Cloning, expression and polyclonal antibodies production against recombinant VP19 envelope and VP26 capsid proteins of white spot syndrome virus (WSSV) in black tiger shrimp (Penaeus monodon)

Abstract

White spot syndrome virus (WSSV) is the causative agent of white spot syndrome in black tiger shrimp (P. monodon) cultured worldwide, including Thailand. This virus was first reported in the 1980s, but major epidemics in Thailand were reported in the 1990s. WSSV transmission can occur both horizontally and vertically. Early detection of WSSV in infected P. monodon is an effective way to control WSSV epidemics in shrimp culture. The objective of this study was to produce polyclonal antibodies (PAbs) to detect white spot syndrome virus. Structural gene of envelope protein VP19 was cloned into expression vector (pMAL-C2) and expressed in E. coli (BL21) as a fusion protein, MBP-VP19 with a M.W. of 55.5 kDa. For WSSV capsid protein VP26, fusion protein with 6-histidine tag, 6 x His- VP 26, was used to generate polyclonal antibodies. The bacterial expression system allowed the production of 10 mg of purified recombinant proteins per liter of bacterial culture. Both recombinant proteins were used to immunize Swiss albino mice to produce polyclonal antibody. The WSSV-specific polyclonal antibodies were characterized by Western blot and immunohistochemistry and detected WSSV in both native and denatured forms. The MBP-VP19 and 6xhis-VP 26 proteins obtained from this project are useful for future vaccine development to prevent WSSV infection in shrimp farms. The recombinant protein will be used to produce monoclonal antibodies (MAbs) and used together with previous WSSV VP28 MAb to confirm and increase detection sensitivity in various immunoassays, including a simple immuno-based test kit (strip- test) and ELISA.

ไวรัสตัวแดงดวงขาว (WSSV) เป็นสาเหตุหลักของโรคตัวแดงดวงขาวในกุ้งกุลาดำ (P. monodon) ซึ่งพบการแพร่กระจายของโรคดังกล่าวในฟาร์มเพาะเลี้ยงกุ้งทั่วโลก ไวรัสชนิดนี้ถูกค้นพบในปี 1980 และพบการแพร่ระบาดอย่างรุนแรงในประเทศไทยในปี 1990 โดยสามารถติดต่อได้หลายช่องทางทั้งจากพ่อแม่พันธุ์ และ สัตว์ที่ติดเชื้อสู่กุ้งโดยตรง การวินิจฉัยการติดเ"