Anti-cd155 protein monoclonal antibody and preparation method and application thereof

Abstract

The present application relates to a kind of monoclonal antibody that can identify human CD155 antigen, its preparation method and its use in immunodetection.The present application provides a kind of anti-CD155 protein rabbit 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.1;The amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.2.The antibody has high specificity, sensitivity, and can specifically recognize the cell expressing CD155 protein, is suitable for immunological detection, especially immunohistochemical detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical engineering, and particularly relates to an anti-CD155 protein monoclonal antibody and a preparation method and application thereof. BACKGROUND

[0002] CD155 (cluster of differentiation 155) is an immunoglobulin-like adhesion molecule, a single transmembrane cell surface protein, which participates in tumor cell adhesion and migration as a cell adhesion molecule, and also mediates the immunity of NK and T cells as an immunoregulatory molecule. The CD155 gene is located on human chromosome 19, consists of 8 exons and 417 amino acids, has two transmembrane selective splicing subtypes (CD155δ and CD155α) and two splicing subtypes lacking transmembrane region (CD155β and CD155γ). CD155β and CD155γ are likely to be potential markers for cancer development and progression.

[0003] CD155 is one of the few ligands that activate and inhibit receptor recognition on immune cells. There are three ligands of CD155, DNAX auxiliary molecule-1 (CD226) is expressed on most immune cells, including T cells, B cells, NK cells and monocytes. T cell immunoglobulin (TIGIT) is an inhibitory receptor expressed on NK cells, activated and memory T cells and Tregs. CD155 also interacts with CD96 of the immunoglobulin superfamily. As an immunoregulatory molecule, CD155 affects the outcome of immune responses by interacting with activating and inhibitory receptors.

[0004] Studies have shown that CD155 is rarely expressed or not expressed in normal tissues, but is highly expressed in various human malignancies including pancreatic cancer, cholangiocarcinoma, colorectal cancer, non-small cell lung cancer, bladder cancer and breast cancer. The expression level of CD155 is closely related to the pathological parameters such as histological differentiation degree, TNM stage and lymph node metastasis of tumor. Studies have shown that overexpression of CD155 is significantly related to poor prognosis of patients with cervical cancer, uroepithelial carcinoma and lung cancer. SUMMARY

[0005] The present application provides an anti-CD155 protein 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 NO. 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 2.

[0006] SEQ ID NO. 1:

[0007] QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYAMIWVRQAPGQGLEYIGFTYIGGPIYYASWAKGRFTISKTSTTVDLKMTSLTTEDTATYFCASGHGIWGPGTLVTVSS

[0008] SEQ ID NO. 2:

[0009] QVLTQTPSPVSAAVGGTVTINCQASQSVYNNKNLAWFQQKPGQPPKRLIYAASNLASGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCLGEFSCSSADCLAFGGGTEVVVK

[0010] Further, the coding DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO. 3, and the coding DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO. 4.

[0011] SEQ ID NO. 3:

[0012] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTAGTTATGCAATGATCTGGGTCCGCCAGGCTCCAGGGCAGGGGCTGGAATACATCGGATTCACGTATATTGGTGGTCCCATATATTACGCGAGCTGGGCGAAAGGCCGGTTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGTGGCCATGGCATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0013] SEQ ID NO. 4:

[0014] CAAGTGCTGACCCAGACTCCATCCCCTGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAACTGCCAGGCCAGTCAGAGTGTTTATAATAACAAAAATTTAGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTATGCTGCATCCAATCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGAATTTAGTTGTAGTAGTGCTGATTGTTTGGCTTTCGGCGGAGGGACCGAGGTGGTCGTCAAA

[0015] Further, the monoclonal antibody specifically recognizes the CD155 protein.

[0016] Further, the monoclonal antibody is a rabbit monoclonal antibody.

[0017] Further, the clone number of the rabbit monoclonal antibody is 18C1.

[0018] The inventors also provide a method for preparing an anti-CD155 protein monoclonal antibody, and the antigen for immunizing the rabbit is a recombinant protein, which is expressed by E. coli.

[0019] Further, the recombinant protein comprises a CD155 protein fragment and a HIS protein tag.

[0020] Further, the CD155 protein fragment is a fragment of 21-343 sites, which is an amino acid sequence shown in SEQ ID NO. 5.

[0021] SEQ ID NO. 5:

[0022] WPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGMSRN

[0023] Further, the plasmid vector selected in the recombinant process is pET-28a-sumo.

[0024] The inventor also provides a CD155 protein immunological detection reagent, which contains the above-mentioned anti-CD155 protein monoclonal antibody as an effective component.

[0025] Differing from the prior art, the beneficial technical effects of the present application are: the above-mentioned technical solution provides an anti-CD155 protein rabbit 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. 1; the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 2. The antibody has high specificity and sensitivity, can specifically recognize cells expressing CD155 protein, and is suitable for immunological detection, especially immunohistochemical detection. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is a comparison chart of lung adenocarcinoma immunohistochemical staining results (left is the rabbit monoclonal antibody CD155 of the present application, and right is the commercially available CD155).

[0027] Figure 2 It is a comparison chart of liver immunohistochemical staining results (left is the rabbit monoclonal antibody CD155 of the present application, and right is the commercially available CD155). DETAILED DESCRIPTION

[0028] In order to explain the possible application scenarios, technical principles, specific implementation schemes, and the purposes and effects of the present application in detail, the following will be described in detail in combination with the specific embodiments listed and the accompanying drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, but cannot limit the protection scope of the present application.

[0029] In this paper, the term "embodiment" means that the specific features, structures or properties described in combination with the embodiment can be included in at least one embodiment of the present application. The term "embodiment" appearing at various places in the specification does not necessarily refer to the same embodiment, and does not particularly limit the independence or association between other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, each technical feature mentioned in each embodiment can be combined in any way to form a corresponding implementable technical solution.

[0030] Unless otherwise defined, the meanings of the technical terms used herein are the same as those commonly understood by those skilled in the art to which the present application belongs; the use of related terms herein is only for the purpose of describing specific embodiments, and is not intended to limit the present application.

[0031] In the description of the present application, the phrase "and / or" is a description of the logical relationship between the objects, which means that there can be three relationships, for example, A and / or B, which means that there are three cases: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in this paper generally represents that the associated objects before and after are a kind of "or" logical relationship.

[0032] In the present application, the terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual quantity, primary and secondary or order relationship between the entities or operations.

[0033] Without more limitations, in the present application, the phrases "include", "contain", "have" or other similar expressions used in the sentence are intended to cover non-exclusive inclusion, and these expressions do not exclude the presence of other elements in the process, method or product including the described elements, so that the process, method or product including a series of elements can not only include those limited elements, but also include other elements not explicitly listed, or also include the elements inherent to such process, method or product.

[0034] The same as the understanding in the "Examination Guidelines", in this application, "greater than", "less than", "exceed" and the like are understood as not including the number; "above", "below", "within" and the like are understood as including the number. In addition, in the description of the embodiments of the present application, the meaning of "multiple" is two or more (including two), and similar expressions related to "multiple" are also understood in this way, for example, "multiple groups", "multiple times" and the like, unless otherwise explicitly and specifically limited.

[0035] Preparation of recombinant CD155 protein fragment of Example 1

[0036] I. Gene optimization and synthesis

[0037] According to the protein sequence in the Uniprot database with accession number P15151, the protein fragment of 21-343 sites was selected and directly optimized into a gene fragment suitable for expression in E. coli Rosetta (DE3), and cloned into the pET-28a-sumo vector.

[0038] The expression vector was used to transform E. coli competent cells E. coli Rosetta (DE3), and the clones on the plate were inoculated and subjected to bacterial liquid PCR identification. The clones with positive PCR results were selected for sequencing analysis, and the clones with completely correct sequences were used.

[0039] Selecting different antigens for immunization can produce antibodies with different binding characteristics. The molecule has multiple variants caused by variable splicing, which ultimately leads to different recognition ability and mode of different antibodies to the cells expressing the antigen. According to the published sequence, the CD155 molecule was analyzed, and based on the structure, antigenicity, hydrophilicity and hydrophobicity of the constituent amino acids, and the secondary structure, a region suitable for soluble expression and good immunogenicity was selected for recombinant expression. The gene sequence of 21-343 sites of CD155 was selected for codon optimization, and the molecular weight was about 62.2 kDa. The CD155 protein was obtained by sequence optimization design using prokaryotic expression gene sequence. The recombinant immunogen is composed of CD155 protein fragment with antigenicity and protein tag for purification of recombinant protein, and the protein tag is HIS.

[0040] II. Protein expression and purification

[0041] The single colony culture was transferred to 100 mL LB medium at a ratio of 1:100, and ampicillin was added at a final concentration of 100 μg / mL. The culture was incubated at 37°C with shaking until the OD600 was 0.6-0.8. Then, 0.8 mmol / L IPTG was added, and the culture was incubated at 37°C for 4 h. After the bacteria were collected, the cells were disrupted by ultrasonic treatment. The recombinant protein with a histidine tag was purified by affinity chromatography using a nickel column. The protein was eluted with 150 mmol / L imidazole, and the eluate was analyzed by SDS PAGE. The pET-28a-sumo-CD155 (21-343 aa) protein was expressed in the supernatant, and the concentration of the purified protein was 4 mg / mL. The purity of the protein reached 80%, which met the requirements for animal immunization and antibody screening and identification.

[0042] Example 2: Single B cell sorting for producing rabbit anti-human CD155 monoclonal antibodies

[0043] I. Immunization and ELISA detection

[0044] The recombinant CD155 protein in Example 1 was emulsified with Freund's complete adjuvant, and six rabbits were selected for immunization at a dose of 300 μg / rabbit. The rabbits were boosted once on day 23, day 37, and day 52, and the antigen was emulsified with Freund's incomplete adjuvant at a dose of 150 μg / rabbit. After the third immunization, the serum ELISA titer was detected, and the A rabbit had an immune titer of greater than 1:2.56K, which met the requirements for immunization. The serum of four rabbits after the fourth immunization was subjected to IHC detection. According to the results (i.e., the positive control photograph corresponding to the antibody was used for detection, and immunohistochemical staining was observed at the corresponding detection site in the positive control photograph), one rabbit (numbered: A) was selected for subsequent monoclonal antibody screening.

[0045] II. Spleen cell separation and B lymphocyte sorting

[0046] The selected rabbit was subjected to monoclonal antibody preparation. The spleen was removed 3 days after the boost immunization, and the rabbit spleen was placed in RMPI basic medium containing 100 U / mL penicillin and 100 μg / mL streptomycin. The spleen was cut into pieces with a surgical blade and transferred to a 100 μm cell screen for grinding. The obtained cell suspension was filtered to remove large cell clumps and tissue envelopes. After centrifugation at 400 g for 5 minutes, the supernatant was removed, and the spleen cell clumps were retained. The spleen cell clumps were resuspended with a hypotonic solution, and the red blood cells were lysed. The spleen cells were again centrifuged at 400 g for 5 minutes, and the spleen cells were retained. The spleen cells were resuspended with RMPI basic medium containing 100 U / mL penicillin and 100 μg / mL streptomycin, centrifuged at 400 g for 5 minutes, and resuspended with complete medium (RMPI basic medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) for use.

[0047] The specific steps of B lymphocyte sorting are described in Chinese Patent 201910125091.4 "Method for efficiently separating single antigen-specific B lymphocytes from spleen cells".

[0048] About 2000 single B cell clones were sorted and cultured, and positive clones specifically recognizing the recombinant CD155 protein in Example 1 were screened by ELISA. According to the ELISA data, 50 supernatants from high to low were selected for IHC verification.

[0049] III. Detection of positive clones of B cell culture

[0050] Single B cell culture supernatants were subjected to IHC verification using a multi-tumor tissue chip and a normal tissue chip containing CD155 positive and negative. Among them, the IHC results of 13 clone culture supernatants were good, and the LEM supernatant was prepared for IHC verification to determine the clone (18C1) with excellent sensitivity and specificity.

[0051] The screening criteria for sensitivity and specificity are as follows: the positive control photograph corresponding to the antibody is used for detection, and immunohistochemical staining can be observed on the corresponding detection site in the positive control photograph, but no immunohistochemical staining is observed on the non-detection site. Compared with the control antibody, the staining intensity reaches or is even higher than that of the control antibody.

[0052] IV. Cloning of rabbit monoclonal antibody gene (18C1) and construction of rabbit monoclonal antibody expression plasmid

[0053] After the cells of the positive clone were collected and lysed, RNA was extracted and reverse transcribed into cDNA. Using PCR method, the naturally paired rabbit monoclonal antibody light and heavy chain variable region genes were amplified from the cDNA corresponding to the positive clone, and sequenced. The above naturally paired rabbit monoclonal antibody light and heavy chain variable region gene sequences were used to construct rabbit monoclonal antibody expression vector plasmids.

[0054] Example 2 was completed by Cugene Biotech (Wuhan) Co., Ltd. on behalf of Fujian Mayxin Biotechnology Development Co., Ltd., in which immunohistochemical screening was performed by Fujian Mayxin Biotechnology Development Co., Ltd.

[0055] Example 3 CD155 rabbit monoclonal antibody expression

[0056] I. Plasmid amplification and extraction

[0057] Take out a tube (100 μl) of competent bacteria (DH5α), insert into ice, ice bath for 5-10 min; add 5 μl of plasmid, gently shake and place on ice for 30 min; gently shake and place in 42°C water bath for 90 s for heat shock, then quickly put back on ice, stand for 5 min; add 800 μl of LB medium (note: without antibiotics) to the above in the clean bench, mix gently, and fix on the shaker at 37°C for 1 h; take 50-100 μl of the above transformation mixture in the clean bench, add to the labeled solid LB plate with Amp, and evenly spread with a glass spreader (sterilized); first stand in a 37°C incubator for 30 min, let the surface bacteria completely penetrate into the medium, then invert and place in a 37°C incubator for overnight culture. Use a gun head to pick a single colony and punch into 4 ml of LB medium (containing 2 uL of 200 mg / ml Amp), and culture at 37°C on a shaker for 16 h at 220 rpm.

[0058] Add 1 ml of bacterial solution to 100 ml of LB medium (containing 50 μl of 200 mg / ml Amp), and culture at 37°C on a shaker for 16 h; use SanPrep endotoxin-free plasmid DNA extraction kit (Shenguo), and extract plasmid according to the instructions.

[0059] II. Transfection

[0060] Adjust the concentration of 293F cells to 2.5-3×10 6 viable cells / ml with Expi293FTM Expression Medium, and culture overnight.

[0061] 1) Count with a hemocytometer, and the viable cell concentration is about 4.5-5.5×10 6 viable cells / ml, and the number of viable cells needs to meet the requirements of the expression system;

[0062] 2) Dilute the cell concentration to 3×10 6 viable cells / ml with Expi293FTM Expression Medium;

[0063] 3) Add plasmid DNA to Opti-MENTM I Reduced Serum Medium, gently blow and mix;

[0064] 4) Gently invert ExpiFectamine™ 293 Reagent 4-5 times, mix ExpiFectamine™ 293 Reagent and Opti-MEM™ I Reduced Serum Medium, gently pipette and invert 2-3 times, and let stand at room temperature for 5 min;

[0065] 5) Mix the solutions from steps 3) and 4), gently pipette and invert 2-3 times, and mix well;

[0066] 6) Let the solution from step 5) stand at room temperature for 10-20 min;

[0067] 7) Slowly pipette the mixture into the cell culture medium, and gently shake the flask;

[0068] 8) Incubate at 37°C in a 8% CO2 incubator for 5-7 days;

[0069] 9) After 18-22 hours, add ExpiFectamine™ 293 Transfection Enhancer 1 and ExpiFectamine™ 293 Transfection Enhancer 2 (Note: mix before use), and gently shake to mix well, and continue incubation.

[0070] III. Purification of Monoclonal Antibodies

[0071] Purify the antibodies from the supernatant using HiTrap rProtein A FF affinity chromatography according to the manufacturer's instructions. Determine the purity by SDS-PAGE and the concentration by Bradford method. Store the purified antibodies at -20°C.

[0072] Example 4. Immunohistochemical Tissue Chip Staining and Identification

[0073] I. Chip Preparation Procedure

[0074] HE staining was performed on each sample to determine the tumor site. The tumor target site was circled and prepared for punching. When making the blank recipient wax block, a plastic frame was placed on the mold, and melted wax (melting point at 55-58°C) was poured into the mold. After cooling to room temperature, the mold was placed in a -20°C refrigerator for 6 min, and the wax block was removed from the mold. On the tissue sample machine, a 1 mm diameter sample needle was selected to punch a hole in the recipient wax block, with a hole depth of 3-4 mm. Another 1 mm diameter punching needle was used to punch a hole in the marked site of the wax block to collect a tissue core, with a length of about 0.1 mm shallower than the hole depth of the recipient wax block. The collected tissue core was directly inserted or carefully clamped with tweezers into the empty hole of the recipient wax block. This was repeated until all sample points were prepared. Finally, a glass slide was used to flatten all the tissue cores, making the tissue core block flat and smooth. The prepared tissue core block was placed in the wax block making mold and placed in a 60°C oven for 15 min to fuse the tissue core with the wax of the recipient wax block. Then the mold was gently removed from the oven, and the semi-melted wax was allowed to cool at room temperature for about 30 min. The tissue core block was then placed in a -20°C refrigerator for 6 min, and then removed from the mold. The tissue core block was sectioned or stored in a 4°C refrigerator for future use. After trimming, continuous sections were cut with a thickness of 3 μm. The continuous sections were floated in cold water to naturally expand, and then transferred to warm water at 45°C for 30 seconds. The sections were mounted on slides treated with polylysine, and the prepared tissue core block was placed in a 65°C oven for 2 hours. The oven was removed and allowed to cool at room temperature, and then stored in a -4°C refrigerator.

[0075] II. IHC Staining and Analysis

[0076] Routine xylene de-waxing was performed 3 times for 6 minutes each, with hydration in 100%, 100%, 95%, and 85% ethanol for 3 minutes each, and finally rinsed with tap water. Antigen retrieval was performed, and then the sections were placed in a wet box and rinsed with PBS for 3 x 3 minutes. 3% H2O2 was added and incubated for 10 minutes, and then rinsed with PBS for 3 x 3 minutes. The sections were spun dry, and the appropriate dilution of primary antibody was added (the first dilution was designed according to the concentration of the antibody). The sections were incubated at room temperature (25°C) for 1 hour, rinsed with PBS for 3 x 3 minutes, and then the secondary antibody was added and incubated at room temperature for 15-30 minutes. The sections were rinsed with PBS for 3 x 3 minutes, spun dry, and then developed with freshly prepared DAB developing solution for 3-10 minutes. Hematoxylin counterstaining was performed for 25 seconds, and then rinsed with PBS for 30 seconds. The sections were dehydrated in an alcohol gradient of 85% (3 minutes), 95% (3 minutes), 100% (3 minutes), and 100% (3 minutes), and finally cleared with xylene for 3 minutes, and then mounted with neutral resin.

[0077] The immunohistochemical staining results are classified as positive and negative. Positive expression must be at the site of cell- and tissue-specific antigens to be considered positive. In the case of clear distribution of tissue staining and accurate cell localization, the staining results are further classified according to the difference in staining intensity, as follows:

[0078] 1. The sample is weakly positive; marked as "+".

[0079] 2. The sample is moderately positive; marked as "++".

[0080] 3. The sample is highly positive; marked as "+++".

[0081] 4. The sample is negative, marked as "-".

[0082] III. Data Statistics

[0083] 1. Tumor tissue chip detection results:

[0084] The present antibody CD155 (18C1) and the commercially available antibody CD155 (rabbit polyclonal antibody) were used to synchronously detect 89 cases of lung adenocarcinoma and the detection results were compared. The immunohistochemical results of CD155 were statistically analyzed. The entire test process adopted a double-blind design, and the statistical results are shown in the following table:

[0085]

[0086] The results show that the rabbit monoclonal antibody CD155 (18C1) has accurate staining localization, clear staining and no non-specific staining, and a clean background. In the immunohistochemical detection, the positive rate is comparable to that of the commercially available antibody, and the positive intensity is higher than that of the commercially available antibody, indicating that the sensitivity of the rabbit monoclonal antibody CD155 (18C1) is higher than that of the commercially available antibody.

[0087] Figure 1 Comparison of immunohistochemical staining results of lung adenocarcinoma (left: rabbit monoclonal antibody CD155 of the present application; right: commercially available CD155 rabbit polyclonal antibody).

[0088] 2. Normal tissue chip detection results:

[0089] The normal tissue chip includes 30 normal tissue samples, which are mainly selected from fresh and timely fixed surgical specimens; each type of tissue includes 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectum, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (infant), skin, bone marrow, peripheral nerve, lung, mesothelial cells.

[0090] The rabbit monoclonal antibody CD155 (18C1) and the commercial CD155 antibody were synchronously detected on a normal tissue chip, and the positive and negative detection results were consistent, indicating that the specificity of the antibody in normal tissues was equivalent to that of the commercial antibody.

[0091] Figure 2 A comparison chart of the results of immunohistochemical staining of the liver (left: rabbit monoclonal antibody CD155 of the application, right: commercial rabbit polyclonal antibody CD155).

[0092] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of the present application, the patent protection scope of the present application should not be limited thereby. Any technical solution obtained by replacing or modifying the equivalent structure or equivalent process based on the essential concept of the present application, using the content described in the specification and drawings of the present application, and directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, etc., are all included in the patent protection scope of the present application.

Claims

1. An anti-CD 155 protein monoclonal antibody, characterized by, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

2.

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

4.

3. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody specifically recognizes CD155 protein.

4. The monoclonal antibody of claim 1, wherein, The monoclonal antibody is a rabbit monoclonal antibody.

5. A CD 155 protein immunoassay test reagent, characterized by, The immunodetection reagent contains the anti-CD155 protein monoclonal antibody of claim 1 as an effective component.

6. The immunoassay test reagent of claim 5, wherein, The immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

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