Process for producing ocean mussel adhesion protein water-proof biological adhesive agent

A mussel adhesive protein and bioadhesive technology, which is applied in the field of preparation of marine mussel adhesive protein waterproof bioadhesive, can solve the problem that it is difficult to meet the needs, less adhesive protein, and expensive adhesive protein products and other issues, to achieve the effect of low cost, simple operation and simple process

Inactive Publication Date: 2009-05-13
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the amount of Adhesin secreted by mussels is very small, and only 1 mg of Adhesin can be extracted from about 10,000 mussels,

Method used

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  • Process for producing ocean mussel adhesion protein water-proof biological adhesive agent
  • Process for producing ocean mussel adhesion protein water-proof biological adhesive agent
  • Process for producing ocean mussel adhesion protein water-proof biological adhesive agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Preparation of marine mussel adhesive protein waterproof bioadhesive Mcfp-1-12

[0037] 1. Obtain the target gene fragment by PCR and connect it to the cloning vector: excise the foot from the live mussel M.coruscus, extract the total RNA, and reverse transcribe it into cDNA. According to the sequence of the Mcfp-1 gene (sequence from Genbank No.D63777) of the thick-shelled mussel M.coruscus, the 12 amino acid residues from the 692nd to the 811th amino acid residues (sequence from Genbank No.Q25434) of the coding Mcfp-1 Design PCR primers (synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.) from the DNA fragment of the continuous decapeptide repeat sequence. When designing the primers, add EcoR I and Sal I restriction sites to the ends of the forward primer and reverse primer, respectively. , the primer sequences are as follows:

[0038] Primer F: 5'-ccgaattctataaacctaagaaaacttatcc-3',

[0039] Primer R: 5'-atgtcgacttacgttggtggatagcg...

Embodiment 2

[0090] Example 2: Adhesion of the adhesive Mcfp-1-12 on different material surfaces in a humid environment

[0091] Add 10 μl of the adhesive Mcfp-1-12 with a concentration of 1.44 mg / ml to the surfaces of cleaned plastic, glass, metal titanium and aluminum, and at the same time add bovine serum albumin (BSA, bovine serum) of the same concentration albumin), the fusion polypeptide GST-Mcfp-1-12 and CELL-TAK without thrombin digestion were used as negative and positive controls, respectively, and the proteins added in this group of experiments were tyrosine-modified and unmodified, respectively. Histones were used as controls. Place it in a humid environment at 25°C for 12 hours, take it out and dry it, rinse it with distilled water for 2 hours; then stain it with Coomassie brilliant blue, such as Figure 4 As shown, the results show that on glass slide A and metal titanium C, the adhesive Mcfp-1-12 has similar adhesion ability to CELL-TAK, while on plastic Petri dish D and me...

Embodiment 3

[0092] Embodiment 3: the mensuration of adhesive agent Mcfp-1-12 adsorption amount on silicon gold sheet

[0093] Add the adhesive Mcfp-1-125μl with a concentration of 1.44mg / ml dropwise to the surface of the Pico EQCM quartz wafer (Outlet (Tianjin) Scientific Instrument Technology Co., Ltd.), and at the same time add the same concentration of BSA, without thrombin The digested fusion polypeptide GST-Mcfp-1-12 and CELL-TAK were used as negative and positive controls, respectively. In addition, the proteins added in this group of experiments were tyrosine-modified and unmodified as controls. Place it in a humid environment at 25°C for 12 hours, take it out and dry it, and rinse it with distilled water for 2 hours; after vacuum drying, measure its frequency change with a QCM instrument to obtain the value of its adhesion. Such as Figure 5 , the results showed that the adhesive Mcfp-1-12 had stronger adhesive ability than other control groups.

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Abstract

The invention provides a method for preparing a marine mussel adhesive protein waterproof biological adhesive, and relates to a biological adhesive. The invention provides the method for preparing marine mussel-Mytilus coruscus adhesive protein waterproof biological adhesive with degradability, water resistance and good biological compatibility by a method of biological gene engineering. The method comprises the following steps: extracting total RNA from the foot part of alive Mytilus coruscus to be reversely transcribed into cDNA; designing a PCR primer according to an Mcfp-1 gene sequence of the Mytilus coruscus; connecting the obtained DNA fragment in a prokaryotic expression vector pGEX-4T through PCR amplification to construct a recombinant plasmid; then transforming the recombined plasmid in a colon bacillus BL21 bacterial strain; and finally, purifying and modifying the expressed polypeptide product. The preparation method is simple, and the expression product is soluble, can be easily purified with low cost, and has similar or better effect compared with CELL-TAK<TM> cell and tissue adhesive without cytotoxicity. The adhesive accords with the requirement of scientific research, and can be used as a biological medical material.

Description

technical field [0001] The invention relates to a bioadhesive, in particular to a preparation method of a marine mussel adhesion protein waterproof bioadhesive. Background technique [0002] Marine mussels belong to the mollusk Phylumbranchia, and are a kind of organisms commonly found in coastal and offshore areas. Their silk glands can secrete silk to attach to the hard substrate, so that they can still adhere tightly under the washing of huge waves. on the reef without separation. The main component of this silk viscose, the silk adhesive protein, has high strength, high toughness and water resistance. At present, BD Bioscience in the United States has extracted adhesion proteins from the feet of blue mussels (Mytilus edulis), which are used as cell and tissue adhesion agents. The product has been commercialized, and the name and article number are respectively BD CELL-TAK TM CELL AND TISSUE ADHESIVE, Catalog No. 354240, 354241. This product can be used to coat glass ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/70C07K14/47C07K1/107C09J189/00
Inventor 金利华张其清刘加鹏
Owner XIAMEN UNIV
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