The porcine interferon-gamma (PoIFN-gamma) gene, in which the sequence encoding signal peptide was replaced by that of the alpha-factor of Saccharomyces cerevisiae, was cloned into Pichia pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-alpha-PoIFN-gamma was then transformed into Pichia pastoris GS115 cells by electroporation and stable multicopy recombinant Pichia pastoris strains were selected by G418 resistance. Two recombinants of multiple inserts were obtained. SDS-PAGE and Western blot assays of culture broth from a methanol-induced expression strain demonstrated that recombinant PoIFN-gamma, 17kD and 23kD proteins, were secreted into the culture medium. Target proteins, 60% of total proteins, were obtained in the culture medium at the concentration of 108 mg/L. This is the first secreted expression of porcine interferon-gamma gene in Pichia pastoris.
Animals ; Electroporation ; Interferon-gamma ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Swine ; genetics

The total RNA was extracted from peripheral blood mononuclear cells (PBMC) which was isolated from Meishan porcine and induced with concanavaline A (ConA), then the porcine interferon gamma gene (PoIFNgamma, 501bp) was amplified by RT-PCR. The result of sequencing demonstrated that the amplified PoIFNgamma had 100% nucleotide homology with the other porcine IFNgamma sequence published on GenBank. The objective gene (PoIFNgamma) was inserted into adenoviral shuttle vector, pShuttle-CMV, to construct recombinant plasmid pSh-PoIFNgamma. And it was co-electrotransformated with adenoviral skeletal vector pAdEasy-1 into competent cells of BJ5183. The transforms were cultured at 37 degrees C for 24h on kanamycin resistance plate and selected for smaller colonies. Then, the extracted recombinant plasmid was named pAd-Sh-PoIFNgamma, which was confirmed by Pac I digestion, and transformed into XL10-Glod(r) for copious preparation. pAd-Sh-PoIFNgamma linearized with Pac I was co-transfected with liposome into 293 package cell-line. After 7d-10d, the typical cytopathic effect indicated that recombinant adenoviral genome (deleted with E1 and E3 genes) carrying PoIFNgamma was successfully packaged into intact virion. The recombinant virion was successively seeded to the 10th generation and the viral genome was extracted from each generation by PCR. The antiviral activity of PoIFNgamma was tested by CPE50 method. The results showed that the PoIFNgamma expressed by adenovirus had high antiviral activity, which was 1.3 x 10(6) U/mL against VSV in MDBK cells. The results demonstrated that the recombinant adenovirus carrying PoIFNgamma could be stably passaged.
Adenoviridae ; genetics ; Animals ; Antiviral Agents ; pharmacology ; Interferon-gamma ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; pharmacology ; Swine

In order to study PBD-2 and PoIFNgamma, the chimeric gene PBD-2-PoIFNgamma was synthesized by overlap extension PCR, and amplified PoIFNgamma on the basis of this sequence, then cloned into yeast expression vector pPICZalphaA separately to get the recombinant plasmid pPICZalphaA-PBD-2-PoINFgamma and pPICZalphaA-PoINFgamma. The recombinant plasmid was digested by Sac I and introduced into Pichia pastoris X33 cells by electroporation. Positive clones were screened and cultivated in BMMY medium containing 0.5% methanol for 72 h. SDS-PAGE and Western blotting analysis showed that the screened recombinant could secrete PBD-2-PoINFgamma and PoINFgamma separately. The activity of fusion protein was not detected by cytopathic effect inhibition assay and agar diffusion assay, but detected obvious antiviral activity of PoINFgamma. The helix and random coil contents was showed vary greatly between PoIFNgamma and PBD-2-PoLNFgamma by circular dichroism analysis. It was speculated that the fusion protein was not correctly folded and may affect the activity of PBD-2-PoINFgamma.
Animals ; Genetic Vectors ; genetics ; Interferon-gamma ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; Swine ; beta-Defensins ; biosynthesis ; genetics

To investigate the role of Interleukin-17 (IL-17), Interferon-gamma (IFN-gamma), and macrophage inflammatory protein-3 alpha (MIP-3alpha) in the pathogenesis of psoriasis, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to semi-quantitatively analyze the mRNA expression of IL-17, IFN-gamma, and MIP-3alpha in 31 psoriatic lesions and 16 normal skin tissues. The results showed that the mRNA of the three cytokines was present in all specimens. And the expression level of IL-17 mRNA in skin lesions was 1.1416 +/- 0.0591, which was significantly higher than that in normal controls (0.8788 +/- 0.0344, P<0.001). The expression levels of IFN-gamma mRNA were 1.1142 +/- 0.0561 and 0. 9050 +/- 0.0263, respectively, with significant difference (P<0.001). And the expression levels of MIP-3alpha mRNA in psoriatic lesions was 1.1397 +/- 0.0521, which was markedly higher than that in normal controls (0.8681 +/- 0.0308, P<0.001). These findings indicate that up-regulated expression of IL-17, IFN-gamma, and MIP-3alpha might be involved in the pathogenesis of psoriasis.
Adolescent ; Adult ; Aged ; Chemokine CCL20 ; Chemokines, CC ; biosynthesis ; genetics ; Female ; Humans ; Interferon-gamma ; biosynthesis ; genetics ; Interleukin-17 ; biosynthesis ; genetics ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; Male ; Middle Aged ; Psoriasis ; metabolism ; RNA, Messenger ; biosynthesis ; genetics

Interferons (IFNs) are natural proteins produced by wide variety of cells in response to viral infection or other biological inducers, and they execute diversified functions as antiviral defense, immune activation and cell growth regulation. Four genes encoding porcine interferons (PoIFN), PoIFN-alpha, PoIFN-gamma, PoIFN-alphagamma or PolFN-omega, were cloned and sequenced. The four types of porcine interferon genes were subcloned into the pET-His vector, and expressed in Escherichia coli Rosetta (DE3). The recombinant products were purified and renaturalized from inclusion bodies to obtain a native state of well biological activity. Antiviral activity assays for porcine interferons were performed and evaluated by standard procedures in following cell/virus test systems: Marc-145/PRRSV, Marc-145/VSV, PK-15/VSV, Vero/VSV or MDBK/VSV. The data showed that both PoIFN-alpha and PoIFN-alpagamma demonstrated significant antiviral activities, and the titer of them against PRRSV was up to 10(8) U/mg. PoIFN-gamma had approximately half or one-thirds antiviral activity of PoIFN-alpha. PoIFN-omega showed inconspicuous antiviral activity.
Amino Acid Sequence ; Animals ; Antiviral Agents ; pharmacology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Interferon Type I ; biosynthesis ; genetics ; pharmacology ; Interferon-gamma ; biosynthesis ; genetics ; pharmacology ; Molecular Sequence Data ; Porcine respiratory and reproductive syndrome virus ; drug effects ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Swine

BACKGROUNDStudies of highly exposed persistently seronegative (HEPS) individuals may provide valuable information on mechanisms of protection and on vaccine design. Cellular immune responses play a critical role in containing human immunodeficiency virus. However, the cellular immune responses in HEPS individuals have not been thoroughly assessed at the entire viral genome level.

METHODSTen HEPS Chinese with a history of frequent penetrative vaginal intercourse (mean frequency, at least once a week), with some unprotected sexual contact occurring in the weeks or days immediately before enrollment, 25 HIV-1 seropositive individuals, 10 HIV-1-seronegative healthy individuals with low-risk sexual behavior and no history suggestive of exposure to HIV-1 infection were enrolled. HIV-1-specific T cell responses were comprehensively analyzed by an interferon-gamma Elispot assay against 770 overlapping peptides spanning all HIV-1 proteins.

RESULTSHIV-1-specific T-cell responses of interferon-gamma secretion were identified in 3 (30%) out of 10 HEPS individuals; the specific cytotoxic T lymphocytes were targeted at Pol (2/10), Env (2/10), and Tat (1/10). HIV-1-specific T-cell responses of interferon-gamma secretion were identified in 20 (80%) out of 25 seropositive intravenous drug users (IDUs), revealing that all HIV-1 proteins and protein subunits could serve as targets for HIV-1-specific CD8(+) T cell responses with 85% recognizing Gag, 80% recognizing Nef, 75% recognizing Pol, 60% recognizing Env, 55% recognizing Vpu, 45% recognizing Vpr, 20% recognizing Vif, 20% recognizing Tat and 15% recognizing Rev in these seropositive individuals. None of the seronegative healthy individuals gave the positive T-cell responses.

CONCLUSIONSAbout 30% of HEPS Chinese mounted HIV-1 specific T cell immune responses. Cell-mediated immunity against HIV-1 may be developed through non-productive infections.

Adult ; Female ; HIV Seronegativity ; immunology ; HIV-1 ; immunology ; Humans ; Interferon-gamma ; biosynthesis ; Male ; Receptors, CCR5 ; genetics ; T-Lymphocytes, Cytotoxic ; immunology

The present study was purposed to investigate the relation and difference of expression phase between class II transactivator (CIITA) and HLA-DR antigens after IFN-gamma induction, and the inhibition of CIITA and HLA-DR by STAT1-alpha antisense oligonucleotides (STAT1-alpha AS); and to explore the potential effect and significance of CIITA and STAT1-alpha AS in transplantation immunity. T lymphocytes from peripheral blood of healthy subjects were incubated with IFN-gamma at different doses. RT-PCR was used to detect CIITA mRNA and Western blot was used to analyze HLA-DR antigen. Then the optimum dose of IFN-gamma was chosen for the experiment. CIITA mRNA and HLA-DR antigen were detected at various time points. Different doses of STAT1-alpha AS and sense oligonucleotides (STAT1-alpha S) were added to T lymphocytes followed by IFN-gamma. After incubation with IFN-gamma, the expression of CIITA mRNA and HLA-DR was detected once again. The results showed that CIITA mRNA was detectable at 5 hours after IFN-gamma incubation and reached the peak at 14 hours, then declined, but the CIITA mRNA was still found at 23 hours. HLA-DR antigen was detectable at 28 hours after IFN-gamma incubation and reached a peak at 52 hours, then declined. CIITA mRNA expression was positively correlated to HLA-DR expression, and was earlier than the latter. The expression of CIITA mRNA in the AS groups was significantly lower than that in the control group after 5 micromol/L, 10 micromol/L and 20 micromol/L STAT1-alpha AS treatment (P < 0.01). The expression of CIITA mRNA in the S groups was higher than that in the AS groups (P < 0.01), but there was no significant difference between the S group and the control group. The expression of HLA-DR antigen was significantly inhibited by STAT1-alpha AS, and the expression level of HLA-DR protein in the AS group was about 64.3% of that in the control group (P < 0.01), while there was no significant difference in HLA-DR expression between the S group and the control group. The changes in HLA-DR expression were similar to those in CIITA expression after STAT1-alpha AS treatment. It is concluded that CIITA expression is positively correlated with HLA-DR expression, and was detectable earlier than that of latter after IFN-gamma incubation. Stat1-alpha antisense oligonucleotides may have a sequence-specific inhibiting effect on the expression of CIITA and HLA-DR antigen after IFN-gamma incubation in vitro culture, and can prevent T lymphocyte activation. CIITA may play an important role in pathogenesis of transplantation immunity.
Cells, Cultured ; HLA-DR Antigens ; biosynthesis ; genetics ; Humans ; Interferon-gamma ; pharmacology ; Nuclear Proteins ; biosynthesis ; genetics ; Oligonucleotides, Antisense ; antagonists & inhibitors ; RNA, Messenger ; biosynthesis ; genetics ; STAT1 Transcription Factor ; antagonists & inhibitors ; T-Lymphocytes ; cytology ; Trans-Activators ; biosynthesis ; genetics ; Transplantation Immunology ; immunology

In order to investigate the effect of T-bet on malignant cells, we selected SGC-7901, a kind of human gastric carcinoma cell line, and used gene clone technique and adeno-associated virus (AAV) packing technology, thus obtaining a recombinant rAAV-eGFP-T-bet and T-bet gene-transfected SGC-7901 cells. Then the function of T-bet gene-infected SGC-7901 cells was researched by detecting the levels of IFN-gamma and T-bet production. The results showed: (1) It was verified that rAAV-T-bet's packing was completed; (2) After SGC-7901 cells was transfected by rAAV-eGFP-T-bet, a green fluorescence was found in about 30%-40% SGC-7901s, and the gene of 1670 bp (T-bet) and 388 bp (IFN-gamma) were generated from SGC-7901s cells; (3) The proteins of IFN-gamma and T-bet secreted by SGC-7901 cells were also detected. These reveal that SGC-7901 cell is efficiently infected by rAAV encoding T-bet, which can induce transfected cells to secret IFN-gamma. It may be useful in the researches on cancer immune therapy of transfecting T-bet gene.
Cell Line, Tumor ; Dependovirus ; genetics ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; Humans ; Interferon-gamma ; biosynthesis ; Recombinant Proteins ; biosynthesis ; genetics ; Stomach Neoplasms ; genetics ; metabolism ; T-Box Domain Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo observe the in vitro effects of gamma-interferon (IFNgamma) on gene expression of collagen I (Col I), III (Col III) and tissue inhibitor of metalloprotenase 1 (TIMP1) of HSC-T6 cells.

METHODSCultured HSC-T6 cells were exposed to IFNgamma at concentrations of 0.1, 1, 10, 10(2), 10(3), 10(4), 2.5 x 10(5), 5 x10(5) U/ml for 48 hours. 4,5-simethylthiazaoly colormetric assay was used to evaluate the effect of IFNgamma on HSC-T6 cell proliferation. After incubating with IFNgamma (1 U/ml, 10(2) U/ml and 10(4) U/ml) for 48 hours, HSC-T6 cells were harvested to detect Col I, Col III and TIMP1 steady state mRNA levels by quantitative reverse-transcription polymerase chain reaction (RT-PCR).

RESULTSThe Col I, Col III and TIMP1 mRNA levels of the control group were 2.86+/-0.21, 2.00+/-0.23 and 3.90+/-0.81, respectively. Col I and Col III mRNA levels in HSC-T6 cells treated by different concentrations of IFNgamma were lower than that of the controls (P < 0.01). There was no significant difference in TIMP1 mRNA levels between IFNgamma groups and controls.

CONCLUSIONIFNgamma suppresses expression of Col I and Col III whereas it has no effect on TIMP1 mRNA expression. The antifibrotic mechanism of IFNgamma may be partly due to its down-regulation of Col I and Col III mRNA levels in HSC-T6 cells.

Animals ; Cell Line ; Collagen Type I ; biosynthesis ; genetics ; Collagen Type III ; biosynthesis ; genetics ; Hepatocytes ; cytology ; metabolism ; Interferon-gamma ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics

In order to effectively cure hepatitis B virus (HBV), we studied on fusion protein HBscFv-IFNgamma, which was connected with single-chain Fv against HBV surface antigen(HBscFv) and gamma-interferon(IFNgamma) of being used in clinic against HBV. Adopting overlap PCR, the hbscfv and the ifngamma were connected into hbscfv-ifngamma. Then the pPICZalphaA/(hbscfv-ifngamma)(1,2,4) of multi-copy recombinant plasmid were constructed and transformed into Pichia pastoris x33. The engineering strain x4 was screened from transformed x33 and could secretively express HBscFv-IFNgamma. The preliminary verification indicates that HBscFv-IFNgamma has the bioactivity of HBscFv and IFNgamma by SDS-PAGE, Western blotting and ELISA. The supernatant of culturing X4 was purified by 14F7 affinity chromatography to HBscFv-IFNgamma with purity of 95%-98%. The HBscFv-IFNgamma is able to bind 27.9% HBV surface antigen (HBsAg) in the serum of HBV transgenic mice, which shows the antibody of HBscFv-IFNgamma has bioactivity in vivo. Therefore HBscFv-IFNgamma can shed light on the development of a new promising HBV-targeted drug.
Chromatography, Affinity ; Genetic Engineering ; Genetic Vectors ; Hepatitis B Antibodies ; biosynthesis ; genetics ; Hepatitis B Surface Antigens ; immunology ; Immunoglobulin Fragments ; genetics ; Immunoglobulin Variable Region ; genetics ; Interferon-gamma ; biosynthesis ; genetics ; Organisms, Genetically Modified ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification