Mouse anti-rabbit cd5 monoclonal antibodies and uses thereof

Abstract

The application provides a mouse anti-rabbit CD5 monoclonal antibody and application thereof, and relates to the technical field of biology.The mouse anti-rabbit CD5 monoclonal antibody provided by the application has a variable region comprising complementarity determining regions CDR1-VH, CDR2-VH, CDR3-VH, CDR1-VL, CDR2-VL and CDR3-VL with specific amino acid sequences.The inventor finds that the monoclonal antibody has high specificity and sensitivity, can specifically recognize a CD5+ T cell group, and is used for sorting the CD5+ T cell group.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a mouse anti-rabbit CD5 monoclonal antibody and its applications. Background Technology

[0002] Flow cytometry offers high-throughput cell sorting capabilities using various fluorescent labels, enabling the sorting of hundreds of thousands to tens of millions of cell populations in a short time. Target B cells selected through screening using B cell surface membrane-bound immunoglobulins and specific fluorescently labeled antigens are then isolated, and in vitro single-cell RNA extraction and amplification yield recombinant antibody sequence information. This significantly increases the speed at which specific antigen-antibody sequences can be obtained from immunized animals. Therefore, antibody development protocols based on flow cytometry for single B cell isolation can improve antibody development efficiency.

[0003] Currently, in vitro culture of single B cells and screening of single B cells using microfluidic technology are two commonly used single B cell sorting methods. Neither of these methods requires screening T cells to obtain antigen-binding positive B cells. Therefore, developing an antibody development protocol for rabbit single B cell isolation 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, while mouse anti-rabbit CD5 monoclonal antibodies have been reported, these are primarily controlled by foreign companies (such as BD and Bio-Rad). Therefore, there is an urgent need to develop an independent anti-rabbit CD5 antibody based on mouse hybridoma technology.

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

[0005] The primary objective of this invention is to provide a mouse anti-rabbit CD5 monoclonal antibody.

[0006] The second objective of this invention is to provide the application of the above-mentioned mouse anti-rabbit CD5 monoclonal antibody in the sorting of CD5+ T cell populations.

[0007] A third objective of this invention is to provide a marker for CD5+ T cell populations.

[0008] The fourth objective of this invention is to provide a hybridoma cell.

[0009] The fifth objective of this invention is to provide a method for preparing a mouse anti-rabbit CD5 monoclonal antibody.

[0010] In a first aspect, the present invention provides a mouse anti-rabbit CD5 monoclonal antibody, wherein the variable region of the mouse anti-rabbit CD5 monoclonal antibody comprises: a complementarity-determining region CDR1-VH having the amino acid sequence shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH having the amino acid sequence shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH having the amino acid sequence shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL having the amino acid sequence shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL having the amino acid sequence shown in SEQ ID NO.5, and a complementarity-determining region CDR3-VL having the amino acid sequence shown in SEQ ID NO.6.

[0011] 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.

[0012] 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.

[0013] Secondly, the present invention provides the application of the above-mentioned mouse anti-rabbit CD5 monoclonal antibody in the sorting of CD5+ T cell populations.

[0014] As a further technical solution, the CD5+ T cell population includes the CD5+ T cell population in rabbit peripheral blood lymphocytes.

[0015] Thirdly, the present invention provides a marker for CD5+ T cell population, the marker comprising the above-mentioned mouse anti-rabbit CD5 monoclonal antibody and fluorescent dye;

[0016] The mouse anti-rabbit CD5 monoclonal antibody was conjugated with a fluorescent dye.

[0017] As a further technical solution, the fluorescent dye includes iFluor488 or iFluor594.

[0018] Fourthly, the present invention provides a hybridoma cell expressing the above-mentioned mouse anti-rabbit CD5 monoclonal antibody.

[0019] As a further technical solution, the hybridoma cells are obtained by fusing mouse spleen cells and mouse myeloma cells;

[0020] The mouse spleen cells expressed the mouse anti-rabbit CD5 monoclonal antibody.

[0021] Fifthly, the present invention provides a method for preparing the above-mentioned mouse anti-rabbit CD5 monoclonal antibody, comprising: culturing the above-mentioned hybridoma cells in the peritoneal cavity of a mouse, and then isolating the mouse anti-rabbit CD5 monoclonal antibody.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention provides a mouse anti-rabbit CD5 monoclonal antibody. The inventors have found that this monoclonal antibody has high specificity and sensitivity, and can specifically recognize CD5+ T cell populations, and thus be used for the sorting of CD5+ T cell populations. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 The SDS-PAGE results provided in Example 1;

[0026] Figure 2 The WB results for the rabbit CD4 overexpressing cell line provided in Example 4;

[0027] Figure 3 The WB results for the rabbit CD5 overexpressing cell line provided in Example 4;

[0028] Figure 4 The results of FC for the rabbit CD4 overexpressing cell line provided in Example 4;

[0029] Figure 5 The results of FC for the rabbit CD5 overexpressing cell line provided in Example 4;

[0030] Figure 6 The flow cytometry results provided in Example 5;

[0031] Figure 7 The results of the flow cytometry competition experiment provided in Example 9;

[0032] Figure 8 The results of the flow cytometry valence experiment provided in Example 9. Detailed Implementation

[0033] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0034] It should be noted that the "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domain of the heavy chain can be referred to as "VH," and the variable domain of the light chain as "VL." These domains are typically the most variable parts of the antibody and contain antigen-binding sites. The variable region of the light or heavy chain consists of a framework region interrupted by three hypervariable regions called "complementarity-determining regions" or "CDRs." The framework region of the antibody, that is, the framework region of the combination of the light and heavy chains, plays a role in locating and aligning the CDRs, which are primarily responsible for binding to the antigen.

[0035] The “framework” or “FR” region refers to the region outside of those defined as CDRs of the antibody variable domain. Each antibody variable domain framework can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3, and FR4).

[0036] Typically, the variable regions VL / VH of heavy and light chains can be obtained by connecting the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0037] In this invention, CDR1-VH, CDR2-VH and CDR3-VH refer to the three highly variable regions of the heavy chain variable region, and correspondingly, CDR1-VL, CDR2-VL and CDR3-VL refer to the three highly variable regions of the light chain variable region.

[0038] In a first aspect, the present invention provides a mouse anti-rabbit CD5 monoclonal antibody, wherein the variable region of the mouse anti-rabbit CD5 monoclonal antibody comprises: a complementarity-determining region CDR1-VH having the amino acid sequence shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH having the amino acid sequence shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH having the amino acid sequence shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL having the amino acid sequence shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL having the amino acid sequence shown in SEQ ID NO.5, and a complementarity-determining region CDR3-VL having the amino acid sequence shown in SEQ ID NO.6.

[0039] The sequences of the variable regions mentioned above are shown in Table 1:

[0040] Table 1

[0041] CDR2-VH YISNGGGSTYYSDTVKG SEQ ID NO.2 CDR3-VH SYYGHYTVDY SEQ ID NO.3 CDR1-VL RASKSVSTSGYSYMH SEQ ID NO.4 CDR2-VL LVSNLES SEQ ID NO.5 CDR3-VL AQNLELPWT SEQ ID NO.6

[0042] The inventors discovered that the mouse anti-rabbit CD5 monoclonal antibody has high specificity and sensitivity, and can specifically recognize CD5+ T cell populations, thus enabling its use in the sorting of CD5+ T cell populations.

[0043] 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.

[0044] The amino acid sequence of the heavy chain variable region VH in the variable region is as follows:

[0045] EVQLQESGGGLVQPGGSLKLSCAASGFTFSTYTMSWVRQTPEKRLEWVAYISNGGGSTYYSDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARSYYGHYTVDYWGQG TTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCK PCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTK GRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQID NO.7).

[0046] 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.

[0047] The amino acid sequence of the light chain variable region VL in the variable region is as follows:

[0048] DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQ

[0049] PPRLLIYLVSNLESGVPARFSGSGSGTDFTLRISRVEAEDVGVYYCAQNLE

[0050] LPWTFGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDI

[0051] NVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSY

[0052] TCEATHKTSTSPIVKSFNRNEC (SEQ ID NO. 8).

[0053] Secondly, the present invention provides the application of the above-mentioned mouse anti-rabbit CD5 monoclonal antibody in the sorting of CD5+ T cell populations.

[0054] The mouse anti-rabbit CD5 monoclonal antibody provided by this invention has high specificity and sensitivity, and can specifically recognize CD5+ T cell populations. Therefore, it can be used for the sorting of CD5+ T cell populations.

[0055] In some preferred embodiments, the CD5+ T cell population includes CD5+ T cell populations from rabbit peripheral blood lymphocytes, rabbit spleen, rabbit lymph nodes, and other related tissue immune cells.

[0056] Thirdly, the present invention provides a marker for CD5+ T cell population, the marker comprising the above-mentioned mouse anti-rabbit CD5 monoclonal antibody and fluorescent dye;

[0057] The mouse anti-rabbit CD5 monoclonal antibody was conjugated with a fluorescent dye.

[0058] The markers provided by this invention can be used for specific labeling of CD5+ T cell populations.

[0059] In some preferred embodiments, the fluorescent dye includes, but is not limited to, iFluor488 or iFluor594, or other fluorescent dyes known to those skilled in the art.

[0060] Fourthly, the present invention provides a hybridoma cell expressing the above-mentioned mouse anti-rabbit CD5 monoclonal antibody.

[0061] These hybridoma cells can be used to produce mouse anti-rabbit CD5 monoclonal antibodies.

[0062] In some preferred embodiments, the hybridoma cells are obtained by fusing mouse spleen cells and mouse myeloma cells;

[0063] The mouse spleen cells expressed the mouse anti-rabbit CD5 monoclonal antibody.

[0064] Fifthly, the present invention provides a method for preparing the above-mentioned mouse anti-rabbit CD5 monoclonal antibody, comprising: culturing the above-mentioned hybridoma cells in the peritoneal cavity of a mouse, and then isolating the mouse anti-rabbit CD5 monoclonal antibody.

[0065] The preparation method of this mouse anti-rabbit CD5 monoclonal antibody is simple, efficient, and low in cost.

[0066] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0067] Example 1: Immunogen Preparation

[0068] CD5 protein is a single-transmembrane glycoprotein, mainly expressed on the surface of T cell membranes. In rabbit CD5, segments 24-488 are extracellular regions. Full-length extracellular rabbit CD5 protein with concentrations and purity meeting immunological requirements was obtained through eukaryotic expression.

[0069] Cell transfection and protein purification:

[0070] Reagent: Expi293 TM Expression system kit (Thermo, A14635), nickel NTA agarose gel, PBS buffer.

[0071] Consumables: 96-well deep well plate, pipette tips.

[0072] Equipment: constant temperature shaker, electric pipette.

[0073] Step a): Incubate the Expi293F medium in a 37°C water bath beforehand. Observe the cell state and growth of Expi293F cells under a microscope. Determine the passage ratio for the cell expansion experiment based on the cell growth volume and subsequent transfection requirements. Generally, the transfection density is controlled at 2x10⁻⁶ cells / day the day before transfection. 6 The cell density is approximately 1 / mL. Remove the cell slurry from the CO2 cell shaker, count the cells, and calculate the required transfer volume. Select the optimal cell shake flask size based on the desired transfer volume; generally, the culture volume should not exceed 1 / 3 of the flask volume and should not be less than 1 / 5. Add preheated Expi 293 medium to achieve a final cell density of 2 x 10⁻⁶ cells / mL. 6 Approximately 100 live cells / mL, for use in the transfection procedure the following day.

[0074] Step b): On the day of transfection, preheat the Expi293 medium to room temperature in the dark. Determine the amount of cells to be transfected and prepare the corresponding number of cell shake flasks. Determine the cell density according to cell counting procedures, and dilute the cells with the preheated medium to a final cell density of 3 x 10⁻⁶ cells / mL. 6For live cells / mL, aliquot 1ml / well into deep-well plates using a pipette and carefully place them on a cell culture shaker: 37℃, 8% CO2, 900rpm, 80% humidity. Take the appropriate number of cell culture flasks and aliquot 1ug DNA + 60ul Opti-MEM (cold reagent) into the culture plates using a pipette to mix thoroughly (recommended antibody ratio HC:LC = 1:2). Separately, take the optimal volume of centrifuge tube and dilute sufficient transfection reagent to 3.2ul Expifectamine 293 + 60ul Opti-MEM (cold reagent) per ml of cells. Gently invert to mix and incubate at room temperature for 5 minutes. After 5 minutes, transfer the diluted transfection reagent to a disposable reservoir and pipette into the corresponding wells of a 96-well cell culture plate, incubating for 10-20 minutes. Using a pipette, add the settled ExpiFectamine 293 / rabbit CD5 expression plasmid complex to the cell suspension and carefully place the mixture on a shaker: incubate at 37°C, 8% CO2, 900 rpm, and 80% humidity. 18-22 hours post-transfection, add 6 μL of Enhancer 1 and 60 μL of Enhancer 2 to 1 ml of the solution. Harvest the supernatant after 4-7 days for recombinant protein purification.

[0075] Step c): The recombinant protein contains a histidine tag (His-tag) and is purified using a nickel column: After passing the cell supernatant through a 0.45 μM filter, a nickel-NTA agarose gel is added and incubated at 4°C for binding. The eluted recombinant protein is then replaced with PBS buffer via ultrafiltration or dialysis and identified by SDS-PAGE staining (e.g., ...). Figure 1 The rabbit CD5 recombinant protein showed a distinct specific band in the PAGE gel, with a molecular weight around 55 kDa, and the protein purity and concentration met the requirements for immunization.

[0076] Example 2: Mouse immunization and serum collection

[0077] Mouse immunization procedures:

[0078] Reagent: adjuvant, 75% alcohol.

[0079] Consumables: Syringes.

[0080] Sample: Rabbit CD5 recombinant protein.

[0081] Step a): Animal selection: Use BALB / c female rabbits, weighing about 18-22g, and around 6-8 weeks old. Select healthy animals with smooth fur and free movement.

[0082] Step b): Preparation before the experiment: Mark the mice.

[0083] Step c): Remove the antigen from the -20°C freezer and thaw it at room temperature, avoiding repeated freeze-thaw cycles. Label the syringe with the project number and animal number.

[0084] Step d): Extract the antigen (the antigen is completely mixed), the antigen concentration is 0.5 mg / mL, and the mouse immunization dose is 0.1 mL / mouse.

[0085] Step e): Draw the adjuvant, with a 1:1 volume ratio of adjuvant to antigen. Use complete adjuvant for the first immunization, and incomplete adjuvant for the second and third immunizations. Ensure the adjuvant is thoroughly mixed before drawing it into the syringe.

[0086] Step f): Connect the two syringes with the syringe connecting tube and emulsify them completely. The emulsification standard is: the emulsified immunogen is qualified if it does not disperse when dropped into 37°C water.

[0087] Step g): Immunization: Mice were administered multiple subcutaneous injections. Immunization time: The immunization interval was 14 days. Blood samples were collected from mice on day 7 after three immunizations for titer testing.

[0088] Step h): The procedure for collecting a small serum sample: The person collecting the blood uses their left thumb and forefinger to press down on both sides of the mouse's neck, holding the mouse firmly from the back to cause congestion of the posterior orbital venous plexus. When holding the mouse as a whole, ensure its body remains in a straight position. With the right hand, hold the blood collection device at a 45° angle to the mouse's face, inserting it from the inner corner of the eye, with the bevel of the needle initially facing the eyeball. After insertion, rotate the needle 180 degrees so that the bevel faces the posterior border of the orbit. The insertion depth is approximately 2–3 mm. Stop advancing when resistance is felt. If there is no bleeding, gently rotate the blood collection device. Collect 200 μL of blood at once. After blood collection, wipe the wound with sterile cotton and apply gentle pressure to stop the bleeding. Only return the mouse to its cage after confirming that its condition is normal.

[0089] Step i): Centrifuge the collected serum sample for 10 minutes at 10,000 rpm. After centrifugation, collect the supernatant and freeze it at -20°C for later use.

[0090] Example 3: Serum titer detection

[0091] The immunogenicity of mouse serum was detected by indirect ELISA. Mice with an OD450nm value exceeding 1.0 under a serum dilution of 1:8100 were considered immunized and could proceed to the next step.

[0092] Serum-stage indirect ELISA procedure:

[0093] Reagents: Goat anti-mouse HRP (Huaan Biotechnology: HA1006), TMB substrate (Sigma: T2885), Tris (Shanghai Sangon Biotech: A501492), glycine (Shanghai Sangon Biotech: GB0235), BSA (Shanghai Sangon Biotech: A500023-0100), Tween-20 (Shanghai Sangon Biotech: A600560), NaHCO3 (Shanghai Sangon Biotech: 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 Sangon Biotech, domestically produced analytical grade.

[0094] Consumables: Microplate (Hangzhou Shengyou).

[0095] Equipment: Electric thermostatic incubator (Shanghai Senxin: DRP-9162), ELISA reader (MD: Cmax plus).

[0096] Step a): Coating: Dilute rabbit CD5 recombinant protein to 1 μg / mL with coating buffer, add 50 μL / well to the microplate, cover and coat overnight at 4°C.

[0097] Step b): Blocking: Shake off the liquid in the wells, add 1% BSA / TBS to the microplate at a rate of 100 μL / well, and place it in a 37°C thermostatic incubator for 1 hour for blocking.

[0098] Step c): Sample addition: After removing the liquid from the wells, add 50 μL / well of serum at different dilution ratios to the microplate. Seal the plate and incubate at 37°C for 45 min.

[0099] Step d): Add secondary antibody: Shake off the primary antibody mixture, add washing buffer (1×TBST) to the microplate at a rate of 180 μL / well, and wash the microplate twice. Dilute goat anti-mouse-HRP to the working concentration (1:10000) with 1% BSA, add 50 μL / well to the microplate, cap it, and incubate at 37°C for 30 min.

[0100] Step e): Color development, termination and reading: Discard the liquid in the wells, add washing buffer to the microplate at a rate of 180 μL / well, and wash the microplate 3 times; add 50 μL of freshly prepared TMB chromogenic substrate to each reaction well, and incubate at 37°C for 10 min; then add 50 μL / well of stop solution to terminate the reaction, and measure the OD value at 450 nm on the microplate reader.

[0101] The results (as shown in Table 2) showed that the titers (1:8100) of all three mice met the requirements.

[0102] Table 2

[0103]

[0104]

[0105] Example 4: Obtaining Rabbit CD4;CD5 overexpressing cell lines using lentiviral infection technology and cell line validation.

[0106] The purpose of antibody screening is to obtain monoclonal cell lines that can be used for flow cytometry. Therefore, a rabbit CD5 overexpressing cell line was constructed using lentiviral infection technology, and a rabbit CD4 overexpressing cell line was also constructed for screening to obtain hybridoma cell lines that secrete CD5-specific antibodies.

[0107] Construction of overexpression monoclonal cell lines

[0108] Reagents: Penicillin and streptomycin (double antibiotic) 100x (Shanghai Yuanpei); High glucose DMEM (Shanghai Yuanpei); Opti-MEM TM IReduced Serum Medium (Thermo); Fetal Bovine Serum (Sijiqing); VigoFect High-Efficiency Eukaryotic Transfection Reagent; 50mM Cell Culture Grade Dimethyl Sulfoxide (DMSO) (Sorbonne); Polygluconine; Trypsin-EDTA Digestion Solution (0.25%) with Phenol Red (HycLone); Phosphate Buffered Sodium (PBS) (Shanghai Yuanpei).

[0109] Consumables: 6-well cell culture plate (thermo-Labserv); 96-well cell culture plate (thermo-Labserv); 24-well cell culture plate (thermo-Labserv); 100mm cell culture dish (thermo-Labserv); 1.5mL cryovials (AVANTECH); 15mL centrifuge tubes (BD); 50mL centrifuge tubes (BD); GLUTAMX (GIBCO); 0.22µm disposable low-adsorption filter tip (yellow) (Milipore); 2mL disposable syringe (domestic); 1.5mL centrifuge tubes (EP); 10µL, 200µL, and 1000µL pipette tips.

[0110] Equipment: CO2 incubator (Thermo: BB150); 12-well pipette (Eppendorf); biosafety cabinet (Boke Biotechnology: BSC-1500IIA2-X); 4℃ freezer (Zhongke Meiling: YC-260L); water bath (Boxun: HHS-21-4); centrifuge (Thermo: ST16).

[0111] Step a): Construct overexpression plasmids for the full-length flag tags of rabbit CD4 and rabbit CD5. The vector selected is PQCXIP, and the full-length sequence is shown below.

[0112] Rabbit CD4-Flag:

[0113] NRRIYFQCLLLVLPLALLPAATWGKTVVRGKAGAIVELPCQSSQKRNSVFNWKHANQVKILGNQGSSSSSFWLKGNSPLSNRVESKKNMWDQGSFPLVIKDLRMDDSGTYICEVGDK KMEVELLVFRLTANPNTRLLHGQSLTLTLEGPSVGSPSVQWKSPENKIIETGPTCSMPKLRLQDSGTWSCHLSFQDQNKLELDIKIIVLGFPKASATVYKKEGEQVEFSFPLNFEDES LSGELMWQVDGASSAQSWVSFSLEDRKVSVQKILPDLKIQMSKGLPLSLTLPQALHRYAGSGNLSLTLDKGKLHQQVSLVMLKVTQVKNKLTCEVLGPIDPKMKLSLKLEDQEAKVS TQKMVQVLDPKAGTWQCLLSSGDQVLLESKADVLATGLSHQQPTLLAGALGGTAGLVLFAGLCIYCCVKCRHRRHQAQRMSQIKKLLSEKKTCQCPHRLQKTYNLLDYKDDDDK(SEQ ID NO.9).

[0114] Rabbit CD5-Flag:

[0115] GSQPPPLAAVSLLGMLVTSCLGWSSWDEPGFLANLTNSHSPCQGQLEVYTTGSWHTVCSRSWGMNSEGWKDPWKASKLCQQLHCGEALAVGPFPHFNKPRNQLFCMGLPGSFANCSRISQCHSL GLVCLEPRKTTPPPTSPPPETTPQPTAPPRLQLVPGPRGLHCAGVVEFYRGSLGGTICAEAQDKNEDLGKFVCATLQCGSLKEVTAVEAAGELGGRRPLPIRWGIQNASCTSLEQCFRRIQPQDG RRALALVCSDFQPKVQSRLVGGSSICEGTAEVRQGPRWAALCHNSSAKGTARWEELCQEQQCGIVNSYYVLDTGKKAAWGFSCPQEKLSQCHELREKKANCKRVFVTCQDPNPAGPAAKAVASI ILALVLLAVLLVVCGPLAYRKLVKKFRQKKQRQWIGPTEMSQNMSFHRNHTATTVRSQAGNPTASHVDNEYSQPPRNSRLSAYPALEGALHRSSTQPDNSSDSDYDLHAAQRLDYKDDDDK(SEQ ID NO.10).

[0116] Step b): Lentiviral packaging experiment: 24 hours before the transfection experiment, 293T cells were passaged into 6-well plates. On the day of the experiment, VigoFect working solution was added to a plasmid containing a total amount of 2.5 μg, and gently mixed. The resulting transfection working solution was incubated at room temperature for 15 min. After gentle mixing, it was added dropwise to the cell culture medium and incubated at 37°C in a 5% CO2 incubator.

[0117] Step c): Lentiviral infection assay: 24 hours before the lentivirus infection experiment, HeLa (human cervical cancer cells) were passaged into 6-well plates. On the day of the lentivirus infection experiment, fresh culture medium (+polybrene) and viral supernatant were added to the cells.

[0118] Step d): Puromycin selection experiment: 48 hours after lentiviral infection, cells were treated with puromycin. Cells were observed daily. After two days, a large number of cells died. The medium (selection medium containing puromycin) was changed according to the amount of dead cells. The control cell line died completely after puromycin treatment. In addition, a large number of cells survived in the infection wells of the experimental group and grew to a certain density before cell monocloning.

[0119] Step e): Monoclonalization assay: Resuspend and count polyclonal cell lines, perform 2-fold serial dilutions (3 columns per dilution), and select 12 wells with monoclonal cells within one week. Once the cells in the 96-well plate reach 70%-90% confluence, trypsin-digest the 12 clones, resuspend them, and transfer them to 24-well plates. Once the cells in the 24-well plate reach 70%-90% confluence, passage them twice. One passage is used for monoclonal identification, and the other is used to select clones for cryopreservation after the identification results are available.

[0120] Step f): Perform single-clonal validation experiments using Western blotting (WB) and flow cytometry (FC).

[0121] WB: Remove the supernatant, wash twice with PBS, add 200 μL of loading solution to 100% cells, stir with a pipette tip until fully mixed, collect in an EP tube for WB detection, the primary antibody is a Flag tag antibody.

[0122] 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.

[0123] Based on cell status and WB / FC test results, HeLa-rCD4-Flag 5# and HeLa-rCD5-Flag 6# were ultimately selected for subsequent clonal validation (WB results are as follows). Figure 2 and Figure 3 ;FC results are as follows Figure 4 and Figure 5 ).

[0124] It should be noted that, Figures 2-5 In the table, 1#-12# are group numbers.

[0125] Example 5: Obtaining Mouse Hybridoma Monoclonal Cell Lines Using Cell Fusion Technology

[0126] Mouse spleen cells injected with the antigen and mouse myeloma cells (SP2 / 0, ATCC) were mixed at a ratio of 7:1 in serum-free IMDM medium. After centrifugation at 1,500 rpm for 3 min, the medium was removed. The mixed cells were then placed in a 37°C water bath with 1 mL of PEG (molecular weight 1500) fusion agent and fused for 1 min. The fusion was terminated with serum-free IMDM medium and centrifuged at 1,200 rpm for 3 min. The supernatant was discarded, and the pellet was resuspended in HAT medium. The pellet was then aliquoted into 96-well cell culture plates and cultured in a 37°C, 5% CO2 cell culture incubator.

[0127] After culturing in a cell culture incubator for 3 days, the medium was changed once with HAT medium. On day 7, HT medium was added. When the confluent cells covered 5%-20% of the bottom of the wells, positive wells were screened using a conventional indirect ELISA method with rabbit CD5 recombinant protein as the coating antigen, yielding 9 positive wells. Flow cytometry was performed on the supernatant from the cloning in the 9 wells, and 8 specific cell lines were screened out. Limiting dilution clones were then used to obtain a hybridoma cell line B1 that could secrete a relatively good anti-rabbit CD5 specific monoclonal antibody. After expansion culture, it was used for ascites preparation and liquid nitrogen preservation.

[0128] Indirect ELISA method using cell supernatant stage:

[0129] Reagents: Goat anti-mouse HRP (Huaan Biotechnology: HA1006), TMB substrate (Sigma: T2885), Tris (Shanghai Sangon Biotech: A501492), glycine (Shanghai Sangon Biotech: GB0235), BSA (Shanghai Sangon Biotech: A500023-0100), Tween-20 (Shanghai Sangon Biotech: A600560), NaHCO3 (Shanghai Sangon Biotech: 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 Sangon Biotech, domestically produced analytical grade.

[0130] Consumables: Microplate (Hangzhou Shengyou).

[0131] Equipment: Electric thermostatic incubator (Shanghai Senxin: DRP-9162), ELISA reader (MD: Cmax plus).

[0132] Indirect ELISA operation

[0133] Step a): Dilute the recombinant rabbit CD5 protein to 1 μg / mL with coating buffer, add 50 μL / well to the microplate, cover and coat overnight at 4°C.

[0134] Step b): Shake off the liquid in the wells, add 1% BSA / TBS to the microplate at a rate of 100 μL / well, and incubate in a 37°C thermostatic incubator for 1 hour.

[0135] Step c): Shake off the liquid in the wells, add 50 μL / well of the fused hybridoma cell supernatant to the ELISA plate, using IV serum as a positive control. Seal the plate and incubate at 37°C for 30 min.

[0136] Step d): Shake off the primary antibody mixture, add washing buffer (1×TBST) to the microplate at a rate of 180 μL / well, and wash the microplate twice. Dilute goat anti-mouse-HRP to the working concentration (1:10000) with 1% BSA, add 50 μL / well to the microplate, cap it, and incubate at 37°C for 30 min.

[0137] Step e): Color development, termination and reading: Discard the liquid in the wells, add washing buffer to the microplate at a rate of 180 μL / well, and wash the microplate 3 times; add 50 μL of freshly prepared TMB chromogenic substrate to each reaction well, and incubate at 37°C for 5 min; then add 50 μL / well of stop solution to terminate the reaction, and measure the OD value at 450 nm on the microplate reader.

[0138] Flow cytometry detection procedure in the cell supernatant stage:

[0139] Reagents: Goat anti-mouse iFluor488 (Huaan Biotechnology: HA1006), phosphate buffer (PBS), pH 7.2 (Shanghai Yuanpei).

[0140] Consumables: 96-well cell culture plate (thermo-Labserv); 15mL centrifuge tubes (BD); 1.5mL centrifuge tubes (EP); 10uL, 200uL, and 1000uL pipette tips.

[0141] Equipment: Flow cytometer (Agilent); 12-well pipette (Eppendorf); 4℃ freezer (Zhongke Meiling: YC-260L); centrifuge (Thermo: ST16).

[0142] Step a): Collect cells and determine the total number of cells and cell viability (usually cell viability is around 95% and not less than 90%).

[0143] Step b): Wash the cells twice with pre-cooled PBS at 1500 rpm for 5 min at 4°C, then spin dry.

[0144] Step c): Add a certain amount of pre-cooled PBS to resuspend the cells, ensuring the cell density is between 5x10⁵ and 2.5x10⁵. 6 Cells / ml, added to 200 μL / well of a 96-well plate. Incubate at 1500 rpm for 5 min at 4°C, then spin dry.

[0145] Step d): Add 50 μL of cell supernatant or the primary antibody diluted appropriately with pre-cooled PBS, and incubate at 4°C for 1 h.

[0146] Step e): Wash the cells twice with pre-cooled PBS at 1500 rpm for 5 min at 4°C, then spin dry.

[0147] Step f): Add fluorescent secondary antibody diluted 1:1000 with pre-cooled PBS and incubate at 4°C in the dark for 30 min.

[0148] Step g): Wash the cells 3 times with pre-cooled PBS at 1500 rpm for 5 min at 4°C, then spin dry.

[0149] Step h): Resuspend the cells in 200uL PBS and analyze them using a flow cytometer.

[0150] Refer to cell state and flow cytometry results (e.g.) Figure 6 Ultimately, B1 was selected for ascites preparation.

[0151] Example 6: Identification of Anti-CD5 Monoclonal Antibody Subtypes

[0152] The cell supernatant was subjected to an enzyme-linked immunosorbent assay (ELISA) with Sigma-Aldrich anti-BALB / c mouse IgG1, IgG2a, IgG2b, IgG3, and IgM antibodies to detect the heavy and light chain subtypes in the cell supernatant.

[0153] Cell supernatant subtype detection procedure

[0154] Reagents: Genotyping secondary antibody (Sigma), TMB substrate (Sigma: T2885), Tris (Shanghai Sangon Biotech: A501492), glycine (Shanghai Sangon Biotech: GB0235), BSA (Shanghai Sangon Biotech: A500023-0100), Tween-20 (Shanghai Sangon Biotech: A600560), NaHCO3 (Shanghai Sangon Biotech: A610482-0500); Na2CO3, Na2HPO4·12H2O, NaH2PO4·2H2O, citric acid, glycerol, DMSO, and concentrated sulfuric acid were purchased from Hangzhou Shuangmu Chemical Co., Ltd.; hydrogen peroxide and EDTA were purchased from Shanghai Sangon Biotech Co., Ltd., and were of domestic analytical grade.

[0155] Consumables: Microplate (Hangzhou Shengyou).

[0156] Equipment: Electric thermostatic incubator (Shanghai Senxin: DRP-9162), ELISA reader (MD: Cmax plus).

[0157] Step a): Dilute the recombinant rabbit CD5 protein to 1 μg / mL with coating buffer, add 50 μL / well to the microplate, cover and coat overnight at 4°C.

[0158] Step b): Shake off the liquid in the wells, add 1% BSA / TBS to the microplate at a rate of 100 μL / well, and incubate in a 37°C thermostatic incubator for 1 hour.

[0159] Step c): Shake off the liquid in the wells and add 50 μL / well of B1 clone cell supernatant to the microplate. Cover and incubate at 37°C for 30 min.

[0160] Step d): Shake off the primary antibody mixture. Add washing buffer (1×TBST) to the microplate at a rate of 180 μL / well and wash the microplate twice. Dilute the typing secondary antibody to the working concentration (1:3000) with 1% BSA and add 50 μL / well to the microplate. Cover the plate and incubate at 37°C for 30 min.

[0161] Step e): Color development, termination and reading: Discard the liquid in the wells, add washing buffer to the microplate at a rate of 180 μL / well, and wash the microplate 3 times; add 50 μL of freshly prepared TMB chromogenic substrate to each reaction well, and incubate at 37°C for 5 min; then add 50 μL / well of stop solution to terminate the reaction, and measure the OD value at 450 nm on the microplate reader.

[0162] The test results showed that the anti-rabbit CD5 recombinant protein monoclonal antibody subtypes secreted by hybridoma cell line B1 were IgG1 heavy chain and Kappa light chain (as shown in Table 3).

[0163] Table 3

[0164]

[0165]

[0166] Example 7: Collection and Purification of Anti-CD5 Monoclonal Antibody

[0167] BALB / c mice around 8 weeks old were injected intraperitoneally with 0.5 mL of liquid paraffin, and 7-10 days later, approximately 1 × 10⁻⁶ mL was injected intraperitoneally. 6 After 7-10 days, the abdomen of the mouse was significantly enlarged by a number of hybridoma cells. Ascites fluid was collected using a blood collection needle, centrifuged at 10,000 rpm for 3 minutes, and the supernatant was collected as monoclonal antibody ascites. The collected monoclonal antibody ascites was then purified to obtain anti-rabbit CD5 recombinant protein monoclonal antibody.

[0168] Purification method:

[0169] Reagents: 1×PBS (pH 7.4), 0.2M glycine (pH 2.7), 1M sodium bicarbonate, 20% ethanol, 1M NaCl.

[0170] Consumables: 1.5mL centrifuge tubes, 50mL centrifuge tubes, 50mL syringes, pH test strips (1-14), sealing film, 8000-14000Da dialysis bags (clamps).

[0171] Equipment: Dual-channel micro-injection pump, computer-controlled nucleic acid and protein detector, 4℃ refrigerator, nucleic acid and protein analyzer.

[0172] Sample: Ascites fluid to be purified.

[0173] Ascites purification steps:

[0174] Step a): Wash the Protein G column thoroughly with 20 mL of 1×PBS (pH 7.4) at a flow rate of 70 mL / h.

[0175] Step b): Take 6 mL of the ascites fluid to be purified into six 1.5 mL centrifuge tubes (1 mL each), and centrifuge at 12000 rpm for 5 min. Take the supernatant into a 50 mL centrifuge tube and dilute it to 15 mL with 1×PBS.

[0176] Step c): Load the diluted ascites fluid sample at a flow rate of 40 mL / h, and repeat once.

[0177] Step d) Wash the column with 40 mL of 1×PBS (pH 7.4) at a flow rate of 70 mL / h, connect the protein analyzer, turn on the chromatogram collector on the computer, edit the sample collection information (project number, clone number) and start. Once the chromatogram baseline has stabilized, transfer the column to a syringe containing glycine solution (pH 2.7, 0.2 M) and elute the antibody at a rate of 40 mL / h. Begin collecting the antibody when the instrument reading starts to rise.

[0178] Step e): During antibody collection, adjust the pH of the antibody to 7.5 with 1M sodium bicarbonate in a timely manner, and record the highest peak value of the elution peak in the purification logbook.

[0179] Step f): After collecting the antibody, adjust the pH to 7.5 and record the volume of eluted antibody in the purification logbook. Dialyze the collected antibody in 2L 1×PBS. Transfer the dialyzed antibody to the original 50mL centrifuge tube and measure the antibody concentration on a nucleic acid protein analyzer. Store at 4℃.

[0180] Purified antibody titer detection: Using rabbit CD5 recombinant protein as antigen, the titer of the monoclonal antibody was detected by indirect ELISA. The criteria for judging a positive result were: a P / N value (OD value of positive well / OD value of negative well) greater than or equal to 2.1 was considered positive. The analysis results showed that the titer of the monoclonal antibody reached 32 ng / mL (as shown in Table 4).

[0181] Table 4

[0182]

[0183] Example 8: Validation of Antibody Fluorescent Conjugation and Direct Labeling Antibody

[0184] The experimental protocol involved conjugating a fluorescein to a monoclonal antibody, which was then used for cell sorting experiments after being coated with the fluorescent dye.

[0185] Reagents: B1 antibody, anhydrous DMSO, 1×PBS (pH=7.4), 1M NaHCO3 (pH8.75), iFluor488 / 594.

[0186] Consumables: 15mL and 50mL imported centrifuge tubes, 1.5mL EP tubes, pipette tips, 3KD / 30KD ultrafiltration tubes.

[0187] Equipment: Electronic analytical balance, pipette, 4℃ freezer, -20℃ freezer, sample mixer, ultra-micro spectrophotometer, centrifuge.

[0188] Fluorescent labeling of antibodies:

[0189] Step a): Receive the antibody to be labeled and confirm its suitability for application. Verify that the antibody buffer information indicates it is PBS only. If the buffer meets the requirements, proceed. If there are other amino compounds besides the antibody, it needs to be purified again before labeling.

[0190] Step b): Measure the antibody concentration and adjust it to about 2.5 mg / mL (if the concentration is too high, it can be diluted; if the concentration is too low, it needs to be concentrated using an ultrafiltration tube).

[0191] Step c): Take a centrifuge tube of appropriate volume and weigh it with a balance as m1. Transfer the antibody to be labeled into the weighed centrifuge tube with a pipette and weigh it again as m2. Then record the volume of the antibody as (m2-m1) mL. Add 2.5*(m2-m1) / 100 of 1M NaHCO3 and mix well for later use.

[0192] Step d): Remove the fluorescent dye from the -20°C freezer and allow it to return to room temperature for 20-30 minutes. Add anhydrous DMSO and mix thoroughly (105.81 uL for iFluor488; 86.17 uL for iFluor594).

[0193] Step e): Add the dissolved dye (10 times the total amount of antibody) to the antibody solution and place it on a mixer to incubate in the dark for 2 hours.

[0194] Step f): Centrifuge the antibody-dye mixture at 4000 rpm using a 30 kDa ultrafiltration tube to remove excess free dye until the centrifuged solution is transparent.

[0195] Step g): Transfer the centrifuged labeled antibody to a light-proof tube and measure the absorbance values ​​of A280, A495, A588, and A656 using an ultra-micro spectrophotometer; calculate the true concentration of the antibody and adjust the concentration of the labeled antibody to 2 mg / mL.

[0196] Step h): Add an equal volume of protective agent at a volume ratio of 1:1 to quantify the antibody to 1 mg / mL.

[0197] Step i): DOL value control: Calculate the DOL value of the labeled antibody. For 488 dye, the DOL value should be controlled between 6 and 9; for 594 dye, it should be controlled between 3.5 and 5.5. If the DOL value meets the standard, add an equal volume of fluorescently labeled antibody protection solution, place it on a mixer and mix for 30 minutes, then store it in a -20°C or 4°C freezer.

[0198] Example 9: Direct-labeled antibody flow cytometry verification

[0199] To determine whether the fluorescently conjugated antibody can recognize the CD5+ T cell population in rabbit peripheral blood lymphocytes, and to assess the sensitivity and specificity of the antibody, flow cytometry is required for verification.

[0200] Reagents: Rabbit peripheral blood lymphocyte separation kit (Solarbio: P8760), red blood cell lysis buffer (Solarbio: R1010), β1-iFluor488, β1-iFluor594, CD5 Antibody-FITC, phosphate-buffered saline (PBS), pH 7.2 (Shanghai Yuanpei).

[0201] Consumables: 15mL centrifuge tubes (BD); 1.5mL centrifuge tubes (EP); 10uL, 200uL, and 1000uL pipette tips.

[0202] Equipment: Flow cytometer (Agilent); 12-well pipette (Eppendorf); 4℃ freezer (Zhongke Meiling: YC-260L); centrifuge (Thermo: ST16).

[0203] Step a): Rabbit PBMCs were obtained by separating fresh rabbit anticoagulated blood using a rabbit peripheral blood lymphocyte separation kit. After lysing the red blood cells, the cells were ready for use.

[0204] Step b): Wash the cells twice with pre-cooled PBS at 1500 rpm for 5 min at 4°C, then spin dry.

[0205] Step c): Add a certain amount of pre-cooled PBS to resuspend the cells, ensuring a cell density of 1x10⁻⁶. 8 cells / ml, added to EP tubes at a rate of 100uL / well.

[0206] Step d): Dilute B1-iFluor488 according to the specified ratio and add it to the cell suspension. At the same time, dilute B1-iFluor594 according to the specified ratio and mix it with CD5 Antibody-FITC and add it to the cell suspension. Incubate at room temperature for 20 min.

[0207] Step e): Wash the cells twice with pre-cooled PBS at 1500 rpm for 5 min at 4°C, then spin dry.

[0208] Step h): Resuspend the cells in 200uL PBS and analyze them using a flow cytometer.

[0209] Competition experiments with CD5-FITC positive antibodies and isotype control iFluor488 showed that B1 and the positive antibody recognize the same cell population (e.g., ...). Figure 7 Additionally, B1-iFluor488 at a concentration of 1:2000 is the best concentration for use (e.g., Figure 8 ).

[0210] Example 10: Sequencing of the variable region of the heavy and light chains

[0211] The antibody gene of mouse hybridoma single-cell clone B1 was sequenced to obtain the variable region sequence of the heavy and light chains of the clone.

[0212] Reagents: TAE buffer, agarose, nucleic acid dye.

[0213] Consumables: pipette tips.

[0214] Equipment: Nucleic acid electrophoresis instrument, nucleic acid imaging instrument.

[0215] Heavy and light chain variable region sequencing:

[0216] The variable region sequence of the cloned B1 heavy and light chains was obtained by sequencing the PCR (2nd PCR) product.

[0217] Step a): The target bands were observed by nucleic acid electrophoresis of the heavy and light chain PCR products;

[0218] Step b): After gel extraction and recovery, the samples were sent to a gene sequencing company to obtain the variable region nucleic acid and protein sequences of the heavy and light chains of clone B1.

[0219] The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.8.

[0220] Further, the high-variable region sequences were obtained as shown in Table 1.

[0221] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 CD5 monoclonal antibody, characterized in that, The variable region of the mouse anti-rabbit CD5 monoclonal antibody includes: the complementarity-determining region CDR1-VH of the amino acid sequence shown in SEQ ID NO.1, the complementarity-determining region CDR2-VH of the amino acid sequence shown in SEQ ID NO.2, the complementarity-determining region CDR3-VH of the amino acid sequence shown in SEQ ID NO.3, the complementarity-determining region CDR1-VL of the amino acid sequence shown in SEQ ID NO.4, the complementarity-determining region CDR2-VL of the amino acid sequence shown in SEQ ID NO.5, and the complementarity-determining region CDR3-VL of the amino acid sequence shown in SEQ ID NO.

6.

2. The application of the mouse anti-rabbit CD5 monoclonal antibody according to claim 1 in the sorting of CD5+ T cell population.

3. The application according to claim 2, characterized in that, The CD5+ T cell population includes CD5+ T cells from rabbit peripheral blood lymphocytes.

4. A marker for CD5+ T cell population, characterized in that, The marker comprises the mouse anti-rabbit CD5 monoclonal antibody and fluorescent dye as described in claim 1; The mouse anti-rabbit CD5 monoclonal antibody was conjugated with a fluorescent dye.

5. The marker according to claim 4, characterized in that, The fluorescent dyes include iFluor488 or iFluor594.

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