家蝇幼虫抗菌肽diptericin_2基因的克隆及原核表达-鉴定  

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

作　　者：杨小蓉[1] 金小宝[2] 丁彩屏[1] 朱家勇[2] 

机构地区：[1]广东药学院附属第一医院检验科,广州510080 [2]广东药学院基础学院生物活性物质研究所

出　　处：《现代预防医学》2012年第11期2791-2793,共3页Modern Preventive Medicine

基　　金：国家自然科学基金(30672670)

摘　　要：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 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.

关 键 词：家蝇抗菌肽 diptericin2基因 5′-FullRACE 原核表达鉴定 

分 类 号：R394[医药卫生—医学遗传学]