摘要
目的:构建多样性良好的人源天然噬菌体抗体库。方法:从正常人外周血中分离淋巴细胞,以RT-PCR和半巢式PCR扩增重链可变区VH基因和轻链可变区VL基因,以重叠延伸PCR将VH、VL组装成scFv基因,并将其克隆入噬菌粒载体pCANTAB-5E中。以pCANTAB-5E电转化大肠杆菌TG1,构建人源天然噬菌体抗体库,测序分析抗体基因的家族信息和多样性,并用多种抗原对其进行筛选。结果:获得了库容为2×108的人源天然噬菌体抗体库。分别用5种抗原对其进行筛选,均可获得特异性噬菌体抗体的富集。结论:成功地构建了一个多样性良好的人源天然噬菌体抗体库,可用于制备具有应用前景的人源抗体。
AIM： To construct a human natural phage single-chain antibody （scFv） library with diversity. METHODS： VH and VL genes were amplified by RT-PCR and hemi-PCR from peripheral blood lymphocytes of healthy persons. The V genes were assembled to form scFv by overlap PCR and cloned into phagemid pCANTAB-SE, and then transformed into E. coli TGI by electroporation to construct a human natural phage scFv library. The diversity and gene family of antibody gene were analysed by sequencing and the specific antibodies against various antigens were screened through bio-panning. RESULTS： A human natural phage scFv library with diversity and 2 × 10^8 sink size was constructed successfully. The specific human scFvs against 5 antigens were obtained by bio-panning. CONCLUSION： A human natural phage scFv library with diversity is constructed successfully and can be applied to human antibody preparation.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第6期786-789,793,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
福建省科技重大项目(2002F013)
国家"十五"创新药物博士基金资助项目(2003AA2Z3539)
福建省自然科学基金资助项目(C031005)
厦门市科技计划项目(3502Z20055002)
