1型鸭甲肝病毒在鸭胚成纤维细胞中的增殖特性及病毒VP0蛋白免疫原性的初步研究
发布时间：2019-03-18 17:26
【摘要】：1型鸭甲肝病毒(duck hepatitis A virus type 1, DHAV-1)主要引起4周龄以内的雏鸭发病,死亡率高达90%以上,给养鸭业带来严重的损失。为了建立DHAV-1的体外培养模型,探究病毒结构蛋白VP0的免疫原性,本研究对DHAV-1在鸭胚成纤维细胞(DEF)中的适应特性及病毒结构蛋白VP0进行了一系列的研究,获得如下结果。1、用实验室分离保存的野毒株DHAV-1-X的尿囊液接种DEF,盲传3代后DEF细胞出现轻微病变,第8代后出现明显、稳定的细胞病变(CPE)。采用RT-PCR、间接免疫荧光、鸭胚接种试验等一系列的方法证明DHAV-1-X能够在DEF细胞内良好增殖。荧光定量RT-PCR检测增殖规律,结果表明,病毒含量在接毒后12h开始升高,48h达到高峰；48h-96h维持在一定的水平,病毒的含量基本趋于稳定。2、将DHAV-1-X结构蛋白VP0全基因序列(VP0-F)和部分序列(VP0-P)分别克隆到表达载体pET-32a (+)和pGEX-4T-1中,构建原核表达质粒pET-32a (+)/VPO-F和pGEX-4T-1/VP0-P并转入大肠杆菌BL21中,成功表达出VP0全序列和部分序列蛋白。两种重组蛋白分别用亲和层析和切胶回收的方法获得纯度较高的重组蛋白,且都能被兔抗DHAV-1血清识别。3、用纯化的VP0-P蛋白免疫健康的家兔,制备兔抗VP0-P高免血清,琼脂扩散实验检测血清效价达到1：16；鸡胚中和试验检测兔抗VP0-P高免血清的中和效价达到1：146；间接免疫荧光试验表明兔抗VP0-P高免血清能识别DHAV-1.4、将纯化的重组蛋白VP0-F和VP0-P作为包被抗原,分别建立基于VP0-F和VP0-P蛋白的间接ELISA方法。基于VP0-F蛋白的间接ELISA方法的最佳条件为：以1.67μg/ml的VP0-F重组蛋白37℃孵育1h后4℃包被过夜；5%明胶封闭0.5h；被检血清1：160稀释,37℃孵育1h；HRP标记的羊抗鸭IgG进行1：400稀释,37℃孵育1h；TMB避光显色10min，，测定OD450/OD630值,阳性阈值为0.375。基于VP0-P蛋白的间接ELISA方法的最佳条件为：以1.67μg/ml的VP0-P重组蛋白37℃孵育2h包被；5%明胶封闭2h；被检血清1：80稀释,37℃孵育1h; HRP标记的羊抗鸭IgG进行1：400稀释,37℃孵育1h;TMB避光显色5min,测定OD450/OD630值,阳性阈值为0.317。两种ELISA方法都具有较强的特异性和重复性,不与禽流感病毒、鸭沙门氏菌、鸭疫里氏杆菌、鸭瘟病毒、肿头败血症病毒、鸭大肠杆菌以及鸭沙门氏菌的阳性血清发生交叉反应,与以DHAV-1作为包被抗原的ELISA方法的符合率分别为90%和93.3%。5、用VP0-F和VP0-P蛋白免疫1日龄雏鸭,不同时间点采血,用于检测抗体水平和CD4、CD8、IL-4、IFN-γ的水平变化。数据分析表明,雏鸭免疫后15d抗体水平达到高峰,之后有不同程度的下降,VP0-F蛋白免疫组的抗体水平明显高于VP0-P蛋白免疫组；CD4、CD8、IL-4、IFN-γ的含量在免疫后7d达到最大值,VP0-F免疫组细胞因子含量的变化较为显著,而VP0-P免疫组细胞因子的含量虽然也有变化蛋白但是明显低于VP0-F免疫组,与对照组显著性不显著。
[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
本文编号：2443061
[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
本文编号：2443061
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