藻蓝蛋白对Hela细胞CD59基因表达调控作用的研究

探讨了钝顶螺旋藻藻蓝蛋白(PC)对Hela细胞CD59基因表达的调控作用.以正常人CD59cDNA基因为模板,经PCR扩增后重组入真核表达质粒载体pALTER-MAX,然后利用阳离子脂质体(Lipfectamine-2000)将重组质粒和PcDNA共转染人子宫颈癌细胞(Hela)和对照用正常中国仓鼠卵巢细胞(Chinese hamster ovary,CHO)进行表达.用不同浓度的钝顶螺旋藻藻蓝蛋白作用于转染细胞,通过核酸分子杂交技术、免疫荧光标记法和ELISA法对细胞中CD59分子的表达进行检测.结果表明:成功构建了重组质粒pALTER-MAX-CD59,并将其导入真核细胞(Hela,CHO),经G418筛选获得了CD59分子高效表达的细胞克隆.用藻蓝蛋白作用于筛选出的转基因细胞,证实藻蓝蛋白可促进Hela细胞表面CD59蛋白的表达并抑制Hela细胞的增殖,而对于正常CHO细胞无明显作用.     

人膜补体调节蛋白MCP、CD59在稳定转染的细胞中的共表达及功能研究

采取内核糖体进入位点(IRES)策略构建含人的膜补体调节蛋白基因MCP和CD59的cDNA的双顺反子真核表达载体pcDNA3-MCPIRESCD59,以磷酸钙沉淀法转染NIH3T3细胞,用 G418筛选阳性克隆,并研究MCP和CD59双基因在稳定细胞系中的共表达及保护功能.PCR实验结果显示双基因稳定整合在异源细胞NIH3T3的染色体上,RT-PCR及Western印迹实验分别从 RNA水平和蛋白质水平证实了人补体调节蛋白分子MCP和CD59在细胞系中皆获得同步表达.检测连续传代30次的NIH3T3 pcDNA3-MCPIRESCD59,结果表明人MCP和CD59基因仍稳定整合在细胞基因组中,并未随着传代而丢失,为稳定的转双基因细胞系.补体依赖的细胞毒反应表明,pcDNA3-MCPIRESCD59转染细胞由于MCP和CD59的共表达获得了高于MCP或CD59单一表达时所提供的保护功效,能更好地抑制人补体依赖的细胞毒作用的发生,保护宿主细胞免受人补体的攻击.以上结果表明,所构建的双基因重组表达载体实现了不同人补体调节蛋白基因高效转移和高水平共表达,在克服超急性排斥反应的基因治疗中有潜在的应用价值.     

利用Cre/lox重组系统在转基因动物中筛选高效表达外源基因的"友好"基因座

本实验的目的是利用Cre／lox重组系统进行“友好”基因座的筛选,即利用一个两端带有lox位点的标记基因去转染动物细胞或生产转基因动物,获得可以高效表达外源基因的“友好”基因座。一旦筛选到好的基因座,就建成了一个可以在动物体稳定地高效表达外源基因的技术平台。为此,利用野生型loxP位点和含有一个点突变的lox511位点分别构建两个载体pSL-loxGFP和pSL-loxIFN。首先将含有GFP和新霉素基因的质粒pSL-loxGFP DNA转染牛胎儿成纤维细胞,并用G418筛选,挑选出发绿色荧光的细胞克隆。经增殖培养之后,再将含有鸡γ-干扰素基因的质粒pSL-loxIFN和含有Cre重组酶基因的质粒pBS185 DNA共转染发荧光的细胞克隆,筛选出发生重组后不再发光的细胞克隆。用PCR法检测了两个不发荧光的细胞克隆及发荧光的细胞克隆,并使用质粒pSL-loxIFN、质粒pSL-loxGFP及牛基因组作为对照。检测结果显示,在Cre重组酶的作用下,两个不发荧光的细胞克隆中,一个发生了基因置换,另一个发生了基因删除。以上实验表明,巧妙地用Cm／lox重组系统,可以构建一套使外源基因在动物体内定点地高效表达的技术体系。     

牙鲆淋巴囊肿病毒主要衣壳蛋白基因片段真核表达载体的构建、表达与免疫效果评价

用淋巴囊肿病毒LCDV-cn感染牙鲆鳃细胞系FG-9307,提取细胞总RNA,用RTPCR法获得了LCDV-cn主要衣壳蛋白(MCP)0.6kb基因片段.将该LCDV-cn-MCP0.6kb片断克隆入真核表达载体pEGFP-N2,得到重组质粒pEGFP-N2-LCDV-cn-MCP 0.6.采用脂质体法将重组质粒转染入牙鲆鳃细胞系FEC,并进行瞬时表达.通过荧光显微镜观察和特异性RT-PCR检测,证实重组质粒pEGFP-N2-LCDV-cn-MCP 0.6kb已成功转染到FEC细胞,并得到了初步表达.将重组质粒肌注入牙鲆体内,检测牙鲆外周血、肠、脾脏、前肾和淋巴细胞的增殖反应、呼吸爆发活性及抗体产生水平.结果表明,构建的核酸疫苗pEGFP-N2-LCDV-cn-MCP 0.6可诱导牙鲆特异性体液免疫和细胞免疫,具有明显的免疫保护作用.     

小鼠对HIV-2嵌合结构基因重组鸡痘病毒的免疫反应

将HIV-2嵌合结构基因gag-gp105插入到转移载体pUTA2复合启动子ATI-P7.5×20下游,构建出鸡痘病毒重组转移质粒pA-gg;经转染和BrdU加压筛选,以基因组PCR和Western blot法鉴定重组病毒;将获得的重组病毒大量制备并纯化后,肌肉注射免疫BALB/c小鼠,ELISA法检测小鼠血清HIV-2抗体,流式细胞仪测定CD4 、CD8 T淋巴细胞亚类数量,乳酸脱氢酶(LDH)释放法检测脾特异性CTL杀伤活性.结果表明,成功筛选出一株可稳定表达HIV-2嵌合蛋白Gag-gp105的重组鸡痘病毒rFPV-gg;该重组病毒免疫组小鼠的血清出现HIV-2抗体,脾T细胞亚类数量增加,并产生针对HIV-2靶细胞的特异性CTL杀伤活性.本研究提示,HIV-2 gag-gp105重组病毒能诱导小鼠产生特异性细胞和体液免疫应答.     

固氮斯氏假单胞菌四碳二羧酸转运调控基因dctB的功能研究

为研究固氮斯氏假单胞菌（Pseudomonas stutzeri）A1501四碳二羧酸转运相关基因dctB的生物学功能,构建了携带功能完整的dctB基因片段的广宿主质粒pLL2922,通过三亲接合将该质粒转移至dctB突变株及野生型菌株A1501,并对其以琥珀酸、延胡索酸、苹果酸等四碳二羧酸为唯一碳源的培养基中的生长情况与固氮活性进行了测定。结果表明：dctB突变株不能利用琥珀酸和延胡索酸进行生长和固氮,但能以苹果酸为唯一碳源进行生长和固氮。携带功能完整dctB的广宿主质粒pLL2922,可以互补dctB位点的突变,并部分恢复突变株的四碳二羧酸转运和固氮能力。将该质粒转入A1501野生型菌株可以提高A1501的固氮活性,在琥珀酸,延胡索酸,苹果酸为唯一碳源的培养基上固氮活性比野生型菌株提高62．8％,33．9％,27．8％。表明dctB基因对于四碳二羧酸的转运及固氮能力均具有重要作用。     

骨形成蛋白-2转染MG-63细胞增加其对壳聚糖膜的粘附

为了观察壳聚糖膜对转染载有骨形成蛋白基因（bone morphogenetic protein,BMPDNA）的成骨样细胞（MG 63）粘附及增殖的影响,本研究构建重组BMP-2质粒并转染入MG 63,分别经酶切、PCR、Western、免疫组化鉴定;制备壳聚糖膜（chitosan,CS）,然后将转染细胞与该材料分别培...     

重组质粒pcFlaA在青石斑鱼肌肉中的表达研究

在哈维氏弧菌TS-628菌株鞭毛丝蛋白FlaA基因末端加上一段编码Flag短肽的核苷酸序列作为检测标记后,将该基因克隆到真核表达载体pcDNA3.1( ),酶切、PCR扩增及重组质粒测序证实基因片段插入正确,将该重组质粒命名为pcFlaA.将pcFlaA以肌肉注射方式免疫青石斑鱼.免疫后第7天开始检测鞭毛丝蛋白在石斑鱼肌肉中的表达状况,之后每隔1周检测1次,共检测4次.首先采用PCR技术在DNA水平检测重组质粒转染石斑鱼肌肉细胞的情况,再以RT-PCR法在mRNA水平上检测转染质粒在鱼肌肉中的转录,最后以免疫组化染色技术在蛋白质水平上检测目的蛋白的表达.结果在DNA及mRNA水平上均可检测到目的条带,在蛋白质水平上可检测到明显阳性位点,由此证实pcFlaA可以转染石斑鱼肌肉细胞并可在其中进行表达,而且质粒在鱼体内持续表达的时间至少1个月.     

路邓葡萄球菌单宁酶基因的克隆、表达、纯化与改造

为提高路邓葡萄球菌(Staphylococcus lugdunensis)单宁酶(Sl-tan)的活性,该文利用化学合成方法获得Sltan基因,将该基因连接到重组表达质粒pET43.1-A,再转化到大肠杆菌感受态细胞BL21-DE3中进行表达,通过亲和层析柱纯化,以没食子酸甲酯为底物进行酶活性测定以及酶学性质分析,并基于生物信息学分析,结合定点突变技术对Sl-tan进行人工改造。结果显示,获得的重组单宁酶产量明显增高,最高可达42 mg/L发酵液;酶学性质研究显示该酶在pH 8.0,温度40℃时获得的活性最高(40 U/mg);Ala460突变为Pro460后的Sl-tan活性可提高82.5%。     

嵌合抗CD20抗体分子在CHO细胞中的表达

利用PCR方法从抗CD20抗体Fab′片段的表达载体上扩增抗CD20抗体的轻链和重链可变区，将轻链和重链可变区组装到含有人抗体恒定区的表达载体中，构建抗CD20抗体IgG的轻链和重链表达载体PK100VL和PGL05VH，并用质脂体法共转染CHO细胞，RT－PCR和ELISA检测结果证实了抗CD20抗体IgG全分子在CHO细胞中的分泌表达。免疫结合结果表明CHO细胞分泌表达的抗CD20抗体IgG全分子可与CD20阳性的B淋巴瘤Raji细胞结合。     

Np(VI) Reduction with Diformylhydrazine in Perchloric Acid Solutions

Radiochemistry - The stoichiometry of the reaction of Np(VI) with diformylhydrazine N2H2(CHO)2 (DFH), in 0.01 and 0.1 M HClO4 solutions was studied by spectrophotometry. At an excess of Np(VI), 1...     

Surfactant-assisted synthesis of Keggin-type polyoxometalates nanorods

It was demonstrated that Keggin-type polyoxometalates nanorods (POMs-nanorods) were prepared from a solution containing a surfactant polyethylene glycol (PEG), when heteropolyanions [X(n+)M12O40](-(8-n)) (X(n+) = P5+ and Si4+; M = W and Mo) were precipitated by ammonia ion obtained by hydrolysis of urea at 373 K. In this method, PEG was used as a template and ammonia ion (NH4+) was a counterion. In an oil bath kept at 373 K without stirring, three kinds of ammonium salts of POMs, i.e., (NH4)3PW12O40, (NH4)4SiW12O40, and (NH4)3PMo12O40 nanorods were obtained. The POMs-nanorods were characterized by transmission electron microscopy (TEM), selected area electron diffraction (SAED), infrared spectra (IR), inductively coupled plasma atomic emission spectrometry (ICP-AES) and X-ray powder diffraction (XRD). TEM showed that nanorods were 0.5-1.5 microm in length, and 50-150 nm in diameter. The results of IR, ICP-AES and XRD proved the existence of Keggin structure. The formation process of POMs-nanorods, (NH4)(8-n) [X(n+)M12O40](-(8-n)) (X(n+) = P5+ and Si4+; M = W and Mo) in a H2O/PEG system was discussed.     

Tracking a new cell-penetrating (W/R) nonapeptide, through an enzyme-stable mass spectrometry reporter tag

We have designed a mass stable reporter (msr) tag with m/z over 500, trifluoroacetyl(alpha,alpha-diethyl)Gly-Lys(Nepsilonbiotin)-(D)Lys-Cys, for the quantification of the uptake and study of the degradation processes of cell-penetrating peptides (CPP), by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This tag was found stable in cell lysis conditions. Using a quantitative MALDI-TOF mass spectrometry analysis based method, an accurate tracking of a new CPP and of its degradation products could be done. (1) The new msr(W/R) nonapeptide (H-RRWWRRWRR-NH2) enters chinese hamster ovary (CHO) K1 cells with a kinetic reaching a steady state after 30-60 min of incubation. This plateau was stable for 4 h and decreased slowly afterward. (2) The peptide msr(W/R) nonapeptide was not cytotoxic over 48 h incubation with CHO cells. (3) After 1 h incubation, the msr(W/R) nonapeptide accumulated with a 3-fold higher concentration than the extracellularly added concentration (7.5 microM). (4) The intracellular quantification was accurate with less than 3% of the quantified peptide being potentially membrane-bound. (5) There was no leakage of the full-length CPP outside the cells. And, finally, (6) analysis of the degradation process of this new CPP suggests that the peptide did not traffick to lysosomes.     

Synthesis, characterization and photocatalytic activity of nanotitania loaded W-MCM-41

Nanocrystalline titanium oxide (TiO(2)) is a promising material as a photocatalyst for photodecomposition of hazardous organic pollutants under illumination, because it is cheap, safe, environmentally benign, and chemically stable. However, the control of particle size and monodispersity of TiO(2) is a challenging task. The use of MCM-41, an inorganic template of uniform pore size (2-10?nm), can overcome this difficulty and produce stable nanoparticles of uniform size and shape. In an attempt to extend light absorption of the TiO(2)-based photocatalyst towards the visible light range and eliminate the rapid recombination of excited electrons/holes during photoreaction, a new photocatalyst (25%TiO(2)-loaded W-MCM-41) powder was prepared. W-MCM-41, with different ratios of Si to W (Si/W = 25, 50, 75), was synthesized by a hydrothermal method and loaded with 25?wt% TiO(2) utilizing a sol-gel method. In order to compare the photocatalytic activity of our sample, titania-loaded plain MCM-41 was also prepared. These materials were characterized by various physiochemical techniques such as UV-visible absorption spectroscopy, x-ray diffraction, nitrogen adsorption-desorption isotherm measurement, Fourier transform infrared (FT-IR) spectroscopy, and transmission electron microscopy. The photocatalytic activity of the prepared samples was evaluated using methyl orange as a model organic compound. It was found that the photodegradation ability of 25% TiO(2)-loaded W-MCM-41 was highly related to the amount of W atoms present in the sample; the optimum atomic ratio of Si to W was 25. It has been confirmed that the recombination rate of electrons/holes in 25%TiO(2)/W-MCM-41 declined due to the existence of W atoms in the sample.     

Determination of binding constants of cyclodextrins in room-temperature ionic liquids by near-infrared spectrometry

Near-infrared spectrometry has been successfully used to determine association binding constants between phenol and alpha-, beta- and gamma-cyclodextrin (CD) in [butylmethylimidazolium][chloride] room-temperature ionic liquid (RTIL). It was found that adding CD into the RTIL solution of phenol resulted in an enhancement in the absorption coefficient of the stretching overtone of the aromatic C-H groups. However, the enhancement induced by CDs in RTIL is much lower (order of magnitude) than those corresponding in D20. The binding constants in RTIL are also much lower than those in D2O ((11 +/- 2), (16 +/- 2), and (40 +/- 6) M(-1) for phenol and alpha-, beta- and gamma-CD, respectively, as compared to 87 and 214 M(-1) for a- and beta-CD in D2O). The results obtained seem to suggest that in ionic liquid, the main interaction between phenol and CDs may not be inclusion complex formation but rather external adsorption. A variety of reasons may be responsible for relatively weaker interactions and lower binding constants in the ionic liquid, including differences in the polarity and viscosity of RTIL and D20. However, the main reason may be due to the possibility that the 1-butyl-3-methylimidazolium cation of the ionic liquid may form inclusion complexes with CDs either through its imidazolium moiety or its butyl group. Such complex formation would prevent phenol from being included in the cavity of the CDs.     

Coupled Electron- and Proton-Transfer Processes in the Reduction of α-[P(2)W(18)O(62)](6)(-) and α-[H(2)W(12)O(40)](6)(-) As Revealed by Simulation of Cyclic Voltammograms

Quantitative analysis of the complex problem of coupled electron- and proton-transfer steps during reduction of the polyoxo anions α-[P(2)W(18)O(62)](6)(-) and α-[H(2)W(12)O(40)](6)(-) in aqueous NaCl (0.5 M) has been achieved by simulation of cyclic voltammograms (Rudolph, M.; Reddy, D. P.; Feldberg, S. W. Anal. Chem. 1994, 66, 589A) over wide ranges of anion concentration, pH, and scan rate. Since there are too many unknown parameters to attempt a one-step global form of simulation, a systematic, stepwise approach has been adopted by progressively accessing regimes of increasing voltammetric complexity. This protocol allows experimental behavior in each system over 5 orders of magnitude in proton concentration to be simulated by estimation of three protonation constants combined with experimentally determined reversible half-wave potentials for the two one-electron processes involved. Fast electron transfer and protonation kinetics are assumed. The importance of the values chosen for the diffusion coefficients of the proton and polyoxo anion species is considered. The simulations account for the fact that pairs of one-electron processes coalesce to give an apparent two-electron process in the pH range 1-6 for reduction of both anions.     

Spark plasma sintered Sm(2)Co(17)-FeCo nanocomposite permanent magnets synthesized by high energy ball milling

Nanocomposite Sm(2)Co(17)-5?wt% FeCo magnets were synthesized by high energy ball milling followed by consolidation into bulk shape by the spark plasma sintering technique. The evolution of magnetic properties was systematically investigated in milled powders as well as in spark plasma sintered samples. A high energy product of 10.2?MGOe and the other magnetic properties of M(s) = 107?emu?g(-1), M(r) = 59?emu?g(-1), M(r)/M(s) = 0.55 and H(c) = 6.4?kOe were achieved in a 5?h milled and spark plasma sintered Sm(2)Co(17)-5?wt% FeCo nanocomposite magnet. The spark plasma sintering was carried out at 700?°C for 5?min with a pressure of 70?MPa. The nanocomposite showed a higher Curie temperature of 955?°C for the Sm(2)Co(17) phase in comparison to its bulk Curie temperature for the Sm(2)Co(17) phase (920?°C). This higher Curie temperature can improve the performance of the magnet at higher temperatures.     

利用多重PCR同时检测WSSV和MBV两种对虾病毒的研究

研究了检测斑节对虾(Penaeus monodon)的主要致病病原——对虾白斑综合征病毒(white spot syndrome virus,WSSV)及斑节对虾杆状病毒(monodon baculovirus,MBV)的技术。用多重PCR检测方法,设计了两对特异性引物,从不同虾池中收集斑节对虾,提取DNA模板,同时检测两种对虾病毒。研究结果表明：该方法检测灵敏度高、特异性好,可检测至每毫克组织100个病毒粒子;从对虾组织中提取的DNA模板对病毒DNA的扩增无抑制,适合于对虾中两种病毒的同时检测。