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Serpin and minimum functional fragment and immunosuppression effect thereof

A serine protease and functional fragment technology, applied in the field of biomedicine, can solve the problems of lack of vaccines for ticks and tick-borne diseases, lack of safe and efficient repellent drugs, etc., and achieve the effect of easy absorption, small molecular weight, and poor immunogenicity

Active Publication Date: 2019-10-15
上海景晗生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most important way to control ticks is to use chemical insecticides. Due to the long-term use of insecticides, ticks develop drug resistance, and there is a lack of safe and efficient repellent drugs.
At the same time, relevant anti-tick vaccines are being studied at home and abroad, and there is a lack of effective vaccines against ticks and tick-borne diseases.

Method used

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  • Serpin and minimum functional fragment and immunosuppression effect thereof
  • Serpin and minimum functional fragment and immunosuppression effect thereof
  • Serpin and minimum functional fragment and immunosuppression effect thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Provide a kind of serine protease inhibitor SA, its protein expression process is:

[0043] (1) Clone the SA gene into the prokaryotic expression vector pGEX-6P-2

[0044] Extract the RNA of the longhorned blood tick, prepare cDNA, amplify the protein coding region fragment of the target gene from the cDNA library through gene-specific primers, and connect the coding region of the target gene to the Escherichia coli expression vector pGEX-6P- 2 in.

[0045] (2) Recombinant expression of serine protease inhibitor SA in Escherichia coli

[0046] The constructed recombinant pGEX-6P-2 target gene expression plasmid was transformed into Escherichia coli BL21 strain, and IPTG was used to induce the expression of the recombinant target protein.

[0047] (3) The method of column chromatography purifies recombinant serine protease inhibitor SA protein

[0048] Use GST medium to purify the recombinant target protein, and use specific protease to digest on the column to remove ...

Embodiment 2

[0051] Activity Inhibition of Serine Protease Inhibitor SA

[0052] 1. The identification experiment of serine protease inhibitor SA on the active inhibitory effect of cathepsin G (cathepsin G) and blood coagulation factor X (FXa), including steps:

[0053] (a) The total reaction system is 200ul, Cathepsin G (6.6nM) and FXa (0.33nM) were incubated with SA (0.1uM, 0.4uM) at room temperature for 30min.

[0054] (b) Add substrates at a final concentration of 250uM: Cathepsin G, methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide; FXa, chromogenic substrate S2765. 37°C, total reaction 90min, use enzyme labeling every 30min Instrument measurement, A = 405nm.

[0055] Such as Figure 4 and Figure 5 As shown, it was found that serine protease inhibitor SA inhibited the enzymatic activity of Cathepsin G and FXa in a dose-dependent manner.

[0056] 2. Identification experiment of the inhibitory effect of serine protease inhibitor SA on the activity of cathepsin B (cathepsin B)

[0057...

Embodiment 3

[0074] The effect of the serine protease inhibitor SA on the secretion of inflammatory factors by BMDMs or BMDCs stimulated by bacterial endotoxin lipopolysaccharide LPS

[0075] Stimulate BMDMs or BMDCs with bacterial endotoxin lipopolysaccharide (LPS), and detect the effect of SA on the expression of inflammatory factors at the mRNA and protein levels by real-time fluorescent quantitative PCR and enzyme-linked immunosorbent assay (ELISA) experiments.

[0076] Sample preparation and cell experiment steps are as follows:

[0077](a) BMDMs or BMDCs cells were plated in a 24-well plate, and three groups were set up: NC (blank control), PBS (negative control), and SA.

[0078] (b) PBS and endotoxin-depleted SA were incubated with cells for two hours.

[0079] (c) Cells in PBS and SA groups were stimulated with 1 μg / ml bacterial lipopolysaccharide (LPS).

[0080] (d) After being stimulated for 12 hours, cellular RNA was extracted and reverse-transcribed into cDNA, which was used...

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Abstract

The invention discloses a serpin and a minimum functional fragment and immunosuppression effect thereof. The serpin SA from haemaphysalis longicornis has activity restraining effects on cathepsin B, cathepsin G, papain and blood coagulation X factors, a minimum functional fragment SA-RCL having the immunosuppression activity is determined and has restraining activity on the cathepsin G, the immunosuppression effects of the SA and the SA-CRL to immunocyte are verified, and the treatment effects of the SA and the SA-CRL in a mouse arthritis model are verified. In the manner, the preparation costof the SA is low, the minimum functional fragment SA-RCL is small in molecular weight, stable in nature, easy to absorb, poor in immunogenicity and not liable to neutralize and degrade by hosts, theSA and the SA-RCL can be used for development of vaccines for resisting tick-borne diseases and treatment of anti-inflammation diseases.

Description

technical field [0001] The invention relates to the field of biomedical application technology, in particular to a serine protease inhibitor and its minimum functional fragment and immunosuppressive effect. Background technique [0002] Ticks are blood-sucking arthropods that parasitize on the surface of humans and animals. They are important vectors of a variety of zoonotic infectious diseases and can cause huge economic losses. Ticks transmit the pathogenic microorganisms they carry to the human body through saliva during blood sucking. Most tick-borne diseases are natural foci diseases, and it is difficult to completely eliminate them, and the distribution, natural foci and epidemic rules of tick-borne diseases are still unclear. At present, the most important way to prevent and control ticks is to use chemical insecticides. Due to the long-term use of insecticides, ticks develop drug resistance, and there is a lack of safe and efficient repellent drugs. At the same tim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/81C12N15/12C12N15/70A61K38/57A61P19/02A61P29/00
CPCC07K14/8121C12N15/70A61P19/02A61P29/00A61K38/00
Inventor 陈静姚远林微微
Owner 上海景晗生物科技有限公司
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