抗人CD80单链抗体的构建、表达及与抗原结合的特性分析  

Construction and expression of anti-CD80-ScFv for feature analysis of its binding with antigen

作　　者：陈昌友[1] 郭静雅[1] 陈永井[1] 陈明心[2] 孙杰[3] 张彦军[1] 黄赛男[1] 朱华亭[1] 邱玉华[1] 

机构地区：[1]苏州大学医学部免疫学系,苏州215123 [2]河南中医学院第一附属医院检验科,郑州450000 [3]中国科学院苏州纳米研究所,苏州215123

出　　处：《中国免疫学杂志》2011年第2期153-157,共5页Chinese Journal of Immunology

基　　金：国家科技重大专项资助项目(2009ZX09103-705)

摘　　要：目的:构建及表达抗人CD80单链抗体(ScFv),并初步研究其生物学功能。方法:采用RT-PCR法从分泌鼠抗人CD80mAb的杂交瘤细胞株(4E5)中克隆VH和VL基因。用重叠延伸拼接PCR方法构建具有前导肽的L-VH-Linker-VL单链抗体基因,并亚克隆至pIRES2-EGFP表达载体,脂质体法转染中华仓鼠卵巢细胞(CHO),G418加压筛选。纯化抗CD80-ScFv,并分析其对膜型CD80的识别。竞争抑制实验分析抗CD80-ScFv与相应抗原的结合能力。MTT法体外分析抗CD80-ScFv对CD80介导的共刺激信号的阻断作用。结果:构建的抗CD80-ScFv基因全长为828 bp,经测序含有信号肽和连接肽。实验获得稳定表达细胞株SA-Ⅱ,其培养上清与L929-CD80细胞的阳性结合率达98%以上。抗体纯化后产率约为15.12 mg/L,其能够识别Raji和Daudi细胞天然表达的CD80分子,结合率分别为96.6%和95.0%。抗CD80-ScFv对鼠源亲本抗体4E5与抗原结合的竞争抑制率达98.67%,并能阻断CD80介导的共刺激信号转导,抑制PBMC的增殖,增殖率下降43.48%。结论:成功建立了抗CD80-ScFv CHO细胞的表达株(命名为SA-Ⅱ),该抗体能够特异识别细胞表面膜型CD80分子并介导相应的生物学功能。Objective：To construct and express the single chain variable fragment（ScFv） of monoclonal antibody against human CD80,in order to study the biological functions of the costimulatory molecule.Methods：The VH and VL genes were cloned by RT-PCR from a murine hybridoma cell line 4E5,which produced the monoclonal antibody（mAb） against human CD80 antigen.Splicing of overlapping extension PCR（SOE-PCR） was used to splice the VH and VL genes to construct CD80-ScFv,which was subcloned into the eukaryotic expressing vector pIRES2-EGFP.CHO cells were transfected by pIRES2-EGFP/ScFv plasmids by the means of liposome-mediated methods and then were selected by G418.Anti-CD80-ScFv was purified from culture supernatant.Then the experiment identification of ScFv binding to membrane CD80 was performed through competitive inhibition assay to analyze the binding ability of anti-CD80-ScFv with the corresponding antigen.The effect of anti-CD80-ScFv on costimulatory signals mediated by CD80 was detected by MTT methods.Results：The ScFv genes consisted of 828 bp and included the signal peptide and linker.The stable cells with highly secretion of ScFv harvested.The cultural supernatant of the cells was 98.0% above in analysis of the combination of the ScFv antibody to L929-CD80 cells.About 15.12 milligrams of CD80-ScFv antibodies were purified from one liter of the culture supernatant,and its positive binding rate with Raji and Daudi was 96.6% and 95.0%,respectively.The competitive inhibition rate of anti-CD80-ScFv from murine parent antibody 4E5 was 98.67%,and anti-CD80-ScFv could make the proliferation rate of PBMC decline by 43.48% via blocking costimulation signal mediated by CD80.Conclusion：Anti-CD80-ScFv has been successfully expressed in CHO cells（names as SA-Ⅱ） and the antibody could identify membrane CD80 molecule in the cell surface and mediate the related biological functions.

关 键 词：CD80 单链抗体 真核表达 

分 类 号：R392.11[医药卫生—免疫学]