【摘要】：KLHL(Kelch-like)蛋白家族是一類在進(jìn)化中非常保守的蛋白質(zhì),其典型的特征是都含有一個(gè)N端的BTB結(jié)構(gòu)域、5-6個(gè)羧基端的Kelch結(jié)構(gòu)域,以及介于它們之間的BACK結(jié)構(gòu)域。該家族蛋白的蛋白質(zhì)序列在進(jìn)化中均非常保守,提示其在機(jī)體內(nèi)可能具有非常重要的生理功能。已有的研究表明,KLHL家族蛋白在細(xì)胞內(nèi)多通過(guò)與Cu13蛋白相互結(jié)合而形成E3泛素連接酶,催化底物蛋白質(zhì)的泛素化修飾,在機(jī)體的炎癥、氧化應(yīng)激、細(xì)胞有絲分裂、胚胎發(fā)育以及淋巴生成等多種生命活動(dòng)中發(fā)揮著重要的作用,但某些家族成員在細(xì)胞中還具有與催化蛋白質(zhì)泛素化修飾無(wú)關(guān)的其他功能。作為KLHL蛋白家族中的一員,KLHL21蛋白含有一個(gè)BTB結(jié)構(gòu)域、BACK結(jié)構(gòu)域與5個(gè)Kelch結(jié)構(gòu)域。2009年,MaerkiS等率先報(bào)道了,KLHL21可通過(guò)與Cullin3蛋白相互結(jié)合形成有功能的E3泛素連接酶,催化蛋白激酶Aurora B的泛素化修飾,在細(xì)胞有絲分裂后期參與調(diào)控染色體過(guò)客復(fù)合體(Chromosomal passenger complex,CPC)從染色體向紡錘體中間區(qū)(spindle midzone)的轉(zhuǎn)運(yùn),采用siRNA抑制其在細(xì)胞中的表達(dá)可導(dǎo)致有絲分裂過(guò)程中胞質(zhì)分離(cytokinesis)的異常。來(lái)自該研究團(tuán)隊(duì)最新的研究結(jié)果表明,KLHL21還可通過(guò)催化EB1蛋白的泛素化修飾而參與對(duì)細(xì)胞運(yùn)動(dòng)的調(diào)控。而我們前期的研究揭示,KLHL21是NF-κB信號(hào)通路中關(guān)鍵的IKK激酶復(fù)合物的特異性結(jié)合蛋白,可通過(guò)與IKKβ的激酶結(jié)構(gòu)域結(jié)合而抑制其激活,差異性地調(diào)控NF-κB下游靶基因的誘導(dǎo)表達(dá),且該過(guò)程不依賴其E3泛素連接酶活性。在前期的研究工作中,我們?cè)囉昧藖?lái)源自多個(gè)生物公司的KLHL21抗體,但普遍存在靈敏性與特異性不高的問(wèn)題,為了后續(xù)研究的需要,本研究嘗試采用基因組編輯技術(shù)在HEK293T細(xì)胞內(nèi)源性表達(dá)的KLHL21蛋白的羧基端敲入mCherry與FLAG雙蛋白質(zhì)標(biāo)簽。目前在實(shí)驗(yàn)室中常用的基因組編輯技術(shù)有ZFN(zinc finger endonuclease,ZFN)、TALEN(transcription activator-like effector nuclease)CRISPR/Cas(Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated)等三種,其中以 2013 年才首次報(bào)道的 CRISPR/Cas系統(tǒng)因其操作相對(duì)簡(jiǎn)單、低成本與基因組編輯的高效性而備受大家的關(guān)注。CRISPR/Cas系統(tǒng)是存在于細(xì)菌和古生菌的一種適應(yīng)性免疫防御系統(tǒng),通過(guò)序列特異性的RNA介導(dǎo),切割并降解噬菌體、質(zhì)粒等外源入侵的遺傳物質(zhì)。當(dāng)病毒首次入侵時(shí),細(xì)菌將病毒基因的一段DNA序列整合進(jìn)自身的CRISPR間隔序列區(qū),其在病毒再次入侵時(shí)轉(zhuǎn)錄生成crRNA(CRISPR RNA)前體,前體crRNA再經(jīng)過(guò)加工后形成與外源性基因DNA序列匹配的成熟crRNA,通過(guò)與病毒基因組的同源DNA序列的識(shí)別而介導(dǎo)Cas蛋白切割病毒DNA將其沉默。目前已經(jīng)被鑒定的CRISPR/Cas系統(tǒng)共有3種類型,其中Ⅱ型CRISPR/Cas系統(tǒng)僅依賴于單一的Cas蛋白Cas9即可完成免疫防御,由于其操作簡(jiǎn)單、要求低而被廣泛應(yīng)用于基因組編輯。CRISPR/Cas9 系統(tǒng)由5'端反式激活核糖核酸(trans-activating CRISPR RNA,tracrRNA)序列區(qū)、中間部分的Cas9基因編碼序列和3'端CRISPR間隔序列區(qū)在DNA鏈上線性排列組成。CRISPR間隔序列區(qū)由高度保守的回文序列/反向重復(fù)序列R(Repeats)和長(zhǎng)度約為30nt的間隔序列S(Spacers)有規(guī)律的間隔排列組成。Spacers經(jīng)轉(zhuǎn)錄和加工形成小干擾RNA分子crRNA,通過(guò)堿基互補(bǔ)配對(duì)識(shí)別靶DNA序列并引導(dǎo)Cas核酸酶對(duì)其進(jìn)行切割。crRNA與靶DNA的互補(bǔ)配對(duì)區(qū)長(zhǎng)度通常為20nt,這一過(guò)程中需要tracrRNA的參與,其通過(guò)與crRNA的部分序列互補(bǔ)配對(duì)而募集Cas9蛋白。在實(shí)際應(yīng)用中,可以將crRNA與tracr-RNA編碼序列整合在一起,采用RNA轉(zhuǎn)錄酶Ⅲ啟動(dòng)子如H1與U6進(jìn)行表達(dá),從而獲得向?qū)NA(guideRNA,gRNA),此外也可以通過(guò)轉(zhuǎn)染體外合成或體外轉(zhuǎn)錄的gRNA來(lái)介導(dǎo)對(duì)靶DNA的切割。在gRNA識(shí)別的靶DNA序列的下游中含有一個(gè)PAM序列(protospacer adjacent motifs),通常為NGG三核苷酸序列(N代表任意核苷酸),是gRNA識(shí)別靶DNA序列所必需的。Cas9蛋白具有兩個(gè)核酸酶活性中心HNH與RuvC,它們分別切割靶DNA雙鏈中的一條鏈,導(dǎo)致DNA雙鏈的平末端斷裂,繼而通過(guò)細(xì)胞的DNA同源重組或非同源性末端連接修復(fù)機(jī)制對(duì)斷裂的DNA進(jìn)行修復(fù),導(dǎo)致靶DNA序列的缺失與插入,引起靶基因讀碼框移位而達(dá)到敲除其在細(xì)胞內(nèi)表達(dá)的目的;或者在外源同源重組模板存在時(shí)對(duì)靶DNA序列進(jìn)行替換、或者敲入一段外源DNA序列,通過(guò)這種對(duì)靶基因DNA序列進(jìn)行的定點(diǎn)編輯可實(shí)現(xiàn)對(duì)細(xì)胞內(nèi)源性表達(dá)的靶基因功能或表達(dá)水平的調(diào)控,促進(jìn)對(duì)靶基因功能與表達(dá)調(diào)控機(jī)制的深入研究。研究目的:利用CRISPR/Cas9基因編輯技術(shù)構(gòu)建KLHL21蛋白羧基端敲入蛋白標(biāo)簽的HEK293T穩(wěn)定細(xì)胞株。研究方法:1、構(gòu)建含有mCherry-FLAG雙蛋白質(zhì)標(biāo)簽的KLHL21基因同源重組模板質(zhì)粒,以及靶向人KLHL21基因終止密碼子附近DNA序列的gRNA表達(dá)載體;2、將線性化的KLHL21基因同源重組模板質(zhì)粒、gRNA表達(dá)質(zhì)粒和人源化的hCas9表達(dá)載體共轉(zhuǎn)染入HEK293T細(xì)胞中;3、采用Blasticidin抗生素篩選獲得敲入目標(biāo)蛋白質(zhì)標(biāo)簽的HEK293T陽(yáng)性細(xì)胞株;4、采用PCR、Western Blot和免疫沉淀技術(shù)驗(yàn)證篩選獲得的陽(yáng)性HEK293T細(xì)胞株;5、構(gòu)建Cre重組酶表達(dá)載體pCDNA3.1-Cre,將其轉(zhuǎn)染篩選獲得的敲入了目標(biāo)蛋白質(zhì)標(biāo)簽的HEK293T細(xì)胞株,利用Cre/Loxp位點(diǎn)特異性重組系統(tǒng)將兩端含有同向Loxp位點(diǎn)的Blasticidin篩選標(biāo)記基因序列切除。研究結(jié)果:1、成功構(gòu)建了用于人KLHL21蛋白羧基端敲入mCherry與FLAG蛋白標(biāo)簽的載體pKCFD。2、成功構(gòu)建了靶向人KLHL21基因終止密碼子附近序列的gRNA表達(dá)載體 pHG4-21A 與 pHG4-21B。3、利用CRISPR/Cas9系統(tǒng)成功將mCherry與FLAG蛋白標(biāo)簽敲入了內(nèi)源性KLHL21蛋白的羧基端。4、采用抗生素Blasticidin成功篩選到在內(nèi)源性表達(dá)的KLHL21蛋白羧基端敲入目標(biāo)蛋白質(zhì)標(biāo)簽mCherry-FLAG的HEK293T陽(yáng)性細(xì)胞株。5、通過(guò)Cre-Loxp位點(diǎn)特異性重組系統(tǒng)成功切除兩端含有同向Loxp位點(diǎn)的Blasticidin篩選標(biāo)記基因序列。結(jié)論:我們成功地采用CRISPR/Cas9基因編輯技術(shù)在HEK293T細(xì)胞中內(nèi)源性表達(dá)的KLHL21蛋白羧基端敲入mCherry與FLAG蛋白標(biāo)簽,為后續(xù)KLHL21蛋白質(zhì)的功能研究提供了便利,同時(shí)也建立了一個(gè)研究其他內(nèi)源性表達(dá)基因功能的技術(shù)平臺(tái)。
[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，

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