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
分类号: Q78 编号:
本科生基因工程实验论文
指导教师： X X X
学 生： X X
专 业： 生 物 科 学
年 级： 2009
2012 年 8 月 24 日
纳豆激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2012年本科生基因工程大实验论文
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目 录
摘要 ...................................................................................................................... .2
绪论 ....................................................................................................................... 3
材料与方法 ........................................................................................................... 4
1材料 ............................................................................................................. 4
1.1试剂及仪器 ....................................................................................... 4
1.2菌种 .................................................................................................. 5
1.3质粒 .................................................................................................. 5
1.4常用溶液的配制 .............................................................................. 5
2方法 ............................................................................................................. 6
2.1引物的设计 ...................................................................................... 6
2.2 NK基因的PCR扩增 ........................................................................ 7
2.3 DH5a和BL21(DE3)感受态的制备 ................................................. 7
2.4 pMD19-T-NK的构建及转化和鉴定 ................................................ 8
2.5 pET-trx、pET-NK、pUC-Nk的酶切与回收 .................................. 9
2.6 pET- trx-NK的构建及转化和检测 ............................................. 11
2.7 pET-trx-NK的诱导表达 .............................................................. 12
2.8 pET-trx-NK表达的SDS-PAGE检测 ............................................ 13
结果 ..................................................................................................................... 15
1. NK基因的PCR扩增 ................................................................................ 15
2. pMD19-T-trx的构建与鉴定 .................................................................. 16
3. pET-trx、pET-NK、pUC-Nk的酶切与回收 .......................................... 16
4. pET-trx-NK的构建与检测 .................................................................... 18
5. pET-trx-NK的诱导表达与SDS-PAGE检测 .......................................... 19
讨论 ..................................................................................................................... 20
参考文献 ............................................................................................................. 21
致谢 ..................................................................................................................... 21
感想 ..................................................................................................................... 22 内蒙古大学生命科学学院生物系2012年本科生基因工程大实验论文
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纳豆激酶基因表达载体的构建及在大肠杆菌中的表达
摘要
纳豆激酶是一种枯草杆菌蛋白激酶。经研究发现纳豆激酶具有纤溶活性和溶栓能力,
可治疗和预防血栓病。此外，纳豆激酶还具有降低血液黏度、降血脂、降胆固醇, 改善血液循环状况, 维持血细胞的正常形态和功能等作用。本文主要介绍利用基因工程实验技术操作纳豆激酶基因，将已经连接在pMD18-T载体上的纳豆激酶酶源基因用PCR扩增出837bp的纳豆激酶的基因片段，与T载体连接后导入感受态的DH5α菌中筛选鉴定。用双酶切切下纳豆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-trx，导入 BL(21)DE3感受态菌中诱导表达纳豆激酶蛋白质，用SDS-PAGE鉴定。
关键词：纳豆激酶，基因表达，载体，大肠杆菌
Constructing Expression Vector and Expressing nattokinase in E.coli
ABSTRACT
Nattokinase is a kind of subtilopeptidase. The study found that Nattokinase
has activity of fibrinoltic which can treat and prevent thrombosis. In addition,
Nattokinase can also lower blood viscosity, blood fat, cholesterol, improve blood
circulation and maintain normal blood cells form and function and so on. Main of
this article describe the gene engineering operation in nottokinase gene which has
already connected with pMD-18-T vector amplified by PCR in which can get
837bp nattokinase gene fragments ,then linking 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-trx. At last, induced the protein expression of nattokinase
in the expression of E.coli BL (21) DE3. Eventually , identify that by SDS-PAGE.
KEY WORDS: nattokinase, gene expression, vector，E. coli
