Deubiquitination mutant of mda-7/IL-24

Abstract

The invention relates to a lysine site mutant of conjugated ubiquitin of mda-7/IL-24 and application thereof, belonging to the field of genetic engineering. In the invention, lysine codons of 63rd, 69th, 78th, 119th, 123rd, 136th, 179th, 189th, 203rd and 206th potential conjugated ubiquitin sites of the mda-7/IL-24 are mutated into arginine codons by utilizing a site-directed mutation PCR (Polymerase Chain Reaction) technology and cloned to pcDNA3.1(+) plasmids; after the mutant plasmids are transfected with cervical carcinoma Hela cells and hepatoma carcinoma HepG2 cells, the expressions of mda-7/IL-24 protein and ubiquitination mda-7/IL-24 protein are respectively detected. A result shows that the quantity of the mda-7/IL-24 protein expressed by a 123rd lysine mutant of the mda-7/IL-24 is remarkably increased, and the quantity of the ubiquitination mda-7/IL-24 protein is remarkably decreased. The invention proves that the 123rd lysine of the mda-7/IL-24 is an ubiquitination site, and mda-7/IL-24 mutants generated by the mutant plasmids are difficult to be degraded by the in-vivo ubiquitin, thereby enhancing the expression and the action of the mda-7/IL-24 in tumour cells.

Description

technical field [0001] The invention belongs to the field of genetic engineering. Specifically, an mda-7 / IL-24 mutant. The present invention relates to the use of PCR site-directed mutation technology to mutate the 123rd lysine in the mda-7 / IL-24 amino acid sequence into arginine, so that it loses the function of binding with ubiquitin, so that it is not affected by ubiquitin- Degradation by the proteasome system. The mda-7 / IL-24 mutant polypeptide or its expression carrier acts on tumor cells, enabling mda-7 / IL-24 to efficiently and continuously exert an anti-tumor effect. Using this mda-7 / IL-24 can treat various tumor diseases. Background technique [0002] (1) Discovery and structure of mda-7 / IL-24 [0003] In 1995, Fisher et al. used β-interferon and protein kinase C activator Mezerin to induce human melanoma cell HO-1, and identified and cloned melanoma differentiation associated gene-7 (melanoma differentiation associated gene-7, mda-7), and found that mda-7 produ...

AI Technical Summary

Problems solved by technology

[0015] Pancreatic cancer progresses rapidly, has a high mortality rate, and currently has no effective treatment

2001 Su et al. [18] Ad.mda-7 was used to treat pancreatic cancer, and it was found that it could not inhibit the growth of pancreatic cancer cells

Due to the wide variety of E3 enzymes, the lack of sequence homology among different types of E3, the composition of various E3s varies greatly, some are monomeric proteins, and some are large multi-subunit complexes, so it is difficult to inhibit E3 ligases

Although some structural domains in the E3 enzyme have been identified, these domains all play a role in binding to E2, but this specificity is much smaller than that of E3 for substrate selection

At the same time, studies have confirmed that the post-translational modification of some substrate proteins will have a greater impact on the binding of E3-substrates

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