The Preliminary Study Of The IHHNV Capsid Protein Receptors On The Gill Membrane Of Litopenaeus Vannamei

Infectious Hypodermal and Haematopoietic Necrosis(IHHN)is one of the main viral diseases of penaeid shrimp.In recent years,it has circulated around the world,causing serious economic loss.The World Organization for Animal Health(OIE) listed IHHNV as a key crustacean virus that must be declared.At present,many studies have been undertaken including epidemiological surveys and establishing diagnostic methods,but the pathogenic mechanism of IHHNV infection are still relatively unknown.This study mainly focus on the gill cell membrane receptors of the IHHNV capsid protein.To scan out the receptors,VOPBA,Pull Down,the phage dodecapeptides display library were used.This study could provide novel insights into the pathogenesis of IHHNV.Prokaryotic expression of IHHNV CP was conducte firstly,using the primers according to the Sheyang isolate(Accession KF214742),after DN A of gill tissue was extracted.The protein was primarily expressed in precipitate.After purified by HisTrapTM HP,CP was urea dialysed and then used to immune the New Zealand white rabbits to prepare polyclonal rabbit antibody.After three times of immune,the titer was up to 1:102400.The gill tissue from Litopenaeus vannamei uninfected by IHHNV was choosen to prepare the gill membrane proteins.Uniform distribution of gill membrane proteins were acquired after tissue homogenization and ultracentrifugation.Signal transducer and activator of transcription,Heat shock protein90,Prophenoloxidase-2,Na+/K+-ATPase alpha subunit,NM23 protein were scaned out using VOPBA and HIS Pull Down.The NM23 gene was cloned into the pGEX-4t-1 vector and prokaryotic expressed.After purified by Glutathione SepharoseTM 4B,the NM23 VOPBA and NM23 GST Pull Down confirmatory experiments were proceeded.The results shown that the NM23 could interact with IHHNV CP.Then IHHNV CP binding peptides were scanned out by the phage dodecapeptides display library.After four times of the circulatory affinity,elution,amplification and affinity,six kinds of dodecapeptides were scanned out,which could interact with IHHNV CP.They were A(SWSSWVYRDPQT),B(HSFKWLDSPRLR),C(SSFKWLDSPRLR),D(HRSKWVYSDPQR),E(YWSKWVDWHPQR),F(SSCKWVDWD*AE).Among these dodecapeptides,B and C differed by the first amino acid,B was H,C was S.Both D and E contained SKWV and PQR,which probably the characteristic binding peptides of IHHNV CP.SW*Y(*meaned Ⅴ or Ⅰ)also possibly the characteristic binding peptides of IHHNV CP after the contrast between sequence A(SWSSWVYRDPQT)and the amino acids sequence of NM23 protein.By means of the NM23 VOPBA and NM23 GST Pull Down confirmatory experiments,the interaction between IHHNV CP and NM23 protein was further proofed.