Recombinant mussel adhesive proteins and their encoding genes and applications

Abstract

This invention relates to recombinant mussel adhesive proteins, the nucleic acid molecules encoding them, nucleic acid constructs, expression vectors, recombinant engineered strains, pharmaceutical compositions, preparation methods, purification methods, and applications. This invention assembles and splices partial functional sequences from different mussels and type III collagen to obtain several novel mussel adhesive protein sequences. Further screening yielded recombinant mussel adhesive proteins Mfp5, Mfp55, Mfp131, and Mfp151, suitable for large-scale industrial production and exhibiting high expression levels. Efficacy experiments demonstrated that these proteins significantly promote cell migration, indicating their excellent cell repair function and broad application prospects.

Claims

1. A recombinant mussel adhesive protein, characterized in that, The amino acid sequences of the recombinant mussel adhesive protein are shown in SEQ ID NO.4, 1, 2, 3, 5, 6, 7, 8, 9, 10 or 11, respectively.

2. The recombinant mussel adhesive protein according to claim 1, characterized in that, The recombinant mussel adhesive protein further includes a functional element at its N-terminus or C-terminus; preferably, the functional element is selected from a tag sequence; more preferably, the tag sequence is selected from an HIS tag; most preferably, the amino acid sequences of the recombinant mussel adhesive protein containing the HIS tag are shown in SEQ ID NO. 15, 12, 13, 14, 16, 17, 18, 19, 20, 21 or 22 respectively.

3. A gene encoding a recombinant mussel adhesive protein, characterized in that, It encodes the recombinant mussel adhesive protein as described in claim 1 or 2; preferably, the nucleic acid sequences of the encoding gene are shown in SEQ ID NO. 26, 23, 24, 25, 27, 28, 29, 30, 31, 32, and 33, respectively.

4. A recombinant gene expression cassette, characterized in that, It includes the coding gene as described in claim 3.

5. A recombinant vector, characterized in that, The recombinant vector comprises the coding gene of claim 3 or the recombinant gene expression cassette of claim 4; preferably, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector; more preferably, the prokaryotic vector comprises an Escherichia coli vector; most preferably, the Escherichia coli vector comprises a pET vector, a pGEX vector, a pMAL vector, a pBAD vector, a pUC vector, or a pBR vector; optionally, the pET vector comprises pET-28a, pET-200, or pET-32a.

6. A recombinant host cell, characterized in that, The recombinant host cell comprises the coding gene of claim 3, the recombinant gene expression cassette of claim 4, or the recombinant vector of claim 5; preferably, the recombinant host cell comprises a eukaryotic cell or a prokaryotic cell; more preferably, the prokaryotic cell comprises *Escherichia coli* cells, *Bacillus subtilis* cells, or *Pseudomonas* cells; most preferably, the *Escherichia coli* cells comprise... E. coli BL21(DE3), E. coli Origami B(DE3) E. coli One of the following: Rosetta Blue (DE3), DH5α, TOP10, and Rosetta.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the recombinant mussel adhesive protein of claim 1 or 2, or the encoding gene of claim 3, or the recombinant gene expression cassette of claim 4, or the recombinant vector of claim 5, or the recombinant host cell of claim 6; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable vector.

8. A method for preparing the recombinant mussel adhesive protein according to claim 1 or 2, characterized in that, The method includes fermenting and purifying the host cells described in claim 6 to prepare recombinant mussel adhesive protein; preferably, the fermentation includes primary seed culture, secondary seed culture, and fermentation culture; more preferably, the fermentation culture includes culturing the recombinant Escherichia coli host cells described in claim 6 at a temperature of 16-37°C, followed by inducing expression at a temperature of 16-37°C, and adding the inducer IPTG to induce the expression of mussel adhesive protein, with a final IPTG concentration of 0.1-2.0 mM.

9. A method for purifying the recombinant mussel adhesive protein according to claim 1 or 2, characterized in that, The fermentation supernatant containing the recombinant mussel adhesive protein of claim 1 or 2 is filtered and clarified. The recombinant mussel adhesive protein in the filtered and clarified liquid is captured by a washed cation exchange chromatography medium. The chromatography medium is equilibrated and then linear elution is performed to obtain the purified recombinant mussel adhesive protein eluent.

10. The use of the recombinant mussel adhesive protein of claim 1 or 2, or the encoding gene of claim 3, or the recombinant gene expression cassette of claim 4, or the recombinant vector of claim 5, or the recombinant host cell of claim 6, or the pharmaceutical composition of claim 7, or the recombinant mussel adhesive protein prepared by the preparation method of claim 8, or the recombinant mussel adhesive protein purified by the purification method of claim 9, in any of the following: (1) Preparation of wound repair drugs; preferably, the wound repair includes burns, scalds, surgical incision repair, chronic ulcer treatment, post-laser repair, persistent ulcers and skin damage; (2) Preparation of medical bioadhesives and medical coating materials; (3) Preparing medical aesthetic products; preferably, the medical aesthetic products include post-operative repair products; more preferably, the post-operative repair products include microneedling and mesotherapy post-operative repair products; or, (4) Preparation of cosmetics; preferably, the cosmetics include anti-aging cosmetics, barrier repair cosmetics, problem skin cosmetics, and protective cosmetics.

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