鴨1型甲肝病毒亞型VP3蛋白間接ELISA方法的建立及其單克隆抗體的制備
發(fā)布時間：2018-08-17 15:37
【摘要】：近些年,DHAV-1出現(xiàn)了新的致病特點,主要表現(xiàn)為胰腺出血和發(fā)黃,不同于經(jīng)典型肝臟出血的特征性病變,其發(fā)病率為42.8%,病死率達77.8%。對2011年從浙江采集的典型樣品進行了病原分離純化鑒定、病毒全基因組序列測定與分析、致病性試驗,確定其病原為鴨1型甲肝病毒,我們又通過鴨胚血清交叉中和試驗測定并分析其與經(jīng)典的DHAV-1的抗原相關(guān)性,結(jié)果表明胰腺炎型與經(jīng)典的DHAV-1的R值為0.62,因此證明胰腺炎型DHAV-1屬于鴨1型甲肝病毒的亞型(DHAV-1a)。本實驗根據(jù)GenBank上已發(fā)表的DHAV-1a基因序列設(shè)計一對特異性引物,以DHAV-1a MPZJ1206株為模板,采用RT-PCR方法擴增VP3基因,將VP3基因克隆至pEASY-Blunt Zero載體上,轉(zhuǎn)化大腸桿菌Trans5α,用BamHⅠ和XhoⅠ雙酶切鑒定及PCR鑒定,篩選出陽性重組克隆質(zhì)粒,并測序。對測序結(jié)果進行分析,發(fā)現(xiàn)DHAV-1a VP3基因與鴨1型甲肝病毒ZJ株(EF382778.1)的核苷酸同源性為94.8%,氨基酸同源型為99.2%;與鴨1型甲肝病毒CE3(EU687756.1)的核苷酸同源性為94.7%,氨基酸同源性為97.9%。將構(gòu)建的DHAV-1a VP3基因陽性重組克隆質(zhì)粒,進行雙酶切,回收VP3基因片段,與pET-32a(+)表達載體連接,重組成pET-32a-VP3,轉(zhuǎn)化E.coli BL21(DE3)感受態(tài)細胞,用濃度為1.0 mmol/L IPTG于37℃誘導(dǎo)表達,經(jīng)SDS-PAGE分析表明VP3蛋白于大腸桿菌中成功表達,融合蛋白分子量約47 kDa。經(jīng)融合蛋白的可溶性分析知VP3蛋白以包涵體的形式存在。使用GE health公司生產(chǎn)的His Trap HP純化柱純化VP3蛋白。Western-blot分析知純化的VP3蛋白能被鴨1型甲肝病毒亞型的陽性高免血清所識別,表明鴨1型甲肝病毒亞型的VP3蛋白具有良好的反應(yīng)原性。以純化的VP3蛋白作為抗原包被ELISA板,建立檢測鴨1型甲肝病毒亞型抗體的間接ELISA診斷方法。實驗結(jié)果表明:以VP3蛋白為抗原的最佳包被濃度為1:800(蛋白濃度1.5 ug/mL),血清最佳稀釋度為1:160,最佳血清反應(yīng)時間為90 min,HRP標(biāo)記的羊抗鴨酶標(biāo)二抗最佳稀釋濃度為1:4000,HRP標(biāo)記的羊抗鴨酶標(biāo)二抗最佳反應(yīng)時間為90 min,最佳顯色時間為20 min。經(jīng)特異性試驗、重復(fù)性試驗知所建立的ELISA檢測方法具有特異性強、重復(fù)性良好的特點。利用所建立的ELISA檢測方法對84份鴨血清進行檢測,陽性檢出率為32.14%,與以純化濃縮鴨1型甲肝病毒為包被抗原的間接ELISA方法相比,兩者的符合率為87.1%。以純化的DHAV-1a VP3蛋白為抗原,免疫6只Blab/c雌性小鼠,用于細胞融合。細胞融合后,用VP3蛋白所建立的間接ELISA方法篩選,再進行4次亞克隆后篩選到2株陽性雜交瘤細胞,命名為1C9和2C9,亞類鑒定均為IgG1(κ),腹水效價分別為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 偽 was transformed into E. coli. The recombinant plasmid was identified by BamH 鈪，
本文編號：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 偽 was transformed into E. coli. The recombinant plasmid was identified by BamH 鈪，
本文編號：2188097
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