人基质金属蛋白酶抑制剂Ⅰ原核核表达系统的构建及单克隆抗体的制备  

Cloning and expressing of tissue inhibitor of metalloproteinases Ⅰ gene fragment and preparation of monoclonal antibodies against the recombinant protein

作　　者：侯俊 张健[4] 郭静霞[2] 陈霖[2] 赵静[2] 刘佳[2] 徐军[2] 刘爱霞[2] 宋永继[2] 貌盼勇[3] 李伯安[2] 毛远丽[2] 

机构地区：[1]解放军医学院/解放军总医院,北京100853 [2]解放军第三0二医院临检中心 [3]解放军第三0二医院实验技术保障中心 [4]解放军第三0二医院肝纤维化中心

出　　处：《中华实验和临床病毒学杂志》2013年第3期231-233,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：解放军第三○二医院重点课题

摘　　要：目的 制备针对人基质金属蛋白酶抑制剂Ⅰ （TIMP-Ⅰ）融合蛋白的单克隆抗体.方法 用逆转录聚合酶链反应法从纤维化的肝组织中扩增TIMP-Ⅰ编码序列,产物克隆入pQE31载体,转化大肠埃希菌BL21,诱导表达后通过6×His标签进行纯化,用纯化后的蛋白免疫BALB/c小鼠,采用常规的细胞融合技术制备单克隆抗体,并进行鉴定.结果 成功构建了TIMP-Ⅰ原核表达体系,并获得了重组蛋白.筛选出4株能稳定分泌抗TIMP-Ⅰ的单克隆抗体杂交瘤细胞株,免疫球蛋白类型有3株为IgGl类.Western Blot印迹显示这些单抗能特异结合TIMP-1蛋白.结论 成功构建了人基质金属蛋白酶抑制剂Ⅰ原核表达体系,并获得了单克隆抗体.Objective To prepare the monoclonal antibody （mAb） against tissue inhibitor of metalloproteinases Ⅰ （TIMP-Ⅰ）fusion protein.Methods TⅠMP-Ⅰ gene was amplified from fibrotic human liver tissue by RT-PCR,then ligated with pQE31 to form recombinant plasmid pQE-TIMP-Ⅰ and transformed into E.coli BL21.The protein induced by IPTG was purified by 6 × His-tag and used to immunize the BALB/c mice.The specific monoclonal antibodies （mAbs） were prepared by the cell fusion technique.Western Blot were used to detect specificity of mAbs.Results The prokaryotic plasmid expressing the recombinant protein was constructed,and the TIMP-Ⅰ recombinant protein was expressed and purified.Four hybridoma cell lines that secreted anti-TIMP-Ⅰ mAbs were obtained.3 of 4 mAbs were the IgG1 subtype.Western Blot indicated the mAbs showed specific combination with TIMP-Ⅰ protein.Conclusion The TIMP-Ⅰ recombinant protein is highly purified and has strong antigenicity.The anti-TIMP-Ⅰ mAbs were prepared successfully.

关 键 词：间质胶原酶 克隆 分子 抗体 单克隆 

分 类 号：R[医药卫生]