《外源基因表达》PPT课件.ppt

第七章 外源基因表达,一、表达机制1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）2MRNA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3MRNA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）4Pr稳定性：构建融合pr、分泌pr、包涵体pr、pr水解酶缺陷型受体细胞,第七章 基因表达系统,外源基因表达系统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1E.coli表达系统：常用lac&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，PL&PR：以phage溶原/溶菌循环T7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，IPTG,第七章 基因表达系统,外源pr表达形式：包涵体pr：融合pr：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,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茎环结构稀有codon tRNA，外源mRNA稳定性、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 vector,Most important elements in the vectorPromoter-35:TTGACA(E.coli)-10:TATAAT(E.coli)Terminator(hairpin)Ribosome binding site(RBS)Selection markerVector replication sequence(ori)Polylinker(MCS)In the gene/vectorStart(ATG)Stop(1-2 TAA,(TAG,TGA)Signal peptide,tac promoter,Hybrid promoter fusion from lac and trp promoters+Combines strong RNA polymerase binding sequences+5 x stronger than lac promoter+20-30%of total protein+Induced by IPTG,lPL promoter,Isolated from l bacteriophage-repressor(cI)binds to lPL promoter+/-cI 857 variant heat inducible 30 degrees,expression+/-Alternatively use special strains with repressor under control of trp promoter which is induced by Trp addition+Tightly regulated-good for toxic proteins,Repressor gene,trp promoter,T7 system(pET-system),T7 RNA polymerase gene controlled by lac promoterIPTG induces T7 RNA polymerase productionT7 RNA polymerase transcribes gene controlled by T7 promoterTarget gene can not be activated by host RNA polymeraseSpecial strain neededlacO in pET and pLysS reduces leakiness,GAL system,Gene cloned under control ofthe GAL4 promoterInduction by galactose as thesole carbon sourceStronger induction by PGAL1Gal4p transcriptional activator-1-5%of total protein-Leaky promoter,赵子昂,