人源抗狂犬病病毒单克隆抗体的构建及验证

Construction and validation of humanized anti-rabies virus monoclonal antibodies

目的利用人源抗狂犬病病毒ScFv噬菌体抗体库,构建单克隆抗体(单抗)轻、重链质粒,瞬时表达,纯化后验证具有高中和活性的单抗。方法以从ScFv噬菌体抗体库中筛选获得特异性抗体为模板,PCR扩增抗体轻、重链可变区序列,构建人源全分子抗体瞬时转染质粒;轻、重链质粒混合后转染HEK293 EBNA1细胞,瞬时表达全分子抗体。采用Mabselect SuRe介质手动亲和纯化抗体,Nano Vue超微量分光光度计测定蛋白质的质量浓度,快速荧光灶抑制试验(rapid fluorescent focus inhibition test, RFFIT)进行体外中和效价验证。结果成功构建22个轻链质粒,15个重链质粒。通过真核细胞瞬时表达后,经手动亲和纯化后,获得22株单抗。除1株抗体外,其余21株抗体的蛋白质量浓度均>300μg/mL;部分抗体纯度均在90%以上。中和活性结果显示,5株在1 500~1 800 IU/mg之间;8株在800~1 500 IU/mg之间;7株在500~800 IU/mg之间;其余2株在500 IU/mg以下。结论成功构建并验证获得20株中和活性>500 IU/mg的人源抗狂犬病病毒单抗,为单抗混合制剂的研究奠定了基础。

Objective Utilizing humanized anti-rabies virus ScFv phage antibody library, to construct plasmids of light and heavy chains of monoclonal antibodies, after express the constructs and purification the preparations to validate the monoclonal antibodies with high neutralizing activity. Methods Based on the sequences of specificity of antibody which was selected from humanized anti-rabies virus ScFv phage antibody library as template, PCR amplified the variable region sequence of light and heavy chain of antibody,the plasmids with different sequences of variable regions of anti-rabies antibody light and heavy chains were constructed, and the plasmids were transiently expressed in HEK-293 EBNA1 cells. The antibodies were purified manually by Mabselect SuRe affinity. The protein concentration was determined by a micro-spectrophotometer, and the neutralization activity was verified by a rapid fluorescent focus inhibition test(rapid fluorescent focus inhibition test, RFFIT). Results A total of 22 light chain plasmids and 15 heavy chain plasmids were successfully constructed. After transient expression by eukaryotic cells and manually affinity purification, 22 monoclonal antibodies were prepared. The antibody level was measured by the micro-spectrophotometer, only one strain was <300 μg/mL and the other 21 antibodies >300 μg/mL. The purity of some antibodies was >90%.The neutralization activity of the antibody was detected by RFFIT, and the specific activity of 5 monoclonal antibodies was between 1 500-1 800 IU/mg, 8 strains between 800-1 500 IU/mg, 7 strains between 500-800 IU/mg, 2 strains below 500 IU/mg. Conclusion We constructed and verified 20 strains of humanized anti-rabies monoclonal antibodies with a neutralizing activity greater than 500 IU/mg successfully, laying a foundation in search for the mixed formulation of humanized anti-rabies virus monoclonal antibodies.