| 张姝，胡蓉，黄健，吴建伟，国果，付萍，修江帆，尚小丽,2018,不同载体表达的家蝇β-葡萄糖苷酶生物学活性比较[J].环境昆虫学报,40(4):908-916 |
| 不同载体表达的家蝇β-葡萄糖苷酶生物学活性比较 |
| Biological activities of Musca domestica-derived beta-glucosidase expressedwith different vectors |
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| DOI： |
| 中文关键词: 家蝇 β-葡萄糖苷酶 异源性表达 酶活性 |
| 英文关键词:Musca domestica β-glucosidase heterogenous express enzymatic activity |
| 基金项目:贵州省教育厅培育项目\[(2009)0137\]；国家科技支撑计划课题（2011BAC06B12）；国家自然科学基金（30960343）；国家教育部博士点专项基金\[(2008)220\]； |
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| 中文摘要: |
| 克隆家蝇内源性β-葡萄糖苷酶（beta-glucosidase，BG）基因，建立两种原核表达体系和一种真核表达体系，分别检测表达产物的活性，比较不同表达体系对其表达水平及重组蛋白酶学性质的影响，为进一步研究和利用家蝇BG酶奠定基础。本研究根据BG酶基因的cDNA序列设计引物扩增BG酶基因成熟肽的基因片段，分别采用表达载体pET-28a(+)和pEGX-4T-1构建原核表达体系并在大肠杆菌BL21(DE3)中诱导表达。在65 kDa附近均出现特异性蛋白条带，进行Western Blotting鉴定证实重组质粒在其宿主菌E.coli BL21(DE3)中成功表达，命名为MDBG-1蛋白和MDBG-2蛋白。同时选用真核表达载体pPICZaA构建酵母分泌型表达体系在酵母细胞GS115中获得稳定的表达，将其命名为MDBG-3蛋白。采用七叶苷平板法和DNS法对三种表达体系所重组表达的蛋白进行酶活性鉴定和检测，显示3种表达体系所表达的融合蛋白均具有BG酶活性，其酶活力有所不同，且真核表达体系所表达的蛋白酶活性最高。本研究对家蝇β-葡萄糖苷酶基因表达的重组蛋白特性进行初步研究，为发现新的有效纤维素酶体系提供基础数据。 |
| 英文摘要: |
| Musca domestica-derived endogenous beta-glucosidase (BG) gene was cloned, and two prokaryotic expression systems and one eukaryotic expression system were constructed. The activities of expression products were detected to compare the effects of different systems on BG expression level and recombinant protein enzymatic properties, and to pave the way for studying and utilizing M. domestica-derived BG. According to the cDNA sequences of BG gene, we herein designed primers to amplify the gene fragments of mature peptide. Prokaryotic expression systems were constructed by using vectors pET-28a(+) and pEGX-4T-1 to induce expressions in Escherichia coliBL21(DE3). Specific protein bands all appeared at approximately 65 kDa. Western blotting verified that recombinant plasmids were successfully expressed in the host bacterium E.coli BL21(DE3), which were referred to as MDBG-1 protein and MDBG-2 protein respectively. Meanwhile, a secretory expression system of yeast was constructed by employing eukaryotic vector pPICZaA to induce stable expression in yeast cells GS115, which was referred to as MDBG-3 protein. The enzymatic activities of recombinant proteins expressed by the three systems were identified and detected by the aesculin agar test and DNS method. These proteins all exhibited various BG enzymatic activities, being highest in the eukaryotic expression system. We preliminarily studied the characteristics of recombinant proteins expressed by M.domestica-derived BG gene. The results provide valuable basic data for finding novel effective cellulase systems. |
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