CN113105547A

CN113105547A - anti-CD 5 protein monoclonal antibody and cell strain, preparation method and application thereof

Info

Publication number: CN113105547A
Application number: CN202110550992.5A
Authority: CN
Inventors: 杨清海; 陈惠玲; 王小亚; 李婷; 赵普
Original assignee: Fuzhou Maixin Biotechnology Development Co ltd
Current assignee: Fuzhou Maixin Biotechnology Development Co ltd
Priority date: 2021-05-18
Filing date: 2021-05-18
Publication date: 2021-07-13
Anticipated expiration: 2041-05-18
Also published as: CN113105547B

Abstract

The invention relates to a monoclonal antibody capable of identifying human CD5 antigen, a secretory cell strain, a preparation method thereof and application thereof in immunoassay. According to the technical scheme, amino acids from 398 th site to 495 th site at the C tail end of the CD5 protein are selected as antigen peptides to perform codon optimization to form a gene fragment suitable for being expressed in escherichia coli BL21, and finally the obtained recombinant protein comprises a CD5 protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 11H3 capable of efficiently secreting the anti-CD 5 protein monoclonal antibody and the anti-CD 5 protein 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 CD5 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Description

anti-CD 5 protein monoclonal antibody and cell strain, preparation method and application thereof

Technical Field

The invention relates to the field of biomedical engineering, in particular to a CD5 protein resistant monoclonal antibody, a cell strain, a preparation method and application thereof.

Background

The human CD5 gene is located on chromosome 11q12.2, the CD5 molecule is a 67KD transmembrane glycoprotein expressed in a subset of T cells and virginal B cells, CD5 is a member of a family of receptors with a cysteine-rich (SRCR) domain homeodomain of the macrophage scavenger receptor, and these CD5 positive B cells can produce autoantibodies and can also spread in autoimmune diseases and malignancies of lymphoid tissues such as leukemia, MCL and CD5 positive DLBCL. Unlike mouse B1 cells expressing CD5, human B1 cells do not have expression of CD 5. Experiments on a nonfunctional egg lysozyme transgenic model of cells with mouse and chicken CD5 gene knocked out show that in vitro experiments, CD5 expression promotes cells to resist autoreactive antigens, and the deletion of CD5 molecules can enhance the proliferation reaction of the cells.

CD5 has been recognized as a negative regulator of B cell receptor signaling, altering the mobilization of intracellular calcium ions, and modulating B cell function by activating a range of signaling pathways including the ERK1/2, PI3K, and the calcineurin pathway. In addition, CD5 promotes B cell survival by stimulating the production of autocrine IL-10, another study showed that CD5 is phosphorylated in CLL B cells and that certain genes are overexpressed in CLL B cells, such as BCL2, certain chemokines, and cytokines. Furthermore, CLL B cells positive for CD5 were shown to promote CLL malignant cell phenotype and metabolic pathways. These results indicate that expression of CD5 in B cells affects malignant lymphoma cells in a number of different ways. The exact mechanism that leads to over-expressed CD5 in DLBCL is not clear. Most studies on the effects of CD5 on B cell function were performed on cells from patients with autoimmune disease and Chronic Lymphocytic Leukemia (CLL).

Disclosure of Invention

The inventor provides an anti-CD 5 monoclonal antibody, wherein 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.

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 CD5 protein.

Further, the monoclonal antibody is a mouse IgG1 kappa subtype monoclonal antibody.

Further, the hybridoma cell line is produced by a hybridoma cell line with the preservation number of CGMCC NO 20767.

The inventor also provides a preparation method of the anti-CD 5 protein monoclonal antibody, the antigen used for immunizing mice is recombinant protein, the recombinant protein is expressed by escherichia coli recombination, and comprises a CD5 protein fragment and a histidine protein tag; the CD5 protein fragment is the amino acid fragment from 398 th position to 495 th position of the CD5 protein, and the amino acid sequence of the CD5 protein fragment is the amino acid sequence shown in SEQ ID NO. 1.

The inventor also provides a hybridoma cell strain secreting anti-CD 5 protein molecules, wherein the cell strain is a mouse hybridoma cell strain 11H3, the cell strain is preserved in China general microbiological culture Collection center (CGMCC NO 20767) at 9 and 17 months of 2020, and the accession number is CGMCC NO 20767, and the cell strain is the institute of microbiology of China academy of sciences No. 3 of the North West Lu No.1 Hopkins of the sunward Yang ward region in Beijing.

The inventor also provides the application of the monoclonal antibody in immunodetection of the CD5 protein.

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

The inventor finally provides a CD5 protein immunohistochemical detection reagent, wherein the immunohistochemical detection reagent contains an amino acid sequence of which the amino acid sequence of a heavy chain variable region is shown as SEQ ID 4; 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, amino acids from 398 th site to 495 th site at the C tail end of the CD5 protein are selected as antigen peptides to perform codon optimization to form a gene fragment suitable for being expressed in escherichia coli BL21, and finally the obtained recombinant protein comprises a CD5 protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 11H3 capable of efficiently secreting the anti-CD 5 protein monoclonal antibody and the anti-CD 5 protein 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 CD5 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a graph comparing the results of immunohistochemical staining of T cell lymphoma (left 11H 3-secreted CD5, right commercial CD 5).

FIG. 2 is a graph comparing the results of immunohistochemical staining of T cell lymphoma (left 11H 3-secreted CD5, right commercial CD 5).

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 CD5 protein fragment

First, gene optimization and synthesis

CD5 selects a protein fragment at position 398-495 as an amino acid sequence shown in SEQ ID NO.1 according to a protein sequence with an accession number of O43866 in a Uniprot database; 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 CD5 molecule was analyzed according to published sequences, and based on the structure, antigenicity, hydrophilicity and hydrophobicity of the constituent amino acids and secondary structure on the cell membrane, a region suitable for soluble expression and good immunogenicity was selected for recombinant expression, and amino acid residues 398-495 of CD5 were selected for codon optimization, with a molecular weight of approximately 36 kDa. The CD5 protein is obtained by using prokaryotic expression gene sequence through sequence optimization design. The recombinant immunogen consists of a CD5 protein fragment with antigenicity and a protein tag for recombinant protein purification, 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.

EXAMPLE 2 establishment of hybridoma cell lines

Immunization

The recombinant protein of example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), and 4-6 week-old female SPF-grade mice (purchased from Beijing Wintolite laboratory animals technologies, Ltd.) were immunized and injected subcutaneously at 6 spots into the abdomen at a dose of 60. 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 20 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.C₂Culturing in an incubator.

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. The decanted solution 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 a murine monoclonal antibody of the IgG1 kappa type.

Second, determination of affinity constant

Weighing the CD5 prepared in example 1Histone, coating concentration of 2. mu.g/ml, 100. mu.l/well, 4 ℃ coated overnight, PBS-T washing 3 times. 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 CD5 recombinant protein prepared in example 1 was used to detect the recognition specificity of the monoclonal antibody of the present invention by immunoblotting, 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 ℃. CD5 monoclonal antibody (1: 1000 dilution) secreted by 11H3 hybridoma was 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 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 CD5(11H3) and the commercial antibody CD5(SP19) were tested simultaneously on different human tumor tissue chips (35 cases of T-cell lymphoma and 20 cases of B-cell lymphoma) and the test results were compared. Immunohistochemical results for CD5 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-CD 5 protein secreted by the 11H3 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 a part of samples is higher than that of the commercially available antibody, so that the sensitivity is higher, and the false negative result is effectively avoided.

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 (11H3) 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.

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.

Sequence listing

<110> Fuzhou mai New Biotechnology development Co., Ltd

<120> anti-CD 5 protein monoclonal antibody and cell strain, preparation method and application thereof

<130> 2021

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 98

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 1

Gly Pro Leu Ala Tyr Lys Lys Leu Val Lys Lys Phe Arg Gln Lys Lys

1 5 10 15

Gln Arg Gln Trp Ile Gly Pro Thr Gly Met Asn Gln Asn Met Ser Phe

20 25 30

His Arg Asn His Thr Ala Thr Val Arg Ser His Ala Glu Asn Pro Thr

35 40 45

Ala Ser His Val Asp Asn Glu Tyr Ser Gln Pro Pro Arg Asn Ser His

50 55 60

Leu Ser Ala Tyr Pro Ala Leu Glu Gly Ala Leu His Arg Ser Ser Met

65 70 75 80

Gln Pro Asp Asn Ser Ser Asp Ser Asp Tyr Asp Leu His Gly Ala Gln

85 90 95

Arg Leu

<210> 2

<211> 339

<212> DNA

<213> Artificial sequence (Artificial)

<400> 2

gaggtccagc tgcaggagtc aggacctagc ctggtgaagc ctggggcctc agtgaagatt 60

tcctgcaagg cttctggcta tgcattcagt ggctcctggc tgaactgggt gaagcagagg 120

cctggaaagg gtcttgagtg gattggacgg atttatcctg gagatggaca tattgactac 180

aatgggaagt tcaagggcaa ggccacactg actgttgaca aatcctccaa cacagcctat 240

attcaactca gcagcctgac atctgaagac tctgcggtct acttctgttc aagaacgttt 300

gcttactggg gccaagggac tctggtcact gtctctgca 339

<210> 3

<211> 336

<212> DNA

<213> Artificial sequence (Artificial)

<400> 3

gatatcttga tgacccaatc tccactctct ttgtctgtta ccattggaca gccagtttcc 60

atttcttgta agtcaagtca gagcctccta tatagtgatg gaagaaccta tttgaattgg 120

ttattacaga gtccaggcca gtctccaaag ctcctaatct atctggtgtc tacactggat 180

tctggagtcc ctgacagatt cagtggcagt ggatcaggga cagattttac actgaaaatc 240

agcagagtgg aggctgagga tttgggaatt tattactgcg tgcaagttac acatttccct 300

catacgttcg gatcggggac caagctggaa ataaaa 336

<210> 4

<211> 113

<212> PRT

<213> Artificial sequence (Artificial)

<400> 4

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

1 5 10 15

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

20 25 30

Trp Leu Asn Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Tyr Pro Gly Asp Gly His Ile Asp Tyr Asn Gly Lys Phe

50 55 60

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

65 70 75 80

Ile Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

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

100 105 110

Ala

<210> 5

<211> 112

<212> PRT

<213> Artificial sequence (Artificial)

<400> 5

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

1 5 10 15

Gln Pro Val Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Asp Gly Arg Thr Tyr Leu Asn Trp Leu Leu Gln Ser Pro Gly Gln Ser

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Claims

1. An anti-CD 5 monoclonal antibody, wherein 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 of claim 1, wherein the monoclonal antibody specifically recognizes the CD5 protein.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG1 kappa subtype monoclonal antibody.

5. The monoclonal antibody of claim 1, which is produced by a hybridoma cell line with a collection number of CGMCC NO 20767.

6. A preparation method of anti-CD 5 protein monoclonal antibody is characterized in that an antigen used for immunizing a mouse is recombinant protein, the recombinant protein is expressed by escherichia coli in a recombinant mode, and comprises a CD5 protein fragment and a histidine protein tag; the CD5 protein fragment is the amino acid fragment from 398 th position to 495 th position of the CD5 protein, and the amino acid sequence of the CD5 protein fragment is the amino acid sequence shown in SEQ ID NO. 1.

7. A hybridoma cell strain capable of secreting anti-CD 5 protein molecules is a mouse hybridoma cell strain 11H3, is preserved in China general microbiological culture Collection center (CGMCC), and has the preservation number: CGMCC NO 20767.

8. Use of the monoclonal antibody of any one of claims 1-5 in an immunoassay for CD5 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 CD5 protein, comprising the anti-CD 5 monoclonal antibody according to claim 1 as an active ingredient.