医学分子生物学杂志
醫學分子生物學雜誌
의학분자생물학잡지
FOREIGN MEDICAL SCIENCES
2007年
1期
20-26
,共7页
徐建华%朱家勇%金小宝%许琴英
徐建華%硃傢勇%金小寶%許琴英
서건화%주가용%금소보%허금영
家蝇%抗菌肽Attacin%基因克隆%生物信息学分析%原核表达
傢蠅%抗菌肽Attacin%基因剋隆%生物信息學分析%原覈錶達
가승%항균태Attacin%기인극륭%생물신식학분석%원핵표체
Musca domestica%antibacterial peptide Attacin%gene cloning%bioinformatic analysis%prokaryotic expression
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.
目的 剋隆傢蠅幼蟲Attacin抗菌肽基因,構建原覈融閤錶達載體,建立Attacin體內抗菌活性檢測繫統,優化錶達和純化Attacin目的蛋白,併初步研究其抗菌生物學功能.方法 以pUCm-T/Attacin重組質粒為模闆,設計特異性引物,PCR擴增Attacin編碼區序列,分彆剋隆至原覈錶達載體pET30a(+)和pGEX-4T-1,構建原覈重組質粒,轉化大腸埃希菌,錶達重組Attacin蛋白,併在大腸埃希菌中體內檢測Attacin的抗菌活性.利用親和層析柱純化重組融閤蛋白Attacin,SDS-PAGE進行純度分析,瓊脂糖平闆抑菌試驗鑒定其生物活性.結果 pET30a(a+)/Attacin和pGEX-4T-1/Attacin重組質粒分彆轉化大腸埃希菌後,以IPTG誘導錶達,與未誘導對照相比,含有重組質粒的宿主菌生長受到抑製,從pET30a(+)/Attacin重組質粒的錶達宿主菌中未能穫得His-Attacin融閤蛋白,而從pGEX-4T-1/Attacin重組質粒轉化菌種穫得GST-Attacin融閤蛋白.SDS-PAGE分析錶明Attacin重組蛋白分子量與預期結果一緻,瓊脂糖平闆抑菌試驗顯示重組Attacin具有抗菌活性.結論 Attacin基因在原覈繫統中成功錶達,併且純化後具有抑菌活性,為下一步研究Attacin的生物學功能及其應用開髮奠定瞭基礎.
목적 극륭가승유충Attacin항균태기인,구건원핵융합표체재체,건립Attacin체내항균활성검측계통,우화표체화순화Attacin목적단백,병초보연구기항균생물학공능.방법 이pUCm-T/Attacin중조질립위모판,설계특이성인물,PCR확증Attacin편마구서렬,분별극륭지원핵표체재체pET30a(+)화pGEX-4T-1,구건원핵중조질립,전화대장애희균,표체중조Attacin단백,병재대장애희균중체내검측Attacin적항균활성.이용친화층석주순화중조융합단백Attacin,SDS-PAGE진행순도분석,경지당평판억균시험감정기생물활성.결과 pET30a(a+)/Attacin화pGEX-4T-1/Attacin중조질립분별전화대장애희균후,이IPTG유도표체,여미유도대조상비,함유중조질립적숙주균생장수도억제,종pET30a(+)/Attacin중조질립적표체숙주균중미능획득His-Attacin융합단백,이종pGEX-4T-1/Attacin중조질립전화균충획득GST-Attacin융합단백.SDS-PAGE분석표명Attacin중조단백분자량여예기결과일치,경지당평판억균시험현시중조Attacin구유항균활성.결론 Attacin기인재원핵계통중성공표체,병차순화후구유억균활성,위하일보연구Attacin적생물학공능급기응용개발전정료기출.
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 pUCm-T/Attacin by PCR with specific primers and then cloned into pET30a (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-1/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 pGEX-4T-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.
