Expression And Antiserum Preparation Of RRSV Genome S10 And S8

Rice ragged stunt virus (RRSV) is the type member of the oryzavirus group of the family Reoviridae. RRSV causes a severe disease in rice which occurs in Southeastern, Eastern and Southern Asia. The genome of RRSV comprises 10 segments of double-stranded RNA (dsRNA), and here we study cloning and expression of genome Segment 8(S8) and Segment 10(S10) and antiserum preparation of Pns 10 fusion protein.The dsRNA was extracted and purified from RRSV infected rice tissue by using CF-11 column and further purified through agarose gel electrophoresis, and total RNA was extracted with Trizol. Two sets of PCR primers were designed according to the ORF( nt 142-1035 ) in the genome segment 10 and the ORF ( nt 23-1810 ) in the genome segment 8 of RRSV-T (Thailand isolate), respectively. To enable directional cloning, each of the forward PCR primers contains the sequence, CACC, at the 5' end of the primer.cDNA of S10 ORF and S8 ORF of RRSV-F (Shaxian isolate) were amplified using M-MuLV Reverse Transcriptase and Taq DNA polymerase by RT-PCR, the sticky-end PCR products were cloned into pMD18-T, sequences of the cloned PCR products were analysed.Comparison of S10 nucleotide and deduced amino acid sequences with RRSV-T showed 99.66% and 99.83% identity, respectively.Comparison of S8 nucleotide and deduced amino acid sequences with RRSV-P showed 99.72% and 99.87% identity.respectively; While Comparison of S8 nucleotide and deduced amino acid sequences with RRSV-T showed 99.61% and 99.75% identity,respectively.Phylogenetic trees were drawn based on the coat proteins of NLRV and some of phytopathogenic reovirus at amino acid level,it showed that RRSV was more related to Fijiviruses than to Phytoreoviruses.To express the protein directionally, cDNA of RRSV S10 ORF and S8 ORF were amplified using M-MuLV Reverse Transcriptase and Pfu DNA polymerase by RT-PCR, the blunt-end PCR products were cloned into pET100/D-TOPOand expression plasmids were constructed. PnslO, the 35kD product of S10, and P8,the 70kD product of S8,were expressed in BL21star(DE3) E.coli.The antiserum against Pns10 was produced in big-ear white rabbits at a high...