烟曲霉纤维二糖水解酶cbh基因在大肠杆菌中的表达  

Expression of the cellobiohydrolase gene of Aspergillus fumigatus in Escherichia coli

作　　者：杨海峰 魏亚琴[2] 杨宇泽 万学瑞[4] 孙康永杰 成述儒[1] 王川[4] YANG Haifeng;WEI Yaqin;YANG Yuze;WAN Xuerui;SUN Kangyongjie;CHENG Shuru;WANG Chuan(College of Animal Science and Technology,Gansu Agricultural University,Lanzhou 730070,Gansu,China;Center of Anaerobic Microbes,Institute of Biology,Gansu Academy of Sciences,Lanzhou 730000,Gansu,China;Beijing Animal Husbandry Station,Beijing 100011,China;College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,Gansu,China)

机构地区：[1]甘肃农业大学动物科学技术学院,甘肃兰州730070 [2]甘肃省科学院生物研究所厌氧微生物中心,甘肃兰州730000 [3]北京市畜牧总站,北京100011 [4]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《草业科学》2020年第12期2457-2462,共6页Pratacultural Science

基　　金：甘肃省科技计划项目重点研发计划(18YF1NA077);国家自然科学基金(31500067)。

摘　　要：为构建高效表达的纤维素酶基因工程菌,本研究以烟曲霉(Aspergillus fumigatus)基因组DNA为模板,通过PCR扩增出纤维二糖水解酶cbh(cellobiohydrolase)基因,将其连接到原核生物表达载体pET-28a中,并转化至大肠杆菌(Escherichia coli)菌株BL21(DE3)中诱导表达蛋白CBH(cellobiohydrolase),同时采用刚果红染色、3,5-二硝基水杨酸(3,5-Dinitrosalicylic,DNS)法测定重组菌株酶活力。结果表明:成功构建表达载体pET-28a::cbh,在异丙基-β-D-硫代半乳糖苷(isopro-pyl-β-D-1-thiogalactopyranoside,IPTG)终浓度为1 mmol·L−1,诱导温度为28℃,诱导时间为14 h,纯化后的蛋白经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)得到一条75 kDa的目的蛋白,通过羧甲基纤维素钠(carboxymethyl cellulose-Na,CMC-Na)刚果红平板检测到pET-28a::cbh/E.coli可以产生水解圈,表明获得的重组菌株具有酶活性,其酶活力为0.0563 U·mL−1。本研究构建的重组纤维素酶菌株,可为改善牧草青贮品质和提高反刍动物生产性能奠定研究基础。In this study,the genomic DNA of Aspergillus fumigatus was used as a template to construct a genetically engineered cellulase strain with high efficiency.The cbh gene was amplified by PCR,ligated into the prokaryotic expression vector pET-28a,and transformed into Escherichia coli BL21(DE3)to induce the expression of cellobiohydrolase(CBH)protein.The enzyme activity of the recombinant strain was determined by Congo red staining and 3,5-Dinitrosalicylic acid(DNS).The results showed that the expression vector pET-28a::cbh was constructed successfully.The final concentration of isopropyl-β-D-1-thiogalactopyranoside(IPTG)was 1 mmol·L−1,the induction temperature was 28℃and induction time was 14 h,and the purified protein of 75 kDa was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The pET-28a::cbh/E.coli hydrolysis loops were detected by the Congo red plate with carboxymethyl cellulose-Na(CMC-Na).The results showed that the obtained recombinant strain had enzyme activity of 0.0563 U·mL−1.The recombinant cellulase strain constructed in this study can lay a foundation to improve the quality of forage silage and production performance of ruminants.

关 键 词：烟曲霉 纤维素 纤维素酶 纤维二糖水解酶 大肠杆菌 酶活 克隆与表达 

分 类 号：Q78[生物学—分子生物学] Q93