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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。
Construction and expression of anti-human CD80 dimeric antibody (dimeric antibody fragment, diabody), and preliminary study of its biological function. PCR method to obtain light and heavy chain variable region gene, construct expression vector pIRES2-EGFP / diabody. The pIRES2-EGFP / diabody was transfected into Chinese hamster ovary cells (CHO), and G418 was pressurized to screen stable and highly expressed cell lines. The supernatant was expanded and purified by a nickel column. The cells were denatured and denatured by SDS-PAGE Antibody molecules were identified for their bodystrophy. Immunofluorescence and flow cytometry (FCM) were used to analyze the binding activity of the antibody to the membrane-type CD80 molecule and the competitive inhibition with the murine parental antibody 4E5. MTT assay was used to analyze the in vitro proliferation of Raji cells with high expression of CD80 influences. RESULTS: Anti-human CD80-diabody expression vector was successfully constructed and contained sequences encoding signal peptide and Gly4Ser linker. (SA-Ⅲ) was obtained. The yield of purified antibody in the culture supernatant was 50 mg / L and the result of PAGE showed that it was a dimer. Mr was about 53 000 Human CD80-ScFv Mr is about 27,000). The positive binding rates of anti-CD80-diabody to L929-CD80, Raji and Daudi were 96.5%, 98.1% and 93.0%, respectively. The inhibitory rate of this antibody to the binding of mouse parental antibody 4E5 to antigen was 97.11% The inhibition rate reached 31.27%. Compared with single-chain antibodies, affinity activity increased significantly. The recombinant strain of CD80-diabody CHO cells has been successfully established, and the secreted antibodies have good biological activity.