重组鲎C因子酶原的高效表达及其在内毒素检测中的应用

High level expression of recombinant horseshoe crab factor C enzymogen and its application in endotoxin detection

摘要:

目的 利用重组鲎C因子酶原(recombinant horseshoe crab factor C enzymogen, rFC)开发一种低成本、高灵敏度的内毒素检测试剂及相应检测方法。方法 采用Bac-to-Bac杆状病毒表达系统在昆虫细胞Sf9中表达rFC，并通过终点荧光法测定其酶活性。优化蛋白表达条件，采用离子交换进行初步纯化。优化rFC的反应条件，进而建立基于终点荧光法的内毒素检测方法，并将该方法与传统鲎试剂(limulus amebocyte lysate, LAL)进行等效性验证。结果 重组鲎C因子酶原的表达量为110.42 mg/L，提高了4.75倍。该内毒素检测方法在0.005-1.000 EU/mL范围内具有良好的线性，反应时间为1 h，检测限为0.005 EU/mL。该方法在实际样本中的适用率为92.45%，检测值一致性为83.67%，且89.80%的样本检测限值与LAL法一致。结论 本研究成功实现了重组鲎C因子酶原的高效表达，建立了低成本、灵敏度高于普通鲎试剂的内毒素检测方法，具有良好的应用潜力。

Abstract:

Objective To develop a low-cost and highly sensitive endotoxin detection reagent and detection method with recombinant horseshoe crab factor C enzymogen (rFC).Methods The Bac-to-Bac baculovirus expression system was used to express rFC in Sf9 cells and the activity of rFC was measured by the end-point fluorescence assay with endotoxin. The conditions of protein expression were optimized, and ion exchange was used for crude enzyme separation. An endotoxin detection method with rFC based on end-point fluorescence assay was established after the reaction conditions were optimized. Furthermore, the established method was compared with the conventional limulus amebocyte lysate (LAL).Results The expression level of rFC was 110.42 mg/L, increasing by 4.75 times. The linear range of endotoxin detection was 0.005-1.000 EU/mL in 1 h, with a good linearity and the limit of detection being 0.005 EU/mL. The applicability rate of this method for actual samples was 92.45%. The consistency of the detection results was 83.67%, and 89.80% of the samples had consistent detection limits with LAL.Conclusion This study achieves the efficient expression of rFC and establishes an endotoxin detection method with higher sensitivity than LAL, which has great potential for application.