White Spot Syndrome Virus (WSSV) VP12、VP14、VP51、VP136C Interact With Proteins Of Virus And Host

White spot syndrome virus （WSSV） has become a major pathogen in the shrimpaquaculture worldwide, and causes up to100%mortality within2to5days from theonset of clinical signs. The structural proteins often play vital roles in cell targeting,virus entry, assembly, and budding, as well as triggering host antiviral defenses. Soanalysis of protein structure, function, and interaction or mechanism of differentproteins, to understand the pathogenic mechanism of WSSV infection mechanism and,to look for ways to effectively prevent the WSSV infection plays an important role.Part1. VP12was found to be present exclusively in the envelope fraction. Itcorresponds to open reading frame432of the WSSV-CN isolate, and its gene encods apolypeptide of102amino acids （aa） with a theoretical mass of12kDa. Apair of primerswas designed according to the sequence of vp12gene of White spot syndrome virus（WSSV） in the GenBank. The vp12DNA fragment was amplified by PCR and clonedinto E. coli expression vector pBAD/gIIIA successfully. Then pBAD/gIIIA–vp12wastransformed into E.coli-Top10cells. After L-Arabinose induction at37℃, the fusionprotein with19kDa was expressed, which was conformed by SDS-PAGE andWestern-blot analysis. The envelope protein VP12was purified by Co²⁺-column affinitychromatography and used to produce anti-rabbit antibody.The transcription analysis of VP12showed that vp12was an early gene byRT-PCR assay. Fluorescence assay showed that significant fluorescence were visible inhemocytes incubated with fluorescence-labeled VP12. This result further identified thatVP12can bind to shrimp cells. A competitive inhibition assay was performed toinvestigate the role of RGD motifs in adhesion. Results showed that motif of RGD maynot play key role for the attachment of VP12to shrimp haemocytes.The interaction between purified protein VP12and WSSV structure protein wasanalyzed by far-western blot, ELISA. It is concluded that VP12could interact withVP24and VP26. Furthermore, one protein was selected by incubating DIG-labeled VP12with gills membrane protein of L.vannamei and a protine of LvANT wasidentified by means of MALDI-MS assay.Part2. LvANT recombinant was induced with L-Arabinose at37℃. rLvANTwas purified by Co²⁺-column affinity chromatographyd and rabbit anti-rLvANTantibody was parped. ELISA and indirect immunofluorescence assay confirmed thatVP12interacted with LvANT. Cellular localization of LvANT protein in shrimphemocytes showed that LvANT was primarily located on the surface and cytoplasm ofhemocytes which was colocalized with mitochondria. Tissue distribution of adeninenucleotide translocase mRNA showed that it was commonly spread in all the tissues ofhemocytes,gills, muscle, lymphoid, hepatopancreas, stomach and intestine.Neutralization assay in vivo showed that LvANT retard WSSV infection in the earlystage.Part3. A pair of primers was designed according to the sequence of vp14gene ofWSSV in the GenBank. The vp14DNA fragment was amplified by PCR and cloned into E.coli expression vector pBAD/gIIIA. Then pBAD/gIIIA–vp14was transformed into E.coli-Top10cells. After L-Arabinose induction at37℃, the fusion protein with15kDa wasexpressed, which was conformed by SDS-PAGE and Western-blot analysis. The envelopeprotein VP14was purified by Co²⁺-column affinity chromatography and mouse anti-VP14antibody was prepared.Indirect fluorescence assay showed that VP14can bind to shrimp cells.Matrix-assisted laser desorption ionization （MALDI） MS assays showed that VP14couldinteract with VP26, VP28. Further evidence for interaction between VP14and VP26,VP28C, VP37, VP39was provided by far-western blot. Furthermore, a new shrimp protein（Arginine kinase, AK） was selected by incubating DIG-VP14with gills membrane proteinof L.vannamei.Part4. A pair of primers was designed according to the sequence of LvAK gene inthe GenBank. The vp14DNA fragment was amplified by PCR and cloned into E. coliexpression vector pBAD/gIIIA. Then recombinant was transformed into E.coli-Top10cells. After L-Arabinose induction at37℃, the fusion protein with45kDa was expressed.The LvAK was purified by Co²⁺-column affinity chromatography and rabbit anti-LvAKantibody was prepared.Far-western and ELISA confirmed that VP14interacted with AK. Indirect immunofluorescence assay indicated that VP14and LvAK co-localized on the surfaceof hemoptes. Tissue distribution of AK mRNA showed that AK gene was spreadcommonly in all the tissues detected and present in very large quantities in shrimpmuscle tissue. Cellular localization of AK protein in shrimp hemocytes showed that AKwas primarily located on the periphery and of hemocytes and was scarcely detectable inthe nucleus. The transcriptional expression of AK after challenge with WSSV was testedin hemocytes and muscle, which indicated that AK might be involved in a transientsystemic immune response to the stimulation inflicted by WSSV.Part5. VP51was found to be present exclusively in the nucleocapsid fraction.Itcorrespond to open reading frame308of the WSSV-CN isolate, and its gene encoded apolypeptide of467amino acids （aa） with a theoretical mass of51.7kDa. A pair of primerswas designed according to the sequence of vp51gene of WSSV in the GenBank. The vp51DNA fragment was amplified by PCR and cloned into E. coli expression vectorpBAD/gIIIA successfully. Then pBAD/gIIIA–vp51was transformed into E.coli-Top10cells. After L-Arabinose induction at37℃, the fusion protein with55kDa was expressed,which was conformed by SDS-PAGE and Western-blot analysis. The envelope proteinVP51was purified by Co²⁺-column affinity chromatography.The interaction between DIG-labeled VP51and gills membrane protein ofL.vannamei was analyzed by Far-western blot. Two protein （LvANT, LvRPL7） wereselected by incubating DIG-labeled VP51with gills membrane protein of L.vannamei.Part6. Apair of primers was designed according to the sequence of LvRPL7. TheDNA fragment was amplified by PCR and cloned into E. coli expression vectorpBAD/gIIIA. After induction with L-Arabinose, a band corresponding to37kDa wasshowed by SDS-PAGE and Western-blot analysis. The protein was purified by Co²⁺column affinity chromatography and rabbit anti-LvRPL7serum was prepared. ELISAand far-Western assay confirmed that VP51interacted with RPL7. Tissue distribution ofRPL7mRNA showed that RPL7gene was spread commonly in all the tissues detectedand present in very large quantities in shrimp muscle then gill tissue. Cellularlocalization of RPL7protein in shrimp hemocytes showed that RPL7was primarilylocated on the surface of hemocytes. The transcriptional expression of RPL7afterchallenge with WSSV was tested in hepatopancreas and muscle, which indicated thatRPL7might be involved in a transient systemic immune response to the WSSV infection.Part7. VP136was found to be present exclusively in the nucleocapsid fraction.VP136C contained RGD motif is part of VP136. It encoded a polypeptide of373aminoacids （aa） with a theoretical mass of40kDa. A pair of primers was designed accordingto the sequence of vp136C gene of White spot syndrome virus （WSSV） in the GenBank.The DNA fragment was amplified by PCR and cloned into E. coli expression vectorpBAD/gIIIA successfully. Then pBAD/gIIIA–vp136C was transformed into E.coli-Top10cells. After L-Arabinose induction at37℃, the fusion protein with45kDa wasexpressed, which was conformed by SDS-PAGE and Western-blot analysis. The proteinVP136C was purified by Co²⁺-column affinity chromatographyd.Fluorescence assay showed that significant fluorescence were visible in hemocytesincubated with fluorescence-VP136C. This result further identified that VP136C canbind to shrimp cells. The interaction between purified protein VP136C and WSSVstructure protein was analyzed by far-western blot and ELISA. It is concluded thatVP136Ccould interact with VP26. Furthermore, several proteins were selected byincubating DIG-VP136C with gills membrane protein of L.vannamei. ATP/ADPtranslocase, mitochondrial F0-ATP synthase, b-chain beta-actin and hemocyanin wereidentified by means of MALDI-MS assay.