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,SP-DP),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.
Abstract
Immunomagnetic beads based on nanobodies(Nbs)and bacterial magnetic particles(BMPs)have shown great potential for the separation and analysis of targets,exhibiting low cost and high separa-tion efficiency.However,the influence of different lengths of flexible linker on the performance of immu-nomagnetic beads has not been explored.In order to enhance the performance of immunomagnetic beads,anti-sulfadimethoxine(SDM)Nb was used to generate different lengths of flexible linker.The recombi-nant plasmids pET28a-SDM-Nb-(G4S)1-Cys and pET28a-SDM-Nb-(G4S)4-Cys were constructed and transformed into E.coli BL21(DE3)for expression,respectively.Then,Nb-(G4S)1-Cys and Nb-(G4S)4-Cys were conjugated with BMPs using N-succinimidyl-3-(2-pyridyldithiol)propionate(SPDP).The coupling was verified by Western blot,and the coupling conditions were optimized.The hydrated particle size,Zeta potential,and dispersibility of the immunomagnetic beads were analyzed by transmis-sion electron microscopy and Zeta potential analyzer.The results demonstrated that Nb-(G4S)1-Cys and Nb-(G4S)4-Cys could be immobilized on the surface of BMPs.The coupling efficiency of Nb-(G4S)1-Cys and BMPs was higher than that of Nb-(G4S)4-Cys and BMPs.Characterization revealed that BMP-(G4S)1-Nb exhibited a higher absolute value of Zeta potential,smaller hydrated particle size,and lower polydispersibility index,indicating enhanced colloidal stability in aqueous systems.Taken together,the immunomagnetic beads constructed with BMPs and Nb-(G4S)1-Cys showed better performance than those constructed with BMPs and Nb-(G4S)4-Cys.This study provides theoretical support for the selec-tion of appropriate linker lengths in the construction of efficient immunomagnetic beads for the separation and analysis of SDM pollutants.
关键词
磺胺间二甲氧嘧啶/磁小体/连接肽/纳米抗体/免疫磁珠
Key words
sulfadimethoxine(SDM)/bacterial magnetic particles(BMPs)/linker/nanobody(Nb)/immunomagnetic beads
维普
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