Selection And Diagnostic Evaluation Of Aptamers Against Arabinogalactan And Mycobacterium Tuberculosis Neoantigen Peptide

PART Ⅰ Selection and diagnostic evaluation of aptamers against arabinogalactanBackground and objectiveTuberculosis(TB)is a chronic infectious disease caused by Mycobacterium tuberculosis(MTB).The specific cell wall structure of MTB is essential for its survival and replication,which is composed of mycolic acid(MA),arabinogalactan(AG)and peptidoglycan(PGN)by covalent bonds.AG plays an important role in maintaining the integrity of cell wall owing to linking MA in the outer cell wall and PGN in the inner cell wall,and is special to mycobacterium.AG antibody is not available until now.Moreover,it’s notoriously difficult to generate antibody against carbohydrates such as AG.Aptamers isolated by a method called systematic evolution of ligands by exponential enrichment(SELEX)that bind to targets including proteins,virus and cells with high affinity.Therefore,the purpose of this study was to screen the aptamers of mycobacterial AG by SELEX technology and use it for the detection of MTB.MethodsThe 78 bp random single-stranded DNA library was synthesized in vitro.SELEX technology was applied for the screening of aptamer library against mycobacterial AG from the random single-stranded DNA library.The enriched aptamers were cloned and sequenced.DNAMAN package was used to analyze the secondary structures of the aptamers,MEGA software was used to analyze the phylogeny tree of the aptamers and enzyme-linked oligonucleotide sorbent assay(ELOSA)was employed to determine the binding affinity between aptamers and AG,the dominant aptamers were selected.48 bacteria strains including mycobacterial strains and nonmycobacterial bacteria were used to assess the binding specificity of FITC-labeled aptamer against AG by fluorescent microscopy observation.ResultAfter seven rounds of aptamer selection,33 aptamers against AG were obtained.One preponderant group and five high-affinity aptamers were obtained by analyzing the dynamic evolution of aptamers and the affinity of aptamers to AG.FITC-labeled aptamer MAA5 was further used for the detection of selected bacterial strains.The results demonstrated that aptamer MAA5 bound with all MTB strains instead of nonmycobacterial bacteria.ConclusionThe aptamers against mycobacterial AG was successfully screened by SELEX technology and showed potential value for the identification and detection of MTB.PART Ⅱ Screening and diagnostic evaluation of mycobacterium tuberculosis neoantigen peptideBackground and objective At present,the epidemic situation of tuberculosis is still very serious.The vast majority of groups in the society is the potential target of Mycobacterium tuberculosis(MTB)infection,and the occurrence of drug-resistant TB is also increasing continuously.Therefore,the prevention and cure of tuberculosis is still an arduous task.The research and development of new diagnostic techniques,drugs and vaccines are very important to control the current and future Tuberculosis(TB)epidemic.The screening of MTB neoantigen peptide provides a basis for the new diagnosis of tuberculosis and the development of TB vaccine.Therefore,the purpose of this study is to screen neoantigen peptide of MTB,evaluate its diagnostic efficacy through clinical sample testing,and identify the dominant antigen peptides with good performance for TB diagnosis.Methods The dissected granuloma tissues of pulmonary tuberculosis patients were lysed,and the human leukocyte antigen(HLA)-peptide complex was co-precipitated with HLA antibody by immunoprecipitation.The sequences of the peptides derived from MTB in the HLA complex were identified by high performance liquid chromatography-mass spectrometry(HPLC-MS),and the identified antigen peptides were synthesized.Whole blood samples of pulmonary TB patients and patients with other pulmonary diseases and healthy subjects were collected,antigen peptides were grouped,and the diagnostic efficacy of antigen peptides was evaluated using the TBIGRA kit and compared with the diagnostic efficacy of the commercial kit.Result A total of 57 peptides were identified to be derived from MTB by HPLC-MS,the antigen peptide synthesis and grouped,TB-IGRA kit and antigenic peptide were used to detect 103 whole blood samples and evaluate the detection effect.The results show that the sensitivity and specificity of antigenic peptides in 103 samples were 34.37% and 74.35%,respectively,and the sensitivity and specific of the kit in 103 samples were 93.75%,64.10% respectively.Subsequently,the 25-57 antigen peptide presented by HLA-Ⅱ were regrouped,and a total of 130 samples were detected.The results showed that the sensitivity and specificity of antigen peptide in 130 samples were 51.43% and 93.33%,respectively,and the sensitivity and specificity of kit in 130 samples were 85.71% and 91.67%,respectively.In addition,a dominant antigen peptide group was screened in the second screening,with sensitivity and specificity of 51.16% and 94.92%,respectively.Conclusion The neoantigen peptide of MTB was identified from the granuloma of tuberculosis patients,which showed potential diagnostic value for tuberculosis.Among them,the dominant antigen peptide group has good application value,which can be further optimized for tuberculosis diagnosis or vaccine development.