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Polypeptide binding with tumor necrosis factor, and applications thereof

A tumor necrosis factor and polypeptide inhibitor technology, applied in the field of biomedicine, can solve the problems of good curative effect, aggravation of cardiac insufficiency, high drug cost, etc., and achieve the effect of inhibiting cell apoptosis, reducing effective concentration, and reducing treatment cost

Active Publication Date: 2015-08-12
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Drugs that directly target TNF-α are mainly recombinant protein drugs of biotechnology, which have the characteristics of small side effects, strong specificity, and good curative effect. The main disadvantages are that repeated administration is required, the dosage is large, and the price is expensive
[0004] However, the current biological agents targeting TNF-α still face the following problems: first, the cost of monoclonal antibody drugs is very high, and some researchers pointed out that the early use of biological agents for RA patients does not have a cost advantage; In the safety study of -alpha treatment, it was found that this type of drug will produce some side effects after medium and long-term use. Increased, aggravated cardiac insufficiency, hypersensitivity, nerve damage and malignant tumors have also been reported

Method used

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  • Polypeptide binding with tumor necrosis factor, and applications thereof
  • Polypeptide binding with tumor necrosis factor, and applications thereof
  • Polypeptide binding with tumor necrosis factor, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] The determination of the affinity of embodiment 1 polypeptide to TNF-α

[0076] (1) Prepare the polypeptide with 1×PBS to a solution with a concentration of 1 mg / mL, and prepare the anti-TNF-α polypeptide to a solution with a concentration of 100 μg / mL, take 2.5 μL and drop it on a carboxylated SPR chip (purchased from Plexera company, Kx5 type SPR standard substrate), and the polypeptide was immobilized on the SPR chip through the specific reaction of streptavidin and biotin. Then the TNF-α solution (diluted in 1×PBS) with a concentration of 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, and 5 μg / mL was passed through the chip surface as the mobile phase, the binding time was 150 s, and the dissociation time was 150 s. The respawn time is 250s. 1×PBS was used as the dissociation solution, and 0.5% phosphoric acid was used as the reconstitution solution. Record the binding-dissociation curve of the polypeptide and TNF-α on the K×5 SPR instrument (Plexera), such as figure 1 show...

Embodiment 2

[0080] Example 2 The MTT method evaluates the effect of the polypeptide on the cytotoxicity induced by TNF-α

[0081] The mouse fibroblast cell line L929 was used as a research model, and the cells were cultured in RPMI-1640 medium (containing 10% North American fetal bovine serum, 1% penicillin and streptomycin). In a 96-well cell culture plate (Corning), each well was cultured with 1×10 4 12 cells, after 12 hours in the cell plate, add a series of concentration gradient TNF-α and the polypeptide in the culture plate (the specific concentration can be seen figure 2 ). The final volume of culture medium in each well was 200 μL. After incubation for 48 hours, 20 μL of thiazolium blue (MTT) 1×PBS solution with a concentration of 5 mg / mL was added to each well of the culture plate. React at 37°C for 4 hours. The solution in the culture plate was sucked away, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan precipitate. In a continuous sp...

Embodiment 3

[0083] Example 3 Observation of the Effect of the Polypeptide on TNF-α-Induced Cell Quantity Reduction Using an Optical Microscope

[0084] The cell culture method was as described in Example 2, except that a 6-well plate was used for cell culture, and the final volume of culture medium in each well was 2 mL.

[0085] After TNF-α and the polypeptide were added to the cells and incubated for 48 hours, observed and photographed under an optical microscope, as shown in image 3 shown. (A) is the untreated cells, (B) is the cells treated with 10ng / mL TNF-α. Compared with the normal cultured cells, it can be found that in the same area, the number of cells is greatly reduced, and apoptosis can be seen. Apoptotic bodies; (C-E) were co-incubated with TNF-α at a concentration of 0.1ng / mL, 10ng / mL, and 1000ng / mL, and it was found that 0.1ng / mL of polypeptide had no significant effect on cell proliferation , when the concentration of the polypeptide increased to 10ng / mL, the number of...

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Abstract

The present invention relates to a polypeptide binding with tumor necrosis factor (TNF-alpha), and applications thereof. According to the present invention, the polypeptide and TNF-alpha have high affinity, and the polypeptide can be bound with the excess TNF-alpha to make the apoptosis promoting protein Bid in the apoptosis-related mitochondrion apoptosis pathway be down-regulated while make the anti-apoptosis protein Bcl-2 be up-regulated in the apoptosis-related mitochondrion apoptosis pathway so as to inhibit the apoptosis caused by TNF-alpha, such that the polypeptide can be used for preparation of TNF-alpha-related disease treating drugs. The present invention further provides a method for screening at the molecular and cellular level and researching the influence of the polypeptide antagonist on the TNF-alpha physiological effect, and applications thereof so as to provide the effective method for development of the low-cost anti-TNF-alpha drug.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a polypeptide and its application, in particular to a polypeptide with a very high affinity with tumor necrosis factor (tumor necrosis factor, TNF-α), a reagent and its application. Background technique [0002] Tumor necrosis factor (TNF-α) is a cytokine closely related to systemic inflammation, mainly secreted by macrophages, and its biological functions are complex, and it is closely related to infectious diseases, autoimmune diseases, organ failure directly related to many diseases. For example, rheumatoid arthritis (RA) is a long-term chronic inflammation caused by an autoimmune disorder in which the immune system attacks the joints. This inflammation deforms the joint and can lead to loss of mobility for the patient due to joint pain and wear and tear. Since a large amount of TNF-α can be detected in the synovial fluid of RA patients, and TNF-α can mediate inflammator...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N15/11C12N15/63A61K38/16A61P19/02A61P29/00A61P17/06
CPCC07K14/00
Inventor 杨延莲张敏王琛王晨轩
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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