蛋白转导域介导BCR/ABL抗原对CML患者T细胞的活化作用

目的：研究蛋白转导域(PTD)介导的BCR／ABL抗原对慢性髓细胞白血病(CML)患者T细胞的特异性活化作用。方法：利用基因工程技术，将PTD基因与CML b3a2 bcr／abl基因融合并原核表达。将纯化的PTD—BCR／ABL融合蛋白与CML患者外周血单个核细胞(PBMC)体外共孵育，用流式细胞仪分别检测CD4^ 、CD8^ T细胞上活化抗原CD25的表达。结果：终浓度为100mg／L的PTD—BCR／ABL抗原体外刺激4d后，10例CML患者中，5例表现为CD8^ T细胞活化，2例表现为CD4^ T细胞活化，其中有1例CD8^ 和CD4^ T细胞同时活化；而作为对照的BCR／ABL抗原刺激组无一例表现为CD8^ 或CD4^ T细胞活化。结论：PTD能将外源性BCR／ABL抗原转导入抗原呈递细胞内，加工呈递后激活抗原特异性CD8^ 及CD4^ T细胞，为CML特异性CD8^ 、CD4^ T细胞的体外活化及细胞免疫治疗开辟一条新的途径。     

小鼠精子Sp17与IL-5融合蛋白的构建、表达及其免疫原性鉴定

目的 构建和表达小鼠精子蛋白Sp17与白介素5(IL-5)融合蛋白,并对其进行纯化和免疫原性鉴定.方法 采用PCR技术从质粒pGEM-1-IL-5中扩增IL-5基因片段,将其克隆至pET/Sp17原核表达载体中与Sp17基因融合,构建重组质粒pET/Sp17-IL-5,经酶切鉴定后转化大肠杆菌B121(DE3),IPTG诱导表达,采用Ni2+-NTA Agarose 纯化,SDS-PAGE检测、N端测序及Western blot;重组蛋白Sp17.IL-5免疫小鼠后ELISA检测小鼠血清特异性抗体.结果 成功构建小鼠精子蛋白Sp17与IL-5融合蛋白的高效表达质粒pET/Sp17-IL-5,重组工程菌pET-28a(+)-sp17-IL-5/BL21经IPTG诱导目的蛋白表达率约28%,PAGE初步测定目的蛋白相对分子量(Mr)约39 000,纯化后蛋白纯度达91%,免疫后小鼠血清中检测到特异性抗体.结论 Sp17-IL-5蛋白经基因克隆获得了较高的表达量,并初步显示了较好的免疫活性,为新型Sp17避孕疫苗的研制奠定基础.     

PTD—bcr／abl融合癌蛋白片段的表达及其跨膜转运

目的 探讨蛋白转导结构域（PTD）介导的转导bcr/abl癌蛋白片段的方法,为进行免疫治疗提供实验依据。方法 合成编码PTD的基因片段,并与PCR扩增的慢性粒细胞白血病癌蛋bcr/abl基因片段融合,在胸有成竹肠杆菌中表达,将纯化的融合蛋白PTD-bcr/abl与HL-60细胞共孵育,用Western blot分析融合蛋白的跨膜转运。结果 PTD基序可介导bcr/abl蛋白由细胞外跨膜转导进行细胞内。结论 这一结果可能为应用外源蛋白负载（loading)抗原提呈细胞（APC）提供新的途径。     

结核分枝杆菌小分子热休克蛋白16.3蛋白的原核表达及功能检测

目的进行结核分枝杆菌小分子热休克蛋白MTB Hsp16.3原核表达载体的构建、表达、纯化并初步观察其生物学效应。方法提取临床H37Rv分离株基因组DNA,PCR扩增Hsp16.3基因,将其重组到原核表达载体Pet28a中,构建原核表达载体Pet28a-Hsp16.3,进行双酶切及测序鉴定。将测序正确的重组质粒转化至E.coli BL21(DE3)中,经IPTG诱导表达后,对表达产物进行SDS-PAGE检测,同时通过镍柱纯化试剂盒纯化Hsp16.3,测定纯化后蛋白浓度,并进行Western blot法检测。将不同浓度纯化后蛋白作用小鼠腹腔巨噬细胞,实时定量PCR(qRT-PCR)检测巨噬细胞IL-10和IFN-γ的表达,同时设定空白对照组及阳性对照组。结果成功构建重组质粒Pet28a-Hsp16.3,并在E.coli BL21(DE3)中获得成功表达,通过镍柱纯化系统得到纯化Hsp16.3融合蛋白。qRT-PCR检测结果显示,不同浓度纯化后Hsp16.3蛋白作用小鼠腹腔巨噬细胞,可促进IFN-γ的产生而抑制IL-10的产生。结论成功克隆、表达和纯化了MTB Hsp16.3蛋白,Hsp16.3能促进小鼠腹腔巨噬细胞产生IFN-γ,抑制IL-10的产生。     

Tat-p53融合蛋白的表达、纯化及其转导活性

目的:原核表达野生型p53与TatPTD(proteintransductiondomain)的融合蛋白,检测TatPTD介导p53进入肝细胞的效率。方法:利用RT-PCR方法从A549细胞系中分离野生型p53基因,将该基因分别克隆入pTAT-HA和pET-32a原核表达载体,在大肠杆菌BL21(DE3)LysS内诱导表达并进行纯化。以纯化的p53蛋白经腹腔免疫BALB/c小鼠,制备高效价的抗血清。将Tat-p53融合蛋白加入HepG2细胞培养上清,利用间接免疫荧光法检测Tat-p53融合蛋白导入HepG2细胞的效率。结果:成功地构建了含有野生型p53基因的原核表达载体,表达纯化了Tat-p53融合蛋白及p53蛋白,制备p53特异的抗血清,证实Tat-p53可以高效的转入HepG2细胞内。结论:Tat-p53融合蛋白的表达纯化及活性分析,为应用Tat-p53融合蛋白治疗肝癌的实验研究奠定了基础。     

幽门螺杆菌UreB-Omp11融合蛋白的重组疫苗候选株构建和融合蛋白的表达、纯化及免疫学活性

目的:构建幽门螺杆菌(Hp)UreB-Omp11融合蛋白的重组疫苗候选株,在大肠杆菌中表达UreB-Omp11融合蛋白,并检测其免疫学活性。方法:用PCR方法扩增郑州分离Hp菌株MEL-HP27的ureB和omp11基因并用重叠延伸PCR法获得ureB-omp11融合基因,将融合基因ureB-omp11插入原核表达载体pET30a( )、pET28a( )及pMAL-c2X中,筛选出合适的表达系统并进行融合蛋白的表达,采用Western blot对表达产物进行鉴定,并用Amylose亲和层析法纯化融合蛋白,应用SDS-PAGE方法对纯化产物进行分析,纯化的融合蛋白辅以免疫佐剂皮下免疫小鼠,Western blot对免疫小鼠血清进行检测。结果:特异PCR法、酶切鉴定并经测序分析后证实融合基因ureB-omp11克隆入表达载体pET30a( )、pET28a( )与pMAL-c2X中;重组菌TB1(pMAL-ureB-omp11)经诱导获得了高效表达的MBP-UreB-Omp11融合蛋白,该融合蛋白可以被Hp免疫小鼠血清和Hp阳性患者血清中的相应抗体所识别,纯化后的融合蛋白纯度达90%以上。通过大肠杆菌抗原吸收法纯化免疫小鼠血清后,与纯化的融合蛋白进行杂交,结果显示在Mr134000处出现特异杂交带,融合蛋白具有良好的免疫原性和免疫反应性。结论:成功地构建并筛选出了HpMELHP27融合蛋白UreB-Omp11的重组疫苗候选株TB1(pMAL-ureB-omp11),为Hp蛋白质疫苗和核酸疫苗的研制奠定了基础。     

蛋白转导在基因治疗中的应用

蛋白转导是近几年生命科学领域发现的一种独特现象,具有蛋白转导功能的蛋白通过几个短的碱性小肽组成的蛋白转导域(proteintransductiondomain,PTD)的介导,能将与其共价连接的DNA、多肽或蛋白质以及其他大分子物质通过非经典途径穿过细胞膜甚至血脑屏障,并在细胞间自由传递。PTD这种能携带融合蛋白自由进入细胞的独特功能为人类一些疾病的基因治疗提供了一种新的运载工具,在基因治疗中有着美妙的应用前景。     

人survivin蛋白的原核表达、纯化及抗原活性鉴定

目的 构建人肿瘤抗原survivin的原核表达载体,优化在大肠杆菌中的表达条件,并对survivin/His融合蛋白进行纯化和抗原活性鉴定.方法 设计针对survivin基因序列的特异引物,通过聚合酶链式反应(PCR)扩增人survivin全长基因序列(538 bp)克隆至原核表达载体pET28a(+),构建重组表达载体pET28 a-survivin,并将该载体转化大肠杆菌BL21(DE3),经IPTG诱导表达survivin/His融合蛋白,并采用Ni亲和层析凝胶纯化重组蛋白.纯化后的重组蛋白经Western blot法、ELISA鉴定其抗原活性.结果 重组表达载体经BamH Ⅰ和HindⅢ鉴定正确;IPTG诱导后经SDS-PAGE分析表明获得了相对分子质量(Mr)24000大小的重组蛋白;纯化后的蛋白纯度达到90％.Western blot法和ELISA检测证实纯化的survivin蛋白能够与特异性抗体发生反应,表明其具有良好的抗原活性.结论 成功构建了原核表达载体pET28 a-survivin,利用大肠杆菌表达系统实现了融合蛋白的可溶性表达并进行纯化,纯化后survivin蛋白经鉴定具备较高的抗原活性.     

抗多囊蛋白1氨基端单克隆抗体的制备和鉴定

目的 :用杂交瘤技术制备抗多囊蛋白 1胞外区氨基端的单克隆抗体 (mAb) ,并对其特异性进行鉴定。方法 :以肾组织总RNA为模板 ,用RT PCR扩增多囊蛋白 1胞外区氨基端的编码基因PKD1cDNA。将该基因克隆到融合蛋白表达载体pQE3 0中 ,转染大肠杆菌M15。以异丙基硫代半乳糖苷 (IPTG)诱导表达多囊蛋白 1胞外区氨基端的组氨酸融合蛋白 (PC1 e2 ) ,用亲和层析法纯化。以纯化的融合蛋白作为抗原免疫BALB/c小鼠 ,取小鼠脾细胞与小鼠骨髓瘤细胞株Sp2 / 0进行细胞融合 ,间接ELISA筛选阳性克隆 ,有限稀释法进行单克隆化。mAb的特异性用间接ELISA和Westernblot鉴定。结果 :克隆到两个编码多囊蛋白 1氨基端的cDNA片段 (50 2bp和 471bp)。构建的表达质粒经酶切和DNA测序证实 ,为所需要的质粒。表达出相对分子质量 (Mr)分别为 1980 0和 1890 0的融合蛋白 ,经Westernblot鉴定 ,均为多囊蛋白 1的融合蛋白。用Mr 为 1890 0的融合蛋白免疫小鼠 ,得到杂交瘤细胞株 7B1。Westernblot分析表明 ,该细胞株分泌的mAb能特异地与多囊蛋白 1氨基端结合。结论 :本实验表达了PKD1多拷贝区所编码的PC1 e2 ,成功地制备了抗多囊蛋白 1胞外区N端的mAb。     

结核分枝杆菌小分子热休克蛋白16．3蛋白的原核表达及功能检测北大核心CSCD

目的进行结核分枝杆菌小分子热休克蛋白MTB Hsp16.3原核表达载体的构建、表达、纯化并初步观察其生物学效应。方法提取临床H37Rv分离株基因组DNA,PCR扩增Hsp16.3基因,将其重组到原核表达载体Pet28a中,构建原核表达载体Pet28a-Hsp16.3,进行双酶切及测序鉴定。将测序正确的重组质粒转化至E.coli BL21(DE3)中,经IPTG诱导表达后,对表达产物进行SDS-PAGE检测,同时通过镍柱纯化试剂盒纯化Hsp16.3,测定纯化后蛋白浓度,并进行Western blot法检测。将不同浓度纯化后蛋白作用小鼠腹腔巨噬细胞,实时定量PCR(qRT-PCR)检测巨噬细胞IL-10和IFN-γ的表达,同时设定空白对照组及阳性对照组。结果成功构建重组质粒Pet28a-Hsp16.3,并在E.coli BL21(DE3)中获得成功表达,通过镍柱纯化系统得到纯化Hsp16.3融合蛋白。qRT-PCR检测结果显示,不同浓度纯化后Hsp16.3蛋白作用小鼠腹腔巨噬细胞,可促进IFN-γ的产生而抑制IL-10的产生。结论成功克隆、表达和纯化了MTB Hsp16.3蛋白,Hsp16.3能促进小鼠腹腔巨噬细胞产生IFN-γ,抑制IL-10的产生。     

蛋白质与蛋白质相互作用的研究进展

通过研究蛋白质与蛋白质的相互作用,人们能更好地注释蛋白质的功能,解码生命现象,特别是在新药物的设计上具有极大的实用价值,这是后基因组研究的重要任务之一。蛋白质结构的剖析、计算机分子模拟技术的发展和分子生物学技术更新为研究蛋白质间相互作用创造了各种有利条件。     

Eosinophil cationic protein and eosinophil protein X in the nasal lavage of children during the first 4 weeks of life. SPACE Collaborative Study Team

Eosinophil cationic protein (ECP) and eosinophil protein X (EPX) are well established as markers of eosinophil activation. We analyzed ECP and EPX concentrations in nasal lavage fluids (NALF) of 378 neonates during their first 4 weeks of life. Inclusion criteria were a positive history of parental allergy and a positive skin prick test or specific IgE (RAST class > or = 2) against at least one out of a panel of common aeroallergens in one or both parents. Twenty-four infants with no history of parental allergy were used as controls. A volume of 2 ml of 0.9% saline was instilled into each nostril and immediately recovered by a suction device. ECP and EPX were analyzed by radioimmunoassay. In 65 samples of three consecutive lavages, EPX was detected in nine samples (13.8%) in the control group, whereas it was detected in 197/360 samples (54.7%) in the study population. The corresponding figures for ECP were 17/65 (26.2%) in the control group and 173/365 (47.4%) in the study group. Both proteins showed a skewed distribution (median/5-95th percentiles for ECP: 0 microg/l [0-69.4] and EPX: 6.6 microg/l [0-73.2]). The differences between the control group and the study group were statistically significant, regardless of the allergic disease of the parents. In children of allergic parents, activation proteins of the eosinophil granulocyte are released on the nasal mucosal surface in about 50% of the studied population at the age of 4 weeks. This early onset of eosinophil activation in the nasal respiratory epithelium may reflect a genetic predisposition to allergy or early exposure to allergens.     

Characterization of the NSP6 protein product of rotavirus gene 11

The 12 kDa non-structural protein 6 (NSP6) is the least studied of the rotavirus proteins. In an attempt to further characterize this protein mono-specific antisera was generated using purified protein expressed in E. coli. Pulse/chase radio-labeling of virus infected cells was used to show that it is expressed at a steady but low rate throughout the virus replication cycle. In contrast to the other rotavirus non-structural proteins, NSP6 was found to have a high rate of turnover, being completely degraded within 2 h of synthesis. NSP6 tagged with GFP was used to probe the intracellular distribution of the protein, perinuclear aggregates were observed in the cytoplasm of transfected cells. Following virus infection of these transfected cells the aggregates were seen to redistribute to the viroplasms. Consistent with its localization to the site of viral genome replication and packaging, NSP6 was found to be a sequence independent nucleic acid binding protein, with similar affinities for ssRNA and dsRNA.     

Mapping and sequence of the gene encoding protein p37, A major structural protein of African swine fever virus

The gene encoding protein p37, one of the major structural proteins of African swine fever (ASF) virus has been mapped and sequenced. Protein p37 was obtained from purified virions and the first 27 amino acids from its NH₂-terminal end were identified by automatic Edman degradation. To map the gene encoding protein p37, a mixture of 20-mer deoxyoligonucleotides based upon a part of this amino acid sequence was hybridized to cloned ASF virus restriction fragments. This allowed localization of the gene in fragmentKpnIF/HindIII G₁ of the African swine fever virus genome. An analysis of the DNA sequence from this region revealed an open reading frame encoding 418 amino acids. In this sequence, the 27 NH₂-terminal amino acids determined by sequence analysis of protein p37 are preceded by a stretch of 132 amino acids residues, indicating that protein p37 is synthesized as a polypeptide of higher molecular weight and then posttranslationally processed by cleavage of a Gly-Ala bond. This processing event accounts for the antigenic relationship of protein p37 to a virus-induced, nonstructural protein with a relative molecular weight of 60 kD.     

线粒体相关凋亡蛋白的研究进展

 细胞凋亡是细胞的主动死亡过程，该过程与肿瘤、神经退行性等疾病的发生发展密切相关。目前研究证实，线粒体是细胞凋亡发生的核心，线粒体通路是细胞凋亡的重要通路之一。与线粒体相关的一些重要蛋白如：Bcl-2家族蛋白、凋亡诱导因子、细胞色素C等在细胞凋亡过程中各司其职，共同调控着凋亡的发生发展，对细胞凋亡产生重要的作用。本文就线粒体相关的主要凋亡蛋白及其最新研究进展进行综述。     

Tamm-Horsfall protein outside the kidney

Tamm-Horsfall protein is the main protein in normal urine and readily precipitates. Previously there has been little study of the protein outside the kidney. An antiserum to the protein was used in an immunohistological investigation of human tissues. No Tamm-Horsfall protein was detected in normal organs other than the kidney. The protein was detected outside the kidney in abnormal organs in three general sites: (i) in ulcers of epithelial surfaces exposed to urine, (ii) deep in the wall of a bladder that had had previous surgery and (iii) in lymph nodes at the hilum of kidneys that had been obstructed or contained extra-tubular deposits of the protein. There was little evidence of a cellular response to the deposits of Tamm-Horsfall protein. The antiserum to Tamm-Horsfall protein is a good immunohistological marker of extravasated urine and is useful in the study of such conditions as fistulas of the urinary tract, operations on the urinary tract and reflux of urine into lymphatics and lymph nodes.     

Uncoupling the functions of a multifunctional protein: the isolation of a DNA pilot protein mutant that affects particle morphogenesis

Defective øX174 H protein-mediated DNA piloting indirectly influences the entire viral lifecycle. Faulty piloting can mask the H protein's other functions or inefficient penetration may be used to explain defects in post-piloting phenomena. For example, optimal synthesis of other viral proteins requires de novo H protein biosynthesis. As low protein concentrations affect morphogenesis, protein H's assembly functions remain obscure. An H protein mutant was isolated that allowed morphogenetic effects to be characterized independent of its other functions. The mutant protein aggregates assembly intermediates. Although excess internal scaffolding protein restores capsid assembly, the resulting mutant H protein-containing particles are less infectious. In addition, nonviable phenotypes of am(H) mutants in Su⁺ hosts, which insert non-wild-type amino acids, do not always correlate with a lack of missense protein function. Phenotypes are highly influenced by host and phage physiology. This phenomenon was unique to am(H) mutants, not observed with amber mutants in other genes.     

Identification of a novel receptor-like protein kinase that interacts with a geminivirus nuclear shuttle protein

Despite extensive studies in plant virus-host interactions, the molecular mechanisms of geminivirus movement and interactions with host components remain largely unknown. A tomato kinase protein and its soybean homolog were found to interact specifically with the nuclear shuttle protein (NSP) of Tomato golden mosaic virus (TGMV) and Tomato crinkle leaf yellows virus (TCrLYV) through yeast two-hybrid screening and in vitro protein binding assays. These proteins, designated LeNIK (Lycopersicon esculentum NSP-Interacting Kinase) and GmNIK (Glycine max NIK), belong to the LRR-RLK (leucine rich-repeat receptor-like kinase) family that is involved in plant developmental processes and/or resistance response. As such, NIK is structurally organized into characteristic domains, including a serine/threonine kinase domain with a nucleotide binding site at the C-terminal region, an internal transmembrane segment and leucine-rich repeats (LRR) at the N-terminal portion. The potential significance of the NSP-NIK interaction is discussed.