无乳链球菌nrdD基因缺失株的构建及其对小鼠的致病性检测OA北大核心CSTPCD

To investigate the effect of the nrdD gene on the virulence of Streptococusagalactiae,in this study,primers were designed by referring to the nrdD whole gene sequence of S.agalactiae GD201008-001(Accession Number:NC_018646)in GenBank.The upstream and downstream homologous arm sequences of nrdD gene were amplified and fused by PCR.The nrdD gene containing promoter sequence was also amplified by PCR.The upstream and downstream homologous arm fusion fragment of nrdD gene and nrdD gene containing promoter sequence were cloned into the Streptococcus-Escherichia coli shuttle plasmids pSET4s and pSET2 respectively.The recombinant plasmids were electro-transformed into GD201008-001 competent cells and ΔnrdD competent cells to construct ΔnrdD and CΔnrdD respectively.PCR and gene sequencing showed success in constructing the nrdD gene deleted and complementary strains.The effects of nrdD gene on the virulence of S.agalactiae were evaluated by morphological observation,growth rate,proliferation ability in mouse serum,and bacterial load in infected mouse tissues.Compared with the wild type strain GD201008-001,ΔnrdD stain showed a significant increase in chain length on bacterial staining morphology but did not affect the formation of the capsule.Under the same culture conditions,ΔnrdD strain showed no significant differences in growth with GD201008-001.In murine serum survival assay,the viability of ΔnrdD strain was significantly decreased compared with that in TSB,and the growth rate was lower compared with wild and complemented strains.At 14 h post infection of mice,the bacterial loads of ΔnrdD strain in their blood,spleens,and brains were significant lower than those in the wild-type strain,whereas the bacterial loads of CΔnrdD in tissues did not differ significantly from the wild-type strain.In addition,the survival time of mice in ΔnrdD injection group was significantly longer than that of wild strains.Therefore,these findings demonstrated that the nrdD gene of S.agalactiae exerted a significant effect on bacterial virulence and provided a foundation for further studies on the function of nrdD in S.agalactiae.