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Adeno-associated virus for silencing expression of Ras guanyl nucleotide releasing protein 1 (RASGRP1) in intestinal tract of mice as well as preparation method and application of adeno-associated virus

A virus and mouse technology, which is used in the field of adeno-associated virus that silences the expression of RASGRP1 in the intestinal tract of mice and its preparation

Active Publication Date: 2019-03-01
SHUGUANG HOSPITAL AFFILIATED WITH SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there is no report on the adeno-associated virus that silences the expression of RASGRP1 in the intestinal tract of mice according to the present invention

Method used

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  • Adeno-associated virus for silencing expression of Ras guanyl nucleotide releasing protein 1 (RASGRP1) in intestinal tract of mice as well as preparation method and application of adeno-associated virus
  • Adeno-associated virus for silencing expression of Ras guanyl nucleotide releasing protein 1 (RASGRP1) in intestinal tract of mice as well as preparation method and application of adeno-associated virus
  • Adeno-associated virus for silencing expression of Ras guanyl nucleotide releasing protein 1 (RASGRP1) in intestinal tract of mice as well as preparation method and application of adeno-associated virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 RasGRP1 overexpression plasmid construction

[0048] 1. Experimental process

[0049] PCR amplification of the target gene. After digestion of the expression vector, the vector fragment was recovered by gel. Transform E.coli competent cells after homologous recombination between the target gene and the vector fragment. Transformants were identified by colony PCR, and positive clones were sent for sequencing. The clones with correct sequencing were subjected to plasmid extraction. The experimental process is as follows figure 1 shown.

[0050] 2. Experimental materials

[0051] Reagent name

Reagent source

cat. No.

tool carrier

Sangon Bioengineering (Shanghai) Co., Ltd.

/

PrimeSTAR

Takara

DR010A

Taq enzymes and dNTPs

Takara

DR001B

Seamless Cloning Kit

Sangon Bioengineering (Shanghai) Co., Ltd.

/

NheI-HF

NEB

R3131L

HindIII-HF

NEB

R3104L

DH5a Compe...

Embodiment 2

[0097] Example 2RasGRP1shRNA interference vector construction (3 targets)

[0098] 1. Experimental process

[0099] PCR amplification of the target gene. After digestion of the expression vector, the vector fragment was recovered by gel. Transform E.coli competent cells after homologous recombination between the target gene and the vector fragment. Transformants were identified by colony PCR, and positive clones were sent for sequencing. The clones with correct sequencing were subjected to plasmid extraction.

[0100] 2. Experimental materials

[0101]

[0102]

[0103] 3. Vector map

[0104] Carrier map such as Figure 8 shown.

[0105] 4. Digestion of vector

[0106] The tool vector was digested with BspEI and NheI-HF, and the 5719bp vector fragment was recovered. The restriction map is as follows: Figure 9 shown.

[0107] 5.siRNA design

[0108] Viral vector construction framework:

[0109] No.

5’

STEMP

Loop

STEMP

3’

pAAV...

Embodiment 3

[0145] Example 3 Co-transfection of 293T cells with overexpression and interference plasmids

[0146] (1) One day before transfection, the cells were plated on a well plate.

[0147] (2) Cells can be transfected 12 hours after plating.

[0148] (3) After 12 hours of cell plating, prepare the corresponding plasmid / Lipo2000.

[0149] (4) The plasmid (1ug for overexpression, 3ug for interference and interference control) was mixed with 250ul of OPTI-MEM and incubated for 5min.

[0150] (5) While performing 4, mix 10ul of Lipo2000 and 250ul of OPTI-MEM and incubate for 5min.

[0151] (6) After five minutes, mix the two well-mixed solutions together and incubate for 15 minutes.

[0152] (7) While incubating, suck out the original culture medium in the plate laid out yesterday, and add 1ml of OPTI-MEM.

[0153] (8) After the incubation is completed, add solution 6 into the well plate and shake gently.

[0154] (9) Place the orifice plate in a 37 degree incubator for cultivation...

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Abstract

The invention discloses an adeno-associated virus for silencing expression of Ras guanyl nucleotide releasing protein 1 (RASGRP1) in the intestinal tract of mice as well as a preparation method and application of the adeno-associated virus. Three pairs of siRNA sequences are designed for an RASGRP1 gene, and a RasGRP1 shRNA interference vector is constructed; screen target results of a quantitative polymerase chain reaction (qPCR) show that PAAVE2113, PAAVE2114 and PAAVE2115 all have a silencing effect; the PAAVE2115 has the best silencing effect, the silencing efficiency is about 74%, which is consistent with a WB result. The interference vector and helper plasmid are co-transfected into the engineering cells so as to obtain the adeno-associated virus for silencing the expression of the RASGRP1 in the intestinal tract of the mice. The animal experiments confirm that when the adeno-associated virus is used for interfering with the C57 mice, the expression of the RasGRP1 in the small intestine tissues of the mice is reduced; an intraperitoneal injection has the best interference effect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an adeno-associated virus for silencing the expression of RASGRP1 in the intestinal tract of mice and its preparation method and application. Background technique [0002] RNA interference refers to the highly conserved phenomenon of highly efficient and specific degradation of homologous mRNA induced by double-stranded RNA in the evolution process. It is a protective type for organisms to resist infections such as external viruses and maintain genome stability mechanism. When exogenous genes such as viral genes, artificially transferred genes, and transposons are randomly integrated into the host cell genome and transcribed by the host cell, some dsRNA is often produced. Host cells respond rapidly to these dsRNAs, and Dicer of the RNaseIII ribozyme family in the cytoplasm cuts dsRNAs into multiple small fragments of RNA (siRNA) with specific length and structure. siRNA is unzipped...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N15/864A01K67/027
CPCA01K67/0275C12N15/113C12N15/85C12N15/86C12N2310/141C12N2750/14143A01K2227/105A01K2217/058
Inventor 李俊燕陆灏陶枫陈清光徐隽斐侯瑞芳韩煦顾逸梦杨雪蓉金昕章丽琼陶乐维
Owner SHUGUANG HOSPITAL AFFILIATED WITH SHANGHAI UNIV OF T C M
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