人源天然抗TNF-α ScFv抗体的筛选及性质分析  

Screening and characterizing anti-TNF-α antibody from human naive ScFv phage display library

作　　者：乔玉玲 卢卫嘉 陈继军 秦海艳 王建锋 张超 熊颖 毛晓燕 QIAO Yu-ling;LU Wei-jia;CHEN Ji-jun;QIN Hai-yan;WANG Jian-feng;ZHANG Chao;XIONG Ying;MAO Xiao-Yan(Forth Department of Research, Lanzhou Institute of Biological Products Co., Ltd., Center Jbr Gansu Provincial Vaccine Engineering Research, Lanzhou 730046, Gansu Province, C)

机构地区：[1]兰州生物制品研究所有限责任公司第四研究室甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2018年第2期20-26,共7页Progress In Microbiology and Immunology

基　　金：兰州市重大专项(2017-2-1)

摘　　要：目的以人肿瘤坏死因子(human tumour necrosis factor-α,human TNF-α)为靶抗原,从构建好的人源天然单链抗体(single-chain antibody fragment,ScFv)噬菌体抗体库中筛选对应的ScFv抗体,验证其中和活性后测定动力学常数(kinetic dissociation,KD)。方法以TNF-α梯度稀释后包被免疫管,对人源天然ScFv噬菌体抗体库进行3轮吸附-洗脱-扩增的富集筛选,制备TNF-α单克隆噬菌体抗体颗粒,经ELISA试验筛选阳性克隆后测序并分析;将正确的阳性抗体序列亚克隆到pET-26b表达载体上,原核表达后用Ni Sepharose 6 Fast Flow介质进行纯化;用MTT比色法对筛选的ScFv进行中和活性验证,并测定其中和抗体的KD。结果通过3轮筛选共得到18个正确的抗体氨基酸序列,对其原核表达及纯化后得到的6株可溶性表达的抗TNF-αScFv进行中和活性验证,并对其中有中和活性的4株ScFv抗体测定KD。结论从人源天然ScFv噬菌体抗体库中成功地筛选出4株抗人TNF-α的ScFv中和抗体,其中1株ScFv抗体的KD为4.80×10^(-8),细胞毒中和率达到48.9%,达到了药用级抗人TNF-α中和抗体的研发要求,为全分子抗体药物的构建及稳定细胞株的筛选奠定了一定的基础。Objective To select anti- TNF-α ScFv antibody from human naive phage display library by using human TNF- a as a target antigen, the KD of ScFv antibody was determined after the related neutralization activity had been verified. Methods The human naive ScFv phage display library was planned for three screening rounds of absorbing-elution-ampli- fieation with human TNF-α which was in gradient dilutions. The anti-TNF-α monoclonal phage antibodies were obtained and analyzed by ELISA. The correct sequences of positive clones were obtained and subcloned into pET-26b. His-TNF-α fusion proteins were expressed in E. coli and purified by Ni Sepharose 6 Fast Flow media. Theirs neutralization activities were test- ed by MTT staining and KDs were determined by Biacore. Results A total of 18 SeFvs were obtained through three rounds of screening, of which amino acid sequences had been validated. Thus, 6 SeFvs were purifed by soluble prokaryotie expres- sion and theirs neutralization activities were verified. The KDs of 4 neutralizing ScFvs were determined. Conlusion A total of 4 neutralizing SeFvs against human TNF-α were obtained from human nffive SeFv phage display library. One of the ScFv antibodies, KD was 4.80×10^-8, while the neutralization activity was 48.9%. It has been achieved the development require- ments of the pharmaceutical grade about anti human TNF-α neutralizing antibody, which laid a foundation for the screening of stable cell lines and the construction of the next full molecular antibody drugs.

关 键 词：噬菌体抗体库 人肿瘤坏死因子Α 筛选 中和活性 亲和力 

分 类 号：R392[医药卫生—免疫学]