HSP60, HSP70and HSP90of Shrimp Litopenaeus Vannamei Interact With White Spot Syndrome Virus

White Spot Syndrome Virus (WSSV) is causing a terrible damage anda great loss in shrimp industry. In order to realize the mechanism of WSSV,the study is investigated about the interaction of HSP60, HSP70andHSP90with WSSV. The main contents are as follows:(1) In order to research the HSP60in WSSV infection, HSP60genesequence was ligated to the expression vector pBAD/gIIIA to constituterecombinants pBAD/gIIIA-HSP60, then were transformed into E.coliTop10, and induced by L-arabinose about4hours. HSP60expression wasconfirmed by SDS-PAGE and Western Blot analysis. Then it was purifiedby using Co2+affinity chromatography. HSP60was sent to a company toprepare anti-LvHSP60antibody, and was purified to determine the titer. FarWestern, ELISA and indirect immunofluorescent assay indicated that therewas an interaction of VP12, VP24, VP26, VP39and VP90with shrimpHSP60. Anti-LvHSP60antibody neutralization assay revealed that it hadprotective effection on shrimps at the early stage of WSSV infection.Neutralization experiments with HSP60combined with Real-Time PCRassay of STAT mRNA expression and WSSV copies showed that HSP60may have protective effect on shrimps at the early stage of WSSVinfection.(2)In order to research the role of HSP70in WSSV infection, HSP70was divided into two parts, HSP70A and HSP70P. HSP70A and HSP70Pgene sequences were ligated to the expression vector pBAD/gIIIA to constitute recombinants pBAD/gIIIA-HSP70A and pBAD/gIIIA-HSP70P,then were transformed into E.coli Top10, and induced by L-arabinose.HSP70A and HSP70P were confirmed by Western Blot analysis. HSP70Aand HSP70P were purified using Co2+affinity chromatography. FarWestern and ELISAassay indicated HSP70interacted with VP24and VP26.The interaction of VP24and VP26with HSP70reduced the ATPase activity.The study provides a basis for exploring the function of HSP70in WSSVinfection.(3) In order to research the role of HSP90in WSSV infection, HSP90was divided into two parts, HSP90N and HSP90C. The gene sequence wasligated to the expression vector pBAD/gIIIA, then were transformed intoE.coli Top10, and induced by L-arabinose. HSP90N and HSP90C wereconfirmed by SDS-PAGE and Western Blot analysis. Then it was purifiedby Co2+affinity chromatography. HSP90C was sent to a company toprepare anti-LvHSP90C antibody, and was purified to determine the titer.ELISA and indirect immunofluorescent study showed that there was aninteraction of VP12, VP24, VP26and VP90with HSP90N and HSP90C.Neutralization experiments with HSP90combined with Real-Time PCRassay showed that HSP90may have protective effect on shrimps at theearly stage of WSSV infection.(4) The mRNA expression levels of HSP60and HSP90in tissues ofgill, intestine, hepatopancreas, muscle, hemocyte and lymphoid wereassayed by quantitative Real-Time PCR. Results showed that HSP60andHSP90were expressed in the detected tissues. WSSV infection could affectthe transcription levels of HSPs mRNA. WSSV infection inhibited theexpression of HSP60in the tissues of gill, intestine, muscle and thehemocyte, and promoted the expression of HSP90in the tissues of gill, intestine, hepatopancreas, muscle, hemocyte and the lymphoid. Thisreflected that HSP60and HSP90took part in immune responses.