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Specific polypeptide related to rheumatoid arthritis and application thereof

A specific, arthritic technology for rheumatoid arthritis detection that addresses inefficiencies and unsatisfactory results

Active Publication Date: 2020-10-30
GUANGZHOU LDEBIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0018] The existing RA antigen design is mainly through the selection of specific antigen regions in natural proteins, the efficiency is relatively low, and the results are not satisfactory

Method used

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  • Specific polypeptide related to rheumatoid arthritis and application thereof
  • Specific polypeptide related to rheumatoid arthritis and application thereof
  • Specific polypeptide related to rheumatoid arthritis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1: Design and synthesis of polycitrulline similar amino acid repeat structure (Multi-Cirtrullin Similar Motif, MCSM) polypeptide:

[0092] The designed peptide sequence is as follows:

[0093] P01: HQDQG(Cit)S(Cit)NRAA (SEQ ID NO.: 1)

[0094] P02: DCDGQW(Cit)GP(Cit)SVE(Cit)HQSAKCK (SEQ ID NO.: 2)

[0095] P03: KCKQFRN(Cit)G(Cit)SPRAADCD (SEQ ID NO.: 3)

[0096] P04: HQCHQESTRGRSRGRCGRSGS (SEQ ID NO.: 4)

[0097] P05: SHQEST(Cit)G(Cit)SRG(Cit)SG(Cit)SGS (SEQ ID NO.: 5)

[0098] P06: HQDNQ(Cit)S(Cit)QNAA (SEQ ID NO.: 6)

[0099] P07: HQGQG(Cit)G(Cit)SQGQGRA (SEQ ID NO.: 7)

[0100] P08: HNGNG(Cit)G(Cit)SNGRGNA (SEQ ID NO.: 8)

[0101] P10: HANN(Cit)T(Cit)SNNGA (SEQ ID NO.: 9)

[0102] P11: RSQFNW(Cit)S(CIT)SRPR (SEQ ID NO.: 10)

[0103] P12: HQFRN(Cit)Q(Cit)RSRNHA (SEQ ID NO.: 11)

[0104] P13: HQYNFQW(Cit)S(Cit)GRPR (SEQ ID NO.: 12)

[0105] P14: GRFQM(Cit)H(Cit)RLIRH (SEQ ID NO.: 13)

[0106] P15: RSQFNF(Cit)P(Cit)SRPR (SEQ ID NO.: 14)

[0107] P16:...

Embodiment 2

[0112] Example 2: Comparison of different citrullinated and MSCM polypeptides to detect differences in anti-citrullinated antibodies in human serum

[0113] Experimental Materials:

[0114] ELISA plate: NUNC446469

[0115] Streptavidin: Hangzhou New Dragon rc-SA

[0116] Coating buffer: PBS, pH7.4

[0117] Washing solution: PBS+Triton X-100, pH7.4

[0118] Blocking solution: PBS+1%BSA, pH7.4

[0119] Sample diluent: PBS+0.1%Triton X-100+1%BSA

[0120] Enzyme-labeled antibody: HRP-labeled mouse anti-human IgG (for 1:15000)

[0121] Enzyme-labeled antibody diluent: PBS+1%BSA+0.1%Triton X-100, pH7.4

[0122] Different biochemical peptides:

[0123] P04 (no citrullination)

[0124] P05 (with citrullination, without MSCM characteristic conformation)

[0125] P11 (citrullinated, MSCM characteristic conformation)

[0126] P18 (with citrullination, MSCM characteristic conformation)

[0127] Omon CCP

[0128] sample:

[0129] Healthy control: 20 cases

[0130] RA, Eumont CC...

Embodiment 3

[0175] Example 3: Synthetic polypeptides were cross-linked with BSA, after purification, rabbits were immunized to produce polyclonal antibodies

[0176] 1) The synthetic polypeptide is coupled to the carrier protein BSA or OVA by EDC method or glutaraldehyde cross-linking method;

[0177] 2) Select two healthy 6-week-old New Zealand white rabbits (about 2Kg);

[0178] 3) Fully mix 1ml of Freund's complete adjuvant with the prepared 1ml of antigen, and it becomes milky white;

[0179] 4) 400ug antigen for the first immunization, 100ug each time for subsequent immunization; subcutaneous injection on the back;

[0180] 5) Immunize once every two weeks, a total of 4 to 5 times, and finally collect blood after verifying that the antibody concentration in the serum reaches the standard;

[0181] 6) Antibodies in serum were purified using ProteinA / G affinity packing.

[0182] The purified polyclonal antibody combined with one or more antibodies against vimentin, fibrinogen, type ...

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Abstract

A specific polypeptide related to rheumatoid arthritis and application thereof are provided. The specific polypeptide includes citrulline, wherein at least one side of the citrulline is connected witha citrulline-like amino acid, and a spacer amino acid is also provided between the citrulline and the citrulline-like amino acid. The specific polypeptide provided has high sensitivity and specificity in identifying autoimmune antibodies of patients with rheumatoid arthritis, and can be directly used for the detection of RA autoimmune antibodies, and can also be used to immunize animals to produce polyclonal antibodies or monoclonal antibodies that can be used to detect citrullinated proteins, and to produce identification kits for citrullinated proteins in rheumatoid arthritis serum, joint fluid and body fluids.

Description

technical field [0001] The present invention relates to technologies related to the detection of rheumatoid arthritis, in particular to a polypeptide design of a multi-citrulline similar amino acid repeating structure (Multi-Cirtrullin Similar Motif, MCSM) and its use in the development of a detection kit related to rheumatoid arthritis Applications. Background technique [0002] Rheumatoid arthritis (rheumatoid factor, RA) is a chronic systemic autoimmune disease, my country adopts the RA classification criteria revised by the American College of Rheumatology (ACR) in 1987, its specificity is 89%, and its sensitivity is 91% to 94%. %, but this standard is easy to miss some early or atypical patients. Rheumatoid factor (rheumatoid factor, RF) is an antibody against the epitope of the Fc fragment of human or animal IgG molecules, and is an autoantibody targeting denatured IgG. RF was first discovered by Rose et al. (1984) in the serum of patients with rheumatoid arthritis (R...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K14/00C07K16/44G01N33/577G01N33/564
CPCC07K7/08C07K14/001C07K16/44G01N33/564G01N33/577G01N2800/102
Inventor 杨翔楼建荣
Owner GUANGZHOU LDEBIO TECH CO LTD
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