摘要
目的:构建elafin基因高效表达重组腺病毒载体Ad-elafin。方法:抽提人肺总RNA进行逆转录多聚酶链反应(RT-PCR)扩增elafin基因,PCR产物双酶切后亚克隆至穿梭质粒pAdTrack-CMV上;在BJ5183细菌内与pAdEasy-1同源重组,筛选阳性克隆,酶切、PCR及测序鉴定;PacⅠ酶切线性化后脂质体法转染293细胞进行包装,获得腺病毒载体Ad-elafin;继之在293细胞内扩增,利用报告基因GFP监测病毒滴度和感染效率,Western blot检测elafin蛋白的表达,ELISA鉴定elafin蛋白对弹性蛋白酶活性拮抗作用。结果:酶切、PCR、蛋白表达测定及拮抗弹性蛋白酶活性初步鉴定证实弹性蛋白酶特异性抑制因子elafin基因重组腺病毒载体Ad-elafin构建成功。结论:成功构建了重组腺病毒载体Ad-elafin,为进一步深入研究慢性阻塞性肺疾病的发病机制奠定了技术基础。
Objective：To construct the highly efficient expression recombinant adenovirus that expresses elafin protein for further study. Methods：The elafin gene was amplified by RT-PCR from lung tissues ,then the fragment was inserted into the multiple clone site of pAdTrack-CMV. The linearized shuttle plasmid was homogenously recombined with pAdEasy-1 in B J5183. The candidate clone was further analyzed by restriction endonuclease digestion, PCR, and sequence scanned. Then the recombined adenovirus plasmid was digested with Pac I and transfected into 293 cells for packaging and amplifying. Infection titer and rate were monitored by green fluorescent protein（GFP） expression. The expression of elafin protein was detected by Western blot and ELISA to appraise the function of elafin protein for oppressing the activity of elastic protease. Results： Digestion with restriction endonuclease, PCR, Western blot and ELISA confirmed that elafin gene was cloned into the adenovirus vector successfully. Conclusion ：The recombined adenovirus Ad-elafin is constructed successfully, which will be benefit for future study.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2007年第6期495-499,共5页
Chinese Journal of Immunology
基金
重庆市应用基础研究资助项目(200332)
关键词
elafin基因
腺病毒
慢性阻塞性肺疾病
elafin gene
Recombinant adenovirus
Chronic obstructive pulmonary disease （COPD）
