Expression of ChIFN-α in Pichia pastoris and Optimization of Its Expression Condition

Because yeast codon preferred to synthesize the gene sequence of ChIFN-α mature peptide, Pichia pastoris expression system was connected into vector pPICgK, to construct recombinant plasmid pPIC9K-α. After digestion and linearization, the recombinant vector was cloned into P. pastor/s GS115, high copy transformant and its methanol utilization type were screened, and its expression conditions were optimized. The results showed that ChlFN-α successfully expressed in P. pastor/s, the expression amount of recombinant protein was the largest after induced expression in 0.5 % methanol containing 2% acid hydrolyzed casein at 28 ℃ for 72 h, and its antiviral activity was the highest.

Association of Host Interferon-γ Gene Polymorphism with Toxoplasma gondii Infection in Pregnant Women of Bangladesh

Human toxoplasmosis is caused by the intracellular protozoan parasite Toxoplasma gondii. Although T. gondii infection is generally asymptomatic for most of the immunocompetent adults, severe complications may occur particularly in pregnant women and immunocompromised individual. Host cell immunity plays a critical role in parasite differentiation and persistence in the host. Therefore, genetic polymorphism in the host immune genes, for instance interferon-γ gene could be linked with possibility of T. gondii infection. The objective of the study was to verify the link between the single nucleotide polymorphisms (SNPs) in the IFN-γ gene of pregnant women and T. gondii infection through correlating with anthropometric and sociodemographic parameters. In this study, ninety-two (N = 92) pregnant women (16 - 40 years) and healthy controls (N = 95) with similar age ranges were included. Among them, 25% (n = 23) pregnant women were seropositive for T. gondii IgG antibodies by Rapid Test Assay. Allelic and genotypic frequencies of IFN-γ +874T/A (rs2430561) SNPs were evaluated by using ARMS-PCR. The distribution of the A and T alleles in the specific position of the IFN-γ gene in the T. gondii-infected pregnant women and the control groups did not differ significantly, according to the data. However, we found a higher frequency (13.04%) of A/A genotype in T. gondii infected pregnant women as compared to non-infected individuals (8.70%), demonstrating that T. gondii infection susceptibility may be increased by homozygosity for the A allele. Further studies are to be needed to find out the link between host gene polymorphism and T. gondii infection in Bangladesh.

Interferon-α response in chronic hepatitis B-transfected HepG2.2.15 cells is partially restored by lamivudine treatment

AIM：To characterize the IFN-response and its modulation by the antiviral compound lamivudine in HBV- transfected HepG2.2.15 cells. METHODS： HepG2.2.15 and HepG2 cells were stimulated with various concentrations of IFN-α 2a in the presence or absence of lamivudine. Then, total RNA was extracted and analysed by customised cDNA arrays and northern blot for interferon-inducible genes （ISGs）. In addition, cellular proteins were extracted for EMSA and western blot. HBV replication was assessed by southern blot or ELISAs for HBsAg and HBeAg. RESULTS： Two genes （MxA, CigS） with completely abolished and 4 genes （IFITM1, -2, -3, and 6-16） with partially reduced IFN-responses were identified in HepG2.2.15 cells. In 2 genes （IFITM1, 6-16）, the response to IFN-α could be restored by treatment with lamivudine. This effect could not be explained by a direct modulation of the Jak/Stat signalling pathway since EMSA and western blot experiments revealed no suppression of Statl activation and ISGF3 formation after stimulation with IFN-α in HepG2.2.15 compared to HepG2 cells. CONCLUSION： These results are consistent with the assumption that chronic hepatitis B may specifically modulate the cellular response to IFN by a selective blockage of some ISGs. Antiviral treatment with lamivudine may partially restore ISG expressionby reducing HBV gene expression and replication.

Repression of interferon-γ expression in T cells by Prosperorelated Homeobox protein

Interferon-gamma （IFN-γ） is a major proinflammatory effector and regulatory cytokine produced by activated T cells and NK cells. IFN-γ has been shown to play pivotal roles in fundamental immunological processes such as inflammatory reactions, cell-mediated immunity and autoimmunity. A variety of human disorders have now been linked to irregular IFN-γ expression. In order to achieve proper IFN-γ-mediated immunological effects, IFN-γ expression in T cells is subject to both positive and negative regulation. In this study, we report for the first time the negative regulation of IFN-γ expression by Prospero-related Homeobox （Proxl）. In Jurkat T cells and primary human CD4＋ T cells, Proxl expression decreases quickly upon T cell activation, concurrent with a dramatic increase in IFN-γ expression. Reporter analysis and chromatin immunoprecipitation （CHIP） revealed that Proxl associates with and inhibits the transcription activity of IFN-γ promoter in activated Jurkat T cells. Co-immunoprecipitation and GST pull-down assay demonstrated a direct binding between Proxl and the nuclear receptor peroxisome proliferator-activated receptor gamma （PPARγ）, which is also an IFN-γ repressor in T cells. By introducing deletions and mutations into Proxl, we show that the repression of IFN-γ promoter by Proxl is largely dependent upon the physical interaction between Proxl and PPARγ. Furthermore, PPARγ antagonist treatment removes Proxl from IFN-γ promoter and attenuates repression of IFN-γ expression by Proxl. These findings establish Proxl as a new negative regulator of IFN-γ expression in T cells and will aid in the understanding of IFN-γ transcription regulation mechanisms.

Analysis Tissue Expression of IFN-γin IL-12 and/or IL-18 Gene Ablated Na(?)ve Mice

Interleukin 12(IL-12)and/or interleukin 18(IL-18)gene ablated mice were applied for the investigation of the tissue expression of interferon γ(IFN-γ).For IL-12^(-/-),IL-18^(-/-),IL-12^(-/-)/18^(-/-) and wt mice,reproductive performance were recorded and IFN-γ concentrations in heart,lung,liver,spleen,kidney and serum were quantified by ELISA. There were no significant differences of IFN-γ in heart,lung and kidney between 4 strains although control group was higher.It was observed that for IL-12^(-/-) mice,compared with other 3 groups,IFN-γ in liver and spleen were decreased(p<0.05)and reproductive performance appeared to be impaired.Serum IFN-γ level of IL-12^(-/-)/18^(-/-) mice was significantly higher(p<0.05).It was showed that IFN-γ productions under the normal condition were independent upon IL-12 and IL-18,its expressions in various tissues were different,and optimal IFN-γ is necessary for the normal growth and development of mammals.This study is helpful for clinical cytokines therapy.Cellular & Molecular Immunology.2005;2(1):68-72.

(-)-Epigallocatechin-3-gallate enhances poly I:C-induced interferon-λ1 production and inhibits hepatitis C virus replication in hepatocytes

AIM To investigate the effect of(-)-epigallocatechin-3-gallate(EGCG) on polyinosinic-polycytidylic acid(poly I:C)-triggered intracellular innate immunity against hepatitis C virus(HCV) in hepatocytes. METHODS A cell culture model of HCV infection was generated by infecting a hepatoma cell line, Huh7, with HCV JFH-1 strain(JFH-1-Huh7). Poly I:C with a high molecular weight and EGCG were used to stimulate the JFH-1-Huh7 cells. Real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of intracellular m RNAs and of intracellular and extracellular HCV RNA. Enzyme-linked immunosorbent assay was used to evaluate the interferon(IFN)-λ1 protein level in the cell culture supernatant. Immunostaining was used to examine HCV core protein expression in Huh7 cells.RESULTS Our recent study showed that HCV replication could impair poly I:C-triggered intracellular innate immune responses in hepatocytes. In the current study, we showed that EGCG treatment significantly increased the poly I:C-induced expression of Toll-like receptor 3(TLR3), retinoic acid-inducible gene I, and IFN-λ1 in JFH-1-Huh7 cells. In addition, supplementation with EGCG increased the poly I:C-mediated antiviral activity in JFH-1-Huh7 cells at the intracellular and extracellular HCV RNA and protein levels. Further investigation of the mechanisms showed that EGCG treatment significantly enhanced the poly I:C-induced expression of IFN-regulatory factor 9 and several antiviral IFNstimulated genes, including ISG15, ISG56, myxovirus resistance A, and 2'-5'-oligoadenylate synthetase 1, which encode the key antiviral elements in the IFN signaling pathway. CONCLUSION Our observations provide experimental evidence that EGCG has the ability to enhance poly I:C-induced intracellular antiviral innate immunity against HCV replication in hepatocytes.

Identification of new type I interferon- stimulated genes and investigation of their involvement in IFN-β activation

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 expres- sions 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 Sendal virus （SeV）-induced IFN-13 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 iden- tify PIM1 as an ISG that participates in the regulation of virus-induced type I interferon production.

Screening and identification of binding proteins to interferon-α from a cDNA library by yeast-two hybrid system

The aim of this study is to screen proteins interacting with interferon-α （IFN-α）. The IFN-α gene was amplified by polymerase chain reaction （PCR） and cloned into pGBKT-/vector, then the resulted pGBKT-/-IFN-α vector was transformed into yeast strain AH109. The transformed yeast AH109 was mated with yeast Y187 containing liver cDNA library plasmid in 2 × YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade and SD/-Trp-Leu-His） con- taining X-α-gal for selection. After plasmid extraction and enzyme cutting analysis, the blue colonies were subjected to sequence analysis and the results were analyzed by bioinformatics. The results showed that IFN-α was successful cloned into the pGBKT7 vector. IFN-α was expressed and there was no selfactivation and toxicity in AH109. Thirty-four positive colonies were obtained after yeast-two hybrid technique screening. After sequence analysis, eight clones were found to have a binding effect with IFN-α protein. IFN-α was successfully cloned into the pGBKT7 vector. IFN-α protein was expressed and there was no self-activation and toxicity in AH109. Eight proteins that interacted with IFN-α, including vitronectin, fibrinogen A alpha polypeptide, HIV-1 Tat interactive protein 2, arginase, NADH dehydrogenase 1 beta subcomplex, transferrin receptor 2 alpha （TFR2）, HCC-1, alcohol dehydrogenase IB （ADHIB） have been identified as IFN-α-binding proteins.

Preliminary Study on Mouse Interleukin-21 Application in Tumor Gene Therapy

lnterleukin-21(IL-21)is a recently characterized T cell-derived cytokine with a significant homology to IL-2, IL-4 and IL-15.To determine whether IL-21 has broad immunoregulatory activity and can stimulate durable antitumour responses,we constructed mouse IL-21(mIL-21) recombinant plasmid and evaluated its antitumor efficacy.Mouse IL-21 cDNA was amplified from Con A-activated mouse T cells by RT-PCR.Recombinant pcDNA3.1/mIL-21 was constructed and transfected into Sp2/0 cells.Mouse IL-21 expression was analyzed by Western blotting and its activities were detected by ~3H-TdR incorporation and MTT assay.The recombinant pcDNA3.1/mIL-21 was injected s.c.into tumor lump.Tumor size,weight,the activities of CTLs,NK cells and LAK cells and serum IFN-γ,level were measured for evaluating mIL-21 mediated antitumor responses.The results indicated that mIL-21 was correctly expressed in Sp2/0 cells and it can improve the proliferation of T cells and B cells,and enhance NK cytotoxic activity in vitro.The activities of CTL and NK cells,and serum IFN-γlevel were significantly improved,furthermore the tumor growth was obviously suppressed in pcDNA3.1/mIL-21 treated mice.However,the LAK activity did not alter significantly.Taken together,this study suggests that the injection with recombinant plasmid containing mIL-21 is a potential approach for tumor gene therapy.Cellular & Molecular Immunology.2004;1(6):461-466.

High expression of synthetic human interferon-γ cDNA in E.coli

Human interferon-γ(IFN-γ)cDNA was synthesized,and it makes the usage of favorable codons in E.coli.The authors got 9 different expression plasmids which contain the synthetic IFN-γ-cDNA and have different spaces between SD sequence and ATG.The free energies G_(f298)~0 in the formation of stable secondary structure in the translation initiation region(TIR)are different in various expression plasmids.One of them, pLY_4-γ_5,can highly yield INF-γ which will be about 60%—80% of the total bacterial proteins,such a high expression was hardly noted in literature.The reasons of high expression in this work are optimal spaces be- tween SD and ATC,favorable △G_(f298)~0,favourable codons usage for E.coli. A simple and rapid method of purification of IFN-γ was developed.The purity of IFN-γ will be over 95%,and the specific activity will be 2.0×10~7 IU/mg after islotation of inclusion body and one step chromatography.

EXPRESSION OF HUMAN INTERFERON-α cDNA IN TRANSFORMED TOBACCO PLANT

Human interferon-α cDNA has been successfully introduced into tobacco plant cells. And some plants producing active IFN-α have been obtained. By blunt end ligation of DNA, human IFN-α cDNA was inserted into the BamHI site between the promotor of transcripton 1 and the tailing signal of transcripton 7 in the plant expression plasmid pAP2304, and so a recombinant plasmid pIG3031 was constructed, and the recombinant plasmid was introduced into the T-region of the Ti plasmid vector pGV3850. By means of agrobacterium infection to the leaf disc of general tobacco plant 1551, some transformed plants resistant to kana-mycin were obtained. DNA Southern hybridization showed that the human IFN-α gene together with T-DNA was integrated into the genome of tobacco cells. The assay of neomycin phosphate transferase Ⅱ indicated that the ability of resistance to kanamycin in the transformed plants resulted from the expression of the NPT Ⅱ gene. The assay of the biological activity of IFN-α showed that active IFN-α could be produced from the transformed plants.