CN113265003B - anti-TdT protein monoclonal antibody, cell strain thereof, preparation method and application - Google Patents

Abstract

The invention relates to a monoclonal antibody capable of identifying human TdT antigen, a secretory cell strain, a preparation method thereof and application thereof in immunoassay. In the technical scheme, 1-150 amino acids at the C terminal of the TdT protein are selected as antigen peptides, codon optimization is carried out, a gene fragment suitable for expression in escherichia coli BL21(DE3) is obtained, and finally the obtained recombinant protein comprises the TdT protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 32D5 capable of efficiently secreting the anti-TdT protein monoclonal antibody and the anti-TdT 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 TdT protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Description

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

Technical Field

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

Background

Terminal deoxynucleotidyl transferase (TdT) is an enzyme in the nucleus, which is a template-free DNA polymerase that catalyzes the binding of deoxynucleotides to the 3' -OH end of a DNA molecule. The molecular weight of TdT is 32000 dalton, and the TdT is composed of alpha peptide chain and beta peptide chain, and the molecular weights of the two are 8000 dalton and 24000 dalton respectively.

The TdT gene is located on human chromosome 10q23-q24 and on murine chromosome 19. TdT has various functions, and the main functions are as follows: TdT can be used as a DNA polymerase without a template and can catalyze the combination of deoxynucleotide to the 3 '-OH end of a DNA molecule, for example, the 3' -OH end of a DNA strand break is fluorescently labeled by using exogenous terminal deoxynucleotidyl transferase; the research of Motea E A and the like finds that: during antibody gene rearrangement, TdT may add ribonucleotides to the exonic regions of T and B cell receptors V, D and J; TDT can be expressed in thymus gland tissue under normal conditions, and can also be expressed in tonsil and lymph node of children and young people in small amount.

TdT has important significance in diagnosing lymphohematopoietic tumors, is expressed negatively in tumor cells of acute myeloid leukemia, is expressed positively in tumor cells of acute lymphocytic leukemia, and can be used as a marker for identifying acute lymphocytic leukemia and acute myeloid leukemia. In addition, TdT may be expressed on naive T and B lymphocytes, expressed in T and B lymphoblastic lymphomas, and commonly used in the pathological diagnosis of thymomas and T, B lymphoblastic lymphomas.

In addition, TdT may also be expressed in a variety of tumors, including sebaceous gland tumors, neuroendocrine tumors, urinary system tumors, and the like. Buresh CJ et al found that TdT could be expressed in Merkel cell carcinoma with a positive rate of 73%. Sidiropoulos M et al found that TdT was 70% (28/40) positive in Merkel cell carcinoma, 25% of which was moderately strong positive, and only 7% positive in small cell lung carcinoma (2/30). Kolhe R et al found that TdT was expressed in Merkel cell carcinoma and small cell lung carcinoma at 78% (21/27) and 90% (9/10), respectively. The studies by Brobeil A et al found that TdT was positive (moderate to strong positive) in 10 cases of seminal cell tumors.

Disclosure of Invention

The invention provides an anti-TdT protein monoclonal antibody, which is produced by a hybridoma cell strain with the preservation number of CGMCC NO 20777.

Further, the monoclonal antibody specifically recognizes the TdT protein.

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 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 monoclonal antibody is mouse IgG_2bSubtype monoclonal antibodies.

The inventor also provides a preparation method of the anti-TdT protein monoclonal antibody, and the antigen used for immunizing a mouse is recombinant protein which is expressed by escherichia coli in a recombinant mode and comprises a TdT protein fragment and a histidine protein tag.

Further, the TdT protein fragment is an amino acid fragment from 1 st to 150 th positions of the TdT protein, and the amino acid sequence of the TdT protein fragment is the amino acid sequence shown in SEQ ID NO. 1.

The inventor also provides a hybridoma cell strain secreting TdT-resistant protein molecules, wherein the cell strain is a mouse hybridoma cell strain 32D5 which is preserved in China general microbiological culture Collection center (CGMCC) at 9 and 17 days 2020, has the preservation number of CGMCC NO 20777 and is addressed to the institute of microbiology of China academy of sciences No. 3 of the North West Lu 1 Hospital of the rising area in Beijing.

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

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

Different from the prior art, the invention has the beneficial technical effects that: in the technical scheme, the 1 st to 150 th amino acids at the N tail end of the TdT protein are selected as antigen peptides, codon optimization is carried out, a gene fragment suitable for being expressed in escherichia coli BL21 is obtained, and finally the obtained recombinant protein comprises the TdT protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 32D5 capable of efficiently secreting the anti-TdT protein monoclonal antibody and the anti-TdT 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 TdT protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a graph of purified pectin of recombinant TdT protein, wherein M represents Marker; 1 is a supernatant sample after ultrasonic treatment; and 2 is a post-sonication sedimentation sample.

FIG. 2 shows the result of immunoblotting detection of recombinant TdT protein.

FIG. 3 is a graph comparing immunohistochemical staining results for lymphoma; the left is TdT secreted by 32D5, and the right is TdT sold on the market.

FIG. 4 is a graph comparing the results of immunohistochemical staining of thymus; the left is TdT secreted by 32D5, and the right is TdT sold on the market.

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 TdT protein fragments

First, gene optimization and synthesis

TdT was directly optimized into a gene fragment suitable for expression in E.coli BL21(DE3) by selecting a protein fragment with amino acids 1-150 according to the protein sequence with accession number P04053 in the Unit prot database. EcoR I and XhoI cleavage sites were added at the 5 'and 3' ends of the gene, respectively, during PCR.

And separating and recovering the PCR product through agarose gel electrophoresis, carrying out EcoR 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. The ligation product was transformed into E.coli competent cells BL21(DE3), and the colonies on the plate were picked and inoculated for PCR identification of the bacterial suspension. 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. TdT molecules are analyzed according to published sequences, regions which are suitable for soluble expression and have good immunogenicity are selected for recombinant expression according to the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and secondary structure on cell membranes, amino acid residues 1-150 of the TdT are selected for codon optimization, and the molecular weight is about 27 kDa. The TdT protein is obtained by using prokaryotic expression gene sequence through sequence optimization design. The recombinant immunogen consists of a TdT 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.

FIG. 1 is a diagram of purified pectin of recombinant TdT 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), and 4-6 week old female ICR mice (purchased from Beijing Wintolite laboratory animals technologies, Inc.) were immunized and injected ventrally at 6 o' clock 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 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, I) diluted 2000gG2b, IgG3, IgA) antibody (Southern Biotech) 0.1ml per well was added to each appropriate well and incubated at 37 ℃ for 1 h. 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 TdT recombinant protein prepared in example 1 was coated at a 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. Drawing a curve of OD value corresponding to the dilution factor of the antibody, finding the dilution factor A corresponding to half of the maximum binding OD value, and calculating the affinity constant of the antibody to be 1.92 multiplied by 10 by using the following formula⁹。

Reaction specificity and application effect of monoclonal antibody

The TdT 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 ℃. Monoclonal antibodies (1: 1000 dilution) of the antibody secreted by the 32D5 hybridoma 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).

FIG. 2 is a diagram showing the result of immunoblotting detection of recombinant TdT protein, and the molecular weight is 58 kDa.

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 TdT (32D5) and the commercial antibody TdT (SEN28) were tested simultaneously on different human tumor tissue chips (including 14 cases of lymphoma and 100 cases of other tumors) and the results were compared.

The immunohistochemical results of TdT 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-TdT protein secreted by the 32D5 cell strain has accurate staining positioning, clear staining, no non-specific staining and clean background. In the immunohistochemical detection, the positive rate is equivalent to that of the commercial antibody, wherein the positive intensity of 2 cases of lymphoma is higher than that of the commercial antibody, which indicates that the sensitivity is higher, and the false negative result is effectively avoided.

FIG. 3 is a comparison of immunohistochemical staining results for lymphoma (TdT, 32D5 on the left and commercial TdT 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 (32D5) 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.

FIG. 4 is a comparison of the results of thymocyte immunohistochemical staining (TdT (32D5) on the left and TdT on the market 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.

SEQUENCE LISTING

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

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

<130> 2021

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 150

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 1

Met Asp Pro Pro Arg Ala Ser His Leu Ser Pro Arg Lys Lys Arg Pro

1 5 10 15

Arg Gln Thr Gly Ala Leu Met Ala Ser Ser Pro Gln Asp Ile Lys Phe

20 25 30

Gln Asp Leu Val Val Phe Ile Leu Glu Lys Lys Met Gly Thr Thr Arg

35 40 45

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

50 55 60

Asn Glu Leu Ser Asp Ser Val Thr His Ile Val Ala Glu Asn Asn Ser

65 70 75 80

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

85 90 95

Ser Gln Pro Glu Leu Leu Asp Val Ser Trp Leu Ile Glu Cys Ile Arg

100 105 110

Ala Gly Lys Pro Val Glu Met Thr Gly Lys His Gln Leu Val Val Arg

115 120 125

Arg Asp Tyr Ser Asp Ser Thr Asn Pro Gly Pro Pro Lys Thr Pro Pro

130 135 140

Ile Ala Val Gln Lys Ile

145 150

<210> 2

<211> 339

<212> DNA

<213> Artificial sequence (Artificial)

<400> 2

gaggtccagc tgcagcagtc tggggctgag cttgtgaggc caggggcctc agtcaagttg 60

tcctgcacaa cttctgactt taacattcaa gacgactata tgtactgggt gaaacagagg 120

cctgaacagg gcctggagtg gattggatgg attgatcctg aaaatggtga tactgcatat 180

gcctcgaagt tccagggcaa ggccactata acagcagaca cattttccaa cacagcctac 240

ctgcagctca gcagcctgac atctgaggac tctgccgtct attactgtgc tgcaggagga 300

ggggactggg gccaaggcac cactctcaca gtctcctca 339

<210> 3

<211> 336

<212> DNA

<213> Artificial sequence (Artificial)

<400> 3

gatatcttga cgacccaaac tccactctct ttgtcggtta ccattggaca accagcctct 60

atctcttgca agtcaagtca gagcctctta tatagtgatg gaaagacata tttgaattgg 120

ttacaacaga ggccaggcca gtctccaaag cgcctaatgt atcaggtgtc caaagtggac 180

cctggcatcc ctgacaggtt cagtggcagt ggatcacaga cacattttac acttaaaatc 240

agcagagtgg aggctgagga tttgggagtt tattactgct tgcaaggttc tttttatccg 300

tggacattcg gtggaggcac caagctggaa atcaaa 336

<210> 4

<211> 113

<212> PRT

<213> Artificial sequence (Artificial)

<400> 4

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

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Thr Ser Asp Phe Asn Ile Gln Asp Asp

20 25 30

Tyr Met Tyr Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Phe Ser Asn Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Ala Gly Gly Gly Asp Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

100 105 110

Ser

<210> 5

<211> 112

<212> PRT

<213> Artificial sequence (Artificial)

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

Pro Lys Arg Leu Met Tyr Gln Val Ser Lys Val Asp Pro Gly Ile Pro

50 55 60

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

65 70 75 80

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

85 90 95

Ser Phe Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Claims (8)

1. An anti-TdT monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC NO 20777.

2. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes the TdT protein.

3. 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.

4. The monoclonal antibody according to claim 1, 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.

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

6. A hybridoma cell strain secreting TdT-resistant protein molecules is a mouse hybridoma cell strain 32D5, and is preserved in the China general microbiological culture Collection center with the preservation number: CGMCC NO 20777.

7. Use of the monoclonal antibody of any one of claims 1-5 in the preparation of an immunodetection reagent for TdT protein.

8. The use according to claim 7, wherein said immunodetection comprises immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay.

Images