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
Objective To investigate the antiviral activity of recombinant interferonα-2b suppository(IFNα-2b)in vivo and in vitro.Methods The cytopathic-effect inhibition assay was applied in this study to investigate the anti...Objective To investigate the antiviral activity of recombinant interferonα-2b suppository(IFNα-2b)in vivo and in vitro.Methods The cytopathic-effect inhibition assay was applied in this study to investigate the antiviral activity of this drug as well as yingtelong and axiluowei as positive control.The guinea pig model of vaginitis and skin infection caused by HSV-2 infection were established,treated with IFNα-2b suppository at dosages of 60000、180000、540000 IU,using IFNα-2b injection 180000 IU·kg-1 as controls.Score the pathological changes of appearance and skin,the virus activities of vaginal secretion and tissue sections of viginae were assayed after treatment.Results The TD50 of IFN α-2b and yingtelong for Vero cells was(>100)μg·mL-1 and(>100000)IU·mL-1,respectively.The IC50 of IFN α-2b and yingtelong and axiluowei for Herpes virus type 1 was(0.29±0.08)μg·mL-1 and(185.0±28.8)IU·mL-1 and(0.19±0.03)μg·mL-1,respectively.The mean scores for vaginal and skin lesion of the treated groups were lower than those of untreated group.Among these concentrations,the IFNα-2b suppository of 540000 IU·kg-1 group.Showed highest anti-viral activity.The virus activity in vaginal secretion of treated group was lower than that of untreated group too(P<0.01 or P<0.05).Tissue sections of viginae after treatment with IFNα-2b suppository showed significantly therapeutical effects on the degrees of vaginal lesion.At the same dosage,The anti-HSV activity of IFNα-2b suppository was also compared with IFNα-2b injection,the results showed that the activity of suppository of 540000 IU·kg-1 group was similar to that of the injection.Conclusions The IFNα-2b suppository has anti-viruses function both in vivo and in vitro.展开更多
Objective To investigate a new approach of the combined use of trichosanthin (TCS) andrecombinant interferon alpha - 2b (rIFN α- 2b) against digestive system cancer cells. Methods Detect separatelythe cytotoxicity of...Objective To investigate a new approach of the combined use of trichosanthin (TCS) andrecombinant interferon alpha - 2b (rIFN α- 2b) against digestive system cancer cells. Methods Detect separatelythe cytotoxicity of TCS, rIFN α- 2b and their combination against digestive system cancer cell SGC- 7901.Results In the experiment in vitro, TCS, rIFN α- 2b both had direct, dose dependent cytotoxicity againstSGC - 7901. Their combined use demonstrated a toxicity signijicantly higher than that of the two drugs used alone,showing a signilicant synergic effect. This synergic cytotoxicity was confirmed in the animal experiment.Conclusion Combined use of TCS and rIFN α - 2b decreases the therapeutic dose of TCS and its toxic adverseellect, and this synergic effect is favorable to the clinical use of TCS protein against gastric cancer.展开更多
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
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
Objective: To study the effects of anti-HPV bioprotein dressing combined with interferon α-2b therapy on the malignant molecule expression in patients with cervical intraepithelial neoplasia (CIN) Ⅲ complicated by h...Objective: To study the effects of anti-HPV bioprotein dressing combined with interferon α-2b therapy on the malignant molecule expression in patients with cervical intraepithelial neoplasia (CIN) Ⅲ complicated by high-risk HPV positive. Methods: Patients who were diagnosed with CINⅢ and high-risk HPV positive and underwent conization in the 3201 Hospital Affiliated to Xi'an Jiaotong University between June 2014 and February 2017 were selected and randomly divided into the observation group who received preoperative anti-HPV bioprotein dressing combined with interferon α-2b therapy and the control group who received no special treatment. CIN lesion was collected to determine the expression of pro-proliferation molecules, pro-apoptosis molecules and epithelial-mesenchymal transition molecules. Results: Rsf1, Piwil2, TOPK, p38MAPK, ERK, Snail, Twist, N-cadherin and Vimentin mRNA expression in cervical intraepithelial neoplasia lesions of observation group were greatly lower than those of control group whereas LRIG3, SARI, IEX-1, FHIT and E-cadherin mRNA expression were greatly higher than those of control group. Conclusion: Anti-HPV bioprotein dressing combined with interferon α-2b therapy can inhibit the proliferation and invasive growth of tumor cells in patients with CINⅢ complicated by high-risk HPV positive.展开更多
目的:观察薄芝糖肽和胸腺五肽分别联合重组人干扰素α2b治疗慢性乙型肝炎的疗效及安全性。方法:选择2014年1月-2015年1月我院90例慢性乙型肝炎患者,随机分为A、B、C组,各30例。A组患者给予注射用重组人干扰素α2b(假单胞菌)500万IU皮下...目的:观察薄芝糖肽和胸腺五肽分别联合重组人干扰素α2b治疗慢性乙型肝炎的疗效及安全性。方法:选择2014年1月-2015年1月我院90例慢性乙型肝炎患者,随机分为A、B、C组,各30例。A组患者给予注射用重组人干扰素α2b(假单胞菌)500万IU皮下注射,qod;B组患者在A组基础上加用薄芝糖肽注射液4 m L加入5%葡萄糖注射液250 m L中,ivgtt,qd;C组患者在A组基础上加用注射用胸腺五肽2 mg加入5%葡萄糖注射液250 m L中,ivgtt,qd。3组患者均治疗24周。比较3组患者治疗4、8、12、24周丙氨酸转氨酶(ALT)复常率、HBeAg阴转率、HBeAg/抗HBeAg血清转换率(以下简称"HBeAg转换率")、HBV-DNA阴转率、乙肝表面抗原(HBsAg)和HBV-DNA下降量,治疗24周时的HBsAg阴转率,并记录不良反应的发生情况。结果:治疗4、8、12周,3组患者ALT复常率、HBeAg阴转率、HBeAg转换率、HBsAg下降量比较,差异均无统计学意义(P>0.05)。治疗4周,B组和C组患者HBV-DNA转阴率显著高于A组,C组患者HBV-DNA下降量显著大于A组和B组,差异有统计学意义(P<0.05)。治疗8、12周,B组和C组患者HBV-DNA阴转率和HBV-DNA下降量显著高于A组,差异有统计学意义(P<0.05),但B组与C组间比较,差异无统计学意义(P>0.05)。治疗24周,3组患者ALT复常率、HBeAg转换率、HBsAg下降量及HBsAg阴转率比较,差异均无统计学意义(P>0.05);B组和C组患者HBeAg阴转率、HBV-DNA阴转率和HBV-DNA下降量显著高于A组,差异有统计学意义(P<0.05),但B组与C组间比较,差异无统计学意义(P>0.05)。3组患者不良反应发生率比较,差异无统计学意义(P>0.05)。结论:薄芝糖肽和胸腺五肽分别联合重组人干扰素α2b对慢性乙型肝炎具有较好的抑制病毒增殖作用,且在ALT复常率、HBeAg转换率、HBsAg下降量及HBsAg阴转率方面效果相当。展开更多
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Objective To investigate the antiviral activity of recombinant interferonα-2b suppository(IFNα-2b)in vivo and in vitro.Methods The cytopathic-effect inhibition assay was applied in this study to investigate the antiviral activity of this drug as well as yingtelong and axiluowei as positive control.The guinea pig model of vaginitis and skin infection caused by HSV-2 infection were established,treated with IFNα-2b suppository at dosages of 60000、180000、540000 IU,using IFNα-2b injection 180000 IU·kg-1 as controls.Score the pathological changes of appearance and skin,the virus activities of vaginal secretion and tissue sections of viginae were assayed after treatment.Results The TD50 of IFN α-2b and yingtelong for Vero cells was(>100)μg·mL-1 and(>100000)IU·mL-1,respectively.The IC50 of IFN α-2b and yingtelong and axiluowei for Herpes virus type 1 was(0.29±0.08)μg·mL-1 and(185.0±28.8)IU·mL-1 and(0.19±0.03)μg·mL-1,respectively.The mean scores for vaginal and skin lesion of the treated groups were lower than those of untreated group.Among these concentrations,the IFNα-2b suppository of 540000 IU·kg-1 group.Showed highest anti-viral activity.The virus activity in vaginal secretion of treated group was lower than that of untreated group too(P<0.01 or P<0.05).Tissue sections of viginae after treatment with IFNα-2b suppository showed significantly therapeutical effects on the degrees of vaginal lesion.At the same dosage,The anti-HSV activity of IFNα-2b suppository was also compared with IFNα-2b injection,the results showed that the activity of suppository of 540000 IU·kg-1 group was similar to that of the injection.Conclusions The IFNα-2b suppository has anti-viruses function both in vivo and in vitro.
文摘Objective To investigate a new approach of the combined use of trichosanthin (TCS) andrecombinant interferon alpha - 2b (rIFN α- 2b) against digestive system cancer cells. Methods Detect separatelythe cytotoxicity of TCS, rIFN α- 2b and their combination against digestive system cancer cell SGC- 7901.Results In the experiment in vitro, TCS, rIFN α- 2b both had direct, dose dependent cytotoxicity againstSGC - 7901. Their combined use demonstrated a toxicity signijicantly higher than that of the two drugs used alone,showing a signilicant synergic effect. This synergic cytotoxicity was confirmed in the animal experiment.Conclusion Combined use of TCS and rIFN α - 2b decreases the therapeutic dose of TCS and its toxic adverseellect, and this synergic effect is favorable to the clinical use of TCS protein against gastric cancer.
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.
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.
基金Natural Science Foundation Project of Shaanxi Province No:2013CM.
文摘Objective: To study the effects of anti-HPV bioprotein dressing combined with interferon α-2b therapy on the malignant molecule expression in patients with cervical intraepithelial neoplasia (CIN) Ⅲ complicated by high-risk HPV positive. Methods: Patients who were diagnosed with CINⅢ and high-risk HPV positive and underwent conization in the 3201 Hospital Affiliated to Xi'an Jiaotong University between June 2014 and February 2017 were selected and randomly divided into the observation group who received preoperative anti-HPV bioprotein dressing combined with interferon α-2b therapy and the control group who received no special treatment. CIN lesion was collected to determine the expression of pro-proliferation molecules, pro-apoptosis molecules and epithelial-mesenchymal transition molecules. Results: Rsf1, Piwil2, TOPK, p38MAPK, ERK, Snail, Twist, N-cadherin and Vimentin mRNA expression in cervical intraepithelial neoplasia lesions of observation group were greatly lower than those of control group whereas LRIG3, SARI, IEX-1, FHIT and E-cadherin mRNA expression were greatly higher than those of control group. Conclusion: Anti-HPV bioprotein dressing combined with interferon α-2b therapy can inhibit the proliferation and invasive growth of tumor cells in patients with CINⅢ complicated by high-risk HPV positive.
文摘目的:观察薄芝糖肽和胸腺五肽分别联合重组人干扰素α2b治疗慢性乙型肝炎的疗效及安全性。方法:选择2014年1月-2015年1月我院90例慢性乙型肝炎患者,随机分为A、B、C组,各30例。A组患者给予注射用重组人干扰素α2b(假单胞菌)500万IU皮下注射,qod;B组患者在A组基础上加用薄芝糖肽注射液4 m L加入5%葡萄糖注射液250 m L中,ivgtt,qd;C组患者在A组基础上加用注射用胸腺五肽2 mg加入5%葡萄糖注射液250 m L中,ivgtt,qd。3组患者均治疗24周。比较3组患者治疗4、8、12、24周丙氨酸转氨酶(ALT)复常率、HBeAg阴转率、HBeAg/抗HBeAg血清转换率(以下简称"HBeAg转换率")、HBV-DNA阴转率、乙肝表面抗原(HBsAg)和HBV-DNA下降量,治疗24周时的HBsAg阴转率,并记录不良反应的发生情况。结果:治疗4、8、12周,3组患者ALT复常率、HBeAg阴转率、HBeAg转换率、HBsAg下降量比较,差异均无统计学意义(P>0.05)。治疗4周,B组和C组患者HBV-DNA转阴率显著高于A组,C组患者HBV-DNA下降量显著大于A组和B组,差异有统计学意义(P<0.05)。治疗8、12周,B组和C组患者HBV-DNA阴转率和HBV-DNA下降量显著高于A组,差异有统计学意义(P<0.05),但B组与C组间比较,差异无统计学意义(P>0.05)。治疗24周,3组患者ALT复常率、HBeAg转换率、HBsAg下降量及HBsAg阴转率比较,差异均无统计学意义(P>0.05);B组和C组患者HBeAg阴转率、HBV-DNA阴转率和HBV-DNA下降量显著高于A组,差异有统计学意义(P<0.05),但B组与C组间比较,差异无统计学意义(P>0.05)。3组患者不良反应发生率比较,差异无统计学意义(P>0.05)。结论:薄芝糖肽和胸腺五肽分别联合重组人干扰素α2b对慢性乙型肝炎具有较好的抑制病毒增殖作用,且在ALT复常率、HBeAg转换率、HBsAg下降量及HBsAg阴转率方面效果相当。
