CN113072642A - Monoclonal antibody of anti-OCT 3/4 protein and cell strain, preparation method and application thereof - Google Patents

Abstract

The invention relates to a monoclonal antibody capable of identifying human OCT3/4 antigen, a secretory cell strain, a preparation method thereof and application thereof in immunoassay. In the technical scheme, 1-65 amino acids at the C terminal of OCT3/4 protein are selected as antigen peptides to perform codon optimization to form a gene fragment suitable for expression in Escherichia coli BL21(DE3), and finally the obtained recombinant protein comprises a TRX protein tag, an OCT3/4 protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 41B1 capable of efficiently secreting the anti-OCT 3/4 protein monoclonal antibody and the anti-OCT 3/4 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 identify cells expressing OCT3/4 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Description

Monoclonal antibody of anti-OCT 3/4 protein and cell strain, preparation method and application thereof

Technical Field

The invention relates to the field of biomedical engineering, in particular to an anti-OCT 3/4 protein monoclonal antibody, a cell strain thereof, a preparation method and application.

Background

Oct3/4 is a transcription factor of the POU family, encoded by the Pousn gene. Oct3/4 is a marker of cell totipotency, and is capable of promoting the formation of an inner cell mass, maintaining an undifferentiated state of an embryonic stem cell, and promoting the proliferation thereof. The gene knockout test of Oct3/4 shows that Oct3/4 plays an important role in the process of embryonic development. Niwa et al have established that Oct3/4 plays an important role in maintaining normal development of embryos by promoting or inhibiting Oct3/4 expression in Es cells. The results indicate that the expression level of Oct3/4, which is critical for maintaining normal self-renewal of embryonic stem cells at an accurate level, is critical to the expression level of Oct3/4, which determines three different fates of embryonic stem cells.

The results of the in vivo experiments corresponded well to those of the in vitro experiments. These results show that the high or low energy of Oct3/4 expression controls the growth and development of embryonic stem cell, and that its up-regulation or down-regulation makes the embryonic stem cell tend to differentiate and dedifferentiate. The expression of the target gene of Oct3/4 in different tissues of the embryo regulates the expression of these genes so that the normal development of the embryo can be maintained.

OCT3/4 has been proved to have higher clinical application value in the diagnosis and typing of germ cell tumor. It is known that the generation of germ cells is regulated by signaling pathways such as BMP, Kit/KL, FGFs, and TGF-activin/nodal, while Oct3/4 is involved in the regulation of signaling pathways such as BMP and FGF as a transcription factor. Therefore, the scholars speculate that the overexpression of Oct3/4 is related to the differentiation abnormality of germ cells and the formation of GCTs. Jones et al selected 64 mixed GCTs and performed Oct3/4 immunohistochemical analysis, with strong positive expression of Oct3/4 in all cells of embryonal carcinoma and seminoma components, and negative expression in yolk sac, teratoma and choriocarcinoma.

Disclosure of Invention

The inventor provides an anti-OCT 3/4 protein monoclonal antibody, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 5.

Furthermore, the DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO.2, and the DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO. 3.

Further, the monoclonal antibody specifically recognizes the OCT3/4 protein.

Further, the monoclonal antibody is mouse IgG_2bSubtype monoclonal antibodies.

Furthermore, the monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC NO 20779.

The inventor also provides a preparation method of the anti-OCT 3/4 protein monoclonal antibody, the antigen used for immunizing mice is recombinant protein, the recombinant protein is expressed by escherichia coli recombination, and comprises a GST protein tag, an OCT3/4 protein fragment and a histidine protein tag.

Furthermore, the OCT3/4 protein fragment is the amino acid fragment from the 1 st to the 65 th positions of the OCT3/4 protein, and the amino acid sequence of the OCT3/4 protein fragment is the amino acid sequence shown in SEQ ID NO. 1.

The inventor also provides a hybridoma cell strain secreting anti-OCT 3/4 protein molecules, wherein the cell strain is a mouse hybridoma cell strain 41B1 which is preserved in China general microbiological culture Collection center (CGMCC NO 20779) at 9 and 17 days 2020, and the preservation number is CGMCC NO 20779, and the address is the institute of microbiology of China academy of sciences No.3 of the Navy region, North Jing city, North Cheng West Lu No.1 institute of China.

The inventor also provides the application of the monoclonal antibody in OCT3/4 protein immunoassay.

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

The inventor finally provides an OCT3/4 protein immunohistochemical detection reagent, wherein the immunohistochemical detection reagent contains an amino acid sequence of a heavy chain variable region, which is an amino acid sequence shown in SEQ ID NO. 4; the monoclonal antibody of anti-OCT 3/4 protein with the amino acid sequence of the light chain variable region as shown in SEQ ID NO.5 is the effective component.

Different from the prior art, the invention has the beneficial technical effects that: in the technical scheme, 1-65 amino acids at the C terminal of OCT3/4 protein are selected as antigen peptides to perform codon optimization to form a gene fragment suitable for expression in Escherichia coli BL21(DE3), and finally the obtained recombinant protein comprises a TRX protein tag, an OCT3/4 protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 41B1 capable of efficiently secreting the anti-OCT 3/4 protein monoclonal antibody and the anti-OCT 3/4 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 identify cells expressing OCT3/4 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a graph of purified pectin of example 1 fusion histidine-tagged recombinant OCT3/4 protein, where M represents Marker; 1 is a supernatant sample after ultrasonic treatment; and 2 is a post-sonication sedimentation sample.

FIG. 2 is a graph comparing immunohistochemical staining results of seminoma; the left is OCT3/4 secreted by 41B1, and the right is commercial OCT 3/4.

FIG. 3 is a graph comparing the immunohistochemical staining results of clonal cell tumors; the left is OCT3/4 secreted by 41B1, and the right is commercial OCT 3/4.

FIG. 4 is a graph comparing immunohistochemical staining of testis; the left is OCT3/4 secreted by 41B1, and the right is commercial OCT 3/4.

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 OCT3/4 protein fragment

First, gene optimization and synthesis

OCT3/4 selects protein fragments of 1-65 amino acids according to protein sequence with accession number Q01860 in Uniprot database, and directly optimizes into gene fragments suitable for expression in Escherichia coli BL21(DE 3). 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. The OCT3/4 molecule is analyzed according to published sequence, based on 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 1-65 amino acid residue of OCT3/4 for codon optimization, and the molecular weight is about 31 kDa. The OCT3/4 protein is obtained by using prokaryotic expression gene sequence through sequence optimization design. The recombinant immunogen consists of an OCT3/4 protein fragment with antigenicity and a protein tag for purifying recombinant protein, wherein the protein tag is TRX and 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 of recombinant OCT3/4 protein fused with histidine tag. 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 recombinant protein of example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), and 4-6 week-old female ICR mice (purchased from Beijing Wintolite laboratory animals technologies, Ltd.) were immunized and injected subcutaneously at 6-point(s) in the abdomen 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 Co., F5506) at a dose of 30. 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.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: 2000 dilution of HRP-labeled goat anti-mouse (Ig)M, IgG1, IgG2a, IgG2b, IgG3, IgA) antibodies (Southern Biotech) were added to each well at 0.1ml per well and incubated 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

OCT3/4 recombinant protein 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 3 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 OCT3/4 recombinant protein prepared in example 1 was taken, the recognition specificity of the monoclonal antibody of the invention was examined 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 41B1 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).

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 OCT3/4(41B1) and the commercial antibody OCT3/4(C-10) are synchronously detected in 25 cases of gastric adenocarcinoma, and the detection results are compared.

The immunohistochemical results of OCT3/4 were statistically analyzed. 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-OCT 3/4 protein secreted by the 41B1 cell strain has accurate staining positioning, clear staining, no non-specific staining and clean background. In the immunohistochemical detection, the positive rate was comparable to the commercial antibody, but the positive intensity was higher than the commercial antibody. In 1 case of seminoma, the staining result of OCT3/4 secreted by 41B1 cell line was neutral positive, while the staining result of commercial OCT3/4 was negative (the number of stained cells was too small due to its low sensitivity, and the false judgment was made). The OCT3/4 secreted by the 41B1 cell strain is higher in sensitivity, and the false negative result is effectively avoided.

FIG. 2 is a graph comparing immunohistochemical staining results of seminoma (41B1 secreted OCT3/4 on the left and commercial OCT3/4 on the right).

FIG. 3 is a graph comparing the immunohistochemical staining results of clonal cell tumors (41B1 secreted OCT3/4 on the left and commercial OCT3/4 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 (41B1) and a commercial antibody (commercial Ks20.8) are synchronously detected on a normal tissue chip, and the negative and positive detection results are consistent, which shows that the specificity of the antibody in the normal tissue is equivalent to that of the commercial antibody.

FIG. 4 is a graph comparing immunohistochemical staining of testis; the left is OCT3/4 secreted by 41B1, and the right is commercial OCT 3/4.

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 (10)

1. The monoclonal antibody for resisting the OCT3/4 protein is 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 NO. 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 5.

2. The monoclonal antibody according to claim 1, wherein the coding DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No.2, and the coding DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No. 3.

3. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes OCT3/4 protein.

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

5. An anti-OCT 3/4 monoclonal antibody is produced by hybridoma cell strain with preservation number of CGMCC NO 20779.

6. The preparation method of the anti-OCT 3/4 protein monoclonal antibody is characterized in that an antigen used for immunizing a mouse is a recombinant protein, and the recombinant protein is expressed by escherichia coli in a recombinant mode and comprises a GST protein tag, an OCT3/4 protein fragment and a histidine protein tag.

7. The preparation method of claim 6, wherein the OCT3/4 protein fragment is the amino acid fragment from 1 st to 65 th of OCT3/4 protein, and the amino acid sequence is the amino acid sequence shown in SEQ ID NO. 1.

8. A hybridoma cell strain capable of secreting anti-OCT 3/4 protein molecules is a mouse hybridoma cell strain 41B1, is preserved in the China general microbiological culture Collection center (CGMCC), and has the preservation number: CGMCC NO 20779.

9. Use of the monoclonal antibody of any one of claims 1-5 in an OCT3/4 protein immunoassay.

10. An OCT3/4 protein immunohistochemical detection reagent, characterized in that the immunohistochemical detection reagent contains the anti-OCT 3/4 protein monoclonal antibody of claim 1 as an active ingredient.

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