Arresten在烟草中的表达及其生物学活性分析

采用5'端引入His-tag的引物从携带有Arresten基因的质粒pCA中扩增血管生成抑制因子Arresten编码基因,构建其植物表达载体pCAMBIAarr并通过冻融法转化根癌农杆菌LBA4404,获得携带目的基因的重组农杆菌.采用叶盘法以重组农杆菌转化烟草,在50 μg/mL潮霉素B为选择压力下获得再生烟草植株,经过Southern杂交、RT-PCR和Western blotting检测,获得稳定整合有Arresten编码基因的烟草转基因植株.牛血管内皮细胞BCE增殖抑制实验表明,采用镍离子螯合次氨基三乙酸亲和层析法从转基因烟草叶片中分离纯化的重组Arresten蛋白具有明显的抑制牛血管内皮细胞增殖的生物活性.     

重组腺伴随病毒载体表达人白细胞介素12的研究

白细胞介素12(IL-12)具有广谱抗肿瘤、抗感染的作用,是由两个亚单位40kD(p40)和35kD(p35)通过二硫键构成的杂合体,有可能通过分别表达亚单位的方式来表达有功能活性的IL-12.该实验尝试利用重组腺伴随病毒(rAAV)载体进行人IL-12(hIL-12)双亚基共表达,将hIL-12的两个亚基分别克隆到AAV载体质粒pSNAV中,构建成pSNAV-IL12-p35和pSNAV-IL12-p40质粒,经转染、G418筛选后建立了rAAV载体细胞株.采用先前建立的rAAV的生产方法,获得了rAAV-IL12-p35和rAAV-IL12-p40,在体外将两种rAAV共同感染BHK-21细胞,48h后收集细胞培养上清进行免疫学和生物学活性检测.经ELISA检测,产生的hIL-12 p70的含量为10.185pg/ml;在体外促进IFN-γ分泌实验中,加入hIL-12的PBMC分泌的IFN-γ含量为37.2mg/ml.实验结果说明:采用两个AAV载体分别表达亚单位的方法可以表达具有功能活性的hIL-12,为IL-12的基因治疗提供了一条新的途径.     

The Transgenic Rabbit as Model for Human Diseases and as a Source of Biologically Active Recombinant Proteins

Until recently, transgenic rabbits were produced exclusively by pronuclear microinjection which results in additive random insertional transgenesis; however, progress in somatic cell cloning based on nuclear transfer will soon make it possible to produce rabbits with modifications to specific genes by the combination of homologous recombination and subsequent prescreening of nuclear donor cells. Transgenic rabbits have been found to be excellent animal models for inherited and acquired human diseases including hypertrophic cardiomyopathy, perturbed lipoprotein metabolism and atherosclerosis. Transgenic rabbits have also proved to be suitable bioreactors for the production of recombinant protein both on an experimental and a commercial scale. This review summarizes recent research based on the transgenic rabbit model.     

Purification to Homogeneity of Biologically Active Human Interleukin 1

Human interleukin 1 (IL-1) produced by lipopolysaccharide-stimulated monocytes was purified to homogeneity with retention of biological activity. IL-1 was measured by its ability to enhance the proliferative response of thymocytes to phytohemagglutinin. The purification procedure included hydrophobic affinity chromatography on phenyl Sepharose, gel filtration through Ultrogel AcA54 and preparative isoelectric focusing. Both charged species of IL-1, pi 5.1 and 6.8 have a molecular weight of 14,500 as determined by SDS-polyacrylamide gel electrophoresis. The complete purification resulted in a recovery of approximately 0.01% of IL-1 protein and if protection against losses by denaturation and adsorption in the final purification step was provided by bovine serum albumin, approximately 11% of IL-1 activity can be recovered.     

Large-Scale Preparation of Biologically Active Recombinant Chicken Obese Protein (Leptin)

Prokaryotic expression vector pMON3401 encoding full size A(−1) chicken leptin (AF012727) was prepared by PCR of previously described cDNA.Escherichia colicells transformed with this vector overexpressed large amounts of chicken leptin upon induction with nalidixic acid. The expressed protein found in the inclusion bodies was refolded and purified to homogeneity on a Q-Sepharose column, yielding two electrophoretically pure fractions (leptin-1 and leptin-2), eluted from the column by 100 and 125 mM NaCl. Both fractions showed a single band of the expected molecular mass of 16 kDa and were composed of over 95% of monomeric protein. The biological activity of both fractions, resulting from proper renaturation, was further evidenced by their ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin–receptor construct and by lowering the food intake of starved chicken following intravenal or intraperitoneal injections.     

Recombinant Protein Expression Plasmids Optimized for Industrial E. coli Fermentation and Plant Systems Produce Biologically Active Human Insulin-like Growth Factor-1 in Transgenic Rice and Tobacco Plants

Human insulin-like growth factor-1 (hIGF-1) is a growth factor with clinical significance in medicine. The therapeutic potential of recombinant hIGF-1 (rthIGF-1) stems from the fact that hIGF-1 resembles insulin in many aspects of physiology. The expression of hIGF-1 in transgenic tobacco and rice plants using different expression cassettes is reported here. In the present study, two coding sequences were tested, one with the original human sequence, but partially optimized for expression in E. coli and the other with a plant-codon-optimized sequence that was expected to give a higher level of expression in plant systems. Three different hIGF-1 recombinant expression constructs were generated. All expression constructs utilized the maize ubiquitin 1 promoter with or without a signal sequence. Analyses conducted using a hIGF-1 specific ELISA kit showed all transgenic plants produced hIGF-1 and the accumulated hIGF-1 increased from the E. coli codon bias to higher levels when the hIGF-1 coding sequence was codon-optimized to match that of the maize zeamatin protein – the most transcribed gene in maize endosperm suspension cells. Further analyses that compared the functionality of the bacterial signal peptide Lam B in plants showed that this leader peptide led to lower expression levels when compared to transgenic plants that did not contain this sequence. This indicated that this expression construct was functional without removal of the bacterial signal sequence. The maize ubiquitin 1 promoter was found to be more active in rice plants than tobacco plants indicating that in this case, there was a class preference that was biased towards a monocot host. Biological analyses conducted using protein extracts from transgenic plants showed that the rthIGF-1 was effective in stimulating the in vitro growth and proliferation of human SH-SY5Y neuroblastoma cells. This indicated that the plant-produced rthIGF-1 was stable and biologically active. As some plants have been reported to express an endogenous insulin-like protein, we also looked for any effect of the human growth factor in transgenic plants, but no developmental or morphological differences with wild type tobacco or rice plants were detected. Since insulin and hIGF-1 share some overlapping roles, hIGF-1 may become a substitute therapeutic agent in subjects with certain defects in their insulin receptor signaling. Hence, if the full beneficial potential of rthIGF-1 is achieved, it is expected that in the future the demand will likely increase significantly.     

Generation and Phenotypic Analysis of a Transgenic Line of Rabbits Secreting Active Recombinant Human Erythropoietin in the Milk

Production of recombinant human erythropoietin (rhEPO) for therapeutic purposes relies on its expression in selected clones of transfected mammalian cells. Alternatively, this glycoprotein can be produced by targeted secretion into the body fluid of transgenic mammals. Here, we report on the generation of a transgenic rabbits producing rhEPO in the lactating mammary gland. Transgenic individuals are viable, fertile and transmit the rhEPO gene to the offspring. Northern blot data indicated that the expression of the transgene in the mammary gland is controlled by whey acidic protien (WAP) regulatory sequences during the period of lactation. While the hybridization with total RNA revealed the expression only in the lactating mammary gland, the highly sensitive combinatory approach using RT-PCR/hybridization technique detected a minor ectopic expression. The level of rhEPO secretion in the founder female, measured in the period of lactation, varied in the range of 60–178 and 60–162 mIU/ml in the milk and blood plasma, respectively. Biological activity of the milk rhEPO was confirmed by a standard [³H]-thymidine incorporation test. Thus, we describe the model of a rhEPO-transgenic rabbit, valuable for studies of rhEPO glycosylation and function, which can be useful for the development of transgenic approaches designed for the preparation of recombinant proteins by alternative biopharmaceutical production.     

The Preparation of Biologically Active Messenger RNA from Human Postmortem Brain Tissue

Abstract: Messenger RNA (mRNA) was extracted from human postmortem brain tissue by alkaline phenol extraction of polysomes followed by oligo (dT)-cellulose chromatography. The mRNA preparations stimulated protein synthesis in a cell-free system containing wheat germ homogenate. The products of protein synthesis were analyzed by one- and two-dimensional gel electrophoresis. These analyses indicated that numerous polypeptides, including tubulin subunits and actin isomers, were synthesized by the human mRNA. The molecular weight range of polypeptides synthesized by human mRNA fractions from two brain specimens were identical, and analysis by two-dimensional gel electrophoresis indicated qualitatively similar products. The yield of mRNA extracted per gram of human tissue was less than the yield obtained with rat forebrains from animals sacrificed immediately before brain removal and mRNA purification. A decrease in the amount of polysomes isolated from human tissue relative to rat brain tissue was a major factor contributing to the low yield. The molecular weight distribution of polypeptides synthesized by human and rat brain mRNA fractions in wheat germ homogenate was similar; thus, there was no indication for selective breakdown or inactivation of high molecular weight mRNA species in the human tissue. Our studies indicate that it is possible to utilize postmortem tissue for molecular biological investigations of human brain mRNA.     

Biologically Active Oligosaccharides from Pectins of Pisum sativum L. Seedlings Affecting Root Generation

Two physiologically active oligosaccharide fractions were isolated from pectin of Pisum sativum L. cell wall after its partial acid hydrolysis. These fractions displayed stimulating and inhibiting effects on root formation in thin-layer explants. The subsequent separation of these fractions by gel permeation and anion-exchange chromatography resulted in fractions with effective concentrations two orders of magnitude lower than the concentrations of the initial fractions. The resulting oligosaccharides displayed their effect on the earliest stage of the rhizogenesis associated with formation of root primordias. The rhizogenesis-inhibiting fraction suppressed cell division by 30-50%. The stimulating fraction mainly contained fragments of xyloglucan and galactan, and the inhibiting fraction contained fragments of xyloglucan, galactan, and arabinan. The polymerization degrees of the stimulating and of the inhibiting oligosaccharides were 10-11 and 5-6, respectively.     

Cytoprotective Effect of Recombinant Human Erythropoietin Produced in Transgenic Tobacco Plants

Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO^M) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO^P) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO^P bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO^P (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO^M (21%). The cytoprotective effect of the asialo-rhuEPO^P was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.     

Molecular Cloning and Expression of Biologically Active Human Glia Maturation Factor-β

Glia maturation factor-beta, a protein found in the brains of all vertebrates thus far examined, appears to play a role in the differentiation, maintenance, and regeneration of the nervous system. Using oligonucleotide probes based on the sequences of three tryptic peptides derived from bovine glia maturation factor-beta, we screened a human brainstem cDNA library in lambda gt11. A 0.7-kb clone was isolated, sequenced in its entirety, and found to encode a polypeptide of 142 amino acids which contained regions identical to the three bovine peptides. This polypeptide, human recombinant glia maturation factor-beta, has been expressed in Escherichia coli and found to possess structural characteristics and biological activity indistinguishable from those of the native bovine protein.     

Biologically Active Natural 2'-Hydroxychalcones

Russian Journal of Bioorganic Chemistry - The study provides modern data from the literature on the results of research in the field of flavonoids of medicinal plants. Systematization was carried...     

Recombinant Hamster Oviductin Is Biologically Active and Exerts Positive Effects on Sperm Functions and Sperm-Oocyte Binding

Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1) in human embryonic kidney 293 (HEK293) cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1. Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP) of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and 83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/ml of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-HamOVGP1 antibody. The successful production and purification of a biologically active rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function.     

Secretion of Biologically Active Human Granulocyte Colony-Stimulating Factor (G-CSF) in Milk of Transgenic Mice

Two constructs were devised, containing the full-length gene of the human granulocyte colony-stimulating factor (G-CSF) fused with the 5′ and 3′ flanking promoter sequences of bovine alpha-S₁-casein gene (CSN1S1). Both constructs contained a 1518-bp fragment that included exons 18 and 19 and 320 bp of the 3′ flanking region of bovine gene CSN1S1, but differed in size of the 5′ flanking sequences, which were of 721 bp, and exon 1 in construct pGCm1 and 2001 bp and exon 1 and intron 1 in construct pGCm2. With both constructs, transgenic mice were produced. The transgene expression was assessed using RT-PCR and immunochemically from the production of human G-CSF in milk of lactating females. Secretion of human G-CSF into the milk varied in a wide range, from 0.8 μg/ml to over 1 mg/ml, in mice with construct pGCm1 and was low (up to 60 μg/ml) or absent in mice with construct pGCm2. G-CSF glycosylation was incomplete in mice with transgene pGCm1 and complete in mice with pGCm2. G-CSF of transgenic mouse milk was shown to stimulate the formation and growth of granulocyte-containing colonies in human umbilical blood cell culture and be close or identical in physiological activity to the natural human G-CSF. __________ Translated from Genetika, Vol. 41, No. 10, 2005, pp. 1331–1337. Original Russian Text Copyright ? 2005 by Dvoryanchikov, Serova, Andreeva, Dias, Azevedo, Serov.     

Secretion of Human Interleukin-2 in Biologically Active Form by Bacillus brevis Directly into Cultute Medium

We constructed an efficient system for synthesis and secretion of human interleukin-2 (IL-2) by Bacillus brevis. The secretion vector we constructed had strong promoters and contained the region coding for the signal peptide of the gene for B. brevis 47 cell-wall protein, followed directly by the gene encoding mature IL-2. Modification of the signal peptide and use of a protease-deficient mutant of B. brevis HPD31 increased productivity. When the signal peptide was more basic near its amino terminal and more hydrophobic in the middle region, IL-2 production increased 20 fold. Production by the mutant harboring the secretion vector was four fold that of the parent harboring the same plasmid. The yield of IL-2 increased further to 0.12 g/liter, when cultural conditions were made optimal, such by the addition of Tween 40 to the medium. The IL-2 produced by B. brevis had the same biological activity as authentic IL-2. Biologically active human IL-2 was produced efficiently and secreted directly into the medium by B. brevis.     

Recombinant Rhodostomin Substrates Induce Transformation and Active Calcium Oscillation in Human Platelets

Platelet activation has been a focus of numerous studies in normal and abnormal states. Morphological changes and calcium signals found with activated platelets in vitro have been well characterized. However, the rate of cell spreading on substrates and the frequency of calcium oscillation within individual platelets upon activation have not yet been reported. In this study, we first examined the ability of a recombinant fusion protein of rhodostomin (GST-rhodostomin), a snake disintegrin containing an Arg-Gly-Asp (RGD) motif, to activate platelets when GST-rhodostomin served as a substrate. Four aspects of platelet activities induced by immobilized GST-rhodostomin and fibrinogen were analyzed in parallel. Examinations of (1) translocation of P-selectin from intracellular compartments to the plasma membrane, (2) platelet adhesion to and spreading on substrates, (3) platelet contact pattern on substrates, and (4) the degree of phosphorylation of focal adhesion kinase in platelets indicated that GST-rhodostomin was a better substrate for platelet activation than fibrinogen. Analysis of the rate of platelet spreading on GST-rhodostomin was examined by time-lapsed video microscopy. The spreading rate averaged 0.43 micrometer/minute, while cell spreading averaged 0.22 micrometer/minute when platelets were plated on fibrinogen and treated with thrombin. A newly developed method, using time-lapsed microscopy and the Metamorph program, was used to analyze calcium signals within platelets. We found that platelets on GST-rhodostomin evoked calcium oscillation at a frequency of 4.77 spike/cell/minute vs 2.76 spike/cell/minute on fibrinogen. The results of cell spreading and calcium oscillation were consistent with the results of microscopic and biochemical assays. We therefore conclude that the determination of the rate of platelet spreading and the frequency of calcium oscillation within platelets performed in this study provides more quantitative parameters for measuring platelet activities. Our results also suggest that GST-rhodostomin might potentially be used as a probe to dissect the molecular mechanisms underlying the kinetic processes of platelet activation.     

Defective Vaccinia Virus as a Biologically Safe Tool for the Overproduction of Recombinant Human Secretory Proteins

We have described recently the construction of a defective vaccinia virus (VV) lacking the essential D4R open reading frame and have shown furthermore the selection of a complementing cell line providing the essential D4R gene product. The D4R gene belongs to the group of early transcribed vaccinia genes preventing a virus defective in D4R from entering into the intermediate and late phase of replication under noncomplementing conditions. Here we show that this property, which is unique among the group of so called nonreplicating poxviruses, is helpful for the production of (secretable) recombinant human proteins. Recombinant VV based on a D4R-defective parental strain expressing cDNAs coding for the human blood coagulation factors VII and XI produced significantly more recombinant protein than the corresponding recombinants based on wild-type VV. Moreover, the complementing cell line RK-D4R-44.20 was a more effective production cell system for both vD4 and wild-type VV recombinants compared to wild-type RK-13 cells. Surprisingly, recombinant human factor VII was more efficiently produced with the defective vaccinia recombinant even under noncomplementing conditions, suggesting that persistence of the early phase of vaccinia replication in combination with a delayed host shutoff is advantageous for the overproduction of certain recombinant proteins using the VV expression system.     

Endotoxin-free Biologically Active Component of Escherichia coli

The proteinaceous component of gram-negative bacteria, which has been termed “protodyne,” enhances nonspecific host resistance while eliciting a slight pyrogenic response equivalent to 0.2% that of a typical endotoxin. Since this material still contains small amounts of carbohydrate and lipid, it was imperative to establish that its biological activities are not the result of endotoxin contamination. Evidence that the protective activity of protodyne does not result from endotoxin contamination has now been obtained by an evaluation of the Pronase digestion products of this substance. These digestion products were found to be nonpyrogenic and to contain no measurable amount of 2-keto-3-deoxyoctonate, an essential component of bacterial lipopolysaccharides.     

Rice Phytochrome Is Biologically Active in Transgenic Tobacco

To investigate the mechanisms of phytochrome action in vivo, we have overexpressed rice phytochrome in transgenic tobacco plants. A full-length rice phytochrome cDNA was fused to the cauliflower mosaic virus 35S promoter and transferred to tobacco. The progeny of some of the transgenic plants contain large amounts of rice phytochrome mRNA in green leaves. Extracts prepared from overexpressing plants contain twofold to fivefold more spectrophotometrically detectable phytochrome than extracts from control plants. Species-specific, anti-phytochrome monoclonal antibodies were used in immunoblots to discriminate between rice and tobacco phytochrome apoproteins in fractions eluted from a DEAE-Sepharose column. Red minus far-red difference spectra of the partially purified rice phytochrome from the transgenic plants indicate that the rice phytochrome assembles with chromophore and is photoreversible. Analysis of the circadian pattern of Cab mRNA levels in transgenic plants versus controls demonstrates that the overproduction of rice phytochrome extends the duration of the free-running rhythm of Cab gene expression. The rice phytochrome is, therefore, biologically active in the transgenic tobacco plant, which establishes a system for in vivo functional analysis of phytochrome.