摘要
目的在中国仓鼠卵巢(CHO)细胞中表达抗鼠疫耶尔森菌荚膜抗原F1嵌合抗体,并对其活性进行分析。方法将具有保护活性的鼠源单克隆抗体(mAb)的轻、重链可变区基因,分别与人κ型轻链恒定区、IgG1重链恒定区基因融合后构建轻重链嵌合抗体基因,克隆到T载体上进行序列测定确证。然后分别构建含有嵌合轻、重链基因的表达载体pcDNA3.1-L和pcDNA3.1-H。构建完成的表达载体电转化CHO-S细胞,G418筛选获得稳定表达细胞株。扩大培养、收集上清用MabSelect Sure亲和层析填料纯化。纯化后的抗体进行SDS-PAGE、ELISA、Western blot分析以及小鼠体内保护活性评价。结果 PCR鉴定及测序结果表明pcDNA3.1-H和pcDNA3.1-L构建完成,序列正确;Dot blot结果表明获得表达量高的稳转株细胞;SDSPAGE及Western blot法证明抗体纯化获得成功,ELISA结果表明该抗体具有结合与F1抗原的活性;小鼠体内攻击实验表明其保护活性与鼠mAb大致相同。结论成功在CHO-S细胞中表达了具有中和活性的抗F1人-鼠嵌合抗体。
Objective To express human-mouse chimeric antibody against Yersinia pestis F1 capsular antigen （ F1antigen） and analyze its biological activities. Methods The heavy chain gene of the chimeric antibody was obtained byfusing the variable region gene of the mouse mAb heavy chain with human IgG1 constant region gene. The light chain geneof the chimeric antibody was obtained by fusing the variable region gene of the mouse mAb light chain with the human kappaconstant region gene. Both the heavy and light chain genes of the chimeric antibody were further verified by sequencing. Thechimeric antibody heavy and light chain genes were inserted into EcoR I /Not I of pcDNA3.1 （ ＋ ） to construct expressionplasmids termed pcDNA3.1-L and pcDNA3.1-H, respectively. Then, two plasmids were mixed and transfected into CHO-Scells. Finally, the stable cell clone secreting chimeric antibody was obtained by G418 selection. The culture supernatants ofserum-free medium were collected and the chimeric antibody was purified by MabSelect SuRe affinity chromatography. Thepurified chimeric antibody was analyzed by SDS-PAGE, Western blotting, ELISA and evaluated in the protective effect invivo. Results PCR and sequencing analysis proved that plasmids pcDNA3.1-H and pcDNA3.1-L were correctly constructed. Dot blot showed that a cell line with high-level expression of chimeric antibody was obtained. SDS-PAGE and western blotshowed that the chimeric antibody was successfully purified. ELISA showed that the chimeric antibody could specifically bindto F1 antigen. In vivo activity assay showed that 80% BALB/c mice treated with the chimeric antibody survived from 36 MLDvirulent Yersinia pestis. Conclusion The chimeric antibody against F1 antigen with neutralizing activity was successfullyexpressed in CHO-S cells, which laid a foundation for the preparation of anti-plague passive immunity agents.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2013年第12期1299-1302,1306,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81000710)
"重大新药创制"科技重大专项(2013ZX09304103)
