本文選題：CXCR1　切入點：腺病毒　出處：《重慶醫(yī)科大學》2012年碩士論文

【摘要】：CXCR1是只含有一條肽鏈的糖蛋白，為α亞族趨化性細胞因子受體。CXCR1可與IL-8特異性結合，介導細胞信號轉導的啟動，通過信號傳導過程，決定相應配體趨化功能的實現(xiàn)，與細胞的活化、增殖、遷徙及刺激血管形成和釋放血管形成前體物質等有關，因此CXCR1涉及到多種疾病的發(fā)生發(fā)展。為進一步研究CXCR1在人體中各種疾病的發(fā)生發(fā)展中的作用，我們構建CXCR1基因腺病毒表達載體，使得CXCR1基因在細胞內得以表達。 目的：應用AdEasy腺病毒載體構建人CXCR1基因的重組腺病毒pAd-CXCR1。 方法：1.根據(jù)GenBank已收錄的CXCR1cDNA全長設計引物并送合成，采用PCR方法擴增CXCR1基因。2.將目的基因的擴增片段克隆至穿梭質粒pAdTrace，構建穿梭質粒pAdTrace-CXCR1，并用KpnI和HindIII雙酶切進行鑒定。3.PemI酶切pAdTrace-CXCR1使之線性化，用電轉化法將線性質粒與腺病毒骨架質粒pAdEasy-1轉入BJ5183細菌內進行同源重組，，構建重組腺病毒質粒pAd-CXCR1。4.重組腺病毒用酶切及PCR方法進行鑒定。5.重組腺病毒質粒用PacI線性化后轉染HEK-293細胞，進行腺病毒的包裝，并在HEK-293細胞中進行腺病毒擴增。 結果：通過腺病毒構建系統(tǒng)及酶切、連接、轉化等技術，成功的構建了穿梭質粒pAdTrace-CXCR1。將線性化的穿梭質粒質粒與腺病毒骨架質粒pAdEasy-1于BJ5183細菌內進行同源重組，最終成功構建重組腺病毒質粒pAd-CXCR1。將pAd-CXCR1質粒于HEK-293細胞內擴增，得到高滴度人CXCR1基因的腺病毒載體。 結論：成功構建了人CXCR1基因的腺病毒載體，得到高滴度的腺病毒，為進一步研究CXCR1蛋白的生物功能及其在臨床各種相關疾病中的作用奠定了基礎。
[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.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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相關期刊論文 前2條

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