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目的 :构建和表达一种可与卵巢癌细胞和淋巴细胞结合的双特异抗体。方法 :利用PCR分别扩增抗人CD3单链抗体 (singlechainvariablefragment,ScFv)重链可变区 (variableregionoftheheavychain ,VH)和轻链可变区 (variableregionofthelightchain ,VL) ,重组抗人CD3ScFv ,经测序后将其克隆入有链间连接肽基因序列的载体pALM中 ,再将抗人卵巢癌COC183B2ScFv克隆至紧邻链间连接肽前形成COC183B2 /抗CD3单链双特异抗体(single-chainbispecificantibody ,scBsAb)的重组。最后将COC183B2 /抗CD3scBsAb克隆入表达载体 pTMFC中进行表达 ,同时分别表达抗人卵巢癌单抗COC183B2和抗人CD3单抗的ScFv作为对照组。用ELISA、流式细胞学方法和玫瑰花环实验对scBsAb进行免疫学活性测定。结果 :成功构建抗人卵巢癌COC183B2 /抗CD3scBsAb ;其表达的蛋白链相对分子质量约 6 0 ;ELISA结果显示scBsAb可与抗原OC183B2结合 ,流式细胞学结果显示scBsAb可与抗原CD3结合 ,花环实验显示scBsAb在体外可引导效应细胞聚集在靶细胞周围。结论 :构建和表达抗人卵巢癌COC183B2 /抗CD3scBsAb成功 ,且具有与抗原OC183B2、CD3结合的免疫学活性
Objective: To construct and express a bispecific antibody that binds to ovarian cancer cells and lymphocytes. Methods: The anti-human CD3ScFv was reconstructed by PCR using variable region of hepatitis C (VH) and variable region of light chain (VL) of anti-human CD3 single chain antibody (ScFv) Was cloned into the vector pALM containing the interchain peptide gene sequence, and then the anti-human ovarian cancer COC183B2ScFv was cloned to form a recombination of the COC183B2 / anti-CD3 single-chain bispecific antibody (scBsAb) immediately before the interchain linker peptide. Finally, the COC183B2 / anti-CD3scBsAb was cloned into the expression vector pTMFC for expression, and the anti-human ovarian cancer monoclonal antibody COC183B2 and the anti-human CD3 monoclonal antibody were respectively scFv as the control group. The immunological activity of scBsAb was determined by ELISA, flow cytometry and rosette assay. Results: The anti-human ovarian cancer COC183B2 / anti-CD3scBsAb was constructed successfully. The relative molecular mass of the expressed protein was about 60%. ELISA showed that scBsAb could bind to antigen OC183B2. Flow cytometry showed that scBsAb could bind antigen CD3. ScBsAb was shown to direct the recruitment of effector cells around target cells in vitro. CONCLUSION: The construction and expression of anti-human ovarian cancer COC183B2 / anti-CD3scBsAb is successful and has the immunological activity of binding to the antigens OC183B2 and CD3