EB病毒自身融合衣壳抗原的研制和应用

发布时间：2018-05-19 00:41

本文选题：EB病毒 + 病毒衣壳抗原　；参考：《青岛大学》2007年硕士论文

【摘要】： 目的原核表达EB病毒(Epstein-Barr virus,EBV)衣壳蛋白p23-p18融合蛋白,研制EBV特异性衣壳抗原(viralcapsid antigen,VCA)抗体ELISA检测试剂。方法①选择p23编码基因BLRF2(氨基酸1～162)和p18编码基因BFRF3的C端(氨基酸105～176)基因序列,设计特异性引物,以EBV标准株B95-8为模板,采用重叠延伸PCR技术,将两段基因通过多肽接头(Gly_4Ser)_3 DNA序列连接以获得融合基因p23-p18。②融合基因经测序证实序列准确无误后,导入表达载体pThioHis A构建原核表达克隆,转染E.coli BL21后IPTG诱导表达,重组蛋白经镍-敖合物琼脂糖树脂柱亲和层析纯化获得融合抗原,Western blot鉴定其特异性。③用标准阳性血清滴定抗原效价,选择最适浓度包被ELISA反应板,配套商品化的辣根过氧化酶标羊抗人IgM、IgG及其它一系列试剂,反复优化反应条件,组装成EBV特异性VCA IgM和IgG间接ELISA诊断试剂。④选择血清标本进行检测,并与“金”标准即间接免疫荧光(IFA)和NOVATEC公司生产的VCAIgM和IgG ELISA检测试剂盒进行比较,计算该试剂的敏感性、特异性、约登指数、符合率、阳性预测值和阴性预测值等指标,同时检测诊断试剂的重复性和稳定性。结果p23和p18融合基因成功构建并有效表达,Western blot显示融合抗原具有较高的特异性和敏感性。与IFA比较,采用融合衣壳抗原制备的EBVVCAIgG诊断试剂盒的敏感性、特异性、约登指数、符合率、阳性预测值和阴性预测值分别为97.3%、97.4%、0.947、97.3%、99.8%和77.6%;IgM ELISA诊断试剂盒的上述各项指标分别为94.8%、99.5%、0.943、98.7%、97.3%和98.9%。融合抗原ELISA与NOVATEC公司同类产品有较高的符合率(IgG为97.7%,IgM为96.6%),且具有良好的重复性和稳定性。结论采用p23-p18融合蛋白制备的EBV VCA IgG和IgM ELISA检测试剂敏感特异,简便快速,重复性和稳定性好,进一步完善后可用于临床EBV感染的诊断及流行病学调查。
[Abstract]:Objective to express the p23-p18 fusion protein of Epstein-Barr virus (EBV) capsid protein in prokaryotic expression and to prepare a ELISA assay for EBV specific capsid antigens. Methods (1) the sequence of p23 gene BLRF2 (amino acid 1: 162) and p18 gene BFRF3 (amino acid 105h176) were selected. Specific primers were designed. The standard EBV strain B95-8 was used as template, and the overlapping extension PCR technique was used. The fusion gene p23-p18.2 fusion gene was cloned into the expression vector pThioHis A to construct the prokaryotic expression clone after the sequence of the fusion gene p23-p18.2 fusion gene was confirmed by sequencing. The expression of IPTG was induced by transfection of E.coli BL21. The recombinant protein was purified by agarose resin affinity chromatography. The fusion antigen was identified by Western blot. The titrated antigen titrated with standard positive serum was used to determine the specificity of the recombinant protein. The optimal concentration was chosen to cover the ELISA reaction plate. The commercial horseradish peroxidase labeled goat anti-human IgMN IgG and a series of other reagents were repeatedly optimized and assembled into EBV specific VCA IgM and IgG indirect ELISA diagnostic reagent .4 to select serum samples for detection. The sensitivity, specificity, Yorden index, coincidence rate, positive predictive value and negative predictive value of the reagent were calculated by comparing with the "gold" standard (IFA) and the VCAIgM and IgG ELISA test kits produced by NOVATEC Company, and the sensitivity, specificity, Yorden index, coincidence rate, positive predictive value and negative predictive value of the reagent were calculated. The reproducibility and stability of diagnostic reagents were also detected. Results the fusion gene p23 and p18 were successfully constructed and expressed effectively. Western blot showed that the fusion antigen had high specificity and sensitivity. Compared with IFA, the sensitivity, specificity, Yorden index, coincidence rate, positive predictive value and negative predictive value of EBVVCAIgG diagnostic kit prepared by fusion capsid antigen were 99.8% and 99.8%, respectively, and 998.98%, respectively. The fusion antigen ELISA has a high coincidence rate with that of the similar products of NOVATEC company. It has a good reproducibility and stability. Conclusion EBV VCA IgG and IgM ELISA prepared by p23-p18 fusion protein are sensitive and specific, simple, rapid, reproducible and stable, and can be used in the diagnosis and epidemiological investigation of clinical EBV infection.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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