【摘要】：豬流行性腹瀉(Porcine epidemic diarrhea,PED)是由豬流行腹瀉病毒(PEDV)引起的一種臨床上以新生仔豬急性腹瀉、嘔吐、脫水及高死亡率為特征的高度接觸性傳染病,給全球養(yǎng)豬國(guó)家造成了巨大經(jīng)濟(jì)損失。目前有關(guān)本病的診斷方法多偏重于分子生物學(xué)方法,如RT-PCR。雖然RT-PCR方法具有特異、敏感、準(zhǔn)確等特點(diǎn),但該方法成本昂貴、難以應(yīng)用于大規(guī)模臨床樣品的診斷和流行病學(xué)調(diào)查。因此,迫切需要建立一種特異、敏感、快速簡(jiǎn)便診斷PED的方法。本實(shí)驗(yàn)應(yīng)用PCR擴(kuò)增出PEDV-BS-14毒株的纖突糖蛋白S基因的部分序列,并將其克隆到原核表達(dá)載體p GEX-4T-1中,構(gòu)建出p GEX-4T-1-S表達(dá)載體。應(yīng)用IPTG誘導(dǎo)表達(dá)重組S蛋白,并將純化的重組蛋白以弗氏完全/不完全佐劑乳化后免疫小鼠,制備抗PEDV S蛋白的單克隆抗體。以制備的抗體建立了檢測(cè)PEDV抗原的雙抗體夾心ELISA方法,并確定、優(yōu)化了所建立的ELISA反應(yīng)條件。捕獲抗體的最佳包被量為0.6μg,酶標(biāo)抗體的最佳稀釋倍數(shù)為1:1000,最佳封閉液為2%BSA,最佳封閉時(shí)間為1 h,酶標(biāo)抗體最佳作用時(shí)間為1 h,最佳顯色時(shí)間為10 min。以建立的雙抗體夾心ELISA和RT-PCR方法對(duì)PEDV糞便樣品進(jìn)行了比較檢測(cè),兩者的符合率為100%。應(yīng)用ELISA檢測(cè)了吉林省疑似PED流行豬場(chǎng)的糞便樣品,發(fā)現(xiàn)PEDV陽(yáng)性檢出率為68%。綜上所述,本研究成功表達(dá)出重組PEDV S蛋白,制備抗PEDV的單克隆抗體,建立了特異、敏感、快速檢測(cè)PEDV病毒抗原的雙抗體夾心ELISA方法,并對(duì)吉林省豬群感染PEDV進(jìn)行了病原流行病學(xué)的研究,發(fā)現(xiàn)豬群的PEDV陽(yáng)性感染率為68%,該研究結(jié)果將為PEDV診斷及流行病學(xué)研究提供技術(shù)手段和流行病學(xué)理論依據(jù)。
[Abstract]:Porcine epidemic diarrhea (PEDV) is a highly contagious disease caused by porcine epidemic diarrhea virus (PEDV), which is characterized by acute diarrhea, vomiting, dehydration and high mortality in newborn piglets. At present, the diagnosis of this disease is mainly focused on molecular biological methods, such as RT-PCR. Although RT-PCR has the characteristics of specificity, sensitivity and accuracy, it is difficult to be used in the diagnosis and epidemiological investigation of large-scale clinical samples because of its high cost. Therefore, it is urgent to establish a specific, sensitive, rapid and simple method for the diagnosis of PED. In this experiment, the partial sequence of fibrin S gene of PEDV-BS-14 strain was amplified by PCR, and cloned into prokaryotic expression vector pGEX-4T-1 to construct pGEX-4T-1-S expression vector. IPTG was used to induce the expression of recombinant S protein. The purified recombinant protein was emulsified with Freund's complete / incomplete adjuvant to prepare monoclonal antibody against PEDV S protein. A sandwich Elisa method for the detection of PEDV antigen was established by using the prepared antibodies, and the conditions of Elisa reaction were optimized. The best encapsulation amount of the capture antibody is 0.6 渭 g, the best dilution multiple of the enzyme labeled antibody is 1: 1000, the best blocking liquid is 2BSA, the best blocking time is 1 hour, the best time of the enzyme labeled antibody is 1 hour, the best color reaction time is 10 min. Double antibody sandwich Elisa and RT-PCR were used to detect PEDV fecal samples and the coincidence rate was 100. The fecal samples of suspected PED epidemic pig farm in Jilin province were detected by Elisa, and the positive rate of PEDV was 68%. In conclusion, the recombinant PEDV S protein was successfully expressed in this study, and the monoclonal antibody against PEDV was prepared. A sandwich Elisa was established for the specific, sensitive and rapid detection of PEDV virus antigen. The pathogenic epidemiology of PEDV in swine herd in Jilin Province was studied. It was found that the positive infection rate of PEDV in swine herd was 68. The results of the study would provide technical means and epidemiological theoretical basis for PEDV diagnosis and epidemiological study.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S858.28

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