无乳链球菌dnaJ基因缺失株的构建及其致病性研究

Study on Construction of dnaJ Gene Knock-out Mutant of Streptococcus agalactiae and Its Pathogenicity

为研究dnaJ基因序列与细菌致病力的关系,本研究构建了dnaJ基因缺失株和互补株,并对其致病性进行验证。参照GenBank中无乳链球菌GD201008-001(登录号:NC_018646)目的基因序列分别设计引物,PCR克隆dnaJ基因上、下游同源臂基因序列,通过融合PCR将两个片段连接到一起,构建dnaJ基因上、下游同源臂融合片段,PCR克隆含有启动子序列的dnaJ基因。将dnaJ基因上、下游同源臂融合片段和含有启动子序列的dnaJ基因分别连接链球菌-大肠杆菌穿梭质粒pSET4s和pSET2,电转化GD201008-001感受态细胞,构建dnaJ基因缺失株ΔdnaJ和互补株CΔdnaJ。通过分析ΔdnaJ和CΔdnaJ的遗传稳定性与形态学变化对dnaJ上、下游基因转录的影响,以及生长速率和斑马鱼攻毒试验评价dnaJ基因对无乳链球菌毒力的影响。经PCR鉴定和测序证明,ΔdnaJ和CΔdnaJ构建成功;与野生株GD201008-001相比,ΔdnaJ和CΔdnaJ在细菌染色形态上均无明显差异,但ΔdnaJ在液体培养基中的生长速度明显减缓;dnaJ基因的缺失未对相邻基因的转录造成影响;ΔdnaJ对斑马鱼的毒力明显下降,对斑马鱼的LD 50为5.68×10 4 CFU,约是野生株的241倍。dnaJ基因对无乳链球菌的毒力有显著影响,本试验结果为进一步探究无乳链球菌dnaJ基因的功能提供了参考依据。

To study the role of dnaJ gene in the pathogenicity of piscine S.agalactiae,isogenic mutant(ΔdnaJ)and complementation strain(CΔdnaJ)of dnaJ gene were respectively constructed.The primers were designed according to the target gene sequence in GenBank of S.agalactiae GD201008-001(accession No.:NC_018646).The upstream and downstream homologous arm sequences of dnaJ gene were amplified and fused by PCR.dnaJ gene containing promoter sequence was also amplified by PCR.The upstream and downstream homologous arm fusion fragment of dnaJ gene and dnaJ gene containing promoter sequence were cloned into the Streptococcus-Escherichia coli shuttle plasmids pSET4s and pSET2,respectively.The recombinant plasmid was electrotransformed into GD201008-001 competent cells to constructΔdnaJ and CΔdnaJ.By analyzing genetic stability and morphological changes ofΔdnaJ,CΔdnaJ and wild-type strain,effects of deletion of dnaJ gene on the transcription of downstream and downstream genes,growth rate and bacterial virulence in a zebrafish infection model,the effects of dnaJ gene on the virulence of Streptococcus agalactis were evaluated.PCR identification and sequencing showed thatΔdnaJ and CΔdnaJ were successfully constructed.ΔdnaJ and CΔdnaJ showed no significant differences in bacterial morphology compared with the wild-type strain,but the growth speed ofΔdnaJ decreased significantly.The deletion of dnaJ gene had no effect on the transcription of upstream and downstream genes.Furthermore,the 50%lethal dose ofΔdnaJ(5.68×10 4 CFU)was increased up to 241-fold of the parental strain in a zebrafish infection model.These findings demonstrated that the dnaJ gene of piscine S.agalactiae exerted a significant effect on bacterial virulence.This results provided a reference basis for further exploration of dnaJ gene of S.agalactiae.