重组人CXCR1腺病毒的构建

发布时间：2018-03-31 09:21

本文选题：CXCR1　切入点：腺病毒　出处：《重庆医科大学》2012年硕士论文

【摘要】：CXCR1是只含有一条肽链的糖蛋白，为α亚族趋化性细胞因子受体。CXCR1可与IL-8特异性结合，介导细胞信号转导的启动，通过信号传导过程，决定相应配体趋化功能的实现，与细胞的活化、增殖、迁徙及刺激血管形成和释放血管形成前体物质等有关，因此CXCR1涉及到多种疾病的发生发展。为进一步研究CXCR1在人体中各种疾病的发生发展中的作用，我们构建CXCR1基因腺病毒表达载体，使得CXCR1基因在细胞内得以表达。目的：应用AdEasy腺病毒载体构建人CXCR1基因的重组腺病毒pAd-CXCR1。方法：1.根据GenBank已收录的CXCR1cDNA全长设计引物并送合成，采用PCR方法扩增CXCR1基因。2.将目的基因的扩增片段克隆至穿梭质粒pAdTrace，构建穿梭质粒pAdTrace-CXCR1，并用KpnI和HindIII双酶切进行鉴定。3.PemI酶切pAdTrace-CXCR1使之线性化，用电转化法将线性质粒与腺病毒骨架质粒pAdEasy-1转入BJ5183细菌内进行同源重组，，构建重组腺病毒质粒pAd-CXCR1。4.重组腺病毒用酶切及PCR方法进行鉴定。5.重组腺病毒质粒用PacI线性化后转染HEK-293细胞，进行腺病毒的包装，并在HEK-293细胞中进行腺病毒扩增。结果：通过腺病毒构建系统及酶切、连接、转化等技术，成功的构建了穿梭质粒pAdTrace-CXCR1。将线性化的穿梭质粒质粒与腺病毒骨架质粒pAdEasy-1于BJ5183细菌内进行同源重组，最终成功构建重组腺病毒质粒pAd-CXCR1。将pAd-CXCR1质粒于HEK-293细胞内扩增，得到高滴度人CXCR1基因的腺病毒载体。结论：成功构建了人CXCR1基因的腺病毒载体，得到高滴度的腺病毒，为进一步研究CXCR1蛋白的生物功能及其在临床各种相关疾病中的作用奠定了基础。
[Abstract]:CXCR1 is a glycoprotein containing only one peptide chain, is a subfamily of chemokine receptor.CXCR1 binds specifically to IL-8 mediated signal transduction, cells start, through the signal transduction process, determine the corresponding ligand to achieve function chemotaxis, cell activation and proliferation, angiogenesis, and release of precursor substances the formation of migration and stimulate the blood vessels, so CXCR1 is related to the occurrence and development of many diseases. For the role in the occurrence and development of the further study of CXCR1 in human diseases, we construct the expression vector of CXCR1 gene of adenovirus, the CXCR1 gene is expressed in cells.
Objective: to construct recombinant adenovirus pAd-CXCR1. of human CXCR1 gene by using AdEasy adenovirus vector
Methods: the full-length CXCR1cDNA primers were designed according to GenBank 1. have been included and sent to synthesis, amplification of the CXCR1 gene.2. gene amplification fragment was cloned into the shuttle plasmid pAdTrace by PCR method, and construct the shuttle plasmid pAdTrace-CXCR1, KpnI and HindIII were identified by double enzyme digestion of linear.3.PemI digestion to pAdTrace-CXCR1, the linear plasmid and adenovirus virus plasmid pAdEasy-1 for homologous recombination in bacteria into BJ5183 by electroporation method, recombinant adenovirus plasmid pAd-CXCR1.4. recombinant adenovirus by restriction enzyme digestion and PCR method for identification of.5. recombinant adenovirus plasmid transfected into HEK-293 cells by PacI after linearization of adenovirus, and adenovirus amplification in HEK-293 cells.
Results: through the construction of recombinant adenovirus system and enzyme digestion, connection, transformation technology, successfully constructed the shuttle plasmid pAdTrace-CXCR1. linearized shuttle plasmid plasmid and adenovirus backbone plasmid pAdEasy-1 for homologous recombination in bacteria BJ5183, finally successfully constructed the recombinant adenovirus plasmid pAd-CXCR1. pAd-CXCR1 plasmid in HEK-293 cell amplification, get gland high titer virus vector of human CXCR1 gene.
Conclusion: the adenovirus vector of human CXCR1 gene has been successfully constructed, and the high titer of adenovirus has been obtained, which laid a foundation for further studying the biological function of CXCR1 protein and its role in various clinical diseases.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

【参考文献】