Screening Of Affinity DNA Aptamer Binding To The Penicillin-antibiotic By SELEX And Preliminary Exploration Of Utilization Method

The penicillin antibiotic is a kind of highly effective medicine of anti-Gram-positive bacteria.Because they are used frequently by ultra dosage,it makes residues in animal source food,and hazards health of the people and the import-export trade seriously.It is significant to develop the penicillin antibiotic determination which is instant-view,accurate,economic in the production and the life.Currently,there are a number of major methods of the microorganism,analysis by instrument,and the kit of immunosorbent assay,which have been used in daily inspection and have advantages and disadvantages.The microoraganism-assay is economic and applicable,but time consuming,as well as,the specificity and the sensitivity are poor.The instrument-assay is high specific and sensitive,however the procedure is troublesome,and it additionally needs the expensive equipment.The kit of immunosorbent assay has the characteristics of the test instent,the specificity well and so on,but its price is exorbilant.In our study,the DNA aptamer binding to the 6-APA has been screened by SELEX though the nucleic acid library huge volume 3×10¹⁴and we have initially explored its method of utilization,to lay the groundwork for studying aptamer-sensor. The thesis consists of two parts:First,78nt-ssDNA library including 35 random sequences has been constructed. 6-APA has been coupled by Epoxy Activated Sepharose FF solid phase separating medium.The ligand has been made affinity to the said ssDNA library,some of which have heen eluted by some buffer,and some of which have been remaided used to PCR, the products of which have been the library of next affinity,so relapse.Among them, the inverse screen is done once in 3 turns,the glycine coupling in Epoxy Activated Sepharose FF to dispose of take the ssDNA stiching the sepharose specifically by affinity.During the said screen,the intensity of the eluting buffer enhanced,and the DNA aptamer binding to the 6-APA has been screened by 25 turns SELEX.The last turn PCR product and the T Vector have been connected,transformed and cloned.10 positive colonies have been got,by selected random 12 white colonies to PCR.9 DNA sequances have been defined by sequencing,and their,first and second structures have been analyzed by the software DNASYS V2.5,which the aptamers has been divided into A,B,C,D,E five classes.Second,it is preliminarily definited D aptamer contained the the best affinity in the five classes by means of the examination to binding AMP,AMO and ceftriaxone connected to the Epoxy Activated Sepharose FF.Then,the aptamer detection range of penicillin antibiotic below 2.5μg/kg,have been definited by D group test.Eventually,it has been proved the aptamer-test to be practicable,in examination to penicillin antibiotic residue in chicken and the recovery test by microoraganism-assay.