内部核糖体结合位点介导的三顺反子抗人肿瘤坏死因子-α单克隆抗体表达质粒的构建及在CHO细胞中的表达

Construction of internal ribosome entry site-mediated tricistronic vector for expression of monoclonal antibody against human tumor necrosis factor-α in CHO cells

目的构建内部核糖体结合位点(internal ribosome entry site,IRES)介导的三顺反子抗人肿瘤坏死因子-α(human tumor necrosis factor-α,hTNF-α)单克隆抗体的表达质粒,并在CHO细胞中进行表达。方法采用重叠延伸PCR(gene splicing by overlap extension PCR,SOE-PCR)技术扩增hTNF-α抗体的轻、重链基因,并与IRES基因连接,将连接产物LC-IRES-HC基因克隆至pOptiVEC^(TM)-TOPO~?TA载体,构建单表达载体pOptiVEC-LIH。将单表达载体pOptiVEC-LIH及共转染载体pOptiVEC-HC和pcDNA3.3-LC分别电转至CHODG44细胞,经氨甲喋呤(methotrexate,MTX)加压筛选稳定转染细胞,有限稀释法筛选高表达单克隆细胞株,并采用3 L反应罐对高表达株进行补料批次发酵培养。结果经测序鉴定,单表达载体pOptiVEC-LIH构建正确。瞬时转染后,单表达载体pOptiVEC-LIH转染组的抗体表达量明显高于共转染载体p Opti VEC-HC和pcDNA3.3-LC(P<0.05);经稳定转染有限稀释筛选后,共获得132个克隆,其中A值>2.0的克隆有18株,应用3 L反应器补料批次发酵培养,其表达量可达250 mg/L。结论成功构建了三顺反子抗hTNF-α单克隆抗体的表达质粒,并在CHO细胞中进行了表达。

Objective To construct the internal ribosome entry site（IRES）-mediated tricistronic vector for expression of monoclonal antibody（McAb） against human tumor necrosis factor（hTNF-α） and express in CHO cells. Methods The light and heavy chain genes of hTNF-α antibody were amplified by gene splicing by overlap extension PCR（SOE PCR）and ligated to IRES gene. The obtained LC-IRES-HC gene was cloned into vector pOptiVEC^（TM）-TOPO~？ TA vector. The constructed recombinant plasmid pOptiVEC-LIH as well as co-transfection vectors pOptiVEC-HC and pcDNA3. 3-LC were transfected to CHODG44 cells by electrotransfection. The stably transfected cells were subjected to pressure screening with methotrexate（MTX）, from which the monoclonal cell lines for high expression were screened by limiting dilution method, and subjected to fed-batch fermentation in 3L bioreactor. Results Sequencing proved that recombinant plasmid pOptiVEC-LTH was constructed correctly. The expression level of antibody in the cells after transient transfection with pOptiVEC-LIH was significantly higher than those with pOptiVEC-HC and pcDNA3. 3-L（P 0. 05）. A total of 132 clones were screened by limited dilution, of which 18 were with A values of more than 2. 0. The expression level of antibody was up to 250 mg/L by fed-batch fermentation in 3 L bioreactor. Conclusion The tricistronic vector for expression of monoclonal antibody against hTNF-α was successfully constructed and expressed in CHO cells.