Prokaryotic expression method of oncoprotein

Abstract

The present invention discloses a prokaryotic expression method of oncoprotein. The method comprises the steps of: extracting total RNA in liver cancer cells HepG2 and conducting reverse transcription to obtain cDNA pool; designing specific oligonucleotide primers; conducting PCR amplification by using the cDNA pool as a template and the designed primers, and then by enzyme digestion and connection to clone part of the MDM2 gene into a pET28a (+) expression vector to obtain a recombinant expression vector; introducing the recombinant expression vector into E. coli expression strains to construct genetically engineered bacteria containing the recombinant expression vector; culturing the genetically engineered bacteria at 37 DEG C to OD600 at about 0.6-0.8, and inducing the expression of the recombinant MDM2 gene in the genetically engineered bacteria; centrifuging, collecting thalli, washing twice, centrifuging, discarding the supernatant and collecting thalli. The invention greatly increases the experiment efficiency of laboratory research based on MDM2 protein, and greatly reduces experiment cost.

Description

technical field [0001] The invention relates to the field of biology, in particular to a method for prokaryotic expression of oncoproteins. Background technique [0002] Tumor is one of the major high-incidence diseases that seriously threaten human health, and has become the first cause of death of residents in my country. The outline of my country's science and technology development plan has listed the basic and applied research of malignant tumors as the key to public health and the prevention and treatment of important diseases Scientific question. Therefore, the research of novel anti-tumor drugs is not only a compelling need for clinical tumor treatment and improving the health level of our people, but also has great significance for the development of my country's pharmaceutical industry and society. Studies have found that the p53 gene is the most relevant tumor suppressor gene for human tumors. It plays an important role in DNA transcription, cell growth and prolif...

AI Technical Summary

Problems solved by technology

This negative feedback pathway keeps the ratio of p53 and MDM2 protein in cells constant. However, in human tumor cells, MDM2 protein will be overexpressed and accumulated for various reasons. Once MDM2 is overexpressed, the negative feedback regulation between p53 and MDM2 will be out of balance

In some tumor cells, the p53 activation mechanism due to external stress is considered insufficient to overcome the negative feedback transcriptional activation domain downstream regulation of MDM2, resulting in insufficient growth arrest and apoptosis