文档介绍：目录
摘要 1
1前言 2
果蝇精巢的基本特征 2
fasⅢ基因 2
3
抗菌素的抗性工作原理 3
2 实验材料与方法 3
3
实验仪器 3
4
5
5
5
5
酶切 6
7
DN***段的体外连接(目的基因导入质粒载体) 7
8
8
9
PET28a-fasⅢ质粒的少量制备 9
10
三、实验结果与分析 11
11
12
琼脂糖凝胶DNA回收试剂盒回收DNA 12
菌落PCR鉴定 12
质粒制备的鉴定 13
重组质粒双酶切鉴定 13
四、讨论 14
参考文献 14
致谢 15
果蝇精巢fasⅢ基因原核表达载体的构建
摘要:果蝇fasⅢ基因是蛋白编码类基因,在精巢中fasⅢ基因主要在hub cell中表达,本实验运用基因克隆技术,以PET28a质粒为载体,选用BamHI、HindⅢ
两种酶做双酶切,将目的基因fasⅢ导入质粒载体PET28a,从而构建原核表达载体PET28a-fasⅢ,再将重组质粒转化到大肠杆菌TOP10中,实现fasⅢ基因的原核表达。实验所用质粒载体用卡那抗性(Kanr )标记,只有转化成功的大肠杆菌才能在涂有卡那霉素的LB培养基中生长。最终目的是实现fasⅢ蛋白的原核表达与纯化,为以后制备多克隆抗体奠定基础。
关键词:基因克隆;fasⅢ基因;原核表达载体;原核表达;
The construction of fas III prokaryotic expression vectors
Abstract: Drosophila Fas gene is protein coding gene in testis, mainly expressed in hub cell. In this experiment, by means of gene cloning technique, the target gene FasIII was inserted into the plasmid vector PET28a which was digested by two enzymes of BamHI and HindⅢ. so as to construct the prokaryotic expression vector PET28a-fasⅢ, then the binant plasmid was transformed into Escherichia coli TOP10, achieve Fas III gene prokaryotic expression. The plasmid vector was marked with kanamycin resistant (Kanr) , Only was transformed essfully Escherichia coli can grow in LB medium which was coated with kanamycin. The ultimate aim is to realize the prokaryotic expression and purification of Fas III protein, for the future lay the foundation for preparing polyclonal antibody.

Key words: Gene cloning;fasⅢ;the prokaryotic expression vector;
prokaryotic expression;
1前言
果蝇精巢的基本特征
有两个精巢,形状为长而卷曲状的管状,图示1-2 [3]显示出了精巢前端的结构。精巢里有两类干细胞:生殖干细胞GSC和体节干细胞SSC。GSC有7-9个,位于精巢的顶端,与Hub cell(HC)直接接触,HC是精巢GSC微环境的重要组成部分[4],SSC包裹在GSC周围,和HC共同构成了GSC的“niche”[5]。同卵巢GSCs一样,精巢的GSCs也通过不对称分裂产生2个子代细胞,一个仍然处于微环境中,与HC直接接触,成为新的GSC,另一个远离微环境,发育为GB(