摘要
几丁质酶是昆虫几丁质降解过程中的重要酶类。本研究旨在利用大肠杆菌(Escherichia coli)原核表达系统获得高纯度的MDCht2重组蛋白,并从几丁质酶活性及抗菌活性两方面来初步探讨MDCht2的生物学功能。MDCht2的cDNA序列设计包含酶切位点Eco RⅠ、HindⅢ和6×His标签的引物,以家蝇3龄幼虫的cDNA为模板进行PCR扩增,以pET28a(+)为载体构建重组质粒,并转化到大肠杆菌Transetta (DE3)中,经IPTG诱导,表达产物用SDS-PAGE凝胶电泳鉴定,利用Ni离子亲和层析技术纯化MDCht2重组蛋白,Western Blot技术及质谱分析鉴定纯化蛋白。以4MU-(GlcNAc)3为底物,测定重组蛋白的酶活性;采用微量液体稀释法分析重组蛋白对金黄色葡萄球菌、大肠杆菌、白假丝酵母菌的抗菌活性。成功克隆MDCht2基因,构建了具有正确序列的原核重组表达质粒,将重组质粒导入Transetta(DE3)中,经镍柱纯化获得带His标签的重组MDCht2蛋白,质谱分析显示重组蛋白与MDCht2蛋白序列一致。酶活分析表明,不同浓度的MDCht2重组酶均有几丁质酶活性,呈现一定的量效关系;该重组蛋白的最适pH值为4.0,在pH=8.0时稳定性最好;最适温度为35℃;金属离子和Tris对MDCht2酶活均有抑制作用。抗菌活性结果显示,MDCht2蛋白对金黄色葡萄球菌,大肠杆菌均无抑菌效果,但对白假丝酵母菌有抑制作用,MIC值为225μg/m L,MBC(Minimum bactericidal concentration)值为450μg/mL。本研究成功获得了MDCht 2重组蛋白,体外检测有较强的几丁质酶活性和抗真菌活性,为进一步研究该酶的功能奠定一定基础。
Chitinase is an important enzyme in the process of insect chitin degradation. This study aims to obtain high purity MDCht2 recombinant protein by E. coli prokaryotic expression system, and to preliminarily explore the biological functions of MdCht2 in terms of chitinase activity and antibacterial activity. The primers containing the restriction enzyme sites Eco R Ⅰ, Hind Ⅲ and 6×His were designed according to the cDNA sequence of MDCht2.The cDNA of housefly 3 rd instar larvae was used as a template for PCR amplification, and pET28 a(+) was used as a vector to construct a recombinant plasmid, which was transformed into E. coli Transetta(DE3). The expression bacteria were induced by IPTG, and the expression products were identified by SDS-PAGE gel electrophoresis.The MdCht2 was purified by Ni ion affinity chromatography technology, and the purified protein was identified by Western Blot technology and mass spectrometry. The enzyme activity of the recombinant protein was determined by using 4 MU-(GlcNAc)3 as substrate. The antibacterial property of the recombinant protein against Staphylococcus aureus, Escherichia coli and Candida albicans was analyzed by micro-liquid dilution method. The MDCht2 gene was successfully cloned and the prokaryotic recombinant expression plasmid with the correct sequence was constructed. The recombinant plasmid was introduced into Transetta(DE3). The His-tagged recombinant MDCht2 protein was obtained by nickel column purification. The mass spectrometric analysis showed that the recombinant protein was consistent with the MDCht2 sequence. Enzyme activity analysis showed that different concentrations of recombinant MDCht2 had chitinase activity, showing a dose-effect relationship;The optimum pH value of the recombinant protein was 4.0, and the stability was best at pH=8.0;The optimum temperature was at 35 ℃;metal ions and Tris have inhibitory effects on MDCht2 enzyme activity. The results of antibacterial activity showed that MDCht2 protein had no antibacterial effect against Staphylococcus aureus and Escherichia coli, but had inhibitory effect on Candida albicans. The MIC value was 225 μg/mL and the MBC value was 450 μg/mL. In this study, MDCht 2 recombinant protein was successfully obtained, and it had strong chitinase activity and antifungal activity in vitro, which laid a foundation for further study of the function of the enzyme.
作者
苏佩佩
赵欣宇
马慧玲
李妍
杨隆兵
国果
Su Peipei;Zhao Xinyu;Ma Huiling;Li Yan;Yang Longbing;Guo Guo(The Key and Characteristic Laboratory of Modern Pathogen Biology,School of Basic Medical Sciences,Guizhou Medical University,Guiyang,550025;Key Laboratory of Environmental Pollution Monitoring and Disease Control of Ministry of Education,Guizhou Medical University,Guiyang,550000)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2021年第1期72-80,共9页
Genomics and Applied Biology
基金
国家自然科学基金项目(No.81560337,No.81760647)资助。
作者简介
通信作者:国果,guoguojsc@163.com。
