Human methyl guanine methyl transferase gene eucaryon expression plasmid and construction method and application thereof

Abstract

The invention discloses a method for constructing a eukaryotic expression plasmid containing a human O6-methylguanine-DNA methyltransferase (MGMT) gene and application of the eukaryotic expression plasmid to the research of drug-resistant genes. The method concretely comprises the following steps: RT-PCR is utilized to obtain cDNA of an MGMT gene from a human liver tissue, and the cDNA is cloned into a T vector and performs restriction enzyme recombination with the eukaryotic expression plasmid containing enhanced fluorescent protein to construct the eukaryotic expression plasmid containing the human MGMT gene. The eukaryotic expression plasmid containing the human MGMT gene is transfected into a K562 cell and is subjected to pressure selection to construct a steady K562/MGMT transfection cell system. The invention also comprises application of the plasmid to the change of the drug resistance of tumor cells and normal cells. The method for constructing the plasmid is based on the prior experimental conditions of cell and molecular biology, does not need large-scale and complicated experimental facilities, has simple and convenient operation, and is safe and high-efficiency. The constructed plasmid has good physical and chemical stability, is easy to detect after the plasmid is transfected into the cell, and has high abundance expression. The method provides new experimental resources and means for MGMT-related research of the drug-resistant genes as well as gene treatment and research of tumor, and is worthy to be promoted and applied.

Description

technical field [0001] The invention relates to the cloning of a gene, the construction of a novel eukaryotic expression carrier and its application in the research of blood system diseases and tumors. Specifically, it involves cloning the "methylguanine methyltransferase" gene from human liver tissue, constructing a eukaryotic expression plasmid containing fluorescent protein, and applying it to the study of drug resistance in cells. Background technique [0002] Gene therapy has become the most eye-catching research hotspot in the field of tumor biotherapy, and it is another promising treatment mode after surgery, radiotherapy, chemotherapy and immunotherapy. Tumor gene therapy includes a variety of technologies and methods, such as antisense nucleic acid blocking technology, suicide gene introduction and so on. A relatively new strategy is to introduce drug resistance gene (Drug Resistance Gene) into normal tissue cells through a suitable carrier, so as to enhance the re...

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Problems solved by technology

While killing tumor cells, chemotherapeutic drugs also have varying degrees of toxic effects on

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