水痘-带状疱疹病毒糖蛋白N的单克隆抗体制备及初步研究

发布时间：2018-06-25 04:06

本文选题：水痘-带状疱疹病毒 + 糖蛋白N　；参考：《病毒学报》2017年03期

【摘要】：水痘-带状疱疹病毒(VZV)是水痘和带状疱疹的共同致病原,其糖蛋白N(gN)在子代病毒的组装、成熟和出胞过程中发挥重要作用。但是目前关于VZV gN的研究还很少,其蛋白性质和具体分子功能仍不清楚。本研究目的是制备抗VZV gN的单克隆抗体,对该蛋白在VZV感染细胞和人皮肤组织内的表达和分布情况进行初步探究。本研究选择抗原性和亲水性较强的gN区段基因,构建rGST-gN融合蛋白的原核表达质粒,将鉴定正确的重组质粒转化大肠杆菌后用IPTG诱导融合蛋白表达,经亲和层析柱纯化rGST-gN融合蛋白后使用PSP酶切除GST标签,将获得的重组蛋白rgN免疫BALB/c小鼠,使用杂交瘤技术制备其单克隆抗体;共获得24株稳定分泌抗VZV gN单克隆抗体的杂交瘤细胞株,选取其中一株性能最优的12E10进行单克隆抗体生产纯化和抗体鉴定;单克隆抗体12E10为IgG2a亚型,其ELISA效价达到1∶10 000;通过Western blot、免疫荧光及免疫组织化学染色方法鉴定,该抗体能够特异性识别细胞和组织中表达的VZV gN;本研究应用该抗体首次确定了gN在VZV感染细胞中的反式高尔基体定位,并且确定了gN在VZV感染人皮肤组织中的表达。以上工作为今后深入研究VZV gN功能奠定了基础。
[Abstract]:Varicella zoster virus (VZV) is the common cause of varicella and herpes zoster, and its glycoprotein N (gN) plays an important role in the assembly, maturation and exocytosis of the progeny virus. But there are few studies on VZV gN, and its protein properties and specific molecular functions are still not clear. The purpose of this study is to prepare monoclonal anti VZV gN. In this study, the expression and distribution of the protein in the VZV infected cells and human skin tissues were preliminarily explored. This study selected the gN region genes with strong antigenicity and hydrophilicity, and constructed the prokaryotic expression plasmid of the rGST-gN fusion protein. The correct recombinant plasmid was identified and the fusion protein was induced by IPTG to induce the expression of the fusion protein after the transformation of the recombinant plasmid. After purifying rGST-gN fusion protein by chromatography column, the GST tag was excised by PSP enzyme, and the recombinant protein rgN was immunized with the BALB/c mouse, and the monoclonal antibody was prepared by hybridoma technology. 24 hybridoma cell lines that secreted the anti VZV gN monoclonal antibody were obtained, and one of the best 12E10 to produce the monoclonal antibody was produced and purified. The monoclonal antibody 12E10 was IgG2a subtype and its ELISA titer reached 1: 10000. The antibody was identified by Western blot, immunofluorescence and immunohistochemical staining. The antibody could identify the VZV gN expressed in cells and tissues. The anti body of gN in VZV infected cells was first identified in this study. Matrix localization and the expression of gN in VZV infected human skin tissues were determined. The above work laid the foundation for further study of VZV gN function in the future.
【作者单位】：国家传染病诊断试剂与疫苗工程技术研究中心厦门大学生命科学学院;分子疫苗学和分子诊断学国家重点实验室厦门大学公共卫生学院;微生物学与分子遗传学系罗格斯大学新泽西医学院;
【基金】：国家自然科学基金(项目号:81601762),题目:人补体调节蛋白CD59在水痘-带状疱疹病毒免疫逃逸中的作用研究
【分类号】：R392

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