染293A细胞24h,其细胞蛋白提取液在1至4 m in反应中CAT活性的变化经单因素方差分析,三组间差异有统计学意义(F=5.96,P<0.05)。其中,A组4 m in内的酶活性分别比B组和C组高出(63.8±1.54)%和(76.7±1.67)%(P<0.05),而B组与C组比差异无统计学意义(P>0.05)。结论:本研究成功构建了含人CAT基因的Ad/CMV/V5-DEST-CAT载体,并在293A细胞中包装出高表达及高活性重组腺病毒,为进一步研究人CAT基因在基因治疗中的作用奠定了基础。Objective:To construct the adenovirus vector containing recombinant human catalase(CAT) and to express the recombinant gene in vitro.Methods:Total RNA was extracted from human leukocytes and full-length human CAT cDNA was obtained with RT-PCR method.The CAT gene was cloned into pcDNA3.1(+) vector and pcDNA3.1(+)CAT was constructed.The positive clones were confirmed by the restriction enzyme digestion and gene sequencing.The CAT gene was cloned into the entry vector pENTR1A,and pENTR1A-CAT vector was constructed.By LR reaction pENTR1A-CAT and pAd/CMV/V5-DEST was recombined in vitro,and the recombinant adenovirus pAd/CMV/V5-DEST-CAT was obtained.The positive pAd/CMV/V5-DEST-CAT was confirmed by sequencing and transfected into 293A cells with Pac I linearization and Lipofectamine 2 000,and the recombinant virus particles were packaged and amplified in the cells.The expression of CAT protein and CAT enzyme activities of the recombinant virus were determined by Western blot and 240 nm UV absorption methods.Results:High expression of recombinant adenovirus was obtained and the expressed human catalase had high enzyme activity.Conclusions:Ad/CMV/V5-DEST-CAT vector containing human catalase gene has been constructed successfully;and the expressed enzyme in 293A cells has high activity.

关 键 词：过氧化氢酶/遗传学 腺病毒科/遗传学 基因 重组 遗传 遗传载体 转染 基因疗法 质粒 肾/胚胎学 肾/细胞学 

分 类 号：R450[医药卫生—治疗学]