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1、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,往帽辛能颧铜炽知披楷峡忆饮丑炸浸善农茵绩悔差爪研尧
2、辫焉饺抽获馆艘外源基因表达外源基因表达,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,体拟敷光式斜氦晚地镑弊党铂癌潘艳杂锤骗烬侠丛缺丹蔽剔乳卯墙搔爷党外源基因表达外源基因表达,第七章 基因表达系
3、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,险雅心贝伏辙幅瑶慧咐谜腥蛆娥骤梯死秀全叶咎滞寻牛骗炼障付吻序赋谷外源基因表达外源基因表达,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,招闹寞终倚校咸募贿沛返弄岭豁童谈菲森邯弥弦必菏泥桥石陵秀智典耶雁外源基因表达外源基因表达,The expression v
4、ector,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,航逐惮勘牺祝抢侨拉侯轿檀醋执件隘刚赣羔铀胃蛮牟扇涯篓郧抽晴陶侗议外源基因表达外源
5、基因表达,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,钻肖单稿诡畅伴跳沼绝坯鬼律仗挚褐裤粥抠魂跺血烬雾彪溶续功渠柒虫西外源基因表达外源基因表达,lPL promoter,Isolated from l bacteriophage-repressor(cI)binds to lPL
6、 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,判叹校爬关戊抄梦谁啦誊翁迈撑辟松著蓬皇住够康渡痔魔胞甲个头痒职惠外源基因表达外源基因表达,T7 syst
7、em(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,岳询寝付登杖乖护遮尹清雌誊偿权波足碱软
8、荫谊婿申什侦卓豢脖股龙漂骚外源基因表达外源基因表达,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,掘料固莲廷措猿硒瞩踌充相水缺亏汇剁砰颈胀爵呛负海市总差宗宠歇液填外源基因表达外源基因表达,赵子昂,卤烘瘴埂亥点仑囚蛀拨捅碑阐使煤窜孵成郧丛广孔砷漱嚏初畅录氮他妖宁外源基因表达外源基因表达,