抗H3N2亚型犬流感病毒重组犬源化抗体的制备及特性分析  

Preparation and Characterization of Recombinant Caninized Antibody scFv-Fc Against H3N2 Subtype Canine Influenza Virus

作　　者：韩德敏 赵丹 李思宇 刘永杰[1] HAN Demin;ZHAO Dan;LI Siyu;LIU Yongjie(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院,南京210095

出　　处：《病毒学报》2020年第3期484-492,共9页Chinese Journal of Virology

基　　金：国家重点研发计划项目(项目号:2017YFD0501101),题目:动物重大疫病病原广谱性中和抗体技术研究;科技部国际合作专项(项目号:2014DFG32770),题目:犬流感病毒中韩分离株跨种致病机制的合作研究。

摘　　要：由于异源抗体在宠物临床治疗中易引起宿主免疫排斥反应,因此制备基于本动物源的基因工程抗体显示了更大的发展优势和前景。为制备H3N2亚型犬流感病毒(Canine influenza virus,CIV)的重组犬源化抗体scFv-Fc,采集CIV感染犬外周血淋巴细胞提取总RNA,利用PCR技术扩增IgG重链(VH)、轻链(VL)和Fc片段。利用重叠延伸PCR将VH和VL通过弹性肽链linker连接成单链抗体(scFv)片段,并转化大肠杆菌,挑取阳性菌测序。经分析筛选后,将目标scFv与Fc片段链接,构建scFv-Fc重组犬源抗体片段。经诱导表达,获得分子量约为50 kD的scFv和约为89 kD的重组抗体scFv-Fc。抗体活性测定结果表明,scFv能与CIV特异性结合,1 mg/mL scFv的ELISA效价为1∶210,血凝抑制(HI)效价为1∶27,中和抗体效价为1∶20。1 mg/mL scFv-Fc的HI效价为1∶26,ELISA效价1∶25,中和抗体效价为1∶20。本研究成功制备了针对H3N2亚型犬流感病毒的重组犬源scFv-Fc,为该病毒感染的治疗提供了物质基础。Heterologous antibodies are likely to cause immune rejection in the host upon clinical treatment of pets. Hence,genetically engineered antibodies of animal origin show advantages and prospects. To prepare recombinant"caninized"antibody scFv-Fc of the H3N2 subtype of the canine influenza virus(CIV),total RNA was extracted from the CIV-infected peripheral blood lymphocytes of dogs. The heavy chain(VH),light chain(VL)and Fc fragment of immunoglobulin(Ig)G were amplified by polymerase chain reaction(PCR). VH and VL were ligated by an elastic peptide-chain linker using overlap extension PCR,and then transformed into Escherichia coli. Positive colonies were verified by PCR and gene sequencing. After screening,the target scFv was caninized and a scFv-Fc recombinant caninized antibody fragment was constructed. Protein analyses indicated that a single-chain antibody(scFv)of size ~50 kD and a recombinant antibody(scFv-Fc)of size ~89 kD were obtained. An assay to measure antibody activity showed that scFv could bind specifically to the CIV,and that 1 mg/mL of scFv showed an enzyme-linked immunosorbent assay(ELISA) titer of 1∶ 210,hemagglutination inhibition(HI)titer of 1∶27,and neutralizing antibody titer of 1∶20. The HI titer of 1 mg/mL scFv-Fc was 1∶26,the ELISA titer was 1∶25,and the neutralizing antibody titer was 1∶20. Hence,we prepared recombinant scFv-Fc against H3N2 subtype CIV,providing a material basis for treatment of this virus infection.

关 键 词：犬流感病毒 犬 单链抗体scFv 重组抗体scFv-Fc 免疫学活性 

分 类 号：S852[农业科学—基础兽医学]