内蒙古大学生物技术基因工程实验论文2.docVIP

分类号: Q78 编号: 本科生基因工程实验论文 指导教师：王 郑 学 生： 专 业： 生物科学 年 级： 2009级 2012年月日 ……………………………………………………………………………1 1 绪论…………………………………………………………………………2 2 材料与方法 ...2 2.1 材料2 2.1.1 菌株与质粒 ………………………………………………2 2.1.2主要试剂及其配制………………………………………… 2 试剂…………………………………………………2 试剂与培养基的配制………………………………2 主要仪器……………………………………………5 2.2 方法5 2.2.1纳豆激酶（NK)引物的设计 5 2.2.2 NK基因的PCR扩增及电泳检测 5 2.2.3 DH5a和BL21(DE3)感受态的制备 7 2.2.4 pMD19-T-NK的构建及转化和鉴定 8 2.2.5 pET-Trx及pMD19-T-NK的酶切与回收 9 2.2.6 pET-Trx-NK的构建及转化和检测 9 2.2.7 pET-Trx-NK的诱导表达及SDS检测 9 3 结果与分析 11 3.1NK基因的PCR扩增 11 3.2. pMD19-T-NK的构建与鉴定 11 3.3 pET-Trx及pMD19-T-NK的酶切和回收 12 3.4 pET-Trx-NK的构建与检测 12 3.5 pET-Trx-NK的诱导表达与SDS检测 13 结论与讨论 13 参考文献 14 致谢与感想 16  纳豆激酶基因表达载体的构建及在大肠杆菌中的表达表达Constructing Expression Vector and Expressing NK in E.coli Author： Supervisor: Wang Zheng ABSTRACT Nattokinase(NK) is a good natural protease thrombolytic substance. It is Gene length about for 837bp. This experiment through building Nattokinase gene expression carrier, make it smoothly expression in Escherichia coli. We acquired Nattokinase gene from clonened in PMD-18-T carrier, with PCR technology expansion increased , we obtained the 837bp of DNA fragment , and connected it with PET-trx carrier , then, we transferred it to DH5a bacteria , filtered out recombinants. We got nattokinase gene fragment resulting from cutting the recombinants by Restriction endonucleaset , inserted the gene fragment into expression vector puck-NK, Transferred it into DH5a bacteria again , filtered out recombinants, and then ,we Transferred the recombinant gene into BL21 (DE3) bacteria, through induced by IPTG , nattokinase Induction products measureed by SDStechnologies. KEY WORDS: Nattokinase, gene expression, vector，E.co弃上清，并将离心管倒置以倒尽上清液。加入 1mL 冰冷的