人呼吸道合胞病毒F蛋白非复制型重组腺病毒的构建与鉴定

发布时间：2018-01-25 16:00

  本文关键词： 人呼吸道合胞病毒 真核表达 融合糖蛋白 非复制型重组腺病毒　出处：《安徽医科大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的：人呼吸道合胞病毒(Human Respiratory Syncytial Virus，RSV)广泛分布于世界各地，是导致婴幼儿严重下呼吸道感染最重要的病毒病原。病毒感染机体后的免疫保护机制尚未明确，也无特异性防治方法。RSV融合糖蛋白(fusionglycoprotein，F)和吸附蛋白(attachment glycoprotein，G)是RSV仅有的两种中和抗原。RSV只有一种血清型，主要由于G蛋白抗原性的差异，可进一步分为A和B两利种抗原亚型。F蛋白在RSV不同亚型间具有高度的抗原同源性，是主要的交叉保护性抗原。本研究拟克隆RSV F基因，采用非复制型腺病毒载体，，利用同源重组方法，构建含有F基因的非复制型腺病毒重组体，获得一株可表达RSV F的非复制型重组腺病毒FGAd／HRSVF，用于体内免疫效果及免疫保护作用的研究。 方法：本文根据F基因碱基序列，设计并合成引物，以F基因逆转录产物cDNA为模板，采用高保真DNA聚合酶，PCR扩增，得到F基因编码序列。然后克隆到pGEM-3zf载体中，序列测定正确后，将其亚克隆到真核表达载体pcDNA3.1(+)上，酶切鉴定，脂质体法转染COS-7细胞，应用Western blot方法分析F基因表达情况。将F基因亚克隆于腺病毒穿梭载体pShuttle-CMV，Pme Ⅰ线性化重组穿梭载体，与腺病毒骨架载体pAdEasy-1在E．coli BJ5183细胞内同源重组得到重组腺病毒DNA质粒。以Pac Ⅰ酶切获得的重组腺病毒DNA分子，脂质体法转染293细胞，产生基因组结构均一的重组腺病毒。Western blot鉴定目的基因表达。 结果：采用RT-PCR法扩增获得F蛋白基因编码序列。核酸序列分析显示没有发生无义突变。转染COS-7细胞后，利用Western blot方法检测到了F蛋白的特异性条带。进一步克隆至腺病毒穿梭载体pShuttle CMV，在Ecoli BJ5183内和腺病毒骨架载体pAdeasy-1实现同源重组，获得克隆有RSV F的重组腺病毒质粒pFGAd／HRSVF，经Pac Ⅰ线性化产生重组腺病毒DNA分子，转染293细胞。观
[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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