缩短的人巨噬细胞集落刺激因子（M－CSF）在大肠杆菌中的表达

本文用聚合酶链反应（ＰＣＲ）获得了一个缩短的人巨噬细胞集落刺激因子（编码３～１４９氨基酸）ｃＤＮＡ基因,并克隆在质粒ｐＥＴ３ｄ中,在Ｔ７启动子指导下,在大肠杆菌ＢＬ２１（ＤＥ３）ＬｙｓＥ中获得了和一个６组氨酸短肽标签的融合表达。重组的融合ｍ－ＣＳＦ表达量占菌体总蛋白的１２％,表达产物一部分以不溶性包涵体形式存在,另一部分则以可溶性蛋白存在。经过金属螫合亲和层析一步纯化,所得的融合（Ｈｉｓ）６－Ｍ－ＣＳＦ在还原型ＳＤＳ－ＰＡＧＥ上基本呈一条均一的蛋白质条带。     

重组人粒细胞-巨噬细胞集落刺激因子复性和纯化方法的比较

比较重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)在用疏水色谱(HIC)、离子交换色谱(IEC)和体积排阻色谱(SEC)3种液相色谱进行复性和纯化,选择较好的复性和纯化方法.通过对比rhGM-CSF在液相色谱上复性和纯化的主要指标,包括比活、纯度和质量回收率,结果发现采用HIC和IEC对rhGM-CSF进行复性和纯化时,其比活可以达到国家标准,纯度和质量回收率也比较高,而采用SEC,其比活、纯度和质量回收率远低于HIC和IEC.     

重组人睫状神经营养因子的聚乙二醇化修饰简

目的：研究重组人睫状神经营养因子（rhCNTF）突变体的聚乙二醇（PEG）化修饰,对rhCNTF的PEG化产物进行初步分离纯化及相关生物活性检测。方法：采用分子生物学技术经点突变得到rhCNTF的突变体cNm通过实验设计研究CN10的最佳PEG化条件;采用分子筛层析方式对偶联产物进行初步纯化,最后用ELISA和小鼠体重增长抑制法检测PEG化后的CN。。蛋白的生物活性。结果：能运用mPEG—MAL对CN,。进行定点修饰,PEG化后用Superdex200能够分离CN10;PEG化后的CN10每2d腹腔注射1次,对小鼠体重的增长抑制率可达50％,与rhCNTF每天注射2次的体重增长抑制作用相当。结论：CN10蛋白在PEG化修饰后,其减重效应持续时间明显延长。     

Use of the Recombinant 38-kDa Antigen of Mycobacterium tuberculosis as an Immunogen for Specific Antisera Production

The Mycobacterium tuberculosis 38-kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B-cell and T-cell levels. Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays. This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system. During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E. coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid-phase nitrocellulose membrane and 4) emulsified with an adjuvant. All strategies yielded specific antisera as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.     

Biochemical Characterization of Recombinant Human Nerve Growth Factor

Recombinant human nerve growth factor (rhNGF) was expressed and secreted by Chinese hamster ovary cells and purified to homogeneity using ion-exchange and reversed-phase (RP) chromatography. The isolated product was shown to be consistent with a 120-amino-acid residue polypeptide chain by amino acid composition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), RP-HPLC, and mass spectrometry and with an N-terminal sequence consistent with that expected from the cDNA for human nerve growth factor. By size-exclusion chromatography, rhNGF behaves like a noncovalent dimer. Limited enzymatic digests of the 120-residue monomer produced additional species of 118 (trypsin, removal of the C-terminal Arg119-Ala120 sequence) and 117 (trypsin plus carboxypeptidase B, removal of the C-terminal Arg118-Arg119-Ala120 sequence) residues. Each of these species was isolated by high-performance ion-exchange chromatography and characterized by amino acid and N-terminal sequence analyses, SDS-PAGE, RP-HPLC, and mass spectrometry. All three species were present in the digests as both homodimeric and heterodimeric combinations and found to be equipotent in both the chick dorsal root ganglion cell survival and rat pheochromocytoma neurite extension assays.     

重组人粒细胞集落刺激因子发酵工艺研究

通过对大肠杆菌（E.coli）表达重组人粒细胞集落刺激因子（rhG-CSF）工程菌DH5a的发酵工艺的研究,对影响工程菌生长和目标蛋白表达条件如：发酵培养基配方,pH值,诱导时间,分批补加营养物质等进行了优化。结果表明,采用优化的条件发酵后,工程菌得率达30g/L以上;目标蛋白表达量为30%以上。     

Radioimmunoassay of Bovine,Ovine and Porcine Luteinizing Hormone with a Monoclonal Antibody and a Human Tracer

A radioimmunoassay for bovine (bLH), ovine (oLH) and porcine (pLH) luteinizing hormone was developed using a human 125 ILH tracer from a commercial kit and a monoclonal antibody (518B7) specific for LH but with low species specificity. Standard curves demonstrated similar binding kinetics when bLH, oLH and pLH were incubated with tracer and antibody for 2 h at room temperature. A 30-min delay in the addition of the tracer gave sufficient sensitivity when analysing pLH. Separation of antibody-bound LH from free hormone was achieved by using second antibody-coated micro Sepharose beads. The assay was validated and the performance compared with that of an RIA currently in use for determination of bLH and oLH (coefficient of correlation: 0.99 and 0.98). Regardless of the standards used, intra-assay coefficients of variation were <10% for LH concentrations exceeding 1 µg/L. The inter-assay coefficients of variation were <15%. The assay was used for clinical evaluation demonstrating the pre-ovulatory LH surge in two cyclic cows, LH pulsatility in an oophorectomized ewe and LH response to GnRH injection in a boar.     

Keratinocyte Growth Factor Administration Attenuates Murine Pulmonary Mycobacterium tuberculosis Infection through Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF)-dependent Macrophage Activation and Phagolysosome Fusion

Augmentation of innate immune defenses is an appealing adjunctive strategy for treatment of pulmonary Mycobacterium tuberculosis infections, especially those caused by drug-resistant strains. The effect of intranasal administration of keratinocyte growth factor (KGF), an epithelial mitogen and differentiation factor, on M. tuberculosis infection in mice was tested in prophylaxis, treatment, and rescue scenarios. Infection of C57BL6 mice with M. tuberculosis resulted in inoculum size-dependent weight loss and mortality. A single dose of KGF given 1 day prior to infection with 10⁵ M. tuberculosis bacilli prevented weight loss and enhanced pulmonary mycobacterial clearance (compared with saline-pretreated mice) for up to 28 days. Similar effects were seen when KGF was delivered intranasally every third day for 15 days, but weight loss and bacillary growth resumed when KGF was withdrawn. For mice with a well established M. tuberculosis infection, KGF given every 3 days beginning on day 15 postinoculation was associated with reversal of weight loss and an increase in M. tuberculosis clearance. In in vitro co-culture experiments, M. tuberculosis-infected macrophages exposed to conditioned medium from KGF-treated alveolar type II cell (MLE-15) monolayers exhibited enhanced GM-CSF-dependent killing through mechanisms that included promotion of phagolysosome fusion and induction of nitric oxide. Alveolar macrophages from KGF-treated mice also exhibited enhanced GM-CSF-dependent phagolysosomal fusion. These results provide evidence that administration of KGF promotes M. tuberculosis clearance through GM-CSF-dependent mechanisms and enhances host defense against M. tuberculosis infection.     

聚乙二醇化重组人粒细胞刺激因子及其药品通用名称命名

有越来越多的聚乙二醇修饰人粒细胞刺激因子研发上市。为适应这类制品的发展,中国药品通用名命名原则也需要不断更新修订。简要介绍了聚乙二醇修饰蛋白技术以及WHO国际非专利药品名(INN)命名委员会对这类制品的命名情况,讨论了我国对这类制品的药品通用命名原则和方法,建议对聚乙二醇化不同的修饰形式在名称上适当加以区分,并注意加强与INN命名委员会的交流合作。     

Heterologous Radioimmunoassay for Llama and Alpaca Luteinizing Hormone with a Monoclonal Antibody,an Equine Standard and a Human Tracer

A radioimmunoassay for llama and alpaca LH was developed using a human I125LH tracer from a commercial kit, equine LH diluted in human LH free serum as standard, and a monoclonal antibody (518B7) specific for LH but with low species specificity. A 60-min delay in the addition of the tracer and overnight incubation gave a sensitivity of 0.8 μg L−1. The intra-assay coefficient of variation was 37% at 1 μg L−1, declined to 15% at 4 pg L−1 and was below 6% for concentrations up to 32 μg L−1. The inter-assay coefficients of variation for 3 control samples were 20% (2.8 μg L−1), 16% (7.1 μg L−1) and 9.8% (19 μg L−1). In an attempt to increase sensitivity, all tubes were preincubated for 4 h at room temperature before adding the tracer, and the sample volume was increased from 50 μL to 100 μL· (in the standard curve the increased volume was compensated for by human LH free serum). With this protocol, the assay sensitivity was 0.5 μg L−1. The assay was validated clinically and demonstrated increased concentrations of LH after mating in llamas and alpacas. Furthermore, the assay was used to monitor LH responses to a single dose of GnRH in llamas (adult males and females at different ages).     

人巨噬细胞集落刺激因子（hM－CSF）基因的合成及表达

按照ｈＭ－ＣＳＦ基因的序列,适当采用大肠杆菌的优选密码子,人工合成了Ｍ－ＣＳＦ的基因。在合成中我们采用了分段克隆和顺次克隆的方法,在次级克隆中我们成功地使用了多片段分组连接和多片段一次克隆战略,还尝试了多种单链战术。在表达载体的构建中,充分利用人工合成基因的灵活性,通过对Ｎ端６个氨基酸编码的变换及ＳＤ序列－ＡＴＧ间距离的改变,获得了在大肠杆菌中高效表达的重组体,表达的蛋白量占菌体总蛋白的２９％。目的蛋白在表达中形成的包含体形式,简化了产物的纯化。     

重组人睫状神经营养因子肽图分析

建立重组人睫状神经营养因子(recombinant human ciliary neurotrophic factor,rhCNTF)的肽图分析方法,用于rh-CNTF的质量控制。胰蛋白酶对rhCNTF进行酶切后,利用RP-HPLC方法对酶切液进行分析,以获得胰蛋白酶切最佳条件及色谱条件,并对连续3批样品进行分析。rhCNTF的胰蛋白酶最佳酶切条件为37℃酶切24h,以A(0.1%TFA-H2O)、B(0.1%TFA-CH3CN)为流动相,采用梯度洗脱的方法对酶切液进行分析,结果连续3批rhCNTF制备产品的肽图完全一致,且其检出峰数目与理论推测值相符。3批产品肽图的一致性为rhCNTF产品的结构同一性提供了有利证据,同时,建立了rhCNTF产品质量控制的一项指标。     

Mammalian Granulocyte–Macrophage Colony-stimulating Factor Receptor Expressed in Primary Avian Hematopoietic Progenitors: Lineage-specific Regulation of Proliferation and Differentiation

The cytokine Granulocyte–Macrophage Colony-Stimulating Factor (GM-CSF) regulates proliferation, differentiation, and apoptosis during myelopoiesis and erythropoiesis. Structure–function relationships of GM-CSF interactions with its receptor (GM-R), the biochemistry of GM-R signal transduction, and GM-CSF action in vivo are relatively well understood. Much less is known, however, about GM-R function in primary hematopoietic cells. In this paper we show that expression of the human GM-R in a heterologous cell system (primary avian erythroid and myeloid cells) confirms respective results in murine or human cell lines, but also provides new insights how the GM-R regulates progenitor proliferation and differentiation. As expected, the hGM-CSF stimulated myeloid progenitor proliferation and differentiation and enhanced erythroid progenitor proliferation during terminal differentiation. In the latter cells, however, the hGM-R only partially substituted for the activities of the erythropoietin receptor (EpoR). It failed to replace the EpoR in its cooperation with c-Kit to induce long-term proliferation of erythroid progenitors. Furthermore, the hGM-R α chain specifically interfered with EpoR signaling, an activity neither seen for the β_c subunit of the receptor complex alone, nor for the α chain of the closely related Interleukin-3 receptor. These results point to a novel role of the GM-R α chain in defining cell type–specific functions of the GM-R.     

异种器官移植用血管内皮细胞特异表达人DAF重组基因载体的构建

PCR插入法将人ICAM－2启动子、人DAF－intron1和DAFcDNA克隆到pGEM-7zf质粒中,进行序列测定及分析,成功构建异种器官移杆用血管内皮细胞特异表达人DAF重组基因,提供了人DAF重组基因用于转基因动物的研究。     

Expression, Purification, and Characterization of Recombinant Human Factor X

A system is described for producing recombinant factor X with properties very similar to human plasma factor X. Optimization of the expression system for factor X resulted in the finding that human kidney cells (293 cells) are superior to the widely utilized baby hamster kidney cells (BHK cells) for the expression of functional factor X. It was also determined that production of factor X by 293 cells requires the substitution of the −2 residue (Thr → Arg) which affords the removal of the factor X propeptide. Purification of recombinant and plasma factor X is accomplished using a calcium-dependent monoclonal antibody directed against the gla domain. The proteins are comparable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rate and extent of activation by the factor X coagulant protein from Russell's viper venom and by factors IXa and VIIIa are similar; activation of the recombinant protein by VIIa and tissue factor is mildly faster. The activated enzymes have the same activity toward a chromogenic substrate and the biologic substrate, prothrombin. Both enzymes have the same apparent affinity for the activated platelet surface as judged by their ability to activate prothrombin. Finally, inhibition by antithrombin, with or without heparin, and inhibition by the tissue factor pathway inhibitor are equivalent. Recombinant factor X produced by this method is therefore well suited for probing structure–function relationships by mutational analysis.     

重组人粒细胞集落刺激因子的纯化

重组人粒细胞集落刺激因子（ｒｈＧ－ＣＳＦ）在工程菌ｐＣＧ－１／ｒｈＧ－ＣＳＦ／ＤＨ５ａ中以无活性的包涵体形式大量表达。经过菌体破碎分离包涵体、包涵体变性复性后,ｒｈＧ－ＣＳＦ的活性得到恢复。用离子交换和疏水层析纯化了ｒｈＧ－ＣＳＦ,比活性达１．５７×１０８ｕ／ｍｇ,纯度大于９８％。     

重组人粒细胞集落刺激因子的复性研究

通过小试研究对重组人粒细胞集落刺激因子(rhG-CSF)的复性条件,如氧化剂和还原剂比例、操作方法、时间和蛋白浓度,进行了优化选择,并在此基础上进行了中试放大试验的验证.试验结果表明,采用优化后的复性方法复性液中rhG-CSF的效价可达到1.8×107U/ml以上,比活性达到0.9×108/mg以上.     

重组人睫状神经营养因子的表达及其临床前药效学研究

目的:人睫状神经营养因子(hCNTF)是神经营养因子(NGF)家族以外的一种神经营养因子,具有明显的减轻体重的作用。为了将hCNTF的研究逐步推向应用,开展了hCNTF减肥作用的临床前相关研究。方法:依据国内减肥药临床前药效学指导原则,设计并实施了动物实验,建立了各种指标与人类肥胖最为相似的肥胖SD大鼠模型,分别测定了重组hCNTF用药后大鼠的体重、血清三酰甘油、总胆固醇、肾周围脂肪重量和肥胖指数。结果:在200μg/(kg·d)剂量下,重组hCNTF对减轻肥胖SD大鼠体重、减轻其右肾周围脂肪及改善体型都有一定的效果;400μg/(kg·d)以上剂量的重组hCNTF,对减轻肥胖SD大鼠体重、改善体型、降低血清三酰甘油都有极其显著的效果;惟有剂量达到1600μg/(kg·d),重组hCNTF才能降低血清总胆固醇含量。结论:所获得的重组hCNTF在肥胖SD大鼠动物模型上具有十分良好的减肥效果。     

重组人KGF制备工艺和活性检测方法的建立

目的:在毕赤酵母中实现了KGF的高效表达,并初步建立了生物学活性检测方法.方法:合成5'端缺失69个核苷酸的KGF基因序列,克隆入pPIC9并转化毕赤酵母菌株GS115中,经诱导表达.发酵液上清采用脱盐层析和阳离子交换层析进行分离纯化.利用貂肺上皮细胞(Mv-1-Lu)检测其生物学活性.结果:表达水平达到了110mg/L发酵液;表达产物经一步离子交换层析就能得到有效分离,总收率在50mg/L发酵液以上;纯化的rhKGF生物学活性与KepivanceTM相当.结论:rhKGF制备工艺和检测方法的建立将为该因子的规模化生产和进一步的临床应用提供良好基础.