CAJ | 학술논문

目的构建副溶血弧菌生物膜相关基因突变株并进行验证.方法采用PCR方法扩增得到靶基因的上下游同源臂片段,然后以上下游同源臂片段为模板,PCR扩增得到同源臂融合片段.将同源臂融合片段经酶切处理后克隆到自杀质粒pDS132中.通过结合转移的方式将携带有同源臂融合片段的重组质粒转入副溶血弧菌RIMD 2210633中,利用同源重组得到突变株.用PCR方法筛选和鉴定突变株,同时对突变株进行表型分析,从而在分子水平和表型试验上验证突变株是否构建成功.结果构建得到了分别携带副溶血弧菌生物膜相关基因vbfR、crp、hns、swrZ、swrT、cpsR融合同源臂片段的重组质粒,通过PCR扩增,分别得到了大小为1190、1128、1136、953、1242、1112 bp的片段；用重组质粒构建了相应的突变株(ΔvbfR、Δcrp、△hns、ΔswrZ、ΔswrT和ΔcpsR),进行PCR鉴定时,突变株得到了大小分别为1190、1128、1136、953、1242、1112 bp的扩增产物,比阳性对照分别小610、739、421、542、427、1367 bp;用靶基因内部的引物进行扩增时,无扩增产物；选其中一株突变株Δhns进行生物膜形成能力表型分析,结果表明突变株Δhns生物膜形成量与野生株相比明显增加.结论构建得到了6株副溶血弧菌生物膜相关基因突变株,并从分子和表型试验上验证了突变株构建正确.
목적 구건부용혈호균생물막상관기인돌변주병진행험증.방법 채용PCR방법확증득도파기인적상하유동원비편단,연후이상하유동원비편단위모판,PCR확증득도동원비융합편단.장동원비융합편단경매절처리후극륭도자살질립pDS132중.통과결합전이적방식장휴대유동원비융합편단적중조질립전입부용혈호균RIMD 2210633중,이용동원중조득도돌변주.용PCR방법사선화감정돌변주,동시대돌변주진행표형분석,종이재분자수평화표형시험상험증돌변주시부구건성공.결과 구건득도료분별휴대부용혈호균생물막상관기인vbfR、crp、hns、swrZ、swrT、cpsR융합동원비편단적중조질립,통과PCR확증,분별득도료대소위1190、1128、1136、953、1242、1112 bp적편단；용중조질립구건료상응적돌변주(ΔvbfR、Δcrp、△hns、ΔswrZ、ΔswrT화ΔcpsR),진행PCR감정시,돌변주득도료대소분별위1190、1128、1136、953、1242、1112 bp적확증산물,비양성대조분별소610、739、421、542、427、1367 bp;용파기인내부적인물진행확증시,무확증산물；선기중일주돌변주Δhns진행생물막형성능력표형분석,결과표명돌변주Δhns생물막형성량여야생주상비명현증가.결론 구건득도료6주부용혈호균생물막상관기인돌변주,병종분자화표형시험상험증료돌변주구건정학.
Objective To construct the mutants of biofilm related genes in Vibrio parahaemolyticus and confirm the mutants.Methods The homologous upstream and downstream flanking fragments of target gene were amplified by using PCR,and the fusion homologous fragment was amplified by using the two flanking fragments as template.Then the fusion homologous fragment was digested by restriction enzyme and cloned into suicide plasmid pDS132.The recombinant plasmid was transferred into Vibrio parahaemolyticus RIMD 2210633 through conjugation.The mutants were screened and identified by PCR and the phenotype of one mutant was analyzed in order to verify that the mutants were constructed successfully.Results Six recombinant plasmids carrying the fusion homologous fragments of genes vbfR,crp,hns,swrZ,swrT and cpsR respectively were constructed and identified by PCR.The amplification products of 1190,1128,1136,953,1242 and 1112 bp were obtained respectively.The six mutants (ΔvbfR,Δcrp,Δhns,ΔswrZ,ΔswrT and ΔcpsR) were constructed using recombinant plasmids.Verified by PCR,the size of amplification products of mutants (1190,1128,1136,953,1242 and 1112 bp respectively) was less (610,739,421,542,427 and 1367 bp respectively) than the corresponding positive control.Meanwhile,none of the products was amplified using the primers locating on the target gene.One mutant Δhns was selected to test the ability of biofilm formation.The result showed that the ability of biofilm formation of mutant Δhns was increased compared with the wild type.Conclusion Six mutants of biofilm related genes in Vibrio parahaemolyticus were constructed and tested by molecular and phenotype experiment to confirm that the mutants were constructed successfully.