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Adeno-associated virus recombinant vector for knocking out Egr3 gene, and construction method and application thereof

A technology for recombining vectors and viruses, applied in the direction of viruses/bacteriophages, using vectors to introduce foreign genetic material, viruses, etc., can solve the problems of lack of effective treatment methods and methods for liver cancer genes

Inactive Publication Date: 2019-02-01
汉恒生物科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Adeno-associated virus recombinant vector for knocking out Egr3 gene, and construction method and application thereof
  • Adeno-associated virus recombinant vector for knocking out Egr3 gene, and construction method and application thereof
  • Adeno-associated virus recombinant vector for knocking out Egr3 gene, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] 1. pHBAAV-CMV-Sacas9-U6-gRNA carrier (structure such as figure 1 ) was digested with BsaI, and the enzyme digestion system was as follows:

[0048]

[0049] 2. After the enzyme digestion of the carrier is completed, the gel is recovered (see the appendix for the recovery steps)

[0050] 3. Preparation of Egr3-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 instrument or a water bath to mix the above components, incubate at 95°C for 4 minutes, then take it out and place it at room temperature to cool.

[0055] 3. The processed target fragment and carrier ligation reaction system (20μl):

[0056] table 3

[0057]

[0058] The above reaction system is placed in a warm bath at 22°C for 20 minutes. If the follow-up operation cannot be performed immediately af...

Embodiment 2

[0063] Injection of Egr3 knockout adeno-associated virus into liver cancer model mice inhibits tumor growth and angiogenesis in mice

[0064] 1. Experimental materials

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

[0066] 2. Experimental method

[0067] 2.1 Mouse tumor modeling

[0068] Each of 24 nude mice was subcutaneously inoculated with 1*10^6 HepG2 cells in the anterior axilla. After 2 weeks, 24 mice were randomly divided into 3 groups, PBS group (blank control group), AAV-GFP group and AAV-EGR3-KO group. The growth of nude mice was observed every day. 2.2 Injection of AAV-GFP group and AAV-EGR3-KO group, control adeno-associated virus and PBS group

[0069] Aspirate 5 ul of liquid and insert the needle at a distance of 5 mm from the tumor with a microinjection needle, subcutaneously migrate to the site of the liver cancer model, and inject slowly. Inject again every 2 days. The ...

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PUM

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Abstract

The invention provides an adeno-associated virus recombinant vector for knocking out Egr3 gene, and a construction method and application thereof. The vector includes at least the following operably connected sequence elements: a 5' end inverted repetitive sequence, a CMV promoter, a sacas9 coding sequence, a polyA signal sequence, a U6 promoter sequence, a gRNA sequence and a 3' end inverted repetitive sequence. The adeno-associated virus recombinant vector can efficiently knock out Egr3. Through researches, the inventors found that the Egr3 gene knockout vector can effectively inhibit the growth of transplanted tumors and tumor angiogenesis of a HepG2 cell hepatoma model. This shows that the technical scheme of the invention can be applied to the treatment of liver cancer diseases.

Description

technical field [0001] The invention relates to an adeno-associated virus recombinant vector for knocking out the Egr3 gene, a construction method and application thereof, and belongs to the field of biotechnology. Background technique [0002] Egr3 is a member of the Egr (Early Growth Response Factor) family, an immediate response factor belonging to the transcription factor family, and is mainly involved in the regulation of cell growth, proliferation, differentiation and apoptosis [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 herpes virus). It encodes cap and rep genes in two terminal inverted repeats (ITRs). The cap gene encodes the viral capsid protein, and the rep gene is involved in the replication and integration of the virus. There are many...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N5/10A61K48/00A61P35/00
CPCA61K48/005A61P35/00C07K14/47C12N15/86C12N2750/14143
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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