14-3-3σ干扰逆转录病毒载体的构建及其稳定转染HaCat细胞系的建立

目的：构建14-3-3σ干扰逆转录病毒载体,建立稳定转染的HaCat细胞系。方法：人工合成14-3-3σ基因干扰序列并定向插入到pSuper-retro-neo-EGFP质粒,并在STBL3菌内进行质粒扩增,刷选阳性克隆,酶切测序鉴定,转染293FT细胞进行病毒包装、扩增、纯化、获取逆转录病毒载体,将逆转录病毒载体感染HaCat细胞后Western免疫印迹法、Real-timePCR法检测14-3-3σ的表达情况。结果：连接重组后经酶切和测序筛选出pSuper-retro-neo-EGFP-si14-3-3σ;干扰质粒稳定转染的HaCat细胞系在倒置荧光显微镜下呈绿色荧光,Western免疫印迹法和Real-timePCR法表明14-3-3σ表达明显抑制。结论：成功构建了14-3-3σ干扰的逆转录病毒载体,并构建了其稳定转染的HaCat细胞系。     

人GST-14-3-3σ融合蛋白的原核表达、纯化及活性检测

目的:构建人14-3-3σ基因的原核表达载体,获得其原核表达产物,并对融合蛋白进行纯化及活性检测。方法:采用PCR技术从人乳腺文库中扩增14-3-3σ基因编码序列,将其克隆到pGEX-KG载体中,重组质粒转化大肠杆菌Rossate后表达重组蛋白,利用GST-Sepharose 4B亲和珠对原核表达产物进行纯化,并通过SDS-PAGE和Western印迹检测融合蛋白的表达,采用GST pull-down技术检测已纯化的蛋白与已知体外相互作用蛋白AKT之间的相互作用。结果:从人乳腺文库中扩增获得约750 bp的DNA片段,并成功克隆至pGEX-KG载体上,经双酶切鉴定得到与预期片段大小相符的外源基因插入片段,测序与目的基因序列完全一致;在Rossate菌株中诱导表达出相对分子质量约52 000的目的蛋白,SDS-PAGE和Western印迹结果表明融合蛋白表达成功,并纯化得到GST-14-3-3σ融合蛋白;通过GST pull-down技术检测证实GST-14-3-3σ融合蛋白可以和AKT在体外结合,并证实其具有生物学活性。结论:获得了原核表达的活性较好的GST-14-3-3σ蛋白,为后续研究细胞周期蛋白调控机制奠定了实验基础。     

人FOXO3a基因真核表达载体的构建及其功能验证

目的:构建带myc标签的人FOXO3a基因真核表达载体,并对其功能进行初步检测。方法:采用PCR技术,从乳腺文库中扩增人FOXO3a基因,并将其正确插入pXJ-40-myc载体;将重组质粒与空载体分别转染人乳腺癌细胞系ZR75-1、MCF-7后,通过Western印迹检测其表达情况,并用CCK8法测定细胞生长曲线。结果:双酶切和测序鉴定表明myc-FOXO3a真核表达质粒构建成功,转染乳腺癌ZR75-1、MCF-7细胞后目的基因成功表达;细胞生长曲线结果显示,转染myc-FOXO3a的乳腺癌细胞较空载体细胞生长较慢。结论:构建了带myc标签的人FOXO3a基因真核表达载体,为进一步研究FOXO3a在乳腺癌中的功能奠定了基础。     

人LC3B基因的真核表达及鉴定

目的:构建带HA标签的重组人LC3B基因的真核表达载体,获得HA-LC3B融合蛋白,并初步检测其生物学功能。方法:从人乳腺文库中通过PCR技术扩增LC3B基因编码序列,将其插入pc DNA3.0-HA载体,获得HA-LC3B真核表达载体;将重组质粒与空载体分别转染人胚肾293T细胞,通过Western印迹检测转染细胞的表达情况,免疫共沉淀实验证实其生物学活性。结果:双酶切和测序结果表明HA-LC3B真核表达质粒构建成功,Western印迹表明重组质粒转染293T细胞后获得表达;免疫共沉淀实验显示HA-LC3B融合蛋白可以和Atg4B蛋白相互作用,具有较好的生物学活性。结论:在真核表达系统中表达了带HA标签的人LC3B,为进一步研究细胞自噬奠定了基础。     

人sTNFR1基因的克隆以及在NIH3T3细胞中的稳定表达

以He1a细胞的总RNA为模板,用RT—PCR方法扩增sTNFR1全编码区基因片段,构建含有目的片段的T载体克隆及真核表达载体pcDNA3．1（-）重组质粒亚克隆,将重组质粒和脂质体共同转染NIH3T3细胞系,G418筛选稳定转染细胞株．经核苷酸序列测序和酶切鉴定,成功构建了pcDNA3．1（-）-sTNFR1真核表达质粒,脂质体法建立了高效表达sTNFRI的稳定转染细胞系,并经RT—PCR和Western Blotting鉴定．人sTNFR1基因能在NIH3T3细胞系中稳定表达,为今后的研究打下了基础．     

INTU-siRNA质粒的构建及在人甲状腺Nthy-ori-3-1细胞中的筛选

目的：构建人源靶向特异INTU-si RNA质粒,转染人源性甲状腺Nthy-ori-3-1细胞以构建INTU基因沉默的Nthy-ori-3-1细胞模型,为探索Graves病的发病机制提供合适的细胞模型。方法：构建四对人源INTU序列（编号分别为INTU-29、INTU-31、INTU-33、INTU-35）si RNA质粒和空载体质粒,将空载体和四对INTU-si RNA质粒分别转染Nthy-ori-3-1细胞培养24 h,通过倒置荧光显微镜观察质粒转染细胞后的荧光表达,使用多功能酶标仪检测荧光强度,通过荧光定量PCR法验证INTU基因的沉默效率,通过蛋白质免疫印迹法验证各组细胞内INTU蛋白的表达水平,筛选沉默INTU基因效率最佳的质粒。结果：空载体和INTU-si RNA质粒转染入Nthy-ori-3-1细胞24 h后,倒置荧光显微镜和荧光酶标仪检测结果显示空载体和四组INTU-si RNA组呈现绿色的荧光表达,与正常组相比,空载体与四组INTU-si RNA组的荧光强度均显著高于正常组（P<0.01）,提示质粒均转染成功。荧光定量PCR结果显示,与正常组和空载体组相比,四组...     

人EYA3基因真核表达载体的构建及其活性检测

目的：构建带myc标签的人EYA3基因真核表达载体,获得myc-EYA3融合表达蛋白,并对其功能进行初步检测。方法：采用PCR技术从乳腺文库中扩增人EYA3基因,并将其正确插入pXJ-40-myc载体;将重组质粒与空载体分别转染人乳腺癌细胞系ZR75-1后,Western印迹检测表达情况,并进行生长曲线实验。结果：双酶切和测序鉴定表明,myc-EYA3真核表达质粒构建成功,转染ZR75-1细胞后成功表达;生长曲线实验结果表明,EYA3可促进乳腺癌细胞的生长。结论：构建了带myc标签的人EYA3基因真核表达载体,myc-EYA3能在乳腺癌细胞ZR75-1中表达,且能促进该细胞的生长,本实验为进一步研究EYA3在乳腺癌中的功能奠定了基础。     

人IL-6受体胞外区真核表达载体的构建及体外表达

目的构建人IL-6受体（IL-6R）胞外区真核表达载体,检测其在体外培养细胞中的表达。方法利用PCR扩增IL-6R胞外区,克隆到pcDNA3．1（＋）中,用双酶切、测序鉴定。重组质粒通过脂质体转染HL-60细胞,用G418进行筛选,利用Western印迹检测IL-6R蛋白表达。结果PCR扩增出1218bp的目的片段,双酶切和测序结果显示重组质粒正确。Western印迹结果显示转染细胞能够表达目的蛋白。结论成功构建了人IL-6R胞外区真核表达载体,并且能够在真核细胞中表达。     

人Tim-3 基因真核表达载体的构建及表达

目的:为了构建可在真核细胞中高效表达人Tim-3(hTim-3)的真核表达质粒,以便用于hTim-3肿瘤免疫治疗研究。方法:取健康人外周血的单个核细胞,用高保真聚合酶扩增hTim-3基因,先进行hTim-3基因的亚克隆,用BglⅡ和SalⅠ限制性内切酶切下带有酶切位点的hTim-3基因,最终构建hTim-3基因的真核表达质粒pEGFP-N1-hTim-3。用脂质体方法转染质粒至肝癌细胞SMMC7721和巨噬细胞U937中。48h后荧光显微镜观察转染细胞绿色荧光表达情况,初步判断转染效率。结果:酶切和测序鉴定证实目的基因hTim-3正确插入到真核表达载体pEGFP-N1中,转染肝癌细胞SMMC7721和巨噬细胞U937后,在荧光显微镜下观察到绿色荧光蛋白的表达。结论:成功构建了可在真核细胞中高效表达hTim-3基因的真核表达质粒pEGFP-N1-hTim-3,为进一步研究hTim-3的肿瘤免疫治疗奠定了基础。     

Rhox5基因真核表达质粒的构建及其在NIH3T3细胞中的稳定表达

目的:构建同源异性框基因Rhox5的真核表达质粒,转染NIH3T3细胞,建立稳定过表达Rhox5的细胞系。方法:PCR方法扩增Rhox5的全长cDNA序列,PCR产物双酶切后和人工合成的HA抗原表位标签共同克隆至pcDNA3.1(-)哺乳动物细胞表达载体中,构建pcDNA-Rhox5-HA融合表达质粒。脂质体法将经过测序成功的pcDNA-Rhox5-HA融合质粒和pcDNA3.1空载体分别转染NIH3T3细胞,潮霉素B筛选后建立阴性对照pcDNA3.1 in NIH3T3和稳定过表达Rhox5的Rhox5-HA in NIH3T3细胞系。RT-PCR和western blotting方法检测Rhox5-HA在稳定转染细胞系中的表达情况。结果:成功构建了pcDNA-Rhox5-Myc重组质粒,获得稳定过表达Rhox5的NIH3T3细胞系。RT-PCR和Western blotting结果表明,构建的稳定细胞系中成功表达Rhox5-HA融合蛋白。结论:Rhox5基因真核表达质粒的构建及其在NIH3T3细胞中的稳定表达为进一步体外研究Rhox5蛋白单独的功能及其与其他分子间功能性相互作用奠定了实验基础。     

14-3-3 蛋白

介绍了14－3－3蛋白的基本结构和功能,并简要概述了14－3－3蛋白在信号转导,细胞周期调控以及前体蛋白的折叠与运输过程中的作用机理。     

Cloning of Schistosoma japonicum 14-3-3 epsilon (Sj14-3-3 epsilon), a new member of the 14-3-3 family of proteins from schistosomes

A new member of the 14-3-3 protein family from Schistosoma japonicum has been identified. Phylogenetic analysis showed that this member belongs to the epsilon subfamily of the 14-3-3 proteins, and it is therefore named Sj14-3-3 epsilon. Consistent with the findings for the previously reported S. japonicum 14-3-3 protein (Sj14-3-3), Southern analysis suggested the presence of more than one gene, and/or introns or allelic polymorphism in this epsilon isoform. By RT-PCR, Sj14-3-3 epsilon was shown to be stage-specifically transcribed, being abundant in adults, present in sporocysts but absent in cercariae. Furthermore, mRNA of the epsilon isoform seemed to be much less abundant in the sporocyst stage, compared with Sj14-3-3. This suggests varying requirements of the different 14-3-3 isoforms at different stages of the life cycle.     

14-3-3 proteins--an update

14-3-3 is a highly conserved acidic protein family, composed of seven isoforms in mammals. 14-3-3 protein can interact with over 200 target proteins by phosphoserine-dependent and phosphoserine-independent manners. Little is known about the consequences of these interactions, and thus are the subjects of ongoing studies. 14-3-3 controls cell cycle, cell growth, differentiation, survival, apoptosis, migration and spreading. Recent studies have revealed new mechanisms and new functions of 14-3-3, giving us more insights on this fascinating and complex family of proteins. Of all the seven isoforms, 14-3-3σ seems to be directly involved in human cancer. 14-3-3σ itself is subject to regulation by p53 upon DNA damage and by epigenetic deregulation. Gene silencing of 14-3-3σ by CpG methylation has been found in many human cancer types. This suggests that therapy-targeting 14-3-3σ may be beneficial for future cancer treatment.     

The 14-3-3s

Multiple members of the 14-3-3 protein family have been found in all eukaryotes so far investigated, yet they are apparently absent from prokaryotes. The major native forms of 14-3-3s are homo- and hetero-dimers, the biological functions of which are to interact physically with specific client proteins and thereby effect a change in the client. As a result, 14-3-3s are involved in a vast array of processes such as the response to stress, cell-cycle control, and apoptosis, serving as adapters, activators, and repressors. There are currently 133 full-length sequences available in GenBank for this highly conserved protein family. A phylogenetic tree based on the conserved middle core region of the protein sequences shows that, in plants, the 14-3-3 family can be divided into two clearly defined groups. The core region encodes an amphipathic groove that binds the multitude of client proteins that have conserved 14-3-3-recognition sequences. The amino and carboxyl termini of 14-3-3 proteins are much more divergent than the core region and may interact with isoform-specific client proteins and/or confer specialized subcellular and tissue localization.     

Survival-promoting functions of 14-3-3 proteins

The 14-3-3 proteins are a family of phosphoserine/phosphothreonine-binding molecules that control the function of a wide array of cellular proteins. We suggest that one function of 14-3-3 is to support cell survival. 14-3-3 proteins promote survival in part by antagonizing the activity of associated proapoptotic proteins, including Bad and apoptosis signal-regulating kinase 1 (ASK1). Indeed, expression of 14-3-3 inhibitor peptides in cells is sufficient to induce apoptosis. Interestingly, these 14-3-3 antagonist peptides can sensitize cells for effective killing by anticancer agents such as cisplatin. Thus, 14-3-3 may be part of the cellular machinery that maintains cell survival, and targeting 14-3-3-ligand interactions may be a useful strategy to enhance the efficacy of conventional anticancer agents.     

14-3-3蛋白家族及其临床应用研究进展

14-3-3蛋白家族是一组高度保守的可溶性酸性蛋白质,分子量在28～33kD之间,广泛分布于各种真核生物之中。该蛋白能够特异地结合含有磷酸化丝氨酸或苏氨酸的肽段,参与多种信号转导途径。14-3-3蛋白调节着许多重要细胞生命活动,如:新陈代谢、细胞周期、细胞生长发育、细胞的存活和凋亡以及基因转录,该蛋白家族异常与疾病的发生密切相关,尤其是14-3-3蛋白在脑脊液中的分布与一些神经系统疾病密切相关。14-3-3蛋白已成为一些疾病的临床诊断指标,其作为疾病治疗的靶点也在研究之中。主要阐述了14-3-3蛋白的结构、功能、及其在疾病治疗中的应用。     

14-3-3蛋白家族及其临床应用研究进展

14-3-3蛋白家族是一组高度保守的可溶性酸性蛋白质,分子量在28～33kD之间,广泛分布于各种真核生物之中。该蛋白能够特异地结合含有磷酸化丝氨酸或苏氨酸的肽段,参与多种信号转导途径。14-3-3蛋白调节着许多重要细胞生命活动,如：新陈代谢、细胞周期、细胞生长发育、细胞的存活和凋亡以及基因转录,该蛋白家族异常与疾病的发生密切相关,尤其是14-3-3蛋白在脑脊液中的分布与一些神经系统疾病密切相关。14-3-3蛋白已成为一些疾病的临床诊断指标,其作为疾病治疗的靶点也在研究之中。主要阐述了14-3-3蛋白的结构、功能、及其在疾病治疗中的应用。     

14-3-3蛋白研究进展

14-3-3蛋白是高度保守的、所有真核生物细胞中都普遍存在的、在大多数生物物种中由一个基因家族编码的一类蛋白调控家族。它几乎参与生命体所有的生理反应过程,人们在各种组织细胞中发现了各种不同的14-3-3蛋白。作为与磷酸丝氨酸／苏氨酸结合的第一信号分子,14-3-3蛋白在细胞的信号转导中起着至关重要的作用,尤其是它直接参与调节蛋白激酶和蛋白磷酸化酶的活性,被称为蛋白质与蛋白质相互作用的”桥梁蛋白”;它可以与转录因子结合形成复合体,调节相关基因的表达。一些研究表明,14-3-3蛋白调控机制的紊乱可以直接导致疾病的发生,在临床上14-3-3蛋白常常可以作为诊断的标志物。     

14-3-3 proteins in neurodegeneration

Among the first reported functions of 14-3-3 proteins was the regulation of tyrosine hydroxylase (TH) activity suggesting a possible involvement of 14-3-3 proteins in Parkinson's disease. Since then the relevance of 14-3-3 proteins in the pathogenesis of chronic as well as acute neurodegenerative diseases, including Alzheimer's disease, polyglutamine diseases, amyotrophic lateral sclerosis and stroke has been recognized. The reported function of 14-3-3 proteins in this context are as diverse as the mechanism involved in neurodegeneration, reaching from basal cellular processes like apoptosis, over involvement in features common to many neurodegenerative diseases, like protein stabilization and aggregation, to very specific processes responsible for the selective vulnerability of cellular populations in single neurodegenerative diseases.Here, we review what is currently known of the function of 14-3-3 proteins in nervous tissue focussing on the properties of 14-3-3 proteins important in neurodegenerative disease pathogenesis.