CN113234155A

CN113234155A - anti-Calponin protein monoclonal antibody, cell strain thereof, preparation method and application

Info

Publication number: CN113234155A
Application number: CN202110518497.6A
Authority: CN
Inventors: 付利利; 陈滢; 杨清海; 陈惠玲; 王小亚
Original assignee: Fuzhou Maixin Biotechnology Development Co ltd
Current assignee: Fuzhou Maixin Biotechnology Development Co ltd
Priority date: 2021-05-12
Filing date: 2021-05-12
Publication date: 2021-08-10
Anticipated expiration: 2041-05-12
Also published as: CN113234155B

Abstract

The invention relates to a monoclonal antibody capable of identifying a human Calponin antigen, a secretory cell strain, a preparation method thereof and application thereof in immunoassay. According to the technical scheme, the 1 st to 297 th amino acids at the C-terminal of the Calponin protein are selected as the antigen peptide, codon optimization is carried out, a gene fragment suitable for expression in escherichia coli BL21 is obtained, and the finally obtained recombinant protein comprises the Calponin protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 24H5 capable of efficiently secreting the anti-Calponin monoclonal antibody and the anti-Calponin monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing the Calponin protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Description

anti-Calponin protein monoclonal antibody, cell strain thereof, preparation method and application

Technical Field

The invention relates to the field of biomedical engineering, in particular to an anti-Calponin protein monoclonal antibody, a cell strain, a preparation method and application thereof.

Background

Calponin (CaP), the first protein found in chicken stomach smooth muscle by Takahashi et al in 1986, with a molecular weight of about 34-kDa (292-330 amino acids), bound to the filaments and involved in smooth muscle contraction as an actin-binding protein. The molecular structure of the polypeptide mainly consists of single-copy or multi-copy tandem repeat sequences of amino-terminal single CH (CH) structural domain and carboxyl-terminal calponin-like repeat motif (CLR). The Cap is a differentiation marker protein, is expressed in a large amount in differentiated smooth muscle cells, participates in actin cytoskeleton reconstruction by combining with a cytoskeletal protein actin, has multiple biological functions, and the expression change of the Cap is closely related to the occurrence and development of a plurality of diseases.

Calponin is expressed in both smooth muscle cells and myofibroblasts and myoepithelial cells and is often used to test the integrity of myoepithelial cells in proliferative disorders such as breast, salivary glands and prostate. The tumor cells are always positive for Calponin, but the malignant tumor shows more decreased Calponin. The level of residual Calponin is positively correlated with disease prognosis. Compared with the parental cell PC3, the expression of the Calponin 2 in the PC3-M is obviously reduced, the proliferation and migration speed of the PC3-M are increased, and the expression level of the Calponin 2 is increased when the PC3-M is transfected to correct the phenotype. Similarly, increased levels of Calponin 1 expression by transfection in human fibrosarcoma, leiomyosarcoma, synovial sarcoma and osteosarcoma cells significantly reduced anchorage-independent cell proliferation and in vivo tumorigenesis, indicating that Calponin functions as a tumor suppressor. This may be associated with a decrease in the level of Calponin expression leading to a decrease in the stability of the actin cytoskeleton of the tumor cells, an increase in motility. Therefore, the Calponin can be used as one of the main means for identifying benign and malignant tumors.

Disclosure of Invention

The inventor provides an anti-Calponin monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown as SEQ ID 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown as SEQ ID 5.

Further, the DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID2, and the DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID 3.

Further, the monoclonal antibody specifically recognizes the Calponin protein.

Further, the monoclonal antibody is mouse IgG_2bSubtype monoclonal antibodies.

Further, the hybridoma cell line is produced by a hybridoma cell line with the preservation number of CGMCC NO 20775.

The inventor also provides a preparation method of the anti-Calponin protein monoclonal antibody, the antigen used for immunizing mice is recombinant protein, the recombinant protein is expressed by escherichia coli in a recombinant mode, and the Calponin protein fragment and the histidine protein tag are contained; the protein fragment of the callonin is the amino acid fragment from the 1 st position to the 297 th position of the callonin protein, and the amino acid sequence of the protein fragment is the amino acid sequence shown in SEQ ID NO. 1.

The inventor also provides a hybridoma cell strain secreting the anti-calcinin protein molecule, wherein the cell strain is a mouse hybridoma cell strain 24H5 which is preserved in China general microbiological culture Collection center (CGMCC NO 20775) at 9 and 17 months of 2020, and the accession number is CGMCC NO 20775, and the microbial research institute is No. 3 of China academy of sciences in North West Lu 1 of the sunward area in Beijing.

The inventor also provides the application of the monoclonal antibody in the immune detection of the Calponin protein.

Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

The inventor finally provides a reagent for immunohistochemical detection of Calponin protein, wherein the reagent for immunohistochemical detection contains an amino acid sequence shown as SEQ ID4 in an amino acid sequence of a heavy chain variable region; the amino acid sequence of the variable region of the monoclonal antibody light chain is the amino acid sequence shown in SEQ ID 5.

Different from the prior art, the invention has the beneficial technical effects that: according to the technical scheme, the 1 st to 297 th amino acids at the C-terminal of the Calponin protein are selected as the antigen peptide, codon optimization is carried out, a gene fragment suitable for expression in escherichia coli BL21 is obtained, and the finally obtained recombinant protein comprises the Calponin protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 24H5 capable of efficiently secreting the anti-Calponin monoclonal antibody and the anti-Calponin monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing the Calponin protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a graph of purified pectin from recombinant Calponin protein fused with histidine tag in example 1, wherein M represents Marker; 1 is a whole fungus sample after ultrasonic treatment; 2, a supernatant sample after ultrasonic treatment; 3, precipitating a sample after ultrasonic treatment; 4 is an uninduced control sample.

FIG. 2 is a graph comparing immunohistochemical staining results for uterine fibroids; the left is the secreted Calponin of 24H5, and the right is the commercial Calponin.

FIG. 3 is a graph comparing the results of immunohistochemical staining of liver tissues; the left is the secreted Calponin of 24H5, and the right is the commercial Calponin.

Detailed Description

To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.

Example 1 preparation of recombinant Calponin protein fragment

First, gene optimization and synthesis

According to the protein sequence with the accession number of P51911 in the Unit database, Calponin selects the protein fragment at the 1 st-297 th position, which is the amino acid sequence shown in SEQ ID NO. 1; directly optimizing into a gene fragment suitable for being expressed in Escherichia coli BL 21. BamH I and XhoI cleavage sites were added to the 5 'and 3' ends of the gene during PCR, respectively.

And separating and recovering the PCR product through agarose gel electrophoresis, carrying out BamH I and XhoI enzyme digestion on the recovered fusion protein gene and a plasmid vector Pet30a for expression respectively, carrying out electrophoresis recovery again, and connecting with T4 DNA ligase. And transforming the connecting product into escherichia coli competent cells BL21, selecting clones on a plate for inoculation, and performing PCR identification on bacterial liquid. And selecting clones with positive PCR results for sequencing analysis, and using the clones with completely correct sequences.

The selection of different antigens for immunization makes it possible to prepare antibodies with different binding characteristics, the molecules present simultaneously a plurality of variants due to variable cleavage, and finally the recognition ability and pattern of different antibodies on antigen-expressing cells are different. The molecule of Calponin is analyzed according to published sequence, according to structure, antigenicity, hydrophilicity and hydrophobicity of amino acids and secondary structure on cell membrane, selecting region with suitable soluble expression and good immunogenicity for recombination expression, selecting amino acid residue 1-297 of Calponin for codon optimization, and molecular weight is about 40 kDa. The Calponin protein is obtained by using a prokaryotic expression gene sequence through sequence optimization design. The recombinant immunogen consists of antigenic protein fragments of the callonin and a protein tag for purifying the recombinant protein, wherein the protein tag is HIS.

II, protein expression and purification

The overnight strain cultured by a single colony is transferred to 100mL LB culture medium according to the proportion of 1:100, kanamycin with the final concentration of 10 mug/mL is added, shaking culture is carried out at 37 ℃ until OD600 is 0.6-0.8, 1mmol/L IPTG is added, shaking culture is carried out at 16 ℃ for overnight, and the strain is collected and then is crushed by ultrasound. The recombinant protein is provided with a histidine tag, and affinity purification of the protein is performed by using a nickel column. Elution was carried out with 500mmol/L imidazole and SDS PAGE separation was carried out.

FIG. 1 is a graph of purified pectin from recombinant Calponin protein. The protein concentration is 0.5mg/mL, and the protein can be used for requirements of animal immunization and antibody screening and identification.

EXAMPLE 2 establishment of hybridoma cell lines

Immunization

The crosslinked polypeptides of example 1 were emulsified with Freund's complete adjuvant (Sigma, F5881), 4-6 week old female SPF-grade mice (purchased from Beijing Wintolite laboratory animals technologies, Inc.) were immunized and injected ventrally subcutaneously at 6 spots per mouse at a dose of 20. mu.g/mouse. The booster was administered every 14 days and the antigen was emulsified with Freund's incomplete adjuvant (Sigma, F5506) at a dose of 20. mu.g/mouse. 7 days after 3 times of booster immunization, the titer of multiple antibodies of the anti-immunogen in the serum of the mice is detected by indirect ELISA (wavelength of 450nm), the mice with the highest titer are injected by tail vein for impact immunization, and the antigen is uniformly mixed by normal saline, and the dosage is 50 mu g/mouse.

Second, cell fusion

A mouse spleen cell suspension with qualified immunity is prepared aseptically, mixed with mouse myeloma cell sp2/0(ATCC number CRL-8287) at a ratio of 5:1, and centrifuged at 1500rpm for 5 min. The supernatant was discarded and the tube was placed in a 37 ℃ water bath, 1ml of PEG1500 (Roche) was added slowly over 1 minute, and the cells were agitated. After standing in warm water for 1min, 10ml of serum-free IMDM (Sigma Co.) was added, mixed well, and centrifuged at 1000rpm for 5 min. After discarding the supernatant, 10ml of serum (PAA) was added to the supernatant, the cells were carefully blown up, 5ml of thymocytes mixed with 10XHAT (Sigma) was added, and the mixture was mixed well. Then, 25ml of a semi-solid medium containing 2.1% nitrocellulose (Sigma) was added thereto, mixed well, and poured into 20 cell culture dishes. Placing the cell culture dish into a wet box, and adding 5% CO at 37 deg.C₂Culture in incubatorAnd (5) nourishing.

Cloning and ELISA screening of positive hybridoma cells

The size and density of the clone cell mass are moderate 7 days after fusion, and the round, solid and large clone mass is sucked under a dissecting mirror and is injected into a 96-hole culture plate which is prepared with a culture medium in advance, and the culture is carried out in a 5% CO2 incubator at 37 ℃. After 3 days, the cell mass was approximately 2/3 basal areas, and 100. mu.L of the supernatant was screened by ELISA using the immunogen and the synthetic polypeptide, respectively. Positive clones were completely replaced and 200. mu.L of complete medium containing feeder cells and 1% HT (Sigma) was added. Two days later, a second ELISA screening was performed and positive clones were transferred to 24-well plates previously prepared in medium (containing feeder cells and HT). After five days, 100 μ l of supernatant was subjected to a third ELISA screening, and the positive clones were transferred to 6-well plates and cell culture flasks successively for expanded culture and frozen.

EXAMPLE 3 preparation of monoclonal antibody by ascites Induction method

First, preparation of ascites

Cells in logarithmic growth phase were washed with serum-free medium and suspended, and counted at about 5X 10⁵And 1 ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The ascites fluid taken out was centrifuged at 4000rpm at 4 ℃ for 10 min. The ascites fluid in the middle is carefully aspirated and collected in a centrifuge tube and stored at 4 ℃ or-20 ℃.

Secondly, purification of monoclonal antibody

Antibodies were purified from ascites fluid by HiTrap rProtein A FF (GE) affinity chromatography as described. Purity was assessed on SDS-PAGE gels and concentration was determined by Bradford method. Purified antibody was stored at-20 ℃.

EXAMPLE 4 characterization of monoclonal antibodies

First, subclass identification

Coated goat anti-mouse IgG (Beijing China fir Jinqiao Biotechnology Co., Ltd.) was diluted to 0.5. mu.g/ml with 100. mu.l/well at 4 ℃ overnight in 100mM PBS (pH 7.4). The liquid was decanted, washed 3 times with PBS containing 0.05% Tween (PBS-T), 200. mu.l of blocking solution (PBS containing 2% BSA and 3% sucrose) was added to each well, and incubated at 37 ℃ for 1 h. The liquid was decanted and washed 3 times with PBS-T. 0.1ml of hybridoma supernatant was added to each well and incubated at 37 ℃ for 1 h. EmptyingThe liquid was washed 3 times with PBS-T. Using a confining liquid 1: 1000 dilution of HRP-labeled goat anti-mouse (κ, λ) antibody or 1: HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibodies (Southern Biotech) were diluted at 2000 in 0.1ml per well and added to the appropriate wells, followed by incubation at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. Add 50. mu.l of 0.15% ABTS (Southern Biotech) and 0.03% H per well₂O₂The reaction was performed in the citric acid buffer (pH4.0), and the OD value at a wavelength of 405nm was measured within 10 to 20 min.

The results show that the monoclonal antibody of the invention is IgG_2bType murine monoclonal antibodies.

Second, determination of affinity constant

The recombinant protein of Calponin prepared in example 1 was taken at a coating concentration of 2. mu.g/ml, 100. mu.l/well, coated overnight at 4 ℃ and washed 3 times with PBS-T. Add 200. mu.l of blocking solution to each well and block for 2h at 37 ℃ and wash 3 times with PBS-T. The monoclonal antibody purified in example 4 was prepared from 1: 200 began a 2-fold gradient dilution, and finally 1 well was blank, incubated at 37 ℃ for 1h, and washed 3 times with PBS-T. HRP-labeled goat anti-mouse secondary antibody 1: 20000 dilution, 100 μ l per well, incubation at 37 ℃ for 1h, PBS-T wash 3 times. Mu.l of a buffer containing 0.1% TMB (Sigma) and 0.03% H was added to each well₂O₂The reaction mixture was developed in a citric acid-phosphoric acid buffer for 10min, and 50. mu.l of a 0.5M sulfuric acid solution was added thereto to terminate the reaction. And measuring the light absorption value with the wavelength of 450nm by using a microplate reader. The OD value is plotted against the dilution factor of the antibody, the dilution factor A corresponding to half of the maximum binding OD value is found, and the affinity constant of the antibody is calculated to be 7.68X 10 by using the following formula⁹。

Reaction specificity and application effect of monoclonal antibody

The identification specificity of the monoclonal antibody of the present invention was determined by immunoblotting using the recombinant Calponin protein prepared in example 1, and 12% polyacrylamide gel electrophoresis was performed. The gel protein bands were transferred to PVDF membranes (Millipore) in a Bio-Rad electrotransfer system according to the conventional method. The membrane was placed in TBS-T blocking solution containing 5% skimmed milk powder overnight at 4 ℃. Monoclonal antibodies to the antibody secreted by the 24H5 hybridoma (1: 1000 dilution) were added and incubated overnight at 4 ℃. After washing the membrane with TBS-T, add 1: a5000-diluted goat anti-mouse secondary antibody (Beijing Zhonghua Jinqiao Biotech Co., Ltd.) was incubated at room temperature for 1 hour. Washing the membrane with TBST again, adding ECL (Beijing prilley Gene technology Co., Ltd.), and collecting chemiluminescence image data with ChemiDocMP multicolor fluorescence imaging system (Bio-Rad).

Example 5 determination of variable region sequences of antibodies

Collecting fresh hybridoma cells, collecting supernatant, performing antigen binding property verification to confirm that the cell strain for cloning can indeed secrete required antibody, and centrifuging to collect 10⁶The hybridoma cells described above. Total RNA of hybridoma cells was extracted by Trizol method, and 9. mu.L of total RNA was added with 2.5. mu.L of oligo (dT) 12-18 primer (10mM) and 5. mu.L of dNTPs, mixed well, incubated at 70 ℃ for 5 minutes and then placed on ice for 5 minutes, or denatured according to the reverse transcriptase used. Then, 5. mu.L of RT buffer (5X), 2.5. mu.L of DTT (0.1M) and 1. mu.L of reverse transcriptase were added and reacted at 42 ℃ for 1 hour. The reaction was terminated by incubation at 70 ℃ for 15 minutes, and the obtained cDNA was stored at-20 ℃. The first strand cDNA thus obtained was subjected to PCR amplification, and 25pmol each of primers was added to a 50. mu.L reaction system, and the sequences of the primers for amplification of the heavy chain variable region and the light chain variable region were designed and synthesized based on the sequence of the murine monoclonal antibody primer as described in "recombinant antibody" by Hippo Seiyaku (science publishers, 2005 publications).

The rest dNTPs and buffer are added according to the conventional method, and finally 1 mu L of cDNA template and 1U of hot start Taq DNA polymerase are added. Setting PCR amplification program as 94 deg.c for 40 sec, 52 deg.c for 40 sec, 72 deg.c for 40 sec, 20-25 cycles, final extension at 72 deg.c for 3 min, and setting the product at 4 deg.c for use or direct electrophoresis. 20 μ L of PCR product was analyzed by electrophoresis, separated on 1.5% agarose gel, the length of light chain (. kappa.light chain) was between 320-340bp and the length of heavy chain was between 340-370bp, and the region-specific product was recovered by gel cutting and cloned into T-vector or expression vector for sequencing.

Example 6 immunohistochemical tissue chip staining and identification

Chip preparation process

HE sections were first stained for each sample to identify tumor sites. The tissue chip was produced using a fully automatic tissue chip machine of 3 DHISTECH. And putting the prepared tissue chip wax block into a wax block manufacturing mold, putting the mold into an oven at 68 ℃ for 10 minutes to enable the tissue core and the wax of the receptor wax block to be fused into a whole, then slightly taking the mold out of the oven, cooling the paraffin in a semi-molten state for about 30 minutes at room temperature, putting the mold into a refrigerator at-20 ℃ for freezing for 6 minutes, taking the tissue chip wax block out of the mold, and slicing or putting the tissue chip wax block into the refrigerator at 4 ℃ for storage for later use. And (3) trimming, continuously slicing, setting the thickness to be 3 mu m, floating the continuous slices in 40% alcohol, naturally unfolding, transferring the separated slices into warm water at 50 ℃ for 30 seconds, pasting the slices with a glass slide treated by polylysine, baking the prepared tissue chip in an oven at 68 ℃ for 2 hours, taking out, cooling at room temperature, and storing in a refrigerator at-4 ℃.

IHC staining and analysis

Conventional xylene dewaxing was performed 3 times for 6 minutes each, 100%, 95%, 85% gradient ethanol hydration for 3 minutes each, and finally tap water rinse. Antigen retrieval was performed and the sections were then placed in a wet box and washed 3 x 3 min with PBS. Dropwise adding 3% H₂O₂Incubate for 10min and wash with PBS for 3 × 3 min. Spin-drying the slices, dripping primary antibody diluted in a proper proportion (the dilution proportion of the antibody is designed according to the concentration of the antibody in the primary dilution) and incubating for 1 hour at room temperature (25 ℃), washing for 3X 3 minutes by PBS, dripping secondary antibody and incubating for 15-30 minutes at room temperature, washing for 3X 3 minutes by PBS, throwing off the PBS, and developing for 3-10 minutes by using a freshly prepared DAB developing solution. Hematoxylin counterstain for 25 seconds, PBS bluing for 30 seconds. Dehydration was carried out in a gradient of 85% (3 min) -95% (3 min) -100% (3 min) alcohol, finally xylene was transparent for 3 min, and the gel was blocked with neutral gum.

The immunohistochemical staining results were divided into: positive and negative. Positive expression must be at a specific antigenic site in cells and tissues to be considered positive. Under the condition that the tissue staining distribution is clear and the cell positioning is accurate, the staining result is further divided according to the difference of staining intensity, which is as follows:

1. the sample is weakly positive; marked "+";

2. the sample is moderately positive; marked "+";

3. the sample is highly positive; marked as "+ + +".

4. The sample was negative and marked "-".

Data statistics

1. Tumor tissue chip detection results:

the antibody Calponin (24H5) and the commercial antibody Calponin (CALP) are synchronously detected on different human tumor tissue chips and the detection results are compared. The immunohistochemical results of Calponin were counted. The whole experimental process adopts a double-blind design, and the statistical results are as follows:

the result shows that the monoclonal antibody of the anti-Calponin protein secreted by the 24H5 cell strain has accurate staining positioning, clear staining, no non-specific staining and clean background. In immunohistochemical detection, the positive rate is equivalent to that of a commercially available antibody, and the positive intensity of most samples is higher than that of the commercially available antibody, so that the sensitivity is higher, and false negative results are effectively avoided.

FIG. 2 is a graph comparing the results of immunohistochemical staining of uterine fibroids (Calponin (24H5) on the left and commercial Calponin on the right).

2. Detection results of the normal tissue chip:

the normal tissue chip comprises 30 normal tissue samples, and the normal tissue samples are mainly selected from fresh and timely fixed operation specimens; each tissue included 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectal, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (young child), skin, bone marrow, peripheral nerve, lung, mesothelial cells.

The antibody (24H5) and the commercial antibody (TA347) were detected simultaneously on the normal tissue chip, and the negative and positive detection results were consistent, indicating that the specificity of the antibody in the normal tissue was equivalent to that of the commercial antibody. Meanwhile, the commercial antibody has background staining in liver tissues, but the antibody does not, which indicates that the specificity of the antibody is higher.

FIG. 3 is a graph comparing the results of immunohistochemical staining of liver tissues (Calponin (24H5) on the left and commercial Calponin on the right).

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.

It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.

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Claims

1. An anti-Calponin monoclonal antibody, characterized in that the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown as SEQ ID 5.

2. The monoclonal antibody of claim 1, wherein the DNA sequence encoding the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID2, and the DNA sequence encoding the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID 3.

3. The monoclonal antibody according to claim 1, wherein the monoclonal antibody specifically recognizes a Calponin protein.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG_2bSubtype monoclonal antibodies.

5. The monoclonal antibody of claim 1, which is produced by a hybridoma cell line with a collection number of CGMCC NO 20775.

6. A preparation method of an anti-Calponin protein monoclonal antibody is characterized in that an antigen used for immunizing a mouse is recombinant protein, and the recombinant protein is expressed by escherichia coli in a recombinant mode and comprises a Calponin protein fragment and a histidine protein tag; the protein fragment of the callonin is the amino acid fragment from the 1 st position to the 297 th position of the callonin protein, and the amino acid sequence of the protein fragment is the amino acid sequence shown in SEQ ID NO. 1.

7. A hybridoma cell strain capable of secreting anti-calcinin protein molecules is a mouse hybridoma cell strain 24H5, and is preserved in China general microbiological culture Collection center with the preservation number: CGMCC NO 20775.

8. Use of the monoclonal antibody of any one of claims 1 to 5 in an immunoassay for a Calponin protein.

9. The use according to claim 8, wherein said immunodetection comprises immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay.

10. An immunohistochemical detection reagent for Calponin protein, comprising the anti-Calponin protein monoclonal antibody according to claim 1 as an active ingredient.