Cloning Analysis Of RHDV RdRp Gene And Its Protein Function Preliminary Study

Rabbit hemorrhagic disease(RHD),also known as rabbit plague,is an acute and highly infectious disease caused by rabbit hemorrhagic disease virus(RHDV)of the Caliciviridae genus.RNA-dependent RNA polymerase(RdRp)is an important non-structural protein of RHDV which has some additional functions besides replicase activity.In order to further understand the biological function of RHDV RdRp,on the basis of cloning GI.1 and GI.2RHDV RdRp,this study have detected the effect of RdRp on RNA transcription and expression of different types of RHDV viruses in RS-17 cells through the construction of eukaryotic expression plasmid of GI.1 RHDV RdRp gene.The main research contents are as follows:1.By PCR,connecting conversions and sequencing,two GI.1(SCH04 strains)and GI.2(SCCN03 strains)RHDV RdRp genes were cloned for comparison and bioinformatics analysis.The results of bioinformatics showed that the full length of RdRp gene sequence of both strains was 1548 bp,encoding 516 amino acids.The nucleotide sequence similarity was84.95%.The amino acid sequence similarity was 96.71%,and there were 17 amino acid difference sites in the encoded protein.There is no significant difference in secondary structure and structure between them.RdRp of SCCN03 strain had four more phosphorylation sites and one more N-glycosylation site than SCH04 strain.2.The SCH04 strain RdRp gene was cloned by digestion,ligation transformation and sequencing,and the prokaryotic expression plasmid p ET-32a-RdRp was constructed.The recombinant protein of RHDV RdRp was expressed and its antiserum was prepared by IPTG-induced,nickel column affinity chromatography,Western blot and animal experiment.The results showed that the titer of serum ELISA prepared with RdRp recombinant protein of75.7 k Da was 1: 512000.It can provide experimental materials for serological detection of cell lines expressing RdRp protein which will be constructed subsequently.3.The eukaryotic expression plasmid p EGFP-N1-RdRp was constructed through PCR,purification,connecting conversions,digestion and sequencing identification,and it was transfected into RS-17 cell line of rabbit spleen fibroblasts.RS-17-RdRp cell line was successfully constructed by G418 screening,RT-PCR,Western blot and indirect immunofluorescence assay(IFA).The viral RNA of SCH04 and SCCN03 strains were respectively transfected into RS-17-RdRp and common RS-17 cells,and the cells were collected at different time points for q PCR and IFA.The results showed that gene copies of SCH04 strain major capsid protein VP60 significantly increased in RS-17-RdRp cells,and was upregulated 170-fold after 24 hours of transfection.However,the copies of the VP60 gene in SCCN03 strain was slightly upregulated after 6 hours of RNA transfection.The IFA results showed that after 24 hours of viral RNA transfection,the expression level of SCH04 strain major capsid protein VP60 in RS-17-RdRp cells is higher than that in ordinary RS-17 cells,while the expression level of VP60 protein in SCCN03 strain did not show significant changes in both types of cells,consistent with the results of q PCR.In conclusion,the effect of RdRp on the transcriptional expression of RHDV genome in RS-17 cells was studied on the basis of RHDV RdRp gene cloning analysis.The results showed that RdRp of GI.1 RHDV(SCH04 strain)could significantly promote the replication of its RHDV genome,but the effect on GI.2 RHDV(SCCN03 strain)was not obvious,indicating that although the tested RHDV strains RdRp was relatively stable,its function may vary in specificity.Whether the functions of both type GI.1 and type GI.2 RHDV RdRp exist type-specificity needs to be further studied and verified.This study provides a scientific reference for a deep understanding of the biological activity of RHDV RdRp and the genetic evolution of RHDV.