Development Of Rapid Detection Methods Based On Dihydropteroate Synthase For Sulfonamides

Sulfonamides (SAs) are a group of synthetic antibiotic agents that are widely used in veterinary practice for the treatment of infectious diseases and as growth-promoting feed additives. Illegal use of SAs or non-compliance of animal-treatment protocols may cause the residues of antibiotics in animal-derived food. It seriously infects food safety and human health, so it is necessary to monitor these chemical residues in food samples in order to obtain a better food safety assurance. For SAs residues, the screening methods must be precise, high sensitive and cheap, moreover, these methods must be simple, quick and broad-spectrum as it possible. In this paper, Dihydropteroate Synthase (DHPS) was produced and rapid detection assay based-on DHPS was developed for SAs in milk.The gene flop encoding DHPS from Streptococcus pneumonia R6and Escherichia coli ATCC25922was ampilied using PCR and inserted in expression vector pET28b, respectively. After optimization, expression was carried out at20℃for14～17h after0.5mM Isopropyl β-D-1-Thiogalactopyranoside (IPTG) was added. Purification of DHPS required two steps of Ni2+chelation affinity chromatography (100mM imidazole in elution buffer) and ion exchange.To identify the ideal Horseradish Peroxidase (HRP)-conjugate matched with DHPS, seven haptens were conjugated to HRP by DCC (dicyclohexylcarbodiimide) method, respectively. Among these conjugates, CS-HRP showed the highest affinity to DHPS and the highest sensitivity to detect sulphamethazine (SM2), and was thus used to develop and optimize the direct competitive assay. We develop a microplate assay based on CS-HRP and DHPS from Streptococcus pneumonia R6for SAs, and after optimization29SAs used in the study could be detected with IC50values ranging from2.95ng mL-1to56.22ng mL-1which were under the Maximum Residue Limit (MRL). IC50values for29SAs ranged from6.16ng mL-1to182.27ng mL-1in the microplate assay based on CS-HRP and DHPS from Escherichia coli ATCC25922. DHPS from Streptococcus pneumonia R6was slected due to high sensitivity obtained. Five SAs, sulfamethoxazole (SMZ), sulfadimethoxine (SDM), sulfaquinoxaline (SQX), sulfamonomethoxine (SMM), and SM2were spiked in whole milk and skim milk samples with simple pretreatment. The recoveries were83.3%～108.8%with coefficient of variation (CV) of1.1%～15.8%and72.7%～129.3%with CV of3.0%～16%in whole milk and skim milk, respectively. In order to simplify the assay, PPi (pyrophosphate) was used to replace the first substrate of DHPS,6-hydroxymethyl-7,8-dihydropterin-pyrophosphate (DHPPP). Under optimized conditions,9SAs and PABA could be detected below100ng mL-1and28SAs used in the study could be detected with IC50values ranging from426ng mL-1to50000ng mL-The best fluorescent tracer CS-BDF was produced and a fluorescence polarization immunoassay (FPIA) based on DHPS from Streptococcus pneumonia R6for SAs in milk was developed. The IC50values of29SAs ranged from4.90ng mL-1to108.24ng mL-1and the lowest IC50value (3.80 ng mL-1) was for PABA. Analysis of SMZ, SDM, SQX, SMM, and SMX fortified milk samples by the FPIA showed average recoveries ranging from53.3%to101.9%with CV0.29～19.7%.In order to increase the sensitivity of the assay of fast detection further more and provide the theoretic foundation for vitro protein evolution, we investigated the binding between6SAs and DHPS using Surflex-X-2.0. The correlation between relative binding affinitys (RBAs) and docking score was investigated, and good correlation was observed. Ile272and Asp221were the important amino acid sites in the interaction between DHPS and SAs, and hydrophobic effect was a decisive factor in the interaction between DHPS and SAs. In conclusion, we produed DHPS with high affinity and broad-spectrum activity for SAs and ordinary antibody could be replaced by DHPS for SAs detection.