鸭1型甲肝病毒亚型VP3蛋白间接ELISA方法的建立及其单克隆抗体的制备
发布时间：2018-08-17 15:37
【摘要】：近些年,DHAV-1出现了新的致病特点,主要表现为胰腺出血和发黄,不同于经典型肝脏出血的特征性病变,其发病率为42.8%,病死率达77.8%。对2011年从浙江采集的典型样品进行了病原分离纯化鉴定、病毒全基因组序列测定与分析、致病性试验,确定其病原为鸭1型甲肝病毒,我们又通过鸭胚血清交叉中和试验测定并分析其与经典的DHAV-1的抗原相关性,结果表明胰腺炎型与经典的DHAV-1的R值为0.62,因此证明胰腺炎型DHAV-1属于鸭1型甲肝病毒的亚型(DHAV-1a)。本实验根据GenBank上已发表的DHAV-1a基因序列设计一对特异性引物,以DHAV-1a MPZJ1206株为模板,采用RT-PCR方法扩增VP3基因,将VP3基因克隆至pEASY-Blunt Zero载体上,转化大肠杆菌Trans5α,用BamHⅠ和XhoⅠ双酶切鉴定及PCR鉴定,筛选出阳性重组克隆质粒,并测序。对测序结果进行分析,发现DHAV-1a VP3基因与鸭1型甲肝病毒ZJ株(EF382778.1)的核苷酸同源性为94.8%,氨基酸同源型为99.2%;与鸭1型甲肝病毒CE3(EU687756.1)的核苷酸同源性为94.7%,氨基酸同源性为97.9%。将构建的DHAV-1a VP3基因阳性重组克隆质粒,进行双酶切,回收VP3基因片段,与pET-32a(+)表达载体连接,重组成pET-32a-VP3,转化E.coli BL21(DE3)感受态细胞,用浓度为1.0 mmol/L IPTG于37℃诱导表达,经SDS-PAGE分析表明VP3蛋白于大肠杆菌中成功表达,融合蛋白分子量约47 kDa。经融合蛋白的可溶性分析知VP3蛋白以包涵体的形式存在。使用GE health公司生产的His Trap HP纯化柱纯化VP3蛋白。Western-blot分析知纯化的VP3蛋白能被鸭1型甲肝病毒亚型的阳性高免血清所识别,表明鸭1型甲肝病毒亚型的VP3蛋白具有良好的反应原性。以纯化的VP3蛋白作为抗原包被ELISA板,建立检测鸭1型甲肝病毒亚型抗体的间接ELISA诊断方法。实验结果表明:以VP3蛋白为抗原的最佳包被浓度为1:800(蛋白浓度1.5 ug/mL),血清最佳稀释度为1:160,最佳血清反应时间为90 min,HRP标记的羊抗鸭酶标二抗最佳稀释浓度为1:4000,HRP标记的羊抗鸭酶标二抗最佳反应时间为90 min,最佳显色时间为20 min。经特异性试验、重复性试验知所建立的ELISA检测方法具有特异性强、重复性良好的特点。利用所建立的ELISA检测方法对84份鸭血清进行检测,阳性检出率为32.14%,与以纯化浓缩鸭1型甲肝病毒为包被抗原的间接ELISA方法相比,两者的符合率为87.1%。以纯化的DHAV-1a VP3蛋白为抗原,免疫6只Blab/c雌性小鼠,用于细胞融合。细胞融合后,用VP3蛋白所建立的间接ELISA方法筛选,再进行4次亚克隆后筛选到2株阳性杂交瘤细胞,命名为1C9和2C9,亚类鉴定均为IgG1(κ),腹水效价分别为1:1024000和1:512000。
[Abstract]:In recent years, a new pathogenicity of DHAV-1 has emerged, which is mainly characterized by pancreatic hemorrhage and yellowing, which is different from the characteristic pathological changes of classical liver hemorrhage. The incidence of DHAV-1 is 42.8, and the mortality is 77.8%. The typical samples collected from Zhejiang Province in 2011 were isolated and purified, the whole genome sequence and pathogenicity of the virus were determined to be duck hepatitis A virus. We also determined and analyzed the correlation between duck embryo serum cross-neutralization test and classical DHAV-1 antigen. The results showed that the R value of pancreatitis type and classical DHAV-1 was 0.62, so it was proved that pancreatitis type DHAV-1 belongs to the subtype of duck type 1 hepatitis A virus (DHAV-1a). A pair of specific primers were designed according to the DHAV-1a gene sequence published on GenBank. The VP3 gene was amplified by RT-PCR using DHAV-1a MPZJ1206 strain as template, and the VP3 gene was cloned into pEASY-Blunt Zero vector. E. coli Trans5 伪 was transformed into E. coli. The recombinant plasmid was identified by BamH 鈪，
本文编号：2188097
[Abstract]:In recent years, a new pathogenicity of DHAV-1 has emerged, which is mainly characterized by pancreatic hemorrhage and yellowing, which is different from the characteristic pathological changes of classical liver hemorrhage. The incidence of DHAV-1 is 42.8, and the mortality is 77.8%. The typical samples collected from Zhejiang Province in 2011 were isolated and purified, the whole genome sequence and pathogenicity of the virus were determined to be duck hepatitis A virus. We also determined and analyzed the correlation between duck embryo serum cross-neutralization test and classical DHAV-1 antigen. The results showed that the R value of pancreatitis type and classical DHAV-1 was 0.62, so it was proved that pancreatitis type DHAV-1 belongs to the subtype of duck type 1 hepatitis A virus (DHAV-1a). A pair of specific primers were designed according to the DHAV-1a gene sequence published on GenBank. The VP3 gene was amplified by RT-PCR using DHAV-1a MPZJ1206 strain as template, and the VP3 gene was cloned into pEASY-Blunt Zero vector. E. coli Trans5 伪 was transformed into E. coli. The recombinant plasmid was identified by BamH 鈪，
本文编号：2188097
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