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Coding sequence of fibronectin mutant with high expression quantity and strong activity and application of coding sequence

A technology of fibronectin and coding sequence, which is applied in the field of coding sequence of fibronectin mutants, can solve the problems of inability to obtain soluble protein, protein misfolding and aggregation, and low expression level, so as to improve cell adhesion activity and facilitate purification , The effect of simple preparation process

Active Publication Date: 2021-06-11
广州启点生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, when recombinant proteins are expressed heterologously, the expression level is often low, or expressed in the form of inclusion bodies, which is another key technology that restricts the application of fibronectin
[0006] The latest research shows that although different DNA can be translated into the same amino acid, the speed of protein production (translation speed) is not constant, and it will be slower in some segments. This phenomenon is called translational pause (translational pausing or translational attenuation) The translation pause site is highly related to protein folding. If the translation pause site is incorrect, the place that should be slow is fast, or the place that should be fast is slow, it will lead to protein misfolding and aggregation, and functional soluble proteins cannot be obtained.

Method used

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  • Coding sequence of fibronectin mutant with high expression quantity and strong activity and application of coding sequence
  • Coding sequence of fibronectin mutant with high expression quantity and strong activity and application of coding sequence
  • Coding sequence of fibronectin mutant with high expression quantity and strong activity and application of coding sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction of recombinant fibronectin mutant vector

[0036] We selected the functional domain of fibronectin to promote cell proliferation and adhesion, and designed a new fibronectin mutant, whose amino acid sequence is as follows:

[0037] MAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTGGGGSGGGGSILDVPSTVQKTPFVTHPGYDTGNGIQLPGTSGQQPSVGQQMIFEEHGFRRTTPPTTATP。

[0038] The nucleotide sequences of the fibronectin mutants were optimized according to the codon bias of E. coli and by translation pause theory.

[0039] The nucleotide sequence of the unoptimized fibronectin mutant (FN-M) is as follows:

[0040]ATGGCGGTTCCGCCGCCGACCGATCTGCGTTTCACCAACATCGGCCCAGACACGATGCGTGTTACGTGGGCGCCACCACCGAGCATCGATCTGACCAATTTCCTCGTGCGCTACAGTCCGGTGAAAAACGA...

Embodiment 2

[0047] Example 2: Expression and purification of recombinant fibronectin mutants

[0048] (1) Screening of engineering bacteria expressing fibronectin:

[0049] The fibronectin expression plasmid pET-28a-FN was transformed into Escherichia coli competent cell BL21, containing 50 μg / ml kanamycin, and LB solid medium plate was used to select positive clones.

[0050] Pick positive clones into liquid medium containing 5ml LB, wait for OD 600 = 0.8, add 1mmol / L IPTG to induce expression, identify the expression of the protein by SDS-PAGE electrophoresis, and select engineering bacteria with high expression to preserve the species.

[0051] (2) Induced expression and solubility analysis of fibronectin

[0052] The expression strain pET-28a-Fibronectin obtained in step (1) was inoculated into 50 mL of LB medium containing 50 μg / mL kanamycin content, cultivated at 37 ° C and 180 rpm, when OD 600 When = 0.8, add IPTG with a final concentration of 1 mmol / L, induce expression at 37°C...

Embodiment 3

[0062] Example 3: Determination of the Proliferative Activity of Recombinant Fibronectin Mutants

[0063] (1) BALB / c 3T3 cells were seeded in 96-well cell culture plates (5000 cells / well), 37°C, 5% CO 2 The cells were cultured in an incubator for 24 hours.

[0064] (2) Replace with DMEM serum-free medium and continue culturing for 12 hours.

[0065] (3) Add fibronectin mutant FN-M, recombinant fibronectin mutant FN-M1 and PBS (negative control group) respectively, and continue culturing for 48-72 hours.

[0066] (4) Add 10 μL CCK-8 reagent to each well, 37°C, 5% CO 2 Remove from the cell culture incubator after 2 hours of incubation.

[0067] (5) Read the absorbance values ​​of the 96-well plate at 450nm and 630nm with a microplate reader, take 630nm as the reference wavelength, measure the absorbance at 450nm, and record the measurement results. Relative cell proliferation-promoting rate=(absorbance value at 450 nm of the experimental group−absorbance value at 450 nm of t...

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Abstract

The invention discloses a coding sequence of a fibronectin mutant with high expression quantity and strong activity and application of the coding sequence. The coding sequence of the fibronectin mutant provided by the invention is as shown in SEQ ID NO. 1. The coding sequence is obtained by selecting an active structural domain for promoting cell proliferation and cell adhesion on fibronectin and replacing a codon with a relatively high translation speed in the last 30 codons of the recombinant fibronectin with a codon with a relatively low translation speed by utilizing a translation pause theory under the condition of not changing an amino acid sequence of the recombinant fibronectin. The coding sequence obviously promotes the soluble expression of the fibronectin mutant in escherichia coli. The fibronectin mutant is obtained by constructing a recombinant vector, expressing and purifying the coding sequence, and the function of promoting cell proliferation activity and adhesion activity is obviously improved.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a coding sequence of a fibronectin mutant with high expression and strong activity and application thereof. Background technique [0002] Fibronectin (FN) is a multifunctional glycoprotein that is abundant in plasma and widely distributed in the extracellular matrix. Fibronectin regulates the adhesion, diffusion, migration, proliferation and differentiation of various cells, thus playing an important role in embryonic development and tissue repair. Fibronectin forms a matrix for cell movement and positioning, and mediates fibroblasts, macrophages, etc. to participate in damage repair. [0003] Fibronectin plays an important physiological function by binding to receptors expressed by cells. It is an essential substance in embryonic development and can guide cell adhesion and migration during embryonic development; for example, it binds to integrin receptor a5β1 expressed by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70C07K14/78A61K38/39A61P43/00
CPCC07K14/78C12N15/70A61P43/00A61K38/00Y02A50/30
Inventor 陈伟熊盛李尚浪苏志旭王一婷熊灿柳耀平
Owner 广州启点生物科技有限公司
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