抗犬细小病毒VP2蛋白单链抗体的制备与中和活性研究

Preparation and Neutralization Activity of Anti-Canine Parvovirus VP2 Protein Single-chain Antibody

为了减轻鼠源单克隆抗体(McAb)异源性引起的宿主免疫排斥反应,同时克服生产McAb成本高及费时费力等缺点,利用原核表达制备具有抗犬细小病毒(CPV)鼠源抗体可变区基因的单链抗体(single chain antibody fragment,ScFv)。提取实验室前期制备筛选的分泌具有良好中和活性抗CPV VP2蛋白McAb的杂交瘤细胞株总RNA,从反转录cDNA中扩增抗体重链可变区(VH)基因和轻链可变区(VL)基因并克隆到表达载体中,构建重组质粒p OPE101-ScFv;将重组质粒转化大肠杆菌进行诱导表达,通过蛋白免疫印迹(Western blot)实验、酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)和间接免疫荧光试验(indirect immunofluorescence assay,IFA)证明,利用大肠杆菌表达系统获得的ScFv具有和CPV特异性结合的能力,且具有中和活性,效价为1:20(0.028μg/ml),为CPV的临床免疫治疗提供基础。

Objective:Single chain fragment variable(ScFv)is the smallest functional structural unit with the antigen-binding specificity of the parent antibody.Due to its affinity and low immunogenicity,ScFv has broad application prospects in medical treatment and diagnosis.To reduce the host immune rejection during clinical treatment with heterologous murine monoclonal antibody(McAb)in canine,ScFv were prepared against canine parvovirus(CPV)using a prokaryotic expression system.Methods:Total RNA was extracted from hybridoma cell lines specific for CPV,amplification of the antibody heavy chain variable region(VH)gene and the light chain variable region(VL)gene from the reverse transcription cDNA into the expression vector pOPE101.The recombinant plasmid was transformed into E.coli for expression,and the expressed protein was identified by Western blot.The activity of the ScFv was detected by ELISA,and the ScFv purified by affinity chromatography was identified by virus neutralization test.Results:The recombinant plasmid p OPE101-ScFv was successfully constructed.The correct expression of single-chain antibody in E.coli was determined by western blot.The ability of the fusion protein to specifically bind to the virus was verified by ELISA and virus neutralization test.The potency was 1∶40(0.014μg/ml).Conclusion:A ScFv with neutralizing activity was obtained using an E.coli expression system,which provides a basis for clinical immunotherapy for CPV disease.