Rabbit monoclonal antibody for resisting human Parkin protein as well as application and product of rabbit monoclonal antibody

Abstract

The invention provides an anti-human Parkin protein rabbit monoclonal antibody as well as application and a product thereof, and relates to the technical field of biology. The rabbit monoclonal antibody for resisting the human Parkin protein, provided by the invention, can specifically recognize the human Parkin protein, is high in specificity and sensitivity, can be used for detecting the human Parkin protein, and plays an important role in researching the action mechanism of Parkin and researching or diagnosing diseases.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a rabbit monoclonal antibody against human Parkin protein and applications and products thereof. Background Art

[0002] Mitochondria are key hubs of energy metabolism, and their quantity, quality, and functional integrity play an important role in maintaining the metabolic balance of cells and tissues. The Parkin protein encoded by the PRKN gene is an E3 ubiquitin ligase that plays a vital role in regulating mitochondrial autophagy.

[0003] Studies have shown that PRKN gene deletion or gene site mutation is associated with the development of autosomal recessive Parkinson's disease. Parkinson's disease is a neurodegenerative disease caused by the gradual death of dopamine-producing neurons in the substantia nigra compacta of the midbrain. The inactivation of PRKN mutations causes abnormal aggregation of its substrates in dopamine neurons, resulting in neurotoxicity, which is the cause of Parkinson's disease. In addition to causing neurodegenerative diseases, it has been reported that Parkin can also serve as a key tumor suppressor pathway and a stress-activated effector, antagonizing the ability of malignant cells to proliferate and metastasize by interfering with the ability of cancer cells to reprogram their metabolism. In addition, it has been reported that Parkin can play a role in balancing mitochondrial autophagy and mitochondrial biogenesis in white adipose tissue, thereby affecting the development of obesity.

[0004] Given the important role Parkin plays in different diseases, the development of highly sensitive and specific Parkin antibodies is of great significance for the diagnosis and treatment of Parkin-related diseases. Clinically, the expression of related proteins at the tissue and cell levels is measured by immunological means and related techniques, such as immunoblotting, immunohistochemistry, and immunofluorescence. The core of these techniques is high-affinity antibodies that bind to specific antigens. Therefore, the development of highly sensitive and high-affinity rabbit monoclonal antibodies against Parkin protein plays an important role in studying the mechanism of Parkin and in its disease research or diagnosis.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The first purpose of the present invention is to provide a rabbit monoclonal antibody against human Parkin protein to solve the problem of detecting human Parkin protein.

[0007] A second object of the present invention is to provide a biomaterial.

[0008] The third object of the present invention is to provide a method for preparing the above-mentioned rabbit monoclonal antibody against human Parkin protein.

[0009] The fourth object of the present invention is to provide the use of the above-mentioned rabbit monoclonal antibody against human Parkin protein in the preparation of a detection product for human Parkin protein.

[0010] The fifth object of the present invention is to provide a marker for human Parkin protein.

[0011] The sixth object of the present invention is to provide a kit for detecting human Parkin protein.

[0012] In order to achieve the above objectives, the following technical solutions are adopted:

[0013] In a first aspect, the present invention provides a rabbit monoclonal antibody against human Parkin protein, wherein the variable region of the rabbit monoclonal antibody against human Parkin protein comprises: a complementary determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO.1, a complementary determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO.2, a complementary determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO.3, a complementary determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO.4, a complementary determining region CDR2-VL having an amino acid sequence as shown in SEQ ID NO.9, and a complementary determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO.5.

[0014] As a further technical solution, the amino acid sequence of the heavy chain variable region VH of the monoclonal antibody is shown in SEQ ID NO.6.

[0015] As a further technical solution, the amino acid sequence of the light chain variable region VL of the monoclonal antibody is shown in SEQ ID NO.7.

[0016] In a second aspect, the present invention provides a biomaterial, wherein the biomaterial is selected from any one of ac:

[0017] a. Nucleotides, comprising a nucleotide sequence encoding the above-mentioned rabbit monoclonal antibody against human Parkin protein;

[0018] b. a vector carrying the nucleotides in a;

[0019] c. A cell, wherein the cell carries the nucleotide in a, or contains the vector in b, or expresses the above-mentioned rabbit monoclonal antibody against human Parkin protein.

[0020] In a third aspect, the present invention provides a method for preparing the above-mentioned rabbit monoclonal antibody against human Parkin protein, which is obtained by fermentation of the above-mentioned cells.

[0021] In a fourth aspect, the present invention provides the use of the above-mentioned rabbit monoclonal antibody against human Parkin protein in the preparation of a detection product for human Parkin protein.

[0022] In a fifth aspect, the present invention provides a marker of human Parkin protein, comprising the above-mentioned rabbit monoclonal antibody against human Parkin protein and a marker;

[0023] The rabbit monoclonal antibody against human Parkin protein is coupled to a label.

[0024] As a further technical solution, the marker includes an enzyme, a fluorescent molecule marker, a fluorescent microsphere, a colored microsphere, colloidal gold, biotin or streptavidin.

[0025] In a sixth aspect, the present invention provides a kit for detecting human Parkin protein, the kit comprising the above-mentioned rabbit monoclonal antibody against human Parkin protein or the above-mentioned marker of human Parkin protein.

[0026] As a further technical solution, the kit includes an immunochromatographic detection kit or an ELISA detection kit.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides a rabbit monoclonal antibody against human Parkin protein, which can specifically recognize human Parkin protein with strong specificity and high sensitivity, and can be used for detecting human Parkin protein, and plays an important role in studying the action mechanism of Parkin and in disease research or diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 The results of Western blot detection of rabbit monoclonal antibody 1A9-1 against human Parkin protein in different cell samples;

[0031] Figure 2 The results of immunohistochemical detection of rabbit monoclonal antibody 1A9-1 against human Parkin protein in mouse brain tissue;

[0032] Figure 3The results of immunocytochemistry detection of rabbit monoclonal antibody 1A9-1 against human Parkin protein in human neuroblastoma cell line SH-SY5Y;

[0033] Figure 4 The results of flow cytometry detection of rabbit monoclonal antibody 1A9-1 against human Parkin protein in human neuroblastoma cell line SH-SY5Y;

[0034] Figure 5 The results of immunoprecipitation detection of anti-human Parkin protein rabbit monoclonal antibody 1A9-1 in human neuroblastoma cell line SH-SY5Y. DETAILED DESCRIPTION

[0035] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0036] Generally, the nomenclature and its technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods as well-known in the art, and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as generally achieved in the art, or as described herein. The nomenclature and its laboratory procedures and technology used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0037] The "variable region" or "variable domain" of an antibody refers to the domain at the amino terminus of the heavy chain or light chain of the antibody that recognizes and binds to the antigen. The composition and arrangement of the amino acids in this segment determine the specificity of the antibody in recognizing the antigen. The heavy chain variable region can be referred to as "VH". The light chain variable region can be referred to as "VL". These domains are usually the most variable parts of the antibody and contain antigen binding sites. The variable regions of the heavy and light chains are each composed of three complementarity-determining regions (CDRs) (also called hypervariable regions) connected by four framework regions (FRs). The CDRs in each chain are held together closely by FRs to form the variable region. Typically, the variable regions VL / VH of the heavy and light chains can be obtained by connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0038] The term "vector" refers to a nucleic acid delivery vehicle into which a nucleotide can be inserted. When a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell.

[0039] The vector is well known to those skilled in the art, including but not limited to: plasmid; phagemid; cosmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as lambda phage or M13 phage and animal virus, etc. Animal viruses that can be used as vectors include but are not limited to retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papillomavirus, papillomavirus. In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0040] In a first aspect, the present invention provides a rabbit monoclonal antibody against human Parkin protein, wherein the variable region of the rabbit monoclonal antibody against human Parkin protein comprises: a complementary determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO.1, a complementary determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO.2, a complementary determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO.3, a complementary determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO.4, a complementary determining region CDR2-VL having an amino acid sequence as shown in SEQ ID NO.9, and a complementary determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO.5.

[0041] The amino acid sequences of SEQ ID NO.1-SEQ ID NO.5 and SEQ ID NO.9 are shown in Table 1.

[0042] Table 1

[0043] Highly variable region sequence serial number CDR1-VH GFSFSAGYD SEQ ID NO.1 CDR2-VH IYADGSGST SEQ ID NO.2 CDR3-VH AREGADGPDYGYAAFSL SEQ ID NO.3 CDR1-VL QSISNY SEQ ID NO.4 CDR2-VL KAS SEQ ID NO.9 CDR3-VL QSFYGSVTSDYGGFA SEQ ID NO.5

[0044] In some optional embodiments, the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.6:

[0045] QSLEESGGDLVKPGASLTLTCTASGFSFSAGYDMCWVRQAPGKGLEWIACIYADGSGSTYYANWAKGRFTISLASSSTTVTLQMTGLTAADTATYFCAREGADGPDYGYAAFS LWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCS KPTCPPPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISK ARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO.6).

[0046] In some optional embodiments, the amino acid sequence of the light chain variable region VL is as shown in SEQ ID NO.7:

[0047] DVVMTQTPASVEAAVGGTVTIKCQASQSISNYFSWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTEFTLTISDLECADAATYYCQSFYGSVTSDYGGFAFGGGTEVV VKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQID NO.7).

[0048] In a second aspect, the present invention provides a biomaterial, wherein the biomaterial is selected from any one of ac:

[0049] a. Nucleotides, comprising a nucleotide sequence encoding the above-mentioned rabbit monoclonal antibody against human Parkin protein;

[0050] b. a vector carrying the nucleotides in a;

[0051] c. A cell, wherein the cell carries the nucleotide in a, or contains the vector in b, or expresses the above-mentioned rabbit monoclonal antibody against human Parkin protein.

[0052] In a third aspect, the present invention provides a method for preparing the above-mentioned rabbit monoclonal antibody against human Parkin protein, which is obtained by fermentation of the above-mentioned cells.

[0053] The preparation method is simple and efficient, and a large amount of rabbit monoclonal antibodies against human Parkin protein can be obtained through fermentation.

[0054] In a fourth aspect, the present invention provides the use of the above-mentioned rabbit monoclonal antibody against human Parkin protein in the preparation of a detection product for human Parkin protein.

[0055] The rabbit monoclonal antibody against human Parkin protein provided by the present invention can specifically recognize human Parkin protein, and thus can be used for the detection of human Parkin protein.

[0056] In a fifth aspect, the present invention provides a marker of human Parkin protein, comprising the above-mentioned rabbit monoclonal antibody against human Parkin protein and a marker;

[0057] The rabbit monoclonal antibody against human Parkin protein is coupled to a label.

[0058] The marker can specifically bind to the human Parkin protein, thereby achieving specific labeling of the human Parkin protein.

[0059] In some optional embodiments, the marker includes but is not limited to enzymes, fluorescent molecule markers, fluorescent microspheres, colored microspheres, colloidal gold, biotin or streptavidin, or other markers known to those skilled in the art.

[0060] In a sixth aspect, the present invention provides a kit for detecting human Parkin protein, the kit comprising the above-mentioned rabbit monoclonal antibody against human Parkin protein or the above-mentioned marker of human Parkin protein.

[0061] The kit uses rabbit monoclonal antibodies against human Parkin protein to detect human Parkin protein.

[0062] In some optional embodiments, the kit includes an immunochromatographic detection kit or an ELISA detection kit.

[0063] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0064] Example 1 Preparation of rabbit monoclonal antibody against human Parkin protein

[0065] 1. Preparation of immunogen

[0066] In this example, human Parkin protein (Uniprot protein number: O60260) 1-144aa was selected as the immunogen after prokaryotic expression in E. coli. The amino acid sequence of human Parkin protein 1-144aa is:

[0067] MIVFVRFNSSHGFPVEVDSDTSIFQLKEVVAKRQGVPADQLRVIFAGKELRNDWTVQNCDLDQQSIVHIVQRPWRKGQEMNATGGDDPRNAAGGCEREPQSLTRVDLSSSVLPGDSVGLAVILHTDSRKDSPPAGSPAGRSIYN (SEQ ID NO. 8).

[0068] 2. Rabbit Immunization

[0069] Reagents: Adjuvant, 75% alcohol.

[0070] Consumables: Syringe.

[0071] Sample: Human Parkin protein immunogen.

[0072] The steps for rabbit immunization are as follows:

[0073] 1) Animal selection: New Zealand white rabbits are used, weighing about 2.5 kg, young and strong. The animals should be healthy animals with shiny fur and free movement. After selecting the animals, raise them for about 2 weeks to adapt to the environment.

[0074] 2) Prepare for the experiment and mark the rabbits.

[0075] 3) Take the antigen out of the -20℃ freezer and dissolve it at room temperature to avoid repeated freezing and thawing. Mark the syringe with the project number and animal number.

[0076] 4) Extract antigen (mix antigen completely), the antigen concentration for the first immunization is 1 mg / ml, and the amount for each rabbit is 0.5 ml. The amount of antigen for the second to fifth immunizations is halved.

[0077] 5) Draw out the adjuvant, with the adjuvant and antigen in a 1:1 volume ratio. Use complete adjuvant for the first immunization, and incomplete adjuvant for the second to fifth immunizations. When drawing, the adjuvant should be fully mixed before being drawn into the syringe.

[0078] 6) The two syringes are connected with a syringe connecting tube for complete emulsification. The emulsification standard is: the emulsified immunogen is qualified if it does not disperse when dropped into 37°C water.

[0079] 7) Rabbits are given multiple subcutaneous injections, with 0.2 ml at each point. The second immunization is given on the 14th day after the first immunization, and the interval between the second and third immunizations is 7 days. After the fourth immunization, booster immunization is performed based on the animal serum titer, generally once a month. After the rabbits have undergone five immunizations, blood is collected for titer testing.

[0080] 8) Procedure for collecting small serum samples: secure the experimental rabbit in a fixed frame, tap the ear with your hand to dilate the central artery of the ear. Disinfect with 75% alcohol, fix the rabbit's ear with your left hand, hold the syringe with your right hand, insert the needle into the central artery of the ear 1 / 3 of the distance from the end, parallel to the artery, and pierce the artery in the centripetal direction. Take 8 ml of blood at a time, and use cotton balls to press and stop bleeding after blood collection.

[0081] 3. Rabbit serum titer test

[0082] The rabbit serum was tested for the immunogen potency by indirect ELISA method. Rabbits with OD450 nm values ​​exceeding 0.6 under the serum dilution condition of 1:64000 were considered to have qualified potency and could proceed to the next step.

[0083] Reagents: goat anti-rabbit-HRP (Hua'an Biotechnology: HA1001), 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, 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.

[0084] Consumables: ELISA plate (Hangzhou Shengyou).

[0085] Equipment: Electric constant temperature incubator (Shanghai Senxin: DRP-9162), ELISA instrument (MD: Cmax plus).

[0086] The indirect ELISA procedure is as follows:

[0087] 1) Dilute the antigen to 2 μg / ml with coating buffer, add 50 μl / well to the ELISA plate, cover the plate, and coat overnight at 4°C.

[0088] 2) Shake off the liquid in the wells, add 1% BSA / TBS to the ELISA plate at a volume of 150 μl / well, and place in a 37°C electric thermostatic incubator for 1 hour.

[0089] 3) Shake off the liquid in the wells, add 50 μl / well of serum with different dilution ratios to the ELISA plate, cover the plate, and incubate in a 37°C incubator for 30 minutes.

[0090] 4) Dry the primary antibody mixture, add washing solution (1×TBST) to the ELISA plate at a volume of 180 μl / well, and wash the ELISA plate twice. Dilute the goat anti-rabbit-HRP to a working concentration (1:30000) with 1% BSA, add 50 μl / well to the ELISA plate, cover the lid, and incubate in a 37°C constant temperature incubator for 45 minutes.

[0091] 5) Color development, termination and reading, discard the liquid in the wells, add washing solution to the ELISA plate at a volume of 180 μl / well, and wash the ELISA plate 3 times; add 100 μl of freshly prepared TMB color development substrate to each reaction well, incubate at 37°C for 10 minutes; then add 90 μl / well of stop solution to terminate the reaction, and measure the OD value at 450nm on the ELISA reader.

[0092] The OD450 nm values ​​of the two rabbit sera were both over 0.6 at a dilution of 1:64000, and the titers were qualified. The test results are shown in Table 2. Rabbit 2 had a higher titer, so Rabbit 2 was selected for the next step.

[0093] Table 2 Rabbit serum indirect ELISA antibody titer test results

[0094]

[0095] 4. Isolation of rabbit peripheral blood lymphocytes (PBMC)

[0096] Reagents: Rabbit peripheral blood lymphocyte separation kit (Solarbio: P8760), red blood cell lysis solution (Solarbio: R1010), PBS (BasalMedia: B310KJ-500 ml).

[0097] Consumables: 50 ml centrifuge tube (BD: 352070), 10 ml pipette (Hangzhou Lanjieke: 352070).

[0098] Equipment: biological safety cabinet (Sujing: BSC-1300IIB2), centrifuge (Thermo: THM#75004530).

[0099] The isolation steps of rabbit peripheral blood lymphocytes (PBMC) are as follows:

[0100] 1) Take 20 ml of anticoagulated blood from the rabbit with the best titer after the fifth immunization, place it on ice, and take an equal volume of PBS to dilute the whole blood.

[0101] 2) Add an equal volume of rabbit peripheral lymphocyte separation fluid into the centrifuge tube and spread the diluted blood evenly above the surface of the separation fluid.

[0102] 3) Centrifuge at 1800 rpm in a swing-out rotor at room temperature for 30 minutes.

[0103] 4) After centrifugation, there will be obvious stratification: the top layer is the diluted plasma layer, the middle layer is the transparent separation liquid layer, the white film layer between the plasma and the separation liquid is the lymphocyte layer, and the bottom of the centrifuge tube is the red blood cells and granulocytes. Carefully pipette the white film layer cells into a 50 ml clean centrifuge tube, and wash the white film layer cells with 10 ml PBS. Centrifuge at 1500rpm for 5 minutes.

[0104] 5) Discard the supernatant, resuspend the cells in 20 ml of PBS, and centrifuge at 1500 rpm for 5 minutes. Repeat the washing process and discard the supernatant for later use.

[0105] 6) Add 5 ml of red blood cell lysis buffer and gently vortex or invert to mix. Incubate at 37°C for 5 minutes. After the red blood cells are lysed, the solution should be clear and transparent.

[0106] 7) Centrifuge at 1500 rpm for 5 minutes to precipitate leukocytes and carefully discard the supernatant.

[0107] 8) Resuspend the cells in 20 ml of PBS and centrifuge at 1500 rpm for 5 minutes.

[0108] 9) Repeat step 8) and discard the supernatant, and separate the peripheral blood lymphocytes for later use.

[0109] 5. Antigen-specific B cell sorting

[0110] Antigen-positive B cells are negatively selected by T cell surface markers and positively selected by B cell surface immunoglobulin, and then specific B cells are sorted from peripheral blood mononuclear cells using fluorescent dye-labeled sorting antigens.

[0111] Reagents: PBS (Basal Media: B310KJ-500 ml), fetal bovine serum (Sijiqing: 11011-8611), mouse anti-rabbit CD4 antibody-FITC, mouse anti-rabbit IgG-PE, mouse anti-rabbit IgM-Biotin, streptavidin-PE / Cy7, Brilliant Violet 421 TM -Streptavidin, Alexa 647-streptavidin, biotin-labeled m6A-BSA.

[0112] Consumables: 15 ml centrifuge tube (BD: 352097), EP tube (AXYGEN: MCT-150-C).

[0113] Equipment: biological safety cabinet (Sujing: BSC-1300IIB2), flow sorter (Sony: MA900), centrifuge (Thermo: THM#75004530).

[0114] The steps for sorting human Parkin protein immunogen-specific B cells are as follows:

[0115] 1) Set the flow cytometry indexes (IgG, IgM, m6A-BSA) and experimental groups (blank cell control group, compensation single positive tube control group, minus one fluorescence control group, experimental group) for this experiment.

[0116] 2) IgM and antigen positive indicators need to firstly react IgM-Biotin and antigen-Biotin with streptavidin-PE / Cy7 and Brilliant Violet 421 respectively. TM / Alexa After pre-incubation with 647-streptavidin for 20 minutes, the cells were blocked with excess BSA-Biotin for 20 minutes before use for sorting and detection, and then added to the corresponding experimental groups in step 5).

[0117] 3) Peripheral blood lymphocytes were collected and resuspended in 10 ml PBS. After counting the cells, the cells were divided into 1.5 ml EP tubes and centrifuged at 1500 rpm for 5 minutes.

[0118] 4) Carefully discard the supernatant and resuspend with 100 μl PBS.

[0119] 5) Add the corresponding antibodies according to the experimental groups set in step 1) and vortex gently to mix.

[0120] 6) After incubation at room temperature for 20 minutes, add 800 μl of PBS for re-suspending and centrifuge at 1500 rpm for 5 minutes.

[0121] 7) Carefully discard the supernatant, resuspend the cells in 1 ml of PBS, and centrifuge at 1500 rpm for 5 minutes.

[0122] 8) Repeat step 7 and discard the supernatant carefully. Add 1 ml of PBS containing 2% FBS to the experimental group and resuspend the cells. Add 200 μl of PBS containing 2% FBS to the other groups and resuspend them.

[0123] 9) Place on ice and load onto the machine.

[0124] 10) Adjust the fluorescence compensation on the flow cytometer through the blank cell control group and the compensation single positive tube control group, and gate through the minus one fluorescence control group. Finally, add the experimental group samples and sort out the antigen double positive cell population.

[0125] Antigen-specific binding B cell sorting: refer to the patent "m6A binding protein and its application" (publication number: CN115975038A).

[0126] 6. Cloning and production of rabbit monoclonal antibodies

[0127] In order to obtain rabbit monoclonal antibodies that recognize Parkin protein, the positive B cells obtained by sorting are reverse transcribed to obtain cDNA, which is then sequenced, and the selected rabbit monoclonal antibody heavy chain variable region (VH) and light chain variable region (VL) are constructed into expression vectors (wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7). Finally, it is produced by mammalian cell expression.

[0128] Reagent: Expi293 TM Expression system kit (Thermo, A14635).

[0129] Consumables: 96-well deep-well plates, pipette tips.

[0130] Equipment: Constant temperature shaker, electronic pipette.

[0131] Antibody plasmid cell transfection:

[0132] 1) Place the Expi293F culture medium in a 37°C water bath for incubation. Observe the cell status and cell growth under a microscope. Determine the cell expansion experiment subculture ratio on the day based on the cell growth on the day and the needs of the subsequent transfection experiment. Generally, the transfection density is controlled at 2×10 6 About 100 living cells / ml. Take out the cell solution in the CO2 cell shaker, count it, and calculate the amount of cells to be transferred. 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 2×10 6 About 100 living cells / ml, used for transfection operation on the next day.

[0133] 2) On the day of transfection, preheat the Expi293 medium at room temperature in the dark. Determine the number of cells to be transfected and prepare the corresponding number of 96-well deep-well plates. Determine the cell density based on the cell counting operation, add the preheated medium to dilute the cells so that the final cell density is 3×10 6 Live cells / ml, use a pipette to dispense into a deep-well plate at 1 ml / well, and carefully place it in a cell shaker: 37°C, 8% CO2, 900rpm, 80% humidity. Take the corresponding number of 96-well cell culture plates, add 1 μg DNA + 60 μl Opti-MEM (cold reagent) per 1 ml of cells and dispense into the culture plate, and mix well with a pipette (antibody recommended ratio HC: LC = 1:2). Take another centrifuge tube with the most suitable volume, dilute the transfection reagent in the ratio of 3.2 μl Expifectamine 293 + 60 μl Opti-MEM (cold reagent) per 1 ml of cells, gently invert and mix, and let stand at room temperature for 5 minutes. After 5 minutes, move the diluted transfection reagent to a disposable reservoir, use a pipette to move to the corresponding position of the 96-well cell culture plate, and let stand for 10-20 minutes. Use a pipette to add the resting Expifectamine293 / DNA complex to the cell suspension and carefully place it in a shaker: 37°C, 8% CO2, 900 rpm, 80% humidity. 18-22 hours after transfection, add 6 μl Enhancer 1 and 60 μl Enhancer 2 to 1 ml.

[0134] 3) After 4-7 days, the supernatant was harvested and purified. The antibody in the supernatant was named anti-human Parkin protein rabbit monoclonal antibody 1A9-1.

[0135] Cloning and production of rabbit monoclonal antibodies: refer to the patent "m6A binding protein and its application" (publication number: CN115975038A).

[0136] Example 2 Identification of the Immunological Application of Rabbit Monoclonal Antibody 1A9-1 Against Human Parkin Protein

[0137] 1. Western Blot (WB) analysis

[0138] In order to detect the recognition specificity of the rabbit monoclonal antibody of the present invention to Parkin protein, human neuroblastoma cell line SH-SY5Y, human embryonic kidney cell line 293T, African green monkey kidney cell line COS-1, mouse neuroblastoma cell line Neuro-2a, rat glioma cell line C6, mouse brain and rat brain expressing Parkin protein were selected as samples for detection.

[0139] The experimental steps are as follows: the above cells were lysed separately, the proteins were extracted, and 4-20% polyacrylamide gel electrophoresis was performed. Subsequently, the proteins in the gel were transferred to the PVDF membrane according to the conventional method. The PVDF membrane was placed in a TBST blocking solution containing 5% skimmed milk powder and incubated at room temperature for 1 hour. Then, anti-Parkin protein rabbit monoclonal antibody 1-A9-1 was added at a dilution ratio of 1:2000 and incubated overnight at 4°C. Subsequently, the membrane was washed with TBST, and then goat anti-rabbit secondary antibody was added and incubated at room temperature for 1 hour. The membrane was washed with TBST again, and then ECL supersensitive color development solution was added for development. The results are as follows. Figure 1 shown.

[0140] 2. Immunohistochemistry (IHC)

[0141] Before staining paraffin sections (mouse brain tissue), dewaxing is required to ensure smooth staining of the sections. Procedure: xylene I for 10 minutes; xylene II for 10 minutes; xylene III for 10 minutes; anhydrous ethanol I for 3 minutes; anhydrous ethanol II for 3 minutes; 95% alcohol for 3 minutes; rinse with running water for 3 minutes.

[0142] Antigen repair: Pour about 1 liter of 1×Tris-EDTA (pH 9.0) into a high-temperature container and heat it with an induction cooker to boil for 3-5 minutes. Make sure there are no bubbles at low heat. Put the tissue slices into the container, adjust the induction cooker to low power and maintain the temperature at 90-95℃ for 20 minutes, cover the pot. Cool at room temperature for 10 minutes, then rinse with running water and cool to room temperature.

[0143] Immunohistochemical staining:

[0144] 1) Wash the sections three times with pure water, 5 minutes each time.

[0145] 2) Blocking: Place the slices in a 3% hydrogen peroxide solution and incubate for 10 minutes.

[0146] 3) Wash the sections twice with pure water, 5 minutes each time.

[0147] 4) After the slices are slightly dried, use filter paper to wipe off the water around the tissue slices, use a histochemical pen to draw a circle around the tissue, add 10% negative goat serum prepared with 1% BSA into the circle, cover the tissue, and block at room temperature for 1 hour.

[0148] 5) Remove the blocking solution, then add the primary antibody diluted in the recommended antibody diluent to each section (usually 100 μl / tissue) and incubate in a humidified chamber at room temperature for 1 hour.

[0149] 6) Remove the antibody solution and wash with 1×TBST three times, 5 minutes each time.

[0150] 7) After patting the slices dry, add goat anti-rabbit or mouse secondary antibody (Hua'an Biotechnology, Cat. No. HA1119) in the circle to cover the tissue and incubate in a humidified box at room temperature for 20 minutes.

[0151] 8) Wash three times with 1× TBST, 5 minutes each time.

[0152] 9) DAB color development: After patting the slices dry, add freshly prepared DAB color development solution in the circle. Control the color development time under a microscope. The positive color is brown-yellow. Observe until a positive signal appears or the color development takes 5 minutes. Rinse the slides with a washing bottle filled with tap water or a special beaker for DAB to stop the color development. Rinse with running tap water for 5-10 minutes.

[0153] 10) Re-staining and dehydration: The process is hematoxylin for 10 minutes; running water washing for 3 minutes; blueing for 1 minute; running water washing for 3 minutes; 95% alcohol I for 1 minute; 95% alcohol II for 1 minute; 95% alcohol III for 1 minute; anhydrous ethanol I for 1 minute; anhydrous ethanol II for 1 minute; xylene I for 1 minute; xylene II for 1 minute; oven at 65℃ for 2 minutes. Then seal the slides with neutral gum. Finally, scan the slides and determine the results.

[0154] The results of immunohistochemical staining showed that the rabbit monoclonal antibody 1A9-1 against human Parkin protein was accurately localized in the cytoplasm with a clean background. Figure 2 shown.

[0155] 3. Immunocytochemistry (IF-cell)

[0156] 1) Prepare SH-SY5Y, a human neuroblastoma cell line with a cell confluence of 70%, remove the culture medium, add 250 μl of 4% paraformaldehyde, and fix at room temperature for 15 minutes.

[0157] 2) Aspirate the fixative solution, add 1× PBS, and wash twice, 5 minutes each time.

[0158] 3) Add 0.1% Triton X-100 cell permeabilization agent, permeabilize the membrane for 15 minutes at room temperature, and wash twice with 1× PBS, each time for 5 minutes.

[0159] 4) Add 10% goat serum blocking solution and block at room temperature for 1 hour.

[0160] 5) Add anti-Parkin protein rabbit monoclonal antibody 1-A9-1 diluted with antibody diluent and place in a humidified box at 4°C overnight.

[0161] 6) The next morning, wash three times with 1×PBST, 5 minutes each time.

[0162] 7) Add fluorescent secondary antibody (Hua'an Biotechnology, HA1121) diluted with antibody diluent and incubate at room temperature in the dark for 1 hour.

[0163] 8) Wash three times with 1× PBST, 5 minutes each time.

[0164] 9) After sealing with an anti-fluorescence quencher containing DAPI, observe and photograph under a fluorescence microscope.

[0165] The results of immunocytochemical staining showed that the rabbit monoclonal antibody 1A9-1 against human Parkin protein was accurately localized in the cytoplasm of the human neuroblastoma cell line SH-SY5Y with a clean background. Figure 3 shown.

[0166] 4. Flow Cytometry (FC)

[0167] 1) Prepare a single cell suspension of human neuroblastoma cell line SH-SY5Y, wash twice with 1×PBS, add 200 μl of 4% paraformaldehyde, and fix at room temperature for 15 minutes.

[0168] 2) Centrifuge at 500 g for 5 minutes and discard the supernatant; wash twice with 1× PBS.

[0169] 3) Add permeabilization buffer and incubate at room temperature for 15 minutes, wash twice with 1× PBS, centrifuge at 500 g, and discard the supernatant.

[0170] 4) Blocking: Add blocking solution (10% goat serum) and block at room temperature for 15 minutes.

[0171] 5) Add anti-Parkin protein rabbit monoclonal antibody 1-A9-1 diluted in 1× PBS and incubate at room temperature for 15 minutes;

[0172] 6) Add 1×PBS and wash 1-2 times, centrifuge at 500 g, and discard the supernatant.

[0173] 7) Add fluorescent secondary antibody (Hua'an Biotechnology, HA1121) and incubate at room temperature in the dark for 15 minutes.

[0174] 8) Add 1×PBS and wash 1-2 times, centrifuge at 500 g, and discard the supernatant.

[0175] 9) Add 100 μl of 1× PBS to resuspend the cells and analyze the cells using flow cytometry.

[0176] The flow cytometry results showed that compared with the rabbit IgG control (green peak), the fluorescence peak of the rabbit monoclonal antibody 1A9-1 against human Parkin protein in the human neuroblastoma cell line SH-SY5Y was significantly shifted to the right (red peak). Figure 4 shown.

[0177] 5. Immunoprecipitation (IP)

[0178] 1) Prepare 2 low-adsorption EP tubes, add 30 μl of Protein A / G magnetic beads, and then add 2 μg of 1A9-1 antibody and rabbit IgG control antibody respectively, fill up to 0.5 ml volume with TBST, place all EP tubes on a rotating rack, and rotate at room temperature for 1 hour.

[0179] 2) During this period, prepare about 1 mg of lysate of human neuroblastoma cell line SH-SY5Y.

[0180] 3) After the antibody and magnetic beads are incubated, remove the EP tube and place it on a magnetic rack for 1 minute, aspirate the liquid, and add 0.5 ml TBST to wash twice.

[0181] 4) Add 200 μg of the lysate prepared above to each of the two tubes, place on a rotating rack, and rotate at room temperature for 1 hour.

[0182] 5) Remove the EP tube and place it on a magnetic rack for 1 minute, remove the liquid, and add 0.5 ml TBST to wash three times.

[0183] 6) Add 20 μl of 2×SDS loading buffer, resuspend, and boil at 95°C for 10 minutes.

[0184] 7) Place the EP tube on the magnetic rack and let it stand for 1 minute. Transfer the liquid to a new EP tube.

[0185] 8) The following operations refer to WB operations.

[0186] Immunoprecipitation results showed that the anti-human Parkin protein rabbit monoclonal antibody 1A9-1 can significantly enrich the Parkin protein in the human neuroblastoma cell line SH-SY5Y. Figure 5 shown.

[0187] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned 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 rabbit monoclonal antibody against human Parkin protein, characterized in that: The variable region of the rabbit monoclonal antibody against human Parkin protein includes: a complementary determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO.1, a complementary determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO.2, a complementary determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO.3, a complementary determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO.4, a complementary determining region CDR2-VL having an amino acid sequence as shown in SEQ ID NO.9, and a complementary determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO.

5.

2. The rabbit monoclonal antibody against human Parkin protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region VH of the monoclonal antibody is shown in SEQ ID NO.

6.

3. The rabbit monoclonal antibody against human Parkin protein according to claim 1, characterized in that: The amino acid sequence of the light chain variable region VL of the monoclonal antibody is shown in SEQ ID NO.

7.

4. Biomaterial, characterized in that The biological material is selected from any one of ac: a. Nucleotides, comprising a nucleotide sequence encoding a rabbit monoclonal antibody against human Parkin protein according to any one of claims 1 to 3; b. a vector carrying the nucleotides in a; c. A cell, wherein the cell carries the nucleotide in a, or contains the vector in b, or expresses the rabbit monoclonal antibody against human Parkin protein according to any one of claims 1 to 3.

5. The method for preparing the rabbit monoclonal antibody against human Parkin protein according to any one of claims 1 to 3, characterized in that: Obtained by fermentation of the cell described in claim 4.

6. Use of the rabbit monoclonal antibody against human Parkin protein according to any one of claims 1 to 3 in the preparation of a detection product for human Parkin protein.

7. A marker for human Parkin protein, characterized in that A method comprising a rabbit monoclonal antibody against human Parkin protein according to any one of claims 1 to 3 and a marker; The rabbit monoclonal antibody against human Parkin protein is coupled to a label.

8. The marker of human Parkin protein according to claim 7, characterized in that The markers include enzymes, fluorescent molecular markers, fluorescent microspheres, colored microspheres, colloidal gold, biotin or streptavidin.

9. A kit for detecting human Parkin protein, characterized in that: The kit comprises the rabbit monoclonal antibody against human Parkin protein according to any one of claims 1 to 3 or the marker of human Parkin protein according to claim 7 or 8.

10. The kit according to claim 9, characterized in that The kit includes an immunochromatographic detection kit or an ELISA detection kit.

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