人白细胞弹性蛋白酶抑制因子elafin的克

发布时间：2018-03-24 12:55

本文选题：Elafin　切入点：腺病毒　出处：《重庆医科大学》2006年博士论文

【摘要】： 第一部分人白细胞弹性蛋白酶抑制因子elafin的克隆及重组腺病毒表达载体的构建、鉴定与表达目的:克隆人白细胞弹性蛋白酶(NE)抑制因子elafin基因cDNA,应用最新的腺病毒载体构建系统AdEasy System构建含elafin基因重组腺病毒载体Ad-elafin,为进一步应用其进行气道上皮细胞黏蛋白(MUC)分泌调控的研究打下基础。方法:(1)抽提人肺总RNA进行逆转录反应,经PCR扩增目的基因片段,elafin目的片段经纯化双酶切后亚克隆至穿梭质粒pAdTrack-CMV上,在BJ5183细菌内和pAdEasy-1行同源重组,筛选阳性克隆,酶切、PCR及测序鉴定,Pac I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。结果:(1)经RT-PCR获得377bp的产物,经酶切、DNA测序,确定所扩增片段为人elafin基因cDNA。(2)质粒经酶切及PCR鉴定证实elafin基因重组腺病毒载体Ad-elafin构建成功,Western-blot检测到elafin蛋白的表达。结论:获得编码elafin的基因片段,并成功构建了腺病毒表达载
[Abstract]:Part I cloning of human leukocyte elastase inhibitor elafin and construction, identification and expression of recombinant adenovirus expression vector. Objective: to clone the elafin gene of human leucocyte elastase inhibitor, and construct the recombinant adenovirus vector Ad-elafin-containing elafin gene by using the newest adenovirus vector AdEasy System. The study on the regulation of MUC) secretion has laid a foundation for the study. Methods the total RNA of human lung was extracted by reverse transcription reaction. The target gene fragment was amplified by PCR and subcloned into shuttle plasmid pAdTrack-CMV after double enzyme digestion. Homologous recombination was carried out in BJ5183 bacteria and pAdEasy-1 to screen positive clones. The recombinant adenovirus vector Ad-elafinwas obtained and then amplified in 293 cells. The reporter gene GFP was used to detect the viral titer and infection efficiency. The expression of elafin protein was detected by Western-blot. Results the product of 377bp was obtained by RT-PCR. The amplified fragment of human elafin gene cDNA.t2) was confirmed by restriction endonuclease digestion and PCR identification. The recombinant adenovirus vector Ad-elafin of elafin gene was constructed successfully and the expression of elafin protein was detected by Western-blot. Conclusion: the gene fragment encoding elafin was obtained and adenovirus was successfully constructed.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R346

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