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A lipopolysaccharide-binding protein polypeptide and its pharmaceutical use

A technology that combines proteins and lipopolysaccharides, applied in the field of biomedicine, can solve the problems of unclear LPS hijacking host proteins and high LPS concentration, and achieve the effects of inhibiting the production of inflammatory factors, good solubility, and reducing mortality

Active Publication Date: 2019-06-25
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during sepsis, LPS concentrations in the blood remain high, suggesting that LPS escapes clearance mechanisms by binding to host proteins, but to date, it remains unclear which host proteins are hijacked by LPS

Method used

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  • A lipopolysaccharide-binding protein polypeptide and its pharmaceutical use
  • A lipopolysaccharide-binding protein polypeptide and its pharmaceutical use
  • A lipopolysaccharide-binding protein polypeptide and its pharmaceutical use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Screening the functional polypeptide DHG15 from plasma HK protein and detecting its binding performance.

[0024] The HK protein in plasma has 6 structural domains, among which the first to third domains (D1~D3) form the heavy chain of HK (Heavy chain, HC), and the fourth to sixth domains (D4~D6) form HK Light chain (Light chain, LC). The heavy chain and light chain proteins of HK were prepared using the Bac-to-Bac insect cell expression system, and the purified HC (55KD) and LC (35KD) proteins were identified by Western blotting. The results are as follows: figure 1 shown.

[0025] In order to study the binding site of HK protein and LPS, 200 ng of HK, HC, LC proteins were incubated with LPS or PBS (4°C, 30 min). Pipette 30 μl of mixed Polymyxin-B (Sigma) into a 1.5 ml centrifuge tube, centrifuge to remove the supernatant, wash twice with 1 ml PBS (7000 rpm, 2 min). Incubate various protein samples with the treated Polymyxin-B (4°C, 5min), centrifuge to r...

Embodiment 2

[0033] Example 2: LPS binds to HK through sugar chains.

[0034] LPS is mainly composed of sugar chains and lipid A. In order to detect which part of LPS binds to HK through its structure, 2 μg of lipid A, defatted LPS, LPS or BSA (inner Contains 0.1M Na 2 CO 3 , pH=9.6), coated at 4°C overnight. After washing the well plate with PBS, 1% gelatin was added and blocked at 37°C for 1 h. After washing the plate with PBS, FITC-labeled HK protein (100 nM) was added to each well and incubated at 37°C for 1 h. After aspirating the liquid, use a microplate reader to detect the fluorescence intensity in each well (excitation light: 494 nm, emission light: 518 nm).

[0035] Such as Figure 7 As shown, the binding ability of defatted LPS to HK is the strongest, which is significantly different from that of lipid A and HK, suggesting that the sugar chain on LPS is the main site for the binding of LPS to HK.

Embodiment 3

[0036] Example 3: Investigation of the activity of the DHG15 polypeptide.

[0037] In order to study whether the DHG15 polypeptide can improve the death of mice caused by LPS, the mice were randomly divided into two groups, administered by intraperitoneal injection, and one group was injected with LPS co-incubated with irrelevant polypeptide (200 g / mouse) group), the other group was injected with LPS co-incubated with DHG15 polypeptide (200 g / mouse) (treatment group), and the survival rate of the two groups of mice was observed. It was found that after treatment with DHG15 polypeptide, the mortality rate of mice caused by LPS could be significantly improved.

[0038] In order to investigate whether DHG15 affects the levels of major inflammatory factors in plasma, the levels of major inflammatory factors in plasma were compared between control mice and DHG15-treated mice. Such as Figure 9 As shown, compared with the mice in the control group, the levels of TNF-a, IL-1b and I...

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Abstract

The invention discloses a lipopolysaccharide-binding protein polypeptide and its pharmaceutical use. Specifically, the lipopolysaccharide-binding protein polypeptide (DHG15) of the present invention comprises 15 amino acids, and its amino acid sequence is shown in SEQ ID NO:1. The lipopolysaccharide-binding protein polypeptide DHG15 of the present invention has good solubility, and its sequence is derived from human body's own HK protein, and has no cytotoxicity. After intraperitoneal injection, it can reduce the death rate of endotoxemia caused by LPS in mice, and inhibit the production of inflammatory factors. After DHG15 combines with the polysaccharide chain of LPS, it blocks the combination of LPS and HK protein; at the same time, the lipid A part of LPS is still exposed, and LBP, sCD14, CTBP and other lipoproteins can still be combined with it, and It is presented to the liver for detoxification, so it has a double protective effect.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a lipopolysaccharide-binding protein polypeptide containing 15 amino acids and its use in preparing medicines for preventing and / or treating sepsis caused by Gram-negative bacteria. Background technique [0002] Sepsis is an acute systemic infection caused by pathogenic bacteria or opportunistic pathogenic bacteria invading the blood circulation to grow and reproduce, producing toxins and other metabolites. Clinically, it is characterized by chills, high fever, rash, arthralgia, and hepatosplenomegaly. Some may have septic shock and migratory lesions. There are two reasons for sepsis: 1. Human factors: When the skin and mucous membranes are damaged or suppurative inflammation occurs, bacteria can easily invade the body; the immune response of the human body can be divided into two types: non-specific immune response and specific immune response. It can be divided ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/81A61K38/57A61P31/04
CPCA61K38/10C07K7/08
Inventor 武艺谢展利阳艾珍
Owner SUZHOU UNIV
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