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Mytilus coruscus foot adhesive protein as well as encoding sequence and preparation method thereof

A technology of thick-shelled mussel and adhesive protein, which is applied in the field of coding sequence and preparation of the thick-shelled mussel's silk adhesion protein, can solve the problem that it is difficult to meet the needs of the industry, the price is expensive, and the secretion of natural mussel adhesive protein is low. And other issues

Inactive Publication Date: 2011-01-19
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the very little secretion of natural mussel adhesion protein, the bioadhesive products developed based on it are very rare in the market, and the price is extremely expensive, which is difficult to meet the needs of the industry

Method used

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  • Mytilus coruscus foot adhesive protein as well as encoding sequence and preparation method thereof
  • Mytilus coruscus foot adhesive protein as well as encoding sequence and preparation method thereof
  • Mytilus coruscus foot adhesive protein as well as encoding sequence and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0067] Example: construction of eukaryotic expression vector.

[0068] The feet were excised from the fresh mussel M. coruscus, the outer pigment epithelium and part of the muscle layer were removed, the foot gland tissue was selected to extract total RNA, reverse transcribed into cDNA and used as a template. According to the mature peptide sequence of the open reading frame (ORF) in the mcofp-3 gene sequence of the thick-shelled mussel Mytilus coruscus, PCR primers (synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.) were designed, and P3F was the forward primer. Two protective bases, CG and XbaI restriction site, were added to the end; followed by a T to prevent the shift of ORF, and then the codon AAGAGA, which encodes two amino acids of KR, so that the precursor processing enzyme Kex2 secreted by yeast itself It can cleave the following mature peptide and secrete it into the medium; P3R is a reverse primer, and three protective bases CCG and HindIII...

Embodiment 2

[0096] Example 2: Eukaryotic expression of mussel thick shell adhesion protein mfp-3.

[0097] 1) Transfer the expression plasmid into competent cells of Saccharomyces cerevisiae S78, spread it on YSD plate medium, and culture at 30°C for 4-6 days;

[0098] 2) Pick the monoclonal strain and expand it into YSD liquid medium for 12 hours, then dilute it with YSD liquid medium at a ratio of 1:25, culture it at 30°C and 250 rpm for 12 hours, and finally transfer it to YPD liquid medium at a ratio of 1:25 30°C constant temperature, 250rpm fermentation for 3 to 4 days;

[0099] 3) Centrifuge the fermentation broth at 12000rpm for 15min, collect the supernatant and freeze-dry it, then redissolve it in deionized water for separation and purification

Embodiment 3

[0100] Example 3: Purification, modification and identification of the expression product of mussel thick shell filum adhesion protein mfp-3.

[0101] The expression supernatant was filtered with a suction filter bottle, passed through a 0.45 μM filter membrane, and the obtained filtrate was collected. The expression filtrate was mixed with 1M NaAc (pH 4.2) prepared in advance to make the final concentration of NaAc 0.1M. Then directly put on the CM-sepharose cation exchange column (5cm×50cm), then eluted with 0.1M NaAc buffer solution containing 0.1M, 0.3M and 0.5M NaCl respectively, and collected each elution peak. The solution collected from each elution peak was desalted on a preparative C18 reverse-phase column (4.6×250 mm) of Waters Company, and the elution peak obtained by reverse phase was identified by SDS-PAGE electrophoresis and then lyophilized and stored.

[0102] The expression product was identified by discontinuous SDS-PAGE vertical plate electrophoresis, the ...

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Abstract

The invention discloses mytilus coruscus foot adhesive protein and encoded polynucleotide thereof, and further discloses a preparation method of the mytilus coruscus foot adhesive protein. Compared with the prior art, the invention has the advantages that: the mytilus coruscus recombinant foot adhesive protein i.e. a waterproof biological adhesive can be rapidly obtained, the functional peptide segment of the foot adhesive protein can be continually expressed through saccharomyces cerevisiae S78 after the recombinant plasmid of an adhesive protein functional fragment is obtained, and the modified mytilus coruscus recombinant foot adhesive protein has better adhesion performance. A polypeptide product has the advantages of simple modifying method and simple operation, and only a small amount of tyrosinase is needed.

Description

technical field [0001] The invention relates to a thick-shell mussel silk adhesion protein, and also relates to a coding sequence and a preparation method of the thick-shell mussel silk adhesion protein. Background technique [0002] Marine periphytons can permanently or temporarily adhere to various solid surfaces in the water environment, such as reefs, ship hulls and other biological surfaces. At present, there are about 5,000 species of marine periphytons, and the development of new, efficient bioadhesives that can be used in water environments from the adhesive substances secreted by marine periphytons is one of the current research hotspots in biomaterials. Among them, mussel silk protein is the focus of this research field. Mussels belong to the mollusk Phylobranchia, which is a common marine bivalve mollusc. It can form a firm connection with solid surfaces such as reefs and ship hulls through the silk secreted by its podocytes, and can withstand repeated washing by...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/81C07K1/20C07K1/18C12Q1/68G01N27/62
Inventor 廖智李楠楠王健鑫鲁涛石戈王日昕
Owner ZHEJIANG OCEAN UNIV
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