猪溶菌酶在大肠杆菌中的表达及其复性  

The expression of Sus scrofa lysozyme in E. coli and its refolding

作　　者：朱德伟[1] 蔡国林[1] 陆健[1] 

机构地区：[1]江南大学工业生物技术教育部重点实验室,粮食发酵工艺与技术国家工程实验室,生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2016年第10期23-28,共6页Food and Fermentation Industries

基　　金：973项目(2013CB733602);中央高校基本科研业务费专项资金资助(JUSRP51302A);江苏高校优势学科建设工程资助项目

摘　　要：作为C型溶菌酶的一种,猪溶菌酶(Sus scrofa lysozyme,SSL)是猪体内抵抗外源性疾病的一道重要屏障。鉴于猪在畜牧行业中的重要地位,尤其是在饲用抗生素的使用严重受限的今天,SSL的生产显得尤为迫切。化学合成得到了SSL的编码基因,通过Bam HⅠ和HindⅢ双酶切之后与载体p ET-28a(+)连接,转化至大肠杆菌BL21(DE3)中诱导表达。在25℃、200 r/min条件下,利用0.1 mmol/L的IPTG诱导8 h,离心发酵液获得菌体。对其进行超声破碎获得重组蛋白包涵体,然后对包涵体进行复性,最终获得了具有生物活性的目的蛋白,并对其酶学性质以及抗菌活性进行了研究。超声破碎后获得了181.05 mg/L的重组蛋白包涵体,对包涵体进行复性后,比酶活力可达8 032.78 U/mg,其最适温度为35℃,最适p H为6.0,与其理论值基本一致,而其抗菌活性与标准品溶菌酶的作用效果也比较类似,为其规模化生产与进一步应用奠定了理论基础。As a kind of C-type lysozyme,Sus scrofa lysozyme（ SSL） plays an important role in anti-exterior microorganisms. Considering the important role of pig in stock farming,especially that the feed antibiotic is severely limited,the production of SSL is urgently demanded. The CDS of SSL gene was synthesized and inserted into vector p ET-28a（ ＋） after digestion with Bam H Ⅰ and Hind Ⅲ. Then the recombinant plasmid was transformed into BL21（ DE3）. Under the condition of 25 ℃,200 r/min,0. 1 mmol/L IPTG was added for 8 h induction. With the process of refolding,SSL with natural activity was obtained. Furthermore,the characterization of the recombinant protein was carried out. After centrifugation and ultrasonic disruption,181. 05 mg / L inclusion body of recombinant protein was harvested. After refolding,SSL with the specific enzyme activity of 8032. 78 U / mg was obtained. In the study of enzymatic property,the optimal temperature and p H was 35 ℃ and 6,respectively,which were the same as their theoretical values. In the antimicrobial activity analysis,recombinant SSL had a similar result to standard lysozyme. The results will lay the foundation for the industrial production of SSL and its further applications.

关 键 词：猪溶菌酶 大肠杆菌 包涵体 复性 

分 类 号：S816[农业科学—饲料科学]