Epidemiological Investigation Of Virulent Diseases In Shrimp And Screening WSSV Structural Proteins Interacting With NM23 Protein

With the continuous development of the economy and the improvement of people's consciousness,high-protein aquatic products have been widely loved by people.So the aquaculture industry has made great progress.And shrimp farming has gradually transformed into intensive farming model.Due to the high density of shrimp farming and the control measures are relatively backward,making the virulent diseases of shrimp emerge in endlessly.So far,viral diseases are still the main factors threatening shrimp farming.The virus with large harm is WSSV,IHHNV,TSV and YHV.These several kinds of shrimp virus diseases have the characteristics of rapid propagation and high incidence which make the shrimp farming economy suffered serious economic losses.At present,there is still no cure for virulent diseases of shrimp,so detection and epidemic prevention is the main means of healthy shrimp culture.In this study,we collected 1436 shrimp samples from May 2015 to December 2016,We use the PCR detection methods recommended by Office International des Epizooties(OIE)to investigate the epidemiology of White Spot Disease(WSD),Infectious hypodermal and hematopoietic necrosis(IHHN)and Taura Syndrome(TS)in Jiangsu province.The WSSV,IHHNV and TSV were detected by nested PCR,one-step PCR and reverse transcription PCR.Results showed the positive rate of WSSV infection was 17.2%,0%in TSV infection and 39.4%in IHHNV infection where shrimp samples collected from Nanjing,Dongtai,Dafeng,Ganyu,and Sheyang.The IHHNV positive shrimp from five regions of Nanjing,Dafeng,Sheyang,Qidong and Dongtai were selected as the research object.The coding region of IHHNV in five regions of Jiangsu was obtained by PCR,T/A cloning,sequence sequencing and sequence splicing.The five strains of IHHNV were analyzed by gene sequence.The data showed that the five strains belonged to type I infection strains,and the evolution relationship was similar to that of Korean strain JN377975.Some researchers found that WSSV infected prawn shows a lower level mortality in the condition of infected by IHHNV.Our laboratory has demonstrated that capsid protein(CP)in IHHNV has the ability to combine with the NM23 protein in prawn's gill membrane in an earlier time.NM23 is a kind of nucleoside diphosphate kinase(NDPK),it is mainly located in the cytoplasm and nucleus,and is also visible in the cell membrane.It has various biological functions.So this experiment aimed at exploring whether WSSV and IHHNV can compete shrimp gill NM23 protein on the cell membrane.In this study,we purified total proteins of WSSV by using sucrose gradient centrifugation,then total proteins were interaction with NM23 protein by using virus overlay protein binding assay(VOPBA)experiment.Three suspected positive proteins were selected by LC-MS/MS analysis after the experiment.They were WSSV013,Wsv497 and Wsv035.Then we constructed prokaryotic expression vectors of these three proteins for purifying WSSV013,Wsv497 and Wsv035 in order to interact with NM23 protein by using virus overlay protein binding assay and Co-Immunoprecipitation respectively.The result shows that Wsv497 and Wsv035 has the ability to interact with NM23 protein,while WSSV013 shows an invalid effect with NM23 protein.According to the result,we find out that WSSV and IHHNV may have the ability to compete with NM23 protein receptor in the cell membrane of prawn gill.This study will provide the theoretical foundation about the mechanism of competing the receptor in the cell membrane of prawn gill between WSSV and IHHNV and lay a certain foundation for further studies of protein interactions between WSSV and prawn.