摘要
目的:构建天然噬菌体单域重链抗体文库,淘选可应用于食品安全检测的单域重链抗体。方法:以未经免疫的健康羊驼(Lama pacos)外周血为起始材料,提取RNA反转录为cDNA,根据重链抗体保守序列设计引物,通过半巢式PCR法扩增获得全套重链抗体可变区编码基因,将其克隆至噬菌粒pHEN1,电转化大肠杆菌TG1得到初级抗体库,辅助噬菌体KM13感染后得到噬菌体展示库。采用固相淘选法分别对3种人工抗原进行淘选。结果:单域重链抗体编码基因得到有效扩增,经10次电转化获得初级文库,命名为SNAL,实际库容量达到1.6×107个独立克隆,菌落PCR鉴定结果表明,克隆效率约为87%,辅助噬菌体救援后得到的展示文库命名为SNA-PDL,滴度达1013CFU/mL。对3种不同人工抗原DON-MBSA、NOR-BSA和AFB1-OVA的淘选均有富集现象。结论:构建了天然噬菌体单域重链抗体文库,文库的多样性较好,可以用于后续淘选。
Objective：To construct a single-domain heavy chain antibody phage display library for food safety detection. Methods：Total RNA was purified from peripheral lymphocytes of two healthy non-immune alpaca （Lama pacos） and directly used for complementary DNA （cDNA） synthesis. The repertoire of VHH encoding DNAs was amplified by semi-nested PCR,and the PCR products were cloned into the phagemid vector pHEN1. Through the electroporation of E. coli TG1,a primary library was established and then rescued by the helper phage KM13 to generate a phage display library. The generated phage display library was used to pan three different artificial antigens by solid phage biopanning. Results：The VHH encoding DNAs was amplified. After electroporating for 10 times,the primary library named as SNAL was generated containing more than 107 independent clones. The cloning efficiency was up to 87%. The titter of the phage display library obtained after the rescue with KM13 named as SNA-PDL was up to 1013 CFU/mL. All antigen screening data exhibited a significant enrichment in phage particles. Conclusion：A naive single-domain antibody phage display library has been successfully constructed. This library exhibits good diversity and can be used to the recovery of binders.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2010年第19期299-303,共5页
Food Science
基金
国家中小企业创新基金项目(09C26213604449)
江西省教育厅青年科学基金项目(GJJ09442)
江西省自然科学基金项目
关键词
单域重链抗体
VHH抗体
噬菌体文库
羊驼
半抗原
single-domain heavy chain antibody
VHHs
phage display library
alpaca
hapten
