HSVⅠ感染人成纤维细胞诱导的SR-15蛋白生物学功能分析

人单纯疱疹病毒Ⅰ型(Herpes simplex virusⅠ, HSVⅠ)结合人成纤维细胞相应受体以启动感染的过程,能够引起细胞产生特异性蛋白分子的表达,其中之一的SR-15蛋白经酵母双杂交系统分析证明可与细胞内多个具有KRAB结构的锌指蛋白产生特异性相互作用,其作用部位为锌指结构域,该作用能够影响该类蛋白之一ZNF268在细胞内的转录抑制作用.     

一个由HSVⅠ诱导的类SR蛋白新基因的克隆

 根据mRNA差异显示分析 ,由单纯疱疹病毒 (HSV)Ⅰ型结合人成纤维细胞膜受体后 2h诱导产生的早期基因cDNA库中 ,分离到一个编码具有SR蛋白结构特征的新基因 ,该基因长 90 4bp .编码产生的蛋白含 12 1个氨基酸残基 ,分子量 14 9kD ,具有RS重复区和PPLP结构域 ,但不具备SR蛋白家族所特有的RNA识别区域 (RRM) .主要分布于细胞质内 ,仅在细胞膜相应受体与HSVⅠ结合后特异产生     

HSV—1感染人成纤维细胞中HTRP基因的生物信息学分析

依靠生物信息技术对HSV I型病毒刺激KMB－17细胞后克隆得到的差异基因HTRP和其编码蛋白序列的特征进行了分析和预测,初步推测了该基因的转录调控序列和编码蛋白的结构特点,为深入研究病毒刺激后细胞基因表达所编码序列的功能提供了基本数据。     

三元基序家族蛋白15敲低诱导肝癌细胞周期阻滞

三元基序家族蛋白15 (tripartite motif-containing protein 15,TRIM15)是TRIM家族成员,该家族是一类具有E3泛素连接酶活性的蛋白质.TRIM15在肿瘤中的功能鲜有报导.本研究意在阐释TRIM15在肝细胞癌(hepatocellular carcinoma,HCC)中的作用...     

成纤维细胞活化蛋白的研究进展

成纤维细胞活化蛋白(fibroblast activation protein,FAP)是一种Ⅱ型跨膜丝氨酸蛋白水解酶,在多种内源性多肽及多肽类药物的代谢中起着至关重要的作用.在过去十年中,FAP已经被多个领域的研究人员广泛关注,包括生物化学、药学、临床医学等.随着对FAP生理功能及其与临床相关疾病发生发展关系研究的深入,FAP在人体内正常和相关疾病状态下的调控方式已经越发清晰.研究表明,FAP既可以作为肿瘤的潜在靶点,又可以作为肿瘤、类风湿性关节炎等疾病早期诊断的生物学标志物.因此,多种FAP的检测方法和抑制剂被相继报道.本综述总结近年来FAP的相关研究,立足于FAP结构特点、催化性能、内源底物特征及外源底物偏好性、组织分布和组织特异性、生物功能,分析当前FAP体外和体内的检测方法和抑制剂开发的优势与不足,总结FAP检测底物的结构特征和抑制剂的潜在构效关系.本综述将对FAP特异性检测方法和强效抑制剂的开发提供重要的参考价值.     

miR15a靶蛋白依赖性的功能研究进展

miRNAs是一类内源性的、非编码蛋白的、长度约为21²3碱基的单链RNA.它们是生物体中一类重要的调控因子,广泛参与多种生物学活动,包括细胞增殖分化和凋亡、个体发育和肿瘤发生等.miR15a通过与其靶基因mRNA的3′UTR互补结合而调控靶蛋白的表达.通过对人源性miR15a靶蛋白依赖性的功能研究进展予以综述,以期对miR15a功能和相对应的生物学机制的进一步研究提供基础.     

HSVⅠ感染L-02细胞对核不均一性核糖核蛋白H2（hnRNP H2）表达影响

单纯疱疹病毒（HSVⅠ）对宿主细胞转录及mRNA修饰翻译通路的调控是一个系统的、复杂的体系,探索其具体机制将有助于了解病毒复制过程及与宿主细胞之间的相互作用。基于2-DE分析,人胎肝细胞 L-02细胞核不均一性核糖核蛋白H2（heterogeneous nuclear ribonucleoprotein H2,hnRNP H2）在感染HSVⅠ前后存在表达差异,通过Western blot ,Northern blot等技术方法验证其在病毒复制过程中不同时段对其表达影响。     

人Pokemon蛋白锌指结构域的表达与纯化

旨在原核表达Pokemon基因的锌指结构域,纯化获得GST-Zinc finger的融合蛋白。以人胶质瘤T98G细胞的c DNA为模板,利用PCR扩增带有Bam H I和Sal I酶切位点的人Pokemon基因的锌指结构域,然后将其克隆到p GEX-4T-1原核表达载体中。将正确的重组载体转入大肠杆菌BL21(DE3),用IPTG诱导表达,再利用Magne GST particles亲和纯化Zinc finger融合蛋白,最后通过Western blot鉴定此融合蛋白。结果显示,成功构建p GEX-4T-1-Zinc finger原核表达载体;30℃条件下,0.2 mmol/L的IPTG能诱导出大量的可溶性GST-Zinc finger蛋白;经Magne GST particles纯化的GST-Zinc finger蛋白可被识别Pokemon锌指结构域的抗体特异识别。纯化的GST-Zinc finger蛋白可用于后续的生物学研究。     

检测人Ⅰ型干扰素生物学活性的新方法

为建立一种更简便、安全、有效的检测Ⅰ型干扰素 (IFN)的方法 ,将可被Ⅰ型IFN诱导的人 6 - 16基因启动子与表达产物容易被检测的 β -半乳糖苷酶基因相连 ,构建成重组质粒 ,然后转染人羊膜传代细胞系 ,筛选阳性克隆 ,最终建立了一种适用于检测Ⅰ型IFN的简便、快速、敏感、特异和重复性好的新型方法 ,其敏感性同标准的细胞病变抑制法相同。     

重组人白细胞介素15融合蛋白的表达、纯化及免疫原性检测

目的:以白细胞介素15(IL-15)为靶点,研制类风湿性关节炎免疫治疗蛋白疫苗。方法:将N端融合破伤风类毒素(TT)表位的人IL-15基因克隆至带有His标签的原核表达载体pQE-30上,转化大肠杆菌M15,经IPTG诱导表达,获得重组人TT-IL-15融合蛋白(简称rtIL-15);目的蛋白经镍柱亲和层析纯化后,用Western印迹和HPLC进行鉴定;将rtIL-15与氢氧化铝佐剂混合,免疫BALB/c小鼠,检测疫苗的免疫原性。结果:双酶切鉴定和核苷酸序列测定结果表明重组融合表达质粒pQE-30-TT-IL-15构建正确,重组蛋白的表达量达到菌体总蛋白的20%,主要以包涵体形式表达,经过纯化、复性后,目的蛋白纯度达到95%以上;蛋白疫苗免疫小鼠后,能诱导高滴度的特异性的IL-15抗体。结论:在大肠杆菌中表达了重组人IL-15融合蛋白疫苗,并具有良好的免疫原性。     

在HSV I感染细胞中类PEBP蛋白基因的克隆及分析

在细胞对病毒感染后的基因反应研究中,从克隆的HSVI感染后细胞特异cDNA文库中筛选出一个705bp克隆基因-HSP-714,基因测序分析表明为一新的全基因序列(Gene Bank Accession NumberAF372624),含有完整的ORF框架,编码126个氨基酸;信息生物学分析结果表明该蛋白含有磷脂酰乙醇胺结合蛋白(PEBP)结构域,与植物、哺乳动物等多物种类CEN家族产物有高度的同源性,此外该基因的进化分析结果表明此基因更接近于植物的类CEN家族.据此推测,该基因可能是人PEBP基因家族中具有特殊意义的一个新成员.     

HSV I感染L-02细胞对核不均一性核糖核蛋白H2(hnRNP H2)表达影响

单纯疱疹病毒(HSV I)对宿主细胞转录及mRNA修饰翻译通路的调控是一个系统的、复杂的体系,探索其具体机制将有助于了解病毒复制过程及与宿主细胞之间的相互作用.基于2-DE分析,人正常肝细胞L-02细胞核不均一性核糖核蛋白H2(heterogeneous nuclear ribonucleoprotein H2,hnRNP H2)在感染HSV I前后存在表达差异,通过 Western blot,Northernblot等技术方法验证其在病毒复制过程中不同时段对其表达影响.     

Human FGF-21 Is a Substrate of Fibroblast Activation Protein

FGF-21 is a key regulator of metabolism and potential drug candidate for the treatment of type II diabetes and other metabolic disorders. However, the half-life of active, circulating, human FGF-21 has recently been shown to be limited in mice and monkeys by a proteolytic cleavage between P171 and S172. Here, we show that fibroblast activation protein is the enzyme responsible for this proteolysis by demonstrating that purified FAP cleaves human FGF-21 at this site in vitro, and that an FAP-specific inhibitor, ARI-3099, blocks the activity in mouse, monkey and human plasma and prolongs the half-life of circulating human FGF-21 in mice. Mouse FGF-21, however, lacks the FAP cleavage site and is not cleaved by FAP. These findings indicate FAP may function in the regulation of metabolism and that FAP inhibitors may prove useful in the treatment of diabetes and metabolic disorders in humans, but pre-clinical proof of concept studies in rodents will be problematic.     

Analysis of Colony-Forming Ability of Human Fibroblast Strains by Linear Regression

A linear regression approach is presented for the statistical analysis of dose-response curves obtained by measuring the colony-forming ability of human fibroblast strains. The crucial determination of the dose range in which the linear model can be assumed is achieved by a combination of statistical criteria and biological claims. As a basic quantitative parameter we investigate the slope of the regression line and, by taking reciprocals, we retransform it into the biologically established parameter D₀. Several methods for the combination of estimates are presented.     

HSV-1感染人成纤维细胞中HTRP基因的生物信息学分析

依靠生物信息技术对HSVI型病毒刺激KMB-17细胞后克隆得到的差异基因HTRP和其编码蛋白序列的特征进行了分析和顶测,初步推测了该基因的转录调控序列和编码蛋白的结构特点,为深入研究病毒刺激后细胞基因表达所编码序列的功能提供了基本数据.     

在HSVI感染细胞中类PEBP蛋白基因的克隆及分析

在细胞对病毒感染后的基因反应研究中 ,从克隆的HSVI感染后细胞特异cDNA文库中筛选出一个 70 5bp克隆基因—HSP 714,基因测序分析表明为一新的全基因序列 (GeneBankAccessionNumber:AF372 6 2 4) ,含有完整的ORF框架 ,编码 12 6个氨基酸 ;信息生物学分析结果表明该蛋白含有磷脂酰乙醇胺结合蛋白 (PEBP)结构域 ,与植物、哺乳动物等多物种类CEN家族产物有高度的同源性 ,此外该基因的进化分析结果表明此基因更接近于植物的类CEN家族。据此推测 ,该基因可能是人PEBP基因家族中具有特殊意义的一个新成员     

Cleavage of Dicer Protein by I7 Protease during Vaccinia Virus Infection

Dicer is the key component in the miRNA pathway. Degradation of Dicer protein is facilitated during vaccinia virus (VV) infection. A C-terminal cleaved product of Dicer protein was detected in the presence of MG132 during VV infection. Thus, it is possible that Dicer protein is cleaved by a viral protease followed by proteasome degradation of the cleaved product. There is a potential I7 protease cleavage site in the C-terminus of Dicer protein. Indeed, reduction of Dicer protein was detected when Dicer was co-expressed with I7 protease but not with an I7 protease mutant protein lack of the protease activity. Mutation of the potential I7 cleavage site in the C-terminus of Dicer protein resisted its degradation during VV infection. Furthermore, Dicer protein was reduced dramatically by recombinant VV vI7Li after the induction of I7 protease. If VV could facilitate the degradation of Dicer protein, the process of miRNA should be affected by VV infection. Indeed, accumulation of precursor miR122 was detected after VV infection or I7 protease expression. Reduction of miR122 would result in the suppression of HCV sub-genomic RNA replication, and, in turn, the amount of viral proteins. As expected, significant reduction of HCVNS5A protein was detected after VV infection and I7 protease expression. Therefore, our results suggest that VV could cleave Dicer protein through I7 protease to facilitate Dicer degradation, and in turn, suppress the processing of miRNAs. Effect of Dicer protein on VV replication was also studied. Exogenous expression of Dicer protein suppresses VV replication slightly while knockdown of Dicer protein does not affect VV replication significantly.     

Synthesis of Chymotrypsin Inhibitor I Protein in Potato Leaflets Induced by Detachment

Chymotrypsin inhibitor I is a protein that can be induced to accumulate in potato leaflets within hr in the light when leaflets are detached from intact plants and supplied with water. This increase in inhibitor I is not accompanied by an increase in all proteins in the detached leaflets. The accumulation is polar, proceeding from the base of the leaflets toward the tip. Low inhibitor I levels can be effectively maintained in detached leaflets by supplying them either water in the dark or solutions of indole acetic acid or 2,4-dichlorophenoxy acetic acid in the light. Inhibitor I from leaflets is not identical to inhibitor I isolated from potato tubers as determined by immunochemical analyses.