| Objective:Rheumatoid arthritis(RA)is a chronic,inflammatory and systematic systemic autoimmune disease,as one of the joint motor diseases that are common,frequent and difficult to treat in clinic called"immortal cancer".There is a high disability rate,with50%in 2 years and 70%in 3 years.The occasion of treatment of RA is closely related to the prognosis.The disability rate of early treatment is much lower than that of late treatment,so early diagnosis and timely treatment is very important to reduce or prevent the subsequent injury.At present,the serological detection of RA mainly includes rheumatoid factor(RF)and anti-citrullinated protein antibody(ACPA),but it is easy to be missed or misdiagnosed in patients with early or atypical RA.Therefore,finding new serum markers with higher sensitivity and specificity and establishing new diagnostic methods have become one of the urgent problems to be solved in the diagnosis and treatment of the disease.In this study,serum in patients with RA was used as biological sample,and RA highly specific serum nucleic acid aptamers were screened by systematic evolution of ligands by exponential enrichment(SELEX).The target protein was separated and extracted by the aptamer.the corresponding detection method was established to evaluate its diagnostic value.The objective of this study is to provide experimental basis for the target protein to become a serum marker of RA,so as to provide the experimental foundation for the establishment of a new and efficient diagnostic method for RA.Methods and results:1.Design and synthesis of ssDNA libraries and primers,optimization of reactionsymmetric conditions for PCR amplificationDesign of aptamer libraries and primers:the full length of ssDNA aptamer sequence was 90 nt,both ends were 18 nt fixed sequences with primer binding sites,the middle random sequence was 54 nt,and the upstream and downstream primers were P1 and P2,respectively.Optimization of reaction conditions for PCR amplification:In the PCR reaction system,Optimization PCR reaction conditions were by changing the number of cycles(8,10,12,15,18,20)and annealing temperature(54?-60?).Finally,the optimum cycle number of symmetrical PCR amplification reaction was 12 cycles and the optimum annealing temperature was 56.4?.2.Aptamer library selectionThe mixed serum of healthy persons,patients with systemic lupus erythematosus,patients with psoriatic arthritis and patients with ankylosing spondylitis were used as anti-screening substances,and subtractive SELEX was carried out for 3 rounds.With the increase of screening cycles,the number of oligonucleotide fragments binding to the anti-screening material decreased gradually,thus achieving the purpose of removing a large number of non-specific oligonucleotide fragments.The mixed serum of patients with RA was used as positive screening material for forward SELEX screening.With the continuous increase of screening times and screening pressure,the oligonucleotide fragments with weak affinity were removed,and the serum RA specific aptamer library was obtained at the 9th round of screening.3.Aptamer cloning and bioinformatics analysisThe selected serum RA specific aptamer library was cloned and sequenced commissioned to Sangon Biotech.After that,30 clones were randomly selected for sequencing.Aptamer homology analysis:the aptamer sequence homology analysis was carried out by DNAMAN software.The results showed that there were 12 repetitive sequences in 30 clones and there was no common conserved sequence.Finally,a total of 18specific aptamers were identified,which were named Rheumatoid Arthritis Aptamer 1-18(RA-Ap1-18).Secondary structure prediction of aptamer:the secondary structure of aptamer sequence was predicted and analyzed by DNAMAN V5.2.2 software.The results showed that all 18 aptamers were stem-loop structures of different forms.It was speculated that this different form of stem-loop structure may be the structural basis of specific binding with serum targets.4.Analysis of affinity and specificity of aptamerThe affinity and specificity of aptamer were detected by fluorescence spectrophotometry.The results of affinity test showed that the dissociation constant Kd had reached the concentration of nmol/L,and the aptamers RA-Ap5,RA-Ap10,RA-Ap13,RA-Ap14,RA-Ap17 and RA-Ap18 showed high affinity.The results of specific detection showed that aptamers RA-Ap5 and RA-Ap18had significant differences in normal serum,serum of systemic lupus erythematosus,ankylosing spondylitis and psoriatic arthritis(P<0.01).According to the results of affinity and specificity analysis,RA-Ap5 and RA-Ap18can be used as highly specific aptamers in rheumatoid arthritis serum.5.Preparation and identification of target proteinsThe target protein was separated and extracted by aptamer-biotin-streptavidin magnetic bead method,and the electrophoretic pure product was purified by SDS-PAGE method,and its molecular weight was about between 25 KDa and 25 KDa-32KDa.Results of ultraviolet spectrum scanning showed that there was a maximum absorption peak at 280nm,which proved the protein chemical properties of the target material.The target protein extracted by aptamer RA-Ap5 has a single electrophoretic band with a molecular weight of about 25 KDa;the target protein extracted by aptamer RA-Ap18 has two bands,one of which was about 25 KDa and the other was between25 KDa-32 KDa,indicating that both aptamers can bind to 25 KDa target protein,which suggested that:(1)the target protein of 25 KDa has two specific binding sites;(2)RA-Ap5 has higher specificity and can be used as a potential reagent for the detection of target protein.6.Establishment and methodological evaluation of fluorescence detection method for target proteinAptamer RA-Ap5,as RA specific aptamer,can indirectly detect target protein content according to the change of fluorescence intensity caused by the binding of aptamer to target protein,that is,target protein content?the difference of fluorescence intensity when aptamer binding to target protein in blank control.The corresponding fluorescence detection method of target protein was established and its methodology was evaluated.The results showed that there was a linear correlation between target protein content and the fluorescence intensity value of blank control-the fluorescence intensity value of aptamer binding with target protein(R~2=0.97).The optimum aptamer amount was 8 pmol/?l,the serum amount was 20?l,the detection range was 19.41ng/?l<X<2000 ng/?l,the intra-batch and inter-batch coefficients of variation were all less than 5,and the minimum serum amount detected by this method was 10?l.7.Evaluation for diagnostic value of target protein fluorescence detection in RAAccording to the established target protein fluorescence detection method,a small sample was used to evaluate the diagnostic value of the method.The results showed that the sensitivity of target protein fluorescence detection method in RA was 83.33%,with the specificity of 91.67%and the accuracy of 88.89%.The AUC of this method for the diagnosis of RA can reach 0.911>0.9,indicating that this method has good diagnostic value for RA.The evaluation results of large samples showed that the sensitivity,specificity and accuracy of target protein fluorescence detection for RA were 79.17%,88.54%and83.85%,respectively.The AUC of this method for the diagnosis of RA was0.871(0.7<AUC<0.9),which further proved that this method had certain diagnostic value for RA.conclusion:1.Nucleic acid aptamers RA-Ap5 and RA-Ap18 with high specificity and high affinity for RA were screened.2.The aptamer RA-Ap5 and RA-Ap18 electrophoretic pure serum target proteins were successfully extracted,and the two aptamer target proteins contained a protein with a molecular weight of about 25 KDa,suggesting that there were two binding sites in the protein around 25 KDa.The aptamer RA-Ap5 has higher specificity and can be used as a potential reagent for the detection of target protein.3.Aptamer-based target protein fluorescence detection has a certain diagnostic value in RA. |
PDF Full Text Request