本文關(guān)鍵詞： 人呼吸道合胞病毒 真核表達(dá) 融合糖蛋白 非復(fù)制型重組腺病毒　出處：《安徽醫(yī)科大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：人呼吸道合胞病毒(Human Respiratory Syncytial Virus，RSV)廣泛分布于世界各地，是導(dǎo)致嬰幼兒嚴(yán)重下呼吸道感染最重要的病毒病原。病毒感染機(jī)體后的免疫保護(hù)機(jī)制尚未明確，也無(wú)特異性防治方法。RSV融合糖蛋白(fusionglycoprotein，F(xiàn))和吸附蛋白(attachment glycoprotein，G)是RSV僅有的兩種中和抗原。RSV只有一種血清型，主要由于G蛋白抗原性的差異，可進(jìn)一步分為A和B兩利種抗原亞型。F蛋白在RSV不同亞型間具有高度的抗原同源性，是主要的交叉保護(hù)性抗原。本研究擬克隆RSV F基因，采用非復(fù)制型腺病毒載體，，利用同源重組方法，構(gòu)建含有F基因的非復(fù)制型腺病毒重組體，獲得一株可表達(dá)RSV F的非復(fù)制型重組腺病毒FGAd／HRSVF，用于體內(nèi)免疫效果及免疫保護(hù)作用的研究。 方法：本文根據(jù)F基因堿基序列，設(shè)計(jì)并合成引物，以F基因逆轉(zhuǎn)錄產(chǎn)物cDNA為模板，采用高保真DNA聚合酶，PCR擴(kuò)增，得到F基因編碼序列。然后克隆到pGEM-3zf載體中，序列測(cè)定正確后，將其亞克隆到真核表達(dá)載體pcDNA3.1(+)上，酶切鑒定，脂質(zhì)體法轉(zhuǎn)染COS-7細(xì)胞，應(yīng)用Western blot方法分析F基因表達(dá)情況。將F基因亞克隆于腺病毒穿梭載體pShuttle-CMV，Pme Ⅰ線性化重組穿梭載體，與腺病毒骨架載體pAdEasy-1在E．coli BJ5183細(xì)胞內(nèi)同源重組得到重組腺病毒DNA質(zhì)粒。以Pac Ⅰ酶切獲得的重組腺病毒DNA分子，脂質(zhì)體法轉(zhuǎn)染293細(xì)胞，產(chǎn)生基因組結(jié)構(gòu)均一的重組腺病毒。Western blot鑒定目的基因表達(dá)。 結(jié)果：采用RT-PCR法擴(kuò)增獲得F蛋白基因編碼序列。核酸序列分析顯示沒(méi)有發(fā)生無(wú)義突變。轉(zhuǎn)染COS-7細(xì)胞后，利用Western blot方法檢測(cè)到了F蛋白的特異性條帶。進(jìn)一步克隆至腺病毒穿梭載體pShuttle CMV，在Ecoli BJ5183內(nèi)和腺病毒骨架載體pAdeasy-1實(shí)現(xiàn)同源重組，獲得克隆有RSV F的重組腺病毒質(zhì)粒pFGAd／HRSVF，經(jīng)Pac Ⅰ線性化產(chǎn)生重組腺病毒DNA分子，轉(zhuǎn)染293細(xì)胞。觀
[Abstract]:Objective: human Respiratory Syncytial virus (RSVV) is widely distributed all over the world. It is the most important virus pathogen that causes severe lower respiratory tract infection in infants. The mechanism of immune protection after virus infection is not clear. There is also no specific prevention and treatment method. RSV fusion glycoprotein fusionglycoprotein FV and adsorption protein attachment glycoprotein. G) is the only two neutralizing antigens of RSV. RSV has only one serotype, mainly due to the difference of G protein antigenicity. It can be further divided into A and B antigenic subtypes. The protein has high antigenic homology among different subtypes of RSV and is the main cross-protective antigen. In this study, RSV F gene was cloned. Non-replicating adenovirus recombinant containing F gene was constructed by homologous recombination method using non-replicating adenovirus vector. A non-replicative recombinant adenovirus FGAd-HRSVF expressing RSVF was obtained, which was used to study the immunological effect and protective effect of FGAd-HRSVF in vivo. Methods: according to the base sequence of F gene, primers were designed and synthesized. CDNA, a reverse transcription product of F gene, was amplified by high fidelity DNA polymerase chain reaction. The F gene encoding sequence was obtained and cloned into pGEM-3zf vector. After the sequence was determined correctly, the gene was subcloned into eukaryotic expression vector pcDNA3.1 () and identified by restriction endonuclease digestion. COS-7 cells were transfected with liposome and the expression of F gene was analyzed by Western blot. F gene was subcloned into adenovirus shuttle vector pShuttle-CMV. Pme 鈪

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