抗结核杆菌人源噬菌体单链抗体库的构建及筛选

Construction and Screening of Human Single-chain Antibody Library of Anti-Mycobacterium Smegmatis

目的:构建人源噬菌体展示单链抗体(ScFv)库,筛选抗结核杆菌特异性、高亲和力的ScFv.方法:从结核患者的外周血淋巴细胞中,提取RNA并通过RT-PCR扩增出VH和VL基因,并采用SOE-PCR构建ScFv基因,将其克隆入噬粒载pCANTAB5E中,转化于大肠杆菌TG1,通过辅助噬菌体M13K07援救构建噬菌体单链抗体库.结果:初级库库容量为3.5×106,在大肠杆菌TG1中重组后得到2.6×106的次级抗体库.结论:成功构建噬菌体展示ScFv库并获得人源抗结核杆菌特异性ScFv,为进一步研制抗抗结核杆菌的高特异性、高亲和力的基因工程抗体奠定了基础.

Aim： To construct a human phage-displayed single-chain Fv antibody（ScFv） library and screen specific ScFv against Mycobacterium Smegmatis.Methods： The total RNA was extracted from peripheral blood lymphocytes in patients with Mycobacterium Smegmatis,first strand cDNA synthesis was performed using a cDNA synthesis kit and an oligo（dT）-18 primer.To human immunoglobulin H chain and L chain variable region of degenerate primers,cDNA first strand as a template,were amplified H chain and L chain variable region genes.SOE-PCR method to VH and VL fragments were assembled into ScFv fragments and then cloned into pCANTAB5E ScFv vector and transformed into competent TG 1 electric bacteria by helper phage rescue M13K07 get Mycobacterium Smegmatis single chain antibody phage display library.Results： A primary library of 3.5×106 and a second library of 2.6×106 were constructed.Conclusion： The results lay a solid foundation for preparation of human engineering antibody to Mycobacterium Smegmatis reported herein with higher affinity.