目的根据构建的人致病性纳尔逊海湾病毒(NBVs)M3基因片段质粒,纯化融合蛋白制备NBVs M3多克隆抗体。方法利用构建的纳尔逊海湾病毒(NBVs)M3基因质粒Pris His MB-M3,转化至大肠埃希菌(E.coil)BL21(DE3)感受态细胞中,异丙基-β-D-硫代半...目的根据构建的人致病性纳尔逊海湾病毒(NBVs)M3基因片段质粒,纯化融合蛋白制备NBVs M3多克隆抗体。方法利用构建的纳尔逊海湾病毒(NBVs)M3基因质粒Pris His MB-M3,转化至大肠埃希菌(E.coil)BL21(DE3)感受态细胞中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达,产物经过镍柱亲和层析法获得Pris His MB-M3融合蛋白;将纯化的融合蛋白作为抗原免疫家兔,获得NBVs M3多克隆抗体;蛋白免疫印迹检测(Western blot)蛋白准确性以及多克隆抗体抗性。结果 SDS-PAGE电泳考马斯亮蓝染色检测显示:25℃、IPTG浓度为0.3 mmol/L,诱导12 h,Pris His MB-M3融合蛋白表达量最高;在咪唑浓度为MCAC-20、MCAC-40条件时洗脱下目的蛋白。用纯化的融合蛋白免疫家兔制备抗体,Western blot验证成功制备出NBVs M3多克隆抗体。结论成功获得了灵敏性及特异性较高的纳尔逊海湾病毒(NBVs)M3多克隆抗体,为进一步研究该病毒的致病性提供了很高的应用价值。展开更多
目的制备纳尔逊海湾病毒(NBV)S3多克隆抗体。方法首先将构建好的Pris His MB-S3重组基因质粒,转化到大肠杆菌(E.coil)BL21(DE3)感受态细胞中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达。镍柱亲和层析法纯化目的蛋白以获得大量融合重...目的制备纳尔逊海湾病毒(NBV)S3多克隆抗体。方法首先将构建好的Pris His MB-S3重组基因质粒,转化到大肠杆菌(E.coil)BL21(DE3)感受态细胞中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达。镍柱亲和层析法纯化目的蛋白以获得大量融合重组蛋白,以此为抗原免疫大鼠制备抗S3多克隆抗体并进行鉴定。结果 Pris His MB-S3蛋白的相对分子质量(Mr)约39 000,以包涵体形式存在,通过免疫大鼠成功制备出NBV S3多克隆抗体。间接ELISA测定抗体效价达1∶64000。间接免疫荧光实验证明S3蛋白成功在细胞内表达并以颗粒状分布在细胞质内。结论成功获得高反应性和高特异性的S3蛋白多克隆抗体。展开更多
Abstract Objectives To identify the effects of tuberculin purified protein derivative (PPD) sensitization on attenuating pulmonary T helper 2 (Th2) reaction and eosinophil infiltration in ovalbumin sensitized mice, a...Abstract Objectives To identify the effects of tuberculin purified protein derivative (PPD) sensitization on attenuating pulmonary T helper 2 (Th2) reaction and eosinophil infiltration in ovalbumin sensitized mice, and to search for the possibility of its clinical use in the management of asthma in a new way. Methods Sixty C57BL/6 mice were sensitized with PPD and then with ovalbumin and aluminum hydroxide, and randomized into 4 groups: ovalbumin (OVA), pre PPD, post PPD and control groups. Aerosol PPD were administered 3 h before or after ovalbumin challenge in the pre PPD and post PPD groups respectively, and control group received aerosol PPD only. IL 4, IL 5 expression was detected by immunocytochemistry in situ hybridization. Lung slides were stained with eosin and hemotoxylin, and pathological changes were observed. Results Ovalbumin aerosol inhalation caused a mixed inflammatory infiltration dominated by CD4 + T lymphocytes and eosinophils in the lung of sensitized mice. 87.5%-89.7% and 89.0%-89.2% of the CD4 + T lymphocytes were IL 4 mRNA + and IL 5 mRNA + respectively. 88.7%-91.2% of IL 4 mRNA + cells and 89.8%-90.6% of IL 5 mRNA + cells were CD4 + T lymphocytes in OVA group. Aerosol administration of PPD markedly suppressed IL 4 and IL 5 expression, and lung eosinophil infiltration. It was more effective in pre PPD group. 76.6%-78.0% of IL 4 mRNA + and 73.8%-79.7% of IL 5 mRNA + cells were CD4 + and 78.1%-84.9% and 78.4%-85.3% of the CD4 + cells were IL 4 mRNA + or IL 5 mRNA + respectively in pre PPD group, both were markedly lower than that of OVA group. CD4 + percentage of IL 4 mRNA + and IL 5 mRNA + cells were 80.7%- 82.0% and 78.0%-83.9% in post PPD group, which were markedly lower than that of OVA group. Conclusions Sensitization with PPD by intraperitoneal injection and then challenged by PPD inhalation markedly suppressed IL 4, IL 5 expression and eosinophil infiltration, and attenuated pulmonary Th2 reaction in ovalbumin sensitiz展开更多
Natural products with significant biological activities continuously act as rich sources for drug discovery and development.To harness the potential of these valuable compounds,robust methods need to be developed for ...Natural products with significant biological activities continuously act as rich sources for drug discovery and development.To harness the potential of these valuable compounds,robust methods need to be developed for their rapid and sustainable production.Cell-free biosynthesis of pharmaceutical natural products by in vitro reconstruction of the entire biosynthetic pathways represents one such solution.In this review,we focus on in vitro biosynthesis of two important classes of natural products,polyketides(PKs)and nonribosomal peptides(NRPs).First,we summarize purified enzyme-based systems for the biosynthesis of PKs,NRPs,and PK/NRP hybrids.Then,we introduce the cell-free protein synthesis(CFPS)-based technology for natural product production.With that,we discuss challenges and opportunities of cell-free synthetic biology for in vitro biosynthesis of natural products.展开更多
文摘目的根据构建的人致病性纳尔逊海湾病毒(NBVs)M3基因片段质粒,纯化融合蛋白制备NBVs M3多克隆抗体。方法利用构建的纳尔逊海湾病毒(NBVs)M3基因质粒Pris His MB-M3,转化至大肠埃希菌(E.coil)BL21(DE3)感受态细胞中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达,产物经过镍柱亲和层析法获得Pris His MB-M3融合蛋白;将纯化的融合蛋白作为抗原免疫家兔,获得NBVs M3多克隆抗体;蛋白免疫印迹检测(Western blot)蛋白准确性以及多克隆抗体抗性。结果 SDS-PAGE电泳考马斯亮蓝染色检测显示:25℃、IPTG浓度为0.3 mmol/L,诱导12 h,Pris His MB-M3融合蛋白表达量最高;在咪唑浓度为MCAC-20、MCAC-40条件时洗脱下目的蛋白。用纯化的融合蛋白免疫家兔制备抗体,Western blot验证成功制备出NBVs M3多克隆抗体。结论成功获得了灵敏性及特异性较高的纳尔逊海湾病毒(NBVs)M3多克隆抗体,为进一步研究该病毒的致病性提供了很高的应用价值。
文摘目的制备纳尔逊海湾病毒(NBV)S3多克隆抗体。方法首先将构建好的Pris His MB-S3重组基因质粒,转化到大肠杆菌(E.coil)BL21(DE3)感受态细胞中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达。镍柱亲和层析法纯化目的蛋白以获得大量融合重组蛋白,以此为抗原免疫大鼠制备抗S3多克隆抗体并进行鉴定。结果 Pris His MB-S3蛋白的相对分子质量(Mr)约39 000,以包涵体形式存在,通过免疫大鼠成功制备出NBV S3多克隆抗体。间接ELISA测定抗体效价达1∶64000。间接免疫荧光实验证明S3蛋白成功在细胞内表达并以颗粒状分布在细胞质内。结论成功获得高反应性和高特异性的S3蛋白多克隆抗体。
文摘Abstract Objectives To identify the effects of tuberculin purified protein derivative (PPD) sensitization on attenuating pulmonary T helper 2 (Th2) reaction and eosinophil infiltration in ovalbumin sensitized mice, and to search for the possibility of its clinical use in the management of asthma in a new way. Methods Sixty C57BL/6 mice were sensitized with PPD and then with ovalbumin and aluminum hydroxide, and randomized into 4 groups: ovalbumin (OVA), pre PPD, post PPD and control groups. Aerosol PPD were administered 3 h before or after ovalbumin challenge in the pre PPD and post PPD groups respectively, and control group received aerosol PPD only. IL 4, IL 5 expression was detected by immunocytochemistry in situ hybridization. Lung slides were stained with eosin and hemotoxylin, and pathological changes were observed. Results Ovalbumin aerosol inhalation caused a mixed inflammatory infiltration dominated by CD4 + T lymphocytes and eosinophils in the lung of sensitized mice. 87.5%-89.7% and 89.0%-89.2% of the CD4 + T lymphocytes were IL 4 mRNA + and IL 5 mRNA + respectively. 88.7%-91.2% of IL 4 mRNA + cells and 89.8%-90.6% of IL 5 mRNA + cells were CD4 + T lymphocytes in OVA group. Aerosol administration of PPD markedly suppressed IL 4 and IL 5 expression, and lung eosinophil infiltration. It was more effective in pre PPD group. 76.6%-78.0% of IL 4 mRNA + and 73.8%-79.7% of IL 5 mRNA + cells were CD4 + and 78.1%-84.9% and 78.4%-85.3% of the CD4 + cells were IL 4 mRNA + or IL 5 mRNA + respectively in pre PPD group, both were markedly lower than that of OVA group. CD4 + percentage of IL 4 mRNA + and IL 5 mRNA + cells were 80.7%- 82.0% and 78.0%-83.9% in post PPD group, which were markedly lower than that of OVA group. Conclusions Sensitization with PPD by intraperitoneal injection and then challenged by PPD inhalation markedly suppressed IL 4, IL 5 expression and eosinophil infiltration, and attenuated pulmonary Th2 reaction in ovalbumin sensitiz
文摘Natural products with significant biological activities continuously act as rich sources for drug discovery and development.To harness the potential of these valuable compounds,robust methods need to be developed for their rapid and sustainable production.Cell-free biosynthesis of pharmaceutical natural products by in vitro reconstruction of the entire biosynthetic pathways represents one such solution.In this review,we focus on in vitro biosynthesis of two important classes of natural products,polyketides(PKs)and nonribosomal peptides(NRPs).First,we summarize purified enzyme-based systems for the biosynthesis of PKs,NRPs,and PK/NRP hybrids.Then,we introduce the cell-free protein synthesis(CFPS)-based technology for natural product production.With that,we discuss challenges and opportunities of cell-free synthetic biology for in vitro biosynthesis of natural products.
