摘要
目的构建人源抗狂犬病病毒单链抗体(Single-chain fragment variable,scFv)噬菌体抗体库,并进行筛选。方法以接种过狂犬病疫苗的21份高效价的健康人外周血为原料,提取淋巴细胞总RNA,PCR扩增VH、Vκ、Vλ基因,重叠延伸PCR扩增获得scFv基因,克隆入噬菌粒pS100,电转化E.coli TG1,建立scFv初级抗体库,并进行基因序列分析及鉴定。以灭活的狂犬病病毒为抗原,进行3轮scFv噬菌体抗体库的富集筛选,随机挑取单克隆,制备噬菌体抗体颗粒,并进行ELISA分析,阳性克隆进行序列分析,采用快速荧光灶抑制试验(Rapid fluorescent facus inhibition test,RFFIT)检测噬菌体抗体颗粒的中和活性。结果获得了库容为1.7×108的人源抗狂犬病病毒scFv噬菌体抗体库,该抗体库具有较好的序列正确率和多样性,经3轮筛选后,ELISA分析显示,共获得24个序列各不相同的针对狂犬病病毒的阳性克隆,对其中7个克隆的噬菌体抗体颗粒进行RFFIT检测,均具有中和活性。结论已成功构建了人源抗狂犬病病毒scFv噬菌体抗体库,并建立了筛选方法。
Objective To construct a human anti-rabies virus scFv phage antibody library and develop a method for screening.Methods Total RNAs of lymphocytes were extracted from 21 high titer peripheral blood samples of healthy donors immunized with rabies vaccine,with which VH、Vκ and Vλ genes were amplified by PCR and scFv gene by overlap and extension PCR and cloned into phagemid pS100.Primary antibody library were established by transformation of E.coli TG1 with the recombinant phagemid by electroporation,then sequenced and identified.Three rounds of enrichment and screening were performed using inactivated rabies virus as antigen.Single clones were randomly selected,prepared into phage antibody particles and analyzed by ELISA.The positive clones were sequenced and determined for neutralizing activity of phage antibody particles by rapid fluorescent focus inhibition test（RFFIT）.Results A human anti-rabies virus scFv phage display library with a capacity of 1.7 × 108 individual clones were successfully constructed,which showed high correct rate of sequence and high diversity.After three rounds of screening,ELISA showed that 24 positive clones with different sequences were obtained,of which 7 were determined by RFFIT and showed neutralizing activity.Conclusion A human anti-rabies virus scFv phage antibody library was successfully constructed,and a method for screening was developed.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第12期1578-1582,共5页
Chinese Journal of Biologicals
