目录
摘要...................................................................................................................……4
AbStract....................................................................................................................……5
第一篇:论文综述部分...............................................·...·····································……6
第一部分:刀一1，4一半乳糖基转移酶的研究进展.....................................................……6
前言..................................................................................................................……7
1、刀一1，4一半乳糖基转移酶家族的发现与克隆...............................................……8
2、两类介1，4一半乳糖基转移酶的区别....……，...............................................……8
3、刀一1，4一半乳糖基转移酶的蛋白分子结构...................................................……9
4、刀一1，4一半乳糖基转移酶的生物学功能.....................................................……10
参考文献...............................................................................................................……11
第二部分:毕赤酵母外源基因表达系统的应用及研究进展...........................……12
前言............................................................……，.............................................……13
1、毕赤酵母表达宿主菌.....................................................................……14
2、毕赤酵母表达载体.……，..................................····..··························……16
3、重组表达载体与毕赤酵母的转化及整合......................................……19
4、外源蛋白在其中的表达..................................................................……21
5、毕赤酵母表达系统与大肠杆菌表达系统的比较..........................……23
6、毕赤酵母表达系统的应用及研究进展..........................................……26
参考文献...............................................................................................................……29
第二篇:论文实验部分.............................................··········································……31
户1，4一半乳糖基转移酶在毕赤酵母中的克隆、表达及其活性的研究..............……31
前言...........................................……，..............................................................……32
实验流程图:............................................................................................····……33
1
.
实验材料和仪器....................................................···································……34
口一1，4一GT在毕赤酵母中的克隆及表达
1
.
1菌种..................................................................................................……34
1
.
2质粒..................................................................................................……34
1
.
3酶及蛋白制剂.…，，.........……，.…，..……，.........................................……34
1
.
4各种试剂盒.、................................……‘.............................................……34
1
.
5其他试剂..…，....................................................................................……35
1.6常用溶液及缓冲液..........................................................................……35
1
.
7培养液和固体培养基......................................................................……37
1.8各种电泳胶的制备..........................................................................……38
1
.
9主要仪器..........................................................................................……39
实验方法............................................................................……、................……40
2
.
1引物的设计与合成........................................................................……40
2
.
2刀一1，4一GT基因克隆片段的制备....................................................……40
2
.
3改造后的声1，4一GT基因片段的回收...........................................……40
2
.
4pUCm一GT的构建及鉴定...................……，....................................……41
2
.
5测序及序列分析............................................................................……42
2
.
6pPICgK一GT表达质粒的构建及鉴定...........................................……42
2
.
7PPCIgK一GT表达工程菌的制备...................................................……43
2
.
8Mu+t表型重组酵母的诱导表达实验............................................……45
2
.
9刀一1，4一GT的纯化............................................................................……46
2
.
10刀一1，4一GT的活性测定.....................................................................……47
2.11胞外分泌及胞内保留的介1，4一GT的酶活比较...........................……48
实验结果...................................................................................................……49
3
.
1刀一1，4一GT基因的改造和克隆.……。................................................……49
3
.
2阳性克隆的筛选及酶切鉴定...……，..............................................……50
3
.
3Fe一SOD基因的序列及分析..........................................................……51
3
.
5pPICgK一GT质粒的构建...............................................................……52
p一1，4一GT在毕赤酵母中的克隆及表达
3
.
6pPICgK一GT基因在毕赤酵母Gsl巧中的表达..........................……53
3
.
7刀一1，4一GT的纯化...........................................................……，..........……55
3
.
8刀一1，4一GT的活性测定..................................................……，........……56
3
.
9胞外分泌及胞内保留的介1，4一GT的活性比较...........................……58
4
.
讨论........................……，................……‘...……，........……，......................……62
4
.
1重组质粒pPICgK一GT线性化原因................................................……62
42巴斯德毕赤酵母的转化方法的选择..............................................……62
4
.
3表达系统的选择..............................……，.........................................……62
44毕赤酵母发酵培养基的选择..........……，.........................................……63
4
.
5活性测定..........................................................................................……63
5
.
总结.............................................................................……，......................……64
参考文献..............................................................................................................……65
附录一:(英文缩写词表).....................................................................……、.....……67
附录二:(质粒pPICgK图谱)..............................................................……，.....……68
附录三:碱提取质粒方法...................................................................................……69
附录四:LICI转化法..................……，.................................................................……70
致谢.....................................................................................................................……71

下载整篇论文发邮件至：lunwen163@126.com  附上标题 1日内回复