Recombinant fibronectin with anti-wrinkle repairing function and preparation method and application of recombinant fibronectin

Abstract

The invention belongs to the technical field of biology. The invention relates to a recombinant fibronectin with anti-wrinkle repairing functions. The recombinant fibronectin comprises at least one RGD structural domain capable of being combined with a cytointegrin receptor, and at least one sodium ion channel binding peptide fragment capable of acting on a sodium ion channel, the RGD structural domain and the sodium ion channel binding peptide fragment are connected through a polypeptide connecting fragment containing 0-300 amino acid residues, the capability of combining a sodium ion channeland an integrin receptor is realized, the effects of promoting skin repair and metabolic renewal and enhancing freckle fading and wrinkle reducing are achieved, and the process for the preparation thereof, high-density fermentation, super-tolerant metal affinity chromatography purification, specific protease label excision and ion column refining purification methods are adopted. The preparationmethod utilizes high-density fermentation, super-tolerant metal affinity chromatography purification, specific protease excision label and ion column refining purification method. The obtained recombinant fibronectin rFN does not contain a purification label, is high in purity, high in specific activity, simple and efficient in preparation process, low in cost and suitable for large-scale production, and further relates to an application of the recombinant fibronectin rFN in in-vitro adherent cell culture, skin injury repair, wrinkle removal and spot fading.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to recombinant fibronectin with anti-wrinkle repair function, its preparation method and application. Background technique [0002] Conopeptides are secreted by the venom tubes of the marine gastropod mollusc Cono snails and the venom glands on the inner wall of the poison sac. The main components of Conopeptides are some active peptides with high specificity for different ion channels and nerve receptors. compound. Cono peptides can specifically act on various receptor subtypes of acetylcholine receptors and other neurotransmitters, and can also act on various ion channels such as calcium, sodium, and potassium. Conopeptide has analgesic and nervous tension-relieving effects, and is not addictive at the same time, so it is a research hotspot in the field of medicine. In recent years, it has begun to be used in the field of beauty, and its main function is to reduce wrinkl...

AI Technical Summary

Problems solved by technology

[0008] Purification using metal affinity packing usually requires the introduction of 6 histidine tags on the target protein, that is, the His-tag tag, which has a minimum of 6 amino acid residues, good water solubility, and usually does not affect the structure and function of the protein , In terms of pharmaceuticals or other applications where His6-tag has adverse effects, it is necessary to remove the His-tag tag, but removing the His-tag is a huge difficulty. The conventional solution is to introduce a gap between the His6-tag and the target protein The enzymatic cleavage site of protease, the fusion protein after rough purification can be processed by specific protease, which can cut off the label, usually using thrombin (Thrombin), activated coagulation factor ten (FactorXa), tobacco mosaic virus protease TEV, etc. to cut, but These enzymes often have insufficient specificity, and will cut or even leave redundant amino acid residues in the target protein sequence; in recent years, it has been found that the eukaryotic ubiquitin-like protein SUMO can be specifically recognized by a space Specific protease ULP1 processing, this process has the highest substrate specificity and cleavage efficiency, if SUMO is connected to the N-terminal of the target protein, after ULP1 protease cleavage, it can perfectly solve the cleavage efficiency, cleavage specificity and redundant residues base question

[0009] At present, there is no strategy of fusion expression of conopeptide and FN functional fragment of fibronectin to express conopeptide, improve the effect of fibronectin in relaxing wrinkles and blemishes, and produce recombinant fibronectin on a large scale report

[0010] And there is no strategy to purify recombinant fibronectin using the third-generation ultra-tolerant nickel column to simplify the purification process and improve purification efficiency, nor to use the SUMO / ULP1 system to construct fusion recombinant fibronectin and then prepare recombinant fibronectin protein strategy

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