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
AIM: To evaluate the antifibrotic effect of different doses of recombinant human Gamma-Interferon (IFN-gamma) in two rat models of hepatic fibrosis, and to observe its effect on moderate chronic hepatitis B virus fibr...AIM: To evaluate the antifibrotic effect of different doses of recombinant human Gamma-Interferon (IFN-gamma) in two rat models of hepatic fibrosis, and to observe its effect on moderate chronic hepatitis B virus fibrosis. METHODS: Hepatic fibrosis was successfully induced in 150 and 196 rats by subcutaneous injection of carbon tetrachloride (CCl4) and intraperitoneal injection of dimethylnitrosamine (DMN), respectively. Each of the two model groups was divided into: (1) fibrotic model group; (2) colchicine treatment group (0.1 mg/kg/day, gastrogavage for 8 weeks); (3) high-dose IFN-gamma group (15 MU/kg per day, i.m. for 8 weeks); (4) medium-dose IFN-gamma group (5 MU/kg daily, i.m. for 8 weeks); and (5) Y low-dose IFN-gamma group (1.67 MU/kg daily, i.m. for 8 weeks). Another group of 10 rats without any treatment was used as normal controls. At the end of the experiment, semi-quantitative histopathological scores of inflammation and fibrosis, liver alpha smooth muscle actin (alpha-SMA) expression level, liver hydroxyl proline content and serum hyaluronic acid levels were compared. And 47 medium chronic hepatitis B viral fibrosis patients were studied. They were given IFN-gamma treatment, 100 MU/day i.m. for the first three months and 100 MU qod i.m. for the next six months. Semi-quantitative pathological scores of inflammation and fibrosis and serum hepatic fibrosis indices were compared within the 9 months. RESULTS: In animal experiment, the pathological fibrosis scores and liver hydroxyl proline content were found to be significantly lower in rats treated with different doses of IFN-gamma as compared with rats in fibrotic model group induced by either CCl4 or DMN, in a dose-dependent manner. For CCl4-induced model, pathological fibrosis scores in high, medium and low doses IFN-gamma groups were 5.10 +/- 2.88, 7.70 +/- 3.53 and 8.00 +/- 3.30, respectively, but the score was 14.60 +/- 7.82 in fibrotic model group. Hydroxyl proline contents were 2.83 +/- 1.18, 3.59 +/- 1.22 and 4.80 +/- 1.62, in the three IFN-gamma groups, and 10.01 +/- 3.23 in fibrotic model group. The difference was statistically significant (P【0.01). Similar results were found in DMN-induced model. Pathological fibrosis scores were 6.30 +/- 0.48, 8.10 +/- 2.72 and 8.30 +/- 2.58, in high, medium and low doses IFN-gamma groups, and 12.60 +/- 3.57 in fibrotic model group. Hydroxyl proline contents were 2.72 +/- 0.58, 3.14 +/- 0.71 and 3.62 +/- 1.02, in the three IFN-gamma groups, and 12.79 +/- 1.54 in fibrotic model group. The difference was statistically significant (P【0.01).Serum hepatic fibrosis indices decreased significantly in the 47 patients after IFN-gamma treatment (HA: 433.38 +/- 373.00 vs 281.57 +/- 220.48; LN: 161.22 +/- 41.02 vs 146 +/- 35 +/- 44. 67; PC III: 192.59 +/- 89.95 vs 156.98 +/- 49.22; C-I: 156.30 +/- 44.01 vs 139.14 +/- 34.47) and the differences between the four indices were significant (P 【0.05). Thirty-three patients received two liver biopsies, one before and one after IFN-gamma treatment. In thirty of 33 patients IFN-gamma had better effects according to semi-quantitative pathological scores (8.40 +/- 5.83 vs 5.30 +/- 4.05, P【0.05). CONCLUSION: All the three doses of IFN-gamma are effective in treating rat liver fibrosis induced by either CCl4 or DMN, the higher the dose, the better the effect. And IFN-gamma is effective for patients with moderate chronic hepatitis B viral fibrosis.展开更多
Novel human interferon alpha 2b (hIFNα2b) muteins were developed by substituting cysteine residue (C) at positions 2 and 99 with aspartic acid residues (D). The mutein forms were then studied for pharmacokinetic prof...Novel human interferon alpha 2b (hIFNα2b) muteins were developed by substituting cysteine residue (C) at positions 2 and 99 with aspartic acid residues (D). The mutein forms were then studied for pharmacokinetic profile. In addition, the influence of charge on the protein structure was tested in vivo for the biodistribution pattern. Codon substitutions were performed by Polymerase Chain Reaction (PCR)-based site-directed mutagenesis on a previously constructed synthetic hIFNα2b open reading frame (ORF) cloned in pET32b expression plasmid. The result of nucleotide sequencing analysis confirmed that all codons were replaced successfully without any additional mutation. Three mutant forms of hIFNα2b ORF were overexpressed in Escherichia coli BL21 (DE3) resulted in three muteins: hIFNα2b C2D, hIFNα2b C99D, hIFNα2b C2D C99D. To follow the kinetic and localization of the mutein interferon after intravenous administration, Tc99m was used to label the proteins. In particular of elimination half-life, it was shown that hIFNα2b C2D C99D > hIFNα2bC2D > hIFNα2bC99D > wild type. hIFNα2b C2D C99D mutein showed highest blood accumulation after 30 minutes administration. Taken together, the charge of hIFNα2b seems to be responsible for the fate of hIFNα2b in vivo.展开更多
A case report presents a progression of autoimmune thyroiditis with an abnormal enlargement of the thyroid glands and increased thyreotropin hormone concentration-associated with interferon treatment in human papillom...A case report presents a progression of autoimmune thyroiditis with an abnormal enlargement of the thyroid glands and increased thyreotropin hormone concentration-associated with interferon treatment in human papillomavirus infected patient with the autoimmune thyroiditis and a daily L-thyroxin hormone replacement therapy background. Observation was supplemented with a brief review of literature and discussion. On the basis of this observation and a brief review of literature authors suggested that the potential adverse effects of interferon therapy are overbalanced than its benefits for gynecological patients, therefore any interferon treatment should be recommended with strict indications as well as after screening of conditions and functions of thyroid glands and other interferon target organs to avoid interferon treatment side effects. Practitioners especially gynecologists should inform their patients about pleiotropic interferon effects and its high frequent and wide range side effects before to start such kind of treatment.展开更多
目的观察重组人干扰素α2b(recombinant human interferon-α2b,rhIFN-α2b)和拉米夫定序贯治疗用于乙肝免疫耐受期患儿的效果。方法选择西安医学院第三附属医院2020年4月至2023年4月收治的96例乙肝免疫耐受期患儿为研究对象,按随机数...目的观察重组人干扰素α2b(recombinant human interferon-α2b,rhIFN-α2b)和拉米夫定序贯治疗用于乙肝免疫耐受期患儿的效果。方法选择西安医学院第三附属医院2020年4月至2023年4月收治的96例乙肝免疫耐受期患儿为研究对象,按随机数字法分为研究组与对照组各48例。研究组男29例,女19例;年龄(7.54±1.36)岁;病程(2.25±0.54)年;有乙肝家族史28例。对照组男26例,女22例;年龄(7.63±1.41)岁;病程(2.44±0.51)年;有乙肝家族史26例。对照组口服拉米夫定片,0.1 g/次,1次/d,治疗24周。研究组采用rhIFN-α2b和拉米夫定序贯治疗:前4周单用rhIFN-α2b肌内或皮下注射,5 mIU/(m^(2)·次),1次/2 d,4周后加用拉米夫定,0.1 g/次,1次/d,持续治疗8周后停用rhIFN-α2b仅用拉米夫定,继续治疗12周。比较两组疗效;治疗4周、12周、24周后,记录两组乙肝病毒E抗原(hepatitis B virus E antigen,HBeAg)、乙肝病毒脱氧核糖核酸(hepatitis B virus DNA,HBV-DNA)转阴率和乙肝病毒E抗体(hepatitis B virus E antibody,HBeAb)转换率;治疗前及治疗24周后,比较两组肝功能[谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspertate aminotransferase,AST)、总胆红素(total bilirubin,TBil)]及肝纤维化指标[透明质酸(hyaluronic acid,HA)、层黏蛋白(laminin,LN)、Ⅲ型前胶原肽(type Ⅲ procollagen peptide,PⅢP)、Ⅳ型胶原(type Ⅳ collagen,CⅣ)];记录两组不良反应发生情况。采用t检验、χ^(2)检验进行统计学分析。结果研究组总有效率高于对照组[77.08%(37/48)比52.08%(25/48)](P<0.05)。治疗4周及12周后,研究组与对照组HBeAg、HBV-DNA转阴率比较差异均无统计学意义(均P>0.05),治疗24周后,研究组HBeAg、HBV-DNA转阴率均高于对照组(均P<0.05);两组治疗后不同时间点HBeAb转换率比较差异均无统计学意义(均P>0.05)。治疗24周后,两组ALT、AST、TBil均降低,且研究组[(39.16±7.51)U/L、(41.92±8.26)U/L、(15.13±4.29)μmol/L]均低于对照组[(47.38±8.02)U/L、(52.36±8.73)U/L、(18.67±4.86)μmol/L](均P<0.05)。治疗24周后,两组HA、LN、PⅢP、CⅣ均降低,且研究组[(113.57±30.16)μg/L、(96.41±29.05)μg/L、(78.14±20.96)μg/L、(90.26±26.31)μg/L]均低于对照组[(208.34±64.72)μg/L、(124.27±32.19)μg/L、(104.37±22.48)μg/L、(143.75±33.49)μg/L](均P<0.05)。两组不良反应发生率比较差异无统计学意义(P>0.05)。结论rhIFN-α2b和拉米夫定序贯治疗在乙肝免疫耐受期患儿中疗效确切,能有效减少病毒含量,阻止肝功能恶化及肝纤维化,且安全性较高。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AIM: To evaluate the antifibrotic effect of different doses of recombinant human Gamma-Interferon (IFN-gamma) in two rat models of hepatic fibrosis, and to observe its effect on moderate chronic hepatitis B virus fibrosis. METHODS: Hepatic fibrosis was successfully induced in 150 and 196 rats by subcutaneous injection of carbon tetrachloride (CCl4) and intraperitoneal injection of dimethylnitrosamine (DMN), respectively. Each of the two model groups was divided into: (1) fibrotic model group; (2) colchicine treatment group (0.1 mg/kg/day, gastrogavage for 8 weeks); (3) high-dose IFN-gamma group (15 MU/kg per day, i.m. for 8 weeks); (4) medium-dose IFN-gamma group (5 MU/kg daily, i.m. for 8 weeks); and (5) Y low-dose IFN-gamma group (1.67 MU/kg daily, i.m. for 8 weeks). Another group of 10 rats without any treatment was used as normal controls. At the end of the experiment, semi-quantitative histopathological scores of inflammation and fibrosis, liver alpha smooth muscle actin (alpha-SMA) expression level, liver hydroxyl proline content and serum hyaluronic acid levels were compared. And 47 medium chronic hepatitis B viral fibrosis patients were studied. They were given IFN-gamma treatment, 100 MU/day i.m. for the first three months and 100 MU qod i.m. for the next six months. Semi-quantitative pathological scores of inflammation and fibrosis and serum hepatic fibrosis indices were compared within the 9 months. RESULTS: In animal experiment, the pathological fibrosis scores and liver hydroxyl proline content were found to be significantly lower in rats treated with different doses of IFN-gamma as compared with rats in fibrotic model group induced by either CCl4 or DMN, in a dose-dependent manner. For CCl4-induced model, pathological fibrosis scores in high, medium and low doses IFN-gamma groups were 5.10 +/- 2.88, 7.70 +/- 3.53 and 8.00 +/- 3.30, respectively, but the score was 14.60 +/- 7.82 in fibrotic model group. Hydroxyl proline contents were 2.83 +/- 1.18, 3.59 +/- 1.22 and 4.80 +/- 1.62, in the three IFN-gamma groups, and 10.01 +/- 3.23 in fibrotic model group. The difference was statistically significant (P【0.01). Similar results were found in DMN-induced model. Pathological fibrosis scores were 6.30 +/- 0.48, 8.10 +/- 2.72 and 8.30 +/- 2.58, in high, medium and low doses IFN-gamma groups, and 12.60 +/- 3.57 in fibrotic model group. Hydroxyl proline contents were 2.72 +/- 0.58, 3.14 +/- 0.71 and 3.62 +/- 1.02, in the three IFN-gamma groups, and 12.79 +/- 1.54 in fibrotic model group. The difference was statistically significant (P【0.01).Serum hepatic fibrosis indices decreased significantly in the 47 patients after IFN-gamma treatment (HA: 433.38 +/- 373.00 vs 281.57 +/- 220.48; LN: 161.22 +/- 41.02 vs 146 +/- 35 +/- 44. 67; PC III: 192.59 +/- 89.95 vs 156.98 +/- 49.22; C-I: 156.30 +/- 44.01 vs 139.14 +/- 34.47) and the differences between the four indices were significant (P 【0.05). Thirty-three patients received two liver biopsies, one before and one after IFN-gamma treatment. In thirty of 33 patients IFN-gamma had better effects according to semi-quantitative pathological scores (8.40 +/- 5.83 vs 5.30 +/- 4.05, P【0.05). CONCLUSION: All the three doses of IFN-gamma are effective in treating rat liver fibrosis induced by either CCl4 or DMN, the higher the dose, the better the effect. And IFN-gamma is effective for patients with moderate chronic hepatitis B viral fibrosis.
文摘Novel human interferon alpha 2b (hIFNα2b) muteins were developed by substituting cysteine residue (C) at positions 2 and 99 with aspartic acid residues (D). The mutein forms were then studied for pharmacokinetic profile. In addition, the influence of charge on the protein structure was tested in vivo for the biodistribution pattern. Codon substitutions were performed by Polymerase Chain Reaction (PCR)-based site-directed mutagenesis on a previously constructed synthetic hIFNα2b open reading frame (ORF) cloned in pET32b expression plasmid. The result of nucleotide sequencing analysis confirmed that all codons were replaced successfully without any additional mutation. Three mutant forms of hIFNα2b ORF were overexpressed in Escherichia coli BL21 (DE3) resulted in three muteins: hIFNα2b C2D, hIFNα2b C99D, hIFNα2b C2D C99D. To follow the kinetic and localization of the mutein interferon after intravenous administration, Tc99m was used to label the proteins. In particular of elimination half-life, it was shown that hIFNα2b C2D C99D > hIFNα2bC2D > hIFNα2bC99D > wild type. hIFNα2b C2D C99D mutein showed highest blood accumulation after 30 minutes administration. Taken together, the charge of hIFNα2b seems to be responsible for the fate of hIFNα2b in vivo.
文摘A case report presents a progression of autoimmune thyroiditis with an abnormal enlargement of the thyroid glands and increased thyreotropin hormone concentration-associated with interferon treatment in human papillomavirus infected patient with the autoimmune thyroiditis and a daily L-thyroxin hormone replacement therapy background. Observation was supplemented with a brief review of literature and discussion. On the basis of this observation and a brief review of literature authors suggested that the potential adverse effects of interferon therapy are overbalanced than its benefits for gynecological patients, therefore any interferon treatment should be recommended with strict indications as well as after screening of conditions and functions of thyroid glands and other interferon target organs to avoid interferon treatment side effects. Practitioners especially gynecologists should inform their patients about pleiotropic interferon effects and its high frequent and wide range side effects before to start such kind of treatment.
文摘目的观察重组人干扰素α2b(recombinant human interferon-α2b,rhIFN-α2b)和拉米夫定序贯治疗用于乙肝免疫耐受期患儿的效果。方法选择西安医学院第三附属医院2020年4月至2023年4月收治的96例乙肝免疫耐受期患儿为研究对象,按随机数字法分为研究组与对照组各48例。研究组男29例,女19例;年龄(7.54±1.36)岁;病程(2.25±0.54)年;有乙肝家族史28例。对照组男26例,女22例;年龄(7.63±1.41)岁;病程(2.44±0.51)年;有乙肝家族史26例。对照组口服拉米夫定片,0.1 g/次,1次/d,治疗24周。研究组采用rhIFN-α2b和拉米夫定序贯治疗:前4周单用rhIFN-α2b肌内或皮下注射,5 mIU/(m^(2)·次),1次/2 d,4周后加用拉米夫定,0.1 g/次,1次/d,持续治疗8周后停用rhIFN-α2b仅用拉米夫定,继续治疗12周。比较两组疗效;治疗4周、12周、24周后,记录两组乙肝病毒E抗原(hepatitis B virus E antigen,HBeAg)、乙肝病毒脱氧核糖核酸(hepatitis B virus DNA,HBV-DNA)转阴率和乙肝病毒E抗体(hepatitis B virus E antibody,HBeAb)转换率;治疗前及治疗24周后,比较两组肝功能[谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspertate aminotransferase,AST)、总胆红素(total bilirubin,TBil)]及肝纤维化指标[透明质酸(hyaluronic acid,HA)、层黏蛋白(laminin,LN)、Ⅲ型前胶原肽(type Ⅲ procollagen peptide,PⅢP)、Ⅳ型胶原(type Ⅳ collagen,CⅣ)];记录两组不良反应发生情况。采用t检验、χ^(2)检验进行统计学分析。结果研究组总有效率高于对照组[77.08%(37/48)比52.08%(25/48)](P<0.05)。治疗4周及12周后,研究组与对照组HBeAg、HBV-DNA转阴率比较差异均无统计学意义(均P>0.05),治疗24周后,研究组HBeAg、HBV-DNA转阴率均高于对照组(均P<0.05);两组治疗后不同时间点HBeAb转换率比较差异均无统计学意义(均P>0.05)。治疗24周后,两组ALT、AST、TBil均降低,且研究组[(39.16±7.51)U/L、(41.92±8.26)U/L、(15.13±4.29)μmol/L]均低于对照组[(47.38±8.02)U/L、(52.36±8.73)U/L、(18.67±4.86)μmol/L](均P<0.05)。治疗24周后,两组HA、LN、PⅢP、CⅣ均降低,且研究组[(113.57±30.16)μg/L、(96.41±29.05)μg/L、(78.14±20.96)μg/L、(90.26±26.31)μg/L]均低于对照组[(208.34±64.72)μg/L、(124.27±32.19)μg/L、(104.37±22.48)μg/L、(143.75±33.49)μg/L](均P<0.05)。两组不良反应发生率比较差异无统计学意义(P>0.05)。结论rhIFN-α2b和拉米夫定序贯治疗在乙肝免疫耐受期患儿中疗效确切,能有效减少病毒含量,阻止肝功能恶化及肝纤维化,且安全性较高。
