【摘要】：2010年10月以來,豬流行性腹瀉在中國和世界其他主要養(yǎng)豬國家大面積流行。本論文首先對42株P(guān)EDV(2010年以后的PEDV中國流行株24個(gè),美國流行株株7個(gè),韓國毒株6個(gè),參考毒株5個(gè))S蛋白氨基酸序列進(jìn)行比較,結(jié)果顯示,與參考毒株相比,2010年以后PEDV流行株的S蛋白的22aa~380aa區(qū)域(命名為SA)為突變的集中區(qū)域;流行株親緣關(guān)系較近且在一個(gè)分支,而疫苗株及早期毒株等參考毒株則位于另一個(gè)分支;早期毒株與中國現(xiàn)用疫苗毒株CV777之間的同源性較高,為95.0%~99.1%,而2010年后的流行株與參考毒株間的同源性為92.3%~94.1%,其中與疫苗株CV777的同源性僅為93.3%;對SA區(qū)域進(jìn)行抗原位點(diǎn)與糖基化位點(diǎn)的預(yù)測發(fā)現(xiàn),2010年以后流行株與疫苗株及早期毒株之間在22aa~380aa之間抗原位點(diǎn)差異較大,且在60aa~280aa之間差異最顯著;2010年以后的毒株在62aa~65aa位多出了一個(gè)N糖基化位點(diǎn)。以上結(jié)果提示2010年以后的PEDV流行株與經(jīng)典毒株相比發(fā)生了明顯變異,在對PEDV S基因進(jìn)行序列分析的基礎(chǔ)上,本論文首先表達(dá)了PEDV中國變異株CH-ZMDZY-11與疫苗株CV777的SA區(qū)域,表達(dá)的SA蛋白具有良好的反應(yīng)原性,為建立檢測抗PEDV抗體的間接ELISA方法、分析PEDV流行株與疫苗株S蛋白免疫反應(yīng)性差異,以及PED亞單位疫苗研究奠定了基礎(chǔ)。以純化的PEDV中國變異株S蛋白為包被抗原,建立了檢測PEDV流行株抗體的間接ELISA方法。建立的間接ELISA方法最佳反應(yīng)條件為:抗原最適包被量為1ug/孔,包被時(shí)間為37℃包被1h后4℃過夜;血清工作濃度為1:100,作用時(shí)間為1.5h;二抗選用HRP標(biāo)記的兔抗豬IgG稀釋度為1:4000,作用時(shí)間為1.5h;顯色液TMB作用時(shí)間為15min;判定標(biāo)準(zhǔn)為,樣品S/P值大于等于0.058判定為陽性,小于0.045判定為陰性。所建ELISA方法具有良好特異性和重復(fù)性。對50份疑似豬流行性腹瀉血清樣品進(jìn)行檢測,結(jié)果表明流行株抗體檢測陽性的樣品均為PEDV流行株抗原陽性。本研究所建立ELISA方法可用于PEDV流行株抗體檢測和疫病監(jiān)測。為分析PEDV流行株和疫苗株S基因差異對疫苗免疫效果影響,本研究將在表達(dá)的PEDV變異株CH/ZMDZY/11與疫苗株CV777 SA蛋白免疫小鼠與成年兔子獲得多克隆抗體,并以SA蛋白為抗原,通過Western bloting及間接ELISA的方法檢測兩種蛋白與多克隆抗體的反應(yīng)性差異,并通過兔抗PEDV SA蛋白多克隆抗體與CV777中和情況比較抗S蛋白抗體中和活性的差異。結(jié)果表明,抗PEDV疫苗株SA蛋白(YSA)多抗與疫苗株SA蛋白的反應(yīng)性明顯高于其與流行株SA蛋白(LSA)的反應(yīng)性;與此相反,抗PEDV流行株SA蛋白多抗與流行株SA蛋白的反應(yīng)性明顯高于其與疫苗株SA蛋白的反應(yīng)性;抗體的中和活性比較顯示,抗PEDV變異株及疫苗株SA蛋白多克隆抗體在中和PEDV疫苗毒CV777上存在很大的差異,即疫苗株的抗體中和CV777效價(jià)明顯高于流行株抗體的中和效價(jià)。研究結(jié)果提示,PEDV S蛋白22aa~380aa區(qū)域存在差異性抗原表位,PEDV變異株和疫苗株S基因的差異可能是導(dǎo)致基于CV777疫苗株的PED疫苗免疫效果不好的原因。本研究將為開發(fā)針對PEDV變異株的PED亞單位疫苗,有效控制由變異株導(dǎo)致的PED奠定基礎(chǔ)。
[Abstract]:Porcine epidemic diarrhea has been prevalent in China and other major pig-raising countries in the world since October 2010. In this paper, 42 strains of PEDV (24 strains of PEDV in China, 7 strains of American epidemic strains, 6 Korean strains and 5 S protein amino acid sequences) were compared. Compared with the reference strain, the 22aa ~ 380aa region (named SA) of the S protein of the PEDV popular strain after 2010 is the concentrated region of the mutation; the genetic relationship of the epidemic strain is close and in one branch, and the reference strain such as the vaccine strain and the early strain is located in the other branch; The homology between the early strain and the vaccine strain CV777 was higher, which was 95.0% ~ 99.1%, and the homology between the epidemic strain and the reference strain after 2010 was 92.3% ~ 94.1%, among which the homology with the vaccine strain CV777 was 93.3%. It was found that there was a significant difference in the antigenic sites between the epidemic strain and the vaccine strain and the early strain at 22aa ~ 380aa after 2010, and the difference between 60aa ~ 280aa was the most significant. The strain after 2010 had an N glycosylation site at 62aa ~ 65aa. The results suggest that the PEDV popular strain after 2010 has a significant variation in comparison with the classical strain. On the basis of sequence analysis of the PEDV S gene, this paper first expresses the SA region of the strain CH-ZMDZY-11 of PEDV and the vaccine strain CV777, and the expressed SA protein has good reactivity. In order to establish an indirect ELISA method for detecting anti-PEDV antibody, the difference of immune reactivity between PEDV and vaccine strain S protein was analyzed, and the basis was laid for the research of PED subunit vaccine. An indirect ELISA method for detecting the antibody of PEDV was established by using purified PEDV Chinese mutant S protein as envelope antigen. The optimal reaction conditions of indirect ELISA were as follows: the optimal coating amount of antigen was 1ug/ hole, the coating time was 37 鈩，

本文編號：2289041