| Rabbit hemorrhagic diseases virus(RHDV)is a calicivirus of genus Lagovirus,can causes the typical rabbit hemorrhagic disease in adult rabbits,commonly known as "rabbit plague".Rabbit hemorrhagic disease is caused by rabbit hemorrhagic disease virus and is an acute?septic highly fatal infectious diseases,mortality rate close to 100%,is a devastating impact to the rabbit keeping.Because of lacking efficient cell c?Ltural system,development of molecular biology of RHDV lags behind,expecially,we know less about the molecular pathogenic mechanism of RHDV.It's necessary to strengthen the research on molecular biology of RHDV.RHDV genome is about 7.5 kb and contains two open reading frames(ORF),ORF1 and ORF2,ORF1 encodes a large polyprotein of ca.257 kDa.the polyprotein s cleaved into seven non-structural and a structural protein of VP60;Earlier stage work of our laboratory findings: rabbit hemorrhagic disease virus VPg protein can binding and interaction with the host translation initiation protein eIF4 E,this kind of interaction is RHDV translation initiation required;VP60 is the rabbit hemorrhagic disease virus capsid protein,with 579 amino acids,relative molec?Lar mass is 60 kDa.It has been proved that VP60 is the most important structural protein of RHDV,which play an important role in the infection,replication and immunity of RHDV.Because of lacking in vitro proliferation cell lines,development of the molecular pathogenic mechanism of RHDV lags behind,the research on VP60 interact with cellular proteins is even less.In this study,we aimed to determine the host cellular protein could interact with VP60,and explore the impact of virus-host interaction on the replication of RHDV.There are three sections in this thesis.(1)Using the CRISPR/Cas9 technology built containing hemoglobin beta subunit sgRNA PX459-sgRNA recombinant plasmid,and transfect into RK-13 cells,through puro screening positive cell clones,and extract the cell genome DNA,design primers for sgRNA targets near the site of about 500 bp genomic DNA for PCR amplification,and is verified by sequencing identification and Western blotting assay,the results show that we have successfully established a RK-13 cell line knocked-out HB gene(named RK-?HB cell).This work provides a good the platform for further studying the role of HB in the life cycle of RHDV.(2)To detect the RHDV replication level in host cells,we established a SYBR Green real-time PCR assay which can detect the RHDV replication.We designed a pair of specific primers according to conserved sequence of the capsid protein VP60 for qPCR replication;the melting temperatures of RHDV and RHDV2 were(86.3± 0.1)? and(85.1±0.1)?,respectively,and single specific peaks were observed.The res?Lts indicated that the method developed here was able to quickly identify RHDV and RHDV2.(3)We screened some host cellular proteins including HB that bind to VP60 through CO-IP technology.We further improved that HB protein can interact with vp60 by GST Pull down and subcellular localization experiments.We also found that over expression of HB in RK-13 cells could inhibit the replication of RHDV,however,if we knockout HB gene from RK13 cells,the replication level of RHDV could be improved.In a word,this thesis first proved that hemoglobin subunits ? can interact with RHDV VP60 protein,and the interaction between HB-VP60 can inhibition RHDV replication.However,if we knockout HB gene of RK-13 cells,RHDV replication level could be improved.Our results will be helpful for further studying RHDV pathogenic mechanism as well as virus-host interactions. |
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