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 study the cloning and sequencing of mature fragment of human bone morphogenetic protein 4 gene. Methods The template DNA was obtained from the human osteosarcoma cell line U2OS. By using RT PCR method, th...Objective To study the cloning and sequencing of mature fragment of human bone morphogenetic protein 4 gene. Methods The template DNA was obtained from the human osteosarcoma cell line U2OS. By using RT PCR method, the cDNA coding for the mature fragment of BMP 4 was amplified, cloned into the vector pUC19, and sequenced by Sanger Dideoxy mediated Chain Termination method. Results The mature fragment of BMP4 cDNA was obtained by RT PCR and determined by sequencing. Through the computer search on Genebank, the analysis showed that the homology of nucleotides and amino acids between cDNA of rhBMP4 mature fragment of this study and the published sequence was 99%. Sequence analysis showed that there were two differences, one was at base 1154(201): G→C, which had no influence on the corresponding amino acids(Val). Another was at base1222(269):C→T, the mutation at the base 1222 had the change of Ala to Val. Conclusion The mature fragment of BMP4 gene has been cloned. The results will be of great significance in treatment of skeletal injuries and diseases.展开更多
目的观察重组人红细胞生成素(r Hu EPO)对戊四氮(PTZ)点燃的癫痫持续状态(SE)的SD大鼠海马神经元的影响,应用磷脂酰肌醇3-激酶(PI3K)抑制剂LY294002,观察Bax的变化情况,探讨r Hu EPO为临床处理SE提供实验依据。方法采用PTZ点燃大鼠SE模...目的观察重组人红细胞生成素(r Hu EPO)对戊四氮(PTZ)点燃的癫痫持续状态(SE)的SD大鼠海马神经元的影响,应用磷脂酰肌醇3-激酶(PI3K)抑制剂LY294002,观察Bax的变化情况,探讨r Hu EPO为临床处理SE提供实验依据。方法采用PTZ点燃大鼠SE模型,将大鼠随机分为正常对照组(NS)、PTZ组(PTZ+NS)、r Hu EPO组(PTZ+r Hu EPO)、LY294002组(PTZ+LY294002+r Hu EPO)、二甲基亚砜(DMSO)对照组(PTZ+DMSO+r Hu EPO),检测各组大鼠行为学和脑电图的改变;TUNEL法检测海马神经细胞的凋亡情况;免疫组织化学法观察磷酸化蛋白激酶B(p-PKB/p-Akt)、Bax的表达;反转录多聚酶链反应(RT-PCR)方法检测各组大鼠海马Baxm RNA的表达,Western blot方法检测各组大鼠海马Akt、p-Akt、Bax蛋白的表达。结果 r Hu EPO可以增加p-Akt蛋白的表达、下调Bax蛋白的表达,发挥神经保护作用;加入PI3K抑制剂LY294002,p-Akt的表达较r Hu EPO组减少,海马Bax蛋白、Baxm RNA的表达较r Hu EPO组增加,减弱了r Hu EPO的保护作用(P<0.05),差异具有统计学意义。结论 r Hu EPO的抗凋亡从应用PI3K/Akt激活剂和抑制剂两个方面证实作用可能是通过了PI3K/Akt信号通路,通过对线粒体凋亡途径的相关调控因子Bax的表达进行调控,发挥神经保护作用。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 study the cloning and sequencing of mature fragment of human bone morphogenetic protein 4 gene. Methods The template DNA was obtained from the human osteosarcoma cell line U2OS. By using RT PCR method, the cDNA coding for the mature fragment of BMP 4 was amplified, cloned into the vector pUC19, and sequenced by Sanger Dideoxy mediated Chain Termination method. Results The mature fragment of BMP4 cDNA was obtained by RT PCR and determined by sequencing. Through the computer search on Genebank, the analysis showed that the homology of nucleotides and amino acids between cDNA of rhBMP4 mature fragment of this study and the published sequence was 99%. Sequence analysis showed that there were two differences, one was at base 1154(201): G→C, which had no influence on the corresponding amino acids(Val). Another was at base1222(269):C→T, the mutation at the base 1222 had the change of Ala to Val. Conclusion The mature fragment of BMP4 gene has been cloned. The results will be of great significance in treatment of skeletal injuries and diseases.
文摘目的观察重组人红细胞生成素(r Hu EPO)对戊四氮(PTZ)点燃的癫痫持续状态(SE)的SD大鼠海马神经元的影响,应用磷脂酰肌醇3-激酶(PI3K)抑制剂LY294002,观察Bax的变化情况,探讨r Hu EPO为临床处理SE提供实验依据。方法采用PTZ点燃大鼠SE模型,将大鼠随机分为正常对照组(NS)、PTZ组(PTZ+NS)、r Hu EPO组(PTZ+r Hu EPO)、LY294002组(PTZ+LY294002+r Hu EPO)、二甲基亚砜(DMSO)对照组(PTZ+DMSO+r Hu EPO),检测各组大鼠行为学和脑电图的改变;TUNEL法检测海马神经细胞的凋亡情况;免疫组织化学法观察磷酸化蛋白激酶B(p-PKB/p-Akt)、Bax的表达;反转录多聚酶链反应(RT-PCR)方法检测各组大鼠海马Baxm RNA的表达,Western blot方法检测各组大鼠海马Akt、p-Akt、Bax蛋白的表达。结果 r Hu EPO可以增加p-Akt蛋白的表达、下调Bax蛋白的表达,发挥神经保护作用;加入PI3K抑制剂LY294002,p-Akt的表达较r Hu EPO组减少,海马Bax蛋白、Baxm RNA的表达较r Hu EPO组增加,减弱了r Hu EPO的保护作用(P<0.05),差异具有统计学意义。结论 r Hu EPO的抗凋亡从应用PI3K/Akt激活剂和抑制剂两个方面证实作用可能是通过了PI3K/Akt信号通路,通过对线粒体凋亡途径的相关调控因子Bax的表达进行调控,发挥神经保护作用。
文摘目的 探讨抗幽门螺旋杆菌(Hp)治疗联合重组人血小板生成素(rhTPO)对难治性特发性血小板减少性紫癜(ITP)患者辅助性T细胞1/辅助性T细胞2(Th1/Th2)细胞因子的影响。方法 选择2008年6月至2018年6月收治的71例难治性ITP患者为研究对象进行回顾性研究,按照治疗方案的不同将患者分为对照组(n=36)及观察组(n=35)。对照组给予rhTPO治疗,观察组在对照组治疗的基础上使用抗Hp治疗,两组均连续治疗2周。统计两组患者治疗2周后临床疗效;比较两组患者治疗前与治疗后Th1细胞因子[白细胞介素(IL)-2、γ-干扰素(IFN-γ)、肿瘤坏死因子-α(TNF-α)]和Th2细胞因子(IL-4、IL-10)、B淋巴细胞(CD19^+、B1细胞)与血小板(PLT)计数;比较治疗后1年内两组患者的复发率。结果 治疗2周后,观察组总有效率高于对照组(94.29% vs 69.44%,P<0.01)。治疗2周后,两组IFN-γ、TNF-α、IL-2、IL-2/IL-4、CD19^+、B1水平均较治疗前降低,且观察组低于对照组(P<0.05,P<0.01);IL-4、IL-10、PLT水平均较治疗前升高,且观察组高于对照组(P<0.05,P<0.01)。随访1年内观察组总复发率低于对照组(21.21% vs 48.00%,P<0.05)。结论 抗Hp联合rhTPO治疗难治性ITP患者可显著提高临床疗效,升高PLT,调节Th1/Th2免疫平衡,缓解临床症状,改善预后。
