从大容量噬菌体抗体库中筛选抗Acr蛋白人源单链抗体

目的:从天然的大容量噬菌体抗体库中筛选特异的抗结核分枝杆菌晶体蛋白( alpha-crystallin Acr)的人源抗体.方法:以结核分枝杆菌Acr蛋白包被免疫管,通过对噬菌体抗体库进行4轮“吸附-洗脱-扩增”的过程从大容量抗体库中筛选特异性抗结核分枝杆菌Acr蛋白的抗体,并对可变区序列进行了测序分析.将特异性的噬菌体抗体感染HB2151菌,经IPTG诱导表达,制备了抗结核分枝杆菌Acr蛋白的可溶性单链抗体;对其序列和抗原结合活性进行分析鉴定.结果:经过4轮筛选,获得了43个与结核分枝杆菌Acr蛋白结合的阳性克隆,其中29个特异结合的克隆;测序分析有26不同的可变区片段;通过可溶性单链抗体(scFv)表达筛选到14株特异性结合Acr蛋白的可溶性单链抗体克隆;经过基因测序,分析了可变区基因的亚群.成功制备了可溶性单链抗体.Westren blotting分析证实筛选的人源单链抗体能与天然蛋白结合.结论:利用单链大容量抗体库获得抗结核分枝杆菌Acr蛋白的噬菌体抗体并且成功制备抗结核分枝杆菌Acr天然蛋白的可溶性单链抗体,为今后的研究和应用奠定基础.

Screening of Human Anti-α-crystallin Protein of Mycobacterium Tuberculosis scFv from Large Phage Library

Objetive:To obtain the specific human scFv against Mycobacterium tuberculosis Acr protein using phage antibody library technology.Methods: A human large phage antibody library was panned four times with the purified Acr protein.Soluble scFvs were prepared through infection of E.coli HB2151 with the selected phage antibodies clones and induction with IPTG.The antigen binding activity were determined ELISA and Western blotting,and DNA sequences of these clones were analyzed by Sanger sequencing.Results:43 positive clones were obtained after 4 rounds of panning and 29 clones had binding ability to Mycobacterium tuberculosis Acr protein.DNA sequencing of the 29 clones showed 26 different positive clones.14 specific soluble scFvs were obtained after infection in E.coli HB2151.DNA sequence analysis showed that the variable regions of these scFvs belonged to different subgroups.The Western blotting results suggested that the anti-Acr antibodies had good immuno-reactivity with natural Acr protein.Conclusion:Human anti-Acr scFvs are obtained from a large phage antibody library,which will benefit to the application of anti-Acr of Mycobacterium tuberculosis antibody to clinical research.