急性百草枯中毒患者30d预后影响因素的研究

目的 探讨应用血液净化治疗后,急性百草枯中毒患者30 d预后的影响因素。方法 选取西安交通大学第一及第二附属医院符合纳排标准急性百草枯中毒患者(79例),电话随访服毒30 d时的预后,分为死亡组(n=40)及存活组(n=39);比较两组年龄、性别、既往高血压史、糖尿病史、服毒量、服毒至就诊的时间、中性粒细胞计数/淋巴细胞计数比值、单核细胞计数/淋巴细胞计数比值、尿素氮、肌酐、天冬氨酸转氨酶、丙氨酸转氨酶、凝血酶原时间、凝血酶时间及急性病生理学和长期健康评价(acute physiology and chronic health evaluationⅡ,APACHEⅡ),采用二分类Logistic回归分析预测死亡组的危险因素;采用受试者工作特征曲线下面积(area under curve, AUC)分析服毒量对死亡组的预测价值;应用Kaplan-Meier生存曲线分析服毒量与30 d预后的关系。结果 服毒量及APACHEⅡ为30 d死亡的独立危险因素(P<0.05);服毒量预测死亡组的AUC为0.882(P<0.001),临界值为40 mL(敏感度72.5%,特异度87.18...     

Bre1与Rad6形成的复合物的表达、纯化和结晶

目的：建立Bre1及Rad6的表达和纯化方法,构建Bre1-Rad6的复合物,寻找复合物的结晶条件,为使用X射线晶体学的方法解析复合物的结构奠定基础。方法：使用大肠杆菌表达系统表达蛋白质;使用共纯化的方法组装复合物;使用气象扩散的方法结晶复合物。结果：建立了来源于Lodderomyces elongisporus的Bre1(LeBre1)氮端与Rad6相互作用的结构域（RBD）和Rad6(LeRad6)在大肠杆菌中大量表达的方法;通过使用共纯化的方法,成功组装了LeBre1 RBD-Rad6复合物,并进一步通过两步凝胶过滤层析纯化的方法精细纯化,获得了纯度超过95%的蛋白复合物;使用气象扩散的方法结晶复合物,在18℃通过对1 000多个结晶条件的筛选,确定了复合物的两种结晶条件,进一步对该条件优化,获得了质量相对较好的晶体。结论：成功的构建了LeBre1 RBD-Rad6复合物的表达、纯化和结晶的方法,为使用X射线晶体学方法解析复合物的结构奠定了基础。     

抗人CD3单链抗体的表达及其分离纯化

本研究将构建的抗人ＣＤ３单链抗体基因，克隆到融合蛋白表达载体ｐＧＥＸ－４Ｔ－１中，用ＩＰＴＧ诱导表达ＧＳＴ－ＳｃＦｖ融合蛋白，并以ＳＤＳ－ＰＡＧＥ分析，在Ｍｒ为５２０００左右出现一条新生蛋白带，表达量约占菌体总蛋白的４０％。经初步纯化和复性后，用ＧＳＴ亲和色谱纯化，再经凝血酶水解获得抗人ＣＤ３ＳｃＦｖ，竞争结合抑制实验证明，该表达产物具有ＣＤ３亲和活性     

磷酸盐型低密度脂蛋白亲和吸附剂制备与体外吸附试验研究

研究目的是制备低密度脂蛋白亲和吸附剂,用于去除高脂血症患者血液中高含量的低密度脂蛋白。首先以悬浮分散法制备壳聚糖微球和纤维素微球载体,然后固定磷酸吡哆醛和磷酸盐配基,制备出三种吸附剂,并进行体外静态吸附性能测试。结果表明,壳聚糖-磷酸吡哆醛吸附剂对LDL最大吸附量为1.30mg/mL,磷酸盐型壳聚糖和纤维素吸附剂对LDL的最大吸附量分别为2.72mg/mL和3.12mg/mL,并且吸附量随配基含量的增加呈增大趋势,3种吸附剂对高密度脂蛋白均无明显吸附。对吸附剂的灭菌和储存稳定性的研究表明,磷酸盐型纤维素吸附剂具有较好的稳定性。本研究第一次报道磷酸盐型低密度脂蛋白吸附剂。     

Isolation and purification of a major allergen from Parietaria officinalis pollen

A major allergen of Parietaria officinalis, a species responsible for a large number of respiratory allergies in Mediterranean areas, has been identified and characterized. This allergen (Pol) was found in the fraction which precipitates between 70 and 100% ammonium sulphate saturation. Pol showed a molecular weight of 15,000 daltons as determined by SDS-PAGE and HPLC. The pI of Pol was in the pH region 4-6, IEF showing four major bands. Two major bands were shown by CIE, CRIE and immunoblotting; major contaminants or aggregates were also revealed by the latter technique and by HPLC. Pol showed an allergic specific activity 2 times higher than the crude extract; moreover it was shown to be a major allergen since it inhibited 29 out of 30 sera from allergic patients sensitive to P. officinalis.     

人绒毛膜滋养层细胞的分离纯化及鉴定

目的建立一种简便易行、经济有效的纯化培养滋养层细胞的方法.方法:取孕6-8w的正常绒毛组织10例,采用胰蛋白酶消化法分离细胞,将细胞随机分为两组(各5例),一组用淋巴细胞分层液进行纯化,另一组未纯化,最后用含15%胎牛血清的培养基培养细胞.用免疫细胞化学法进行细胞纯度鉴定.结果本纯化的细胞阳性率为(68.6 2±2.69)%,纯化后的细胞阳性率为(91.60±1.55)%,两者之间的差异具极显著性意度( P<0.001).结论该法简便易行,经济有效,可获得纯度较高、合乎试验要求的细胞滋养层细胞.     

一种快速分离纯化外周血细胞线粒体DNA方法的建立

目的探索快速分离纯化人外周血细胞线粒体DNA(mtDNA)的方法. 方法收集抗凝外周血,首先破红细胞,然后裂解白细胞,去除细胞膜和核DNA后获得mtDNA;再经过去除蛋白和RNA,获得纯mtDNA.用PCR扩增人线粒体ND1基因片段,PCR产物经纯化后测序和核苷酸同源性分析.结果用本研究中建立的方法制备的mtDNA纯度高,每毫升抗凝血可获得100ng左右的mtDNA.经过PCR扩增ND1基因433bp片段,较用细胞总DNA为模板,模板用量少,扩增产物多.测序后经核苷酸同源性分析证实扩增片段为ND1基因.结论本研究中建立的快速分离外周血细胞mtDNA的方法,可制备高纯度的mtDNA用于线粒体相关研究.     

Rapid isolation, characterization, and glycan analysis of Cup a 1, the major allergen of Arizona cypress (Cupressus arizonica) pollen

BACKGROUND: A rapid method for the purification of the major 43-kDa allergen of Cupressus arizonica pollen, Cup a 1, was developed. METHODS: The salient feature was a wash of the pollen in acidic buffer, followed by an extraction of the proteins and their purification by chromatography. Immunoblotting, ELISA, and lectin binding were tested on both the crude extract and the purified Cup a 1. Biochemical analyses were performed to assess the Cup a 1 isoelectric point, its partial amino-acid sequence, and its glycan composition. RESULTS: Immunochemical analysis of Cup a 1 confirmed that the allergenic reactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and allergenic proteins from the Cupressaceae and Taxodiaceae families displaying a similar molecular mass. The purified protein shows one band with an isoelectric point of 5.2. Nineteen out of 33 sera (57%) from patients allergic to cypress demonstrated significant reactivity to purified Cup a 1. MALDI-TOF mass spectrometry indicated the presence of three N-linked oligosaccharide structures: GnGnXF(3) (i.e., a horseradish peroxidase-type oligosaccharide substituted with two nonreducing N-acetylglucosamine residues), GGnXF(3)/GnGXF(3) (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF(3)/Gn(GF)XF(3) (with a Lewisa epitope on one arm) in the molar ratio 67:8:23. CONCLUSION: The rapid purification process of Cup a 1 allowed some fine studies on its properties and structure, as well as the evaluation of its IgE reactivity in native conditions. The similarities of amino-acid sequences and some complex glycan stuctures could explain the high degree of cross-reactivity among the Cupressaceae and Taxodiaceae families.     

大鼠β细胞素蛋白的表达、纯化及生物学活性鉴定

目的:获得大量重组大鼠β细胞素并检测其活性。方法:用PCR法从大鼠肾组织中扩增534bp的β细胞素基因片段,并按读框克隆到原核表达载体pET28a( )中。以构建的重组质粒pET28a-rBTC转化大肠杆菌BL-21(DE3)菌株,在IPTG诱导下表达β细胞素蛋白,表达产物用SDS-PAGE和Westernblot检测。采用镍柱亲和层析法纯化目的蛋白。结果:经IPTG诱导后,在大肠杆菌中可表达相对分子质量Mr为20000的目的蛋白。目的蛋白主要以包涵体的形式表达;表达量约占菌体蛋白总量的20%～30%。表达的目的蛋白与抗His标签抗体具有良好的反应性。结论:大鼠β细胞素基因在PET表达系统中得到高效表达;蛋白复性后能有效地促进NIH3T3细胞的体外增殖。