TIA抗原基因原核表达载体的构建及表达

Construction and expression of prokaryotic expression vector of transient ischemic attack-associated antigens

目的:克隆并构建短暂性脑缺血发作重组cDNA表达克隆血清学鉴定技术(SEREX)抗原基因原核表达载体,重组GST融合蛋白.方法:采用SEREX筛选所获的70个重组cDNA克隆pBluescript质粒,用限制性内切酶(EcoRⅠ/XhoⅠ、BamHⅠ/XhoⅠ或SmaⅠ/XhoⅠ)双酶切,用质量分数为1%琼脂糖凝胶电泳分离回收目的基因片段.应用Ligation和In-Fusion基因重组技术,将目的基因插入原核表达载体质粒pGEX-4T-1,2,3,转化表达菌株E.coli BL-21感受态细胞,并用IPTG诱导GST标签蛋白表达,通过SDS-聚丙烯酰氨凝胶电泳(SDS-PAGE)分析表达产物,再经酶切及DNA测序鉴定,成功重组pGEX-4T载体的插入片段可以作为候选靶抗原.结果:成功获得21个重组pGEX-4T载体的靶抗原目的基因,均能够在原核表达系统中成功表达GST融合目的蛋白.结论:应用Ligation和In-Fusion基因重组技术能够有效构建原核表达载体,并在大肠杆菌中表达重组融合目的蛋白.

Objective:To construct the prokaryotic expressing vectors of cDNA insert fragments for transient ischemic attack-associated antigens(TIA),and to express the recombinant GST-fusion proteins.Methods:The cDNA inserts of 70 clones incorporated in pBluescript were cleaved by EcoRI/BamHI/SmalI and XhoI,and the insert fragments were isolated by 1%agarose gel electrophoresis.The expression plasmids for glutathione-S-transferase(GST)-fused proteins were constructed by recombining the cDNA sequence into pGEX-4T-1,2,3.The inserted DNA fragments were ligated in frame to pGEX-4T vectors by using Ligation or In-Fusion protocol.The reaction mixtures were used to transform ECOS TM competent Escherichia coli BL21 and appropriate recombinants were confirmed by DNA sequencing as well as protein expression through SDS-polyacrylamide gel electrophoresis(SDS-PAGE).The antigenic proteins succeeded in expression as GST-tagged recombinant proteins could be candidate antigens.Results:21 recombinant pGEX-4T expression vectors containing cDNA sequences of target genes were obtained from 70 SEREX antigens,and the expression of the GST-fused proteins were shown on SDS-PAGE gels stained with Coomassie brilliant blue staining.Conclusion:Recombination pGEX-4T expression plasmids could be constructed by Ligation and In-Fusion methods and expressed successfully in E.coli,which provides basis for the extraction and purification of these antigenic proteins aiming to identify serum TIA antibody biomarkers in further study.