Study On Polyclonal Antibody Preparation Of GCRV Genotype ? Nonstructural Protein NS66 And The Expression Of Host Heat Shock Protein In Response To Virus Infection

Grass carp hemorrhagic disease caused by grass carp reovirus has seriously endangered the healthy development of China's freshwater aquaculture industry.The research on the molecular structure and gene function of grass carp reovirus is the basis for clarifying the host mechanism of virus infection,and the study of the interaction between grass carp reovirus protein and host protein is the focus and difficulty of overcoming grass carp hemorrhagic disease.Heat shock protein 70 has the functions of assisting protein folding,promoting the assembly of protein complexes,promoting the distribution of proteins across the membrane,and preventing protein from folding and accumulating incorrectly.In mammals,Hsc70 is highly expressed under normal conditions and the expression level is further increased after stress.Hsp70 is absent or lower expression under normal physiological conditions.However,its expression level increases fast after stimulation,it has a cytoprotective function and is an inducible protein.Unlike Hsc70,mammalian Hsp70 has been identified as a key host factor for promotion of many viruses.However,no research has confirmed whether fish Hsp70 can promote any aquatic reovirus infection.The main contents are as follows:1.Polyclonal antibody preparation and application of GCRV genotype III nonstructural protein NS66s6 gene segment amplified by PCR was cloned into the expression vector p GEX-4T-3,transformed into E.coli BL21 and induced by IPTG.After analysis and identification by SDS-PAGE,the target protein is obtained through purification.The purified recombinant protein were injected to mice to obtain polyclonal antibody.Titers were determined by Western blot,and antibody specificity was identified by Western blot and IFA.The recombinant protein expressed by SDS-PAGE was about66 k Da,the same size as expected.The target protein was mainly present in inclusion bodies.The polyclonal antibody titer prepared by Western blot was more than1:50000.Western blot and IFA showed that the prepared polyclonal antibody recognizes the 104 strain,and the polyclonal antibody recognizes a single band with high specificity.[Conclusion]The study showed that the polyclonal antibody against NS66 protein can specifically recognize GCRV104 virus,which lays a foundation for the establishment of GCRV104 immunodiagnostic method and the study of GCRV-encoded NS66 protein.2.Induction of pro-viral grass carp Ctenopharyngodon idella Hsp70 instead of Hsc70 during infection of grass carp reovirusThe specific antibody which can distinguish Hsp70 from the homologous protein Hsc70 was prepared.In this study,real-time fluorescence quantification and western blot showed that grass carp Hsp70 was shown to be induced by GCRV-104 in different grass carp cell lines,whereas Hsc70 was expressed in a relatively constant level during the infection.The expression patterns of Hsc70 and Hsp70 were similar to their homologs in mammals.HSP family homologous protein HSP30 expression pattern is consistent with HSP70,HSP90 expression pattern is consistent with HSC70.Notably,both inhibitor and over-expression assays indicated that Hsp70 was required for efficient viral replication.Thus,our study supported a novel pro-viral property of Hsp70 besides its reported role in the viral attachment.Results herein presented also suggested that the heat shock response of grass carp might be manipulated by aquareovirus to facilitate its replication in fish cells.3.Possibility of regulating mechanism of grass carp heat shock protein HSPsTo further clarify how grass carp HSP70 protein participates in the regulation of the immune response system,we constructed HSP70 and HSC70 into yeast on the expression vector,and the experimentally constructed p GADT7-HSC70 or p GADT7-HSP70 plasmid does not have self-activation effect in yeast,using them as bait and GCRV104 gene fragment orientation screening did not find positive colonies,indicating that HSP70 and HSC70 may not directly interact with GCRV104.In order to verify the hypothesis that HSP may not be caused by virus-mediated activation of UPR,we detected UPR key indicator factors.The results showed that XBP1 S,ATF4,ATF6,and GRP94 were not significantly induced during the viral infection,and only GRP78 was induced to increase the transcription volume 10-fold at 144 hours of viral infection.In summary,the upregulation of Hsp70 expression in the virus GCRV-104 infection can not activative UPR.