猪传染性胃肠炎病毒ELISA抗体检测方法的建立及胶体金免疫层析试纸条的研制

发布时间：2018-03-22 01:01

本文选题：猪传染性胃肠炎病毒　切入点：ELISA　出处：《中国兽医药品监察所》2017年硕士论文　论文类型：学位论文

【摘要】：猪传染性胃肠炎(transmissible gastroenteritis,TGE)是由猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)引起的一种猪的急性、高度接触性传染病,以腹泻、呕吐、脱水等为主要临床特征,是近年来引起我国及亚洲其他国家腹泻病的主要病原之一,对养殖业造成了严重的经济损失。TGE的快速准确的诊断对于该病的防控十分重要。然而目前我国尚无正式注册的商品化TGE抗原或抗体检测试剂或试剂盒。因此,亟需开展TGEV抗原或抗体检测试剂或试剂盒的研究,以更好地服务我国猪传染性胃肠炎疫情防控。本研究主要开展了以下几个方面的工作:1.TGEV病毒的分离与鉴定将从内蒙古某猪场采集的经胶体金试纸条和RT-PCR鉴定为TGEV阳性的粪便样品,在ST细胞上分离培养后,得到一株能产生明显细胞病变的毒株。经纯净性检测,该毒株无菌生长、无支原体污染及外源病毒污染。特异性检测结果显示:该分离株能被猪传染性胃肠炎病毒特异性阳性血清中和,且能被TGEV FA荧光抗体识别。经对该分离株进行全序列测定并与GenBank登录的13株TGEV毒株进行同源性比较,显示同源性为97.9%～99.9%,与我国TGEV疫苗株华毒株同源性为99.9%。系统进化树表明,该分离毒株与TGEV华毒株强毒株和弱毒株、Miller株、TS株和JS2012株亲缘关系较近。以上结果表明分离到的毒株TGEV,命名为TGEVNMG株。2.TGEV间接ELISA抗体检测方法的建立以浓缩纯化的TGEV灭活病毒作为包被抗原,建立了间接ELISA抗体检测方法。其优化反应条件为:该批抗原最佳包备稀释浓度为1:2560,抗原包被量1.36 μg/孔;最佳包被时间为4℃过夜(约10～16小时);封闭液为5%脱脂牛奶,37℃封闭2h;血清样品最佳稀释度为1:400,37℃作用1h;兔抗猪酶标二抗最佳稀释度为1:20000,37℃作用1h;抗体临界值为S/P≥0.40判为阳性,S/P0.40判为阴性。进一步试验证实,该方法具有较好的敏感性、特异性、重复性和符合率,显示出较好的应用前景。3.TGEV N融合蛋白的原核表达和鉴定成功构建了 TGEVN基因的原核表达载体pET-28a,经转化感受态细胞Rosetta(DE3)、IPTG诱导表达和SDS-PAGE电泳检测,显示重组大肠杆菌能够有效表达TGEV N重组蛋白,目的蛋白大小为50kDa,且以可溶性蛋白表达形式为主。Western blot结果表明,该重组蛋白能被猪抗TGEV特异性阳性血清识别,具有良好的抗原性。4.TGEV N蛋白单克隆抗体的制备与鉴定以纯化的重组TGEVN蛋白为抗原免疫BALB/c小鼠,取免疫小鼠致敏脾细胞与SP2/0骨髓瘤细胞在PEG作用下融合,经克隆培养、间接ELISA方法和IFA方法筛选,获得了 3株能稳定分泌抗TGEVN蛋白的抗体的杂交瘤细胞株,分别命名为MAb-N-10#,MAb-N-17#,MAb-N-18#。生物学鉴定试验表明:3株细胞株抗体类型和亚类均为IgG1,经20代连续继代后细胞培养上清均能与TGEV阳性固定板呈现IFA阳性反应,显示出良好的遗传稳定性。单抗腹水纯度均在90%以上,亲和常数分别为4.3×109,1.22×109,7.52×108,敏感性显示IFA效价均不低于1:1600,且特异性良好,为建立TGEV抗原快速检测试剂盒奠定了物质基础。5.TGEV胶体金免疫层析试纸条的研制应用胶体金免疫层析技术,采用柠檬酸三钠还原法制备20nm的胶体金颗粒,标记单克隆抗体Mab-N-17#后包被于玻璃纤维膜作为金标垫,在硝酸纤维素膜上分别包被羊抗小鼠IgG和单克隆抗体Mab-N-10#株作为质控线和检测线,组装成胶体金检测试纸条。该试纸条具有良好的特异性和敏感性,与RT-PCR检测结果吻合性较好,检测灵敏度约为102.1TCID50/0.1mL,与进口产品的符合率为100%,敏感性优于进口试剂。
[Abstract]:Transmissible gastroenteritis (transmissible gastroenteritis TGE) is a transmissible gastroenteritis virus (transmissible gastroenteritis, virus, TGEV) caused a swine acute, highly contagious disease, diarrhea, vomiting, dehydration as the main clinical features, is one of the main pathogens of diarrhea in China and other Asian countries caused in recent years. For the rapid and accurate diagnosis of aquaculture caused serious economic losses of.TGE is very important for the prevention and control of the disease. However, there is no formal registration of commercial TGE antigen or antibody detection reagent or kit. Therefore, the research of TGEV antigen or antibody detection reagent kit or the need to be done, in order to better serve our country transmissible gastroenteritis epidemic prevention and control. This study mainly includes the following aspects: isolation and identification of 1.TGEV virus collected from a pig farm in Inner Mongolia. The colloidal gold strip and RT-PCR were identified as TGEV positive fecal samples were isolated and cultured in ST cells, obtained strains could produce obvious cytopathic strain. The purity of the virus detection, sterile growth, no mycoplasma contamination and exogenous virus contamination. Specific detection results showed that the isolates can be neutralized transmissible gastroenteritis virus specific positive serum, and can be TGEV FA fluorescent antibody recognition. The isolates were sequenced and 13 strains of TGEV strain and GenBank log homology, showed the homology was 97.9% ~ 99.9%, and the TGEV vaccine strain of Chinese strain homology 99.9%. phylogenetic tree show that the Miller isolates and TGEV Chinese strains of virulent and avirulent strains, strain, TS strain and JS2012 strain is close relationship. The above results showed that the isolated TGEV strains, named TGEVNMG strain ELISA anti.2.TGEV indirect examination A measuring method to concentrate and purify TGEV inactivated virus as antigen to establish the indirect ELISA assay for detecting antibody. The optimum reaction conditions were: the number of the best package by antigen dilution 1:2560, antigen amount of 1.36 g/ hole; the optimal coating time is 4 DEG C for the night (about 10~16 hours); closed solution of 5% skim milk, 37 C closed 2H; serum sample dilution at 1:400,37 DEG 1H; Rabbit anti pig enzyme labeled two anti dilution at 1:20000,37 DEG 1H; antibody critical value S/P = 0.40 were considered positive, S/P0.40 negative. Further tests show that this method has better the sensitivity, specificity, repeatability and accuracy, showing prokaryotic expression and identification of.3.TGEV with good prospect of N fusion protein was successfully constructed the prokaryotic expression vector of pET-28a TGEVN gene, was transformed into competent cell Rosetta (DE3), IPTG and SD induced expression S-PAGE electrophoresis showed that the recombinant Escherichia coli TGEV N recombinant protein expression, protein size is 50kDa, and the soluble protein expression mainly in the form of.Western blot showed that the recombinant protein could be pig anti TGEV specific positive sera, preparation and identification of the purified recombinant TGEVN protein as antigen to immune BALB/c mice with antigenicity.4.TGEV N protein monoclonal antibody was good, immunized mice were sensitized spleen cells were fused with SP2/0 myeloma cells in the presence of PEG by cloning culture, indirect ELISA method and IFA method for screening hybridoma cell lines obtained 3 hybridoma cell lines secreting anti TGEVN antibody, named MAb-N-10#, MAb-N-17# show, biological identification test MAb-N-18#.: 3 cell lines antibody types and subtypes were IgG1, after 20 generations of continuous subculture and cell culture supernatant were TGEV positive plate Are IFA positive, showing good genetic stability. Monoclonal antibody purity was above 90%, the affinity constants were 4.3 * 109,1.22 * 109,7.52 * 108, IFA showed sensitivity titers were not less than 1:1600, and the specificity is good, for the establishment of TGEV rapid antigen detection kit laid the development and application of colloidal gold immunochromatography the material basis of.5.TGEV colloidal gold immunochromatographic test strip, using citric acid three sodium colloidal gold particles prepared by 20nm reduction, Mab-N-17# labeled monoclonal antibody coated on glass fiber membrane as the gold standard pad, nitrocellulose membrane in which are coated by Goat anti mouse IgG monoclonal antibody and Mab-N-10# strain as a control line and test line and assemble the colloidal gold test strip. The strip has good specificity and sensitivity, and the detection results are in good agreement with RT-PCR, the detection sensitivity is about 102.1TCID50/0.1mL, and the The coincidence rate of the oral product is 100%, and the sensitivity is better than that of the import reagent.

【学位授予单位】：中国兽医药品监察所
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.28

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