CN114989303A

CN114989303A - anti-CD 56 recombinant rabbit monoclonal antibody and application thereof

Info

Publication number: CN114989303A
Application number: CN202210614768.2A
Authority: CN
Inventors: 刘杨; 张伟; 巩丽; 李艳红; 封兰兰; 富金; 王圆圆; 张富琴; 宋砚明; 吴纯
Original assignee: Suzhou Baidao Medical Technology Co ltd
Current assignee: Suzhou Baidao Medical Technology Co ltd
Priority date: 2022-05-30
Filing date: 2022-05-30
Publication date: 2022-09-02
Anticipated expiration: 2042-05-30
Also published as: CN114989303B

Abstract

The invention relates to an anti-CD 56 recombinant rabbit monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 4; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 5. Compared with a commercially available anti-CD 56 antibody, the anti-CD 56 recombinant rabbit monoclonal antibody provided by the invention has higher affinity with a CD56 protein, can identify and detect the expression of the CD56 protein on tumor cells or immune cells with high specificity and high sensitivity, can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, western blotting (Westernblotting), antibody chip preparation, flow cytometry and the like, is favorable for obtaining more accurate detection and evaluation results, and reduces the detection cost and the interference of background signals.

Description

anti-CD 56 recombinant rabbit monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of immunochemistry, and particularly relates to an anti-CD 56 antibody and application thereof, in particular to application in immunohistochemical detection.

Background

CD56 is a group of related cell surface glycoproteins that play important roles in embryogenesis and development as well as in neuronal cell interconnections. The CD56 antigen is mainly expressed in neurons, astrocytes, schwann cells, NK cells, and a small fraction of activated T lymphocytes. The antibody mainly determines the diagnosis of tumors derived from neuroectoderm, lung small cell carcinoma and the diagnosis and research of NK cell lymphoma. CD56 is highly expressed in normal thyroid tissue, nodular goiter, follicular adenoma and papillary hyperplasia, but is underexpressed or weakly expressed in thyroid papillary carcinoma, so that the diagnosis of thyroid papillary carcinoma can be assisted by detecting molecular markers such as CD56, and the diagnosis sensitivity, specificity and accuracy are improved. Therefore, the screening of a high-sensitivity and strong-specificity anti-CD 56 antibody has very important effect on recognizing and detecting the expression of CD56 protein on tumor cells or immune cells.

Disclosure of Invention

Technical problem to be solved

In view of the defects and shortcomings of the prior art, the invention provides the anti-CD 56 recombinant rabbit monoclonal antibody which is wide in application and can accurately identify CD56 expression, and the antibody can well detect the expression of CD56 protein on tumor cells or immune cells through immunohistochemical detection of various tissues, and can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, Western blotting (Western blotting), antibody chip preparation, flow cytometry and the like. The invention also relates to a nucleotide sequence, a recombinant plasmid or an expression vector for encoding the anti-CD 56 recombinant rabbit monoclonal antibody, a preparation method and application of the anti-CD 56 recombinant rabbit monoclonal antibody in a CD56 protein detection method or device.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

in a first aspect, the present invention provides an anti-CD 56 recombinant rabbit monoclonal antibody, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is represented by SEQ ID No. 4; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 5.

The anti-CD 56 recombinant rabbit monoclonal antibody (CD56 rabbit source antibody) can be used for immunohistochemical detection, and can identify and detect the expression of CD56 protein on tumor cells or immune cells with high specificity and high sensitivity.

The anti-CD 56 monoclonal antibody is obtained by recombinant expression of mammalian cells. Specifically, the anti-CD 56 recombinant rabbit monoclonal antibody provided by the invention is generated by fusion screening of rabbit hybridomas and 293 cell eukaryotic expression. When the anti-CD 56 monoclonal antibody is prepared, an antigen for an immune rabbit (New Zealand white rabbit) is a synthetic polypeptide, the amino acid sequence of the synthetic polypeptide is shown as SEQ ID No.1, and the synthetic polypeptide is obtained by artificial chemical synthesis. After immunization, cell fusion and clone screening are carried out to obtain a positive hybridoma cell line capable of efficiently secreting the monoclonal antibody, a molecular cloning technology is used for obtaining a nucleotide sequence for coding a heavy chain amino acid sequence and a light chain amino acid sequence of the antibody, the nucleotide sequence is constructed on a eukaryotic expression vector, the eukaryotic expression vector is transfected into a 293 cell line through a transfection reagent, cell supernatant is collected, and the cell supernatant is purified through Protein A column affinity chromatography to obtain the rabbit monoclonal antibody. Immunohistochemical detection showed that the antibody specifically recognized the CD56 protein.

The anti-CD 56 monoclonal antibody can recognize a recombinant CD56 antigen protein and a CD56 molecule on tumor cells and immune cells; the anti-CD 56 monoclonal antibody can also be applied to immunohistochemical pathological diagnosis reagents.

In a second aspect, the present invention provides a coding gene for encoding the recombinant rabbit monoclonal antibody against CD56 described above.

Preferably, the coding gene comprises a DNA sequence shown as SEQ ID No.2, and is used for coding the heavy chain variable region of the anti-CD 56 recombinant rabbit monoclonal antibody; and the DNA sequence shown in SEQ ID No.3 is used for coding the light chain variable region of the anti-CD 56 recombinant rabbit monoclonal antibody.

In a third aspect, the present invention relates to a nucleic acid molecule comprising a coding gene for encoding said recombinant rabbit monoclonal antibody against CD 56.

In a fourth aspect, the invention provides an expression vector or recombinant plasmid comprising a nucleic acid molecule as described above.

In a fifth aspect, the invention provides a preparation method of the anti-CD 56 recombinant rabbit monoclonal antibody, which comprises the steps of transfecting cells by using the expression vector, culturing the transfected cells, collecting cell supernatant and purifying to obtain the anti-CD 56 recombinant rabbit monoclonal antibody.

More preferably, the preparation method comprises the following steps:

(1) immunizing rabbits: analyzing a CD56 protein molecule sequence, selecting and using a proper polypeptide sequence as an immunogen according to the structure, antigenicity, hydrophilicity and hydrophobicity of amino acids and a secondary structure of CD56 on a cell membrane, coupling by KLH or OVA to serve as the immunogen, and immunizing a rabbit; the polypeptide is artificially synthesized polypeptide shown in SEQ ID No. 1;

(2) preparation of hybridoma cell lines: obtaining a positive hybridoma stable cell line capable of efficiently secreting antibodies through cell fusion and cloning screening, and separating total RNA from the hybridoma cell line;

(3) obtaining the antibody sequence: obtaining the nucleotide sequences of the heavy chain variable region and the light chain variable region of the antibody by using a specific primer and a PCR amplification technology;

(4) antibody expression and purification: cloning the nucleotide sequence into an expression vector, transiently transfecting cultured cells by using a transfection method, collecting supernatant after culturing, and purifying the supernatant by using Protein A to obtain the antibody with the purity of more than 95%.

In a sixth aspect, the anti-CD 56 recombinant rabbit monoclonal antibody, the coding gene, the nucleic acid molecule, the expression vector or the recombinant plasmid are applied to the preparation of a CD56 protein molecule detection device. The detection device includes but is not limited to a kit, an antibody chip, and the like.

In a seventh aspect, the present invention also provides a CD56 detection kit, which comprises the above-mentioned anti-CD 56 recombinant rabbit monoclonal antibody and an immunohistochemical detection reagent.

Preferably, the CD56 test kit comprises: the kit comprises an anti-CD 56 recombinant rabbit monoclonal antibody, an HRP enzyme-labeled secondary antibody, an EDTA repair solution, a catalase blocking solution, a DAB concentrated solution, a DAB buffer solution, hematoxylin and a blue returning solution.

When the immune tissue detection is carried out, the detection steps comprise dewaxing, antigen repairing, endogenous peroxidase inactivation, sealing, primary antibody incubation, secondary antibody incubation, DAB coloration, counterstaining, dehydration, mounting, microscopic examination and the like.

(III) advantageous effects

The anti-CD 56 recombinant rabbit monoclonal antibody provided by the invention has high specificity and high sensitivity in combination with CD56 protein molecules, can specifically identify and detect the expression of CD56 protein on tumor cells or immune cells, and shows positive high expression when detecting CD56 protein, so that the antibody can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, immunoblotting (Western blotting), antibody chip preparation, flow cytometry and the like, and is favorable for obtaining accurate evaluation and detection results. The CD56 recombinant rabbit monoclonal antibody cloned from 165A3F9 is easy to score in IHC staining due to the characteristics of good specificity, strong positive signal and the like, and is favorable for accurately detecting and distinguishing cancers.

Drawings

FIG. 1 is a graph showing the results of immunohistochemical detection of 165A3F9 anti-CD 56 monoclonal antibody prepared in the present invention and a commercially available anti-CD 56 clone MRQ-42 antibody in human appendix tissue, wherein the primary antibody was used at a concentration of 0.6. mu.g/mL.

FIG. 2 is a statistical chart of titer detection of the 165A3F9 anti-CD 56 monoclonal antibody of the invention at 7 gradient concentrations.

Fig. 3 is a graph comparing fluorescence signal intensity (MFI) of a blank control and a 165A3F9 anti-CD 56 mab sample obtained by flow cytometry staining analysis after adding a quantitative 165A3F9 anti-CD 56 mab to cells expressing CD56 protein.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer. The human tissue samples are formalin-fixed paraffin-embedded human tissue samples, are pathologically verified, and are informed consent of patients.

Example 1

This example is the preparation and screening of recombinant rabbit monoclonal antibodies against CD56, comprising the steps of:

(1) antigen preparation

The specific sequence of the CD56 antigen is shown as SEQ ID NO. 1.

SEQ ID No.1：VKTVPNDATQTKENESKA。

The polypeptide sequence is selected by analyzing the CD56 molecule sequence according to the structure and antigenicity of CD56 protein molecules on cell membranes, the hydrophilicity and hydrophobicity of the constituent amino acids and the secondary structure. The polypeptide with the sequence shown in SEQ ID NO.1 is artificially synthesized, and the synthesized polypeptide is used as the antigen for immunizing rabbits. In the immunization, the polypeptide with the sequence shown in SEQ ID NO.1 is coupled through KLH or OVA and then used as immunogen to immunize rabbits.

(2) Immunization

The polypeptide sequence (CD56 antigen) of SEQ ID NO.1 is mixed and emulsified with complete Freund's adjuvant (1:1) respectively, a plurality of New Zealand white rabbits are immunized by subcutaneous injection respectively, and the CD56 antigen containing the sequence (polypeptide shown in SEQ ID NO. 1) is emulsified with the complete Freund's adjuvant (1:1) after two weeks to carry out the second and third immunization. After three times of immunization, blood is taken and subjected to gradient dilution by an ELISA method to determine the serum titer; selecting the rabbit with the highest titer of the antigen-antibody with SEQ ID NO.1 to perform the next cell fusion.

(3) Cell fusion

Murine sp2/0 myeloma cells were prepared in advance so that the sp2/0 myeloma cells were in the logarithmic growth phase at the time of fusion. Taking the spleen of the immunized rabbit, and preparing lymphocyte single cell suspension; mixing rabbit spleen lymphocytes and the myeloma cells, dropwise adding 50% PEG1500, adding IMDM culture medium, centrifuging, removing supernatant, adding HAT culture medium, suspending, mixing, diluting to volume of 800mL, subpackaging in 96-well plate, standing at 37 deg.C with 5% CO ₂ Culturing in a constant temperature incubator. Observing the state of fused cells in a 96-well plate after fusion for 6-9 days, and continuously placing the fusion medium in a temperature range of 37 ℃ and 5% CO by using HT for liquid exchange ₂ Culturing in a constant temperature incubator.

(4) Screening and cloning

Cloned cells were screened 7-10 days after fusion by ELISA assay using CD56 antigen (SEQ ID NO: 1). Marking the corresponding cell strain number, and performing limited dilution on the positive well cells until the whole plate result of the 96-well plate is positive by ELISA determination. The monoclonal stable strain with high positive value was selected to obtain hybridoma cell line secreting specific monoclonal antibody, which was recorded as 165A3F 9.

(5) Performing antibody sequencing on the screened hybridoma cell strain

Total RNA was isolated from 165A3F9 hybridoma cells according to the instructions of the reagent TriZol, reverse-transcribed into cDNA according to the instructions of the TIANCcript first strand cDNA Synthesis kit, amplified using specific primers (heavy chain variable region primer, VH-F: AGACTGGGCTGCGCTGGCTTC, VH-R GTGAGGGTGCCCGAG; light chain variable region primer: VK-F ATGGACAYGAGGGCCCCCACTC, VK-R: GGTGGGAAGATGAGGACAGTAGG) to obtain the nucleotide sequences of the antibody heavy chain variable region and the antibody light chain variable region, and then cloned into a eukaryotic expression vector (InvivoGen, pfuse-rchg, pfuse2-rclk1) to prepare for cell transfection.

(6) Cell transfection and selection

293 cells to be transfected are prepared in advance, centrifuged to replace fresh medium and placed in 24-well plates with a density of 3X 10 and a required amount of 1.5ml per well ⁶ One per ml.

Mixing the eukaryotic expression vector and PEI according to the proportion of 1:6, adding the mixture into prepared 293 cells, and placing the cells at 37 ℃ and 5% CO ₂ The shaking table of (4) was cultured. After culturing for 3-5 days, performing ELISA detection on the transfected cell supernatant and the corresponding antigen to screen positive holes, and then performing immunohistochemical detection on the cell supernatant of the positive holes, wherein if the immunohistochemical detection is positive, the detected antibody sequence is correct.

(7) Preparation and purification of cell supernatant monoclonal antibody

And (3) performing mass cell transfection on the positive expression vector, continuously culturing for 3-5 days, collecting cell suspension, centrifuging, taking supernatant, and purifying by using an affinity chromatography method. And (4) measuring the concentration of the purified monoclonal antibody, subpackaging and storing in a refrigerator at 4-8 ℃.

Finally, the amino acid sequence of the heavy chain variable region of the 165A3F9 anti-CD 56 recombinant rabbit monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No.2, and the amino acid sequence of the light chain variable region of the anti-CD 56 recombinant rabbit monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No. 3.

The specific sequence of SEQ ID No.2-3 is as follows:

SEQ ID No.2：

cagtcggtggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagtctctggattctccctcagtagctatggaatgaactgggtccgccaggctccagggaaggggctggaatacatcggaatcattagtattagtggtaacacattctacgcgagctgggcgaaaggccgattcaccatctccagaacctcgaccacggtggatctgaaaatcaccagtccgacaaccgaggacacggccacctatttctgtgccagatcttatactggtagtagtacttatggttttgatccctggggcccaggcaccctggtcaccgtctcctcaa。

SEQ ID No.3：

gcctatgatatgacccagactccagcctctgtggaggtagctgtgggaggcacagtcaccatcaagtgccaggccagtcagagcattagtagctacttagcctggtatcagcagaaaccagggcagcctcccaagctcctgatctacagggcatccactctggcatctggggtcccatcgcggttcaaaggcagtggatctgggacagagttcactctcaccattagcgacctggagtgtgccgatgctgccacttattactgtcaacagacttatgctaatggtgatgttgataatagtttcggcggagggaccgaggtggtggtcaaag。

the obtained base sequence is translated into an amino acid sequence, and the amino acid sequence of the heavy chain variable region of the 165A3F9 anti-CD 56 recombinant rabbit monoclonal antibody is shown as SEQ ID No.4, and the amino acid sequence of the light chain variable region of the anti-CD 56 recombinant rabbit monoclonal antibody is shown as SEQ ID No. 5.

The specific sequences of SEQ ID Nos. 4 to 5 are as follows:

SEQ ID No.4：

QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYGMNWVRQAPGKGLE YIGIISISGNTFYASWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARSY TGSSTYGFDPWGPGTLVTVSS。

SEQ ID No.5:

AYDMTQTPASVEVAVGGTVTIKCQASQSISSYLAWYQQKPGQPP KLLIYRASTLASGVPSRFKGSGSGTEFTLTISDLECADAATYYCQQTYA NGDVDNSFGGGTEVVVK。

example 2

This example is an immunohistochemical assay of the anti-CD 56 recombinant rabbit monoclonal antibody as a primary antibody, the method of which is as follows:

(1) sample section preparation: the human appendix tissue section embedded by formalin-fixed paraffin is baked for 1-2h in a thermostat at 60 ℃ and stored for later use.

(2) Slicing and dewaxing: paraffin slices are firstly placed in fresh dimethylbenzene for dewaxing, and are soaked for 2 times, and each time lasts for 10 min.

(3) Hydration of the slices: soaking in anhydrous ethanol, 95% ethanol, 85% ethanol, and 70% ethanol for 5min for hydration, washing with purified water for 2 times, each for 3 min.

(4) Antigen retrieval: repairing with high temperature heat repairing method for 3min (98 deg.C for 20min if using automatic repairing instrument), cooling the slices to room temperature, looping the tissue with immunohistochemical pen, washing with purified water for 2 times, 3min each time.

(5) Inactivation of endogenous peroxidase: dropping proper amount of endogenous peroxidase blocker to completely cover the tissue, incubating at room temperature for 10min, washing with purified water for 2 times (3 min each time), and washing with PBST once.

(6) Primary antibody incubation: the experiments were divided into two groups, one group with 100. mu.L of 0.6. mu.g/mL 165A3F9 anti-CD 56 recombinant rabbit monoclonal antibody and the other group with a commercial anti-CD 56 cloned MRQ-42 antibody to completely cover the tissue, incubated for 1h at 37 ℃ in an incubator, and washed 3 times with PBST for 5min each.

(7) And (3) secondary antibody incubation: and (4) performing secondary antibody incubation according to the instruction of the DAB staining solution kit of the secondary antibody staining system, after the incubation is finished, washing the PBST by using the washing sheet for 3 times, 5min each time, and washing by using purified water for 1 time.

(8) DAB color development: preparing DAB color developing solution according to the DAB staining solution kit instruction, dripping proper amount of the prepared DAB color developing solution until the tissue is completely covered, stopping staining when the color is not deepened, and washing with purified water for 3 times.

(9) Hematoxylin counterstaining: the sections were counterstained according to the protocol and recommendations of the hematoxylin manufacturer's instructions, washed back to blue with PBST or tap water.

(10) And (3) dehydrating and transparency: soaking in 70%, 85%, 95%, 100%, 100% gradient alcohol for 3min each time; 2 times, 5min each time, xylene was clear.

(11) And (3) sealing: the samples were mounted with neutral gum.

As can be seen from the results in FIG. 1, the CD56 protein showed specific cell membrane staining in human appendix tissue covered with 165A3F9 clone antibody and MRQ-42 antibody, and the staining intensity was higher (the color was darker after gray scale treatment) and the staining range was larger. Therefore, the affinity of the CD56 recombinant rabbit monoclonal antibody cloned from 165A3F9 and the CD56 protein is superior to that of a commercially available MRQ-42 antibody, and the monoclonal antibody has the characteristics of better specificity, stronger positive signal and the like, and is more accurate in detecting and distinguishing cancers.

Example 3

This example is an assay for the affinity of 165A3F9 for recombinant rabbit monoclonal antibody CD56, as follows:

(1) the labeled CD56 polypeptide was removed from 4 ℃ and returned to room temperature. Diluted to a concentration of 1. mu.g/ml, added to a 96-well plate at 100. mu.L/well and incubated overnight at 4 ℃ followed by blocking overnight at 4 ℃ with 2% BSA.

(2) The CD56 recombinant rabbit monoclonal antibody cloned from 165A3F9 was diluted to an initial concentration of 0.5. mu.g/mL, and serially diluted in 2-fold gradients, setting 7 concentration gradients for comparison.

(3) And adding the diluted anti-CD 56 recombinant rabbit monoclonal antibody to a 96-well enzyme label plate with polypeptide according to 100 mu L/well, covering a sealing plate membrane, and incubating at the constant temperature of 37 ℃ for 1h to balance the reaction.

(4) And (3) taking out the enzyme label plate after the reaction is finished, discarding the liquid, washing with purified water for 5 times, and patting to dry the water.

(5) The HRP-labeled goat anti-rabbit IgG was diluted according to the instructions for the use of the secondary antibody, added to the ELISA plate at 100. mu.L/well, and incubated at 37 ℃ for 1h to allow the reaction to reach equilibrium.

(6) And (3) taking out the enzyme label plate after the reaction is finished, discarding the liquid, washing with purified water for 5 times, and patting to dry the water.

(7) TMB developing solution was added to the reaction vessel at a rate of 100. mu.L/well, and the reaction was carried out at room temperature for 6 minutes.

(8) After the reaction was complete, 2M H was added at 50. mu.L/well ₂ SO ₄ The color development was terminated.

(9) OD values were read at 450nm on a microplate reader, and the data were collated and analyzed as shown in FIG. 2.

The results show that in 7 concentration gradient tests, the anti-CD 56 recombinant rabbit monoclonal antibody cloned from 165A3F9 has strong affinity and high sensitivity to CD56 protein molecules, can still reach a higher OD value under the condition of lower antibody concentration, and can save the test and detection cost.

Example 4

This example performs flow cytometric staining analysis of 165A3F9 secreted anti-CD 56 antibody, according to the following protocol:

(1) 1X 10^6 cells expressing the CD56 protein were collected, washed once with 500. mu.L PBS, and resuspended in 100. mu.L PBS.

(2) To the cells, 0.5. mu.g of the anti-CD 56 recombinant rabbit monoclonal antibody cloned in 165A3F9 was added, mixed well with gentle blowing and incubated at room temperature for 15 min.

(3) Cells were incubated with 2 washes of 500. mu.L PBS and resuspended in 100. mu.L PBS.

(4) 0.5 mu g of Alexa fluor 488 fluorescence-labeled goat anti-rabbit fluorescent secondary antibody is added into the cells, and the cells are evenly mixed by light blowing and incubated for 15min at room temperature in a dark place.

(5) The cells were washed 2 times with 500 μ L PBS to wash out residual fluorescent secondary antibody and resuspended in 500 μ L PBS for flow-on-machine detection, and 20000 cells were sampled to analyze the intensity of fluorescent signal in both cases (no antibody and addition of 165A3F9 antibody). The results of fluorescence signal intensity relative values (MFI) are shown in Table 1, and the results of flow assay are shown in FIG. 3.

Table 1:

sample (I)	MFI
		Blank control	25.6
165A3F9 antibody	1732

The above experimental results show that the 165A3F9 anti-CD 56 recombinant rabbit monoclonal antibody of the invention has higher antibody affinity, compared with a blank control group, under the condition of the same cell number, the MFI signal intensity of the 165A3F9 anti-CD 56 antibody group is increased by 67.65 times, which indicates that the 165A3F9 anti-CD 56 recombinant rabbit monoclonal antibody of the invention can obtain very high fluorescence signal intensity.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

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<120> anti-CD 56 recombinant rabbit monoclonal antibody and application thereof

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gggaaggggc tggaatacat cggaatcatt agtattagtg gtaacacatt ctacgcgagc 180

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gccgatgctg ccacttatta ctgtcaacag acttatgcta atggtgatgt tgataatagt 300

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Claims

1. The anti-CD 56 recombinant rabbit monoclonal antibody, characterized in that, it comprises heavy chain variable region and light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 4; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 5.

2. A gene encoding the recombinant rabbit monoclonal antibody to CD56 of claim 1.

3. The coding gene according to claim 2, characterized in that it comprises: a DNA sequence as shown in SEQ ID No.2 for encoding the heavy chain variable region of the anti-CD 56 recombinant rabbit monoclonal antibody, and a DNA sequence as shown in SEQ ID No.3 for encoding the light chain variable region of the anti-CD 56 recombinant rabbit monoclonal antibody.

4. A nucleic acid molecule comprising the coding gene of claim 2 or 3.

5. An expression vector or recombinant plasmid comprising the nucleic acid molecule of claim 4.

6. A preparation method of an anti-CD 56 recombinant rabbit monoclonal antibody, which is characterized in that the expression vector of claim 5 is adopted to transfect cells, the cells are continuously cultured after the transfection, cell supernatant is collected and purified, and the anti-CD 56 recombinant rabbit monoclonal antibody is obtained.

7. The method of claim 6, comprising the steps of:

(1) immunizing rabbits: firstly analyzing the sequence of CD56 molecule, selecting and using proper polypeptide sequence as immunogen according to the structure, antigenicity, hydrophilicity and hydrophobicity of amino acid and secondary structure of CD56 molecule on cell membrane, and taking the immunogen as an immunogen to immunize rabbit after coupling by KLH or OVA; the polypeptide sequence is artificially synthesized polypeptide shown in SEQ ID No. 1;

(4) antibody expression and purification: cloning the nucleotide sequence into a eukaryotic expression vector, transiently transfecting the cultured 293 cells by using a transfection method, collecting supernatant after culturing, and purifying the supernatant by using Protein A to obtain the antibody with the purity of more than 95%.

8. Use of the recombinant rabbit monoclonal antibody against CD56 of claim 1, the coding gene of claim 2 or 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, or the recombinant plasmid for the preparation of a CD56 detection device.

9. A CD56 test kit comprising the anti-CD 56 recombinant rabbit monoclonal antibody of claim 1, and immunohistochemical detection reagents.

10. The CD56 detection kit according to claim 9, characterized in that it comprises: the kit comprises an anti-CD 56 recombinant rabbit monoclonal antibody, an HRP enzyme-labeled secondary antibody, an EDTA repair solution, a catalase blocking solution, a DAB concentrated solution, a DAB buffer solution, hematoxylin and a blue returning solution.