本科生基因工程实验论文

本科生基因工程实验论文

指导教师 王郑苏慧敏

学生 王大伟

专业 生物科学

年级 2012级

2015年8月10日

碱性磷酸酶基因

表达载体的构

建及在大肠杆菌中的表达内蒙古大学生命科学学院生物系本科生基因工程实验论文碱性磷酸酶基因表达载体的构建及在大肠杆菌中的表达 刘帆01011017 生物科学 摘要 碱性磷酸酶广泛分布于人体各脏器器官中 这种酶能催化核酸分子脱掉5’磷酸

基团 从而使DNA或RNA片段的5’-P末端转换成5’-OH末端。碱性磷酸酶在酶联

免疫等生化检测中有重要作用 是许多生化反应试剂盒的检测酶。从大肠杆菌提取的碱

性磷酸酶成本低 热稳定性好 而且能用基因方法实现蛋白质定向标记等。本文主要介

绍利用基因工程实验技术操作碱性磷酸激酶基因 将已经连接在pMD19-T载体上的碱性

磷酸激酶酶源基因用PCR扩增出837bp的碱性磷酸激酶的基因片段 与T载体连接后导

入感受态的DH5α菌中筛选鉴定。用双酶切切下碱性磷酸激酶的成熟基因片段 插入带

有6个组氨酸标签的原核表达载体pET-his 导入BL(21)DE3感受态菌中诱导表达碱性

磷酸激酶蛋白质 用SDS-PAGE鉴定。

关键词 碱性磷酸酶基因表达载体大肠杆菌SDS-PAGE Construction and Expression of Alkaline Phosphatase Expression Vector in

E.coli

ABSTRACT Alkaline phosphatase is a widely distributed in human organs in various

organs , the enzyme catalyzes the nucleic acid molecule off the 5 ' phosphate group , so that

the DNA or RNA fragments of the 5'-P terminal converts 5'-OH ends . Alkaline phosphatase

in ELISA and other biochemical detection plays an important role in many biochemical reactions kit detection enzymes. Extracted from E. coli alkaline phosphatase , low cost, good thermal stability, but also can be used genetic methods to achieve protein orientation markings.Main of this article describe the gene engineering operation in BAP gene which has already connected with pMD-1-T vector amplified by PCR in which can get 837bp

nattokinase gene fragments ,then liBAPing with T vector that transform it into DH5α bacteria which were screened after identification. The mature peptide can be cutted by double-digested and insert with six histidine-tagged prokaryotic expression vector pET-his. At last, induced the protein expression of nattokinase in the expression of E.coli BL (21) DE3. Eventually ,

identify that by SDS-PAGE. KEYWORDS: Bacterial alkaline phosphatase , gene expression, vector E. coli, SDS-PAGE 内蒙古大学生命科学学院生物系本科生基因工程实验论文目录引言 (1)

材料与方法...........................................................................................................

1材料.............................................................................................................

1.1试剂、仪器及实验用具...................................................................

1.2菌种..................................................................................................

1.3质粒..................................................................................................

1.4常用溶液的配制..............................................................................

2方法.............................................................................................................

2.1引物的设计......................................................................................

2.2 碱性磷酸激酶基因的PCR扩增.....................................................

2.3 DH5a和BL21感受态的制备..........................................................

2.4 pMD19-T-BAP的构建及转化和检测..............................................

2.5 pET-his及BAP的酶切和回收......................................................

2.6 pET-his-BAP的构建及转化和检测..............................................

2.7 pET-his-BAP的诱导表达..............................................................

2.8 细菌碱性磷酸酶表达的SDS-PAGE检测.......................................

3结果与分析................................................................................................