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目的分离鼠源抗粘蛋白1(mucin1,MUC1)单克隆抗体可变区基因,构建有结合功能的单链抗体,并初步检测结合活性。方法提取杂交瘤细胞总mRNA,合成cDNA后,PCR扩增鼠特异性抗体可变区编码基因,通过9个氨基酸的连接子构建表达单链抗体的原核重组表达载体,体外诱导表达纯化后,采用Westernblot和流式细胞术方法分析单链抗体与变性或天然状态下抗原的结合活性。结果在杂交瘤细胞中成功分离到抗体轻重链可变区基因,重组链接后形成单链抗体能够在大肠杆菌中分泌表达,Westernblot和流式细胞术分析表明该单链抗体能够与变性或天然状态下的T47D肿瘤细胞表面MUC1特异性结合。结论成功制备具有生物活性的抗肿瘤相关MUC1单链抗体,该单链抗体具有与MUC1特异性结合的生物学活性。
OBJECTIVE: To isolate the variable region gene of murine mucin 1 (MUC1) monoclonal antibody and construct a single chain antibody with binding function and to detect the binding activity. Methods The total mRNA of hybridoma cells was extracted. After the cDNA was synthesized, the variable region of mouse specific antibody was amplified by PCR. The prokaryotic recombinant expression vector expressing the single chain antibody was constructed by 9 amino acid linker. Westernblot and flow cytometry methods were used to analyze the binding activity of the single-chain antibody to the antigen in its denatured or native state. Results The light and heavy chain variable region genes of the antibody were successfully isolated in hybridoma cells. The single chain antibody was expressed secreted in E.coli. Westernblot and flow cytometry analysis showed that the single chain antibody could interact with the degenerate or natural state MUC1 specific binding on T47D tumor cells underneath. Conclusion The biologically active anti-tumor related MUC1 single chain antibody has been successfully prepared and has the biological activity of binding specifically to MUC1.