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Active polypeptide with antagonistic chemotactic factor receptor CCR5 and preparation method and application thereof

A chemokine receptor and active polypeptide technology is applied in the field of active polypeptides that antagonize chemokine receptor CCR5 and the preparation thereof, and can solve the problems of large toxic and side effects, interfering enzyme metabolism, and easy generation of virus drug resistance. , to achieve weak immunogenicity, enhance immune function, and increase the effect of T lymphocyte function

Inactive Publication Date: 2011-08-10
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because these drugs have the disadvantages of interfering with the normal enzyme metabolism of the human body, having relatively large toxic and side effects, and prone to viral drug resistance, human beings urgently need antiviral drugs with new mechanisms to overcome AIDS, the plague of the twentieth century.

Method used

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  • Active polypeptide with antagonistic chemotactic factor receptor CCR5 and preparation method and application thereof
  • Active polypeptide with antagonistic chemotactic factor receptor CCR5 and preparation method and application thereof
  • Active polypeptide with antagonistic chemotactic factor receptor CCR5 and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Peptide Design

[0043] Protein sequences were retrieved from the SWISS-PROT protein database using mature forms of chemokine sequences (http: / / us.expasy.org / sprot).

[0044] The multiple alignment and comparative analysis of protein sequences was completed using the Informax Vector NTI Version 9.0 software package. The AlignX process is used to predict and analyze the residues that affect the binding of chemokine receptors: first select the human chemokine sequence that specifically binds to a certain receptor for structure-based multiple sequence alignment analysis, Residues that exist in all factor sequences or are conservatively substituted may be residues related to the receptor binding (that is, the establishment of a standard binding residue model). Then introduce the sequence of vMIP-II into the standard binding residue model for multiple sequence comparison analysis to obtain the residues related to the binding of vMIP-II to the receptor (ie, the res...

Embodiment 2

[0047] Example 2 Synthesis and purification of polypeptides

[0048] (1) Polypeptide synthesis and purification

[0049] Polypeptides were synthesized on a peptide synthesizer using the Fmoc-solid-phase method. After the reaction bottle was fully cleaned with 100% ethanol, about 40 ml of 5% dimethyldichlorosilane was added and placed in a fume hood overnight to silanize the reaction bottle. After roughly washing with ethanol, place the reaction bottle in a -80°C refrigerator for 30 minutes to cool, wash with ethanol again, and dry it for later use; weigh 0.5mmol Wang's resin 0.7130g and place it in the reaction bottle; add DCM solution 3 times the volume of the resin to swell Drain the liquid after 30 minutes. Then, on the peptide synthesizer, it is carried out from the C-terminal to the N-terminal according to the cycle of coupling amino acid, ninhydrin reaction detection and α-amino deprotection until all amino acid residues are coupled. After the coupling is completed, ...

Embodiment 3

[0054] Example 3 Chemotactic Activity Experiment of Polypeptides

[0055] In the study of the chemotaxis activity of the polypeptide, we first studied the influence of the polypeptide itself on the chemotaxis activity of peripheral blood mononuclear cells, and then studied whether the polypeptide itself has the ability to chemoattract lymphocytes. In the experiment, a blank control group, a positive control group and different concentrations of polypeptide experimental groups were set up.

[0056] (1) Separation of human peripheral blood mononuclear cells (PBMCs) (Ficoll density gradient centrifugation)

[0057] 1. Take 3ml of lymphocyte separation solution, carefully add to the bottom of the centrifuge tube, the volume should not exceed 1 / 4 of the test tube.

[0058] 2. Take 6ml of anticoagulant blood, dilute it with D-Hank’s solution by 1 time, mix well, and use a dropper to slowly superimpose on the layered liquid surface of lymphocyte separation medium along the tube wa...

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Abstract

The invention discloses an active polypeptide with an antagonistic chemotactic factor receptor CCR5, which has an amino acid sequence in SEQ ID NO:1 or an amino acid sequence obtained by carrying out deletion, addition, insertion and substitution of one or more amino acid residues on the amino acid sequence in SEQ ID NO:1. The invention also discloses a preparation method of the polypeptide, which comprises the following steps of: searching related protein sequences in a database; carrying out multiple alignment comparative analysis to screen out a highly conservative region for combination of a vMIP-II receptor; eliminating interference of unrelated residues through analysis simulation means; determining a target peptide sequence; synthetizing the active polypeptide by a solid-phase synthesis method; and finally testing the bioactivity of the polypeptide. The polypeptide can be used as a CCR5 receptor antagonist for the development of an anti-inflammatory drug, can be used as a lead drug for treating AIDS (Acquired Immune Deficiency Syndrome) and can also be used as a medicament with the immune regulation effect or a medicament for improving the functions of an immune system of an organism.

Description

[0001] technical field [0002] The invention relates to the field of biopharmaceuticals, in particular to an active polypeptide antagonizing chemokine receptor CCR5 and its preparation method and application. Background technique [0003] AIDS is an infectious disease that is caused by human immunodeficiency virus (HIV) after invading the human body, destroying the immune function of the human body, causing a series of incurable infections and tumors in the human body, and finally leading to the death of the patient. AIDS is spreading all over the world and on a large scale in my country due to its characteristics of rapid transmission, difficulty in curing and lack of effective drugs. However, there is no effective drug for treating AIDS in the domestic and foreign markets at present, and the existing anti-AIDS drug has relatively large side effects and is expensive. Anti-AIDS drugs currently on the market are mainly inhibitors of related proteases, mainly reverse transcr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435A61K38/17A61P29/00A61P31/18A61P37/02A61P37/04
Inventor 孙晗笑莫雪梅李秀英张光叶石敦何滔汤曼莉
Owner JINAN UNIVERSITY
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