Abstract: Cloning and expression analysis of transcriptional regulatorrovS gene of Stretococcus agalactiae ZQ0910 isolated from tilpia were studied in this study.A pair of primers were designed based on rovS gene sequence published in GenBank.The gene sequence of coding the RovS protein was cloned and then inserted into the pET-28(+)vector to construct prokaryotic expression plasmid pET-28-RovS.The recombinant RovS fusion protein was overexpressed in E.coli Rosetta(DE3)cells in the presence of isopropyl-β-thiogalactopyranoside(IPTG).Sequence analysis revealed that rovS gene is 849 bp and encodes a putative protein of 282 amino acids,and the amino acid sequence of RovS of S.agalactiae ZQ0910 showed highest identity of S.agalactiae 2 603 V and ATCC13813.The His-RovS fusion protein with 34 ku molecular mass was successfully expressed inE.coli Rosetta(DE3).The soluble recombinant protein was highly expressed under induction conditions of exposure to IPTG and successfully purified on Ni²⁺-IDA column.The purified fusion protein was injected into New Zealand white rabbits to produce polyclonal anti-RovS serum.The antibody titer detected by ELISA reached 1∶512 000.In summary,we identified and characterized a novel regulatory gene,termed rovS,encoding a regulator of virulence in S.agalactiae,which is involved in the expression of known and putative virulence genes in the bacteria.