Isolation And Identification Of White Spot Syndrome Virus And Effects Of Related Structural Proteins On Non-specific Immune Factors In Procambarus Clarkii

WSS,a kind of shrimp virus disease caused by WSSV,is one of the most harmful diseases challenging the prawn aquaculture in the world.It’s characterized by the white spot on the shrimp’s carapace.During 10 days after the clinical symptoms are shown,the cumulative mortality rate is so serious and reach 100%.In the early 1990 s,WSS first attacked Taiwan,has swept the world since then and taken a heavy toll on the prawn aquaculture around the world.After shrimps are affected by WSS,no specific medicine can be used to control the virus,which wreaks havoc on the aquaculture.The prevention methods focus on prevention ones.In the study,the cloned VP28,VP24,VP466 gene of WSSV is isolated from the naturally infected procambarus clarkii in Qianjiang in Hubei province to construct recombinant eukaryotic expression vector,and the protective effect on the crayfish is studied in order to lay a foundation for the research on WSSV nucleic acid vaccine.The WSSV genome is a double-stranded circular DNA.According to the VP28,VP24,VP466 gene sequence of WSSV published on Genbank,the specific primer is designed,the VP28,VP24,VP466 gene is amplified,the obtained PCR product is purified and cloned into p MD19-T vector and the recombinant plasmid p MD19-T-VP28,p MD19-T-VP24,p MD19-T-VP466 is obtained through identification.After the recombinant plasmids Bam HI and HindⅢ are conducted through double enzymes restriction,the target gene VP28,VP24,VP466 are linked to the eukaryotic expression vector p EGFP-N1 to construct the recombinant eukaryotic expression plasmid p EGFP-N1-VP28,p EGFP-N1-VP24,p EGFP-N1-VP466.The 50μg of constructed recombinant plasmid is injected into every crayfish’s abdominal muscles,and the p EGFP-N1 group and PBS group are set.The tissues of each group of crayfish are collected after immunization within 0,48,72,168 and 336 hours.The activities of phenol oxidase(PO),superoxide dismutase(SOD),peroxidase(POD)in the crayfish are detected through ELISA method.In the p EGFP-N1 and PBS groups,the activities of PO,SOD,and POD remain unchanged.In the p EGFP-N1-VP28,p EGFP-N1-VP24,p EGFP-N1-VP466 group,the SOD activity in the crayfish begins to increase after 48 hours of immunization,and reaches the highest level within 72 hours.The PO activity in the crayfish in the p EGFP-N1-VP28,p EGFP-N1-VP24,p EGFP-N1-VP466 group is higher within 72 hours and 168 hours,and has significant difference with the PO activity in the crayfish in the control group.The PO activity in the crayfish in the p EGFP-N1-VP28 group is higher within 48 hours and 336 hours,and has significant difference with the PO activity in the crayfish in the control group.After the crayfish is immune to the recombinant plasmid p EGFP-VP24,p EGFP-VP28,p EGFP-VP466 within 12 days,the target gene is detected in the muscle,hepatopancreas,gill,intestine and stomach of the crayfish.That indicates that the recombinant eukaryotic expression plasmid p EGFP-VP24,p EGFP-VP28,p EGFP-VP466 are widely distributed in all tissues of the crayfish.After 7 days of immunization,0.1ml of 10-3.1.m L-1 venom is injected into every crayfish’s abdominal muscles.So the hepatopancreas,intestines and gill tissues of the crayfish after the immunization and challenge are collected to go through the histopathological observation.The results show that each immunized group has protective effect on the tissues of crayfish.The relative percent survival of the p EGFP-N1-VP24 group,p EGFP-N1-VP28 group,p EGFP-N1-VP466 group are 36.84%,47.36% and 31.58%respectively.In this study,the isolation and identification of WSSSV and the influence of eukaryotic expression plasmid constructed by wssv-related structural protein on non-specific immune factors of lobsters were investigated,so as to provide a basis for the subsequent development of candidate nucleic acid vaccine of WSSV.