CN113480646B

CN113480646B - Preparation and application of monoclonal antibody specifically binding H1.4 frameshift mutant protein

Info

Publication number: CN113480646B
Application number: CN202110754911.3A
Authority: CN
Inventors: 李国红; 俞洋; 包新华; 胡明丽; 王胜; 章清萍; 阮海荷
Original assignee: Institute of Biophysics of CAS; Peking University First Hospital
Current assignee: Institute of Biophysics of CAS; Peking University First Hospital
Priority date: 2021-07-01
Filing date: 2021-07-01
Publication date: 2022-03-15
Anticipated expiration: 2041-07-01
Also published as: CN113480646A

Abstract

The invention discloses preparation and application of a monoclonal antibody specifically bound with H1.4 frameshift mutant protein. Specifically disclosed is a detection antibody or antigen-binding portion thereof that specifically binds to a H1.4 frameshift mutein, comprising a heavy chain having the amino acid sequence SEQ ID No.1 and a light chain having the amino acid sequence SEQ ID No. 2. The invention prepares the mouse monoclonal antibody aiming at the H1.4 frameshift mutant protein by utilizing the H1.4 frameshift mutant polypeptide hapten for the first time, and the prepared antibody can be specifically combined with the H1.4 frameshift mutant protein, can quickly, sensitively and accurately detect the H1.4 frameshift mutation, provides an essential experimental material for the research of the generation of an autism mechanism caused by the H1.4 frameshift mutation, provides a detection basis for the diagnosis of an autism patient, and has wide application prospect and important significance in the field of the diagnosis or auxiliary diagnosis of autism and the development of targeted drugs of related diseases.

Description

Preparation and application of monoclonal antibody specifically binding H1.4 frameshift mutant protein

Technical Field

The invention belongs to the technical field of biology, and particularly relates to preparation and application of a monoclonal antibody specifically bound with H1.4 frameshift mutant protein.

Background

Chromatin is a carrier of eukaryotic genetic material. Nucleosomes are the basic structural unit of chromatin, consisting of histones and DNA. Histones are the basic structural proteins of chromatin, H1, H2A, H2B, H3, H4, respectively, with H1 being the earliest discovered histone and the remaining 4 being core histones. DNA in eukaryotic cells does not exist in a naked state, but is wound on a bead structure formed by connecting nucleosomes formed outside a core structure formed by histone octamers (formed by 2 molecules of H2A, H2B, H3 and H4), and the histone H1 does not form the nucleosome but is combined at the inlet and outlet ends of the nucleosome connected with the DNA. The bead structure of the nucleosome is further folded under the action of the connecting histone H1 to form a 30nm chromatin structure, and then the chromosome is formed by gradual compression and stored in a nucleus, the H1-mediated 30nm chromatin structure is always concerned, and as a basic structural protein of the chromosome, the histone H1 plays an important role in aspects of apparent regulation, gene transcription, DNA replication, DNA damage repair, chromosome remodeling and the like.

There are multiple variants of histone H1. 11 variants have been identified in humans and mice, including seven somatic subtypes H1.0, H1.1, H1.2, H1.3, H1.4, H1.5, H1X, three testis-specific subtypes H1T, H1T2, HILS1, and one oocyte-specific subtype H1 oo. The connexin H1 and its variants exert different functions in different biological processes. In recent years, the literature reports that the mutation of the shift-frame at the carboxyl terminal of the variant H1.4 of the connecting histone H1 can cause the autism, and patients simultaneously show symptoms of high hairline, premature senility, intellectual deficit and the like, however, the pathogenic mechanism of the mutants is not clear, and no specific medicine is available for treating the autism caused by the mutation. Since H1.4 is an important chromatin structure regulatory protein, it is necessary to elucidate the possible pathogenic mechanism of H1.4 starting from the effect of H1.4 frameshift mutations on chromatin structure. However, no H1.4 frameshift mutant protein detection antibody exists in the markets at home and abroad at present, and research on H1.4 pathogenesis and diagnosis of patients are greatly limited, so that the specific detection antibody capable of identifying H1.4 frameshift mutation has wide application prospect and important significance in the fields of diagnosis or auxiliary diagnosis of autism patients and targeted drug development of related diseases by exploring and mining.

Disclosure of Invention

The technical problem to be solved by the invention is how to detect the H1.4 frameshift mutant protein quickly, sensitively and specifically and how to diagnose or assist in diagnosing H1.4 frameshift mutant protein related diseases. The technical problem to be solved is not limited to the technical subject as described, and other technical subject not mentioned herein may be clearly understood by those skilled in the art through the following description.

In order to solve the above technical problems, the present invention firstly provides a monoclonal antibody or an antigen binding portion thereof specifically binding to the H1.4 frameshift mutein, wherein the monoclonal antibody or the antigen binding portion thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises three complementarity determining regions with amino acid sequences of 45 th-52 th position of SEQ ID No.1, 70 th-77 th position of SEQ ID No.1 and 116 th-125 th position of SEQ ID No. 1; the light chain variable region comprises three complementarity determining regions having amino acid sequences 46-56 of SEQ ID No.2, 74-76 of SEQ ID No.2, and 113-121 of SEQ ID No.2, respectively. Wherein:

the amino acid sequence of CDR1 of the heavy chain variable region is position 45-52 of SEQ ID No. 1;

the amino acid sequence of CDR2 of the heavy chain variable region is positions 70-77 of SEQ ID No. 1;

the amino acid sequence of CDR3 of the heavy chain variable region is position 116-125 of SEQ ID No. 1;

the amino acid sequence of CDR1 of the light chain variable region is positions 46-56 of SEQ ID No. 2;

the amino acid sequence of CDR2 of the light chain variable region is positions 74-76 of SEQ ID No. 2;

the amino acid sequence of CDR3 of the light chain variable region is position 113-121 of SEQ ID No. 2.

Wherein the antigen binding portion can be a bispecific antibody, a Fab antibody, a Fv antibody, a single chain antibody (ScFv), a single domain antibody, or a Minimal Recognition Unit (MRU).

Further, the amino acid sequence of the heavy chain variable region is SEQ ID No. 14; the amino acid sequence of the light chain variable region is SEQ ID No. 16.

The amino acid sequence of the heavy chain variable region is 20 th to 136 th of SEQ ID No. 1; the amino acid sequence of the light chain variable region is 20 th to 131 th positions of SEQ ID No. 2.

Further, the amino acid sequence of the heavy chain is SEQ ID No. 1; the amino acid sequence of the light chain is SEQ ID No. 2.

Further, the heavy chain variable region and the light chain variable region each comprise a framework region, which may be derived from a mouse.

Further, the monoclonal antibody may be a murine monoclonal antibody.

Further, the monoclonal antibody may be a detectable antibody.

The invention also provides application of the polypeptide or the polypeptide as a hapten in preparing a detection antibody specifically bound with the H1.4 frameshift mutant protein, wherein the amino acid sequence of the polypeptide is SEQ ID No. 3.

In the application, the application comprises the application of the artificial antigen obtained by coupling the hapten and a protein carrier in the preparation of the detection antibody specifically bound with the H1.4 frameshift mutant protein.

The protein carrier can be any one of bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.

The invention also provides a biomaterial, which is any one of the following B1) to B6):

B1) nucleic acid molecules encoding the heavy and/or light chain of the monoclonal antibody or antigen-binding portion thereof;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a cell line containing B1) the nucleic acid molecule, or a cell line containing B2) the expression cassette, or a cell line containing B3) the recombinant vector;

B6) a nucleic acid molecule encoding the heavy chain variable region and/or the light chain variable region of the monoclonal antibody or antigen binding portion thereof.

In the above biological material, the nucleic acid molecule is any one of:

C1) the coding sequence is a heavy chain DNA molecule of SEQ ID No. 4;

C2) the coding sequence is a light chain DNA molecule of SEQ ID No. 5;

C3) the coding sequence is the heavy chain variable region DNA molecule of SEQ ID No. 15;

C4) the coding sequence is the light chain variable region DNA molecule of SEQ ID No. 17;

C5) 75% or greater than 75% identity to a nucleotide sequence defined by C1) -C4) and encodes a heavy chain and/or light chain and/or heavy chain variable region and/or light chain variable region DNA molecule in the monoclonal antibody or antigen binding portion thereof.

The DNA molecule shown in SEQ ID No.4 encodes the heavy chain shown in SEQ ID No. 1;

the DNA molecule shown in SEQ ID No.5 encodes the light chain shown in SEQ ID No. 2;

SEQ ID No.15 is a DNA molecule from position 58 to 408 of SEQ ID No. 4;

SEQ ID No.17 is a light chain variable region DNA molecule from position 58 to 393 of SEQ ID No. 5;

in the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi. Among them, the bacteria may be derived from Escherichia (Escherichia), Erwinia (Erwinia), Agrobacterium (Agrobacterium), Flavobacterium (Flavobacterium), Alcaligenes (Alcaligenes), Pseudomonas (Pseudomonas), Bacillus (Bacillus), etc.

In the above biological material, the cell may be a mammalian cell, an insect cell, a yeast cell or a prokaryotic cell.

The invention also provides a reagent or a kit for detecting H1.4 frameshift mutation, wherein the reagent or the kit contains the monoclonal antibody or the antigen binding part thereof.

The kit can be a chemiluminescence immunoassay kit, an enzyme-linked immunoassay kit, a colloidal gold immunoassay kit or a fluorescence immunoassay kit, but is not limited thereto.

The invention also provides application of the monoclonal antibody or the antigen binding part thereof and/or the biological material in preparing products for diagnosing or assisting in diagnosing H1.4 frameshift mutant protein related diseases.

In the above application, the H1.4 frameshift mutein-related disease may be autism.

As used herein, the term "bispecific antibody" refers to a bispecific antibody that can be obtained in large, uniform and high purity by introducing two sets of light and heavy chain genes into a myeloma cell and selecting appropriate antibody constant regions and Ig classes. In addition, bispecific antibodies can also be obtained by chemical cross-linking techniques or hybrid-hybridoma techniques.

The term "Fab antibody" is a heterodimer of a heavy chain Fd and an intact light chain, which contains only one antigen binding site, bound by a disulfide bond. The encoding genes of the heavy chain Fd and the complete light chain are connected, and the Fab antibody can be expressed in the escherichia coli incretion after the fusion of the bacterial protein signal peptide gene, and has complete three-dimensional folding and intra-chain and inter-chain disulfide bonds. The heavy chain Fd refers to the H chain portion of about 1/2 (about 225 amino acid residues, including V) in Fab_H、C_H1 and a partial hinge region).

The term "Fv antibody" means that V can be constructed separately_HAnd V_LCo-transfecting cells with a gene vector, expressing the cells respectively, and assembling into a functional Fv antibody; v which may also be in a vector_HAnd V_LSetting stop codes between the two small molecular protein fragments, respectively expressing the two small molecular protein fragments, and combining the two small molecular protein fragments through a non-covalent bond to form the Fv antibody.

The term "single chain antibody (ScFv)" refers to the expression of a single polypeptide chain by linking the light and heavy chain variable region genes using an appropriate oligonucleotide linker (linker), and is called single chain antibody (ScFv). The polypeptide chain is capable of spontaneously folding into the natural idea, retaining the specificity and affinity of the Fv.

The term "single domain antibody" refers to an antibody heavy chain V region which is expressed by genetic engineering method to obtain a V-containing antibody_HAn antibody to the fragment. The ability of single domain antibodies to bind antigen and their stability are essentially identical to those of full antibodies.

The term "Minimal Recognition Unit (MRU)" refers to a single CDR structure containing only the variable region, having a molecular mass of only about 1% of that of the whole antibody, and capable of binding to the corresponding antigen.

The invention prepares the mouse monoclonal antibody aiming at the H1.4 frameshift mutant protein by utilizing the H1.4 frameshift mutant polypeptide hapten for the first time, and the prepared antibody can be specifically combined with the H1.4 frameshift mutant protein, so that the H1.4 frameshift mutant protein can be quickly, sensitively and accurately detected, and the effect of recognizing the H1.4 frameshift mutation is unexpectedly good. Provides an indispensable experimental material for the research of the mechanism of the autism caused by the H1.4 frameshift mutation, provides a detection basis for the diagnosis of the autism patients, provides great possibility for the design of targeted drugs of diseases, and has important application value.

Drawings

FIG. 1 shows the specificity of Western Blot to detect monoclonal antibodies (H1.4 frameshift mutant antibodies) that specifically bind to H1.4 frameshift muteins.

FIG. 2 shows the properties of H1.4 frameshift mutant antibody detected by immunofluorescence.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 preparation of monoclonal antibodies that specifically bind to H1.4 frameshift muteins

1. Preparation of antigens

(1) Preparation of haptens

The invention firstly uses the peptide segment (H1.4 frameshift mutant polypeptide hapten) of the last 30 amino acids of the frameshift mutant to prepare a mouse monoclonal antibody aiming at the H1.4 frameshift mutant protein, and the prepared antibody has a good effect of recognizing the H1.4 frameshift mutation.

The H1.4 frameshift mutant polypeptide hapten amino acid sequence is as follows: CWSQKSEKPEKGESSQAKKGAQEPSEGQSS (SEQ ID No.3), hapten polypeptide was synthesized and purified by the Biotech company, Inc., of Bordetella, Wuhan.

(2) Preparation of artificial antigens (immunogens)

Generally, artificially synthesized simple hapten molecules have low immunogenicity, and can induce anti-hapten antibodies only after being coupled with protein carriers. The protein carrier can be any one of bovine serum albumin, ovalbumin, human serum albumin or hemocyanin. In this example, Hemocyanin (KLH) was used as a carrier protein for immunoconjugates and Bovine Serum Albumin (BSA) was used for screening. The specific operation steps are as follows:

respectively coupling the purified hapten polypeptide in the step (1) With hemocyanin (KLH) and Bovine Serum Albumin (BSA), carrying out coupling reaction according to the operation instruction of an Imject maleimid Activated Immunogen coupling Kit With mcKLH and BSA of Pierce company in America, and immediately purifying and packaging through a column after coupling.

The hapten polypeptide (SEQ ID No.3) is respectively connected with carrier protein (KLH) and Bovine Serum Albumin (BSA) according to the steps to obtain a hapten polypeptide-KLH coupling compound (used for immunizing mice) and a hapten polypeptide-BSA coupling compound (used for detecting the titer of the serum antibody of the mice by an ELASA method).

2. Preparation of antibodies

2-1 immunized mice

The immunized mice are Balb/C mice, female mice or male mice can be used, the mice are 6-10 weeks old, the weight is about 20g, the mice are healthy and disease-free, and 5 Balb/C mice are immunized by using immunogen (hapten polypeptide-KLH coupling compound synthesized in the step (2) in the step 1) and adjuvant (incomplete Freund's reagent). And (3) carrying out second immunization three weeks after the first immunization, carrying out boosting immunization two weeks after the second immunization, and carrying out ELISA (enzyme-linked immunosorbent assay) detection on the tail vein serum of the mouse to detect antibody titer and Western Blot (WB) detection on endogenous samples after the third immunization. If the ELISA detection antibody titer reaches more than 1:50000 and WB target bands indicate that the immunity is qualified, 1 mouse with the best detection result is selected to take splenocytes for cell fusion and screening; if the immunity is unqualified, the immunity can be added and then the detection is carried out again;

the experiment set up immune and control groups:

the immunogen of the immunization group is the hapten polypeptide-KLH coupling compound synthesized in the step (2) in the step 1, the immunization dose is 100 mu g/mouse, the adjuvant is incomplete Freund type adjuvant, and 5 mice are immunized;

the immunogen of the control group is normal saline, the immunization dose is 100 mu g/mouse, the adjuvant is incomplete Freund type adjuvant, and 5 mice are immunized.

2-2ELISA method for detecting mouse serum antibody titer

After the three-immunization, the tail vein serum of the mice is subjected to ELISA method to detect the antibody titer.

The titer of the antibody in the serum was determined by indirect ELISA.

a)2 references: the negative reference is pre-taken blood serum, all diluted with primary antibody starting concentration (blocking solution dilution); the positive reference is a previously positive serum or cell supernatant, or primary antibody is directly embedded as an antigen.

b) To a 96-well plate, 50. mu.L of antigen per well, i.e., 100ng, was added. The edge holes should be avoided as much as possible, which reduces the light absorption value and affects the result. At 4 ℃ overnight, time emergency can also be 37 ℃ incubated for 2 hours.

c) The antigen was decanted and 100. mu.L of blocking solution was added to each well. If the carrier protein is BSA, 1% skimmed milk powder is used instead of BSA. Incubate at 4 ℃ overnight or at 37 ℃ for 2 hours.

d) Pouring out the confining liquid, beating on absorbent paper to suck residual liquid as much as possible, washing the plate for 3 times by using a washing solution (0.1M KPi, 0.05% Tween-20, pH7), beating the residual liquid (absorbent paper) as much as possible each time, and airing the plate as much as possible if the plate is placed for a period of time, or drying at 30 ℃ and sealing by using a sealing bag for storage at 4 ℃.

e) 50 μ L of primary antibody per well, if hybridoma cells, is cell supernatant, and if serum from an immunized animal, a series of dilutions (blocking dilution) is required, typically starting at a dilution of 1: 499, followed by a 2-fold dilution. Incubate at 37 ℃ for 1 hour.

f) Wash plate 3 times, as above.

g) Add 50. mu.L per well of 1: HRP-labeled secondary antibodies were diluted 2000 (blocking dilution) and incubated at 37 ℃ for 45 min.

h) Wash plate 3 times, as above.

i) 100 μ L of TMB substrate per well (must be freshly prepared) was added and incubated for 5-20 minutes at room temperature (depending on the rate of color change).

j) mu.L of stop solution (0.5M oxalic acid) was added to each well.

k) Absorbance at 450nm was read on a microplate reader.

The titer results of the antibody detected by ELISA method are shown in Table 1:

TABLE 1 detection of antibody titer by ELISA method

ELISA screening antigen: CWSQKSEKPEKGESSQAKKGAQEPSEGQSS + BSA (conjugate of a polypeptide with BSA, wherein the amino acid sequence of the polypeptide is CWSQKSEKPEKGESSQAKKGAQEPSEGQSS);

coating concentration: 1 mu g/mL, 100 mu L/well;

secondary antibody: peroxidase-conjugated affinity Goat Anti-Mouse IgG (H + L) [ Jackson ImmunoResearch,115-

The secondary antibody dilution ratio: 1:10000

As shown in table 1, the M02 mice immunized produced the highest antibody titer, which was 0.970.

2-3 endogenous sample protein immunoblotting (Western Blot, WB) identification

H1.4K157E frameshift mutants with Flag tag at N-terminal, all on pFLAG-CMV vector, NIH 3T3 cells (3X 10) were transfected with recombinant plasmid of pFLAG-CMV-H1.4K 157E fs⁶) Cells were harvested after 36 hours. The endogenous sample is a plasmid for overexpressing pFLAG-CMV-H1.4K 157E fs in mouse NIH 3T3 cells to obtain a flag-H1.4K 157E protein.

(1) Cells were lysed with 200. mu.L Lysis buffer (10mM Tris,1mM EDTA, 1% SDS).

(2) And (4) performing ultrasonic treatment in a water bath until the sample is not viscous, and separating the protein by sodium dodecyl sulfate polyacrylamide gel.

(3) The sample on the denatured gel was transferred to a nylon membrane.

(4) The milk was blocked for 1 hour at room temperature and washed three times with TBST.

(5) The nylon membrane is incubated overnight at 4 ℃ with primary antibody of H1.4 frameshift mutation

(6) The primary antibody was recovered and the nylon membrane was washed three times with TBST.

(7) The corresponding secondary antibodies were incubated for 1 hour at room temperature and washed three times with TBST.

(8) And (3) incubating the nylon membrane and the luminescent substrate for 3min, and developing.

The WB test showed a distinct band at 25kDa, indicating that antibodies specifically binding to the H1.4 frameshift mutein were produced in the immunized mice.

2-4 cell fusion and monoclonal antibody screening

1 mouse spleen cell which is qualified in immunity is selected to be fused with myeloma cell (SP2/0) to prepare hybridoma, the number of the fused cell plates is 10, 96-hole cell plates are prepared, after the fused cell plates are cultured for 7-10 days, ELISA detection and WB endogenous verification of fused supernatant (culture supernatant of the hybridoma) are carried out by using a screening source (the screening source of ELISA is antigen, and the screening source of WB is H1.4K157E frameshift mutant purified in vitro), and subcloning is carried out by a limiting dilution method according to the detection result.

Screening 1-20 positive monoclonals for subsequent subcloning according to the experimental condition, and finally screening 1-3 positive monoclonals for cell strain determination;

a hybridoma cell strain which can secrete the monoclonal antibody specifically combined with the H1.4 frameshift mutant protein is obtained and named A1.

Injecting the obtained hybridoma cell strain A1 into BALB/c mouse abdominal cavity with cell concentration of 3 × 10⁵One cell/one. Ascites fluid containing the monoclonal antibody was collected after 5 to 10 days. Antibody purification in ascites using Protein G-Sepharose4B adsorption chromatography column gave monoclonal antibody specifically binding to H1.4 frameshift mutein.

2-5 sequence determination of monoclonal antibodies

The monoclonal antibody obtained by sequencing and purifying the Wuhan Eboltach biotech GmbH is entrusted to obtain a corresponding amino acid sequence by utilizing the rule that a triplet codon codes an amino acid on the basis of obtaining a nucleic acid sequence by Sanger sequencing.

Finally, the amino acid sequence of the heavy chain of the monoclonal antibody specifically combined with the H1.4 frameshift mutant protein is determined to be SEQ ID No.1, and the nucleotide sequence of the heavy chain gene is determined to be SEQ ID No. 4; the amino acid sequence of the light chain is SEQ ID No.2, and the nucleotide sequence of the light chain gene is SEQ ID No. 5. Wherein:

the amino acid sequence of the heavy chain variable region is SEQ ID No. 14;

the amino acid sequence of the heavy chain variable region is 20 th to 136 th of SEQ ID No. 1;

the amino acid sequence of the light chain variable region is SEQ ID No. 16;

the amino acid sequence of the light chain variable region is 20 th to 131 th positions of SEQ ID No. 2;

the nucleotide sequence of the heavy chain variable region gene is SEQ ID No. 15;

the nucleotide sequence of the heavy chain variable region gene is 58 th to 408 th of SEQ ID No. 4;

the nucleotide sequence of the light chain variable region gene is SEQ ID No. 17;

the nucleotide sequence of the light chain variable region gene is 58-393 th position of SEQ ID No. 5;

2-6 gene engineering method for preparing antibody

1. Cloning the light chain and heavy chain sequences of the antibody to an expression vector of a eukaryotic cell;

2. transfecting eukaryotic cells by using the cloned vector, and collecting a culture medium after 48 hours;

3. the medium was collected and centrifuged to leave a supernatant, and the antibody was purified by using a Protein G-Sepharose4B adsorption column.

Example 2 detection of antibody specificity by Western immunoblotting (WB)

1. Sample preparation

The following samples were prepared to test the specificity of the monoclonal antibodies:

in vitro purified histone H1.4(WT H1.4)

In vitro purified H1.4 frameshift muteins (H1.4K 157E fs)

Endogenous over-expressed histone H1.4(WT H1.4)

Endogenous over-expressed H1.4 frameshift muteins (H1.4K 157E fs)

Purification of 1-1 Histone H1.4 and H1.4 frameshift muteins

1) The recombinant vectors pET-28a-WT H1.4 and pET-28a-H1.4K157E were introduced into E.coli BL21(DE3), respectively, to obtain a strain containing the recombinant vectors.

The recombinant vector pET-28a-WT H1.4 is a recombinant expression vector obtained by replacing the fragment between the recognition sites Nco I and Xhol I of pET-28a (+) vector with the DNA fragment shown at positions 7-665 of SEQ ID No.6 in the sequence list, and keeping the other sequences of pET-28a (+) vector unchanged. Wherein, the 9 th to 665 th sites of SEQ ID No.6 are nucleotide sequences of histone H1.4 gene (wild type). pET-28a-WT H1.4 contains the fusion gene whose coding sequence is SEQ ID No.6, can express the fusion protein whose amino acid sequence is SEQ ID No.8 in the sequence table, and the 3 rd to 221 th position of SEQ ID No.8 is the amino acid of histone H1.4 (wild type).

The recombinant vector pET-28a-H1.4K157E is a recombinant expression vector obtained by replacing the fragment between the recognition sites Nco I and Xhol I of pET-28a (+) vector with the DNA fragment shown in the 7 th to 590 th positions of SEQ ID No.7 in the sequence list, and keeping the other sequences of pET-28a (+) vector unchanged. Wherein, the 9 th to 590 th positions of the SEQ ID No.7 are the nucleotide sequence of the frame shift mutant Lys157Glu fs 39. pET-28a-H1.4K157E contains the fusion gene whose coding sequence is SEQ ID No.7, can express the fusion protein whose amino acid sequence is SEQ ID No.9 in the sequence table, and the 3-196 th position of SEQ ID No.9 is the amino acid of frameshift mutant Lys157Glu fs 39.

2) A single clone was selected and inoculated into 100mL of LB liquid medium, and cultured at 37 ℃ and 220rpm for 12 hours.

3) And (3) mixing the bacterial liquid according to the proportion of 1:50 were inoculated into 6 LB liquid media (750 mL) and expanded (37 ℃, 220rpm), and when the OD reached 0.6, IPTG (final concentration of 0.5mM) was added and induced for 4 hours.

4) The cell suspension was centrifuged, the supernatant was discarded, 100mL of PBS was used to resuspend the suspension and centrifuge (4 ℃, 4000rpm,30min), and the supernatant was discarded.

5) Breaking the bacteria, adding 100mL lysine Buffer (50mM Tris, pH 7.5, 100mM NaCl, 1mM EDTA), fully suspending the bacteria, performing ultrasonic treatment for 5s and stopping the ultrasonic treatment for 8s, and performing power 200W.

6) After completion of sonication, the mixture was centrifuged at 18,000rpm at 4 ℃ for 20min, and the supernatant was discarded.

7) The precipitate was dissolved in H1.4 by adding 30mL of Unfolding Buffer (1M NaCl, 50mM Tris-HCl, pH 7.5, 5 mM. beta. -Me), and stirred at room temperature for 2 hours.

8) Centrifuge at 18,000rpm at 4 ℃ for 30min and transfer the supernatant to a clean beaker.

9) H1.4 was purified using a HiTrap Heparin HP column, the protein was eluted by hanging the column with low salt Buffer A (50mM Tris, pH 7.5, 500mM NaCl) and mixing the low salt Buffer A with high salt Buffer B (50mM Tris, 2M NaCl, pH 8.0) to form different salt concentrations, the eluate was collected and the results of the purification were checked by SDS-PAGE gel electrophoresis.

10) Removing nucleic acid in protein by using a hydroxyapatite column, hanging the low-salt Buffer A (200mM K2HPO4, KH2PO4 and pH 6.8) on the column, mixing the low-salt Buffer A with the high-salt Buffer B (1M K2HPO4, KH2PO4 and pH 6.8) to form different salt concentrations to elute the protein, collecting eluent, and detecting the purification result by SDS-PAGE gel electrophoresis.

11) The protein of interest was collected and dialyzed into BC100 Buffer (100mM NaCl, 20mM Tris, pH 8.0, 20% Glycerol), and finally the protein was dispensed and stored at-80 ℃.

Finally, the histones H1.4(WT H1.4) and H1.4 frameshift muteins (H1.4K 157 Efs) purified in vitro were obtained.

Overexpression of 1-2 histones H1.4 and H1.4 frameshift muteins

1) pFLAG-CMV-WT H1.4, pFLAG-CMV-H1.4K 157E fs recombinant plasmid 4ug were transfected into NIH 3T3 cells, and the cells were harvested after 36 hours.

Wherein, the recombinant vector pFLAG-CMV-WT H1.4 is a recombinant expression vector obtained by replacing a fragment between EcoRI recognition sites and BamHI recognition sites of the pFLAG-CMV vector with a DNA fragment shown in 88 th to 747 th sites of SEQ ID No.10 in a sequence table and keeping other sequences of the CMV-flag vector unchanged. Wherein, the 88 th to 747 th positions of SEQ ID No.10 are the nucleotide sequence of histone H1.4 gene (wild type). pFLAG-CMV-WT H1.4 contains a fusion gene whose coding sequence is SEQ ID No.10, can express a fusion protein whose amino acid sequence is SEQ ID No.12 in a sequence table, and the 30 th to 248 th positions of SEQ ID No.12 are the amino acids of histone H1.4 (wild type).

The recombinant vector pFLAG-CMV-H1.4K 157E fs is a recombinant expression vector obtained by replacing a fragment between EcoRI and BamHI recognition sites of the pFLAG-CMV vector with a DNA fragment shown in positions 88 to 672 of SEQ ID No.11 in the sequence table and keeping other sequences of the pFLAG-CMV vector unchanged. Wherein, the 88 th to 672 th positions of the SEQ ID No.11 are the nucleotide sequence of the H1.4 frameshift mutant protein (H1.4K 157E fs). pFLAG-CMV-H1.4K 157E fs contains a fusion gene of which the coding sequence is SEQ ID No.11, can express a fusion protein of which the amino acid sequence is SEQ ID No.13 in a sequence table, and the 30 th to 223 th positions of the SEQ ID No.13 are amino acids of H1.4 frameshift mutant protein (H1.4K 157E fs).

2) Cells were lysed with 200. mu.L of Lysis buffer (10mM Tris,1mM EDTA, 1% SDS).

Finally, the endogenously over-expressed histone H1.4(WT H1.4) and H1.4 frameshift mutant protein (H1.4K 157E fs) are obtained.

2. Specificity detection

1) 100ng each of the histones H1.4(WT H1.4) and H1.4 frameshift mutein (H1.4K 157E fs) purified in vitro in step 1-1 was taken, and total protein amount of 50 μ g of cell lysate of histones H1.4(WT H1.4) and H1.4 frameshift mutein (H1.4K 157E fs) endogenously overexpressed in step 1-2 was taken for sodium dodecyl sulfate polyacrylamide gel electrophoresis.

2) The sample on the gel was transferred to a nylon membrane.

3) The milk was blocked for 1 hour at room temperature and washed three times with TBST.

4) The nylon membrane was incubated with the H1.4 frameshift mutated antibody (i.e., the monoclonal antibody of the invention that specifically binds to the H1.4 frameshift mutein) at 4 ℃ overnight.

5) The primary antibody was recovered and the nylon membrane was washed three times with TBST.

6) The nylon membrane was incubated with goat anti-mouse secondary antibody for 1 hour at room temperature and washed three times with TBST.

7) And (3) incubating the nylon membrane and the luminescent substrate for 3min, and developing.

The antibody detection results are shown in fig. 1: the antibody can be well recognized by H1.4K157E fs frameshift mutants which are purified in vitro or overexpressed in cells, however, the antibody cannot be recognized by wild type H1.4 which is purified in vitro or overexpressed in cells, which indicates that the monoclonal antibody specifically binding to the H1.4 frameshift mutant protein can only recognize the H1.4 frameshift mutant protein, and indicates that the antibody specificity of the H1.4 frameshift mutation is very good.

EXAMPLE 3 immunofluorescence detection of monoclonal antibodies

The antibody prepared in example 1 was used for immunofluorescent staining of mouse brain slices. The distribution of H1.4K157E mutant in H1.4K157E frameshift mutant mouse brain tissue section is observed in the experiment, and the steps of mouse brain slice preparation and immunofluorescence staining are as follows:

1) after anesthetizing the mice, the mice were heart perfused with PBS to wash away blood in the brain tissue of the mice.

2) Heart perfusion is carried out by using 4% paraformaldehyde, after the mouse is fixed, the brain tissue of the mouse is carefully taken out and soaked in the 4% paraformaldehyde for more than 48 hours.

3) Brain tissue is placed in 20% sucrose for more than 72 hours, mainly to remove water from the tissue.

4) Brain tissue was further sweetened in 30% sucrose for over 72 hours.

5) Brain tissue is embedded with tissue embedding fluid, and the embedded sample is placed at-20 ℃ for at least 48 hours.

6) Sagittal sections of brain tissue were taken with a cryomicrotome (Leica CM1950) and attached to a glass slide, where hippocampal neurons in the brain tissue were clearly visible, and the slide was stored at-80 ℃.

7) The slide was washed three times with PBS and the embedding solution was removed from the vicinity of the sample.

8) The slide was placed in citric acid buffer (9mM sodium citrate +1mM citric acid), and then the sample was placed in a microwave oven, the buffer was boiled over a high fire for 4min, the buffer was heated over a low fire for 4min, the heating over a low fire was repeated twice, and the slide was cooled at room temperature. In the heating process, a citric acid buffer solution needs to be supplemented in time, the liquid level of the buffer solution cannot be lower than that of the tissue, and the flaking can be caused.

9) PBS was washed three times, and the cells were permeabilized with PBS containing 1% Triton X-100 for 30min at room temperature.

10) PBS was washed three times, 5% BSA blocked samples, room temperature 1 hour.

11) PBS was washed three times, and samples were washed with H1.4 frameshift mutated murine antibody and Nucleolin antibody (Anti-Nucleolin: ab129200), 4 ℃ overnight.

12) PBS was washed three times and the samples were incubated with the corresponding fluorescent secondary antibodies (Alex488 anti-mouse IgG, Genecopoeia, L109A, Alex494 anti-rabbit IgG, Genecopoeia, L120A) for 1 hour at room temperature.

13) PBS washing three times, adding anti-quenching agent, sealing, using fluorescence microscope to observe the result.

14) The results showed that H1.4K157E fs localized in the nucleus and was enriched in the nucleolar region (fig. 2).

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

SEQUENCE LISTING

<110> institute of biophysics of Chinese academy of sciences

First Hospital of Beijing university

<120> preparation and application of monoclonal antibody specifically binding to H1.4 frameshift mutant protein

<160> 17

<170> PatentIn version 3.5

<210> 1

<211> 460

<212> PRT

<213> Mus musculus

<400> 1

Met Gly Trp Val Trp Asn Leu Leu Phe Leu Met Ala Ala Ala Gln Ser

1 5 10 15

Ile Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys

20 25 30

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

35 40 45

Thr Asp Tyr Ser Met Gln Trp Met Lys Gln Ala Pro Gly Lys Gly Leu

50 55 60

Lys Trp Met Gly Trp Ile Asp Thr Gly Thr Gly Glu Pro Ser Tyr Ala

65 70 75 80

Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser

85 90 95

Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr

100 105 110

Tyr Phe Cys Ala Arg Arg Ser Tyr Gly Tyr Phe Ala Phe Trp Gly Gln

115 120 125

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

130 135 140

Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr

145 150 155 160

Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr

165 170 175

Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val

180 185 190

Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser

195 200 205

Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala

210 215 220

Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys

225 230 235 240

Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe

245 250 255

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

260 265 270

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

275 280 285

Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro

290 295 300

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro

305 310 315 320

Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val

325 330 335

Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

340 345 350

Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys

355 360 365

Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp

370 375 380

Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro

385 390 395 400

Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser

405 410 415

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

420 425 430

Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His

435 440 445

His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

450 455 460

<210> 2

<211> 238

<212> PRT

<213> Mus musculus

<400> 2

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

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

20 25 30

Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile

35 40 45

Val His Val Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro

50 55 60

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

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr

85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys

100 105 110

Phe Gln Gly Ser His Val Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu

115 120 125

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

130 135 140

Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu

145 150 155 160

Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly

165 170 175

Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp

195 200 205

Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr

210 215 220

Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

<210> 3

<211> 30

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 3

Cys Trp Ser Gln Lys Ser Glu Lys Pro Glu Lys Gly Glu Ser Ser Gln

1 5 10 15

Ala Lys Lys Gly Ala Gln Glu Pro Ser Glu Gly Gln Ser Ser

20 25 30

<210> 4

<211> 1383

<212> DNA

<213> Mus musculus

<400> 4

atgggttggg tgtggaactt gctattcctg atggcagctg cccaaagtat ccaagcacag 60

atccagttgg tgcagtctgg acctgaactg aagaagcctg gagagacagt caagatctcc 120

tgcaaggctt ctggttacac cttcacagac tattcaatgc agtggatgaa gcaggctcca 180

ggaaagggtt taaagtggat gggctggata gacactggga ctggtgagcc atcatatgca 240

gatgacttca agggacggtt tgccttctct ttggaaacct ctgccagcac tgcctatttg 300

cagatcaaca acctcaaaaa tgaggacacg gctacatatt tctgtgctag acgctcctat 360

ggttattttg ctttctgggg ccaagggact ctggtcgctg tctctgcagc caaaacgaca 420

cccccatctg tctatccact ggcccctgga tctgctgccc aaactaactc catggtgacc 480

ctgggatgcc tggtcaaggg ctatttccct gagccagtga cagtgacctg gaactctgga 540

tccctgtcca gcggtgtgca caccttccca gctgtcctgc agtctgacct ctacactctg 600

agcagctcag tgactgtccc ctccagcacc tggcccagcg agaccgtcac ctgcaacgtt 660

gcccacccgg ccagcagcac caaggtggac aagaaaattg tgcccaggga ttgtggttgt 720

aagccttgca tatgtacagt cccagaagta tcatctgtct tcatcttccc cccaaagccc 780

aaggatgtgc tcaccattac tctgactcct aaggtcacgt gtgttgtggt agacatcagc 840

aaggatgatc ccgaggtcca gttcagctgg tttgtagatg atgtggaggt gcacacagct 900

cagacgcaac cccgggagga gcagttcaac agcactttcc gctcagtcag tgaacttccc 960

atcatgcacc aggactggct caatggcaag gagttcaaat gcagggtcaa cagtgcagct 1020

ttccctgccc ccatcgagaa aaccatctcc aaaaccaaag gcagaccgaa ggctccacag 1080

gtgtacacca ttccacctcc caaggagcag atggccaagg ataaagtcag tctgacctgc 1140

atgataacag acttcttccc tgaagacatt actgtggagt ggcagtggaa tgggcagcca 1200

gcggagaact acaagaacac tcagcccatc atggacacag atggctctta cttcgtctac 1260

agcaagctca atgtgcagaa gagcaactgg gaggcaggaa atactttcac ctgctctgtg 1320

ttacatgagg gcctgcacaa ccaccatact gagaagagcc tctcccactc tcctggtaaa 1380

taa 1383

<210> 5

<211> 717

<212> DNA

<213> Mus musculus

<400> 5

atgggctggt cctgtatcat cctgttcctg gtggctacag ccacaggagt gcatagtgat 60

gttgtgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120

tcttgcagat ctagtcagaa cattgtacat gttaatggaa atacctattt agaatggtac 180

ttgcagaaac caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct 240

ggggtcccag acaggttcag tggcagtgga tcagggacag aattcacact caagatcagc 300

agagtggagg ctgaggatct gggagtttat tattgctttc aaggttcaca tgttcctttc 360

acgttcggtg ctgggaccaa gctggagctg aaacgggctg atgctgcacc aactgtatcc 420

atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 480

aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 540

aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 600

agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 660

actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgttaa 717

<210> 6

<211> 692

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

ccatgggcat gtccgagact gcgcctgccg cgcccgctgc tccggcccct gccgagaaga 60

ctcccgtgaa gaagaaggcc cgcaagtctg caggtgcggc caagcgcaaa gcgtctgggc 120

ccccggtgtc cgagctcatt actaaagctg ttgccgcctc caaggagcgc agcggcgtat 180

ctttggccgc tctcaagaaa gcgctggcag ccgctggcta tgacgtggag aagaacaaca 240

gccgcatcaa gctgggtctc aagagcctgg tgagcaaggg caccctggtg cagaccaagg 300

gcaccggcgc gtcgggttcc ttcaaactca acaagaaggc ggcctctggg gaagccaagc 360

ctaaggctaa aaaggcaggc gcggccaagg ccaagaagcc agcaggagcg gcgaagaagc 420

ccaagaaggc gacgggggcg gccaccccca agaagagcgc caagaagacc ccaaagaagg 480

cgaagaagcc ggctgcagct gctggagcca aaaaagcgaa aagcccgaaa aaggcgaaag 540

cagccaagcc aaaaaaggcg cccaagagcc cagcgaaggc caaagcagtt aaacccaagg 600

cggctaaacc aaagaccgcc aagcccaagg cagccaagcc aaagaaggcg gcagccaaga 660

aaaagctcga gcaccaccac caccaccact ga 692

<210> 7

<211> 617

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

ccatgggcat gtccgagact gcgcctgccg cgcccgctgc tccggcccct gccgagaaga 60

ctcccgtgaa gaagaaggcc cgcaagtctg caggtgcggc caagcgcaaa gcgtctgggc 120

ccccggtgtc cgagctcatt actaaagctg ttgccgcctc caaggagcgc agcggcgtat 180

ctttggccgc tctcaagaaa gcgctggcag ccgctggcta tgacgtggag aagaacaaca 240

gccgcatcaa gctgggtctc aagagcctgg tgagcaaggg caccctggtg cagaccaagg 300

gcaccggcgc gtcgggttcc ttcaaactca acaagaaggc ggcctctggg gaagccaagc 360

ctaaggctaa aaaggcaggc gcggccaagg ccaagaagcc agcaggagcg gcgaagaagc 420

ccaagaaggc gacgggggcg gccaccccca agaagagcgc caagaagacc cccaaagaag 480

gcgaagaagc cggctgcagc tgctggagcc aaaaaagcga aaagcccgaa aaaggcgaaa 540

gcagccaagc caaaaaaggc gcccaagagc ccagcgaagg ccaaagcagt ctcgagcacc 600

accaccacca ccactga 617

<210> 8

<211> 229

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 8

Met Gly Met Ser Glu Thr Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro

1 5 10 15

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

20 25 30

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

35 40 45

Ala Val Ala Ala Ser Lys Glu Arg Ser Gly Val Ser Leu Ala Ala Leu

50 55 60

Lys Lys Ala Leu Ala Ala Ala Gly Tyr Asp Val Glu Lys Asn Asn Ser

65 70 75 80

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

85 90 95

Gln Thr Lys Gly Thr Gly Ala Ser Gly Ser Phe Lys Leu Asn Lys Lys

100 105 110

Ala Ala Ser Gly Glu Ala Lys Pro Lys Ala Lys Lys Ala Gly Ala Ala

115 120 125

Lys Ala Lys Lys Pro Ala Gly Ala Ala Lys Lys Pro Lys Lys Ala Thr

130 135 140

Gly Ala Ala Thr Pro Lys Lys Ser Ala Lys Lys Thr Pro Lys Lys Ala

145 150 155 160

Lys Lys Pro Ala Ala Ala Ala Gly Ala Lys Lys Ala Lys Ser Pro Lys

165 170 175

Lys Ala Lys Ala Ala Lys Pro Lys Lys Ala Pro Lys Ser Pro Ala Lys

180 185 190

Ala Lys Ala Val Lys Pro Lys Ala Ala Lys Pro Lys Thr Ala Lys Pro

195 200 205

Lys Ala Ala Lys Pro Lys Lys Ala Ala Ala Lys Lys Lys Leu Glu His

210 215 220

His His His His His

225

<210> 9

<211> 204

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 9

Met Gly Met Ser Glu Thr Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro

1 5 10 15

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

20 25 30

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

35 40 45

Ala Val Ala Ala Ser Lys Glu Arg Ser Gly Val Ser Leu Ala Ala Leu

50 55 60

Lys Lys Ala Leu Ala Ala Ala Gly Tyr Asp Val Glu Lys Asn Asn Ser

65 70 75 80

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

85 90 95

Gln Thr Lys Gly Thr Gly Ala Ser Gly Ser Phe Lys Leu Asn Lys Lys

100 105 110

Ala Ala Ser Gly Glu Ala Lys Pro Lys Ala Lys Lys Ala Gly Ala Ala

115 120 125

Lys Ala Lys Lys Pro Ala Gly Ala Ala Lys Lys Pro Lys Lys Ala Thr

130 135 140

Gly Ala Ala Thr Pro Lys Lys Ser Ala Lys Lys Thr Pro Lys Glu Gly

145 150 155 160

Glu Glu Ala Gly Cys Ser Cys Trp Ser Gln Lys Ser Glu Lys Pro Glu

165 170 175

Lys Gly Glu Ser Ser Gln Ala Lys Lys Gly Ala Gln Glu Pro Ser Glu

180 185 190

Gly Gln Ser Ser Leu Glu His His His His His His

195 200

<210> 10

<211> 753

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

atgtctgcac ttctgatcct agctcttgtt ggagctgcag ttgctgacta caaagacgat 60

gacgacaagc ttgcggccgc gaattcaatg tccgagactg cgcctgccgc gcccgctgct 120

ccggcccctg ccgagaagac tcccgtgaag aagaaggccc gcaagtctgc aggtgcggcc 180

aagcgcaaag cgtctgggcc cccggtgtcc gagctcatta ctaaagctgt tgccgcctcc 240

aaggagcgca gcggcgtatc tttggccgct ctcaagaaag cgctggcagc cgctggctat 300

gacgtggaga agaacaacag ccgcatcaag ctgggtctca agagcctggt gagcaagggc 360

accctggtgc agaccaaggg caccggcgcg tcgggttcct tcaaactcaa caagaaggcg 420

gcctctgggg aagccaagcc taaggctaaa aaggcaggcg cggccaaggc caagaagcca 480

gcaggagcgg cgaagaagcc caagaaggcg acgggggcgg ccacccccaa gaagagcgcc 540

aagaagaccc caaagaaggc gaagaagccg gctgcagctg ctggagccaa aaaagcgaaa 600

agcccgaaaa aggcgaaagc agccaagcca aaaaaggcgc ccaagagccc agcgaaggcc 660

aaagcagtta aacccaaggc ggctaaacca aagaccgcca agcccaaggc agccaagcca 720

aagaaggcgg cagccaagaa aaagtaggga tcc 753

<210> 11

<211> 678

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

atgtctgcac ttctgatcct agctcttgtt ggagctgcag ttgctgacta caaagacgat 60

gacgacaagc ttgcggccgc gaattcaatg tccgagactg cgcctgccgc gcccgctgct 120

ccggcccctg ccgagaagac tcccgtgaag aagaaggccc gcaagtctgc aggtgcggcc 180

aagcgcaaag cgtctgggcc cccggtgtcc gagctcatta ctaaagctgt tgccgcctcc 240

aaggagcgca gcggcgtatc tttggccgct ctcaagaaag cgctggcagc cgctggctat 300

gacgtggaga agaacaacag ccgcatcaag ctgggtctca agagcctggt gagcaagggc 360

accctggtgc agaccaaggg caccggcgcg tcgggttcct tcaaactcaa caagaaggcg 420

gcctctgggg aagccaagcc taaggctaaa aaggcaggcg cggccaaggc caagaagcca 480

gcaggagcgg cgaagaagcc caagaaggcg acgggggcgg ccacccccaa gaagagcgcc 540

aagaagaccc ccaaagaagg cgaagaagcc ggctgcagct gctggagcca aaaaagcgaa 600

aagcccgaaa aaggcgaaag cagccaagcc aaaaaaggcg cccaagagcc cagcgaaggc 660

caaagcagtt aaggatcc 678

<210> 12

<211> 248

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 12

Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Ala Asp

1 5 10 15

Tyr Lys Asp Asp Asp Asp Lys Leu Ala Ala Ala Asn Ser Met Ser Glu

20 25 30

Thr Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro Ala Glu Lys Thr Pro

35 40 45

Val Lys Lys Lys Ala Arg Lys Ser Ala Gly Ala Ala Lys Arg Lys Ala

50 55 60

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

65 70 75 80

Lys Glu Arg Ser Gly Val Ser Leu Ala Ala Leu Lys Lys Ala Leu Ala

85 90 95

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

100 105 110

Leu Lys Ser Leu Val Ser Lys Gly Thr Leu Val Gln Thr Lys Gly Thr

115 120 125

Gly Ala Ser Gly Ser Phe Lys Leu Asn Lys Lys Ala Ala Ser Gly Glu

130 135 140

Ala Lys Pro Lys Ala Lys Lys Ala Gly Ala Ala Lys Ala Lys Lys Pro

145 150 155 160

Ala Gly Ala Ala Lys Lys Pro Lys Lys Ala Thr Gly Ala Ala Thr Pro

165 170 175

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

180 185 190

Ala Ala Gly Ala Lys Lys Ala Lys Ser Pro Lys Lys Ala Lys Ala Ala

195 200 205

Lys Pro Lys Lys Ala Pro Lys Ser Pro Ala Lys Ala Lys Ala Val Lys

210 215 220

Pro Lys Ala Ala Lys Pro Lys Thr Ala Lys Pro Lys Ala Ala Lys Pro

225 230 235 240

Lys Lys Ala Ala Ala Lys Lys Lys

245

<210> 13

<211> 223

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 13

Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Ala Asp

1 5 10 15

Tyr Lys Asp Asp Asp Asp Lys Leu Ala Ala Ala Asn Ser Met Ser Glu

20 25 30

Thr Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro Ala Glu Lys Thr Pro

35 40 45

Val Lys Lys Lys Ala Arg Lys Ser Ala Gly Ala Ala Lys Arg Lys Ala

50 55 60

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

65 70 75 80

Lys Glu Arg Ser Gly Val Ser Leu Ala Ala Leu Lys Lys Ala Leu Ala

85 90 95

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

100 105 110

Leu Lys Ser Leu Val Ser Lys Gly Thr Leu Val Gln Thr Lys Gly Thr

115 120 125

Gly Ala Ser Gly Ser Phe Lys Leu Asn Lys Lys Ala Ala Ser Gly Glu

130 135 140

Ala Lys Pro Lys Ala Lys Lys Ala Gly Ala Ala Lys Ala Lys Lys Pro

145 150 155 160

Ala Gly Ala Ala Lys Lys Pro Lys Lys Ala Thr Gly Ala Ala Thr Pro

165 170 175

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

180 185 190

Ser Cys Trp Ser Gln Lys Ser Glu Lys Pro Glu Lys Gly Glu Ser Ser

195 200 205

Gln Ala Lys Lys Gly Ala Gln Glu Pro Ser Glu Gly Gln Ser Ser

210 215 220

<210> 14

<211> 117

<212> PRT

<213> Mus musculus

<400> 14

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Trp Ile Asp Thr Gly Thr Gly Glu Pro Ser Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Arg Ser Tyr Gly Tyr Phe Ala Phe Trp Gly Gln Gly Thr Leu

100 105 110

Val Ala Val Ser Ala

115

<210> 15

<211> 351

<212> DNA

<213> Mus musculus

<400> 15

cagatccagt tggtgcagtc tggacctgaa ctgaagaagc ctggagagac agtcaagatc 60

tcctgcaagg cttctggtta caccttcaca gactattcaa tgcagtggat gaagcaggct 120

ccaggaaagg gtttaaagtg gatgggctgg atagacactg ggactggtga gccatcatat 180

gcagatgact tcaagggacg gtttgccttc tctttggaaa cctctgccag cactgcctat 240

ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc tagacgctcc 300

tatggttatt ttgctttctg gggccaaggg actctggtcg ctgtctctgc a 351

<210> 16

<211> 112

<212> PRT

<213> Mus musculus

<400> 16

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Val

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 17

<211> 336

<212> DNA

<213> Mus musculus

<400> 17

gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gaacattgta catgttaatg gaaataccta tttagaatgg 120

tacttgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagaattcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tattattgct ttcaaggttc acatgttcct 300

ttcacgttcg gtgctgggac caagctggag ctgaaa 336

Claims

1. A monoclonal antibody or an antigen-binding portion thereof that specifically binds to an H1.4 frameshift mutein, wherein said monoclonal antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising three complementarity determining regions having amino acid sequences respectively at positions 45-52 of SEQ ID No.1, 70-77 of SEQ ID No.1 and position 116-125 of SEQ ID No.1 and a light chain variable region; the light chain variable region comprises three complementarity determining regions having amino acid sequences 46-56 of SEQ ID No.2, 74-76 of SEQ ID No.2, and 113-121 of SEQ ID No.2, respectively.

2. The monoclonal antibody, or antigen-binding portion thereof, of claim 1, wherein the amino acid sequence of the heavy chain variable region is SEQ ID No. 14; the amino acid sequence of the light chain variable region is SEQ ID No. 16.

3. The monoclonal antibody, or antigen-binding portion thereof, of claim 1 or 2, wherein the amino acid sequence of the heavy chain is SEQ ID No. 1; the amino acid sequence of the light chain is SEQ ID No. 2.

4. The monoclonal antibody, or antigen-binding portion thereof, of any one of claims 1-3, wherein the heavy chain variable region and the light chain variable region each comprise a framework region, and the framework region is derived from a mouse.

5. The application of the polypeptide or the polypeptide as hapten in preparing a detection antibody specifically binding with H1.4 frameshift mutant protein is characterized in that the amino acid sequence of the polypeptide is SEQ ID No. 3.

6. A biomaterial, characterized in that it is any one of the following B1) to B6):

B1) nucleic acid molecules encoding the heavy and light chains of the monoclonal antibody, or antigen-binding portion thereof, of any one of claims 1-4;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B6) a nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody or antigen binding portion thereof of any one of claims 1-4.

7. The biomaterial according to claim 6, wherein the nucleic acid molecule is any of:

C1) the coding sequence is a heavy chain DNA molecule of SEQ ID No. 4;

C2) the coding sequence is a light chain DNA molecule of SEQ ID No. 5;

C4) the coding sequence is the light chain variable region DNA molecule of SEQ ID No. 17.

8. A reagent or kit for detecting a H1.4 frameshift mutation, comprising the monoclonal antibody or antigen binding portion thereof of any one of claims 1-4.