腺病毒55型抗原表位分析及评价

目的:筛选腺病毒55型(Ad55)抗原表位,为研制腺病毒免疫诊断试剂提供基础。方法:采用生物信息学方法预测Ad55六邻体、纤突的B细胞抗原表位;利用大肠杆菌优势密码子获得相应基因,插入载体pBVIL-1克隆表达后获得重组Ad55表位抗原;用临床疑似腺病毒呼吸道感染患者血清对重组Ad55表位抗原活性进行检测,用ROC曲线分析重组Ad55抗原的诊断意义;采用ClustalX软件进行多序列比较。结果:Ad55六邻体含有6个主要抗原表位,纤突含有2个主要抗原表位;采用退火延伸法获得上述8种表位基因,片段长度为90~180 bp;获得上述8种重组基因工程抗原,相对分子质量为18×103~21×103;血清学检测的ROC曲线分析显示,270~320、410~460和135~165氨基酸残基抗原表位的AUC面积超过0.75,具有一定的诊断意义(P0.05);序列比较结果显示,上述3种抗原表位与腺病毒11型序列高度同源,与3型存在较大差异。结论:获得了3个Ad55主要抗原,对研制通用性呼吸道传播腺病毒免疫诊断试剂具有一定的意义。     

Ad.RGD-ING4-PTEN对MEG01人白血病细胞的抑制作用

目的:构建RGD修饰的ING4和PTEN双基因共表达重组腺病毒载体(Ad.RGD-ING4-PTEN),研究其对人白血病细胞MEG01的抑制作用。方法:采用AdEasyTM腺病毒重组系统,在本科室已成功构建的pAdTrack-CMV-ING4-polyA-promoter、pAdTrack-CMV-polyA-promoter腺病毒转移质粒的基础上,在多克隆酶切位点的Not I、Xho I间插入PTEN片段,得到pAdTrack-CMVING4-polyA-promoter-PTEN重组转移载体,与RGD-4C修饰的腺病毒骨架质粒pAdEasy-1(RGD)同源重组后,经包装和扩增获得ING4和PTEN双基因共表达重组腺病毒Ad.RGD-ING4-PTEN。将Ad.RGD-ING4-PTEN体外感染人白血病细胞MEG01,检测腺病毒对MEG01细胞的感染效率,Western blot法检测外源基因ING4和PTEN在MEG01细胞中的表达,CCK8法检测外源基因的导入对MEG01细胞的生长抑制作用,流式细胞仪检测外源基因导入对MEG01细胞的凋亡和周期的影响。结果:鉴定结果显示,成功构建了Ad.RGD-ING4-PTEN双基因共表达腺病毒,其能有效地感染MEG01细胞。Western blot检测到ING4和PTEN在MEG01细胞中的表达;ING4和PTEN基因能明显抑制MEG01细胞的生长,诱导其凋亡并产生G2/M期阻滞作用,且双基因联合作用较单基因作用更明显。结论:成功构建了RGD-4C修饰的ING4和PTEN双基因共表达腺病毒载体Ad.RGD-ING4-PTEN。收获的腺病毒能有效感染人白血病MEG01细胞。Ad.RGD-ING4-PTEN具有抑制MEG01细胞的生长、诱导其凋亡及G2/M期阻滞的作用。     

纤维顶球改造增强了人5型腺病毒载体对造血细胞的感染

基于人5型腺病毒(Human adenovirus type 5,HAdV-5)的腺病毒载体对造血细胞的基因转导效率低,将病毒fiber基因替换为HAdV-11p的同源基因F11p后,载体对造血细胞的感染效率增强.本研究拟在F11p纤维顶球(knob)结构域添加RGD4C多肽或HIV包膜糖蛋白(gp120)的V3结构域,观察重组HAdV-5对造血细胞感染效率的变化.在前期构建的pKAd5f11p153R-EPG腺病毒质粒基础上,结合限制性酶切和DNA组装技术,在F11p 153 aa后(knob AB loop,153位)、228位(FG loop)以及300位(IJ loop)插入 RGD4C 肽或者 gp120的 V3肽,构建了共6种重组腺病毒载体(F153RGD-EG、F228RGD-EG、F300RGD-EG、F153CV-EG、F228CV-EG和 F300CV-EG),以fiber未改造的HAdV5-EG和改造为F11p的F11p-EG病毒作对照,观察了其对4种造血细胞系U937、K562、Jurkat和HL60以及人原代T细胞的感染效率.结果显示,对于U937细胞,当感染复数(MOI,vp/cell)为100时,HAdV5-EG感染效率最低,为2％;其次为F228CV-EG,感染率为45％;F300RGD-EG、F153CV-EG和F300CV-EG感染率为85％～90％;F153RGD-EG、F228RGD-EG高于阳性对照病毒F11p-EG,三者分别为99％、99％、95％.各病毒对于Jurkat细胞的感染率均较高,但HAdV5-EG明显低于F11p-EG、F153RGD-EG和F228RGD-EG,当MOI为100时分别为75％、93％、93％和96％.感染K562细胞的情况与U937细胞类似.各病毒对于HL60细胞感染效率最低,MOI为500时,F300RGD-EG和F300CV-EG的转导效率为28％和33％,是F11p-EG的10倍.对于人原代T细胞,F153RGD-EG和F228RGD-EG优于F11p-EG,当MOI为1000时,感染率分别为87％、90％和84％.研究结果表明,F11p knob插入RGD4C比单独F11p替换的HAdV-5对造血细胞的感染效率高,同时,本研究还发现HAdV-11p fiber knob的AB、FG或IJ loop可插入外源多肽,为腺病毒嗜向性改造增加了新靶点.     

NIPSNAP3A短发夹RNA腺病毒载体的构建及病毒包装

目的:构建靶向NIPSNAP3A的短发夹RNA(shRNA)腺病毒载体,干扰HEK293A细胞中NIPSNAP3A的表达。方法:设计靶向NIPSNAP3A的shRNA,插入穿梭质粒pENTRY,通过Gateway法获得腺病毒颗粒pAd-NIPSNAP3A-shRNA,转染HEK293A细胞,在细胞内包装获得腺病毒。结果:重组腺病毒载体经酶切鉴定正确,制备的病毒感染效率高,能显著抑制NIPSNP3A蛋白的表达。结论:干扰HEK293A细胞NIPSNAP3A表达的shRNA重组腺病毒载体构建成功。     

RGD序列修饰的MS2噬菌体样颗粒的构建及表达

目的:构建并表达衣壳蛋白表面展示有精氨酸-甘氨酸-天冬氨酸(RGD)序列的MS2噬菌体样颗粒,以期获得肿瘤细胞靶向性RNA递送载体。方法:用分子克隆技术将RGD4C编码序列插入MS2衣壳蛋白编码基因,然后构建到p ETDuet-1质粒多克隆位点MCS1上;将含有MS2噬菌体包装位点的pre-let-7编码基因构建到p ETDuet-1质粒多克隆位点MCS2上;重组质粒转化大肠杆菌,表达产物纯化后经琼脂糖凝胶电泳、电子显微镜鉴定。结果和结论:采用分子克隆技术和原核细胞单质粒双表达系统获得表面展示有RGD序列,内部包装有pre-let-7 RNA的MS2噬菌体样颗粒,为体内和体外研究其肿瘤细胞靶向递送和功能奠定了基础。     

针对Oct-4和Survivin基因的双靶向shRNA腺病毒载体的构建及其对肝癌细胞的抑制作用

转录因子Oct-4和Survivin是细胞增殖的关键调控因子,构建针对Oct-4和Survivin基因的双靶向shRNA腺病毒载体Ad5-Dual-shRNA,并研究其对肝癌细胞及移植瘤的生长抑制作用。合成Oct-4和Survivin基因的shRNA序列,插入腺病毒穿梭载体pDC312,含有shRNA的穿梭载体与腺病毒骨架载体pBHGloxdeltaE13Cre共转染HEK293细胞,经Cre/LoxP位点特异性重组获得重组腺病毒Ad5-Dual-shRNA;腺病毒Ad5-Dual-shRNA感染肝癌细胞系EHBH-H1,经Western blotting检测Oct-4和Survivin基因的表达情况,用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐染色法(MTT实验)和裸鼠荷瘤实验检测对肿瘤细胞生长的影响。研究结果显示,双靶向重组腺病毒Ad5-Dual-shRNA感染肝癌细胞系EHBH-H1能够有效沉默Oct-4与Survivin基因的表达,并且在MTT实验和裸鼠荷瘤试验中都显示出较单一靶向的shRNA腺病毒载体Ad5-Surv-shRNA、Ad5-Oct4-shRNA具有更为明显的肿瘤细胞生长抑制作用。实验结果表明,特异性双靶向shRNA腺病毒载体Ad5-Dual-shRNA是一种更为高效的靶向肿瘤基因治疗载体。     

腺病毒4型和7型重组乙肝疫苗在犬体内的免疫效果

<正>近年来,应用感染性哺乳动物病毒载体,特别是腺病毒(Ad)和牛痘病毒,作为有潜能的疫苗,日益受到重视。腺病毒尤其受人青睐,有下列原因: 1.腺病毒感染产生的大量腺病毒蛋白有助于异源基因的高水平表达。 2.切去无意义的E_3区,可供插入大片段的外源基因,以建立有活力的不依赖辅助物的腺病毒载体。     

ω-6脂肪酸脱氢酶基因在乳腺癌细胞内的表达和作用

为探讨ω- 6脂肪酸脱氢酶基因fat -1在人类乳腺癌细胞MCF- 7中表达和对其生长的作用,将fat -1基因插入到腺病毒载体中,构建腺病毒重组载体(Ad·GFP·fat1) .通过包装细胞系(2 93)产生重组腺病毒,感染MCF 7细胞.用核糖核酸酶保护性分析技术,检测fat -1基因在MCF- 7细胞内的表达,细胞增殖试剂盒(MTT)和凋亡染色试剂盒染色分析fat 1基因对MCF- 7细胞增殖和凋亡的影响,用酶联免疫分析花生酸类(eicosanoids)前列腺素E2 (prostaglandinE2 )的含量.结果显示,腺病毒介导的fat- 1基因能在MCF- 7细胞内有效异源表达,抑制MCF -7细胞的增殖且导致凋亡,前列腺素的含量也明显地减少.结果说明,fat- 1基因在乳腺癌的基因治疗中具有良好利用价值.     

含口蹄疫病毒VP1基因与结核杆菌HSP70基因的重组腺病毒转移载体的构建

根据GenBank已发表的结核杆菌HSP70的基因序列及O型口蹄疫病毒VP1基因序列,应甩Primer5．0软件设计了2对引物,其中一对引物用于扩增结核杆菌基因组中的HSP70完整基因,另一对引物用于以pMD-D为模板,扩增其中的VP1完整基因序列。将所扩增的2段基因串联插入腺病毒转移载体．pAdenovator-CMV5-IRES-GFP的BgⅢ位点,经PCR方法和限制性酶切方法鉴定2基因已成功插入了转移载体并且插入方向正确,获得了重组腺病毒转移载体pAdenovator-CMV5-IRES-GFP-VP1-HSP,此转移载体可与腺病毒骨架载体进行细菌内同源重组,以产生重组腺病毒载体。     

表达猪γ干扰素的重组腺病毒的构建和活性鉴定

以刀豆素A(ConA)诱导的梅山猪外周血淋巴细胞中提取的总RNA为模板,采用RT-PCR方法克隆扩增出全长猪γ干扰素基因(PoIFNγ,501bp)。测序结果证实扩增得到的PoIFNγ与GenBank上所报道的猪γ干扰素基因同源性为100%。将PoIFNγ插入腺病毒穿梭载体pShuttle-CMV,与腺病毒骨架载体pAdEasy-1同源重组。在293A包装细胞系中成功包装成完整的病毒粒子。将此重组后的腺病毒连续接种传至第10代,每代提取病毒基因组,PCR均能扩增出目的片段,以细胞病变抑制法(CPE50)测定干扰素生物活性达1.3×106U/mL,证明该种表达猪γ-干扰素的重组腺病毒能稳定传代。     

The activity of membranes reconstituted from HVJ envelope proteins and lipids to induce hemolysis and fusion between liposomes and erythrocytes

A simple method for preparation of lipid-free envelope proteins (HN protein and F protein) of HVJ (Sendai virus) was developed. Reconstituted 'envelopes' were then prepared from envelope proteins and various lipids by the detergent dialysis method, and the activity to induce hemolysis and fusion between liposome and erythrocyte was studied. Lipid-free envelope protein aggregates could induce hemolysis and liposome-erythrocyte fusion. The activity was however greatly augmented by incorporation of envelope proteins into membrane of viral total lipids. Hemolytic and fusogenic activity was somewhat augmented by incorporation of envelope proteins into dipalmitoylphosphatidylcholine/cholesterol (1:1, molar ratio) and dimyristoylphosphatidylcholine/cholesterol (1:1), though the augmentation was lower than that observed with viral total lipids. When 'envelopes' were reconstituted with the proteins and viral total lipids supplemented with phosphatidylethanolamine, two kinds of 'envelopes' were prepared; one was permeable to Dextran (Mr 75000) and hemolytic, and the other was impermeable to Dextran and nonhemolytic. The latter acquired hemolytic activity after subjection to freezing and thawing, and its barrier function was lost concomitantly. The study suggests that envelope proteins (HN protein and F protein) could function without lipids but their activity was greatly influenced by not only the composition of additional lipids but also mode of arrangement of components on the reconstituted membranes.     

Unfolding and refolding of porcine odorant binding protein in guanidinium hydrochloride: equilibrium studies at neutral pH

Unfolding and refolding studies on porcine odorant binding protein (pOBP) have been performed at pH 7 in the presence of guanidinium hydrochloride (GdnHCl). Unfolding, monitored by following changes of protein fluorescence and circular dichroism (CD), was found to be a reversible process, in terms of recovered structure and function. The equilibrium transition data were fitted by a simple two-state sigmoidal function of denaturant concentration and the thermodynamic folding parameters, derived from the two techniques, were very similar (average values: C(1/2) approximately 2.4 M, m approximately 2 kcal mol(-1) M(-1), DeltaG(unf,w)(0) approximately 4.7 kcal mol(-1)). The transition was independent of protein concentration, indicating that only monomeric species are involved. Only a minor protective effect by the fluorescent ligand 1-amino-anthracene (AMA) against protein unfolding was detected, whereas dihydromyrcenol (DHM) stabilised the protein to a larger extent (DeltaC(1/2) approximately 0.5 M). Refolding was complete, when the protein, denatured with GdnHCl, was diluted with buffer. On the other hand, refolding by dialysis was largely prevented by concomitant aggregation. The present results on pOBP are compared with those on bovine OBP (bOBP) [Biochim. Biophys. Acta 1599 (2002) 90], where subunit folding is accompanied by domain swapping. We finally suggest that the generally observed two-state folding of many lipocalins is probably favoured by their beta-barrel topology.     

(1-->6)-Beta-D-glucan as the cell wall binding site for Debaryomyces hansenii killer toxin

AIMS: The aims of this study were to characterize the cell wall binding site of Debaryomyces hansenii killer toxin to provide a simple purification method and to determine some characteristics of this toxin. METHODS AND RESULTS: Various linear (1-->6)-beta-D-glucans of different origins were effective competitive inhibitors of the toxin action. Periodate oxidation and 1H-NMR was used to determine the receptor nature. Affinity chromatography on pustulan-Sepharose column was used to purify D. hansenii killer toxin, probably a 23-kDa protein. The killer toxin character was cureless. CONCLUSIONS: The investigation revealed that the killer toxin was mainly adsorbed by (1-->6)-beta-D-glucans. This is a low molecular weight protein, probably encoded by chromosomal genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The specificity of the killer toxin for its receptor provides an effective means to purify the killer toxin. This study is the first to identify the cell wall binding site of this killer toxin, a toxin with properties of industrial relevance.     

Co-determination of ATP and proteins in Triton X 100 non-ionic detergent-opened monolayer cultured cells.

Human monolayer cells (HEp-2 and Hep G2) were cultured in 96-well plates. A modified Triton X 100 nonionic detergent extraction method was used for releasing intracellular ATP and protein in one step. The detergent technique was compared to perchloric acid (PCA) extraction. ATP was determined by the firefly bioluminescence method and ATP values were referred to cell protein (ATP:protein ratio). There was no significant difference in ATP data between detergent and PCA treatments. The ATP:protein ratio seems to be a sensitive tool for characterizing the metabolic activity of monolayer tissue culture cells. The protein-mobilizing capability of Triton X 100 depends on the type of cell culture used. Our modified extraction gives reliable ATP:protein values with one simple extraction step.     

Oxidation of sperm whale oxymyoglobin, catalyzed by ferrocyanide ions: kinetics and mechanisms

Specific catalytic oxidation of sperm whale oxymyoglobin by small amounts of potassium ferri- and ferrocyanide, from 1 to 20% in relation to the protein concentration, was studied. The mechanism of catalysis was shown to involve specific binding of the ferrocyanide anion to the protein. The influence of pH and ionic strength of the medium, the [Fe(CN)6]4- concentration and of chemical modification of Mb histidines by bromoacetate, as well as the effect of the Mb complexing with redox-inactive zinc ion on the rate of reaction was examined. The zinc ion forms a stable complex with His 119(GH1) on the Mb surface at the equimolar Zn2+ concentration. The kinetic scheme of the reaction was analyzed, and the equilibrium and kinetic parameters were obtained. It was first shown that the strong oxidant such as potassium ferricyanide is able to react with the same protein by two distinct mechanisms: (i) a simple outer sphere electron transfer over the heme edge and (ii) electron transfer after the specific binding of [Fe(CN)6]4- to oxyMb in the His 119(GH1) region, thus catalyzing the protein oxidation.     

Lipoprotein lipase-catalyzed hydrolysis of phospholipid monolayers: effect of fatty acyl composition on enzyme activity

The phospholipase A1 activity of lipoprotein lipase (LpL) was determined with monomolecular phospholipid films. Rates of phospholipid hydrolysis were dependent on apolipoprotein C-II (the activator protein for LpL) phospholipid fatty acyl composition, and lipid-packing density. In sphingomyelin: cholesterol (2:1, molar) monolayers containing 5 mol % disaturated phosphatidylcholines (PC) and at a surface pressure of 22 mNm-1, rates of LpL hydrolysis of diC14:0PC, diC16:0PC, and diC18:0PC were 74, 207, and 65 nmol h-1 mg LpL-1, respectively. At 22 mNm-1, phospholipids containing unsaturated fatty acyl chains were hydrolyzed at rates 5-10 times greater than saturated lipids. At higher lipid packing densities, the difference in hydrolysis rates between saturated and unsaturated lipids was less apparent. Comparison of molecular areas indicate no simple dependency between the rate of LpL catalysis and phospholipid fatty acyl chain length and saturation/unsaturation.     

THE PROTEIN COMPOSITION OF ISOLATED MYELIN1

—Three fractions, each containing markedly different proteins, was obtained from myelin: (1) The first fraction was obtained as an insoluble residue when myelin was extracted with neutral chloroform-methanol (CM, 2:1, v/v). It was digestible with trypsin and had an amino acid composition similar to that of the acidic proteolipid protein of Wolfgkam (1966). (2) The second fraction was obtained as a precipitate by the addition of various electrolytes (KCl, NaCl, CaCl₂, MgCl₂ or HCl) to the CM (2:1 v/v) extract. This fraction consisted mainly of a basic protein which exhibited an electrophoretic mobility and amino acid composition indistinguishable from those of the basic protein obtained from white matter (Martensson and LeBaron, 1966). This procedure provided for a simple and rapid isolation of the basic protein from myelin. Depending on the conditions of precipitation, this fraction was either free of lipid or contained tri- and diphosphoinositide. The effects of different ions at differing concentrations and the yield and nature of the precipitate have been studied. (3) A third fraction remained in solution in CM (2:1, v/v) after the addition of the electrolyte. It comprised the bulk of the myelin lipids and a protein fraction which was resistant to digestion with trypsin and had an amino acid composition similar to the classical proteolipid protein of Folch-Pi and Lees (1951). The possibility of a salt-type bonding between the basic protein and the polyphosphoinositides is discussed, and values for tri- and diphosphoinositide in bovine myelin are given.     

重组类胰岛素样生长因子-Ⅰ的纯化与复性

目的 获得高纯度和高活性的胰岛素样生长因子（Insulin-like growth factor, IGF-1）;方法 构建好的BL21大肠杆菌工程菌经IPTG诱导,以融合一段截短型半乳糖苷酶及His-tag形式表达IGF-1融合蛋白(约15,000Da),超声破碎,提取包涵体经镍柱亲和层析后, 用羟氨切割纯化的融合蛋白,纯化后的蛋白质在小分子保护剂及GSH/GSSG的存在下复性。结果 经Ni2＋柱亲和层析, IGF-1纯度达90％以上,复性后得到有较高生物活性的IGF-1。结论 IGF-1发酵及纯化和复性方法的建立为大量生产IGF-1打下了基础。     

Purification and Chemical Characterization of a W2 Protein from Brain Myelin

Abstract: Starting from a pellet of beef brain myelin insoluble in chloroform/ methanol (2:1, vol/vol) (Wolfgram protein fraction), a pure W2 protein with apparent molecular weight of 52,000 was isolated by a simple preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. A comparative chemical analysis was carried out between purified W2 and a standard tubulin. Glutamic acid and arginine were the N-terminals detected. Similar peptide maps and amino acid composition were also found in both proteins. Immunological cross-reactivity was detected when W2 protein was tested against antitubulin serum. These results suggest that W2 protein could have a tubulin-like protein nature that is associated with the myelin membrane and could play a role in the myelination process.     

Application of atomic force microscopy and grating coupler for the characterization of biosensor surfaces

Atomic force microscopy (AFM) and an optical grating coupler system were used to improve the understanding of the biosensing layer on a Ta(2)O(5)-light-guiding surface. Exemplary, we investigated the immobilization of the protein avidin, the subsequent binding of biotinylated oligonucleotides and hybridization of a complementary 12-mer. The AFM measurements revealed the height of approximately 1.6 nm for a single avidin molecule, while the thickness of the avidin layer on the biosensor surface seemed to be 2.8-3.0 nm. This result lead to the conclusion that the protein was not forming a simple monolayer. However, the thickness of the avidin layer could not be determined directly, but only after shifting of protein by the tip of the AFM leading to grooves of 1 micro m(2) and approximately 3 nm depth. As the height of oxide particles forming the waveguide surface was also in the range of 1.5 nm, the depth of these grooves could also be a result of the deposition of proteins on top of the oxide particles. This was consistent with the increased roughness of the surface after protein binding. Thus, investigations with the grating coupler were used to determine quantitatively the amount of immobilized avidin. On a biotinylated surface the amount of immobilized avidin lead to the assumption of a complete monolayer, whereas simple adsorption proved to be less efficient. A binding ratio of 1:1.3 for avidin and a biotinylated oligonucleotide was achieved. Up to 83% of the bound single strand were accessible for a subsequent hybridization reaction with a 12-mer. These results supported the model of avidin being deposited mainly on top of the oxide particles leading to the picture of a 'rough' complete protein monolayer, which was postulated from the AFM investigations.