人源抗创伤弧菌溶血素蛋白单链抗体的筛选及初步鉴定  

Screening and initial identification of human anti-vibrio vulnificus haemolysin ScFv

作　　者：谢学渊[1] 付凯飞[1] 王欲晓[1] 吴成林[1] 周丽君[1] 

机构地区：[1]海军总医院,北京100048

出　　处：《中华航海医学与高气压医学杂志》2016年第2期137-140,共4页Chinese Journal of Nautical Medicine and Hyperbaric Medicine

基　　金：国家自然科学基金项目（31170008,31400107）;全军医药卫生科研基金项目（062007）

摘　　要：目的 从天然单链（scFv）大容量噬菌体抗体库中筛选特异性抗创伤弧菌溶血素蛋白（vibrio vulnificus haemolysin,VVH）的scFv并进行初步鉴定.方法 以表达纯化后的VVH为靶抗原,采用酸洗脱的方法从天然大容量噬菌体抗体库中筛选人源抗VVH噬菌体单链抗体,并将噬菌体抗体的上清梯度稀释后感染HB2151菌,经异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达后获得抗VVH蛋白可溶性单链抗体,ELISA检测其与抗原结合活性,DNA序列测定鉴定抗体序列.结果 经过4轮筛选,共筛选到22株可以与VVH结合的噬菌体阳性克隆,其中8株获得可溶性表达,DNA序列分析表明获得3株可变区基因型不同的单链抗体.结论 利用大容量噬菌体抗体库筛选技术获得抗创伤弧菌溶血素蛋白的可溶性抗体,为创伤弧菌感染的治疗奠定了基础.Objective To screen specific human antibodies against Vibrio vulnificus hemolysin （VVH ） from natural single stranded large capacity phage antibody library and to perform initial identification of the antibodies,for the treatment of vibrio vulnificus infection.Methods The specific antibody against purified VVH was screened from large natural phage antibody library by 4 rounds of ＂ adsorption-elution-amplification＂ screening process.Soluble ScFvs were prepared through infecting E coli.HB2151 and inducing expression with IPTG.The antigen binding activity and DNA sequence of the antibodies against VVH were identified by ELISA and DNA fingerprint analysis.Results After 4 rounds of panning,22 positive clones with specific binding ability to VVH were obtained,of which 8 obtained soluble expressions.DNA sequence analyses showed that the variable region genes of the clones belonged to different subgroups of immunoglobulin gene family.Conclusions The acquirement of human anti-VVH ScFv from large phage antibody library has laid a good foundation for the treatment of vibrio vulnificus infection.

关 键 词：大容量噬菌体抗体库 创伤弧菌 溶血素蛋白 可溶性抗体 

分 类 号：R392[医药卫生—免疫学]