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.
Animals ; Recombinant Proteins/genetics* ; Horseshoe Crabs/chemistry* ; Baculoviridae/metabolism* ; Endotoxins/analysis* ; Protein C/biosynthesis* ; Genetic Vectors/genetics* ; Arthropod Proteins/genetics* ; Enzyme Precursors ; Serine Endopeptidases

Objective To determine alkylglycerol (AKG) contents and variation in breast-milk of lactating women. Methods Five cases of healthy lactating women with term delivery were selected from June 2011 to June 2012. Breast-milk samples were collected at 1, 2, 3, 4, 8, 12, 16, 20 and 24 weeks postpartum. Breast milk samples were extracted, saponificated and derivatized. AKGs composition in breast-milk was quantitatively analyzed by GC chromatography. Results Mean 16C:0 AKG content in breast-milk decreased from(17.31 ± 3.59)× 10-3g/L to(11.14 ± 1.83)× 10-3g/L. Mean 18C:0 AKG content de-creased from(14.95±6.00)×10-3g/L to(9.68±2.51)×10-3g/L. Mean 18C:1 AKG content fluctuated between(4.64±0.91)×10-3g/L and(3.95±0.68)×10-3g/L. Conclusions 16C:0, 18C:0 and 18C:1 AKG contents exist in Chinese breast-milk through determina-tion by GC chromatography, and the concentrations vary among different stages of lactation.