Preparation method of superoxide dismutase with membrane crossing capability and high stability

Abstract

The invention belongs to the technical field of gene modification of enzyme preparations, and particularly relates to a preparation method of superoxide dismutase with membrane crossing capability and high stability. The method is characterized by comprising the following steps: extracting mRNA from geobacillus stearothermophilus, synthesizing cDNA by utilizing a reverse transcription means, designing a specific primer to massively amplify coding regions of the cDNA, linking the coding regions to an E.coli expression vector, and then transforming the E.coli expression vector into an engineering bacterium BL21 (DE3). The stability of the superoxide dismutase is enhanced by utilizing a point mutation technology, and a flexible polypeptide connexin GGGSGGGS is designed, so that the soluble fusion expression of the transmembrane peptide YGRKKRRQRRR and the superoxide dismutase is successfully realized. The method has the beneficial effects that the thermal stability of the superoxide dismutase obtained by the method can be improved to the greatest extent; and a CPP sequence YGRKKRRQRRR (i.e., a trans-activating transcriptional activator sequence) with the membrane crossing capability on an HIV-1 peptide fragment and superoxide dismutase are subjected to coupled expression, and the transmembrane performance is given to the superoxide dismutase.

Description

technical field [0001] The invention belongs to the technical field of gene modification of enzyme preparations, and in particular relates to a preparation method of superoxide dismutase with transmembrane ability and high stability. Background technique [0002] Superoxide free radicals are the culprit of human diseases and aging. Under normal circumstances, the amount of free radicals produced in the body is relatively small, which can be removed in time by its own antioxidant system. However, when the body is strongly stimulated by external machines, microbial invasion, illness, staying up late, environmental pollutants, ultraviolet radiation, ionizing radiation, etc., free radicals cannot be removed in time, and the body will be attacked by free radicals. resulting in oxidative damage. [0003] Based on the above phenomenon, it is necessary to rationally use exogenous superoxide dismutase at an appropriate time to block the invasion of free radicals to the human body, w...

AI Technical Summary

Problems solved by technology

However, at present, there are four bottleneck problems in the application of superoxide dismutase: the first is that the activity is not high enough, and it does not have the feasibility of adding end products; the second is that the stability is not good, even when it is just added to the end product It has a certain activity, but with the passage of time in storage, transportation and other links, the activity gradually decreases, and when it reaches the consumer, it is no longer active; the third is that it is impossible to realize the liquid supply. Currently, all superoxide dismutase on the market is Provided in the form of freeze-dried powder, it is extremely inconvenient to quantitatively reconstitute during use; fourth, superoxide dismutase itself is a biological macromolecule, and even the molecular weight of the subunit is above 20,000, and the cell permeability is not good , not easy to be absorbed by cells, and not easy to scavenge superoxide free radicals inside cells

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