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

There are many E. coli rare codons in the gene of porcine interferon alpha-1. In order to obtain high expression of poIFN-alpha1 in E. coli, the cDNA encoded poIFN-alpha1 mature protein was synthesized using biased codons of E. coli without changing the original amino acid sequence and the terminator was changed as TAA. At the same time, Adenine and Thymine were used to the largest extent near the 5' terminus of poIFN-alpha1 mature protein gene. The synthesized gene was inserted into the Eco RI and Sal I site of the expression vector pRLC resulting pRLC-poIFN-alpha1. The poIFN-alpha1 is highly expressed in E. coli DH5alpha when the induction was carried out at 42 degrees C . The expressed poIFN-alpha1 account for 24.5% of the total cellular proteins and existed as inclusion body. The poIFN-alpha1 inclusion body was dissolved in 6mol/L guanidine chloride contained DTT and subsequently the denatured poIFN-alpha1 was re-natured by dilution in refolding buffer containing GSH and GSSH. In the present study it was found that the denatured poIFN-alpha1 was most efficiently re-natured in refolding buffer containing 1 mol/L guanidine chloride. In order to obtain pure protein, the concentrated re-natured poIFN-alpha1 was purified by Sephacryl S-200 chromatography. As a result, the purified poIFN-alpha1 is verified to be of high cytokine activity by inhibiting the cytopathic effect of vesicular stomatitis virus in MDBK cells, which is about 6.4 x 10(6) u/mg. This study paved the way for large-scale production of recombinant poIFN-alpha1 and its usage in virus disease control of pigs.
Animals ; Codon ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Interferon-alpha ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Swine ; Transduction, Genetic

The study was aimed to investigate the synergistically effect of interferon-&alpha; (IFN-&alpha;) and homoharringtonine (HHT) on the proliferation, apoptosis, cell cycle of K562 cells and the expression of β-catenin. The proliferation, apoptosis, cell cycle and β-catenin mRNA expression of K562 cells treated with IFN-&alpha; and/or HHT were assayed with MTT, flow cytometry or RT-PCR respectively. The results showed that HHT alone, but not IFN-&alpha; alone, displayed a proliferation inhibition, apoptosis induction, G(0)/G(1) phase block and down-regulation of β-catenin expression in K562 cells with concentration- and time-dependent manners. The expression level of β-catenin mRNA after being treated with HHT was 0.5576 ± 0.0373, which were lower than that in control group (0.9369 ± 0.0142). The down-regulation of β-catenin expression in group of IFN-&alpha; combined with HHT was higher significantly than that in HHT group (0.3737 ± 0.0529 vs 0.5576 ± 0.0373, P < 0.05). Otherwise, HHT combined with IFN-&alpha; did not demonstrate obvious toxicologic effect on bone marrow mononuclear cells. It is concluded that IFN-&alpha; combined with HHT can enhance the cytotoxic effect of HHT on K562 cells, which may be associated with down-regulation of β-catenin expression.
Cell Proliferation ; drug effects ; Harringtonines ; pharmacology ; Humans ; Interferon-alpha ; pharmacology ; K562 Cells ; beta Catenin ; genetics ; metabolism

OBJECTIVETo screen enhancer-like sequences from vaccinia virus genome, to construct an expression vector harboring prokaryotic enhancer-like sequence and study the effect of interferon gene expression.

METHODSEnhancer-like element from vaccinia virus genome was obtained by using the chloramphenicol acetyl-transferase cat gene as reporter gene. An expression vector harboring prokaryotic enhancer-like sequence VV1 from vaccinia virus was constructed. Interferon was expressed and assayed.

RESULTSEighteen enhancing sequences were found. From them two enhancer-like sequences with distance and orientation independence property were screened and named VV1 and VV16 respectively. Quantification test showed that the direct and reverse orientation of VV1 could increase the activity of beta-galactosidase with 10.9 and 3.8 times and those of VV16 could increase by 9.0 times and 4.1 times respectively. The enhancing activity of the element was on transcription level. An expression vector harboring prokaryotic enhancer-like sequence VV1 was constructed. Using this vector the antiviral activity of interferon alpha-2b was increased by 2.6 times in comparison with the original expression plasmid.

CONCLUSIONTwo enhancer-like sequences were screened from vaccinia virus genome. Interferon gene was highly expressed by using an expression vector harboring enhancer-like sequences.

Enhancer Elements, Genetic ; Genetic Vectors ; genetics ; Interferon-alpha ; biosynthesis ; genetics ; pharmacology ; Plasmids ; Recombinant Proteins ; Vaccinia virus ; genetics

OBJECTIVETo construct a recombinant bacillus Calmette-Guérin vaccine (rBCG) secreting human interferon-alpha 2b (IFN alpha-2b).

METHODSBCG Ag85B signal sequence and IFN alpha-2b gene were amplified from the genome of BCG and of human peripheral blood by polymerase chain reaction (PCR), respectively. IFN alpha-2b gene was cloned in E. coli-BCG shuttle-vector pMV261 to get pMV261-IFN alpha-2b. A new recombinant plasmid pMV261-IFN alpha-2b was constructed by inserting BCG Ag85B signal sequence into pMV261-Ag85B-IFN alpha-2b. Then, BCG was transformed with this recombinant plasmid by electroporation, and designated as rBCG-IFN alpha-2b. The DNA and protein expressions of IFN alpha-2b gene in rBCG were determined by PCR and Western blot respectively. Also the quantity of IFN alpha-2b protein secreted by rBCG in culture supernatants was determined by enzyme linked immunosorbent assay (ELISA).

RESULTSBy partial nucleotide sequencing, the DNA sequences of human IFN alpha-2b and BCG Ag85B were consistent with that in the Gene Bank, and were correctly inserted into the shuttle expression vector pMV261 to construct recombinant plasmid pMV261-Ag85B-IFN alpha-2b. BCG was successfully transformed with this recombinant plasmid by electroporation and the recombinant BCG (rBCG-IFN alpha-2b) was capable of synthesizing and secreting cytokine IFN alpha-2b. The concentration of IFN alpha-2b in culture supernatants was quantified by ELISA and calculated to be approximately 301.45 pg/ml.

CONCLUSIONSRecombinant BCG secreting human IFN alpha-2b (rBCG-IFN alpha-2b) was constructed successfully and the specific IFN alpha-2b protein can be expressed highly and steadily by rBCG vaccine.

BCG Vaccine ; genetics ; immunology ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Interferon-alpha ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Proteins ; Transformation, Bacterial

Porcine interferon-alpha (pIFN-alpha) fermentative production by recombinant Pichia pastoris was carried out in a 10-L bioreactor to study its metabolism changes and effects on fermentation under different inducing strategies, by analyzing the change patterns of the corresponding metabolism and energy regeneration. The results show that the specific activities of alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH) largely increased when reducing temperature from 30 degrees C to 20 degrees C under pure methanol induction, leading significant enhancements in methanol metabolism, formaldehyde dissimilatory energy metabolism and pIFN-alpha antiviral activity. The highest pIFN-alpha antiviral activity reached 1.4 x 10(6) IU/mL, which was about 10-folds of that obtained under 30 degrees C induction. Using methanol/sorbitol co-feeding strategy at 30 degrees C, the major energy metabolism energizing pIFN-alpha synthesis shifted from formaldehyde dissimilatory energy metabolism pathway to TCA cycle, formaldehyde dissimilatory pathway was weakened and accumulation of toxic intermediate metabolite-formaldehyde was relieved, and methanol flux distribution towards to pIFN-alpha synthesis was enhanced. Under this condition, the highest pIFN-alpha antiviral activity reached 1.8 x 10(7) IU/mL which was about 100-folds of that obtained under pure methanol induction at 30 degrees C. More important, enhanced pIFN-alpha production with methanol/sorbitol co-feeding strategy could be implemented under mild conditions, which greatly reduced the fermentation costs and improved the entire fermentation performance.
Animals ; Energy Metabolism ; Fermentation ; Interferon-alpha ; biosynthesis ; genetics ; Methanol ; pharmacology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Sorbitol ; pharmacology ; Swine

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

BACKGROUNDRegulated on activation, normal T-cell expressed and secreted (RANTES) plays a critical role in T-lymphocyte activation and proliferation. The process is involved in both acute and chronic phases of inflammation. The present study was to ascertain the possible correlations between chronic hepatitis B virus (HBV) infection and the RANTES gene polymorphisms and their expression.

METHODSThe study included 130 HBV negative healthy donors and 152 patients with chronic hepatitis B (CHB) virus infection. The polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLPs) were used to detect RANTES gene single nucleotide polymorphisms (SNPs). RANTES levels in the platelet depleted plasma were detected by enzyme linked immunosorbent assay (ELISA).

RESULTSRANTES alleles -403G, -28C and In1.1T were the predominant alleles in the subjects studied. No significant correlation was found between CHB infection and the RANTES alleles, while a significant correlation was found between CHB infection and increased RANTES expression in platelet depleted plasma (P < 0.05).

CONCLUSIONSSNPs in RANTES gene do not affect chronic HBV infection or the outcome of interferon-alpha treatment in patients positive for HBV "e" antigen (HBeAg+). However, patients with CHB infection express the higher levels of plasma RANTES, which is thus associated with CHB infection.

Alleles ; Chemokine CCL5 ; genetics ; Genotype ; Hepatitis B, Chronic ; drug therapy ; genetics ; Humans ; Interferon-alpha ; therapeutic use ; Polymorphism, Single Nucleotide

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