SLC2A4基因启动子区rs5418位点变异对基因表达的影响

目的: 研究葡萄糖转运蛋白4(SLC2A4)基因启动子区rs5418多态位点G→A变异对基因表达的影响。方法: PCR法扩增SLC2A4基因核心启动子区序列。采用基因重组、定点突变等技术,构建rs5418位点含有不同等位基因的SLC2A4基因启动子重组表达载体。脂质体转染法将重组质粒转入HEK293T细胞,采用双萤光素酶报告系统,观察携带不同等位基因的重组质粒中下游报告基因的表达活性。结果: PCR扩增获得长度为716 bp的SLC2A4基因核心启动子序列,成功构建pGL3-SLC2A4-prom(A)和pGL3-SLC2A4-prom(G)重组表达载体。双萤光素酶报告基因活性检测结果显示,携带A等位基因的载体启动下游报告基因表达的相对活性(19.49±4.41)比携带G等位基因的载体(13.04±4.45)强,两者存在显著差异(P<0.05)。结论: SLC2A4基因启动子区rs5418位点的G→A突变能显著增强启动子活性,从而影响SLC2A4基因表达活性。     

多发性硬化症相关蛋白Mayven全长及结构域在酵母双杂交系统中的表达及对报告基因激活作用的检测

目的 探索Mayven在多发性硬化症病程中的作用以及寻找能与之发生相互作用的蛋白.方法 通过PCR扩增得到Mayven基因全长P1及4个不同长度的Mayven基因片段,构建出包含Mayven全长P1及4个不同结构域片段的酵母双杂交诱饵载体,然后将其导入酵母菌株MAV203中,并检测其表达产物在酵母细胞中对报告基因的激活作用.结果 实验结果表明全长P1、片段P3、P4不能激活报告基因HIS3和LacZ的表达,而片段P7和P8可以激活报告基因的表达,Mayven的C末端可能存在一个转录激活区.结论 提示在筛选相互作用蛋白时可以使用全长P1及片段P3、P4,但转录激活是否是Mayven的生理功能仍有待进一步研究确证.     

重组人角质细胞生长因子在毕赤酵母中的表达、纯化及活性测定

目的:利用巴斯德毕赤酵母(Pichiapastoris)重组表达人角质细胞生长因子(humankeratinocytegrowthfactor,hKGF),为实现规模化生产重组hKGF奠定基础。方法:根据已报道的hKGF成熟肽序列,人工合成由毕赤酵母偏好密码子组成的编码hKGF的DNA片段后连接到分泌型表达质粒pPICZαA中,获得重组表达质粒并转化毕赤酵母X-33中进行表达。优化发酵条件,并利用5L生物反应器进行中试发酵。发酵上清通过超滤及层析方法分离纯化重组蛋白,并利用MMT法检测其对恒河猴肺上皮细胞的促增殖活性。结果:在20℃,甲醇诱导60h后,获得分子量约为28kD的目的蛋白,表达量约占菌体上清总蛋白的14.1%。发酵液经肝素亲和层析和SephardexG-25分子筛分离获得纯度为95.0%以上的重组蛋白,得率为12mg/L。该重组hKGF具有糖基化修饰,能显著促进恒河猴肺上皮细胞增殖,其ED₅₀约为57μg/L。结论:利用毕赤酵母成功表达出糖基化修饰及具促恒河猴肺上皮细胞增殖活性的重组hKGF。     

雷奈酸锶通过上调骨形态发生蛋白2的表达促进骨髓间充质干细胞分化为成骨细胞

目的: 探讨雷奈酸锶(strontium ranelate,Sr)是否可以通过上调骨形态发生蛋白 2(bone morphogenetic protein 2,BMP-2)的表达而促进大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)分化为成骨细胞。方法: 对大鼠BMSCs进行分离、纯化、培养及向成骨细胞的定向诱导分化,并在诱导培养时,根据实验目的加入不同浓度Sr以及BMP-2的拮抗剂noggin。用酶标法检测成骨细胞分化和功能成熟的早期标志物——碱性磷酸酶(alkaline phosphatase,ALP)活性的变化,用茜素红染色检测细胞钙化水平,用Western blotting法检测BMP-2蛋白的表达水平。结果: 应用0.1~7 mmol/L Sr处理细胞7 d均可使细胞ALP活性显著增加,其中浓度为3 mmol/L时效果最显著;应用3 mmol/L Sr处理细胞21 d可使细胞钙化结节明显增多;应用0.1~7 mmol/L Sr处理细胞7 d后细胞内BMP-2蛋白水平明显增高;在Sr处理BMSCs前,应用BMP-2拮抗剂noggin预处理细胞2 h不仅抑制Sr对BMP-2表达的上调作用,还拮抗Sr对ALP活性及钙化结节形成的促进作用。结论: 上调BMP-2的表达可能是Sr促进大鼠BMSCs分化为成骨细胞的作用机制之一。     

应用酵母双杂交系统筛选与乙型流感病毒BM2相互作用的蛋白质

目的　筛选与乙型流感病毒BM2蛋白相互作用的蛋白质。方法　应用酵母双杂交系统,以BM2 (2 6 10 9)插入载体pGBKT7作为诱铒,在四缺培养基上筛选人KidneyMATCHMAKERcDNA文库,寻找与BM2蛋白相互作用的宿主蛋白。结果　筛选得到6个阳性AD 文库质粒,并用酵母双杂交实验验证了阳性AD 文库质粒与BM2的相互作用。将阳性AD 文库质粒测序并对测序结果做BLAST分析,发现它们分别是N 乙酰神经氨酸酶丙酮酸裂解酶,Angiopoietin 3、锌指蛋白2 5 1、核糖体蛋白S2 0、蛋白精氨酸N 甲基转移酶1(PRMT)、转录因子样1(TCFL1)。结论　BM2能与这些转录翻译功能相关的蛋白质相互作用,表明BM2蛋白在病毒生活周期中发挥重要作用。     

siRNA特异性沉默septin 2基因抑制大肠癌LoVo细胞迁移

目的:检测新型骨架蛋白septin2在不同转移能力的人结直肠癌细胞株中的表达水平,探究其在结直肠癌细胞迁移中的作用。方法:采用实时荧光定量RT-PCR及Westernblotting检测septin2基因及其蛋白在高转移潜能细胞株LoVo和低转移潜能癌细胞株HT-29、SW480、HCT-116中的表达;用siRNA干扰方法沉默高表达的septin2基因,实时荧光定量RT-PCR检测干扰效率,免疫荧光共聚焦实验观测干扰前后septin2蛋白表达的变化,划痕实验检测干扰前后细胞的迁移能力。结果:septin2在LoVo细胞的表达水平显著高于HT-29、SW480及HCT-116细胞(P<0.05)。沉默septin2基因后,LoVo细胞septin2mRNA表达降低(P<0.05);septin2与纤维状肌动蛋白(F-actin)存在共表达;F-actin表达减弱,应力纤维减少,片状伪足细而短;细胞迁移能力减弱。结论:septin2基因在LoVo细胞高表达,干扰其表达能导致细胞骨架重构,从而降低细胞迁移能力。     

hCDC14A与BRAP2相互作用的初步研究

目的： 筛选hCDC14A(human cell division cycle gene 14A)的相互作用蛋白，探索功能联系。方法： 酵母双杂交筛选hCDC14A的可能相互作用蛋白，Pulldown实验和免疫共沉淀实验验证蛋白质间相互作用，免疫荧光显微镜观察目的基因在细胞中的表达和定位，体内泛素化实验提示可能的相互作用机制。结果： 酵母双杂交筛查到BRAP2(BRCA1 associated protein 2)为hCDC14A的可能相互作用蛋白，两者有共同定位，BRAP2可以增强hCDC14A的泛素化修饰。结论： BRAP2可以与hCDC14A相互作用，可能是hCDC14A的泛素连接酶。     

14-3-3γ对烧伤及LPS诱导所致大鼠心肌损伤的保护作用

目的: 研究14-3-3γ在烧伤及脂多糖(LPS)引起的心肌损伤中的保护作用。方法: 体内实验建立大鼠烧伤及LPS损伤模型,检测3、6、12、24 h各时点心功能及心肌细胞14-3-3γ蛋白表达水平的改变;体外实验采用新生大鼠心肌细胞,构建pFLAG-14-3-3γ质粒,于LPS损伤前24 h转染至心肌细胞,实验结束后检测培养液中乳酸脱氢酶(LDH) 活性,MTT法测定细胞存活率,流式细胞术检测细胞凋亡,线粒体肿胀实验检测线粒体通透性转换孔(mPTP)开放。结果: 烧伤及LPS损伤模型大鼠心电图ST段均有明显改变,心肌细胞14-3-3γ蛋白表达水平均显著升高。转染了pFLAG-14-3-3γ质粒的心肌细胞能明显对抗随后的LPS损伤,与未转染组比较,14-3-3γ能明显提高细胞存活率,降低LDH活性,减少细胞凋亡,抑制mPTP的开放。结论: 14-3-3γ可能通过抑制mPTP的开放对烧伤及LPS所致的心肌损伤起保护作用。     

应用酵母双杂交技术筛选与RANK蛋白新基序IVVY相互作用的蛋白

目的: 应用酵母双杂交技术筛选小鼠巨噬细胞中与核因子κB受体激活因子(RANK)蛋白上新基序IVVY相互作用的蛋白,以探寻RANKL/RANK系统中介导破骨细胞形成的RANK下游新信号转导蛋白。方法: 应用GAL4酵母双杂交系统3,构建仅包含编码新基序⁵³⁵IVVY⁵³⁸ 的一小段RANK的cDNA片段为诱饵质粒pGBKT7-IVVY,并与巨噬细胞cDNA文库质粒pGADT7-library共转化AH109酵母,筛选与RANK蛋白新基序IVVY相互作用的蛋白,通过回复性杂交实验验证其可靠性,并对阳性克隆进行测序和基因同源性分析。结果: 筛选出4个可能与IVVY基序有相互作用的蛋白,包括Ring1和YY1结合蛋白(RYBP)、ATP结合盒、E2F转录因子和热休克蛋白8,其中表达RYBP的阳性克隆出现频率高、速度快。结论: 应用酵母双杂交技术成功地筛选出4个可能与IVVY基序相互作用的候选蛋白,其中RYBP可能性最大。     

Link between macrophage migration inhibitory factor and cellular redox regulation

Macrophage migration inhibitory factor (MIF) is an evolutionary conserved 12.5-kDa protein mediator with multiple functions in innate and acquired immunity. Upon leaderless secretion, MIF acts as a typical inflammatory cytokine, but there is no structural homology between MIF and any of the known cytokine protein families. Also, MIF is unique among cytokines in that it exhibits certain endocrine properties and has enzymatic activity. The catalytic thiol-protein oxidoreductase (TPOR) activity of MIF is mediated by a Cys-Ala-Leu-Cys active site between residues 57 and 60 that can undergo reversible intramolecular disulfide formation. Such a redox motif is typically found in TPORs of the thioredoxin (Trx) family of proteins. MIF seems to act as a disulfide reductase, and structure-function analyses of the redox site indicate that this activity is not only observed in vitro, but plays a role in cellular redox homeostasis, apoptosis inhibition, MIF-mediated monocyte/macrophage activation, and possibly the modulation of the activity of MIF-binding proteins. In this Forum review, the biochemical and biological evidence for a role of the TPOR activity for various MIF functions is summarized and discussed. In particular, the marked functional homologies with Trx proteins, the MIF redox/MHC II link, and recent attempts to discern the intra- versus extracellular roles of the MIF TPOR activity are dealt with.     

Filarial Nematode Parasites Secrete a Homologue of the Human Cytokine Macrophage Migration Inhibitory Factor

Filarial nematode parasites establish long-term chronic infections in the context of an antiparasite immunity that is strongly biased toward a Th2 response. The mechanisms that lead to this Th2 bias toward filarial antigens are not clear, but one possibility is that the parasites produce molecules that have the capacity to proactively modify their immunological environment. Here we report that filarial parasites of humans secrete a homologue of the human proinflammatory cytokine macrophage migration inhibitory factor (MIF) that has the capability of modifying the activity of human monocytes/macrophages. A cDNA clone isolated from a Brugia malayi infective-stage larva expression library encoded a 12.5-kDa protein product (Bm-MIF) with 42% identity to human and murine MIF. MIF homologues were also found to be expressed in the related filarial species Wuchereria bancrofti and Onchocerca volvulus. Bm-mif was transcribed by adult and larval parasites, and the protein product was found in somatic extracts and in the parasite’s excretory-secretory products. Immunohistocytochemistry revealed that Bm-MIF was localized to cells of the hypodermis/lateral chord, the uterine wall, and larvae developing in utero. Unexpectedly, the activities of recombinant Bm-MIF and human MIF on human monocytes/macrophages were found to be similar. When placed with monocytes/macrophages in a cell migration assay, Bm-MIF inhibited random migration. When placed away from cells, Bm-MIF induced an increase in monocyte/macrophage migration that was specifically inhibited by neutralizing anti-Bm-MIF antibodies. Bm-MIF is the first demonstration that helminth parasites produce cytokine homologues that have the potential to modify host immune responses to promote parasite survival.     

表达载体介导的反义RNA抑制人巨噬细胞移动抑制因子的表达

目的：研究表达载体介导的反义RNA对人巨噬细胞移动抑制因子(MIF)表达的抑制作用。 方法:用亚克隆技术构建可转录MIF反义RNA的真核表达载体pcDNA3-antiMIF。用lipofectamine2000分别将pcDNA3、pcDNA3-antiMIF转染可表达MIF的HEK293(293-MIF)细胞，用Real-time定量PCR鉴定MIF mRNA表达水平。将pcDNA3-antiMIF转化人脐静脉血管内皮细胞(HUVECs)，建立可表达MIF反义RNA的HUVECs(HUVECs-antiMIF)细胞。将MIF的真核表达载体pSecTag-MIF转染HUVECs-antiMIF，用Real-time定量PCR鉴定MIF mRNA的表达水平。 结果:正确构建了MIF反义RNA的表达载体pcDNA3-antiMIF。MIF 反义RNA对293-MIF细胞中MIF表达的抑制水平达32%（P<0.05）。建立稳定表达MIF反义RNA的HUVECs-antiMIF细胞株。HUVECs-antiMIF中MIF的表达受到抑制，表达水平降低40%（P<0.05）。 结论:表达载体介导的反义RNA能有效地抑制MIF的表达，建立了稳定表达MIF反义RNA的HUVECs。     

NALP3富含亮氨酸重复序列结构域识别人细胞周期蛋白H和禽冠状病毒蛋白

 目的：寻找与NALP3富含亮氨酸重复序列（leucine-rich repeat, LRR）结构域相互作用的蛋白质分子。方法：克隆NALP3富含亮氨酸重复序列（NALP3-LRR）结构域的DNA序列并经测序检验。应用酵母双杂交技术,构建以NALP3-LRR结构域为诱饵基因的酵母双杂交载体,对人胚肺cDNA文库进行杂交筛选,经酵母回交实验验证蛋白质在酵母细胞内的相互作用并对阳性克隆的DNA进行序列测定和生物信息学分析。将筛选到的阳性克隆进一步用免疫共沉淀实验验证NALP3-LRR结构域与阳性蛋白之间相互作用的可靠性。结果：成功克隆NALP3-LRR结构域的DNA序列并经测序检验正确。用酵母双杂交技术对人胚肺cDNA文库进行酵母杂交筛选共获得4个阳性克隆。免疫共沉淀实验证实能与NALP3-LRR结构域发生相互作用的阳性蛋白是人细胞周期蛋白H和禽传染性支气管炎病毒株Cal99的ORF1a。结论：NALP3的富含亮氨酸重复序列结构域能与人细胞周期蛋白H和禽冠状病毒蛋白发生相互作用。     

NMDA受体亚单位NR2D与MOCA相互作用的鉴定

目的:寻找N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受体亚单位NR2D的结合蛋白,为探讨NR2D在视网膜兴奋性毒性损伤中的作用提供依据。方法:构建了包含NR2D细胞内C末端的cDNA片段为诱饵质粒,应用酵母双杂交技术筛选小鼠脑cDNA文库,并用免疫共沉淀实验进一步验证NR2D与其结合蛋白之间的相互作用,免疫荧光显微镜观察NR2D和目的蛋白在视网膜中的共表达。结果:酵母双杂交筛选到细胞黏附修饰因子(modifier of cell adhesion,MOCA)为NR2D可能的相互作用蛋白,两者在视网膜有共定位。结论:MOCA能特异结合谷氨酸受体NR2D,这为进一步研究谷氨酸的兴奋性毒性参与视网膜退行性变的机制奠定了实验基础。     

PTEN与NHERF-1相互作用

目的筛选与PTEN结合的含PDZ结构域的蛋白,并利用蛋白质组学方法对筛选出的蛋白质的相互作用予以鉴定。方法利用PDZ蛋白质芯片筛选PTEN的结合蛋白。对所发现的PTEN与NHERF-1的相互作用,应用GST融合蛋白沉降和免疫共沉淀实验等多种方法予以鉴定。结果发现了几种新的与PTEN结合的含PDZ结构域的蛋白质。并对PTEN/NHERF-1的相互作用进行了进一步研究,发现PTEN的羧基端可与NHERF-1的第一个PDZ结构域(PDZ1)结合。将PTEN羧基最末端的4个氨基酸(I-T-K-V)中任何一个突变为丙氨酸,都明显抑制PTEN与NHERF-1的结合。免疫共沉淀结果显示,在细胞内完整的蛋白质分子PTEN与NHERF-1也可以结合。结论PTEN与NHERF-1之间存在相互作用,该相互作用是由PTEN的羧基末端与NHERF-1的PDZ1结构域结合而实现的,PTEN羧基最末端的4个氨基酸对于维持它们之间的结合有十分重要的作用。     

MIF from mussel: coding sequence, phylogeny, polymorphism, 3D model and regulation of expression

Three macrophage migration inhibitory factor (MIF)-related sequences were identified from a Mytilus galloprovincialis EST library. The consensus sequence included a 5′-UTR of 32 nucleotides, the complete ORF of 345 nucleotides, and a 3′-UTR of 349 nucleotides. As for other MIFs, M. galloprovincialis ORF does not include any signal or C-terminus extensions. The translated sequence of 115 amino acids possesses a molecular mass of 12,681.4, a pI of 6.27 and a stability index of 21.48. Its 3D structure resembles human MIF except for one shorter α-helix. Although evolutionary separated from ticks and vertebrates, Mg-MIF appeared to be closely related to Pinctada fucata and Haliotis, but not to Chlamys farreri and Biomphalaria glabrata. Numerous mutation points were observed within the Mg-MIF ORF, defining 11 amino acid variants within the mussels from Palavas-France and 14 amino acid variants within the mussels from Palermo-Italy. The 2 major variants from Palavas were identical to 2 of the 4 major variants from Palermo. In all the 18 Mg-MIF variants, residues involved in tautomerase and in oxidoreductase activities were conserved. Generally, one mussel expressed 2 Mg-MIF amino acid sequences but with different frequencies of occurrence. Mg-MIF is constitutively expressed principally in hemocytes and in the mantle. In contrast to other animal models, Mg-MIF expression was always down regulated following challenge by bacteria and fungi, confirming previous data obtained with microarray. Down regulation started as soon as 1 h and Mg-MIF expression returned to background 9-48 h after the challenge. Exception was regarding the yeast, Candidaalbicans, down-regulation between 9 and 72 h, suggesting yeast and bacteria-filamentous fungi trigger different mechanisms of elimination.     

Macrophage migration inhibitory factor increases neuronal delayed rectifier K+ current

Macrophage migration inhibitory factor (MIF) has widespread actions in the immune, endocrine, and nervous systems. Previously, we reported that increases in the intracellular levels of MIF depress the firing of hypothalamus/brain stem neurons in culture, including the chronotropic actions of angiotensin II. The objective of this study was to investigate the effects of MIF on delayed rectifier K+ current (I(Kv)), one of the component currents whose activity contributes to neuronal firing. Intracellular perfusion of MIF (80 nM) into Sprague-Dawley rat neuronal cultures caused a significant increase in I(Kv), as measured by patch-clamp recordings. This effect was apparent by 3 min, and was maximal after 20-30 min. I(Kv) current density (pA/pF) increased from 31.58 +/- 2.36 in controls to 41.88 +/- 3.76 in MIF-treated neurons (mean +/- SE; n = 9; P < 0.01). MIF that had been inactivated by boiling did not alter I(Kv), and MIF-neutralizing antibodies abolished the action of recombinant MIF (rMIF). The stimulatory effect of MIF on I(Kv) current density was mimicked by intracellular application of either P1S-MIF (80 nM) or the peptide MIF-(50-65) (0.8-8 microM), both of which harbor the thiol-protein oxidoreductase (TPOR) activity of the MIF molecule. Conversely, neither C60S-MIF (80 nM) nor the MIF homologue D-dopachrome tautomerase (80 nM), both of which lack TPOR activity, altered I(Kv). Finally, the increase in I(Kv) produced by rMIF was abolished by the superoxide scavenger Tiron (1 mM). These studies indicate that the neuronal action of MIF includes a stimulatory action on I(Kv) that may be mediated by a TPOR/superoxide-scavenging mechanism.     

The polymerase (L) protein of rinderpest virus interacts with the host cell protein striatin

Rinderpest virus (RPV) is a morbillivirus that causes a highly contagious disease affecting members of the order Artiodactyla. The viral L protein is the catalytic subunit of the RNA-dependent RNA polymerase. To search for host cell proteins with which L interacts, a library screen was performed using the yeast two-hybrid system. Several host cell proteins were recovered from the library screen as putative L-interactors; one of these was identified as striatin. A direct interaction between RPV L and striatin was confirmed using both co-immunoprecipitation assays and co-localisation studies using confocal microscopy. Striatin was also shown to co-localise with the RPV L protein in infected cells. The L proteins of morbilliviruses consist of three long highly conserved domains separated by short unconserved stretches of amino acids. The L domain with which striatin interacts was investigated by co-immunoprecipitation and striatin was shown to interact primarily with the central conserved domain.     

Evidence of association of macrophage migration inhibitory factor gene polymorphisms with systemic lupus erythematosus

The aim of this study was to evaluate the potential association of functional polymorphisms of macrophage migration inhibitory factor with systemic lupus erythematosus. Our study includes 711 systemic lupus erythematosus (SLE) patients and 755 healthy controls. We genotyped the migration inhibitory factor (MIF) -173G/C using a polymerase chain reaction (PCR) system with predeveloped TaqMan allelic discrimination assay and the MIF -794 CATT(n) microsatellite polymorphism using a PCR-fluorescent method. A statistically significant difference in the distribution of the MIF -173(*)C allele between SLE patients and controls (P=0.004, OR=1.34, 95% CI=1.05-1.27) was observed. In addition, the frequency of the MIF -173(*)C/C genotype was higher in SLE patient (P=0.002, OR=2.58, 95% CI=1.32-5.10). No differences in the distribution of CATT(n) were found. However, the haplotypes analyses showed that only the CATT(7)-MIF -173(*)C haplotype was associated with a higher susceptibility to SLE (P=0.001, OR 1.84, 95% CI 1.35-2.79). No association with clinical features was detected in any case. These results suggest that both, MIF -173(*)C allele and CATT(7)-MIF -173(*)C haplotype, confer susceptibility to SLE in our population.