禽腺病毒六邻体基因的原核表达

发布时间：2018-09-08 06:19

【摘要】：1953年,Rowe等在健康人群腺样组织的细胞培养过程中发现了一种病毒,被国际病毒命名委员会定名为腺病毒(Adenovirus,ADV),原因是该病毒喜欢感染上皮细胞。而禽腺病毒的最早发现报道是在1987的巴基斯坦安卡拉地区附近,故又称之为安卡拉病。禽腺病毒(Fowl adenovirus,FAV)是一种双链DNA病毒,共分三个亚群:I群、II群、III群。Ⅰ群禽腺病毒共有12个血清型(FAV1~12),并且它们具备共同的群抗原,在这之中的Ⅰ群4型禽腺病毒被认为是引发鸡安卡拉病的主要病原。FAVⅠ病毒粒子的直径是70~90nm,无囊膜,结构为球形、二十面体对称,有252个病毒壳粒,在这之中的二十面体的顶角壳粒是12个五邻体,另外240个非顶角壳粒为六邻体(hexon)。Hexon是FAVⅠ最主要的结构蛋白,和五邻体蛋白以及纤维蛋白共同构成FAVⅠ的外壳,hexon蛋白包含了主要的属和亚属特异性抗原决定簇,以及次要的种特异性抗原决定簇,是中和抗体的靶目标和主要保护性抗原基因,与致病性有紧密的关联,是以在被当作诊断抗原方面拥有很好的远景。本研究通过对湖北仙桃鸡场送检病鸡进行一系列观察,剖检发现心包积液、肝脏泛黄肿大等病变特征;病理切片通过H.E染色后镜检观察,可以发现肝脏组织脂肪变性;PCR结果与预期扩增片段大小一致;测序分析对比后发现该毒株序列同源性与I群4型禽腺病毒同源性最高,达到99.2%,跟其他血清型相似性对比则相对较低;用盲传3代病毒液接种1日龄雏鸡亦能导致发病,确定本次发生的禽安卡拉病病原为禽腺病毒I群4型。对临床上分离的I群4型禽腺病毒hexon基因进行PCR扩增并克隆到pMD19-T Simple载体上。重组克隆pMD19-T Simple-hexon经双酶切后,将hexon基因插入到pET-28a载体上,构建重组质粒pET-28a-hexon。将重组表达质粒转化BL21,经由含卡那霉素的LB培养基培育筛选,挑取阳性菌送测序。用IPTG诱导表达鉴定正确的菌液,继而对菌体进行超声破碎,通过SDS-PAGE分析其可溶性,利用亲和层析分离纯化目的蛋白,运用western-blot分析重组蛋白的免疫原性,以盲传3代含禽腺病毒尿囊液灭活后制备免疫原,以0.2mL/只的剂量采用多点注射的方式分三次免疫小鼠,三免过后采血后分离血清,制备鼠多克隆抗体,最后由Western-blot实验最终检测重组六邻体蛋白特异性。结果成功对六邻体蛋白进行了重组表达,诱导表达温度为37℃、IPTG浓度为1mM、诱导时间为4h。可溶性分析表明重组六邻体主要以包涵体的形式进行表达,利用亲和层析分离纯化目的蛋白,目的蛋白在10mM、20mM、50mM、100mM、150 mM、250 mM、500 mM等7个浓度的咪唑中均可洗脱。Westeron-blot分析结果证明,重组六邻体蛋白可以与禽腺病毒阳性血清产生特异性结合,能显示出特异性条带,说明重组六邻体蛋白具有很好的反应原性。对小鼠三免过后分离的血清进行Westeron-blot鉴定,结果表面成功制备了鼠抗重组六邻体蛋白多克隆抗体。研究表明,重组六邻体蛋白以包涵体的形式进行表达,具有较好的抗原性,与FAV病毒3免小鼠血清反应性良好,为进一步制备禽腺病毒新型疫苗及相关研究奠定了基础。
[Abstract]:In 1953, Rowe et al. discovered a virus in the cell culture of adenoid tissues of healthy people. It was named Adenovirus (ADV) by the International Committee for the Nomenclature of Viruses because it likes to infect epithelial cells. Cara disease. Avian adenovirus (FAV) is a double-stranded DNA virus, divided into three subgroups: group I, group II, and group III. Avian adenovirus group I has 12 serotypes (FAV1-12) and they share a common group antigen. Among them, group I-4 avian adenovirus is considered to be the main pathogen causing chicken Ankara disease. Hexon is the most important structural protein of FAV I, and the pentahedral protein and fibrin together form the outer shell of FAV I. Proteins contain major genera and subgenera specific antigenic determinants and minor species specific antigenic determinants. They are the target and major protective antigenic genes of neutralizing antibodies. They are closely related to pathogenicity and have a good prospect of being used as diagnostic antigens. After a series of observations, pericardial effusion and hepatic yellowing and enlargement were found, fatty degeneration of liver tissue could be found in pathological sections by H.E staining and microscopic examination; PCR results were consistent with the expected amplified fragment size; sequencing analysis and comparison showed that the sequence homology of the strain was the highest with that of avian adenovirus type I 4, and reached the highest level. By 99.2%, the similarity with other serotypes was relatively low. Blind inoculation with 3-generation virus solution could also cause the disease in 1-day-old chickens. Avian adenovirus group I type 4 was identified as the pathogen of this disease. The hexon gene of Avian adenovirus group I type 4 was amplified by PCR and cloned into pMD19-T Simple vector. The recombinant plasmid pET-28a-hexon was constructed by inserting the hexon gene into the pET-28a vector. The recombinant plasmid was transformed into BL21. The positive bacteria were selected and sequenced through cultivation and screening on the LB medium containing kanamycin. The correct bacterial liquid was identified by IPTG induction, and then the bacterial body was broken by ultrasound. The recombinant plasmid was transformed into SDS-P-hexon. AGE was used to analyze its solubility, affinity chromatography was used to isolate and purify the target protein, Western-blot was used to analyze the immunogenicity of the recombinant protein. Immunogen was prepared after 3 passages of inactivated allantoic fluid containing avian adenovirus. The mice were immunized three times at a dose of 0.2 mL per mouse by multi-point injection. The specificity of the recombinant hexa-neighbor protein was detected by Western-blot. Results The recombinant hexa-neighbor protein was successfully expressed at 37 C, IPTG concentration was 1 mM and induction time was 4 h. Solubility analysis showed that the recombinant hexa-neighbor protein was expressed mainly in the form of inclusion body and purified by affinity chromatography. The results of Westeron-blot analysis showed that the recombinant hexagonal protein could bind specifically to avian adenovirus-positive serum and display specific bands, indicating that the recombinant hexagonal protein had a good reactant. The results showed that the recombinant hexagonal protein was expressed in the form of inclusion body and had good antigenicity. The recombinant hexagonal protein had good reactivity with the serum of mice immunized with FAV-3 and could be used for further preparation of poultry glands. The new vaccine and related research laid the foundation.
【学位授予单位】：长江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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