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Method for constructing shRNA eukaryon expression vector for inhibiting mouse B7-DC gene expression

A eukaryotic expression vector, B7-DC technology, applied in the field of molecular biology, can solve the problem of enhancing the immune response and achieve the effect of convenient application, simple operation and low cost

Inactive Publication Date: 2011-02-02
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, inhibition of the negative regulatory signaling molecule B7-DC may enhance the effect of dendritic cell-induced immune responses

Method used

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  • Method for constructing shRNA eukaryon expression vector for inhibiting mouse B7-DC gene expression
  • Method for constructing shRNA eukaryon expression vector for inhibiting mouse B7-DC gene expression
  • Method for constructing shRNA eukaryon expression vector for inhibiting mouse B7-DC gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The construction process of shRNA eukaryotic expression vector that inhibits the expression of mouse B7-DC gene is as follows figure 1 As shown, the specific operations are as follows:

[0033] A) According to the selection principle of siRNA target sequence, according to the gene sequence of mouse B7-DC provided by Genebank (NM_021396), select the 361-379th nucleotide sequence GCTTCTTACATGAGGATAG from the coding region as the target sequence, design and synthesize primers ( Synthesized by Shanghai Shenggong Biological Engineering Technology Service Co., Ltd.), the primers are:

[0034] siRNA1-Oligo1: 5′-GATCCCGCTTCTTACATGAGGATAGTTCAAGAGACTATC

[0035] CTCATGTAAGAAGCTTTTTGGAAA-3′

[0036] siRNA1-Oligo2: 5′-AGCTTTTCCAAAAAGCTTCTTACATGAGGATAGTCTCTT

[0037] GAACTATCCTCATGTAAGAAGCGG-3′

[0038] siRNA2-Oligo1: 5′-GATCCCCTTCAGCTGCATGTTCTGGTTCAAGAGACCAGA

[0039] ACATGCAGCTGAAGTTTTTGGAAA-3′

[0040] siRNA2-Oligo2: 5′-AGCTTTTCCAAAAACTTCAGCTGCATGTTCTGGTCTCTT

[...

Embodiment 2

[0052] The eukaryotic expression vector shRNA-B7-DC constructed in Example 1 was applied to inhibit the expression of B7-DC gene in dendritic cells derived from mouse bone marrow in vitro, and the specific steps are as follows:

[0053] ①Prepare a large amount of shRNA-B7-DC high-purity plasmid;

[0054] ②Preparation of dendritic cells from mouse bone marrow: according to the conventional method introduced by Inaba et al.;

[0055] ③Inoculate 5×10 on the day of transfection 5 Two well-growing mouse bone marrow-derived dendritic cells are placed in a six-well plate. When their abundance reaches 70-80%, refer to the instructions of Lipofecter liposome transfection reagent (Biyuntian Institute of Biotechnology). Plasmid shRNA-B7-DC was transfected into dendritic cells, where: plasmid DNA (μg): Lipofecter liposome transfection reagent (μl) was 1.5μg: 4μl;

[0056] ④48 hours after transfection, centrifuge to collect cells, every 5-10×10 6 Add 1ml Trizol (Invitrogen, USA) to the cells to ly...

Embodiment 3

[0060] The eukaryotic expression vector shRNA-B7-DC constructed in Example 1 was applied in vitro to enhance the proliferation of T cells by dendritic cells derived from mouse bone marrow, and the specific steps are as follows:

[0061] ①Prepare a large amount of shRNA-B7-DC high-purity plasmid;

[0062] ②Preparation of dendritic cells from mouse bone marrow: according to the conventional method introduced by Inaba et al.;

[0063] ③Inoculate 5×10 on the day of transfection 5 Two well-growing mouse bone marrow-derived dendritic cells are placed in a six-well plate. When their abundance reaches 70-80%, refer to the instructions of Lipofecter liposome transfection reagent (Biyuntian Institute of Biotechnology). Plasmid shRNA-B7-DC and empty vector pAS were transfected into dendritic cells, wherein: plasmid DNA (μg): Lipofecter liposome transfection reagent (μl) was 1.5μg: 4μl;

[0064] ④ After transfection for 48 hours, add mitomycin C (final concentration of 30μg / ml) to each well. Afte...

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Abstract

The invention discloses a constructing method of eucaryon expression vector shRNA for restraining the gene expression of a mouse B7-DC. The method has the constructing processes that the positions from 361 to 379 of a nucleotide sequence in a code area are selected to be a target sequence, and are designed and combined to form a primer according to the gene order of the mouse B7-DC provided by Genebank; through the annealing, the primer is connected to an interference vector pAS, so that the eucaryon expression vector that is shRNA-B7-DC which is formed by the method is obtained. The eucaryonexpression vector which is formed by the method not only can target specifically and restrain the gene expression of B7-DC in dendritic cells which are the marrow sources of the mouse, but also can augment greatly and can clone the valid parts thereof onto a better vector; the constructing method has the advantages of simple operation, convenient application, small toxic and side effects, low cost, etc.; in addition, the constructing method can provide an effective tool for the application research of researching on the gene functions of the B7-DC and increasing the immune response of dendritic cell induction.

Description

Technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to a method for constructing a shRNA eukaryotic expression vector that inhibits the expression of mouse B7-DC genes. Background technique [0002] In recent years, a large number of studies have shown that T cell activation is regulated by the balance between positive and negative signals. Co-stimulatory or co-inhibitory receptors or ligands on T cells interact with the corresponding ligands or receptors on antigen-presenting cells to maintain this balance and ultimately determine the activation of T cells. B7-DC is a member of the B7 family of costimulatory molecules and has recently been identified as a ligand for the immunosuppressive receptor programmed death protein 1 (PD-1). B7-DC is mainly expressed on monocytes and selectively expressed on dendritic cells. Peripheral monocytes are induced by GM-CSF and IL-4, and the dendritic cells produced can highly express B7-DC. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/79C12N15/113C12N15/66
Inventor 江文正樊燕闻洁君郝文丽
Owner EAST CHINA NORMAL UNIV
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