Gene coding alpha-conch toxin peptide and uses thereof

Abstract

The invention discloses a gene encoding alpha-conch toxin peptide (Vc1.1) and a utilization method thereof. belong to the biological field. Vc1.1 is a 16-peptide with 2 pairs of disulfide bonds. The present invention discloses a gene encoding this peptide and its genetic engineering. The utilization method of the gene includes recombining it into an expression vector, transforming the host cell, cloning and expressing the fusion protein in it, and obtaining a protein with the activity of antagonizing neuronal nicotinic acetylcholine receptors through affinity chromatography, renaturation and purification. Vc1.1 peptide. The above method has the advantages of few steps, stable quality, high product purity, low preparation cost, few by-products, and no environmental pollution. The prepared Vc1.1 also has important value in receptor research and drug development.

Description

technical field [0001] The invention relates to the genetic engineering of polypeptide compound alpha-conch toxin peptide Vc1.1 and its application in pharmacy, belonging to the field of biology. Background technique [0002] The clinical use of morphine and other drugs to relieve cancer pain, neuralgia and other intractable pain will cause tolerance, short-term drug effect and addiction, so it has great limitations. Since Ziconotide, the first analgesic drug with an analgesic effect 1000 times stronger than morphine, was successfully launched, its active ingredient omega conch toxin MVII A has attracted great attention from the biomedical community. In addition, several other conch toxins also have non-addictive, low-tolerance analgesic effects. For example, alpha-conch toxin Vc1.1, which is a polypeptide composed of sixteen amino acids, has the activity of antagonizing neuronal nicotinic acetylcholine receptors (nAChr). This receptor is related to the transmission of pai...

AI Technical Summary

Problems solved by technology

Chemical synthesis has many reaction steps, many by-products, complex purification technology, great environmental pollution, and adverse health of workshop workers, so it has high manufacturing cost

Although the genetic engineering method can overcome many shortcomings of chemical synthesis, it still faces many challenges in the process: (1) Vc1.1 is a small peptide of 16 amino acids with a molecular weight of only about 1.7kD. In the process, there is no dialysis bag or ultrafiltration membrane for this molecular weight cut-off; (2) There is no simple and easy method to identify and detect it: due to its small molecular weight, it is difficult to aggregate into the SDS-PAGE electrophoresis A narrow band, even Tricine electrophoresis is not successful every time. In addition, protein markers in small molecular weight regions are difficult to purchase and expensive; (3) Vc1.1 is a peptide rich in disulfide bonds, which makes renaturation more difficult , and the current alternative renaturation method must use a low molecular weight cut-off dialysis bag or ultrafiltration membrane, which is difficult to achieve as mentioned above; (4) due to the small molecular weight, the induced expression is easily absorbed by the bacterial intracellular Degraded by proteases, it is difficult to express alone, so tandem expression or fusion expression must be considered, these methods need to cut the expression product to obtain the target molecule

At present, the main ways to cut proteins in genetic engineering are cyanogen bromide cleavage or thrombin cleavage, etc.: cyanogen bromide specifically cleaves the peptide bond at the methionine in the protein, but the reagent itself is highly toxic. The use is strictly limited, and the next step requires a higher cost to remove cyanogen bromide; thrombin is more expensive, has poor specificity of enzyme digestion and is easy to denature and inactivate

[0004] Probably due to the above-mentioned technical bottlenecks, there is no report on the preparation of Vc1.1 or similar small peptide compounds rich in disulfide bonds by genetic engineering

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