作　　者： ; ; ;

机构地区： 黑龙江八一农垦大学

出　　处： 《中国生物制品学杂志》 2013年第10期1388-1391,共4页

摘　　要： 目的克隆、表达枯草芽孢杆菌耐热丝氨酸蛋白酶基因,并检测其酶学性质。方法从枯草芽孢杆菌染色DNA中,PCR扩增枯草芽孢杆菌耐热丝氨酸蛋白酶基因Sapr,插入载体pET-28a(+)中,构建重组分泌型表达质粒pET-28a-Sapr,热激转化大肠埃希菌(E.coli)BL21(DE3),25及37℃条件下,IPTG诱导重组蛋白表达,表达的重组蛋白经10%SDS-PAGE分析,并采用Folin法测定酶活性。结果重组表达质粒经双酶切及PCR鉴定,证明构建正确,测序结果表明与GenBank中登录的序列同源性达100%;表达的重组蛋白相对分子质量约39 600,最佳诱导温度为25℃,主要以可溶性形式表达,表达量约占菌体总蛋白的12%;重组菌发酵产物酶活性为322 U/ml。结论在大肠埃希菌中成功表达的特异蛋白具有生物学活性,为进一步研究改性分离蛋白的功能特性奠定了基础。 Objective To clone and express the heat-resistant serine protease gene of Bacillus subtilis and determine the enzymological property of expressed product. Methods The heat-resistant serine protease gene Sapr of B. subtilis was amplified from chromosome DNA of B. subtilis by PCR and inserted into vector pET-28a （＋）. The constructed recombinant secretory expression vector pET-28a-Sapr was transformed to E. coli BL21 （DE3） by heat shock, and induced with IFFG at 25℃ and 37. The expressed recombinant protein was analyzed by SDS-PAGE and determined for activity by Folin method. Results Both restriction analysis and PCR proved that recombinant plasmid pET-28a-Sapr was constructed cor- rectly. Sequencing result proved that the homology of nueleotide sequence of target gene was 100% to that reported in GenBank. The optimal temperature for induction was 25 ℃. The expressed recombinant protein, with a relative molecular mass of about 39 600, mainly existed in a soluble form and contained about 12% of total somatic protein. The enzyme ac- tivity of fermentation product of recombinant E. coli was 322 U / ml. Conclusion The specific protein successfully ex- pressed in BL21 / pET-Sapr showed biology activity, which laid a foundation of further study on function and property of modified protein isolate.

关 键 词： 枯草芽孢杆菌 丝氨酸蛋白酶 大肠埃希菌 克隆 表达

领　　域： [生物学] [生物学]