Dissertation > Medicine, health > Clinical > Diagnostics > Laboratory diagnosis > Immunology Examination

Screening Aptamers to Penicilloin-Binding Protein2a and Primary Application

Author MaHongYu
Tutor LanXiaoPeng
School Second Military Medical University
Course Clinical Laboratory Science
Keywords methicillin resistant staphylococcus aureus (MRSA) penicillin bindingprotein2a (PBP2a) systematic evolution of ligands by exponential enrichment (SELEX) aptamer dot filter assay
CLC R446.6
Type Master's thesis
Year 2013
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[Objective]Hospital infection rates has increased year by year due to methicillin-resistant Staphylococcus aureus(MRSA), as its main mechanism of resistance,Penicillin-binding protein2a(PBP2a), has become the focus of attention and the direction of the global anti-MRSA.In this study, We put the transpeptidase domain of PBP2a as target, to screen its high specificity, high affinity binding aptamer, and establish a method of rapid detection of PBP2a with screened aptamers.[Methods]1. The gene fragment which encodes the transpeptidase domain of PBP2a was amplified by PCR, and then the objective gene fragment was cloned into pET-His plasmid. After being identified by enzyme digestion and sequencing, the recombinant plasmid was transformed into E. coli BL21(DE3)plysS. The expression was induced by IPTG and the expressed product was purified by Ni " afinity chromatography" and analyzed by Western-blot。2. The research started with the synthesis of a96nt random oligonucleotides DNA library containing a central region of60random nucleotides flanked by a5’ and a3’ region of constant sequence. The storage capacity of the library is about1016the ssDNA pool was subjected to13rounds of SELEX selection.The ssDNA aptamers specifically binding to PBP2a were obtained through selection.3. PCR products of the13th round of selection were cloned and sequenced. Relative softwares were employed to analyze the primary structure and prediction secondary structure of the aptamers.4. we experimented the preparation of the aptamers labelled with colloidal gold which is used as probes to established a dot immuno filtration assay, and to assess the specificity of the aptamers,according to Digoxigenin-anti-digoxigenin-AP system. The dissociation constant (Kd) was obtained through softwares that analysised the binding capabilitie of aptamers in the different concentration.[Results]1. The corresponding prokaryotic expression vector was successfully constructed, The recombinant expression vector was dentified by enzyme digestion and the Production was at the expected size of the strip.99.8%of the sequencing results was same as the sequence of mecA genea published in GenBank, no frameshift mutations.The expressed PBP2a was identified by SDS-PAGE electrophoresis and Western blot, Chu, Mr38000were seen a new protein bands at Mr38000, and the transpeptidase domain of PBP2a was successfully expressed and purified.2. With the increasing of the screening round, a significant growth of the binding capabilities was seen, After13rounds of selection, the binding ability of the aptamers ascended from2.9%to40.4%, and maintained a steady state.3. We cloned and sequenced the production of the13th round selection. Multiple sequence alignments using ClustaX and Meg5.3revealed that four paires of sequences in the sixty three sequences were mostly enriched. RNAstructure4.3software was used to predict the secondary structures of the aptamers. we found that the main secondary structures were stem-loop、hairpin and G-quadruplex, The number size and location of stem-loop of each family and each sequence were different.4.We applied a dot aptamers labelled with colloidal gold filtration assay to detect specificity of aptamers, the aptamers were provided to be with highly specificitied. We use Digoxin-alkaline phosphatase anti digoxin antibody system as a bridge to detect the adhesion between target proteinand aptamer with different concentrations. Through the software analysis,we know the dissociation constants of PBP2a Kd value can be as low as21.9nm.[Conclusion]The corresponding prokaryotic expression vector was successfully constructed, and the transpeptidase domain of PBP2a was successfully expressed and purified. By using Systematic Evolution of Ligands by Exponential enrichment(SELEX) technology, We have successfully selected a suit of aptamers with high affinity and specificity to PBP2a after13rounds of selection. A colloidal gold labelled aptamers dot filtration assay was used to detect specificity of aptamers.and The Digoxigenin-anti-digoxigenin-AP system was used to analysise the binding capabilitie of aptamers with PBP2a in different concentration. Confirmed that screening aptamers with specificity and high affinity to PBP2a turn to.this study.which established the foundation for further investigation.

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