CN111560362B

CN111560362B - Antigen M2-3E-GS and product and application thereof

Info

Publication number: CN111560362B
Application number: CN202010562355.5A
Authority: CN
Inventors: 庄兰芳; 胡志强; 储迅涛
Original assignee: Zhuhai Livzon Diagnostics Inc
Current assignee: Zhuhai Livzon Diagnostics Inc
Priority date: 2020-06-18
Filing date: 2020-06-18
Publication date: 2022-03-25
Anticipated expiration: 2040-06-18
Also published as: CN111560362A

Abstract

The invention relates to the technical field of biology, and particularly provides an antigen M2-3E-GS, and a product and application thereof. The antigen M2-3E-GS comprises a protein fragment 1, a protein fragment 2 and a protein fragment 3 which are connected through a connecting peptide to form a linear fragment. The antigen avoids the problem of steric hindrance in detection of the antigen by AMA-M2 in the prior art, obviously reduces the positive omission ratio, effectively improves the accuracy and specificity, and has good stability and small batch difference in the production process. The antigen and the biological material related to the antigen can be used for preparing products for diagnosing PBC or detecting AMA-M2, and the stability, the accuracy and other properties of the products are effectively improved.

Description

Antigen M2-3E-GS and product and application thereof

Technical Field

The invention relates to the technical field of biology, and particularly relates to an antigen M2-3E-GS, and a product and application thereof.

Background

Primary Biliary Cirrhosis (PBC) is an autoimmune liver disease with unknown etiology, is characterized by progressive small bile duct destruction in the liver, inflammation and fibrosis around a junction area and a junction, and early diagnosis of the primary biliary cirrhosis is a key point of treatment, and provides an important basis for diagnosis of various autoantibodies through detection. In PBC patients, the serological features were mainly high titers of autoimmune AMA-M2 (anti-mitochondrial antibodies-type M2) antibodies.

In vitro diagnosis, it is most important to select an antigen with strong antigenicity and good specificity. With the development of molecular biotechnology, many main antigenic genes of AMA-M2 were clonally expressed and purified for PBC antibody detection, including pyruvate dehydrogenase complex E2 subunit (PDC-E2), branched diketonate dehydrogenase complex E2 subunit (bcadc-E2), 2-ketoglutarate dehydrogenase complex E2 subunit (OGDC-E2), dihydroformamide dehydrogenase E3 binding protein (E3BP), pyruvate dehydrogenase complex E1 α (PDC-E1 α), and pyruvate dehydrogenase complex E1 β subunit (PDC-E1 β). The antibody positive rates of PBC patients to the PDC-E2, BCOADC-E2 and OGDC-E2 target antigens are 90-95%, 50-80% and 20-60%, respectively, while the antibody positive rates to the E3BP, PDC-E1 alpha and PDC-E1 beta target antigens are lower.

The clinical detection methods for AMA-M2 include enzyme-linked immunosorbent assay (ELISA), Linear Immunoblotting (LIA), chemiluminescence immunoassay (CLIA), immunofluorescence assay (IF), etc. The target antigen used in the current commercial kit is mostly a fusion recombinant protein (M2-3E) of the lipoyl region of three proteins, namely PDC-E2, BCOADC-E2 and OGDC-E2, or a combination of M2-3E and pyruvate dehydrogenase complex (PDH). Detection with PDC-E2 alone as the target antigen missed approximately 15% of AMA-M2 positive patients. If a plurality of antigen mixtures are coated on the solid phase carrier for detection, the coating is not uniform, and the difference between batches is large.

M2-3E is a fusion recombinant protein obtained by extracting and splicing the lipoyl groups of PDC-E2, BCOADC-E2 and OGDC-E2 together, and the omission factor is reduced by linearly connecting the three groups, however, the spliced fusion protein is easy to cause steric hindrance, the specific site is blocked, and the specificity is deteriorated.

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

Disclosure of Invention

The first purpose of the invention is to provide an antigen M2-3E-GS. The second object of the present invention is to provide a biomaterial related to the above antigen M2-3E-GS. The third objective of the invention is to provide a method for preparing the antigen M2-3E-GS. A fourth object of the invention is to provide the use of the antigen M2-3E-GS or biomaterial. A fifth object of the present invention is to provide a product for diagnosing PBC.

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

an antigen M2-3E-GS, wherein M2-3E-GS comprises a protein fragment 1, a protein fragment 2 and a protein fragment 3 which are connected through a connecting peptide to form a linear fragment;

the protein fragment 1 is an amino acid sequence shown in SEQ ID NO.2, or a protein which has homology of more than 90% and has the same function with the protein fragment;

the protein fragment 2 is an amino acid sequence shown in SEQ ID NO.1, or a protein which has homology of more than 90% and has the same function with the protein fragment;

the protein fragment 3 is an amino acid sequence shown in SEQ ID NO.3, or a protein which has homology of more than 90% and has the same function with the protein fragment;

the connecting peptide is (GGGGSGGGS)_nWherein n is an integer of 1-5, and the connecting peptides between each of the protein fragment 1, the protein fragment 2 and the protein fragment 3 are the same or different.

Further, the linker peptide between protein fragment 1 and protein fragment 2 was (GGGGSGGGS)_nWherein n is 4 or 5;

preferably, the connecting peptide between protein fragment 2 and protein fragment 3 is (GGGGSGGGS)_nWherein n is 2, 3 or 4;

preferably, the connecting peptide between protein fragment 1 and protein fragment 3 is (GGGGSGGGS)_nWherein n is 1 or 4.

Further, the M2-3E-GS is connected in any one of the following ways:

(1) protein fragment 2-linker peptide-protein fragment 1-linker peptide-protein fragment 3;

(2) protein fragment 2-linker peptide-protein fragment 3-linker peptide-protein fragment 1;

(3) protein fragment 3-linker peptide-protein fragment 1-linker peptide-protein fragment 2;

(4) protein fragment 3-linker peptide-protein fragment 2-linker peptide-protein fragment 1.

Further, the amino acid sequence of M2-3E-GS is any one of SEQ ID NO.4-SEQ ID NO. 7.

A biomaterial related to the above antigen M2-3E-GS, said biomaterial being any one of the following:

(a) a nucleic acid molecule encoding the antigen M2-3E-GS;

(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).

Further, the nucleic acid molecule is (A) or (B) as follows:

(A) the nucleotide sequence is any one of SEQ ID NO.8-SEQ ID NO. 11;

(B) and (B) a nucleic acid molecule which has a homology of 90% or more with (A) and encodes a protein having the same function.

The method for preparing the antigen M2-3E-GS comprises the steps of introducing a nucleic acid molecule encoding the antigen M2-3E-GS into a host to obtain a recombinant expression system, and expressing the recombinant expression system to obtain the antigen M2-3E-GS.

Further, a nucleic acid molecule encoding the antigen M2-3E-GS and a pET28a vector construct a recombinant vector, and the recombinant vector is transfected into escherichia coli to express the antigen M2-3E-GS.

The antigen M2-3E-GS or the biological material can be applied to the preparation of PBC diagnosis or AMA-M2 detection products.

A product for diagnosing PBC, which employs the above antigen M2-3E-GS as a detection antigen.

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

the invention provides an antigen M2-3E-GS, which comprises a protein fragment 1, a protein fragment 2 and a protein fragment 3, wherein the three are connected through a connecting peptide to form a linear fragment. The antigen avoids the problem of steric hindrance in detection of the antigen by AMA-M2 in the prior art, obviously reduces the positive omission ratio, effectively improves the accuracy and specificity, and has good stability and small batch difference in the production process. The antigen and the biological material related to the antigen can be used for preparing products for diagnosing PBC or detecting AMA-M2, and the stability, the accuracy and other properties of the products are effectively improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a SDS-PAGE result of B4P protein in example 1 of the present invention, wherein M: protein marker; 2: 500mM imidazole eluent;

FIG. 2 is a SDS-PAGE result of the P1O protein in example 1 of the present invention, wherein M: protein marker; 2: 500mM imidazole eluent;

FIG. 3 is a SDS-PAGE result of B4O protein in example 1 of the present invention, wherein M: protein marker; 2: 500mM imidazole eluent;

FIG. 4 is a SDS-PAGE result of B4P1O protein in experimental example 2 of the present invention, wherein M: proteinmarker; 2: 500mM imidazole eluent.

Detailed Description

The invention provides an antigen M2-3E-GS, wherein the M2-3E-GS comprises a protein fragment 1, a protein fragment 2 and a protein fragment 3, and the three are connected through a connecting peptide to form a linear fragment. Wherein the protein fragment 1 is an amino acid sequence shown in SEQ ID NO.2, or,a protein having a homology of 90% or more and having the same function; the protein fragment 2 is an amino acid sequence shown in SEQ ID NO.1, or a protein which has more than 90 percent of homology with the protein and has the same function; the protein fragment 3 is an amino acid sequence shown in SEQ ID NO.3, or a protein which has more than 90 percent of homology with the protein fragment and has the same function; the connecting peptide is (GGGGSGGGS)_nWherein n is an integer of 1-5, and the connecting peptides between each of the protein fragment 1, the protein fragment 2 and the protein fragment 3 are the same or different.

QVVQFKLSDIGEGIREVTVKEWYVKEGDTVSQFDSICEVQSDKASVTITSRYDGVIKKLYYNLDDIAYVGKPLVDIETEALKDSEEDVVETPAVSHDEHTHQEIKGRKTLATPAVRR(SEQ ID NO.1)。

KNYTLDSSAAPTPQAAPAPTPAATASPPTPSAQAPGSSYPPHMQVLLPALSPTMTMGTVQRWEKKVGEKLSEGDLLAEIETDKATIGFEVQEEGYLAKILVPEGTRDVPLGTPLCII(SEQ ID NO.2)。

KDDLVTVKTPAFAESVTEGDVRWEKAVGDTVAEDEVVCEIETDKTSVQVPSPANGVIEALLVPDGGKVEGGTPLFTLRKTG(SEQ ID NO.3)。

The antigen avoids the problem of steric hindrance in detection of the antigen by AMA-M2 in the prior art, obviously reduces the positive omission ratio, effectively improves the accuracy and specificity, and has good stability and small batch difference in the production process. The antigen and the biological material related to the antigen can be used for preparing products for diagnosing PBC or detecting AMA-M2, and the stability, the accuracy and other properties of the products are effectively improved.

The structure of the antigen M2-3E-GS of the present invention may be, for example, protein fragment 1-linker peptide-protein fragment 2-linker peptide-protein fragment 3, protein fragment 2-linker peptide-protein fragment 3-linker peptide-protein fragment 1, alternatively, protein fragment 1-linker peptide-protein fragment 3-linker peptide-protein fragment 2, and the like, meanwhile, it is understood that, for example, in "protein fragment 1-linker peptide-protein fragment 2-linker peptide-protein fragment 3", the "linker peptide" of "protein fragment 1-linker peptide-protein fragment 2" and the "linker peptide" of "protein fragment 2-linker peptide-protein fragment 3" may be the same, for example, the "linker peptide" of "protein fragment 1-linker peptide-protein fragment 2" is (GGGGSGGGS).₃"protein fragment 2-linkThe "linker peptide" in linker-protein fragment 3 "is also (GGGGSGGGS)₃(ii) a Or may be different, for example, the "linker peptide" of "protein fragment 1-linker peptide-protein fragment 2" is (GGGGSGGGS)₄"linker peptide" in "protein fragment 2-linker peptide-protein fragment 3" is also (GGGGSGGGS)₂。

In some embodiments, if the structure of antigen M2-3E-GS:

the structure of protein fragment 1-linker peptide-protein fragment 2 exists, and the linker peptide is (GGGGSGGGS)_nWherein n is preferably 4 or 5.

The structure of protein fragment 2-linker peptide-protein fragment 3 is present, then the linker peptide is (GGGGSGGGS)_nWherein n is preferably 2, 3 or 4.

The structure of protein fragment 1-linker peptide-protein fragment 3 exists, and the linker peptide is (GGGGSGGGS)_nWherein n is preferably 1 or 4.

In a preferred embodiment, M2-3E-GS is linked by either:

In a more preferred embodiment, the amino acid sequence of M2-3E-GS is any one of SEQ ID No.4-SEQ ID No. 7.

QVVQFKLSDIGEGIREVTVKEWYVKEGDTVSQFDSICEVQSDKASVTITSRYDGVIKKLYYNLDDIAYVGKPLVDIETEALKDSEEDVVETPAVSHDEHTHQEIKGRKTLATPAVRRGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSKNYTLDSSAAPTPQAAPAPTPAATASPPTPSAQAPGSSYPPHMQVLLPALSPTMTMGTVQRWEKKVGEKLSEGDLLAEIETDKATIGFEVQEEGYLAKILVPEGTRDVPLGTPLCIIGGGGSGGGSKDDLVTVKTPAFAESVTEGDVRWEKAVGDTVAEDEVVCEIETDKTSVQVPSPANGVIEALLVPDGGKVEGGTPLFTLRKTG(SEQ ID NO.4)。

QVVQFKLSDIGEGIREVTVKEWYVKEGDTVSQFDSICEVQSDKASVTITSRYDGVIKKLYYNLDDIAYVGKPLVDIETEALKDSEEDVVETPAVSHDEHTHQEIKGRKTLATPAVRRGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSKDDLVTVKTPAFAESVTEGDVRWEKAVGDTVAEDEVVCEIETDKTSVQVPSPANGVIEALLVPDGGKVEGGTPLFTLRKTGGGGGSGGGSKNYTLDSSAAPTPQAAPAPTPAATASPPTPSAQAPGSSYPPHMQVLLPALSPTMTMGTVQRWEKKVGEKLSEGDLLAEIETDKATIGFEVQEEGYLAKILVPEGTRDVPLGTPLCII(SEQ ID NO.5)。

KDDLVTVKTPAFAESVTEGDVRWEKAVGDTVAEDEVVCEIETDKTSVQVPSPANGVIEALLVPDGGKVEGGTPLFTLRKTGGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSQVVQFKLSDIGEGIREVTVKEWYVKEGDTVSQFDSICEVQSDKASVTITSRYDGVIKKLYYNLDDIAYVGKPLVDIETEALKDSEEDVVETPAVSHDEHTHQEIKGRKTLATPAVRRGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSKNYTLDSSAAPTPQAAPAPTPAATASPPTPSAQAPGSSYPPHMQVLLPALSPTMTMGTVQRWEKKVGEKLSEGDLLAEIETDKATIGFEVQEEGYLAKILVPEGTRDVPLGTPLCII(SEQ ID NO.6)。

KDDLVTVKTPAFAESVTEGDVRWEKAVGDTVAEDEVVCEIETDKTSVQVPSPANGVIEALLVPDGGKVEGGTPLFTLRKTGGGGGSGGGSKNYTLDSSAAPTPQAAPAPTPAATASPPTPSAQAPGSSYPPHMQVLLPALSPTMTMGTVQRWEKKVGEKLSEGDLLAEIETDKATIGFEVQEEGYLAKILVPEGTRDVPLGTPLCIIGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSQVVQFKLSDIGEGIREVTVKEWYVKEGDTVSQFDSICEVQSDKASVTITSRYDGVIKKLYYNLDDIAYVGKPLVDIETEALKDSEEDVVETPAVSHDEHTHQEIKGRKTLATPAVRR(SEQ ID NO.7)。

The specificity and sensitivity of M2-3E-GS in the preferred embodiment are further improved.

The invention also provides biological materials related to the antigen M2-3E-GS, which comprise a nucleic acid molecule for coding the antigen M2-3E-GS, an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant eukaryotic cell or a recombinant prokaryotic cell. Among them, the nucleic acid molecule is preferably any one of SEQ ID NO.8-SEQ ID NO.11, or a nucleic acid molecule which has more than 90% homology with the nucleic acid molecule and encodes the same functional protein.

CAGGTTGTTCAGTTCAAGCTCTCAGACATTGGAGAAGGGATTAGAGAAGTAACTGTTAAAGAATGGTATGTAAAAGAAGGAGATACAGTGTCTCAGTTTGATAGCATCTGTGAAGTTCAAAGTGATAAAGCTTCTGTTACCATCACTAGTCGTTATGATGGAGTCATTAAAAAACTCTATTATAATCTAGACGATATTGCCTATGTGGGGAAGCCATTAGTAGACATAGAAACGGAAGCTTTAAAAGATTCAGAAGAAGATGTTGTTGAAACTCCTGCAGTGTCTCATGATGAACATACACACCAAGAGATAAAGGGCCGAAAAACACTGGCAACTCCTGCAGTTCGTCGCGGGTGGTGGTGGTTCCGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTTCCAAAAATTATACACTGGATTCCTCAGCAGCACCTACCCCACAAGCGGCCCCAGCACCAACCCCTGCTGCCACTGCTTCGCCACCTACACCTTCTGCTCAGGCTCCTGGTAGCTCATATCCCCCTCACATGCAGGTACTTCTTCCTGCCCTCTCTCCCACCATGACCATGGGCACAGTTCAGAGATGGGAAAAAAAAGTGGGTGAGAAGCTAAGTGAAGGAGACTTACTGGCAGAGATAGAAACTGACAAAGCCACTATAGGTTTTGAAGTACAGGAAGAAGGTTATCTGGCAAAAATCCTGGTCCCTGAAGGCACAAGAGATGTCCCTCTAGGAACCCCACTCTGTATCATTGTAGAAAAAGAGGCAGATATATCAGCATTTGCTGACTATAGGCCAACCGAAGTAACAGATTTAGGTGGTGGTGGTTCCGGTGGTGGTTCCAATGATGTGATTACAGTCCAGACCCCAGCGTTTGCAGAGTCTGTCACAGAGGGAGATGTCAGGTGGGAGAAAGCTGTTGGAGATGCAGTTGCAGAAGATGAAGTGGTGTGTGAGATTGAGACAGACAAGACTTCTGTGCAGGTTCCATCACCAGCAAATGGCATCATTGAAGCTCTTTTGGTACCCGATGGGGGCAAAGTTGAAGGAGGAACTCCTCTATTCACACTCAGGAAAACCGGTGCTGAG(SEQIDNO.8)。

CAGGTTGTTCAGTTCAAGCTCTCAGACATTGGAGAAGGGATTAGAGAAGTAACTGTTAAAGAATGGTATGTAAAAGAAGGAGATACAGTGTCTCAGTTTGATAGCATCTGTGAAGTTCAAAGTGATAAAGCTTCTGTTACCATCACTAGTCGTTATGATGGAGTCATTAAAAAACTCTATTATAATCTAGACGATATTGCCTATGTGGGGAAGCCATTAGTAGACATAGAAACGGAAGCTTTAAAAGATTCAGAAGAAGATGTTGTTGAAACTCCTGCAGTGTCTCATGATGAACATACACACCAAGAGATAAAGGGCCGAAAAACACTGGCAACTCCTGCAGTTCGTCGCGGGTGGTGGTGGTTCCGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTTCCAATGATGTGATTACAGTCCAGACCCCAGCGTTTGCAGAGTCTGTCACAGAGGGAGATGTCAGGTGGGAGAAAGCTGTTGGAGATGCAGTTGCAGAAGATGAAGTGGTGTGTGAGATTGAGACAGACAAGACTTCTGTGCAGGTTCCATCACCAGCAAATGGCATCATTGAAGCTCTTTTGGTACCCGATGGGGGCAAAGTTGAAGGAGGAACTCCTCTATTCACACTCAGGAAAACCGGTGCTGAGGGTGGTGGTGGTTCCGGTGGTGGTTCCAAAAATTATACACTGGATTCCTCAGCAGCACCTACCCCACAAGCGGCCCCAGCACCAACCCCTGCTGCCACTGCTTCGCCACCTACACCTTCTGCTCAGGCTCCTGGTAGCTCATATCCCCCTCACATGCAGGTACTTCTTCCTGCCCTCTCTCCCACCATGACCATGGGCACAGTTCAGAGATGGGAAAAAAAAGTGGGTGAGAAGCTAAGTGAAGGAGACTTACTGGCAGAGATAGAAACTGACAAAGCCACTATAGGTTTTGAAGTACAGGAAGAAGGTTATCTGGCAAAAATCCTGGTCCCTGAAGGCACAAGAGATGTCCCTCTAGGAACCCCACTCTGTATCATTGTAGAAAAAGAGGCAGATATATCAGCATTTGCTGACTATAGGCCAACCGAAGTAACAGATTTA(SEQIDNO.9)。

AATGATGTGATTACAGTCCAGACCCCAGCGTTTGCAGAGTCTGTCACAGAGGGAGATGTCAGGTGGGAGAAAGCTGTTGGAGATGCAGTTGCAGAAGATGAAGTGGTGTGTGAGATTGAGACAGACAAGACTTCTGTGCAGGTTCCATCACCAGCAAATGGCATCATTGAAGCTCTTTTGGTACCCGATGGGGGCAAAGTTGAAGGAGGAACTCCTCTATTCACACTCAGGAAAACCGGTGCTGAGGGTGGTGGTGGTTCCGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTTCCCAGGTTGTTCAGTTCAAGCTCTCAGACATTGGAGAAGGGATTAGAGAAGTAACTGTTAAAGAATGGTATGTAAAAGAAGGAGATACAGTGTCTCAGTTTGATAGCATCTGTGAAGTTCAAAGTGATAAAGCTTCTGTTACCATCACTAGTCGTTATGATGGAGTCATTAAAAAACTCTATTATAATCTAGACGATATTGCCTATGTGGGGAAGCCATTAGTAGACATAGAAACGGAAGCTTTAAAAGATTCAGAAGAAGATGTTGTTGAAACTCCTGCAGTGTCTCATGATGAACATACACACCAAGAGATAAAGGGCCGAAAAACACTGGCAACTCCTGCAGTTCGTCGCGGGTGGTGGTGGTTCCGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTTCCAAAAATTATACACTGGATTCCTCAGCAGCACCTACCCCACAAGCGGCCCCAGCACCAACCCCTGCTGCCACTGCTTCGCCACCTACACCTTCTGCTCAGGCTCCTGGTAGCTCATATCCCCCTCACATGCAGGTACTTCTTCCTGCCCTCTCTCCCACCATGACCATGGGCACAGTTCAGAGATGGGAAAAAAAAGTGGGTGAGAAGCTAAGTGAAGGAGACTTACTGGCAGAGATAGAAACTGACAAAGCCACTATAGGTTTTGAAGTACAGGAAGAAGGTTATCTGGCAAAAATCCTGGTCCCTGAAGGCACAAGAGATGTCCCTCTAGGAACCCCACTCTGTATCATTGTAGAAAAAGAGGCAGATATATCAGCATTTGCTGACTATAGGCCAACCGAAGTAACAGATTTA(SEQIDNO.10)。

AATGATGTGATTACAGTCCAGACCCCAGCGTTTGCAGAGTCTGTCACAGAGGGAGATGTCAGGTGGGAGAAAGCTGTTGGAGATGCAGTTGCAGAAGATGAAGTGGTGTGTGAGATTGAGACAGACAAGACTTCTGTGCAGGTTCCATCACCAGCAAATGGCATCATTGAAGCTCTTTTGGTACCCGATGGGGGCAAAGTTGAAGGAGGAACTCCTCTATTCACACTCAGGAAAACCGGTGCTGAGGGTGGTGGTGGTTCCGGTGGTGGTTCCAAAAATTATACACTGGATTCCTCAGCAGCACCTACCCCACAAGCGGCCCCAGCACCAACCCCTGCTGCCACTGCTTCGCCACCTACACCTTCTGCTCAGGCTCCTGGTAGCTCATATCCCCCTCACATGCAGGTACTTCTTCCTGCCCTCTCTCCCACCATGACCATGGGCACAGTTCAGAGATGGGAAAAAAAAGTGGGTGAGAAGCTAAGTGAAGGAGACTTACTGGCAGAGATAGAAACTGACAAAGCCACTATAGGTTTTGAAGTACAGGAAGAAGGTTATCTGGCAAAAATCCTGGTCCCTGAAGGCACAAGAGATGTCCCTCTAGGAACCCCACTCTGTATCATTGTAGAAAAAGAGGCAGATATATCAGCATTTGCTGACTATAGGCCAACCGAAGTAACAGATTTAGGTGGTGGTGGTTCCGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTTCCCAGGTTGTTCAGTTCAAGCTCTCAGACATTGGAGAAGGGATTAGAGAAGTAACTGTTAAAGAATGGTATGTAAAAGAAGGAGATACAGTGTCTCAGTTTGATAGCATCTGTGAAGTTCAAAGTGATAAAGCTTCTGTTACCATCACTAGTCGTTATGATGGAGTCATTAAAAAACTCTATTATAATCTAGACGATATTGCCTATGTGGGGAAGCCATTAGTAGACATAGAAACGGAAGCTTTAAAAGATTCAGAAGAAGATGTTGTTGAAACTCCTGCAGTGTCTCATGATGAACATACACACCAAGAGATAAAGGGCCGAAAAACACTGGCAACTCCTGCAGTTCGTCGCG(SEQIDNO.11)。

The invention also provides a preparation method of the antigen M2-3E-GS, wherein the antigen M2-3E-GS is obtained by introducing a nucleic acid molecule encoding the antigen M2-3E-GS into a host to obtain a recombinant expression system, and the antigen M2-3E-GS is obtained by expression of the recombinant expression system. Among them, the expression vector of the nucleic acid molecule is preferably pET28a vector, and the host is preferably Escherichia coli.

The invention also protects the application of the antigen M2-3E-GS or biological material in the preparation of PBC diagnosis or AMA-M2 detection products. Specifically, the product for diagnosing PBC adopts the antigen M2-3E-GS as a detection antigen. The detection principle of the product can be enzyme-linked immunosorbent assay (ELISA), Linear Immunoblotting (LIA), chemiluminescence immunoassay (CLIA), Immunofluorescence (IF) and the like.

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.

CAGGTTGTTCAGTTCAAGCTCTCAGACATTGGAGAAGGGATTAGAGAAGTAACTGTTAAAGAATGGTATGTAAAAGAAGGAGATACAGTGTCTCAGTTTGATAGCATCTGTGAAGTTCAAAGTGATAAAGCTTCTGTTACCATCACTAGTCGTTATGATGGAGTCATTAAAAAACTCTATTATAATCTAGACGATATTGCCTATGTGGGGAAGCCATTAGTAGACATAGAAACGGAAGCTTTAAAAGATTCAGAAGAAGATGTTGTTGAAACTCCTGCAGTGTCTCATGATGAACATACACACCAAGAGATAAAGGGCCGAAAAACACTGGCAACTCCTGCAGTTCGTCGCG(SEQ ID NO.12)。

AAAAATTATACACTGGATTCCTCAGCAGCACCTACCCCACAAGCGGCCCCAGCACCAACCCCTGCTGCCACTGCTTCGCCACCTACACCTTCTGCTCAGGCTCCTGGTAGCTCATATCCCCCTCACATGCAGGTACTTCTTCCTGCCCTCTCTCCCACCATGACCATGGGCACAGTTCAGAGATGGGAAAAAAAAGTGGGTGAGAAGCTAAGTGAAGGAGACTTACTGGCAGAGATAGAAACTGACAAAGCCACTATAGGTTTTGAAGTACAGGAAGAAGGTTATCTGGCAAAAATCCTGGTCCCTGAAGGCACAAGAGATGTCCCTCTAGGAACCCCACTCTGTATCATTGTAGAAAAAGAGGCAGATATATCAGCATTTGCTGACTATAGGCCAACCGAAGTAACAGATTTA(SEQ ID NO.13)。

AATGATGTGATTACAGTCCAGACCCCAGCGTTTGCAGAGTCTGTCACAGAGGGAGATGTCAGGTGGGAGAAAGCTGTTGGAGATGCAGTTGCAGAAGATGAAGTGGTGTGTGAGATTGAGACAGACAAGACTTCTGTGCAGGTTCCATCACCAGCAAATGGCATCATTGAAGCTCTTTTGGTACCCGATGGGGGCAAAGTTGAAGGAGGAACTCCTCTATTCACACTCAGGAAAACCGGTGCTGAG(SEQ ID NO.14)。

Example 1

Step 1: synthesis of target Gene and construction of recombinant vector of the invention

By adopting a gene synthesis method, the SEQ ID NO.12(BCOADC-E2 protein, B for short) coding the SEQ ID NO.1, the SEQ ID NO.13(PDC-E2 protein, P for short) coding the SEQ ID NO.2 and the SEQ ID NO.14(OGDC-E2 protein, O for short) coding the SEQ ID NO.3 are connected to a pET28a vector through connecting peptides according to a structure shown in the following table 1 to construct a recombinant vector.

TABLE 1

B0P	SEQ ID NO.12—SEQ ID NO.13
		B1P	SEQ ID NO.12—GGGGSGGGSGGGGSGGGS—SEQ ID NO.13
B2P	SEQ ID NO.12—(GGGGSGGGSGGGGSGGGS)₂—SEQ ID NO.13
		B3P	SEQ ID NO.12—(GGGGSGGGSGGGGSGGGS)₃—SEQ ID NO.13
B4P	SEQ ID NO.12—(GGGGSGGGSGGGGSGGGS)₄—SEQ ID NO.13
		B5P	SEQ ID NO.12—(GGGGSGGGSGGGGSGGGS)₅—SEQ ID NO.13
P0O	SEQ ID NO.13—SEQ ID NO.14
		P1O	SEQ ID NO.13—GGGGSGGGSGGGGSGGGS—SEQ ID NO.14
P2O	SEQ ID NO.13—(GGGGSGGGSGGGGSGGGS)₂—SEQ ID NO.14
		P3O	SEQ ID NO.13—(GGGGSGGGSGGGGSGGGS)₃—SEQ ID NO.14
P4O	SEQ ID NO.13—(GGGGSGGGSGGGGSGGGS)₄—SEQ ID NO.14
		P5O	SEQ ID NO.13—(GGGGSGGGSGGGGSGGGS)₅—SEQ ID NO.14
B0O	SEQ ID NO.12—SEQ ID NO.14
		B1O	SEQ ID NO.12—GGGGSGGGSGGGGSGGGS—SEQ ID NO.14
B2O	SEQ ID NO.12—(GGGGSGGGSGGGGSGGGS)₂—SEQ ID NO.14
		B3O	SEQ ID NO.12—(GGGGSGGGSGGGGSGGGS)₃—SEQ ID NO.14
B4O	SEQ ID NO.12—(GGGGSGGGSGGGGSGGGS)₄—SEQ ID NO.14
		B5O	SEQ ID NO.12—(GGGGSGGGSGGGGSGGGS)₅—SEQ ID NO.14

Step 2: induced expression purification and application of antigen

1. Transferring the recombinant vector in the step 1 into escherichia coli E.coli Rosetta (DE3) competent cells, culturing on an LB culture plate containing 50 ug/ml kanamycin and 50 ug/ml chloramphenicol, and culturing at 37 ℃ for 14-16 h; positive recombinant bacteria were selected, and a single colony was inoculated into 5ml of LB medium containing 50. mu.g/ml kanamycin and 50. mu.g/ml chloramphenicol, and cultured overnight.

2. The overnight-cultured bacterial solution was inoculated at 1% inoculum size into LB medium containing 50. mu.g/ml kanamycin and 50. mu.g/ml chloramphenicol, cultured at 37 ℃ and 250rpm until the bacterial solution OD600 became 1.0-1.3, and then induced with IPTG at a final concentration of 0.5mM-1.0mM at 25 ℃ and 250rpm for 3-4 hours.

3. Coli e.coli Rosetta cells obtained in 2 were collected by centrifugation, and 20ml of lysine buffer (ph7.5) was added to ice to resuspend the solid until no clumpy cells were visible to the naked eye.

4. Ultrasonic crushing: and (4) breaking the bacteria according to the conditions of ultrasonic power of 400W, ultrasonic for 4s and stopping for 3s and circulation for 99 times until the bacteria liquid is clear and transparent. After the sonication was completed, the supernatant and the precipitate were separated by centrifugation at 12000rpm for 30min at 4 ℃.

5. The supernatant separated in step 4 was added with balanced 1ml of Ni resin and placed in an ice box and incubated with shaking for 1 hour.

6. Pouring the incubated mixture into a gravity column, collecting the flow-through liquid by using a clean EP tube, and adding 15mL of lysine buffer to wash out the residual flow-through liquid in the pipeline after the flow-through liquid completely flows out.

7. And (3) elution: the Ni column was eluted with 50mM, 100mM and 500mM imidazole, and the eluates were collected by peak separation tube and 20. mu.l was left.

8. The results of SDS-PAGE detection of the samples retained in the above steps are shown in FIGS. 1 to 3, in which M: protein marker; 2: 500mM imidazole eluent. The obtained protein is named as BnP, PnO, BnO and the like, wherein n is 0, 1, 2, 3, 4, 5, the result of B4P is shown in figure 1, the result of P1O is shown in figure 2, and the result of B4O is shown in figure 3.

Test example 1: liquid phase chip coating and reactivity test

Each of the proteins prepared in example 1 was coated on a solid support.

Step 1, preparation of activation buffer solution

The numbering reagent name concentrations are as in table 2 below:

TABLE 2

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

The numbering reagent name concentrations are as in table 3 below:

TABLE 3

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), oscillating at room temperature for reaction for 50mim, 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.

Step 4, fluorescent marker

Phycoerythrin-labeled goat anti-human IgG-PE: the concentration is 0.5mg/ml, and the solution is diluted to the working concentration of 0.5ug/ml by using the diluent of the coating solution.

Step 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.

Proteins prepared according to the method of test example 1 from B0P, B1P, B2P, B3P, B4P, B5P, P0O, P1O, P2O, P3O, P4O, P5O, B0O, B1O, B2O, B3O, B4O and B5O were coated on a liquid phase chip, and specific antibodies in sample serum were detected and compared for specificity, and the experimental results are shown in the following tables 4-6:

TABLE 4

Envelope antigens	Type of sample	(number of positive test/total number of test samples)	Specificity (%)
				B0P	Positive sample	15/54	27.8
B1P	Positive sample	17/54	31.5
				B2P	Positive sample	17/54	31.5
B3P	Positive sample	16/54	29.6
				B4P	Positive sample	21/54	38.9
B5P	Negative sample	19/54	35.2

The optimal n-4 is chosen, i.e. the number of linkers between B and P is 4.

TABLE 5

The optimal n is 1, i.e. the number of linkers between P and O is 1.

TABLE 6

Envelope antigens	Type of sample	(number of positive test/total number of test samples)	Specificity (%)
				B0O	Positive sample	12/54	22.2
B1O	Positive sample	12/54	22.2
				B2O	Positive sample	15/54	27.8
B3O	Positive sample	15/54	27.8
				B4O	Positive sample	16/54	29.6
B5O	Negative sample	14/54	25.9

The optimum n-4 is chosen, i.e. the number of linkers between B and O is 4.

Test example 2: B. p, O sequence order

B, P, O were concatenated together by a linker peptide, the order was interchangeable, but if B was linked to O, the number of linkers was 4, and if P was linked to O, the number of linkers was 1, and if B was linked to P, the number of linkers was 4, designated B4P1O, B4O1P, O4B4P, O1P4B, P1O4B and P4B4O, respectively, and M2-3E-GS was prepared according to the method of example 1, wherein the results for the B4P1O protein are shown in FIG. 4.

According to the method of test example 1, each M2-3E-GS was subjected to liquid phase chip coating and performance test, and specific antibodies in sample serum were detected to compare sensitivity and specificity, and the experimental results are shown in Table 7 below:

TABLE 7 analysis of sensitivity and specificity of M2-3E-GS test results

As a result, the specificity and sensitivity of B4P1O to sample detection were high, and B4P1O was abbreviated as M2-3E-GS (B4P 1O).

Test example 3 comparative test for sensitivity and specificity

Liquid-phase chip coating and performance tests were carried out on M2-3E-GS (B4P1O), M2-3E (BPO), M2-3E + PDH, and the specificity and sensitivity of the antibody in the sample serum were measured according to the method of test example 1, in which the control kit: IgG anti-M2-3E antibody detection kit (enzyme-linked immunosorbent assay) (EA 1622-9601G: 96 parts/cassette, EUROIMMUN, above commercial products including M2-3E and PDH samples). The results are shown in table 8 below:

TABLE 8 PBC antigen and antigen immune serological test results

The specificity and sensitivity of M2-3E-GS (B4P1O) prepared by the scheme are respectively improved by 32.4 percent and 5.2 percent compared with M2-3E and are improved by 6.5 percent and 17.5 percent compared with M2-3E + PDH. In terms of nonspecific and false positive probability, M2-3E-GS (B4P1O) prepared by the method is reduced from 31.5% to 9.7% in terms of nonspecific and from 9.5% to 4.8% in terms of false positive probability compared with M2-3, is reduced from 15.8% to 9.7% in terms of nonspecific and from 19.0% to 4.8% in terms of false positive probability compared with M2-3E + PDH.

Test example 4: precision survey

Liquid phase chip coating and performance testing were performed on M2-3E-GS (B4P1O), M2-3E (BPO), M2-3E + PDH, and the antibody in the sample serum was examined to calculate the coefficient of variation, i.e., precision, according to the method of test example 1, in which the control kit: IgG anti-M2-3E antibody detection kit (enzyme-linked immunosorbent assay) (EA 1622-9601G: 96 parts/cassette, EUROIMMUN, above commercial products including M2-3E and PDH samples). The results are shown in table 9 below:

TABLE 9 precision investigation

The same sample is tested for 10 times, the detection result of the coating of the liquid phase chip coated with the M2-3E-GS (B4P1O) antigen has the variation coefficient (namely the precision) within 4 percent, the precision of the M2-3E-GS (B4P1O) prepared by the method is improved by 39.6 percent compared with that of M2-3E and is improved by 65.7 percent compared with that of M2-3E + PDH, and the test repeatability of the M2-3E-GS (B4P1O) prepared by the method is better.

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

<110> Zhuhaili bead reagent GmbH

<120> antigen M2-3E-GS and product and application thereof

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 117

<212> PRT

<213> Artificial sequence

<400> 1

Gln Val Val Gln Phe Lys Leu Ser Asp Ile Gly Glu Gly Ile Arg Glu

1 5 10 15

Val Thr Val Lys Glu Trp Tyr Val Lys Glu Gly Asp Thr Val Ser Gln

20 25 30

Phe Asp Ser Ile Cys Glu Val Gln Ser Asp Lys Ala Ser Val Thr Ile

35 40 45

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

50 55 60

Asp Ile Ala Tyr Val Gly Lys Pro Leu Val Asp Ile Glu Thr Glu Ala

65 70 75 80

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

85 90 95

Asp Glu His Thr His Gln Glu Ile Lys Gly Arg Lys Thr Leu Ala Thr

100 105 110

Pro Ala Val Arg Arg

115

<210> 2

<211> 117

<212> PRT

<213> Artificial sequence

<400> 2

Lys Asn Tyr Thr Leu Asp Ser Ser Ala Ala Pro Thr Pro Gln Ala Ala

1 5 10 15

Pro Ala Pro Thr Pro Ala Ala Thr Ala Ser Pro Pro Thr Pro Ser Ala

20 25 30

Gln Ala Pro Gly Ser Ser Tyr Pro Pro His Met Gln Val Leu Leu Pro

35 40 45

Ala Leu Ser Pro Thr Met Thr Met Gly Thr Val Gln Arg Trp Glu Lys

50 55 60

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

65 70 75 80

Thr Asp Lys Ala Thr Ile Gly Phe Glu Val Gln Glu Glu Gly Tyr Leu

85 90 95

Ala Lys Ile Leu Val Pro Glu Gly Thr Arg Asp Val Pro Leu Gly Thr

100 105 110

Pro Leu Cys Ile Ile

115

<210> 3

<211> 81

<212> PRT

<213> Artificial sequence

<400> 3

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

1 5 10 15

Thr Glu Gly Asp Val Arg Trp Glu Lys Ala Val Gly Asp Thr Val Ala

20 25 30

Glu Asp Glu Val Val Cys Glu Ile Glu Thr Asp Lys Thr Ser Val Gln

35 40 45

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

50 55 60

Gly Gly Lys Val Glu Gly Gly Thr Pro Leu Phe Thr Leu Arg Lys Thr

65 70 75 80

Gly

<210> 4

<211> 360

<212> PRT

<213> Artificial sequence

<400> 4

Gln Val Val Gln Phe Lys Leu Ser Asp Ile Gly Glu Gly Ile Arg Glu

1 5 10 15

Val Thr Val Lys Glu Trp Tyr Val Lys Glu Gly Asp Thr Val Ser Gln

20 25 30

Phe Asp Ser Ile Cys Glu Val Gln Ser Asp Lys Ala Ser Val Thr Ile

35 40 45

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

50 55 60

Asp Ile Ala Tyr Val Gly Lys Pro Leu Val Asp Ile Glu Thr Glu Ala

65 70 75 80

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

85 90 95

Asp Glu His Thr His Gln Glu Ile Lys Gly Arg Lys Thr Leu Ala Thr

100 105 110

Pro Ala Val Arg Arg Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Ser Lys Asn Tyr Thr Leu Asp Ser

145 150 155 160

Ser Ala Ala Pro Thr Pro Gln Ala Ala Pro Ala Pro Thr Pro Ala Ala

165 170 175

Thr Ala Ser Pro Pro Thr Pro Ser Ala Gln Ala Pro Gly Ser Ser Tyr

180 185 190

Pro Pro His Met Gln Val Leu Leu Pro Ala Leu Ser Pro Thr Met Thr

195 200 205

Met Gly Thr Val Gln Arg Trp Glu Lys Lys Val Gly Glu Lys Leu Ser

210 215 220

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

225 230 235 240

Phe Glu Val Gln Glu Glu Gly Tyr Leu Ala Lys Ile Leu Val Pro Glu

245 250 255

Gly Thr Arg Asp Val Pro Leu Gly Thr Pro Leu Cys Ile Ile Gly Gly

260 265 270

Gly Gly Ser Gly Gly Gly Ser Lys Asp Asp Leu Val Thr Val Lys Thr

275 280 285

Pro Ala Phe Ala Glu Ser Val Thr Glu Gly Asp Val Arg Trp Glu Lys

290 295 300

Ala Val Gly Asp Thr Val Ala Glu Asp Glu Val Val Cys Glu Ile Glu

305 310 315 320

Thr Asp Lys Thr Ser Val Gln Val Pro Ser Pro Ala Asn Gly Val Ile

325 330 335

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

340 345 350

Leu Phe Thr Leu Arg Lys Thr Gly

355 360

<210> 5

<211> 360

<212> PRT

<213> Artificial sequence

<400> 5

Gln Val Val Gln Phe Lys Leu Ser Asp Ile Gly Glu Gly Ile Arg Glu

1 5 10 15

Val Thr Val Lys Glu Trp Tyr Val Lys Glu Gly Asp Thr Val Ser Gln

20 25 30

Phe Asp Ser Ile Cys Glu Val Gln Ser Asp Lys Ala Ser Val Thr Ile

35 40 45

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

50 55 60

Asp Ile Ala Tyr Val Gly Lys Pro Leu Val Asp Ile Glu Thr Glu Ala

65 70 75 80

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

85 90 95

Asp Glu His Thr His Gln Glu Ile Lys Gly Arg Lys Thr Leu Ala Thr

100 105 110

Pro Ala Val Arg Arg Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Ser Lys Asp Asp Leu Val Thr Val

145 150 155 160

Lys Thr Pro Ala Phe Ala Glu Ser Val Thr Glu Gly Asp Val Arg Trp

165 170 175

Glu Lys Ala Val Gly Asp Thr Val Ala Glu Asp Glu Val Val Cys Glu

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Gly Ser Lys Asn Tyr Thr Leu Asp Ser Ser Ala Ala Pro Thr Pro

245 250 255

Gln Ala Ala Pro Ala Pro Thr Pro Ala Ala Thr Ala Ser Pro Pro Thr

260 265 270

Pro Ser Ala Gln Ala Pro Gly Ser Ser Tyr Pro Pro His Met Gln Val

275 280 285

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

290 295 300

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

305 310 315 320

Glu Ile Glu Thr Asp Lys Ala Thr Ile Gly Phe Glu Val Gln Glu Glu

325 330 335

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

340 345 350

Leu Gly Thr Pro Leu Cys Ile Ile

355 360

<210> 6

<211> 387

<212> PRT

<213> Artificial sequence

<400> 6

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

1 5 10 15

Thr Glu Gly Asp Val Arg Trp Glu Lys Ala Val Gly Asp Thr Val Ala

20 25 30

Glu Asp Glu Val Val Cys Glu Ile Glu Thr Asp Lys Thr Ser Val Gln

35 40 45

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

50 55 60

Gly Gly Lys Val Glu Gly Gly Thr Pro Leu Phe Thr Leu Arg Lys Thr

65 70 75 80

Gly Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

85 90 95

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Ser Gln Val Val Gln Phe Lys Leu Ser Asp Ile Gly

115 120 125

Glu Gly Ile Arg Glu Val Thr Val Lys Glu Trp Tyr Val Lys Glu Gly

130 135 140

Asp Thr Val Ser Gln Phe Asp Ser Ile Cys Glu Val Gln Ser Asp Lys

145 150 155 160

Ala Ser Val Thr Ile Thr Ser Arg Tyr Asp Gly Val Ile Lys Lys Leu

165 170 175

Tyr Tyr Asn Leu Asp Asp Ile Ala Tyr Val Gly Lys Pro Leu Val Asp

180 185 190

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

195 200 205

Pro Ala Val Ser His Asp Glu His Thr His Gln Glu Ile Lys Gly Arg

210 215 220

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

225 230 235 240

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly

245 250 255

Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Lys Asn

260 265 270

Tyr Thr Leu Asp Ser Ser Ala Ala Pro Thr Pro Gln Ala Ala Pro Ala

275 280 285

Pro Thr Pro Ala Ala Thr Ala Ser Pro Pro Thr Pro Ser Ala Gln Ala

290 295 300

Pro Gly Ser Ser Tyr Pro Pro His Met Gln Val Leu Leu Pro Ala Leu

305 310 315 320

Ser Pro Thr Met Thr Met Gly Thr Val Gln Arg Trp Glu Lys Lys Val

325 330 335

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

340 345 350

Lys Ala Thr Ile Gly Phe Glu Val Gln Glu Glu Gly Tyr Leu Ala Lys

355 360 365

Ile Leu Val Pro Glu Gly Thr Arg Asp Val Pro Leu Gly Thr Pro Leu

370 375 380

Cys Ile Ile

385

<210> 7

<211> 360

<212> PRT

<213> Artificial sequence

<400> 7

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

1 5 10 15

Thr Glu Gly Asp Val Arg Trp Glu Lys Ala Val Gly Asp Thr Val Ala

20 25 30

Glu Asp Glu Val Val Cys Glu Ile Glu Thr Asp Lys Thr Ser Val Gln

35 40 45

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

50 55 60

Gly Gly Lys Val Glu Gly Gly Thr Pro Leu Phe Thr Leu Arg Lys Thr

65 70 75 80

Gly Gly Gly Gly Gly Ser Gly Gly Gly Ser Lys Asn Tyr Thr Leu Asp

85 90 95

Ser Ser Ala Ala Pro Thr Pro Gln Ala Ala Pro Ala Pro Thr Pro Ala

100 105 110

Ala Thr Ala Ser Pro Pro Thr Pro Ser Ala Gln Ala Pro Gly Ser Ser

115 120 125

Tyr Pro Pro His Met Gln Val Leu Leu Pro Ala Leu Ser Pro Thr Met

130 135 140

Thr Met Gly Thr Val Gln Arg Trp Glu Lys Lys Val Gly Glu Lys Leu

145 150 155 160

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

165 170 175

Gly Phe Glu Val Gln Glu Glu Gly Tyr Leu Ala Lys Ile Leu Val Pro

180 185 190

Glu Gly Thr Arg Asp Val Pro Leu Gly Thr Pro Leu Cys Ile Ile Gly

195 200 205

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

210 215 220

Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

225 230 235 240

Gly Gly Ser Gln Val Val Gln Phe Lys Leu Ser Asp Ile Gly Glu Gly

245 250 255

Ile Arg Glu Val Thr Val Lys Glu Trp Tyr Val Lys Glu Gly Asp Thr

260 265 270

Val Ser Gln Phe Asp Ser Ile Cys Glu Val Gln Ser Asp Lys Ala Ser

275 280 285

Val Thr Ile Thr Ser Arg Tyr Asp Gly Val Ile Lys Lys Leu Tyr Tyr

290 295 300

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

305 310 315 320

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

325 330 335

Val Ser His Asp Glu His Thr His Gln Glu Ile Lys Gly Arg Lys Thr

340 345 350

Leu Ala Thr Pro Ala Val Arg Arg

355 360

<210> 8

<211> 1147

<212> DNA

<213> Artificial sequence

<400> 8

caggttgttc agttcaagct ctcagacatt ggagaaggga ttagagaagt aactgttaaa 60

gaatggtatg taaaagaagg agatacagtg tctcagtttg atagcatctg tgaagttcaa 120

agtgataaag cttctgttac catcactagt cgttatgatg gagtcattaa aaaactctat 180

tataatctag acgatattgc ctatgtgggg aagccattag tagacataga aacggaagct 240

ttaaaagatt cagaagaaga tgttgttgaa actcctgcag tgtctcatga tgaacataca 300

caccaagaga taaagggccg aaaaacactg gcaactcctg cagttcgtcg cgggtggtgg 360

tggttccggt ggtggttccg gtggtggtgg ttccggtggt ggttccggtg gtggtggttc 420

cggtggtggt tccggtggtg gtggttccgg tggtggttcc aaaaattata cactggattc 480

ctcagcagca cctaccccac aagcggcccc agcaccaacc cctgctgcca ctgcttcgcc 540

acctacacct tctgctcagg ctcctggtag ctcatatccc cctcacatgc aggtacttct 600

tcctgccctc tctcccacca tgaccatggg cacagttcag agatgggaaa aaaaagtggg 660

tgagaagcta agtgaaggag acttactggc agagatagaa actgacaaag ccactatagg 720

ttttgaagta caggaagaag gttatctggc aaaaatcctg gtccctgaag gcacaagaga 780

tgtccctcta ggaaccccac tctgtatcat tgtagaaaaa gaggcagata tatcagcatt 840

tgctgactat aggccaaccg aagtaacaga tttaggtggt ggtggttccg gtggtggttc 900

caatgatgtg attacagtcc agaccccagc gtttgcagag tctgtcacag agggagatgt 960

caggtgggag aaagctgttg gagatgcagt tgcagaagat gaagtggtgt gtgagattga 1020

gacagacaag acttctgtgc aggttccatc accagcaaat ggcatcattg aagctctttt 1080

ggtacccgat gggggcaaag ttgaaggagg aactcctcta ttcacactca ggaaaaccgg 1140

tgctgag 1147

<210> 9

<211> 1147

<212> DNA

<213> Artificial sequence

<400> 9

caggttgttc agttcaagct ctcagacatt ggagaaggga ttagagaagt aactgttaaa 60

gaatggtatg taaaagaagg agatacagtg tctcagtttg atagcatctg tgaagttcaa 120

agtgataaag cttctgttac catcactagt cgttatgatg gagtcattaa aaaactctat 180

tataatctag acgatattgc ctatgtgggg aagccattag tagacataga aacggaagct 240

ttaaaagatt cagaagaaga tgttgttgaa actcctgcag tgtctcatga tgaacataca 300

caccaagaga taaagggccg aaaaacactg gcaactcctg cagttcgtcg cgggtggtgg 360

tggttccggt ggtggttccg gtggtggtgg ttccggtggt ggttccggtg gtggtggttc 420

cggtggtggt tccggtggtg gtggttccgg tggtggttcc aatgatgtga ttacagtcca 480

gaccccagcg tttgcagagt ctgtcacaga gggagatgtc aggtgggaga aagctgttgg 540

agatgcagtt gcagaagatg aagtggtgtg tgagattgag acagacaaga cttctgtgca 600

ggttccatca ccagcaaatg gcatcattga agctcttttg gtacccgatg ggggcaaagt 660

tgaaggagga actcctctat tcacactcag gaaaaccggt gctgagggtg gtggtggttc 720

cggtggtggt tccaaaaatt atacactgga ttcctcagca gcacctaccc cacaagcggc 780

cccagcacca acccctgctg ccactgcttc gccacctaca ccttctgctc aggctcctgg 840

tagctcatat ccccctcaca tgcaggtact tcttcctgcc ctctctccca ccatgaccat 900

gggcacagtt cagagatggg aaaaaaaagt gggtgagaag ctaagtgaag gagacttact 960

ggcagagata gaaactgaca aagccactat aggttttgaa gtacaggaag aaggttatct 1020

ggcaaaaatc ctggtccctg aaggcacaag agatgtccct ctaggaaccc cactctgtat 1080

cattgtagaa aaagaggcag atatatcagc atttgctgac tataggccaa ccgaagtaac 1140

agattta 1147

<210> 10

<211> 1228

<212> DNA

<213> Artificial sequence

<400> 10

aatgatgtga ttacagtcca gaccccagcg tttgcagagt ctgtcacaga gggagatgtc 60

aggtgggaga aagctgttgg agatgcagtt gcagaagatg aagtggtgtg tgagattgag 120

acagacaaga cttctgtgca ggttccatca ccagcaaatg gcatcattga agctcttttg 180

gtacccgatg ggggcaaagt tgaaggagga actcctctat tcacactcag gaaaaccggt 240

gctgagggtg gtggtggttc cggtggtggt tccggtggtg gtggttccgg tggtggttcc 300

ggtggtggtg gttccggtgg tggttccggt ggtggtggtt ccggtggtgg ttcccaggtt 360

gttcagttca agctctcaga cattggagaa gggattagag aagtaactgt taaagaatgg 420

tatgtaaaag aaggagatac agtgtctcag tttgatagca tctgtgaagt tcaaagtgat 480

aaagcttctg ttaccatcac tagtcgttat gatggagtca ttaaaaaact ctattataat 540

ctagacgata ttgcctatgt ggggaagcca ttagtagaca tagaaacgga agctttaaaa 600

gattcagaag aagatgttgt tgaaactcct gcagtgtctc atgatgaaca tacacaccaa 660

gagataaagg gccgaaaaac actggcaact cctgcagttc gtcgcgggtg gtggtggttc 720

cggtggtggt tccggtggtg gtggttccgg tggtggttcc ggtggtggtg gttccggtgg 780

tggttccggt ggtggtggtt ccggtggtgg ttccaaaaat tatacactgg attcctcagc 840

agcacctacc ccacaagcgg ccccagcacc aacccctgct gccactgctt cgccacctac 900

accttctgct caggctcctg gtagctcata tccccctcac atgcaggtac ttcttcctgc 960

cctctctccc accatgacca tgggcacagt tcagagatgg gaaaaaaaag tgggtgagaa 1020

gctaagtgaa ggagacttac tggcagagat agaaactgac aaagccacta taggttttga 1080

agtacaggaa gaaggttatc tggcaaaaat cctggtccct gaaggcacaa gagatgtccc 1140

tctaggaacc ccactctgta tcattgtaga aaaagaggca gatatatcag catttgctga 1200

ctataggcca accgaagtaa cagattta 1228

<210> 11

<211> 1147

<212> DNA

<213> Artificial sequence

<400> 11

aatgatgtga ttacagtcca gaccccagcg tttgcagagt ctgtcacaga gggagatgtc 60

aggtgggaga aagctgttgg agatgcagtt gcagaagatg aagtggtgtg tgagattgag 120

acagacaaga cttctgtgca ggttccatca ccagcaaatg gcatcattga agctcttttg 180

gtacccgatg ggggcaaagt tgaaggagga actcctctat tcacactcag gaaaaccggt 240

gctgagggtg gtggtggttc cggtggtggt tccaaaaatt atacactgga ttcctcagca 300

gcacctaccc cacaagcggc cccagcacca acccctgctg ccactgcttc gccacctaca 360

ccttctgctc aggctcctgg tagctcatat ccccctcaca tgcaggtact tcttcctgcc 420

ctctctccca ccatgaccat gggcacagtt cagagatggg aaaaaaaagt gggtgagaag 480

ctaagtgaag gagacttact ggcagagata gaaactgaca aagccactat aggttttgaa 540

gtacaggaag aaggttatct ggcaaaaatc ctggtccctg aaggcacaag agatgtccct 600

ctaggaaccc cactctgtat cattgtagaa aaagaggcag atatatcagc atttgctgac 660

tataggccaa ccgaagtaac agatttaggt ggtggtggtt ccggtggtgg ttccggtggt 720

ggtggttccg gtggtggttc cggtggtggt ggttccggtg gtggttccgg tggtggtggt 780

tccggtggtg gttcccaggt tgttcagttc aagctctcag acattggaga agggattaga 840

gaagtaactg ttaaagaatg gtatgtaaaa gaaggagata cagtgtctca gtttgatagc 900

atctgtgaag ttcaaagtga taaagcttct gttaccatca ctagtcgtta tgatggagtc 960

attaaaaaac tctattataa tctagacgat attgcctatg tggggaagcc attagtagac 1020

atagaaacgg aagctttaaa agattcagaa gaagatgttg ttgaaactcc tgcagtgtct 1080

catgatgaac atacacacca agagataaag ggccgaaaaa cactggcaac tcctgcagtt 1140

cgtcgcg 1147

<210> 12

<211> 352

<212> DNA

<213> Artificial sequence

<400> 12

caggttgttc agttcaagct ctcagacatt ggagaaggga ttagagaagt aactgttaaa 60

gaatggtatg taaaagaagg agatacagtg tctcagtttg atagcatctg tgaagttcaa 120

agtgataaag cttctgttac catcactagt cgttatgatg gagtcattaa aaaactctat 180

tataatctag acgatattgc ctatgtgggg aagccattag tagacataga aacggaagct 240

ttaaaagatt cagaagaaga tgttgttgaa actcctgcag tgtctcatga tgaacataca 300

caccaagaga taaagggccg aaaaacactg gcaactcctg cagttcgtcg cg 352

<210> 13

<211> 414

<212> DNA

<213> Artificial sequence

<400> 13

aaaaattata cactggattc ctcagcagca cctaccccac aagcggcccc agcaccaacc 60

cctgctgcca ctgcttcgcc acctacacct tctgctcagg ctcctggtag ctcatatccc 120

cctcacatgc aggtacttct tcctgccctc tctcccacca tgaccatggg cacagttcag 180

agatgggaaa aaaaagtggg tgagaagcta agtgaaggag acttactggc agagatagaa 240

actgacaaag ccactatagg ttttgaagta caggaagaag gttatctggc aaaaatcctg 300

gtccctgaag gcacaagaga tgtccctcta ggaaccccac tctgtatcat tgtagaaaaa 360

gaggcagata tatcagcatt tgctgactat aggccaaccg aagtaacaga ttta 414

<210> 14

<211> 246

<212> DNA

<213> Artificial sequence

<400> 14

aatgatgtga ttacagtcca gaccccagcg tttgcagagt ctgtcacaga gggagatgtc 60

aggtgggaga aagctgttgg agatgcagtt gcagaagatg aagtggtgtg tgagattgag 120

acagacaaga cttctgtgca ggttccatca ccagcaaatg gcatcattga agctcttttg 180

gtacccgatg ggggcaaagt tgaaggagga actcctctat tcacactcag gaaaaccggt 240

gctgag 246

Claims

1. An antigen M2-3E-GS, wherein the amino acid sequence of M2-3E-GS is SEQ ID NO. 4.

2. The biological material related to the antigen M2-3E-GS of claim 1, wherein the biological material is any one of the following:

(a) a nucleic acid molecule encoding the antigen M2-3E-GS of claim 1;

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

3. The method for producing the antigen M2-3E-GS according to claim 1, wherein the antigen M2-3E-GS is obtained by introducing a nucleic acid molecule encoding the antigen M2-3E-GS according to claim 1 into a host to obtain a recombinant expression system and expressing the recombinant expression system.

4. The method for preparing the recombinant plasmid of claim 3, wherein the antigen M2-3E-GS is obtained by transfecting E.coli with a recombinant vector constructed by combining a nucleic acid molecule encoding the antigen M2-3E-GS with pET28a vector.

5. Use of the antigen M2-3E-GS of claim 1 or the biomaterial of claim 2 for the preparation of a diagnostic PBC or for the detection of AMA-M2 products.

6. A product for diagnosing PBC, which employs the antigen M2-3E-GS of claim 1 as a detection antigen.