【摘要】 囊膜病毒与宿主细胞的膜融合是病毒入侵宿主细胞的第一步. 禽副粘病毒-2 (APMV-2) 囊膜表面糖蛋白有2种,与宿主受体结合的血凝素神经氨酸酶(HN) 及介导膜融合的融合糖蛋白(F). HN蛋白的茎部区域(stalk region) 与球状头部区域(globular head region) ,以及F蛋白的3段七肽重复区域(heptad repeat,HR) 都可能与膜融合直接相关,将5段多肽进行基因的构建与表达研究. 根据已发表的禽副粘病毒-1 (APMV-1) 氨基酸序列,应用BLAST程序进行同源性分析,以确定APMV-2相应区域,使用搭桥PCR或普通PCR方法构建基因,分别克隆入表达载体pGEX-6p-Ⅰ获得重组质粒,阳性重组质粒转化入大肠杆菌BL21 (DE3),表达后获得可溶性融合蛋白,3C蛋白酶酶切后的蛋白质混合物经Glutathione-Sepharose 4B亲和层析纯化,最终获得可溶性、高纯度的5段多肽. 应用LearnCoil-VMF软件与ExPASy系列软件对蛋白质结构与功能进行预测与分析. 分子筛实验结果表明,F蛋白的HR1与HR2可形成六聚体结构,圆二色谱实验结果则表明,六聚体蛋白富含琢螺旋结构.更多还原

【Abstract】 Membrane fusion between the virus envelope and host cells is the first step of the enveloped virus entry into the host cells. This process involves the interaction of viral envelope proteins and their cellular receptors (proteins or sialic aids), which leads to the conformational changes of the envelope proteins. Avian paramyxovirus-2 (APMV-2) has the hemagglutinin- neuraminidase (HN) glycoprotein in which there are the stalk and globular head regions, and the fusion (F) glycoprotein in which there are the heptad repeat 1 (HR1) and heptad repeat 2 (HR2) and heptad repeat 3 (HR3) regions. To construct and express the correlative genes with membrane fusion of APMV-2,the relative sequences basing on the published sequences of avian paramyxovirus-1 (APMV-1) and using BLAST bio-software were ensured, then constructed genes by PCR and cloned genes into the BamHⅠ-XhoⅠ restriction sites of the GST fusion expression vector pGEX-6P-I, in which there is a rhinovirus 3C protease cleavage site for the fusion protein. E. coli strain BL21 (DE3) was transformed with the recombinant GST fusion plasmids. The supernatants lysed by sonication and clarified by centrifugation were passed over Glutathione-Sepharose 4B column for purifying, respectively. The GST fusion proteins were then cleaved by GST-fusion rhinovirus 3C protease and then were purified by the affinity chromatography. The LearnCoil-VMF and ExPASy bio-softwares were used for predict and analysis the structure and function of five peptides. And the results of Gel-filtration and circular dichroism (CD) showed that the HR1 and HR2 form a six-helix structure.更多还原