In Vitro Selection Of Pepsin Aptamers

Gastric hemorrhage is a common and critical gastrointestinal disease.Due to its special focus,the gastric juice and pepsin prevent thrombin from hemostasis.Therefore,inhibiting pepsin activity is the key to treating this type of disease.In addition,the content of pepsin in the serum of patients with gastric hemorrhage is much higher than that of normal people.So,detecting the changes of serum pepsin is also an effective method to diagnose gastric hemorrhage painlessly.Aptamers are oligonucleotide chains of single-stranded DNA or RNA with three-dimensionil structure.Recently,they have been used to diagnose and treat various diseases,for they could bind to definded molecular target with high specificity and high affinity.The purpose of this study is to screen and identify pepsin aptamers from 80nt(nucleotide)DNA random oligonucleotides,in vitro,with SELEX(systematic evolution of ligands by exponential enrichment)technique.Then,the aptamers would be labeled with biotin to establish ELADA(Enzyme-linked aptamer direct assay)method for pepsin.It may be useful for clinical diagnosis and treatment of gastric hemorrhage in the future.The results were followed.1.The pepsin was linked to Dynal(Dynabeads M-280 Carboxylic Acid)magnetic bead.The initial concentration of pepsin was 1mg/MI.After coating,the concentration changed to 0.0345 mg/mL.So 98.6%pepsin linked to beads,it provded the fundtaion for the screening of apatmer.2.The 80 mer random oligonucleotide library was synthesized,the agarose electrophiresis band of 80nt ssDNA was observed near the band of 50 bp band of dsDNA ladder.After PCR,the band of 100 bp dsDNA was by the 80 bp of dsDNA ladder.3.In symmetric PCR,the appropriate heat cycles were 30 and annealing temperature is 58?.In asymmetric PCR,the proportion of two primers was 100:1 and the heat cycles were 40.The direct asymmetric PCR amplification did not get ssDNA but by-products.4.The pepsin coated on the magnetic beads was used as the target to screen its aptamer,the combined and unbound ssDNA was separated by magnetic frame,and the pepsin aptamer was screened by increasing the screening intensity(shortening the binding time,increasing the vibration frequency,etc.)and using SELEX technology such as reverse screening.In the first round of screening,the binding rate of ssDNA and pepsin was only 1.48%.With the increase of screening rounds,the binding rate of ssDNA and pepsin increased gradually.The highest binding rate of the 13th round of aptamer was 73.57%,indicating that the candidate ssDNA aptamer of pepsin was successfully screened5.Amplification,purification,TA cloning,blue and white spot selection and sequencing were carried out for the 13th round of screening products,and 27 sequences were detected.After removing the sequence with less than 40 bases from the intermediate random sequence,eight possible pepsin aptamers(No.l,No.5,no.6,No.9,No.11,No.15,No.21,No.26)were successfully obtained.6.The primary structure and secondary structure of the 8 aptamers were analyzed by related software.The results showed that the primary structure homology of the 8 aptamers was only 43.3%,and the secondary structure was mainly stem ring structure.7.The results showed that only three of the eight aptamers(NO.5,NO.6,NO.21)had a high inhibitory effect on pepsin activity.Measure the half inhibitory concentration of three aptamers,it was found that the inhibition of NO.21 was strongest.8.The selected aptamers were labeled with Biotin.According to the reacti on steps of pepsin-aptamer-biotin-avidin-horseradish-peroxidase-substrate binding,ELADA method was used to detect the binding specificity of aptamer with different types of proteins.The results showed that three aptamers only had high specificity for pepsin(P<0.01),and the specificity of the 21 was highest.So,in this study,in order to obtain the aptamers for pepsin with SELEX technique,the magnetic system was used as the separation system and non-specific binding reverse screening as improved method.After thirteen rounds of screening,ssDNA aptamers for pepsin were screened firstly.Then,the pharmacopoeia 2000 method was used to detect the inhibitory effect of aptamers on pepsin and the aptamer specific detection method was established.All of the results verified that the selected aptamer 21 has the highest specificity and inhibition rate.It provided a basis for further improving the specificity and affinity of aptamers and target substances,and would be benefit for the later clinical utilization of pepsin ssDNA aptamers in the treatment and diagnosis of gastric hemorrhage.