抗H5N1禽流感病毒M1蛋白全人源胞内抗体的制备及纯化  

Preparation and purification of whole human intracellular antibody against M1 protein of H5N1 avian influenza virus

作　　者：蔄弘扬 孙赫 田园[2] 吴广谋 张国利 邱月 王瑜 张文慧[1] 岳玉环[2] MAN Hong-yang;SUN He;TIAN Yuan;WU Guang-mou;ZHANG Guo-li;QIU Yue;WANG Yu;ZHANG Wen-hui;YUE Yu-huan(College of Life Sciences,Jilin Agricultural University,Changchun 130117,Jilin Province,China)

机构地区：[1]吉林农业大学生命科学学院,吉林长春130117 [2]中国农业科学院长春兽医研究所生物技术应用研究室,吉林长春130117

出　　处：《中国生物制品学杂志》2022年第10期1200-1204,共5页Chinese Journal of Biologicals

基　　金：吉林省重点研发计划(20200404113YY);军队后勤项目(2019SWAQ05-6-8)。

摘　　要：目的 制备抗H5N1禽流感病毒(avian influenza virus,AIV)M1蛋白全人源胞内抗体TAT-ScFv M1-mFc,并进行纯化。方法 从pET28(a)-TAT-ScFv M1质粒中通过PCR扩增目的基因片段TAT-ScFv M1,将该片段与表达载体pET32(a)-HisTag-Sumo-mFc连接,构建重组表达质粒pET32(a)-HisTag-Sumo-TAT-ScFv M1-mFc,转化大肠埃希菌BL21(DE3)中,IPTG诱导表达目的蛋白。通过金属螯合层析、Sumo酶切及亲和层析等方法对目的蛋白进行纯化,ELISA法检测抗体的亲和活性。结果 重组表达质粒经双酶切及测序鉴定证明构建正确。重组蛋白以包涵体形式表达,相对分子质量约80 000。制备的胞内抗体具有明显结合M1蛋白的活性。结论 获得了具有亲和活性的抗H5N1AIV M1蛋白的全人源胞内抗体蛋白TAT-ScFv M1-mFc,为进一步开发针对AIV H5N1的抗体药物奠定了基础。Objective To prepare and purify the whole human intracellular antibody TAT-ScFv M1-mFc against M1 protein of H5N1 avian influenza virus(AIV). Methods The target gene fragment TAT-ScFv M1 was amplified from plasmid pET28(a)-TAT-ScFv M1 by PCR,and inserted into expression vector pET32(a)-HisTag-Sumo-mFc. The constructed recombinant expression plasmid pET32(a)-HisTag-Sumo-TAT-ScFv M1-mFc was transformed to E. coli BL21(DE3) and induced by IPTG. The expressed target protein was purified by metal chelation chromatography,Sumo digestion and affinity chromatography,and determined for affinity activity by ELISA. Results Restriction analysis and sequencing proved that the recombinant expression plasmid was constructed correctly. The recombinant protein was expressed in a form of inclusion body,with a relative molecular mass of about 80 000. The prepared intracellular antibody showed obvious binding activity to M1 protein. Conclusion A whole human intracellular antibody protein TAT-ScFv M1-mFc with affinity activity against M1 protein of H5N1 AIV was obtained,which laid a foundation of further development of antibody drugs against H5N1 AIV.

关 键 词：H5N1禽流感病毒 M1蛋白 全人源抗体 胞内抗体 

分 类 号：S831.7[农业科学—畜牧学]