Prokaryotic expression of S5-2 gene and cloning of S5 segment from Anhui isolate of Southern rice black-streaked dwarf virus

Objective To study genetic characteristics of the isolates of Southern rice black-streaked dwarf virus(SRBSDV) from Anhui province, and to obtain P5-2 protein by prokaryotic expression.

Method The S5 segment of SRBSDV was amplified by RT-PCR, and it was cloned, sequenced and analyzed. Gene S5-2 was inserted into prokaryotic expression vector. The recombinant vector was transformed into Escherichia coli and was induced by IPTG. The fusion protein was purified by Ni²⁺-NTA affinity column. The expression of P5-2 protein was analyzed by SDS-PAGE.

Result The S5 segment from Anhui isolate of SRBSDV(SRBSDV-AnHui-HN2) was 3 167 bp in full length and contained a 612 bp S5-2 gene encoding 204 amino acids. Sequence comparison showed that the S5 segment of SRBSDV-AnHui-HN2 shared high sequence similarity(99.0%-99.7%) with other SRBSDV isolates, while had relatively low sequence similarity(38.0%-71.3%) to other Fijivirus members. The phylogenetic tree based on S5 segment sequences showed that SRBSDV and RBSDV clustered into a branch, and six isolates of SRBSDV clustered into a sub-branch. The recombinant protein with approximately 47 000 relative molecular mass was obtained by prokaryotic expression. Western blot analysis revealed that GST monoclonal antibody could specifically bind to the fusion protein.

Conclusion All isolates of SRBSDV are closely related, and they have relatively far relationship to other Fijivirus members. The fusion protein obtained by prokaryotic expression is the target protein.