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1.
Acta Pharmaceutica Sinica B ; (6): 942-954, 2023.
Article in English | WPRIM | ID: wpr-971761

ABSTRACT

The extraordinary advantages associated with mRNA vaccines, including their high efficiency, relatively low severity of side effects, and ease of manufacture, have enabled them to be a promising immunotherapy approach against various infectious diseases and cancers. Nevertheless, most mRNA delivery carriers have many disadvantages, such as high toxicity, poor biocompatibility, and low efficiency in vivo, which have hindered the widespread use of mRNA vaccines. To further characterize and solve these problems and develop a new type of safe and efficient mRNA delivery carrier, a negatively charged SA@DOTAP-mRNA nanovaccine was prepared in this study by coating DOTAP-mRNA with the natural anionic polymer sodium alginate (SA). Intriguingly, the transfection efficiency of SA@DOTAP-mRNA was significantly higher than that of DOTAP-mRNA, which was not due to the increase in cellular uptake but was associated with changes in the endocytosis pathway and the strong lysosome escape ability of SA@DOTAP-mRNA. In addition, we found that SA significantly increased the expression of LUC-mRNA in mice and achieved certain spleen targeting. Finally, we confirmed that SA@DOTAP-mRNA had a stronger antigen-presenting ability in E. G7-OVA tumor-bearing mice, dramatically inducing the proliferation of OVA-specific CLTs and ameliorating the antitumor effect. Therefore, we firmly believe that the coating strategy applied to cationic liposome/mRNA complexes is of potential research value in the field of mRNA delivery and has promising clinical application prospects.

2.
Frontiers of Medicine ; (4): 275-289, 2023.
Article in English | WPRIM | ID: wpr-982567

ABSTRACT

The abnormal activation of HER family kinase activity is closely related to the development of human malignancies. In this study, we used HER kinases as targets for the treatment of nasopharyngeal carcinoma (NPC) and explored the anti-tumor effects of the novel pan-HER inhibitor HM781-36B, alone or in combination with cisplatin. We found that HER family proteins were positively expressed in tumor tissues of some NPC patients, and the high levels of those proteins were significantly related to poor prognosis. HM781-36B inhibited NPC in vitro and in vivo. HM781-36B exerted synergistic effects with cisplatin on inhibiting proliferation and promoting apoptosis of NPC cells. In NPC xenograft models in nude mice, HM781-36B and cisplatin synergistically inhibited tumor growth. Downregulating the activity of HER family proteins and their downstream signaling pathways and regulating tumor microenvironment may explain the synergistic anti-tumor effects of HM781-36B and cisplatin. In conclusion, our study provides evidence for HER family proteins as prognostic biomarkers and potential therapeutic targets for NPC. The pan-HER inhibitor HM781-36B alone or in combination with cisplatin represents promising therapeutic effects for the treatment of NPC patients, which provides a new idea for the comprehensive treatment of NPC.


Subject(s)
Humans , Animals , Mice , Cisplatin/therapeutic use , Antineoplastic Agents/therapeutic use , Nasopharyngeal Carcinoma/drug therapy , Mice, Nude , Nasopharyngeal Neoplasms/pathology , Tumor Microenvironment
3.
Acta Pharmaceutica Sinica B ; (6): 3935-3949, 2021.
Article in English | WPRIM | ID: wpr-922451

ABSTRACT

A significant proportion of non-small cell lung cancer (NSCLC) patients experience accumulating chemotherapy-related adverse events, motivating the design of chemosensitizating strategies. The main cytotoxic damage induced by chemotherapeutic agents is DNA double-strand breaks (DSB). It is thus conceivable that DNA-dependent protein kinase (DNA-PK) inhibitors which attenuate DNA repair would enhance the anti-tumor effect of chemotherapy. The present study aims to systematically evaluate the efficacy and safety of a novel DNA-PK inhibitor M3814 in synergy with chemotherapies on NSCLC. We identified increased expression of DNA-PK in human NSCLC tissues which was associated with poor prognosis. M3814 potentiated the anti-tumor effect of paclitaxel and etoposide in A549, H460 and H1703 NSCLC cell lines. In the four combinations based on two NSCLC xenograft models and two chemotherapy, we also observed tumor regression at tolerated doses

4.
Protein & Cell ; (12): 11-16, 2016.
Article in English | WPRIM | ID: wpr-757172

ABSTRACT

Mitochondrion is known as the energy factory of the cell, which is also a unique mammalian organelle and considered to be evolved from aerobic prokaryotes more than a billion years ago. Mitochondrial DNA, similar to that of its bacterial ancestor’s, consists of a circular loop and contains significant number of unmethylated DNA as CpG islands. The innate immune system plays an important role in the mammalian immune response. Recent research has demonstrated that mitochondrial DNA (mtDNA) activates several innate immune pathways involving TLR9, NLRP3 and STING signaling, which contributes to the signaling platforms and results in effector responses. In addition to facilitating antibacterial immunity and regulating antiviral signaling, mounting evidence suggests that mtDNA contributes to inflammatory diseases following cellular damage and stress. Therefore, in addition to its well-appreciated roles in cellular metabolism and energy production,mtDNA appears to function as a key member in the innate immune system. Here, we highlight the emerging roles of mtDNA in innate immunity.


Subject(s)
Animals , Humans , DNA, Mitochondrial , Genetics , Immunity, Innate , Allergy and Immunology , Mitochondria , Genetics , Allergy and Immunology , Signal Transduction
5.
Acta Laboratorium Animalis Scientia Sinica ; (6): 88-92, 2014.
Article in Chinese | WPRIM | ID: wpr-452655

ABSTRACT

Objective To reduce the animal component contamination for human embryonic stem cells ( hESCs ) and to simplify hESCs culture process , we develop a new coating substrate which can support the hESCs growth without dif -ferentiation, and is easy to store and use. Methods Mouse embryonic fibroblasts(MEF)were fixed on the surface of plate by methanol.hESCs were cultured on this new substrate and were passaged every 5 to 6 days.After 10 passages, we checked the cell morphology , alkaline phosphatase expression , embryonic specific markers and the differentiation ability in vitro.Results After 10 passages , the hESCs grew well on this new substrate and maintained the typical hESCs morpholo -gy.Alkaline phosphatase staining was positive .Immunofluorescence staining showed that the expressions of Oct 4, SSEA4, Tra-1-60 were positive .The cells formed embryoid body in vitro .Conclusions This methanol-fixed MEF substrate can support the growth of undifferentiated hESCs .The coating material can be produced in large scale and stored for a long time.It provides a new and relatively easy way to amplify hESCs .

6.
Acta Pharmaceutica Sinica ; (12): 322-8, 2014.
Article in Chinese | WPRIM | ID: wpr-448762

ABSTRACT

In order to obtain the lead compound for treatment of rheumatoid arthritis (RA), in this study, therapeutic efficacy of three bispecific antibodies (BsAB-1, BsAB-2 and BsAB-3) against both hIL-1beta and hIL-17 were compared on CIA model mice. First, by ELISA method we compared the binding capacity of the three bispecific antibodies to the two antigens. The results showed that all three antibodies could simultaneously bind both antigens, among these antibodies, BsAB-1 was superior over BsAB-2 and BsAB-3. CIA model was established with chicken type II collagen (CII) and developed RA-like symptoms such as ankle swelling, skin tight, hind foot skin hyperemia. The CIA mice were treated with three antibodies once every two days for total of 29 days. Compared with the CIA model mice, the RA-like symptoms of the antibody treated-mice significantly relieved, while the BsAB-1 treated-mice were almost recovered. CII antibody level in the serum and cytokines (IL-2, IL-1beta, IL-17A and TNF-alpha) expression in the spleen were examined. Compared with the CIA model mice, all three antibodies could significantly reduce CII antibody and cytokine expression levels. BsAB-1 antibody was more potent than BsAB-2 and BsAB-3. In summary, BsAB-1 is superior over BsAB-2 and BsAB-3 in amelioration of RA symptoms and regulation of CII antibody production and pro-inflammatory cytokine expression, therefore, BsAB-1 can be chosen as a lead compound for further development of drug candidate for treatment of RA.

7.
Chinese Journal of Biotechnology ; (12): 955-964, 2013.
Article in Chinese | WPRIM | ID: wpr-233184

ABSTRACT

To enhance the penetration of P53 into tumor cells by fusion it with the cell penetrating peptide 9R. The fusion gene of 9R-p53 was cloned into the expression vector. The fusion protein, CPPs-P53, was expressed and purified. We detected the rate of cell growth inhibition and apoptosis by MTT and Annexin-V-FITC/PI double stained method respectively for measuring its effect on tumor cells. CPPs-P53 and P53 were successfully expressed and purified, the purity of both proteins reached up to 90%. MTT assay showed that the cell growth inhibition by CPPs-P53 was more efficient than P53, and the rate of cell growth inhibition is dose-dependent. The apoptosis experiment showed that P53 could induce apoptosis of tumor cells. Compared with the P53, CPPs-P53 had a more significant effect in inducing cell apoptosis (**P < 0.01). The CPPs-P53 shows more significant effects than P53 in inhibiting cell growth and inducing apoptosis on tumor cells.


Subject(s)
Humans , Apoptosis , Cell Line, Tumor , Cell-Penetrating Peptides , Pharmacology , Tumor Suppressor Protein p53 , Pharmacology
8.
Chinese Journal of Cancer ; (12): 507-518, 2012.
Article in English | WPRIM | ID: wpr-295878

ABSTRACT

The growing demand for new therapeutic strategies in the medical and pharmaceutic fields has resulted in a pressing need for novel druggable targets. Paradoxically, however, the targets of certain drugs that are already widely used in clinical practice have largely not been annotated. Because the pharmacologic effects of a drug can only be appreciated when its interactions with cellular components are clearly delineated, an integrated deconvolution of drug-target interactions for each drug is necessary. The emerging field of chemical proteomics represents a powerful mass spectrometry (MS)-based affinity chromatography approach for identifying proteome-wide small molecule-protein interactions and mapping these interactions to signaling and metabolic pathways. This technique could comprehensively characterize drug targets, profile the toxicity of known drugs, and identify possible off-target activities. With the use of this technique, candidate drug molecules could be optimized, and predictable side effects might consequently be avoided. Herein, we provide a holistic overview of the major chemical proteomic approaches and highlight recent advances in this area as well as its potential applications in drug discovery.


Subject(s)
Humans , Chromatography, Affinity , Drug Delivery Systems , Methods , Drug Design , Drug Discovery , Methods , Mass Spectrometry , Proteome , Chemistry , Proteomics , Methods , Small Molecule Libraries , Chemistry
9.
Experimental & Molecular Medicine ; : 146-152, 2011.
Article in English | WPRIM | ID: wpr-171914

ABSTRACT

Our previous report has demonstrated that 5-formylhonokiol (FH), a derivative of honokiol (HK), exerts more potent anti-proliferative activities than honokiol in several tumor cell lines. In present study, we first explored the antiangiogenic activities of 5-formylhonokiol on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) for the first time in vitro. Then we investigated the in vivo antiangiogenic effect of 5-formylhonokiol on zebrafish angiogenesis model. In order to clarify the underlying molecular mechanism of 5-formylhonokiol, we investigated the signaling pathway involved in controlling the angiogenesis process by western blotting assay. Wound-healing results showed that 5-formylhonokiol significantly and dose-dependently inhibited migration of cultured human umbilical vein enthothelial cells. The invasiveness of HUVEC cells was also effectively suppressed at a low concentration of 5-formylhonokiol in the transwell assay. Further F-actin imaging revealed that inhibitory effect of 5-formylhonokiol on invasion may partly contribute to the disruption of assembling stress fiber. Tube formation assay, which is associated with endothelial cells migration, further confirmed the anti-angiogenesis effect of 5-formylhonokiol. In in vivo zebrafish angiogenesis model, we found that 5-formylhonokiol dose-dependently inhibited angiogenesis. Furthermore, western blotting showed that 5-formylhonokiol significantly down-regulated extracellular signal-regulated kinase (ERK) expression and inhibited the phosphorylation of ERK but not affecting the total protein kinase B (Akt) expression and related phosphorylation, suggesting that 5-formylhonokiol might exert anti-angiogenesis capacity via down-regulation of the ERK signal pathway. Taken together, these data suggested that 5-formylhonokiol might be a viable drug candidate in antiangiogenesis and anticancer therapies.


Subject(s)
Animals , Humans , Actins/metabolism , Angiogenesis Inhibitors/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Biphenyl Compounds/pharmacology , Blotting, Western , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Drugs, Chinese Herbal , Embryo, Nonmammalian/drug effects , Endothelium, Vascular/drug effects , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Lignans/pharmacology , Neovascularization, Physiologic/drug effects , Signal Transduction/drug effects , Umbilical Veins/cytology , Wound Healing , Zebrafish/embryology
10.
Journal of Biomedical Engineering ; (6): 1121-1125, 2008.
Article in Chinese | WPRIM | ID: wpr-318201

ABSTRACT

In this paper, a series of low-molecular-weight PEG-PCL-PEG triblock copolymers were successfully synthesized by ring-opening polymerization method, and were characterized using 1H-NMR and FTIR. The aqueous solution displayed specific thermosensitive gel-sol transition when the concentration was above corresponding critical gel concentration (CGC). The gel-sol phase diagram was recorded using test tube-inverting method, which was depended on the hydrophilic/hydrophobic balance in macromolecular structure, as well as heating history. As a result, the gel-sol transition temperature range could be altered, which might be very useful for its application as injectable drug delivery system.


Subject(s)
Biocompatible Materials , Chemistry , Drug Carriers , Chemistry , Drug Delivery Systems , Hydrogel, Polyethylene Glycol Dimethacrylate , Chemistry , Polyesters , Chemistry , Polyethylene Glycols , Chemistry , Spectroscopy, Fourier Transform Infrared , Temperature
11.
Journal of Biomedical Engineering ; (6): 157-167, 2008.
Article in Chinese | WPRIM | ID: wpr-291276

ABSTRACT

The extracellular domain 2-4 loop cDNA of quail vascular endothelial growth factor receptor quek1 (qVEGFR2) was obtained from plasmid carrying qVEGFR2 by PCR. Then it was cloned into expression vector pPICZalphaA of Pichia pastoris. To construct recombinant expression plasmid pPICZalphaA-qVEGFR2, linearized pPICZalphaA-qVEGFR2 with SacI was transformed to electroporated Pichia pastoris GS115. Subsequently, positive clone was selected by PCR and its phenotype was determined. SDSPAGE and Western blot assays of culture medium from a methanol-induced expression strain demonstrated that recombinant qVEGFR2 proteins were expressed and secreted into the culture medium. These results could provide a basis for further researches on tumor protein vaccine as well as for the preparation of tumor protein vaccine with Pichia pastoris.


Subject(s)
Animals , Cancer Vaccines , DNA, Complementary , Genetics , Genetic Vectors , Genetics , Pichia , Genetics , Metabolism , Polymerase Chain Reaction , Quail , Receptors, Neurotransmitter , Genetics , Recombinant Proteins , Genetics , Vascular Endothelial Growth Factor Receptor-2 , Genetics
12.
Journal of Biomedical Engineering ; (6): 866-869, 2007.
Article in Chinese | WPRIM | ID: wpr-346053

ABSTRACT

A DNA fragment encoding human interleukin 4 was obtained by PCR from pORF-hIL4 plasmid. The amplified fragment was inserted into prokaryotic expression vector PQE60 and recombinant protein was expressed in E. Coli M15 by adding isopropyl-beta-D-thiogalactoside (IPTG). The hIL-4 protein was present as insoluble inclusion bodies in the bacterial extract. After denaturation of inclusion bodies with 5 mol/L guanidine hydrochloride, the supernate was diluted to get renaturized. Then dialysis and Ni chelating chromatography were used for purification. TF-1 proliferation assay of recombinant human interleukin 4 was performed, and then rhIL-4 was fit to be used for proliferation of human dendritic cells from monocyte in vitro.


Subject(s)
Humans , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Inclusion Bodies , Metabolism , Interleukin-4 , Genetics , Protein Folding , Recombinant Proteins , Genetics
13.
Journal of Biomedical Engineering ; (6): 191-195, 2007.
Article in Chinese | WPRIM | ID: wpr-331367

ABSTRACT

After the preparation of cationic liposomes composed of DDAB/DOPE, cationic liposome-DNA complexes and lipid-polycation-DNA (LPD) complexes were formulated, respectively. Gel retardation assay was employed to select appropriate ratios of cationic liposomes to DNA of the liposome-DNA complexes. The morphology of LPD and liposome-DNA complexes was observed by transmission electron microscopy. The diameter and surface charge of LPD and liposome-DNA complexes were measured by photon correlation spectroscopy (PCS). Their transfection efficiencies in Chang cells and HepG2 cells were evaluated by beta-gal assay kit. It was found that LPD and liposome-DNA complexes had a regular spherical surface. However, compared with liposome-DNA complexes, LPD had rather smaller particle size and much higher transfection efficiency in Chang cells and HepG2 cells in vitro. LPD could be prepared easily with small particle sizes and high transfection activities. LPD may be a good non-viral gene delivery vehicle for applications in gene delivery.


Subject(s)
Humans , Cations , DNA , Genetics , Genetic Therapy , Genetic Vectors , Lipids , Chemistry , Liposomes , Chemistry , Liver Neoplasms , Metabolism , Pathology , Protamines , Chemistry , Transfection , Tumor Cells, Cultured
14.
Journal of Biomedical Engineering ; (6): 1289-1293, 2006.
Article in Chinese | WPRIM | ID: wpr-331428

ABSTRACT

The purpose of this study was to clone human HSP90beta cDNA and construct its eukaryote expression vector . The total RNA was isolated by TRIzol Reagent (Invitrogen) from human NPC and its cDNA was gained by RT-PCR. The purified RT-PCR products and PGEM-T Easy Vector were ligated and transformed into XL1-blue E. coli bacteria. The white clones were selected and the plasmid was purified, which was further identified by double enzyme digestion and sequenced. PGEM-hHSP90beta and pcDNA3.1(+) DNA were digested by AflII and Xbal respectively. After purification, the two fragments obtained were ligased by using T4 DNA ligase (Fermentas). This recombinant DNA was then transformed into E. coli Competent Cells XL1-blue and positive clones were selected on the LB agarose plate containing Ampr (100 microg/ml). Single clones were identified by double digestion with AflII and Xbal, and two fragments with the size 5.4 kb and 2.1 kb were produced as expected. The hHSP90beta gene was successfully inserted into the eukaryote expression vector pcDNA3.1(+) by the recombination technique in vitro.


Subject(s)
Humans , Cloning, Molecular , DNA, Complementary , Genetics , Escherichia coli , Genetics , Metabolism , Genetic Vectors , HSP90 Heat-Shock Proteins , Genetics , Plasmids , Genetics , Recombinant Proteins , Genetics
15.
Journal of Biomedical Engineering ; (6): 844-847, 2006.
Article in Chinese | WPRIM | ID: wpr-320469

ABSTRACT

To obtain the expression of Isoleucine Zipper modified soluble CD40L (IZ-sCD40L) in Pichia pastoris, firstly, DNA fragment of IZ-sCD40L was obtained by PCR and over-lap PCR . Then the expression vector pPICZaA-IZ-sCD40L was constructed. Nucleotide sequencing analysis indicated that the DNA fragment of IZ-sCD40L was correctly inserted into the pPICZaA vector. Linearized pPICZ(alpha)A-IZ-sCD40L was introduced into Pichia pastoris GS115. Positive clone was selected by PCR and its phenotype was determined. The positive clone was introduced with methanol. The results of SDS-PAGE and Western blot showed that product was recombinant Isoleucine Zipper modified soluble CD40L fusion protein.


Subject(s)
Angiotensin II , CD40 Ligand , Genetics , Cloning, Molecular , Isoleucine , Genetics , Pichia , Genetics , Metabolism , Plasmids , Polymerase Chain Reaction , Methods , Recombinant Fusion Proteins , Genetics , Recombination, Genetic
16.
Chinese Journal of Lung Cancer ; (12): 501-503, 2005.
Article in Chinese | WPRIM | ID: wpr-313315

ABSTRACT

<p><b>BACKGROUND</b>It has been known that the growth of solid tumors is dependent on angiogenesis, and neoangiogeneses of tumor become main target to control tumor growth. The aims of this study are to investigate the inhibition effect of replicate-deficient adenovirus encoding the soluble form of mouse vascular endothelial growth factor receptor 1 (sFlt1-Adv) on angiogenesis and tumor growth in established tumor model.</p><p><b>METHODS</b>Mouse Lewis lung cancer cells were inoculated subcutaneously into C57 mice. sFlt1-Adv, GFP-Adv and normal saline were injected twice intravenously after establishing Lewis cancer model. Diameters of tumors were measured every other day. Tumors were resected, weighed and fixed in 3% paraformadehyde. Microvessel density of tumors was determined by immunohistochemical staining with anti-CD31 antibody.</p><p><b>RESULTS</b>The planted tumor volume and weight in sFlt1-Adv group were significantly lower compared with the two controls (P < 0.01). Its inhibition rate was 71.8%. The microvessel density in sFlt1-Adv group decreased markedly compared with that of the control groups (P < 0.01).</p><p><b>CONCLUSIONS</b>sFlt1-Adv can inhibit the growth of tumor through the inhibition of tumor angiogenesis. sFlt1-Adv may be potentially valuable for clinical treatment of solid tumor.</p>

17.
Journal of Biomedical Engineering ; (6): 374-376, 2005.
Article in Chinese | WPRIM | ID: wpr-354295

ABSTRACT

The immune system of mammalian has developed sophisticated mechanism to deal with diverse unknown antigens by random rearrangement of V, D and J antigen gene fragments. The immune self-tolerance is the mechanism to avoid self-destruction by the gene rearrangement generated autoreactive lymphocytes. Until recently it was believed that autoreactive lymphocytes are either deleted or rendered unable to respond by the supposed cell or clone selection mechanism. However, recent findings from a number of laboratories suggest instead of cell selection but receptor selection plays an essential role in immune self-tolerance. Receptor selection is carried out by secondary or nested rearrangement of antigen receptor gene fragments, and can occur at different stages of lymphocyte differentiation. Furthermore, secondary rearrangement of receptor gene also plays an important role in shaping immune response after the interaction of receptor with antigen by altering its specificity.


Subject(s)
Humans , Autoimmune Diseases , Allergy and Immunology , B-Lymphocytes , Allergy and Immunology , Gene Rearrangement, B-Lymphocyte , Allergy and Immunology , Genes, Immunoglobulin , Immune Tolerance , Receptors, Antigen, B-Cell , Allergy and Immunology , Self Tolerance , Allergy and Immunology
18.
Journal of Biomedical Engineering ; (6): 535-539, 2005.
Article in Chinese | WPRIM | ID: wpr-354256

ABSTRACT

Interferon gamma-inducible protein 10, a member of the family of CXC chemokines, is secreted by interferon gamma-stimulated, monocytes, endothelial cells and keratinocytes. Interferon gamma-inducible protein 10 plays an important role in recruiting activated T cells into sites of tissue inflammation. In this experiment, PCR products of Interferon gamma-inducible protein 10 were cloned into prokaryote expression vector pET 32(a) to generate recombinant pET-IP10 with S-Tag at the N-terminus, and expressed successfully in E. coli BL21 (DE3). The total expressed products amounted to 25.3% in all bacterion proteins. pET-IP10 mainly formed inclusion body in E. coli. Soluble recombinant protein accounted for 20% among IP-10 fusion protein. The soluble recombinant proteins were purified by using S-Tag affinity chromatography effectively with purity of over 90%. The chemotaxis biological activity of purified Interferon gamma-inducible protein 10 could specifically exhibit the directional migration of stimulated T cells at concentration of 100 ng/ml. The results indicated that the strategy we used in this experiment was effective for recombinant Interferon gamma-inducible protein 10 production with biological activity.


Subject(s)
Humans , Chemokine CXCL10 , Escherichia coli , Metabolism , Genetic Vectors , Recombinant Proteins , T-Lymphocytes , Cell Biology , Thioredoxins
19.
Journal of Biomedical Engineering ; (6): 251-254, 2004.
Article in Chinese | WPRIM | ID: wpr-291138

ABSTRACT

A DNA fragment encoding mouse B Lymphocyte Chemoattractant (BLC, MV10Kda) was obtained by PCR. The amplified fragment was inserted into prokaryotic expression vector PQE30. Recombinant protein was expressed in E. Coli XL-1 blue and purified by affinity chromatography on a nickel-nitrilotriacetic acid gel matrix. Then it was identified by sequence analysis and Western blot analysis. The fragment inserted into prokaryotic expression vector PQE30 was identified to be BLC gene fragment by sequence analysis. And a specfic band was shown by Western blot analysis. These findings provide the evidence that the recombinant protein obtained and purified in this study using gene engineering method is mouse B Lymphocyte Chemoattractant.


Subject(s)
Animals , Mice , Chemokine CXCL13 , Chemokines, CXC , Genetics , Escherichia coli , Genetics , Genetic Vectors , Mice, Inbred BALB C , Plasmids , Genetics , Prokaryotic Cells , Metabolism , Recombinant Proteins , Genetics , Sequence Analysis, DNA
20.
Journal of Biomedical Engineering ; (6): 897-900, 2004.
Article in Chinese | WPRIM | ID: wpr-342585

ABSTRACT

Mouse colon cancer cells CT26 were transfected with constructed plasmid expressing mouse soluble B lymphocyte stimulator (msBlyS). Single cell clones were selected with 100 microg/ml Zeosin and subcloned by serial limiting dilution. Eight resistant transfectants were isolated and expanded, and five of them displayed the desirable msBlyS cDNA band amplified by semi-quantitative RT-PCR assay. Western blot analysis showed that only msBlyS molecules of the expected size were detected in the cell lysates from transfectants. The supernatant of transfectants could costimulate B cell proliferation in standard costimulation assay. Thus we have successfully screened the stable transfectants expressing high levels of msBlyS in CT26 cells, which could be used as cancer vaccines for further anti-tumor immunotherapy.


Subject(s)
Animals , Mice , Antibodies, Monoclonal , B-Cell Activating Factor , B-Lymphocytes , Allergy and Immunology , Cancer Vaccines , Genetics , Cloning, Molecular , Colonic Neoplasms , Pathology , DNA, Complementary , Genetics , Epitopes, B-Lymphocyte , Genetics , Membrane Proteins , Genetics , Recombinant Proteins , Genetics , Recombination, Genetic , Transfection , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha , Genetics
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