生物工程技术制备人源抗-HBs Fab 片段

目的：用生物工程技术制备人源性抗-HBsFab。方法：将从抗体文库中筛选出的人源抗-HBsFab基因克隆入pBAD/gⅢA载体，进而转化Tpo10大肠杆菌，对重组质粒菌发酵表达后，利用Ni-NTA-Agarose螯合层析柱纯化周质腔可溶性Fab蛋白。对所得包涵体依次变性，溶解，纯化后，利用透析进行复性，用Western blot检测Fab蛋白的特异性，Dot blot测定其生物学活性。结果：经Ni-NTA-Agarose柱纯化的周质腔可溶性Fab蛋白，有较好的生物学活性，并且总量达到80mg/L。对所获包涵体进行透析复性后，也可得到少量有活性的蛋白，但比例很小。结论；用pBAD/gⅢA-Top10表达系统表达人源抗-HBsFab片段，发酵培养后，经有效纯化可得到生物学活性较好的可溶性蛋白。为人源抗-HBsFab片段的大量制备提供了有效手段。     

Calcitonin gene-related peptide in cardiovascular tissues of the rat

The distribution of calcitonin gene-related peptide immunoreactivity in the cardiovascular system of the rat was investigated by radioimmunoassay and immunocytochemistry. The nature of the immunoreactivity was studied by gel permeation and high performance liquid chromatography. Immunocytochemistry demonstrated the existence of calcitonin gene-related peptide-containing nerve fibres throughout the cardiovascular system. These were present in all regions of the heart, particularly in association with the coronary arteries, within the papillary muscles and within the sinoatrial and atrioventricular nodes. Calcitonin gene-related peptide-containing fibres were found mainly in the adventitia of the arteries and veins. Calcitonin gene-related peptide concentrations were high in major arteries and veins but comparatively low in the heart, aortic arch and thoracic aorta. Chromatography showed that approximately 70% of the total immunoreactivity was identical to synthetic calcitonin gene-related peptide. Calcitonin gene-related peptide concentrations in the blood vessels of rats treated neonatally with capsaicin were not found to be significantly different from those in control animals although capsaicin caused significant reductions of calcitonin gene-related peptide levels in certain other tissues. The results of this study suggest that calcitonin gene-related peptide-containing fibres are likely to be of importance in the innervation of vascular tissues and raise the possibility that these fibres are different in character from calcitonin gene-related peptide-containing fibres found in other tissues.     

Significance of methods for purification of oligodeoxyribonucleotide probes for the efficiency of gene diagnosis by real-time PCR

Analysis of the use of real-time PCR with fluorescent registration of results for gene diagnosis of infectious diseases showed that the sensitivity and reliability of quantitative evaluation of DNA targets directly depended on the method of purification of oligonucleotide probes. Chromatographic behavior of synthetic probes carrying various fluorophores and fluorescence quenchers was analyzed. Approaches to optimization of purification methods are proposed enabling elimination of previously undetectable admixtures. The importance of these studies is explained by the need in extending the armory of methods for the development and production of diagnosticums for detection of infectious and hereditary diseases, identification of genetically modified organisms, and for a wide spectrum of research in molecular biology and medicine. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 3, pp. 280–284, March, 2008     

Recognition of an extensive range of IgE-reactive proteins in cod extract

Allergy to fish is one of the most common food allergies. Gad c 1 is the only fish allergen which has been purified and characterized. Other allergens have been detected by Western blot in cod extracts. We have now improved the Western-blot procedure in order to characterize fish IgE-reactive proteins from extracts prepared under different conditions: pre-rigor mortis and postrigor mortis. EDTA addition or not. and DEAE ion-exchange chromatography. Several IgE-reactive protein bands have been identified over a wide molecular-weight range. In particular, the 104- and 130-kDa IgEreactive protein bands were detected. These new bands may correspond to aggregates, as EDTA increased the relative amount of the 60-, 67-, 104-, and 130-kDa IgE-reactive protein bands in Western blot. All these bands were also detected by an antiparvalbumin monoclonal antibody, specific to the first calcium-binding site. The longer period of storage increased the relative amounts of the 41-, 80-, 104-. and 130-kDa IgE-reactive protein bands. The 18-kDa band was detected only in fish stored for several days. In conclusion, we have described IgE-reactive protein bands over a wide molecular-weight range (12–130 kDa) in Western blot of cod extract, and shown that EDTA and storage conditions may influence the relative distribution of IgE-reactive protein bands.     

The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.     

人血管抑制因子的原核表达、纯化及活性研究

目的：利用大肠杆菌表达系统表达人血管抑制因子，并利用镍金属螯合层析法进行纯化，探讨其抑制血管内皮细胞增殖的活性。方法：采用RT-PCR技术从人肝脏组织中获取人血管抑制因子的cDNA，将其克隆至原核表达载体pQE30中进行IPTG诱导表达。表达产物经SDS-PAGE分析，并利用镍金属螯合层析法进行纯化。^3H-TdR法检测纯化的血管抑制因子对血管内皮细胞增殖的抑制作用。结果：利用pQE30表达载体表达的含6个组氨酸尾的vasostafin蛋白，在SDS-PAGE上表现出一条约2lkD的阳性条带，经镍金属螯合层析纯化后的蛋白经肽指纹图谱分析鉴定为目的蛋白，在体外可抑制人脐静脉血管内皮细胞的增殖。结论：人血管抑制因子可在大肠杆菌中以包涵体形式高水平表达，并具有抑制血管内皮细胞增殖的活性．     

Characterization of Porcine Zona Pellucida Antigens by Immunoaffinity Chromatography and by High-Pressure Liquid Chromatography

ABSTRACT: In the present work, 500 and 50,000 porcine zonae pellucidae were solubilized using Lithium-3,5-diiodosalicylate. The zona antigens were purified by immunoaffinity chromatography (IAC) on immobilized antizona immunoglobulin G (IgG). The antizona-IgG was raised by immunization of female rabbits with 500 heat-solubilized porcine zonae. Four antigens could be detected following IAC: ZP I/1 (M_r = 42,000), ZP II/1 (M_r = 67,000), ZP II/2 (M_r = 32,000), ZP III/1 (M_r = 17,000). In a parallel experiment, 50,000 zonae were solubilized in a similar manner and the mixture was analyzed by high-pressure liquid chromatography (HPLC) using a protein column. Altogether, 9 protein peaks that contained the antigens ZP I/1, ZP II/1, ZP II/2, and ZP III/1 could be detected following HPLC. The carbohydrate composition is characteristic for O-glycosidic-glycoproteins. ZP II/1 and ZP II/2 are probably in close association within the zona. Based on the reaction of the antigens with antibodies induced by intact and heat-solubilized zonae, it is postulated that only ZP I/1 and ZP II/l are expressed on the surface in intact zonae.     

Multidimensional Chromatographic and Hyphenated Techniques for Hydrophilic Copolymers, 1

Summary: The chromatographic analysis of hydrophilic copolymers is complicated due to the fact that in most cases aqueous eluents must be used. In aqueous eluents different polar and ionic effects may disturb the selective interactions between the macromolecules and the stationary phase making it impossible to separate such copolymers with regard to chemical composition. Therefore, 2D chromatography combining a separation according to composition with a separation according to molar mass has been applied mostly to polymers that are soluble in organic solvents. The present contribution describes experimental approaches to analyze such hydrophilic copolymers by 2D‐chromatography. For a model polymer system resulting from the copolymerization of methacrylic acid and a poly(ethylene glycol) macromonomer, it is shown that different analytical techniques including SEC, LC‐CC, MALDI‐TOF MS and 2D chromatography can be used to analyze the different parameters of molecular heterogeneity of such copolymers.

2D separation of poly(MPEG‐MM 2), 1^st dimension: LC‐CC, 2^nd dimension: SEC.     

运用变性高效液相色谱对肺炎克雷伯菌产ESBL进行基因分型

目的 通过运用变性高效液相色谱（DHPLC）技术对前期研究已确认产超广谱β-内酰胺酶（ESBL）的肺炎克雷伯菌临床分离株TEM型质粒进行基因分型,试图建立一种方便快捷的用于ESBL分子诊断及其流行病学监测的新方法.方法 利用PCR技术从肺炎克雷伯菌临床分离株中扩增出TEM型质粒的编码序列,扩增产物运用DHPLC技术进行分析,分析提示,异常的样本通过测序确定其基因突变的类型,最后通过比对确定其基因型.结果 共分析了101例肺炎克雷伯菌临床分离株,全部样本均扩增出TEM型质粒的编码序列,经过DHPLC分析,52例（51.4%）样本表现为单一的洗脱峰,其形态与TEM-1标准菌株的峰型相一致,测序确定它们的碱基序列亦相一致,不存在变异,为TEM-1型;49例（48.6%）样本表现为异常的洗脱峰,它们均为双峰,形态一致,但异源双链峰的高度有差异,测序结果表明它们均存在四种相同的基因突变,在NCBI网站比对后确定为TEM-116;测序结果还提示,部分样本中TEM-1和TEM-116混合存在,其比例的不同表现为DHPLC时异源双链峰高度的差异;文献检索表明,本次确定的TEM-116为一新的基因亚型,为国内首次报道.结论 DHPLC具有简便快捷、高通量和自动化的特点,重复性好,不仅可对已知突变作出即时诊断,还可发现新的基因亚型,不失为一种较好的ESBL分子诊断方法及其流行病学监测手段.     

人初乳分泌片的纯化和兔抗人分泌片免疫血清的制备及应用

目的：获得足够量的分泌片（SC）及其相应的抗血清，用于研究SC的功能与SC在人和某些物种黏膜组织内的分布。方法：在已有的凝胶过滤和离子交换层析的基础上，进一步通过亲和层析和凝胶过滤分离纯化人的SC并进行相应的鉴定。用免疫组织化学技术检测了SC在部分小鼠组织中的表达。结果：获得了免疫纯的SC，并制备了相应的兔抗人SC的免疫血清；免疫组化显示，SC在小鼠小肠和子宫内膜处有表达。结论：利用改进的方法可以获得免疫纯的SC，进一步证实人和小鼠SC之间的交叉反应性。