基于杆状病毒表达系统的鲎C因子表达及其活性检测

Baculovirus expression system-based expression of horseshoe crab factor C and its activity

摘要:

内毒素是常见外致热原，内毒素超标会导致脓毒血症、感染性休克甚至死亡等严重后果，因此其检测在医疗、制药和食品领域至关重要。鲎试剂检测法的广泛应用导致鲎数量急剧下降，且该方法存在批间差异和难以定量等限制。重组C因子检测法批次间稳定、灵敏度高且可定量，可代替鲎试剂，但高成本和复杂操作限制了该方法的推广。为简化制备重组C因子的步骤，同时降低重组C因子的生产成本，本研究利用高产抗凋亡载体qBac-IIIG、优选信号肽和优化密码子的策略，使用杆状病毒蛋白表达系统制备重组C因子，分泌至细胞培养上清液中的重组C因子无需纯化，可直接用细胞培养上清液检测内毒素。表达重组C因子的1 L发酵体系中发酵液的内毒素检测灵敏度可达到0.05 EU/mL，每L发酵液可以支持50万个检测反应。本研究提高了重组C因子的产量和活性，节省了蛋白纯化步骤，降低了生产成本，为重组C因子内毒素检测法的推广应用奠定基础。

Abstract:

Endotoxins are common exogenous pyrogens. Excessive endotoxins in medical devices and injections can lead to serious consequences such as sepsis, septic shock, and even death. Therefore, endotoxin detection plays a crucial role in medical, pharmaceutical, and food sectors. The wide application of Limulus amebocyte lysate (LAL) has led to a sharp decline in the number of horseshoe crabs. Moreover, the LAL assay has limitations such as interbatch variations and difficulty in quantification. The recombinant factor C (rFC) assay is stable between batches, highly sensitive, and capable of quantitation, and thus it can be used as an alternative for the LAL assay. However, the high cost and complex procedures involved in producing recombinant factor C have limited the widespread application of this method. In order to simplify the preparation and reduce the production cost of recombinant factor C, this study focuses on the production of recombinant factor C based on the baculovirus expression system. Multiple measures such as a high-yield and anti-apoptotic vector qBac-IIIG, the optimal signal peptide, and the optimized codon were used to reach the goal of endotoxin detection with cell supernatant. This method simplifies the steps of protein purification. The sensitivity of the supernatant reached 0.05 EU/mL in a 1-L fermentation system, and 500 000 detecting reactions can be supported per liter of fermentation broth. This study increases the yield and activity of recombinant factor C, simplifies the procedures of protein purification, and reduces the cost, laying a foundation for the promotion and application of recombinant factor C in endotoxin detection.