摘要
为构建欧洲鳗鲡噬菌体天然单链抗体(scFv)库,筛选鳗鲡病原菌特异性scFv,本研究采用PCR扩增健康欧洲鳗鲡重链可变区(VH)与轻链可变区(VL)基因,通过重叠延伸PCR(SOE-PCR)技术扩增完整的scFv基因(VH-Linker-VL)序列,与T7噬菌体载体连接,经体外包装后接种于宿主菌BLT5403,增殖后经PCR扩增获得噬菌体scFv库。进而以嗜水气单胞菌重组外膜蛋白为抗原,对噬菌体scFv库进行4轮生物淘洗,采用阻断ELISA选取抗体活性最高的scFv,构建pGEX-4T-2-OMP-scFv重组表达载体,转化大肠杆菌BL21进行原核表达。结果显示:本研究得到了完整的scFv基因序列,并以此构建的噬菌体scFv原始库容为1.26×10~6pfu,经扩增后初级库滴度为2.8×10~9pfu/mL;淘洗获得的嗜水气单胞菌重组外膜蛋白scFv抑制率最高达到81.7%,并实现了该scFv在大肠杆菌中的表达,SDS-PAGE检测其蛋白分子量约为53 ku。本研究构建的欧洲鳗鲡噬菌体天然scFv库及淘选获得的特异性嗜水气单胞菌scFv,为养殖鳗鲡病原菌的检测与免疫防治奠定了基础。
To construct phage display single-chain antibody variable fragment(sc Fv)library for healthy Anguilla Anguilla and screen its specific sc Fv against pathogen,this study amplified the heavy chains variable region(VH)and light chains variable region(VL)genes by PCR,and amplified the full-length sc Fv genes by SOE-PCR.Then the sc Fv gene library was ligated to the T7 phage vector,and the T7-sc Fv phage library was obtained by amplification in the host strain BLT5403 after packaging in vitro.The phage antibody library was subjected to 4 rounds of biopanning using the recombinant outer membrane protein of Aeromonas hydrophila as antigen,and the sc Fv with the highest activity was selected by ELISA blocking test.The recombinant expression vector p GEX-4 T-2-OMP-sc Fv was constructed and transformed into E.coli BL21 for prokaryotic expression.The results showed that the full-length sc Fv gene was successfully obtained.The original antibody storage capacity was 1.26×10~6 pfu,and the primary librarytiter after amplification was 2.8×10~9 pfu/m L.After screening,the highest inhibition rate of anti-recombinant OMP sc Fv against Aeromonas hydrophila was 81.7%.The SCFV was successfully expressed in E.coli,and the molecular weight of recombinant sc Fv was about 53 ku identified with SDS-PAGE.In conclusion,the phage display sc Fv library for healthy Anguilla Anguilla was constructed and the specific sc Fv against Aeromonas hydrophila was obtained through biopanning.These results laid the foundation for the detection and immunotherapy of pathogens in eel aquaculture.
作者
徐元凯
林鹏
王艺磊
彭欣慰
冯建军
XU Yuan-kai;LIN Peng;WANG Yi-lei;PENG Xin-wei;FENG Jian-jun(Fisheries College,Jimei University,Engineering Research Centre of Eel Modern Technical Industry,Ministry of Education,Key Laboratory of Healthy Mariculture for the East China Sea,Ministry of Agriculture,Xiamen 361021,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2020年第4期401-406,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
福建省自然科学基金项目(2017J01637)
鳗鲡现代产业技术教育部工程研究中心开放基金(RE201905)
集美大学自然科学基金(ZP2020017)
关键词
欧洲鳗鲡
T7噬菌体
单链抗体库
外膜蛋白
原核表达
Anguilla anguilla
T7 phage
scFv library
outer membrane protein(OMP)
prokaryotic expression
