The human interferon α 2b（hIFN-α2b） gene was cloned into binary vector pBI121 to obtain plant expression vector pBIFN. The recombinant plasmid pBIFN was transferred into Agrobacterium tumefaciens strain LBA4404. T...The human interferon α 2b（hIFN-α2b） gene was cloned into binary vector pBI121 to obtain plant expression vector pBIFN. The recombinant plasmid pBIFN was transferred into Agrobacterium tumefaciens strain LBA4404. Then the hIFN-α2b gene was introduced into ginseng callus cells via Agrobacterium-mediated transformation and the ginseng cell line carrying hIFN-α2b gene was selected on G418-containing medium. The presence of target gene in transformed cells was confirmed by PCR and RT-PCR. The results indicate that hIFN-α2b gene has been integrated into the ginseng cells＇ genome, with transcription products, hIFN-α2b expressed by the transgenic ginseng cells was detected by Western blot. It was shown that a specific protein band at 19000 could be observed. Cytopathic effect（CPE） inhibition assay using the W1SH-VSV system shows that the mean antiviral activity of expressed hlFN-a2α was 6.0× 10^4 IU/mL.展开更多
Recombinant human interferon a2b(rhIFNa2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C.The current identification test for rhIFNa2b is complex.In this study,an anti-rhI...Recombinant human interferon a2b(rhIFNa2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C.The current identification test for rhIFNa2b is complex.In this study,an anti-rhIFNa2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNa2b.RhIFNa2b was used to immunize an alpaca,which established a phage nanobody library.After five steps of enrichment,the nanobody I22,which specifically bound rhIFNa2b,was isolated and inserted into the prokaryotic expression vector pET28a.After subsequent purification,the physicochemical properties of the nanobody were determined.A semiquantitative detection and rapid identification assay of rhIFNa2b was developed using this novel nanobody.To develop a rapid test,the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips.The developed rhIFNa2b detection assay had a limit of detection of 1 mg/mL.The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products.The principle of this novel assay is generally applicable for the rapid testing of other commercial products,with a great potential for routine use in detecting counterfeit recombinant protein products.展开更多
A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling ...A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling efficiency and specific activity of IFN—α_A-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α_A (rIFN-α_A) rose up to 1.03×10~7IU/mg protein and the purification efficiency is appoximately 100 times.展开更多
E.coli cells expressing recombinant human interferon-γ was disrupted by sonication anddissolved in 7mol·L<sup>-1</sup> guanidine hydrochloride.The extract obtained was then renaturated by 70 folddilu...E.coli cells expressing recombinant human interferon-γ was disrupted by sonication anddissolved in 7mol·L<sup>-1</sup> guanidine hydrochloride.The extract obtained was then renaturated by 70 folddilution with PBS.HulFN γ was purified by affinity chromatography with monoclonal antibody fromthe renaturated crude feed solution.After washing the column with PBS,the adsorbed HulFN γ waseluted with PBS containing 0.5mol·L<sup>-1</sup> NaCl.The column was regenerated with 2mol·L<sup>-1</sup> GuHClfor reuse.After one step of affinity purification the purity of interferon-γ was over 95%.and thespecific activity of the HulFN-γ reached 1.2×10<sup>7</sup> IU·mg<sup>-1</sup> protein.92.8% of recovery was obtainedin the elution step.Total recovery of HulFN γ activity in the affinity chromatography was 78%.展开更多
Establishing the hepatoma cell-specific expression of human interferon gene mediated by retroviral vectors. Methods: Human interferon-β complementary DNA (IFN-β cDNA) was inserted into polylinker site of pMNSM retro...Establishing the hepatoma cell-specific expression of human interferon gene mediated by retroviral vectors. Methods: Human interferon-β complementary DNA (IFN-β cDNA) was inserted into polylinker site of pMNSM retroviral vector to construct recombinant retroviral vector pMNSIFNB, where the transcription of IFN-β gene was driven by SV40 early region promoter, and MNAIFNB, where the transcription of IFN-β gene was driven by SV40 early region promoter regulated by α-fetoprotein enhancer. The retroviral constructs were respectively introduced into PA317 amphotropic packaging cells by means of lipofectamine mediated gene transfer procedure. The plasmids transfection efficiency was among (4-25)ｘ103 colonies/μg DNA/106 PA317 cells. The retrovirus infection efficiency was among (4. 5-500)ｘ104 Colony Forming Units (CFU)/ml. The recombinant retroviruses were used to infect human hepatoma cells, renal cell carcinoma cells and melanoma cell lines in the presence of 4 μg/ml polybrene. Results: Dot hybridization of total RNA from the neomycin resistant colonies and interferon expression assay indicated that human α-fetoprotein enhancer induced efficient and specific transcription and expression of IFN-β gene driven by the promoter of different origin in human hepatoma cells by which α-fetoprotein was highly produced. Conclusion: Cis-active element of α-fetoprotein gene can drive IFN-β gene specifically expressed in human hepatoma cells, which presents some valuable materials for the hepatoma-specific immune gene therapy.展开更多
Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells...Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells still harbored the vector after 50-generations growth in non-selective medium, which confirmed the existence of a non-functional region in 2 μm plasmid. The human interferon αA (IFN αA) gene expression-secretion cassette was inserted into pHC11, and the yeast transformant was cultured in complex medium. Tbe data showed that the expressed product was 36.8% of the total protein amount in the culture supernatant and the IFN αA biological activity was 2.6×10^(10) units per liter, demonstrating that high-level expression and secretion of IFN αA were achieved in yeast by using the stable vector pHC11.展开更多
Human interferon-αlc/86D (IFNαlc/86D) was functionally displayed on the surface of the filamentous bacteriophage using a phagemid vector system (pCANTAB5E). The key amino acid residues involved in the receptor bindi...Human interferon-αlc/86D (IFNαlc/86D) was functionally displayed on the surface of the filamentous bacteriophage using a phagemid vector system (pCANTAB5E). The key amino acid residues involved in the receptor binding were further defined with phage displayed 6-mer peptide library and two neutralizing antibodies against linear epitopes on the IFN-αlb, indicating that residues 30, 33, 34, (Mi-loop) and residues 124, 126, 127 (D helix, DE-loop) were more critical than the adjacent residues for recognition of receptor. In addition, a cassette mutagenesis library was generated by fully randomizing the sequence of the four positions 29, 31, 32 and 35 in AB-loop, and used to select phage-IFN variants with WISH-based panning method. Three phage-IFN variants were isolated to possess more antiviral activity in the range of 4-16-fold than parental phage-IFN after IPTG-induced soluble expression. The results suggest that phage displayed phage-IFN αlc/86D variants with increased specific activity might be obtained after purification procedures.展开更多
Previous work from our laboratory has demonstrated that T lymphocyte-derived cytokine,interferon-gamma(IFN-γ) may play a role in human luteal regression by inhibiting luteal progesterone production.Prostaglandin F2α...Previous work from our laboratory has demonstrated that T lymphocyte-derived cytokine,interferon-gamma(IFN-γ) may play a role in human luteal regression by inhibiting luteal progesterone production.Prostaglandin F2αhas been known as an important luteolytic factor in a wide range of mammalian species.It was of interest to investigate the effects of IFN-γon prostaglandin synthesis and their possible interaction with the inhibition on human luteal steroidogenesis.Human luteal cells were cultured for four days in the presence or absence of IFN-γ.Simultaneously, the productions of progesterone,prostaglandin F2α(PGF2α),Prostaglandin E2(PGE2),and 6-ketoprostaglandin F1α(PGF1α) were evaluated.Concomitant with the inhibition of progesterone production induced by IFN-γ,αbiphasic pattern of response of prostaglandin synthesis was observed,i.e.a slight decrease of PGF2αand PGF1αafterα48 h exposure to IFN-γ while an increase of PGE2 after 96 h. In a separate experiment,a luteotropic action of PGE2 and PGF2a on human luteal cells from different stages was observed during 48 and 96 h periods of culture.In addition,while indomethacin(INDO) treatment markedly blocked the prostaglandin synthesis, the hasal as well as hCG stimulated progesterone production was still inhibited by IFN-γas usual.These results suggested that prostaglandins appeared to be not responsible for the observed inhibition Of progesterone production since the inhibitory effect was not influenced by concurrent treatment with INDO which suppressed prostaglandin synthesis.展开更多
Objective： To investigate the apoptosis of human pancreatic carcinoma PC3 cells induced by the combination of all-trans retinoic acid （ATRA） with interferon alpha （IFN-α）. Methods： PC3 cells were treated with ...Objective： To investigate the apoptosis of human pancreatic carcinoma PC3 cells induced by the combination of all-trans retinoic acid （ATRA） with interferon alpha （IFN-α）. Methods： PC3 cells were treated with ATRA and IFN-α. The inhibitory rate of PC3 cell proliferation was detected using MTT method. Cellular apoptosis was determined with flow cytometry. The percentage of PC3 cell apoptosis was assayed using TUNEL methods. Results： ATRA and IFN-α could inhibit cellular proliferation and induces cellular apoptosis of PC3 cells. The inhibitory effect was stronger when the ATRA and IFN-α were combined as a therapy. Conclusion： ATRA inhibits the proliferation of PC3 cells and induce the apoptosis of PC3 cells. The combination of IFN-α with ATRA may enhance these effects on PC3 cells.展开更多
目的研究宫颈高危型HPV感染应用重组人干扰素α2b阴道泡腾胶囊治疗的作用。方法选取100例宫颈高危型HPV感染患者,根据随机数字表法分为研究组与一般组,各50例。一般组定期随访不做任何处理,研究组采用重组人干扰素α2b阴道泡腾胶囊治疗...目的研究宫颈高危型HPV感染应用重组人干扰素α2b阴道泡腾胶囊治疗的作用。方法选取100例宫颈高危型HPV感染患者,根据随机数字表法分为研究组与一般组,各50例。一般组定期随访不做任何处理,研究组采用重组人干扰素α2b阴道泡腾胶囊治疗。比较两组转阴情况、治疗前后生活质量评分。结果研究组转阴率高于(62.00 vs 22.00)%一般组(P<0.05)。治疗后,研究组生活质量评分(75.98±10.52 vs 85.98±11.03)分高于一般组(P<0.05)。结论宫颈高危型HPV感染患者实施重组人干扰素α2b阴道泡腾胶囊治疗,可有效提高转阴率,改善患者生活质量,值得临床推广。展开更多
基金Supported by the Science and Technology Development Planning Foundation of Jilin Province of China(No.20030405)
文摘The human interferon α 2b（hIFN-α2b） gene was cloned into binary vector pBI121 to obtain plant expression vector pBIFN. The recombinant plasmid pBIFN was transferred into Agrobacterium tumefaciens strain LBA4404. Then the hIFN-α2b gene was introduced into ginseng callus cells via Agrobacterium-mediated transformation and the ginseng cell line carrying hIFN-α2b gene was selected on G418-containing medium. The presence of target gene in transformed cells was confirmed by PCR and RT-PCR. The results indicate that hIFN-α2b gene has been integrated into the ginseng cells＇ genome, with transcription products, hIFN-α2b expressed by the transgenic ginseng cells was detected by Western blot. It was shown that a specific protein band at 19000 could be observed. Cytopathic effect（CPE） inhibition assay using the W1SH-VSV system shows that the mean antiviral activity of expressed hlFN-a2α was 6.0× 10^4 IU/mL.
基金support was provided by the National Science and Technology Major Project(Grant No.:2015ZX09501008)。
文摘Recombinant human interferon a2b(rhIFNa2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C.The current identification test for rhIFNa2b is complex.In this study,an anti-rhIFNa2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNa2b.RhIFNa2b was used to immunize an alpaca,which established a phage nanobody library.After five steps of enrichment,the nanobody I22,which specifically bound rhIFNa2b,was isolated and inserted into the prokaryotic expression vector pET28a.After subsequent purification,the physicochemical properties of the nanobody were determined.A semiquantitative detection and rapid identification assay of rhIFNa2b was developed using this novel nanobody.To develop a rapid test,the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips.The developed rhIFNa2b detection assay had a limit of detection of 1 mg/mL.The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products.The principle of this novel assay is generally applicable for the rapid testing of other commercial products,with a great potential for routine use in detecting counterfeit recombinant protein products.
文摘A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling efficiency and specific activity of IFN—α_A-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α_A (rIFN-α_A) rose up to 1.03×10~7IU/mg protein and the purification efficiency is appoximately 100 times.
文摘E.coli cells expressing recombinant human interferon-γ was disrupted by sonication anddissolved in 7mol·L<sup>-1</sup> guanidine hydrochloride.The extract obtained was then renaturated by 70 folddilution with PBS.HulFN γ was purified by affinity chromatography with monoclonal antibody fromthe renaturated crude feed solution.After washing the column with PBS,the adsorbed HulFN γ waseluted with PBS containing 0.5mol·L<sup>-1</sup> NaCl.The column was regenerated with 2mol·L<sup>-1</sup> GuHClfor reuse.After one step of affinity purification the purity of interferon-γ was over 95%.and thespecific activity of the HulFN-γ reached 1.2×10<sup>7</sup> IU·mg<sup>-1</sup> protein.92.8% of recovery was obtainedin the elution step.Total recovery of HulFN γ activity in the affinity chromatography was 78%.
文摘Establishing the hepatoma cell-specific expression of human interferon gene mediated by retroviral vectors. Methods: Human interferon-β complementary DNA (IFN-β cDNA) was inserted into polylinker site of pMNSM retroviral vector to construct recombinant retroviral vector pMNSIFNB, where the transcription of IFN-β gene was driven by SV40 early region promoter, and MNAIFNB, where the transcription of IFN-β gene was driven by SV40 early region promoter regulated by α-fetoprotein enhancer. The retroviral constructs were respectively introduced into PA317 amphotropic packaging cells by means of lipofectamine mediated gene transfer procedure. The plasmids transfection efficiency was among (4-25)ｘ103 colonies/μg DNA/106 PA317 cells. The retrovirus infection efficiency was among (4. 5-500)ｘ104 Colony Forming Units (CFU)/ml. The recombinant retroviruses were used to infect human hepatoma cells, renal cell carcinoma cells and melanoma cell lines in the presence of 4 μg/ml polybrene. Results: Dot hybridization of total RNA from the neomycin resistant colonies and interferon expression assay indicated that human α-fetoprotein enhancer induced efficient and specific transcription and expression of IFN-β gene driven by the promoter of different origin in human hepatoma cells by which α-fetoprotein was highly produced. Conclusion: Cis-active element of α-fetoprotein gene can drive IFN-β gene specifically expressed in human hepatoma cells, which presents some valuable materials for the hepatoma-specific immune gene therapy.
文摘Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells still harbored the vector after 50-generations growth in non-selective medium, which confirmed the existence of a non-functional region in 2 μm plasmid. The human interferon αA (IFN αA) gene expression-secretion cassette was inserted into pHC11, and the yeast transformant was cultured in complex medium. Tbe data showed that the expressed product was 36.8% of the total protein amount in the culture supernatant and the IFN αA biological activity was 2.6×10^(10) units per liter, demonstrating that high-level expression and secretion of IFN αA were achieved in yeast by using the stable vector pHC11.
基金Project supported by the China National Expert Committee for Biotechnology Development.
文摘Human interferon-αlc/86D (IFNαlc/86D) was functionally displayed on the surface of the filamentous bacteriophage using a phagemid vector system (pCANTAB5E). The key amino acid residues involved in the receptor binding were further defined with phage displayed 6-mer peptide library and two neutralizing antibodies against linear epitopes on the IFN-αlb, indicating that residues 30, 33, 34, (Mi-loop) and residues 124, 126, 127 (D helix, DE-loop) were more critical than the adjacent residues for recognition of receptor. In addition, a cassette mutagenesis library was generated by fully randomizing the sequence of the four positions 29, 31, 32 and 35 in AB-loop, and used to select phage-IFN variants with WISH-based panning method. Three phage-IFN variants were isolated to possess more antiviral activity in the range of 4-16-fold than parental phage-IFN after IPTG-induced soluble expression. The results suggest that phage displayed phage-IFN αlc/86D variants with increased specific activity might be obtained after purification procedures.
文摘Previous work from our laboratory has demonstrated that T lymphocyte-derived cytokine,interferon-gamma(IFN-γ) may play a role in human luteal regression by inhibiting luteal progesterone production.Prostaglandin F2αhas been known as an important luteolytic factor in a wide range of mammalian species.It was of interest to investigate the effects of IFN-γon prostaglandin synthesis and their possible interaction with the inhibition on human luteal steroidogenesis.Human luteal cells were cultured for four days in the presence or absence of IFN-γ.Simultaneously, the productions of progesterone,prostaglandin F2α(PGF2α),Prostaglandin E2(PGE2),and 6-ketoprostaglandin F1α(PGF1α) were evaluated.Concomitant with the inhibition of progesterone production induced by IFN-γ,αbiphasic pattern of response of prostaglandin synthesis was observed,i.e.a slight decrease of PGF2αand PGF1αafterα48 h exposure to IFN-γ while an increase of PGE2 after 96 h. In a separate experiment,a luteotropic action of PGE2 and PGF2a on human luteal cells from different stages was observed during 48 and 96 h periods of culture.In addition,while indomethacin(INDO) treatment markedly blocked the prostaglandin synthesis, the hasal as well as hCG stimulated progesterone production was still inhibited by IFN-γas usual.These results suggested that prostaglandins appeared to be not responsible for the observed inhibition Of progesterone production since the inhibitory effect was not influenced by concurrent treatment with INDO which suppressed prostaglandin synthesis.
文摘Objective： To investigate the apoptosis of human pancreatic carcinoma PC3 cells induced by the combination of all-trans retinoic acid （ATRA） with interferon alpha （IFN-α）. Methods： PC3 cells were treated with ATRA and IFN-α. The inhibitory rate of PC3 cell proliferation was detected using MTT method. Cellular apoptosis was determined with flow cytometry. The percentage of PC3 cell apoptosis was assayed using TUNEL methods. Results： ATRA and IFN-α could inhibit cellular proliferation and induces cellular apoptosis of PC3 cells. The inhibitory effect was stronger when the ATRA and IFN-α were combined as a therapy. Conclusion： ATRA inhibits the proliferation of PC3 cells and induce the apoptosis of PC3 cells. The combination of IFN-α with ATRA may enhance these effects on PC3 cells.
文摘目的研究宫颈高危型HPV感染应用重组人干扰素α2b阴道泡腾胶囊治疗的作用。方法选取100例宫颈高危型HPV感染患者,根据随机数字表法分为研究组与一般组,各50例。一般组定期随访不做任何处理,研究组采用重组人干扰素α2b阴道泡腾胶囊治疗。比较两组转阴情况、治疗前后生活质量评分。结果研究组转阴率高于(62.00 vs 22.00)%一般组(P<0.05)。治疗后,研究组生活质量评分(75.98±10.52 vs 85.98±11.03)分高于一般组(P<0.05)。结论宫颈高危型HPV感染患者实施重组人干扰素α2b阴道泡腾胶囊治疗,可有效提高转阴率,改善患者生活质量,值得临床推广。
