ABSTRACT
AIM: To prokaryotically express and purify fusion protein containing extracellular region of human BTLA and prepare the antiserum of it. METHODS: Human BTLA (hBTLA) gene was amplified by PCR, digested with enzymes, ligated and subcloned into a his-tagged prokaryotic expression vector to generate a recombinant plasmid named pET28a-hBTLA. Then pET28a-hBTLA was transformed into E.coli BL21 (DE3). The hBTLA fusion protein was obtained upon IPTG induction, purified by Ni-NTA Purification System, and analyzed by SDS-PAGE. Rabbit anti-hBTLA antiserum was prepared and identified. RESULTS: The pET28a-hBTLA plasmid was confirmed to carry the correct hBTLA gene by sequencing. It expressed a 15.7kD protein that could be purified by Ni-NTA purification system. The titer of the multiclonal antibody was 1:16 detected by double diffusion test, and 1:20 000 by ELISA, respectively. The anti-hBTLA antibodies can bind to hBTLA specifically shown by Western blot. CONCLUSION: The prokaryotic expression vector has been constructed successfully, leading to highly purified hBTLA protein. The antiserum of hBTLA has been prepared, with high titer and specificity.
Subject(s)
Immune Sera/immunology , Receptors, Immunologic/genetics , Animals , Escherichia coli/genetics , Humans , Plasmids , Rabbits , Receptors, Immunologic/immunology , Recombinant Proteins/isolation & purificationABSTRACT
The significance of BmK CT, a key chlorotoxin-like peptide isolated from the scorpion venom of Buthus martensii Karsch, is a novel blocker of the chloride ion channel and matrix metalloproteinase-2 (MMP-2). Site-directed mutagenesis of BmK CT, wound healing assay, gelatin zymography assay and computational simulation highlight the importance of electrostatic contribution to BmK CT-MMP-2 catalytic domain complex and a model of BmK CT-MMP-2 catalytic domain complex is therefore proposed. This is the first documentation of the structural mechanism of in the inhibition of glioma cell migration by BmK CT and may lead to the molecular design of specific inhibitors of MMP-2.
Subject(s)
Antineoplastic Agents/metabolism , Cell Movement/drug effects , Enzyme Inhibitors/metabolism , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase Inhibitors , Scorpion Venoms/metabolism , Antineoplastic Agents/chemistry , Enzyme Inhibitors/chemistry , Models, Molecular , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Protein Structure, Tertiary , Scorpion Venoms/chemistry , Scorpion Venoms/genetics , Wound Healing/drug effectsABSTRACT
AIM: To study the clinical significance of determination of serum B7-H4 in patients with malignant hematologic diseases. METHODS: Serum B7-H4 levels were determined in 65 patients with leucemia, 34 patients with lymphoma, 12 patients with multiple myeloma as well as in 50 healthy controls. RESULTS: The serum B7-H4 levels in patients with lymphoma [(38.81+/-10.34) kappag/L] were significantly higher than healthy controls [(31.62+/-9.850) kappag/L] (P<0.01). But there are no significant difference of B7-H4 levels in serum among patients with leucemia, patients with multiple myeloma and healthy controls. CONCLUSION: These results suggest that the B7-H4 may correlated with lymphoma, but uncorrelated with leucemia and multiple myeloma. Measurement of serum B7-H4 level provide useful information for distinctive diagnosis of different kinds of malignant hematologic diseases.
Subject(s)
B7-1 Antigen/blood , Hematologic Diseases/blood , Adolescent , Adult , Aged , Child , Child, Preschool , Enzyme-Linked Immunosorbent Assay , Female , Humans , Infant , Leukemia/blood , Lymphoma/blood , Male , Middle Aged , Multiple Myeloma/blood , Young AdultABSTRACT
Previous studies demonstrate that both membrane B7-H4 and B7-H4-Ig fusion protein could inhibit T-cell responses. In the present study, we explored the potential effect of B7-H4-Ig on liver injury in a hepatitis mouse model induced by concanavalin A (ConA). A B7-H4-Ig construct was introduced into animals by the hydrodynamic gene delivery approach. It was found that ectopic expression of B7-H4-Ig could inhibit ConA-induced elevation of serum levels of ALT and AST, suppress liver necrosis and even mortality of mice. Furthermore, we observed that pretreatment of B7-H4-Ig dramatically decreased serum levels and the expression of mRNA for IL-2, IFN-gamma and IL-4, but increased IL-10 in ConA-treated mice. Our results suggest that B7-H4-Ig may protect animals from liver injury induced by ConA, which could be associated with reduced serum levels for IL-2, IFN-gamma and IL-4 as well as enhanced IL-10 production.
Subject(s)
B7-1 Antigen/therapeutic use , Chemical and Drug Induced Liver Injury/prevention & control , Concanavalin A/pharmacology , Genetic Therapy/methods , Immunoglobulin Fc Fragments/genetics , Immunoglobulin G/genetics , Recombinant Fusion Proteins/therapeutic use , Alanine Transaminase/blood , Animals , Aspartate Aminotransferases/blood , B7-1 Antigen/blood , B7-1 Antigen/genetics , B7-1 Antigen/metabolism , Chemical and Drug Induced Liver Injury/blood , Chemical and Drug Induced Liver Injury/pathology , Gene Expression/drug effects , Gene Expression/genetics , Gene Transfer Techniques , Humans , Immunoglobulin Fc Fragments/blood , Interferon-gamma/blood , Interferon-gamma/genetics , Interleukin-10/blood , Interleukin-10/genetics , Interleukin-2/blood , Interleukin-2/genetics , Interleukin-4/blood , Interleukin-4/genetics , Leukocytes, Mononuclear/metabolism , Liver/drug effects , Liver/metabolism , Liver/pathology , Mice , Mice, Inbred BALB C , Necrosis/chemically induced , Necrosis/pathology , Recombinant Fusion Proteins/blood , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Survival Analysis , V-Set Domain-Containing T-Cell Activation Inhibitor 1ABSTRACT
OBJECTIVE: To investigate the in-vitro interferon-gamma (IFN-gamma) release assay based on three different Mycobacterium tuberculosis antigens in the diagnosis of tuberculosis and Mycobacterium tuberculosis infections. METHODS: The peripheral blood mononuclear cells (PBMC) were collected from the patients with tuberculosis (tuberculosis group, n = 57), patients with lung cancer (lung cancer group, n = 29), and healthy controls (healthy control group 2, n = 27). The PBMCs were co-cultured for 5 days with different antigens: purified protein derivatives (PPD) of tuberculin, early secretary antigenic target 6,000 protein (ESAT6) and 38,000 antigen. The protein levels of IFN-gamma were detected by ELISA, and the results were compared to those with the tuberculin skin test (TST). RESULTS: (1) For healthy controls, the TST was positively related to the history of BCG vaccination and the closeness of contact with sputum-positive tuberculosis patients (P = 0.047, P = 0.041 respectively). The ESAT6 based IFN-gamma release assay was only significantly related to the closeness of contact with sputum-positive tuberculosis patients (P = 0.005), but not to the history of BCG vaccination. (2) There was no significant difference of the TST results among the three groups (P > 0.05). (3) The receiver operating characteristic (ROC) curve analysis indicated that the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of the IFN-gamma release assay based on 38,000 antigen were 64.9%, 89.3%, 77.0%, 86.0%, and 71.0% respectively for the diagnosis of tuberculosis. CONCLUSIONS: IFN-gamma release assay based on ESAT6 appears to be better than TST in the diagnosis of infection of Mycobacterium tuberculosis, while IFN-gamma release assay based on 38,000 may be helpful for the diagnosis of tuberculosis.