Substrate protein SNVP, and coding gene and application thereof

Abstract

The invention discloses a clostridial neurotoxin substrate protein, and a coding gene and an application thereof. The clostridial neurotoxin substrate protein is a protein (a) composed of an amino acid sequence represented by sequence 2 in a sequence table, or is a protein (b) obtained through substituting and / or deleting and / or adding one or more amino acid residues to the amino acid sequence represented by the sequence 2, related to the clostridial neurotoxin detection and derived from the sequence 2. The substrate protein SNVP provided by the invention has seven toxin serotype botulinum and tetanus toxin enzyme hydrolysis sites, can specifically identify A, B, E, C, D, F and G type botulinum toxins and tetanus toxins, and can be used for the detection and the parting discrimination of the botulinum toxins and tetanus toxins as a core detection reagent.

Description

technical field [0001] The present invention relates to a Clostridium neurotoxin substrate protein and its coding gene and application, in particular to a Clostridium neurotoxin (botulinum toxin and tetanus) obtained by fusing SNAP-25 protein and VAMP-2 protein Toxin) substrate protein and its coding gene and application. Background technique [0002] Botulinum toxin (botulinum neurotoxin, BoNT) is the most toxic substance known in the world. It is produced by Clostridium botulinum under anaerobic conditions. There are 7 serotypes (A-G), of which human The main pathogens are A and B types, which can cause severe poisoning symptoms, the mortality rate is as high as 40%, and the half-lethal dose LD50 of the human body is only 0.1-1ng / kg. Poisoning usually manifests as paralysis of eye and throat muscles, and then develops into general skeletal muscle paralysis, and finally dies due to respiratory failure. In recent years, botulism incidents have occurred frequently. Although...

AI Technical Summary

Problems solved by technology

Although the results of traditional gold standard analysis are true and reliable for most samples, there are still many limitations, mainly in high cost (high requirements for animal feeding conditions and high professional skills of operators), and long time-consuming (requiring 4 days) ), the large amount of samples required, difficult to promote, etc., cannot meet the requirements of emergency analysis of samples in public health emergencies, so there is an urgent need for simple, fast and sensitive new technology methods to effectively replace the traditional gold standard

[0005] In recent years, many scholars at home and abroad have carried out relevant research on the analysis methods of botulinum toxin, and some new analysis methods have come out. In addition to the traditional gold standard mentioned above, they mainly include: (1) Endopeptide Enzyme activity assay (endopeptidase activity assay), can use artificially synthesized or recombinantly expressed peptides containing specific serotype enzyme cleavage sites as substrates, and use methods such as detection of labeled fluorescent signals to analyze toxins in the test sample; ( 2) Mass spectrometry, which directly detects the substrate products of toxin enzymatic hydrolysis, can achieve even higher sensitivity than mouse analysis, but it relies on special equipment, and its results are easily interfered by other substances in the sample to be tested; (3 ) cell analysis method can simulate the process of botulism in vitro, which solves the problem of using a large number of animals in the mouse analysis method, but the operation is more complicated and time-consuming

At present, most of these methods and supporting reagent products are for the detection and analysis of specific serotype toxins, and there is no detection system that can easily apply a single detection product to cover all serotype toxins

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