【作者】 康雪燕；

【导师】 姜世金；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2010， 硕士

【摘要】 猪传染性胃肠炎(Transmissible gastroenteritis, TGE)是一种急性、高度接触性传染病,以呕吐、严重腹泻、脱水为特征,各种年龄的猪只都可以发病,5周龄以下仔猪的病死率最高,给养猪业造成了较大的损失。TGE的病原体为猪传染性胃肠炎病毒(Transmissible gastroenteritis virus of swine, TGEV),该病毒是冠状病毒科冠状病毒属成员。在众多对TGEV研究的基础上,本研究就TGEV在猪睾丸细胞(ST)上的增殖特点的认识、花青素体外抗TGEV的试验研究、TGEV纤突糖蛋白在毕赤酵母中的分泌表达及免疫原性分析三个部分对TGEV展开研究。第一部分:猪传染性胃肠炎病毒的增殖特点的认识。本试验复苏和传代培养了TGEV宿主细胞-ST,测定TGEV的半数细胞培养物感染剂量(TCID50),以便于病毒感染毒性的定量,显微镜下观察TGEV吸附ST细胞后引起的细胞病变现象,免疫过氧化物酶单层试验(IPMA)进一步认识TGEV的增殖规律。细胞病变观察结果显示,TGEV侵染ST细胞后可引起具有“种”的特异性的细胞病变,引起细胞核变形或碎裂,甚至导致细胞的完全破坏,IPMA证明TGEV蛋白质的合成在胞浆内。第二部分:花青素体外抗猪传染性胃肠炎病毒的试验研究。在已测定TGEV的TCID50的基础上,利用细胞形态学观察方法,具体研究了花青素对ST细胞的毒性及花青素对TGEV的直接灭活、治疗作用。结果表明:花青素在ST细胞上最大安全浓度为0.002g/ml(纯度5%),即1mg/ml为实际花青素的最大安全浓度;花青素在ST细胞上对抗TGEV的最佳用量为0.002g/ml 500μl;起到治疗作用的花青素用量为0.002g/ml 500μl、375μl,但是花青素只是在ST细胞感染TGEV一定时间内对该病毒起到了治疗作用,而且体外试验结果不能完全等同于体内的治疗效果,所以体内抗病毒效果有待于进一步试验加以验证。第三部分:猪传染性胃肠炎病毒纤突糖蛋白在毕赤酵母中的分泌表达及免疫原性分析。根据GenBank上猪传染性胃肠炎病毒纤突糖蛋白S全基因序列设计一对引物,并在5’引物和3’引物中引入EcoR I、Not I酶切位点,经PCR扩增出长2.2kb的目的基因S(其中包含A、B、C、D四个主要抗原位点),克隆于pBS-T载体,克隆载体TS经EcoR I、Not I双酶切,S基因与pPIC9k载体连接,测序验证阳性克隆,Sal I线性化重组穿梭质粒pPIC9k-S,电转化GS115感受态,G418筛选和PCR鉴定得到阳性重组子,1%甲醇诱导表达,诱导后上清经SDS-PAGE电泳、免疫印迹和薄层凝胶扫描分析,并利用离子交换技术和膜超滤方法纯化目的蛋白,将纯化后的蛋白免疫两周龄BALB/c小鼠。SDS-PAGE电泳表明,S蛋白以可溶性形式分泌表达于胞外,且表达产物的分子量约为82 000,与理论推测的分子量一致。免疫印迹结果证明,S蛋白可被TGEV猪阳性血清识别,且具有良好的免疫原性。纯化蛋白免疫小鼠后,获得了抗S蛋白的特异性血清抗体,ELISA效价最高可达1:100。更多还原

【Abstract】 Transmissible gastroenteritis (TGE) has been reported in many parts of the world, including America, Asia and Europe. The disease is spread by the faeco-oral route and is characterised by vomiting, diarrhoea and high mortality in piglets. The causative agent is a coronavirus (TGEV), which is a member of the Coronaviridae family and belongs to the order Nidovirales. This study includes three parts.PartⅠThe acquaintance of the proliferation of TGEV in ST cellsAs the host of the TGEV, ST cell was revived and cultured. At fist, the tissue culture infective dose (TCID50) of the TGEV was determinated to measure the cytotoxicity. To confirmed the proliferation of TGEV, cytopathic effect (CPE) and Immunoperoxidase monolayer assay was observed by the Optical Microscopy. The CPE result showed that nucleus had been denatured or integrity was fully destroied. The IPMA confirmed the place of the sythesis of the protein was cytoplasm.PartⅡExperimental study on anthocyanin resisting transmissible gastroenteritis virus in vitroIn the patrtⅠ, the TCID50 of TGEV had been determinated. By the observation of the morphological changes, the cytotoxic influence, directly inactivited action and treatment action of the procyanidins were studied. The results showed that the safe concentration was not more than 0.002 gram per milliliter, in fact 1 milligram per milliliter. The optimal dose of directly inactivited action was confirmed to be 500 microliter and the treatment dose be 500-375 microliter. This part is only about the study in vitro, since the result is not identical to the study of animals. More animal experiment on procyanidins resisting TGEV needs to be done further. PartⅢSecretory expression of the spike glycoprotein of transmissible gastroenteritis virus in yeast Pichia pastoris and analysis on Immunogenicity of the recombinant proteinIn this study, A pair of primers were disigned according to TGEV S gene sequence in Genbank. After amplified by Polymerase Chain Reaction, a 2.2 kb fragment of the amino terminal half of the S gene containing all four major antigenic sites (A, B, C and D) was sub-cloned into pBS-T vector, then S gene was ligated with vector pPIC9K after cleaved from T-vector,.the recombinant shuttle plasmids pPIC9k-S were linearized by SalI and transformed into competent Pichia pastoris GS115 by electroporation and positive clones were screened with G418 and PCR amplification and induced with 1% methanol. At last, the supernatant of medium was analyzed by SDS-PAGE and Western-blotting and purified by ion exchange chromatography and ultrafiltration. A group of 2-week old BALB/c mouse were injected with the purified protein and the serum was detected by ELISA. The results showed that the partial fragment of spike protein (S), a approximately molecular weight 82 000 soluble protein, was secreted into the culture supernatant. The western-blotting results indicated that the protein could be recognized by the positive swine’s serum of TGEV. After diluted into 1:100, the specific antibody against S protein could be detected by ELISA.更多还原