应用毕赤酵母分泌表达筛选人抗狂犬病毒抗体scFv-Fc

Expression and selection of single-chain Fv-Fc(scFv) antibody fragment against antigen of rabies virus in Pichia pastoris

目的:利用毕赤酵母人抗狂犬病毒抗体分泌表达文库获得具特异性狂犬病毒抗原结合活性的分泌型小分子抗体(scFv-Fc)。方法:RT-PCR方法扩增获得一组轻链和重链可变区基因。利用重叠延伸PCR方法组装scFv基因后克隆入毕赤酵母scFv-Fc抗体库通用表达质粒pPICZα/Fc后,电转化X33酵母菌,甲醇诱导表达后进行ELISA筛选、基因序列分析及免疫印迹分析。结果:构建了抗狂犬病毒毕赤酵母分泌型scFv-Fc库,获得了12株阳性菌株。对其中2株阳性克隆进行了序列分析证实为新的抗体可变区基因。ELISA、Western blotting分析,证实该scFv-Fc具有特异性狂犬病毒抗原结合活性,相对分子质量为56 000。结论:通过毕赤酵母人抗狂犬病毒抗体scFv-Fc分泌表达文库筛选获得具较高亲和力的新scFv-Fc抗体。

Objective To clone and express functional recombination human scFv-Fc antibody against rabies virus in methylotropic yeast Pichia pastoris. Methods The genes of antibodies against rabies virus were amplifies from the donors vaccinated by vaccine of PM strain produced in Vero cell line. The human recombinant scFv antibody genes were prepared by SOE PCR and inserted into yeast expression vector pPICZα carrying the human IgG Fc region. The scFv Fc antibodies were selected from a human scFv-Fc Pichia pastoris secretory expression library. Results After three rounds of selection against the antigen of the rabies virus using ELISA assay, 12 clones recognized the rabies antigen. The variable region genes of the heavy and light chains of two scFv-Fc antibodies to rabies virus were sequenced and identified as new genes. The specificity of the resulting scFv-Fc molecules for rabies antigen was established by ELISA and Western blotting analyses. It＇ s relative molecular mass was 56 000. Oonclusion The results demonstrate that functional antibodies can be screened from the human scFv-Fc Pichia pastoris secretory expression library.