Activated protein C up-regulates IL-10 and inhibits tissue factor in blood monocytes

The protective effect of recombinant activated protein C therapy in patients with severe sepsis likely reflects the ability of recombinant activated protein C to modulate multiple pathways implicated in sepsis pathophysiology. In this study, we examined the effects of recombinant activated protein C on the anti-inflammatory cytokine IL-10 and on the procoagulant molecule tissue factor (TF) in LPS-challenged blood monocytes. Treatment of LPS-stimulated monocytes with recombinant activated protein C resulted in an up-regulation of IL-10 protein production and mRNA synthesis. The up-regulation of IL-10 required the serine protease activity of recombinant activated protein C and was dependent on protease-activated receptor-1, but was independent of the endothelial protein C receptor. At the intracellular level, p38 MAPK activation was required for recombinant activated protein C-mediated up-regulation of IL-10. We further observed that incubation of LPS-stimulated monocytes with recombinant activated protein C down-regulated TF Ag and activity levels. This anticoagulant effect of recombinant activated protein C was dependent on IL-10 since neutralization of endogenously produced IL-10 abrogated the effect. In patients with severe sepsis, plasma IL-10 levels were markedly higher in those treated with recombinant activated protein C than in those who did not receive recombinant activated protein C. This study reveals novel regulatory functions of recombinant activated protein C, specifically the up-regulation of IL-10 and the inhibition of TF activity in monocytes. Our data further suggest that these activities of recombinant activated protein C are directly linked: the recombinant activated protein C-mediated up-regulation of IL-10 reduces TF in circulating monocytes.     

重组人E1A激活基因阻遏子/myc-His融合糖蛋白的纯化及功能鉴定

目的:纯化重组人E1A激活基因阻遏子(hCREG)糖蛋白,验证重组hCREG糖蛋白具有抑制体外培养的人胸廓内动脉平滑肌细胞(HITASY)增殖的生物学功能。方法:根据6×His亲和层析原理,应用Ni-NTA柱纯化重组hCREG蛋白,纯化后蛋白通过HiTrap脱盐柱脱盐。用流式细胞周期分析研究添加0.5μg/ml、1μg/ml及2μg/ml重组hCREG/myc-His融合糖蛋白对体外培养HITASY细胞增殖的影响。用BrdU掺入方法研究重组蛋白对体外培养HITASY细胞增殖的影响。结果:根据6×His亲和层析原理用Ni-NTA纯化重组hCREG蛋白,浓缩并脱盐后的重组hCREG蛋白浓度为1.6mg/ml,纯度为92%,此蛋白仍保留了糖基化形式。流式细胞仪细胞周期分析结果提示重组hCREG蛋白可抑制体外培养的HITASY细胞增殖,且低浓度组的抑制效应要高于高浓度组。BrdU掺入实验结果提示,添加2μg/ml重组hCREG蛋白组与对照组相比可显著抑制体外培养的HITASY细胞增殖,组间比较有统计学差异(P0.05)。结论:具有生物学功能的重组hCREG/myc-His糖蛋白的成功纯化,为hCREG蛋白的功能研究及生物工程制备提供了实验平台。     

重组葡激酶工程菌的构建方法

目的：利用N端缺失10个氨基酸的葡激酶（recombinant staphylokinase,rSaK）重组质粒,构建了表达可溶性rSaK126蛋白的工程菌,并研究不同条件下工程菌诱导表达目的蛋白含量的差异及纯化途径。 方法：采用细菌活化和培养方法诱导目的蛋白,并用SDS-PAGE测其含量,应用层析技术纯化蛋白。 结果：成功构建表达重组葡激酶的工程菌,表达的重组葡激酶蛋白约占菌体总蛋白的50％,经纯化后回收率为60%,纯度达99%以上。结论：成功构建高效表达重组葡激酶的工程菌,并获得了高含量、高纯度的目的蛋白。     

免疫A型呼吸道合胞病毒G75-225蛋白的小鼠对病毒感染的抗性

目的:评价A型呼吸道合胞病毒(RSV)G75-225蛋白(G151)与二硫键异构酶(DsbA)的重组蛋白抗原DsbA-G151的免疫活性。方法:采用PCR方法从A型RSVG蛋白扩增G151基因片段,插入表达载体pET-DsbA,经E.coli表达、亲和层析纯化制备DsbA-G151蛋白;将其免疫BALB/c小鼠后获得相应的抗血清,利用ELISA方法、保护性实验检测蛋白免疫活性。结果:构建了表达载体pET-DsbA-G151,表达、纯化获得了重组蛋白DsbA-G151。ELISA检测表明,DsbA-G151能在小鼠体内产生高滴度的特异性IgG;保护性实验显示该蛋白能有效保护BALB/c小鼠不被RSV感染。结论:经ELISA检测、保护性实验,表明DsbA-G151具有良好的免疫原性。     

Expression, secretion, and glycosylation of the 45- and 47-kDa glycoprotein of Mycobacterium tuberculosis in Streptomyces lividans

The gene encoding the 45/47 kDa glycoprotein (Rv1860) of Mycobacterium tuberculosis was expressed in Streptomyces lividans under its own promoter and under the thiostrepton-inducible Streptomyces promoter PtipA. The recombinant protein was released into the culture medium and, like the native protein, migrated as a double band at 45 and 47 kDa in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels. However, in contrast to the native protein, only the 47-kDa recombinant protein could be labeled with concanavalin A (ConA). Carbohydrate digestion with jack bean alpha-D-mannosidase resulted in a reduction in the molecular mass of the recombinant protein upper band and completely eliminated ConA binding. Two-dimensional gel electrophoresis revealed only one isoelectric point for the recombinant protein. Comparative fingerprinting analysis of the individually purified upper and lower recombinant protein bands, treated under the same conditions with specific proteases, resulted in similar peptide patterns, and the peptides had the same N-terminal sequence, suggesting that migration of the recombinant protein as two bands in SDS-PAGE gels could be due to differences in glycosylation. Mass spectrometry analysis of the recombinant protein indicated that as in native protein, both the N-terminal and C-terminal domains of the recombinant protein are glycosylated. Furthermore, it was determined that antibodies of human tuberculosis patients reacted mainly against the carbohydrate residues of the glycoprotein. Altogether, these observations show that expression of genes for mycobacterial antigens in S. lividans is very useful for elucidation of the functional role and molecular mechanisms of glycosylation in bacteria.     

抗菌肽CM4与人可溶性B淋巴细胞因子hsBAFF在大肠杆菌中的融合表达

为进一步探讨抗菌肽CM4的原核表达及其生物学功能,本实验研究了抗菌肽CM4与人可溶性B淋巴细胞刺激因子hsBAFF的融合表达及抗菌肽CM4的生物学活性。运用PCR把B淋巴细胞因子hsBAFF和家蚕抗菌肽CM4进行基因融合,构建了融合表达载体pET28a (+)/CM4-hsBAFF,并在大肠杆菌中获得高可溶性表达的融合靶蛋白,且存在于超声破碎后的上清,经分子筛Sephadex G-75纯化后的重组融合蛋白用SDS-PAGE和Western blot分析鉴定.SDS-PAGE分析表明：可以通过分子筛一步纯化得到融合蛋白,该重组融合蛋白的分子量约22.0 KDa。Western blot结果显示该重组蛋白能与鼠抗人hsBAFF的抗体发生特异性反应.运用基因工程的方法获得CM4-hsBAFF重组融合蛋白,并具有很好的抑菌生物学活性。     

Protection against Atlantic halibut nodavirus in turbot is induced by recombinant capsid protein vaccination but not following DNA vaccination

Fish nodaviruses (betanodaviruses) are small, non-enveloped icosahedral single-stranded positive-sense RNA viruses that can cause viral encephalopathy and retinopathy (VER) in a number of cultured marine teleost species, including Atlantic halibut (Hippoglossus hippoglossus). A recombinant protein vaccine and a DNA vaccine were produced, based on the same capsid-encoding region of the Atlantic halibut nodavirus (AHNV) genome, and tested for protection in juvenile turbot (Scophthalmus maximus). Vaccine efficacy was demonstrated in the fish vaccinated with recombinant capsid protein but not in the DNA-vaccinated fish, despite the fact that in vivo expression of the DNA vaccine-encoded antigen was confirmed by RNA in situ hybridisation and immunohistochemistry. Combined DNA and recombinant vaccine administration did not improve the effect of the latter. Surprisingly, fish vaccinated with 50 microg recombinant protein demonstrated a threefold lower survival rate than the two groups that received 10 microg recombinant protein. Neither the recombinant protein vaccine nor the DNA vaccine induced anti-viral antibodies 9 weeks after immunisation, while antibodies reactive with the recombinant protein were detectable mainly in fish vaccinated with 50 microg recombinant protein. The study also demonstrates evidence of viral replication inside the myocytes of intramuscularly challenged fish.     

兔多杀性巴氏杆菌C51-3株黏附蛋白的表达、纯化及其抗原性检测

应用PCR从兔多杀性巴氏杆菌C51-3株基因组DNA中扩增出编码36 kD黏附蛋白的cp36基因, 将其克隆到pMD18-T载体并对插入片段进行测序。以重组质粒pMD18-cp36为模板, 用PCR扩增得到编码信号肽除外的成熟黏附蛋白基因cpm36, 并克隆到原核表达质粒pQE30中, 得到重组质粒pQE30-cpm36, 转化大肠杆菌M15, 在IPTG诱导下表达融合蛋白CPM36, 经Ni2+-NTA亲和层析纯化。DNA测序结果表明cp36基因片段大小为1032 bp, 与已报道的16个血清型多杀性巴氏杆菌cp36基因的核苷酸序列比较, 同源性在76.9%~100%之间。SDS-PAGE结果显示, 表达分子量约为37 kD的带有6×His标签的CPM36蛋白, 与预期分子量相符。Western blotting结果表明, 抗重组蛋白抗体分别能与CPM36蛋白和多杀性巴氏杆菌36 kD蛋白发生特异性反应, 证明原核表达蛋白具有抗原性, 为进一步开展多杀性巴氏杆菌免疫保护性抗原的研究奠定了基础。     

SARS-CoV S蛋白受体结合区的重组表达及免疫原性分析

为了表达SARS-CoV的S蛋白的受体结合区并对其免疫原性进行分析,用PCR方法扩增S蛋白的受体结合区基因片段,克隆至原核表达质粒pET-F32a＋并在大肠杆菌中表达,应用Western—blot鉴定表达的目的蛋白,而后以该蛋白作为诊断抗原包被酶联卡反来检测20份SARS病人血清和28份健康人血清,结果原核表达的S蛋白能够和所用的SARS病人血清反应。这提示表达的S重组蛋白具有良好的抗原性。将变性纯化的重组蛋白和复性蛋白分别皮下免疫小鼠,第三次免疫一周后收集抗血清,用ELISA测定抗体和同时测定中和抗体活性。用变性的抗原免疫的小鼠血清均无中和活性;而用复性的蛋白免疫的小鼠产生了中和抗体。实验表明,S蛋白受体结合区无线性中和表位,中和抗体的产生是由构象表位诱导的。提示该蛋白有可能应用于亚单位疫苗的研究。     

Expression of ovine gamma interferon in Escherichia coli and Corynebacterium glutamicum.

Bacteria of two species, Escherichia coli and Corynebacterium glutamicum, were used as hosts to express recombinant ovine gamma interferon as a fusion protein with glutathione S-transferase. The recombinant gamma interferon produced by both bacteria was biologically active in vitro and was recognized by anti-gamma interferon monoclonal antibodies. E. coli produced large amounts of soluble recombinant protein which could be purified by a simple affinity chromatography method. Only a small fraction of the recombinant protein made by C. glutamicum was recovered by this method. Expression of recombinant protein in C. glutamicum was unstable but could be controlled by increased regulation of the tac promoter. Both hosts expressed ovine gamma interferon at high levels, with the recombinant protein making up a significant proportion of the cellular protein content.     

SARS-CoV核衣壳蛋白的表达与免疫研究

依据GenBank中SARS基因组序列,采用人工合成的方法合成编码SARS病毒N蛋白的全基因(1296bp)序 列,再与设计的CTL特异性表位基因(195bp)重组后,克隆到pET-28a( )质粒中,重组质粒转化到大肠杆菌BL21 (DE3)中诱导表达。利用SARS患者恢复期阳性血清,鉴定表达蛋白。进一步纯化后免疫马,并用ELISA方法检 测血清抗体效价。结果显示SDS-PAGE表明所表达的蛋白相对分子质量约为55000 Da,与预计大小相符;Western blot显示表达的蛋白具有良好的抗原性和特异性。对表达蛋白形成的包涵体进行洗涤,得到的蛋白纯度可达到 70．1％。切胶纯化免疫马后,获得的抗SARS抗体滴度达1:2560。为亚单位疫苗研制和精制抗SARS抗体免疫制 剂提供基础。     

MMP-2结合肽-蜂毒素杂合基因的表达纯化及活性测定

为开发抗癌导向多肽药物,用从噬菌体十二肽库中筛选的与MMP-2具有高度亲和性的十二肽与蜂毒素连接,采用基因工程方法在大肠杆菌中进行了高效表达;并分别通过亲和层析、肠激酶酶切、凝胶层析获得高纯度的多肽(相对分子质量45000),经体外抑瘤作用测定该融合蛋白具有明显抑制肿瘤细胞的生长。该研究对肿瘤的导向治疗和临床应用提供了一定的帮助。     

Genetic immunization for the recovery and purification of recombinant proteins

A system that uses genetic immunization for recombinant protein recovery and purification is described. The genetic sequence encoding a target protein is subcloned into both a eukaryotic and a prokaryotic vector. With the eukaryotic construct, a rabbit is genetically immunized and specific polyclonal antibodies to the encoded protein raised. The prokaryotic construct is used for bacterial transformation and expression of recombinant protein. Recovery and purification of target recombinant protein are obtained by passing the lysate of expressing bacteria through an immunoaffinity column prepared with the polyclonal antibodies raised in the genetically immunized animal. This method allows purification of recombinant protein without fusion tails and can be applied to purify any protein whose encoding genetic sequence is known.     

禽多杀性巴氏杆菌C48-3株成熟黏附蛋白的表达及其免疫原性检测

目的：在大肠杆菌原核表达系统中高效表达禽多杀性巴氏杆菌成熟黏附蛋白Cpm39,并检测其免疫原性。方法：通过BamHⅠ和SalⅠ双酶切重组载体pMD18-cpm39获得cpm39基因片段,将该基因片段克隆至表达载体pMAL-p2X上,构建表达质粒pMAL-p2X-cpm39,转化至大肠杆菌BL21（DE3）,在IPTG诱导下表达融合蛋白,用禽多杀性巴氏杆菌C48-3株Cp39天然粘附蛋白的免疫血清经Western印迹检测其免疫原性。结果：SDS-PAGE结果显示表达的融合蛋白相对分子质量为78×103,与预期结果相符,而Western印迹结果表明诱导表达的融合蛋白MBP-Cpm39能与Cp39天然黏附蛋白抗体发生特异性反应。结论：构建了表达质粒pMAL-p2X-cpm39,获得了具有免疫原性的重组融合蛋白,为进一步研究禽多杀性巴氏杆菌成熟黏附蛋白的免疫保护功能奠定了基础。     

Expression of human VEGF165 in silkworm (Bombyx mori L.) by using a recombinant baculovirus and its bioactivity assay

Silkworm larva has a lot of advantages as a "biofactory" to produce recombinant protein. A recombinant baculovirus, carrying cDNA encoding the 165 amino-acid long isoform of human vascular endothelial growth factor (VEGF) was successfully constructed for the large-scale production of this protein using silkworms as an in vivo host. The fifth-instar silkworm larvae were inoculated with the recombinant virus. Time-course expression analysis indicated that the expression level was highest at around 80 h post-infection and the recombinant protein was found mainly in the haemolymph. Therefore, the hemolymph was collected from the infected larvae and the recombinant protein was purified by using Nickel affinity chromatography under native condition. The expression level was estimated to be as high as approximately 426 microg per larva. Furthermore, the recombinant protein was characterized and was found biologically active in inducing endothelial cell proliferation in vitro.     

重组人蛋白激酶CK 2α亚基的原核表达、纯化与鉴定

将构建成功的人蛋白激酶 CK2 α亚基 c DNA的重组质粒 ,转化大肠杆菌 BL2 1 ( DE3) ,IPTG诱导后获特异高效表达 ,表达蛋白占菌体总蛋白的 30 % ,但大多数重组蛋白以不溶形式存在 .表达产物依次进行 DE- 5 2、P1 1磷酸纤维素和肝素 - Sepharose柱层析分离 ,最后从 30 9mg可溶性蛋白质中得到 6.1 mg纯化蛋白 . SDS- PAGE显示纯化的蛋白质为一分子量 4 2 k D的单一蛋白带 .Western- blot的结果证明 :纯化的表达产物与抗人 CK2α抗体可发生特异性免疫反应 . CK2α和β亚基等摩尔分子混合可组成有完全活性的全酶 .重组的 CK2全酶的性质和功能与该酶的已知特性一致 .这些结果证明重组蛋白是人蛋白激酶 CK2 α亚基     

表达重组人骨形态发生蛋白-7工程菌的发酵及表达产物的纯化

目的:研究表达重组人骨形态发生蛋白-7工程菌的发酵和表达产物的纯化工艺。方法:利用16L发酵罐发酵培养工程菌,设定了溶氧、搅拌速度、诱导时机、补料和培养基pH值等发酵条件;通过包涵体洗涤、离子交换层析法纯化目的蛋白。结果:工程菌目的蛋白质表达量占菌体总蛋白质的30%以上,纯化后目的蛋白的纯度可达98%。结论:建立了大肠杆菌高效表达人骨形态发生蛋白-7的发酵及纯化工艺。     

Extracellular recombinant protein production from an Escherichia coli lpp deletion mutant

E. coli is one of the most commonly used host strains for recombinant protein production. However, recombinant proteins are usually found intracellularly, in either cytoplasm or periplasmic space. Inadequate secretion to the extracellular environment is one of its limitations. This study addresses the outer membrane barrier for the translocation of recombinant protein directed to the periplasmic space. Specifically, using recombinant maltose binding protein (MalE), xylanase, and cellulase as model proteins, we investigated whether the lpp deletion could render the outer membrane permeable enough to allow extracellular protein production. In each case, significantly higher excretion of recombinant protein was observed with the lpp deletion mutant. Up to 90% of the recombinant xylanase activity and 70% of recombinant cellulase activity were found in the culture medium with the deletion mutant, whereas only 40-50% of the xylanase and cellulase activities were extracellular for the control strain. Despite the weakened outer membrane in the mutant strain, cell lysis did not occur, and increased excretion of periplasmic protein was not due to cell lysis. The lpp deletion is a simple method to generate an E. coli strain to effect significant extracellular protein production. The phenotype of extracellular protein production without cell lysis is useful in many biotechnological applications, such as bioremediation and plant biomass conversion.     

A strategy for generating monoclonal antibodies against recombinant baculovirus-produced proteins: application to the Bcl-2 oncoprotein.

A strategy is described for production of monoclonal antibodies against recombinant proteins that are produced using the baculovirus expression system and that requires no prior purification of the protein of interest. Crude lysates prepared from cultured Sf9 insect cells infected with recombinant or control baculoviruses are absorbed to nitrocellulose filters and used in a dot-immunobinding assay for screening hybridomas. The monoclonal antibody-producing hybridomas are derived by immunization of mice with a synthetic peptide corresponding to a hydrophilic region in the recombinant protein of interest. By using the baculovirus-produced recombinant protein as the screening antigen and by comparing antibody binding to filters containing control Sf9 lysates, hybridomas are identified that produce monoclonal antibodies with specific reactivity for the recombinant protein of interest and that can then subsequently be used to assist in the large-scale purification of the recombinant protein from baculovirus-infected cells. We applied this method to recombinant 26-kDa human Bcl-2 (B-cell lymphoma/leukemia-2), an integral membrane oncoprotein that regulates programmed cell death ("apoptosis") in hematolymphoid cells through unknown mechanisms. Two mouse monoclonal antibodies were produced that specifically bound the recombinant Bcl-2 baculoprotein in both solution and solid-phase assays.