【作者】 王雪；

【导师】 任晓峰；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2013， 硕士

【摘要】 猪传染性胃肠炎(TGE)是由猪传染性胃肠炎病毒(TGEV)引起的以严重的呕吐、脱水和腹泻为主要特征的猪的一种急性高度接触性肠道传染病,该病的流行给养猪业带来了巨大的损失。因此传染性胃肠炎病毒一直是猪病研究的热点之一。纤突蛋白(S)是TGEV表面突起的糖蛋白,可以刺激动物机体产生特异性的中和抗体并能使病毒与细胞膜上的特异性受体结合,参与细胞膜融合等过程,是作为免疫保护性抗原的主要候选基因。故S蛋白在猪传染性胃肠炎病毒诊断试剂及新型疫苗研究领域具有重要价值。本研究将包含S-AD (1102bp-1812bp)基因片段的重组质粒pET30a-S-AD在E.coli Rosetta中进行高效表达。SDS-PAGE结果显示,表达产物分子量为34KDa,与预期大小一致。Western-Blot显示,此蛋白可与TGEV的多抗血清发生特异性反应,说明该重组蛋白具有很好的反应原性。利用噬菌体展示技术,以S-AD重组蛋白为靶分子进行了四轮生物淘选。结果显示,经过四轮生物学筛选后,成功筛选出9种能与靶分子蛋白结合的十二肽。并应用筛选出的噬菌体,建立了一种针对TGEV的快速的,敏感性与特异性均很强且操作简单的噬菌体ELISA检测方法。将亲和力最高的序列合成多肽,命名为T多肽,序列为：-T-L-N-M-H-L-F-P-F-H-T-G-。使用TCID50、噬斑法和间接免疫荧光检测了T多肽的体外抗病毒活性。结果显示TGEV和T多肽37℃孵育1h后,加入ST细胞这种方式可有效的降低病毒的感染。本研究为建立新的TGE诊断方法和TGEV多肽疫苗的研发提供了一定的理论依据。更多还原

【Abstract】 Porcine transmissible gastroenteritis virus (TGEV) caused by porcine transmissible gastroenteritis is an highly contagious enteric diaease of swine, which was characterized acute chordapsus, diarrhea and death in piglets. The disease is widespread around the world, Bring great losses to pig industry.Thus, developing an effective vaccine is an important research topic for prevention of TGEV infection, protein (S) is the TGEV surface glycoproteins,whitch induced animal immune system to produce specific neutralizing antibody And can be mediated virus specific receptors on the cell membrane,involved in cell membrane fusion process.This experiment using recombinant plasmidpET30a-S-AD, whitch contains the S-AD (1102bp-1812bp) highly expressed the fusion protein in Escherichia Coli Rosseta (DE3).SDS-PAGE results showed that the expression of protein molecular weight is34KDa. form the results of the Western-Blot, we geted that the protein can reacted with positive serum of TGEV specificity, indicated that the recombinant protein has a very well biological activity.We compouned the peptide with the best affinity, named T. The Sequence is followed:-T-L-N-M-H-L-F-P-F-H-T-G-. We used the method of that detected the TCID50of virus, detected the fluorescent signal by indirect immunofluorescence, calculated the reduction of plaques number to detect the antiviral activity of the synthetic peptides in vitroantiviral activity. Results showed that the way TGEV and T peptides after1h of incubation at37℃, joined into ST cells had a high effect to inhibit the infectivity of TGEV.The current study may be helpful to design preventive strategies to TGEV infection based on phage display techniques.更多还原