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Anti-inflammatory active peptide Hydrostatin-SN1 derived from Hydrophis cyanocinctus, coding gene thereof and application in pharmacy

A green ring sea snake, coding gene technology, applied in the field of biomedicine, to achieve the effect of strong anti-inflammatory activity, great clinical application value, and simple structure

Active Publication Date: 2013-04-10
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no literature report about the bioactive peptides of Qinghuan sea snakes, and there is no report about the relationship between the bioactive peptides of Qinghuan sea snakes and TNF-α

Method used

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  • Anti-inflammatory active peptide Hydrostatin-SN1 derived from Hydrophis cyanocinctus, coding gene thereof and application in pharmacy
  • Anti-inflammatory active peptide Hydrostatin-SN1 derived from Hydrophis cyanocinctus, coding gene thereof and application in pharmacy
  • Anti-inflammatory active peptide Hydrostatin-SN1 derived from Hydrophis cyanocinctus, coding gene thereof and application in pharmacy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Source and synthesis of Qinghuan sea snake venom active peptide Hydrostatin-SN1.

[0057] Use TNFR1 as the target to pan the Qinghuan sea snake venom gland phage display library: Dilute TNFR1 to a concentration of 1 μg / ml with pH 9.6 carbonate coating buffer, coat a 96-well ELISA plate with 100 μl / well, and Seal each well with a blocking agent; add 100 μl phage to each well of the coated ELISA plate, and incubate at 37°C for 1 hour; wash the plate with TBST solution 4 times, add eluent and incubate at 37°C for 20 minutes, and remove the bacteriophage from the ELISA plate The bound phages were eluted, and the eluted phages were amplified with the host bacterium BLT5403 (purchased from Novagen, provided in the Novagen T7Select Cloning Kit kit, and the modified specific host bacterium) to complete the first round panning.

[0058] The Qinghuan sea snake venom gland phage display library was prepared by the following method:

[0059] 1. Acquisition of the mRNA ...

Embodiment 2

[0072] Example 2: biacore (biomacromolecule interaction analysis technology based on surface plasmon resonance technology) analyzes the interaction between Hydrostatin-SN1 and TNF-α, TNFR1 and TNFR2.

[0073]In order to further verify the interaction between Hydrostatin-SN1 and TNF-α, TNFR1 and TNFR2, the interaction between SN1 and TNF-α, TNFR1 and TNFR2 was analyzed by the surface plasmon resonance (SPR)-based biomacromolecular interaction analyzer BIACORE T100. Interaction between TNFR1. The experimental procedure is as follows: human recombinant TNFR1, TNF-α and TNFR2 (purchased from PeproTech, USA, purity ≥ 98%) were covalently coupled to the surface of the sensor chip using the amino coupling method in BIACORE T100 control software, TNF-α, TNFR1 and TNFR2 coupling amounts were about 8000 ( image 3 ), 3000 ( Figure 4 ) and 200 ( Figure 5 ) response unit (Response Unit, RU); Hydrostatin-SN1 was dissolved in PBS and diluted to a concentration gradient of 24μM, 60μM, 1...

Embodiment 3

[0074] Example 3: Inhibitory effect of Hydrostatin-SN1 on TNF-α killing activity of its sensitive target cells.

[0075] After biacore proved the interaction between Hydrostatin-SN1 and TNFR1, TNF-α-sensitive target cells L929 cells were further used to detect whether Hydrostatin-SN1 inhibited the activity of TNF-α to kill its sensitive target cells.

[0076] The specific experimental steps are as follows: subcultured mouse fibroblast L929 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were digested with trypsin and resuspended in RPMI-1640 containing 10% fetal bovine serum (commercially available, purchased from Thermo Company) for culture Base (commercially available, purchased from Thermo Company), adjust the cell density to 2×105 / ml, inoculate 100 μl in a 96-well cell culture plate, and culture overnight in a CO2 incubator. TNF-α was mixed with actinomycin D and a series of concentrations of Hydrostatin-SN1 and then added to L929 cell 96-well cell ...

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Abstract

The invention belongs to the technical field of biomedicine. So far, no report on bioactive peptides of Hydrophis cyanocinctus exists. The invention provides an anti-inflammatory active peptide Hydrostatin-SN1 derived from Hydrophis cyanocinctus. The anti-inflammatory active peptide Hydrostatin-SN1 derived from Hydrophis cyanocinctus contains a protein having an amino acid sequence shown as SEQ ID NO.1. The invention further provides a coding gene of Hydrostatin-SN1 and the application of anti-inflammatory active peptide Hydrostatin-SN1 derived from Hydrophis cyanocinctus and coding gene thereof for preparing medicaments for treatment of TNF-alpha-related diseases. The TNF-alpha-related diseases may include rheumatoid arthritis, diabetes, coronary heart disease, sepsis, colonitis and other complicated inflammation or immunological diseases. The anti-inflammatory active peptide Hydrostatin-SN1 derived from Hydrophis cyanocinctus and coding gene thereof are significant in clinical applications.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a Qinghuan sea snake anti-inflammatory active peptide Hydrostatin-SN1, its coding gene and its application in pharmacy. Background technique [0002] Tumor necrosis factor (TNF-α, also known as TNF) is a cytokine mainly secreted by activated macrophages and lymphocytes. For different cell types, TNF can cause diverse biological effects, implying that it plays an important regulatory role in a variety of physiological and pharmacological conditions. In addition, TNF-α is also one of the key regulators in the process of apoptosis (programmed cell death). As a pleiotropic cytokine, TNF-α plays a role in biological processes such as cell regeneration, migration, inflammation and apoptosis. With the deepening of research, the key role of TNF-α in rheumatoid arthritis, asthma, sepsis, colitis, diabetes, coronary heart disease and other complex inflammatory diseases or...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/04C12N15/12A61K38/17A61P19/02A61P29/00A61P3/10A61P9/10A61P31/04A61P1/00
Inventor 陆一鸣郑增节王俊杰胡振林厉建中张俊平
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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