A kind of lentiviral single plasmid in vivo biotinylation vector and its preparation method

Abstract

The invention belongs to the field of protein expression and purification and stem cell technical research and particularly relates to a slow virus single-plasmid in-vivo biotinylation vector and a preparation method. The slow virus single-plasmid in-vivo biotinylation vector comprises a framework carrier pCDH-MCS-T2A-PURO-MSCV, an FLAG tag, a biotinylation identification tag AVI, self-splicing peptides P2A, an HA tag and a biotin ligase BirA gene sequence. The in-vivo biotinylation vector is used for preparing biotinylation interest protein, and then protein-protein interaction or protein-nucleic acid interaction is researched through streptavidin-biotin affinity purification. Compared with a traditional immunoprecipitation experiment method, the slow virus single-plasmid in-vivo biotinylation vector has higher specificity and affinity, and more accurate experiment results can be obtained.

Description

technical field [0001] The invention belongs to the field of protein expression and purification and stem cell technology research, and specifically relates to a biotinylated carrier in a single plasmid of lentivirus and a preparation method thereof. Background technique [0002] Co-immunoprecipitation and chromatin immunoprecipitation are the most classic and convincing experiments to study protein interaction and protein-nucleic acid interaction. The principle of the traditional co-immunoprecipitation method is to incubate the protein (antigen) with its specific antibody. While the antibody enriches the corresponding antigen, it also enriches other proteins that interact with the antigen protein molecule, and then analyzes the interacting protein by Western blotting or mass spectrometry. . The traditional chromatin immunoprecipitation also uses the principle of antigen-antibody interaction to enrich the nucleic acid bound to the antigen protein, and then conducts follow-u...

AI Technical Summary

Problems solved by technology

[0005] At present, there are some commercial biotinylation vectors in mammalian cells, but there are the following limitations: 1. The target protein and BirA gene are expressed in two vectors respectively, and the biotin modification level is relatively low; 2. Most of the promoters are Widely expressed promoter without expression specificity; 3. The vector backbone is a common plasmid vector, and it is difficult to obtain stably expressed biotin-modified proteins

4. Expensive and limited use

The price ranges from 5,000 yuan to tens of thousands of yuan, and the manufacturer has linearized the vector, which limits the amplification of the backbone plasmid

Images