基因2b亚型猪流行性腹泻病毒变异株的分离鉴定和疫苗研制

发布时间：2018-06-04 22:20

本文选题：猪流行性腹泻病毒 + G2b亚型　；参考：《扬州大学》2017年硕士论文

【摘要】：猪流行性腹泻病毒(PEDV)是猪流行性腹泻(PED)的致病因子,对养猪业危害巨大。自2010年12月以来,中国各地猪群中暴发大规模的腹泻疾病,其特征是水性腹泻、呕吐、脱水和乳猪高死亡率。研究表明,该疫病是由强毒力的基因2b亚型(G2b亚型)PEDV变异株引起,该型毒株与经典疫苗株之间存在较大的抗原性差异,导致免疫保护效果不理想,因此迫切需要研制相应的疫苗用于该疫病的防控。本研究从江苏某规模化猪场急性腹泻的哺乳仔猪小肠样品中分离到1株PEDV(命名为JSX2014株),该毒株通过细胞传代适应之后可在Vero细胞上稳定增殖并产生细胞病变(CPE)。采用RT-PCR方法对JSX2014株的全基因进行扩增并测序,运用MEGA5.05软件分析比较JSX2014株与其他PEDV毒株的全基因序列及S基因序列遗传进化关系,表明JSX2014毒株属于当前流行的G2b亚型。本研究检测了 G2b亚型PEDV JSX2014毒株在Vero细胞上以不同接毒量及接毒后不同时间点的病毒滴度,以确定该毒株的细胞适应毒株增殖规律。针对PEDV的N基因设计特异性引物,建立实时荧光定量方法,对增殖的JSX2014毒株进行定量。根据《中国兽药典》和《中华人民共和国兽用生物制品质量标准》要求建立了 JSX2014毒株的种子库,经RT-PCR检测未见外源病毒污染。利用Vero细胞转瓶培养工艺增殖JSX2014毒株抗原,浓缩后经甲醛灭活,添加氢氧化铝佐剂,制备了灭活疫苗。用该灭活疫苗免疫产前母猪及50日龄断奶仔猪,利用间接ELISA方法和血清中和试验分别检测疫苗免疫后的抗体消长规律。间接ELISA试验显示,断奶仔猪于免疫后14d已开始产生特异性抗体,免疫后42d表现100%血清抗体转阳。以JSX2014作为指示病毒进行中和实验,显示接种灭活JSX2014疫苗的断奶仔猪于免疫后42天表现最高血清抗体水平,与间接ELISA检测抗体增长规律相一致。免疫母猪分娩后7天的血清、初乳样品的中和抗体效价明显高于G1a型CV777毒株制备的灭活疫苗免疫组。综合所有实验结果本实验制备的JSX2014株灭活疫苗对PEDV G2b型毒株可以提供高效免疫保护。
[Abstract]:Porcine epidemic diarrhea virus (PEDVV) is a pathogenic factor of swine epidemic diarrhea virus (PED), which is harmful to pig industry. Since December 2010, large-scale diarrhoeal diseases have broken out in pig herds across China, characterized by waterborne diarrhea, vomiting, dehydration and high mortality in suckling pigs. The results showed that the disease was caused by the highly virulent gene 2b subtype G2b subtype PEDV variant, and there was a large antigenicity difference between this strain and the classical vaccine strain, which resulted in the poor immune protection effect. Therefore, there is an urgent need to develop a corresponding vaccine for the prevention and control of the epidemic. In this study, a JSX2014 strain was isolated from the small intestine of lactating piglets with acute diarrhea in a large scale pig farm in Jiangsu province. The strain was named JSX2014 strain, which could proliferate stably on Vero cells and produce cytopathic changes after passage adaptation. The whole gene of JSX2014 strain was amplified by RT-PCR and sequenced. The genetic evolution of the whole gene sequence and S gene sequence between JSX2014 strain and other PEDV strains was analyzed by MEGA5.05 software. The results showed that JSX2014 strain belongs to the current prevalent G2b subtype. In this study, the virus titers of G2b subtype PEDV JSX2014 strain on Vero cells at different doses and at different time points after inoculation were determined to determine the cell proliferation of G2b subtype PEDV JSX2014 strain. Specific primers were designed for N gene of PEDV and a real-time fluorescent quantitative method was established to quantify the proliferating JSX2014 strains. According to the requirements of the Chinese Veterinary Pharmacopoeia and the quality Standard of Veterinary Biological products of the people's Republic of China, the seed bank of JSX2014 strain was established, and no exogenous virus contamination was detected by RT-PCR. The inactivated vaccine was prepared by using Vero cell flask culture process to proliferate JSX2014 virus antigen, then concentrated and inactivated by formaldehyde and adding aluminum hydroxide adjuvant. The inactivated vaccine was used to immunize antepartum sows and 50-day-old weaning piglets. Indirect ELISA method and serum neutralization test were used to detect the growth and decline of antibodies after immunization. Indirect ELISA test showed that the weaned piglets began to produce specific antibodies on the 14th day after immunization, and 100% of the serum antibodies turned positive at 42 days after immunization. The neutralization test using JSX2014 as indicator virus showed that weaning piglets inoculated with inactivated JSX2014 vaccine showed the highest serum antibody level 42 days after immunization, which was consistent with the rule of indirect ELISA detection. The neutralizing antibody titer of colostrum samples was significantly higher than that of the inactivated vaccine immunized with G1a CV777 strain 7 days after delivery. Combined with all the experimental results, the inactivated vaccine of JSX2014 strain prepared in this experiment can provide high efficiency immune protection against PEDV G2b strain.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.28

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