Assessment of salivary opiorphin in oral lichen planus

Opiorphin is a pentapeptide, which could be isolated from human fluids and has a decreasing effect on pain. Aim: Since lichen planus is a chronic mucocutaneous disease, which causes pain or burning feeling in the oral mucosa, this study aimed to compare salivary opiorphin levels of oral lichen planus (OLP) patients with healthy subjects. Methods: This case-control study, was performed on 24 patients with OLP lesions and 21 healthy subjects. After collecting unstimulated saliva, opiorphin levels were compared between two groups through statistical analyses. Results: There was not any significant difference between OLP patients and healthy subjects according to salivary opiorphin concentration (p=0.378). Also, in the OLP group, opiorphin concentration was not significantly different between males and females (p=0.601). Analytical analysis could not show any remarkable difference between various severity of OLP lesions regarding to salivary opiorphin levels (p=0.653). Conclusion: In this study, salivary opiorphin levels was not significantly different between patients with OLP and healthy subjects; however, more studies are suggested for better assessment of salivary opiorphin levels in various types of OLP lesions and its correlation with pain severity

Effects of skin care products containing oligopeptides and bio-polysaccharides on sensitive skin barrier function / 中华医学美学美容杂志

Objective:To evaluate the effects and adverse reactions of external skin care products containing oltides and bio-polysaccharides on epidermaligopep barrier function of sensitive skin.Methods:From December 2019 to July 2020, there were 30 sensitive skin volunteers diagnosed and treated in the dermatology clinic of Beijing Tongren Hospital, including 3 males and 27 females, aged 18-57 years, with an average of 34 years, and the course of disease was 1-10 years, with an average of 5.75 years. They were treated once with products containing oligopeptides and biopolysaccharides on the day of enrollment. Before treatment, 1 week and 4 weeks after treatment, we observed and evaluated through VISIA analysis; skin physiological index measurement, subjective and objective improvement assessment, and product safety were evaluated through questionnaire surveys.Results:The VISIA data showed that the red zone was significantly lower than the baseline, and the data at the 4th week and before treatment were significantly improved ( P<0.05). On skin physiology, the test showed that after treatment, the difference between two follow-up visits and the water content before was statistically significant ( P<0.05). TEWL value after 4 weeks of treatment was significantly improved as compared with the baseline ( P<0.05). During the entire study process, no adverse reactions related to the product occurred. Conclusions:This skin care product containing oligopeptides and biopolysaccharides can increase the water content of the sensitive skin, reduce the water loss through the skin, and improve the skin barrier function. Meanwhile, no server adverse reaction is detected through the whole experiment.

Antioxidant function of marine fish oligopeptide vitamin C solid beverage in healthy people / 中华健康管理学杂志

Objective:To evaluated the antioxidant function of marine fish oligopeptide vitamin C solid beverage in healthy people.Methods:From June 1st to December 31st, 2017, 110 volunteers aged 18 to 65 years in good health were recruited by Zhejiang Provincial Center for Disease Control and Prevention, and were randomly divided into control group and test group (55 cases in each group). The test group took the marine fish oligopeptide vitamin C solid beverage (the main components were marine fish oligopeptide powder, vitamin C, acai fruit powder, blueberry fruit powder, pomegranate fruit powder), and the control group took the placebo with the same appearance, package and taste as the solid beverage (the main components were maltodextrin, acai fruit powder, blueberry fruit powder, pomegranate fruit powder). The test samples were both packaged in 5 g/bag and were taken 1 bag daily for 4 months. During the trial, both groups maintained their daily life and dietary habits, and there were no other interventions except for the test samples. During the intervention, 3 cases dropped out, and finally, 53 cases were included in the control group and 54 cases in the test group. Before and after the test, the volunteers were examined for general physical conditions, routine blood, urine and stool tests, and biochemical indicators. At the same time, the levels of blood malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were measured, and the differences of various indicators between the two groups before and after the test were compared by t-test. Results:There was no significant difference in baseline data between the two groups (all P>0.05). After the test, the MDA level in the test group was significantly lower than that before the test [(4.06±1.09) vs (4.73±0.99) μmol/L] ( t=15.160, P<0.001), SOD level was significantly higher than that before the test [(20 987±2 593) vs (18 564±2 194) U/gHb] ( t=-4 338.337, P<0.05), there was no significant change in GSH-Px level before and after the test ( P>0.05). There was no significant differences in the levels of MDA, SOD and GSH-Px in the control group before and after the test (all P>0.05). After trial feeding, the MDA level in the test group was significantly lower than that in the control group [(4.06±1.09) vs (4.63±0.91)] μmol/L] ( t=31.220, P<0.001), SOD level was higher than that in the control group [(20 987±2 593) vs (19 042±2 100) U/gHb] ( t=-3 124.231, P<0.05), there was no significant difference in GSH-Px level between the two groups ( P>0.05). All the test indexes in the two groups were in the normal range before and after the test; there were no abnormal changes in chest radiography, electrocardiogram and abdominal B-ultrasonography; during the trial feeding, no allergy or adverse reaction was found, and no abnormal change of subjective feeling and eating condition was found. Conclusion:Marine fish oligopeptide vitamin C solid beverage can significantly reduce the level of MDA, improve the level of SOD, it has antioxidant function and good safety.

IgY antialérgenos específicos del grupo 1 de ácaros del polvo doméstico inducidos por oligopéptidos sintéticos no glicosilados / Specific IgY anti-group 1 dust mite allergens induced by unglycosylated synthetic oligopeptides

Resumen Introducción. La obtención de anticuerpos específicos capaces de detectar alérgenos del grupo 1 de ácaros del polvo doméstico representa una estrategia potencial de salud pública para reducir la exposición y la sintomatología clínica asociada con el asma y la rinitis alérgica. Objetivo. Producir y purificar anticuerpos aviares antialérgenos específicos del grupo 1 de los ácaros Dermatophagoides sp.y Blomia tropicalis utilizando la tecnología IgY. Materiales y métodos. Se diseñaron y sintetizaron oligopéptidos que evidenciaran epítopes inmunogénicos de los alérgenos Der p1, Der f1 y Blo t1 empleados posteriormente para producir anticuerpos IgY policlonales en gallinas Hy Line Brown. Las IgY presentes en las yemas de los huevos se purificaron mediante cromatografía tiofílica. Su inmunorreactividad y especificidad se determinaron mediante un inmunoensayo ELISA indirecto y Dot Blot. Resultados. Se obtuvo una reactividad elevada de las IgY contra epítopes de alérgenos presentes en extractos de cuerpo entero de D. farinae, D. pteronyssinus y B. tropicalis. Los niveles más altos de IgY se produjeron entre los días 32 y 40 de inmunización. Los anticuerpos mostraron mayor inmunorreactividad y especificidad en el reconocimiento de proteínas de D. farinae, con un límite de detección mayor de 0,03 µg de proteína total delcaroajo las condiciones experimentales analizadas. Las IgY purificadas no mostraron reactividad significativa frente al extracto de Periplaneta americana. Conclusión. La tecnología IgY permitió la producción de anticuerpos específicos contra alérgenos del grupo 1 de los ácaros del polvo al utilizar oligopéptidos sintéticos no glicosilados. Hasta donde se sabe, esta es la primera vez que se usan estos reactivos inmunológicos para la detección de ácaros de importancia médica.

Abstract Introduction: The use of specific antibodies capable of detecting allergens of the group 1 of house dust mites represents a potential strategy to reduce exposure and clinical symptomatology associated with asthma and allergic rhinitis. Objective: To produce and purify chicken antibodies specific for the dust mites Dermatophagoides sp. and B. tropicalis using the IgY technology. Materials and methods: We designed and synthesized oligopeptides showing immunogenic epitopes of Der p1, Der f1, and Blo t1. These were used to produce IgY antibodies in Hy Line Brown chickens. IgY were extracted from egg yolk using thiophilic chromatography. The immunogenicity and specificity were assayed by indirect ELISA and Dot Blot. Results: We obtained high reactivity of IgY antibodies against epitopes of allergens present in whole body mites extracts of D. farinae, D. pteronyssinus, and B. tropicalis. The highest IgY levels were registered between days 32 and 40 after immunization. The antibodies showed high immunoreactivity and specificity towards D. farinae proteins with detection limits above 0.03 µg of mite proteins under the experimental conditions used. Purified IgY did not show significant reactivity when binding to Periplaneta americana extract. Conclusion: The IgY technology allowed the production of specific antibodies against house dust mites group 1 allergens using non-glycosylated synthetic peptides. To our knowledge, this is the first time that this immunochemicals are used in the detection of mites of medical relevance.

Pharmacological mechanism analysis of oligopeptide from Pinctada fucata based on in silico proteolysis and protein interaction network / 中国中药杂志

Pinctada fucata oligopeptide is one of key pharmaceutical effective constituents of P. fucata. It is significant to analyze its pharmacological effect and mechanism. This study aims to discover the potential oligopeptides from P. fucata and analyze the mechanism of P. fucata oligopeptide based on in silico technologies and protein interaction network(PIN). First, main protein sequences of P. fucata were collected, and oligopeptides were obtained using in silico gastrointestinal tract proteolysis. Then, key potential targets of P. fucata oligopeptides were obtained through pharmacophore screening. The protein-protein interaction(PPI) of targets was achieved and implemented to construct PIN and analyze the mechanism of P. fucata oligopeptides. P. fucata oligopeptide database was constructed based on in silico technologies, including 458 oligopeptides. Twelve modules were identified from PIN by a graph theoretic clustering algorithm Molecular Complex Detection(MCODE) and analyzed by Gene ontology(GO) enrichment. The results indicated that P. fucata oligopeptides have an effect in treating neurological diseases, such as Alzheimer's disease. In silico proteolysis could be used to analyze the protein sequences of traditional Chinese medicine(TCM). According to the combination of in silico proteolysis and PIN, the biological activity of oligopeptides could be interpreted rapidly based on the known TCM protein sequence. The study provides the methodology basis for rapidly and efficiently implementing the mechanism analysis of TCM oligopeptides.

Prediction of ETA oligopeptides antagonists from Glycine max based on in silico proteolysis / 中国中药杂志

Oligopeptides are one of the the key pharmaceutical effective constituents of traditional Chinese medicine(TCM). Systematic study on composition and efficacy of TCM oligopeptides is essential for the analysis of material basis and mechanism of TCM. In this study, the potential anti-hypertensive oligopeptides from Glycine max and their endothelin receptor A (ETA) antagonistic activity were discovered and predicted based on in silico technologies.Main protein sequences of G. max were collected and oligopeptides were obtained using in silico gastrointestinal tract proteolysis. Then, the pharmacophore of ETA antagonistic peptides was constructed and included one hydrophobic feature, one ionizable negative feature, one ring aromatic feature and five excluded volumes. Meanwhile, three-dimensional structure of ETA was developed by homology modeling methods for further docking studies. According to docking analysis and consensus score, the key amino acid of GLN165 was identified for ETA antagonistic activity. And 27 oligopeptides from G. max were predicted as the potential ETA antagonists by pharmacophore and docking studies.In silico proteolysis could be used to analyze the protein sequences from TCM. According to combination of in silico proteolysis and molecular simulation, the biological activities of oligopeptides could be predicted rapidly based on the known TCM protein sequence. It might provide the methodology basis for rapidly and efficiently implementing the mechanism analysis of TCM oligopeptides.

Oligopeptides in plant medicines cited in Chinese Pharmacopoeia / 中国中药杂志

In total, 23 plant plant medicined containing oligopeptides were cited in Chinese Pharmacopoeia (1 part) of 2015 version including Rubia cordifolia, Linum usitatissimum, Aster tataricus, Psammosilene tunicoides, Pseudostellaria heterophylla, Stellaria dichotoma, Vaccaria segetalis, Dianthus superbus, Celosia argentea, Lycii Cortex, Citrus medica, C. aurantium, Panax ginseng, Parmx notoginseng, Schisandra chinensis, Sparganium stoloniferum, Euryale ferox, Ophiopogon japonicas, Pinellia ternate, Achyranthes bidentata, Physalis alkekengi, Polygonatum odoratum, and Leonuri Fructus. There were 187 oligopeptides in plant medicines above as reported. Oligopeptides consisted mainly of linear peptides and cyclic peptides. The linear peptides included dipeptides, tripeptides and pentapeptides, and cyclic peptides included cyclic, bicyclic and tricyclic peptides. The number of residues of single cyclic peptides ranged from two to twelve. Bicyclic peptides were isolated mainly from R. cordifolia and C. argentea. Modern pharmacological study showed that oligopeptides had many pharmacological effects, including antitumor, anticoagulant, antibacterial, immune suppression and so on.

Improvement of osteogenic potential of biphasic calcium phosphate bone substitute coated with synthetic cell binding peptide sequences

PURPOSE: The aim of this study was to evaluate the improvement of osteogenic potential of biphasic calcium phosphate (BCP) bone substitute coated with synthetic cell-binding peptide sequences in a standardized rabbit sinus model. METHODS: Standardized 6-mm diameter defects were created bilaterally on the maxillary sinus of ten male New Zealand white rabbits, receiving BCP bone substitute coated with synthetic cell binding peptide sequences on one side (experimental group) and BCP bone substitute without coating (control group) on the other side. Histologic and histomorphometric analysis of bone formation was carried out after a healing period of 4 or 8 weeks. RESULTS: Histological analysis revealed signs of new bone formation in both experimental groups (4- and 8-week healing groups) with a statistically significant increase in bone formation in the 4-week healing group compared to the control group. However, no statistically significant difference in bone formation was found between the 8-week healing group and the control group. CONCLUSIONS: This study found that BCP bone substitute coated with synthetic cell-binding peptide sequences enhanced osteoinductive potential in a standardized rabbit sinus model and its effectiveness was greater in the 4-week healing group than in the 8-week healing group.

The biological effects of fibrin-binding synthetic oligopeptides derived from fibronectin on osteoblast-like cells

PURPOSE: The aim of this study was to investigate the effects of synthetic fibronectin (FN) fragments, including fibrin binding sites from amino-terminal FN fragments containing type I repeats 1 to 5, on osteoblast-like cell activity. METHODS: Oligopeptides ranging from 9 to 20 amino acids, designated FF1, FF3, and FF5, were synthesized by a solid-phase peptide synthesizing system, and we investigated the effects of these peptides on cell attachment and extent of mineralization using confocal microscopy, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and Alizarin red S staining. RESULTS: FF3 and FF5 peptides increased the number of attached human osteoblastic cells, and FF3 administration led to prominent cell spreading. Mineralization was increased in FF3 and FF5 compared to FF1 and the untreated control. CONCLUSIONS: Taken together, it can be concluded that the fibrin-binding oligopeptides FF3 and FF5 enhanced cell attachment and mineralization on osteoblast-like cells. These results indicate that FF3 and FF5 have the potential to increase osteoblast-like cell activity. Performing an in vivo study may provide further possibilities for surface modification of biomimetic peptides to enhance osteogenesis, thus improving the regeneration of destroyed alveolar bone.

Effects of fibrin-binding oligopeptide on osteopromotion in rabbit calvarial defects

PURPOSE: Fibronectin (FN) has been shown to stimulate bone regeneration in animal models. The aim of this study was to evaluate the capacity of bovine bone mineral coated with synthetic oligopeptides to enhance bone regeneration in rabbit calvarial defects. METHODS: Oligopeptides including fibrin-binding sequences of FN repeats were synthesized on the basis of primary and tertiary human plasma FN structures. Peptide coated and uncoated bone minerals were implanted into 10 mm calvarial defects in New Zealand white rabbits, and the animals were sacrificed at 4 or 8 weeks after surgery. After specimens were prepared, histologic examination and histomorphometric analysis were performed. RESULTS: At 4 weeks after surgery, the uncoated groups showed a limited amount of osteoid formation at the periphery of the defect and the oligopeptide coated groups showed more osteoid formation and new bone formation in the center of the defect as well as at the periphery. At 8 weeks, both sites showed increased new bone formation. However, the difference between the two sites had reduced. CONCLUSIONS: Fibrin-binding synthetic oligopeptide derived from FN on deproteinized bovine bone enhanced new bone formation in rabbit calvarial defects at the early healing stage. This result suggests that these oligopeptides can be beneficial in reconstructing oral and maxillofacial deformities or in regenerating osseous bone defects.

Expression and purification of high purity soluble chimeric protein VEGI+ / 第二军医大学学报

Objective: To prepare a novel vascular endothelial growth inhibitor-soluble chimeric protein VEGI+, so as to lay a basis for studying its biological activity. Methods: Chimeric molecule VEGI+ was constructed by grafting oligopeptide CTTH-WGFTLC to extracellular region of VEGI (VEGI23-174). Before ligation into pET30a(+) expression vector, PCR product of the recombinant gene was cloned into pGEM-T vector and verified by restriction enzyme digestion and DNA sequencing, then pET30a-VEGI was used to transfect BL21 (modified E. coli strain). The chimeric protein was purified by metal affinity chromatography. Western blotting and coomassie blue staining were used for protein identification. Results: The chimeric molecule VEGI+ was confirmed by restriction enzyme digestion and DNA sequencing. The constructed pET30a-VEGI was confirmed by enzymatic digestion. The expression was mainly in the form of inclusion body. SDS-PAGE electrophoresis and Western blotting revealed a chimeric protein about 23 000, with a purity of about 90%. Conclusion: We have successfully constructed the recombinant plasmid pET30a-VEGI+ and expressed it in E. coli. And we have obtained high purity of soluble chimeric protein VEGI+ through affinity chromatography.

Correlation between neuronal injury and the expression of caspase-3 and caspase-activated deoxyribonuclease and effect of cnspase-3 particular inhibitor after focal cerebral ischemia-reperfusion in rats / 中华神经科杂志

Objective To investigate the correlation between neuronal injury and the expression of caspase-3 and caspase-activated deoxyribonuclease (CAD) after focal cerebral ischemia-reperfusion in rats, also to study the effect of caspase-3 particular inhibitor. Methods The focal cerebral ischemia model (occluding middle cerebral artery of the rats) was made by using modified inserting thread method and reperfusion after embolizing for one hour. Using HE staining, TUNEL staining and microscopy to observe the morphological changes of ischemic neurons at six different time points including 6,12,24,48 and 72 h, using immunohistochemistry to observe the changes of caspase-3 and CAD protein in two groups (model group and interfere group). Results There was no significant difference between the two groups using HE staining and microscopy. While there was difference of TUNEL staining positive cells in all time points, except 6 h time point; Both the two groups reached the expression peak of caspase-3 in 24 h, and the number was 2. 360± 0. 318 and 0. 804 ± 0. 206 respectively(t' = 10. 039, P < 0. 01), there was statistical significance from 12 h to 48 h between the two groups ; The expression peak time of CAD protein in two groups was 48 h, and the number was 3.061 ± 0. 567 and 0. 812 ± 0. 240 respectively (t' = 8. 960, P < 0. 01), there was statistical significance from 12 to 72 h between two groups. Conclusions Caspase-3-CAD-DNA degradation is one important way of neuronal injury in cerebral ischemia-reperfusion of rats, caspase-3 inhibitor can protect neuron in a certain degree.

Ação da pancreatina na obtenção de hidrolisados protéicos de soro de leite com elevado teor de oligopeptídeos / Action of pancreatin for obtaining whey protein hydrolysates with high oligopeptide contents

Vários hidrolisados enzimáticos de soro de leite foram preparados visando a elevação do teor de oligopeptídeos e a redução de custos para produção em larga escala. Para isso, empregou-se a pancreatina e alguns parâmetros hidrolíticos como o tempo de reação (5, 10 e 15h), a relação E:S (1:100, 2:100 e 4:100) e a concentração do substrato (10 e 15 por cento) foram testados. Os hidrolisados foram fracionados por cromatografia liquida de alta eficiência de exclusão molecular (SEHPLC) e, para a quantificação dos componentes das frações cromatográficas, empregou-se o método rápido da Área Corrigida da Fração. Para os parâmetros estudados, observou-se efeito benéfico sobre o perfil peptídico, sendo que o melhor resultado, associado, principalmente, ao maior teor de oligopeptídeos (49 por cento) e ao menor conteúdo de aminoácidos livres (22 por cento), foi obtido quando se empregou a concentração do substrato a 10 por cento, a relação E:S de 4:100 e o tempo de reação de 10h.

Several enzymatic hydrolysates of whey were prepared with the objective of increasing the oligopeptide content and reducing the cost of large scale production. Pancreatin was used and some hydrolytic parameters such as reaction time (5, 10 and 15h), enzyme:substrate ratio (E:S) (1:100, 2:100 and 4:100) and substrate concentration (10 and 15 percent) were tested. The hydrolysates were fractionated by size-exclusion-HPLC and the rapid corrected fraction area method was used for quantifying the chromatographic fractions. The beneficial effect on the peptide profile was observed in several cases, and it was mainly associated with higher oligopeptide (49 percent) and lower amino acid (22 percent) contents, which were obtained for a substrate concentration of 10 percent, E:S ratio of 4:100 and reaction time of 10h.

Biomimetics of crystal texture in dental enamel prism by self-assembly oligopeptide / 北京大学学报(医学版)

Objective: To evaluate the possibility of self-assembly oligopeptide(T2) for dental enamel biomimetics, especially for the prism’s crystal texture since it could prompt calcium phosphate precipitated in gel carrier. Methods:SEM (Scaning electron microscope) and TEM (Transmission electron microscope) were used to observe the morphologic presentation and ED(Electron diffraction) to crystal texture comparing with the human molar enamel powder. Results: (a) Flake-like and needle-like octacalcium phosphate precipitated in the gel carrier with self-assemble oligopeptide(T2). They transformed into rod-like hydroxyapatite crystals gradually in the following 2-4 weeks. (b) The rod-like hydroxyapatite may arrange or grow into bundles which are similar to the human enamel prisms in both appearance and size. (c) The rod-like hydroxyapatite showed polycrystal while the enamel prisms showed monocrystal under examination of ED. Conclusion:The self-assemble oligopeptide(T2) could regulate the speed of nucleation and crystallization of hydroxyapatite in morphology and crystalline size. Thus, the self-assembly oligopeptide and the gel carrier mineralization system could be primarily applied in biomimetic use for the crystallization of hydroxyaptite in dental prism in vitro.

Trp-Lys-Tyr-Met-Val-Met stimulates phagocytosis via phospho-lipase D-dependent signaling in mouse dendritic cells

Dendritic cells (DCs) play a key role in activating the immune response against invading pathogens as well as dying cells or tumors. Although the immune response can be initiated by the phagocytic activity by DCs, the molecular mechanism involved in this process has not been fully investigated. Trp-Lys-Tyr-Met-Val-Met-NH2 (WKYMVM) stimulates the activation of phospholipase D (PLD) via Ca2+ increase and protein kinase C activation in mouse DC cell line, DC2.4. WKYMVM stimulates the phagocytic activity, which is inhibited in the presence of N-butanol but not t-butanol in DC2.4 cells. Furthermore, the addition of phosphatidic acid, an enzymatic product of PLD activity, enhanced the phagocytic activity in DC2.4 cells. Since at least two of formyl peptide receptor (FPR) family (FPR1 and FPR2) are expressed in DC2.4 as well as in mouse bone marrow-derived dendritic cells, this study suggests that the activation of FPR family by WKYMVM stimulates the PLD activity resulting in phagocytic activity in DC2.4 cells.

A novel peptides, like nerve growth factor, inducing pheochromocytoma PC12 cell differentiation / 吉林大学学报(医学版)

Objective: To isolate the key domain of a novel polypeptide fragment from NGN-β that functions like intact NGF molecule.Methods: NGF-β had been treated with cyanogen bromide and trypsin skillfully. The peptide fragment with the activity inducing PC12 pheochromocytoma cells differentiation was isolated and purified by Sephadex G-50 gel filtration chromatography , DE-52 celluloseion exchange chromatography and C-18 reversed-phase column HPLC after NGF-β cleavaged by CNBr at 9th met then by trypsin at Arg or Lyscleavaged by CNBr at 9th met then by trypsin at Arg or Lys. Amino acid sequencing of this novel peptide fragment was performed by Automatic Amino Acid Analyser and Amino Acid Sequencer. Results: The functional fragment from cleavaged NGF might induce differentiation of PC12 cells. The fragment was consisting of two linear polypeptides . One of them was 16 peptide, GEFSVCDSVSVWVGDK , and other was 14 peptide, HWNSYCTTTHTFVK, linked by a disulphide bridge corresponding to residues 10～25 and 75～88, respectively, of the amino acid sequence of nerve growth factor, the result of biological activity assay in PC12 cells showed that the optimum concentration of this peptide were 0.001～0.1 μg*L-1. Conclusion: A novel peptide inducing differentiation of PC12 cell line of pheochromocytoma cells was obtained in the study. It′s isolation and purification successfully will underlie synthesis or expression of hyperactive neurotrophic small molecular substance although the relationship between the configuration and functions is not clearly.

Expression and purification of high purity soluble chimeric protein VEGI~+ / 第二军医大学学报

Objective:To prepare a novel vascular endothelial growth inhibitor-soluble chimeric protein VEGI~+,so as to lay a basis for studying its biological activity.Methods:Chimeric molecule VEGI~+ was constructed by grafting oligopeptide CTTH- WGFTLC to extraeellular region of VEGI(VEGI_(23-174)).Before ligation into pET30a(+)expression vector,PCR product of the recombinant gene was cloned into pGEM-T vector and verified by restriction enzyme digestion and DNA sequencing,then pET30a-VEGI was used to transfect BL21(modified E.coli strain).The chimeric protein was purified by metal affinity chro- matography.Western blotting and coomassie blue staining were used for protein identification.Results:The chimeric molecule VEGI~+ was confirmed by restriction enzyme digestion and DNA sequencing.The constructed pET30a-VEGI was confirmed by enzymatic digestion.The expression was mainly in the form of inclusion body.SDS-PAGE electrophoresis and Western blotting revealed a chimeric protein about 23000,with a purity of about 90%.Conclusion:We have successfully constructed the recom- binant plasmid pET30a-VEGI~+ and expressed it in E.coll.And we have obtained high purity of soluble chimeric protein VEGI~+ through affinity chromatography.