Establishment and application of wheat germ cell-free protein synthesis system for high level expression of snake venom kininogenase

Abstract

The present invention provides a wheat germ cell-free protein synthesis system for high level expression of recombination snake venom kininogenase. According to the system, snake venom kininogenase gene of agkistrodon halys pallas is cloned through reverse transcription, wheat germ cell-free expression plasmid pCS<2+>/VK is constructed, a wheat germ extract is prepared, an in vitro transcription and translation system is established, the snake venom kininogenase is expressed and the system is optimized. The N terminal of the exogenous protein expressed by the expression system has the His protein tag, and a Ni column elution method is adopted to carry out purification, such that the subsequent purification of the recombinant protein is substantially simplified, the purification efficiency is high, and the cost is low. The exogenous protein further has biological activity, and provides the foundation for the next clinical study.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a wheat germ cell-free protein synthesis system for highly expressing human snake venom kininogenase, its construction method and application. Background technique [0002] Kinikogenase is a proteolytic enzyme in animals that exists in the tissues of many organs. Pancreatic kininogenase in snake venom is the earliest known and most clearly studied. Pancreatic Kallidinogenase, also known as Pancreatic Kallikrein, is a type of endo-proteolytic enzyme that exists in various tissues in mammals, especially in snake venom. It acts on kininogen in the body to release kinin, thus exerting a series of pharmacological effects: dilating microvessels, improving peripheral blood circulation; promoting prostaglandin secretion, dilating small arteries, increasing renal blood flow; activating fibrinolysis The enzyme system hydrolyzes fibrin, reduces blood viscosity, prevents thrombus f...

AI Technical Summary

Problems solved by technology

[0003] At present, the kininogenase used clinically in China is extracted from the pancreas of mammals. The purity is not high and it has certain antigenicity. After entering the human body, it may induce a series of antigen-antibody reactions. It has been reported that the injection of pancreatic hormone Peptidase leads to fixed drug eruption and severe erythema multiforme drug eruption, and intravenous administration cannot be realized, so that the drug cannot exert its maximum effect

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