人博卡病毒HBoV结构蛋白基因的克

发布时间：2018-01-12 11:31

本文关键词：人博卡病毒HBoV结构蛋白基因的克隆、原核表达、纯化及VP1在杆状病毒中的表达　出处：《华中师范大学》2010年硕士论文　论文类型：学位论文

【摘要】：下呼吸道感染能引起儿童高发病率,是导致儿童死亡的重要病因之一。在2005年10月瑞典科学家Allander等从下呼吸道感染婴幼儿的分泌物中发现了一种新的细小病毒,它是人支气管炎的一种病原体,该病毒能引起儿童上、下呼吸道感染,甚至可引起严重的呼吸道疾病。从分子生物学、系统发育树分析和流行病学等多方面综合研究表明,该病毒具有细小病毒亚科的基本特征,并与细小病毒亚科的博卡病毒属成员结构特征相似,从而将其归类为博卡病毒属的新成员,并命名为人博卡病毒。人博卡病毒是一种无包膜的、单链DNA病毒,基因组DNA长度大约5299 nt,它是继细小病毒B19之后发现的第二种对人类致病的细小病毒,到目前为止已有18个国家和地区的研究者证实了人博卡病毒感染在儿童呼吸道疾病中普遍存在,部分研究者还在血清、粪便以及尿液的标本中也检测到了人博卡病毒,推测该病毒与胃肠道疾病有关。人博卡病毒可以单独感染,也可以与其他病毒混合感染,感染可全年发生,但深秋、冬季和早春为感染多发季节。目前对人博卡病毒生物学特性及其在人类疾病中的意义仍不清楚。本文根据我们实验室克隆得到的大片段人博卡病毒序列设计VP1和VP2引物(该序列已经提交GeneBank,登录号为：GU 139423),通过PCR扩增得到结构蛋白基因。并将VP1和VP2基因分别都克隆到原核表达载体pMAL-c2X和pET28a,转化大肠杆菌DH5α和BL21(DE3),经IPTG诱导表达融合蛋白,通过Westen Blot检测目的蛋白的表达。利用Amylose亲和层析柱和Ni柱纯化目的蛋白,并用纯化后得到的目的蛋白免疫新西兰大白兔,制备多克隆抗体,从而为进一步研究该病毒结构蛋白基因的转录和翻译机制提供可靠的工具。此外,为了弄清楚结构蛋白VP1独特区(VP1-u)是否可以单独表达,还是从整个结构蛋白VP1表达后被裂解下来的。本实验将人博卡病毒主要衣壳蛋白基因VP1克隆到杆状病毒表达转移载体(pFastBacHTA),通过转座获得重组Bacmid DNA,用这种DNA转染昆虫细胞Sf9,转染7到10天细胞完全破裂后,扩大培养使重组病毒达到一个比较高的滴度,收获重组病毒粒子,分别应用His-tag抗体和兔抗HBoVVP1-U高价免疫血清进行Western Blot,以检测VP1蛋白除了自身表达外,它所包含的VP1-U蛋白是否也进行了表达。结果表明VP1独特区不能单独表达。
[Abstract]:Lower respiratory tract infections can cause high morbidity in children. In October 2005 Swedish scientist Allander et al found a new parvovirus in the secretions of infants with lower respiratory infections. It is a pathogen of human bronchitis that can cause upper and lower respiratory infections in children and can even cause serious respiratory diseases from molecular biology. Phylogenetic tree analysis and epidemiology show that the virus has the basic characteristics of the subfamily of parvovirus and is similar to the member structure of the genus Boca of the subfamily of parvovirus. Thus it is classified as a new member of the genus Boca virus and named human Boca virus. Human Boca virus is an uncoated, single-stranded DNA virus with a genomic DNA length of about 5299nt.It is the second parvovirus to cause human disease after parvovirus B19. So far, researchers in 18 countries and regions have confirmed the prevalence of human Boca virus infection in children with respiratory diseases, and some researchers are still in the serum. Human Boca virus has also been detected in feces and urine specimens, presumably associated with gastrointestinal diseases. Human Boca virus can be infected alone or in combination with other viruses, which can occur all year round. However, late autumn, winter and early spring are frequently infected seasons. At present, the biological characteristics of human Boca virus and its significance in human diseases are still unclear. In this paper, VP1 and VP2 primers were designed according to the large fragment of human Boca virus sequence cloned in our laboratory (the sequence has been submitted to Gene Bank, accession number is:: GU139423). The structural protein gene was amplified by PCR and the VP1 and VP2 genes were cloned into the prokaryotic expression vector pMAL-c2X and pET28a respectively. E. coli DH5 伪 and BL21 DDE3 were transformed into E. coli. The fusion protein was induced by IPTG. The expression of the target protein was detected by Westen Blot. The target protein was purified by Amylose affinity chromatography and Ni column. The purified protein was used to immunize New Zealand white rabbits. The preparation of polyclonal antibodies provides a reliable tool for the further study of the transcription and translation mechanism of the structural protein gene of the virus. In addition, in order to determine whether the structural protein VP1 unique region of VP1-u) can be expressed alone. The main capsid protein gene VP1 of human Boca virus was cloned into baculovirus expression transfer vector pFastBacHTA. The recombinant Bacmid DNA was obtained by transposition and transfected into insect cell line Sf9 with this DNA. After 7 to 10 days of transfection, the recombinant Bacmid DNA was completely ruptured, and the recombinant virus was expanded to a higher titer. The recombinant virus particles were harvested and Western Blot was carried out by using His-tag antibody and rabbit anti-#en1# high value immunized serum respectively. The results showed that the unique region of VP1 could not be expressed alone.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373;Q78

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