CAJ | 학술논문

用分子克隆手段获得栖热袍菌(Thermotoga neapolitana DSM 4359)中的α-葡糖苷酶基因,构建该基因的原核表达载体,并在大肠杆菌BL21(DE3)中表达.以Thermotoga neapolitana DSM 4359的α-葡糖苷酶基因DNA为模板,通过PCR扩增目的基因,并连接至表达载体中,构建重组质粒pET-28a-glu,然后转化到大肠杆菌中,加IPTG诱导获得重组蛋白.提取粗酶,用葡萄糖测定试剂盒测酶活.序列分析结果表明,该基因的读码框碱基长度为2166bp,编码722个氨基酸;SDS-PAGE电泳结果表明,α-葡糖苷酶基因在大肠杆菌BL21(DE3)中高效表达,蛋白分子量约为62KDa;酶的最适反应温度为70℃,最适pH为5.0.
용분자극륭수단획득서열포균(Thermotoga neapolitana DSM 4359)중적α-포당감매기인,구건해기인적원핵표체재체,병재대장간균BL21(DE3)중표체.이Thermotoga neapolitana DSM 4359적α-포당감매기인DNA위모판,통과PCR확증목적기인,병련접지표체재체중,구건중조질립pET-28a-glu,연후전화도대장간균중,가IPTG유도획득중조단백.제취조매,용포도당측정시제합측매활.서렬분석결과표명,해기인적독마광감기장도위2166bp,편마722개안기산;SDS-PAGE전영결과표명,α-포당감매기인재대장간균BL21(DE3)중고효표체,단백분자량약위62KDa;매적최괄반응온도위70℃,최괄pH위5.0.
We get the α-glucosidase gene from Thermotoga neapolitana by molecular cloning, then the gene was cloned and expressed in E.coli BL21 (DE3). The target gene was amplified by PCR and connected to the expres-sion vector pET-28a. The recombinant plasmid pET-28a-glu was constructed and transferred into E.coli BL21 (DE3). Obtained the recombinant protein and extracted crude enzyme. Sequence analysis indicated that the length of the gene was 2166bp. The result of the SDS-PAGE showed that the gene was effectively expressed in E.coli BL21 (DE3). The enzyme's optimum reaction temperature was at 70℃ and the pH value was about 5.0.