恒河猴CD40L胞外段基因腺病毒载体的构建与鉴定

发布时间：2018-01-10 00:31

本文关键词：恒河猴CD40L胞外段基因腺病毒载体的构建与鉴定　出处：《昆明医学院》2011年硕士论文　论文类型：学位论文

【摘要】：【目的】利用AdEasy腺病毒载体系统,应用细菌内同源重组法构建并鉴定恒河猴CD40L胞外段基因(efCD40L)重组腺病毒载体。【方法】 overlap PCR法合成恒河猴CD40L胞外段基因,琼脂糖凝胶电泳、测序鉴定正确后与高效克隆载体pGEM-T连接,转化进E.coli Top10化学感受态细菌中,克隆扩增pGEM-T-efCD40L,提取质粒；AdEasy腺病毒载体系统中的pShuttle-CMV穿梭质粒与pGEM-T-efCD40L进行双酶切、连接后,得到穿梭质粒pShuttle-CMV- efCD40L。pShuttle-CMV-efCD40L经酶切、琼脂糖凝胶电泳鉴定后,Pmel酶切线性化,再与腺病毒骨架质粒pAdEasy-1共电转化到感受态BJ5183细菌中,进行同源重组产生腺病毒质粒。经过抗性筛选以及PCR鉴定得到阳性的pAd-CMV-efCD40L重组质粒。将pAd-CMV-efCD40L线性化后,利用脂质体2000转染AD293细胞,利用PCR和RT-PCR鉴定正确后,然后进行病毒扩增。【结果】构建了腺病毒穿梭质粒pShuttle-CMV-efCD40L,并进一步应用细菌内同源重组成功构建了含CD基因的重组腺病毒载体pAd-CMV-efCD40L。【结论】应用细菌内同源重组法可构建含恒河猴CD40L胞外段基因的重组腺病毒载体,为后续实验应用于与PDL-1共转染未成熟树突状细胞,进一步研究肝移植免疫耐受奠定了实验基础。
[Abstract]:[purpose] The recombinant adenovirus vector of rhesus monkey CD40L was constructed and identified by using AdEasy adenovirus vector system and homologous recombination method. [methods] The extracellular segment of rhesus monkey CD40L gene was synthesized by overlap PCR method, and agarose gel electrophoresis was performed. The gene was identified correctly by sequencing and ligated with highly efficient clone vector pGEM-T. The plasmid was extracted by cloning and amplifying pGEM-T-efCD40L into E. coli Top10 chemosensitive bacteria. The pShuttle-CMV shuttle plasmid in AdEasy adenovirus vector system was digested with pGEM-T-efCD40L and ligated. The shuttle plasmid pShuttle-CMV- efCD40L.pShuttle-CMV-efCD40L was digested by enzyme and identified by agarose gel electrophoresis. Pmel was digested linearized and then co-transformed with adenovirus skeleton plasmid pAdEasy-1 into competent BJ5183 bacteria. Adenovirus plasmids were produced by homologous recombination. Positive pAd-CMV-efCD40L recombinant plasmids were obtained by resistance screening and PCR identification. After transformation. AD293 cells were transfected with liposome 2000 and identified by PCR and RT-PCR, then the virus was amplified. [results] Adenovirus shuttle plasmid pShuttle-CMV-efCD40L was constructed. Furthermore, the recombinant adenovirus vector pAd-CMV-efCD40Lcontaining CD gene was successfully constructed by homologous recombination in bacteria. [conclusion] Recombinant adenovirus vector containing the extracellular segment of rhesus monkey CD40L gene could be constructed by homologous recombination of bacteria. The recombinant adenovirus vector could be used to co-transfect immature dendritic cells with PDL-1. Further study of liver transplantation immune tolerance laid the experimental foundation.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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