本文選題：豬流行性腹瀉病毒 + G2b亞型�。� 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：豬流行性腹瀉病毒(PEDV)是豬流行性腹瀉(PED)的致病因子,對養(yǎng)豬業(yè)危害巨大。自2010年12月以來,中國各地豬群中暴發(fā)大規(guī)模的腹瀉疾病,其特征是水性腹瀉、嘔吐、脫水和乳豬高死亡率。研究表明,該疫病是由強(qiáng)毒力的基因2b亞型(G2b亞型)PEDV變異株引起,該型毒株與經(jīng)典疫苗株之間存在較大的抗原性差異,導(dǎo)致免疫保護(hù)效果不理想,因此迫切需要研制相應(yīng)的疫苗用于該疫病的防控。本研究從江蘇某規(guī)模化豬場急性腹瀉的哺乳仔豬小腸樣品中分離到1株P(guān)EDV(命名為JSX2014株),該毒株通過細(xì)胞傳代適應(yīng)之后可在Vero細(xì)胞上穩(wěn)定增殖并產(chǎn)生細(xì)胞病變(CPE)。采用RT-PCR方法對JSX2014株的全基因進(jìn)行擴(kuò)增并測序,運(yùn)用MEGA5.05軟件分析比較JSX2014株與其他PEDV毒株的全基因序列及S基因序列遺傳進(jìn)化關(guān)系,表明JSX2014毒株屬于當(dāng)前流行的G2b亞型。本研究檢測了 G2b亞型PEDV JSX2014毒株在Vero細(xì)胞上以不同接毒量及接毒后不同時間點(diǎn)的病毒滴度,以確定該毒株的細(xì)胞適應(yīng)毒株增殖規(guī)律。針對PEDV的N基因設(shè)計特異性引物,建立實時熒光定量方法,對增殖的JSX2014毒株進(jìn)行定量。根據(jù)《中國獸藥典》和《中華人民共和國獸用生物制品質(zhì)量標(biāo)準(zhǔn)》要求建立了 JSX2014毒株的種子庫,經(jīng)RT-PCR檢測未見外源病毒污染。利用Vero細(xì)胞轉(zhuǎn)瓶培養(yǎng)工藝增殖JSX2014毒株抗原,濃縮后經(jīng)甲醛滅活,添加氫氧化鋁佐劑,制備了滅活疫苗。用該滅活疫苗免疫產(chǎn)前母豬及50日齡斷奶仔豬,利用間接ELISA方法和血清中和試驗分別檢測疫苗免疫后的抗體消長規(guī)律。間接ELISA試驗顯示,斷奶仔豬于免疫后14d已開始產(chǎn)生特異性抗體,免疫后42d表現(xiàn)100%血清抗體轉(zhuǎn)陽。以JSX2014作為指示病毒進(jìn)行中和實驗,顯示接種滅活JSX2014疫苗的斷奶仔豬于免疫后42天表現(xiàn)最高血清抗體水平,與間接ELISA檢測抗體增長規(guī)律相一致。免疫母豬分娩后7天的血清、初乳樣品的中和抗體效價明顯高于G1a型CV777毒株制備的滅活疫苗免疫組。綜合所有實驗結(jié)果本實驗制備的JSX2014株滅活疫苗對PEDV G2b型毒株可以提供高效免疫保護(hù)。
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.28

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