应用毕赤酵母分泌表达筛选人抗狂犬病毒抗体scFv-Fc

doi:国家高技术研究发展计划（863计划）资助课

应用毕赤酵母分泌表达筛选人抗狂犬病毒抗体scFv-Fc

王丁丁^1,2，许天敏³，余丽江⁴，苏曼曼¹，黄树林²，宿晓云¹，陈磊¹，颜炜群¹

(1.吉林大学再生医学科学研究所生物化学研究室，吉林长春130021；2.广东药学院生命科学与生物制药学院生物技术教研室，广东广州 510006；3.吉林大学第二医院妇产科，吉林长春130041；4.吉林省疾病预防控制中

收稿日期:2007-03-14 修回日期:1900-01-01 出版日期:2007-11-28 发布日期:2007-11-28
通讯作者: 颜炜群

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

WANG Ding-ding^1,2,XU Tian-min³,YU Li-jiang⁴,SU Man-man¹,HUANG Shu-lin²,SU Xiao-yun¹,CHEN Lei¹,YAN Wei-qun¹

1.Department of Biochemistry,Institute of Frontier Medical Science,Jilin University,Changchun 130021,China;2.Department of Biotechnology,School of Life Science and Biopharmacology,Guangdong Pharmaceutical University,Guangzhou 510006,China;3.Department of Obstetrics and Gynecology,Second Hospital,Jilin University,Changchun 130041,China;4.Jilin Provincial Center for Disease Control and Prevention,Changchun 130062,China)

Received:2007-03-14 Revised:1900-01-01 Online:2007-11-28 Published:2007-11-28
Contact: YAN Wei-qun

摘要/Abstract

摘要： 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。

关键词: 毕赤酵母, 狂犬病病毒, 分泌表达, 筛选

Abstract: To clone and express functional recombination human scFv-Fc antibody against rabies virus in methylotropic yeast Pichia pastoris.MethodsThe genes of antibodies against rabies virus were amplifies from the donors vaccinatedby 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.ResultsAfter 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’[KG-*3]s relative molecular mass was 56 000. ConclusionThe results demonstrate that functional antibodies can be screened from the human scFv-Fc Pichia pastoris secretory expression library.

Key words: Pichia, rabies virus, secretory expression, screen

中图分类号:

王丁丁，许天敏，余丽江，苏曼曼，黄树林，宿晓云，陈磊，颜炜群. 应用毕赤酵母分泌表达筛选人抗狂犬病毒抗体scFv-Fc[J]. J4, 2007, 33(6): 963-967.

WANG Ding-ding,XU Tian-min,YU Li-jiang,SU Man-man,HUANG Shu-lin,SU Xiao-yun,CHEN Lei,YAN Wei-qun. Expression and selection of single-chain Fv-Fc (scFv) antibody fragment against antigen of rabies virus in Pichia pastoris[J]. J4, 2007, 33(6): 963-967.

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