【作者】 宋蔚；

【导师】 谭锦泉； 何金生；

【作者基本信息】 安徽医科大学 ， 免疫学， 2004， 硕士

【摘要】 人呼吸道合胞病毒G蛋白多肽重组腺病毒的构建、表达与鉴定 目的 人工合成的人呼吸道合胞病毒(hRSV)G蛋白多肽,含有两个二硫键和多个保护性B细胞表位,免疫动物后,能产生高水平的保护性抗体,可作为一种新型RSV疫苗抗原。本文采用非复制型脓病毒载体,利用同源重组方法,获得表达G蛋白多肽的非复制型重组腺病毒。 方法 本文根据G蛋白多肽碱基序列的需要,设计并合成了带有突变核苷酸序列的寡聚核苷酸引物,以G基因RT-PCR产物为模板,采用高保真DNA聚合酶,PCR突变扩增,得到G蛋白多肽编码序列。利用普通Taq DNA聚合酶PCR产物3′末端加“A”特性,将G蛋白多肽编码序列3′平末端加“A”,并与T载体连接。并将外源性基因亚克隆于穿梭载体pShuttle—CMV。PmeⅠ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。PCR方法检测G蛋白多肽在重组腺病毒中的稳定整合。 结果 利用带有突变核苷酸序列的寡核苷酸引物,用PCR突变法成功合成了G蛋白多肽编码序列。在E.coli BJ5183细胞内进行腺病毒骨架质粒与克隆带有外源基因的穿梭载体同源重组,获得了重组腺病毒质粒DNA分子。将获得重组腺病毒质粒DNA分子转染293细胞,可观察到293细胞出现肿胀,圆缩等典型的细胞病变(CPE)。 结论 获得的G蛋白多肽编码序列及其重组腺病毒,为研究免疫效果和研制新型呼吸道合胞病毒基因工程疫苗奠定了基础。更多还原

【Abstract】 Objective: Synthetic G-protein polypeptide comprises two disulfide bridges and multiple protective B cell epitopes. The G-protein polypeptide which immuned the animal models generated a highly protective antibody response against RSV challenge . Therefore, the G-protein polypeptide represents a new potential antigen for an RSV vaccine. We used the replication-defective adenovirus vector and the homologous recombination , so we obtained the recombinant adenovirus by carrying the G-protein polypeptide .Methods: Based on the base sequence of the G-protein polypeptide, the oligonucleotide primers were designed and synthesized which contains the mutational nucleotide sequence. The G-protein polypeptide DNA was obtained by the PCR mutation which the template was the RT-PCR production of G protein and the pfu DNA polymerse was used. So the Taq DNA polymerse added the dATP to the PCR product 3’ end of the G-protein polypeptide with pGEM-T easy vector. The gene of the G-protein polypeptide was cloned into the shuttle vector pShuttle-CMV, thus the recombinant shuttle plasmids were obtained. The resultant constructs were linearized with Pme I and transformed along with the supercoiled adenoviral vector pAdEasy-1 into RecA~+ E.coli strain BJ5183.The recombinant adenoviral constructs were cleaved with Pac I to expose their inverted terminal repeats (ITR) and transfected into 293 packaging cells to generate viruses. The recombinant adenovirus containing the G-protein polypeptide was obtained. The G-protein polypeptide specific DNA was integrated in the viral genome being identified by PCR assay and the viral genomes was stable when tested by PCR更多还原