MOLECULAR CLONING, DUAL EXPRESSION, AND COMPLEX ISOLATION OF TOXA AND TOXB TOXINS CAUSING ACUTE HEPATOPANCREATIC NECROSIS DISEASE (AHPND) IN SHRIMPAbstract views: 204 / PDF downloads: 369
Keywords:AHPND, dual expression, pVA1, ToxA, ToxB
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.
Lightner D.V. (1999): The penaeid shrimp viruses TSV, IHHNV, WSSV, and YHV: current status in the Americas, available diagnostic methods, and management strategies. Journal of Applied Aquaculture 9 (2):27-52.
Seibert CH, Pinto A.R. (2012): Challenges in shrimp aquaculture due to viral diseases: distribution and biology of the five major penaeid viruses and interventions to avoid viral incidence and dispersion. Brazilian Journal of Microbiology 43:857-864.
FAO FAO (2013) Report of the FAO/MARD Technical Workshop on Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis Syndrome (AHPND) of Cultured Shrimp (under TCP/VIE/3304). Hanoi, Viet Nam, 25–27 June 2013. FAO Fisheries and Aquaculture Report No. 1053.
NACA‐FAO (2011): Quarterly Aquatic Animal Disease Report (Asia and Pacific Region), 2011/2, April-June 2011. NACA Bangkok,
Flegel T.W. (2012): Historic emergence, impact and current status of shrimp pathogens in Asia. Journal of Invertebrate Pathology 110 (2):166-173.
Nunan L., Lightner D., Pantoja C., Gomez-Jimenez S. (2014): Detection of acute hepatopancreatic necrosis disease (AHPND) in Mexico. Diseases of aquatic organisms 111 (1):81-86.
Lightner D., Flegel T. (2012): Diseases of Crustaceans–Acute Hepatopancreatic Necrosis Syndrome (AHPNS) (Disease card). Asia Pacific Emergency Regional Consultation on Early Mortality Syndrome (EMS)/Acute Hepatopancreatic Necrosis Syndrome (AHPNS). In: Workshop organized by the Australian Government Department of Agriculture, Fisheries and Forestry and the Network of Aquaculture Centres in Asia-Pacific (NACA) Bangkok, Thailand (August 9-10, 2012).
Dhar A.K,. Piamsomboon P., Caro L.F.A., Kanrar S., Adami Jr R., Juan Y-S. (2019): First report of acute hepatopancreatic necrosis disease (AHPND) occurring in the USA. Dis Aquat Organ 132 (3):241-247.
Hong X., Xu D., Zhuo Y., Liu H., Lu L. (2016): Identification and pathogenicity of Vibrio parahaemolyticus isolates and immune responses of Penaeus (Litopenaeus) vannamei (Boone). Journal of Fish Diseases 39 (9):1085-1097.
Tran L., Nunan L., Redman R.M., Mohney L.L., Pantoja C.R., Fitzsimmons K., Lightner D.V. (2013): Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp. Diseases of aquatic organisms 105 (1):45-55.
Lee C.-T., Chen I.-T., Yang Y.-T., Ko .T-P., Huang Y.-T., Huang J.-Y., Huang M.-F., Lin S.-J., Chen C.-Y., Lin S.-S. (2015): The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin. Proceedings of the National Academy of Sciences 112 (34):10798-10803.
Yang Y.T., Chen I.T., Lee C.T., Chen C.Y., Lin S.S., Hor L.I., Tseng T.C., Huang Y.T., Sritunyalucksana K., Thitamadee S., Wang H.C., Lo C.F .(2014): Draft Genome Sequences of Four Strains of Vibrio parahaemolyticus, Three of Which Cause Early Mortality Syndrome/Acute Hepatopancreatic Necrosis Disease in Shrimp in China and Thailand. Genome Announcements 2 (5): e00816-00814-e00816-00814.
Kondo H., Tinwongger S., Proespraiwong P., Mavichak R., Unajak S., Nozaki R., Hirono I. (2014): Draft genome sequences of six strains of Vibrio parahaemolyticus isolated from early mortality syndrome/acute hepatopancreatic necrosis disease shrimp in Thailand. Genome Announcements 2 (2).
Sirikharin R., Taengchaiyaphum S., Sritunyalucksana K., Thitamadee S., Flegel T.W., Mavichak R., Proespraiwong P. (2014): A new and improved PCR method for detection of AHPND bacteria. Network of Aquaculture Centres in Asia-Pacific (NACA)(http://www enaca org/modules/news/article php.
Dangtip S., Sirikharin R., Sanguanrut P., Thitamadee S., Sritunyalucksana K., Taengchaiyaphum S., Mavichak R., Proespraiwong P., Flegel T.W. (2015): AP4 method for two-tube nested PCR detection of AHPND isolates of Vibrio parahaemolyticus. Aquaculture Reports 2:158-162.
Kongrueng J., Tansila N., Mitraparp-arthorn P., Nishibuchi M., Vora G.J., Vuddhakul V. (2015): LAMP assay to detect Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease in shrimp. Aquaculture international 23 (5):1179-1188.
Wangman P., Chaivisuthangkura P., Sritunyalucksana K., Taengchaiyaphum S., Senapin S. Pengsuk C., Sithigorngul P., Longyant S. (2017): Development of monoclonal antibodies specific to ToxA and ToxB of Vibrio parahaemolyticus that cause acute hepatopancreatic necrosis disease (AHPND). Aquaculture 474:75-81.
Nguyen-Phuoc K.-H., Duong N.-D., Van Phan T., Do K.-Y.T., Nguyen N.-T.T., Tran T.L., Tran-Van H. (2021): Generation and evaluation of polyclonal antibodies specific for ToxA from Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) in shrimp. Molecular Biology Research Communications 10 (1):23.
Duong N.-D., Nguyen-Phuoc K.-H.., Do K-Y.T., Nguyen N.-T.T., Tran T.L,.. Tran-Van H (2021): Production of polyclonal antibody against the recombinant PirB vp protein of Vibrio parahaemolyticus. Journal of Genetic Engineering and Biotechnology 19 (1):1-8.
Lin S.-J., Hsu K.-C,. Wang H..-C (2017): Structural Insights into the Cytotoxic Mechanism of Vibrio parahaemolyticus PirAvp and PirBvp Toxins. Marine Drugs 15 (12):373.
Lin S.-J., Chen Y.-F., Hsu K.-C., Chen Y.-L., Ko T.-P., Lo C.-F., Wang H.-C., Wang H.-C. (2019): Structural insights to the heterotetrameric interaction between the vibrio parahaemolyticus pirAvp and pirBvp toxins and activation of the cry-like pore-forming domain. Toxins 11 (4):233.
Kumar V., Bels L.D., Couck L., Baruah K., Bossier P., Broeck W.Vd. (2019): PirABVP toxin binds to epithelial cells of the digestive tract and produce pathognomonic AHPND lesions in germ-free brine shrimp. Toxins 11 (12):717.
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