We investigated the possibility of producing chicken alpha interferon (ChIFN-alpha) in transgenic plants. The cDNA encoding ChIFN-alpha was introduced into lettuce (Lactuca sativa L.) plants by using an agro-infiltration transient expression system. The ChIFN-alpha gene was correctly transcribed and translated in the lettuce plants according to RT-PCR and ELISA assays. Recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus (VSV) replication on chicken embryonic fibroblast (CEF). The results demonstrate that biologically active avian cytokine with potential pharmaceutical applications could be expressed in transgenic lettuce plants and that it is possible to generate interferon protein in forage plants for preventing infectious diseases of poultry.
Animals ; Chickens ; Interferon Type I ; biosynthesis ; Interferon-alpha ; genetics ; Lettuce ; genetics ; Plants, Genetically Modified ; genetics ; Recombinant Proteins

The effect of induction temperature on aggregation of consensus interferon-alpha expressed by Pichia pastoris was investigated. The cell growth and cIFN level were analyzed and compared when Pichia pastoris was grown at 30,25,20 degrees C during induction phase, using 5.0L fermentor. The result suggested that the cell growth was not affected much under the different induction temperature, but the protein level declined markedly with the decrease of the induction temperature. The total protein ammount induced at 20 degrees C was 67.8 percent of that at 30 degrees C. SDS-PAGE and native-PAGE as well as Western blotting analysis were further conducted. The electrophoresis results revealed that cIFN formed aggregates after secreted into media when protein was induced at 30 degrees C but this problem can be restored by decreasing the induction temperature to 20 degrees C. cIFN monomer in supernatant arrived at 570mg/L and bioactivity of fermentation broth reached 1.05 x 10(9) IU/mL at 20 degrees C of induction temperature. The amount of cIFN monomer and bioactivity in supernatant elevated 7.2 and 38.7 times, respectively, when the induction temperature was controlled at 20 degrees C instead of conventional 30 degrees C.
Fermentation ; Humans ; Interferon Type I ; biosynthesis ; genetics ; Interferon-alpha ; Pichia ; genetics ; growth & development ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Temperature

Hydrophobic interaction chromatography was used to separate correctly refolded and mis-refolded consensus interferon. The effects of ligand types, salt concentration, pH and flow rate were investigated. The best result could be obtained by using Butyl Sepharose 4 Fast Flow, 0.8 mol/L of ammonium sulfate, pH 8.3 and 90cm/h of linear flow rate. Reverse-phase HPLC analysis showed the purity of the pooled fraction was as high as 99.6%. The specific activity of purified consensus interferon was 2.3 x 10(9) IU/mg, The mass recovery of targeth protein was 36.7%.
Chromatography, Liquid ; methods ; Hydrophobic and Hydrophilic Interactions ; Interferon Type I ; chemistry ; isolation & purification ; Interferon-alpha ; Protein Conformation ; Protein Folding ; Recombinant Proteins

BACKGROUNDTo study the anti-SARS virus activities of different recombinant human interferons on the cell culture system.

METHODSAnti-SARS virus activities of interferons were determined by using CPE inhibition test in human skeletal muscle sarcoma (Rda) cell culture.

RESULTSThe average minimum amount of interferon alpha 2b, alpha 1b, beta 1b or omega 1b to inhibit 50% CPE in Rda cell culture was (160.5+/-129.5) IU/ml, (149.0+/-71.7) IU/ml, (69.5+/-61.5) IU/ml, (87.3+/-47.1) IU/ml, respectively or (0.6+/-0.5) ng/ml, (10.6+/-5.1) ng/ml, (3.5+/-3.1) ng/ml, (0.9+/-0.5) ng/ml, respectively.

CONCLUSIONAll the tested recombinant interferons showed anti-SARS virus activities on the Rda cell culture with different sensitivities.

Antiviral Agents ; pharmacology ; Cell Line, Tumor ; Humans ; Interferon Type I ; pharmacology ; Interferon-alpha ; pharmacology ; Recombinant Proteins ; SARS Virus ; drug effects ; Severe Acute Respiratory Syndrome ; drug therapy ; virology

Both IFN-omega and IFN-alpha belong to type I interferon and have antiviral, antiproliferative, immunomodulatory activities, but their bioactivities are usually different. FeIFN-omega gene was amplified by PCR. FeIFN-alpha gene was synthesized based on the published sequences of GenBank. Then the two types of feline interferon genes were subcloned into the pET-His vector, and expressed in Escherichia coli Rosetta (DE3). Recombinant interferons were purified by affinity chromatography with immobilized nickel chelating NTA (Ni-NTA) and their antiviral activity was estimated according to the ability of IFNs to inhibit the cytopathic effects (CPE) of virus on cells. Results showed that the antiviral activities against various viruses of FeIFN-omega were higher than those of FeIFN-alpha. Against H9N2 subtype avian influenza virus (AIV) and canine distemper virus (CDV), the antiviral activities of FeIFN-omega were 160 folds and 4 folds higher than those of FeIFN-alpha.
Animals ; Antiviral Agents ; pharmacology ; Cats ; Distemper Virus, Canine ; drug effects ; Influenza A Virus, H9N2 Subtype ; drug effects ; Interferon Type I ; biosynthesis ; genetics ; pharmacology ; Interferon-alpha ; biosynthesis ; genetics ; pharmacology

OBJECTIVETo explore the therapeutic mechanisms of interferon-beta (IFN-beta) and intravenous immunoglobulin (IVIG) for experimental peripheral neuropathy induced by Campilobacter jejuni (Cj) lipopolysaccharide (LPS).

METHODForty healthy Wistar rats weighing 205 - 230 g were divided into IFN-beta, IVIG, IFN-beta plus IVIG and control groups. After the immune neuropathy was induced in the rats by Cj LPS, IFN-beta (1.3 microg/kg) was given by subcutaneous injection to the rats every other day for 6 weeks; IVIG [400 mg/(kg x d)] was given to the rats for five days, every other week for two times and IFN-beta [1.3 microg/(kg x d)] and IVIG [400 mg/(kg x d)] were given to the rats on the same days. Meanwhile, the control group was given PBS. The sera were collected in the 2nd, 4th and 6th week after therapy, the titers of anti-GM(1) IgG, MMP-9 and TNF-alpha in sera of immunized rats were measured by ELISA; histological study of sciatic nerve was performed and IgG on sciatic nerve was detected by immunohistochemistry in the 6th week.

RESULTS(1) There were no significant differences in titers of anti-GM(1) IgG, MMP-9 and TNF-alpha among the 3 therapeutic groups and control group after therapy for 2 weeks (P > 0.05). (2) The titers of anti- GM(1) IgG, MMP-9 or TNF-alpha in the control group were much higher than those of the IFN-beta group, the IVIG group or the IFN-beta and IVIG group after therapy for 4 weeks (P > 0.01) and there were no significant differences in titers of antibody among the 3 therapeutic groups (P > 0.05); the titers of MMP-9 or TNF-alpha in the IFN-beta and IVIG group were lower than those of the IFN-beta group or the IVIG group (P < 0.05). (3) The titers of anti-GM(1) IgG, MMP-9 or TNF-alpha in the control group were much higher than those of the IFN-beta group, the IVIG group or the IFN-beta with IVIG group after therapy for 6 weeks (P > 0.01); the IFN-beta with IVIG group had much lower levels of all indexes than the IFN-beta group or the IVIG group (P < 0.01).

CONCLUSIONIFN-beta and IVIG showed therapeutic effects on immune peripheral neuropathy through inhibiting the humoral and cellular immunity simultaneously in the peripheral neuropathy induced by CJ LPS, treatment with combined IFN-beta and IVIG was more effective.

Animals ; Enzyme-Linked Immunosorbent Assay ; Immunoglobulins, Intravenous ; therapeutic use ; Immunotherapy ; Interferon Type I ; therapeutic use ; Interferon-beta ; immunology ; therapeutic use ; Lipopolysaccharides ; pharmacology ; Peripheral Nervous System Diseases ; therapy ; Rats ; Rats, Wistar ; Recombinant Proteins ; Sciatic Nerve ; drug effects ; Tumor Necrosis Factor-alpha ; immunology

BACKGROUNDBased on the earlier mapping of the epitope recognized by neutralizing antibody, the authors directly replaced binding domain of IFN in AB-loop for enhancement of biological activity.

METHODSTwo unique restriction sites (EcoR? and BsE?) were created into region flanking LoopAB. Casette mutagesis, restriction enzyme digestion, DNA sequencing, antiviral activity assay and antiproliferative activity assay have been used in the project.

RESULTSThe mutated residues M31?D,D32?P of LoopAB in parent IFN were produced. The recombinant phagemid pCANTAB5E/3132IFN 1c/86D and expression plasmid PBV322-132IFN 1c/86D were constructed respectively by replacing the corresponding LoopAB with DNA fragment mutated in the residues M31?D,D32?P, which have been confirmed. The recombinant protein has been expressed in E.coli JM103. The crude 3132IFN 1c/86D has been assayed on human WISH cells challenged with VSV and on HeLa cells by colorimetric MTT. 3132IFN 1c/86D showed 8-old antiviral activity compared to that of parent IFN 1c/86D, while IFN?induced growth inhibition of both types had no difference.

CONCLUSIONSThe authors concluded that a mutant IFN with enhanced antiviral activity can be obtained via a targeted replacement of receptor binding domain in AB-loop.

Amino Acid Substitution ; Antibodies, Viral ; Antiviral Agents ; pharmacology ; Cells, Cultured ; DNA Mutational Analysis ; Humans ; Interferon Type I ; genetics ; pharmacology ; Interferon-alpha ; Mutagenesis, Site-Directed ; Mutation ; Peptide Fragments ; genetics ; pharmacology ; Plasmids ; genetics ; Receptors, Interferon ; metabolism ; Recombinant Proteins

Virus infection induces the production of type I interferons (IFNs). IFNs bind to their heterodimeric receptors to initiate downstream cascade of signaling, leading to the up-regulation of interferon-stimulated genes (ISGs). ISGs play very important roles in innate immunity through a variety of mechanisms. Although hundreds of ISGs have been identified, it is commonly recognized that more ISGs await to be discovered. The aim of this study was to identify new ISGs and to probe their roles in regulating virus-induced type I IFN production. We used consensus interferon (Con-IFN), an artificial alpha IFN that was shown to be more potent than naturally existing type I IFN, to treat three human immune cell lines, CEM, U937 and Daudi cells. Microarray analysis was employed to identify those genes whose expressions were up-regulated. Six hundred and seventeen genes were up-regulated more than 3-fold. Out of these 617 genes, 138 were not previously reported as ISGs and thus were further pursued. Validation of these 138 genes using quantitative reverse transcription PCR (qRT-PCR) confirmed 91 genes. We screened 89 genes for those involved in Sendai virus (SeV)-induced IFN-β promoter activation, and PIM1 was identified as one whose expression inhibited SeV-mediated IFN-β activation. We provide evidence indicating that PIM1 specifically inhibits RIG-I- and MDA5-mediated IFN-β signaling. Our results expand the ISG library and identify PIM1 as an ISG that participates in the regulation of virus-induced type I interferon production.
Cells, Cultured ; Gene Library ; Humans ; Interferon Type I ; metabolism ; Interferon-beta ; genetics ; metabolism ; Proto-Oncogene Proteins c-pim-1 ; genetics ; Up-Regulation

The full length of chicken interferon alpha (ChIFN-alpha) gene was amplified by the polymerase chain reaction (PCR) from total liver genome of Sanhuang meat-chicken and sequenced. The amplified gene was about 582bp. The coding region for mature protein (489bp) was subcloned into pET-28a(+). The recombinant plasmid pET-28a(+)-IFNalpha was identified by enzyme digestion and DNA sequencing. Data of SDS-PAGE and Western-blot indicated that a 22kD fusion protein was expressed in the form of inclusion bodies with good immunity. The purity of inclusion bodies was above 70% and that of protein purified by nickel affinity chromatography was 95%. The recombinant protein could inhibit H9N2 avian influenza virus (H9N2 AIV) replication on chick embryo fibroblast. 2 microg of recombinant IFN-alpha could completely protect Chick embryo from H9N2 AIV infection. The recombinant IFN-alpha can also delay Newcastle disease virus (NDV) replication on chick embryo for 12 approximately 48h. Chicken administered recombinant IFN-alpha can resist the H9N2 AIV infection. The bioactivities of recombinant IFN-alpha purified by affinity chromatograph were 20 times higher than that of inclusion bodies.
Animals ; Antiviral Agents ; pharmacology ; Blotting, Western ; Chick Embryo ; Cloning, Molecular ; Influenza A Virus, H9N2 Subtype ; drug effects ; Interferon Type I ; biosynthesis ; isolation & purification ; pharmacology ; Interferon-alpha ; genetics ; Newcastle disease virus ; drug effects ; Plasmids ; Recombinant Proteins

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