摘要
构建及表达抗人CD80双价抗体(dimeric antibody fragment,diabody),并初步研究其生物学功能。PCR法获得轻重链可变区基因,构建表达载体pIRES2-EGFP/diabody。将pIRES2-EGFP/diabody转染中华仓鼠卵巢细胞(CHO),G418加压,筛选稳定高表达细胞株,扩大培养并收集上清,经镍柱纯化,并经变性及非变性SDS-PAGE电泳对抗体分子的双体性进行鉴定。采用免疫荧光及流式细胞术(FCM)分析抗体与细胞膜型CD80分子的结合活性及与鼠源性亲本抗体4E5的竞争抑制效应;MTT法分析该抗体对天然高表达CD80的Raji细胞体外增殖的影响。结果:成功构建抗人CD80-diabody表达载体并含有编码信号肽和Gly4Ser连接肽的序列。获得了稳定分泌双价抗体的细胞株(命名为SA-Ⅲ),培养上清中纯化抗体的得率为50 mg/L,PAGE结果显示其为一个二聚体,Mr约为53 000(抗人CD80-ScFv Mr约为27 000)。抗CD80-diabody与L929-CD80、Raji及Daudi的阳性结合率分别为96.5%、98.1%及93.0%;该抗体对鼠源亲本抗体4E5与抗原结合的抑制率为97.11%,对Raji细胞体外增殖的抑制率达31.27%。与单链抗体相比,亲和活性明显提高。成功建立了抗CD80-diabody CHO细胞的表达株,其分泌的抗体具有良好的生物学活性。
To construct and express anti-human CD80 bivalent dimmer（diabody） and to study its biological functions,VH and VL genes were cloned by PCR,and then subcloned into the eukaryotic expression vector pIRES2-EGFP to construct recombinant pIRES2-EGFP/diabody.Chinese hamster ovary（CHO） cells was transfected by this recombinant plasmid through liposome-mediated methods and selected by G418.The anti-human CD80-diabody was purified from culture supernatants by Ni catridges.Reducing and non-reducing SDS-PAGE was used to analyze its bivalent property.The identification of diabody to membrane CD80 and competitive inhibition effect against the parent antibody 4E5 were performed via immunofluorescence and FCM.Meanwhile,the effect of anti-human CD80-diabody on the proliferation of Raji cells expressing CD80 highly and naturally was detected by MTT method.By using these methods,the expression vector for pIRES2-EGFP/diabody against CD80 was constructed successfully.This vector concluded a sequence of 798 bp in length,part of which encoded signal peptide and Gly4Ser linker peptide.One cell line stably secreting diabody was obtained and named as SA-Ⅲ.Appromately,50 milligram of CD80-diabody could be purified from one liter of culture supernatant.The molecular weight（Mr） of this dimer was about 53 000,while Mr of ScFv was about 27 000.The positive binding rates of anti-human CD80-diabody with L929-CD80,Raji and Daudi cells were 96.5%,98.1% and 93% respectively.This diaboby could inhibit not only the combination of the parent antibody 4E5 with CD80 antigen in an inhibition rate of 97.11%,but also the in vitro proliferation of Raji cells with a corresponding rate of 31.27%.Conclusively,a CHO cell line secreting anti-human CD80-diabody was generated and its secreted diabody shows good biological function.
出处
《现代免疫学》
CAS
CSCD
北大核心
2011年第3期192-196,共5页
Current Immunology
基金
国家科技部科技重大专项资助项目(2009ZX09103-705)
