氧化固醇结合蛋白结构、功能与应用

氧化固醇结合蛋白(oxysterol binding protein,OSBP)是存在于真核细胞内的一类参与脂质代谢的非囊泡运输蛋白质,在哺乳动物中被称为氧化固醇结合蛋白相关蛋白质(oxysterol binding protein-related proteins,ORPs),而在酵母中被称为氧化固醇结合蛋白同源物质(oxysterol-binding protein homologues,OSH）。近年来人们对氧化固醇结合蛋白的研究不断深入,特别是对其同源蛋白质（例如,ORP5/8、Osh3/4、ORP4L等）的结构功能差异和其在信号转导中的作用的相关研究,以及在生物医药方面的应用更成为了本领域的热点。本文综述了关于OSBP及其同源蛋白质结构和功能的相关研究,指出了该领域存在的一些关键问题。与此同时,对OSH和ORPs在细胞内的膜接触位点（membrane contact sites,MCS）进行对比,以及对今后OSBP的研究方向做了展望。     

人热休克蛋白转录因子1 DNA结合结构域的表达、纯化和晶体生长

目的:获得大量热休克转录因子1(HSF1)DNA结合结构域(DBD)蛋白,用于晶体生长的三维结构解析。方法:将DBD基因片段克隆至原核表达载体pGEX-6P-1中并获得高效表达,经过Glutathione SepharoseTM 4B亲和层析、ResourceQ纯化后,蛋白纯度达到95%以上。结果:圆二色谱仪分析蛋白质的二级结构结果显示α螺旋占33%,β折叠占15%;采用悬滴气相扩散法得到了针状DBD晶体。结论:纯化的蛋白质与同源性达68%的Kluyveromyceslactis的DBD有相似的空间构象。获得的蛋白质晶体为进一步的三维结构解析奠定了基础。     

膜转运蛋白的功能性超表达和纯化

膜转运蛋白结构和功能的研究是功能膜蛋白质组研究中的一个重要内容,而大量蛋白质的分离纯化是进行蛋白质的结构和功能研究的基础.目前,结构和功能膜蛋白质组学相关研究的瓶颈,在于不能有效地超量表达和纯化具有生物活性的膜转运蛋白.影响膜转运蛋白超量表达和纯化的关键因素,包括目标蛋白的拓扑学结构分析和去垢剂的选择.进行膜转运蛋白拓扑学结构的分析,对于构建用于活体表达的重组膜转运蛋白具有指导意义.去垢剂能够稳定去膜状态的膜蛋白,在膜转运蛋白的离体表达和亲和纯化以及包涵体的处理过程中具有重要的作用.本文就目前功能膜蛋白质组学研究中所涉及的有关膜转运蛋白功能性超表达和分离纯化策略及关键技术作一简述.     

外源H2S影响黄瓜幼苗响应高盐胁迫的蛋白质组学分析

为了阐明外源H₂S对高盐胁迫下黄瓜蛋白质表达的影响,以盐敏感型黄瓜栽培种‘春夏秋王’为材料,通过双向电泳（2 DE）技术分离叶片总蛋白质,采用基质辅助激光解析飞行时间串联质谱（MALDI TOF/TOF MS）技术对差异表达蛋白质进行质谱鉴定,并利用NCBI及SwissProt数据库检索进行差异表达蛋白质功能注释和代谢通路分析。结果表明：(1) 共检测到约2 490个蛋白质,其中蛋白质表达差异在1.5倍以上且差异显著(P<0.05)的有45个,其中有24个蛋白质表达上调,21个蛋白质表达下调。(2) 成功鉴定蛋白质26个,这些蛋白质参与了光合作用（26.92%）、蛋白质代谢（23.08%）、能量与碳水化合物代谢（11.54%）、氨基酸生物合成（11.54%）、细胞结构相关蛋白（7.69%）、抗氧化作用（3.85%）、信号转导（3.85%）及未知功能蛋白（11.54%）。(3) GO注释表明差异表达的蛋白质分子功能主要以蛋白质结合与水解酶活性为主,生物学过程涉及应激反应、有机物代谢过程、胁迫响应和细胞分化等,细胞组成集中分布在细胞部分和一些胞内细胞器。KEGG代谢通路分析表明,差异表达蛋白质主要参与了肌动蛋白细胞骨架调控、细胞凋亡、碳代谢和光和调控等代谢途径。研究表明,外源H₂S诱导了高盐胁迫下黄瓜叶片蛋白质的差异表达,为后续探索外源H₂S对黄瓜的抗盐分子机制研究提供了理论依据。     

人源Fank1蛋白质FN3结构域多肽的原核表达，纯化及晶体筛选

目的纯化人源Fank1（fibronectin type Ⅲ and ankyrin repeat domain1）蛋白质N端FN3（fibronectin typeⅢ,Ⅲ型纤黏连蛋白）结构域蛋白,用于晶体生长的三维结构分析。方法将FN3结构域基因片段克隆至原核表达载体pGEX-6P-1中,将菌落PCR和测序鉴定正确的重组质粒转化E.coli BL21（DE3）后获得表达菌株。该菌株经IPTG诱导高效表达出带有GST标签的可溶性的融合蛋白,经过Glutathione Sepha-rose^TM 4B亲和层析、Hiload16／60 superdex200分子筛层析纯化后,蛋白纯度达到95％以上。结果纯化蛋白采用悬滴气相扩散法得到棒状晶体。结论成功制备了高纯度FN3蛋白,获得FN3蛋白质晶体,为进一步的三维结构解析及Fank1功能研究奠定了基础。     

重组人可溶性B淋巴细胞刺激因子细菌表达、纯化及其对B淋巴细胞的增殖作用

采用PCR法从人胎盘cDNA文库中扩增出人B淋巴细胞刺激因子（hBLyS）的cDNA;再用巢式PCR法（Nest-PCR）扩增出hBLyS的膜外可溶性功能部分hBLyS,152aa）的DNA片段,经过纯化及测序鉴定后,双酶切法定向插入原核表达载体pET-30a（＋）;然后转化λDE3溶原宿主菌并诱导其高效表达（分析得知靶蛋白约占总蛋白质的46％）出带有His-tag的重组融合蛋白。采用本室自行研制的能与His-tag特异性结合的固定化Ni^2 亲和层析胶（Ni^2-IDA）纯化重组融合蛋白,经SDS－PAGE鉴定为单一区带。生物学功能检测得知,在anti-hIgM的协同作用下,hsBLyS具有明显地刺激人B淋巴细胞的增殖作用。     

表达重组人骨形态发生蛋白-7工程菌的发酵及表达产物的纯化

目的:研究表达重组人骨形态发生蛋白-7工程菌的发酵和表达产物的纯化工艺。方法:利用16L发酵罐发酵培养工程菌,设定了溶氧、搅拌速度、诱导时机、补料和培养基pH值等发酵条件;通过包涵体洗涤、离子交换层析法纯化目的蛋白。结果:工程菌目的蛋白质表达量占菌体总蛋白质的30%以上,纯化后目的蛋白的纯度可达98%。结论:建立了大肠杆菌高效表达人骨形态发生蛋白-7的发酵及纯化工艺。     

Mn-SOD与抗癌胚抗原单链抗体基因的融合及高效表达

将Ｍｎ－ＳＯＤ与抗癌胚抗原（ＣＥＡ）单链抗体基因（Ｓｃ－Ｆｖ ｇｅｎｅ）融合,重组到含Ｔ７启动子的表达载体ｐＥＴ－２２ｂ（＋）中,构建表达质粒ｐＥＴＭｎ－ＳＯＤ－ＳｃＦｖ,并转化大肠杆菌ＢＬ２１（ＤＥ３）,进行高效表达,表达物占菌体可溶性总蛋白的２４％。ＳＤＳ－ＰＡＧＥ和蛋白质和迹图谱显示表达物分子量为４５ｋＤ与融合基因编码蛋白质的理论值相符。该蛋白质在大肠杆菌中为泌型表达有利于纯化。ＲＩＡ测定表     

大肠杆菌UvrB基因的克隆、表达与功能研究

[目的]在大肠杆菌表达系统表达UvrB蛋白,优化其表达和纯化的条件,并初步研究其对抗紫外线伤害的功能。[方法]克隆大肠杆菌MG1655菌株UvrB编码序列,采用酶切连接的方法构建表达载体p Waldo-GFP-UvrB,并转入BL21(DE3)表达菌株诱导蛋白质表达,采用Ni-NTA亲和层析以及分子筛层析的方法纯化蛋白质,采用紫外光刺激的方法研究其细胞内功能。[结果]成功构建UvrB表达载体,获得UvrB在BL21(DE3)中重组菌株,其表达条件为22℃,0. 2 mmol/L IPTG诱导20 h,UvrB以可溶蛋白的形式存在。SDS-PAGE结果显示UvrB蛋白质分子量约76 k Da与预期相符;经纯化后UvrB蛋白质得率约为0. 6 mg/L培养基,纯度 85%,为其进一步研究奠定了基础。[结论]研究克隆并构建了大肠杆菌UvrB蛋白的原核表达体系,优化了其纯化方案,得到纯度 85%的可溶UvrB蛋白质,为进一步研究UvrB蛋白质功能奠定了基础。     

大肠杆菌表达的单链抗体柱复性的研究

对包含体表达的抗乙肝病毒表面抗原（HBsAg）的单链抗体（ScFv）纯化复性进行了探索.尝试了利用金属螯合亲和层析和凝胶层析柱进行柱上在位复性的可行性. 对包含体表达的ScFv进行透析复性与柱复性,比较其相对复性率及蛋白质回收率,发现柱上复性效果优于传统的透析复性.抗HBsAg ScFv经凝胶色谱Sephacyl S-200柱复性的相对复性率为98%, 蛋白质回收率为81%.由于将纯化复性同步进行,简化了操作程序,提高了产品的回收率.     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin

The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke.     

Synthesis of hapten and conjugates of coumestrol and development of immunoassay

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。     

Effect of soil salinity and water stresses on growth and content of nitrogen,chloride and phosphate of wheat and triticale

Summary Three wheats and one triticale were grown, up to flowering stage, in pots on calcareous soil adjusted to a range of salinities (S₁=3.5, S₂=6, S₃=8.5, and S₄=11 mmhos/cm, 20°C, soilpaste extract) by adding solution consisting of 3∶2∶1 of Na-, Ca- and Mg chlorides in chemical equivalent amounts. Moisture in the pots was kept at 100% (W₁), 40% (W₂) and 20% (W₃) of the available water. The vegetative growth, nitrogen and phosphate were affected by S and W treatments, chloride was affected only by S. The interaction S×W affected only dry weight. Varietal effect was observed between wheat as a group and triticale. Multiple quadratic regression equations of these properties on salinity and water revealed that the higher the available water the wider the range of tolerable salinity. Triticale was relatively more tolerant to water stress. Salinity increases Cl and decreases N, whereas water stress enhances N accumulation to a certain extent. However, in triticale at S₃ and S₄ the effect of water stress on N was overshadowed by the excessive salinity. This did not occur for the wheat (Florence). P trends were described. R² for P was low (0.7435–0.3603) which made interpretations rather difficult.     

放牧对牧草光合作用、呼吸作用和氮、碳吸收与转运的影响

研究放牧对草地植物生理活动的影响,对于揭示草地放牧演替的生理机制有重要意义.大量研究表明,家畜放牧对牧草光合作用、呼吸作用以及C和N吸收与转运的影响,可以分为生理伤害和生理恢复2个阶段.放牧通过改变草地冠层结构影响牧草光合作用,净光合作用速率短期内迅速下降,随着叶面积指数增加又逐渐上升,呼吸作用有相似的变化趋势.牧草放牧后再生长所需的C和N最初主要来自根系和留茬中的贮藏物质,此后随着牧草生长恢复逐渐由同化作用供给,C代谢与土壤N水平负相关.放牧后牧草生理活动变化与牧草遗传特性、种间竞争、家畜放牧特征、非生物环境等因素密切相关.