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Recombinant human fibronectin III1-C, and preparation method and application thereof

A technology of human fibronectin and III1-C, applied in the biological field, can solve the problems of long time, loss of function, complicated technology, etc., and achieve the effect of low production cost, short production cycle and easy purification

Active Publication Date: 2020-06-02
ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This preparation process is complicated and takes a long time, and the activity of the protein is easily damaged, resulting in the loss of the corresponding function.
[0004] In the current existing technology, there is no published rhFNⅢ1-C development technology and its application method

Method used

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  • Recombinant human fibronectin III1-C, and preparation method and application thereof
  • Recombinant human fibronectin III1-C, and preparation method and application thereof
  • Recombinant human fibronectin III1-C, and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0036] The preparation method of rhFNⅢ1-C comprises the following steps:

[0037] (1) With the amino acid sequence shown in SEQUENCE LISTING 400, the human fibronectin FNⅢ1-C gene fragment of the gene sequence shown in SEQUENCE LISTING 400, by adjusting the GC ratio in the gene sequence, adjusting The preferred codons of human fibronectin in Escherichia coli expression system optimize and synthesize the human fibronectin FNⅢ1-C gene fragment, and the optimized human fibronectin FNⅢ1-C gene sequence is shown as SEQUENCE LISTING 400 As shown, the upstream and downstream primer sequences of the optimized rhFNⅢ1-C gene are: upstream primer: 5'-GGATCCAACGCCCCTCAGCCG-3', downstream primer: 5'-CGGCTGAGGGGCGTTGGATCC-3';

[0038] (2) Insert the optimized rhFNⅢ1-C gene into pET-32a to construct the recombinant plasmid rhFNⅢ1-C / pET32-a;

[0039] (3) Transform the recombinant plasmid into the expression vector BL21 (DE3), and screen the positive clone bacteria on the LB plate containing ...

Embodiment 2

[0057] RhFNⅢ1-C promotes cell adhesion test, the specific steps are as follows:

[0058] (a) The purified rhFNⅢ1-C obtained in Example 1 and the positive control fibronectin (purchased from Shanghai Fibrolink Biotechnology Co., Ltd.) were diluted 10 times and 100 times with PBS buffer respectively, and added to the 96-well cell plate Dilute according to 2-fold gradient; in order to avoid the edge effect, the surrounding edge wells were not used for experiments, and a negative control was set up (only 100 μL PBS buffer was added to the negative control); incubate overnight at 4°C;

[0059] (b) 100 μL of PBS was added to each well to wash for a total of 3 times; after washing, 1% BSA was added to each well to block, that is, it was incubated in a 37° C. incubator for 1 h. After the incubation is complete, discard the liquid in the plate;

[0060] (c) Preparation of MDBK single cell suspension in good growth state. Adjust the density of MDBK single cell suspension to 1.0×10 / mL,...

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Abstract

The invention discloses a recombinant human fibronectin III1-C (rhFNIII1-C), and a preparation method and application thereof. The preparation method comprises the following steps: optimizing a targetgene segment of the target protein according to codon expression preference, and inserting the obtained gene segment into pET-32a to construct a recombinant plasmid; transforming and introducing therecombinant plasmid into an expression vector BL21(DE3), and carrying out screening to obtain positive clone bacteria capable of realizing efficient soluble expression; carrying out enlarging fermentation culture on the positive clone bacteria, carrying out induced expression, performing crushing and centrifuging to obtain a supernatant, and carrying out purification through elution with a His-tagaffinity chromatography column, dialysis, filtration and other steps to obtain a high-quality rhFNIII 1-C solution with a protein concentration of 1.0 mg / mL or above and a purity of 90% or above. According to detection and observation results of cell adhesion promoting tests, the rhFNIII1-C has the performance of obviously promoting adherence and adhesion of MDBK cells and Balb / c / 3T3 cells and rapid division and growth of the cells, which indicates that the rhFNIII1-C has huge potential of being used as a raw material and a finished product for cosmeceuticals and medical skincare.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant human fibronectin III 1-C protein and its preparation method and application. Background technique [0002] Human skin is composed of epidermis, dermis, subcutaneous tissue and skin appendages, and has functions such as protection, temperature regulation, metabolism and sensation. The state of the epidermis and dermis is closely related to the state of the skin, and the homeostasis of the cellular environment composed of cells and extracellular matrix is ​​related to the state of the skin. Fibronectin (FN) is a high molecular weight (about 440kDa) dimeric glycoprotein of the extracellular matrix, which binds to the integrin transmembrane receptor protein and consists of two almost identical monomers through a pair of Linked by disulfide bonds, its gene is located on human chromosome 2 and consists of three homologous sequences of type I, type II, and type I...

Claims

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Application Information

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IPC IPC(8): C07K14/78C12N15/70C07K1/36C07K1/34C07K1/22C12N5/071A61K8/64A61Q19/00A61Q19/02A61Q19/08
CPCC07K14/78C12N15/70C12N5/0602A61K8/64A61Q19/00A61Q19/004A61Q19/02A61Q19/08C12N2533/52
Inventor 王明丽徐艳艳蒋敏之吴博周炜何志远夏兵兵
Owner ANHUI MEDICAL UNIV
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