Cloning And Expression Of Main Structure Protein Genes Of Taura Syndrome Virus In P. Vannamei Shrimp

Penaeus vannamei is one of the principal penaeid shrimp farmed all over the world and Taura syndrome vims is the most important vims affecting penaeid shrimp production. Since the discovery of Taura syndrome in 1992 on shrimp farms in Ecuador, the disease has disseminated into virtually all major shrimp-growing areas in the Americas. Shrimp that die from Taura syndrome disease have multifocal acute necrosis of the cuticle epidermis and underlying tissue. Diagnosis of the disease is made by histopathological demonstration of the pathogonomic lesions in the integument of acutely moribund shrimp or by demonstration of TSV in necrotic foci by in situ hybridization using a commercially available cDNA gene probe. But these methods of diagnosis need too much time and could not fit the request of rapid quarantine, so we need a new method for rapid quarantine.According to the complete genome sequences of Taura Syndrome Vims (TSV) published on GenBank, three pairs of primers were designed to amplify the main structure protein genes (vp1,vp2,vp3) of TSV respectively. These amplified products were 1164bp,849bp and 507bp. They were constructed to the expression vector pET-28a and named pET-VP1,pET-VP2,pET-VP3. The recombinant plasmids were transformed into E.cloi BL21(DE3) after the identification of the nuclear restriction enzymes cutting and sequencing. After IPTG induction and SDS-PAGE analysis , the fusion proteins of about 48KDa,36KDa and 24KDa in molecule weight were detected. These isolated recombinant proteins were purified by nickel pillars, and then were inoculated into the KM mice to get the polyclonal antibodies. The western blot and the dot-elisa tests indicate that the polyclonal antibodies can take specific reactions with the corresponding fusion proteins.TSV is a major cause of mortality and morbidity in shrimp, and has a profound economic impact on commercial shrimp farming all over the world. The results of this research should make a great foundation for developping a effective way to detect TSV rapidly.