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。     

中国版纳猪MHCI类P1分子全长的原核表达与纯化

目的：获得原核表达的中国版纳猪ＳＬＡＩ类Ｐ１蛋白质分子。方法：ＰＣＲ扩增去信号肽的ＳＬＡＩ类Ｐ１ｃＤＮＡ序列，亚克隆至ｐＧＥＭＴ载体，测序。将亚克隆的Ｐ１　ｃＤＮＡ片段插入表达载体ｐＥＴ４２ｂ（＋），构建重组表达质粒ｐＥＴ－４２ｂ（＋）／ｓｌａ－ｐｌ，转化Ｅ·ｃｏｌｉ表达菌 ＢＬ２１－ＣｏｄｏｎＰｌｕｓ（ＤＥ３）－ＲＩＬ，ＩＰＴＧ诱导 Ｐ１－８ ｘ ｈｉｓ融合蛋白表达，经包涵体洗涤，８ ｍｏｌ／Ｌ尿素变性溶解，Ｎｉ２＋亲和层析，梯度透析后，定量保存。ＳＤＳ－ＰＡＧＥ、ｗｅｓｔｅｒｎ－ｂｌｏｔｔｉｎｇ鉴定目的蛋白的表达与纯化。结果：目的蛋白（分子量３９．５ ｋＤ）表达量占细菌总蛋白 １５％，每升表达菌获得纯度９５％的目的蛋白 ４０ ｍｇ～６０ｍｇ。结论：成功建立猪 ＳＬＡ分子全长原核表达、纯化体系，为建立间接识别猪移植抗原ＳＬＡＩ类分子的人Ｔ细胞系及表位分析打下基础。     

Preparation of Aminated Macroporous Polyvinyl Alcohol Resins and Evaluation for Bilirubin Adsorption

In the present study we prepared macroporous polyvinyl alcohol beads. A series of bilirubin adsorbents were generated by immobilization of eight amine agents to the beads as ligands. The adsorption of bilirubin was evaluated by in vitro static and dynamic adsorption tests. The results show that these adsorbents have excellent adsorption efficiency and capacity. Among the eight ligands, trimethylamine (TMA), triethylamine (TEA) and 1,6- hexanediamine(HDA) showed the highest adsorption capacity. The adsorption equilibrium can be achieved in half an hour, and the adsorption percentage of bilirubin was up to 80%. Static electricity and hydrophobic interaction played the main role in bilirubin adsorption, and the adsorption was found to match the monolayer model. The excellent adsorption of these adsorbents indicates their potential in clinical treatment.     

Preparation of a proteolytic enzyme mixture containing trypsin,elastase, and chymotrypsin for use in tissue disaggregation

Summary We describe a purification method for tissue culture-grade trypsin that yields an enzyme mixture with reproducible activities of trypsin, elastase, and chymotrypsin and eliminates amylase and lipase.     

高效凝胶色谱一步法制备级纯化单克隆抗体

作者采用ＰｈａｒｍａｃｉａＳｅｐｈａｃｒｙｌＳ—３００凝胶色谱柱，建立了ＩｇＧ类ＭｃＡｂ的一步法制各级纯化方法。该法是将ＭｃＡｂ腹水直接上样，用ｐＨ７．４１０ｍｍｏｌ／ＬＰＢＳ洗脱（流速０．５ｍｌ／ｍｉｎ），即得到纯化的ＭｃＡｂ。一次上样量４０～５０ｍｌ腹水，回收率为８５％－９０％．整个纯化周期４ｈ。纯化的ＭｃＡｂ经ＳＤＳ—ＰＡＧＥ测定，纯度＞９０％，免疫组化ＡＢＣ法测定活性为１：８００００（７．８×１０－１１ｍｏｌ／Ｌ）。该法操作简单、快速，只要有一台核酸／蛋白检测仪，便可进行制备级水平的纯化。     

生物组织微阵列芯片自动制备仪的研制

介绍了一种新型生物组织微阵列芯片自动制备仪的研制。分析了组织微阵列制备过程中的操作任务和实现目标,进行了制备仪的结构设计和各功能模块研究开发。制备仪从结构上分为蜡块承载定位模块和三工位操作模块,控制系统的组成有操作空间精密定位子系统,组织蜡块图像识别子系统,蜡块打孔填埋作业子系统等。研制成功的制备仪样机具备了图像自动识别、精密定位、自动打孔填埋等功能,实现了生物组织微阵列芯片的自动化制备。     

非受体型酪氨酸激酶-Syk蛋白的提取与纯化

目的 :从转染syk基因的Sf2 1细胞中提取、纯化免疫相关因子Syk蛋白。方法 :将syk基因转染Sf2 1细胞 ,于 2 8℃培养 48h ,收集细胞 ,用超声波破碎仪裂解细胞 ,提取裂解液中总蛋白 ,用Yellow 3凝胶和Toyopearl AF Heptin 650M凝胶层析柱分离、纯化。层析液中的Syk蛋白存在和性质 ,用SDS PAGE、免疫印迹实验和等电聚焦实验鉴定。结果 :从 2 5亿个Sf2 1细胞裂解液中提取了含有Syk的 2 2 5mg蛋白质。经Yellow 3凝胶层析分离 ,得到两个亚种的Syk蛋白 ,相对分子质量 (Mr)均为72× 10 3 。进一步用Toyopearl AF Heptin 650M凝胶层析纯化后 ,得到两个纯的Syk蛋白 ,SDS PAGE、免疫印迹实验结果显示 ,两种Syk的Mr 均为 72× 10 3,与Syk的理论相对分子质量吻合。但等电聚焦实验显示 ,这两种Syk蛋白成分具有不同的pI值。结论 :从 2 5亿个转染syk基因的Sf2 1细胞中纯化出8mgSyk蛋白 ,纯度高于 95%。这两种Syk的Mr 虽然相同 ,但具有不同的pI值 ,是两个亚种。这些Syk可用于研究Syk的作用机制、抗Syk抗体的制备和Syk诊断试剂盒的制备等     

Isolation and characterization of liver-derived hepatitis B e antigen

Two subpopulations of hepatitis B e antigen (HBeAg) were isolated from a human liver infected with hepatitis B virus. HBeAg extracted from liver homogenate subsequent to treatment with buffered 3 M NaSCN or 0.5 M MgCl2 banded at the density of 1.13 g/cm3 in CsCl and was polydispersed on gel filtration. In contrast, HBeAg released with phosphate-buffered saline (PBS) was detected mainly at a density of 1.20 g/cm3 in a CsCl gradient and consisted of low molecular weight species on gel chromatography. Polypeptides of 40,000 and 45,000 daltons were found in NaSCN and PBS-released HBeAg preparations, respectively. The results are interpreted as suggestive that liver HBeAg is a dimer of the major core particle polypeptide in different physicochemical forms.