Abstract: Objective To construct pilus backbone gene SSU0474 knock-out mutant of a strong virulent strain of Strepococcus suis serotype 2(S.suis 2)(05ZYH33) isolated in China and to analyze its biological characteristics.Methods The pilus backbone gene SSU0474 knock-out mutant strain was constructed with homologic recombination method and the mutant strains were identified with PCR and reverse transciption PCR.Gram’s stain and electroic microscope observation were used to examine the difference bwtween SSU 0474 mutant srain and wild strain.The virulent change of the mutant strain was tested with experiment in mice.Results Results of biological characteristics showed that there were no differences in dyeing properties and growth kinetics between the mutant SSU0474 strain and the wild type strain 05ZYH33.The abolishment of the SSU0474 expression had influence on cell wall upon cultivation in standard laboratory in vitro assay.Furthermore,mutants were impaired in virulence when evaluated in a murine model of S.suis 2.Conclusion These results suggest that SSU0474 plays an important role in the S.suis 2 05ZYH33,which laid the foundation for the study of the mechanisms of pilus assembly and biological function in the S.suis 2.