本文關(guān)鍵詞：人博卡病毒HBoV結(jié)構(gòu)蛋白基因的克隆、原核表達(dá)、純化及VP1在桿狀病毒中的表達(dá)　出處：《華中師范大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 呼吸道疾病 人博卡病毒 結(jié)構(gòu)蛋白基因 蛋白表達(dá)純化 桿狀病毒表達(dá)系統(tǒng)

【摘要】：下呼吸道感染能引起兒童高發(fā)病率,是導(dǎo)致兒童死亡的重要病因之一。在2005年10月瑞典科學(xué)家Allander等從下呼吸道感染嬰幼兒的分泌物中發(fā)現(xiàn)了一種新的細(xì)小病毒,它是人支氣管炎的一種病原體,該病毒能引起兒童上、下呼吸道感染,甚至可引起嚴(yán)重的呼吸道疾病。從分子生物學(xué)、系統(tǒng)發(fā)育樹分析和流行病學(xué)等多方面綜合研究表明,該病毒具有細(xì)小病毒亞科的基本特征,并與細(xì)小病毒亞科的博卡病毒屬成員結(jié)構(gòu)特征相似,從而將其歸類為博卡病毒屬的新成員,并命名為人博卡病毒。 人博卡病毒是一種無包膜的、單鏈DNA病毒,基因組DNA長度大約5299 nt,它是繼細(xì)小病毒B19之后發(fā)現(xiàn)的第二種對人類致病的細(xì)小病毒,到目前為止已有18個國家和地區(qū)的研究者證實(shí)了人博卡病毒感染在兒童呼吸道疾病中普遍存在,部分研究者還在血清、糞便以及尿液的標(biāo)本中也檢測到了人博卡病毒,推測該病毒與胃腸道疾病有關(guān)。人博卡病毒可以單獨(dú)感染,也可以與其他病毒混合感染,感染可全年發(fā)生,但深秋、冬季和早春為感染多發(fā)季節(jié)。目前對人博卡病毒生物學(xué)特性及其在人類疾病中的意義仍不清楚。 本文根據(jù)我們實(shí)驗(yàn)室克隆得到的大片段人博卡病毒序列設(shè)計(jì)VP1和VP2引物(該序列已經(jīng)提交GeneBank,登錄號為：GU 139423),通過PCR擴(kuò)增得到結(jié)構(gòu)蛋白基因。并將VP1和VP2基因分別都克隆到原核表達(dá)載體pMAL-c2X和pET28a,轉(zhuǎn)化大腸桿菌DH5α和BL21(DE3),經(jīng)IPTG誘導(dǎo)表達(dá)融合蛋白,通過Westen Blot檢測目的蛋白的表達(dá)。利用Amylose親和層析柱和Ni柱純化目的蛋白,并用純化后得到的目的蛋白免疫新西蘭大白兔,制備多克隆抗體,從而為進(jìn)一步研究該病毒結(jié)構(gòu)蛋白基因的轉(zhuǎn)錄和翻譯機(jī)制提供可靠的工具。 此外,為了弄清楚結(jié)構(gòu)蛋白VP1獨(dú)特區(qū)(VP1-u)是否可以單獨(dú)表達(dá),還是從整個結(jié)構(gòu)蛋白VP1表達(dá)后被裂解下來的。本實(shí)驗(yàn)將人博卡病毒主要衣殼蛋白基因VP1克隆到桿狀病毒表達(dá)轉(zhuǎn)移載體(pFastBacHTA),通過轉(zhuǎn)座獲得重組Bacmid DNA,用這種DNA轉(zhuǎn)染昆蟲細(xì)胞Sf9,轉(zhuǎn)染7到10天細(xì)胞完全破裂后,擴(kuò)大培養(yǎng)使重組病毒達(dá)到一個比較高的滴度,收獲重組病毒粒子,分別應(yīng)用His-tag抗體和兔抗HBoVVP1-U高價免疫血清進(jìn)行Western Blot,以檢測VP1蛋白除了自身表達(dá)外,它所包含的VP1-U蛋白是否也進(jìn)行了表達(dá)。結(jié)果表明VP1獨(dú)特區(qū)不能單獨(dú)表達(dá)。
[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 偽 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.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R373;Q78

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