OBJECTIVETo investigate the anti-HBV effect of fusion protein thymosin alpha1-interferon alpha (TA1-IFN) in vitro and to compare its effect with a combination of interferon alpha and thymosin alpha1.

METHODSAfter 2.2.15 cells were seeded for 24 hours, drugs of five serial concentrations (8000, 4000, 2000, 1000, 500 U/ml) were added to the wells, then the medium was changed every three days. After 2.2.15 cells were treated with drugs for 6 days, the medium was collected. The inhibitory rates on HBsAg and HBeAg were determined using Abbot kit, and the cytotoxicity of different drugs by means of MTT colorimetric assays was also observed.

RESULTSThe inhibitory rate of fusion protein on HBsAg, HBeAg was dose-dependent and reached the maximum at 8000 U/ml concentration. In the meantime, the inhibitory rates of fusion protein on HBsAg and HBeAg were 72.2% +/- 0.8% and 60.4% +/- 1.1% respectively, and the cell survival rate was 85.2% +/- 2.0%; In the corresponding concentration, the inhibitory rates of combination thymosin alpha 1 and interferon alpha on HBsAg and HBeAg were 40.0% +/- 0.7%, 34.5% +/- 3.2% respectively. The results showed significant statistical differences between them; cell survival rate 70.0% +/- 1.9%, and the difference of the results was also significant. Cytotoxicity of fusion protein was weaker than a combination of thymosin alpha 1 and interferon alpha.

CONCLUSIONFusion protein TA1-IFN exerted stronger anti-HBV effects in vitro. Its anti-HBV effects in vitro were stronger than the combination of thymosin alpha and interferon alpha, and its cytotoxicity was weaker than the combination of thymosin alpha and interferon alpha. Our studies provided important evidence for clinical research on TA1-IFN, and also brought new hope for hepatitis B therapy.

Antiviral Agents ; pharmacology ; Hepatitis B virus ; drug effects ; Humans ; Interferon-alpha ; biosynthesis ; genetics ; pharmacology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Thymosin ; biosynthesis ; genetics ; pharmacology

The epithelial growth factor receptor interference (EGFRi) was obtained by synthetic primers. Overlapping PCR was used to produce EGFRi-IL-24 fusion gene, which is linked by Gly4Ser3. After sequence analysis, EGFRi-IL-24 was cloned into expression vector pPIC9k; EGFRi-IL-24/pPIC9k was linearized with SacI,and then transformed to electroporated pastoris GS115. Subsequently, positive clone was selected by G418 and PCR, and its phenotype was determined by SDS-PAGE and MTT assay. The results demonstrated that EGFRi-IL-24 protein was expressed and shown to have the potential for use in researches of its biological function and in clinical application.
Antineoplastic Agents ; pharmacology ; Genetic Vectors ; genetics ; Humans ; Interleukins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

The inductive conditions for the flask-shaking of E.coli BL21-pET-hBD3-Bra had been optimized, at the same time, the expressed protein had been purified and analyzed. The effect of three factors which were IPTG concentration, induction time and temperature on growth of strain and on the yield of hBD3-Bra was analyzed in detail. The result indicated that the concentration of IPTG had little effect on the growth and the expression of target protein between 0.2-1 mmol/L, Biomass would be improved as time passed, but the target protein didn't increase obviously as the same time, temperature was the most important factor, the expressed level of hBD3-Bra, as high as about 35% of total cell protein, could be gained when strain was induced by IPTG under 30 degrees C. Further analysis showed the best temperature for growth was 30 degrees C-32 degrees C and for expression protein was 30 degrees C.The purified hBD3-Bra has a weak antimicrobial activity, but is 200 times sweeter than that of sucrose. After digested by thrombin and purified by affinity column, the natural des-pGlul-Brazzein also has 600-time sweetness of sucrose, and the recombinant hBD3 has a high antimicrobial activity again E. coli and S. aureus.
Anti-Infective Agents ; Escherichia coli ; genetics ; metabolism ; Humans ; Plant Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Sweetening Agents ; Temperature ; beta-Defensins ; biosynthesis ; genetics

In this study, we expressed and purified supercharged green fluorescent protein (+36GFP) that we used to study its combination with nucleic acid and its cell transduction efficiency as carrier of DNA. We transformed pET+36GFP-HA2 plasmid into Escherichia coli BL21 (DE3), then expressed and purified the target protein. We used the protein to transduce a variety of mammalian cell lines including B16 cells, 293 cells, A549 cells and HepG2 cells at specified protein concentrations. Transduction efficiency of the protein was analyzed by flow cytometry. Under laser scanning confocal microscope, we observed visually transduction efficiency of +36GFP protein (100 nmol/L) to A549 cells. We incubated +36GFP with plasmid DNA and analyzed their binding ability with gel mobility shift assay. Then we transduced cells with the mixture of plasmid DNA/+36GFP protein at various ratio and detected the expression of reporter gene by using laser scanning confocal microscope and flow cytometry. The experimental results demonstrate that +36GFP had high transduction efficiency, and as the concentration increased, the efficiency improved in a dose-dependent manner. Gel mobility shift assay indicates that +36GFP could bind to plasmid DNA, blocking the migration of DNA in the gel in a concentration-dependent manner. After the plasmid wrapped by +36GFP penetrated into cells, the cells could express target protein efficiently, proving that +36GFP had the ability to carry nucleic acids into cells. Sucussful expression and purification of +36GFP protein confirms its high efficiency of cell transduction and its ability as carrier to deliver exogenous nucleic acids into cells.
Cell Line, Tumor ; DNA ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; genetics ; pharmacology ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Transduction, Genetic ; methods ; Transfection

The expression of major human apurinic/apyrimidinic DNA endonuclease (APEX) from its cDNA in E. coli (DH5 alpha) was attempted in order to obtain a biologically active recombinant APEX. E. coli cells were transformed by a prokaryotic translation vector (pGEX-4T-3) harboring APEX cDNA. GST-APEX fusion protein with a molecular weight of 6.3 KDa was induced by IPTG (1.0 mM) treatment. Western blot immunodetection identified the induced protein as the GST-APEX fusion protein. The survival rate of E. coli cells (DH5 alpha) transformed with pGEX-4T-3-APEX increased when the cells were treated with N-diethyl-N-nitrosamine (DENA) or 3'-methyl-4-monomethylaminoazobenzene (3'-MeMAB), indicating that APEX expression had a protective effect on the cytotoxicity of these carcinogens. The fusion protein extracted from E. coli cells and purified by GSH-agarose gel affinity chromatography exhibited APEX activity. Treatment of thrombin to the GST-APEX fusion protein and affinity purification followed by Sephacryl S-100 gel filtration resulted in APEX peptide with MW 36 KDa, which exhibited AP DNA repair activity (8,7000 EU/mg protein). N-ethylmaleimide (0.1 mM) or AMP (0.98 mM) inhibited APEX activity by 50% and kinetic analysis indicated that the recombinant APEX (rAPEX) had a Km value of 0.022 microM (AP sites for AP DNA) and the Ki value was 0.48 mM for AMP. These results indicated that E. coli cells expressing biologically active GST-APEX were resistant to the cell damage caused by chemical carcinogens and that rAPEX purified from E. coli cells transformed with APEX cDNA-inserted translation vector was similar to native APEX in some properties.
Carbon-Oxygen Lyases/biosynthesis* ; Diethylnitrosamine/pharmacology ; Escherichia coli/genetics ; Escherichia coli/drug effects ; Human ; Recombinant Fusion Proteins/biosynthesis*

Natural hirudin extracted from the secretion of medical leech salivary gland is a single-chain peptide containing 65 aminoacid residues with molecular weight of 7000 D, and exists in three isomers of HV1, HV2 and HV3. Hirudin possesses three disulfide bridges forming the structure of core cyclic peptides, which binds to the catalytic site of thrombin so as to inhibit the catalysis of thrombin. Its c-terminus rich in acidic aminoacid residues possesses hydrophilicity, and is free on the molecular surface, and can bind with fibrin recognition site of hirudin. The minimal segment of 12 - 16 C-terminal acidic residues keeps the minimal activity of anti-thrombosis. Thus, hirudin, as a potent and specific inhibitor of thrombin, can be used to protect from and to treat clinically thrombosis. As it has some disadvantages such as short half-life, bleeding side-effect and mono-function, and so on, hirudin has been fused with some other functional proteins in recent years. The obtained fusion proteins can prolong the half life of hirudin, or relieve it bleeding side effect, or bring new functions, such as thrombolysis, inhibiting the platelet aggregation, targeting specifically. The research progress in hirudin fusion protein was summarized in this review.
Anticoagulants ; pharmacology ; Delayed-Action Preparations ; Drug Delivery Systems ; Glucokinase ; biosynthesis ; genetics ; pharmacology ; Hirudins ; biosynthesis ; genetics ; pharmacology ; Humans ; Platelet Aggregation Inhibitors ; pharmacology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Urokinase-Type Plasminogen Activator ; biosynthesis ; genetics ; pharmacology

OBJECTIVETo construct pBV-BPI600-Fcgamma1(700) recombinant expression vector, to transform it into Escherichia coli DH5alpha, and to induce the expression of BPI23-Fcgamma1 anti-bacterial recombinant protein.

METHODSGenes coding for BPI23 and Fcgamma1 were amplified by RT-PCR from mRNA extracted from HL-60 cell and normal human leukocytes; recombinant cloning vector and recombinant expression vector were then constructed. pBV-BPI600-Fcgamma1(700) recombinant expression vector was transformed into the competent Escherichia coli DH5alpha and BPI23-Fcgamma1 recombinant protein was expressed by a temperature-induced method.

RESULTS(1) Expected amplified products BPI600hp and Fcgamma1(700bp) were obtained by RT-PCR method. (2) pUC18-BPI180, pUC18-BPI420 and pUC18-Fcgamma1(700) recombinant cloning vector were successfully constructed, and sequences were identical with the reported ones. 3) pBV-BPI600-Fcgamma1(700) recombinant expression vector was successfully constructed, and the enzyme digestion analysis showed an expected result. (4) The expression level of BPI23-Fcgamma1 recombinant protein accounted for 20% of total bacterial proteins. (5) The renatured BPI23-Fcgamma1 recombinant protein showed bacteriocidal activity and biological function of complement fixation, and opsonization.

CONCLUSIONpBV-BPI600-Fcgamma1(700) recombinant expression vector was successfully constructed, and BPI23-Fcgamma1 recombinant protein with double biological activity of BPI and IgGFc was expresed in Escherichia coli.

Antimicrobial Cationic Peptides ; Blood Bactericidal Activity ; drug effects ; Blood Proteins ; biosynthesis ; genetics ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; pharmacology ; Cell Adhesion Molecules ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; HL-60 Cells ; Humans ; Membrane Proteins ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

In recent years, Bacterial resistance is more and more serious for the irrational use of antibiotics produces resistant strains and other reasons. Human are trying to solve the problem from different ways, including the study of antimicrobial peptides. Defensin is one of the most important of antimicrobial peptides. A novel antimicrobial peptide, human beta-defensin 3, was isolated and demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes. The total RNA was extracted from human tonsil and the hbetaD-3 specific cDNA sequence was amplified with RT-PCR. After sequenced, the target DNA fragment was cloned into pQE-80L vector together with the DNA fragment encoding carrier protein DHFR. The recombinant vectors were transformed into E. coli M15 and the expression was induced based on the optimal values of the IPTG concentration incubation temperature and induction time determined in the previous section. The expressed proteins were analyzed by SDS-PAGE and Western-blotting. The mass of the recombinant protein was about 40% of total bacteria protein. Isolate and purify the target protein. The recombinant hbetaD-3 fusion proteins possess the antimicrobial activity to staphylococcus aureus, multiresistant staphylococcus aureus (only vancomycin-sensitive) and Candida albicans in the assay of drug susceptibility. Advanced study can be continued based on our experiments.
Cloning, Molecular ; Escherichia coli ; genetics ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; isolation & purification ; pharmacology ; Staphylococcus aureus ; drug effects ; Tetrahydrofolate Dehydrogenase ; genetics ; beta-Defensins ; biosynthesis ; genetics ; pharmacology

To get the mature peptide of human-derived neurotrophin-6 (NT-6), NT-6 gene encoding mature peptide was amplified by PCR, using the NT-6 cDNA that had been cloned as templet. The gene encoding mature peptide of NT-6 gene was cloned into pGEX1-lambdaT plasmid to construct the fusion expression vector. Expression of fusion protein in Escherichia coli was defected after induction by isopropyl beta-D-thiogalactoside(IPTG). The mature peptide of NT-6 was collected with GST fusion protein purifying kit. It was shown that a fragment of 460bp was gained by PCR. With the techniques of double-cleave and electrophoresis, the recombinant vector was identified as pGEX1-NT-6. The recombinant vector pGEX1-NT-6 transformed Escherichia coli expressed fusion protein of 41KD after induction by IPTG. Cleaved by thrombin, the mature peptide of NT-6 was obtained; its molecular weight was about 15KD. The cloning and expression of human-derived NT-6 gene encoding mature protein has provided a basis for further studies on the function and clinical application of NT-6.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Isopropyl Thiogalactoside ; pharmacology ; Nerve Growth Factors ; biosynthesis ; genetics ; Peptides ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

This study aimed to obtain recombinant fusion protein of thymosin alphal(TM-alpha1) and consensus IFNalpha (IFNalpha-con) which have bath TM-alpha1 and IFNalpha-con activities. The DNA sequence for the fusion protein was cloned into expression vector of pET-22b (+) and expressed in BL21 (DE3)-Codon plus-RP-X. The expressed product (TM-alpha1-IFN-con) was soluble, and amounted to more than 20% in total proteins of E. coli. By precipitation of (NH4)2SO4, hydrophobic interaction chromatography (HIC, Phenyl Sepharose 6 Fast Flow), anion-exchange chromatography (Q Sepharose Fast Flow), cation-exchange chromatography (SP Sepharose Fast Flow) and gel filtration (Sephadex G-75), it was purified to more than 96% purity. The activity of fusion protein for antivirus was tested by cytopathic-effect inhibition assay and activity for promoting lymphocyte proliferation was tested by cell proliferative assay. The activity for antivirus was higher than commercial IFNalpha1b and IFNalpha2a and activity for promoting lymphocyte proliferation was similar to commercial TM-alpha1. The fusion protein had better effect for anti-HBV in vitro, its effect was stronger than combination of IFNalpha and TM-alpha1 and cell toxicity was less than combination of IFNalpha and TM-alpha1. The above results show that it has effect bath antivirus of IFNalpha and promoting lymphocyte proliferation of the soluble fusion protein expressed in E. coli.
Amino Acid Sequence ; Animals ; Antiviral Agents ; pharmacology ; Base Sequence ; Escherichia coli ; genetics ; metabolism ; Humans ; Interferon Type I ; biosynthesis ; genetics ; Interferon-alpha ; Mice ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Recombinant Proteins ; Thymosin ; analogs & derivatives ; biosynthesis ; genetics