Biosynthesis of staphylococcal enterotoxin A by genetic engineering technology and determination of staphylococcal enterotoxin A in water by HPLC-ESI-TOF

Abstract

Staphylococcal enterotoxin A (SEA) was the major virulence factor of Staphylococcus aureus and a biomarker of S. aureus. To establish a fast, low cost, high accuracy, reliable, and simple method for detecting S. aureus, SEA was analyzed by HPLC-ESI-TOF. SEA was not yet commercially available in universal, so SEA was prepared before it was analyzed by HPLC-ESI-TOF. The result showed that high purified SEA was successfully prepared and SEA has normal distribution in mass spectra. A large amount of recombinant SEA (rSEA) was obtained by engineering technology and was purified by Ni affinity chromatography column, and the expression and purity of rSEA and SEA were analyzed by SDS-PAGE. The factors effected on ionization of SEA were studied, and the qualitative analysis of SEA by HPLC-ESI-TOF. The result showed that large amount of SEs expressed within a short time at 28 °C or thereabouts, and there was no impurity bands in electrophorogram after rSEA was purified by Ni affinity chromatography column. In addition, the SEA which had homologous AA sequence with wild SEA was made by rSEA. The retention of SEA in column and ionization of SEA in ESI-TOF were studied for qualitative analysis of S. aureus. The result showed that the content of formic acid in mobile phase was an important factor for ionization of SEs in ESI-TOF. And the result provided theoretical foundation for qualitative detection of S. aureus. [SEs + nH + mNH] was shown on ESI-TOF spectra when SEA was detected by ESI-TOF in positive ion mode, and the numerical value of n+m was less than or equal to the number of basic amino acids in SEs. This method was applied to determine SEA in water samples preliminarily, and the detection limit of SEA in spiked water sample was 3 mg/kg. The limit of detection of 3 mg/kg was low sensitivity for low molecular weight matters, but it was high sensitivity for SEA which had a high molecular weight of 27 kDa. Of SEA, 3 mg/kg was equivalent to 10 mmol/kg of SEA. This study can provide evidence for establishing method to determine SEA in real samples.

Staphylococcal Enterotoxin A Induces Intestinal Barrier Dysfunction and Activates NLRP3 Inflammasome via NF-κB/MAPK Signaling Pathways in Mice

“…Preparation of SEA toxin protein was completed by Escherichia coli expression system and Ni-NTA affinity chromatography [45,46]. In short, the sea gene was amplified by polymerase chain reaction (PCR) from S. aureus.…”

Semi‐captive Przewalski's gazelles cope with winter adversity by adjusting their gut bacterial communities

“…Clostridium perfringens can lead to neurological lesions and enteritis in animals or humans (Uzal and Kelly 1998, Uzal et al 2002, Kiu and Hall 2018; Table ). It has been reported that high temperatures in summer can increase the occurrence of intestinal pathogen infections in animals (Li et al 2023). Therefore, we concluded that Przewalski's gazelles might be at risk of infection with potential pathogens and were more vulnerable to potential pathogens in summer.…”