Yeast fermentation small-molecular recombinant fibronectin protein peptide and preparation method and application thereof

A fibronectin and yeast fermentation technology, applied in the field of bioengineering, can solve the problems of low stability and low yield, and achieve the effects of good heat resistance, high yield and high degree of glycosylation

Active Publication Date: 2019-09-06
MELLGEN SHENZHEN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the first object of the present invention is to provide a yeast-fermented small molecule recombinant fibronectin peptide to solve the problems of low yield and low stability obtained by the existing Escherichia coli expression system

Method used

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  • Yeast fermentation small-molecular recombinant fibronectin protein peptide and preparation method and application thereof
  • Yeast fermentation small-molecular recombinant fibronectin protein peptide and preparation method and application thereof
  • Yeast fermentation small-molecular recombinant fibronectin protein peptide and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 constructs the expression plasmid encoding Chimeric FN protein

[0037] This embodiment uses the commercial carrier pPIC9K (such as figure 1 shown), purchased from Wuhan Sanying Biotechnology Co., Ltd., according to figure 1 Relevant sequence positions are designed to select restriction enzyme cutting sites EcoR I and Not I, and the gene sequence encoding Chimeric FN is artificially optimized with preferred codons of Pichia pastoris and obtained by artificial synthesis. The 5' and 3' ends of the synthesized recombinant fibronectin full-length DNA fragment each have a restriction endonuclease site, corresponding to EcoR I and Not I, respectively. The target fragment of recombinant fibronectin will be inserted between these two restriction sites to obtain the expression plasmid encoding Chimeric FN protein. Wherein, the amino acid sequence of recombinant fibronectin is:

[0038]ACSPPHSKSHCGGGGSIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQL ISIQQYGH...

Embodiment 2

[0044] Embodiment 2 Expression purification and electrophoresis identification method of recombinant fibronectin

[0045] 1) Preparation of yeast clones. Genomic DNA of the expression vector Chimeric FN was extracted and digested with nuclease to obtain linearized DNA, which was dissolved in 5-10 ulTE (purchased from Neopro Biotech). Take 80ul of commercially competent Pichia pastoris GS115 (purchased from Tiangen Biology) mixed with 10ug of linearized DNA, and transfer it into a pre-cooled 0.2cm electroporation cup. Place on ice for 5 min. Set the machine parameters, immediately add 1ml of pre-cooled 1M sorbitol to the cup, transfer the contents to a sterilized centrifuge tube, divide it into 200ul aliquots, spread it on the MD plate, and incubate the plate at 30°C until the clones are produced, due to transformation The vector contains the mut gene, and only the successfully transformed strains can be screened by the mut phenotype, so the Mut+ / Muts phenotype strains are sc...

Embodiment 3

[0054] Example 3 Recombinant fibronectin promotes cell adhesion and growth test

[0055] The recombinant fibronectin purified in Example 2 was formulated into various concentrations (1, 6, 9, 15, 24 ug / ml), coated on a 96-well plate for 30 minutes, and washed twice with PBS. Add 1% BSA and block at 37°C for 30 minutes, add rat fibroblasts (cultivated with serum-free medium), gently suck the medium in the well after 1 hour, gently rinse the unadsorbed cells with PBS, and use CCD8 The number of living cells adsorbed on the bottom of the well plate was detected by the method to verify the activity of recombinant fibronectin. For the results, please refer to Figure 5 and Figure 6 .

[0056] Figure 5 The results showed that the cell attachment activity of the recombinant fibronectin was better than that of the natural structure fibronectin. Yeast-fermented proteins were significantly more effective in cell attachment than E. coli-fermented proteins due to the important role ...

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Abstract

The invention discloses a yeast fermentation small-molecular recombinant fibronectin protein peptide comprising at least one sodium-potassium ATPase beta subunit binding domain, and the sodium-potassium ATPase beta subunit binding domain has the amino acid sequence shown in SEQ ID NO:2. The invention also discloses a preparation method of the yeast fermentation small-molecular recombinant fibronectin protein peptide and an application of the yeast fermentation small-molecular recombinant fibronectin protein peptide. The yeast fermentation small-molecular recombinant fibronectin protein peptidecan be effectively absorbed by skin and has excellent healing and repairing effects on traumatic skin lesions or subcutaneous lesions with intact keratin.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a yeast fermented small molecule recombinant fibronectin peptide and its preparation method and application. Background technique [0002] Fibronectin (FN) is a macromolecular glycoprotein with a sugar content of 4.5% to 9.5% and a molecular weight of about 450kd. The molecule binds to 11 integrin receptors and plays extremely important functions in cell-cell and cell-matrix interactions. A large number of studies have found that fibronectin (FN) is involved in wound healing, tissue repair, embryonic differentiation, immune response, tumor differentiation and metastasis, childbirth and other processes, and is closely related to many diseases. Fibronectin matrix polymerization also promotes type I collagen deposition and enhances collagen-based tissue architecture. [0003] Fibronectin is a protein dimer composed of two nearly identical monomers linked by a pair of disu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/78C12N15/12C12N15/81A61K38/39A61K8/64A61Q19/00A61P17/00
CPCC07K14/78C12N15/815A61K8/64A61Q19/00A61P17/00A61K2800/85A61K38/00C07K2319/01C07K2319/70C12N9/14C07K2319/00C12R2001/84C12N9/00A61K9/0014C07K14/39
Inventor 阮仁全温龙平
Owner MELLGEN SHENZHEN BIOTECHNOLOGY CO LTD
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