CN111849930B

CN111849930B - Mi-2 recombinant antigen and preparation method and application thereof

Info

Publication number: CN111849930B
Application number: CN202010835800.0A
Authority: CN
Inventors: 胡志强; 况承钰; 曾敏霞; 张晨光
Original assignee: Zhuhai Livzon Diagnostics Inc
Current assignee: Zhuhai Livzon Diagnostics Inc
Priority date: 2020-08-18
Filing date: 2020-08-18
Publication date: 2022-01-25
Anticipated expiration: 2040-08-18
Also published as: CN111849930A

Abstract

The invention relates to the technical field of biology, and particularly provides an Mi-2 recombinant antigen and a preparation method and application thereof. The invention provides a Mi-2 recombinant antigen which comprises at least one of SEQ ID NO.1, SEQ ID NO.5 or SEQ ID NO.7, and does not contain SEQ ID NO.2 and/or SEQ ID NO. 6. The antigen improves the sensitivity and specificity of Mi-2 antibody detection, is used for Mi-2 antibody detection or multiple myositis diagnosis, can greatly improve the accuracy, and provides a new technical support for production application.

Description

Mi-2 recombinant antigen and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, and particularly relates to an Mi-2 recombinant antigen and a preparation method and application thereof.

Background

Polymyositis (PM) is a common autoimmune disease, and the Mi-2 antibody is one of myositis-specific antibodies, and has important significance for diagnosis of myositis. The target antigen for the anti-Mi-2 antibody is the Mi-2 antigen protein located within the nuclear nucleoplasm of the cell nucleus with a relative molecular mass of 218 kD.

The method for obtaining the natural Mi-2 antigen is usually to extract from the thymus of rabbits, and has higher extraction difficulty and low yield. The Mi-2 antigen protein is composed of 1,912 amino acids, the relative molecular weight of the protein is large, and the full-length Mi-2 antigen protein expressed by insect cell recombination is easy to obtain, but the immunogenicity is low, so that the usability of the diagnostic reagent cannot be met.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide an Mi-2 recombinant antigen.

The second purpose of the invention is to provide the biological material related to the Mi-2 recombinant antigen.

The third purpose of the invention is to provide a preparation method of the Mi-2 recombinant antigen.

The fourth purpose of the invention is to provide the application of the Mi-2 recombinant antigen.

The fifth purpose of the invention is to provide a product for detecting polymyositis.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a Mi-2 recombinant antigen comprising at least one of SEQ ID No.1, SEQ ID No.5 or SEQ ID No.7 and not comprising SEQ ID No.2 and/or SEQ ID No. 6.

Further, the Mi-2 recombinant antigen does not contain at least one of SEQ ID No.3, SEQ ID No.4 or SEQ ID No. 8.

Further, the Mi-2 recombinant antigen includes any one of (a) - (d):

(a) SEQ ID NO.1 and SEQ ID NO. 5;

(b) SEQ ID NO.1 and SEQ ID NO. 7;

(c) SEQ ID NO.5 and SEQ ID NO. 7;

(d) SEQ ID NO.1, SEQ ID NO.5 and SEQ ID NO. 7;

and the sequences in any one of (a) - (d) are connected through a connecting peptide;

preferably, the linker peptide is GGGGSGGGG.

Further, the Mi-2 recombinant antigen is any one of SEQ ID NO.9 or SEQ ID NO. 10.

A biomaterial associated with the above Mi-2 recombinant antigen, said biomaterial being any one of the following:

(a) nucleic acid molecules encoding the above-described Mi-2 recombinant antigens;

(b) an expression cassette comprising the nucleic acid molecule of (a);

(c) a recombinant vector comprising the nucleic acid molecule of (a) or the expression cassette of (b);

(d) a recombinant eukaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c);

(e) a recombinant prokaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c).

The preparation method of the Mi-2 recombinant antigen comprises the steps of introducing nucleic acid molecules for coding the Mi-2 recombinant antigen into a host to obtain a recombinant expression system, and expressing to obtain the Mi-2 recombinant antigen.

Further, the nucleic acid molecule encoding the Mi-2 recombinant antigen and the pET-28a vector construct a recombinant vector, the recombinant vector is introduced into escherichia coli, and the Mi-2 recombinant antigen is obtained through expression.

Application of the Mi-2 recombinant antigen in detection of Mi-2 antibody or preparation of products for detection of Mi-2 antibody.

Application of Mi-2 recombinant antigen in preparation of products for detecting polymyositis.

A product for detecting polymyositis adopts the Mi-2 recombinant antigen as a detection antigen.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a Mi-2 recombinant antigen which is obtained by optimizing an Mi-2 antigen protein (UniProtKB-Q14839(CHD4_ HUMAN)), so that the problems of high extraction difficulty, low yield and high cost of a natural Mi-2 antigen protein in the prior art are solved, and the defect of low immunogenicity of the recombinant full-length Mi-2 antigen protein is overcome. The antigen remarkably improves the steric hindrance problem of Mi-2 antibody detection, the antigen sites are more fully exposed, and the sensitivity and specificity of Mi-2 antibody detection are improved. The antigen of the invention is used for Mi-2 antibody detection or multiple myositis diagnosis, can greatly improve the accuracy and provide new technical support for production application.

Drawings

FIG. 1 shows the SDS-PAGE results of the NCM fusion protein in the examples of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

The invention provides a Mi-2 (anti-histone acetyltransferase complex helicase component) recombinant antigen, which comprises at least one of SEQ ID NO.1, SEQ ID NO.5 or SEQ ID NO.7, and does not contain SEQ ID NO.2 and/or SEQ ID NO. 6.

The antigen is obtained by optimizing the Mi-2 antigen protein (UniProtKB-Q14839(CHD4_ HUMAN)), so that the problems of high extraction difficulty, low yield and high cost of the natural Mi-2 antigen protein in the prior art are solved, and the defect of low immunogenicity of the recombinant full-length Mi-2 antigen protein is overcome. The antigen remarkably improves the steric hindrance problem of Mi-2 antibody detection, the antigen sites are more fully exposed, and the sensitivity and specificity of Mi-2 antibody detection are improved. The inventor finds in experiments that compared with natural Mi-2 antigen protein, the detection sensitivity of the Mi-2 recombinant antigen can be improved by more than 3 times, the specificity can be improved by more than 1.5 times, and the effect is obvious.

In the present invention, the "Mi-2 antibody" refers to a protein, polypeptide or other substance that can specifically bind to Mi-2 protein or polypeptide to produce an "antigen-antibody", and may also be referred to as an "anti-Mi-2 antibody"; "Mi-2 antigen" or "Mi-2 recombinant antigen" refers to a protein or polypeptide that can produce an "antigen-antibody" specific structure with Mi-2 antibodies.

It should be noted that, when the Mi-2 recombinant antigen contains two or three amino acid sequences, the fusion protein constructed by the antigen can be a protein containing SEQ ID No.1 and SEQ ID No.5, SEQ ID No.1 and SEQ ID No.7, SEQ ID No.5 and SEQ ID No. 7; alternatively, SEQ ID NO.1, SEQ ID NO.5 and SEQ ID NO. 7. The sequence of the amino acid sequences of the sequence numbers of any one of the fusion proteins is not particularly limited, and for example, the fusion proteins comprising SEQ ID NO.1 and SEQ ID NO.5 may comprise SEQ ID NO.1 and SEQ ID NO.5 in sequence, or may comprise SEQ ID NO.5 and SEQ ID NO.1 in sequence.

In a preferred embodiment, the Mi-2 recombinant antigen further does not contain at least one of SEQ ID No.3, SEQ ID No.4 or SEQ ID No. 8.

In a preferred embodiment, in order to reduce steric hindrance while fully exposing the antigen site and shortening the length of the antigen sequence, when the Mi-2 recombinant antigen contains two or three amino acid sequences of sequence number, the fusion proteins constructed by the Mi-2 recombinant antigen are connected by a connecting peptide. For example, the fusion protein may be of the structure: SEQ ID NO. 1-linker peptide-SEQ ID NO.5, SEQ ID NO. 5-linker peptide-SEQ ID NO.1, SEQ ID NO. 1-linker peptide-SEQ ID NO. 5-linker peptide-SEQ ID NO.7, SEQ ID NO. 5-linker peptide-SEQ ID NO. 1-linker peptide-SEQ ID NO.7, SEQ ID NO. 1-linker peptide-SEQ ID NO. 7-linker peptide-SEQ ID NO.5, or SEQ ID NO. 7-linker peptide-SEQ ID NO. 1-linker peptide-SEQ ID NO.5, etc., wherein in the fusion protein comprising the three-part sequence number amino acid sequence, the two "linker peptides" may be the same or different.

In a more preferred embodiment, the linker peptide may be GGGGSGGGG.

The invention simultaneously protects Mi-2 recombinant antigen with amino acid sequence as any one of SEQ ID NO.9 or SEQ ID NO. 10.

The invention also protects biological materials related to the Mi-2 recombinant antigens, such as nucleic acid molecules, expression cassettes, recombinant vectors, recombinant eukaryotic cells, recombinant prokaryotic cells and the like. It is understood that the above-mentioned biological materials are all biological materials encoding or expressing Mi-2 recombinant antigen, and can be used as biological modules directly in molecular biological operations.

A "nucleic acid molecule" may be DNA, such as cDNA or the like, or RNA, such as mRNA or the like.

An "expression cassette" comprises a polynucleotide sequence encoding the Mi-2 recombinant antigen to be expressed and sequences controlling its expression such as a promoter and optionally enhancer sequences, including any combination of cis-acting transcriptional control units. Sequences that control the expression of a gene (i.e., its transcription and translation of the transcription product) are often referred to as regulatory units. Most of the regulatory units are located upstream of and operably linked to the coding sequence of the gene. The expression cassette may also contain a downstream 3' untranslated region comprising a polyadenylation site.

The vector of the "recombinant vector" may be a plasmid, a phage or a virus.

The host cell of the "recombinant eukaryotic cell" may be a yeast, mammalian cell, insect cell, or the like.

The host cell of the "recombinant prokaryotic cell" may be a bacterium or the like.

The invention also provides a preparation method of the Mi-2 recombinant antigen, wherein a nucleic acid molecule for coding the Mi-2 recombinant antigen is introduced into a host to obtain a recombinant expression system, and the Mi-2 recombinant antigen is obtained through expression. The method greatly reduces the production cost of the Mi-2 recombinant antigen, can obtain a large amount of Mi-2 recombinant antigen, has a relatively mature expression system, and is simple and convenient to operate. Preferably, the nucleic acid molecule encoding the Mi-2 recombinant antigen and the pET-28a vector construct a recombinant vector, the recombinant vector is introduced into escherichia coli, and the Mi-2 recombinant antigen is obtained through expression.

The Mi-2 recombinant antigen is used for Mi-2 antibody detection or multiple myositis diagnosis, can greatly improve the accuracy and provides a new technical support for production application.

The invention finally protects a product for multiple myositis diagnosis or Mi-2 antibody detection by adopting Mi-2 recombinant antigen as detection antigen.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Example 1

Mi-2 was expressed in E.coli as 8 fragments by bioinformatics analysis of the epitope of the Mi-2 antigen protein (UniProtKB-Q14839(CHD4_ HUMAN), 1912 aa). The corresponding amino acid sequences SEQ ID NO.1(amino 1-239), SEQ ID NO.2(amino 240-448), SEQ ID NO.3(amino 449-677), SEQ ID NO.4(amino 678-948), SEQ ID NO.5(amino 949-1209), SEQ ID NO.6(amino 1210-1498), SEQ ID NO.7(amino 1499-1696) and SEQ ID NO.8(amino 1697-1912), respectively.

Plasmid construction and protein expression

【1】 Primers for 8 fragments were as follows:

the primers of the SEQ ID NO.1 fragment are:

F1:5’-atggcgtcgggcctgggc-3’(SEQ ID NO.11)；

R1:5’-gctctccaccacagctac-3’(SEQ ID NO.12)；

the primers of the SEQ ID NO.2 fragment are:

F2:5’-atggtgacagccactgaggttg-3’(SEQ ID NO.13)；

R2:5’-atggtggtcatcctcctcttcg-3’(SEQ ID NO.14)；

the primers for the fragment of SEQ ID NO.3 are:

F3:5’-atggaattctgtcgggtctgcaagg-3’(SEQ ID NO.15)；

R3:5’-taactccctgtgattcc-3’(SEQ ID NO.16)；

the primers for the fragment of SEQ ID NO.4 are:

F4:5’-atgaggggtgaggaaggccg-3’(SEQ ID NO.17)；

R4:5’-gtcatgcagtttttttatctg-3’(SEQ ID NO.18)；

the primers for the fragment of SEQ ID NO.5 are:

F5:5’-atgctggggccgcacatgttgc-3’(SEQ ID NO.19)；

R5:5’-agatccagtcttggagc-3’(SEQ ID NO.20)；

the primers for the fragment of SEQ ID NO.6 are:

F6:5’-atgtccaaacaggagcttgatg-3’(SEQ ID NO.21)；

R6:5’-aacaccaattctagtaaggac-3’(SEQ ID NO.22)；

the primer of the SEQ ID NO.7 fragment is

F7:5’-atgtctttgattcgcaag-3’(SEQ ID NO.23)；

R7:5’-gaaacgttgtttaatatttttc-3’(SEQ ID NO.24)；

The primer of the SEQ ID NO.8 fragment is

F8:5’-atgtttaacattgcagatgg-3’(SEQ ID NO.25)；

R8:5’-ctgctgctgggctacctgc-3’(SEQ ID NO.26)；

And (3) respectively carrying out PCR reaction by using DNA (deoxyribonucleic acid) for coding the full-length Mi-2 antigen protein as a template and the 8 groups of primers to obtain 8 target fragments.

The PCR reaction conditions are as follows: pre-denaturation: at 98 ℃ for 2 min; deformation, annealing and extension: 10s at 98 ℃; 60 ℃ for 10 s; 72 ℃ for 20 s; circulating for 30 times; and (3) heat preservation: 72 ℃ for 4 min.

The PCR product is respectively subjected to agarose gel electrophoresis, gel cutting recovery and purification to obtain the nucleic acid fragment of the code SEQ ID NO. 1-8.

【2】 Respectively inserting 8 sections of PCR products in [ 1 ] into a modified pET-28a vector (the modified vector inserts MBPtag between enzyme cutting sites Nco I and BamH I of the pET-28a vector, and TEV enzyme cutting sites are added), wherein the enzyme cutting sites of the PCR products are BamH I and Hind III.

【3】 Respectively transforming 8 recombinant plasmids in [ 2 ] into E.coli DH5 alpha competent cells, carrying out inverted culture on an LB agar culture plate at 37 ℃ for overnight, selecting bacteria for colony PCR verification and sequencing, carrying out enlarged culture after the sequencing is correct, and extracting plasmids.

【4】 The plasmids with the correct sequencing were transformed into E.coli Rosseta (DE3) competent cells, respectively, and cultured to OD at 37 ℃ in LB liquid medium_600nmWhen the concentration was 0.6 to 0.8, IPTG was added to a final concentration of 1mM, and the cells were induced at 25 ℃ for 4 hours and collected.

Protein purification

【1】 The collected cells were removed, lysed by adding lysis Buffer (20 mM Hepes,1M NaCl, 5% Glycerol, pH8.0), and resuspended in ice.

【2】 Ultrasonic crushing: and (4) breaking the bacteria according to the conditions of ultrasonic power of 400W, ultrasonic 4S and stopping 3S, and circulating for 200 times until the bacteria liquid is clear and transparent.

【3】 After the completion of sonication, the supernatant was separated from the precipitate by centrifugation at 12000rpm for 30min at 4 ℃ and the supernatant was filtered through a 0.22 μ M membrane.

【4】 And (3) taking 10ml of MBP resin, balancing the column by using a lysis Buffer for 5CV, then adding the MBP resin into the filtered supernatant, uniformly mixing, and placing the mixture in an ice box for shaking and incubating for 40-60 min.

【5】 Pouring the incubated mixture into a gravity column, collecting the flow-through liquid by using a clean centrifugal tube, and adding a 10CV lysis Buffer to clean the residues in the pipeline after the flow-through liquid completely flows out.

【6】 And (3) elution: elution was carried out with 20ml of elution Buffer (elution Buffer formulation: 20mM Hepes, 50mM NaCl, 5% Glycerol, 10mM Maltose, pH8.0) and the eluate was collected and desalted by G25.

【7】 Enzyme digestion: adding TEV enzyme according to the mass ratio of 1:20, and carrying out enzyme digestion at 4 ℃ overnight for 16 h.

【8】 Adding MBP resin into the solution after enzyme digestion again, pouring the solution into a gravity column after incubation, and collecting the flow-through liquid to obtain the target protein.

【9】 And (3) verifying the purity of the target protein in the eluent by protein electrophoresis, wherein the purity is more than or equal to 90 percent and can be used for verifying the antigen.

Liquid phase chip coating and reactivity test

Coating each prepared protein on a solid phase carrier.

Step 1, preparation of activation buffer solution

Taking 2- (N-morpholino) ethane sulfonic acid sodium salt (C)₆H₁₂NO₄SNa)21.7g, adding a proper amount of ultrapure water, after complete dissolution, adjusting the pH of the solution to 6.0 + -0.2 at 25 + -1 ℃, adding 0.5mL of Proclin300, mixing well, and diluting to 1000 mL.

Step 2, preparation of coating liquid

Collecting potassium dihydrogen phosphate (KH)₂PO₄)0.24g disodium hydrogen phosphate (Na)₂HPO₄·12H₂O molecular weight 357.96)2.89g, NaCl 8g and KCl 0.2g, adding a proper amount of ultrapure water, after completely dissolving, adjusting the pH of the solution to 7.4 +/-0.2 under the condition of 25 +/-1 ℃, adding 0.5mL of Proclin300, mixing uniformly, and adding to a constant volume of 1000 mL.

Step 3, coating of liquid phase chip BMB

The solid phase carrier is a Digital Liquid crystal Chip (Digital Liquid Chip) of Applied BioCode company, which has superparamagnetism and is composed of biocompatible high molecular polymers (BMB), and each Chip has a unique 12-bit binary Digital code. BMB surface covalent cross-linking has abundant carboxyl.

The coating process is as follows:

sucking up the BMB suspension with the required volume;

washing with an activation buffer solution for several times;

adding a certain amount of EDC (1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride)/NHS (N-hydroxysuccinimide Chinese name), oscillating at room temperature for 50mim reaction, and activating carboxyl;

washing the coating solution for several times;

adding the prepared antigen, and oscillating and incubating for 60min at room temperature;

blocking BMB for 60min with blocking solution (coating solution containing 1% BSA and 0.1% Tween 20);

the coating solution containing 1% BSA bovine serum albumin was resuspended.

4. Fluorescent marker

Phycoerythrin-labeled goat anti-human IgG-PE: the concentration was 0.5mg/ml, diluted with coating diluent to working concentration 0.5 ug/ml.

5. Reaction scheme

Adding diluted samples to 50. mu.l of BMB per well, and incubating for 15min at 37 ℃;

washing with a magnetic plate washing machine for 3 times, and sucking away residual liquid;

adding 50 mul of fluorescent marker goat anti-human IgG-PE into each hole, and incubating for 15min at 37 ℃;

adding 100 μ l of detection solution into each well, sucking and stirring with 50 μ l gun, defoaming, and standing for 30s for detection.

Test examples

1. Screening for Positive sample binding segments

The 8 proteins expressed in example 1 and the full-length Mi-2 antigen protein expressed by the DNA encoding the full-length Mi-2 antigen protein were verified separately.

The method comprises the steps of firstly coating the antigen to be verified on a liquid phase chip, then adding a known negative or positive serum sample for reaction, carrying out closed washing, finally carrying out incubation by using a secondary antibody with fluorescein Phycoerythrin (PE), detecting a corresponding fluorescence value under a fluorescence discriminator after washing again, and judging the negative or positive according to the fluorescence value.

The expressed antigens were verified separately, with full-length Mi-2 antigen protein as a control. 53 positive samples and 45 negative samples are selected in the verification experiment, and the positive detection rate of each protein is verified to be sensitivity, and the negative detection rate is verified to be specificity.

From the above test results, it can be seen that: the sensitivity of SEQ ID NO.1, SEQ ID NO.5 and SEQ ID NO.7 is greatly improved compared with that of the full-length Mi-2 antigen, and 3 fragments of the SEQ ID NO.1, SEQ ID NO.5 and SEQ ID NO.7 comprise main antigenic sites. For convenience of description, SEQ ID NO.1 is named N, SEQ ID NO.5 is named M, and SEQ ID NO.7 is named C. By modifying the protein sequence, the antigenic sites are exposed more fully, so that the specificity and the sensitivity of the anti-Mi-2 antibody are enhanced.

2. Screening and negative sample binding segments

Based on the full-length Mi-2 antigen protein, 8 recombinant proteins (recombinant proteins a-h) are respectively constructed, the 8 recombinant proteins lack SEQ ID NO.1-8 sequences relative to the full-length Mi-2 antigen protein, and then the expressed 8 recombinant proteins (recombinant proteins a-h) and the full-length Mi-2 antigen are verified respectively.

The antigens expressed above were verified separately, with full-length Mi-2 as a control. 53 positive samples and 45 negative samples are selected in the verification experiment, and the positive detection rate of each protein is verified to be sensitivity, and the negative detection rate is verified to be specificity.

From the above test results, it can be seen that:

compared with the full-length Mi-2 antigen, after the deletion of SEQ ID NO.6, SEQ ID NO.5 and SEQ ID NO.2, the sensitivity and specificity of the recombinant protein f, the recombinant protein e and the recombinant protein b are improved compared with the full-length Mi-2 antigen. The data analysis shows that the SEQ ID NO.6, SEQ ID NO.5 and SEQ ID NO.2 have interval structures which are easy to combine the full-length Mi-2 antigen in a negative sample, and the sequence length is reduced compared with the full-length Mi-2 antigen, so that the steric hindrance of the anti-Mi-2 antibody is reduced, and the specificity is improved.

Compared with the full-length Mi-2 antigen, when SEQ ID No.4 and SEQ ID No.8 are respectively rejected, the sensitivity and specificity of the recombinant protein d and the recombinant protein h are not greatly changed compared with the full-length Mi-2 antigen, so that the protein structures of SEQ ID No.4 and SEQ ID No.8 are presumed to have no great effect on identifying and binding the anti-Mi-2 antibody and binding a negative sample.

Compared with the full-length Mi-2 antigen, the sensitivity of the recombinant protein c with the SEQ ID NO.3 rejected is improved, but the specificity is reduced, and the protein structure of the SEQ ID NO.3 is supposed to be favorable for being combined with a negative sample.

Combining the specificity and sensitivity results of SEQ ID NO.1, SEQ ID NO.5 and SEQ ID NO.7, it can be speculated that the main antigenic sites are contained in the SEQ ID NO.1 and the SEQ ID NO.7, no binding sites beneficial to negative samples exist, the main antigenic sites are contained in the SEQ ID NO.5, and the binding sites beneficial to negative samples also exist. For convenience of description, SEQ ID NO.1 is named N, SEQ ID NO.5 is named M, and SEQ ID NO.7 is named C.

3. Expression and validation of fusion proteins

In order to enhance the specificity and sensitivity to anti-Mi-2, on the premise of minimizing the length of an antigen sequence, reducing steric hindrance during combination and more fully exposing an antigen site, N, M, C fragments are spliced together by adding a linker (GGGGSGGGG) between every two fragments to construct 6 new fusion proteins, wherein the fusion proteins are respectively as follows: fusion protein NM (structure is SEQ ID NO.1-GGGGSGGGG-SEQ ID NO.5), fusion protein NC (structure is SEQ ID NO.1-GGGGSGGGG-SEQ ID NO.7), fusion protein MC (structure is SEQ ID NO.5-GGGGSGGGG-SEQ ID NO.7), fusion protein MN (structure is SEQ ID NO.5-GGGGSGGGG-SEQ ID NO.1), fusion protein CN (structure is SEQ ID NO.7-GGGGSGGGG-SEQ ID NO.1), and fusion protein CM (structure is SEQ ID NO. 7-GGGGGGSGGGG-SEQ ID NO. 5).

6 expression plasmids corresponding to fusion proteins are respectively constructed according to the scheme, and a linker, namely GGGGSGGGG, is added between the fragments. Coli, and then verifying the antigen sensitivity and specificity of the 6 fusion proteins, respectively, as follows:

from the above test results, it can be seen that: the sensitivity and specificity of the fusion protein NC are the best, and are greatly improved compared with the N, C protein. The specificity of NM, MN, MC, CM is much lower than that of NC, CN because M has binding sites favorable for negative samples.

With reference to the above scheme, the NC protein and the M protein are expressed in a fusion manner, and 2 expression plasmids are respectively constructed: MNC (SEQ ID NO.9) and NCM (SEQ ID NO.10) expression plasmids, linker: GGGGSGGGG was added between the fragments. Coli, wherein the SDS-PAGE detection result of the fusion protein NCM is shown in fig. 1, and then the antigen sensitivity and specificity of the 2 fusion proteins are verified respectively, and the verification results are as follows:

from the above test results, it can be seen that: compared with Mi-2, the fusion protein NCM reduces steric hindrance when being combined with an anti-Mi-2 antibody, improves exposure to an antigen site, greatly improves sensitivity of the anti-Mi-2 antibody from 28.3% to 90.6% (by over 3 times), improves specificity from 53.3% to 88.9% (by over 1.5 times), meets the requirement of a detection reagent on antigen performance, and is improved compared with NC protein, so that the recombinant fusion protein NCM can replace Mi-2 to be used as the detection reagent antigen.

Meanwhile, the sensitivity and specificity of NCM are superior to those of MNC, probably because the antigen site on M is positioned at the rear end of the sequence, and the binding site favorable for negative samples is positioned at the front end of the sequence. Compared with MNC, the NCM can expose antigen sites more sufficiently, increase the steric hindrance when combined with a negative sample, and improve the sensitivity and specificity of the NCM.

The protein structures of SEQ ID NO.4 and SEQ ID NO.8 have no great effect on identifying and combining the anti-Mi-2 antibody and the negative sample, and the protein structures of SEQ ID NO.6, SEQ ID NO.5, SEQ ID NO.3 and SEQ ID NO.2 have interval structures which are easy to combine the full-length Mi-2 antigen in the negative sample. To enhance specificity and sensitivity against Mi-2, the NCM is the optimal Mi-2 antigen sequence on the premise of minimizing the length of the Mi-2 antigen sequence, reducing steric hindrance when combined with an anti-Mi-2 antibody and fully exposing the antigen site.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

SEQUENCE LISTING

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<212> PRT

<213> Artificial sequence

<400> 3

Met Glu Phe Cys Arg Val Cys Lys Asp Gly Gly Glu Leu Leu Cys Cys

1 5 10 15

Asp Thr Cys Pro Ser Ser Tyr His Ile His Cys Leu Asn Pro Pro Leu

20 25 30

Pro Glu Ile Pro Asn Gly Glu Trp Leu Cys Pro Arg Cys Thr Cys Pro

35 40 45

Ala Leu Lys Gly Lys Val Gln Lys Ile Leu Ile Trp Lys Trp Gly Gln

50 55 60

Pro Pro Ser Pro Thr Pro Val Pro Arg Pro Pro Asp Ala Asp Pro Asn

65 70 75 80

Thr Pro Ser Pro Lys Pro Leu Glu Gly Arg Pro Glu Arg Gln Phe Phe

85 90 95

Val Lys Trp Gln Gly Met Ser Tyr Trp His Cys Ser Trp Val Ser Glu

100 105 110

Leu Gln Leu Glu Leu His Cys Gln Val Met Phe Arg Asn Tyr Gln Arg

115 120 125

Lys Asn Asp Met Asp Glu Pro Pro Ser Gly Asp Phe Gly Gly Asp Glu

130 135 140

Glu Lys Ser Arg Lys Arg Lys Asn Lys Asp Pro Lys Phe Ala Glu Met

145 150 155 160

Glu Glu Arg Phe Tyr Arg Tyr Gly Ile Lys Pro Glu Trp Met Met Ile

165 170 175

His Arg Ile Leu Asn His Ser Val Asp Lys Lys Gly His Val His Tyr

180 185 190

Leu Ile Lys Trp Arg Asp Leu Pro Tyr Asp Gln Ala Ser Trp Glu Ser

195 200 205

Glu Asp Val Glu Ile Gln Asp Tyr Asp Leu Phe Lys Gln Ser Tyr Trp

210 215 220

Asn His Arg Glu Leu

225

<210> 4

<211> 271

<212> PRT

<213> Artificial sequence

<400> 4

Met Arg Gly Glu Glu Gly Arg Pro Gly Lys Lys Leu Lys Lys Val Lys

1 5 10 15

Leu Arg Lys Leu Glu Arg Pro Pro Glu Thr Pro Thr Val Asp Pro Thr

20 25 30

Val Lys Tyr Glu Arg Gln Pro Glu Tyr Leu Asp Ala Thr Gly Gly Thr

35 40 45

Leu His Pro Tyr Gln Met Glu Gly Leu Asn Trp Leu Arg Phe Ser Trp

50 55 60

Ala Gln Gly Thr Asp Thr Ile Leu Ala Asp Glu Met Gly Leu Gly Lys

65 70 75 80

Thr Val Gln Thr Ala Val Phe Leu Tyr Ser Leu Tyr Lys Glu Gly His

85 90 95

Ser Lys Gly Pro Phe Leu Val Ser Ala Pro Leu Ser Thr Ile Ile Asn

100 105 110

Trp Glu Arg Glu Phe Glu Met Trp Ala Pro Asp Met Tyr Val Val Thr

115 120 125

Tyr Val Gly Asp Lys Asp Ser Arg Ala Ile Ile Arg Glu Asn Glu Phe

130 135 140

Ser Phe Glu Asp Asn Ala Ile Arg Gly Gly Lys Lys Ala Ser Arg Met

145 150 155 160

Lys Lys Glu Ala Ser Val Lys Phe His Val Leu Leu Thr Ser Tyr Glu

165 170 175

Leu Ile Thr Ile Asp Met Ala Ile Leu Gly Ser Ile Asp Trp Ala Cys

180 185 190

Leu Ile Val Asp Glu Ala His Arg Leu Lys Asn Asn Gln Ser Lys Phe

195 200 205

Phe Arg Val Leu Asn Gly Tyr Ser Leu Gln His Lys Leu Leu Leu Thr

210 215 220

Gly Thr Pro Leu Gln Asn Asn Leu Glu Glu Leu Phe His Leu Leu Asn

225 230 235 240

Phe Leu Thr Pro Glu Arg Phe His Asn Leu Glu Gly Phe Leu Glu Glu

245 250 255

Phe Ala Asp Ile Ala Lys Glu Asp Gln Ile Lys Lys Leu His Asp

260 265 270

<210> 5

<211> 261

<212> PRT

<213> Artificial sequence

<400> 5

Met Leu Gly Pro His Met Leu Arg Arg Leu Lys Ala Asp Val Phe Lys

1 5 10 15

Asn Met Pro Ser Lys Thr Glu Leu Ile Val Arg Val Glu Leu Ser Pro

20 25 30

Met Gln Lys Lys Tyr Tyr Lys Tyr Ile Leu Thr Arg Asn Phe Glu Ala

35 40 45

Leu Asn Ala Arg Gly Gly Gly Asn Gln Val Ser Leu Leu Asn Val Val

50 55 60

Met Asp Leu Lys Lys Cys Cys Asn His Pro Tyr Leu Phe Pro Val Ala

65 70 75 80

Ala Met Glu Ala Pro Lys Met Pro Asn Gly Met Tyr Asp Gly Ser Ala

85 90 95

Leu Ile Arg Ala Ser Gly Lys Leu Leu Leu Leu Gln Lys Met Leu Lys

100 105 110

Asn Leu Lys Glu Gly Gly His Arg Val Leu Ile Phe Ser Gln Met Thr

115 120 125

Lys Met Leu Asp Leu Leu Glu Asp Phe Leu Glu His Glu Gly Tyr Lys

130 135 140

Tyr Glu Arg Ile Asp Gly Gly Ile Thr Gly Asn Met Arg Gln Glu Ala

145 150 155 160

Ile Asp Arg Phe Asn Ala Pro Gly Ala Gln Gln Phe Cys Phe Leu Leu

165 170 175

Ser Thr Arg Ala Gly Gly Leu Gly Ile Asn Leu Ala Thr Ala Asp Thr

180 185 190

Val Ile Ile Tyr Asp Ser Asp Trp Asn Pro His Asn Asp Ile Gln Ala

195 200 205

Phe Ser Arg Ala His Arg Ile Gly Gln Asn Lys Lys Val Met Ile Tyr

210 215 220

Arg Phe Val Thr Arg Ala Ser Val Glu Glu Arg Ile Thr Gln Val Ala

225 230 235 240

Lys Lys Lys Met Met Leu Thr His Leu Val Val Arg Pro Gly Leu Gly

245 250 255

Ser Lys Thr Gly Ser

260

<210> 6

<211> 289

<212> PRT

<213> Artificial sequence

<400> 6

Met Ser Lys Gln Glu Leu Asp Asp Ile Leu Lys Phe Gly Thr Glu Glu

1 5 10 15

Leu Phe Lys Asp Glu Ala Thr Asp Gly Gly Gly Asp Asn Lys Glu Gly

20 25 30

Glu Asp Ser Ser Val Ile His Tyr Asp Asp Lys Ala Ile Glu Arg Leu

35 40 45

Leu Asp Arg Asn Gln Asp Glu Thr Glu Asp Thr Glu Leu Gln Gly Met

50 55 60

Asn Glu Tyr Leu Ser Ser Phe Lys Val Ala Gln Tyr Val Val Arg Glu

65 70 75 80

Glu Glu Met Gly Glu Glu Glu Glu Val Glu Arg Glu Ile Ile Lys Gln

85 90 95

Glu Glu Ser Val Asp Pro Asp Tyr Trp Glu Lys Leu Leu Arg His His

100 105 110

Tyr Glu Gln Gln Gln Glu Asp Leu Ala Arg Asn Leu Gly Lys Gly Lys

115 120 125

Arg Ile Arg Lys Gln Val Asn Tyr Asn Asp Gly Ser Gln Glu Asp Arg

130 135 140

Asp Trp Gln Asp Asp Gln Ser Asp Asn Gln Ser Asp Tyr Ser Val Ala

145 150 155 160

Ser Glu Glu Gly Asp Glu Asp Phe Asp Glu Arg Ser Glu Ala Pro Arg

165 170 175

Arg Pro Ser Arg Lys Gly Leu Arg Asn Asp Lys Asp Lys Pro Leu Pro

180 185 190

Pro Leu Leu Ala Arg Val Gly Gly Asn Ile Glu Val Leu Gly Phe Asn

195 200 205

Ala Arg Gln Arg Lys Ala Phe Leu Asn Ala Ile Met Arg Tyr Gly Met

210 215 220

Pro Pro Gln Asp Ala Phe Thr Thr Gln Trp Leu Val Arg Asp Leu Arg

225 230 235 240

Gly Lys Ser Glu Lys Glu Phe Lys Ala Tyr Val Ser Leu Phe Met Arg

245 250 255

His Leu Cys Glu Pro Gly Ala Asp Gly Ala Glu Thr Phe Ala Asp Gly

260 265 270

Val Pro Arg Glu Gly Leu Ser Arg Gln His Val Leu Thr Arg Ile Gly

275 280 285

Val

<210> 7

<211> 198

<212> PRT

<213> Artificial sequence

<400> 7

Met Ser Leu Ile Arg Lys Lys Val Gln Glu Phe Glu His Val Asn Gly

1 5 10 15

Arg Trp Ser Met Pro Glu Leu Ala Glu Val Glu Glu Asn Lys Lys Met

20 25 30

Ser Gln Pro Gly Ser Pro Ser Pro Lys Thr Pro Thr Pro Ser Thr Pro

35 40 45

Gly Asp Thr Gln Pro Asn Thr Pro Ala Pro Val Pro Pro Ala Glu Asp

50 55 60

Gly Ile Lys Ile Glu Glu Asn Ser Leu Lys Glu Glu Glu Ser Ile Glu

65 70 75 80

Gly Glu Lys Glu Val Lys Ser Thr Ala Pro Glu Thr Ala Ile Glu Cys

85 90 95

Thr Gln Ala Pro Ala Pro Ala Ser Glu Asp Glu Lys Val Val Val Glu

100 105 110

Pro Pro Glu Gly Glu Glu Lys Val Glu Lys Ala Glu Val Lys Glu Arg

115 120 125

Thr Glu Glu Pro Met Glu Thr Glu Pro Lys Gly Ala Ala Asp Val Glu

130 135 140

Lys Val Glu Glu Lys Ser Ala Ile Asp Leu Thr Pro Ile Val Val Glu

145 150 155 160

Asp Lys Glu Glu Lys Lys Glu Glu Glu Glu Lys Lys Glu Val Met Leu

165 170 175

Gln Asn Gly Glu Thr Pro Lys Asp Leu Asn Asp Glu Lys Gln Lys Lys

180 185 190

Asn Ile Lys Gln Arg Phe

195

<210> 8

<211> 216

<212> PRT

<213> Artificial sequence

<400> 8

Met Phe Asn Ile Ala Asp Gly Gly Phe Thr Glu Leu His Ser Leu Trp

1 5 10 15

Gln Asn Glu Glu Arg Ala Ala Thr Val Thr Lys Lys Thr Tyr Glu Ile

20 25 30

Trp His Arg Arg His Asp Tyr Trp Leu Leu Ala Gly Ile Ile Asn His

35 40 45

Gly Tyr Ala Arg Trp Gln Asp Ile Gln Asn Asp Pro Arg Tyr Ala Ile

50 55 60

Leu Asn Glu Pro Phe Lys Gly Glu Met Asn Arg Gly Asn Phe Leu Glu

65 70 75 80

Ile Lys Asn Lys Phe Leu Ala Arg Arg Phe Lys Leu Leu Glu Gln Ala

85 90 95

Leu Val Ile Glu Glu Gln Leu Arg Arg Ala Ala Tyr Leu Asn Met Ser

100 105 110

Glu Asp Pro Ser His Pro Ser Met Ala Leu Asn Thr Arg Phe Ala Glu

115 120 125

Val Glu Cys Leu Ala Glu Ser His Gln His Leu Ser Lys Glu Ser Met

130 135 140

Ala Gly Asn Lys Pro Ala Asn Ala Val Leu His Lys Val Leu Lys Gln

145 150 155 160

Leu Glu Glu Leu Leu Ser Asp Met Lys Ala Asp Val Thr Arg Leu Pro

165 170 175

Ala Thr Ile Ala Arg Ile Pro Pro Val Ala Val Arg Leu Gln Met Ser

180 185 190

Glu Arg Asn Ile Leu Ser Arg Leu Ala Asn Arg Ala Pro Glu Pro Thr

195 200 205

Pro Gln Gln Val Ala Gln Gln Gln

210 215

<210> 9

<211> 716

<212> PRT

<213> Artificial sequence

<400> 9

Met Leu Gly Pro His Met Leu Arg Arg Leu Lys Ala Asp Val Phe Lys

1 5 10 15

Asn Met Pro Ser Lys Thr Glu Leu Ile Val Arg Val Glu Leu Ser Pro

20 25 30

Met Gln Lys Lys Tyr Tyr Lys Tyr Ile Leu Thr Arg Asn Phe Glu Ala

35 40 45

Leu Asn Ala Arg Gly Gly Gly Asn Gln Val Ser Leu Leu Asn Val Val

50 55 60

Met Asp Leu Lys Lys Cys Cys Asn His Pro Tyr Leu Phe Pro Val Ala

65 70 75 80

Ala Met Glu Ala Pro Lys Met Pro Asn Gly Met Tyr Asp Gly Ser Ala

85 90 95

Leu Ile Arg Ala Ser Gly Lys Leu Leu Leu Leu Gln Lys Met Leu Lys

100 105 110

Asn Leu Lys Glu Gly Gly His Arg Val Leu Ile Phe Ser Gln Met Thr

115 120 125

Lys Met Leu Asp Leu Leu Glu Asp Phe Leu Glu His Glu Gly Tyr Lys

130 135 140

Tyr Glu Arg Ile Asp Gly Gly Ile Thr Gly Asn Met Arg Gln Glu Ala

145 150 155 160

Ile Asp Arg Phe Asn Ala Pro Gly Ala Gln Gln Phe Cys Phe Leu Leu

165 170 175

Ser Thr Arg Ala Gly Gly Leu Gly Ile Asn Leu Ala Thr Ala Asp Thr

180 185 190

Val Ile Ile Tyr Asp Ser Asp Trp Asn Pro His Asn Asp Ile Gln Ala

195 200 205

Phe Ser Arg Ala His Arg Ile Gly Gln Asn Lys Lys Val Met Ile Tyr

210 215 220

Arg Phe Val Thr Arg Ala Ser Val Glu Glu Arg Ile Thr Gln Val Ala

225 230 235 240

Lys Lys Lys Met Met Leu Thr His Leu Val Val Arg Pro Gly Leu Gly

245 250 255

Ser Lys Thr Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Met Ala

260 265 270

Ser Gly Leu Gly Ser Pro Ser Pro Cys Ser Ala Gly Ser Glu Glu Glu

275 280 285

Asp Met Asp Ala Leu Leu Asn Asn Ser Leu Pro Pro Pro His Pro Glu

290 295 300

Asn Glu Glu Asp Pro Glu Glu Asp Leu Ser Glu Thr Glu Thr Pro Lys

305 310 315 320

Leu Lys Lys Lys Lys Lys Pro Lys Lys Pro Arg Asp Pro Lys Ile Pro

325 330 335

Lys Ser Lys Arg Gln Lys Lys Glu Arg Met Leu Leu Cys Arg Gln Leu

340 345 350

Gly Asp Ser Ser Gly Glu Gly Pro Glu Phe Val Glu Glu Glu Glu Glu

355 360 365

Val Ala Leu Arg Ser Asp Ser Glu Gly Ser Asp Tyr Thr Pro Gly Lys

370 375 380

Lys Lys Lys Lys Lys Leu Gly Pro Lys Lys Glu Lys Lys Ser Lys Ser

385 390 395 400

Lys Arg Lys Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp Ser Lys

405 410 415

Glu Pro Lys Ser Ser Ala Gln Leu Leu Glu Asp Trp Gly Met Glu Asp

420 425 430

Ile Asp His Val Phe Ser Glu Glu Asp Tyr Arg Thr Leu Thr Asn Tyr

435 440 445

Lys Ala Phe Ser Gln Phe Val Arg Pro Leu Ile Ala Ala Lys Asn Pro

450 455 460

Lys Ile Ala Val Ser Lys Met Met Met Val Leu Gly Ala Lys Trp Arg

465 470 475 480

Glu Phe Ser Thr Asn Asn Pro Phe Lys Gly Ser Ser Gly Ala Ser Val

485 490 495

Ala Ala Ala Ala Ala Ala Ala Val Ala Val Val Glu Ser Gly Gly Gly

500 505 510

Gly Ser Gly Gly Gly Gly Met Ser Leu Ile Arg Lys Lys Val Gln Glu

515 520 525

Phe Glu His Val Asn Gly Arg Trp Ser Met Pro Glu Leu Ala Glu Val

530 535 540

Glu Glu Asn Lys Lys Met Ser Gln Pro Gly Ser Pro Ser Pro Lys Thr

545 550 555 560

Pro Thr Pro Ser Thr Pro Gly Asp Thr Gln Pro Asn Thr Pro Ala Pro

565 570 575

Val Pro Pro Ala Glu Asp Gly Ile Lys Ile Glu Glu Asn Ser Leu Lys

580 585 590

Glu Glu Glu Ser Ile Glu Gly Glu Lys Glu Val Lys Ser Thr Ala Pro

595 600 605

Glu Thr Ala Ile Glu Cys Thr Gln Ala Pro Ala Pro Ala Ser Glu Asp

610 615 620

Glu Lys Val Val Val Glu Pro Pro Glu Gly Glu Glu Lys Val Glu Lys

625 630 635 640

Ala Glu Val Lys Glu Arg Thr Glu Glu Pro Met Glu Thr Glu Pro Lys

645 650 655

Gly Ala Ala Asp Val Glu Lys Val Glu Glu Lys Ser Ala Ile Asp Leu

660 665 670

Thr Pro Ile Val Val Glu Asp Lys Glu Glu Lys Lys Glu Glu Glu Glu

675 680 685

Lys Lys Glu Val Met Leu Gln Asn Gly Glu Thr Pro Lys Asp Leu Asn

690 695 700

Asp Glu Lys Gln Lys Lys Asn Ile Lys Gln Arg Phe

705 710 715

<210> 10

<211> 716

<212> PRT

<213> Artificial sequence

<400> 10

Met Ala Ser Gly Leu Gly Ser Pro Ser Pro Cys Ser Ala Gly Ser Glu

1 5 10 15

Glu Glu Asp Met Asp Ala Leu Leu Asn Asn Ser Leu Pro Pro Pro His

20 25 30

Pro Glu Asn Glu Glu Asp Pro Glu Glu Asp Leu Ser Glu Thr Glu Thr

35 40 45

Pro Lys Leu Lys Lys Lys Lys Lys Pro Lys Lys Pro Arg Asp Pro Lys

50 55 60

Ile Pro Lys Ser Lys Arg Gln Lys Lys Glu Arg Met Leu Leu Cys Arg

65 70 75 80

Gln Leu Gly Asp Ser Ser Gly Glu Gly Pro Glu Phe Val Glu Glu Glu

85 90 95

Glu Glu Val Ala Leu Arg Ser Asp Ser Glu Gly Ser Asp Tyr Thr Pro

100 105 110

Gly Lys Lys Lys Lys Lys Lys Leu Gly Pro Lys Lys Glu Lys Lys Ser

115 120 125

Lys Ser Lys Arg Lys Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp

130 135 140

Ser Lys Glu Pro Lys Ser Ser Ala Gln Leu Leu Glu Asp Trp Gly Met

145 150 155 160

Glu Asp Ile Asp His Val Phe Ser Glu Glu Asp Tyr Arg Thr Leu Thr

165 170 175

Asn Tyr Lys Ala Phe Ser Gln Phe Val Arg Pro Leu Ile Ala Ala Lys

180 185 190

Asn Pro Lys Ile Ala Val Ser Lys Met Met Met Val Leu Gly Ala Lys

195 200 205

Trp Arg Glu Phe Ser Thr Asn Asn Pro Phe Lys Gly Ser Ser Gly Ala

210 215 220

Ser Val Ala Ala Ala Ala Ala Ala Ala Val Ala Val Val Glu Ser Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Met Ser Leu Ile Arg Lys Lys Val

245 250 255

Gln Glu Phe Glu His Val Asn Gly Arg Trp Ser Met Pro Glu Leu Ala

260 265 270

Glu Val Glu Glu Asn Lys Lys Met Ser Gln Pro Gly Ser Pro Ser Pro

275 280 285

Lys Thr Pro Thr Pro Ser Thr Pro Gly Asp Thr Gln Pro Asn Thr Pro

290 295 300

Ala Pro Val Pro Pro Ala Glu Asp Gly Ile Lys Ile Glu Glu Asn Ser

305 310 315 320

Leu Lys Glu Glu Glu Ser Ile Glu Gly Glu Lys Glu Val Lys Ser Thr

325 330 335

Ala Pro Glu Thr Ala Ile Glu Cys Thr Gln Ala Pro Ala Pro Ala Ser

340 345 350

Glu Asp Glu Lys Val Val Val Glu Pro Pro Glu Gly Glu Glu Lys Val

355 360 365

Glu Lys Ala Glu Val Lys Glu Arg Thr Glu Glu Pro Met Glu Thr Glu

370 375 380

Pro Lys Gly Ala Ala Asp Val Glu Lys Val Glu Glu Lys Ser Ala Ile

385 390 395 400

Asp Leu Thr Pro Ile Val Val Glu Asp Lys Glu Glu Lys Lys Glu Glu

405 410 415

Glu Glu Lys Lys Glu Val Met Leu Gln Asn Gly Glu Thr Pro Lys Asp

420 425 430

Leu Asn Asp Glu Lys Gln Lys Lys Asn Ile Lys Gln Arg Phe Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Met Leu Gly Pro His Met Leu Arg Arg

450 455 460

Leu Lys Ala Asp Val Phe Lys Asn Met Pro Ser Lys Thr Glu Leu Ile

465 470 475 480

Val Arg Val Glu Leu Ser Pro Met Gln Lys Lys Tyr Tyr Lys Tyr Ile

485 490 495

Leu Thr Arg Asn Phe Glu Ala Leu Asn Ala Arg Gly Gly Gly Asn Gln

500 505 510

Val Ser Leu Leu Asn Val Val Met Asp Leu Lys Lys Cys Cys Asn His

515 520 525

Pro Tyr Leu Phe Pro Val Ala Ala Met Glu Ala Pro Lys Met Pro Asn

530 535 540

Gly Met Tyr Asp Gly Ser Ala Leu Ile Arg Ala Ser Gly Lys Leu Leu

545 550 555 560

Leu Leu Gln Lys Met Leu Lys Asn Leu Lys Glu Gly Gly His Arg Val

565 570 575

Leu Ile Phe Ser Gln Met Thr Lys Met Leu Asp Leu Leu Glu Asp Phe

580 585 590

Leu Glu His Glu Gly Tyr Lys Tyr Glu Arg Ile Asp Gly Gly Ile Thr

595 600 605

Gly Asn Met Arg Gln Glu Ala Ile Asp Arg Phe Asn Ala Pro Gly Ala

610 615 620

Gln Gln Phe Cys Phe Leu Leu Ser Thr Arg Ala Gly Gly Leu Gly Ile

625 630 635 640

Asn Leu Ala Thr Ala Asp Thr Val Ile Ile Tyr Asp Ser Asp Trp Asn

645 650 655

Pro His Asn Asp Ile Gln Ala Phe Ser Arg Ala His Arg Ile Gly Gln

660 665 670

Asn Lys Lys Val Met Ile Tyr Arg Phe Val Thr Arg Ala Ser Val Glu

675 680 685

Glu Arg Ile Thr Gln Val Ala Lys Lys Lys Met Met Leu Thr His Leu

690 695 700

Val Val Arg Pro Gly Leu Gly Ser Lys Thr Gly Ser

705 710 715

<210> 11

<211> 18

<212> DNA

<213> Artificial sequence

<400> 11

atggcgtcgg gcctgggc 18

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence

<400> 12

gctctccacc acagctac 18

<210> 13

<211> 22

<212> DNA

<213> Artificial sequence

<400> 13

atggtgacag ccactgaggt tg 22

<210> 14

<211> 22

<212> DNA

<213> Artificial sequence

<400> 14

atggtggtca tcctcctctt cg 22

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence

<400> 15

atggaattct gtcgggtctg caagg 25

<210> 16

<211> 17

<212> DNA

<213> Artificial sequence

<400> 16

taactccctg tgattcc 17

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<400> 17

atgaggggtg aggaaggccg 20

<210> 18

<211> 21

<212> DNA

<213> Artificial sequence

<400> 18

gtcatgcagt ttttttatct g 21

<210> 19

<211> 22

<212> DNA

<213> Artificial sequence

<400> 19

atgctggggc cgcacatgtt gc 22

<210> 20

<211> 17

<212> DNA

<213> Artificial sequence

<400> 20

agatccagtc ttggagc 17

<210> 21

<211> 22

<212> DNA

<213> Artificial sequence

<400> 21

atgtccaaac aggagcttga tg 22

<210> 22

<211> 21

<212> DNA

<213> Artificial sequence

<400> 22

aacaccaatt ctagtaagga c 21

<210> 23

<211> 18

<212> DNA

<213> Artificial sequence

<400> 23

atgtctttga ttcgcaag 18

<210> 24

<211> 22

<212> DNA

<213> Artificial sequence

<400> 24

gaaacgttgt ttaatatttt tc 22

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence

<400> 25

atgtttaaca ttgcagatgg 20

<210> 26

<211> 19

<212> DNA

<213> Artificial sequence

<400> 26

ctgctgctgg gctacctgc 19

Claims

1. The Mi-2 recombinant antigen is characterized by being SEQ ID NO.9 or SEQ ID NO. 10.

2. Biomaterial associated with the Mi-2 recombinant antigen of claim 1, characterized in that it is any of the following:

(a) a nucleic acid molecule encoding the Mi-2 recombinant antigen of claim 1;

(b) an expression cassette comprising the nucleic acid molecule of (a);

3. The method for preparing Mi-2 recombinant antigen of claim 1, wherein the nucleic acid molecule encoding the Mi-2 recombinant antigen is introduced into a host to obtain a recombinant expression system, and the expression system is used to obtain the Mi-2 recombinant antigen.

4. The method of claim 3, wherein the nucleic acid molecule encoding the Mi-2 recombinant antigen is combined with pET-28a to construct a recombinant vector, and the recombinant vector is introduced into E.coli and expressed to obtain the Mi-2 recombinant antigen.

5. Use of the Mi-2 recombinant antigen of claim 1 for the preparation of a product for detecting Mi-2 antibodies.

6. Use of the Mi-2 recombinant antigen of claim 1 in the preparation of a product for detecting polymyositis.

7. A product for detecting polymyositis, which employs the Mi-2 recombinant antigen of claim 1 as a detection antigen.