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Human Fc<gamma>R II linear ligand binding epitope

An epitope and linear technology, applied in the field of Fc receptors, can solve the problem of low affinity, achieve the effect of small side effects, solve autoimmune diseases, and high curative effect

Inactive Publication Date: 2012-02-01
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the affinity of small molecular peptides to receptors is far lower than that of complete antibody molecules, and the improvement of peptide affinity has yet to be resolved.

Method used

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  • Human Fc&lt;gamma&gt;R II linear ligand binding epitope
  • Human Fc&lt;gamma&gt;R II linear ligand binding epitope
  • Human Fc&lt;gamma&gt;R II linear ligand binding epitope

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Design of huFcγRII polypeptide

[0029] Using DNASIS (Ver.2.5, Hitachi) software, the amino acid sequences of huFcγRIIA (NP_067674) and boFcγRII (NP_776964), huFcγRIIB (NP_003992) and moFcγRIIB (NP_034317) were compared and analyzed ( figure 1 ), and referring to the crystal structures of huFcγRIIA (Maxwell et al., 1999; Sondermann et al., 2001) and huFcγRIIB (Sondermann et al., 1999), six huFcγRII polypeptides were designed and synthesized, covering A-B, B-C, C-C', C'-E, E-F and F-G loops, except for huRII2 and huRII6 polypeptides that contain Cys at the N-terminus, Cys is added to the N-terminus of the other four polypeptides for carrier protein coupling (see Table 1).

[0030] Table 1 Characteristics of huFcγRII polypeptides and reactivity with human IgG

[0031]

[0032] a For subsequent linking of the N-terminal brackets of the polypeptide sequence is an additional cysteine ​​residue, b Dot-blot detection of the binding of each peptide to human IgG...

Embodiment 2

[0033]Example 2: Synthesis procedure and screening procedure of huFcγRII polypeptide

[0034] Using the Symphony12-channel peptide synthesizer (Protein Technologies Inc.) on Fmoc-Amino Acids attached to Wang Resin (Fmoc-Amino Acids attached to Wang Resin, Shanghai Jier) with solid-phase peptide synthesis (solid-phase peptide synthesis) to synthesize peptides, peptides Synthesis was performed according to standard operating procedures. The specific process is as follows:

[0035] Design and synthesize peptide sequence→peptide program analysis→design synthesis program→calculate and weigh Fmoc-AA-Wang-resin or Rink resin according to the substitution value of the resin→DMF swelling resin→ * Add 20% piperidine to remove Fmoc protection, stir for 6min→ * Add the next amino acid and HBTU for acylation reaction, N 2 Stir the reaction for 30min→ * Test the completion of the reaction by the Kaiser method or the TNBS method → * Wash 5 times with DMF, 1 min each time → repeat with ...

Embodiment 3

[0037] Example 3: Coupling of huFcγRII polypeptide

[0038] Application of heterobifunctional reagent Sulfo-SMCC (MW: 436.37, Spacer Arm Length: 11.6 , Pierce) by adding -NH on the carrier protein BSA 2 It is connected with the -SH of the N-terminal Cys of the polypeptide to form the artificial binding antigen polypeptide BSA-Pep. The coupling steps are:

[0039] (1) 4 mg of IgG-free bovine serum albumin (IgG-free BSA, Sigma-Aldrich) was weighed and dissolved in 0.5 ml of coupling buffer (0.1M PB pH7.2, 0.15M NaCl, 1 μM EDTA). (2) Dissolve 1 mg Sulfo-SMCC (MW: 436.37, Spacer arm length: 11.6 , Pierce), add BSA solution, mix thoroughly, react at room temperature for 1 h or 30 min at 37°C, and mix from time to time. (3) Dialyze the coupling buffer solution overnight at 4°C, and change the solution 3 times to remove excess coupling agent. The solution is SMCC-activated BSA carrier protein (SMCC-BSA), and the protein concentration is adjusted to 5 mg / ml with coupling buffer...

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Abstract

The present invention relates to a linear ligand-binding epitope of human Fc Gamma RII, the amino acid sequence of which is Cys-Thr-Gly-Asn-Ile-Gly-Tyr-Thr-Leu-Phe-Ser-Ser-Lys-Pro-Val-Thr-Ile-Thr-Val. The present invention utilizes technologies, such as bioInformatics, polypeptide synthesis, cellular immunology and molecular biology, to chemically synthesize a linear ligand-binding epitope polypeptide of human Fc Gamma RII and conducts analysis and identification. An experiment conducted on the inhibition effect of the polypeptide on human IgG-soluble receptor binding and human IgG-cell surface receptor binding indicates that the linear epitope polypeptide can show good inhibition effect on human Fc Gamma RII and human IgG, so the linear epitope polypeptide is significant for the in-depth understanding of the molecular foundation of Fc Gamma R-IgG interaction and provides a new approach for the molecular design of human FcR target drugs.

Description

1. Technical field: [0001] The present invention relates to Fc receptors in the field of molecular immunology, in particular to human FcγRII (Fc GammaReceptor II) linear ligand-binding epitopes. 2. Background technology: [0002] Fc receptor (FcR) is a cell surface molecule with specific affinity for the Fc fragment of immunoglobulin (Ig). There are three types of human FcγR, namely FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Similar in structure, both contain two (FcγRII and FcγRIII) or three (FcγRI) Ig-like domains, belonging to the Ig supergene family, but there are significant differences in the transmembrane and intracellular regions, and Fc receptors are widely expressed in immune adjuvant Cells and effector cells have many important physiological functions, are the link between humoral immunity and cellular immunity, and play a key role in the immune regulation of the body. Antibody-mediated inflammatory responses can be regulated by activating and inhibitory FcR...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C12N15/12
Inventor 张改平席俊郭军庆王选年赵东杨艳艳张利娜苗现伟乔松林张红郅玉宝邓瑞广
Owner HENAN ACAD OF AGRI SCI
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