抗A型产气荚膜梭菌α毒素全人源单分子抗体的表达、纯化及鉴定  

Expression,purification and identification of fully human monomolecular antibody against Clostridium perfringens type A α-toxin

作　　者：邱玥 孙赫 王瑜 蔄弘扬[1,2] 张国利 岳玉环 吴广谋 苏玉春 QIU Yue;SUN He;WANG Yu;MAN Hongyang;ZHANG Guoli;YUE Yuhuan;WU Guangmou;SU Yuchun(College of Life Science,Jilin Agricultural University,Changchun 130118,Jilin Province,China;不详)

机构地区：[1]吉林农业大学生命科学学院,吉林长春130118 [2]中国农业科学院长春兽医研究所,吉林长春130112

出　　处：《中国生物制品学杂志》2024年第7期849-854,共6页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划(20190304038YY)。

摘　　要：目的表达及纯化抗A型产气荚膜梭菌α毒素的全人源单分子抗体,并进行鉴定,以期为防治该毒素引起的各类疾病奠定基础。方法将抗A型产气荚膜梭菌全人源单链抗体(single-chain fragment variable,Sc Fv)基因与人抗体轻、重链恒定区基因以不同组合方式连接,构建多种抗产气荚膜梭菌α毒素单分子抗体重组表达质粒,于感受态E.co Li BL21(DE3)中表达,间接ELISA法检测各单分子抗体的免疫结合活性。将免疫结合活性最大的单分子抗体表达产物通过Sepharo Se 4 FF及r Protein-A FF亲和层析进行纯化,纯化产物经12%SDS-PAGE分析。结果共构建5种重组表达质粒,分别为PTS-Sc Fv-CL-CH_(2)-CH_(3)、PTS-Sc Fv-CH_(2)-CH_(3)、PTS-Sc Fv-CL-CH_(2)、PTS-Sc Fv-CH_(2)、PTS-Sc Fv-CL,经E.coli BL21(DE3)表达后,获得相应单分子抗体,分别为Sc Fv-CL-CH_(2)-CH_(3)、Sc Fv-CH_(2)-CH_(3)、Sc Fv-CL-CH_(2)、Sc Fv-CH_(2)、Sc Fv-CL,其中单分子抗体Sc Fv-CH_(2)-CH_(3)的免疫结合活性最大,A_(450)达3.9,远高于其他4种单分子抗体,浓度约为1 mg/m L,纯度约为86%。结论获得1株亲和力高、免疫原性低且具有内化活性的全人源单分子抗体Sc Fv-CH_(2)-CH_(3),为治疗性抗体的进一步深入研究奠定了基础。Objective To express,purify and identify human monomolecular antibody against Clostridium perfringens type A α-toxin,in order to lay a foundation for the prevention and treatment of various diseases caused by this toxin.Methods The fully human single-chain fragment variable(ScFv) gene against Clostridium perfringens type A was linked with the constant region of light chain and heavy chain of human antibody in different combinations to construct multiple monomolecular antibody expression plasmids against Clostridium perfringens α-toxin,which were expressed in competent E.coLi BL21(DE3).Indirect ELISA was used to detect the immunobinding activity of the monomolecular antibodies,and the monomolecular antibody with the highest immunobinding activity was purified by SepharoSe 4 FF and rProtein-A FF affinity chromatography,The purified products were analyzed by 12% SDS-PAGE.Indirect ELISA was used to detect the immune binding activity of each monomolecular antibody.Results Five recombinant plasmids,PTS-ScFv-CL-CH_2-CH_3,PTS-ScFv-CH_2-CH_3,PTS-ScFv-CL-CH_2,PTS-ScFvCH_2,and PTS-ScFv-CL,were constructed.After transfection into E.coli BL21(DE3) and purification,the corresponding monomolecular antibodies,ScFv-CL-CH_2-CH_3,ScFv-CH_2-CH_3,ScFv-CL-CH_2,ScFv-CH_2,and ScFv-CL,were obtained,which had the relative molecular mass of about 60 000,and the concentrations of about 1 mg/mL.Among them,ScFv-CH_2-CH_(3showed) the highest immune binding activity,and the A_(450) value reached 3.9,much higher than the other four monomolecular antibodies,with the concentration of about 1 mg/mL and the purity about 86%.Conclusion A fully human monomolecular antibody ScFv-CH_2-CH_(3) with high affinity,low immunogenicity and internalization activity was obtained,which lays a foundation for the further study of therapeutic antibody against CPA.

关 键 词：产气荚膜梭菌α毒素 单分子抗体 基因克隆 亲和纯化 

分 类 号：R373.9[医药卫生—病原生物学]