外源基因表达

1、第七章第七章 外源基因表达外源基因表达n一、表达机制表达机制n1起始转录起始转录：转录起始的速率是gene表达的限速步骤，用诱导型启动子（lac、trp、 pl/pr、 tac、组成型启动子：t7）n2mrnamrna延伸与稳定延伸与稳定：正常终止序列、减少dna反向转录产生反义mrna、polya掺入对3正确加工n3mrnamrna有效翻译有效翻译：aug首选起始codon、sd序列与16srrna互补结合，含有至少aggagg中4个碱基、sd与atg间距离3-9bp、翻译起始区周围不形成明显二级结构、codon选择性相近（没有明显偏好性不同）n4prpr稳定性稳定性：构建融合pr、分泌pr

2、、包涵体pr、pr水解酶缺陷型受体细胞第七章第七章 基因表达系统n外源基因表达系统外源基因表达系统n泛指目的基因与表达载体重组后，导入合适受体细胞，并在其中有效表达，产生目的基因产物n1e. colie. coli表达系统表达系统：n常用常用lac & taclac & tac：以lac操纵子调控机制为基础：lacp laco 结构基因（lacz, y, a），laci 阻遏pr，iptg诱导，npl & prpl & pr：以phage溶原/溶菌循环nt7t7启动子启动子：t7rna聚合酶识别序列在pet中mcs上端，rna聚合酶位于bl21(de3)基因组中，上端为lac启动子调控，ipt

3、g第七章第七章 基因表达系统n外源外源prpr表达形式表达形式：n包涵体pr：n融合pr：n寡聚型pr：多表达单元重组、多顺反子、多编码序列第七章高效表达策略第七章高效表达策略n优化表达载体设计优化表达载体设计：启动子和mrna中sd序列优化，强启动子，sdatg间距，rbs茎环结构n稀有codon trna，n外源mrna稳定性、n外源pr稳定性：pr分泌胞外，pr e缺失，pr e敏感序列修饰，pr稳定性因子n优化发酵过程：工艺 生物学：菌体生长，表达条件，产物总量the expression vectormost important elements in the vectoruprom

4、oter f-35: ttgaca (e. coli )f-10: tataat (e. coli )uterminator (hairpin)uribosome binding site (rbs)uselection markeruvector replication sequence (ori)upolylinker (mcs)in the gene/vectorustart (atg)ustop (1-2 taa, (tag, tga)usignal peptidetac promoterhybrid promoter fusion from lac and trp promoters

5、+ combines strong rna polymerase binding sequences+ 5 x stronger than lac promoter+ 20-30% of total protein+ induced by iptg l lpl promoterisolated from l bacteriophage - l repressor (ci) binds to lpl promoter+/- ci 857 variant heat inducible 30 degrees, expression+/-alternatively use special strain

6、s with l repressor under control of trp promoter which is induced by trp addition+tightly regulated - good for toxic proteins target protein target genelplrepressor geneci repressortrp promotertrp+t7 system (pet-system)nt7 rna polymerase gene controlled by lac promoterniptg induces t7 rna polymerase productionnt7 rna polymerase transcribes gene controlled by t7 promoterntarget gene can not be activated by host rna polymerasenspecial strain needednlaco in pet and plyss reduces leakinessgal systemgene cloned under control ofthe gal4 promoterinduction by galactose as thesole carbon