CAJ | 학술논문

目的：构建猪链球菌2型强毒株05ZYH33 cAMP结合蛋白（CRP）编码基因敲除突变株及基因回复互补株，并探究CRP基因的缺失对细菌生物学特性及毒力的影响。方法：构建中间为壮观霉素抗性基因（Spcr）、两侧为CRP编码基因上下游同源序列的基因敲除质粒，通过同源重组筛选CRP编码基因敲除突变株ΔCRP；构建CRP编码基因的互补质粒，通过电转化敲除株ΔCRP，筛选CRP的基因回复互补株CΔCRP；比较分析突变株、野生株和回复互补株的基本生物学特征的差异，并以小鼠作为动物感染模型对突变株、互补株及野生株的毒力进行评估分析。结果：应用组合PCR和基因测序分析，证实构建了CRP的突变株ΔCRP，并筛选出CRP的回复互补株CΔCRP；逆转录PCR证实在突变株ΔCRP中CRP在转录水平缺失，而在回复互补株CΔCRP中其转录回复；在丰富营养情况下，突变株ΔCRP与野生株的溶血活性、生长速率及对小鼠的致病力均无显著性差异，但突变株的成链能力减弱。结论：CRP编码基因的缺失并未显著改变野毒株05ZYH33的基本生物学特性和毒力，提示CRP可能不是猪链球菌的关键毒力决定因子，其参与碳源代谢等功能有待进一步研究。
목적：구건저련구균2형강독주05ZYH33 cAMP결합단백（CRP）편마기인고제돌변주급기인회복호보주，병탐구CRP기인적결실대세균생물학특성급독력적영향。방법：구건중간위장관매소항성기인（Spcr）、량측위CRP편마기인상하유동원서렬적기인고제질립，통과동원중조사선CRP편마기인고제돌변주ΔCRP；구건CRP편마기인적호보질립，통과전전화고제주ΔCRP，사선CRP적기인회복호보주CΔCRP；비교분석돌변주、야생주화회복호보주적기본생물학특정적차이，병이소서작위동물감염모형대돌변주、호보주급야생주적독력진행평고분석。결과：응용조합PCR화기인측서분석，증실구건료CRP적돌변주ΔCRP，병사선출CRP적회복호보주CΔCRP；역전록PCR증실재돌변주ΔCRP중CRP재전록수평결실，이재회복호보주CΔCRP중기전록회복；재봉부영양정황하，돌변주ΔCRP여야생주적용혈활성、생장속솔급대소서적치병력균무현저성차이，단돌변주적성련능력감약。결론：CRP편마기인적결실병미현저개변야독주05ZYH33적기본생물학특성화독력，제시CRP가능불시저련구균적관건독력결정인자，기삼여탄원대사등공능유대진일보연구。
Objective: To construct a gene knock-out mutant of cAMP receptor protein(CRP) and its complemen?tary strains in Streptococcus suis type 2 virulent strain 05ZYH33, and analyze the virulence as well as biological characteristics of the mutant strain. Methods: Based on the principle of homologous recombination, recombinant gene konck-out vector was constructed consisting of Spcr cassette with the flanking homology regions to the target gene and the isogenic CRP-deficient mutant was screened by allelic replacement. The complementary plasmid of CRP coding gene was also constructed and the complementary strain CΔCRP was obtained through electric conver?sion. The basic biological characteristics difference between the mutant and the wild type strain was analyzed. The virulence difference was also analyzed with murine model. Results: PCR analysis and gene sequencing results con?firmed that the gene knock-out mutant strain ΔCRP was successfully constructed, and the CRP complementary strain CΔCRP was also successfully screened. Reverse transcription PCR confirmed the absence of CRP in theΔCRP strain and the restoration in the CΔCRP strain at the level of transcription. Analysis of biological character?istics and the virulence showed that there were no significant differences between the mutant and the wild type strain in terms of hemolytic activity, growth kinetics and the mouse pathogenicity under the condition of rich nutri?tion. The mutant strain grew as much shoter chains than the wild type strain. Conclusion: The CRP gene knock-out mutant strain ΔCRP as well as the complementary strain CΔCRP were successfully constructed.The knocking out of CRP encoding gene did not change the virulence of wild strains 05ZYH33, showing that CRP is not the vir?ulence decision factor of S.suis. But the CRP was likely involved in the regulation of the chain ability in S.suis.