Screening Of Gene Of Proteins Intacting With VP7Protein In Grass Carp Reovirus

Grass carp reovirus （GCRV） is an important pathogen causing hemorrhagicdisease of grass carp （ctenopharyngodon idella） and threatening the aquacμLtureindustry in our country. There is an important significance to research the interactionsbetween virus and susceptible host. It is helpful to prevention and treatment of viraldiseases and the understanding of viral pathogenic mechanism in the molecuLar level.It is also useful to the developmental research in the novel antiviral medicine. Theresearch utilizes the Yeast Two-hybrid technology to preliminary screen the gene ofinteractive proteins.The virus used in this study was named GCRV096conserved in the laboratory.The Fusion vector of presumably VP7meditating viral particles enter into host cellswas constructed. In this study, vp7ORF （open reading frame） were cloned usionghomology cloning technique, meanwhile introduced the endonuclease site EcoR I，BanH I in vp7. After digestion with endonuclease, the products were ligated intopGBKT7and transformed E.coli DH5α. The recombinant plasmids were identified bythe means of double digests, clony PCR and sequencing. Extracting recombinantplasmids, and then transformed competent yeast cells of Y2HGold. The resuLtidentified by dropout substrates is that the recombinants did not activate the reportergenes of HIS3and ADE2, but activate genes of MEL1and AUR1-C. HIS3and ADE2could be used in this study.A full-length cDNA library of Grass carp kidney cells was constructed byhomologous recombination. The dscDNA and pGADT7-Rec vector were ligationthrough homologous recombination method, and then transformed competent yeastcells of Y187. Spread transformed cells on SD-Leu ager plates, incubate a certaintime, and then harvest500mL Transformants. According to calculating by bloodcorpuscle count method, the library titer was3.5×108cfu/mL, so it is couLd be usedfor the library screening.Mating the recombinants Y2HGlod[pGBKT7-vp7] and1mL library strain. Thenscreening yeast diplods by DDO, QDO medium. At last,2positive clones interacting with VP7was obtained. By sequencing, the size of cDNA fragments is298bp and534bp, respectively named M1and M2. Comparing with GeneBank Database using Blastprogram, it was found that the fragment M1has89%homologous to channel catfish（Ictalurus punctatus）40S ribosomal protein S20and82%homologous to Chlamysfarreri ribosomal protein S20. And the M2has92%homologous to predictedzebrafish （Danio rerio） eukaryotic translation initiation factor3（XM₀01335444）.The analyzing of bioinformatics indicated that the two cDNA sequences were notcomplete, but providing valuable information to find the complete cDNA sequence.We infered that the two cDNA fragment is40S ribosomal protein S20and eukaryotictranslation initiation factor eIF3b of the ctenopharyngodon idella. Analysis forecastedVP7through interacting with S20and eIF3b to help the virus synthetizing viralproteins continuously in the host cell.