KLHL21蛋白羧基端蛋白质标签的敲入及初步分析
发布时间：2019-06-27 08:49
【摘要】：KLHL(Kelch-like)蛋白家族是一类在进化中非常保守的蛋白质,其典型的特征是都含有一个N端的BTB结构域、5-6个羧基端的Kelch结构域,以及介于它们之间的BACK结构域。该家族蛋白的蛋白质序列在进化中均非常保守,提示其在机体内可能具有非常重要的生理功能。已有的研究表明,KLHL家族蛋白在细胞内多通过与Cu13蛋白相互结合而形成E3泛素连接酶,催化底物蛋白质的泛素化修饰,在机体的炎症、氧化应激、细胞有丝分裂、胚胎发育以及淋巴生成等多种生命活动中发挥着重要的作用,但某些家族成员在细胞中还具有与催化蛋白质泛素化修饰无关的其他功能。作为KLHL蛋白家族中的一员,KLHL21蛋白含有一个BTB结构域、BACK结构域与5个Kelch结构域。2009年,MaerkiS等率先报道了,KLHL21可通过与Cullin3蛋白相互结合形成有功能的E3泛素连接酶,催化蛋白激酶Aurora B的泛素化修饰,在细胞有丝分裂后期参与调控染色体过客复合体(Chromosomal passenger complex,CPC)从染色体向纺锤体中间区(spindle midzone)的转运,采用siRNA抑制其在细胞中的表达可导致有丝分裂过程中胞质分离(cytokinesis)的异常。来自该研究团队最新的研究结果表明,KLHL21还可通过催化EB1蛋白的泛素化修饰而参与对细胞运动的调控。而我们前期的研究揭示,KLHL21是NF-κB信号通路中关键的IKK激酶复合物的特异性结合蛋白,可通过与IKKβ的激酶结构域结合而抑制其激活,差异性地调控NF-κB下游靶基因的诱导表达,且该过程不依赖其E3泛素连接酶活性。在前期的研究工作中,我们曾试用了来源自多个生物公司的KLHL21抗体,但普遍存在灵敏性与特异性不高的问题,为了后续研究的需要,本研究尝试采用基因组编辑技术在HEK293T细胞内源性表达的KLHL21蛋白的羧基端敲入mCherry与FLAG双蛋白质标签。目前在实验室中常用的基因组编辑技术有ZFN(zinc finger endonuclease,ZFN)、TALEN(transcription activator-like effector nuclease)CRISPR/Cas(Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated)等三种,其中以 2013 年才首次报道的 CRISPR/Cas系统因其操作相对简单、低成本与基因组编辑的高效性而备受大家的关注。CRISPR/Cas系统是存在于细菌和古生菌的一种适应性免疫防御系统,通过序列特异性的RNA介导,切割并降解噬菌体、质粒等外源入侵的遗传物质。当病毒首次入侵时,细菌将病毒基因的一段DNA序列整合进自身的CRISPR间隔序列区,其在病毒再次入侵时转录生成crRNA(CRISPR RNA)前体,前体crRNA再经过加工后形成与外源性基因DNA序列匹配的成熟crRNA,通过与病毒基因组的同源DNA序列的识别而介导Cas蛋白切割病毒DNA将其沉默。目前已经被鉴定的CRISPR/Cas系统共有3种类型,其中Ⅱ型CRISPR/Cas系统仅依赖于单一的Cas蛋白Cas9即可完成免疫防御,由于其操作简单、要求低而被广泛应用于基因组编辑。CRISPR/Cas9 系统由5'端反式激活核糖核酸(trans-activating CRISPR RNA,tracrRNA)序列区、中间部分的Cas9基因编码序列和3'端CRISPR间隔序列区在DNA链上线性排列组成。CRISPR间隔序列区由高度保守的回文序列/反向重复序列R(Repeats)和长度约为30nt的间隔序列S(Spacers)有规律的间隔排列组成。Spacers经转录和加工形成小干扰RNA分子crRNA,通过碱基互补配对识别靶DNA序列并引导Cas核酸酶对其进行切割。crRNA与靶DNA的互补配对区长度通常为20nt,这一过程中需要tracrRNA的参与,其通过与crRNA的部分序列互补配对而募集Cas9蛋白。在实际应用中,可以将crRNA与tracr-RNA编码序列整合在一起,采用RNA转录酶Ⅲ启动子如H1与U6进行表达,从而获得向导RNA(guideRNA,gRNA),此外也可以通过转染体外合成或体外转录的gRNA来介导对靶DNA的切割。在gRNA识别的靶DNA序列的下游中含有一个PAM序列(protospacer adjacent motifs),通常为NGG三核苷酸序列(N代表任意核苷酸),是gRNA识别靶DNA序列所必需的。Cas9蛋白具有两个核酸酶活性中心HNH与RuvC,它们分别切割靶DNA双链中的一条链,导致DNA双链的平末端断裂,继而通过细胞的DNA同源重组或非同源性末端连接修复机制对断裂的DNA进行修复,导致靶DNA序列的缺失与插入,引起靶基因读码框移位而达到敲除其在细胞内表达的目的;或者在外源同源重组模板存在时对靶DNA序列进行替换、或者敲入一段外源DNA序列,通过这种对靶基因DNA序列进行的定点编辑可实现对细胞内源性表达的靶基因功能或表达水平的调控,促进对靶基因功能与表达调控机制的深入研究。研究目的:利用CRISPR/Cas9基因编辑技术构建KLHL21蛋白羧基端敲入蛋白标签的HEK293T稳定细胞株。研究方法:1、构建含有mCherry-FLAG双蛋白质标签的KLHL21基因同源重组模板质粒,以及靶向人KLHL21基因终止密码子附近DNA序列的gRNA表达载体;2、将线性化的KLHL21基因同源重组模板质粒、gRNA表达质粒和人源化的hCas9表达载体共转染入HEK293T细胞中;3、采用Blasticidin抗生素筛选获得敲入目标蛋白质标签的HEK293T阳性细胞株;4、采用PCR、Western Blot和免疫沉淀技术验证筛选获得的阳性HEK293T细胞株;5、构建Cre重组酶表达载体pCDNA3.1-Cre,将其转染筛选获得的敲入了目标蛋白质标签的HEK293T细胞株,利用Cre/Loxp位点特异性重组系统将两端含有同向Loxp位点的Blasticidin筛选标记基因序列切除。研究结果:1、成功构建了用于人KLHL21蛋白羧基端敲入mCherry与FLAG蛋白标签的载体pKCFD。2、成功构建了靶向人KLHL21基因终止密码子附近序列的gRNA表达载体 pHG4-21A 与 pHG4-21B。3、利用CRISPR/Cas9系统成功将mCherry与FLAG蛋白标签敲入了内源性KLHL21蛋白的羧基端。4、采用抗生素Blasticidin成功筛选到在内源性表达的KLHL21蛋白羧基端敲入目标蛋白质标签mCherry-FLAG的HEK293T阳性细胞株。5、通过Cre-Loxp位点特异性重组系统成功切除两端含有同向Loxp位点的Blasticidin筛选标记基因序列。结论:我们成功地采用CRISPR/Cas9基因编辑技术在HEK293T细胞中内源性表达的KLHL21蛋白羧基端敲入mCherry与FLAG蛋白标签,为后续KLHL21蛋白质的功能研究提供了便利,同时也建立了一个研究其他内源性表达基因功能的技术平台。
[Abstract]:The KLHL (Kelch-like) family of proteins is a class of very conserved proteins in evolution, typically characterized by a BTB domain at the N-terminus, a Kelch domain at the 5-6 antigen base, and a BACK domain between them. The protein sequence of the family protein is very conservative in the evolution, suggesting that it may have a very important physiological function in the body. The research has shown that the KLHL family protein can form the E3 ubiquitin ligase by binding to the Cu13 protein in the cell, and the ubiquitin modification of the substrate protein is catalyzed, and the inflammation, the oxidative stress and the cell mitosis of the body are carried out on the body. Some of the family members also have other functions unrelated to the catalytic protein ubiquitination modification in the cell. As a member of the KLHL protein family, the KLHL21 protein has a BTB domain, a BACK domain and five Kelch domains. In 2009, MaerkiS and the like were the first to report that the KLHL21 can catalyze the ubiquitination modification of the protein kinase Aurora B by forming a functional E3 ubiquitin ligase with the Cullin3 protein, In the later stage of cell mitosis, it is involved in the transfer of the chromosome-passing complex (CPC) from the chromosome to the middle zone of the spindle, and the use of siRNA to inhibit its expression in the cell can lead to the abnormal cytokinesis in the process of mitosis. The latest research from the research team showed that KLHL21 could also be involved in the regulation of cellular movement by catalyzing the ubiquitination modification of the EB1 protein. In our earlier studies, KLHL21 is a specific binding protein of the key IKK kinase complex in the NF-B signal pathway, which can inhibit the activation and differential regulation of the induction expression of the downstream target gene of NF-B B by binding to the kinase domain of IKK antigen, And the process does not rely on its E3 ubiquitin ligase activity. In the previous research work, we have tried the KLHL21 antibody from multiple biological companies, but there is a problem of low sensitivity and specificity, for the need of follow-up research, In this study, an attempt was made to use a genomic editing technique to knock at the proximal end of the KLHL21 protein, which is endogenously expressed by the HEK293T cells, into the mCherry and FLAG double-protein labels. There are three kinds of genome editing techniques commonly used in the laboratory, such as ZFN (ZFN) and TALEN (Clustered Regularized Interspaced Short Palindromic Repeats/ CRISPR-associated), among which the CRISPR/ Cas system, which was first reported in 2013, has attracted much attention because of its relatively simple operation, low cost and high efficiency of genome editing. The CRISPR/ Cas system is an adaptive immune defense system for bacteria and archaea, which can cut and degrade the foreign-invasive genetic material such as the bacteriophage, the plasmid and the like through the sequence-specific RNA. when the virus is first invaded, the bacteria fully integrate a length of the DNA sequence of the viral gene into a CRISPR interval sequence region of the virus, and the precursor crRNA is transcribed to generate crRNA (CRISPR RNA) precursor when the virus is again invaded, and the precursor crRNA is processed to form a mature crRNA matched with the exogenous gene DNA sequence, The Cas protein-cut viral DNA is silenced by the identification of the homologous DNA sequence of the viral genome. There are three types of CRISPR/ Cas systems that have been identified, and the type 鈪，
本文编号：2506658
[Abstract]:The KLHL (Kelch-like) family of proteins is a class of very conserved proteins in evolution, typically characterized by a BTB domain at the N-terminus, a Kelch domain at the 5-6 antigen base, and a BACK domain between them. The protein sequence of the family protein is very conservative in the evolution, suggesting that it may have a very important physiological function in the body. The research has shown that the KLHL family protein can form the E3 ubiquitin ligase by binding to the Cu13 protein in the cell, and the ubiquitin modification of the substrate protein is catalyzed, and the inflammation, the oxidative stress and the cell mitosis of the body are carried out on the body. Some of the family members also have other functions unrelated to the catalytic protein ubiquitination modification in the cell. As a member of the KLHL protein family, the KLHL21 protein has a BTB domain, a BACK domain and five Kelch domains. In 2009, MaerkiS and the like were the first to report that the KLHL21 can catalyze the ubiquitination modification of the protein kinase Aurora B by forming a functional E3 ubiquitin ligase with the Cullin3 protein, In the later stage of cell mitosis, it is involved in the transfer of the chromosome-passing complex (CPC) from the chromosome to the middle zone of the spindle, and the use of siRNA to inhibit its expression in the cell can lead to the abnormal cytokinesis in the process of mitosis. The latest research from the research team showed that KLHL21 could also be involved in the regulation of cellular movement by catalyzing the ubiquitination modification of the EB1 protein. In our earlier studies, KLHL21 is a specific binding protein of the key IKK kinase complex in the NF-B signal pathway, which can inhibit the activation and differential regulation of the induction expression of the downstream target gene of NF-B B by binding to the kinase domain of IKK antigen, And the process does not rely on its E3 ubiquitin ligase activity. In the previous research work, we have tried the KLHL21 antibody from multiple biological companies, but there is a problem of low sensitivity and specificity, for the need of follow-up research, In this study, an attempt was made to use a genomic editing technique to knock at the proximal end of the KLHL21 protein, which is endogenously expressed by the HEK293T cells, into the mCherry and FLAG double-protein labels. There are three kinds of genome editing techniques commonly used in the laboratory, such as ZFN (ZFN) and TALEN (Clustered Regularized Interspaced Short Palindromic Repeats/ CRISPR-associated), among which the CRISPR/ Cas system, which was first reported in 2013, has attracted much attention because of its relatively simple operation, low cost and high efficiency of genome editing. The CRISPR/ Cas system is an adaptive immune defense system for bacteria and archaea, which can cut and degrade the foreign-invasive genetic material such as the bacteriophage, the plasmid and the like through the sequence-specific RNA. when the virus is first invaded, the bacteria fully integrate a length of the DNA sequence of the viral gene into a CRISPR interval sequence region of the virus, and the precursor crRNA is transcribed to generate crRNA (CRISPR RNA) precursor when the virus is again invaded, and the precursor crRNA is processed to form a mature crRNA matched with the exogenous gene DNA sequence, The Cas protein-cut viral DNA is silenced by the identification of the homologous DNA sequence of the viral genome. There are three types of CRISPR/ Cas systems that have been identified, and the type 鈪，
本文编号：2506658
本文链接：https://www.wllwen.com/yixuelunwen/jichuyixue/2506658.html
