离子色谱法检测食品中硼酸及硼酸盐

通过优化试验对离子色谱法检测食品中硼酸及硼酸盐的工艺进行了研究,探讨了取样量、超声提取时间、滤膜的选择、OnGuard RP柱和Ag柱的活化条件为：分析柱的选择、淋洗液的选择、进样体积和流速的选择,确定了离子色谱法检测食品中硼酸及硼酸盐的最佳检测条件,淋洗液：甲基磺酸（3mmol/L）/甘露醇（60mmol/L）;再生液：四甲基氢氧化铵（25mmol/L）/甘露醇（15mmol/L）;色谱柱：IonPac-borate分析柱,250*9mm或等效柱。检测器：电导检测器。抑制器：AMMS-ICE300,4mm微膜抑制器;柱温：25℃;流速：0.8mL/min;进样量：300μL。     

免疫亲和层析法分离纯化含次黄嘌呤核苷mRNA

采用免疫亲和层析法分离纯化含次黄嘌呤核苷的mRNA(I-mRNA）。将Poly-I与BSA交联,并用Poly-I-BSA交联体免疫家兔,获取抗次黄呤核苷抗血清,斑点印迹法检测到该抗体滴度高、选择性强,将抗次黄嘌呤核苷抗体与蛋白A Sapharose交联,并制备抗体亲和层析柱。将小鼠肺mRNA上样于层析柱,淋洗层析柱后,用洗脱液洗脱I－mRNA,并以斑点印迹法在洗脱液中检测到很强的I－mRNA阳性信号;淋洗液中I－mRNA阳性信号的强度则随淋洗液体积的增加而减弱。以上结果表明,应用本方法可以有效分离得到含次黄嘌呤核苷的mRNA。     

枯草杆菌可溶性YlyA蛋白的诱导表达与纯化研究

YlyA是枯草杆菌一种功能未知蛋白.本研究旨在建立可溶性YlyA的诱导表达体系和纯化方法,为其功能研究奠定基础.PCR扩增ylyA序列,将其克隆到pETMCSⅢ中构建表达载体pNG252,用IPTG诱导6×His-YlyA融合蛋白在大肠杆菌BL21(DE3)中表达,对表达产物进行分析,最后对可溶性YlyA重组蛋白进行Ni2+-WTA亲和层析加以纯化.结果表明pNG252中ylyA的插入方向正确,序列无突变;用0.5 mmol/L IPTG,37℃诱导3 h时,YlyA虽高效表达,但为包涵体形式;调整诱导条件至0.05 mmoL/L IPTG,25℃,5 h时,高效表达的YlyA部分转为可溶性蛋白.纯化后的YlyA浓度达204.2119 μmoL/L;调整洗涤液中的咪唑浓度至15 mmol/L,pH至9,可使纯化蛋白的产量大幅提高.本文成功构建了YlyA高效可诱导表达载体,建立了可溶性蛋白的诱导表达条件,确立了Ni2+-NTA亲和层析纯化方法,所得的6×His-YlyA融合蛋白,可用于YlyA晶体结构和与功能分析.     

蚕豆叶片下表皮ABA结合蛋白的分离纯化

将ＡＢＡ通过一个１０ 碳原子的臂高效率地（６～８ ｍｍｏｌ／Ｌ凝胶）偶联到琼脂糖凝胶４Ｂ上,制成亲和层析柱,用以纯化蚕豆（Ｖｉｃｉａｆａｂａ Ｌ．） 叶片下表皮ＡＢＡ 结合蛋白（ＡＢＡ＿ＢＰ）。样品上柱后,通过ＮａＣｌ 盐梯度洗脱去掉大部分非特异结合的杂蛋白后,用１ ｍｍｏｌ／ＬＡＢＡ亲和洗脱竞争亲和柱中的ＡＢＡ＿ＢＰ,纯化到一种ＡＢＡ特异结合蛋白,纯化了１１２ 倍。纯化蛋白与ＡＢＡ 最大结合为５８．３３ ｎｍｏｌ／ｇ ｐｒｏｔｅｉｎ,Ｋｄ 值为２１ ｎｍｏｌ／Ｌ,亚基分子量为４４ ．２ ｋＤ,纯度约为９０ ％ 。纯化的ＡＢＡ结合蛋白具有典型的蛋白质紫外吸收光谱,在２８０ ｎｍ 处有明显吸收峰     

Anti-IgE单链抗体纯化工艺研究及活性鉴定

目的:建立anti-IgE单链抗体纯化工艺并对其活性进行鉴定。方法:根据protein L亲和层析填料和Ni亲和层析填料的特点分析,经初筛后选用protein L填料作为第一步初纯化填料,Ni亲和层析填料作为第二步精细纯化填料。通过对这两种纯化方式的上样条件和洗脱条件进行研究,建立了anti-IgE单链抗体的纯化工艺。结果:protein L亲和层析的初纯化工艺:最佳杂质洗涤pH=3.5,最佳目标蛋白洗脱pH=2.0,并且pH=2.0洗脱的目标蛋白收集在第二步Ni亲和层析的上样缓冲液中,可直接进行第二步Ni亲和层析纯化。所建立的Ni亲和层析精细纯化工艺:最佳杂质洗涤为50mmol/L咪唑,最佳目标蛋白洗脱为500mmol/L咪唑。经两步亲和纯化,目标产物在SDS-PAGE上纯度为99.0%,CE-SDS上纯度为99.5%,两步总收率为80.0%。纯化后的蛋白经竞争ELISA和Biacore检测,证实该产品特异性识别IgE靶标,与IgE靶标的亲和力达到8.59e~(-9)M,并且竞争Biacore结果显示该抗体对IgE有良好的中和活性,其抑制IgE的EC50值为70n M。结论:建立了一种高效、简洁的大肠杆菌表达的anti-IgE单链抗体纯化工艺,为其进一步规模放大工艺的建立奠定了基础。同时证明了该新型小分子anti-IgE抗体对靶标具有良好的特异性、亲和力以及中和活性,并展示出其在医用中的应用价值。所建立的小分子抗体纯化工艺技术对其他小分子单链抗体的纯化具有参考价值。     

克伦特罗单克隆抗体的纯化及其生物学特性的研究

目的：将自制克伦特罗（CL）单克隆抗体纯化并研究其生物学特性,进行性质鉴定并建立检测标准曲线。方法：用ELISA法测定克伦特罗单克隆抗体的亲和常数和抗体活性,ELJSA测定单克隆抗体与BSA的交叉反应及与几种结构和功能类似物的交叉反应,然后采用间接竞争ELISA方法建立检测标准曲线。将制备的含克伦特罗单克隆抗体的小鼠腹水用盐析法和免疫亲和柱层析法进行抗体纯化。结果：经ELISA法测定,单克隆抗体亲和常数为2．90×10mmol／L,抗体效价最高达10^6。单克隆抗体对BSA无反应,对几种结构和功能类似物的交叉反应率均小于0．005％。建立的标准曲线R2=0．9812,最低检测限为1．0ng／ml。结论：建立了间接竞争ELISA检测cL的标准曲线。自制的克伦特罗单克隆抗体亲和力好,特异性高。为以后实际样品的检测及制备CL免疫检测试纸条和试剂盒奠定了基础。     

2,5-DKG还原酶Ⅰ的分离纯化与性质研究

欧文氏菌ER97高效表达了从棒状杆菌SCB3058克隆的2,5-二酮基-D-葡萄糖酸（2,5-DKG）还原酶Ⅰ基因,5L罐发酵后,收集菌体破碎,将胞内可溶性的蛋白通过硫酸铵分级沉淀、DEAE—Sepharose CL-6B离子交换柱层析和Phenyl Sepharose CL-4B疏水柱层析后分离纯化到了2,5-DKG还原酶Ⅰ,纯化了5倍,得率27％,比活力为3,418U／mg。测定了该酶的一些特性参数：分子量为34kD,等电点为6．0,它以NADPH为辅酶,将2,5-DKG还原为2-酮基-L-古龙酸（2-KLG）,对NADPH和2,5-DKG底物的Km值分别是0．29mmol／L和14．7mmol／L,1mmol／L Cu^2＋、Zn^2＋等有强烈抑制作用,EDTA和巯基乙醇对该酶没有抑制作用,酶的最适pH为7．0,最适反应温度为40℃。     

腺苷减少豚鼠心室肌细胞内游离钙浓度

目的：研究腺苷对豚鼠心室肌细胞内游离钙浓度（[Ca^2＋]i）的影响并探讨其可能机制。方法：用激光共聚焦显微镜探测细胞内游离钙浓度,结果用相对荧光强度（（FI-FI0）／FI0,％;FI0：对照;FI：给药）表示。结果：①在正常台氏液和无钙台氏液中,腺苷（10,50,100μmol／L）浓度依赖性地降低[Ca^2＋];。②含30mmol／L KCl的台氏液（高钾台氏液）能够增加[Ca^2＋]i。腺苷（10,50,100μmol／L）能够显著抑制KCl引起的[Ca^2＋]i的增加。③预先应用选择性腺苷AI受体拮抗剂DPCPX（1μmol／L）,可大部分取消腺苷（100μmol／L）在高钾台氏液中的作用。腺苷（100μmol／L）在高钾台氏液的作用也可被预先应用一氧化氮（No）合酶抑制剂L-NAME（1mmol／L）所部分减弱。④腺苷（100μmol／L）能明显抑制无钙台氏液中由低浓度ryanodine引起的[Ca^2＋];增加。⑤当细胞外液钙浓度由1mmol／L增加到10mmol／L而诱发心室肌细胞钙超载时,部分心室肌细胞产生可传播的钙波,腺苷（100μmol／L）可降低钙波发生的频率和持续时间,最终阻断钙波并降低[Ca^2＋];。结论：腺苷可通过抑制外钙内流和减少肌浆网内钙释放从而降低[Ca^2＋],其减少外钙内流可能是由于腺苷A1受体介导的电压依赖性Ca^2＋通道的抑制,NO可能参与这一过程。     

RNA编辑蛋白OTP82的表达与纯化

摘要 目的：利用原核系统表达RNA编辑蛋白OTP82,通过蛋白变复性的方法得到全长蛋白,并优化实验条件提高OTP82的收率。方法：利用原核表达系统表达OTP82全长融合蛋白（HSO）,表达后的菌体用高压细胞破碎仪匀浆后收集包涵体并用包涵体洗涤液重复洗涤两遍,然后用含有8 M尿素的变性缓冲液将包涵体搅拌溶解得到蛋白原始液。将蛋白原始液复性后采用镍柱亲和纯化,通过SDS-PAGE和Western-blot检测等方法对HSO的变复性结果进行筛选。结果：通过对复性缓冲液中盐浓度、谷胱甘肽的浓度和比例以及小分子添加剂的探索,得到了OTP82融合蛋白适合的变复性条件（pH 8.5 100 mM Tris,400 mM NaCl,200 mM 精氨酸,5 mM GSH,0.5 mM GSSG,6 mM β-环糊精,2 mM EDTA,1 mM PMSF）复性率达到2.73%（获得蛋白0.42 mg/L）。结论：通过变复性的方式能够在体外得到OTP82的粗蛋白,为揭示RNA编辑蛋白作用机制提供基础实验依据,为后续利用PPR蛋白进行工程蛋白设计奠定了基础。     

Daxx过表达对氧化应激诱导的肝肿瘤细胞凋亡的影响

死亡结构域相关蛋白Daxx可以敏化多种肿瘤细胞的凋亡过程,但对于肝肿瘤细胞株HepG2的影响未见报道．为了研究Daxx增加肝HepG2细胞对药物敏感性的影响及机制,为开发药物新的药理作用提供理论依据,分别转染pEGFP-C1和pEGFP-C1-Daxx这两个载体到HepG2细胞．实验分组如下：（1）正常对照组（未转染细胞组）;（2）pEGFP-C1空载体转染组（HepG2/GFP细胞）;（3）pEGFP-C1-Daxx表达载体转染组（nepG2/GFP-Daxx细胞）．筛选稳定细胞株,用逆转录聚合酶链反应检测mRNA的表达;用过氧化氢孵育24h诱导细胞凋亡,采用MTT法和流式细胞术检测细胞凋亡率,Western blot检测蛋白质的表达．经G418筛选稳定的细胞运用RT-PCR技术分析其mRNA,结果显示,转染绿色荧光蛋白Daxx表达载体的细胞Daxx的mRNA明显上调：用荧光显微镜观察到Daxx蛋白主要定位于细胞核．用过氧化氢诱导HepG2细胞凋亡,观察到过氧化氢呈浓度依赖性地抑制HepG2细胞活性．正常对照细胞、HepG2／GFP、HepG2／GFP-Daxx 3组细胞的IC50值分别是0．72、0．76、0．49mmol／L．并且运用流式细胞仪检测到HepG2／GFP-Daxx组细胞凋亡率明显高于转染空载体质粒组与未转染组（（42．9±8．42）vs（27．3±6．38）or（28．5±4．71））．提示HepG2/GFP-Daxx细胞对过氧化氢的反应性较未转染细胞和HepG2/GFP敏感．还运用Western-blot检测到活化的caspase3在Daxx转染组细胞表达最强,达到（204．66±19．68）％,而未转染和HepG2/GFP组细胞分别是（100±3．1）％、（107．39±20．1）％,进一步说明了Daxx可以增加HepG2细胞对于过氧化氢的敏感性．同时,观察到过氧化氢处理24h后,Daxx转染组细胞磷酸化的JNK表达明显高于空载体转染组和未转染细胞组．上述结果表明：a．Daxx可以增加肝HepG2细胞对过氧化氢诱导的细胞凋亡敏感性;b．Daxx蛋白敏化过氧化氢诱导的HepG2细胞凋亡可能与协同增加JNK活性有关．     

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.     

A simplified purification procedure for recombinant human granulocyte macrophage-colony stimulating factor from periplasmic space of Escherichia coli

Cytoplasmic expression is commonly used for production of recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) which most often comes with inclusion body formation. We expressed rhGM-CSF in periplasmic space of Escherichia coli and optimized its extraction by osmotic shock and purification by anion exchange chromatography. Our works show that MgCl2 at 2 mM in osmotic shock buffer improves extraction of the protein and reduces contamination with other proteins. To achieve a simplified purification procedure for rhGM-CSF, efforts were focused on the adjustment of pH of the buffers and application of proper concentration of salt. Following to measurement of the pI of 5.4 for rhGM-CSF by isoelectric focusing, the pH of dialysis buffer and buffers used in anion exchange chromatography were adjusted to 6.5 for optimal binding of the protein to the column and removal of proteins with higher pIs during washing of the column. In addition, it was found that appliance of NaCl at a concentration of 20 mM in dialysis and column washing buffers prior to elution with elution buffer containing 120 mM NaCl significantly improves purification of the protein. Starting with specific amount of total proteins obtained by osmotic shock, it was possible to recover 95% of which following to purification with a purification yield of 72% for rhGM-CSF along with appropriate biological activity.     

Characterization of fractionated polyclonal antibodies for immunoaffinity chromatography

Polyclonal anti-BSA antibodies were ractionated by stepwise elution from an immobilized BSA column by decreasing pH or increasing the concentration of NaSCN. The binding affinities of each fraction and original globulin under physiological conditions and their dependence on pH and ionic environments were compared. Fractions with high association constant under physiological conditions did not necessarily show antigen binding affinity over a wide pH range, but they retained a high affinity at higher ionic strength of NaSCN. Consequently, by combining these two fractionation procedures, a fraction with high affinity and which dissociated at moderate pH was obtained. It is clear that high affinity is not always incompatible with ease of dissociation accompanying a change in conditions.     

Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH

While phosphoproteins have attracted great interest toward the post-genome research (e.g. clinical diagnosis and drug design), there have been few procedures for the specific enrichment of native phosphoproteins from cells or tissues. Here, we describe a simple and efficient protocol to enrich phosphoproteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on immobilized metal affinity chromatography using a phosphate-binding tag molecule (i.e. a dinuclear zinc(II) complex) attached on a highly cross-linked agarose. The binding, washing, and elution processes were all conducted without a detergent or a reducing agent at pH 7.5 and room temperature. An additive, 1.0 M CH3COONa, was necessary in the binding and washing buffers (0.10 M Tris-CH3COOH, pH 7.5) to prevent the nonphosphorylated protein from binding. The absorbed phosphoproteins were eluted using a mixed buffer solution (pH 7.5) consisting of 0.10 M Tris-CH3COOH, 10 mM NaH2PO4-NaOH, and 1.0 M NaCl. In this study, we demonstrate a typical example of phosphate-affinity chromatography using an epidermal growth factor-stimulated A431 cell lysate. The total time for the column chromatography (1 mL gel scale) was less than 1 h. The strong enrichment of the phosphoproteins into the elution fraction was evaluated using SDS-PAGE followed by Western blotting analysis.     

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%–60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.     

Lagenin, a novel ribosome-inactivating protein with ribonucleolytic activity from bottle gourd (Lagenaria siceraria) seeds

The seeds of Lagenaria siceraria (Family Cucurbitaceae) were extracted with water and the extract was lyophilized. The lyophilized extract was chromatographed on a DEAE-cellulose column in 10 mM Tris-HCl buffer (pH 7.2). The unadsorbed fraction was applied to an Affi-gel Blue gel column previously equilibrated with the same buffer. After removal of unadsorbed materials, the adsorbed proteins were eluted with 1.5 M NaCl in the Tris-HCl buffer. After dialysis the adsorbed fraction was loaded on a CM-Sepharose CL-6B column which had been equilibrated with and was eluted with the same buffer. After elution of unadsorbed proteins, the column was eluted with a gradient of 0-1 M NaCl in 10 mM Tris-HCl buffer (pH 7.2). The fraction eluting at about 0.55 M NaCl, which represented pure ribosome inactivating protein (RIP), inhibited cell-free translation in a rabbit reticulocyte system with an IC50 of 0.21 nM and exerted ribonuclease activity on yeast tRNA with an activity of 45 U/mg. The RIP was designated lagenin. It possessed a molecular weight of 20 kDa, smaller than the range of 26-32 kDa reported for other RIPs. The N-terminal sequence of lagenin exhibited a lesser extent of similarity to those of other Cucurbitaceae RIPs, characterized by a deletion of the first three amino acid residues and a replacement of the 4th (Phe), 17th (Phe), 18th (Ile) and 22nd (Arg) residues which are invariant in other RIPs.     

新型柱前衍生试剂分析草甘膦的高效液相色谱研究

以2,5-二甲氧基苯磺酰氯(DMOSC)为柱前衍生化试剂,建立了柱前衍生草甘膦的紫外检测反相高效液相色谱法,并优化了衍生化条件,得最佳条件:衍生温度35℃,时间15 min,pH 10.0,草甘膦与DMOSC的摩尔比为1∶6。HPLC分析条件:采用Kromasil C18柱,流速1.0 mL/min,柱温30℃,检测波长220 nm,流动相为甲醇-乙腈-磷酸盐缓冲溶液(0.02 mol/L、pH 5.5),三者的体积比为15∶5∶80。结果表明:草甘膦质量浓度在5~100μg/mL范围内线性关系良好,相关系数为0.996 2,检测限为0.067μg/mL。实验表明该方法反应条件温和,灵敏度高,衍生产物稳定。     

Hexokinases of tobacco leaves: Subcellular localization and characterization

Subeellular localization of the enzymes which phosphorylate hexoses was studied in photosynthesizing tobacco leaves by means of differential centrifugation and centrifugation in sucrose gradient. More than 80 % of the total hexokinase activity of leaf tissues were found to be associated with the particulate fraction of mitochondria; however, the ratio of the particulate hexokinase fraction to the soluble fraction was influenced by the extraction medium applied. The particulate hexokinases showed a high affinity to glucose (Km = 26.8 μM) and a relatively low affinity to fructose (Km = 17.6 mM). They had a broad pH optimum, because 81 % of the phosphorylating activity obtained for glucose and 75 % of the activity obtained for fructose occurred in pH range from 7.9 to 9.1 (Tris-HCl buffer). The hexokinases were Mg2+ dependent with the highest activity occurring at equimolar Mg2+ and ATP concentrations. Their activity was enhanced by KC1, NaCl, and (NH4)2SO4 at 30 to 120 mM concentrations.     

Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.