摘要
目的基于噬菌体抗体库技术制备抗诺如病毒(norovirus,NoV)全人源单链抗体(single-chain antibody fragment,scFv)。方法从GⅡ.6 NoV感染者恢复期外周血中分离得到外周血单核细胞(peripheral blood mononuclear cells,PBMCs),提取总RNA,RT-PCR法分别扩增scFv的重链和轻链可变区基因片段,通过Overlap PCR技术将两者随机拼接成scFv基因片段。将scFv基因克隆至噬菌体质粒pCANTAB-5E,转化至感受态E.coli TG1,获得全人源scFv噬菌体抗体库。以GⅡ.6 P蛋白为固相抗原对抗体库进行4轮生物筛选,以选取的特异性抗NoV阳性噬菌体为模板,扩增scFv基因后,进行原核表达及纯化,并鉴定其特异性及中和性。结果人源噬菌体scFv抗体库库容约为8.8×106PFU/mL。4轮生物筛选共获得10株scFv,均能与GⅡ.4、GⅡ.6和GⅡ.17 NoV P蛋白发生特异性结合,且对3株NoV毒株均有不同程度的阻断作用。结论利用GⅡ.6 NoV感染者恢复期的PBMCs构建了全人源scFv噬菌体抗体库,筛选出的10株抗NoV scFv对NoV具有一定的中和作用。
Objective To prepare fully human single-chain antibody fragment(scFv)against norovirus(NoV)based on phage antibody library technology.Methods Peripheral blood mononuclear cells(PBMCs)of patients infected with GⅡ.6NoV were isolated at the convalescent phase,of which total RNA were extracted.The heavy chain(VH)and light chain(VL)variable region genes of scFv were separately amplified by RT-PCR,and then linked randomly into scFv gene sequences by Overlap PCR.The obtained scFv gene was cloned into phage plasmid pCANTAB-5E and transformed to E.coli TG1 to obtain a fully human scFv phage antibody library which was subjected to four rounds of biopurification using GⅡ.6 P protein as the solid phase antigen.Using the obtained specific anti-NoV positive phage as template,scFv gene was amplified,expressed prokaryotically and purified,and then identified for the specificity and neutrality.Results The scFv antibody library of human phage was about 8.8×10~6PFU/mL.A total of 10 strains of scFv were obtained through four rounds of biopurification,all of which bound specifically to the NoV P proteins GⅡ.4、GⅡ.6 and GⅡ.17,and showed various degrees of blocking effect on the three NoV strains.Conclusion A fully human scFv phage antibody library was constructed by using PBMCs from patients infected with GII.6 NoV at the convalescence phase,from which 10 strains of anti-NoV scFv were obtained and showed neutralization effect on NoV.
作者
周飞园
王璐
余京蓉
梁芷妍
林璧慧
张绪富
戴迎春
ZHOU Fei-yuan;WANG Lu;YU Jing-rong;LIANG Zhi-yan;LIN Bi-hui;ZHANG Xu-fu;DAI Ying-chun(不详;Department of Epidemiology,School of Public Heath,Southern Medical University,Guangzhou 510515,Guangdong Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2022年第12期1458-1464,1476,共8页
Chinese Journal of Biologicals
基金
国家自然科学基金(317710017)
广东省自然科学基金(2019A1515010951)
广东省重点领域研发计划资助(2022B1111020002)。
关键词
诺如病毒
噬菌体抗体库
单链抗体
Norovirus(NoV)
Phage antibody library
Single-chain antibody fragment(scFv)
