CARP编码蛋白的相互作用蛋白筛选

目的 :筛选与CARP编码蛋白相互作用的蛋白 ,为其功能研究奠定基础。方法 :将编码全长CARP的DNA序列插入到pGBKT7 BD载体中 ,转化AH1 0 9酵母 ,然后与含有已克隆到pACT2载体上的人心脏cDNA文库的Y1 87酵母融合。CARP与相应的人心脏cDNA片段编码的蛋白发生相互作用后 ,可激活报告基因的表达。阳性克隆的质粒进行测序分析。同源性检索搜寻GenBank中与之相同或相似的序列。结果 :共筛选约 3× 1 0 7个cDNA ,筛选出 9个阳性克隆 ,其中包括FLNa (actin bindingprotein 2 80 )。结论 :CARP编码蛋白在酵母中可以特异性的结合FLNa ,提示CARP可能通过与FLNa相互作用参与调控细胞增殖     

CARP基因重组腺病毒载体的构建

目的：利用Adeasy-1系统，构建并鉴定CARP基因重组腺病毒载体。方法：PCR扩增含有CARP全长cDNA的片段，经测序验证无误后，亚克隆至pAdTrack-CMV穿梭质粒，再与pAdEasy．1质粒在大肠杆菌BJ5 183中进行同源重组产生腺病毒载体质粒。经过抗性筛选以及酶切鉴定得到阳性的重组质粒，再在293细胞中进行包装扩增，利用Adeasy系统上的绿色荧光蛋白标签鉴定病毒表达。结果：测序证实PCR产物为CARP全长cDNA；抗性筛选及酶切鉴定均表明重组腺病毒载体构建成功；转染293细胞3天后可见绿色荧光，回收病毒可以重复感染293细胞，证明病毒包装成功。     

犬新孢子虫ENO1蛋白的表达及亚细胞定位

目的为研究犬新孢子虫烯醇化酶(ENO1)的生物学功能,对ENO1进行原核表达,蛋白纯化后进行抗原性鉴定和亚细胞定位。方法根据GenBank公布的犬新孢子虫ENO1序列设计引物,以犬新孢子虫速殖子cDNA为模板,PCR扩增ENO1基因。将ENO1基因片段与原核表达载体pGEX-4T-1连接,连接产物转入表达感受态细胞Rosetta DE3a,经IPTG诱导表达ENO1并进行SDS-PAGE分析。重组蛋白经纯化后免疫小鼠,制备多克隆抗体,ELISA检测抗体效价,Western blot鉴定其反应原性,通过间接免疫荧光(IFA)和免疫电镜(IEM)试验进行亚细胞定位。结果 PCR扩增得到1 428 bp大小的ENO1基因片段,构建的原核表达载体pGEX-4T-1-ENO1鉴定正确。重组载体转化DE3a后经IPTG诱导高效表达重组蛋白,相对分子质量大小为78×10~3,该蛋白主要存在于重组菌超声破碎上清中。Western blot显示重组蛋白能被感染犬新孢子虫小鼠血清识别,即具有反应原性,可作为犬新孢子虫病的诊断抗原或疫苗候选靶标;IFA和IEM试验显示,ENO1在虫体的细胞质和细胞核内都有分布。结论成功构建了原核表达载体pGEX-4T-1-ENO1,ENO1在虫体的细胞质和细胞核内都有分布,且具有反应原性,为ENO1作为犬新孢子病的诊断抗原、药物靶标和疫苗候选分子提供了试验依据。     

人CSF3蛋白的原核表达和纯化及其亚细胞定位

目的 粒细胞集落刺激因子(CSF3)作为免疫调控中的重要细胞因子,在中性粒细胞增殖分化的调控方面发挥关键作用。然而,在病原菌感染中CSF3的调控机制仍不清楚。本研究旨在通过原核表达人CSF3,并观察其在HEK-293T细胞中的亚细胞定位,为重组CSF3药物开发以及揭示其在病原菌感染和免疫调控中的作用机制奠定基础。方法 基于人CSF3的生物信息学分析,分别通过QuikChange和基因克隆获得CSF3 pGlO1和CSF3-pmCherry两种重组质粒。前者在大肠埃希菌原核表达系统中表达,并通过镍离子亲和层析和凝胶过滤层析进行蛋白纯化。后者通过jetPEI试剂转染到HEK-293T细胞中,48 h后通过荧光共聚焦显微镜观察,确定其亚细胞定位。结果 在原核系统中成功表达人CSF3蛋白,其相对分子质量为18.8×10³。该蛋白的溶解性良好,PP酶酶切后蛋白浓度较高,但蛋白性质相对不稳定,对pH值和温度敏感,容易发生聚集和降解。将荧光融合质粒转染到HEK-293T细胞中48 h后观察,人CSF3-pmCherry蛋白定位在细胞质内,呈现出特定的定位和分布模式。结论 成功构...     

人肝脏胆汁酸转运蛋白基因的克隆表达及其亚细胞定位

胆汁酸在脂肪和维生素消化吸收中发挥重要作用,当被排入小肠后约95%通过顶端钠依赖胆汁酸转运蛋白(ABST)被重吸收,其中的80%可通过牛磺胆汁酸钠共转运多肽(NTCP)被摄入肝细胞内并再次被分泌到胆汁中形成胆汁酸的肠肝循环[1].     

人肝脏胆汁酸转运蛋白基因的克隆表达及其亚细胞定位

Not Abstract. [Chinese FullText URL http://zhgz.chinajournal.net.cn].     

SHR血管平滑肌PDGF—A链,TGFβ1及其受体mRNA表达的研究

目的：在自发型高血压和正常血压的Ｖｉｓｔａｒ－ｋｙｏｔｏ大鼠血管壁平滑肌细胞（ＳＨＲ－ＶＳＭＣ和ＷＫＹ－ＶＳＭＣ）,比较几种内源性生长因子及它们受体的表达水平,从ＶＳＭＣ自分泌和旁分泌产生骨源性生长因子的角度,阐述ＳＨＲ－ＶＳＭＣ和ＷＫＹ－ＶＳＭＣ的差异。方法：以定量ＲＴ－ＰＣＲ技术,在二株系ＶＳＭＣ,检测长型ＰＤＧＦ－Ａ ,ＴＧＦβ１及它的受体ｍＲＮＡ的表达水平。结果：（１）在ＳＨＲ－ＶＳＭＣ,     

外源HCV核心基因表达载体在HepG2中蛋白表达的亚细胞定位

目的研究HCV全长（天然）核心蛋白（C蛋白）和成熟C蛋白在HepG2细胞内的亚细胞定位，为进一步研究C蛋白与宿主细胞蛋白因子相互作用提供实验依据。方法设计HCV全长和成熟核心基因引物，以pBRTM／HCV1-3011为真核表达载体为模版行PCR；将PCR扩增产物酶切纯化后定向插入pEGFP-C1的BeglⅡ和EcoRⅠ位点，得到能表达全长HCV核心蛋白（C191aa．）和成熟核心蛋白（C173aa．）的真核表达载体pEGFP-C1-C191、pEGFP-C1-C173；将扩增pEGFP-C1-C191、pEGFP-C1-C173质粒转染到HepG2细胞中，28h、5d在荧光显微镜下观察两种C蛋白的亚细胞定位。5d后再用免疫细胞化学法观察两种C蛋白在HepG2细胞中着色现象。结果HCV核心基因PCR产物和构建的pEGFP-C1-C191、pEGFP-C1-C173经扩增测序证实。pEGDP-C1-C191转染HepG2细胞28h后融合荧光全长C蛋白C191主要出现在核膜和胞质中，少部分出现在胞核及其膜中；而pEGDP-C1-C173转染HepG2细胞28h后主要定位在核中和核膜上。但pEGDP-C1-C191转染HepG2细胞5d后，其表达的融合荧光蛋白主要出现在核内和核膜上，少量在胞质中；而成熟C蛋白C173则仍然在胞核内及核膜上。pEGDP-C1-C转染HepG2细胞5d后进行免疫细胞化学显示，C191蛋白和C173蛋白主要在核中、核膜着色，胞质有少量着色。结论HCV全长C蛋白的亚细胞定位是一动态过程，先出现在胞质中和核膜上，最后定位在核中和核膜上；成熟C蛋白主要定位在核中和核膜上。这种现象为解释瞬时转染HCV不同长度C基因对p53／p21通路作用出现相反结果奠定基础。     

乙型肝炎病毒核心蛋白在HepG2.2.15细胞中的亚细胞定位及转移

目的观察HepG2．2．15细胞内HBV核心蛋白的亚细胞定位及转移，了解核心蛋白的入核机制。方法2％二甲亚砜或（和）1μmol／LBay414109处理HepG2．2．15细胞4d；荧光共聚焦显微镜观察HBsAg和HBcAg在细胞内的定位；Westernblot检测胞质和胞核中的HBcAg水平；选择性PCR检测胞核内HBV共价闭合环状DNA（cccDNA）水平。结果二甲亚砜处理提高了胞质和胞核内的HBcAg表达及核内的cccDNA水平；Bay414109处理后胞质中HBcAg水平下降但胞核中HBcAg水平上升，cccDNA水平下降；联合应用二甲亚砜和Bay41—4109处理后HBsAg在胞质内呈条索状分布，胞质中HBcAg水平下降，但胞核内HBcAg明显上升，cccDNA水平下降。结论HepG2．2．15细胞中存在HBV核心颗粒入核障碍，游离核心蛋白易于通过核孔，二甲亚砜可促进核心蛋白进入细胞核，并有助于cccDNA的形成。     

缺氧及一氧化碳对大鼠血管平滑肌细胞的作用

目的 探讨缺氧及低浓度一氧化碳（ＣＯ）对大鼠血管平滑肌细胞（ＶＳＭＣ）的作用机制。方法 应用改良的Ｌｏｗｒｙ法测ＶＳＭＣ蛋白质含量,Ｆｕｒａ－２荧光指标剂测ＶＳＭＣ内的钙浓度（〔Ｃａ＾２＋〕）,放射免疫环腺苷酸（ｃＡＭＰ）、环一磷酸鸟苷（ｃＧＭＰ）药盒测ｃＡＭＰ、ｃＧＭＰ浓度。结果 （１）缺氧组ＶＳＭＣ内质网扩张,胞浆内出现髓鞘样结构,线粒体肿胀、空泡化。低浓度ＣＯ复合缺氧组ＶＳＭＣ的损害减轻,仅     

Molecular Characterizations of Subcellular Localization Signals in the Nucleocapsid Protein of Porcine Epidemic Diarrhea Virus

The nucleolus is a dynamic subnuclear structure, which is crucial to the normal operation of the eukaryotic cell. The porcine epidemic diarrhea virus (PEDV), coronavirus nucleocapsid (N) protein, plays important roles in the process of virus replication and cellular infection. Virus infection and transfection showed that N protein was predominately localized in the cytoplasm, but also found in the nucleolus in Vero E6 cells. Furthermore, by utilizing fusion proteins with green fluorescent protein (GFP), deletion mutations or site-directed mutagenesis of PEDV N protein, coupled with live cell imaging and confocal microscopy, it was revealed that, a region spanning amino acids (aa), 71–90 in region 1 of the N protein was sufficient for nucleolar localization and R87 and R89 were critical for its function. We also identified two nuclear export signals (NES, aa221–236, and 325–364), however, only the nuclear export signal (aa325–364) was found to be functional in the context of the full-length N protein. Finally, the activity of this nuclear export signal (NES) was inhibited by the antibiotic Lepomycin B, suggesting that N is exported by a chromosome region maintenance 1-related export pathway.     

Left-right asymmetric ventricular expression of CARP in the piglet heart: regional response to experimental heart failure

BACKGROUND AND AIM: Cardiac ankyrin repeat protein (CARP), whose expression is down-regulated in response to doxorubicin (Dox) in vitro, has been proposed to be a marker of experimentally-induced cardiac hypertrophy in rodent models. In piglets, the rapid hypertrophy rate of the left ventricle (LV) as compared to that of the right ventricle (RV) represents a natural model of asymmetric ventricular enlargement. We tested whether CARP expression correlates with postnatal ventricular hypertrophy and to what extent CARP can be sensitive to Dox treatment in vivo. METHODS: CARP mRNA and protein levels were quantified (by Northern blot hybridization, semi-quantitative RT-PCR and Western blot) in the piglet heart, both during early postnatal development and upon Dox-induced cardiomyopathy (Dox-CM). RESULTS: The study revealed: (1) significantly augmented CARP mRNA and protein levels in the LV compared to the RV resulting in left vs. right asymmetry in ventricular CARP expression throughout early postnatal development; (2) dose- and chamber-dependent CARP mRNA and protein enrichment in ventricular myocardium in response to Dox; and (3) abolishment of asymmetric patterns of ventricular CARP expression at heart failure resulting from Dox-CM. CONCLUSIONS: (1) CARP is differentially regulated in the LV and RV during both postnatal development and disease; and (2) monitoring of ventricular CARP expression patterns can be used for further analysis of transition from compensated to overt heart failure.     

Clinical trials update from the American Heart Association meeting: ACORN-CSD, primary care trial of chronic disease management, PEACE, CREATE, SHIELD, A-HeFT, GEMINI, vitamin E meta-analysis, ESCAPE, CARP, and SCD-HeFT cost-effectiveness study

This article provides information and a commentary on landmark trials presented at the American Heart Association meeting held in November 2004, relevant to the pathophysiology, prevention, and treatment of heart failure. An open trial of the ACORN Cardiac Support Device (CSD) showed encouraging preliminary results in patients with severe heart failure. The PEACE (Prevention of Events with Angiotensin-Converting Enzyme inhibition) study supports data from previous studies showing that ACE inhibitors reduce vascular events in patients at increased risk. The CREATE (clinical trial of metabolic modulation in acute MI treatment evaluation) study of patients with acute myocardial infarction (MI) showed no mortality benefit of a glucose/insulin/potassium regimen, but treatment with reviparin reduced the incidence of death, MI, or stroke. Azimilide was not associated with a significant reduction in shocks, but reduced the shocks or episodes of markedly symptomatic ventricular tachycardia terminated by pacing in the SHIELD (Shock Inhibition Evaluation with Azimilide) study. The addition of isosorbide dinitrate plus hydralazine to standard therapy improved survival in black heart failure patients in the A-HeFT (African-American Heart Failure Trial) study. In an investigation of hypertensive patients with diabetes, carvedilol had fewer adverse effects on diabetic control than metoprolol. A meta-analysis of high-dose vitamin E supplementation suggested an association with increased mortality. The ESCAPE (Evaluation Study of CHF and Pulmonary Artery Catheterisation Effectiveness) study showed no benefit of pulmonary artery catheterisation over clinical management in patients with severe heart failure. Routine prophylactic coronary revascularisation for stable coronary disease prior to major vascular surgery showed no benefit in the CARP (Coronary Artery Revascularization Prophylaxis) study. Analysis of data from SCD-HeFT supports the cost-effectiveness of ICDs in heart failure, although overall cost implications may be prohibitive.     

安氏隐孢子虫热休克蛋白编码基因的克隆、表达和分析

目的 克隆、表达和分析安氏隐孢子虫Mr 70 000热休克蛋白（CaHSP70） 的部分编码基因。 方法 依据公布的CaHSP70基因序列设计引物，以江苏徐州安氏隐孢子虫（XZ-BOV）总RNA为模板，反转录PCR（RT-PCR）扩增目的编码基因。PCR产物经TA克隆后，亚克隆入pET28a原核表达载体，构建重组质粒pET28a-CaHSP70，转化感受态大肠埃希菌BL21（DE3），异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达并获得纯化的重组蛋白（简称为rCaHSP70）。用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、蛋白质印迹（Western blotting）和ELISA对该重组蛋白进行分析和鉴定。采用相关生物信息学软件对序列进行分析。 结果 根据克隆的目的基因序列推导的氨基酸序列与GenBank登录的CaHSP70一致。SDS-PAGE和Western blotting分析显示，重组蛋白（rCaHSP70） Mr约为43 000（含6个组氨酸），以包涵体的形式存在，可被辣根过氧化物酶标记的抗组氨酸抗体、安氏隐孢子虫感染的小鼠血清、微小隐孢子虫感染的儿童血清和rCaHSP70免疫小鼠血清识别。rCaHSP70存在多个功能位点和潜在的抗原决定簇。种系发生分析表明XZ-BOV与安氏隐孢子虫进化关系最近。ELISA检测结果表明，rCaHSP70免疫的C57BL/6小鼠与BALB/c小鼠血清特异性抗体滴度均显著高于免疫前。 结论 XZ-BOV HSP70部分编码基因的克隆获得成功，研究获得的重组蛋白具有一定的免疫原性和免疫反应性。