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Recombinant gene, vector and exosome for double-way antagonism of PCSK9 as well as preparation method and application of exosome

A technology for recombining genes and exosomes, applied in the field of genetic engineering, can solve the problems of limited antagonism efficiency, adverse reactions, and high production costs, and achieve the effects of low cost, reduced degradation, and low immunogenicity

Active Publication Date: 2021-11-02
AIR FORCE MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many lipid-lowering drugs, long-term use often has adverse reactions
At present, PCSK9 has become a new lipid-lowering target, but most of the PCSK9 antagonists are monoclonal antibodies, RNA interference drugs, and mimetic peptides, etc., which have high production costs, unknown side effects, poor stability, and limited antagonism efficiency. The problem

Method used

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  • Recombinant gene, vector and exosome for double-way antagonism of PCSK9 as well as preparation method and application of exosome
  • Recombinant gene, vector and exosome for double-way antagonism of PCSK9 as well as preparation method and application of exosome
  • Recombinant gene, vector and exosome for double-way antagonism of PCSK9 as well as preparation method and application of exosome

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Experimental program
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preparation example Construction

[0028] The present invention provides a method for preparing the above-described exosomes, comprising the steps of:

[0029] After the above-described recombinant expression vector transfected into human mesenchymal stem cells (the MSC), incubated to obtain exosomes, namely EGFA modified exosomes.

[0030] In the present invention, comprising the transfection of the recombinant expression vector and transfection reagent mixing and the MSC, the recombinant expression vector mass volume and transfection reagent is preferably from (2 ~ 4) μg: (4 ~ 8 ) μL, more preferably (2.5 ~ 3.5) μg: (5 ~ 7) μL.

[0031] In the present invention, the incubation is preferably carried out in a cell incubator; the incubation temperature is preferably 37 [deg.] C; during incubation, CO 2 The volume percentage of preferably 5%; incubating the incubation comprises a first and a second incubation; preferably in the absence of the first double-antibody incubation medium (DMEM, Gibco, USA; FBS, Gibco compa...

Embodiment 1

[0039] Construction of recombinant expression vector: the recombinant expression vector is done by Nanjing GenScript Corporation;

[0040] (1) selection of restriction sites XhoI and HindⅢ;

[0041] (2) 3 'end to the 5' end is connected Kozak sequence fragment (as shown in SEQ ID No.3), CD63 first segment (e.g. as shown in SEQ ID No. 4), outer-EGF-A fragment (e.g., SEQ FIG ID No.5), CD63 fragment of a second gene (e.g., SEQ IDNo.6 shown), inner-EGF-A fragment (as shown in SEQ ID) and No.7 FLAG tag (e.g., as SEQ ID No. 8 shown), giving a recombinant gene, the nucleotide sequence shown in sequence Listing SEQ ID Nos. 1;

[0042] (3) The above recombinant gene loaded pcDNA3.1 (+) and XhoⅠ HindⅢ between restriction sites vector successfully humanized CD63-2 × EGF-A motif loaded into pcDNA3.1 (+) on a support , recombinant expression vectors, e.g. figure 1 Indicated.

[0043] (4) 2 ~ 4μg recombinant expression vector with 4 ~ 8μL after transfection reagent mixture, after standing for 3...

Embodiment 2

[0049] The resulting secreted outside the body westernblot detecting FLAG tag.

[0050] The extraction method of the exosomes precipitate after centrifugation exosomes, vacuum suction residual liquid was added 50μL RIPA lysis buffer (Pik days, China), by pipetting with a pipette to completely dissolve exosomes, placed on ice 30min, so that sufficient exosomes proteins cleavage. For protein quantification (Thermo, USA) by doubling dilution and protein sample preparation. 12% of the gel formulation itself (Pik days, China), was added after exosomes protein samples were electrophoresed. Setting electrophoresis voltage 90V, 30min, start electrophoresis. When the protein samples into the underlying separation gel, the electrophoresis voltage is adjusted to 120V, 90min. Protein samples were observed position when the position reaches the electrophoresis can be stopped, in turn be transferred to a membrane, to set current 200mA, expected time 120min. After antibody incubation. The antibo...

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Abstract

The invention relates to the field of gene engineering, in particular to a recombinant gene, a vector and an exosome for double-way antagonism of PCSK9 as well as a preparation method and application of the exosome. The invention provides a recombinant gene for double-way antagonism of PCSK9. The recombinant gene comprises a Kozak segment, a first segment of CD63(N-CD63), an outer-EGF-A segment, a second segment of CD63(C-CD63), an inner-EGF-A segment and an FLAG tag which are sequentially connected from the 3' end to the 5' end, wherein the Kozak segment, the first segment of CD63(N-CD63) , the outer-EGF-A segment, the second segment of CD63(C-CD63), the inner-EGF-A segment and the FLAG tag are sequentially connected. The recombinant gene can effectively treat hyperlipidemia and atherosclerosis, and has the advantages of small side effect, low cost, high stability and high antagonism efficiency.

Description

Technical field [0001] The present invention relates to the field of genetic engineering, particularly to a recombinant gene antagonizing PCSK9 in a dual way, the carrier, the exosomes and preparation method and application. Background technique [0002] Department of Human hyperlipidemia lipid metabolism disorders caused mainly refers to the serum total cholesterol (TC), triglyceride (TG) levels are too high, too high blood low-density lipoprotein cholesterol (LDL-C) levels or high blood density lipoprotein cholesterol (HDL-C) levels are too low, it is an important factor leading to atherosclerosis, and is recognized as hypertension, coronary heart disease and cerebrovascular accident a major risk factor. [0003] Prevention and treatment of hyperlipidemia has been an important topic in the medical field. Lipid-lowering therapy to avoid or reduce the occurrence of atherosclerosis, thereby reducing the incidence of coronary heart disease and stroke. Although currently more lipid-...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N5/10C12N15/85A61K35/28A61P3/06A61P9/10
CPCC07K14/485C07K14/70596C12N15/85C12N5/0662A61K35/28A61P3/06A61P9/10C07K2319/43C12N2510/00
Inventor 袁丽君王辰杨国栋韦梦影
Owner AIR FORCE MEDICAL UNIV
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