枯草芽孢杆菌β-葡聚糖酶基因的克隆和表达  

Cloning and expression of β-glucanase gene from Escherichia coli Bacillus subtilis

作　　者：孙珂[1] 王亚楠[1] 张亚南[1] 朱文华[1] 程殿林[1] 

机构地区：[1]青岛大学生命科学学院,山东省青岛市266071

出　　处：《中国组织工程研究》2016年第B05期49-52,共4页Chinese Journal of Tissue Engineering Research

基　　金：山东省优秀中青年科学家奖励基金（BS20IOSW007）

摘　　要：背景：β-葡聚糖已广泛用于保健和临床治疗。通过酶水解的方法得到分子量大小适中的β-葡聚糖，这一研究日益受到重视。目的：实验旨在构建高表达β-葡聚糖酶的工程菌。并对该酶活性及热稳定性做出分析，为今后能够利用到临床医学奠定基础。方法：通过克隆得到枯草芽孢杆菌β-葡聚糖酶的基因，利用分子生物学技术将β-葡聚糖酶基因与表达载体pET28b＋构建重组质粒后导入大肠杆菌中构建工程菌株。利用异丙基-β-D-硫代半乳糖苷诱导其表达目的蛋白，通过镍离子螯合树脂纯化得到目的蛋白。结果与结论：实验成功编码枯草芽孢杆菌β-葡聚糖酶的基因被克隆到大肠杆菌中，并能高效的表达。根据酶活力测定发现，该酶在50-60℃有较高的酶活性，在50℃最高。β-葡聚糖酶在30—40℃保持较高的热稳定性。实验初步发现，β-葡聚糖酶的工程菌表达产物能分解它的底物，但是否适用于临床治疗需要进行更多的研究。BACKGROUND： β-glucan has been widely used for healthcare and clinical treatment. 13-glucan with reasonably sized molecular weight obtained by enzymatic hydrolysis have been increasingly addressed. OBJECTIVE： To analyze the activity and thermo-stability of an engineering strain secreting high-expression β-glucanase, providing a basis for its clinical application. METHODS： β-glucanase gene of Bacillus subbtilis was obtained by cloning. Recombinant plasmids containing β-glucanase gene and pET28b（＋） were introduced into Escherichia coil to construct engineering strain. The expression of the target protein was induced using IPTG, and the protein was purified by nickel ion chelating resin. RESULTS AND CONCLUSION： Gene encoding β-glucanase in Bacillus subtilis was cloned into Escherichia coli and expressed efficiently. β-glucanase activity was high between 50 ℃ and 60 ℃ and reached the highest at 50 ℃ as determined by the enzymatic assay. The relative high-thermostability of β=glucanase under the temperature of 30-40℃ was found. β-glucanase expressed by the engineering strain can decompose its substrate. However further study is needed for its application in the clinic.

关 键 词：组织工程 枯草芽孢抒菌 大肠杆菌 质粒 Β-葡聚糖酶 临床医学 保健 克隆 表达 酶活性 治疗 

分 类 号：R378.21[医药卫生—病原生物学]