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。
Preparation and Identification of Sulfadimethoxine Nanobody Immunomagnetic Beads
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.
sulfadimethoxine(SDM)bacterial magnetic particles(BMPs)linkernanobody(Nb)immunomagnetic beads
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