人端粒酶催化亚单位hTERT基因重组腺病毒载体的构建与鉴定  

CONSTRUCTION AND IDENTIFICATION OF hTERT cDNA RECOMBINANT ADENOVIRUS EXPRESSION VECTOR

作　　者：廖亚平[1] 张洁[1] 周俊宜[1] 汪华侨[1] 

机构地区：[1]中山大学基础医学院解剖学和脑研究室,广州510080

出　　处：《神经解剖学杂志》2006年第6期609-613,共5页Chinese Journal of Neuroanatomy

基　　金：国家重点基础研究发展计划(973计划):神经变性病的机制和防治的基础研究(2006cb500700);国家自然科学基金(No.30470904);广东省自然科学基金(No.04009356)资助项目

摘　　要：为构建人端粒酶催化亚单位(hTERT)重组腺病毒载体,采用PCR法从pCI-neo-hTERT中钓取hTERT全长cDNA,定向克隆到pADTrack-CMV穿梭质粒。通过分步转化把pADEasy-1和重组穿梭质粒(pADTrack-hTERT)转化入BJ5183菌进行同源重组。hTERT重组腺病毒骨架质粒(pAD-hTERT)转染HEK293细胞,荧光显微镜观察绿色荧光蛋白(GFP)的表达,收获重组腺病毒(AD-hTERT)提取DNA行PCR鉴定并扩增。经PCR、酶切和测序鉴定证实hTERT的cDNA正确插入穿梭质粒且与pADEasy-1正确同源重组;PCR鉴定及报告基因分析说明hTERT重组腺病毒包装正确且具有较好的感染活力。结果表明本研究已成功构建了hTERT重组腺病毒,为hTERT的神经保护作用研究奠定了基础。To construct human telomerase reverse transcriptase （hTERT） cDNA recombinant adenovirus expression vector, hTERT cDNA was amplified from pCI-neo-hTERT plasmid by PCR and was cloned to pADTrack-CMV plasmid, pADEasy-1 and recombinant shuttle plasmid （pADTrack-hTERT） were transfected to E. coli BJ5183 step by step and homologous recombinate with each other in it. The recombinant adenoviral backbone vectors （pAD- hTERT）was packaged into infectious recombinant adenoviral particles by transfecting HEK293 cells. Green fluorescent protein （GFP） expression were observed by fluorescent microscope. Recombinant adenovirus hTERT （AD-hTERT） were collected to extract DNA for identification by PCR amplification, then was amplified. The identification results by PCR, enzyme digestion and DNA sequence analysis show that hTERT cDNA was cloned to pADTrack-CMV correctly and homologous recombinate with pADEasy-1 successfully and AD-hTERTs were packaged successfully. The results indicate that hTERT and AD-hTERT were constructed successfully,which may provide the bases for studying neuroprotective effect of hTERT.

关 键 词：ALZHEIMER病 端粒酶 人端粒酶催化亚基 重组腺病毒 

分 类 号：R346[医药卫生—基础医学]