人源抗戊型肝炎病毒单克隆抗体的获得与鉴定  

Screening and identification of a human monoclonal antibody to ORF2 protein of hepatitis E virus

作　　者：张建琼[1] 魏华[1] 鲁晓萱[1] 鲍海逊[1] 孟继鸿[2] 谢维[1] 

机构地区：[1]东南大学医学院遗传学研究中心 [2]东南大学医学院微生物与免疫学教研室

出　　处：《中华微生物学和免疫学杂志》2004年第3期198-201,共4页Chinese Journal of Microbiology and Immunology

基　　金：江苏省科委自然科学基金资助项目 (BK2 0 0 0 2 2 )

摘　　要：目的　获得人源中和性抗戊型肝炎病毒 (HEV)单克隆基因工程抗体。方法　采用五轮筛选法 (逐轮降低抗原包被量 ,严格洗脱条件 ) ,以固相化的 4种含中和抗原表位的HEV代表株ORF2重组混合抗原淘筛本室构建的HEV患者外周血源的噬菌体抗体库。获得有特异结合活性的噬菌体抗体 ,酶切鉴定抗体基因插入。切去噬菌粒上衣壳蛋白gⅢ基因构建表达载体 ,在大肠杆菌中可溶性表达抗HEV抗体Fab段 ,ELISA、Westernblot检测抗体免疫学活性 ;测序分析抗体基因。结果　筛选出 4株与含中和抗原表位的HEV代表株ORF2重组混合抗原特异结合而与BSA、HBsAg无交叉结合反应的噬菌体抗体 ,可能为中和抗体。切去gⅢ基因 ,在大肠杆菌中表达可溶性抗体Fab段 ,表达量在细菌培养液上清和细菌裂解液中差异无显著性。可溶性抗体与含中和抗原表位的HEVORF2重组混合抗原有特异结合活性 ,与BSA、HBsAg无交叉结合反应。Westernblot显示在相对分子质量 (Mr)略大于 4 5× 10 3 处有一明显条带 ,与Mr4 8× 10 3 的Fab大小一致。DNA测序分析表明 ,Fab段VH 属于IgGVH3基因家族 ,VL 属于Vκ1基因家族。结论　利用噬菌体抗体库技术成功获得抗HEV的人源单克隆抗体。Objective To screen and identify human neutralizing antibody to hepatitis E virus ORF2 protein. Methods Phage display technique was employed in the screening and identification of human antibody to HEV by using the recombinant HEV ORF2 protein as coated antigen. Results and Conclusion After 5 rounds of biopanning of Fab antibody phage library, 4 phage clones were selected from 30 clones according to highest A value in the ELISA identification. The HEV ORF2 protein binding phage particles were treated by a 30 fold concentration. Expression vector was successfully constructed and transformed into E.coli XL1 Blue to produce anti HEV soluble antibody Fab. Soluble antibody has high A value in ELISA and meagle cross binding to the BSA and HBsAg . Western blotting using rabbit anti human IgG(H+L) HRP demonstrated the size of the protein as 48kD. DNA sequence data showed that V H was a very immige of human V H3 family of gene line segment and V L has the most identity with the human Vκ1 family.

关 键 词：戊型肝炎病毒 单克隆抗体 鉴定 免疫学活性 外周血 

分 类 号：R392[医药卫生—免疫学]