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目的扩增出全长diptericin2基因,并构建表达pGEX-4T-1/diptericin2重组蛋白。方法运用经典的5′末端扩增法(5′-Full RACE)法,通过双酶切、连接反应,将目的基因片段定向插入到表达载体pGEX-4T-1上,转化大肠埃希菌BL21,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析diptericin2重组蛋白的表达情况,蛋白质印迹实验(Western-blotting)对其进行了鉴定。结果获得目的片段468bp的家蝇抗菌肽diptericin2基因(GENBANK登录号FJ795370),双酶切鉴定及DNA测序结果显示,目的基因diptericin2已成功连接到表达载体pGEX-4T-1上,SDS-PAGE结果显示表达产物相对分子质量约为27 kDa。结论扩增出了全长diptericin2基因并成功构建pGEX-4T-1/diptericin2重组蛋白原核表达系统,融合蛋白以包涵体形式在大肠埃希菌BL21中表达,为下一步研究其生物活性打下初步的基础。
Objective To amplify full-length diptericin2 gene and construct pGEX-4T-1 / diptericin2 recombinant protein. Methods The target gene fragment was inserted into the expression vector pGEX-4T-1 by double restriction enzyme digestion and 5’-Full RACE method. The recombinant plasmid was transformed into Escherichia coli BL21, The expression of diptericin2 recombinant protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after induced by isopropyl-β-D-thiogalactoside (IPTG). Western blotting (Western-blotting) were identified. Results The diphtheria diptericin2 gene (GENBANK accession number FJ795370) with a 468bp fragment was obtained. Double enzyme digestion and DNA sequencing showed that the target gene diptericin2 was successfully ligated into the expression vector pGEX-4T-1. The results of SDS-PAGE The relative molecular weight of the expressed product was about 27 kDa. Conclusion The full-length diptericin2 gene was amplified and the recombinant prokaryotic expression system pGEX-4T-1 / diptericin2 was successfully constructed. The fusion protein was expressed in inclusion bodies in Escherichia coli BL21, which laid the foundation for the further study of its biological activity basis.