论文总字数：21136字

摘 要

基因工程是70年代发展起来的一项具有革命性的研究技术，在遗传学、分子生物学、生物化学、微生物学、等诸多学科的基础上发展而来，可概括为∶分、切、连、转、选。最终目的在于通过相应技术手段，将目的基因导入寄主细胞，在宿主细胞内目的基因被大量的复制。基因是细胞内DNA分子上具有遗传效应的特定核苷酸序列的总称，是具有遗传效应的DNA分子片段。并被称为DNA重组技术，核心基因工程技术是基因表达的技术。该技术为创建任何一种生物接受另一种生物的遗传物质并为细胞提供定性遗传稳和功能提供可能，同时也为我们对遗传物质的操作，改变基因功能，基因的调节表达。基因工程技术已被广泛应用于基础生命科学研究中各学科，成为微生物学，生物学，甚至宏观强有力的研究工具。农业，医药，食品，化工，环境方面还承担诸多卓有成效的应用。

棒状杆菌的设计为研究对象，选择一个常用的绿色荧光蛋白GFP基因工程进行实验，它具有以下优点：易于检测，荧光稳定，无毒害，通用性强，易于构建的载体，可用于体内定位。通过对基因组序列分析及谷氨酸棒杆菌操纵子基因启动子序列的引物和靶向基因操纵子的启动子序列，gRNA载体构建，转化为谷氨酸棒状杆菌感受正常细胞，细菌培养和抗生素药物筛选候选基因的克隆、测序和PCR技术，利用荧光蛋白凝胶电泳测定候选克隆分析和不同培养条件的筛选，对粉刺的绿色荧光蛋白修饰的表达构建实验方案的优化谷氨酸棒杆菌。

关键词：谷氨酸棒杆菌 表达载体 分泌蛋白

Construction of an expression vector for secretory production of proteins in Corynebacterium glutamicum

Abstract

Genetic engineering is developed in the 70 s a revolutionary research techniques, in genetics, molecular biology, biochemistry, microbiology, and develops on the basis of many disciplines, such as can be summarized as: points, cut, even, turn, optional. Final purpose is to through the corresponding technical means, the purpose gene into host cells, within the host cells by a large number of duplicate gene. Gene is on of DNA molecules in a cell with the floorboard of the specific nucleotide sequence of genetic effect, is a genetic effect of segments of the DNA molecule. Is known as recombinant DNA technology, the core of genetic engineering technology is the technology of gene expression. The technology to create any kind of creature to accept another creature's genetic material and provide qualitative cell genetic stability and possible functions, but also to the operation of the genetic material for us, and change the gene function and regulation of gene expression. Genetic engineering has been widely applied in life science research in various disciplines, become a microbiology, biology, even macro powerful research tools. Agriculture, medicine, food, chemical industry, environmental aspects also undertake many successful applications.

Corynebacterium design as the research object, select a common green fluorescent protein, GFP gene engineering experiment it has the following advantages: easy to detect, fluorescent stability, non-toxic, strong commonality, easy to build the carrier, can be used to locate in the body. Through the analysis of genome sequences and glutamic acid bacillus operon gene promoter sequences of primers and targeted gene operon promoter sequences, gRNA carrier construction, into glutamate corynebacterium

feel normal cells, bacterial culture and antibiotics drug screening candidate gene cloning, sequencing and PCR technology, using fluorescent protein gel electrophoresis to determine the candidate cloning analysis and screening of different culture conditions, building of acne expression of green fluorescent protein modification experimental scheme optimization of glutamic acid bacteria.

Keywords: Corynebacterium glutamicum； expression vector；secretory protein

目 录

摘 要 I

第一章 文献综述 1

1.1引言 2

1.2各表达系统 2

1.2.1大肠杆菌表达系统 3

1.2.2 酵母表达系统 3

1.2.3 动物细胞表达系统 3

1.3谷氨酸棒杆菌表达系统 4

1.4重组蛋白应用 4

1.5本课题的主要研究内容 5

1.5.1研究内容 5

1.5.2研究手段 5

第二章 材料与方法 6

2.1材料与仪器 6

2.1.1 实验材料 6

2.1.2 主要试剂 6

2.1.3主要仪器 6

2.2实验方法 7

2.2.1 谷氨酸棒杆菌的培养、质粒转化及质粒提取 7

2.2.2引物的设计与表达质粒载体的构建 9

2.2.3聚合酶链接式反应（PCR） 12

第三章 结果与讨论 14

3.1 3.1 带谷氨酸棒杆菌启动子及分泌信号肽序列的GFP基因的T-克隆

14

3.2pXMJ19-H36GFP构建 16

3.3 pXMJ19-H36GFP转化谷氨酸棒杆菌ATCC13032 17

第四章 总结与展望 18

4.1总结 18

4.2 展望 18

参考文献 19

致 谢 22

第一章 文献综述

1.1引言

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