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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。
OBJECTIVE: To screen specific human anti-Mycobacterium anti-Mycobacterium antibody against natural large-scale phage antibody library. Methods: Immunomagnetic tubes were coated with Acr protein of M. tuberculosis. The phage antibody library was screened for specific anti-Mycobacterium tuberculosis Acr by large-capacity antibody library by 4 rounds of “adsorption-eluting-amplification” process Protein antibodies, and variable region sequences were sequenced. The specific bacteriophage antibody was infected into HB2151 strain and induced by IPTG to prepare soluble single chain antibody against Mycobacterium tuberculosis Acr protein. The sequence and antigen binding activity of HBsAg were analyzed and identified. Results: After four rounds of screening, 43 positive clones that bind to Mycobacterium tuberculosis Acr protein were obtained, of which 29 were specific binding clones. There were 26 different variable region fragments in sequencing analysis. By using soluble single chain antibody (scFv) Fourteen strains of soluble single chain antibody specific for Acr protein were screened and identified. Subgroups of variable region genes were analyzed by gene sequencing. Soluble single chain antibody was successfully prepared. Westren blotting analysis confirmed that the screened human scFv was able to bind to native protein. Conclusion: The single-chain high-capacity antibody library can be used to obtain the phage antibody of Mycobacterium tuberculosis Acr protein and the successful preparation of soluble single chain antibody against Mycobacterium tuberculosis Acr natural protein, which will lay the foundation for future research and application.