枯草芽孢杆菌高效表达系统构建及饲用酶制剂的开发研究

发布时间：2018-05-21 11:31

本文选题：枯草芽孢杆菌 + 分泌　；参考：《中国农业科学院》2016年博士后论文

【摘要】：枯草芽孢杆菌是能形成芽孢的革兰氏阳性芽孢属的模式菌株,是工业、食品、医用和饲用重组蛋白的生产优秀的宿主表达系统之一。本论文主要是研究了饲用酶制剂在枯草芽孢杆菌中的分泌表达和分泌机制,并构建了一套蛋白质量控制体系的系列敲除菌株,高效的分泌重组蛋白。本论文的实验内容主要分为三部分：实验一,利用基因工程的方法优化了蛋白分泌信号系统,建立了一套针对特定目的蛋白CBP21的信号多肽筛选体系,获得了与特定的目的蛋白CBP21表达匹配的信号肽,实现目的蛋白的优化表达。结果显示：YlqB对CBP21的引导分泌能力最优。实验二,研究了淬灭酶在枯草杆菌中的分泌表达和其分泌机制。结果表明AI06BS在没有信号肽的引导下能分泌到胞外。AI06BS在自溶基因lytC和lytD突变和双突变工程菌株中以及自溶抗性工程菌株LM2531的表达分析,表明AIO6BS不是由于细胞的溶解释放到胞外的。另外,实验表明AIO6BS在Sec途径、Tat途径和ABC transporter途径的突变菌的表达模式和野生型菌株中的没有明显的变化。这些结果表明,AIO6BS可能是通过非传统的分泌途径分泌到胞外的。我们对AIO6BS蛋白的cys267进行了饱和突变,分析了所有可能的氨基酸替换对AI06BS的蛋白质的分泌表达影响。位点cys267饱和突变结果表明,位点cys267对淬灭酶的合成与分泌是非常重要的氨基酸。另外,我们把AIO6BS的同源蛋白、非同源蛋白和分泌伴侣融合在其C端。这些结果表明融合蛋白没有输出到胞外,可能AIO6BS不能作为信号来引导其他重组蛋白的跨膜运输。实验三,建了一套高效的分泌重组蛋白的蛋白质量控制体系的系列敲除菌株为重组蛋白的表达提高提供了一套工具。影响重组蛋白分泌的瓶颈之一就是所谓的质量控制体系中的蛋白酶。对于枯草芽孢杆菌的胞外蛋白酶、细胞膜和细胞壁蛋白酶有详尽的研究,但是对枯草芽孢杆菌的胞内的蛋白质量控制系统研究的较少,尤其是关于胞内蛋白酶都重组蛋白的分泌表达的影响较少。本研究基于同源重组的原理方法,构建了胞内蛋白酶的系列敲除株。我们用非传统分泌蛋白AIOBS和Sec途径分泌的CBP21作为报告基因,分别分析了胞内蛋白酶敲除菌株对两种不同类型的重组蛋白的分泌表达影响。与野生型菌株相比较,CBP21在BSΔYlbL和BSΔMlpA菌株没有分泌表达,而在其他的突变株的表达量都有不同程度的增加。AIO6BS在BSΔAlbF菌株不能分泌表达,而在其他的突变株的表达量都有不同程度的增加。
[Abstract]:Bacillus subtilis (Bacillus subtilis) is a model strain of Gram-positive spore forming spores. It is one of the excellent host expression systems for the production of recombinant proteins in industry, food, medicine and feed. In this paper, the secretory expression and secretory mechanism of feed enzyme in Bacillus subtilis were studied, and a series of knockout strains with protein quantity control system were constructed to secrete recombinant protein efficiently. The main contents of this thesis are divided into three parts: experiment 1. The protein secretion signal system was optimized by genetic engineering method, and a signal peptide screening system for specific target protein CBP21 was established. The signal peptide matched with the specific target protein CBP21 expression was obtained, and the optimal expression of the target protein was realized. The results showed that: YlqB had the best ability to guide and secrete CBP21. In experiment 2, the secretory expression and mechanism of quenching enzyme in Bacillus subtilis were studied. The results showed that AI06BS could be secreted into extracellular. AI06BS under the guidance of no signal peptide. The expression analysis of AIO6BS in lytC and lytD mutation and double mutant engineering strains and LM2531 showed that AIO6BS was not released to extracellular cells due to cell dissolution. In addition, the expression pattern of AIO6BS in mutants of Sec pathway tat pathway and ABC transporter pathway and wild-type strain were not significantly changed. These results suggest that AIO 6 BS may be secreted to extracellular via unconventional secretory pathways. The saturation mutation of cys267 of AIO6BS protein was carried out, and the effect of all possible amino acid substitutions on the secretion of AI06BS protein was analyzed. The results of cys267 saturation mutation showed that site cys267 was an important amino acid in the synthesis and secretion of quenched enzymes. In addition, we fused the homologous protein, non-homologous protein and secretory chaperone of AIO6BS at its C terminal. These results suggest that the fusion protein is not exported to the extracellular and AIO6BS may not act as a signal to guide the transmembrane transport of other recombinant proteins. In the third experiment, a series of knockout strains with high efficiency for secreting recombinant proteins were established, which provided a tool for improving the expression of recombinant proteins. One of the bottlenecks affecting the secretion of recombinant proteins is the protease in the quality control system. The extracellular protease, cell membrane and cell wall protease of Bacillus subtilis have been studied in detail, but the protein quantity control system of Bacillus subtilis is less studied. In particular, the secretion and expression of recombinant proteins of intracellular protease have little effect. Based on the principle of homologous recombination, a series of knockout strains of intracellular protease were constructed. We used CBP21 secreted by non-traditional secretory protein AIOBS and Sec as reporter genes to analyze the effects of intracellular protease knockout strains on the secretion and expression of two different types of recombinant proteins. Compared with wild-type strains, CBP21 was not secreted in BS 螖 YlbL and BS 螖 MlpA, while in other mutants, the expression of AIO6BS could not be secreted in BS 螖 AlbF. However, the expression of other mutants increased in varying degrees.
【学位授予单位】：中国农业科学院
【学位级别】：博士后
【学位授予年份】：2016
【分类号】：Q78;S816

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