Antigen polypeptide, antibody prepared from antigen polypeptide and application of antigen polypeptide in detection of CD8

Abstract

The invention discloses an antigen polypeptide, an antibody prepared from the antigen polypeptide and application of the antigen polypeptide in detection of CD8. Specifically, the invention provides an antigen polypeptide, the antigen polypeptide is derived from an N-terminal extracellular region of CD8a, and the N terminal of the antigen polypeptide comprises additional cysteine. The antigen polypeptide is located at the N end of a CD8a molecule, a polyclonal anti-CD8 antibody obtained after a rabbit is immunized can be applied to an immunofluorescence technology after being purified, and a single cell membrane structure can be detected. The polyclonal anti-CD8 antibody disclosed by the invention has the advantages that the epitope is clear, and compared with the polyclonal anti-CD8 antibody positioned at the C terminal of an immunogen, the polyclonal anti-CD8 antibody has a stronger light-emitting signal in human tonsil frozen tissues and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an antigen polypeptide, an antibody prepared therefrom, and an application thereof in detecting CD8. Background Art

[0002] Human CD8 (cluster of differentiation 8) is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR). CD8 binds to major peptide-histocompatibility complex (pMHC) on antigen presenting cells, resulting in antigen-specific activation of naive CD8+ T cells or effector CD8+ T cells. Since properly activated and uninhibited CD8+ cytotoxic T lymphocytes (CTLs) can recognize tumor-associated antigens and kill tumor cells, CTLs are an important component of the immune system.

[0003] Under natural conditions, CD8 usually forms dimers, and the dimer can have two structures. The first is a heterodimer composed of one α chain and one β chain (CD8αβ), and the two chains are encoded by the leu-2a gene and the leu-2b gene respectively. The second is a homodimer composed of two α chains (CD8αα), and both chains are encoded by the leu-2a gene. Among them, the α chain is about 34-37kD in size, while the β chain is 32kD. The CD8α chain is often used as an immunogen for anti-CD8 antibodies.

[0004] CD8 molecules are distributed on the surface of some T cells and thymocytes. Methods for detecting the localization of CD8 molecules in cells or tissues include cell immunofluorescence (ICC), immunohistochemistry (IHC), etc., but there is still a need to develop antibodies that can be applied to tissue immunofluorescence (IF). CD8a is a transmembrane protein. Currently, the immunogens of anti-CD8 antibodies include T cells, CD8a full-length protein, or intracellular peptides. Anti-CD8 antibodies are usually polyclonal antibodies obtained by immunization, or monoclonal antibodies obtained by further screening.

[0005] The sequences targeted by anti-CD8 antibodies obtained using T cells or full-length CD8a proteins as immunogens are unclear, while antibodies obtained using CD8a intracellular segments as immunogens are more suitable for IHC of paraffin sections. Therefore, it is necessary to develop anti-CD8 antibodies for IF of frozen tissues. Summary of the invention

[0006] In order to solve the defects of the IF technology that the immune epitopes of anti-CD8 antibodies obtained in the prior art are unclear and are not conducive to application to frozen tissues, the present invention provides an antigen polypeptide, an antibody prepared therefrom, and its application in detecting CD8. Specifically, the antigen polypeptide provided by the present invention is derived from the N-terminal extracellular region of CD8a, and the polyclonal antibody obtained after immunization has a stronger signal in immunofluorescence applications than the polypeptide located at the C-terminus. The anti-human CD8 rabbit polyclonal antibody obtained by immunizing animals with the antigen polypeptide provided by the present invention can be used for immunofluorescence detection of human tonsil tissue after purification, and can detect single cell membrane structures.

[0007] The first aspect of the present invention provides an antigen polypeptide, which is derived from the N-terminal extracellular region of CD8a, and the N-terminus of the antigen polypeptide includes an additional cysteine.

[0008] In certain embodiments, the antigenic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1. Preferably, the amino acid sequence of the antigenic polypeptide is shown in SEQ ID NO: 1.

[0009] The second aspect of the present invention provides a derivative polypeptide, which can produce the antigen polypeptide as described in the first aspect of the present invention after enzymatic hydrolysis, or the derivative polypeptide has a tag sequence added to the N-terminus or C-terminus of the antigen polypeptide as described in the first aspect of the present invention.

[0010] The third aspect of the present invention provides a fusion protein, which comprises the antigen polypeptide as described in the first aspect of the present invention or the derivative polypeptide as described in the second aspect of the present invention. Preferably, a carrier protein such as KLH or OVA is coupled to the N-terminus of the antigen polypeptide or the derivative polypeptide.

[0011] The fourth aspect of the present invention provides an isolated nucleic acid molecule encoding the antigenic polypeptide as described in the first aspect of the present invention or the derivative polypeptide as described in the second aspect of the present invention or the fusion protein as described in the third aspect of the present invention.

[0012] The fifth aspect of the present invention provides a cell comprising the nucleic acid molecule as described in the fourth aspect of the present invention. Preferably, the cell is a T cell or an antigen presenting cell. More preferably, the antigen presenting cell includes a macrophage, a dendritic cell, a synaptic cell, a Langerhans cell, a B cell, and the like.

[0013] The sixth aspect of the present invention provides an anti-CD8 antibody, which is obtained by immunizing an animal with the antigen polypeptide as described in the first aspect of the present invention, the derivative polypeptide as described in the second aspect of the present invention, or the fusion protein as described in the third aspect of the present invention, and specifically targets the antigen polypeptide as described in the first aspect of the present invention. Preferably, the animal is a mammal such as a rabbit.

[0014] In certain embodiments, the anti-CD8 antibody is a monoclonal antibody or a polyclonal antibody.

[0015] The seventh aspect of the present invention provides the use of the antigen polypeptide as described in the first aspect of the present invention, the derivative polypeptide as described in the second aspect of the present invention, the fusion protein as described in the third aspect of the present invention, the cell as described in the fifth aspect of the present invention, and the anti-CD8 antibody as described in the sixth aspect of the present invention in ELISA, WB, flow cytometry, immunofluorescence, immunohistochemistry detection and / or preparation of detection reagents.

[0016] The eighth aspect of the present invention provides a method for detecting CD8, wherein the anti-CD8 antibody as described in the sixth aspect of the present invention is contacted with a sample, and the sample is characterized to determine whether CD8 is contained in the sample.

[0017] The sample of the present invention may be from a tissue or organ, etc. The tissue may be epithelial tissue, connective tissue, nerve tissue and muscle tissue; the organ may be an organ containing a single cell membrane structure, such as tonsil.

[0018] The ninth aspect of the present invention provides an application of the antigen polypeptide as described in the first aspect of the present invention or the derivative polypeptide as described in the second aspect of the present invention in preparing tumor-specific T cells or antigen-presenting cells, preparing TCR-T cells, preparing tumor diagnostic reagents and / or preparing drugs for preventing or treating tumors.

[0019] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0020] The reagents and raw materials used in the present invention are commercially available.

[0021] The positive and progressive effects of the present invention are:

[0022] The present invention provides a polypeptide for preparing an anti-CD8 antibody and its application in Elisa and IF detection. The polypeptide is located at the N-terminus of the CD8a molecule, and the polyclonal anti-CD8 antibody obtained after immunizing a rabbit can be applied to immunofluorescence technology after purification to detect a single cell membrane structure. The polyclonal anti-CD8 antibody of the present invention has the advantages of clear epitope and stronger luminescent signal in frozen human tonsil tissue compared to the polyclonal anti-CD8 antibody located at the C-terminus of the immunogen. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It shows the structure of the CD8a extracellular region and the location of the polypeptide (dark color).

[0024] Figure 2 Negative control for immunofluorescence detection of human tonsil tissue.

[0025] Figure 3 Immunofluorescence detection of human tonsil tissue using the monoclonal antibody ant527_351_B06 (reference PMID: 18047641) obtained for extracellular domain immunoscreening.

[0026] Figure 4 Immunofluorescence assay of human tonsil tissue immunized with CD8a C-terminal peptide to obtain polyclonal anti-CD8 antibodies.

[0027] Figure 5 Immunofluorescence detection of human tonsil tissue immunized with CD8a N-terminal peptide to obtain polyclonal anti-CD8 antibodies.

[0028] Figure 6 A partial magnified image of immunofluorescence detection of human tonsil tissue obtained by immunization with CD8a N-terminal peptide to obtain polyclonal anti-CD8 antibodies.

[0029] Figure 7 Image J immunofluorescence signal analysis of three anti-CD8 antibodies. DETAILED DESCRIPTION

[0030] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0031] Amino acid sequence of antigenic polypeptide (SEQ ID NO: 1)

[0032] CPKAAEGLDTQRFSGKRLGDTFVLTLSD

[0033] Amino acid sequence of human CD8a (SEQ ID NO: 2)

[0034] MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD IYIWAPLAGTCGVLLLSLVIT LYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV

[0035] The underlined part is the transmembrane region.

[0036] Example 1 Coupling and purification of CD8a polypeptide

[0037] This embodiment provides a new antigen polypeptide, whose amino acid sequence is SEQ ID NO: 1, and the antigen polypeptide is selected from human CD8a, whose amino acid sequence is: SEQ ID NO: 2. The position of the antigen polypeptide in CD8a is as follows Figure 1 Dark color shown.

[0038] Cysteine ​​was added to the N-terminus of the antigen polypeptide to facilitate coupling with the carrier protein KLH for immunization. The antigen polypeptide was coupled and purified with maleimide-activated mcKLH according to the kit instructions. The solvents and consumables were all from ThermoScientific TM Imject TM Maleimide-activated mcKLH is provided in the Centrifugation Kit (Cat. No. 77666).

[0039] The specific steps of coupling are:

[0040] 1. Add 200 μL of ultrapure water to mcKLH to make a 10 mg / mL solution.

[0041] 2. Add 200 μL Conjugation Buffer to 2 mg of the antigen polypeptide.

[0042] 3. Immediately mix the antigen polypeptide and mcKLH, incubate at room temperature (22-26° C.) for 2 h to obtain a coupled sample.

[0043] After coupling, desalting and purification are carried out. The specific operation steps are as follows:

[0044] 1. Preparation of eluent: Add 10 mL of ultrapure water to Imject Purification Buffer Salts.

[0045] 2. Balancing the desalting column: unscrew the knob at the bottom of the centrifugal desalting column, centrifuge at 1000g for 2 minutes, add 1mL purification buffer, and centrifuge at 1000g for 2 minutes to obtain a balanced desalting column.

[0046] 3. Sample desalting and purification: Replace the new receiving tube, add 500 μL of the coupled sample to the balanced desalting column, and centrifuge at 1000 g for 2 minutes. The new receiving tube contains the desalted and purified antigen polypeptide KLH coupled product, which can be used for immunization.

[0047] Example 2 Animal Immunization

[0048] The immunization in this example was performed by CRO company, and two rabbits were immunized with each antigen polypeptide.

[0049] 1. Take 1 ml of blood from the ears of two rabbits respectively, and collect serum for control. Immunize each rabbit with 1 mg of antigen, emulsify with complete adjuvant, dilute 2 mg of antigen into 1.5 ml of PBS, mix 1.5 ml of antigen + 1.5 ml of complete adjuvant, and shake on an oscillator for more than 15 minutes until the oil-in-water state; 1.5 ml is immunized on the back of each rabbit.

[0050] 2. Two weeks later, the second immunization was carried out. Each rabbit was immunized with 0.5 mg of antigen. It was emulsified with incomplete adjuvant. 1 mg of antigen was diluted into 1 ml of PBS. The mixture was 1 ml of antigen + 1 ml of incomplete adjuvant and oscillated on an oscillator for more than 15 minutes until it reached the oil-in-water state. 1 ml was applied to the back of each rabbit.

[0051] 3. One week later, each rabbit was immunized with 0.5 mg of antigen, which was emulsified with incomplete adjuvant. 1 mg of antigen was diluted into 1 ml of PBS, and mixed with 1 ml of antigen + 1 ml of incomplete adjuvant. The mixture was shaken on an oscillator for more than 15 minutes until it reached the oil-in-water state. Each rabbit was immunized with 1 ml on its back.

[0052] 4. One week later, 0.5 mg of antigen was emulsified with incomplete adjuvant for each rabbit, 1 mg of antigen was diluted into 1 ml of PBS, and 1 ml of antigen + 1 ml of incomplete adjuvant were mixed, and shaken on an oscillator for more than 15 minutes until the oil-in-water state; 1 ml was immunized on the back of each rabbit;

[0053] 5. One week later, collect 1 ml of blood from the ear and test the titer by ELISA. If it reaches 1:100,000 or above, perform shock immunization.

[0054] 6. For shock immunization, 1 mg of antigen was immunized per rabbit. Emulsified with complete adjuvant, 2 mg of antigen was diluted into 1.5 ml PBS, and mixed according to 1.5 ml antigen + 1.5 ml complete adjuvant. Oscillate on an oscillator for more than 15 minutes until the oil-in-water state is reached; 1.5 ml was applied to the back of each rabbit for immunization.

[0055] 7. Three days later, blood was collected from the ears. 5 ml of blood was collected from each rabbit for titer measurement. If the titer was in line with expectations (ELISA titer reached over 1:100,000), whole blood was collected in anticoagulant tubes and serum was separated.

[0056] Example 3 Antiserum titer ELISA

[0057] The OVA-coupled antigen peptide was used for post-immunization titer test. The OVA-coupled reagent was ThermoScientific TM , Imject TM The specific operation and purification scheme of maleimide-activated ovalbumin (OVA product number: 77126) are as described in Example 1.

[0058] The specific steps of antiserum titer ELISA detection are as follows:

[0059] 1. Dilute the OVA-coupled polypeptide to 0.1 μg / mL with PBS, add 100 μL to each well of the immunoplate, and incubate overnight in a 4°C refrigerator.

[0060] 2. Wash the ELISA plate 3 times with PBST, 300 μl per well. Pat off any residual liquid on a paper towel for the last wash.

[0061] 3. Add 300 μl of 2% BSA blocking solution to each well and incubate at 37°C for 1 h.

[0062] 4. Wash the ELISA plate three times with PBST, 300 μl per well, and tap off any remaining liquid on a paper towel the last time.

[0063] 5. Dilute the antiserum with PBS by 22,000, 64,000, and 128,000 times, respectively, add 100 μL to each well of the immunoplate, and incubate at 37°C for 1 hour.

[0064] 6. Wash the ELISA plate 6 times with PBST, 300 μl per well. Pat off any residual liquid on a paper towel for the last wash.

[0065] 7. Add 100 μl of Anti-rabbit Antibody (HRP) diluted in PBS to each well and incubate at room temperature for 1 hour.

[0066] 8. Wash the ELISA plate 6 times with PBST, 300 μl per well. Pat off any residual liquid on a paper towel for the last wash.

[0067] 9. Take out Solution A and Solution B of TMB Substrate Solution Set from 4°C, return to room temperature, mix in equal proportions to prepare an appropriate amount of colorimetric solution, add 100 μl to each well of the ELISA plate, and react for 10 minutes in the dark.

[0068] 10. Add 50 μl 1M HCl to each well to terminate the color development reaction and read the OD450 value with a microplate reader.

[0069] The test results are shown in Table 1. The titer after four immunizations was greater than 128,000.

[0070] Table 1 OD450 values ​​after immunization of rabbits

[0071] serial number Dilution OD450 OD450-Blank blank 0.045 Rabbit 1 Four Free 22000 2.539 2.494 Rabbit 1 Four Free 64000 2.228 2.183 Rabbit 1 Four Free 128000 1.980 1.935 Rabbit 1 before immunization 22000 0.046 0.001 Rabbit 1 before immunization 64000 0.044 -0.001 Rabbit 1 before immunization 128000 0.045 0.000 Rabbit 2 Four Free 22000 1.560 1.515 Rabbit 2 Four Free 64000 2.058 2.013 Rabbit 2 Four Free 128000 2.060 2.015 Rabbit 2 before immunization 22000 0.042 -0.003 Rabbit 2 before immunization 64000 0.044 -0.001 Rabbit 2 before immunization 128000 0.047 0.002

[0072] Example 4 Affinity chromatography purification of antiserum

[0073] The magnetic beads (Yisheng Bio, 36417ES03) needed for affinity chromatography (protein A) were washed with PBS, and the serum was added to the balanced magnetic beads for penetration (it can also be placed at 4°C for 1-2 hours and mixed every 10 minutes), washed with PBS (5 times column volume 3 times or more), eluted with eluent (100mM glycine + 20mM arginine, acetic acid adjusted to pH 3.0), and the protein concentration was detected using the IgG mode of the microplate reader. 1M Tris-HCL pH 9.0 was added to neutralize the eluent, and dialyzed into PBS to obtain the polyclonal anti-CD8 antibody after Protein A purification. After aliquoting, it was quickly frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0074] Example 5 Immunofluorescence detection of polyclonal anti-CD8 antibodies

[0075] Place the purchased human tonsil slices on a slide, immerse the tissue with methanol, and place at -20℃ for 30 minutes. Take out and dry. Wash the slices 1-2 times with washing solution (PBS+0.1% Triton X-100), add 100μL of blocking solution (PBS+0.1% TritonX-100+5% serum (sheep serum and bovine serum mixed in a ratio of 1:1; sheep serum, Gibco, 16210064; bovine serum, Gibco, 26050070)), and incubate at room temperature for 15-20 minutes. Use a pipette to suck up the blocking solution, immediately add the polyclonal anti-CD8 antibody (1mg / mL anti-CD8 antibody diluted 1000 times with blocking solution), and incubate at room temperature for 45 minutes. Use a pipette to suck up the anti-CD8 antibody solution, wash the slices 3 times with the above washing solution, and incubate at room temperature for 1 minute each time. Immediately add the diluted anti-RabbitIgG Alexa Fluor TM 488 fluorescent secondary antibody (Thermo, A-21206), incubate at room temperature for 30 minutes. Wash the sections 3 times with the above washing solution, incubate at room temperature for 1 minute each time. Wash the sections 3 times with nuclease-free water. Dry and observe under a microscope. Results The polyclonal anti-CD8 antibody obtained by immunization with the polypeptide can detect single cell membrane structures in immunofluorescence detection of human tonsils, such as Figure 2 shown.

[0076] Example 6 Image J immunofluorescence analysis

[0077] Use Image J software to open the original immunofluorescence images of three anti-CD8 antibodies ( Figure 3 , Figure 4 , Figure 5), convert to 8-bit grayscale (Image>Type>8-bit) and invert black and white (Edit>Invert), select the measurement unit (Analyze>Set scale) as pixels and the measurement items (Analyze>Set measurements) in Area and Integrated density, check Limit to threshold, and click OK. Frame the target area in the image (i.e. Figure 6 Area), measure (Analyze>Measure), the signal value of each area is displayed in the results (Results), record the measured area (Area) and signal value (IntDen), and calculate the unit signal value (Average). The statistical results are shown in Table 2 and the statistical graph is shown in Figure 7 shown.

[0078] Table 2 Measurement area and signal value of different anti-CD8 antibodies

[0079] Anti-CD8 antibody Area IntDen Average NC 19320 579.164 0.029977 ECD monoclonal antibody 19872 625.625 0.031483 C-terminal polyclonal antibody 18216 1110.054 0.060938 N-terminal polyclonal antibody 18144 3253.732 0.179328

[0080] Image J immunofluorescence analysis showed that the polyclonal anti-CD8 antibody obtained after immunization with the antigen peptide derived from the N-terminal extracellular region of CD8a had a stronger signal than other peptides in immunofluorescence applications.

Claims

1. An antigen polypeptide, characterized in that The antigen polypeptide is derived from the N-terminal extracellular region of CD8a, and the N-terminus of the antigen polypeptide includes an additional cysteine.

2. The antigen polypeptide according to claim 1, characterized in that The antigen polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1; Preferably, the amino acid sequence of the antigen polypeptide is as shown in SEQ ID NO:

1.

3. A derivative polypeptide, characterized in that: The derived polypeptide can produce the antigen polypeptide as claimed in claim 1 or 2 after enzymatic hydrolysis, or the derived polypeptide has a tag sequence added to the N-terminus or C-terminus of the antigen polypeptide as claimed in claim 1 or 2.

4. A fusion protein, characterized in that It comprises the antigenic polypeptide according to claim 1 or 2 or the derivative polypeptide according to claim 3; Preferably, a carrier protein such as KLH or OVA is coupled to the N-terminus of the antigenic polypeptide or the derivative polypeptide.

5. An isolated nucleic acid molecule, characterized in that It encodes the antigenic polypeptide according to claim 1 or 2, the derivative polypeptide according to claim 3, or the fusion protein according to claim 4.

6. A cell, characterized in that It comprises the nucleic acid molecule according to claim 5; preferably, the cell is a T cell or an antigen presenting cell.

7. An anti-CD8 antibody, characterized in that It is obtained by immunizing an animal with the antigen polypeptide according to claim 1 or 2, the derivative polypeptide according to claim 3, or the fusion protein according to claim 4, and specifically targets the antigen polypeptide according to claim 1 or 2; Preferably, the animal is a mammal such as a rabbit; and / or the anti-CD8 antibody is a monoclonal antibody or a polyclonal antibody.

8. Use of the antigen polypeptide according to claim 1 or 2, the derivative polypeptide according to claim 3, the fusion protein according to claim 4, the cell according to claim 6, and the anti-CD8 antibody according to claim 7 in ELISA, WB, flow cytometry, immunofluorescence, immunohistochemistry detection and / or preparation of detection reagents.

9. A method for detecting CD8, characterized in that: The anti-CD8 antibody according to claim 7 is contacted with a sample, and the sample is characterized to determine whether it contains CD8.

10. Use of the antigen polypeptide according to claim 1 or 2 or the derivative polypeptide according to claim 3 in preparing tumor-specific T cells or antigen-presenting cells, preparing TCR-T cells, preparing tumor diagnostic reagents and / or preparing drugs for preventing or treating tumors.

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