Engineering strain construction method for improving activity of heparinase I

Abstract

The invention relates to an engineering strain construction method for improving the activity of heparinase I. An amino acid sequence is optimized according to spatial structure analysis on the heparinase I to obtain Hep169 with the activity improved by 48% compared with the heparinase; a Hep169 gene is optimized according to codon preference, a gene DNA is obtained through artificial synthesis and is cloned into an expression vector to perform fusion expression with SUMO and other labels, host cell conversion and screening are performed to establish a soluble genetic engineering production system of the Hep169, analysis results show that a target protein obtains an efficient soluble expression, has very good biological activity and can efficiently crack heparin to generate low-molecular-weight heparin. By the adoption of the method, the highly active HepI is provided, a new method is provided for efficient soluble expression gene engineering production of the heparinase I, the production cost of the low-molecular-weight heparin and other drugs can be effectively reduced, and the engineering strain construction method has wide application prospect.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a highly active heparanase I and a production method for expressing the high-efficiency soluble gene engineering thereof. Background technique [0002] Heparin is a natural anticoagulant substance in animals, which has been used as the main anticoagulant drug, and studies have shown that in addition to anticoagulant activity and related antithrombotic activity, heparin also has the ability to inhibit smooth muscle cell proliferation, anti Biological functions such as inflammation, anti-tumor and anti-virus. Although heparin is still used in clinical treatment, its side effects are becoming more and more obvious. For example, long-term use of heparin can easily cause massive bleeding, induce thrombocytopenia and thrombosis, affect platelet stability and allergic reactions and other symptoms. However, low molecular weight heparin has been widely used clinically due to its good injection a...

AI Technical Summary

Problems solved by technology

[0006] The object of the present invention is to overcome the shortcoming of current low heparanase activity, provide a kind of highly active HepI and its high-efficiency soluble genetic engineering expression method, the engineering bacteria obtained by the present invention can soluble express highly active HepI, therefore not only reduce the inclusion body Enzyme activity loss during the denaturation process shortens the production process, reduces production costs, facilitates better large-scale industrial production of HepI, and can be more efficiently used in the industrial production of low molecular weight heparin

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