Expression Of The Receptor Protein Saxiphilin Of Paralytic Shellfish Posion And Assay Of Its Binding Activity

Paralytic shellfish poisons (PSPs) are natural products produced by somepoisonous algae. Shellfish feeding these toxic algae is harmless for itself and thesePSPs accumulate in its body. After ingestion of these contaminated shellfish byhuman, their PSPs rapidly release and can cause muscle weakness, difficultybreathing, sometimes even be fatal in severe cases. Paralytic shellfish toxin receptorprotein-Saxiphilin (SAX) is a soluble protein widely distributed in amphibians,arthropods, reptiles and fish.In the present study, the sax gene was obtained by reverse transcription frombullfrog liver. sax with histidine-tag in the C-terminus and no signal peptide sequencein the N-terminus was cloned into pET-28a and transformed to BL21(DE3) to obtain astrain BL21(DE3)-sax-p that could abundantly express SAX-p (SAX expressed inprokaryotic expression system). The optimum conditions for expression of SAX-pwere investigated and the results demonstrated that SAX-p was expressed abundantlyin the form of inclusion bodies after cells were induced in23℃,1mmol/L IPTG andOD600=0.415for9.5h. Using high concentrations of guanidine hydrochloride solution,followed by gradually decreasing the concentration of guanidine hydrochloride,SAX-p was refolded and purified using the nickel column chromatography. By usingBac-to-Bac technique and baculovirus expression vector system, sax with histidine-tag in the C-terminus and signal peptide sequence in the N-terminus wascloned into pEASY-T3, then subcloned into the donor plasmid pFastBac1to get thedonor plasmid pFB1-sax-e. The donor plasmid was transformed to DH10Bac and arecombinant baculovirus (vAc-SAX-e) overexpressing SAX-e (SAX expressed ineukaryotic expression system) was constructed. Sf9cells were cultured in serum-freemedium for72h after infected with vAc-SAX-e and the soluble SAX-e was secretedinto the supernatant. SAX-e was first concentrated by ultrafiltration and then purifiedusing the nickel column chromatography.Capillary electrophoresis, also known as high performance capillaryelectrophoresis, is designed to separate particle based on their mobility or distributionbehavior, moving in the interior of a small capillary driven by an electrolyte. Thistechnique is widely applied to the study of the protein-protein interactions. In thiswork, capillary electrophoresis was adopted to study the interaction between SAX-eand PSPs. The conditions of capillary electrophoresis were investigated and theresults showed that the optimal condition were injection time of20sec and separationvoltage of15kv. The STX (saxitoxin), dcSTX (decarbamoyl saxitoxin) and neoSTX(neosaxitoxin) were separated in the optimal separation conditions. Capillaryelectrophoresis was performed with the running buffer including differentconcentrations of SAX-e and PSPs as the injected samples to study the bindingactivity of SAX-e and PSPs. By analysis of the migration time, the correspondingregression equation was obtained, and the binding affinity of STX, dcSTX andneoSTX with SAX-e was calculated, which was in the order of STX> neoSTX>dcSTX.The conclusion indicated that SAX-e protein expressed in baculovirus expressionvector system had the binding activity with PSPs, and it could be further used for thedetection of PSPs toxins in food.