抗 AEG-1单链可变区抗体的原核表达及纯化

摘要

Objective To construct anti-astrocyte elevated gene-1(AEG-1)single-chain variable antibody (V23)prokaryotic ex-pression 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 Pst1 and correct DNA se-quencing,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 expres-sion plasmid PRsetC/V23,the single enzyme digestion and sequencing analysis displayed that the constructed V23 gene was com-pletely consistent to the designing sequences.After IPTG induction,SDS-PAGE electrophoresis showed an apparent protein band at 31×103 ,the Western blot detection showed a specific AEG-1 response band at 80 ×103 ,the ELISA test showed the positive re-sults.Conclusion The PRsetC/V23 prokaryotic expression plasmid and the V23 prokaryotic expression engineering strain are suc-cessfully constructed,this engineering strain can express anti-AEG-1 single-chain variable region antibody protein,and the protein has good immune activity.%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。