發(fā)布時間：2019-03-18 17:26

【摘要】：1型鴨甲肝病毒(duck hepatitis A virus type 1, DHAV-1)主要引起4周齡以內(nèi)的雛鴨發(fā)病,死亡率高達90%以上,給養(yǎng)鴨業(yè)帶來嚴重的損失。為了建立DHAV-1的體外培養(yǎng)模型,探究病毒結(jié)構(gòu)蛋白VP0的免疫原性,本研究對DHAV-1在鴨胚成纖維細胞(DEF)中的適應(yīng)特性及病毒結(jié)構(gòu)蛋白VP0進行了一系列的研究,獲得如下結(jié)果。1、用實驗室分離保存的野毒株DHAV-1-X的尿囊液接種DEF,盲傳3代后DEF細胞出現(xiàn)輕微病變,第8代后出現(xiàn)明顯、穩(wěn)定的細胞病變(CPE)。采用RT-PCR、間接免疫熒光、鴨胚接種試驗等一系列的方法證明DHAV-1-X能夠在DEF細胞內(nèi)良好增殖。熒光定量RT-PCR檢測增殖規(guī)律,結(jié)果表明,病毒含量在接毒后12h開始升高,48h達到高峰；48h-96h維持在一定的水平,病毒的含量基本趨于穩(wěn)定。2、將DHAV-1-X結(jié)構(gòu)蛋白VP0全基因序列(VP0-F)和部分序列(VP0-P)分別克隆到表達載體pET-32a (+)和pGEX-4T-1中,構(gòu)建原核表達質(zhì)粒pET-32a (+)/VPO-F和pGEX-4T-1/VP0-P并轉(zhuǎn)入大腸桿菌BL21中,成功表達出VP0全序列和部分序列蛋白。兩種重組蛋白分別用親和層析和切膠回收的方法獲得純度較高的重組蛋白,且都能被兔抗DHAV-1血清識別。3、用純化的VP0-P蛋白免疫健康的家兔,制備兔抗VP0-P高免血清,瓊脂擴散實驗檢測血清效價達到1：16；雞胚中和試驗檢測兔抗VP0-P高免血清的中和效價達到1：146；間接免疫熒光試驗表明兔抗VP0-P高免血清能識別DHAV-1.4、將純化的重組蛋白VP0-F和VP0-P作為包被抗原,分別建立基于VP0-F和VP0-P蛋白的間接ELISA方法�；赩P0-F蛋白的間接ELISA方法的最佳條件為：以1.67μg/ml的VP0-F重組蛋白37℃孵育1h后4℃包被過夜；5%明膠封閉0.5h；被檢血清1：160稀釋,37℃孵育1h；HRP標記的羊抗鴨IgG進行1：400稀釋,37℃孵育1h；TMB避光顯色10min，，測定OD450/OD630值,陽性閾值為0.375�；赩P0-P蛋白的間接ELISA方法的最佳條件為：以1.67μg/ml的VP0-P重組蛋白37℃孵育2h包被；5%明膠封閉2h；被檢血清1：80稀釋,37℃孵育1h; HRP標記的羊抗鴨IgG進行1：400稀釋,37℃孵育1h;TMB避光顯色5min,測定OD450/OD630值,陽性閾值為0.317。兩種ELISA方法都具有較強的特異性和重復(fù)性,不與禽流感病毒、鴨沙門氏菌、鴨疫里氏桿菌、鴨瘟病毒、腫頭敗血癥病毒、鴨大腸桿菌以及鴨沙門氏菌的陽性血清發(fā)生交叉反應(yīng),與以DHAV-1作為包被抗原的ELISA方法的符合率分別為90%和93.3%。5、用VP0-F和VP0-P蛋白免疫1日齡雛鴨,不同時間點采血,用于檢測抗體水平和CD4、CD8、IL-4、IFN-γ的水平變化。數(shù)據(jù)分析表明,雛鴨免疫后15d抗體水平達到高峰,之后有不同程度的下降,VP0-F蛋白免疫組的抗體水平明顯高于VP0-P蛋白免疫組；CD4、CD8、IL-4、IFN-γ的含量在免疫后7d達到最大值,VP0-F免疫組細胞因子含量的變化較為顯著,而VP0-P免疫組細胞因子的含量雖然也有變化蛋白但是明顯低于VP0-F免疫組,與對照組顯著性不顯著。
[Abstract]:Duck hepatitis A virus type 1 (duck hepatitis A virus type-1, DHAV-1) mainly causes the disease of ducklings under 4 weeks old, and the mortality rate is more than 90%, which brings serious losses to duck industry. In order to establish the culture model of DHAV-1 in vitro and explore the immunogenicity of viral structural protein VP0, the adaptive characteristics of DHAV-1 in (DEF) of duck embryo fibroblasts and the viral structural protein VP0 were studied. The following results were obtained: 1. DEF cells showed slight pathological changes after being inoculated with the allantoic fluid of the wild strain DHAV-1-X isolated and preserved in the laboratory after 3 passages of blind passage of DEF, and obvious and stable cytopathic effects of (CPE). Appeared after the 8th generation. A series of methods such as RT-PCR, indirect immunofluorescence and duck embryo inoculation test were used to prove that DHAV-1-X could proliferate well in DEF cells. The rule of proliferation was detected by fluorescence quantitative RT-PCR. The results showed that the virus content began to increase at 12 h after exposure and reached its peak at 48 h. 48h-96h remained at a certain level, and the virus content tended to be stable. 2, The full gene sequence (VP0-F) and partial sequence (VP0-P) of DHAV-1-X structural protein VP0 were cloned into expression vector pET-32a () and pGEX-4T-1, respectively. The prokaryotic expression plasmids pET-32a () / VPO-F and pGEX-4T-1/VP0-P were constructed and transformed into E. coli BL21. The full sequence and partial sequence proteins of VP0 were successfully expressed. The recombinant proteins with high purity were obtained by affinity chromatography and gelatinization respectively, and could be recognized by rabbit anti-DHAV-1 serum. 3. Healthy rabbits were immunized with purified VP0-P protein. Rabbit anti-VP0-P high immuno-serum was prepared, and the titer of serum was up to 1? 16 by Agar diffusion test. The neutralization titer of rabbit anti-VP0-P high immune serum was 1? 146 by chicken embryo neutralization test. Indirect immunofluorescence assay showed that the rabbit anti-VP0-P high immune serum could recognize the purified recombinant proteins VP0-F and VP0-P as the coated antigen and establish the indirect ELISA method based on VP0-F and VP0-P proteins respectively. The optimal conditions of indirect ELISA method based on VP0-F protein were: 1. 67 渭 g / ml VP0-F recombinant protein was incubated at 37 鈩

本文編號：2443061