Study On The Interaction Between Grass Carp Reovirus Non-structural Protein And Grass Carp Host Protein

Grass carp（Ctenopharyngodon Idella）has an annual output of more than 5million tons and is widely distributed throughout my country.The annual output value of grass carp aquaculture is about 70 to 80 billion yuan and it is one of the main freshwater fish species in China.Grass carp hemorrhagic disease,as a second-category animal disease recognized by the Ministry of Agriculture and Rural Affairs of my country,brings huge economic losses to the grass carp breeding industry in our country every year,and seriously endangers the green and sustainable development of grass carp breeding.Grass carp reovirus（Grass carp reovirus,GCRV）is the main viral pathogen of grass carp hemorrhagic disease.So far,dozens of grass carp reoviruses have been isolated and identified.According to the homology of the S6 segment,they can be divided into three genotypes,type I,type II,and type III.The representative strains are GCRV-873,GCRV-HZ08 and GCRV-HZ08 respectively^[1].GCRV-104.Grass carp type Ⅰ reovirus S7 fragment encodes the non-structural protein NS12 as the newly discovered viral protein,and its specific biological function is not yet clear.Proteasomes are widely present in the protein complexes of eukaryotic cells and are the main engine of protein degradation.Intracellular pathogens are the targets of their enzyme activity,and many animal viruses are known to interfere with the activity of these enzymes.Myosin-9 is involved in cell division,cell movement,cell morphology maintenance,cell phagocytosis and apoptosis.Myosin-9 is inextricably linked to the occurrence and development of various diseases,and participates in the progress of some human cancers.This study uses yeast two-hybrid technology,GST-Pulldown and co-immunoprecipitation to discover the potential interactions between grass carp PSMB7 and grass carp Myosin-9 and the non-structural protein NS12 of grass carp type Ⅰ reovirus,and use a variety of molecular biology.The technology verified the interaction between the grass carp type Ⅰ reovirus non-structural protein NS12 and the above two host proteins.The specific research content is as follows:1.Using yeast two-hybrid technology to screen the host protein that interacts with the non-structural protein NS12 of grass carp type Ⅰ reovirusThe yeast two-hybrid technique was used to screen the host protein that can interact with the non-structural protein NS12 of grass carp type Ⅰ reovirus.PCR technology was used to amplify the GCRV-JX01NS12 gene fragment to construct the bait plasmid p GBKT7-NS12;auto-activate the recombinant plasmid and screen the interacting host protein in the grass carp yeast library with NS12 as the bait,and extract and screen the positive yeast Plasmid and sequenced.The results showed that the bait plasmid p GBKT7-NS12 had no self-activation reaction;the bait plasmid p GBKT7-NS12 was screened to obtain 10 positive clones,which coded forβ-actin,F-actin capping protein subunitα1,TAR DNA binding protein 43,N-Acylphosphatidylethanolamine hydrolyzed phospholipase D,grp E protein homolog 2,RNA binding protein 48,nuclear receptor coactivator 4,protease receptor subunitβ7,melanotransferrin and NADH-ubiquinone oxidoreductase 75 k Da subunit.2.The interaction between the non-structural protein NS12 of grass carp type Ⅰ reovirus and Proteasome subnit beta type 7,PSMB7Using targeted yeast two-hybrid technology,GST-pulldown,intracellular co-localization and other technologies to explore the interaction between NS12 and PSMB7;using liposome-mediated transfection technology in the grass carp ovarian cells（GCO）overexpression of PSMB7 after infection GCRV-JX01,using realtime RT-PCR to detect the expression level of NS12 transcription level in GCO cells overexpressing PSMB7 after virus infection.The results showed that PSMB7interacted with the non-structural protein NS12 of grass carp type Ⅰ reovirus;overexpression of PSMB7 could up-regulate the transcript level of NS12 in cells infected with GCRV-JX01.3.The interaction between grass carp type Ⅰ reovirus non-structural protein NS12and grass carp myosin-9（Myosin-9）Use GST-pulldown,Co-immunoprecipitation（Co-immunoprecipitation,CO-IP）,protein mass spectrometry and other technologies to screen the interacting host proteins in grass carp ovary cells（GCO）;perform protein mass spectrometry sequencing on the extracted extracts;Western blot and realtime RT-PCR were used to detect the expression of Myosin-9 in GCO cells after virus infection at the translation level and transcription level;RNAi interference technology was used to knock down Myosin-9 in grass carp ovarian cells（GCO）The amount of expression and viral infection.The results showed that Myosin-9 was down-regulated at the transcriptional level after GCRVJX01 infection.Knockdown of Myosin-9 could down-regulate the expression of NS12 at the transcriptional and translational levels in host cells.In this study,the yeast two-hybrid technology and GSTpulldown-CO-IP were used to screen the host protein that interacts with the non-structural protein NS12encoded by GCRV-JX01.The soluble prokaryotic expression of NS12 was performed.Various experimental techniques were used to verify PSMB7 and PSMB7.The interaction between Myosin-9 and the non-structural protein NS12 of grass carp type Ⅰ reovirus was investigated.The expression level of PSMB7 and Myosin-9 changed at the m RNA level after viral infection and the effect of PSMB7 and Myosin-9 on the viral non-structural protein NS12 were investigated.The influence of expression changes in cells provides a theoretical basis for further understanding of the mechanism of GCRV infection.