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ELIS; A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V; ZV gN功能奠定了基础。Varicella-zoster virus(VZV)is the causative agent for both; varicella(chicken pox)and herpes zoster(shingles).Glycoprotein; N(gN)plays an important role in VZV virion assembly,maturation and; egress.However,properties and the precise molecular functions of VZV gN; are still unclear.This research is to prepare monoclonal; antibodies(mAbs)specific for VZV gN,and study its expression and; localization in VZV-infected cells and human skin; tissues.Firstly,apotentially antigenic and hydrophilic region of VZV gN; was selected to construct its prokaryotic expression plasmid.Then,the; correct recombinant plasmid was transformed into E.coli,and IPTG was; used to induce the expression of the fusion protein rGST-gN.After; purification by affinity chromatography,rGST-gN was cut with PreScission; protease(PSP)to remove GST tag,and the resulting protein rgN was used to; immunize BALB/c mice.MAbs were prepared by hybridoma technique.A total; of 24 hybridoma cell lines secreting mAbs against VZV gN were; obtained,and one of them,12E10,was selected for mAb production and; characterization.The results showed that the Ig subclass of 12E10 was; IgG2 a,and it exhibited an ELISA titer of 1∶10,000.12E10 can be used to; detect expression of VZV gN in cell lines and tissues in Western; blot,immunofluorescence and immunohistochemistry.In addition,our study; for the first time determined the trans-Golgi network(TGN)localization; of gN in VZV-infected cells,and we confirmed the expression of gN in; VZV-infected human skin tissues.Overall,the above work laid the; foundation for further research on the function of VZV gN.国家自然科学基
