耐甲氧西林金黄色葡萄球菌PBP2a的原核表达及多克隆抗体的制备

目的 研究耐甲氧西林金黄色葡萄球菌(MRSA)青霉素结合蛋白2a (PBP2a)的原核表达,及PBP2a多克隆抗体的制备.方法 根据基因文库登录的mecA基因的编码序列,设计合成了1对寡核苷酸引物,应用PCR法从MRSA基因组中获得编码PBP2a全长的DNA,将此目的 基因片段克隆至pET32a(+)载体,转化E.coli BL21(DE3);经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达后,利用Ni2+亲和层析技术纯化目的 蛋白;用目的 蛋白免疫小鼠3～8次后,收获血清并鉴定.结果 成功构建了PBP2a原核表达载体,并获得了高效表达;利用纯化的蛋白制备了理想的多克隆抗体.结论 利用分子克隆技术,获得了高纯度的PBP2a蛋白并制备了多克隆抗体,为其进一步研究奠定了基础.     

耐甲氧西林金黄色葡萄球菌青霉素结合蛋白2a转肽酶区的克隆、表达及纯化鉴定

目的构建编码耐甲氧西林金黄色葡萄球菌(MRSA)青霉素结合蛋白2a(PBP2a)转肽酶区基因片段的原核表达载体,并表达、纯化及鉴定蛋白。方法从临床标本中分离鉴定MRSA,设计针对编码PBP2a转肽酶区基因片段的引物,采用聚合酶链式反应(PCR)扩增目的基因片段,克隆至pET28a(+)载体,双酶切鉴定并测序,转化大肠杆菌BL21(DE3)plysS株;用0.7mmol/L异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后,利用Ni亲和层析技术纯化目的蛋白;蛋白免疫印迹法(WB)鉴定重组蛋白。结果重组表达载体经BamHⅠ、EcoRⅠ酶切,产物在预期大小处出现条带,测序结果显示有两个碱基突变,无移码突变。所表达的PBP2a蛋白经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和WB鉴定,在相对分子质量38×103处可见一新生蛋白条带。结论成功构建了PBP2a转肽酶区原核表达载体,并获得了高效表达,制备了高纯度的目的蛋白。     

用合成肽单抗检测MRSA的青霉素结合蛋白2’

耐甲氧西林金葡菌（MRSA）对β-内酰胺类药物的耐药性是因其具有青霉素结合蛋白2’（PBP2’）结构。MRSA带有编码PBP2’的mecA。血浆凝固酶阴性葡萄球菌也可有PBP2’及mecA基因。作者等合成了含31个和32个氨基酸的多肽，它们分别相当于PBP2’中的第382个～412个和第414个～443个氨基酸序列。用合成肽免疫小鼠所得脾细胞与骨髓瘤细胞融合产生2种单抗DM－2－12和DM3～1。经Western转印（免疫印迹）分析证实了此2种单抗可与MRSA的PBP2’特异地结合，并建立了免疫放射检测（IRMA）技术。作者等作IRMA检测了医院中分离的56株金…     

胶乳凝集试验快速检测耐甲氧西林金黄色葡萄球菌

耐甲氧西林金黄色葡萄球菌 (MRSA)是重要的医院感染致病菌 ,快速准确检测MRSA是控制感染的关键。MRSA的表型检测受多种因素的影响 ,结果不够准确 ,耗时长。通过聚合酶链反应 (PCR)扩增编码耐药菌特异的青霉素结合蛋白 2a (PBP2a)的mecA基因来鉴定是实验室最可靠的方法之一 ,但受设备、试剂等的限制 ,不易在临床推广应用。现报道一种新的直接测定PBP2a的快速检测方法———MRSA 胶乳凝集试验法 ,并以PCR法为“金标准”评价了胶乳凝集试验法、全自动仪器 (VITEK1)法、琼脂筛选法、纸片扩散法 4种方法测定MRSA的可靠性和临床应…     

耐甲氧西林金黄色葡萄球菌耐药机制及检测方法研究进展

耐甲氧西林金黄色葡萄球菌(MRSA)广泛传播是全球重要的公共卫生问题。葡萄球菌染色体mec基因盒元件(SCCmec)在金黄色葡萄球菌中转移是导致MRSA广泛传播的重要原因。SCCmec中的mecA和mecC基因分别编码PBP2a和PBP2c,其结合β-内酰胺类抗菌药物能力低下,是导致MRSA发生的关键因素,而且mecA和mecC介导的耐药表型存在一定差异。本文就SCCmec的基本结构与功能、mecA与mecC介导的耐药机制与表型以及MRSA的检测方法等方面做以下综述。     

乳胶结合试验快速检测耐甲氧西林金黄色葡萄球菌

目的：评价乳胶结合试验检测耐甲氧西林金黄色葡萄球菌的试验方法。方法：收集81株金黄色葡萄球菌临床分离株，通过药敏试验将其分为耐甲氧西林金黄色葡萄球菌（MRSA）和对甲氧西林敏感的金葡菌（MSSA），应用乳胶结合试验和基因扩增分别检测青霉素结合蛋白2a(PBP2a)和mecA基因。结果：33株mecA基因扩增阳性菌株中的28株PBP2a检测呈阳性，5株为阴性，46株MSSA中，meca基因扩增及PBP2a检测全部为阴性。与聚合酶链反应检测mecA基因相比，乳胶结合试验检测MRSA的特异性和敏感性分别为100％和91．3％（74／81）。结论：乳胶结合试验是一种简便、快速、准确地检测MRSA的方法，适于在普通临床微生物实验室开展。     

甲氧西林耐药金黄色葡萄球菌的实验室诊断

MRSA定义为携带mecA基因的金葡菌和(或)苯唑西林MIC>2mg/L的金葡菌。大部分MRSA菌株携带mecA基因,但是一小部分MRSA不携带mecA基因,存在其他的耐药机制,如青霉素结合蛋白(PBP)改变或高产β内酰胺酶等。以下简述MRSA的实验室检     

耐甲氧西林金黄色葡萄球菌MecA基因的鉴定分析

目的探讨临床分离金黄色葡萄球菌中PCR检测MecA基因用于耐甲氧西林金黄色葡萄球菌(MRSA)鉴定的评价。方法对临床分离的120株金黄色葡萄球菌,根据PBP2a编码基因设计MecA引物采用PCR快速检测MRSA的决定基因MecA。结果MecA基因片段经PCR扩增,可见MRSA扩增出一条310bp大小的DNA片段,而敏感株和金黄色葡萄球菌标准株ATCC25923均未出现特异长度的DNA片段;在临床分离的120株金黄色葡萄球菌中,MecA检测阳性率为45.0%(54/120)。结论采用PCR方法对金黄色葡萄球菌临床分离株中的MecA基因进行检测,对MRSA的快速、及时检出,有效控制MRSA造成的感染,指导临床用药,限制其传播等具有重要的现实意义。     

骨形态发生蛋白诱导基因克隆和表达的实验研究

目的:用基因工程技术在大肠杆菌中表达骨形态发生蛋白诱导基因(BMP-2-inducedgene3kbgene,BIG-3)所编码的蛋白。方法:依据Genbank中BIG-3的基因序列设计并合成引物,从新生小鼠颅骨组织中提取总RNA,通过反转录聚合酶链式反应(RT-PCR)得到BIG-3全长编码序列。将所得到的基因片段克隆至原核表达载体pGEX-4T-2的多克隆位点获得pGEX-4T-2-BIG-3重组表达载体,经测序证实后转化大肠杆菌BL21(DE3)菌株,挑选阳性克隆,经诱导表达后SDS-PAGE鉴定。结果:克隆得到BIG-3全长编码序列并在大肠杆菌中表达了GST-BIG-3融合蛋白,融合蛋白约占菌体总蛋白的45.3%。结论:通过RT-PCR从新生小鼠颅骨中克隆到BIG-3基因并在大肠杆菌中获得GST-BIG-3融合蛋白的高水平诱导表达。     

问：如何准确检测耐甲氧西林的金黄色葡萄球菌和凝固酶阴性葡萄球菌？

答：检测mecA基因和其表达的青霉素结合蛋白（PBP2a）是预测葡萄球菌对苯唑西林耐药的最准确方法。罕见非mecA基因介导的苯唑西林耐药机制，纸片法测定苯唑西林为中介或耐药，应加测苯唑西林的MIC，如果MIC≥4ug／ml，即使mecA基因和PBP2a检测阴性，苯唑西林也应报告耐药。在凝固酶阴性葡萄球菌（CONS）中头孢西丁纸片扩散法的结果与检测mecA／PBP2a的一致性好于苯唑西林纸片，     

Construction of a water-soluble form of penicillin-binding protein 2a from a methicillin-resistant Staphylococcus aureus isolate.

The mecA gene from methicillin-resistant Staphylococcus aureus 27r, which encodes the membrane-bound penicillin-binding protein 2a (PBP 2a), was cloned, sequenced, and expressed in Escherichia coli. PBP 2a is the major factor that mediates methicillin resistance in staphylococci. The DNA sequence of the mecA gene from strain 27r was greater than 99% identical to the DNA sequence of other S. aureus mecA genes and the mecA gene from Staphylococcus epidermidis. Analysis of the deduced amino acid sequence of PBP 2a from strain 27r revealed a hydrophobic region at the amino terminus that possessed characteristics of an uncleaved signal peptide such as those found in type II integral membrane proteins. Site-specific mutagenesis was used to modify the strain 27r mecA gene to permit removal of the region encoding the putative transmembrane region (amino acids 2 to 22). When it was expressed in E. coli, the modified mecA gene from strain 27r encoded a water-soluble form of PBP 2a that was detectable in the cytoplasm of transformants. The water-soluble form of PBP 2a protein from S. aureus 27r retained the same binding efficiency for beta-lactam antibiotics as the unmodified membrane-bound PBP 2a from S. aureus 27r.     

The structure and function of the mecA gene in methicillin-resistant Staphylococcus aureus]

Penicillin binding protein (PBP) 2' is the most important mechanism of the resistance to beta-lactams in methicillin-resistant Staphylococcus aureus (MRSA). And the mecA gene is the coding gene of PBP2', and located in the SmaI fragment G of the chromosome map by Pattle P.A.,. A part of the structure of mecA is similar to that of the penicillinase gene. The resistance of MRSA to beta-lactams were influenced by the presence of penicillinase plasmid and the alternation of femA gene.     

In vitro selection and characterization of ceftobiprole-resistant methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to beta-lactam antibiotics because it expresses penicillin-binding protein 2a (PBP2a), a low-affinity penicillin-binding protein. An investigational broad-spectrum cephalosporin, ceftobiprole (BPR), binds PBP2a with high affinity and is active against MRSA. We hypothesized that BPR resistance could be mediated by mutations in mecA, the gene encoding PBP2a. We selected BPR-resistant mutants by passage in high-volume broth cultures containing subinhibitory concentrations of BPR. We used strain COLnex (which lacks chromosomal mecA) transformed with pAW8 (a plasmid vector only), pYK20 (a plasmid carrying wild-type mecA), or pYK21 (a plasmid carrying a mutant mecA gene corresponding to five PBP2a mutations). All strains became resistant to BPR by day 9 of passaging, but MICs continued to increase until day 21. MICs increased 256-fold (from 1 to 256 microg/ml) for pAW8, 32-fold (from 4 to 128 microg/ml) for pYK20, and 8-fold (from 16 to 128 mug/ml) for pYK21. Strains carrying wild-type or mutant mecA developed six (pYK20 transformants) or four (pYK21 transformants) new mutations in mecA. The transformation of COLnex with a mecA mutant plasmid conferred BPR resistance, and the loss of mecA converted resistant strains into susceptible ones. Modeling studies predicted that several of the mecA mutations altered BPR binding; other mutations may have mediated resistance by influencing interactions with other proteins. Multiple mecA mutations were associated with BPR resistance in MRSA. BPR resistance also developed in the strain lacking mecA, suggesting a role for chromosomal genes.     

DNA vaccination by mecA sequence evokes an antibacterial immune response against methicillin-resistant Staphylococcus aureus

More than 90% of methicillin-resistant Staphylococcus aureus (MRSA) isolates produce a penicillin-binding protein PBP2' (or PBP2a) with low affinity for beta-lactam antibiotics. PBP2' is encoded by the mecA gene, a foreign gene integrated into the chromosome of methicillin-susceptible S. aureus (MSSA). DNA vaccination by injection of transgene-expressing plasmids has been demonstrated to elicit an immune response against transgene-encoded protein. We hypothesized that the application of DNA vaccination with the mecA sequence would elicit protective immunity against MRSA. This immunity was evoked by injection of a mecA-expressing plasmid into BALB/c mice. Anti-PBP2' antibody was detected in the sera obtained from the DNA-vaccinated mice. These sera produced a five-fold increase in phagocytosis of MRSA compared with sera from mice treated with control plasmid. However, there was no difference in phagocytosis of MSSA among these groups. In addition, the in-vivo antibacterial effect of DNA vaccination was demonstrated in mice infected with MRSA. Eight days after iv inoculation of 10(8) cfu of MRSA into mice, the number of bacteria in the kidneys obtained from mice vaccinated with mecA-expressing plasmid (1.48 +/- 0.27 x 10(5) cfu/mg kidney; n = 18) was significantly lower than that from mice vaccinated with negative control plasmid (3.59 +/- 0.57 x 10(5) cfu/mg kidney; n = 17) (P < 0.02) or that from sham-treated mice (3. 43 +/- 0.66 x 10(5) cfu/mg kidney; n = 9) (P < 0.02). Interestingly, PBP2' was found in both the bacterial membrane fraction and the supernatant, thus being accessible to serum antibodies. Together these observations indicate that PBP2' or the mecA sequence may be eligible as a candidate molecule for vaccination against MRSA.     

PBP 2a mutations producing very-high-level resistance to beta-lactams

Resistance to the beta-lactam class of antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is mediated by PBP 2a, a synthetic bacterial cell wall penicillin-binding protein with a low affinity of binding to beta-lactams that is encoded by mecA. Beta-lactams that bind to PBP 2a with a high affinity and that are highly active against MRSA are under development. The potential for the emergence of resistance to such compounds was investigated by passage of homogeneous MRSA strain COL in L-695,256, an investigational carbapenem. A highly resistant mutant, COL52, expressed PBP 2a in which a two-amino-acid deletion mutation and three single-amino-acid substitution mutations were present. To examine the effects of these mutations on the resistance phenotype and PBP 2a production, plasmids carrying (i) PBP 2a with two or three of the four mutations, (ii) wild-type PBP 2a, or (iii) COL52 PBP 2a were introduced into methicillin-susceptible COL variants COLnex and COL52ex, from which the staphylococcus cassette chromosome mec (SCCmec) has been excised, as indicated by the "ex" suffix. Two amino acids substitutions, E-->K(237) within the non-penicillin-binding domain and V-->E(470) near the SDN(464) conserved penicillin-binding motif in the penicillin-binding domain in COL52, were important for high-level resistance. The highest level of resistance was observed when all four mutations were present. The emergence of PBP 2a-mediated resistance to beta-lactams that bind to PBP 2a with a high affinity is likely to require multiple mutations in mecA; chromosomal mutations appear to have a minor role.     

Cloning and expression of methicillin resistance from Staphylococcus epidermidis in Staphylococcus carnosus.

A 6.2-kilobase chromosomal DNA fragment from a methicillin-resistant Staphylococcus epidermidis strain was cloned into Staphylococcus carnosus by using staphylococcal plasmid pCA44 as the vector. The recombinant plasmid obtained, pBBB21, conferred methicillin resistance on its host and was responsible for the synthesis of a low-affinity penicillin-binding protein (PBP), PBP 2'. PBP 2' determined by the S. epidermidis DNA and expressed as a membrane-bound PBP in S. carnosus reacted with monoclonal antibodies directed against PBP 2' of Staphylococcus aureus origin, and the cloned S. epidermidis DNA hybridized to the methicillin (mec)-specific DNA from S. aureus. These findings point to a common origin of the methicillin resistance determinant in staphylococci.     

哺乳动物极性蛋白mInscuteable-C末端肽段/GST融合蛋白的原核表达与纯化

目的:构建哺乳动物极性蛋白mInscuteable C末端257～532位氨基酸结构域与谷胱甘肽巯基转移酶(GST)的融合蛋白GST-mInsc 257～532的原核表达载体,在大肠杆菌中表达并纯化该融合蛋白。方法:将已经构建好的mInsc 257～532位氨基酸序列克隆至原核表达载体pGEX-4T中,构建重组的质粒pGEX-4T/mInsc 257～532;将重组质粒转化感受态细菌BL21,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达GST蛋白;经谷胱甘肽-琼脂糖球珠分离纯化;产物经SDS-PAGE电泳及Western Blot鉴定。结果:获得高表达及纯化的pGEX-4T/mInsc 257～532融合蛋白。结论:成功构建重组pGEX-4T/mInsc 257～532原核表达载体;诱导表达pGEX-4T/mInsc 257～532融合蛋白并纯化。