摘要
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。
Objective: To generate a single-chain bispecific antibody (scBsAb) that could retarget human cytotoxic T lymphocytes to destroy human ovarian carcinoma cells. Methods: First, the heavy chain and light chain of anti-CD3 ScFv, and recombined anti-CD3 ScFv were cloned. Then the scBsAb was constructed by genetic engineering: The ScFvs of anti-human ovarian carcinoma COC183B2 and anti-CD3 were linked by an interlinker. The scBsAb was cloned in the expression vector pTMFC, with the comparison of COC183B2 or anti-CD3 ScFv. Finally, the activity of the scBsAb was detected by ELISA, flow cytometry and resetting assay. Results: The construction of a single-chain bispecific antibody COC183B2/anti-CD3 was successful. And the molecular weight of the scBsAb protein was about 60. The results showed that protein was capable of binding to antigen OC183B2 as detected by ELISA, to human CD3 by flow cytometry, and it had the ability to redirect the affected cells to the target tumor cells in vitro. Conclusion: A single-chain bispecific antibody COC183B2/anti-CD3 is constructed and expressed successfully, which can keep approximately the antigen-binding affinity of the two parent ScFvs.
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2002年第4期358-361,共4页
Journal of Peking University:Health Sciences
基金
国家自然科学基金 ( 3980 0 174)资助~~
