铜绿假单胞菌clpP基因缺陷株的构建
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摘要
目的构建铜绿假单胞菌clpP基因缺陷株。方法分别以质粒pUCGM和铜绿假单胞菌PAO1基因组为模板PCR扩增庆大霉素抗性基因(GM)和铜绿假单胞菌clpP基因及其3′、5′侧翼序列,将clpP基因及其3′、5′侧翼序列克隆至pMD19T载体,EcoRV切除clpP基因37bp~453bp片段后,引入庆大霉素抗性基因,构建重组质粒pCKR2;EcoRⅠ/HindⅢ双酶切重组质粒pCKR2,回收FclpP-GM-clpPR片段,与自杀质粒pEX18Tc连接,得到clpP基因缺陷的同源重组载体pCKR3;将pCKR3质粒转化大肠埃希菌SM10,与铜绿假单胞菌PAO1双亲杂交,庆大霉素筛选得到铜绿假单胞菌clpP基因缺陷株。结果经酶切鉴定同源重组载体pCKR3构建正确;PCR和DNA测序鉴定铜绿假单胞菌clpP基因缺陷株构建成功。结论本研究成功敲除了铜绿假单胞菌clpP基因,为进一步研究clpP基因的生物学功能奠定了基础。
To delete clpPgene of Pseudomonas aeruginosa PAO1,the clpPgene with 3′and 5′flanking sequences was amplified by PCR using the primers clpP-F1 and clpP-R1,and then the PCR product was cloned into pMD19 Tfor construction of pCKR1.A gentamicin resistance cassette amplified from pUCGM was inserted into EcoRV sites of pCKR1 for construction of pCKR2.Then the FclpP-GM-clpPR fragment was obtained by cutting pCKR2 with EcoRⅠ/HindⅢ and gel purification,and inserted it into EcoRⅠ/HindⅢ sites of pEX18 Tc to construct the homologous recombinant vector pCKR3.The P.aeruginosa clpP mutant strain was obtained by conjugations between P.aeruginosa PAO1 and E.coli SM10 transformed with pCKR3.The homologous recombinant vector pCKR3 was verified by enzymatic digestion and the final P.aeruginosa clpP mutant strain was verified by PCR analysis and DNA sequencing.The clpP gene of P.aeruginosa was successfully deleted,which laid a foundation for further study of its biological function.
To delete clpPgene of Pseudomonas aeruginosa PAO1,the clpPgene with 3′and 5′flanking sequences was amplified by PCR using the primers clpP-F1 and clpP-R1,and then the PCR product was cloned into pMD19 Tfor construction of pCKR1.A gentamicin resistance cassette amplified from pUCGM was inserted into EcoRV sites of pCKR1 for construction of pCKR2.Then the FclpP-GM-clpPR fragment was obtained by cutting pCKR2 with EcoRⅠ/HindⅢ and gel purification,and inserted it into EcoRⅠ/HindⅢ sites of pEX18 Tc to construct the homologous recombinant vector pCKR3.The P.aeruginosa clpP mutant strain was obtained by conjugations between P.aeruginosa PAO1 and E.coli SM10 transformed with pCKR3.The homologous recombinant vector pCKR3 was verified by enzymatic digestion and the final P.aeruginosa clpP mutant strain was verified by PCR analysis and DNA sequencing.The clpP gene of P.aeruginosa was successfully deleted,which laid a foundation for further study of its biological function.
引文
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[2]Dougan DA,Mogk A,Bukau B.Protein folding and degradation in bacteria:to degrade or not to degrade?That is the question[J].Cell Mol Life Sci,2002,59(10):1607-1616.
[3]Gottesman S.Proteases and their targets in Escherichia coli[J].Annu Rev Genet,1996,30:465-506.
[4]WANG zhen-hai,SUN ye-qing.Progress in Study on Clp Protease[J].Pharmaceutical Biotechnology,2005,12(6):412-415.王振海,孙野青.Clp蛋白酶研究进展[J].药物生物技术,2005,12(6):412-415.英文对照
[5]Yu AY,Houry WA.ClpP:a distinctive family of cylindrical energy-dependent serine proteases[J].FEBS Lett,2007,581(19):3749-3757.
[6]de Bruijn I,Raaijmakers JM.Regulation of cyclic lipopeptide biosynthesis in Pseudomonas fluorescens by the ClpP protease[J].J Bacteriol,2009,191(6):1910-1923.
[7]Bjarnsholt T,Jensen PO,Fiandaca MJ,et al.Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients[J].Pediatr Pulmonol,2009,44(6):547-558.