Study On The Screening Of Trypsin Aptamer Andthe New Method For Immobilization Of Trypsin

Aptamer is a kind of oligonucleotide sequence with high affinity and high specificity for its target molecules,which is obtained from the random oligonucleotide library by Systematic Evolution of Ligands by Exponential Enrichment(SELEX).Compared with other aptamer screening techniques,the aptamer screening technique based on capillary electrophoresis(CE-SELEX)has the advantages : high separation efficiency,high speed and low sample consumption.With the high-throughput,large-scaleand integrated research in proteomics,online digestion technology become more and more important.And the immobilized enzyme reactor is the key point to achieve online enzymolysis.Compared with traditional enzyme immobilization method,the enzyme aptamer has many advantages,such as strong stability,good regeneration ability,low cost and little effect on enzyme activity.In this paper,we will make use of CE-SELEX to select the aptamer of trypsin,and use the aptamer which is selected to immobilize trypsin to prepare immobilized trypsin.The main contents of this thesis include:(1)A random oligonucleotide library suitable for trypsin aptamer screening was designed.The amplification conditions of this library and the preparation conditions of the secondary library were optimized;(2)A capillary electrophoresis method which is suitable for trypsin aptamer screening was established.The electrophoresis conditions were optimized and the interaction between the library and trypsin was characterized;(3)On the basis of the designed nucleotide library and the established capillary electrophoresis method,the aptamer aptamer of trypsin was screened by capillary electrophoresis based aptamer selection(CE-SELEX)technique;(4)Chemically modified the aptamers,and modified the modified aptamer onto the surface of an amino silica gel by chemical cross-linking and polymerization.Thus,the trypsin was modified onto the surface of silica gel to prepare filler which could be used to immobilize the micro enzyme reactor.In this study,after 4 rounds of screening,the binding efficiency of random oligonucleotide libraries with trypsin reached 83.3%.we successfully obtained 25 aptamers which has high affinity with trypsin,and then immobilized trypsin was prepared based on those aptamers.Two kinds of immobilized method were used in this study,immobilized enzyme loading were 1.7 g/mg and 5.3 g/mg,which is suitable for immobilized enzyme reactors.This study can be used as a reference for the selection of aptamers by CE-SELEX method and the preparation of immobilized enzyme reactors.