The IsoLation And Detection Of An Infectious Hematopoietic Necrosis Virus (IHNV) In Rainbow Trout、Sequence Analysis And Prokaryotic Expression Of The GLycoprotein

The infectious hematopoietic necrosis virus (IHNV) is probabLy one of the most importantfish viraL pathogens causing acute, systemic and often viruLent disease predominantLy in bothwiLd and cuLtured saLmon and trout.IHNV is cLassfied in the famiLy Rhabdoviridae, genusNovirhabdovirus.IHN outbreak caused serious economic Losses.A strain of rhabdovirus wasisoLated from the Larvae of rainbow trout in JiLin province Yanbian autonomous prefecture.The obvious cytopathic effect (CPE) was observed in Fathead minnow (FHM) inocuLated withtissue homogenates，By using eLectron microscope, we found the IHNV particLes。with thespecific primers of IHNV、infectious pancreatic necrosis virus (IPNV),and other aquatic animaLvirus for RT-PCR, the resuLt shows that onLy the IHNV specific primers ampLify out aspecific target band，assigned CJ-13。The compLete genomic sequence of IHNV strain220-90was determined from the DNA ofsix overLapping cLones obtained by RT-PCR ampLification of genomic RNA.The compLetegenome sequence of CJ-13comprises11133bp.From3’ to5’ end, genome structure wasencoding protein: N, P, M, G, NV, L.These structures connected by conservative sequence.Thesequences of structuraL gene of CJ-13was compared with the sequences of other IHNV strainsin GenBank by DNAStar and MEGA5.2software。Sequence anaLysis showed that the Gsequence simiLarity ChYa07and PcKw11was97.5%and97.2%，Data anaLysis showed theamino acid sequence homoLogy was96.1%and96.8%respectiveLy;The N sequence simiLarityHV7601was97.1%, Data anaLysis showed the amino acid sequence homoLogy was95.2%;The P sequence simiLarity HV7601was96.4%, Data anaLysis showed the amino acid sequencehomoLogy was95%; The M sequence simiLarity RB1and HV7601was98.7%, Data anaLysisshowed the amino acid sequence homoLogy was95.7%and96.1%respectiveLy;The Lsequence simiLarity IHNV(GenBank: X89213.1) was94.9%, Data anaLysis showed the aminoacid sequence homoLogy was83.9%.The evoLutionary tree anaLysis shows that the CJ-13strains、Japanese strains and Korea strains had the cLosest genetic reLatives and beLonged tothe same branch,so we infer that the cj-13strain maybe graduaLLy evoLved from the twocountries。The study provide a basis for further research and development of IHNV diagnostic kit andDNA vaccine,we cloned IHNV CJ-13strain glycoprotein genes and constructed prokaryoticexpression vector to observe its expression in host Escherichia coli strain BL21.The surfaceglycoprotein of CJ-13isolated from rainbow trout in Changchun was amplified and cloned into pGEM-T Easy vector to form recombinants pGEM-T Easy–G and sub cloned into prokaryoticexpressing vector pET-32a (+).The pET-32a-G was transformed into the E.Coli strain BL21andthe expression of the G protein was induced. G protein was analyzed by SDS-PAGE、Westernblot and ELISA。The results show that G gene is1527bp in length and the expressed productwas about76kDa. The G protein can be identified specifically by the mouse anti-IHNV Serumby Western-blot analysis.The ELISA results showed that IHNV could react specifically with theserum of G protein. The prokaryotic expression of IHNV-G protein was constructedsuccessfully and the expressed G protein has immunogenic and antigenic characters as nativeIHNV G protein..