Журнал клинической токсикологии
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ISSN: 2161-0495
ISSN: 2161-0495
Nguyen van Khanh, Tran Quoc Dung, Tran Quang Khanh van, Nguyen Quang Linh
Acute Hepatopancreatic Necrosis Disease (AHPND) is a bacterial disease of white leg shrimp, which has a high mortality rate (100%) and economic losses. Our objective was to identify the genes which caused cell and organ toxic damage and applied bioproducts to prevent it. Litopenaeus vannamei shrimp were collected from an infected pond in Central Vietnam and analyzed at the Institute of Biotechnology, Hue University. The PirA gene of Vibrio parahaemolyticus strain K5 was isolated and analyzed for nucleotide sequence, paired with the expression vector pQE30. The expression vector was transformed into E. coli strain M15, the PirA recombinant protein was expressed in the form of 6xHis-PirA fusion protein of about 15 kDa. PirA recombinant protein was purified and determined the PirAvp binding ratio, cloning, and sequencing of PirA gene from Vibrio parahaemolyticus strain K5 causing AHPND by PCR method with specific primers and molecular weights of PirAvp and the PirAvp complex. PirA gene from Vibrio parahaemolyticus strain K5 was cloned into a pGEM-T easy vector (Promega, USA) and screened E. coli TOP10 colonies containing pGEM T easy/PirA recombinant plasmid on LB agar/ampicillin/ IPTG/X-Gal medium. PCR showed a band of about 347 bp, matching the size of the PirA gene and two nucleotide sequences (BamHI and HindIII). The results showed that the PirA gene has a length of 336 bp and is like the PirA gene on GenBank (Code: KU556825.1). The results of protein extracted from E. coli M15 recombinant cells and 6xHis-PirA target protein was collected in elution fractions from EF2 to EF6, showed that the concentration of 6xHis-PirA protein and EF3 elution fraction collected the highest protein concentration (1,586.54 µg/ml). The purified PirA recombinant protein will provide materials for development research to create biological products to prevent and treat AHPND.