作　　者： ; ; ; (徐建华）;

机构地区： 广州中医药大学第二附属医院

出　　处： 《医学分子生物学杂志》 2007年第1期20-26,共7页

摘　　要： 目的克隆家蝇幼虫Attacin抗菌肽基因,构建原核融合表达载体,建立Attacin体内抗菌活性检测系统,优化表达和纯化Attacin目的蛋白,并初步研究其抗菌生物学功能。方法以pUCm-T/Attacin重组质粒为模板,设计特异性引物,PCR扩增Attacin编码区序列,分别克隆至原核表达载体pET30a(+)和pGEX-4T-1,构建原核重组质粒,转化大肠埃希菌,表达重组Attacin蛋白,并在大肠埃希菌中体内检测Attacin的抗菌活性。利用亲和层析柱纯化重组融合蛋白Attacin,SDS-PAGE进行纯度分析,琼脂糖平板抑菌试验鉴定其生物活性。结果pET30a(a+)/Attacin和pGEX-4T-1/Attacin重组质粒分别转化大肠埃希菌后,以IPTG诱导表达,与未诱导对照相比,含有重组质粒的宿主菌生长受到抑制,从pET30a(+)/Attacin重组质粒的表达宿主菌中未能获得His-Attacin融合蛋白,而从pGEX-4T-1/Attacin重组质粒转化菌种获得GST-Attacin融合蛋白。SDS-PAGE分析表明Attacin重组蛋白分子量与预期结果一致,琼脂糖平板抑菌试验显示重组Attacin具有抗菌活性。结论Attacin基因在原核系统中成功表达,并且纯化后具有抑菌活性,为下一步研究Attacin的生物学功能及其应用开发奠定了基础。 Objective To clone the cDNA sequence of Attacin gene from Musca domestica larvae, construct prokaryotic fusion expression vectors, express and purify the Attacin protein, and preliminarily study its antibiotic biological function. Methods The cDNA fragment encoding Attacin was amplified from recombinant plasmid pUC m-T/Attacin by PCR with specific primers and then cloned into pET 30a （ a ＋ ） and pGEX-4T-1 vector separately. These prokaryotic expression recombinant plasmids were transformed into E. coli to express the relative proteins respectively. The antibacterial activity of Attacin in E. coli was monitored with an in vivo system. The expressed recombinant products were purified with affinity-chromatograph column and analyzed with SDS-PAGE. Then the biological activities of Attacin were detected by agarose plate antibiotic assay. Results The 674 bp cDNA of Attacin was obtained by RT-PCR. The sequence has been accepted by GenBank （ Accession number DQ062744）. The pET30a （a ＋ ） /Attacin and pGEX-4T-l/Attacin recombinant plasmids were Confirmed by PCR, restriction endonuclease digestion and DNA sequencing, respectively. Transformation assay of E. coli with the recombinant plasmids revealed that, after IPTG induction and compared to non-induced control, the growth of host cells containing pET30a （ ＋ ） / Attacin was significantly suppressed, with no His-Attacin band on SDA-PAGE as expected. Although the growth of host cells containing pGEX4T-1/Attacin was also inhibited, the GST-Attacin fusion protein was obtained. The GST-Attacin fusion protein then underwent GST affinity-chromatography, and SDS-PAGE analysis confirmed the final purified product had the expected size. The recombinant protein exhibited antibacterial activities when assayed with agarose plates inoculated with bacteria. Conclusion The Attacin expressed in prokaryotic system possesses antibacterial activities, which lays a reliable foundation for further research on biological functions and application of Attaicn.

关 键 词： 家蝇 抗菌肽 基因克隆 生物信息学分析 原核表达

领　　域： [生物学]