耐甲氧西林金黄色葡萄球菌PBP2a相互作用蛋白的筛选与鉴定
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摘要
随着抗生素在临床上长期、广泛的使用,细菌耐药性的出现及日益严重化给临床抗感染治疗带来了严峻挑战。金黄色葡萄球菌是一种重要的条件致病菌,特别是耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA )已成为全球院内感染的首要病原菌,其多重耐药菌株不断涌现,使得临床治疗变得更加困难。万古霉素曾是治疗MRSA感染的理想药物,但随着万古霉素的广泛应用,已出现一些耐万古霉素金黄色葡萄球菌的报道,因而寻找新的治疗方法十分必要。
研究表明,MRSA耐药机制包括靶位改变、钝化酶产生、主动外排等。其中,抗生素作用靶位的改变是最主要的耐药机制。通常,金葡菌细胞壁的肽聚糖合成是由一组称为青霉素结合蛋白(Penicillin-binding proteins, PBPs)的蛋白酶系完成的,PBPs有转糖基酶(glycosyltransferase,TGase)和转肽酶(transpeptidase,TPase) 2种酶学活性,β-内酰胺类抗生素能与PBPs的转肽酶功能域(TPase domain)选择性结合,使其乙酰化而灭活TPase活性,阻断细菌肽聚糖合成,进而杀灭敏感菌。耐药MRSA菌则从一种未知宿主中获得了一mecA基因,编码产生一种新的PBP,称为PBP2a,其TPase domain与β-内酰胺类抗生素的亲和力极低,当正常PBPs被β-内酰胺类药物结合而失去活性时,PBP2a能代替其功能,继续肽聚糖合成,维持金葡菌的生长和成活。也就是说,MRSA通过表达PBP2a,改变抗生素的作用靶点,参与MRSA的耐药性表达。
许多研究表明,在MRSA中,PBP2a是其高水平β-内酰胺类抗生素耐药性所必须的。然而,PBP2a如何参与MRSA的耐药性表达?有否相应的共激活或抑制分子等调节因子存在?目前都不清楚。本研究中,我们将MRSA菌PBP2a TPase domain克隆到pRBR载体上,构建成诱饵质粒,利用细菌双杂交系统(Bacterial two-hybrid system,B2H),从MRSA基因组表达文库中筛选到了九个可能与PBP2a有相互作用的猎物多肽。随后,利用体外Pull-down实验验证了其中一个猎物多肽P2与PBP2a的相互作用。
本研究主要内容和结果包括以下两个部分:
1.利用细菌双杂交系统筛选PBP2a相互作用的蛋白:①以质粒pMD-PBP2a(含有MRSA的PBP2a TPase domain编码序列)为模板,利用PCR扩增MRSA PBP2a蛋白的TPase编码基因,经BamHI/XhoI双酶切,克隆入pRBR质粒载体中,转化入大肠杆菌DH5α,构建了诱饵质粒pBR-PBP2a,并进行PCR、限制性酶切和测序鉴定。②提取了MRSA基因组DNA,Sau3AI部分酶切后克隆入pRAC质粒载体中,成功构建了基因组文库。随机选取50个克隆,用位于pRAC质粒多克隆位点两侧的测序引物进行菌落PCR扩增,检测文库插入片段的分布情况。所构建的MRSA N315株基因组文库覆盖率达9倍,满足后续文库筛选的需要。③将构建正确的诱饵质粒pBR-PBP2a与基因组文库共转化KS1细胞(其为染色体上整合有由λ操纵子控制的报告基因lacZ的报告菌株),接种于含50mg/ml X-gal,100μmol/L IPTG,100μg/ml AMP,50μg/ml Kan,34μg/ml Cl的琼脂平板培养后,挑取深蓝色菌落进行LacZ活性检测。同时以空质粒pRAC转化pBR-PBP2a /KS1细胞为阴性对照,以文献报道的pACλCI-βflap(克隆了沙眼衣原体RNA聚合酶β亚单位的β-flap domain)转化含pBRL28(克隆了沙眼衣原体CT663编码基因)的KS1菌为阳性对照。④通过对200个克隆的LacZ活性检测,将筛选到β-半乳糖苷酶活性明显升高的克隆用B2H进行第二次双杂交验证,LacZ确实升高2倍以上者为猎物质粒。一共获得9个猎物克隆,分别将含有插入序列的pRAC质粒分离并测序,并将9个克隆质粒命名为pAC- p1~ pAC- p9。⑤将9个插入序列用BLAST工具进行序列比对,信息学分析表明它们均来自于MRSA N315基因组,插入片段最长者649 bp,最短者335bp, 9个插入片段中含14个编码基因,其中10个功能未知,1个编码二氢吡啶二羧酸合酶、1个编码二氢吡啶二羧酸还原酶,2个编码染色体解离SMC蛋白。9个克隆中介导与PBP2a相互作用的多肽由14-46氨基酸组成。
2. PBP2a与猎物多肽相互作用的再验证:为证实B2H筛选到的猎物质粒所编码的蛋白与PBP2a TPase domain确实存在相互作用,我们进行了猎物融合蛋白的表达、纯化以及体外Pull-down PBP2a的实验,具体包括①根据B2H检测中β-半乳糖苷酶活性升高程度以及测序结果分析,选择了pAC-p2质粒中的插入片段进行深入探讨。设计并合成一对引物,以质粒pAC-p2为模板,PCR扩增了插入的p2序列,克隆入原核表达载体pGEX-6p-1中,构建了pGEX-p2重组质粒,转化大肠杆菌BL21感受态。②对pGEX-p2/BL21重组菌进行诱导,表达目标融合蛋白,并对表达条件进行摸索,得到最适表达条件。③大量表达融合蛋白,利用GST-Sepharose4B免疫亲和纯化GST-多肽融合蛋白,得到可溶的纯度较高的GST-多肽融合蛋白。同时对含GST基因的pGEX-6p-1空载体进行诱导表达,纯化获得GST蛋白,作为对照。④将GST融合蛋白固化在GST-Sepharose4B树脂上,充当诱饵蛋白,通过蛋白间相互作用,捕获了PBP2-2a蛋白(含有PBP2分子绞链区N-端与PBP2a分子TPase domain的嵌合蛋白),用Western blot鉴定了该多肽与PBP2a蛋白质间存在相互作用。
综上所述,本研究设计并利用细菌双杂交技术从MRSA基因组文库中成功筛选到与PBP2a相互作用的多肽,并成功克隆表达纯化该多肽的融合蛋白,利用体外Pull-down实验验证了两者的相互作用,为下一步研究该多肽对PBP2a活性的影响,深入认识PBP2a介导MRSA耐药性发生机理奠定了坚实的基础。
With the widespread use of the conventional antibiotics in clinic, bacterial resistance to antibiotics has become a challenge to the anti-infection treatments for clinical infectious disease. Staphylococcus aureus is one of the important opportunistic pathogens. With the development of drug-resistance, the methicillin-resistant Staphylococcus aureus (MRSA) has become the most important pathogens of nosocomial infection. Because the multi-resistant strains of MRSA are emerging, treatments have become more difficult. With the extensive use of vancomycin,the vancomycin-resistant Staphylococcus aureus (VRSA) infection have been reported.Thus, finding a new reagent to MRSA infection is necessary. The resistance mechanism of MRSA is complex, including drug-target changes, producing passivation enzymes as well as active effluxing,and so on. Among them, the change of target sites for antibiotics is a major mechanism. Typically, the bacterial cell wall is built by Penicillin-binding proteins (PBPs), a family of enzymes which provide transglycosylase (TGase) and transpeptidase (TPase).Theβ-lactam antibiotics can selectively bind to TPase domain of PBPs,make it acetylated and inactivated, block the synthesis of bacterial peptidoglycan, and then kill the sensitive bacteria. MRSA obtains a mecA gene from an unknown host, which encodes a new PBP,called PBP2a,whose TPase domain has low affinity with theβ-lactam antibiotics.When the normal PBPs is combined with theβ-lactam antibiotics and losses its TPase activity, PBP2a can provide a TPase function for the synthesis of cell peptidoglycan and maintain the growth and survival of Staphylococcus aureus in the existance ofβ-lactam antibiotics.That is, the expression of PBP2a in MRSA is involved in the drug resistance. Many studies have showed that, in MRSA, PBP2a is required toβ-lactam resistance. However, how PBP2a participates in the drug resistance of MRSA? Are there some activation or inhibition regulatory factors? In this study,we cloned the TPase domain of MRSA PBP2a into pRBR vector to construct a bait plasmid to screen a MRSA genomic library constructed in pRAC vector using bacterial two-hybrid system (B2H), and nine candidates were screened out that might interact with PBP2a. One of the candidates (P2) was confirmed to have the interaction with PBP2a by the pull-down assay.The main contents and results are as followings:
1. Screen the proteins that interacts with PBP2a using a bacterial two-hybrid system:①Using the pMD-PBP2a plasmid (carrying the coding sequence of TPase domain of MRSA PBP2a) as template, the fragment encoded PBP2a TPase domain of MRSA strain N315 was amplified by PCR,digested with BamHI / XhoI and cloned into pRBR plasmid vector to obtain a bait plasmid pBR-PBP2a. The bait plasmid was identified by PCR, restriction enzyme digestion and sequencing.②The genomic DNA of MRSA strain N315 was extracted and partially digested with Sau3A I,then the fragments between 500~1500bp were recovered, and ligated into pRAC vector to get a genomic DNA library. 50 clones were selected randomly and the colony PCR amplification was used to detect the distribution of library inserts with pRAC sequencing primers. The constructed MRSA N315 strains genomic library constructed were 9 times of coverage,which meeted the needs for subsequent library screening.③The bait plasmid pBR-PBP2a and genomic library co- transformed KS1 cells (a reporter strain with a genomic integrated lacZ gene under the control ofλoperator) were cultured in agar plates with 50mg/ml X-gal,100μmol/L IPTG,100μg/ml AMP,50μg/ml Kan,34μg/ml Cl and the blue colonies were picked up for LacZ assay. The empty vector pRAC transformed into pBR-PBP2a /KS1 served as a negative control and the KS1 carrying pACλCI-βflap and pBRL28 (with the confirmed interaction between chlamydialβflap and CT663 expressed by pACλCI-βflap and pBRL28 respectively) used as positive control.④The colony with LacZ activities significantly increased after induction of IPTG was selected as the a prey. Then the pRAC plasmid with a candidate insertion was isolated and sequenced. After performing LacZ assay upon 200 colonies, 9 prey plasmids were obtained, named as pAC-p1 ~ pAC-p9.⑤The 9 inserted sequences were subjected to BLAST analysis and we found that all of them came from the MRSA N315 genome.The longest insert was 649bp and the shortest one was 335bp. There were 14 genes related with the 9 inserts and 10 of them encoded proteins with unknow functions. Among the 4 familiar genes, one encoded dihydrodipicolinate synthase,one encoded dihydrodipicolinate reductase and the others encoded chromosome segregation SMC proteins. The peptides that might interact with PBP2a encoded by the 9 prey plasmids were composed of 14~46 amino acids.
2. Identifying the interaction between PBP2a and prey peptide:①According to evaluation ofβ-galactosidase activity and sequence analysis, the peptide encoded by p2 was selected for further in vitro Pull-down experiments.The insert of p2 was amplificated by PCR and cloned into pGEX-6p-1 vector to construct the pGEX-p2 plasmid,then pGEX-p2 was transformed into E. coli BL21 competent cells to screen the recombinants.②The pGEX-p2/BL21 recombinant bacteria was induced for the expression of GST-P2 fusion proteins, and the optimal expression condition was explored. We find that the optimal expression condition of pGEX-p2/BL21 were 6 hours post induction by 500μmol/L IPTG at 28℃.③The recombinant GST-P2 fusion protein was expressed using pGEX-p2/BL21 engineered bacteria, purified by GST-Sepharose4B immunoaffinity chromatography and characterized by Western blotting with the polyclone sera against GST tag. As for negative control, the GST protein was expressed by pGEX-6p-1 and purified with GST-Sepharose4B resin.④The purified GST or GST-P2 proteins were imobilized by GST-Sepharose4B resin and subjected to capture PBP2-2a fusion proteins in a pull down assay. The results showed that the GST-P2 could pull down PBP2-2a determined by Western blot using the polyclone mouse sera against PBP2-2a.In the same condition, the purified GST protein could not pull down PBP2-2a.
In summary, we successfully screened out the proteins that interact with PBP2a from MRSA genomic library using a bacterial two-hybrid system and the interaction between the two proteins was verified by in vitro Pull-down experiments. Our results facilitated further understanding the mechanism of PBP2a mediated the drug-resistance in MRSA.
研究表明,MRSA耐药机制包括靶位改变、钝化酶产生、主动外排等。其中,抗生素作用靶位的改变是最主要的耐药机制。通常,金葡菌细胞壁的肽聚糖合成是由一组称为青霉素结合蛋白(Penicillin-binding proteins, PBPs)的蛋白酶系完成的,PBPs有转糖基酶(glycosyltransferase,TGase)和转肽酶(transpeptidase,TPase) 2种酶学活性,β-内酰胺类抗生素能与PBPs的转肽酶功能域(TPase domain)选择性结合,使其乙酰化而灭活TPase活性,阻断细菌肽聚糖合成,进而杀灭敏感菌。耐药MRSA菌则从一种未知宿主中获得了一mecA基因,编码产生一种新的PBP,称为PBP2a,其TPase domain与β-内酰胺类抗生素的亲和力极低,当正常PBPs被β-内酰胺类药物结合而失去活性时,PBP2a能代替其功能,继续肽聚糖合成,维持金葡菌的生长和成活。也就是说,MRSA通过表达PBP2a,改变抗生素的作用靶点,参与MRSA的耐药性表达。
许多研究表明,在MRSA中,PBP2a是其高水平β-内酰胺类抗生素耐药性所必须的。然而,PBP2a如何参与MRSA的耐药性表达?有否相应的共激活或抑制分子等调节因子存在?目前都不清楚。本研究中,我们将MRSA菌PBP2a TPase domain克隆到pRBR载体上,构建成诱饵质粒,利用细菌双杂交系统(Bacterial two-hybrid system,B2H),从MRSA基因组表达文库中筛选到了九个可能与PBP2a有相互作用的猎物多肽。随后,利用体外Pull-down实验验证了其中一个猎物多肽P2与PBP2a的相互作用。
本研究主要内容和结果包括以下两个部分:
1.利用细菌双杂交系统筛选PBP2a相互作用的蛋白:①以质粒pMD-PBP2a(含有MRSA的PBP2a TPase domain编码序列)为模板,利用PCR扩增MRSA PBP2a蛋白的TPase编码基因,经BamHI/XhoI双酶切,克隆入pRBR质粒载体中,转化入大肠杆菌DH5α,构建了诱饵质粒pBR-PBP2a,并进行PCR、限制性酶切和测序鉴定。②提取了MRSA基因组DNA,Sau3AI部分酶切后克隆入pRAC质粒载体中,成功构建了基因组文库。随机选取50个克隆,用位于pRAC质粒多克隆位点两侧的测序引物进行菌落PCR扩增,检测文库插入片段的分布情况。所构建的MRSA N315株基因组文库覆盖率达9倍,满足后续文库筛选的需要。③将构建正确的诱饵质粒pBR-PBP2a与基因组文库共转化KS1细胞(其为染色体上整合有由λ操纵子控制的报告基因lacZ的报告菌株),接种于含50mg/ml X-gal,100μmol/L IPTG,100μg/ml AMP,50μg/ml Kan,34μg/ml Cl的琼脂平板培养后,挑取深蓝色菌落进行LacZ活性检测。同时以空质粒pRAC转化pBR-PBP2a /KS1细胞为阴性对照,以文献报道的pACλCI-βflap(克隆了沙眼衣原体RNA聚合酶β亚单位的β-flap domain)转化含pBRL28(克隆了沙眼衣原体CT663编码基因)的KS1菌为阳性对照。④通过对200个克隆的LacZ活性检测,将筛选到β-半乳糖苷酶活性明显升高的克隆用B2H进行第二次双杂交验证,LacZ确实升高2倍以上者为猎物质粒。一共获得9个猎物克隆,分别将含有插入序列的pRAC质粒分离并测序,并将9个克隆质粒命名为pAC- p1~ pAC- p9。⑤将9个插入序列用BLAST工具进行序列比对,信息学分析表明它们均来自于MRSA N315基因组,插入片段最长者649 bp,最短者335bp, 9个插入片段中含14个编码基因,其中10个功能未知,1个编码二氢吡啶二羧酸合酶、1个编码二氢吡啶二羧酸还原酶,2个编码染色体解离SMC蛋白。9个克隆中介导与PBP2a相互作用的多肽由14-46氨基酸组成。
2. PBP2a与猎物多肽相互作用的再验证:为证实B2H筛选到的猎物质粒所编码的蛋白与PBP2a TPase domain确实存在相互作用,我们进行了猎物融合蛋白的表达、纯化以及体外Pull-down PBP2a的实验,具体包括①根据B2H检测中β-半乳糖苷酶活性升高程度以及测序结果分析,选择了pAC-p2质粒中的插入片段进行深入探讨。设计并合成一对引物,以质粒pAC-p2为模板,PCR扩增了插入的p2序列,克隆入原核表达载体pGEX-6p-1中,构建了pGEX-p2重组质粒,转化大肠杆菌BL21感受态。②对pGEX-p2/BL21重组菌进行诱导,表达目标融合蛋白,并对表达条件进行摸索,得到最适表达条件。③大量表达融合蛋白,利用GST-Sepharose4B免疫亲和纯化GST-多肽融合蛋白,得到可溶的纯度较高的GST-多肽融合蛋白。同时对含GST基因的pGEX-6p-1空载体进行诱导表达,纯化获得GST蛋白,作为对照。④将GST融合蛋白固化在GST-Sepharose4B树脂上,充当诱饵蛋白,通过蛋白间相互作用,捕获了PBP2-2a蛋白(含有PBP2分子绞链区N-端与PBP2a分子TPase domain的嵌合蛋白),用Western blot鉴定了该多肽与PBP2a蛋白质间存在相互作用。
综上所述,本研究设计并利用细菌双杂交技术从MRSA基因组文库中成功筛选到与PBP2a相互作用的多肽,并成功克隆表达纯化该多肽的融合蛋白,利用体外Pull-down实验验证了两者的相互作用,为下一步研究该多肽对PBP2a活性的影响,深入认识PBP2a介导MRSA耐药性发生机理奠定了坚实的基础。
With the widespread use of the conventional antibiotics in clinic, bacterial resistance to antibiotics has become a challenge to the anti-infection treatments for clinical infectious disease. Staphylococcus aureus is one of the important opportunistic pathogens. With the development of drug-resistance, the methicillin-resistant Staphylococcus aureus (MRSA) has become the most important pathogens of nosocomial infection. Because the multi-resistant strains of MRSA are emerging, treatments have become more difficult. With the extensive use of vancomycin,the vancomycin-resistant Staphylococcus aureus (VRSA) infection have been reported.Thus, finding a new reagent to MRSA infection is necessary. The resistance mechanism of MRSA is complex, including drug-target changes, producing passivation enzymes as well as active effluxing,and so on. Among them, the change of target sites for antibiotics is a major mechanism. Typically, the bacterial cell wall is built by Penicillin-binding proteins (PBPs), a family of enzymes which provide transglycosylase (TGase) and transpeptidase (TPase).Theβ-lactam antibiotics can selectively bind to TPase domain of PBPs,make it acetylated and inactivated, block the synthesis of bacterial peptidoglycan, and then kill the sensitive bacteria. MRSA obtains a mecA gene from an unknown host, which encodes a new PBP,called PBP2a,whose TPase domain has low affinity with theβ-lactam antibiotics.When the normal PBPs is combined with theβ-lactam antibiotics and losses its TPase activity, PBP2a can provide a TPase function for the synthesis of cell peptidoglycan and maintain the growth and survival of Staphylococcus aureus in the existance ofβ-lactam antibiotics.That is, the expression of PBP2a in MRSA is involved in the drug resistance. Many studies have showed that, in MRSA, PBP2a is required toβ-lactam resistance. However, how PBP2a participates in the drug resistance of MRSA? Are there some activation or inhibition regulatory factors? In this study,we cloned the TPase domain of MRSA PBP2a into pRBR vector to construct a bait plasmid to screen a MRSA genomic library constructed in pRAC vector using bacterial two-hybrid system (B2H), and nine candidates were screened out that might interact with PBP2a. One of the candidates (P2) was confirmed to have the interaction with PBP2a by the pull-down assay.The main contents and results are as followings:
1. Screen the proteins that interacts with PBP2a using a bacterial two-hybrid system:①Using the pMD-PBP2a plasmid (carrying the coding sequence of TPase domain of MRSA PBP2a) as template, the fragment encoded PBP2a TPase domain of MRSA strain N315 was amplified by PCR,digested with BamHI / XhoI and cloned into pRBR plasmid vector to obtain a bait plasmid pBR-PBP2a. The bait plasmid was identified by PCR, restriction enzyme digestion and sequencing.②The genomic DNA of MRSA strain N315 was extracted and partially digested with Sau3A I,then the fragments between 500~1500bp were recovered, and ligated into pRAC vector to get a genomic DNA library. 50 clones were selected randomly and the colony PCR amplification was used to detect the distribution of library inserts with pRAC sequencing primers. The constructed MRSA N315 strains genomic library constructed were 9 times of coverage,which meeted the needs for subsequent library screening.③The bait plasmid pBR-PBP2a and genomic library co- transformed KS1 cells (a reporter strain with a genomic integrated lacZ gene under the control ofλoperator) were cultured in agar plates with 50mg/ml X-gal,100μmol/L IPTG,100μg/ml AMP,50μg/ml Kan,34μg/ml Cl and the blue colonies were picked up for LacZ assay. The empty vector pRAC transformed into pBR-PBP2a /KS1 served as a negative control and the KS1 carrying pACλCI-βflap and pBRL28 (with the confirmed interaction between chlamydialβflap and CT663 expressed by pACλCI-βflap and pBRL28 respectively) used as positive control.④The colony with LacZ activities significantly increased after induction of IPTG was selected as the a prey. Then the pRAC plasmid with a candidate insertion was isolated and sequenced. After performing LacZ assay upon 200 colonies, 9 prey plasmids were obtained, named as pAC-p1 ~ pAC-p9.⑤The 9 inserted sequences were subjected to BLAST analysis and we found that all of them came from the MRSA N315 genome.The longest insert was 649bp and the shortest one was 335bp. There were 14 genes related with the 9 inserts and 10 of them encoded proteins with unknow functions. Among the 4 familiar genes, one encoded dihydrodipicolinate synthase,one encoded dihydrodipicolinate reductase and the others encoded chromosome segregation SMC proteins. The peptides that might interact with PBP2a encoded by the 9 prey plasmids were composed of 14~46 amino acids.
2. Identifying the interaction between PBP2a and prey peptide:①According to evaluation ofβ-galactosidase activity and sequence analysis, the peptide encoded by p2 was selected for further in vitro Pull-down experiments.The insert of p2 was amplificated by PCR and cloned into pGEX-6p-1 vector to construct the pGEX-p2 plasmid,then pGEX-p2 was transformed into E. coli BL21 competent cells to screen the recombinants.②The pGEX-p2/BL21 recombinant bacteria was induced for the expression of GST-P2 fusion proteins, and the optimal expression condition was explored. We find that the optimal expression condition of pGEX-p2/BL21 were 6 hours post induction by 500μmol/L IPTG at 28℃.③The recombinant GST-P2 fusion protein was expressed using pGEX-p2/BL21 engineered bacteria, purified by GST-Sepharose4B immunoaffinity chromatography and characterized by Western blotting with the polyclone sera against GST tag. As for negative control, the GST protein was expressed by pGEX-6p-1 and purified with GST-Sepharose4B resin.④The purified GST or GST-P2 proteins were imobilized by GST-Sepharose4B resin and subjected to capture PBP2-2a fusion proteins in a pull down assay. The results showed that the GST-P2 could pull down PBP2-2a determined by Western blot using the polyclone mouse sera against PBP2-2a.In the same condition, the purified GST protein could not pull down PBP2-2a.
In summary, we successfully screened out the proteins that interact with PBP2a from MRSA genomic library using a bacterial two-hybrid system and the interaction between the two proteins was verified by in vitro Pull-down experiments. Our results facilitated further understanding the mechanism of PBP2a mediated the drug-resistance in MRSA.
引文
1. Pantosti A,Venditti M. What is MRSA[J].Eur Respir J, 2009,34(5):1190-1196.
2. Nakou A,Woodhead M,Torres A.MRSA as a cause of community-acquired pneumonia [J]. Eur Respir, 2009,34(5):1013-1014.
3. Frazee B,Lynn J, Charlebois E, et al. High prevalence of methicillin-resistant Staphylococcus aureus in emergency department skin and soft tissue infections [J]. Ann Emerg Med, 2005, 45(3):311-320.
4. M. Willey,T. Mazzμlli, A. McGeer, et al. Evaluation of screening media for detection of methicillin-resistant Staphylococcus aureus (MRSA) from surveillance swabs [J]. American Journal of Infection Control, 2005, 5(33):181.
5. Coburn PS, Pillar CM, Jett BD, et al. Enterococcus faecalis senses target cells and in response expresses cytolysin [J]. Science, 2004, 306(5705):2270-2272.
6. Lim D. and Natalie Strynadk CJ. Structural Basis for theβ-lactam Resistance of PBP2a from Methicillin-resistant Staphylococcus aureus[J]. Nature Structural Biology, 2002,9(5), 870-8.
7. Georgopapadakou NH, Smith SA, Bonner DP. Penicillin-binding proteins in a Staphylococcus aureus strain resistant to specific beta-lactam antibiotics[J]. Antimicrob Agents Chemother. 1982;22(1):172-5.
8. Pinho MG, de Lencastre H, Tomasz A. An acquired and a native penicillin-binding protein cooperate in building the cell wall of drug-resistant staphylococci[J]. Proc Natl Acad Sci USA. 2001;98(19):10886-91.
9. Katayama Y, Zhang HZ, Chambers HF. Effect of disruption of Staphylococcus aureus PBP4 gene on resistance to beta-lactam antibiotics[J]. Microb Drug Resist. 2003;9 (4):329-36.
10. Finan JE, Archer GL, Pucci MJ, Climo MW. Role of penicillin-binding protein 4 in expression of vancomycin resistance among clinical isolates of oxacillin-resistant Staphylococcus aureus[J]. Antimicrob Agents Chemother. 2001;45(11):3070-5.
11. Park Y, Oh EJ, Lee SO, Kim BK. Absence of mutation in the region (nt. 710-1010) ofpbp4 gene in clinical isolates of Staphylococcus aureus with low-level teicoplanin resistance[J]. Diagn Microbiol Infect Dis. 2001;39(4):271-3.
12. Higashi Y, Wakabayashi A, Matsumoto Y, Ohno A. Role of inhibition of penicillin binding proteins and cell wall cross-linking by beta-lactam antibiotics in low- and high-level methicillin resistance of Staphylococcus aureus[J]. Chemotherapy. 1999;45 (1):37-47.
13. Ishikawa T , Mat sunaga N , Tawada H , et al. TA K-599 , a novel N-phosphono type prodrug of anti-MRSA cephalo-sporin T-91825 : synthesis , physicochemical and pharmaco-logical properties[J]. Bioorg Med Chem , 2003 , 11 ( 11 ) :2427-2437.
14. Treitman AN, Yarnold PR, Warren J, et al. Emerging incidence of Enterococcus faecium among hospital isolates [J]. J Clin Microbiol. 2005, 43(1):462-463.
15. Appelbaum P C.The emergence of vancomycin-intermediate and vancomycin-resistant Staphylococcus aureus [J].Clin Microbiol Infect,2008,12(1):16.
16. Rao X, Padraig D, Hua Z, et al. A regμlator from Chlamydia trachomatis modμlates the activity of RNA polymerase through direct interaction with theβsubunit and the primaryσsubunit[J]. Genes Dev, 2009, 23: 1818-1829
17.马学军,舒跃龙.分子克隆实验指南[M].第4版.上海:科学出版社,2006:44-92.
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33. Hua Z, Rao X, Feng X, et al. Mutagenesis of Region 4 of Sigma 28 from Chlamydia trachomatis Defines Determinants for Protein-Protein and Protein-DNA Interactions[J]. J Bacteriol,2009,1: 651-660
34. Surobhi L, Lakshmi P, Nara G. Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein[J]. Biochemical and Biophysical Research Communications,2005,337:677-684
35. Lim D,Strynadka N.Structural basis for the beta lactam resistance of PBP2a from methicillin-resistant Staphylococcus[J]. Nat Struct Biol,2002,9(11):870-876.
36. Rohrer S, Berger B.Fem A. A Link between branched-chain cell wall peptide formation andβ-lactam resistance in gram-positive cocci[J]. Antimicrob Agents Chemother,2005, 47:837-846.
37.杨杰,饶贤才,胡福泉,等. MRSA耐药相关TG-TPase嵌合基因的原核表达与纯化[J].免疫学杂志, 2008, 23:289-294.
38.汪家政,范明主编.蛋白质技术手册[M],北京:科学出版社,2000:29-33.
39.卢圣栋主编,现代分子生物学技术[M],第二版.北京:中国协和医科大学出版社, 1999 :417-429.
40. Phizicky EM,Fields S.Protein-protein interactions:methods fordetection and analysis[J].Microbiol Rev,1995,59:94-123.
41. Frangioni JV,Neel BG..Solubilization and purification of enzymatically active glutathione S-transferase(pGEX)fusion proteins[J].Anal Biochem.1993,Apr,210(1): 179-187.
42. Gribskov M,Burgess RR.Overexpression and purification of the sigma subunit of E.coil RNA polymerase[J].Gene,1983,26:109.
43. Lilie H,Schwarz E,Rudolph R.Advances in refolding of proteins produced in E.coli[J].Curr Opin Biot,1998,9:442.
44. Belin P and.Boquet. P.L Green fluorescent protein as a marker for gene expession [J]. Microbiol, 1994, 140:3337-3348
45. DR马歇尔等著,朱厚础等译.蛋白质纯化与鉴定实验指南[M],北京:科学出版社, 1999 :144-149.
46. Ren L,Chang E,Makky K,et al.Glutathione S-transferase pull-down assays using dehydrated immobilized glutathione resin[J].Anal Biochem,2008, Nov 15,322(2): 164-169.
47. Maggi S, Massidda O, Luzi G. Division protein interaction web: identification of a phylogenetically conserved common interactome between Streptococcus pneumoniae and Escherichia coli[J]. Microbiology-SGM , 2008,154:3042-3052.
48. Baldiek,C.J.,Jr.,andT.Shenk.Protein sassoeiated with Purified human eytomegalovims Partieles[J],Virol,1996,70: 97-105.
49. Fengliang Jin, Xiaoxia Xu, Liexi Wang, et al. Expression of recombinant hybrid peptide cecropinA-magainin2 in Pichia pastoris: Purification and characterization [J]. Protein Expression and Purification, 2006, 50:147-156.
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5. Coburn PS, Pillar CM, Jett BD, et al. Enterococcus faecalis senses target cells and in response expresses cytolysin [J]. Science, 2004, 306(5705):2270-2272.
6. Lim D. and Natalie Strynadk CJ. Structural Basis for theβ-lactam Resistance of PBP2a from Methicillin-resistant Staphylococcus aureus[J]. Nature Structural Biology, 2002,9(5), 870-8.
7. Georgopapadakou NH, Smith SA, Bonner DP. Penicillin-binding proteins in a Staphylococcus aureus strain resistant to specific beta-lactam antibiotics[J]. Antimicrob Agents Chemother. 1982;22(1):172-5.
8. Pinho MG, de Lencastre H, Tomasz A. An acquired and a native penicillin-binding protein cooperate in building the cell wall of drug-resistant staphylococci[J]. Proc Natl Acad Sci USA. 2001;98(19):10886-91.
9. Katayama Y, Zhang HZ, Chambers HF. Effect of disruption of Staphylococcus aureus PBP4 gene on resistance to beta-lactam antibiotics[J]. Microb Drug Resist. 2003;9 (4):329-36.
10. Finan JE, Archer GL, Pucci MJ, Climo MW. Role of penicillin-binding protein 4 in expression of vancomycin resistance among clinical isolates of oxacillin-resistant Staphylococcus aureus[J]. Antimicrob Agents Chemother. 2001;45(11):3070-5.
11. Park Y, Oh EJ, Lee SO, Kim BK. Absence of mutation in the region (nt. 710-1010) ofpbp4 gene in clinical isolates of Staphylococcus aureus with low-level teicoplanin resistance[J]. Diagn Microbiol Infect Dis. 2001;39(4):271-3.
12. Higashi Y, Wakabayashi A, Matsumoto Y, Ohno A. Role of inhibition of penicillin binding proteins and cell wall cross-linking by beta-lactam antibiotics in low- and high-level methicillin resistance of Staphylococcus aureus[J]. Chemotherapy. 1999;45 (1):37-47.
13. Ishikawa T , Mat sunaga N , Tawada H , et al. TA K-599 , a novel N-phosphono type prodrug of anti-MRSA cephalo-sporin T-91825 : synthesis , physicochemical and pharmaco-logical properties[J]. Bioorg Med Chem , 2003 , 11 ( 11 ) :2427-2437.
14. Treitman AN, Yarnold PR, Warren J, et al. Emerging incidence of Enterococcus faecium among hospital isolates [J]. J Clin Microbiol. 2005, 43(1):462-463.
15. Appelbaum P C.The emergence of vancomycin-intermediate and vancomycin-resistant Staphylococcus aureus [J].Clin Microbiol Infect,2008,12(1):16.
16. Rao X, Padraig D, Hua Z, et al. A regμlator from Chlamydia trachomatis modμlates the activity of RNA polymerase through direct interaction with theβsubunit and the primaryσsubunit[J]. Genes Dev, 2009, 23: 1818-1829
17.马学军,舒跃龙.分子克隆实验指南[M].第4版.上海:科学出版社,2006:44-92.
18. F奥斯伯等著,颜子颖,王海林译.精编分子生物学实验指南[M],北京:科学出版社, 1998:22-23.
19. Eiko I,Thomas A.Screening of a Genomic DNA Library and Recombinant Expression: A New Approach to the Question of Allergic Responses to Bacterial Proteins[J].Int Arch Allergy Immunol,2007,124:97-99
20. Cosgrove S E,Sakoμlas G, Perencevich E N. Comparison of mortality associated with methicillin-resistant and methicillin-susceptible S. aureus bacteraemia:a meta -analysis [J]. Clin Infect Dis,36:53-59
21. Lim D. and Natalie Strynadk CJ. Structural Basis for theβ-lactam Resistance of PBP2a from Methicillin-resistant StapHylo-coccus aureus [J]. Nature Structural Biology, 2002,9(5), 870-878.
22. Georgopapadakou NH, Smith SA, Bonner DP. Penicillin-binding proteins in aStaphylococcus aureus strain resistant to specific beta-lactam antibiotics [J]. Antimicrob Agents Chemother. 1982, 22(1):172-175.
23. Brian S,Richard C ,Normen D. Representation of DNA sequences in recombinant DNA Libraries Prepared by restriction enzyme partial digestion[J]. Gene ,2004 ,19 :201-209.
24. Pérez-Ortín,J.E.and Del Olno,M.L.,et al. Making your own gene library[J]. Biochemical Education,1997,25:237-242.
25. Dove S L, Joung J K, Hochschild A. Activation of prokaryotic transcription through arbitrary protein-protein contacts[J]. Nature,1997, 386 (6625): 627-630.
26. Figeys D.Novel approaches to map protein interactions[J].Curr Opin Biotechnol, 2007,14(1),119-125.
27. Dove S L, Hochschild A. A bacterial two-hybrid system based on transcription activation[J].Methods Mol Biol,2004,261:231-246.
28. Joung J K, Ramm E I , Pabo C O. A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions[J]. Proc Natl AcadSci USA, 2000,97:382-387.
29. Hu J C,Komacker M G,Hochschild A.E.coli one-and two-hybrid systems for the analysis and identification of protcin-protein interactions[J].Methods,2000,20:80-94.
30. Drewes G,Bouwmeester T.Global approaches to protein-protein interactions[J].Curt Opin Cell Biol ,2003,15(2):199-205.
31. Seidl K,Stucki M,Ruegg M.Staphylococcus aureus CcpA affects virμlence determinant production and antibiotic resistance[J].Antimicrob Agents Chemother,2006, 50:1183– 1194.
32.符庆瑛,高钰琪,刘昕.大肠杆菌双杂交系统筛选与AMPKα2相互作用的蛋白质[J].第三军医大学学报, 2007, 29(9):820-823.
33. Hua Z, Rao X, Feng X, et al. Mutagenesis of Region 4 of Sigma 28 from Chlamydia trachomatis Defines Determinants for Protein-Protein and Protein-DNA Interactions[J]. J Bacteriol,2009,1: 651-660
34. Surobhi L, Lakshmi P, Nara G. Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein[J]. Biochemical and Biophysical Research Communications,2005,337:677-684
35. Lim D,Strynadka N.Structural basis for the beta lactam resistance of PBP2a from methicillin-resistant Staphylococcus[J]. Nat Struct Biol,2002,9(11):870-876.
36. Rohrer S, Berger B.Fem A. A Link between branched-chain cell wall peptide formation andβ-lactam resistance in gram-positive cocci[J]. Antimicrob Agents Chemother,2005, 47:837-846.
37.杨杰,饶贤才,胡福泉,等. MRSA耐药相关TG-TPase嵌合基因的原核表达与纯化[J].免疫学杂志, 2008, 23:289-294.
38.汪家政,范明主编.蛋白质技术手册[M],北京:科学出版社,2000:29-33.
39.卢圣栋主编,现代分子生物学技术[M],第二版.北京:中国协和医科大学出版社, 1999 :417-429.
40. Phizicky EM,Fields S.Protein-protein interactions:methods fordetection and analysis[J].Microbiol Rev,1995,59:94-123.
41. Frangioni JV,Neel BG..Solubilization and purification of enzymatically active glutathione S-transferase(pGEX)fusion proteins[J].Anal Biochem.1993,Apr,210(1): 179-187.
42. Gribskov M,Burgess RR.Overexpression and purification of the sigma subunit of E.coil RNA polymerase[J].Gene,1983,26:109.
43. Lilie H,Schwarz E,Rudolph R.Advances in refolding of proteins produced in E.coli[J].Curr Opin Biot,1998,9:442.
44. Belin P and.Boquet. P.L Green fluorescent protein as a marker for gene expession [J]. Microbiol, 1994, 140:3337-3348
45. DR马歇尔等著,朱厚础等译.蛋白质纯化与鉴定实验指南[M],北京:科学出版社, 1999 :144-149.
46. Ren L,Chang E,Makky K,et al.Glutathione S-transferase pull-down assays using dehydrated immobilized glutathione resin[J].Anal Biochem,2008, Nov 15,322(2): 164-169.
47. Maggi S, Massidda O, Luzi G. Division protein interaction web: identification of a phylogenetically conserved common interactome between Streptococcus pneumoniae and Escherichia coli[J]. Microbiology-SGM , 2008,154:3042-3052.
48. Baldiek,C.J.,Jr.,andT.Shenk.Protein sassoeiated with Purified human eytomegalovims Partieles[J],Virol,1996,70: 97-105.
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