Screening and identification method for aptamer of T cell immunity check point PD-1 and antitumor application

Abstract

The invention relates to a screening and identification method for an aptamer of a T cell immunity check point PD-1 and antitumor application. The method comprises the following steps: 1) pretreatmentof an ss DNA library of dry powder; 2) positive screening; 3) negative screening; 4) PCR amplification; 5) single stranded DNA preparation; 6) multiple screening; 7) cloning and sequencing; 8) flow cytometry detection; 9) immunofluorescence detection; 10) detection and separation; 11) slicing; and 12) detecting of closeness of Bi-Apt. According to the method, the aptamer in regard to a protein PD-1 is screened from cells, the target protein is expressed on surfaces of cell membranes, stable expression of target molecules PD-1 is achieved through individually constructing a cell line 293T, T cells are selected, immunity-suppressant target points PD-1 of surfaces of the T cells are closed, specific-expressive PD-1 molecules are selected in the aspect of target molecules, and thus, a whole screening process is completed. The method can be tested and applied to antitumor clinics of human bodies and provides a very good method for clinical use in human tumor resisting.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to an aptamer screening and identification method and anti-tumor application of T cell immune checkpoint PD-1. Background technique [0002] Aptamers have a relatively stable 3D structure under certain conditions, and their 3D structure can be affected by the following factors, including the molecular sequence of nucleotides and sequence length (usually less than 100nt), specific environmental conditions (such as temperature, etc.) variety of its structure. For the target molecule, the aptamer changes its own conformation through folding, and forms a shape complementary specific binding site with the target molecule under the three-dimensional structure. In immunotherapy, the application of aptamers has its natural advantages over previous antibody therapy. Antibody therapy often leads to certain immunotoxic effects due to its immunogenicity, namely immune-related side effec...

AI Technical Summary

Problems solved by technology

As we all know, antibodies have poor stability and often require special handling during transportation and storage; and studies have found that short-chain nucleotides can be stored for twenty years in a dry and cool environment

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