Dectin-1 fusion protein expression vector and application thereof

A dectin-1 and fusion protein technology, applied in the field of construction of fusion protein expression vectors, can solve problems such as cumbersome operations

Inactive Publication Date: 2009-12-02
THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is similar to the activation of Limulus coagulation pathway by bacterial endotoxin. Therefore, Gram-yin bacteria containing endotoxin will have a greater impact on this method, and pretreatment is often required to remove it, which makes the operation more cumbersome.

Method used

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  • Dectin-1 fusion protein expression vector and application thereof
  • Dectin-1 fusion protein expression vector and application thereof
  • Dectin-1 fusion protein expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of Dectin-1 Fusion Protein Expression Vector

[0035] 1. Extraction of mouse peritoneal macrophages

[0036] (1) Experimental supplies and instruments:

[0037]Kunming mice (male or female, 22-25g, purchased from the Experimental Animal Center of Chongqing Medical University), 6% starch broth (soluble starch 6g, peptone 1g, beef extract 0.3g, NaCl 0.5g, distilled water 100ml, high-pressure steam bacteria), 1ml syringe, 75% alcohol, cotton swab, beaker, dissecting tray, dissecting scissors, ophthalmic forceps, 20ml syringe (large needle), autoclaved PBS, sterilized 15ml centrifuge tube, DMEM (10% fetal bovine serum , penicillin 100U / ml, streptomycin 100ug / ml), sterilized cell culture bottle, alcohol lamp, ultra-clean workbench, 37 ° constant temperature water bath, 37 ° CO 2 incubator

[0038] (2) Experiment content:

[0039] ① Inject 1ml of sterilized 6% starch broth into Kunming mice for two consecutive days

[0040] ② On the third day, t...

Embodiment 2

[0177] Example 2 Dectin-1 fusion protein expression

[0178] The recombinant plasmid (pSecTag2C-Dectin-1) contains the Igk protein secretory expression gene to ensure protein expression; contains 6×His tag to facilitate protein purification; without green fluorescent protein tag.

[0179] Experimental materials: Antibiotic-free serum-free DMEM, 10% fetal bovine serum antibiotic-free DMEM, Liposome 2000,

[0180] Ni-NTA His-Bind Resin

[0181] (1) Transfection:

[0182] 1. The day before transfection, inoculate 293 cells into three 6-well plates. On the day of transfection, the cells should reach 90% to 95% confluence (the culture medium is DMEM without antibiotics).

[0183] 2. On the day of transfection, within a short time before adding the liposome / DNA mixture, suck off the original medium, wash once with PBS, and replace with 1ml of DMEM medium without antibiotics and serum.

[0184] 3. Prepare the following mixture:

[0185] (1) Dilute the recombinant plasmid (4ug)...

Embodiment 3

[0207] Example 3 Identification of Dectin-1 fusion protein by western blot

[0208] (1) Compounding glue:

[0209]

[0210] After adding the separating gel, add some water to accelerate the coagulation. When a broken line is formed between the water and the gel, the gel has solidified; suck out the water and dry it with absorbent paper, add the concentrated gel, and fill it up as much as possible to avoid air bubbles; after gelling, take out the comb and rinse each hole with electrophoretic solution. That's it.

[0211] (2) Sample loading: protein (16ul)+water (0ul)+5×loading buffer (4ul), 98°C for 10 minutes, centrifuged and loaded. Add protein markers (8ul).

[0212] (3) Electrophoresis: Fill up the electrophoresis liquid in the inner tank, and add the outer tank to the lowest line. First, electrophoresis at 80V for 15-20 minutes, and then at 120V for about 1.5 hours.

[0213] (4) Transfer membrane: pre-cool the transfer fluid. Cut the corners of the PAGE glue to ...

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Abstract

The invention relates to a Dectin-1 fusion protein expression vector and application thereof, and also relates to construction of the fusion protein expression vector. The Dectin-1 fusion protein expression vector is constructed successfully, Dectin-1 fusion protein with high purity is obtained after the Dectin-1 fusion protein expression vector is transfected to recipient cells, and the obtained soluble Dectin-1 fusion protein can be fixed on the metal surface of an SPR biosensor, thus fungi can be monitored quickly, quantitatively and flexibly in real time.

Description

technical field [0001] The invention relates to the construction of a fusion protein expression vector, in particular to a Dectin-1 fusion protein expression vector and application thereof. Background technique [0002] Nosocomial infection (NI) is a major problem facing the medical field today. It not only affects the quality of medical care, but also affects the safety of patients. At present, superinfection caused by irrational use of antibacterial drugs is more common in NI, especially fungal infection. At the same time, with the widespread clinical application of antibiotics, immunosuppressants and corticosteroids, as well as the widespread development of organ transplantation and catheter intubation, the rate of fungal infection is increasing. Especially for immunosuppressed patients and critically ill patients, invasive fungal infection has become a major infectious complication with high morbidity and mortality. [0003] Because the early clinical symptoms and sign...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66C07K19/00G01N33/569G01N33/566G01N33/543
Inventor 刘丁张莉萍孙雯雯王玎陈萍王政王豪成瑶
Owner THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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