天花粉蛋白一级结构的修正及不同产地天花粉蛋白的研究

胰蛋白酶酶解天花粉蛋白, 用高效液相色谱分离酶解肽段, 用顺序仪测定其有关肽段的顺序。用羧肽酶A, B, Y测定了天花粉蛋白C-端和天花粉蛋白溴化氰降解肽CB1的C-端顺序, 修正了我们1985年测定的天花粉蛋白一级结构, 证明天花粉蛋白由246(7)氨基酸残基所组成, 除C-端微观不均一外, 与Collins结果一致。同时比较了芜湖产天花粉蛋白一级结构与平湖产的天花粉蛋白一级结构, 没有发现两者的一级结构有差别。     

径向离子交换色谱柱在生物工程产品下游纯化中的应用

本文利用我们自行研制的径向离子交换色谱柱,对人血浆成分、登革病毒单克隆抗体及基因工 程产物A蛋白及人白介素-2进行了分离纯化。结果表明,以上目标产品均可得到有效的分离,它在流速、处理量方面均优于传统的色谱方法。由于其体积小,易于放大,不失为一种理想的生物工程产品中试至生产规模的初级分离纯化方法。     

径向离子交换色谱柱在生物工程产品下游纯化中的应用

 ]本文利用我们自行研制的径向离子交换色谱柱，对人血浆成分、登革病毒单克隆抗体及基因工程产物A蛋白及人白介素-2进行了分离纯化结果表明，以上目标产品均可得到有效的分离，它在流速、处理量方面均优于传统的色谱方法。由于其体积小，易于放大，不失为一种理想的生物工程产品中试至生产规模的初级分离纯化方法。     

超高效纳升液相色谱-电喷雾串联质谱鉴定重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白

采用超高效纳升液相色谱-电喷雾串联质谱对重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白进行了分析.样品经胰蛋白酶酶切后,进行液相色谱-串联质谱分析和数据库检索.结果表明, 分别有5个和7个肽段匹配于人肿瘤坏死因子受体和人Ig gamma-1 chain C region.比对分析表明,重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白中检测到的人IgG的7个肽段完全与人IgG1的序列匹配,与其它3个亚型只有部分肽段匹配.说明重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白一级结构正确,Fc片段确实为人IgG1的Fc片段.     

蛋白质顺序测定技术(一)

引言自从1955年F.Sanger测定了胰岛素结构以后,促进了蛋白质一级结构测定技术的发展。目前世界上搞清一级结构的蛋白质已有一千多个,其中最大的为β-半乳糖苷酶(β-Galactosidase),它由1021个氨基酸组成。氨基酸自动分析仪,自动顺序仪和高效液相层析技术的出现,更使蛋白质一级结构测定技术突飞猛进。目前仅须几个毫克的样品就能进行一级结构的测定。在我国,蛋白质一级结构的研究比较落后,但目前已有很多单位开展蛋白质一级结构的研究。为了更快促进我们这方面工作的进展,1980年10月西德马-普学会分子遗传研究所的B.Wittmann-Liebold     

血清中可溶性白介素-2受体胶体金免疫层析试纸检测方法研究

快速、灵敏地检测血清中的可溶性白介素-2受体(sIL-2R)对相关疾病的临床诊断至关重要.本研究建立了一种基于胶体金免疫层析试纸条的快速、定量检测实际样品中sIL-2R的方法.在硝酸纤维膜上分别包被羊抗兔二抗(2ndAb)和兔抗鼠sIL-2R单克隆抗体(cAb)作为质控线(C线)和检测线(T线);以兔抗鼠sIL-2R单...     

生物合理设计光系统Ⅱ抑制剂研究(Ⅴ)──取代苯甲酰胺基丙烯酸乙酯和新型嘧啶酮类化合物的合成及其Hil反应抑制活性

基于Dl蛋白结构模型,设计并合成了取代苯甲酰胺基丙烯酸乙酯及有关成环化合物嘧啶酮,通过X射线衍射确定了2-芳基-5-乙氧甲酰基-6-甲硫基-4-嘧啶酮的结构.生物活性测定结果表明,部分化合物显示出一定的Hill反应抑制活性.     

生物合理设计光系统Ⅱ抑制剂研究(Ⅴ)——取代苯甲酰胺基丙烯酸乙酯和新型嘧啶酮类化合物的合成及其Hill反应抑制活性

基于Dl蛋白结构模型,设计并合成了取代苯甲酰胺基丙烯酸乙酯及有关成环化合物嘧啶酮,通过X射线衍射确定了2-芳基-5-乙氧甲酰基-6-甲硫基-4-嘧啶酮的结构.生物活性测定结果表明,部分化合物显示出一定的Hill反应抑制活性.     

桑蚕丝素-RGD融合蛋白的固态结构及其细胞粘附性分析

利用基因工程方法把含有短肽RGD的氨基酸序列连接到桑蚕丝素蛋白的结晶序列GAGAGS上, 通过调节DNA的聚合度, 合成了具有[TGRGDSPA(GVPGV)₂GG(GAGAGS)₃AS]_n一级结构、不同分子量大小的桑蚕丝素-RGD融合蛋白, 并且通过在M9培养基中添加[3-¹³C]Ala的方法进行融合蛋白的稳定同位素标记. ¹³C CP/MAS NMR结果显示, 融合蛋白中的GAGAGS部分具有与天然桑蚕丝素结晶部分相同的分子结构, 即Silk I处理后为均一的分子结构, 而Silk II处理后为不均一的分子结构, 它包含了三种不同的结构成分. 另一方面, 通过对小鼠成纤维细胞BALB/3T3在不同蛋白材料载体上的粘附和增殖性能的测定结果显示, 融合蛋白对细胞的增殖性能与天然胶原蛋白相近, 但表现出了比胶原蛋白更好的细胞粘附性能. 该研究结果显示, 如果对该桑蚕丝素-RGD融合蛋白进行适当加工, 可能适合于组织工程支架材料的应用.     

稀土离子诱导钙调蛋白构象变化后的单克隆抗体制备

采用单克隆抗体技术研究了三价稀土离子与钙调蛋白作用后对其与靶分子识别能力的影响。牛脑钙调蛋白经2.4-二硝基氟苯修饰后再结合三价的铕离子,然后免疫Balb/c小鼠,经过3次免疫后在小鼠血清中检测到相应的抗体,抗体效价为1:12000;用杂交瘤细胞技术制备出一株抗钙调蛋白的单克隆抗体细胞株2C3;酶联免疫吸附法(ELISA)测定结果证实钙调蛋白结合稀土离子前后对该抗体的识别能力存在显着差异,表明该抗体可以用于进一步研究金属离子对钙调蛋白构象变化及其对靶分子识别的影响。     

Simultaneous determination of phenolic acids and flavonoids in rice using solid-phase extraction and RP-HPLC with photodiode array detection

An analytical method based on an optimized solid-phase extraction procedure and followed by high-performance liquid chromatography (HPLC) separation with diode array detection was developed and validated for the simultaneous determination of phenolic acids (gallic, protocatechuic, 4-hydroxy-benzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, sinapic, and cinnamic acids), flavanols (catechin and epicatechin), flavonols (myricetin, quercetin, kaempferol, quercetin-3-O-glucoside, hyperoside, and rutin), flavones (luteolin and apigenin) and flavanones (naringenin and hesperidin) in rice flour (Oryza sativa L.). Chromatographic separation was carried out on a PerfectSil Target ODS-3 (250 mm × 4.6 mm, 3 μm) column at temperature 25°C using a mobile phase, consisting of 0.5% (v/v) acetic acid in water, methanol, and acetonitrile at a flow rate 1 mL min(-1) , under gradient elution conditions. Application of optimum extraction conditions, elaborated on both Lichrolut C(18) and Oasis HLB cartridges, have led to extraction of phenolic acids and flavonoids from rice flour with mean recoveries 84.3-113.0%. The developed method was validated in terms of linearity, accuracy, precision, stability, and sensitivity. Repeatability (n = 5) and inter-day precision (n = 4) revealed relative standard deviation (RSD) <13%. The optimized method was successfully applied to the analysis of phenolic acids and flavonoids in pigmented (red and black rice) and non-pigmented rice (brown rice) samples.     

Changes in the proteome of extracellular vesicles shed by rat liver after subtoxic exposure to acetaminophen

To identify changes in extracellular vesicles (EVs) secreted by the liver following drug-induced liver injury (DILI), rats were treated with a subtoxic dose (500 mg/kg) of the analgesic drug, acetaminophen (APAP). EVs were collected by liver perfusion of sham and APAP-treated rats. Changes in EVs morphology were examined by transmission electron microscopic analysis of negatively stained vesicles. Results from morphometric analysis of EVs revealed striking differences in their size and distribution. Proteome composition of EVs collected by liver perfusion was determined by mass spectrometry using methods of sample preparation that enabled better detection of both highly hydrophobic proteins and proteins with complex post-translational modifications. The collection of EVs after liver perfusion is an approach that enables the isolation of EVs shed not only by isolated hepatocytes, but also by the entire complement of hepatic cells. EVs derived after DILI had a lower content of alpha-1-macroglobulin, ferritin, and members of cytochrome 450 family. Fibronectin, aminopeptidase N, metalloreductase STEAP4, integrin beta, and members of the annexin family were detected only in APAP-treated samples of EVs. These results show that the present approach can provide valuable insights into the response of the liver following drug-induced liver injury.     

TPD study of NH3 adsorption/desorption on the surface of V/Ti, V/Al based catalysts for selective catalytic reduction of Nox by ammonia 2. TPD test of commercial V/W/Ti, V/Al, Pd/V/Al catalysts

The effect of temperature on the adsorption/desorption of ammonia from the air mixture on the surface of commercial binary V/Al and ternary Pd/V/Al, V(0.65 wt.%) /W(6.73 wt.%) /Ti and V(1.8 wt.%) /W(7.3 wt.%) /Ti de-NO_x catalysts has been investigated by temperature-programmed desorption (TPD) method. The ability of the commercial catalysts to adsorb ammonia in the most stable surface species was shown to correlate well with their suppression of the NH₃ oxidation.     

乙酸镍吡啶配合物催化的分子氧氧化反应研究

Ni(OAc)2结合吡啶和叔丁基过氧化氢(TBHP)实现了苄基C-H与苄基醇类化合物在温和条件下(80～90℃,O21 atm)的选择性催化分子氧氧化反应.研究了过氧化物添加剂,配体,溶剂和温度的影响,得到了优化的反应条件.在苄基C-H的氧化中显示了很高的酮/醇选择性.用ESR法进行了Ni(III)的检测,证实了反应机理.竞争实验说明羰基化合物的生成不是因为醇继续氧化.酮可被解释为过氧化氢中间体受金属催化分解的产物.     

乳腺癌代谢物组模式特征发现方法及HPLC/M S/M S分析

提出一种基于单独最优特征组合和BP神经网络的代谢物组模式特征发现方法,并用其寻找到尿样中与乳腺癌最为相关的4种核苷,组成一组特异性检测参数.经HPLC/MS/MS联用法鉴定,它们是乳清酸核苷、1-甲酰化腺苷、S-腺苷-L-蛋氨酸及N²-甲酰化鸟苷.将这4种核苷作为输入变量,用BP神经分类网络建立乳腺癌诊断模型.留一法交叉验证和独立验证结果表明,该模型预测准确率达到90%以上.     

Elucidation of the absolute configuration of rhizopine by chiral supercritical fluid chromatography and vibrational circular dichroism

The absolute configuration of rhizopine, an opine‐like natural product present in nitrogen‐fixing nodules of alfalfa infected by rhizobia, is elucidated using a combination of state‐of‐the‐art analytical and semi‐preparative supercritical fluid chromatography and vibrational circular dichroism spectroscopy. A synthetic peracetylated racemate was fractionated into its enantiomers and subjected to absolute configuration analysis revealing that natural rhizopine exists as a single enantiomer. The stereochemistry of non‐derivatized natural rhizopine corresponds to (1R,2S,3R,4R,5S,6R)‐4‐amino‐6‐methoxycyclohexane‐1,2,3,5‐tetraol.     

Paternal genetic structure of Kyrgyz ethnic group in China revealed by high-resolution Y-chromosome STRs and SNPs

Kyrgyz ethnic group is one of the nomads in China, with the majority in Xinjiang and a small part of them living in Heilongjiang province. Historically, they have went through five migrations westward due to the wars. The name “Kyrgyz” means 40 tribes, originating from the primary groups of Kyrgyz. However, it is a largely understudied population, especially from the Y chromosome. In this study, we used a previously validated high-resolution Y-chromosome single nucleotide polymorphisms (Y-SNPs) and short tandem repeats (Y-STRs) system to study Kyrgyz ethnic group. A total of 314 male samples of Kyrgyz ethnic group were genotyped by 173 Y-SNPs and 27 Y-STRs. After data analysis, the results unveiled that Kyrgyz ethnic group was a population with high percentage of both haplogroup C2a1a3a1d∼-F10091 (91/134) and R1a1a1b2a2-Z2124 (109/134), which has never been reported. This implied that Kyrgyz ethnic group might have gone through bottleneck effects twice, with these two main lineages left. Mismatch analysis indicated that the biggest mismatch number in haplogroup C2a1a3a1d∼-F10091 was 10, while that of haplogroup R1a1a1b2a2-Z2124 was 20. This huge difference reflected the different substructure in two lineages, suggesting that haplogroup C2a1a3a1d∼-F10091 might have the least admixture compared to the other two lineages. After admixture modelling with other datasets, the conclusion could be drawn that Kyrgyz ethnic group had great genetic affinity with Punjabi from Lahore, Pakistan, which supported that Kyrgyz ethnic group in China was close to central Asian.     

Analysis of alendronate in human urine and plasma by magnetic solid-phase extraction and capillary electrophoresis with fluorescence detection

A simple, rapid and sensitive CE-fluorescence (FL) detection method for the analysis of alendronate (ALEN), a bisphosphonate drug, has been developed. Using a buffer solution of 20 mM sodium phosphate (pH 10.0) and a voltage of 24 kV, separation of ALEN in a 55-cm length (35-cm effective length) capillary was achieved in 5 min. FL detection of ALEN was performed via pre-column derivatization with 2,3-naphthalene dicarbox-yaldehyde (NDA). Linear correlation (r=0.9981, n=6) between FL intensity and analyte concentration was obtained in the range of 7-200 ng/mL ALEN. The developed CE-FL method was applied to the analysis of ALEN in human urine and plasma samples. In order to eliminate the interfering matrix components, SPE using magnetic Fe(3) O(4) @Al(2) O(3) nanoparticles as solid sorbents was employed to clean the biological fluids before CE-FL analysis. The linear ranges of ALEN in urine and plasma were 5-100 ng/mL (r = 0.9982, n = 7) and 5-70 ng/mL (r = 0.9954, n = 7), respectively. The LOD and LOQ in both urine and plasma samples were 1.5 and 5 ng/mL ALEN, respectively. Total analysis time including sample pre-treatment and CE separation was less than 1.5 h.     

Simultaneous liquid-liquid extraction of dibenzyl disulfide, 2,6-di-tert-butyl-p-cresol, and 1,2,3-benzotriazole from power transformer oil prior to GC and HPLC determination

2,6-Di-tert-butyl-p-cresol (DBPC), dibenzyl disulfide (DBDS), and 1,2,3-benzotriazole (BTA) are additives that may be found concomitantly in the oil matrix of power transformer. DBPC and DBDS act as antioxidants while, BTA is a corrosion inhibitor that protects copper conductors inside the transformer unit from corrosion. A powerful analytical method is, therefore, required to determine these additives at trace levels in the transformer oil. This work describes a unique single liquid-liquid extraction pretreatment step prior to the determination of the components by gas chromatography (GC) and high-performance liquid chromatography (HPLC) techniques. The optimum volume ratio used in the pretreatment step was determined as 5:2:5 for mineral oil/n-hexane/acetonitrile, respectively. Relatively, the method is simple and quick with a minimal use of solvents. Analytical results indicate that the method is relatively sensitive, accurate, and precise for each of the three components in fresh and used mineral oil. The calibration curves for the three components demonstrate a significant increase in sensitivities. Detection limits found were, 100 mg L(-1) (0.01% w/v), 0.80 mg L(-1) , and 2.04 mg L(-1) for DBPC, DBDS, and BTA, respectively. The Student's t values determined at 95% confidence level indicate that there is no significant difference between the experimental means obtained by this method and the standard method for each component.