Prokaryotic Expression And Bioinformatics Analysis Of The P8 Gene And Pns10 Gene Of Rice Gall Dwarf Virus

Prokaryotic expression and bioinformatics analysis of the P8 gene and Pns10 gene of RGDV (Rice gall dwarf virus) and antiserum preparation of their fusion proteins were studied in this paper.The P8 and PnslO gene of RGDV Xinyi isolate (RGDV-C) was amplified successfully by RT-PCR and cloned into pMD18-T vector. Targeted gene was transfered into pGEX-4T-l prokaryotic expression vector respectively after the recombinated plasmid containing targeted gene was double digested with BamH I and Xho I. The expression vectors were transformed into E.coli BL21(DE3). The nucleotide sequence of the recombinated plasmid pGTP8 and pGTPns 10 were sequenced and the results indicated that the target genes had been inserted into the BamH I/Xho I site of the expression vector in right reading frame. 74kDa protein and 62kDa protein were expressed respectively with the induction of IPTG. The molecular weight of express products accorded with the anticipated molecular weight of pGTP8 and pGTPns10 fusion protein by SDS-PAGE. By indirect ELISA the antiserums against P8 and PnslO were produced in big-ear white rabbits at high titer of 1: 9600 and 1: 8192 respectively, then their specificities were demonstrated by Western blotting.In virtue of biology software and correlative analysis tools online, a series of bioinformatics analysis were performed on the structure and function of P8 and Pns 10 protein including prediction of the physical and chemical properties, domain, motif and dimensional structure etc. The results showed that P8 had a transmembrane helices region with 26 amino acid residues, but neither P8 nor PnslO had any signal peptides and known coiled-coils regions. The specific domain analysis indicated that P8 is a member of the Phytoreo_P8 family whose function was noted as structural molecule activity in Interpro; that Pns10 belongs to the Phyto_Pns9₁0 family with function unknown. On the basis of the major outer capsid protein sequences with the method of the Neighbor-Joining and the Maximum likelihood, Phylogenetics trees were constructed for the Reoviridae family respectively, and the results supported the classification of Reoviridae family well.