Verifying The Interaction Of GPV Structural Protein VP1with Duck Embryo Fibroblast Cell Ribosomal Protein S12and The Effect On GPV Proliferation

Gosling plague is caused by Goose parvovirus (Goose parvovirus, GPV). The diseaseoften occurs in8-30day-old Goslings, Young Muscovy can infect it. Clinical manifestationsinclude systemic septic lesions, focal or diffuse hepatitis, pseudomembranous embolic enteritisand myocarditis embolism. It has features of wide popular range, spreading fast, highmortality and high mortality, so it still is one of the major infectious diseases which endangerthe healthy development of waterfowl breeding currently. Latent infectious adult geese withsubclinical symptoms spread GPV to the susceptible goslings by fertilized eggs in clinicalinfection, resuLting in GPV spreading and epidemic, which has brought great difficuLties forprevention and treatment. Therefore, it is critical that preventing vertical transmission of GPVfor effective control GPV. It has a very important significance to understand the pathogenesisof the GPV, study proliferation and replication mechanism of virus in the host cells, reveal theinteraction of viral structural proteins and host cell proteins, clarify the process of viralinvasion in protein level. Five positive clones were filtered out by Yeast Two-hybrid Systemin goose parvovirus VP1structural protein interaction research in our laboratory. One of thepositive clones is442bp in gene size and found the homology with chicken nuclear ribosomalprotein S12reach to96%by BLAST tool for sequence alignment on the NCBI Web site. Tofurther verify the interaction between S12with VP1and study GPV proliferation in cells, thisstudy and experiments were conducted.The total RNA of duck embryo fibroblasts was extracted and the ribosomal protein S12was amplified by RT-PCR. The S12gene was subcloned into the prokaryotic expressionvector pGEX-4T-1and selected positive plasmids pGEX-4T-1-S12by digestion andsequencing in this study. Expression strains E.coli BL21(DE3) containing the positiveplasmids were induced with IPTG by0.6mmol/L and the expression products were identifiedby SDS-PAGE. The result indicated that the recombinant protein was about41kDa whichconsistent with the theoretical size and demonstrated recombinant proteins containing S12gene expressed successfuLly. Meanwhile, the S12gene was inserted into the eukaryoticexpress plasmid pcDNA-3.0-Flag. Positive plasmids pcDNA-3.0-Flag-S12transfected293Tcells after tested correct. Verify the interaction between expression products collected andGPV structural protein VP1through GST Pull-Down protein technology in vitro. Conjugate products were analyzed by SDS-PAGE gel analyses, the results proved VP1and S12can becombined in vitro.According to the conserved region of GPV major structural protein VP1gene, primerswere designed to establish a rapid detection of GPV real-time fluorescent quantitative PCR(FQ-PCR) method. The reaction system and the conditions of FQ-PCR assay were optimized.The standard curve was established by different dilutions of positive plasmid pGEX-4T-1-VP1.Additional, the sensitivity and repeatability test were also assessed, the results showed that thelinear correlation factor of the standard curve for the FQ-PCR was0.993and the detectivequantity was from2.24×101to2.24×1011copies/uL of GPV DNA, proving that the methodhad high sensitivity. GST-S12fusion protein pGEX-4T-1-S12expressed was purified andseparated by glutathione agarose resin. The protein obtained was mixed and incubated withGPV LS strains laboratory isolated while establishing the control group only GPV, wererespectively inocuLated in goose embryonic fibroblasts. Viral cuLtures were collected after12h and24h respectively and detected by fluorescence quantitative PCR method establishedby relative quantitative analysis. The results showed that the copies of GPV in goose embryofibroblasts were about5times of the copy number GPV which interacted with S12protein.These indicated that duck embryo fibroblast nuclear ribosomal protein S12has someinhibition on the replication of GPV in goose embryo fibroblasts. The study laid a foundationfor further study on mechanism of duck embryo fibroblast nuclear ribosomal protein’sinhibition to the replication of GPV and provided new ideas and strategies for exploringpossible existing inhibitory effect proteins to GPV.