Screening And Study Of Aptamers For Gastric Cancer Cell Secreted Protein Based On The Med-SELEX Technology

Gastric cancer is not only one of the most common cancers in recent years,but also one of the very high mortality cancers.However,due to the lack of obvious symptoms of early gastric cancer,the screening rate is low,leading to the diagnosis of patients with advanced gastric cancer.The treatment effect of gastric cancer is poor,the healing is low,and the survival rate is low.Therefore,early diagnosis and treatment are of great significance for patients with gastric cancer.In addition,nucleic acid aptamers,as chemical antibodies,are widely used in clinical diagnosis and drug research and development because of their low cost,easy modification,good stability,good tissue permeability and no immunogenicity.In this study,the magnetic bead-based Media-SELEX(Med-SELEX)technology was used to screen nucleic acid aptamers that specifically target the secreted proteins of gastric cancer cells.In the screening process,SGC-7901 gastric cancer cell culture medium and SNU-1 gastric cancer cell culture medium were used as positive screening targets,and HFE-145 normal gastric epithelial cell culture medium was used as negative screening targets.The specificity of the aptamer was improved by increasing the screening pressure and replacing the screening target.A total of 12 rounds of screening were conducted.Through Shanghai Sangon high-throughput sequencing,homology analysis,secondary and tertiary structure prediction,and G-quadruplex analysis,4 candidate aptamers was selected for subsequent validation.Through the validation of affinity and specificity,APT-1 was selected as the optimal aptamer.The results of q-PCR showed that the aptamer APT-1 not only had high affinity for gastric cancer cell culture medium,Kd value was about 5 n M,but also had good affinity for gastric cancer serum.The specificity of aptamer APT-1 was verified by magnetic bead coupling method.The RFU value of aptamer APT-1 to gastric cancer cell culture medium was significantly higher than that of other cell cultures,indicating that aptamer APT-1 had good specificity.Further verification by q-PCR and quantum dot method showed that the aptamer had good specificity for gastric cancer serum.The stability test of APT-1 in serum and its effect on cytotoxicity and migration showed that the aptamer APT-1 was gradually degraded after 24 hours in serum,and had no obvious toxicity to cells and did not affect cell migration.In this study,the aptamer APT-1 with good affinity and high specificity for gastric cancer serum was finally obtained through Med-SELEX technology,and the aptamer is not easy to be degraded,and is non-toxic to cells,which has certain application value for the early diagnosis of gastric cancer.