| Gosling plague, also called Derzsy’s disease, is an acute and subacute septic infectious disease caused by Goose Parvovirus(GPV) and only infect young Goose and Muscovy ducks. The disease was first reported by a Chinese scholar Fang Dingyi and characterized by rapid transmission and a high mortality rate of 90%--100%, especially for 3-20 days old goslings and Muscovy duck. The younger, the higher the mortality will be. The total length of GPV genome is about 5 kb, including about 2 open reading frame. One on the left side of genome encodes the structural protein, including NS1 and NS2. The NS1 gene consists of 1 884 nucleotides and plays an important role in pathogenicity, replication, and regulating expression of virus. The phosphorylated NS1 protein can conjugate cytokines involved in regulating the promoter, which has a variety of functions in the early DNA replication process of GPV. NS1 can bind to viral replication initiation sequence with higher affinity, which is necessary to the viral DNA replication. NS1 inhibits cell DNA replication and can be positive control to the promoter of capsid protein, besides, NS1 may have negative regulatory role on P41 promoter. Currently, the hotspot at home and abroad have focused on structural protein, but rarely on the study of the nonstructural protein. Therefore, NS1 expression vector was constructed in this study to carry on the study on interactions between NS1 protein and goose embryo fibroblast protein, aiming to provide scientific basis to elucidate the molecular mechanism of GPV replication.This research is divided into the following four parts:Isolation and identification of Goose parvovirus isolates(Li shu) and NS1 gene sequencing and analysis: A strain of virus was isolated from the clinical epidemic materials by goose embryo. After observation by negative-stain electron microscopy and identification by specific PCR primers of GPV, the virus isolated proved to be goose parvovirus and named strain GPV/2010/LS. The nonstructural protein NS1 gene of GPV/2010/LS was amplified and cloned. Sequence analysis showed that NS1 gene and amino acid sequence of the isolate has high homology with Taiwan82-0321 isolate, Shanghai SHFX1201 isolate, Yangzhou YZ99-6 and Changchun CH isolate, and the NS1 gene is relatively conservative.Selection and identification of interacting protein of goose embryo fibroblast and GPV NS1 protein: The goose embryo fibroblast cDNA library was constructed rapidly and efficiently by SMART method, Clontech company. The nonstructural NS1 gene was used as bait gene to construct the recombinant vector pGBKT7-NS1 through yeast two hybrid technique. Then, goose embryo fibroblast cDNA library was constructed using the bait vector. Positive yeast diploid were selected by multiple filter medium SD/-Trp/-Leu/X-α-Gal/AbA、SD/-Ade /-Trp/-Leu/-His/ X-α-Gal /AbA. The yeast diploid library plasmid were saved and sent to biotech company for sequencing and results comparison analysis. Results showed that goose embryo fibroblast cDNA library was constructed with a concentration of 4×106 pfu/mL, recombinant vector pGBKT7- NS1 was constructed, expressed and validated. the two kinds of protein that can interact with goose parvovirus NS1 protein were selected from goose embryo fibroblast yeast hybrid library, sequenced and identified as an encoding ribosome speed 12 protein of 411 bp(named GP1) and unknown protein(named GP2) of 296 bp by online Blast.GST-pulldown verification of interactions among goose embryo fibroblast GP1, GP2 and GPV NS1 protein: Results showed that the pGEX-4t-1-NS1 prokaryotic expression plasmid was constructed and successfully expressed 95 kDa NS1 protein. Two eukaryotic expression plasmid, pcDNA 3.0- Flag- GP1 and pcDNA 3.0- Flag- GP2, was constructed and successfully expressed. Interactions among goose embryo fibroblast GP1, GP2 and GPV NS1 protein were verified by GST-EEF1A1 pulldown technology, which laid the foundation of proliferation effect of GPV inside and outside the goose embryo fibroblast in later studies.the impact of goose embryo fibroblast(GEF) interacting protein on proliferation of GPV : The experiment has confirmed GPV with goose embryo fibroblast interacting protein GP1 and GP2 by GST- pulldown technology. In order to make a further study of two protein on the effect of GPV replication, we undertake the research from two aspects. One is outside the GEF cells, GPV/2010/LS interact with purified LS GP1, GP2 protein and then infect GEF. Another is inside the GEF cells, GEF cells were transfected with GP1, GP2 gene and then infected with GPV/2010/LS at the same time. GPV proliferation was detected after both experiments to evaluate the impact of two proteins on the absorption of GPV by using SYBR Green I fluorescent quantitative method. TCID50 value of GPV/2010/LS was determined and the proportion of virus and GEF cells was identified as 1 moi values. GST-GP1 and GST-GP2 protein were purified respectively using GST-pulldown technology with concentrations of 0.54 μg/ μL and 0.38 μg/ μL. Outside the GEF cells, with the increase of GP1 and GP2 protein content, the proliferation of the GPV DNA copy decreased, indicating an inhibition effect of the two interacting protein on the adsorption of GPV. Inside GEF cells, after 12 hours’ transfection of GP1 and GP2, detection of DNA copies of GPV proliferation between experimental group and control group has insignificant changes; while changes were significant afer 24 hours’ transfection. The content of nucleic acid GPV copy in GP1 experimental protein group(GPV infection GEF alone) is 2.6 times of control group; The content of nucleic acid GPV copy in GP2 experimental protein group(GPV infection GEF alone) is 0.3 times of control group. After 24 hours’ transfection in GEF cell, GP1 copy number is 1.89 times of control group(including viruses and water), 2.56 times that of GP1 and GP2 group and 7.77 times of GP2 group. In conclusion, this study verified again that after 24 hours’ transfection in GEF cell, GP1 protein had a promoting effect on GPV proliferation, while GP2 protein had an inhibiting effect. |
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