本文選題：豬傳染性胃腸炎病毒　切入點(diǎn)：ELISA　出處：《中國(guó)獸醫(yī)藥品監(jiān)察所》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：豬傳染性胃腸炎(transmissible gastroenteritis,TGE)是由豬傳染性胃腸炎病毒(transmissible gastroenteritis virus,TGEV)引起的一種豬的急性、高度接觸性傳染病,以腹瀉、嘔吐、脫水等為主要臨床特征,是近年來(lái)引起我國(guó)及亞洲其他國(guó)家腹瀉病的主要病原之一,對(duì)養(yǎng)殖業(yè)造成了嚴(yán)重的經(jīng)濟(jì)損失。TGE的快速準(zhǔn)確的診斷對(duì)于該病的防控十分重要。然而目前我國(guó)尚無(wú)正式注冊(cè)的商品化TGE抗原或抗體檢測(cè)試劑或試劑盒。因此,亟需開展TGEV抗原或抗體檢測(cè)試劑或試劑盒的研究,以更好地服務(wù)我國(guó)豬傳染性胃腸炎疫情防控。本研究主要開展了以下幾個(gè)方面的工作:1.TGEV病毒的分離與鑒定將從內(nèi)蒙古某豬場(chǎng)采集的經(jīng)膠體金試紙條和RT-PCR鑒定為TGEV陽(yáng)性的糞便樣品,在ST細(xì)胞上分離培養(yǎng)后,得到一株能產(chǎn)生明顯細(xì)胞病變的毒株。經(jīng)純凈性檢測(cè),該毒株無(wú)菌生長(zhǎng)、無(wú)支原體污染及外源病毒污染。特異性檢測(cè)結(jié)果顯示:該分離株能被豬傳染性胃腸炎病毒特異性陽(yáng)性血清中和,且能被TGEV FA熒光抗體識(shí)別。經(jīng)對(duì)該分離株進(jìn)行全序列測(cè)定并與GenBank登錄的13株TGEV毒株進(jìn)行同源性比較,顯示同源性為97.9%～99.9%,與我國(guó)TGEV疫苗株華毒株同源性為99.9%。系統(tǒng)進(jìn)化樹表明,該分離毒株與TGEV華毒株強(qiáng)毒株和弱毒株、Miller株、TS株和JS2012株親緣關(guān)系較近。以上結(jié)果表明分離到的毒株TGEV,命名為TGEVNMG株。2.TGEV間接ELISA抗體檢測(cè)方法的建立以濃縮純化的TGEV滅活病毒作為包被抗原,建立了間接ELISA抗體檢測(cè)方法。其優(yōu)化反應(yīng)條件為:該批抗原最佳包備稀釋濃度為1:2560,抗原包被量1.36 μg/孔;最佳包被時(shí)間為4℃過(guò)夜(約10～16小時(shí));封閉液為5%脫脂牛奶,37℃封閉2h;血清樣品最佳稀釋度為1:400,37℃作用1h;兔抗豬酶標(biāo)二抗最佳稀釋度為1:20000,37℃作用1h;抗體臨界值為S/P≥0.40判為陽(yáng)性,S/P0.40判為陰性。進(jìn)一步試驗(yàn)證實(shí),該方法具有較好的敏感性、特異性、重復(fù)性和符合率,顯示出較好的應(yīng)用前景。3.TGEV N融合蛋白的原核表達(dá)和鑒定成功構(gòu)建了 TGEVN基因的原核表達(dá)載體pET-28a,經(jīng)轉(zhuǎn)化感受態(tài)細(xì)胞Rosetta(DE3)、IPTG誘導(dǎo)表達(dá)和SDS-PAGE電泳檢測(cè),顯示重組大腸桿菌能夠有效表達(dá)TGEV N重組蛋白,目的蛋白大小為50kDa,且以可溶性蛋白表達(dá)形式為主。Western blot結(jié)果表明,該重組蛋白能被豬抗TGEV特異性陽(yáng)性血清識(shí)別,具有良好的抗原性。4.TGEV N蛋白單克隆抗體的制備與鑒定以純化的重組TGEVN蛋白為抗原免疫BALB/c小鼠,取免疫小鼠致敏脾細(xì)胞與SP2/0骨髓瘤細(xì)胞在PEG作用下融合,經(jīng)克隆培養(yǎng)、間接ELISA方法和IFA方法篩選,獲得了 3株能穩(wěn)定分泌抗TGEVN蛋白的抗體的雜交瘤細(xì)胞株,分別命名為MAb-N-10#,MAb-N-17#,MAb-N-18#。生物學(xué)鑒定試驗(yàn)表明:3株細(xì)胞株抗體類型和亞類均為IgG1,經(jīng)20代連續(xù)繼代后細(xì)胞培養(yǎng)上清均能與TGEV陽(yáng)性固定板呈現(xiàn)IFA陽(yáng)性反應(yīng),顯示出良好的遺傳穩(wěn)定性。單抗腹水純度均在90%以上,親和常數(shù)分別為4.3×109,1.22×109,7.52×108,敏感性顯示IFA效價(jià)均不低于1:1600,且特異性良好,為建立TGEV抗原快速檢測(cè)試劑盒奠定了物質(zhì)基礎(chǔ)。5.TGEV膠體金免疫層析試紙條的研制應(yīng)用膠體金免疫層析技術(shù),采用檸檬酸三鈉還原法制備20nm的膠體金顆粒,標(biāo)記單克隆抗體Mab-N-17#后包被于玻璃纖維膜作為金標(biāo)墊,在硝酸纖維素膜上分別包被羊抗小鼠IgG和單克隆抗體Mab-N-10#株作為質(zhì)控線和檢測(cè)線,組裝成膠體金檢測(cè)試紙條。該試紙條具有良好的特異性和敏感性,與RT-PCR檢測(cè)結(jié)果吻合性較好,檢測(cè)靈敏度約為102.1TCID50/0.1mL,與進(jìn)口產(chǎn)品的符合率為100%,敏感性優(yōu)于進(jìn)口試劑。
[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.

【學(xué)位授予單位】：中國(guó)獸醫(yī)藥品監(jiān)察所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.28

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