摘要
目的:制备人类疱疹病毒8型(HHV-8)包膜糖蛋白K8.1的单克隆抗体,并对其特异性进行鉴定。方法:用异丙基硫代-β-D-半乳糖苷(IPTG)诱导含重组质粒pGEX-6p-1+K8.1的大肠杆菌BL21表达谷胱甘肽-S-转移酶GST/K8.1融合蛋白,将以包涵体形式存在的融合蛋白进行变性、复性、复性后的GlutathioneSepharose4B亲和层析柱纯化。以纯化的GST/K8.1融合蛋白为抗原免疫BALB/c小鼠,常规杂交瘤技术制备单克隆抗体。采用酶联免疫吸附试验(ELISA)筛选分泌K8.1单抗的杂交瘤细胞株。免疫组化染色(IHC)和Westernblot法鉴定单抗的特异性。结果:建立了一株稳定分泌抗K8.1单抗的杂交瘤细胞株,命名为3G10;IHC和Westernblot法显示,3G10株单抗能特异地识别HHV-8K8.1蛋白。结论:成功制备出抗HHV-8包膜糖蛋白K8.1的单克隆抗体。
Objective： To generate monoclonal antibodies against the envelope glycoprotein K8.1 of human herpesvirus 8（HHV-8）, and further identify their specification. Methods： The fusion protein GST/K8.1 was obtained from inclusion bodies expressed by isopropyl-β-D-thiogalactoside（IPTG）-induced pGEX-6p-1 ＋ K8.1 expression plasmid in E.coli using denaturing and refolding techniques. The fusion protein was further purified with glutathione sepharose 4B affinity chromatography and then used to immunize BALB/c mice as the antigen. Furthermore, both hybridization technique and enzyme linked immunosorbent assay （ELISA） were performed to generate cell lines which produce monoclonal antibodies against K8.1 protein. Thereafter, the specificity of monoclonal antibodies was identified by immunohistochemical staining （IHC） and Western blot. Results： The cell line named 3G10 which stably produces monoclonal antibodies against the K8.1 protein was screened successfully by using ELISA assay. Immunohistochemical stain and Western blot analysis demonstrated that the monoclonal antibodies produced by the cell line 3G10 could specifically recognize K8.1 protein of HHV-8. Conclusion： Monochmal antibodies against the envelope glycoprotein K8.1 of HHV-8 was prepared successfully,
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2005年第10期685-688,F0002,共5页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金资助项目(30100160/30271179)
