结核杆菌phoP突变株的构建及鉴定
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摘要
研究背景及目的结核病(tuberculosis,TB)是一种由结核杆菌(Mycobacteriatuberculosis,MTB)引起的可累及全身多个脏器的人兽共患的慢性传染病,近些年来由于MTB耐药菌株出现、艾滋病合并MTB感染以及高危人群的大规模流动,造成TB的在全球流行,使其成为我国乃至全世界危害最严重的疾病之一。长期以来,化疗一直是防治TB的主要措施,但因化疗的疗程较长、费用较高、毒副作用较大及不规律用药造成耐药株的产生,导致治愈率降低和复发率升高,迫切需要研究更有效的防治措施。而目前广泛用于预防TB的唯一疫苗—BCG,存在许多缺陷,因此开发安全有效的新疫苗势在必行。Esat-6和Mpt64均为MTB早期培养滤液中的一种分泌蛋白有较强的细胞免疫活性,而在大多数BCG中又缺乏其编码基因,因此二者均是基因疫苗的候选目的基因。目前众多研究者所关注的phoP基因与结核杆菌的毒力有密切的关系的[25,26],其对应结核杆菌基因组的Rv0757[53]且其编码转录因子被命名为phoPR二元系统(TCS)。研究证明,phoPR是结核杆菌中重要的毒力调控基因[31],本研究采用λ-Red同源重组系统成功构建了phoP基因缺陷型重组菌、Esat-6基因重组菌和Mpt64基因重组菌各一株,旨在寻求具有较强免疫原性且较弱毒性的结核杆菌突变株,为开发新型结核疫苗奠定基础。
材料与方法1、菌株、质粒来源。标准菌株H37Rv由本实验室保存。质粒pKD4、pKD46由中国人民解放军军事医学科学院微生物流行病研究所韩延平博士惠赠。2、菌株培养。采用固体改良罗氏培养基(L-J)和液体苏通培养(sauton)相结合的方法培养。3、线性DNA片段的准备。以H37Rv为模板PCR扩增Esat-6和Mpt64,以pKD4为模板PCR扩增启动目的基因表达的上游片段Kan-p和敲除phoP基因的卡那霉素抗性基因片段phoP-K,用融合PCR方法将Kan-p分别与Esat-6和Mpt64连接组成Kan-Esat-6和Kan-Mpt64融合基因片段。4、电转化。设置电转化参数:2.5KV,25uF,200Ω;取对数生长期的H37Rv菌株制感受态细胞,将质粒pKD46导入H37Rv菌体中,筛选阳性菌株26℃培养至对数生长期,加入L-阿拉伯糖至终浓度为10mmol/L诱导48小时,制感受态细胞,将Kan-Esat-6、Kan-Mpt64、phoP-K导入含有pKD46质粒的H37Rv菌体中,Esat-6和Mpt64基因重组菌株在26℃用普通平板筛选,而phoP基因缺陷型重组菌在26℃用含卡那霉素培养基筛选。5、鉴定。取平板上菌落,煮沸法提取DNA,用引物Homo-arm-F和Homo-arm-R扩增目的基因,琼脂糖凝胶电泳鉴定,切胶测序鉴定。
结果齐-尼氏抗酸染色镜检证实用固体改良罗氏培养基(L-J)和液体苏通(sauton)培养基培养的H37Rv菌株生长良好。琼脂糖凝胶电泳证实了PCR扩增出Kan-Esat-6、Kan-Mpt64融合基因和敲除phoP基因的卡那霉素抗性基因片段phoP-K,大小分别为852bp、1248bp、1678bp。取含50ug/ml氨苄青霉素的L-J培养基平板筛选的阳性菌株经PCR鉴定分析,证实质粒pKD46导入成功。将含50ug/ml卡那霉素的L-J培养基平板筛选的阳性菌株和普通平板上筛选的单克隆菌株经PCR鉴定分析,大小风别为1716bp、890bp、1286bp,序列分析与预期结果一致,确证了phoP基因缺陷型重组菌、Esat-6基因重组菌和Mpt64基因重组菌构建成功。
结论通过融合PCR成功扩增出Kan-Esat-6、Kan-Mpt64融合基因。构建了phoP基因缺陷型重组菌、Esat-6基因重组菌和Mpt64基因重组菌各一株。
Background of study and Objectives Tuberculosis(TB) is a Chronic infectious disease by mycobacterium tuberculosis,which were incidenced in all over the body of human and animals organ. In recently years, drug resistant strain of TB appearance,merging AIDS and MTB and flowing of high-risk group make TB epidemic in global. Chemotherapy were main measure which TB was prevented and therapied, but long time of therapy, high cost, more strengthed side effect and appearance of persister lead to lower healing rate and higher recurrence rate. Therefore BCG has a lot of shortage, which was used widely at present. So it was very essential that new vaccine was exploitated.Esat-6 and Mpt64 were a secretion proteinum with more strengthed immunocompetence in culture filter liquor of earlier period. But they were lacked in BCG, so they were purpose gene in gene vaccine.At present, phoP gene was closely attended by plenty of researcher, which was relation with virulence of Mycobacterium tuberculosis[25,26]. It was corresponded with Rv0757 of Mycobacterium tuberculosis genome[53],and encoded transcription factor was named two-component system. The phoPR was Virulence controlling gene of Mycobacterium tuberculosis[31]. In this study, for seeking a MTB mutant strain of strengthed immunogenicity and hypotoxicity. Genes of phoP were comp lately deleted and Esat-6,Mpt64 gene recombination strain were constructed successfully byλ-Red homologous recombination. For exploitating new generation tuberculosis vaccine settle foundation.
Matetials and methods 1、Strain and Plasmid origin.Standard strain H37Rv: conserved by ourselves laboratory. Plasmid pKD4 and pKD46 were honored by Dr. Han Yanping who is from the Chinese People's Liberation Army 309 hospital entire armed forces institute of microorganism epidemic.2、Culture of Bacterial Strains H37Rv were grown L-J medium modified medium and sauton medium.3、reparation of Linearity DNA frag.The Esat-6 and Mpt64 genes were amplified by PCR from MTB H37Rv. The Kan-p was priming gene and the phoP-K was kanamycin resistance gene with replacing phoP. The Kan-p and phoP-K genes were amplified by PCR from pKD4. The Kan-p and Esat-6、Mpt64 genes were connected by overlapping PCR, and the Kan-Esat-6 and Kan-Mpt64 genes were obtain.4、Electrotransformation.Electrotransforma- tion parameter: 2.5KV,25uF,200Ω; When H37Rv strain were cultured exponential phase of growth, freshly prepared competent cell were electroporated in the presence of 1ug of plasmid pKD46. H37Rv with pKD46 of exponential phase of growth was induced 48 hour with 10mmol/L L-arabopyranose, which was prepared freshly prepared competent cell, and was were electroporated in the presence of 500ng of vector DNA. Gene recombination strain of Esat-6 and Mpt64 were screened by common L-J flat in 26℃and shortcoming strain of phoP gene were screened by L-J flat with kanamycin in 26℃.5、Identify.Strain DNA was obtained By boiling method,production of DNA was amplified by Homo-arm-F and Homo-arm-R primer, and was identified by agarose gel electrophoresis and sequencing.
Results To test under microscope result of H37Rv with culturing L-J and sauton medium Ziehl-Neelsen acid-fast stain.Kan-Esat-6,Kan-Mpt64 fusion gene and phoP-K identified by Agarose gel electrophoresis was about 852bp,1248bp,1678bp bp.It was confirmed that the plasmid pKD46 was successfully introduced into H37Rv by analyzing the resistance selected in L-J medium with 50μg/ml Amp by PCRIt was confirmed that the genes of phoP were comp lately deleted and Esat-6,Mpt64 gene recombination strain were successfully constructed by analyzing the resistance selected in L-J medium with 50μg/ml kanamycin and L-J medium by PCR, which was in accordance with the expected result by sequence analyzing.
Conclusions Genes of phoP are complately deleted and Esat-6,Mpt64 gene recombination strain are constructed successfully.
材料与方法1、菌株、质粒来源。标准菌株H37Rv由本实验室保存。质粒pKD4、pKD46由中国人民解放军军事医学科学院微生物流行病研究所韩延平博士惠赠。2、菌株培养。采用固体改良罗氏培养基(L-J)和液体苏通培养(sauton)相结合的方法培养。3、线性DNA片段的准备。以H37Rv为模板PCR扩增Esat-6和Mpt64,以pKD4为模板PCR扩增启动目的基因表达的上游片段Kan-p和敲除phoP基因的卡那霉素抗性基因片段phoP-K,用融合PCR方法将Kan-p分别与Esat-6和Mpt64连接组成Kan-Esat-6和Kan-Mpt64融合基因片段。4、电转化。设置电转化参数:2.5KV,25uF,200Ω;取对数生长期的H37Rv菌株制感受态细胞,将质粒pKD46导入H37Rv菌体中,筛选阳性菌株26℃培养至对数生长期,加入L-阿拉伯糖至终浓度为10mmol/L诱导48小时,制感受态细胞,将Kan-Esat-6、Kan-Mpt64、phoP-K导入含有pKD46质粒的H37Rv菌体中,Esat-6和Mpt64基因重组菌株在26℃用普通平板筛选,而phoP基因缺陷型重组菌在26℃用含卡那霉素培养基筛选。5、鉴定。取平板上菌落,煮沸法提取DNA,用引物Homo-arm-F和Homo-arm-R扩增目的基因,琼脂糖凝胶电泳鉴定,切胶测序鉴定。
结果齐-尼氏抗酸染色镜检证实用固体改良罗氏培养基(L-J)和液体苏通(sauton)培养基培养的H37Rv菌株生长良好。琼脂糖凝胶电泳证实了PCR扩增出Kan-Esat-6、Kan-Mpt64融合基因和敲除phoP基因的卡那霉素抗性基因片段phoP-K,大小分别为852bp、1248bp、1678bp。取含50ug/ml氨苄青霉素的L-J培养基平板筛选的阳性菌株经PCR鉴定分析,证实质粒pKD46导入成功。将含50ug/ml卡那霉素的L-J培养基平板筛选的阳性菌株和普通平板上筛选的单克隆菌株经PCR鉴定分析,大小风别为1716bp、890bp、1286bp,序列分析与预期结果一致,确证了phoP基因缺陷型重组菌、Esat-6基因重组菌和Mpt64基因重组菌构建成功。
结论通过融合PCR成功扩增出Kan-Esat-6、Kan-Mpt64融合基因。构建了phoP基因缺陷型重组菌、Esat-6基因重组菌和Mpt64基因重组菌各一株。
Background of study and Objectives Tuberculosis(TB) is a Chronic infectious disease by mycobacterium tuberculosis,which were incidenced in all over the body of human and animals organ. In recently years, drug resistant strain of TB appearance,merging AIDS and MTB and flowing of high-risk group make TB epidemic in global. Chemotherapy were main measure which TB was prevented and therapied, but long time of therapy, high cost, more strengthed side effect and appearance of persister lead to lower healing rate and higher recurrence rate. Therefore BCG has a lot of shortage, which was used widely at present. So it was very essential that new vaccine was exploitated.Esat-6 and Mpt64 were a secretion proteinum with more strengthed immunocompetence in culture filter liquor of earlier period. But they were lacked in BCG, so they were purpose gene in gene vaccine.At present, phoP gene was closely attended by plenty of researcher, which was relation with virulence of Mycobacterium tuberculosis[25,26]. It was corresponded with Rv0757 of Mycobacterium tuberculosis genome[53],and encoded transcription factor was named two-component system. The phoPR was Virulence controlling gene of Mycobacterium tuberculosis[31]. In this study, for seeking a MTB mutant strain of strengthed immunogenicity and hypotoxicity. Genes of phoP were comp lately deleted and Esat-6,Mpt64 gene recombination strain were constructed successfully byλ-Red homologous recombination. For exploitating new generation tuberculosis vaccine settle foundation.
Matetials and methods 1、Strain and Plasmid origin.Standard strain H37Rv: conserved by ourselves laboratory. Plasmid pKD4 and pKD46 were honored by Dr. Han Yanping who is from the Chinese People's Liberation Army 309 hospital entire armed forces institute of microorganism epidemic.2、Culture of Bacterial Strains H37Rv were grown L-J medium modified medium and sauton medium.3、reparation of Linearity DNA frag.The Esat-6 and Mpt64 genes were amplified by PCR from MTB H37Rv. The Kan-p was priming gene and the phoP-K was kanamycin resistance gene with replacing phoP. The Kan-p and phoP-K genes were amplified by PCR from pKD4. The Kan-p and Esat-6、Mpt64 genes were connected by overlapping PCR, and the Kan-Esat-6 and Kan-Mpt64 genes were obtain.4、Electrotransformation.Electrotransforma- tion parameter: 2.5KV,25uF,200Ω; When H37Rv strain were cultured exponential phase of growth, freshly prepared competent cell were electroporated in the presence of 1ug of plasmid pKD46. H37Rv with pKD46 of exponential phase of growth was induced 48 hour with 10mmol/L L-arabopyranose, which was prepared freshly prepared competent cell, and was were electroporated in the presence of 500ng of vector DNA. Gene recombination strain of Esat-6 and Mpt64 were screened by common L-J flat in 26℃and shortcoming strain of phoP gene were screened by L-J flat with kanamycin in 26℃.5、Identify.Strain DNA was obtained By boiling method,production of DNA was amplified by Homo-arm-F and Homo-arm-R primer, and was identified by agarose gel electrophoresis and sequencing.
Results To test under microscope result of H37Rv with culturing L-J and sauton medium Ziehl-Neelsen acid-fast stain.Kan-Esat-6,Kan-Mpt64 fusion gene and phoP-K identified by Agarose gel electrophoresis was about 852bp,1248bp,1678bp bp.It was confirmed that the plasmid pKD46 was successfully introduced into H37Rv by analyzing the resistance selected in L-J medium with 50μg/ml Amp by PCRIt was confirmed that the genes of phoP were comp lately deleted and Esat-6,Mpt64 gene recombination strain were successfully constructed by analyzing the resistance selected in L-J medium with 50μg/ml kanamycin and L-J medium by PCR, which was in accordance with the expected result by sequence analyzing.
Conclusions Genes of phoP are complately deleted and Esat-6,Mpt64 gene recombination strain are constructed successfully.
引文
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57 Luis Reinaldo Camacho,Danielle Ensergueix, et al.Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature- tagged transposon mutagenesis[J].Molecular Microbiology, 1999,34 (2):257-267.
58秦珑.整体调控子PhoP直接调控鼠疫耶尔森氏菌包内生存能力的研究.博士学位论文,2006年5月.
59茅凌翔,朱超望,许化溪,等.伤寒沙门氏菌phoP基因缺失变异株的制备[J].江苏大学学报(医学版),2007,17(2):145-149.
60 Datsenko KA, WannerBL. One-step inactivation of chromosomal genes in Escherichia coli K212 using PCR p roducts [J]. Proc Natl Acad Sci USA, 2000, 97(12):6640-6645.
61崔文禹,李山虎,姜飞,等.应用Red重组工程技术建立asd基因缺失的大肠杆菌DH10B菌株[J].生物技术通讯,2006,17(4): 493-495.
62 Muyrers JP,Zhang Y,Testa G, et al. Rapid modification of bacterial artificeal chromosomes by ET-recombination [J]. Nucleic Acids Res, 1999, 27(6):1555-1557.
63 Misulovin Z,Yang XW,Yu W,et al. A rapid method for targeted modific- ation and screening of recombinant bacterial artificial chromosome [J]. J Immunol Methods,2001,257 (1-2): 99-105.
64杨建岭,顾淑萍,陈臣,等.用Red /ET重组酶构建基因打靶载体[J].生物工程学报, 2006, 22 (6): 919-924.
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3 Garcia Vescovi E,Soncini FC,Croisman EA.Mg2+ as an extracellular signal:environmental regulation of Salmonella virulence[J]. cell,1996,84(1):165.
4 Ernst RK,Yi EC,Guo L,et al.Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginasa[J]. science, 1999,286(5444): 1561.
5 Vescovi EG,Ayala YM,Di Cera E,et al.Characterization of the hacterial sensor peotein PhoQ.Evidence for distinct hinding sites for Mg2+ and Ca2+[J].J Biol Chem,1997,272(3):1440.
6 Mollie DW,Tammy I,Eduardo G.Transcriptional regulation of the 4-amino-4-deoxy-1-arabinose biosynthetic genes in Yersinia pestis[J]. JBC,2005,280(15):147-65.
7 Miller SI,Mekalanos JJ.Constitutive expression of the phoP regulon attenuates Salmonella virulence and suevival within macro-phages[J].J Bacteriol,1990,172(5):2485.
8 Gunn JS,Hohmann EI,Miller SI.Transcriptional regulation of Samonella virulence:a PhoQ periplasmic donmain mutation results in increased net phosphotransfer to PhoP[J].J Bacteriol,1996,178(21):6369.
9 Guo I,Lim KB,Gunn JS,et al.Regulation of lipid A modification by Salmonella typhimurium virulence genes phoP-phoQ[J]. Science, 1997,276(5310):250.
10 Gunn JS,Lim KB,Krueger J,et al.PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance[J].Mol Microbiol,1998,27(6):1171.
11 Bishop RE,Gibbons HS,Guina T,et al.Transfer of palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria[J].Embo J,2000,19(19):250.
12 Gunn JS,Ryan SS,van Velkinburgh JC,et al.Genetic and functional analysis of a PmrA-PmrB-regulated locus necessary for lipopolysaccharide modification,antimicrobial peptide resistance,and oral virulence of Salmonella enterica serovar typhimurium[J].Infect Immun,2000,68(11):6139.
13 Matceau M,Sebbane F,Ewann F,et al.The pmrF polymyxin- resistance operon of Yesinia pseudontuberculosis is upregulated by the PhoP-PhoQ two-component system but not by PmrA-PmrB,and is not required for virulence[J].Microbiology, 2004,150(Pt12):3947.
14 Kato A,Latifi T,Groisman EA.Closing the PmrA/PmrB two-component system negatively controls expression of its posttranscriptional actinvator PmrD[J].Proc Natl Acad Sci USA,2003,103(1):113.
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55 Carlos Martin, Ann Williams, Rogelio Hernandez-Pando et al. The live Mycobacterium tuberculosis phoP mutant strain is more attenuated than BCG and confers protective immunity against tuberculosis in mice and guinea pigs[J]. Vaccine 24 (2006) 3408-3419.
56 Esther Perez,Sofia Samper, et al.An essential role for phoP in Mycobacterium tuberculosis virulence[J]. Molecular Microbiology, 2001,41(1):179-187.
57 Luis Reinaldo Camacho,Danielle Ensergueix, et al.Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature- tagged transposon mutagenesis[J].Molecular Microbiology, 1999,34 (2):257-267.
58秦珑.整体调控子PhoP直接调控鼠疫耶尔森氏菌包内生存能力的研究.博士学位论文,2006年5月.
59茅凌翔,朱超望,许化溪,等.伤寒沙门氏菌phoP基因缺失变异株的制备[J].江苏大学学报(医学版),2007,17(2):145-149.
60 Datsenko KA, WannerBL. One-step inactivation of chromosomal genes in Escherichia coli K212 using PCR p roducts [J]. Proc Natl Acad Sci USA, 2000, 97(12):6640-6645.
61崔文禹,李山虎,姜飞,等.应用Red重组工程技术建立asd基因缺失的大肠杆菌DH10B菌株[J].生物技术通讯,2006,17(4): 493-495.
62 Muyrers JP,Zhang Y,Testa G, et al. Rapid modification of bacterial artificeal chromosomes by ET-recombination [J]. Nucleic Acids Res, 1999, 27(6):1555-1557.
63 Misulovin Z,Yang XW,Yu W,et al. A rapid method for targeted modific- ation and screening of recombinant bacterial artificial chromosome [J]. J Immunol Methods,2001,257 (1-2): 99-105.
64杨建岭,顾淑萍,陈臣,等.用Red /ET重组酶构建基因打靶载体[J].生物工程学报, 2006, 22 (6): 919-924.
1 Kier LD,Weppelman RM,Ames BN. Regulation of nonspecific acid phosohatase in Salmonella:phoN and phoP gene[J].J Bactetiol, 1979,138(1):155.
2 Soncini FC,Garcia Vescovi E,Solomon F,et al.Molecular hasis of the magnesium deprivation response in Salmonella typhimurium: identification of PhoP-regulated gene[J] J Bacteriol,1996,178(17),5092.
3 Garcia Vescovi E,Soncini FC,Croisman EA.Mg2+ as an extracellular signal:environmental regulation of Salmonella virulence[J]. cell,1996,84(1):165.
4 Ernst RK,Yi EC,Guo L,et al.Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginasa[J]. science, 1999,286(5444): 1561.
5 Vescovi EG,Ayala YM,Di Cera E,et al.Characterization of the hacterial sensor peotein PhoQ.Evidence for distinct hinding sites for Mg2+ and Ca2+[J].J Biol Chem,1997,272(3):1440.
6 Mollie DW,Tammy I,Eduardo G.Transcriptional regulation of the 4-amino-4-deoxy-1-arabinose biosynthetic genes in Yersinia pestis[J]. JBC,2005,280(15):147-65.
7 Miller SI,Mekalanos JJ.Constitutive expression of the phoP regulon attenuates Salmonella virulence and suevival within macro-phages[J].J Bacteriol,1990,172(5):2485.
8 Gunn JS,Hohmann EI,Miller SI.Transcriptional regulation of Samonella virulence:a PhoQ periplasmic donmain mutation results in increased net phosphotransfer to PhoP[J].J Bacteriol,1996,178(21):6369.
9 Guo I,Lim KB,Gunn JS,et al.Regulation of lipid A modification by Salmonella typhimurium virulence genes phoP-phoQ[J]. Science, 1997,276(5310):250.
10 Gunn JS,Lim KB,Krueger J,et al.PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance[J].Mol Microbiol,1998,27(6):1171.
11 Bishop RE,Gibbons HS,Guina T,et al.Transfer of palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria[J].Embo J,2000,19(19):250.
12 Gunn JS,Ryan SS,van Velkinburgh JC,et al.Genetic and functional analysis of a PmrA-PmrB-regulated locus necessary for lipopolysaccharide modification,antimicrobial peptide resistance,and oral virulence of Salmonella enterica serovar typhimurium[J].Infect Immun,2000,68(11):6139.
13 Matceau M,Sebbane F,Ewann F,et al.The pmrF polymyxin- resistance operon of Yesinia pseudontuberculosis is upregulated by the PhoP-PhoQ two-component system but not by PmrA-PmrB,and is not required for virulence[J].Microbiology, 2004,150(Pt12):3947.
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15 Lesley JA,Waldburger CD.Comparison of the Pseudonmonas aeruginosa and Escherichia coli PhoQ sensor domains:evidence for distinct mchanisms of sigal detection[J].J Biol Chem,2001,276(33):308-27.
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