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(xué)院》2011年碩士論文　論文類型：學(xué)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(yīng)用細(xì)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(cè)序鑒定正確后與高效克隆載體pGEM-T連接,轉(zhuǎn)化進(jìn)E.coli Top10化學(xué)感受態(tài)細(xì)菌中,克隆擴(kuò)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(jìn)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(xì)菌中,進(jìn)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(xì)胞,利用PCR和RT-PCR鑒定正確后,然后進(jìn)行病毒擴(kuò)增。 【結(jié)果】 構(gòu)建了腺病毒穿梭質(zhì)粒pShuttle-CMV-efCD40L,并進(jìn)一步應(yīng)用細(xì)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(yīng)用細(xì)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(shí)驗(yàn)應(yīng)用于與PDL-1共轉(zhuǎn)染未成熟樹突狀細(xì)胞,進(jìn)一步研究肝移植免疫耐受奠定了實(shí)驗(yàn)基礎(chǔ)。
[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(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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