DNA Aptamer Selected Against CD3 Protein And The Study Of Biological Function

Background The tumor has become the main disease of the world.Tumor microenvironment and cell regulation play a very important role in the occurrence and development of cancer.The majority of patients are treated with chemotherapy,radiotherapy and surgery in the treatment of tumors,but the tumor has not yet been effectively controlled.Immune therapy as a new security concept is gradually accepted.Looking for a target has become a new hotspot of tumor biological treatment.The therapy targeting tumor angiogenesis has a good application prospect.The basis of anti-tumor immunity is mainly composed of T lymphocytes.The growth of tumor can be inhibited by immunotherapy combined with anti-tumor angiogenesis therapy.Therefore,T lymphocytes combined with tumor endothelial specific markers become a new therapeutic strategy.Aptamers are single-stranded oligonucleotide(DNA or RNA)molecules,with the high specificity and high affinity and screened through SELEX in random nucleotide library.Aptamer has more advantages,such as a molecular target a wide range,good stability,easy preparation,high affinity and high specificity.It has more widely used than antibodies.Aptamers has a good application in the clinical diagnosis and treatment has,and widely recognized with tumor development.Objective In vitro selection and biological effects detection of DNA aptamers specifically against CD3 to obtain DNA aptamers.We synthesis bispecific aptamer CD3-CD105.We suggest a new method which has potential applications for the targeted diagnosis and treatment of tumor targeting.Methods Application of Cell-SELEX method and the related literature,the42 bp fixed primer sequence synthesis both ends,a 45 bp random sequence,a 87 bp the full-length ss DNA random library.The artificial construction stably transfected CD3 of 293 T cell line(293T-CD3)as a positive screening cell,using293 T cells with the empty vector as control cells.Gradually increase the screening intensity and screening efficiency.When the enrichment degree of random library achieved balance,the PCR products were cloned and sequenced.The sequencing results were enriched in structural analysis and the two level structure of the use of software.We select and synthes oligonucleotide sequences representative with fluorescently labeled nucleic acid.Aptamers were detected by flow cytometry.The use of aptamer with FITC labeled by fluorescence microscopy imaging and the affinity of aptamer was detected.The aptamer with the highest affinity and the strongest specificity was chosen.Finally,the effects of bsa CD3-CD105 on T cell function anti-tumor were investigated in vitro.Results 1.Optimize the annealing temperature and number of cycles to improve the amplification efficiency and the optimal annealing temperature was53 °C.2.After the 15 round screening,Kd value of CD3-4 against 293T-CD3 aptamer is only 18.56 ± 1.49 n M.3.It was detected at different temperatures in4 °C and 37 °C.CD3-4 could bind to CD3 expressed cells,confirmed that these aptamers have a good biological effect,are were not affected by environmental temperature.4.bsa CD3-CD105 can promote T cell activation and proliferation,and induce T lymphocytes to kill Hep G2 cells.5.bsa CD3-CD105 combined with PBMC can inhibit tumor growth in tumor bearing SCID mice,and can prolong the survival time of mice.Conclusion The SELEX screening successfully screened aptamers for the T cell surface molecules CD3 nucleic acid.bsa CD3-CD105 can inhibit tumor growth and prolong the survival time of the tumor bearing mice.It provides new clinical application for the prevention and treatment of cancer.