MOLECULAR CLONING, DUAL EXPRESSION, AND COMPLEX ISOLATION OF TOXA AND TOXB TOXINS CAUSING ACUTE HEPATOPANCREATIC NECROSIS DISEASE (AHPND) IN SHRIMP
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AHPND, dual expression, pVA1, ToxA, ToxBAbstract
Acute Hepatopancreatic Necrosis Disease (AHPND) is a dangerous disease in shrimp farming, which has a mortality rate of up to 100 percent. AHPND is caused by two toxins, ToxA and ToxB, which are expressed from pVA1 plasmid in Vibrio parahaemolyticus. Currently, the pathogenic mechanism of AHPND has not been clearly elucidated, thus drugs development to treat AHPND still faces many difficulties. Individually expressing ToxA and ToxB for experiments may be problematic. Therefore, imitating the structure of pVA1 plasmid to naturally generate ToxA and ToxB in complex form is an ideal approach. In this present study, we constructed a recombinant plasmid pACYACDuet-toxA-toxB encoding for both ToxA and ToxB proteins for the first time. Then, the toxins were dual expressed and evaluated by SDS-PAGE, Western blot, and pull-down assay. As expected, ToxA and ToxB were simultaneously expressed in soluble fraction at 1mM IPTG, and 37°C condition. ToxA and ToxB complex was also collected by pull-down assay using ToxB-antibody conjugated magnetic beads. The data laid a groundwork for further researches on the pathogenicity of AHPND.
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