Mouse anti-rabbit CD4 monoclonal antibody and its application

Abstract

The present invention provides a mouse anti-rabbit CD4 monoclonal antibody and its application, relating to the field of biotechnology. The mouse anti-rabbit CD4 monoclonal antibody provided by the present invention has a variable region comprising complementary determining regions (CDR1-VH, CDR2-VH, CDR3-VH, CDR1-VL, CDR2-VL, and CDR3-VL) having specific amino acid sequences. This monoclonal antibody has strong specificity and high sensitivity, can specifically identify CD4+ T cell populations, and can be used for sorting CD4+ T cell populations.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a mouse anti-rabbit CD4 monoclonal antibody and an application thereof. Background Art

[0002] Flow cytometry allows for high-throughput cell sorting using different fluorescent markers, enabling the sorting of hundreds of thousands to tens of millions of cell populations in a short period of time. Target B cells, identified through screening with immunoglobulins bound to B cell surface membranes and specific fluorescently labeled antigens, are then isolated and subjected to in vitro single-cell RNA extraction and amplification to obtain recombinant antibody sequence information. This can significantly increase the speed of obtaining specific antigen-antibody sequences from immunized animals. Therefore, antibody development protocols based on single B cell isolation using flow cytometry can improve antibody development efficiency.

[0003] At present, the two commonly used single B cell sorting schemes are single B cell culture in vitro and single B cell screening by microfluidic technology. Neither of these methods requires screening T cells to obtain antigen-binding positive B cells. Therefore, the development of an antibody development scheme for separating rabbit single B cells based on flow cytometry requires the use of corresponding rabbit T cell sorting marker antibodies to ensure the purity of the obtained B cells. Secondly, there are also reported mouse anti-rabbit CD4 monoclonal antibodies, but they are basically controlled by foreign companies (such as BD, Bio-Rad, etc.). Therefore, there is an urgent need to develop independent anti-rabbit CD4 antibodies based on mouse hybridoma technology.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The first object of the present invention is to provide a mouse anti-rabbit CD4 monoclonal antibody.

[0006] The second object of the present invention is to provide the use of the above mouse anti-rabbit CD4 monoclonal antibody in the separation of CD4+ T cell populations.

[0007] The third object of the present invention is to provide a marker for CD4+ T cell population.

[0008] The fourth object of the present invention is to provide a gene encoding the mouse anti-rabbit CD4 monoclonal antibody.

[0009] A fifth object of the present invention is to provide a hybridoma cell.

[0010] The sixth object of the present invention is to provide a method for preparing mouse anti-rabbit CD4 monoclonal antibody.

[0011] In a first aspect, the present invention provides a mouse anti-rabbit CD4 monoclonal antibody, wherein the variable regions of the mouse anti-rabbit CD4 monoclonal antibody include: a complementary determining region CDR1-VH having the amino acid sequence shown in SEQ ID NO.1, a complementary determining region CDR2-VH having the amino acid sequence shown in SEQ ID NO.2, a complementary determining region CDR3-VH having the amino acid sequence shown in SEQ ID NO.3, a complementary determining region CDR1-VL having the amino acid sequence shown in SEQ ID NO.4, a complementary determining region CDR2-VL having the amino acid sequence shown in SEQ ID NO.5, and a complementary determining region CDR3-VL having the amino acid sequence shown in SEQ ID NO.6.

[0012] As a further technical solution, the variable region includes a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.7.

[0013] As a further technical solution, the variable region includes a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO.8.

[0014] In a second aspect, the present invention provides the use of the above-mentioned mouse anti-rabbit CD4 monoclonal antibody in sorting CD4+ T cell populations.

[0015] As a further technical solution, the CD4+T cell population includes the CD4+T cell population in rabbit peripheral blood lymphocytes.

[0016] In a third aspect, the present invention provides a marker for a CD4+ T cell population, the marker comprising the mouse anti-rabbit CD4 monoclonal antibody and a fluorescent dye;

[0017] The mouse anti-rabbit CD4 monoclonal antibody is conjugated to a fluorescent dye.

[0018] As a further technical solution, the fluorescent dye includes iFluor488 or iFluor594.

[0019] In a fourth aspect, the present invention provides a gene encoding the mouse anti-rabbit CD4 monoclonal antibody, wherein the gene has the nucleic acid sequence shown as SEQ ID NO.9 and SEQ ID NO.10.

[0020] In a fifth aspect, the present invention provides a hybridoma cell, wherein the hybridoma cell expresses the mouse anti-rabbit CD4 monoclonal antibody.

[0021] As a further technical solution, the hybridoma cells are obtained by fusion of mouse spleen cells and mouse myeloma cells;

[0022] The mouse spleen cells express the mouse anti-rabbit CD4 monoclonal antibody.

[0023] In a sixth aspect, the present invention provides a method for preparing a mouse anti-rabbit CD4 monoclonal antibody, comprising: culturing the hybridoma cells in the peritoneal cavity of a mouse, and then isolating and obtaining the mouse anti-rabbit CD4 monoclonal antibody.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides a mouse anti-rabbit CD4 monoclonal antibody. The monoclonal antibody has strong specificity and high sensitivity, can specifically identify CD4+T cell populations, and can be used for sorting CD4+T cell populations. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 The SDS-PAGE results provided in Example 1;

[0028] Figure 2 The WB results of the rabbit CD4 overexpression cell line provided in Example 4;

[0029] Figure 3 The WB results of the rabbit CD5 overexpression cell line provided in Example 4;

[0030] Figure 4 The FC results of the rabbit CD4 overexpressing cell line provided in Example 4;

[0031] Figure 5 The FC results of the rabbit CD5 overexpression cell line provided in Example 4;

[0032] Figure 6 The flow cytometry results provided in Example 5;

[0033] Figure 7 The flow cytometry competition experiment results provided in Example 9;

[0034] Figure 8 The flow cytometry titer test results provided in Example 9. DETAILED DESCRIPTION

[0035] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0036] It should be noted that the "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domain of the heavy chain can be referred to as "VH". The variable domain of the light chain can be referred to as "VL". These domains are usually the most variable parts of the antibody and contain the antigen binding site. The light or heavy chain variable region is composed of a framework region interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The framework region of an antibody, that is, the combined framework region of the constituent light and heavy chains, plays a role in positioning and aligning the CDRs, which are primarily responsible for binding to the antigen.

[0037] "Framework" or "FR" regions refer to the regions of an antibody variable domain excluding those defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR1, FR2, FR3, and FR4) separated by CDRs.

[0038] Typically, the variable regions VL / VH of the heavy and light chains can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0039] In the present invention, CDR1-VH, CDR2-VH and CDR3-VH refer to the three hypervariable regions of the heavy chain variable region, respectively. Correspondingly, CDR1-VL, CDR2-VL and CDR3-VL refer to the three hypervariable regions of the light chain variable region, respectively.

[0040] In a first aspect, the present invention provides a mouse anti-rabbit CD4 monoclonal antibody, wherein the variable regions of the mouse anti-rabbit CD4 monoclonal antibody include: a complementary determining region CDR1-VH having the amino acid sequence shown in SEQ ID NO.1, a complementary determining region CDR2-VH having the amino acid sequence shown in SEQ ID NO.2, a complementary determining region CDR3-VH having the amino acid sequence shown in SEQ ID NO.3, a complementary determining region CDR1-VL having the amino acid sequence shown in SEQ ID NO.4, a complementary determining region CDR2-VL having the amino acid sequence shown in SEQ ID NO.5, and a complementary determining region CDR3-VL having the amino acid sequence shown in SEQ ID NO.6.

[0041] The sequences of the above variable regions are shown in Table 1:

[0042] Table 1

[0043] CDR1-VH DYAMH SEQ ID NO.1 CDR2-VH VISIHYDKTNYNQKFKG SEQ ID NO.2 CDR3-VH EGIYFDY SEQ ID NO.3 CDR1-VL KSSQSLLESDGKTYLN SEQ ID NO.4 CDR2-VL LVSKLDS SEQ ID NO.5 CDR3-VL WQGTHFPLT SEQ ID NO.6

[0044] The mouse anti-rabbit CD4 monoclonal antibody provided by the present invention has strong specificity and high sensitivity, can specifically identify CD4+T cell populations, and can be used for sorting CD4+T cell populations.

[0045] In some preferred embodiments, the variable region includes a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.7.

[0046] The amino acid sequence of the heavy chain variable region VH of the variable region is as follows:

[0047] QVQLQQSGPELVRPGESVKISCKGSGYTFTDYAMHWVKQSHAKSLEWVGVISIHYDKTNYNQKFKGKATMTVDKSSSTAYLELARLTSEDSAIYYCTREGIYFDYWGQGTTLTVSS (SEQ ID NO. 7).

[0048] In some preferred embodiments, the variable region includes a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO.8.

[0049] The amino acid sequence of the light chain variable region VL of the variable region is as follows:

[0050] DVVMTQTPFTLSVIIGQPASISCKSSQSLLESDGKTYLNWLLQRPGQ SPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGT HFPLTFGAGTKLELK (SEQ ID NO. 8).

[0051] In some preferred embodiments, the binding protein further comprises a light chain constant region and a heavy chain constant region. The constant region and the variable region are combined to obtain a complete antibody.

[0052] In a second aspect, the present invention provides the use of the above-mentioned mouse anti-rabbit CD4 monoclonal antibody in sorting CD4+ T cell populations.

[0053] The mouse anti-rabbit CD4 monoclonal antibody provided by the present invention has strong specificity and high sensitivity, can specifically identify CD4+T cell populations, and therefore can be used for sorting CD4+T cell populations.

[0054] In some preferred embodiments, the CD4+T cell population includes CD4+T cell populations in rabbit peripheral blood lymphocytes, rabbit spleen, rabbit lymph nodes and other related tissue immune cells.

[0055] In a third aspect, the present invention provides a marker for a CD4+ T cell population, the marker comprising the mouse anti-rabbit CD4 monoclonal antibody and a fluorescent dye;

[0056] The mouse anti-rabbit CD4 monoclonal antibody is conjugated to a fluorescent dye.

[0057] The marker of the CD4+ T cell population provided by the present invention can be used for specific labeling of the CD4+ T cell population.

[0058] As a further technical solution, the fluorescent dye includes but is not limited to iFluor488 or iFluor594, or other dyes well known to those skilled in the art.

[0059] In a fourth aspect, the present invention provides a gene encoding the mouse anti-rabbit CD4 monoclonal antibody, wherein the gene has the nucleic acid sequence shown in SEQ ID NO.9 and SEQ ID NO.10:

[0060] CAGGTCCAGCTGCAGCAGTCTGGGCCTGAGCTGGTGAGGCCTGGGGAATCAGTGAAGATTTCCTGCAAGGGTTCCGGCTACACATTCACTGATTATGCTATGCACTGGGTGAAGCAGAGTCATGCAAAGAGTCTAGAGTGGGTTGGAGTTATTAGTATTCATTATGATAAAACAAA CTACAACCAGAAGTTTAAGGGCAAGGCCACAATGACTGTAGACAAATCCTCCAGCACAGCCTATTTGGAACTTGCCAGATTGACATCTGAGGATTCTGCCATCTATTACTGTACAAGAGAGGGCATCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ ID NO.9).

[0061] GATGTTGTGATGACCCAGACTCCATTCACTTTGTCGGTTATCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGAAAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTC TAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ IDNO.10).

[0062] In a fifth aspect, the present invention provides a hybridoma cell, wherein the hybridoma cell expresses the mouse anti-rabbit CD4 monoclonal antibody.

[0063] The hybridoma cell can be used for the preparation of mouse anti-rabbit CD4 monoclonal antibody.

[0064] In some preferred embodiments, the hybridoma cells are obtained by fusion of mouse spleen cells and mouse myeloma cells;

[0065] The mouse spleen cells express the mouse anti-rabbit CD4 monoclonal antibody.

[0066] In a sixth aspect, the present invention provides a method for preparing a mouse anti-rabbit CD4 monoclonal antibody, comprising: culturing the hybridoma cells in the peritoneal cavity of a mouse, and then isolating and obtaining the mouse anti-rabbit CD4 monoclonal antibody.

[0067] The preparation method is simple and convenient.

[0068] The present invention is further described below by means of specific examples and comparative examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0069] Example 1 Immunogen Preparation

[0070] The CD4 protein is a single-pass transmembrane glycoprotein primarily expressed on the surface of helper T cells. CD4 molecules bind to MHC II molecules on helper T cells to present specific antigens. The extracellular region 29-396 of the rabbit CD4 molecule is expressed in eukaryotic cells. Full-length rabbit CD4 extracellular protein is obtained at a concentration and purity that meets immunization requirements.

[0071] Cell transfection and protein purification:

[0072] Reagent: Expi293 TM Expression system kit (Thermo, A14635), nickel NTA agarose gel, PBS buffer.

[0073] Consumables: 96-well deep-well plates, pipette tips.

[0074] Equipment: Constant temperature shaker, electronic pipette.

[0075] Step a): Incubate the Expi293F culture medium in a 37°C water bath in advance. Observe the cell status and cell growth under a microscope. Determine the cell expansion ratio for the day based on the cell growth and the needs of the subsequent transfection experiment. Generally, the cell density is controlled at 2x10 6 About 100 living cells / mL. Take out the cell solution from the CO2 cell shaker, count it, and calculate the amount of cells required for transfer. Select the most suitable cell shake flask according to the amount of cells transferred. Generally, the culture volume should not exceed 1 / 3 of the shake flask volume and should not be less than 1 / 5 of the shake flask volume. Add preheated Expi293 culture medium according to the cell density to make the final cell density at 2x10 6 About 100 living cells / mL, used for transfection operation on the next day.

[0076] Step b): On the day of transfection, pre-warm Expi293 medium at room temperature, away from light. Determine the number of cells to be transfected and prepare the appropriate number of shake flasks. Determine the cell density by cell counting. Dilute the cells with pre-warmed medium to a final density of 3x106 viable cells / mL. Pipette 1 ml / well into a deep-well plate and carefully incubate in a shaker at 37°C, 8% CO2, 900 rpm, and 80% humidity. Dispense the appropriate number of shake flasks into the culture plate at a ratio of 1 μg DNA to 60 μl Opti-MEM (cold reagent) per 1 ml of cells. Mix thoroughly by pipetting (recommended antibody ratio: HC:LC = 1:2). In a separate centrifuge tube of optimal volume, dilute the transfection reagent to a ratio of 3.2 μl Expifectamine 293 to 60 μl Opti-MEM (cold reagent) per 1 ml of cells. Gently invert to mix thoroughly and let stand at room temperature for 5 minutes. After 5 minutes, transfer the diluted transfection reagent to a disposable reservoir and pipette into the appropriate well of a 96-well cell culture plate. Let it sit for 10-20 minutes. Add the ExpiFectamine 293 / rabbit CD4 expression plasmid complex to the cell suspension using a pipette. Carefully incubate the suspension in a shaker at 37°C, 8% CO2, 900 rpm, and 80% humidity. 18-22 hours after transfection, add 6 μl of Enhancer 1 and 60 μl of Enhancer 2 to each ml of the culture medium. Harvest the supernatant 4-7 days later for recombinant protein purification.

[0077] Step c): The recombinant protein contains a histidine tag and is purified by nickel column: the cell supernatant is filtered through a 0.45 μM filter and then added to nickel NTA agarose gel and incubated at 4 degrees for binding. The eluted recombinant protein is replaced with PBS buffer by ultrafiltration or dialysis and identified by SDS-PAGE staining (e.g. Figure 1 ), rabbit recombinant CD4 protein showed obvious specific bands in PAGE gel, with a molecular weight between 40-55KDa, and the protein purity and concentration met the requirements for immunity.

[0078] Example 2 Mouse immunization and serum collection

[0079] Mouse immunization procedures:

[0080] Reagents: adjuvant, 75% alcohol.

[0081] Consumables: Syringe.

[0082] Sample: Rabbit recombinant CD4 protein.

[0083] Step a): Animal selection: BALB / c female rabbits weighing about 18-22 g, aged about 6-8 weeks, with smooth fur and free movement were selected.

[0084] Step b): Preparation before the experiment: The mice are marked.

[0085] Step c): Remove the antigen from the -20°C freezer and dissolve it at room temperature, avoiding repeated freeze-thaw cycles. Label the syringe with the project number and animal number.

[0086] Step d): extract the antigen (mix the antigen completely), the antigen concentration is 0.5 mg / mL, and the immunization dose for mice is 0.1 mL / mouse.

[0087] Step e): Dispense the adjuvant at a 1:1 volume ratio of adjuvant to antigen. Use complete adjuvant for the first dose, and incomplete adjuvant for the second and third doses. Thoroughly mix the adjuvant before drawing it into the syringe.

[0088] Step f): After connecting two syringes with a syringe connecting tube, complete emulsification is performed. The emulsification standard is: the emulsified immunogen is qualified if it does not disperse when dropped into 37° C. water.

[0089] Step g): Immunization: Mice were immunized with multiple subcutaneous injections. Immunization interval: 14 days. Blood samples were collected from mice on day 7 after three immunizations for titer testing.

[0090] Step h): Steps for collecting small samples of serum: The blood collector uses the thumb and index finger of his left hand to press on both sides of the mouse's neck, and hold the mouse's neck tightly from the back to cause congestion of the retro-orbital venous plexus. When holding the mouse as a whole in the hand, make sure that the mouse's body remains in a straight position. Use the right hand to make the blood collector form an angle of 45° with the mouse's face, and insert the needle from the inner corner of the eye, with the bevel of the needle facing the eyeball first. After insertion, turn 180 degrees so that the bevel faces the posterior border of the orbit. The insertion depth is about 2 to 3 mm. Stop pushing when you feel resistance, and slightly turn the blood collector if there is no bleeding. Draw 200uL of blood at a time. After blood collection, wipe the wound with sterile cotton, press slightly to stop bleeding, and put the mouse back into the cage only after confirming that there is no abnormality.

[0091] Step i): Centrifuge the collected serum sample at 10,000 rpm for 10 minutes. Collect the supernatant and freeze it at -20°C for later use.

[0092] Example 3 Serum titer detection

[0093] The immunogen titer of mouse serum was tested by indirect ELISA. Mice with an OD450nm value exceeding 1.0 at a serum dilution of 1:8100 were considered to have qualified for immunization and could proceed to the next step.

[0094] Serum stage indirect ELISA operation:

[0095] Reagents: goat anti-mouse-HRP (Hua'an Biotechnology: HA1006), TMB substrate (Sigma: T2885), Tris (Shanghai Bioengineering: A501492), glycine (Shanghai Bioengineering: GB0235), BSA (Shanghai Bioengineering: A500023-0100), Tween-20 (Shanghai Bioengineering: A600560), NaHCO3 (Shanghai Bioengineering: A610482-0500); Na2CO3, Na2HPO4·12H2O, NaH2PO4·2H2O, citric acid, glycerol, DMSO, and concentrated sulfuric acid were purchased from Hangzhou Shuangmu Chemical; hydrogen peroxide and EDTA were purchased from Shanghai Bioengineering and were domestically produced analytical grade.

[0096] Consumables: ELISA plate (Hangzhou Shengyou).

[0097] Equipment: electric constant temperature incubator (Shanghai Senxin: DRP-9162), enzyme labeling instrument (MD: Cmax plus).

[0098] Step a): Coating: dilute the rabbit recombinant CD4 protein to 1 μg / mL with coating buffer, add 50 μL / well to the ELISA plate, cover the plate, and coat at 4°C overnight.

[0099] Step b): Blocking: Shake off the liquid in the wells, add 1% BSA / TBS to the ELISA plate at a volume of 100 μL / well, and place in a 37° C. electric thermostatic incubator for blocking for 1 hour.

[0100] Step c): Sample addition: Shake wells dry and add 50 μL / well of serum at different dilution ratios to the ELISA plate. Cover the plate and incubate in a 37°C incubator for 45 minutes.

[0101] Step d): Add secondary antibody: Drain the primary antibody mixture and add 180 μL / well of 1× TBST to the ELISA plate. Wash the plate twice. Dilute goat anti-mouse HRP to a working concentration of 1:10,000 in 1% BSA and add 50 μL / well to the ELISA plate. Cover the plate and incubate in a 37°C incubator for 30 min.

[0102] Step e): Color development, termination, and reading: Discard the liquid in the wells, add 180 μL / well of wash solution to the ELISA plate, and wash the plate three times; add 50 μL of freshly prepared TMB chromogenic substrate to each reaction well, incubate at 37°C for 10 minutes; then add 50 μL / well of stop solution to terminate the reaction, and measure the OD value at 450 nm on a microplate reader.

[0103] The results showed (as shown in Table 2) that the titers (1:8100) of the three mice all met the requirements.

[0104] Table 2

[0105]

[0106]

[0107] Example 4: Obtaining Rabbit CD4;CD5 Overexpressing Cell Lines Using Lentiviral Infection Technology and Cell Line Verification

[0108] The purpose of antibody screening is to obtain monoclonal cell lines with flow cytometry applications. Therefore, a rabbit CD4 overexpression cell line was constructed through lentiviral infection technology. At the same time, a rabbit CD5 overexpression cell line was constructed to screen for hybridoma cell lines that secrete CD4-specific antibodies.

[0109] Construction of overexpression monoclonal cell lines:

[0110] Reagents: Penicillin-streptomycin (double antibody) 100x (Shanghai Yuanpei); High-glucose DMEM (Shanghai Yuanpei); Opti-MEM TM Reduced Serum Medium (Thermo); fetal bovine serum (Sijiqing); VigoFect high-efficiency eukaryotic transfection reagent; 50 mM cell culture-grade dimethyl sulfoxide (DMSO) (Suobao); polybrene; trypsin-EDTA digestion solution (0.25%) containing phenol red (HycLone); phosphate-buffered saline (PBS) (Shanghai Yuanpei).

[0111] Consumables: 6-well cell culture plates (thermo-Labserv); 96-well cell culture plates (thermo-Labserv); 24-well cell culture plates (thermo-Labserv); 100mm cell culture dishes (thermo-Labserv); 1.5mL cryotubes (AVANTECH); 15mL centrifuge tubes (BD); 50mL centrifuge tubes (BD); GLutamax (GIBCO); 0.22um disposable low-adsorption filter tips (yellow) (Milipore); 2mL disposable syringes (domestic); 1.5mL centrifuge tubes (EP tubes); 10uL, 200uL, and 1000uL pipette tips.

[0112] Equipment: CO2 incubator (Thermo: BB150); 12-hole gun (Eppendorf); biological safety cabinet (Bocco Bio: BSC-1500IIA2-X); 4°C refrigerator (Zhongke Meiling: YC-260L); water bath (Boxun: HHS-21-4); centrifuge (Thermo: ST16).

[0113] Step a): construct overexpression plasmids of rabbit CD4 and rabbit CD5 full-length Flag tags, and select pQCXIP as the vector. The full-length sequence is shown below.

[0114] Rabbit CD4-Flag

[0115] NRRIYFQCLLLVLPLALLPAATWGKTVVRGKAGAIVELPCQSSQKRNSVFNWKHANQVKILGNQGSSSSSFWLKGNSPLSNRVESKKNMWDQGSFPLVIKDLRMDDSGTYICEVGDK KMEVELLVFRLTANPNTRLLHGQSLTLTLEGPSVGSPSVQWKSPENKIIETGPTCSMPKLRLQDSGTWSCHLSFQDQNKLELDIKIIVLGFPKASATVYKKEGEQVEFSFPLNFEDES LSGELMWQVDGASSAQSWVSFSLEDRKVSVQKILPDLKIQMSKGLPLSLTLPQALHRYAGSGNLSLTLDKGKLHQQVSLVMLKVTQVKNKLTCEVLGPIDPKMKLSLKLEDQEAKVS TQKMVQVLDPKAGTWQCLLSSGDQVLLESKADVLATGLSHQQPTLLAGALGGTAGLVLFAGLCIYCCVKCRHRRHQAQRMSQIKKLLSEKKTCQCPHRLQKTYNLLDYKDDDDK(SEQ ID NO.11).

[0116] Rabbit CD5-Flag

[0117] GSQPPPLAAVSLLGMLVTSCLGWSSWDEPGFLANLTNSHSPCQGQLEVYTTGSWHTVCSRSWGMNSEGWKDPWKASKLCQQLHCGEALAVGPFPHFNKPRNQLFCMGLPGSFANCSRISQCHSL GLVCLEPRKTTPPPTSPPPETTPQPTAPPRLQLVPGPRGLHCAGVVEFYRGSLGGTICAEAQDKNEDLGKFVCATLQCGSLKEVTAVEAAGELGGRRPLPIRWGIQNASCTSLEQCFRRIQPQDG RRALALVCSDFQPKVQSRLVGGSSICEGTAEVRQGPRWAALCHNSSAKGTARWEELCQEQQCGIVNSYYVLDTGKKAAWGFSCPQEKLSQCHELREKKANCKRVFVTCQDPNPAGPAAKAVASI ILALVLLAVLLVVCGPLAYRKLVKKFRQKKQRQWIGPTEMSQNMSFHRNHTATTVRSQAGNPTASHVDNEYSQPPRNSRLSAYPALEGALHRSSTQPDNSSDSDYDLHAAQRLDYKDDDDK(SEQ ID NO.12).

[0118] Step b): Lentivirus packaging experiment: 24 hours before transfection, plate 293T cells in a 6-well plate. On the day of the experiment, add VigoFect working solution to 2.5 μg of plasmid and mix gently. The resulting transfection solution should be incubated at room temperature for 15 minutes. After gentle mixing, add dropwise to the cell culture medium and incubate at 37°C in a 5% CO2 incubator.

[0119] Step c): Lentiviral infection assay: 24 hours prior to the lentiviral infection assay, HeLa (human cervical cancer) cells were plated in a 6-well plate. On the day of the lentiviral infection assay, fresh culture medium (with polybrene) and viral supernatant were added to the cells.

[0120] Step d): Puromycin screening experiment: 48 hours after lentiviral infection, treat with puromycin. Observe cells daily. After two days, significant cell death will occur. Change the medium (selection medium containing puromycin) based on the number of dead cells. Control cell lines will die completely after puromycin treatment. In contrast, cells in the experimental group infected wells survive and grow to a certain density. Cell monoclonal isolation is then performed.

[0121] Step e): Monoclonalization: Resuspend the polyclonal cell line, count it, and perform a 2-fold serial dilution (three columns per serial dilution). Within one week, select 12 wells containing single clones. After the cells in the 96-well plate have grown to 70%-90% confluence, trypsinize the 12 clones, resuspend them, and transfer them to a 24-well plate. After the cells in the 24-well plate have grown to 70%-90% confluence, subculture them twice, one minute apart. One aliquot is used for single clone identification, and the other aliquot is used for clone expansion and cryopreservation, pending identification results.

[0122] Step f): Perform monoclonal verification experiments by WB (Western Blot) and FC (Flow Cytometry):

[0123] WB: remove the supernatant, wash twice with PBS, add 200 μL loading solution to 100% cells, stir with a pipette tip until completely contacted and evenly distributed, collect into EP tube for WB detection, and use Flag tag antibody as the primary antibody.

[0124] FC: Remove the supernatant, wash twice with PBS, resuspend the cells, and incubate with mouse anti-rabbit CD4 / CD5 positive antibodies for flow cytometry detection.

[0125] HeLa-rCD4-Flag 5# and HeLa-rCD5-Flag6# were finally selected for subsequent cloning verification based on the cell status and WB / FC test results (WB results are shown in Figure 2). Figure 2 and Figure 3 ; FC results are as follows Figure 4 and Figure 5 ).

[0126] Example 5 Obtaining Mouse Hybridoma Monoclonal Cell Lines by Cell Fusion Technology

[0127] The above-mentioned mouse spleen cells injected intrasplenically with antigen were mixed with mouse myeloma cells (SP2 / 0, ATCC) at a ratio of 7:1 in serum-free IMDM medium. The mixture was centrifuged at 1,500 rpm for 3 minutes and the medium was removed. The mixed cells were added with 1 mL of PEG (molecular weight 1500) fusion agent in a 37°C water bath and fused for 1 minute. The fusion was terminated with serum-free IMDM medium and centrifuged at 1,200 rpm for 3 minutes. The supernatant was discarded and the precipitate was suspended in HAT medium, distributed into 96-well cell plates, and cultured in a cell culture incubator at 37°C and 5% CO2.

[0128] After 3 days of culture in a cell culture incubator, the medium was replaced with HAT medium. On the 7th day, HT medium was added. When confluent cells covered 5%-20% of the well bottom, positive wells were screened using a conventional indirect ELISA method using rabbit CD4 recombinant protein as the coating antigen. A total of 18 positive wells were obtained. Antibody supernatants from these 18 wells were analyzed by flow cytometry, and five specific cell lines were selected. Cloning was performed by limiting dilution, resulting in a hybridoma cell line, A5, that secreted a highly specific anti-rabbit CD4 monoclonal antibody. After expansion, the cells were used for ascites preparation and storage in liquid nitrogen.

[0129] Indirect ELISA method for cell supernatant:

[0130] Reagents: goat anti-mouse-HRP (Hua'an Biotechnology: HA1006), TMB substrate (Sigma: T2885), Tris (Shanghai Bioengineering: A501492), glycine (Shanghai Bioengineering: GB0235), BSA (Shanghai Bioengineering: A500023-0100), Tween-20 (Shanghai Bioengineering: A600560), NaHCO3 (Shanghai Bioengineering: A610482-0500); Na2CO3, Na2HPO4·12H2O, NaH2PO4·2H2O, citric acid, glycerol, DMSO, and concentrated sulfuric acid were purchased from Hangzhou Shuangmu Chemical; hydrogen peroxide and EDTA were purchased from Shanghai Bioengineering and were domestically produced analytical grade.

[0131] Consumables: ELISA plate (Hangzhou Shengyou).

[0132] Equipment: electric constant temperature incubator (Shanghai Senxin: DRP-9162), enzyme labeling instrument (MD: Cmax plus).

[0133] Indirect ELISA procedure

[0134] Step a): Recombinant rabbit CD4 protein was diluted to 1 μg / mL with coating buffer, and 50 μL / well was added to the ELISA plate. The plate was covered and coated at 4°C overnight.

[0135] Step b): Shake off the liquid in the wells and add 1%

[0136] BSA / TBS was placed in a 37°C electric thermostat incubator and blocked for 1 h.

[0137] Step c): Drain the liquid from the wells and add 50 μL / well of the fused hybridoma cell supernatant to the ELISA plate, using IV serum as a positive control. Cover the plate and incubate in a 37°C incubator for 30 minutes.

[0138] Step d): Drain the primary antibody mixture and add 180 μL / well of 1× TBST to the ELISA plate. Wash the plate twice. Dilute goat anti-mouse HRP to a working concentration of 1:10,000 in 1% BSA and add 50 μL / well to the ELISA plate. Cover the plate and incubate in a 37°C incubator for 30 min.

[0139] Step e): Color development, termination, and reading: Discard the liquid in the wells, add 180 μL / well of wash solution to the ELISA plate, and wash the plate three times; add 50 μL of freshly prepared TMB chromogenic substrate to each reaction well, incubate at 37°C for 5 minutes; then add 50 μL / well of stop solution to terminate the reaction, and measure the OD value at 450 nm on a microplate reader.

[0140] Flow cytometry operation of cell supernatant:

[0141] Reagents: goat anti-mouse-iFluor488 (Hua'an Biotechnology: HA1006), phosphate buffered saline (PBS), pH 7.2 (Shanghai Yuanpei).

[0142] Consumables: 96-well cell culture plates (thermo-Labserv); centrifuge tubes-15mL (BD); 1.5mL centrifuge tubes EP tubes; 10uL, 200uL, and 1000uL pipette tips.

[0143] Equipment: flow cytometer (Agilent); 12-well array gun (Eppendorf); 4°C refrigerator (Zhongke Meiling: YC-260L); centrifuge (Thermo: ST16).

[0144] Step a): Collect cells and determine the total cell number and cell viability (usually the cell viability is around 95% and not less than 90%).

[0145] Step b): Wash the cells twice with pre-cooled PBS, 1500 rpm, 5 min, 4°C, and spin dry.

[0146] Step c): Resuspend the cells in a volume of pre-chilled PBS to a density of 5 x 105-2.5 x 106 cells / ml. Add 200 μL / well to a 96-well plate. Spin at 1500 rpm for 5 min at 4°C, then spin dry.

[0147] Step d): Add 50ul of cell supernatant or primary antibody appropriately diluted in pre-cooled PBS and incubate at 4°C for 1h.

[0148] Step e): Wash the cells twice with pre-cooled PBS, 1500 rpm, 5 min, 4°C, and spin dry.

[0149] Step f): Add fluorescent secondary antibody diluted 1:1000 in pre-cooled PBS and incubate at 4°C in the dark for 30 min.

[0150] Step g): Wash the cells three times with pre-cooled PBS, 1500 rpm, 5 min, 4°C, and spin dry.

[0151] Step h): Resuspend the cells with 200 μL PBS and detect on a flow cytometer.

[0152] Refer to cell status and flow cytometry results (such as Figure 6 )Finally, A5 was selected for ascites preparation.

[0153] Example 6 Identification of anti-CD4 monoclonal antibody subtypes

[0154] The cell supernatant was subjected to enzyme-linked immunosorbent assay with anti-BALB / c mouse IgG1, IgG2a, IgG2b, IgG3, and IgM antibodies produced by Sigma Company to detect the subtypes of heavy and light chains in the cell supernatant.

[0155] Cell supernatant subtype detection operation:

[0156] Reagents: typing secondary antibody (Sigma), TMB substrate (Sigma: T2885), Tris (Shanghai Biotech: A501492), glycine (Shanghai Biotech: GB0235), BSA (Shanghai Biotech:

[0157] A500023-0100), Tween-20 (Shanghai Biotech: A600560), NaHCO3 (Shanghai Biotech:

[0158] A610482-0500); Na2CO3, Na2HPO4·12H2O, NaH2PO4·2H2O, citric acid, glycerol, DMSO, and concentrated sulfuric acid were purchased from Hangzhou Shuangmu Chemical; hydrogen peroxide and EDTA were purchased from Shanghai Shenggong and were of analytical grade.

[0159] Consumables: ELISA plate (Hangzhou Shengyou).

[0160] Equipment: electric constant temperature incubator (Shanghai Senxin: DRP-9162), enzyme labeling instrument (MD: Cmax plus).

[0161] Step a): Recombinant rabbit CD4 protein was diluted to 1 μg / mL with coating buffer, and 50 μL / well was added to the ELISA plate. The plate was covered and coated at 4°C overnight.

[0162] Step b): Shake off the liquid in the wells and add 1%

[0163] BSA / TBS was placed in a 37°C electric thermostat incubator and blocked for 1 h.

[0164] Step c): Drain the liquid from the wells and add 50 μL / well of the A5 clone cell supernatant to the ELISA plate. Cover the plate and incubate in a 37°C incubator for 30 minutes.

[0165] Step d): Drain the primary antibody mixture and add 180 μL / well of 1× TBST to the ELISA plate. Wash the plate twice. Dilute the typing secondary antibody to a working concentration of 1:3000 in 1% BSA and add 50 μL / well to the ELISA plate. Cover the plate and incubate in a 37°C incubator for 30 min.

[0166] Step e): Color development, termination, and reading: Discard the liquid in the wells, add 180 μL / well of wash solution to the ELISA plate, and wash the plate three times; add 50 μL of freshly prepared TMB chromogenic substrate to each reaction well, incubate at 37°C for 5 minutes; then add 50 μL / well of stop solution to terminate the reaction, and measure the OD value at 450 nm on a microplate reader.

[0167] The test results showed that the anti-rabbit CD4 recombinant protein monoclonal antibody subtype secreted by hybridoma cell line A5 was IgG2a heavy chain and Kappa light chain (as shown in Table 3).

[0168] Table 3

[0169]

[0170] Example 7 Collection and purification of anti-CD4 monoclonal antibodies

[0171] BALB / c mice aged about 8 weeks were injected intraperitoneally with 0.5 mL of liquid paraffin. 7-10 days later, approximately 1×10 6 After 7-10 days, the abdomen of the mouse was obviously swollen. Ascites was collected with a blood collection needle and centrifuged at 10,000 rpm for 3 minutes. The supernatant was collected as the monoclonal antibody ascites. The collected monoclonal antibody ascites was then purified to obtain anti-rabbit CD4 recombinant protein monoclonal antibodies.

[0172] Purification method:

[0173] Reagents: 1× PBS (pH 7.4), 0.2 M glycine (pH 2.7), 1 M sodium bicarbonate, 20% ethanol, 1 M NaCl.

[0174] Consumables: 1.5mL centrifuge tube, 50mL centrifuge tube, 50mL syringe, pH test paper (1-14), sealing film, 8000-14000Da dialysis bag (clamp).

[0175] Equipment: dual-channel microinjection pump, computer nucleic acid protein detector, 4°C refrigerator, nucleic acid protein analyzer.

[0176] Sample: Ascites to be purified.

[0177] Ascites purification steps:

[0178] Step a): The Protein G column was thoroughly washed with 20 mL of 1×PBS (pH 7.4) at a flow rate of 70 mL / h.

[0179] Step b): 6 mL of the ascites to be purified was placed in six 1.5 mL centrifuge tubes (1 mL each), centrifuged at 12,000 rpm for 5 min, and the supernatant was placed in a 50 mL centrifuge tube and diluted to 15 mL with 1× PBS.

[0180] Step c): The diluted ascites sample was loaded at a flow rate of 40 mL / h, and the process was repeated once.

[0181] Step d) Wash the column with 40 mL of 1× PBS (pH 7.4) at a flow rate of 70 mL / h. Connect the column to the protein analyzer and start the chromatogram collector on the computer. Edit the sample information (project number, clone number) and begin. Once the chromatogram baseline reaches a stable state, transfer the column to a syringe filled with glycine solution (pH 2.7, 0.2 M) and elute the antibody at a rate of 40 mL / h. Collect the antibody when the instrument reading begins to rise.

[0182] Step e): During the antibody collection process, the pH value of the antibody was promptly adjusted to 7.5 with 1 M sodium bicarbonate, and the highest peak value of the elution peak was recorded in the purification record book.

[0183] Step f): After collecting the antibodies, adjust the pH to 7.5 and record the volume of eluted antibodies in the purification record book. The collected antibodies are dialyzed against 2L of 1× PBS. The dialyzed antibodies are transferred to the original 50mL centrifuge tube and the antibody concentration is determined on a nucleic acid protein analyzer. The antibodies are then stored in a refrigerator at 4°C.

[0184] Purified antibody titer detection: Rabbit CD4 recombinant protein was used as the antigen, and the monoclonal antibody titer was detected by indirect ELISA method. The positive result judgment standard was: a P / N value (positive well OD value / negative well OD value) greater than or equal to 2.1 was considered positive. The analysis results showed that the monoclonal antibody titer reached 32 ng / mL (as shown in Table 4).

[0185] Table 4

[0186]

[0187] Example 8 Antibody Fluorescence Conjugation and Directly Labeled Antibody Verification

[0188] The protocol used in the experiment is to couple fluorescein to monoclonal antibodies, and then use the antibodies to carry fluorescent dyes for cell sorting experiments.

[0189] Reagents: A5 antibody, anhydrous DMSO, 1×PBS (PH=7.4), 1M NaHCO 3 (pH 8.75), iFluor 488 / 594.

[0190] Consumables: 15mL and 50mL imported centrifuge tubes, 1.5mL EP tubes, pipette tips, 3KD / 30KD ultrafiltration tubes.

[0191] Equipment: electronic analytical balance, pipette, 4℃ refrigerator, -20℃ refrigerator, sample mixer, ultramicro spectrophotometer, centrifuge.

[0192] Fluorescent labeling of antibodies

[0193] Step a): Receive the antibody to be labeled and confirm its suitability for use. Determine whether the antibody's buffer is PBS-only. If the buffer meets the requirements, proceed. If the antibody contains any amino acids, repurify the buffer before labeling.

[0194] Step b): Measure the antibody concentration and adjust it to about 2.5 mg / mL (if the concentration is too high, dilute it; if the concentration is too low, concentrate it using an ultrafiltration tube).

[0195] Step c): Take a centrifuge tube of appropriate volume and weigh it as m1 using a balance. Use a pipette to transfer the antibody to be labeled into the weighed centrifuge tube and weigh it again as m2. The volume of the antibody is recorded as (m2-m1) mL. Add 2.5*(m2-m1) / 100 of 1M NaHCO3 and mix well for use.

[0196] Step d): Take out the fluorescent dye from the -20 degree refrigerator and return it to room temperature for 20-30 minutes, add anhydrous DMSO and mix thoroughly (add 105.81 uL for iFluor488 and 86.17 uL for iFluor594).

[0197] Step e): Pipette the dissolved dye (10 times the total amount of the antibody) and add it to the antibody solution, place it on a sample mixer and incubate in the dark for 2 hours.

[0198] Step f): The antibody-dye mixture was centrifuged at 4000 rpm using a 30KD ultrafiltration tube to remove excess free dye until the centrifuged solution became transparent.

[0199] Step g): The centrifuged labeled antibody was transferred to a light-proof tube and the absorbance values ​​of A280, A495, A588, and A656 were measured using an ultra-micro spectrophotometer; the actual concentration of the antibody was calculated and the labeled antibody concentration was adjusted to 2 mg / mL.

[0200] Step h): Add an equal volume of protective agent at a volume ratio of 1:1 and quantify the antibody to 1 mg / mL.

[0201] Step i): DOL value control: Calculate the DOL value of the labeled antibody. The DOL value for 488 dye should be between 6-9; the DOL value for 594 dye should be between 3.5-5.5. If the DOL value meets the standard, add an equal volume of fluorescently labeled antibody protection solution, mix thoroughly on a sample mixer for 30 minutes, and store in a -20°C or 4°C refrigerator.

[0202] Example 9 Flow Cytometry Verification of Directly Labeled Antibodies

[0203] In order to determine whether the fluorescent-conjugated antibody can recognize the CD4+ T cell population in rabbit peripheral blood lymphocytes, as well as the sensitivity and specificity of the antibody, it is necessary to verify it through flow cytometry experiments.

[0204] Reagents: rabbit peripheral blood lymphocyte separation kit (Solarbio: P8760), red blood cell lysis buffer (Solarbio: R1010), A5-iFluor488, A5-iFluor594, CD4 Antibody-FITC, phosphate buffered saline (PBS), pH 7.2 (Shanghai Yuanpei).

[0205] Consumables: centrifuge tubes-15mL (BD); 1.5mL centrifuge tubes EP tubes; 10uL, 200uL, 1000uL pipette tips.

[0206] Equipment: flow cytometer (Agilent); 12-well array gun (Eppendorf); 4°C refrigerator (Zhongke Meiling: YC-260L); centrifuge (Thermo: ST16).

[0207] Step a): rabbit PBMCs are separated from fresh rabbit anticoagulated blood using a rabbit peripheral blood lymphocyte separation kit, and the red blood cells are lysed and the cells are set aside.

[0208] Step b): Wash the cells twice with pre-cooled PBS, 1500 rpm, 5 min, 4°C, and spin dry.

[0209] Step c): Add a certain amount of pre-cooled PBS to resuspend the cells to ensure that the cell density is 1x10 8 cells / ml, and added into EP tube at 100uL / well.

[0210] Step d): A5-iFluor488 was diluted in proportion and added to the cell suspension. Meanwhile, A5-iFluor594 was diluted in proportion and mixed with CD4 Antibody-FITC, and then added to the cell suspension. The mixture was incubated at room temperature for 20 minutes.

[0211] Step e): Wash the cells twice with pre-cooled PBS, 1500 rpm, 5 min, 4°C, and spin dry.

[0212] Step h): Resuspend the cells with 200 μL PBS and detect on a flow cytometer.

[0213] Competition experiments with CD4-FITC positive antibody and isotype control-iFluor488 showed that the cell population recognized by A5 and the positive antibody was consistent (e.g. Figure 7 ), and the best concentration for use is A5-iFluor488 1:200 (e.g. Figure 8 ).

[0214] Example 10 Sequencing of heavy and light chain variable regions

[0215] The antibody gene of the mouse hybridoma single cell clone A5 was sequenced to obtain the heavy and light chain variable region sequences of the clone.

[0216] Reagents: TAE buffer, agarose, nucleic acid stain.

[0217] Consumables: Pipette tips.

[0218] Equipment: nucleic acid electrophoresis instrument, nucleic acid imaging instrument.

[0219] Heavy and light chain variable region sequencing:

[0220] The heavy and light chain variable region sequences of clone A5 were obtained by sequencing the PCR (2nd PCR) products.

[0221] Step a): heavy chain and light chain PCR products are subjected to nucleic acid electrophoresis, and target bands are observed;

[0222] Step b): After recovery by gel excision, the variable region nucleic acid and protein sequences of the heavy and light chains of clone A5 are obtained by a gene sequencing company.

[0223] Among them, the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.7, the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.8, the nucleic acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.9, and the nucleic acid sequence of the light chain variable region VL is shown in SEQ ID NO.10.

[0224] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mouse anti-rabbit CD4 monoclonal antibody, characterized in that: The variable regions of the mouse anti-rabbit CD4 monoclonal antibody include: a complementary determining region CDR1-VH of the amino acid sequence shown in SEQ ID NO.1, a complementary determining region CDR2-VH of the amino acid sequence shown in SEQ ID NO.2, a complementary determining region CDR3-VH of the amino acid sequence shown in SEQ ID NO.3, a complementary determining region CDR1-VL of the amino acid sequence shown in SEQ ID NO.4, a complementary determining region CDR2-VL of the amino acid sequence shown in SEQ ID NO.5, and a complementary determining region CDR3-VL of the amino acid sequence shown in SEQ ID NO.

6.

2. The mouse anti-rabbit CD4 monoclonal antibody according to claim 1, wherein The variable region includes the heavy chain variable region VH having the amino acid sequence shown in SEQ ID NO.7; The variable region includes the light chain variable region VL with the amino acid sequence shown in SEQ ID NO.

8.

3. Use of the mouse anti-rabbit CD4 monoclonal antibody according to claim 1 or 2 in sorting rabbit CD4+ T cell populations.

4. The use according to claim 3, characterized in that The CD4+T cell population includes the CD4+T cell population in rabbit peripheral blood lymphocytes.

5. A marker for a CD4+ T cell population, characterized in that The marker comprises the mouse anti-rabbit CD4 monoclonal antibody according to claim 1 or 2 and a fluorescent dye; The mouse anti-rabbit CD4 monoclonal antibody is conjugated to a fluorescent dye.

6. A gene encoding the mouse anti-rabbit CD4 monoclonal antibody according to claim 1 or 2, characterized in that: The gene has the nucleic acid sequence shown as SEQ ID NO.9 and SEQ ID NO.10.

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