OBJECTIVETo purify recombinant nuclear protein of Hantaan virus.

METHODSThe recombinant plasmid was transformed into E.coli and induced by IPTG. The expressed protein is a fusion protein with GST and existed in inclusion bodies. The inclusion bodies were processed through denaturation and renaturation and were purified with Glutathione Sepharose 4B affinity chromatography column. Then the purified fusion protein and nuclear protein were examined by sandwich ELISA and Western blot.

RESULTSThe expressed fusion protein was separated from the mixture proteins by the first affinity chromatography. GST was cut from the purified fusion protein with thrombin. The nuclear protein was separated from GST by the second affinity chromatography. The crossed peak represents nuclear protein and the elute peak represents GST. The purified fusion protein and nuclear protein were single band by SDS-PAGE. Both of them had available antigen activity.

CONCLUSIONSPurification of nuclear protein of Hantaan virus with Glutathione Sepharose 4B affinity chromatography is an effective method.

Blotting, Western ; Chromatography, Affinity ; Enzyme-Linked Immunosorbent Assay ; Hantaan virus ; Nuclear Proteins ; analysis ; isolation & purification ; Plasmids ; Recombinant Fusion Proteins ; analysis ; isolation & purification ; Viral Proteins ; analysis ; isolation & purification

To establish a refolding process for the protein fused with 12-peptide of hirudin and reteplase (HV12p-rPA), we developed an anion-exchange chromatography assisted method to form correct disulfide bonds. After evaluating various parameters by orthogonal experiments with Q Sepharose XL as refolding medium, we found that urea gradient, sample loading size and L-Arg concentration were three major factors to affect the refolding outcomes, and urea gradient was critical to the recovery yield. Meanwhile, enzymatic activity of the refolded protein was decreased by the increase of sample loading size, and the optimal concentration of L-Arg in the eluting buffer was 1 mol/L. Thus, a dual-gradient of urea and pH on the anion-exchange chromatography resulted in remarkable increase of specific fibrinolytic and anti-coagulative activities of the refolded protein. Compared with the dilution method for refolding HV12p-rPA, the present approach was more effective and advantageous.
Chromatography, Ion Exchange ; methods ; Disulfides ; chemistry ; Fibrinolytic Agents ; analysis ; chemistry ; Hirudins ; analysis ; chemistry ; Protein Refolding ; Recombinant Fusion Proteins ; analysis ; chemistry ; Recombinant Proteins ; analysis ; chemistry ; Tissue Plasminogen Activator ; analysis ; chemistry

BACKGROUND: Several studies reported that sub-clinical rejection (SCR) detected by a protocol biopsy soon after renal transplantation does permanent damage to a renal allograft, contributing to chronic allograft nephropathy (CAN). This article investigated the risk factors involved in SCR and the effects of treating SCR, and evaluated the clinical significance of a protocol biopsy soon after renal transplantation. METHODS: From January 2007 to June 2010, 253 patients received renal transplantation. Patients were divided into two groups according to whether or not they had undergone a protocol biopsy. To analyze the effect of SCR treatments, patients who were diagnosed with SCR were divided into two groups according to whether or not they had been treated with SCR. The patients who did not undertake a protocol biopsy were included in the untreated groups. RESULTS: Among 138 patients who undertook protocol biopsies, 65 patients (47.1%) showed SCR. In univariate analysis, both the number of HLA-DR mismatches (P=0.003) and not using Simulect (P=0.01) were identified as risk factors of SCR. In multivariate analysis, not using Simulect (P=0.006) was identified as an risk factor independent of SCR. deltaGFR, subtracting GFR at 1 week from GFR at that point, showed significant differences between SCR-treated patients and untreated patients at 1, 3, 6, 9, 12, 24, and 36 months with a P value of less than 0.05. CONCLUSIONS: A protocol biopsy can detect SCR, especially in patients with risk factors such as a high number of HLA mismatches or not using Simulect. Treatment of SCR detected by protocol biopsy will help to improve long-term renal function.
Antibodies, Monoclonal ; Biopsy ; HLA-DR Antigens ; Humans ; Kidney Transplantation ; Multivariate Analysis ; Recombinant Fusion Proteins ; Rejection (Psychology) ; Risk Factors ; Transplantation, Homologous

BACKGROUND: Several studies reported that sub-clinical rejection (SCR) detected by a protocol biopsy soon after renal transplantation does permanent damage to a renal allograft, contributing to chronic allograft nephropathy (CAN). This article investigated the risk factors involved in SCR and the effects of treating SCR, and evaluated the clinical significance of a protocol biopsy soon after renal transplantation. METHODS: From January 2007 to June 2010, 253 patients received renal transplantation. Patients were divided into two groups according to whether or not they had undergone a protocol biopsy. To analyze the effect of SCR treatments, patients who were diagnosed with SCR were divided into two groups according to whether or not they had been treated with SCR. The patients who did not undertake a protocol biopsy were included in the untreated groups. RESULTS: Among 138 patients who undertook protocol biopsies, 65 patients (47.1%) showed SCR. In univariate analysis, both the number of HLA-DR mismatches (P=0.003) and not using Simulect (P=0.01) were identified as risk factors of SCR. In multivariate analysis, not using Simulect (P=0.006) was identified as an risk factor independent of SCR. deltaGFR, subtracting GFR at 1 week from GFR at that point, showed significant differences between SCR-treated patients and untreated patients at 1, 3, 6, 9, 12, 24, and 36 months with a P value of less than 0.05. CONCLUSIONS: A protocol biopsy can detect SCR, especially in patients with risk factors such as a high number of HLA mismatches or not using Simulect. Treatment of SCR detected by protocol biopsy will help to improve long-term renal function.
Antibodies, Monoclonal ; Biopsy ; HLA-DR Antigens ; Humans ; Kidney Transplantation ; Multivariate Analysis ; Recombinant Fusion Proteins ; Rejection (Psychology) ; Risk Factors ; Transplantation, Homologous

Pyruvate phosphate dikinase (PPDK; EC 2.7.9.1) is found in certain microorganisms and plants, and catalyzes the conversion of AMP, PPi and phosphoenolpyruvate (PEP) to ATP, Pi and pyruvate. Using the genomic DNA of Microbispora rosea subsp. aerata as the template, a DNA fragment encoding the gene PPDK was amplified by PCR and inserted into the expression vector pET28a(+), yielding pET28a (+)-PPDK. The E. coli BL21 (DE3) was transformed with the pET28a (+)-PPDK. After inducing with IPTG, the E. coli BL21 (DE3) [pET28a (+)-PPDK] expressed recombinant PPDK fused to an N-terminal sequence of 6-His Tag. The molecular weight of PPDK was estimated to be 101 kD by SDS-PAGE. The PPDK was purified by His * Bind Resin affinity chromatography and ultrafiltration using 10 kD cut-off membrane. The successful application of PPDK in pyrosequencing was also demonstrated.
Actinomyces ; enzymology ; Escherichia coli ; genetics ; metabolism ; Pyruvate, Orthophosphate Dikinase ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Sequence Analysis

The porcine IL-18 gene was amplified from recombinant plasmid pGEM-IL-18 by PCR, then the pPIC9K-IL-18 of fusion expression vector was constructed by inserting IL-18 fragment,and was transformed to GS115 by electroporation, multi-copy recombinant strains were screened by G418. The expression of recombinant fusion protein was induced by methanol, SDS-PAGE was used to analyze expression product, fusion protein was purified by Sephadex G-100 column, bioactivity of IL-18 was measured by MTT assays. Experiment results show fusion protein of pIL-18 secreted by GS115,expression reaches the secretion peak of 160 mg/L at 72 h. We have expressed and purified successfully the recombinant pIL-18 with obvious biological activity in Pichia pastoris.
Animals ; Electrophoresis, Polyacrylamide Gel ; Electroporation ; Interleukin-18 ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; analysis ; biosynthesis ; genetics ; Swine

OBJECTIVETo investigate the effect of adiponectin on hepatocyte steatosis.

METHODSL02 cells were transfected with pEGFP-N1-AdipoQ, a plasmid encoding pEGFP-adiponectin fusion protein, or pEGFP-N1. Lipid droplets in the hepatocytes were observed by oil red staining at 72 h. The contents of TG, FFA and glycerol in hepatocytes were measured.

RESULTSCompared to cells transfected with pEGFP-N1-AdipoQ plasmid, much more lipid droplets were observed in cells transfected with pEGFP-N1 plasmid. TG, FFA and glycerol contents in L02 cells and L02/pEGFP-N1 cells were significantly higher than those in L02/pEGFP-N1-AdipoQ cells.

CONCLUSIONSOverexpression of adiponectin prevent hepatocyte steatosis.

Adiponectin ; genetics ; Cell Line ; Fatty Acids, Nonesterified ; analysis ; Fatty Liver ; metabolism ; Genetic Vectors ; Glycerol ; analysis ; Hepatocytes ; cytology ; metabolism ; Humans ; Plasmids ; Recombinant Fusion Proteins ; genetics ; Transfection ; Triglycerides ; analysis

The aim of this study is to develop monoclonal antibody against human hepatocyte growth factor activator inhibitor 1 (HAI-1) for future study of HAI-1. The cDNA fragments of human hepatocyte growth factor activator inhibitor 1 (HAI-1) were subcloned to construct GST-HAI-1 fusion protein expression vectors. The vectors were transformed into E. coli and fusion protein expression was induced by IPTG. The GST-HAI-1 fusion proteins were separated on preparative SDS-PAGE and recovered by electroelution, and used to immunize BALB/c mice. Hybridomas producing monoclonal antibodies against human HAI-1 were prepared by cell fusion technique and characterized by ELISA, Western Blot and immunohistochemical staining. One hybridoma cell line, ZMC6, was obtained, which produces specific antibody against the expressed GST-HAI-1 fusion protein. The monoclonal antibody recognizes both the membrane-type and secretory-type HAI-1 proteins of colorectal tissue. The successful development of anti-HAI-1 antibody provides a powerful tool for further investigation on HAI-1's function.
Animals ; Antibodies, Monoclonal ; immunology ; Blotting, Western ; Glutathione Transferase ; genetics ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; Proteinase Inhibitory Proteins, Secretory ; analysis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; immunology

OBJECTIVETo clone the recombinant fusion gene of Escherichia coli heat-liable enterotoxin B subunit (Ltb) and Actinobacillus actinomycetemcomitans fimbria associative protein (Fap).

METHODSTwo couples of primers were designed for PCR according to the known sequence of ltb and fap. The ltb and fap gene were obtained by amplification PCR technique from plasmid EWD299 of Escherichia coli and Actinobacillus actinomycetemcomitans 310 DNA respectively, and fused them by PCR. The fusion gene ltb-fap were cloning into plasmid pET28a(+). The recombined plasmid pET28a ltb-fap was transformed into Escherichia coli DH5alpha. The recombinant was screened and identified by restriction enzyme and PCR. The cloned gene was sequenced.

RESULTSThe ltb-fap about 531bp in size was obtained successfully, and identified by PCR, restrictive enzyme and sequence analysis.

CONCLUSIONThe vector of pET28a ltb-fap was obtained.

Aggregatibacter actinomycetemcomitans ; Bacterial Toxins ; Cloning, Molecular ; Cloning, Organism ; Enterotoxins ; Escherichia coli ; Escherichia coli Proteins ; Hot Temperature ; Plasmids ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; Sequence Analysis

The delivery of transgenes to the central nervous system (CNS) can be a valuable tool to treat CNS diseases. Various systems for the delivery to the CNS have been developed; vascular delivery of viral vectors being most recent. Here, we investigated gene transfer to the CNS by intravenous injection of recombinant adenoviral vectors, containing green fluorescence protein (GFP) as a reporter gene. Expression of GFP was first observed 6 days after the gene transfer, peaked at 14 days, and almost diminished after 28 days. The observed expression of GFP in the CNS was highly localized to hippocampal CA regions of cerebral neocortex, inferior colliculus of midbrain, and granular cell and Purkinje cell layers of cerebellum. It is concluded that intravenous delivery of adenoviral vectors can be used for gene delivery to the CNS, and hence the technique could be beneficial to gene therapy.
Adenoviruses, Human/isolation & purification* ; Animals ; Blood-Brain Barrier ; Brain/virology* ; Cerebellum/cytology ; Cerebellum/virology ; Comparative Study ; Female ; Genes, Reporter ; Genetic Vectors/administration & dosage ; Genetic Vectors/isolation & purification ; Genetic Vectors/pharmacokinetics* ; Hippocampus/virology ; Inferior Colliculus/virology ; Injections, Intravenous ; Luminescent Proteins/analysis ; Luminescent Proteins/biosynthesis ; Luminescent Proteins/genetics ; Mice ; Mice, Inbred BALB C ; Neuroglia/virology ; Neurons/virology* ; Purkinje Cells/virology ; Pyramidal Cells/virology ; Recombinant Fusion Proteins/analysis ; Recombinant Fusion Proteins/biosynthesis ; Recombinant Fusion Proteins/genetics ; Tail/blood supply ; Tissue Distribution