小鹅瘟病毒保护性抗原的原核表达及免疫原性研究

发布时间：2018-03-05 04:12

本文选题：小鹅瘟病毒　切入点：抗原表位富集区　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：小鹅瘟的病原为小鹅瘟病毒(Goose parvovirus, GPV),主要导致雏鹅发病,该病传播快,死亡率高,制约着鹅业养殖的健康发展。现有弱毒活疫苗和小鹅瘟抗体的广泛应用,在我国小鹅瘟疫病防控过程中发挥了巨大的作用。伴随着分子生物学技术的发展和疫苗产业化工艺的的革新,基因工程亚单位疫苗成为一种新型广泛推广应用的候选疫苗。基因工程亚单位疫苗具有生产成本低、抗原含量高、无外源病毒、无毒力返强的优势。本研究针对小鹅瘟的保护性抗原VP3结构蛋白,选取B细胞抗原表位富集区,开展原核表达、规模化发酵制备及其免疫原性研究工作,以期制备的亚单位抗原在小鹅瘟疫病的综合防控过程中,发挥其应有的作用,积极推进小鹅瘟疫病的净化。在山东地区开展了小鹅瘟的流行病学调查,采集山东地区某规模化养殖场疑似小鹅瘟病料,开展了GPV的分离鉴定、鹅胚中增值特性、3日龄雏鹅攻毒试验、中和试验、GPV VP3基因序列分析等工作；通过一系列的鉴定工作证实证实成功分离一株GPV毒株,该病毒第5代培养物鹅胚增殖死亡的高峰为96 h,其ELD50为10-6.32/0.2 mL,对3日龄雏鹅具有较强的致病性,LD50为10-2.2866/0.2 mL；该分离株克隆测序获得的VP3基因序列,与国内外参比毒株的同源性均在95.8%以上,推导的氨基酸同源性在97.5%以上,命名该GPV毒株为BZ株。为了获得高效表达且具有免疫原性的小鹅瘟亚单位抗原,以GPV BZ株为模板,分析预测GPV结构蛋白VP3的B细胞抗原表位分布,并选中预测的一段抗原表位富集区为研究目标,将其成功克隆构建了pET-32a-VP3原核表达载体,在实验室条件下实现了VP3-2蛋白的可溶性表达,可溶性的VP3-2蛋白免疫SPF鸡制备抗血清,中和效价能够达到10-1.91,说明该重组GPV亚单位蛋白抗原具有很好的中和抗体产生能力。为了实现具有良好免疫原性的GPV VP3-2蛋白的大规模生产,本研究通过细菌发酵罐培养技术,开展VP3-2蛋白的高密度发酵工作。高密度发酵研究证实,溶氧反馈控制补料适合VP3-2蛋白的规模化生产：通过在发酵过程中溶氧反馈补充调节葡萄糖溶液,控制溶氧水平分别在60%(0-4 h)、30%(4-10 h),使乙酸积累量大大降低至0.716g/L,细胞的吸光度(OD600)数值为2.256,VP3-2蛋白包涵体产量为85.64 mg/L。通过“溶氧反馈-分阶段控制”补料方案,实现了GPV VP3-2蛋白高效表达。高密度发酵表达的小鹅瘟亚单位抗原VP3-2蛋白产物为包涵体,为此开展了该蛋白变性复性工艺优化工作,形成了以CBS (pH9.6)为缓冲液、6M的尿素为变性剂、稀释复性的抗原后续处理工艺,质量检验证实该工艺成功制备了具有良好免疫原性的亚单位疫苗抗原；以复性的VP3-2蛋白作为疫苗抗原,配制了小鹅瘟蜂胶亚单位疫苗,进行了常规检验及安全性检验、效力检验、抗体持续期检验等,证实原核表达的VP3-2蛋白能够作为新型小鹅瘟疫苗的候选抗原。
[Abstract]:The pathogen of Gosling plague is Goose parvovirus (GPVV), which mainly causes the disease in goslings. The disease spreads rapidly and the mortality rate is high, which restricts the healthy development of goose breeding. The existing live attenuated vaccine and goose plague antibody are widely used. It has played a great role in the prevention and control of goose plague in China. With the development of molecular biology technology and the innovation of vaccine industrialization technology, Genetic engineering subunit vaccine has become a new candidate vaccine for wide application. It has the advantages of low production cost, high antigen content and no exogenous virus. This study aimed at the protective antigen VP3 structural protein of Gosling plague, selected B cell antigen epitope rich region, carried out prokaryotic expression, large-scale fermentation preparation and immunogenicity study. It was hoped that the prepared subunit antigen could play its due role in the comprehensive prevention and control of goose plague and actively promote the purification of goose plague. The epidemiological investigation of Gosling plague was carried out in Shandong Province. The suspected Gosling blast materials were collected from a large scale breeding farm in Shandong Province, and the isolation and identification of GPV were carried out, the virus attack test of 3-day-old goslings in goose embryo and the sequence analysis of VP3 gene were carried out in the neutralization test. A successful isolation of GPV strain was confirmed by a series of identification work. The peak of embryo proliferation and death was 96 h, the ELD50 was 10-6.32 / 0.2 mL, LD50 was 10-2.2866 / 0.2 mL for 3-day-old goslings, and the cloned sequence of VP3 gene was obtained. The homology of the strain with reference strain at home and abroad was above 95.8%, and the deduced amino acid homology was more than 97.5%. The strain of GPV was named as BZ strain. In order to obtain the highly expressed and immunogenicity goose plague subunit antigen, GPV BZ strain was used as template. The B cell epitope distribution of GPV structural protein VP3 was analyzed and predicted, and a segment of antigen epitope rich region was selected as the research objective. The prokaryotic expression vector of pET-32a-VP3 was successfully cloned and constructed. The soluble expression of VP3-2 protein was realized in laboratory. The soluble VP3-2 protein was immunized with SPF chicken to prepare antiserum. The neutralization titer can reach 10-1.91, which indicates that the recombinant GPV subunit protein antigen has a good ability to produce neutralizing antibody. In order to realize the large-scale production of GPV VP3-2 protein with good immunogenicity, the technique of bacterial fermentor culture was used in this study. High density fermentation of VP3-2 protein. The study of high density fermentation confirmed that the feedstock controlled by dissolved oxygen was suitable for the large-scale production of VP3-2 protein: the glucose solution was supplemented by dissolved oxygen feedback during fermentation. Controlling dissolved oxygen levels at 600-4 h and 30mg / L for 4-10 h, so that the accumulation of acetic acid was significantly reduced to 0.716 g / L, and the cell absorbance was 2.256 渭 g / L, and the protein inclusion body output was 85.64 mg / L. Through the "dissolved oxygen feedback-phased control" feeding scheme, The high efficient expression of GPV VP3-2 protein was achieved. The high density fermentative expression of goose blast subunit antigen VP3-2 protein product was used as inclusion body. The denaturation and renaturation process of the protein was optimized, and urea with CBS pH 9.6) as buffer was formed as denaturant. The process of dilution and renaturation of antigens showed that the subunit vaccine antigen with good immunogenicity was successfully prepared by this process, and the vaccine of goose blast propolis subunit was prepared by renaturation of VP3-2 protein as vaccine antigen. Routine and safety tests, potency tests and antibody duration tests were carried out to confirm that the prokaryotic expressed VP3-2 protein could be used as a candidate antigen for a novel Gosling plague vaccine.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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