抗AEG-1单链可变区抗体的原核表达及纯化  

Prokaryotic expression and purification of anti-AEG-1 single-chain variable antibody

作　　者：路蔓[1] 刘昕阳[1] 龙敏[1] 王琛[1] 刘冲[1] 郜赵伟[1] 欧阳永日[1] 陈曦[1] 张惠中[1] 

机构地区：[1]第四军医大学唐都医院检验科,陕西西安710038

出　　处：《国际检验医学杂志》2015年第1期20-21,24,共3页International Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目(81201525)

摘　　要：目的构建抗星形细胞上调基因1(AEG-1)单链可变区抗体(V23)的原核表达载体,并对表达蛋白进行纯化及免疫活性检测。方法应用Primer5软件设计针对抗AEG-1单链可变区抗体基因序列的引物,构建PRsetC/V23原核表达质粒,经限制性内切酶Pst1酶切以及DNA测序鉴定正确后,将原核表达质粒导入大肠杆菌BL21中,构建含V23基因的原核表达工程茵。经IPTG诱导后,用带His标签的磁珠纯化目的蛋白,SDS-PAGE电泳检测目的蛋白含量,Western blot及ELISA检测抗AEG-1单链可变区抗体的免疫活性。结果构建的原核表达质粒PRsetC/V23经单酶切和测序分析显示,构建的V23基因与设计序列100%一致。IPTG诱导后,SDS-PAGE电泳显示在31×103处出现一条明显蛋白条带,Western blot检测在80×103处出现AEG-1特异反应条带,ELISA检测显示阳性结果。结论成功构建了PRsetC/V23原核表达质粒及V23原核表达工程茵,该工程茵可表达抗AEG-1单链可变区抗体蛋白,且该蛋白具有良好的免疫活性。Objective To construct anti-astrocyte elevated gene-l（AEG-1） single-chain variable antibody （V23） prokaryotic expression vector,and to conduct the protein purification and immunological activity detection. Methods The Primer5 software was applied to design the primers aiming at the gene sequence of the antibody anti-AEG-1 single-chain variable region for constructing the prokaryotic expression plasmid of PRsetC/V23. After the enzyme digestion by the restriction enzyme Pstl and correct DNA sequencing,the prokaryotic expression plasmid was led to E. coli BL21,the prokaryotic expression engineering strain containing the V23 gene was constructed. After the induction with IPTG, the interest protein was purified by the magnetic beads with the HIS tag, and the content of interest protein was determined by the SDS-PAGE electrophoresis. Western blot and ELISA were adopted to detect the immune activity of the nti-AEG-1 single-chain variable region antibody. Results For the constructed prokaryotic expression plasmid PRsetC/V23,the single enzyme digestion and sequencing analysis displayed that the constructed V23 gene was completely consistent to the designing sequences. After IPTG induction, SDS-PAGE electrophoresis showed an apparent protein band at 31×10^3 ,the Western blot detection showed a specific AEG-1 response band at 80 ×10^3, the ELISA test showed the positive results. Conclusion The PRsetC/V23 prokaryotic expression plasmid and the V23 prokaryotic expression engineering strain are successfully constructed,this engineering strain can express anti-AEG-1 single-chain variable region antibody protein, and the protein has good immune activity.

关 键 词：星形细胞上调基因1 单链可变区抗体 原核表达 

分 类 号：R446.6[医药卫生—诊断学]