Dominant epitopes for identification of anti-Titin antibodies and application thereof

Abstract

The invention discloses dominant epitopes for the identification of anti-Titin antibodies. The dominant epitopes have the following amino acid sequences, and are positioned at loci from 103 to 115, 76 to 187 and 13,211 to 13,227, and the amino acid sequences in polypeptide sequences are CEQWVDYNLKWNPD, IVTHFPFDEQNC and EIIVTHFPFDEQNCSMK respectively. The dominant epitopes for the identification of the anti-Titin antibodies are used for preparing medicaments for myasthenia gravis and diagnostic reagent kits. The dominant epitopes which have high effectiveness and are used for the identification of the anti-Titin antibodies are screened by the microarray and chip diagnostic technology, so that corresponding polypeptides can be prepared by a method of chemical synthesis or the recombination of genetic engineering expression, and antibodies are prepared from the polypeptides and are used as probes, and the medicaments for the myasthenia gravis and diagnostic reagents are developed. By the method, the defect that the conventional preparation method for antigen antibodies and the conventional method for screening the medicaments are troublesome is overcome, labor and material resources for performing detection respectively are reduced greatly, the detection error of laboratories is overcome during analysis, and a new path is provided for the treatment of relevant diseases such as the myasthenia gravis and the like, and serodiagnosis.

Description

technical field

[0001] The present invention relates to the dominant epitope recognized by anti-Titin antibody and its application. Background technique

[0002] Among individuals with myasthenia gravis, about 85% of patients have autoantibodies against AChR molecules, and the main binding target of pathogenic autoantibodies is located in the main immunogenic region (main immunogenic region, MIR) on the subunits of AChR molecules. Antibody-dependent complement lysis is the main reason for the destruction of AChR molecules; at the same time, antibody-mediated antigen modulation also accelerates the internalization of AChR molecules and the degradation of lysosomes, reducing the half-life of AChR molecules from 7 days to 2 days. The number of functional AChR molecules on the postsynaptic membrane of nerves is drastically reduced in vivo. In patients with pure extraocular muscle type, only 50% of them have a seropositive rate, and the main binding target of autoantibodies in t...

Examples

Embodiment 1

[0015] Example 1: Peptide Design and Protein Conjugation

[0016] According to the method of bioinformatics, 9 polypeptide sequences targeting the extracellular region of Titin protein are designed, as shown in Table 1. Polypeptides were synthesized by solid-phase synthesis (SPPS), and the synthesized peptides were then succinimide 4-[N-methylmaleic acid]-1-carboxycyclohexane (SMCC) bifunctional cross-linking agent in the peptide thiol group. conjugated to the amino-terminus of bovine serum albumin (BSA).

[0017] Table 1. Synthetic peptide sequences

[0018]

Embodiment 2

[0019] Example 2: protein microarray spot system

[0020] After adjusting the concentration of each protein by the BCD method, use a protein spotter to spot each polypeptide-BSA coupled protein on a nitrocellulose membrane (100ng / spot) in a pattern of 8×12, and repeat the detection of each polypeptide 6 times. β-actin (β-actin) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) internal references were dried and blocked with 5% calf serum for later use.

Embodiment 3

[0021] Example 3: Protein microarray detection

[0022] The diluted serum to be tested (1:100, diluted with 5% calf serum) was reacted with the microarray chip at room temperature for 2 hours, and washed with 0.01Mol / L pH7.4 phosphate buffered saline (PBS) containing 0.5% Tween 20 5 times, then add alkaline phosphatase-labeled secondary antibody for 1 hour at room temperature, wash 4 times with 0.01Mol / L pH7.4 PBS containing 0.5% Tween 20 and 0.01Mol / L pH7.4 PBS without Tween 20 1 time, and finally add CDP-star luminescent substrate, and expose for 1 min to develop color. After converting the microarray detection results into TIFF image format, use Array2.0 version software to analyze, if the reaction signal is more than 1.5 times different from the control, it is positive, otherwise it is negative.

technical field

[0001] The present invention relates to the dominant epitope recognized by anti-Titin antibody and its application. Background technique

[0002] Among individuals with myasthenia gravis, about 85% of patients have autoantibodies against AChR molecules, and the main binding target of pathogenic autoantibodies is located in the main immunogenic region (main immunogenic region, MIR) on the subunits of AChR molecules. Antibody-dependent complement lysis is the main reason for the destruction of AChR molecules; at the same time, antibody-mediated antigen modulation also accelerates the internalization of AChR molecules and the degradation of lysosomes, reducing the half-life of AChR molecules from 7 days to 2 days. The number of functional AChR molecules on the postsynaptic membrane of nerves is drastically reduced in vivo. In patients with pure extraocular muscle type, only 50% of them have a seropositive rate, and the main binding target of autoantibodies in t...

Examples

Embodiment 1

[0015] Example 1: Peptide Design and Protein Conjugation

[0016] According to the method of bioinformatics, 9 polypeptide sequences targeting the extracellular region of Titin protein are designed, as shown in Table 1. Polypeptides were synthesized by solid-phase synthesis (SPPS), and the synthesized peptides were then succinimide 4-[N-methylmaleic acid]-1-carboxycyclohexane (SMCC) bifunctional cross-linking agent in the peptide thiol group. conjugated to the amino-terminus of bovine serum albumin (BSA).

[0017] Table 1. Synthetic peptide sequences

[0018]

Embodiment 2

[0019] Example 2: protein microarray spot system

[0020] After adjusting the concentration of each protein by the BCD method, use a protein spotter to spot each polypeptide-BSA coupled protein on a nitrocellulose membrane (100ng / spot) in a pattern of 8×12, and repeat the detection of each polypeptide 6 times. β-actin (β-actin) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) internal references were dried and blocked with 5% calf serum for later use.

Embodiment 3

[0021] Example 3: Protein microarray detection

[0022] The diluted serum to be tested (1:100, diluted with 5% calf serum) was reacted with the microarray chip at room temperature for 2 hours, and washed with 0.01Mol / L pH7.4 phosphate buffered saline (PBS) containing 0.5% Tween 20 5 times, then add alkaline phosphatase-labeled secondary antibody for 1 hour at room temperature, wash 4 times with 0.01Mol / L pH7.4 PBS containing 0.5% Tween 20 and 0.01Mol / L pH7.4 PBS without Tween 20 1 time, and finally add CDP-star luminescent substrate, and expose for 1 min to develop color. After converting the microarray detection results into TIFF image format, use Array2.0 version software to analyze, if the reaction signal is more than 1.5 times different from the control, it is positive, otherwise it is negative.

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