Preparation method and application of MYC tag fusion expression vector

Abstract

The invention provides a preparation method of an MYC tag fusion expression vector, which comprises the following steps: using a KpnI / XhoI double-enzyme digestion pGL3 Basic vector to obtain a recovered product of the KpnI / XhoI double-enzyme digestion pGL3 Basic vector, using a pRGEB32Bar-Cas9 plasmid as a template to obtain a 2 * 35S promoter PCR recovered product, carrying out a ligation reaction to obtain an intermediate vector pGL-35S, carrying out double-enzyme digestion, connecting an MCS annealing primer to obtain an intermediate vector pGL-35S-MCS, carrying out double-enzyme digestion, connecting a Nos Ter sequence, carrying out double-enzyme digestion, and carrying out purification to obtain the MYC tag fusion expression vector. The MYC tag fusion expression vector is characterized in that an intermediate vector pGL-35S-MCS-Nos is obtained by using an MYC tag as a template, the intermediate vector pGL-35S-MCS-Nos is connected with a connecting peptide Linker primer subjected to denaturation annealing after double enzyme digestion to obtain an intermediate vector pGL-35S-MCS-Linker-Nos, and the intermediate vector pGL-35S-MCS-Linker-Nos is connected with an MYC tag annealing primer after double enzyme digestion to obtain the MYC tag The MYC tag fusion expression vector prepared by the invention has a small framework, has a full length of 4052bp, and is suitable for plant protoplast transformation.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a preparation method and application of a MYC tag fusion expression vector. Background technique [0002] The MYC tag is an epitope tag derived from the proto-oncogene c-Myc. The MYC tag contains 10 amino acids, the sequence is EQKLISEEDL, and the molecular weight is about 1.2KDa. In the research of protein expression, protein interaction, etc., the target gene to be studied can be connected with the MYC tag sequence by means of genetic engineering technology. The MYC tag can be fused to the N-terminal or C-terminal of the target protein, and then the "target gene- The expression vector after MYC" fusion is transferred into bacteria, yeast, animal or plant cells. On the one hand, because the molecular weight of the MYC tag is very small, it will not cover the protein epitope and structural domain in the fusion protein, and it is not easy to change the func...

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Problems solved by technology

Although this method can be used to obtain a large amount of expressed "target gene-MYC" fusion protein, the tobacco seedling cultivation cycle is long, and the transformation steps mediated by Agrobacterium are complicated.

In addition, the tobacco system cannot simulate the environment of different plants, and the research on protein function and protein interaction, especially when using the "target gene-MYC" fusion protein carrier system to find the interaction protein of the target gene in plants, requires "purpose Gene-MYC" fusion vector should be expressed in the target plant under study, at this time, the application of Agrobacterium-mediated transformation methods in other plants such as Arabidopsis, rice, and maize is hindered

[0004] PEG-mediated transformation of plant protoplasts is currently the most widely used fusion vector expression system, but the pCambia1300 framework is too large, the transformation efficiency is low, and the copy number of the pCambia1300 framework is low, which cannot meet the large number of plasmid requirements of the protoplast transformation system

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