抗Ⅱ型单纯疱疹病毒表面抗原gD单克隆抗体的制备及鉴定  

Preparation and Characterization of Monoclonal Antibodies against Glycoprotein D of HSV-2

作　　者：王耀[1,2] 刘武 周洁[1,2] 蔡冉[2] 潘英[2] 李素芹[2] 张庶民[4] 李越希[1,2] 

机构地区：[1]南京医科大学基础医学院生物化学与分子生物学系,江苏南京210029 [2]南京军区军事医学研究所,江苏南京210002 [3]江苏先声卫科生物制药有限公司,江苏常州213032 [4]中国食品药品检定研究院,北京100050

出　　处：《药物生物技术》2012年第6期495-497,502,共4页Pharmaceutical Biotechnology

基　　金：国家"863"重大课题(No.2006AA02A226;No.SS2012AA020905);江苏省自然基金课题(No.BK2008068);江苏省社会发展课题(No.BE2010603)资助

摘　　要：制备抗Ⅱ型单纯疱疹病毒(Simplex Herpes VirusⅡ,HSV-2)表面抗原gD的单克隆抗体(mAb)并对其性质进行鉴定。以基因工程重组制备纯化的HSV2-gD为抗原免疫BALB/c小鼠,采用常规融合制备杂交瘤细胞,通过HAT选择培养、有限稀释法获得单克隆细胞株,用间接ELISA法、Western blot方法筛选和鉴定阳性杂交瘤细胞株。获得了4株可稳定分泌抗HSV2-gD抗体的杂交瘤细胞株,分别命名为1A11C6、1A11F10、4C9D5以及4C9E9,抗体的类型均为IgG1,轻链均为κ型,Western blot结果显示各单抗都可以特异性识别HSV2-gD。用杂交瘤细胞注射小鼠制备腹水,腹水内的抗体效价均大于1×10-5,腹水内的抗体经硫酸铵-辛酸分级沉淀和阴离子交换柱纯化,成功制备了纯度超过95%的高特异性抗体,抗体有较好的特异性。为进一步研究HSV2感染和临床上的预防、诊断及治疗提供了有力的工具。To prepare monoclonal antibodies against recombinant glycoprotein D of Simplex Herpes Virus Ⅱ（HSV2-gD） and to characterize their properties, BALB/c mice were immunized by injection of recombinant HSV2-gD, and the hybridomas which can produce mAbs were prepared by hybridoma technique. Through PEG fusion and screening by limited dilution, four strains of hybridomas obtained were specific to HSV2-gD and were named as 1 A11C6,1A11F10,4C9D5 and 4C9E9 respectively. All of them have Klight chains and were identified as the immunoglobulin IgG1. The mAbs in ascitic fluid were purified by anion exchange, the SDS-PAGE result showed the purity of purified mAb was over 95%. To identify the characters of the mAbs against HSV2-gD,which include spcificity, affinity and isotypes,indirect enzyme-linked immunosorbent assay （ELISA） and Western Blot were performed. The tilters of mAbs in ascitic fluid were all more than 1 ×10^-5. These obtained high-specificity and high-affinity mAbs could provide an useful reagent for either further study of HSV2 or diagnosis of HSV2 infection.

关 键 词：Ⅱ型单纯疱疹病毒 糖蛋白D 单克隆抗体 酶联免疫法 

分 类 号：Q511[生物学—生物化学]