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从分泌鼠抗人CD86单克隆抗体的杂交瘤细胞株(克隆号:1D1)中抽提总RNA,采用简并引物,经RT-PCR扩增单抗VH和VL的DNA编码区基因。通过SMART-PCR扩增VH和VL基因相应的信号肽序列。采用基因重组技术,将单抗VH、VL基因及其相应信号肽序列,与人IgG1的CH基因、Cκ链基因进行拼接,构建人-鼠嵌合抗体基因的表达质粒pIRES/1D1。转染293T细胞进行瞬时表达,采用流式细胞术分析,转染上清与L929-CD86基因转染细胞的阳性结合率为97.6%。既而转染CHO细胞,经G418筛选,获取稳定分泌嵌合抗体的基因转染细胞株(CHO-ch1D1)。收集无血清培养基的培养上清,采用Protein G亲和层析柱分离纯化,经Lowr法定量。从上清中获取的嵌合抗体蛋白的得率为3.066 mg/L。经进一步间接免疫荧光及流式细胞术分析,纯化嵌合抗体与L929-CD86基因转染细胞,及Daudi细胞膜型CD86分子的阳性结合率分别为98.5%和95.7%。将嵌合抗体(终浓度为5μg/ml)加入到Daudi细胞的培养体系中,经显微镜观察及MTT法分析,嵌合抗体对Daudi细胞的生长具有抑制作用(P<0.05)。本研究获取的嵌合抗体在CD86分子的基础和应用研究中具有重要的价值。
Total RNA was extracted from a hybridoma cell line secreting murine anti-human CD86 monoclonal antibody (clone number: 1D1), and degenerate primers were used to amplify DNA coding region genes of monoclonal antibodies VH and VL by RT-PCR. The signal peptide sequences corresponding to the VH and VL genes were amplified by SMART-PCR. The recombinant plasmid pIRES / 1D1 was constructed by the gene recombination technique. The monoclonal antibody VH, VL gene and its corresponding signal peptide sequence were cloned with the CH gene and C kappa chain of human IgG1. 293T cells were transiently transfected, and the positive binding rate of transfected cells with L929-CD86 gene transfected cells was 97.6% by flow cytometry. The CHO cells were transfected with CHO cells and screened by G418 to obtain the CHO-ch1D1 cell line stably secreting the chimeric antibody. The culture supernatant of the serum-free medium was collected and purified by Protein G affinity chromatography and quantified by the Lowr method. The yield of chimeric antibody protein obtained from the supernatant was 3.066 mg / L. After further indirect immunofluorescence and flow cytometry analysis, the positive binding rates of purified chimeric antibody to L929-CD86 gene transfected cells and Daudi cell membrane type CD86 molecules were 98.5% and 95.7%, respectively. The chimeric antibody was added into the culture system of Daudi cells at a final concentration of 5μg / ml. The chimeric antibody had an inhibitory effect on the growth of Daudi cells (P <0.05) by microscope and MTT assay. The chimeric antibodies obtained in this study are of great value in the study of the basis and application of CD86 molecules.