Method for preparing methyl pseudouracil monoclonal antibody based on hybridoma technology

Abstract

The invention discloses a method for preparing a methyl pseudouracil monoclonal antibody based on a hybridoma technology. The method specifically comprises the steps of experimental animal preparation, antigen synthesis, coupling of methyl pseudouracil and carrier protein, animal immunization, antiserum titer detection, cell fusion and subcloning of screened mice with good serum titer, positive clone Dot Blot identification and antibody purification. And carrying out light chain sequencing and heavy chain sequencing on the prepared methyl pseudouracil monoclonal antibody. The invention relates to the technical field of bioengineering, and provides a method for preparing a methyl pseudouracil monoclonal antibody based on a hybridoma technology, and the method adopts a mode that methyl pseudouracil-KLH is used as an immunogen, methyl pseudouracil-BSA is used as a positive screening antigen, and uracil and pseudouracil are used as negative screening antigen. And the monoclonal antibody which can only specifically recognize the methyl pseudouracil is screened out.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a method for preparing methyl pseudouracil monoclonal antibodies based on hybridoma technology. Background Art

[0002] Various modifications of DNA and proteins have always been a hot topic in the scientific research field, but there are fewer studies on the modification of mRNA. Currently, all the studies on the market synthesize mRNA in vitro and directly modify it with methyl pseudouracil to study its function.

[0003] Methyl pseudouracil, as the name implies, adds a methyl group to the N1 position of pseudouracil. Methyl pseudouracil has several analogs in the body, such as uracil and pseudouracil. The chemical structures of pseudouracil, uracil and methyl pseudouracil have not changed, but the connection sites of the nucleotides below are different. To determine the specific location of the methyl pseudouracil antibody modification in the mRNA cell. This project aims to prepare a monoclonal antibody for methyl pseudouracil through hybridoma technology to provide assistance to scientific researchers in the functional research of mRNA. Summary of the invention

[0004] In view of the above situation, in order to make up for the above existing defects, the present invention provides a method for preparing methylpseudouracil monoclonal antibody based on hybridoma technology.

[0005] The present invention provides the following technical solution: The present invention proposes a method for preparing a methylpseudouracil monoclonal antibody based on hybridoma technology, which specifically comprises the following steps:

[0006] 1. Experimental Animal Preparation

[0007] Prepare several 8-week-old healthy female Balb / C mice;

[0008] 2. Antigen synthesis

[0009] Synthesize methylpseudouracil, pseudouracil, and uracil as antigens, which are then used for immunization and screening;

[0010] 3. Coupling of methylpseudouracil to carrier protein

[0011] After oxidizing the glycosidic alcohol in methyl pseudouracil with NaIO4, it was coupled with protein;

[0012] 4. Animal immunization

[0013] The first time, Freund's complete adjuvant was mixed with an equal volume of antigen and injected into the mouse for the main injection. After that, Freund's incomplete adjuvant was mixed with an equal volume of antigen and injected multiple times for animal immunization.

[0014] 5. Antiserum titer detection

[0015] After immunization, blood was collected from the tail vein, and the serum titer of the mice was tested by indirect ELISA method to screen out mice with better serum titer;

[0016] 6. Perform cell fusion and subcloning on the selected mice with better serum titers;

[0017] 7. Dot Blot identification of positive clones and antibody purification, i.e. obtaining monoclonal antibodies that can specifically recognize methyl pseudouracil;

[0018] 8. Perform light chain sequencing and heavy chain sequencing on the prepared methyl-pseudouracil monoclonal antibody.

[0019] Furthermore, the specific steps of coupling methyl pseudouracil with the carrier protein in step 3 are:

[0020] (1) 20 mg of methyl pseudouracil was dissolved in 1 ml of 100 m mol / L NaIO4 solution and reacted at 4°C in the dark for 30 minutes;

[0021] (2) Add 1 drop of ethylene glycol to obtain liquid A;

[0022] (3) Add solution A to the protein solution;

[0023] (4) React at 4 °C for 2 hours, during which the pH is constantly adjusted to 9.0;

[0024] (5) Add 50 mg / ml NaBH4 solution prepared temporarily, the amount is 1 / 10 of the reaction volume, and react at 4°C overnight;

[0025] (6) Dialyze against 50 mmol / L phosphate buffer.

[0026] Furthermore, the preparation steps of the protein solution in step (3) are:

[0027] 20 mg / ml of protein was dissolved in 150 mmol / L NaCl, 10 mmol / L MgCl2 aqueous solution, and the pH was adjusted to 9.0 with 3% K2CO3.

[0028] Furthermore, the phosphate buffer in step (6) contains 10 mmol / L MgCl2, 10 mmol / L 2-mercaptoethanol and 100 mmol / L NaCl.

[0029] Furthermore, the pH of the phosphate buffer in step (6) is 7.4.

[0030] Furthermore, the injection position in step 4 is multiple-point injection on the back.

[0031] Furthermore, in step 4, the main injection amount is 100 ug antigen / experimental mouse, and the booster injection amount is 50 ug antigen / experimental mouse.

[0032] Preferably, the booster injections are given three times.

[0033] Furthermore, the specific steps of cell fusion and subcloning in step 6 are as follows:

[0034] (1) Myeloma cell preparation

[0035] One week before fusion, SP2 / 0 cells were resuscitated and cultured normally to the logarithmic phase;

[0036] (2) Preparation of spleen cells

[0037] The mice to be fused were selected by antiserum titer detection, and on the day of fusion, they were killed by cervical dislocation, and spleens were removed. Splenocytes were collected and counted according to standard procedures;

[0038] (3) Cell fusion

[0039] Myeloma cells and spleen cells were mixed at a ratio of 1:3-1:10, and cell fusion was performed according to the standard procedure. Then, HAT DMEM complete medium was used for culture. Hybridoma cells were visible 3 days after fusion. 1 / 2 HAT complete medium was replaced on the 7th day, and 1 / 2 HT medium was replaced on the 8th day. Screening tests were started about 10 days after fusion.

[0040] After fusion, the cells were cultured in HAT selective medium and observed under a microscope, where multiple growing hybridoma cells were observed, proving that the fusion operation was successful.

[0041] (4) Fusion screening

[0042] Pipette 100ul / well of cell supernatant for indirect ELISA detection;

[0043] According to the ELISA results, determine the positive wells; use a single-channel pipette to pick out the positive wells detected on the entire plate, conduct a second re-test, and further confirm the positive wells;

[0044] (5) Subcloning

[0045] Perform two rounds of subcloning on the cells in the rescreened positive wells;

[0046] For the first subcloning, the cells in the limiting dilution positive wells are transferred to multiple wells, and HT DMEM medium is added for culture. After about 7 days, the wells with clone growth are observed under a microscope, and the wells with high OD values ​​are detected by indirect ELISA. The cells in the positive wells are picked for the second subcloning, and the stable positive hybridoma cell lines are detected as the cells for the final preparation of monoclonal antibodies, and the culture is expanded;

[0047] (6) Monoclonal antibody subtype identification

[0048] Use the monoclonal antibody subtype identification kit to measure the subtype of each supernatant, prepare the strips coated with immunogenic protein, 50ng / well, collect 600ul supernatant from each clone, add them to 6 enzyme-labeled wells of the corresponding protein, 100ul / well, incubate at 37° for 1h, wash three times with PBST, add the diluted typing secondary antibodies against IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 to 6 wells, incubate at 37° for 1h, wash three times with PBST, and develop with TMB. The subtype of the identified secondary antibody corresponding to the well with signal reaction is the subtype of the antibody;

[0049] (7) Screen out preliminary positive clones.

[0050] Furthermore, the sequencing result of the light chain sequencing is:

[0051] CTATAGGGATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATGATATCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAGGTCACCATGACCTGCAGTGCCACCTCAAGTGTAAGTTACATGCACTGGTACCAACAGAAGTCAGGCACTTCCCCCAAAAGATGGATTTACGACACA TCCAAGCTGGCTTCTGGAGTCCCTGGTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAATAACCCGCTCACGTTCGGTGCTGGGACCAAGGTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTCCGCTGAGCAT;

[0052] The protein sequence corresponding to the light chain sequencing is as follows:

[0053] MDFQVQIFSFLLISASVMISRGQIVLTQSPAIMSASPGEKVTMTCSATSSVSYMHWYQQ KSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSNNPLTFGAGTKVELKRADAAPTVSIFPPSSPLS;

[0054] The sequencing result of the heavy chain sequencing is as follows:

[0055] TAGGAGTCAATAGGTTAATACGGGACCACTATAGGGATGAACTTCGGGTTGAGATTGGTTTTCCTTGTCCTTATTTTAAAAAGTGTACAGTGTGAGGTGCACCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGAGGCCCTGAAACTCTCCTGTGAATCCAATGAATACGAATTCCCTTCCCATGACATGTCTTGGGTCCGCAAGACTCCGGAGAAGAGGCTGGAGTTGGTCGCAGCCATTAATAGTGATGGTGGTAGCACCGACTATCCAGACACCATGGAGAGACGACTCATCATCTCCAGAGACAATACCAAGAAGACCCTGTACCTGCAAATGAGGAGTCTGAGGTCTGAGGACACAGCCTTGAAATACTGTGCAAGACCTAGTATGGTTACTTTGGTCCTTCGATGTCTGCGGCGCAGGAACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTTTATCCATTGGTCCCTGGCCGCTGAGCATAAACTAGCAAACTATCTGGAGG;

[0056] The protein sequence corresponding to the heavy chain sequencing is as follows:

[0057] MNFGLRLVFLVLILKSVQCEVHLVESGGGLVQPGEALKLSCESNEYEFPSHDMSWVR KTPEKRLELVAAINSDGGSTDYPDTMERRLIISRDNTKKTLYLQMRSLRSEDTALKYCARPS MVTLVLRCLRRRNHGHRLLSQNDTPICLSIGPWPLSIN。

[0058] The beneficial effects achieved by the present invention using the above structure are as follows: A method for preparing methyl pseudouracil monoclonal antibodies based on hybridoma technology proposed by the present invention has the following advantages:

[0059] Since monoclonal antibodies can only recognize one antigenic determinant, this scheme uses methylpseuduracil-KLH as an immunogen, methylpseuduracil-BSA as a positive screening source, and uracil and pseudouracil as negative screening sources to screen out monoclonal antibodies that can only specifically recognize methylpseuduracil.

[0060] There are currently no mature products of this type on the market. The successful development of this solution will provide support for scientific researchers in the functional research of mRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0062] Figure 1 This is a graph showing the titer of mouse antiserum in an embodiment of the present invention;

[0063] Figure 2 A graph showing the titer of mouse antiserum in an embodiment of the present invention;

[0064] Figure 3 This is a monoclonal antibody screening chart in an embodiment of the present invention;

[0065] Figure 4 The variable region sequence structure diagram of the IgG light chain of the mouse of the present invention;

[0066] Figure 5 The variable region sequence structure diagram of the IgG heavy chain of the mouse of the present invention; DETAILED DESCRIPTION

[0067] The following will combine the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0068] Since methyl pseudouracil is a very small chemical molecule, it is difficult to produce antibodies in the body. Therefore, in this embodiment, methyl pseudouracil is first synthesized, and then KLH or BSA macromolecules are added to increase immunogenicity.

[0069] Secondly, the structures of uracil, pseudouracil, and methylpseudouracil are very similar, so only by preparing monoclonal antibodies can a unique single antigenic determinant be recognized.

[0070] Therefore, in this embodiment, it is considered to use chemical small molecule European Union technology to increase the immunogenicity of the antigen, and hybridoma technology to prepare monoclonal antibodies, and then screen out monoclonal antibodies that recognize methyl pseudouracil.

[0071] This embodiment provides a method for preparing a methylpseudouracil monoclonal antibody based on hybridoma technology, which specifically comprises the following steps:

[0072] (I) Animal immunization and serum titer testing

[0073] 1. Animals

[0074] Five 8-week-old Balb / C mice.

[0075] 2. Antigen synthesis

[0076] Synthesize methylpseudouracil, pseudouracil, and uracil as antigens, which are then used for immunization and screening;

[0077] 3. Coupling of methylpseudouracil to carrier protein

[0078] After oxidizing the glycosidic alcohol in methyl pseudouracil with NaIO4, it is coupled with protein. Since methyl pseudouracil is a small molecule compound and a hapten, it cannot produce immunogenicity in animals, so it needs to be coupled to protein before animal immunization;

[0079] The specific steps of coupling methyl pseudouracil with the carrier protein are as follows:

[0080] (1) 20 mg of methyl pseudouracil was dissolved in 1 ml of 100 mmol / L NaIO4 solution and reacted at 4°C in the dark for 30 minutes;

[0081] (2) Add 1 drop of ethylene glycol to obtain liquid A;

[0082] (3) Add solution A to the protein solution;

[0083] The steps for preparing the protein solution are:

[0084] Dissolve 20 mg / ml of protein in 150 mmol / L NaCl, 10 mmol / L MgCl2 aqueous solution, and adjust the pH to 9.0 with 3% K2CO3;

[0085] (4) React at 4 °C for 2 hours, during which the pH is constantly adjusted to 9.0;

[0086] (5) Add 50 mg / ml NaBH4 solution prepared temporarily, the amount is 1 / 10 of the reaction volume, and react at 4°C overnight;

[0087] (6) Dialysis with phosphate buffer at pH 7.450 mmol / L;

[0088] The phosphate buffer contains 10 mmol / L MgCl2, 10 mmol / L 2-mercaptoethanol and 100 mmol / L NaCl;

[0089] 4. Animal immunization

[0090] The first time, Freund's complete adjuvant was mixed with an equal volume of antigen and injected into the mouse for the main injection. After that, Freund's incomplete adjuvant was mixed with an equal volume of antigen and injected multiple times for booster injection. The injection site was multiple injections at the back to immunize the animal.

[0091] Immunization dose and immunization cycle

[0092] The main injection was 100ug antigen / mouse, and the booster injection was 50ug antigen / mouse.

[0093] operate date dose Adjuvant Main injection 2022.11.30 100ug / piece Freund's complete adjuvant First reinforcement 2022.12.7 50ug / pc Freund's incomplete adjuvant Second reinforcement 2022.12.22 50ug / pc Freund's incomplete adjuvant The third reinforcement 2023.1.4 50ug / pc Freund's incomplete adjuvant Take a serum sample 2023.1.14

[0094] 5. Antiserum titer detection

[0095] After immunization, blood was collected from the tail vein, and the serum titers of five mice (numbered L, R, O, LR, and L2) were detected by indirect ELISA.

[0096] Prepare the strips coated with antigen (100ng / well), add serum 1:500 times starting gradient dilution (500 times, 1000 times, 2000 times, 4000 times...), 100ul / well, incubate at 37° for 1h, wash three times with PBST. Add 100ul diluted goat anti-mouse HRP secondary antibody to each well, incubate at 37° for 1h, wash three times with PBST, develop with TMB, and read the OD value. According to the ELISA results, the dilution ratio corresponding to the OD value ≥0.2 is the mouse titer. The larger the dilution ratio, the better the titer.

[0097] See also Figure 1 and Figure 2 : The experimental results show that the titer of LR mice is better, which is 1:512000.

[0098] 6. Perform cell fusion and subcloning on the selected mice with better serum titers

[0099] (1) Myeloma cell preparation

[0100] One week before fusion, SP2 / 0 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were resuscitated and cultured normally to the logarithmic phase.

[0101] (2) Preparation of spleen cells

[0102] The LR mice to be fused were selected and killed by cervical dislocation on the day of fusion. The spleens were removed and spleen cells were collected and counted according to standard procedures.

[0103] (3) Cell fusion

[0104] Myeloma cells and spleen cells were mixed at a ratio of 1:3-1:10, and cell fusion was performed according to the standard procedure. Then, they were cultured with HATDMEM complete medium. Hybridoma cells were visible 3 days after fusion. On the 7th day, 1 / 2HAT complete medium was replaced, and on the 8th day, 1 / 2HT medium was replaced. Screening tests were started about 10 days after fusion.

[0105] Cell fusion results: After fusion, the cells were cultured in HAT selective medium and observed under a microscope, where multiple growing hybridoma cells were seen, proving that the fusion operation was successful.

[0106] (4) Fusion screening

[0107] Pipette 100ul / well of cell supernatant for indirect ELISA detection. Determine the positive wells based on the ELISA results. Use a single-channel pipette to pick out the positive wells detected on the entire plate and conduct a second re-test to further confirm the positive wells.

[0108] (5) Subcloning

[0109] The rescreened positive well cells were subjected to two rounds of subcloning (because the positive well cell lines obtained in the first subcloning were not yet stable and might contain multiple hybridoma cells, it was generally believed that the hybridoma cells after the second subcloning were a single cell line and were confirmed to be positive).

[0110] For the first subclone, the cells in the limiting dilution positive wells were transferred to multiple wells and cultured with HT DMEM medium. The wells with clone growth were observed under a microscope for about 7 days, and the wells with high OD values ​​were detected by indirect ELISA. The cells in the positive wells were picked for the second subcloning, and the stable positive hybridoma cell lines were detected as the cells for the final preparation of monoclonal antibodies, and the culture was expanded.

[0111] (6) Monoclonal antibody subtype identification

[0112] Use the monoclonal antibody subtype identification kit of Southern Biotech in the United States to measure the subtype of each supernatant. Prepare the strips coated with immunogenic protein, 50ng / well, collect 600ul supernatant from each clone, and add them to 6 enzyme-labeled wells of the corresponding protein, 100ul / well. Incubate at 37° for 1h, wash three times with PBST, add the diluted typing secondary antibodies against IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 to 6 wells, incubate at 37° for 1h, wash three times with PBST, and develop with TMB. The subtype of the secondary antibody corresponding to the well with signal reaction is the subtype of the antibody.

[0113] (7) The final screening results can be found in Figure 3 :

[0114] Figure 3 In the figure, the preliminary positive clones are marked in red.

[0115] 7. Dot Blot identification of positive clones and antibody purification

[0116] Dot Blot identified positive clones: 1A8, 6E9, 1A9

[0117] Positive clone antibody subtype and purification results:

[0118]

[0119]

[0120] Through the above method, three monoclonal antibodies that can specifically recognize methyl pseudouracil were obtained. Currently, there are no similar products on the market.

[0121] 8. Perform light chain sequencing and heavy chain sequencing on the prepared methyl pseudouracil monoclonal antibody. The specific sequencing method is as follows:

[0122] (1) Cultivation of hybridoma cells

[0123] After the hybridoma cell line was revived, it was cultured and when the cell number expanded to about 1×107, the cells were collected by centrifugation at 1000 rpm x 5 min;

[0124] (2) Extraction of cellular RNA

[0125] Under the clean bench environment, add 1ml Trizol reagent to the centrifuged cells, let stand for 5min, add 2mL of chloroform, shake vigorously for 15sec, let stand at room temperature for 3min, 12000rpm x 15min, transfer the upper water layer to a new EP tube, add 0.5mL of isopropanol, let stand at room temperature for 10min. 12000rpm x 10min. Discard the supernatant, add 1mL of 75% ethanol, 7500rpmx5 min, dry the precipitate, and add 50μL of double distilled water. Identify the purity and quantify by agarose electrophoresis, and store at -70℃ for future use;

[0126] (3) Reverse transcription to prepare cDNA

[0127] Total cell RNA 1 μL, RNase Free ddH2O 6 μL, oligodT Primer 0.5 μL, PRIME ScriptRT Enzyme Mix I 0.5 μL, 5x Prime Script Buffer 2 μL, mix well, 37°C for 15 min, 85°C for 5 s;

[0128] (4) Amplification of cDNA

[0129] Use mouse IgG VH VL primer library to amplify the above cDNAs respectively. 5x PrimeStarBuffer 10μL, dNTP 4μL, cDNA 1μL, upstream primer 1μL, downstream primer 1μL, PrimeSTAR 0.5μL, add water to 50μL. Perform PCR reaction according to the following reaction conditions: incubate at 94℃ for 5min, denature at 94℃ for 45s, anneal at 63℃ for 45s, extend at 72℃ for 1min, and extend at 72℃ for 10min after 30 cycles;

[0130] (5) Agarose gel electrophoresis and gel recovery

[0131] The above PCR products were subjected to agarose gel electrophoresis, the electrophoresis results were observed, and the amplified products with a molecular weight of 250-350 bp were sent for sequencing.

[0132] The sequencing results of the light chain sequencing are:

[0133] CTATAGGGATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATGATATCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAGGTCACCATGACCTGCAGTGCCACCTCAAGTGTAAGTTACATGCACTGGTACCAACAGAAGTCAGGCACTTCCCCCAAAAGATGGATTTACGACACA TCCAAGCTGGCTTCTGGAGTCCCTGGTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAATAACCCGCTCACGTTCGGTGCTGGGACCAAGGTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTCCGCTGAGCAT;

[0134] The protein sequence corresponding to the light chain sequencing is as follows:

[0135] MDFQVQIFSFLLISASVMISRGQIVLTQSPAIMSASPGEKVTMTCSATSSVSYMHWYQQ KSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSNNPLTFGAGTKVELKRADAAPTVSIFPPSSPLS;

[0136] Compared with the mouse proteome, the above sequence is the variable region sequence of mouse IgG light chain. Its specific structure is referenced Figure 4 .

[0137] exist Figure 4 middle:

[0138] CDR1:SSVSY

[0139] CDR2:DTS

[0140] CDR3:QQWSNNPLT.

[0141] The sequencing results of the heavy chain sequencing are:

[0142] ;

[0143] The protein sequence corresponding to the heavy chain sequencing is as follows:

[0144] MNFGLRLVFLVLILKSVQCEVHLVESGGGLVQPGEALKLSCESNEYEFPSHDMSWVR KTPEKRLELVAAINSDGGSTDYPDTMERRLIISRDNTKKTLYLQMRSLRSEDTALKYCARPS MVTLVLRCLRRRNHGHRLLSQNDTPICLSIGPWPLSIN.

[0145] Compared with the mouse proteome, the above sequence is the variable region sequence of mouse IgG heavy chain. Its specific structure is referenced Figure 5 .

[0146] exist Figure 5 middle:

[0147] CDR1:EYEFPSHD

[0148] CDR2:INSDGGST

[0149] CDR3:ARPSMVTLVLRC.

[0150] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, material or apparatus including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, material or apparatus.

[0151] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing methylpseudouracil monoclonal antibody based on hybridoma technology, characterized in that: The specific steps include: Step 1. Experimental animal preparation Prepare several 8-week-old healthy female Balb / C mice; Step 2: Antigen synthesis Synthesize methylpseudouracil, pseudouracil, and uracil as antigens, which are then used for immunization and screening; Step 3: Coupling of methylpseudouracil to carrier protein After oxidizing the glycosidic alcohol in methyl pseudouracil with NaIO4, it was coupled with protein; Step 4: Animal immunization The first time, Freund's complete adjuvant was mixed with an equal volume of antigen and injected into the mouse for the main injection. After that, Freund's incomplete adjuvant was mixed with an equal volume of antigen and injected multiple times for animal immunization. Step 5: Antiserum titer detection After immunization, blood was collected from the tail vein, and the serum titer of the mice was tested by indirect ELISA method to screen out mice with better serum titer; Step 6: Perform cell fusion and subcloning on the screened mice with better serum titers; Step 7, Dot Blot identification of positive clones and antibody purification, i.e. obtaining a monoclonal antibody that can specifically recognize methyl pseudouracil; Step 8: Perform light chain sequencing and heavy chain sequencing on the prepared methyl-pseudouracil monoclonal antibody.

2. The method for preparing methylpseudouracil monoclonal antibody based on hybridoma technology according to claim 1, characterized in that: The specific steps of coupling methyl pseudouracil with the carrier protein in step 3 are: (1) 20 mg of methyl pseudouracil was dissolved in 1 ml of 100 m mol / L NaIO4 solution and reacted at 4°C in the dark for 30 minutes; (2) Add 1 drop of ethylene glycol to obtain liquid A; (3) Add solution A to the protein solution; (4) React at 4 °C for 2 hours, during which the pH is constantly adjusted to 9.0; (5) Add 50 mg / ml NaBH4 solution prepared temporarily, the amount is 1 / 10 of the reaction volume, and react at 4°C overnight; (6) Dialyze against 50 mmol / L phosphate buffer.

3. The method for preparing methylpseudouracil monoclonal antibody based on hybridoma technology according to claim 2, characterized in that: The steps for preparing the protein solution in step (3) are: 20 mg / ml of protein was dissolved in 150 mmol / L NaCl, 10 mmol / L MgCl2 aqueous solution, and the pH was adjusted to 9.0 with 3% K2CO3.

4. The method for preparing methylpseudouracil monoclonal antibody based on hybridoma technology according to claim 2, characterized in that: The phosphate buffer in step (6) contains 10 mmol / L of MgCl2, 10 mmol / L of 2-mercaptoethanol and 100 mmol / L of NaCl.

5. The method for preparing methyl-pseudouracil monoclonal antibody based on hybridoma technology according to claim 4, characterized in that: The pH of the phosphate buffer in step (6) is 7.

4.

6. The method for preparing methyl-pseudouracil monoclonal antibody based on hybridoma technology according to claim 1, characterized in that: The injection position described in step 4 is multiple point injection on the back.

7. The method for preparing methyl-pseudouracil monoclonal antibody based on hybridoma technology according to claim 6, characterized in that: In step 4, the main injection amount is 100ug antigen / experimental mouse, and the booster injection amount is 50ug antigen / experimental mouse.

8. The method for preparing methyl-pseudouracil monoclonal antibody based on hybridoma technology according to claim 7, characterized in that: The number of booster injections was three times.

9. The method for preparing methyl-pseudouracil monoclonal antibody based on hybridoma technology according to claim 1, characterized in that: The specific steps of cell fusion and subcloning in step 6 are as follows: (1) Myeloma cell preparation One week before fusion, SP2 / 0 cells were resuscitated and cultured normally to the logarithmic phase; (2) Preparation of spleen cells The mice to be fused were selected by antiserum titer detection, and on the day of fusion, they were killed by cervical dislocation, and spleens were removed. Splenocytes were collected and counted according to standard procedures; (3) Cell fusion Myeloma cells and spleen cells were mixed at a ratio of 1:3-1:10, and cell fusion was performed according to the standard procedure. Then, they were cultured with HATDMEM complete medium. Hybridoma cells were visible 3 days after fusion. On the 7th day, 1 / 2HAT complete medium was replaced, and on the 8th day, 1 / 2HT medium was replaced. Screening tests were started about 10 days after fusion. After fusion, the cells were cultured in HAT selective medium and observed under a microscope, where multiple growing hybridoma cells were observed, proving that the fusion operation was successful. (4) Fusion screening Pipette 100ul / well of cell supernatant for indirect ELISA detection; According to the ELISA results, determine the positive wells; use a single-channel pipette to pick out the positive wells detected on the entire plate, conduct a second re-test, and further confirm the positive wells; (5) Subcloning Perform two rounds of subcloning on the cells in the rescreened positive wells; For the first subcloning, the cells in the limiting dilution positive wells are transferred to multiple wells, and HT DMEM medium is added for culture. After about 7 days, the wells with clone growth are observed under a microscope, and the wells with high OD values ​​are detected by indirect ELISA. The cells in the positive wells are picked for the second subcloning, and the stable positive hybridoma cell lines are detected as the cells for the final preparation of monoclonal antibodies, and the culture is expanded; (6) Monoclonal antibody subtype identification Use the monoclonal antibody subtype identification kit to measure the subtype of each supernatant, prepare the strips coated with immunogenic protein, 50ng / well, collect 600ul supernatant from each clone, add them to 6 enzyme-labeled wells of the corresponding protein, 100ul / well, incubate at 37° for 1h, wash three times with PBST, add the diluted typing secondary antibodies against IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 to 6 wells, incubate at 37° for 1h, wash three times with PBST, and develop with TMB. The subtype of the identified secondary antibody corresponding to the well with signal reaction is the subtype of the antibody; (7) Screen out preliminary positive clones.

10. The method for preparing methyl-pseudouracil monoclonal antibody based on hybridoma technology according to claim 1, characterized in that: The sequencing results of the light chain sequencing are: CTATAGGGATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATGATATCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAGGTCACCATGACCTGCAGTGCCACCTCAAGTGTAAGTTACATGCACTGGTACCAACAGAAGTCAGGCACTTCCCCCAAAAGATGGATTTACGACACA TCCAAGCTGGCTTCTGGAGTCCCTGGTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAATAACCCGCTCACGTTCGGTGCTGGGACCAAGGTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTCCGCTGAGCAT; The protein sequence corresponding to the light chain sequencing is as follows: MDFQVQIFSFLLISASVMISRGQIVLTQSPAIMSASPGEKVTMTCSATSSVSYMHWYQQ KSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSNNPLTFG AGTKVELKRADAAPTVSIFPPSSPLS; The sequencing results of the heavy chain sequencing are: TAGGAGTCAATAGGTTAATACGGGACCACTATAGGGATGAACTTCGGGTTGAGATTGGTTTTCCTTGTCCTTATTTTAAAAAGTGTACAGTGTGAGGTGCACCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGAGGCCCTGAAACTCTCCTGTGAATCCAATGAATACGAATTCCCTTCCCATGACATGTCTTGGGTCCGCAAGACTCCGGAGAAGAGGCTGGAGTTGGTCGCAGCCATTAATAGTGATGGTGGTAGCACCGACTATCCAGACACCATGGAGAGACGACTCATCATCTCCAGAGACAATACCAAGAAGACCCTGTACCTGCAAATGAGGAGTCTGAGGTCTGAGGACACAGCCTTGAAATACTGTGCAAGACCTAGTATGGTTACTTTGGTCCTTCGATGTCTGCGGCGCAGGAACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTTTATCCATTGGTCCCTGGCCGCTGAGCATAAACTAGCAAACTATCTGGAGG; The protein sequence corresponding to the heavy chain sequencing is as follows: MNFGLRLVFLVLILKSVQCEVHLVESGGGLVQPGEALKLSCESNEYEFPSHDMSWVRKTPEKRLELVAAINSDGGSTDYPDTMERRLIISRDNTKKTLYLQMRSLRSEDTALKYCARPSMVTLVLRCLRRRNHGHRLLSQNDTPICLSIGPWPLSIN。

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