Induced expression method of recombinant human endostatin

Abstract

The invention discloses an induced expression method of recombinant human endostatin. The method comprises the following steps: fermenting engineering bacteria until the OD600 value of fermentation liquor is 10-15, adding an inducer isopropyl-beta-d-thiogalactoside (IPTG), a carbon source substance and a nitrogen source substance, so that the concentration of the IPTG in the fermentation liquor is 0.2-0.4mmol / L; cultivating at 30-37 DE C for 2.5-3.5 hours; adding the inducer IPTG, so that the concentration of the IPTG in the fermentation liquor is 0.1-0.3mmol / L, and continuing to cultivate for 3-5 hours. By adopting the induced expression method of the recombinant human endostatin disclosed y the invention, the requirements of large-scale production of the recombinant human endostatin can be met, and efficient induced expression is achieved at high cell concentration.

Description

technical field [0001] The invention belongs to the field of extraction and purification of recombinant human vascular endostatin, and specifically relates to a method for inducing expression of recombinant human vascular endostatin. Background technique [0002] Human vascular endostatin is currently the most effective inhibitor of tumor angiogenesis with the best experimental effect. Experiments have shown that endostatin can inhibit vascular endothelial cells and growing blood vessels, and can better inhibit tumor growth and metastasis. In terms of its mechanism of action, human endostatin inhibits the blood supply of tumor tissue in the human body, causing the tumor to lack nutrients and oxygen and stop growing, and then gradually shrink until death. , has the advantage of no obvious toxic and side effects, and thus has received extensive attention from the medical community. [0003] At present, human endostatin is mainly prepared by obtaining engineering bacteria car...

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Problems solved by technology

[0005] If the above-mentioned method is adopted in large-scale industrial production, a large amount of culture medium and culture solution will be consumed to keep the cell concentration of the bacterial fermentation liquid at a low level, while increasing the cell concentration and synchronously increasing the amount of the inducer. Not only will the engineering bacteria of recombinant human vascular endostatin rapidly increase their tolerance to the inducer, but their sensitivity will decrease significantly, and then the phenomenon of inducing the expression of recombinant human vascular endostatin protein will inevitably decrease, and it will not be possible to obtain At the same time, at the same time, under the high concentration of bacteria, the culture medium and culture solution with higher concentration are also required, and the bacteria must grow faster in this environment, and the nutrients are easy to be exhausted prematurely. Cause premature aging of bacteria and autolysis, unable to obtain ideal expression

In either case, it means that the production efficiency of recombinant human endostatin in large-scale production is extremely low, which will cause a large amount of raw material consumption

Therefore, although the expression amount of the above-mentioned method can reach 38%, the expression amount can only be obtained in a small amount of culture fluid and the OD of the fermentation broth. 600nm The value is realized under the condition of 0.4-0.6. In other words, the above method can only achieve high-efficiency induction expression at low concentration of engineering bacteria in small experiments, and then obtain a very small amount of product, which cannot be applied to industrialization

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