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A kind of recombinant human fibronectin peptide

A technology of human fibronectin and carrier, which is applied in the field of genetic engineering, can solve the problems of poor processability, excessive molecular weight of fibronectin, and carrying pathogens to harm the human body, etc., to achieve small molecular weight, enhance cell metabolism, and increase cell adhesion rate Effect

Active Publication Date: 2021-11-26
BIOPHARM RES & DEV CENT JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the existing fibronectin has poor processability due to its large molecular weight, and is mostly extracted from animal tissues. It is a heterologous protein, which has certain rejection reactions and has the potential risk of carrying pathogens and endangering the human body.

Method used

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  • A kind of recombinant human fibronectin peptide
  • A kind of recombinant human fibronectin peptide
  • A kind of recombinant human fibronectin peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Expression of recombinant human fibronectin peptide in Escherichia coli

[0024] 1 Construct the expression vector for recombinant expression of human fibronectin peptide:

[0025] The DNA fragments (SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4) encoding the recombinant human fibronectin peptide were named BL21(DE3) / pET20b- Fnp-1, the expression strain using sequence SEQ ID NO:3 is named BL21(DE3) / pET20b-Fnp-2, and the expression strain using sequence SEQ ID NO:4 is named BL21(DE3) / pET20b-Fnp-3. BL21 (DE3) / pET20b-Fnp-1, BL21(DE3) / pET20b-Fnp-2, BL21(DE3) / pET20b-Fnp-3 induction results are as follows image 3 As shown, the results showed that the expression levels of BL21(DE3) / pET20b-Fnp-1 were higher than those of BL21(DE3) / pET20b-Fnp-2 and BL21(DE3) / pET20b-Fnp-3. The sequence used in subsequent experiments is SEQ ID NO: 2 by default. ) was sent to Jinweizhi Biotechnology Co., Ltd. for whole gene synthesis, and Nde1 and Hind111 (TAKARA Co.Ltd) restriction sites we...

Embodiment 2

[0029]The gene sequence (SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4) of the artificially synthesized recombinant human fibronectin peptide obtained in Example 1 was introduced into Nde I and Hind 111 at the 5' end and 3' end respectively Restriction site, through Nde I / Hind 111 double digestion, it was connected into pET20b vector, thus obtaining pET20b-Fnp recombinant plasmid. This recombinant plasmid was introduced into competent Escherichia coli BL21 (DE3) for cultivation, and IPTG was used to induce the expression of Fnp protein, and the purified protein was obtained by nickel affinity chromatography, and the molecular weight and immunology of the recombinant protein were analyzed by SDS-PAGE and Western blotting Verify and test its biological activity. Specific steps are as follows:

[0030] (1) Construction and identification of pET20b-Fnp recombinant plasmid: the present invention artificially synthesizes the peptide sequence of fibronectin gene, and introduces Nde I and...

Embodiment 3

[0034] (1) Determination of Fnp protein cell adhesion: HaCat cells were cultured with DMEM containing 10% FBS at 37°C, CO 2 Concentration: 5%; wash once with PBS, then add 0.25% trypsin solution for digestion, collect cells by centrifugation; resuspend with DMEM, control the cell density at 6.2×10 4 cells / mL, the cell suspension was inoculated into the culture dish with Fnp protein film on the bottom layer, and the cell density was controlled at 1.5×10 4 individual / mL. Incubate at 37°C for 5h, maintain CO 2 The concentration was 5%; the non-adherent cells were washed away with PBS; counted under a phase-contrast microscope and compared with the number of cells in each group by the MTT method. Positive control group: covered with commercially available bovine fibronectin. see results Figure 5 , the results show that the commercially available bovine fibronectin can increase the adhesion rate of HaCat (ATCC No: CM-1252) cells, and the adhesion of the patented Fnp between 0....

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Abstract

The invention belongs to the field of genetic engineering, and more specifically discloses a recombinant human fibronectin peptide. The invention discloses a codon-optimized recombinant human fibronectin peptide gene, which is expressed and purified in vitro by a genetic engineering method. The present invention uses Escherichia coli as an expression host cell to provide a recombinant human fibronectin peptide with small molecular weight, which retains the original properties of fibronectin, can promote cell growth, increase cell adhesion rate, and enhance cell metabolism.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a preparation method of recombinant human fibronectin peptide. Background technique [0002] Fibronectin (abbreviated as fibronectin) is also called fibronectin, fibronectin, and fibronectin. The English name is fibronectin, and the English abbreviation is Fn. Fibronectin is mainly produced by liver and vascular endothelial cells, and widely exists in animal tissues and interstitial fluids. It is a macromolecular glycoprotein with a dimer molecular weight of about 450KD and has various biological activities. Fn molecules are highly conserved during evolution, and Fn in various animal body fluids have very similar structures, properties and biological functions, so Fn from different sources can be used interchangeably. Fibronectin can promote cell growth, increase cell adhesion rate, enhance cell metabolism, shorten cell growth time, and increase cell fusion rate in hybridoma t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/78C07K19/00C12N15/70C12N1/21
CPCC07K14/78C07K2319/21C12N15/70C12N15/815C12N2800/22
Inventor 项琪黄亚东程雅婷曹苗苗
Owner BIOPHARM RES & DEV CENT JINAN
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