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DC-SIGNR in-vitro expression plasmid with green fluorescent protein label and construction method thereof

A technology of DC-SIGNR and green fluorescent protein, which is applied in the field of DC-SIGNR expression plasmids in vitro and its construction, can solve the problems of lack, unclear expression regulation mechanism, and difficulties in functional research of DC-SIGNR, and achieve wide application prospects, The effect of efficient expression

Inactive Publication Date: 2010-07-07
FUZHOU HOSPITAL FOR INFECTIOUS DISEASE +1
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  • Claims
  • Application Information

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Problems solved by technology

However, the expression regulation mechanism of DC-SIGNR is still unclear, and there is no related research on the induction mechanism of DC-SIGNR expression at home and abroad. Therefore, the in vitro expression model data of DC-SIGNR are relatively lacking, which has caused difficulties for the functional research of DC-SIGNR. extremely difficult

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  • DC-SIGNR in-vitro expression plasmid with green fluorescent protein label and construction method thereof
  • DC-SIGNR in-vitro expression plasmid with green fluorescent protein label and construction method thereof
  • DC-SIGNR in-vitro expression plasmid with green fluorescent protein label and construction method thereof

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Embodiment Construction

[0036] Below in conjunction with specific implementation mode, content of the present invention is described in detail:

[0037] 1. The construction method of the DC-SIGNR in vitro expression plasmid with green fluorescent protein marker is as follows:

[0038] (A) Total RNA was obtained from PBMC. The general steps can be operated in the following order:

[0039] 1. Add 7.5ml of lymphocyte separation medium Ficoll to 5ml of EDTA anticoagulated whole blood, and centrifuge at 1800 rpm for 20 minutes;

[0040] 2. Aspirate the middle buffy cell layer (PBMC), add 1ml of normal saline, centrifuge at 5000 rpm for 5 minutes, discard the supernatant, then add 1ml of normal saline, centrifuge at 5000 rpm for 5 minutes, discard the supernatant to obtain a precipitate.

[0041] 3. Add 1ml Trizol (manufactured by Invitrogen) to the precipitate obtained in step 2 and leave it at room temperature for 5 minutes;

[0042] 4. Add chloroform to 200ul chloroform / ml Trizol, shake and mix for 1...

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Abstract

The invention relates to DC-SIGNR in-vitro expression plasmid with a green fluorescent protein label and a construction method thereof. The plasmid is the green fluorescent protein expression plasmid DC-SIGNR-GFP which adopts green fluorescent protein peGFP-N1 as a carrier and is constructed by fusing a (DC-Specific Intercellular Adhesion Molecule-3-Grabbing Nonintegrin Related) DC-SIGNR coding sequence with a green fluorescent protein peGFP-N1 sequence on the surface of a dendritic cell. Efficient expression can be carried out in multiple cells through the constructed plasmid after cell transfection without specific induction, and the limit that DC-SIGNR is only expressed in a few internal cells and the limit caused by an expression regulation mechanism are broken through, thereby greatly facilitating correlational researches on DC-SIGNR.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a DC-SIGNR in vitro expression plasmid marked with green fluorescent protein and a construction method thereof. Background technique [0002] Dendritic cells (dendritic cells, DC) play an important role in the infection and immunity of pathogenic microorganisms. Dendritic cells can capture viruses and present them to T cells, inducing the special immunity of T cells. important role in the initiation reaction. Recent studies have found that DC-Specific Intercellular Adhesion Molecule-3-Grabbing Nonintegrin (DC-SIGN) on the surface of dendritic cells and DC-SIGN DC-Specific Intercellular Adhesion Molecule-3-Grabbing Nonintegrin Related (DC-SIGNR) is the adhesion receptor of many pathogenic microorganisms, which can recognize and bind pathogenic microorganisms, and is involved in the pathogenesis and immunity of infectious diseases makes an important impact. [0003] DC...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/65C12N15/12
Inventor 许利军
Owner FUZHOU HOSPITAL FOR INFECTIOUS DISEASE
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