本文關(guān)鍵詞：恒河猴CD40L胞外段基因腺病毒載體的構(gòu)建與鑒定　出處：《昆明醫(yī)學院》2011年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 恒河猴 CD40L胞外段基因 同源重組 BJ5183 腺病毒載體

【摘要】：【目的】 利用AdEasy腺病毒載體系統(tǒng),應用細菌內(nèi)同源重組法構(gòu)建并鑒定恒河猴CD40L胞外段基因(efCD40L)重組腺病毒載體。 【方法】 overlap PCR法合成恒河猴CD40L胞外段基因,瓊脂糖凝膠電泳、測序鑒定正確后與高效克隆載體pGEM-T連接,轉(zhuǎn)化進E.coli Top10化學感受態(tài)細菌中,克隆擴增pGEM-T-efCD40L,提取質(zhì)粒；AdEasy腺病毒載體系統(tǒng)中的pShuttle-CMV穿梭質(zhì)粒與pGEM-T-efCD40L進行雙酶切、連接后,得到穿梭質(zhì)粒pShuttle-CMV- efCD40L。pShuttle-CMV-efCD40L經(jīng)酶切、瓊脂糖凝膠電泳鑒定后,Pmel酶切線性化,再與腺病毒骨架質(zhì)粒pAdEasy-1共電轉(zhuǎn)化到感受態(tài)BJ5183細菌中,進行同源重組產(chǎn)生腺病毒質(zhì)粒。經(jīng)過抗性篩選以及PCR鑒定得到陽性的pAd-CMV-efCD40L重組質(zhì)粒。將pAd-CMV-efCD40L線性化后,利用脂質(zhì)體2000轉(zhuǎn)染AD293細胞,利用PCR和RT-PCR鑒定正確后,然后進行病毒擴增。 【結(jié)果】 構(gòu)建了腺病毒穿梭質(zhì)粒pShuttle-CMV-efCD40L,并進一步應用細菌內(nèi)同源重組成功構(gòu)建了含CD基因的重組腺病毒載體pAd-CMV-efCD40L。 【結(jié)論】 應用細菌內(nèi)同源重組法可構(gòu)建含恒河猴CD40L胞外段基因的重組腺病毒載體,為后續(xù)實驗應用于與PDL-1共轉(zhuǎn)染未成熟樹突狀細胞,進一步研究肝移植免疫耐受奠定了實驗基礎。
[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.
【學位授予單位】：昆明醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前10條

1 鄧珊;胡大偉;;樹突狀細胞在系統(tǒng)性紅斑狼瘡免疫發(fā)病中的作用[J];中華風濕病學雜志;2006年07期

2 陶紹富;李濟宇;;樹突狀細胞在器官移植中誘導移植耐受的研究進展[J];醫(yī)學研究生學報;2010年01期

3 邵海楓;崔蕾蕾;趙曉智;王錦娜;王衛(wèi)萍;;多重PCR技術(shù)檢測肺炎克雷伯菌中質(zhì)粒介導的AmpC酶基因[J];臨床檢驗雜志;2005年06期

4 王繼納;朱同玉;;藥物在誘導臨床移植免疫耐受中的應用[J];中國臨床藥學雜志;2007年05期

5 唐雪明,沈微,方惠英,王正祥,諸葛健;用異丙醇沉淀法快速提取質(zhì)粒DNA[J];生物學通報;2002年01期

6 劉袁媛;范華驊;任亞娜;楊潔;聶曉絢;趙麗華;林俊杰;;小鼠調(diào)節(jié)性樹突狀細胞分泌的外泌體誘導免疫耐受的實驗研究[J];中國實驗血液學雜志;2008年02期

7 程金科,林晨,張雪艷;應用于基因治療的重組體腺病毒的構(gòu)建方法[J];遺傳;1997年04期

8 王家寧,黃永章,王俊峰,王衛(wèi)民,李瑞明,葛永貴,張群林;重組β-半乳糖苷酶腺病毒載體的構(gòu)建及其在血管平滑肌細胞中的表達[J];鄖陽醫(yī)學院學報;2000年04期

9 王家寧,黃永章,王俊峰,王衛(wèi)民,李瑞明,張群林;反義VEGF165腺病毒重組體的構(gòu)建及鑒定[J];鄖陽醫(yī)學院學報;2001年01期

10 孫躍玲,王家寧,涂遠超,黃永章,黃從新;菌體內(nèi)同源重組構(gòu)建pAdEasy-1-Cat重組體[J];鄖陽醫(yī)學院學報;2003年04期

相關(guān)博士學位論文 前1條

1 胡明道;CTLA4-Ig融合蛋白、TGF-β1誘導獼猴同種異體肝移植免疫耐受的實驗研究[D];昆明醫(yī)學院;2008年

，

本文編號：1403175