The wild-type human Fas-associated death domain (FADD) protein was expressed as a His-tag fusion protein in Escherichia coli. Recombinant FADD proteins were purified under the denatured condition. After denatured prot...The wild-type human Fas-associated death domain (FADD) protein was expressed as a His-tag fusion protein in Escherichia coli. Recombinant FADD proteins were purified under the denatured condition. After denatured protein purification, it was refolded and obtained at a yield of about 23 mg/L. Purified FADD exhibited as a homogenous band corresponding to the molecular weight of 31 kDa. Immunization of rabbits against the refolded FADD protein was allowed the production of high titre polyclonal antiserum. This new polyclonal antibody could recognize recombinant FADD protein in Western blot. Immunoreactivity was also observed in immunofluorescence assay. The low cost polyclonal antiserum was applicable to extensive detection of FADD in various immunoassays.展开更多
Human African trypanosomiasis (HAT), commonly known as sleeping sickness is one of the neglected tropical diseases (NTDs), which is fatal if left untreated. Its diagnosis is a challenge since the signs and symptoms of...Human African trypanosomiasis (HAT), commonly known as sleeping sickness is one of the neglected tropical diseases (NTDs), which is fatal if left untreated. Its diagnosis is a challenge since the signs and symptoms of the primary phase are not specific, the existing diagnostic methods have low sensitivity and specificity, and the available drugs have some toxicity. New, robust, and cost-effective techniques are needed for the early identification of parasites. This study aimed to assess the sensitivity and specificity of two different types of polyclonal antibodies against T. b. gambiense using antigen detection ELISA. Polyclonal antibodies against the expressed proteins Tbg I2 and Tbg I17 were produced using New Zealand white rabbits. The antibody titer measured was greater than 32 g/L after the 3<sup>rd</sup> immunization for the expressed protein Tbg I2. For the expressed protein Tbg I17, the antibody titer measured was greater than 32 g/L after the 4<sup>th</sup> immunization. The sensitivity and specificity of the Tbg I2 polyclonal antibody confirmed with Polymerase Chain Reaction (PCR) as gold standard were respectively 89.5% and 80.6%, while for the Tbg I17 polyclonal antibody, the sensitivity and specificity were respectively 92.1% and 88.9%. The area under the curve for the Tbg I2 polyclonal antibody was 0.90 ± 0.032, while for the Tbg I17 polyclonal antibody, the area under the curve was 0.92 ± 0.0. The Tbg I17 polyclonal antibody produced in New Zealand white rabbits has good sensitivity and good specificity;it can be successfully used in the diagnosis of HAT.展开更多
该研究采用高毒力持留基因型单增李斯特菌819-2菌株全菌蛋白免疫SPF级Balb/C小鼠制备抗血清,利用免疫蛋白质组学对菌株胞外蛋白质组进行分析,旨在挖掘筛选单增李斯特菌强免疫原性蛋白作为特异性抗体制备的候选抗原。超声法提取819-2菌...该研究采用高毒力持留基因型单增李斯特菌819-2菌株全菌蛋白免疫SPF级Balb/C小鼠制备抗血清,利用免疫蛋白质组学对菌株胞外蛋白质组进行分析,旨在挖掘筛选单增李斯特菌强免疫原性蛋白作为特异性抗体制备的候选抗原。超声法提取819-2菌株全菌蛋白免疫SPF级Balb/C小鼠制备抗血清,四次免疫后经间接ELISA测定效价达1:512000。脱氧胆酸钠(DOC)-10%(m/V)TCA沉淀法提取单增李斯特菌819-2菌株胞外蛋白,利用免疫蛋白质组学和LC-MS/MS技术挖掘并鉴定具有强免疫反应的蛋白点,结果表明双向电泳图谱成功获得85个蛋白点,并成功鉴定了P60、InlC、MltG、Enolase和假定蛋白YxeA family protein等5个强免疫原性蛋白,研究结果为基于强免疫原性蛋白制备特异性抗体用于食品及其加工环境中单增李斯特菌富集及快速检测技术研制提供了数据基础。展开更多
【目的】维生素D3(vitamin D3,VD3)是一类重要的类固醇激素,参与调控动物体内多种生理活动,具有生物活性的VD3代谢物1,25(OH)2D3通过与维生素D受体(vitamin D receptor,VDR)结合发挥生物学功能。试验旨在克隆鹅VDR基因CDS序列并制备鹅VD...【目的】维生素D3(vitamin D3,VD3)是一类重要的类固醇激素,参与调控动物体内多种生理活动,具有生物活性的VD3代谢物1,25(OH)2D3通过与维生素D受体(vitamin D receptor,VDR)结合发挥生物学功能。试验旨在克隆鹅VDR基因CDS序列并制备鹅VDR多克隆抗体,为后续开展VDR介导VD3调控鹅体内多种生理活动的分子机制研究提供基础支撑。【方法】通过基因克隆方法获得鹅VDR基因CDS序列,并采用生物信息学方法对其编码蛋白进行结构和功能预测分析。通过基因合成和亚克隆方法构建鹅VDR基因重组表达质粒,转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达和镍柱纯化后获得鹅VDR重组蛋白。以纯化的鹅VDR重组蛋白作为免疫原,制备兔抗鹅VDR多克隆抗体,采用间接ELISA法检测抗体效价,采用Western blotting检测抗体特异性。【结果】试验成功克隆了鹅VDR基因编码区序列,长度为1356 bp,编码451个氨基酸残基。该编码蛋白与鸡VDR蛋白的相似性高达91.4%,无信号肽和跨膜结构域,N-端含有核定位序列,且含有DNA结合域和配体结合域2个保守结构域,属于类固醇/甲状腺激素受体超家族成员。试验成功构建了pET-30a(+)-VDR重组质粒,并诱导表达鹅VDR重组蛋白。纯化后的重组蛋白纯度高达90%以上,且大小符合预期(52 ku)。试验制备了兔抗鹅VDR多克隆抗体,其抗体效价>1∶1093500;除肝脏组织外,该多克隆抗体在产蛋鹅肾脏、十二指肠、空肠和回肠组织中均能够识别天然VDR蛋白的2种亚型。【结论】试验成功克隆了鹅VDR基因CDS序列,并制备了兔抗鹅VDR多克隆抗体,该多克隆抗体特异性高,可用于后续鹅体内VDR介导的生物学功能研究。展开更多
猪肺炎支原体的持续性感染问题频发,同源重组介导的抗原变异扮演重要角色。解螺旋酶RuvA调节霍利迪连接体变构是参与同源重组的关键步骤。鉴于此,本研究旨在原核表达猪肺炎支原体解螺旋酶RuvA重组蛋白,鉴定其DNA结合活性并制备多克隆抗...猪肺炎支原体的持续性感染问题频发,同源重组介导的抗原变异扮演重要角色。解螺旋酶RuvA调节霍利迪连接体变构是参与同源重组的关键步骤。鉴于此,本研究旨在原核表达猪肺炎支原体解螺旋酶RuvA重组蛋白,鉴定其DNA结合活性并制备多克隆抗体。试验通过分子克隆构建原核表达质粒pET21a-RuvA,经诱导表达和纯化获得RuvA M hp重组蛋白;免疫家兔制备抗RuvA M hp多克隆抗体,再利用间接ELISA和Western blot试验进行效价测定和特异性验证;利用凝胶迁移试验结合表面等离子共振技术分析RuvA M hp的DNA结合活性;利用凝胶迁移试验结合Western blot试验鉴定RuvA M hp的寡聚特性。结果显示:原核表达的RuvA M hp重组蛋白约为26 ku;制备的抗RuvA M hp多克隆抗体效价为1∶256000,且具有良好的特异性;RuvA M hp具有较强的DNA结合活性,对霍利迪连接体的亲和力高达624.4 pmol·L^(-1)(K D),并且主要以八聚体形式与其形成稳定复合物。通过RuvA M hp的原核表达、多克隆抗体制备及活性鉴定,为后续探索RuvA调解同源重组介导猪肺炎支原体抗原变异的分子机制奠定了基础。展开更多
基金financially supported by the following funds to Zi-Chun Hua: the National Nature Science Foundation of China (No. 30425009, 30600320, 30730030, 30330530, 30270291)Jiangsu Provincial Nature Sciences Foundation (No. BK2007715)Jiangsu Provincial Department of Health (No. H200524, H200742)
文摘The wild-type human Fas-associated death domain (FADD) protein was expressed as a His-tag fusion protein in Escherichia coli. Recombinant FADD proteins were purified under the denatured condition. After denatured protein purification, it was refolded and obtained at a yield of about 23 mg/L. Purified FADD exhibited as a homogenous band corresponding to the molecular weight of 31 kDa. Immunization of rabbits against the refolded FADD protein was allowed the production of high titre polyclonal antiserum. This new polyclonal antibody could recognize recombinant FADD protein in Western blot. Immunoreactivity was also observed in immunofluorescence assay. The low cost polyclonal antiserum was applicable to extensive detection of FADD in various immunoassays.
文摘Human African trypanosomiasis (HAT), commonly known as sleeping sickness is one of the neglected tropical diseases (NTDs), which is fatal if left untreated. Its diagnosis is a challenge since the signs and symptoms of the primary phase are not specific, the existing diagnostic methods have low sensitivity and specificity, and the available drugs have some toxicity. New, robust, and cost-effective techniques are needed for the early identification of parasites. This study aimed to assess the sensitivity and specificity of two different types of polyclonal antibodies against T. b. gambiense using antigen detection ELISA. Polyclonal antibodies against the expressed proteins Tbg I2 and Tbg I17 were produced using New Zealand white rabbits. The antibody titer measured was greater than 32 g/L after the 3<sup>rd</sup> immunization for the expressed protein Tbg I2. For the expressed protein Tbg I17, the antibody titer measured was greater than 32 g/L after the 4<sup>th</sup> immunization. The sensitivity and specificity of the Tbg I2 polyclonal antibody confirmed with Polymerase Chain Reaction (PCR) as gold standard were respectively 89.5% and 80.6%, while for the Tbg I17 polyclonal antibody, the sensitivity and specificity were respectively 92.1% and 88.9%. The area under the curve for the Tbg I2 polyclonal antibody was 0.90 ± 0.032, while for the Tbg I17 polyclonal antibody, the area under the curve was 0.92 ± 0.0. The Tbg I17 polyclonal antibody produced in New Zealand white rabbits has good sensitivity and good specificity;it can be successfully used in the diagnosis of HAT.
文摘该研究采用高毒力持留基因型单增李斯特菌819-2菌株全菌蛋白免疫SPF级Balb/C小鼠制备抗血清,利用免疫蛋白质组学对菌株胞外蛋白质组进行分析,旨在挖掘筛选单增李斯特菌强免疫原性蛋白作为特异性抗体制备的候选抗原。超声法提取819-2菌株全菌蛋白免疫SPF级Balb/C小鼠制备抗血清,四次免疫后经间接ELISA测定效价达1:512000。脱氧胆酸钠(DOC)-10%(m/V)TCA沉淀法提取单增李斯特菌819-2菌株胞外蛋白,利用免疫蛋白质组学和LC-MS/MS技术挖掘并鉴定具有强免疫反应的蛋白点,结果表明双向电泳图谱成功获得85个蛋白点,并成功鉴定了P60、InlC、MltG、Enolase和假定蛋白YxeA family protein等5个强免疫原性蛋白,研究结果为基于强免疫原性蛋白制备特异性抗体用于食品及其加工环境中单增李斯特菌富集及快速检测技术研制提供了数据基础。
文摘猪肺炎支原体的持续性感染问题频发,同源重组介导的抗原变异扮演重要角色。解螺旋酶RuvA调节霍利迪连接体变构是参与同源重组的关键步骤。鉴于此,本研究旨在原核表达猪肺炎支原体解螺旋酶RuvA重组蛋白,鉴定其DNA结合活性并制备多克隆抗体。试验通过分子克隆构建原核表达质粒pET21a-RuvA,经诱导表达和纯化获得RuvA M hp重组蛋白;免疫家兔制备抗RuvA M hp多克隆抗体,再利用间接ELISA和Western blot试验进行效价测定和特异性验证;利用凝胶迁移试验结合表面等离子共振技术分析RuvA M hp的DNA结合活性;利用凝胶迁移试验结合Western blot试验鉴定RuvA M hp的寡聚特性。结果显示:原核表达的RuvA M hp重组蛋白约为26 ku;制备的抗RuvA M hp多克隆抗体效价为1∶256000,且具有良好的特异性;RuvA M hp具有较强的DNA结合活性,对霍利迪连接体的亲和力高达624.4 pmol·L^(-1)(K D),并且主要以八聚体形式与其形成稳定复合物。通过RuvA M hp的原核表达、多克隆抗体制备及活性鉴定,为后续探索RuvA调解同源重组介导猪肺炎支原体抗原变异的分子机制奠定了基础。
