CD20/486 SsDNA Aptamers Screening By SELEX And Its Application In Tumor Markers Protein Microarray Construction

ObjectiveRapid screening and treatment of cancer is a worldwide problem.The purpose of this study is to use the high sensitivity chemiluminescence protein chip technology(iPDMS protein chip)to achieve efficient screening of a variety of tumor-related surface markers with low abvmdance.Various tumor-related surface markers obtained by screening were analyzed,and CD20,which is highly co-expressed on 8 tumor cell membranes,was selected as the research object to design and screen the CD20 nucleic acid aptamer,and to explore its tumor detection effect,s0 as to lay a foundation for the further use of CD20 aptamer in clinical targeted therapy of tumor.Methods1.Preparation and detection of PDMS chip:A surface-initiated polymerization initiator with olefin terminal is added to the traditional polydimethylsiloxane(PDMS)material and fixed into the three-dimensional structure of PDMS through thermal crosslinking(silicon hydrogen bonding)to obtain a new material,namely modified silica gel iPDMS.Poly(OEGMA)polymer brushes are synthesized from active initiation sites by surface-initiated atom transfer radical polymerization(SI-ATRP),i.e.iPDMS materials capable of resisting non-specific adsorption of proteins are obtained.The tumor-associated antigens are high-throughput printed on specific areas of the chip by using spray point printing technology,and 48-hole iPDMS chip microplates are assembled Two rounds of microarray screening were carried out in 1152 patients with common tumors and 192 healthy persons by using the iPDMS chip to detect tumor-associated autoantibodies in serum samples.2.Screening of CD20 aptamers:The ssDNA aptamers with specificity and high affinity binding to CD20 major extramembranous region and cytoplasmic region fragment CD20/486,a molecular marker on the surface of tumor cells,were obtained through screening by systematic evolution of ligands by exponential enrichment(SELEX)9 and the sequence and conformation of the screened CD20/486 aptamers were detected and analyzed;The binding ability of the aptamer to the target molecule and the application of pathological histochemical staining can lay the foundation for further use of CD20/487 aptamer in the targeted therapy and diagnosis of clinical tumors.In this study,the full sequence of human CD20 gene was got from Genebank,and the nucleotide sequence of 486 base pairs near the C terminal,which include all the sequences of the extracellular peptide and cytoplasmic segment were cloned.According to the degeneracy of codons,some codons are modified so that they canbe expressed in prokaryotes.Then CD20/486 was constructed into pGEX--4T,and the fusion protein GST-CD20/486 was expressed in Escherichia coll The next work is to select the specific aptamer that bind to CD20/486 by SELEX,using GST-CD20/486 as a positive target,and GST as a negative target.Firstly,an 80nt single stranded DNA(ssDNA)library was synthesized,with 40 random sequences.The random ssDNA library was amplified to dsDNA by PCR,and conserved as initial template.Before each select round,the ssDNA random library of each round of screening was amplified by asymmetric PCR,and used as screening aptamer library.After 10 rounds of screening,ssDNA was amplified into dsDNA,which was connected to the T vector and sequenced.DNAMAN software was used to analyze the primary and secondary structure of the determined sequences and compare their homology.The binding affinity of the single aptamer with CD20/486 was deteced by ELISA.And the dissociation constant(Kd)between aptamers and target selecting CD20/486 were earied out by FCM.The aptamer with the highest affinity was labeled with biotin and used for histochemxcal staining of clinical tumor pathological specimens,and the color was developed with SABC reagent.Meanwhile,conventional HE staining was used as the control to determine the effect of the aptamer obtained by screening for tumor diagnosis.Results1.iPDMS chip technology for tumor screening was established to detect 1152 clinically confirmed tumor samples.It was found that the expression of vascular endothelial growth factor 1,vascular endothelial growth factor 121,vascular endothelial growth factor and CD20 were differently expressed in 8 common tumor patients.The antibody levels of VEGF1,VEGF121 and VEGF in serum of tumor group were significantly higher,and the anti-CD20 antibody level was significantly lower than that of healthy control group(P<0.05).2.After modification of CD20 protein c-terminal 486bp gene,GST-CD20/486 protein,was successfully expressed and purified in prokaryotic cells,and aptamers that specifically bind CD20/486 were screened by SELEX technology.The aptamer selected in the 10th round was cloned into T vector to obtain the sequencing results of 22 clones,which were named SW1 to SW22.The single aptamer SW11 showed the strongest binding ability with CD20/486,and the dissociation constant(Kd)was 28±6 nM.3.Biotin-labeled single aptamer SW11 can bind to tunor cell CD20 in tumor pathological sections when used as diagnostic molecular for the detection of pathological tissues.Conclusions1.A chemiluminescent labeled protein chip of iPDMS was successfully constructed,which can be used for the screening of tumor-associated antibodies or autoantibodies.The CD20 antibody obtained from tumor screening in this study could be used as a potential tumor marker and provide an entry point for tumor diagnosis and mechanism analysis.2.The ssDNA aptamer SW11 that specifically binds to GST-CD20/486 protein was successfully screened by SELEX technology for the first time,which is expected to be a new diagnostic reagent for tumor detection and lay a foundation for further clinical targeted therapy.