| White spot syndrome virus (WSSV) is one of the main virus causing shrimp viral diseasesT The virus has strong infectivity and wide range of hosts, causing huge economic losses in shrimp farming industry. Yet so far, there are no effective treatments for controlling the virus. Screening healthy shrimp by early detection and diagnosis, establishing effective preventive monitoring system, and cutting off the transmission route as early as possible have become effective ways to prevent this kind of disease. Structural protein is the agent of virus structure and function, and is the main ingredient of a variety of animal toxic reactions. Importing WSSV structural proteins to shrimp can improve the ability of the body to resist WSSV. In this study, the WSSV of Guangxi vannamei shrimp was detected by GB nested PCR, which was rapidly and effectively understood, providing theoretical reference for vannamei farming. Proteins VP24 and VP51 were successfully cloned and expressed, which are the basis of further research about protein purification, antibody preparation and recombinant protein properties and functions, developing a new shrimp-specific immune method, preparing a kind of vaccine formulation-to prevent WSSV infection. The-results are as follows:1 The GB nested PCR method was established as the optimal method for detecting WSSV by comparing the detection results between one-step PCR and nested PCR.-Through this method, a total of 462 shrimp samples collected from 8 counties of 3 cities in Guangxi province from 2013 to 2014 were investigated. The results showed that a total of 83 positive reactions for WSSV were observed, with a total positive rate of 17.97%. Distribution of WSSV-in Beihai region had the highest positive rate of 43.59%. Positive samples were detected in all the investigated regions escept in Tieshangang of Beihai. Detection rate of WSSV in the juveniles/adults shrimp was 29.41%, whereas detection rate of WSSV in postlarvae/hepatopancreatic shrimp was 5.80%, which is lower than that of juveniles/adults shrimp. The WSSV positive rate was 20.19% in 2013, and the positive rate in 2014 was less (13.10%) than that in 2013.2 Sequences of samples from seven counties (WSSV positive) were analyzed by DNAStar software, and the results showed that the nucleotide homologies among these seven strains were 96.8%-99.4%, and they had the highest nucleotide homology (99.8-100%) with Indian strain (JN 165706.1) by comparing with known sequences in Genbank. Phylogenetic evolutionary tree was constructed and analysed, and these seven strains were in the same big branch-, and the sequence difference of species-was small. It is suggested that the WSSV strains isolated from Guangxi had no significant spatio-temporal differences, and had a close affinity with the foreign reference strains (96.8-100%).3 PCR amplification and cloning of vp24 and vp51 genes were employed to gain recombinant plasmid pMD18-T-vp24 and pMD18-T-vp51, and the cloning was succeeded by sequencing and double enzyme digestion detection. The recombinant expression vector pET-His-VP24 and pET-His-VP51 were constructed, and then competent cells of Escherichia coli BL21 were transformed, finally the transformed products were induced with IPTG for target proteins expression. The expression products were subjected for analysis by SDS-PAGE and the results showed that the size of expression products is corresponded to the estimated size of 23 kDa and 29 kDa respectively, suggesting that the target proteins were preliminary successfully expressed. |
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