无乳链球菌dnaJ基因缺失株的构建及其致病性研究

doi:10.16431/j.cnki.1671-7236.2019.09.002

Abstract

Abstract: 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 construct ΔdnaJ and CΔdnaJ.By analyzing genetic stability and morphological changes of ΔdnaJ,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 that ΔdnaJ 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 of ΔdnaJ decreased significantly.The deletion of dnaJ gene had no effect on the transcription of upstream and downstream genes.Furthermore,the 50% lethal dose of ΔdnaJ (5.68×10⁴ 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.

Key words: Streptococcus agalactiae; dnaJ gene; gene knock-out mutant; virulence

CLC Number:

S852.61⁺1

GUO Changming, WU Zhi, WU Shuang, WANG Yongjuan, WANG Anping, LIU Guangjin, LIU Yongjie, ZHU Shanyuan. Study on Construction of dnaJ Gene Knock-out Mutant of Streptococcus agalactiae and Its Pathogenicity[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(9): 2507-2515.

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

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