一种真核表达的抗H5N1-M1入胞单分子抗体的生物活性评价  

Evaluation of biological activity of an eukaryotic expressed anti-H5N1-M1 cell entry single molecule antibody

作　　者：孙赫 王瑜[1,2] 蔄弘扬 邱玥 田园 李泽鸿[2] 岳玉环[1] SUN He;WANG Yu;MAN Hongyang;QIU Yue;TIAN Yuan;LI Zehong;YUE Yuhuan(Biotechnology Application Research Laboratory,Chinese Academy of Agricultural Sciences Changchun Veterinary Institute,Changchun130122,Jilin Province,China;不详)

机构地区：[1]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130122 [2]吉林农业大学生命科学学院,吉林长春130117

出　　处：《中国生物制品学杂志》2023年第9期1047-1053,共7页Chinese Journal of Biologicals

基　　金：吉林省重点研发计划(20200404113YY);军队后勤重大专项(2019SWAQ05-6-9)。

摘　　要：目的评价一种真核表达的抗H5N1-M1入胞单分子抗体(TAT-ScFv-mFc)的生物活性。方法采用Western blot法及局域表面等离子共振技术(localized surface plasmon resonance,LSPR)分别检测TAT-ScFv-mFc与H5N1-M1蛋白的免疫结合活性及亲和力;CCK-8法检测TAT-ScFv-mFc对流感病毒H1N1的抑制作用;免疫荧光法检测TAT-ScFv-mFc对MDCK细胞的穿膜能力。雌性BALB/c小鼠尾静脉注射TAT-ScFv-mFc,200μL/只,共30只,于注射后5、60、120、240及360 min,断尾采血,分离血清,ELISA法检测血清效价,根据Origin 2021软件绘制的半衰期曲线计算半衰期。结果TAT-ScFv-mFc与H5N1-M1蛋白可发生特异性结合,结合数率常数(Ka)达6.67×104[1/(M*s)]。经H1N1感染的MDCK细胞的存活率随着TAT-ScFv-mFc浓度增加而逐渐升高,且呈剂量依赖性,对H1N1的复制有明显抑制作用。TAT-ScFv-mFc可在短时间内穿过MDCK细胞膜,进入细胞内,结合病毒M1蛋白,从而抑制病毒的复制和组装。TAT-ScFv-mFc在小鼠体内的半衰期为212 min。结论TAT-ScFv-mFc具有与H5N1-M1良好的免疫结合活性及亲和力,可有效抑制H1N1的复制,对MDCK细胞膜有良好的穿透能力,且在小鼠体内半衰期较长,为H5N1感染的药物治疗、疫苗研究、预防治疗奠定了基础。Objective To evaluate the biological activity of a eukaryotic expressed anti-H5N1-M1 cell entry single molecule antibody(TAT-ScFv-mFc).Methods The immune binding activity and affinity of TAT-ScFv-mFc to H5N1-M1 protein were detected by Western blot and localized surface plasmon resonance(LSPR)respectively;The inhibitory effect of TAT-ScFv-mFc on influenza virus H1N1 was detected by CCK-8 assay;The membrane penetration ability of TAT-ScFv-mFc to MDCK cells was verified by immunofluorescence assay.A total of 30 female BALB/c mice were injected with TAT-ScFv-mFc via tail vein,200μL per mouse.Blood samples were collected at 5,60,120,240 and 360 min after injection.Serum samples were separated and detected for the titers by ELISA,and the half-life of TAT-ScFv-mFc was calculated according to the half-life curve drawn by Origin 2021 software.Results TAT-ScFv-mFc showed specific binding to H5N1-M1 protein with a binding rate constant of 6.67×10~4[1/(M*s)].The survival rate of MDCK cells infected by H1N1 increased gradually with the increase of TAT-ScFv-mFc concentration in a dose-dependent manner,which obviously inhibited the replication of H1N1.TAT-ScFv-mFc penetrated the cell membrane of MDCK cells in a short time,entered the cell and bound to virus M1protein,thus inhibiting virus replication and assembly.The half-life of TAT-ScFv-mFc in mice was 212 min.Conclusion TAT-ScFv-mFc has good immune binding activity and affinity with H5N1-M1,can effectively inhibit the replication of H1N1,has good penetration ability to MDCK cell membrane,and has a long half-life in mice,which lays a foundation of the drug treatment,vaccine research and preventive treatment of H5N1 infection.

关 键 词：流感病毒H5N1 M1蛋白 单分子抗体 真核表达 生物活性 

分 类 号：S852.43[农业科学—基础兽医学]