Hybrid peptide with functions of regulating immunity, neutralizing and digesting endotoxin and resisting inflammation, and preparation method and application thereof

An immunomodulatory, hybrid peptide technology, applied in the field of genetic engineering and biological preparations, can solve the problems of increasing the complexity and antagonism of clinical drug selection, decreasing immunity and production performance, failing to improve the immune function of the body, and achieving weight reduction. As well as the effects of intestinal damage, tissue damage alleviation, good immunity and anti-inflammatory two-way regulation

Active Publication Date: 2019-07-30
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are also obvious problems in the use of antibiotics or hormonal anti-inflammatory drugs in the treatment of infection and inflammation: although antibiotics can reduce or kill pathogenic bacteria, they cannot improve the immune function of the body. On the contrary, the bacteria killed by antibiotics also produce endotoxin or Exotoxins, which in turn aggravate the inflammatory response and even lead to systemic inflammatory response syndrome (Botwinski, 2001), which can cause fever, anorexia, excessive energy consumption in the body, decomposition of body tissues, and decline in immunity and production performance in animals. Avian mortality (Botwinski, 2001; Zinner, 1999)
Therefore, in the field of traditional medicine, the clear separation of anti-inflammatory drugs and immune-enhancing drugs does not meet the actual needs of the body or the principle of the interaction between anti-inflammatory and immune, and greatly increases the complexity and antagonism of clinical drug selection.

Method used

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  • Hybrid peptide with functions of regulating immunity, neutralizing and digesting endotoxin and resisting inflammation, and preparation method and application thereof
  • Hybrid peptide with functions of regulating immunity, neutralizing and digesting endotoxin and resisting inflammation, and preparation method and application thereof
  • Hybrid peptide with functions of regulating immunity, neutralizing and digesting endotoxin and resisting inflammation, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Preparation of immune anti-inflammatory hybrid peptide LTA

[0060] 1. Obtaining the hybrid peptide sequence

[0061] By studying the sequence, structure and relationship between sequence structure and function of polypeptide LL-37 (sequence shown in SEQ ID NO.2) and thymosin Tα1 (sequence shown in SEQ ID NO.3), protein molecular design technology was used to The hybridization of polypeptide LL-37 and thymosin Tα1 was carried out to obtain multiple candidate hybrid peptides. In this example, the hybrid peptide LTA (sequence shown in SEQ ID NO.1), Tα1-LL-37 (sequence shown in SEQ ID NO.4) and LL-37-Tα1' (sequence shown in SEQ ID NO.5 shown) as an example to illustrate the screening process of immune anti-inflammatory hybrid peptide LTA.

[0062] Take advantage of the properties of myeloid differentiation protein-2 (MD-2) (i.e., MD-2 binds to TLR4, confers TLR4 responsiveness to various ligands including LPS; and MD-2 can promote the expression of TLR4 and TLR...

Embodiment 2

[0076] Example 2 The neutralization effect of hybrid peptide LTA on LPS

[0077] Dissolve and dilute the peptide LTA and its parent peptides LL-37 and Tα1 into solutions of different concentrations (0-64 μg / mL) using pyrogen-free endotoxin testing water, and take 100 μL of the above-mentioned peptide solutions of each concentration and LPS (1EU / mL) respectively. mL) mixed. After incubating at 37°C for 30 min, the neutralization rate of polypeptide LTA, LL-37, and Tα1 to LPS was detected using a chromogenic mechanism Limulus kit, and polymyxin B (PMB) was used as a control. The result is as Figure 5 As shown, the polypeptide LTA has a high LPS neutralization activity, and its LPS neutralization activity is equivalent to that of polymyxin B. When the concentration is 8 μg / mL, the neutralization rate of the polypeptide LTA to LPS is close to 100%, and its neutralization The activity was significantly higher than that of its parent peptide LL-37 and Tα1.

Embodiment 3

[0078] Example 3 Effect of Hybrid Peptide LTA on Survival Rate of Mouse Macrophage Cells

[0079] The macrophage RAW264.7 in the logarithmic growth phase was inoculated in a 96-well plate, and the initial cell culture density was 1×10 4 cells / mL, 100 μL per well, at 37°C, 5% CO 2 After culturing overnight under certain conditions, a series of concentration gradient LTA, LL-37 and Tα1 (0-100μg / mL) solutions were added, and after culturing for 24 hours, the effect of polypeptide LTA on the survival rate of mouse macrophages was detected by CCK8 method . The result is as Image 6 As shown, the cytotoxicity of polypeptide LTA was significantly lower than that of its parent peptide LL-37, and the survival rate of macrophages was greater than 83% in the concentration range of 0-100 μg / mL, indicating that LTA has low cytotoxicity and high security.

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Abstract

The invention relates to the technical field of gene engineering and biological agents, in particular to a hybrid peptide with functions of regulating immunity, neutralizing and digesting endotoxin and resisting inflammation, and a preparation method and application thereof. The hybrid peptide is obtained from antibacterial peptide LL-37 and thymosin T alpha 1 through designing a protein engineering computer, optimizing heterozygosity and re-screening in vivo and in vitro (abbreviated as LTA), wherein the amino acid sequence is shown as SEQ ID NO. 1. The LTA has higher bidirectional immunoregulation activity, can enhance the immunologic function of an organism under the normal or immunosuppression state and protect the damage to the organism caused by immunosuppression; in an inflammatorystate, the LTA can also neutralize endotoxin, inhibit the inflammatory reaction of organisms, relieve the damage of the inflammatory reaction to tissues, has the advantages of low cytotoxicity, high safety, convenient preparation, low cost and the like, can be used as an ideal immune anti-inflammatory regulator, and has good application potential and value.

Description

technical field [0001] The invention relates to the field of genetic engineering and biological preparations, in particular to a hybrid peptide with functions of immune regulation, neutralization and digestion of endotoxin and anti-inflammation, its preparation method and application. Background technique [0002] When animals or humans are young, weak, disease, stress, pathogenic infection, etc., their immunity is often reduced, and then secondary infection (bacterial or viral mixed infection) occurs, causing inflammatory reactions (red, hot, swollen) , pain, etc.). At present, for the treatment of infection and inflammation, the traditionally commonly used prevention and treatment strategy is to use antibiotics and steroid anti-inflammatory drugs (such as hydrocortisone, dexamethasone, etc.), although it can effectively control infectious inflammation and non-infectious inflammation, However, continuous use can cause various side effects: such as serious disturbance of wa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/81C12N1/19A23L33/18A23K20/147A61K38/22A61P29/00A61P39/02A61P37/02A61K38/17C12R1/84
CPCA23V2002/00A61K38/1729A61K38/2292A23K20/147A23L33/18A61P29/00A61P37/02A61P39/02C07K14/4723C07K14/57581C07K2319/00C12N15/815C12N2800/102A23V2200/324A23V2200/30A23V2250/55A61K2300/00
Inventor 张日俊张璐璐卫旭彪黄燕阿曼斯大勇李仲玄程俊豪杜孟思
Owner CHINA AGRI UNIV
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