Adeno-associated virus recombinant vector for knocking out CXCL12 gene and its construction method and application

A recombinant vector, viral vector technology, applied in the direction of virus/bacteriophage, introduction of foreign genetic material, virus using vectors, etc., can solve the problem of lack of effective genetic methods and methods for glioma treatment

Active Publication Date: 2022-06-07
汉恒生物科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The problem in the prior art is the lack of genetic means and methods for effective treatment of glioma

Method used

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  • Adeno-associated virus recombinant vector for knocking out CXCL12 gene and its construction method and application
  • Adeno-associated virus recombinant vector for knocking out CXCL12 gene and its construction method and application
  • Adeno-associated virus recombinant vector for knocking out CXCL12 gene and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. pHBAAV-CMV-Sacas9-U6-gRNA vector (structure as figure 1 ) was digested with BsaI, and the enzyme cleavage system was as follows:

[0047]

[0048]

[0049] 2. The gel is recovered after the enzyme digestion of the vector is completed

[0050] 3. Preparation of CXCL12-gRNA fragment PCR

[0051] The upstream and downstream primers are annealed, wherein the sequence of the forward primer is shown in SEQ ID NO.2, and the sequence of the reverse primer is shown in SEQ ID NO.3.

[0052] Table 2

[0053]

[0054] Use a PCR machine or a water bath to mix the above components, incubate at 95°C for 4 minutes, and then take it out and place it at room temperature to cool.

[0055] 3. Ligation reaction system of the processed target fragment and the vector (20 μl):

[0056] table 3

[0057]

[0058] The above reaction system is placed in a warm bath at 22°C for 20min. After the reaction is completed, if subsequent operations cannot be performed immediately, the ...

Embodiment 2

[0063] Injection of adeno-associated virus recombinant vector for CXCL12 knockout into glioma model mice inhibits tumor growth and angiogenesis in mice

[0064] 1. Experimental materials

[0065] 4-6 weeks old nude mice (the mice can be purchased from Shanghai Slack Laboratory Animal Co., Ltd.), weighing 16-20 g.

[0066] 2. Experimental method

[0067] 2.1 Mouse tumor modeling

[0068] 24 nude mice were inoculated subcutaneously in the anterior axilla with 1*10^8 U87 cells. After 2 weeks, 24 mice were randomly divided into 3 groups, PBS group (blank control group), AAV-GFP group and AAV-CXCL12-KO group. The growth of nude mice was observed every day.

[0069] 2.2 Injection of AAV-GFP group and AAV-CXCL12-KO, control adeno-associated virus and PBS group

[0070] Aspirate 5ul of liquid with a micro-injection needle and insert the needle at a distance of 5 mm from the tumor, transfer it subcutaneously to the glioma site, and inject slowly. Inject again every 2 days. The c...

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Abstract

The present invention provides an adeno-associated virus recombinant vector for knocking out the CXCL12 gene, the recombinant vector at least includes the following sequence elements operably linked: 5' terminal inverted repeat sequence, CMV promoter, sacas9 sequence, polyA signal sequence, U6 promoter sequence, gRNA sequence, 3'-end inverted repeat sequence. The inventors found that the CXCL12 gene knockout vector can effectively inhibit the growth of transplanted tumors and tumor angiogenesis in the U87 cell glioma model, and can effectively inhibit the tumor growth and tumor angiogenesis in the U87 cell subcutaneous glioma model in nude mice. It shows that the technical scheme of the present invention can be applied to the treatment of glioma diseases.

Description

technical field [0001] The invention relates to an adeno-associated virus recombinant vector for knocking out CXCL12 gene, its construction method and application, and belongs to the field of biotechnology. Background technique [0002] CXCL12, also known as stromal cell-derived factor-1 (SDF-1), is a small molecule cytokine that belongs to the chemokine protein family. CXCL12 and its receptor (CXCR4) play an important role in the occurrence and development of many physiology and pathology. In tumors, intracellular signals are changed through different pathways, which can promote the proliferation, movement, adhesion and invasion of tumor cells. Affect the occurrence, development and prognosis of tumors. [0003] Adeno-associated virus (adeno-associated virus, AAV) is a genus of dependent viruses in the Parvoviridae family. The size of the virus particle is about 20-26 nm. The complete life cycle requires the participation of helper virus (usually adeno-associated virus or ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/864C12N5/10C12N15/66A61K35/768A61P35/00
CPCA61P35/00A61K35/768C12N15/66C12N15/86C12N2750/14143Y02A50/30
Inventor 陈意雄蔡晓龙邹杰朱鹏飞白易鑫
Owner 汉恒生物科技(上海)有限公司
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