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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 anti-human CD80 single chain antibody (ScFv) and to study its biological function. Methods: VH and VL genes were cloned by RT-PCR from murine anti-human CD80 mAb-secreting hybridoma cell line (4E5). The L-VH-Linker-VL scFv gene with leader peptide was constructed by overlap extension splicing PCR method and subcloned into pIRES2-EGFP expression vector. Lipofectamine was used to transfect Chinese hamster ovary cells (CHO) filter. Anti-CD80-ScFv was purified and analyzed for its recognition of membrane type CD80. Competitive inhibition assay was used to analyze the binding ability of anti-CD80-ScFv to the corresponding antigen. Inhibition of CD80-mediated costimulation by anti-CD80-ScFv in vitro by MTT assay. Results: The constructed anti-CD80-ScFv gene was 828 bp in length and sequenced to contain signal peptide and linker peptide. The stable expression cell line SA-Ⅱ was obtained experimentally, and the positive binding rate of the culture supernatant to L929-CD80 cells was over 98%. After purification, the yield of antibody was about 15.12 mg / L, which could recognize CD80 molecules naturally expressed in Raji and Daudi cells with the binding rates of 96.6% and 95.0%, respectively. The competitive inhibition rate of anti-CD80-ScFv on murine parental antibody 4E5 binding to antigen reached 98.67%, and blocked the CD80-mediated costimulation signal and inhibited the proliferation of PBMC. The proliferation rate decreased by 43.48%. CONCLUSION: The expression of anti-CD80-ScFv CHO cells (named as SA-Ⅱ) was successfully established, which can specifically recognize CD80 molecules on cell surface and mediate the corresponding biological functions.