家蝇幼虫抗菌肽diptericin2基因的克隆及原核表达-鉴定

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。

Diptericin2 gene cloning expression and identification of antibacterial peptide from musca domestica

OBJECTIVE To amplify the full-length diptericin2 gene and construct expression pGEX-4T-1/diptericin2 recombinant protein.METHODS The 5′-Full RACE was used to amplify the full-length sequence that then digested by double enzymes,and ligated so it was cloned into the pGEX-4T-1 and transformed in E.coliBL21.Analysed the recombinant protein expression by sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)after induced with isopropyl-β-D-thiggalactoside(IPTG)and identified with Western-blotting.RESULTS The target sequence(Genbank accession number:FJ795370)was attained by sequencing and ligated in vetor by double digestion.And the molecular mass was detected by SDS-PAGE.CONCLUSION The full-length diptericin2 gene and pGEX-4T-1/diptericin2 recombinant prokaryotic expression system is successfully amplified and constructed.The fusion protein expression by inclusion bodies in E.coli BL21 provides the bases for further study of biological activity.