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Objective To screen and identify the aptamers of the recombinant transpeptidase domain of PBP2a(penicillin binding protein 2a ,PBP2a) .Methods By using the recombinant transpeptidase domain of PBP2a as the screening target ,oligonucle-otides which were capable of specifically binding to the protein were screened by a random oligonucleotide library through the stem -atic evolution of ligand by exponential enrichment (SELEX )technique .The ssDNA was cloned and sequenced ,and the secondary structure of aptamer clones was predicted with mfold program .Results After 11 cycles of the selection ,the aptamers which were capable of binding to PBP2a with high affinity have been selected .40 clones from the 8 and 10 cycles were selected randomly and se-quenced .The aptamers obtained had no obvious homology according to their sequences by the sequence alignments ,and the 40 aptamers were classified to three groups according to their secondary structures .The aptamer 13 was found to be specific for the target protein with the highest affinity .Conclusion Aptamers for the recombinant transpeptidase domain of PBP2a with high affili-ty and specificity were successfully screened by SELEX ,which lays a foundation for exploring new ways of diagnosis and treatment of MRSA infection .
ABSTRACT
Objective To construct the prokaryontic expression vector of the gene fragment which encodes the transpeptidase domain of penicillin binding protein 2a(PBP2a) of methicillin‐resistant Staphylococcus aureus(MRSA) ,and to express ,purify and i‐dentify the objective protein .Methods Strains of MRSA were isolated and identified from clinical samples ,according to the se‐quence of mecA gene recorded in GenBank ,the primers of mecA fragment which encoded the transpeptidase domain of PBP2a was designed .The gene fragment from MRSA was amplified by using polymerase chain reaction(PCR) and cloned into pET28a(+ ) plasmid .After being identified by enzyme digestion and sequencing ,the recombinant plasmid was transformed into the strain of Escherichia coli BL21(DE3)plysS .The expression of transpeptidase domain of PBP2a was induced by 0 .7 mmol/L IPTG ,the ex‐pressed products were purified by using Ni afinity chromatography ,then were analyzed by using Western blot .Results The recom‐binant expression vector was digested by BamHⅠ and EcoRⅠ ,and the products were at the expected size .The result of sequencing showed two bases undergoing mutation ,while there were no frameshift mutations .The expressed protein was identified by using SDS‐PAGE and Western blot ,a new protein band was visible at the relative molecular mass of 38 × 103 .Conclusion The corre‐sponding prokaryotic expression vector is successfully constructed ,and the transpeptidase domain of PBP2a is successfully ex‐pressed and purified .
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BACKGROUND: This study compared three non-molecular methods for the detection of methicillin-resistance directly from blood cultures containing Staphylococcus aureus: penicillin-binding protein (PBP) 2a latex agglutination (LA), PBP2a immunochromatographic assay (ICA) and MRSA chromogenic medium (CM). METHODS: Fifty methicillin-resistant S. aureus (MRSA) and 50 methicillin-susceptible S. aureus (MSSA) were seeded into blood-culture bottles. When isolates returned a positive signal, 5 mL of culture was added to serum separator tubes and centrifuged at 1,300 g for 10 min. The pellets were then used as the inoculum for the PBP2a LA, MRSA-CM and PBP2a ICA. The pure colony was used for PBP2a LA test, additionally. RESULTS: The respective sensitivities and specificities were 98 and 100% for PBP2a ICA, and 100 and 100% for MRSA-CM in direct detection of MRSA from positive blood culture. The results of PBP2a LA test using pure colony were entirely compatible with those by mecA gene PCR but the PBP2a LA test using the pellets directly isolated from positive blood culture showed sometimes ambiguous agglutination; its sensitivity and specificity were 78 and 100%, if ambiguous results were scored as negative, and were 90 and 92%, if ambiguous results were scored as positive, respectively. CONCLUSION: For direct detection of MRSA in positive blood culture, MRSA-CM and PBP2a ICA provided excellent results. The PBP2a LA test using pure colony also gave excellent results but the PBP2a LA test by the direct method using pellet of positive blood culture was slightly less sensitive than the other two methods.
Subject(s)
Adenosine , Agglutination , Chromatography, Affinity , Latex , Methicillin Resistance , Methicillin-Resistant Staphylococcus aureus , Penicillin-Binding Proteins , Polymerase Chain Reaction , Seeds , Sensitivity and Specificity , StaphylococcusABSTRACT
Objetivo: Determinar si existe asociación entre genes implicados en la codificación de PBP2a con la expresión fenotípica de resistencia a meticilina en cepas de Staphylococcus spp. Diseño: Descriptivo Transversal. Metodologias: e determinó la resistencia y sensibilidad de 67 aislamientos, mediante pruebas fenotípicas (difusión en disco, concentración inhibitoriamínima CIM, producción de PBP2a y pruebas genotípicas para detectar los genes mecA y sus reguladores mecR1 y mecI por Reacción em Cadena de la Polimerasa (PCR). Resultados: De 9 cepas de S. aureus resistentes por difusión en disco solo 1 fue sensible por CIM. De 7 cepas resistentes por CIM, fueron sensibles por difusión en disco. Por el contrario las 7 cepas de Staphylococcus coagulasa negativo sensibles por difusión en disco fueron resistentespor CIM.En cuanto a la prueba de producción de PBP2a, los resultados fueron discordantes con la prueba de difusión en disco en 20..
Objective: Determining the association between genes involved in the codifi cation of Penicillin Binding Proteins 2A (PBP2A) with the phenotypic expression of methicillin resistance in Staphylococcus spp. Strains Design: Descriptive cross sectional Methodology: The sensitivity of 67 isolates was determined by means of a phenotypic test (disk diffusion, minimum inhibitory concentration CIM, production of PBP2a) and genotype tests to detect the mecA gene and its regulatory mecR1 and mecI by Polymerase Chain Reaction (PCR). Results: From 9 S. aureus resistant strains by disk diffusion 1 was sensitive by CIM, 7 CIM resistant strains were sensitive by disk diffusion. The 7 coagulase negative (CNS) sensitive strains by disk diffusion were resistant by CIM. By production of PBP2a, the results were discordant with the disk diffusion test in 20% and 34%with CIM. The genotype, reveals that, from 60 S.aureus strains 10(17%), and 7 S. coagulase negative strains 4 (57%) carry the mecA gene. From 10 S. aureus mecA positive strains, 5 carry the mecR1 gene and 7 carry the mecI gene. Of the 4 strains of S.coagulase negative mecA positive 2 carry the mecR1 and 2 carry the mecI gene. Conclusion: There is no association between genotype and phenotype in Staphylococcus spp. methicillin resistant strains, since, the resistance is due to many factors that the classical phenotypic test does not include.
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Penicillin-Binding Proteins/pharmacology , Penicillin-Binding Proteins/chemistry , Penicillin-Binding Proteins/blood , Penicillin-Binding Proteins/chemical synthesis , Methicillin-Resistant Staphylococcus aureus/genetics , PhenotypeABSTRACT
OBJECTIVE To develop a rapid method for simultaneously identifying Staphylococcus aureus(SA) and its resistance against meticillin. METHODS The target strains authenicated to Staphylococcus by traditional methods(appearance,Gram-stain,catalase) first,then using Slidex~staph.plus to authenticate SA;establishing traditional method or coagulase test and ATB-ID32STAPH system for verification.The penicillin-binding protein 2a(PBP2a) was examined by Slidex MRSA Detection,establishing resistance oxacillin sieving test and mecA gene was examined by PCR for verification. RESULTS The 135 strains were positive and 321 strains were negative for Slidex~staph.plus from 456 strains.The 127 S.aureus strains and eight others were confirmed from 135 positive strains finally,11 SA strains and 310 other strains were confirmed from 321 negative strains,there were 92.0% for sensitivity and 97.5% for specificity in this method.The 52 strains PBP2a positively confirmed to meticillin-resistant S.aureus(MRSA) using Slidex MRSA Detection and 57 MRSA strains were confirmed using resistance oxacillin sieving test or PCR from 138 strains.There were 91.2% for sensitivity and 100% for specifity in this method. CONCLUSIONS The duplex Slidex~-staph monoclonal antibody examinated to SA and confirmed to MRSA has higher sensitivity and specificity.
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OBJECTIVE To screen the anti-meticillin-resistant Staphylococcus aureus(MRSA)traditional Chinese materia medica(TCMM)by biosensor technique,targeted on the soluble penicillin binding protein 2a(PBP2a)of clinical MRSA.METHODS The soluble PBP2a with amino acid sequence from 25 to 668 from clinical MRSA were expressed in Escherichia coli by gene recombination technique.Then,the expressed product was identified and its biological function was analyzed.After the PBP2a was immobilized into the carboxymethyl dextran cuvette(CMD),the anti-MRSA TCMM was screened by means of biosensor.RESULTS The soluble protein PBP2a had been successfully expressed,whose relative molecular mass was 74?103.It was confirmed that the soluble PBP2a had transpeptidase activitiy and ?-lactamase activitiy.Subsequently,10 kinds of anti-MRSA TCMM were screened out by biosensor technique.Moreover,Radix Scutellariae,Rhizoma Coptidis and Spica Prunellae had greater anti-MRSA effect than others.CONCLUSIONS Anti-MRSA TCMM has been successfully screened out by biosensor technique,targeted on the soluble PBP2a of clinical MRSA.