目的对菌源性磁珠(Bacterial Magnetic Particles,BMPs)在溶液中的凝集,及免疫BMPs在免疫检测中的凝集性特征进行分析和评价。方法从磁性细菌体内提取BMPs,以粒径相仿的Fe3O4磁珠为对照,对BMPs在溶液中的凝集,以吸光度半减法进行量化比...目的对菌源性磁珠(Bacterial Magnetic Particles,BMPs)在溶液中的凝集,及免疫BMPs在免疫检测中的凝集性特征进行分析和评价。方法从磁性细菌体内提取BMPs,以粒径相仿的Fe3O4磁珠为对照,对BMPs在溶液中的凝集,以吸光度半减法进行量化比较。将癌胚抗原(Carcinoembryonic Antigen CEA)相关抗体接种于BMPs表面形成免疫BMPs。在CEA溶液中添加免疫BMPs,借助显微镜观察凝集现象;并且对回收的凝结体经0.1MAlbumiHCl缓冲液(pH2.3)处理,再进行同样免疫凝集检测及显微镜观察。结果浓度为50μg/ml的BMPs PBS溶液,测定吸光度半减时间值为126min,Fe3O4磁珠吸光度半减时间值为35min。在一定浓度的CEA溶液中添加免疫BMPs5μg,通过显微镜观察,CEA浓度在100pg/ml以上时,凝集现象非常明显,且CEA浓度增大时凝集体也随之增大。但结合了CEA的免疫BMPs经0.1M Albumin-HCl缓冲液(pH2.3)处理,解开抗原抗体的结合,再进行同样免疫凝集检测及显微镜观察,发现无类似凝集现象出现。表明凝集现象系由抗原抗体所致,而非单纯的磁性体凝集。结论与Fe3O4磁珠相比,BMPs相互间不易凝集,主要表现为对外磁场的感应性凝集。从BMPs的凝集性特征分析,与普通Fe3O4磁珠相比,具有明显的优势,比较适合作为免疫磁珠法检测中的磁性载体。展开更多
基于纳米抗体(nanobody,Nb)和磁小体(bacterial magnetic particles,BMPs)的免疫磁珠在污染物分离分析中具有良好的应用前景,然而,不同长度柔性连接肽(linker)对免疫磁珠性能的影响尚未见相关报道。为了探究柔性连接肽长度对免疫磁珠的...基于纳米抗体(nanobody,Nb)和磁小体(bacterial magnetic particles,BMPs)的免疫磁珠在污染物分离分析中具有良好的应用前景,然而,不同长度柔性连接肽(linker)对免疫磁珠性能的影响尚未见相关报道。为了探究柔性连接肽长度对免疫磁珠的性能影响,本研究使用pET-28a作为载体,在磺胺间二甲氧嘧啶(sulfadimethoxine,SDM)Nb基因上融合了不同长度的柔性连接肽,分别为pET28a-SDM-Nb-(G4S)1-Cys和pET28a-SDM-Nb-(G4S)4-Cys,并使用大肠杆菌BL21(DE3)作为重组工程菌进行表达,最终获得Nb-(G4S)1-Cys和Nb-(G4S)4-Cys重组蛋白质。利用异源双功能试剂3-(2-吡啶二巯基)丙酸N-羟基琥珀酰亚胺酯(N-succinimidyl-3-(2-pyridyldithiol)propionate,SPDP),分别将重组蛋白质与BMPs进行偶联,构建了免疫磁珠。利用免疫印迹对偶联结果进行了初步鉴定,并对偶联条件进行了优化。同时,使用透射电镜和Zeta电位分析仪对免疫磁珠的水合粒径、Zeta电位和分散性进行了分析。研究结果表明,SPDP能有效地将Nb-(G4S)1-Cys和Nb-(G4S)4-Cys定向固定在BMPs表面。通过差值法计算发现,Nb-(G4S)1-Cys与BMPs的偶联效率高于Nb-(G4S)4-Cys与BMPs的偶联效率。进一步表征结果显示,BMP-(G4S)1-Nb的Zeta电位绝对值更高,水合粒径更小,并且具有较低的多分散性指数,说明其在水相体系中具有更强的胶体稳定性。综上所述,利用BMPs和Nb-(G4S)1-Cys构建的免疫磁珠性能优于BMPs和Nb-(G4S)4-Cys构建的免疫磁珠。这为今后选择合适长度的连接肽构建高效的免疫磁珠分离分析SDM提供了理论依据。展开更多
A novel real-time RT-PCR method,BMPs based real-time RT-PCR which integrated with magne-tic separation technique of bacterial magnetic particles(BMPs),was set up for detection of Squash mosaic virus(SqMV).After SqMV p...A novel real-time RT-PCR method,BMPs based real-time RT-PCR which integrated with magne-tic separation technique of bacterial magnetic particles(BMPs),was set up for detection of Squash mosaic virus(SqMV).After SqMV particles in crude sap were concentrated by BMPs,viral RNAs were released and detected by real time RT-PCR.The results indicated that BMPs based real-time RT-PCR was efficient,and the detection sensibility was equivalent to that of the Trizol based real-time RT-PCR,of which Trizol reagent was used for viral RNAs extration.Comparing to Trizol-based method,the BMPs-based method had advantages of simplicity on operation,time saving for RNA extraction and without using noxious organic chemicals.展开更多
文摘基于纳米抗体(nanobody,Nb)和磁小体(bacterial magnetic particles,BMPs)的免疫磁珠在污染物分离分析中具有良好的应用前景,然而,不同长度柔性连接肽(linker)对免疫磁珠性能的影响尚未见相关报道。为了探究柔性连接肽长度对免疫磁珠的性能影响,本研究使用pET-28a作为载体,在磺胺间二甲氧嘧啶(sulfadimethoxine,SDM)Nb基因上融合了不同长度的柔性连接肽,分别为pET28a-SDM-Nb-(G4S)1-Cys和pET28a-SDM-Nb-(G4S)4-Cys,并使用大肠杆菌BL21(DE3)作为重组工程菌进行表达,最终获得Nb-(G4S)1-Cys和Nb-(G4S)4-Cys重组蛋白质。利用异源双功能试剂3-(2-吡啶二巯基)丙酸N-羟基琥珀酰亚胺酯(N-succinimidyl-3-(2-pyridyldithiol)propionate,SPDP),分别将重组蛋白质与BMPs进行偶联,构建了免疫磁珠。利用免疫印迹对偶联结果进行了初步鉴定,并对偶联条件进行了优化。同时,使用透射电镜和Zeta电位分析仪对免疫磁珠的水合粒径、Zeta电位和分散性进行了分析。研究结果表明,SPDP能有效地将Nb-(G4S)1-Cys和Nb-(G4S)4-Cys定向固定在BMPs表面。通过差值法计算发现,Nb-(G4S)1-Cys与BMPs的偶联效率高于Nb-(G4S)4-Cys与BMPs的偶联效率。进一步表征结果显示,BMP-(G4S)1-Nb的Zeta电位绝对值更高,水合粒径更小,并且具有较低的多分散性指数,说明其在水相体系中具有更强的胶体稳定性。综上所述,利用BMPs和Nb-(G4S)1-Cys构建的免疫磁珠性能优于BMPs和Nb-(G4S)4-Cys构建的免疫磁珠。这为今后选择合适长度的连接肽构建高效的免疫磁珠分离分析SDM提供了理论依据。
文摘A novel real-time RT-PCR method,BMPs based real-time RT-PCR which integrated with magne-tic separation technique of bacterial magnetic particles(BMPs),was set up for detection of Squash mosaic virus(SqMV).After SqMV particles in crude sap were concentrated by BMPs,viral RNAs were released and detected by real time RT-PCR.The results indicated that BMPs based real-time RT-PCR was efficient,and the detection sensibility was equivalent to that of the Trizol based real-time RT-PCR,of which Trizol reagent was used for viral RNAs extration.Comparing to Trizol-based method,the BMPs-based method had advantages of simplicity on operation,time saving for RNA extraction and without using noxious organic chemicals.
