In Vitro Selection Of Aptamers To Recombinant Human FcγRI And Application In Laboratory Diagnosis Of Sepsis

【Objective】SELEX(Systematic Evolution of Ligands by Exponential Enrichment,SELEX) protocol was applied to screen aptamers against recombinant human FcγRI protein fragment from a 96 nt ss DNA random library. A diagnosis method for sepsis by fluorophore-labed aptamers direct assay( FLADA) was preliminary established.【Methods】1. A synthetic 96 nt random ss DNA oligonucleotides library with a 60 random nucleotides central region and fixed 18 nt primer sequence of 5’ and 3’ region was used to obtain anti-FcγRI aptamers with high affinity and specificity. Then the ss DNA pool was subjected to 8 rounds of SELEX selection. FLADA was applied to determine the binding capabilities between the ss DNA pool and FcγRIevery one round of selection.2. PCR products of 7th and 8th round of selection were cloned and sequenced. Relative software was employed to analyse biotic infomation of aptamers.3. Three aptamers of 7th round of selection against human FcγRI were further chosen to determine their affinity, specificity and sensitivity by flow cytometer and fluorescence microscope.【Results】1. FLADA showed that an increaed significant signal of binding capabilities of ss DNA pool with the selection. The binding ss DNA aptamers didn’t increase when the epitope of target protein were saturation.2. PCR products of 7th and 8th round of selection were cloned and sequenced. Clustal X 1.83, Mega 5 and RNAstructure 5.6 were employed to analyze the homology of primary sequence structure, clustering analysis and mimicing secondary structures. There are 7 and 5 families of 7th and 8th round of selection respectively in which there are 3 pairs of rich oligonucleotides of 33 aptamers of 7th round selection. RNAstructure 5.6 mimicing secondary structures suggested that stem-loop and G-quartet are the dominated structures of these aptamers.3. Affinity examination of flow cytometric assay(FCA) showed that Kd values of aptamers LW7-1,LW7-9,LW7-27 are 12.76 n M,14.03 n M,and 6.676 n M respectively. Their sensitivity is 2×106 peripheral blood leukocytes of patients in sepsis.4. FCA demonstrated that LW7-1,LW7-9,LW7-27 could bind to human FcγRI of neutrophiles for their fluorescence intensity are more stronger than negative control and normal control.5. The fluorescence microscope demonstrated that LW7-9 was the key aptamer of our research for its much stronger fluorescence intensity. A preliminary diagnosis method of sepsis of FLADA by fluorescence microscope was established and hopeful for identified diagnosis of sepsis.【Conclusions】SELEX was applied to screen a number of DNA aptamers that specifically bind to human FcγRI from ss DNA random library after 8 rounds of selection. The binding affinity, specificity, and sensitivity of some selected aptamers were analyzed. A preliminary diagnosis method of sepsis of FLADA by fluorescence microscope was established and hopeful for identified diagnosis of sepsis. We laid a foundation for the later quantitative and semi- quantitative Point Of Care Testing(POCT) in sepsis.