人鼠嵌合Fab抗体通用表达载体的构建和抗人肝癌相关抗原HAb18G嵌合Fab抗体的表达

Construction of a universal expression vector for human-mouse chimeric Fab antibody and expression of chimeric Fab antibody against human hepatoma associated antigen HAb18G

目的:构建一个在大肠杆菌中表达人鼠嵌合Fab抗体的通用载体,并用于抗人肝癌相关抗原HAb18G人鼠嵌合Fab抗体的表达.方法:利用特异引物PCR扩增人IgG3的CHl和Cκ基因,分别克隆到表达载体pComb3中,从而构建成人-鼠嵌合Fab抗体的通用表达载体pComb3C.然后将扩增的mAbHAb18的VL和VH基因分别组装到通用表达载体中,酶切去除gⅢ片段后,连接成为HAb18嵌合Fab抗体的分泌型表达载体pComb3C/cFab.转化大肠杆菌后诱导其表达,通过ELISA检测产物的表达和定位.最后,利用亲合层析纯化表达产物,进行SDS-PAGE电泳和Western-blot检测,同时采用ELISA和免疫荧光法检测表达产物的亲和力和特异性.结果:测序及酶切鉴定表明,人IgG3的CHl和Ck基因正确插入到pComb3中.大小分别为324bp和333bp,同时mAbHlAb18的嵌合Fab基因也分别获得正确组装和表达.表达产物的分子质量约为45ku,主要位于周质腔中.另外,ELISA和免疫荧光检测证实,表达产物含有人的抗体片段,并具有与相应抗原特异结合的活性,亲和力约为亲本抗体的10%.结论:成功构建了人鼠嵌合Fab抗体的通用表达载体并制备了抗人肝癌小分子嵌合Fab抗体,为其进一步在肝癌诊断与治疗中的应用奠定了基础.

AIM: To construct a universal expression vector for human-mouse chimeric Fab antibody and to use it for construction and expression of human-mouse chimeric Fab antibody against human hepatoma associated antigen HAb18G. METHODS: Human IgG3 CH1 and Ocgenes were amplified by PCR with specific primers and cloned into vector pComb3 to construct a human-mouse chimeric Fab antibody universal expression vector pComb3C. Then, the VK and VH genes of mAb HAb18 were amplified by PCR using specific primers and cloned into the expression vector pComb3-cFab. After gⅢ was cut away, the secretory expression vector pComb3C/cFab containing chimeric Fab antibody gene of HAb18 was constructed and transfected into competent E.Coli. And then, the antibody induction expression by IPTG was conducted. The locaeization of expression products was detected by sandwich ELISA. Finally, The expression product was purified by affinity chromatography and the antigen-binding specificity and affinity of the expression product were tested by ELISA and immunofluorescence staining. RESULTS: Human IgG3 CH1 and Ck genes were correctly inserted into vector pComb3 by nucleotide sequencing and restriction endonucleases digestion, with the size of 324bp and 333 bp, and chimeric Fab gene of mAb HAb18 was successfully constructed and expressed in E.Coli. The molecular mass of expression product was about 45 ku. It was mainly located in the periplasm. The results of ELISA and immunofluorescence staining showed that the expressed chimeric Fab contained human antibody fragment and had specific antigen-binding activity, and its affinity was about 10% of parental antibody HAb18. CONCLUSION: We have successfully constructed a universal expression vector for human-mouse chimeric Fab antibody. Based on this result, a small molecule of chimeric Fab antibody against human hepatoma is prepared, which lays a foundation for its further application into diagnosis and therapy of human hepatocellular carcinoma.