EGF-like protein and construction method thereof, and chimeric protein as well as preparation method and application thereof

A technology of chimeric protein and construction method, applied in the field of bioengineering

Active Publication Date: 2019-08-02
广东格烯生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although macromolecular proteins can enter the brain through peptide substances that penetrate the cell membrane and use specific receptors, the design and development of proteins that can enter the brain are still very urgent

Method used

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  • EGF-like protein and construction method thereof, and chimeric protein as well as preparation method and application thereof
  • EGF-like protein and construction method thereof, and chimeric protein as well as preparation method and application thereof
  • EGF-like protein and construction method thereof, and chimeric protein as well as preparation method and application thereof

Examples

Experimental program
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preparation example Construction

[0052] The present invention also provides a preparation method for the chimeric protein of the above-mentioned technical scheme, comprising the following steps:

[0053] EGF-CTA 2 -TAT protein with (CTB) 5 The protein is acid-base chimerized to obtain the chimeric protein EGF-CTA 2 -TAT / (CTB) 5 .

[0054] In the embodiment of the present invention, the EGF-CTA 2 -TAT protein with (CTB) 5 Protein acid-base chimerization specifically includes:

[0055] EGF-CTA 2 -TAT protein and CTB protein were denatured at pH 2.0-2.5, 23°C-25°C for 15min-30min, and then at pH 8.3-8.5, 4°C for 1h-48h.

[0056] More preferably EGF-CTA 2 -TAT protein with (CTB) 5 The protein was denatured at pH 2.3 and 23°C for 20 minutes, and then refolded at pH 8.5 and 4°C for 1 hour.

[0057] In the embodiment of the present invention, the pH value of denaturation is adjusted by 0.5M citric acid, and the pH value of renaturation is adjusted by 2M Tris.

[0058] In the embodiment of the present inve...

Embodiment 1

[0063] see figure 1 , is pET22b-EGF-CTA in the embodiment of the present invention 2 - Construction map of the TAT plasmid. In this example, the plasmid pET22b-EGF-CTA 2 -TAT was introduced into Escherichia coli BL21 to obtain pET22b-EGF-CTA 2 -TAT / BL21 for protein expression, inclusion bodies were obtained by cracking, freeze-thawing, and EGF-CTA was obtained by dissolving 2 -TAT protein, comprising the steps of:

[0064] 1) Protein expression: Add 7 μL of ampicillin solution (50 mg / mL stock solution, 50 μg / mL final concentration) and 10 μL pET22b-EGF-CTA to 6 test tubes (7 mL) on a clean bench. 2 - TAT / BL21 bacteria solution, place the test tube in a constant temperature culture shaking box at 37° C. at 200 rpm to shake and shake the bacteria and culture overnight for 12 hours. After 12 hours, take out the 6 test tubes that have been cultivated overnight, and transfer them to 350 mL LB liquid medium containing 350 μL ampicillin solution (50 mg / mL stock solution, 50 μg / m...

Embodiment 2

[0070] In this example, the dissolved supernatant of Example 1 was refolded on an affinity chromatography column (nickel column) for protein purification, and lyophilized to obtain EGF-CTA 2 -TAT protein powder, a total of 10 hours. Wherein, protein purification comprises the following steps:

[0071] 1) Column packing: check that the affinity chromatography column is well sealed, put 10mL filler (histidine-labeled gel) into the affinity chromatography column, and at the same time turn on the nucleic acid and protein detector to preheat for 30 minutes, and adjust the peristaltic pump (final rpm1.0, sensitivity 1.0A, baseline 0.02), wash with deionized water for 12h;

[0072] 2) Refolding: adjust the peristaltic pump rpm 3.6, wash with 30ml of 8mol urea-Tris-HCl buffer solution, add 20mL of the dissolved supernatant to the affinity chromatography column, after loading the sample, continue to add 30ml of 8mol urea-Tris - HCl buffer balance, after the peak is stable, use 30ml o...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to EGF-like protein and a construction method thereof, and chimeric protein as well as a preparation method andapplication thereof. The invention provides the EGF-like protein; the EGF-like protein is EGF-CTA2-TAT protein; the nucleotide sequence of the EGF-CTA2-TAT protein is shown in SEQ ID NO: 1. The EGF-CTA2-TAT protein is chimeric with EGF protein with a repair effect, cell-penetrating peptide assisting transdermal absorption, and a cholera toxin A2 subunit (CTA2) with the ability of penetrating cellmembranes; it is shown by a cell activity experiment test result that the EGF-CTA2-TAT protein has obvious effect of promoting the growth of BALB/c 3T3 cell strains, and has bioactivity.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an EGF-like protein, a construction method thereof, a chimeric protein, a preparation method and application thereof. Background technique [0002] Due to the selective opening mechanism of the blood-brain barrier, the blood-brain barrier is a major obstacle to the transport of therapeutic biomacromolecules, which have limited use in the treatment of central nervous system diseases. Although macromolecular proteins can enter the brain through peptide substances that penetrate the cell membrane and use specific receptors to mediate, the design and development of proteins that can enter the brain are still very urgent. Contents of the invention [0003] In view of this, the present invention discloses EGF-like protein and its construction method, chimeric protein and its preparation method and application, which expands the design of brain-into protein. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/22C12N15/62C12N15/70A61K38/18A61K47/64A61P25/00
CPCA61K38/00A61K47/6415A61P25/00C07K14/485C07K2319/10C07K2319/55C12N15/70
Inventor 王华倩何华锋林丹敏孙家杰方颖琳刘倚君任东婷李武填赵肃清张焜
Owner 广东格烯生物科技股份有限公司
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