Monoclonal antibody against chinese shrimp hemocyanin and its application

Abstract

The application relates to the technical field of shrimp molecular immunology, and in particular to an anti-Chinese shrimp hemocyanin monoclonal antibody and application. The anti-Chinese shrimp hemocyanin monoclonal antibody is anti-SHC-1A1, which can be expressed by recombinant Expi293 cells containing a recombinant plasmid pcDNA3.1 of the anti-Chinese shrimp hemocyanin monoclonal antibody gene. The anti-Chinese shrimp hemocyanin monoclonal antibody has good specificity, can be specifically combined with two subunits with molecular weights of 73kDa and 75kDa of Chinese shrimp hemocyanin, and can be applied as a positive internal reference for detecting the content of the anti-Chinese shrimp hemocyanin monoclonal antibody in animals immunized with Chinese shrimp hemocyanin.

Description

A monoclonal antibody against hemocyanin in Chinese shrimp and its application

[0001] This application is a divisional application of the application filed on October 30, 2024, with application number 2024115287370 and invention title "A monoclonal antibody against hemocyanin of Chinese shrimp and its application". Technical Field

[0002] This application relates to the technical field of shrimp molecular immunology, and in particular to a monoclonal antibody against hemocyanin in Chinese shrimp and its application. Background Technology

[0003] The Chinese shrimp is an important marine aquaculture economic animal in my country. Since the outbreak of whitespot syndrome virus (WSSV) in 1993, this disease has been a serious constraint on the development of my country's shrimp farming industry. Therefore, research on the immune mechanism of the Chinese shrimp has become particularly urgent and necessary.

[0004] Shrimp hemocyanin (SHC) is an important component of the hemolymph of crustaceans, possessing a variety of physiological functions. Besides carrying oxygen, hemocyanin also exhibits phenoloxidase activity, antibacterial and antiviral activity, metal ion transport, protein storage, osmotic pressure regulation, molting hormone transport, and participation in epidermal fixation. Therefore, research on crustacean hemocyanin has become a hot topic of interest for scholars both domestically and internationally.

[0005] Crustaceans perform various physiological functions by regulating the concentration and properties of hemocyanin. Therefore, using monoclonal antibodies against hemocyanin in Chinese shrimp can conveniently and rapidly detect and monitor the fluctuations in hemocyanin concentration within the shrimp.

[0006] Currently, the monoclonal antibodies against hemocyanin in Chinese shrimp reported in the literature are mainly expressed through hybridoma cells, but hybridoma cell expression has technical problems of unstable expression. Summary of the Invention

[0007] The purpose of this application is to provide a monoclonal antibody against hemocyanin in Chinese shrimp in order to solve the problem of unstable expression in hybridoma cells. This monoclonal antibody against hemocyanin in Chinese shrimp can be stably expressed in mammalian cells Expi293 and has good specificity. It can be used as a positive internal control for the detection of monoclonal antibodies against hemocyanin in Chinese shrimp.

[0008] The technical solution of this application

[0009] In a first aspect, this application provides a monoclonal antibody against hemocyanin in Chinese shrimp, wherein the monoclonal antibody against hemocyanin in Chinese shrimp is anti-SHC-1A1;

[0010] The light chain amino acid sequence of anti-SHC-1A1 is shown in SEQ ID NO.3, and the light chain amino acid sequence of anti-SHC-1A1 contains a light chain variable region VL and a light chain constant region CL; the heavy chain amino acid sequence of anti-SHC-1A1 is shown in SEQ ID NO.4, and the heavy chain amino acid sequence of anti-SHC-1A1 contains a heavy chain variable region VH and a heavy chain constant region CH1.

[0011] The amino acid sequences of the light chain variable region of the aforementioned anti-SHC-1A1 include CDR1 as shown in SEQ ID NO.5, CDR2 as shown in SEQ ID NO.6, and CDR3 as shown in SEQ ID NO.7; the amino acid sequences of the heavy chain variable region of anti-SHC-1A1 include CDR1 as shown in SEQ ID NO.8, CDR2 as shown in SEQ ID NO.9, and CDR3 as shown in SEQ ID NO.10.

[0012] Secondly, this application provides a nucleic acid that encodes a monoclonal antibody against hemocyanin of Chinese shrimp, anti-SHC-1A1. The nucleotide sequence of the light chain amino acid sequence of the nucleic acid encoding anti-SHC-1A1 is shown in SEQ ID NO.1, and the nucleotide sequence of the heavy chain amino acid sequence of the nucleic acid encoding anti-SHC-1A1 is shown in SEQ ID NO.2.

[0013] By adopting the above technical solution, the antigenic determinant of the obtained monoclonal antibody anti-SHC-1A1 against Chinese shrimp hemocyanin is located on two subunits of shrimp hemocyanin with molecular weights of 73kDa and 75kDa, and has good specificity.

[0014] Thirdly, this application provides a hybridoma cell expressing a monoclonal antibody anti-SHC-1A1 against hemocyanin in Chinese shrimp.

[0015] Fourthly, this application provides a recombinant plasmid pcDNA3.1 containing the above-mentioned nucleic acid.

[0016] Fifthly, this application provides a recombinant Escherichia coli DH5α containing the recombinant plasmid pcDNA3.1.

[0017] Sixthly, this application provides a recombinant Expi293 cell containing the recombinant plasmid pcDNA3.1.

[0018] Seventhly, this application provides a monoclonal antibody against Chinese shrimp hemocyanin as a positive internal control for qualitatively determining whether the immunization of animals immunized with Chinese shrimp hemocyanin was successful, or for quantitatively detecting the content of monoclonal antibody against Chinese shrimp hemocyanin in animals immunized with Chinese shrimp hemocyanin.

[0019] Beneficial technical effects of this application

[0020] The monoclonal antibody anti-SHC-1A1 against hemocyanin of Chinese shrimp, as described in this application, can be stably expressed in recombinant Expi293 cells, and the obtained monoclonal antibody against hemocyanin of Chinese shrimp has a purity of ≥90% after purification.

[0021] Furthermore, the monoclonal antibody anti-SHC-1A1 against hemocyanin of Chinese shrimp in this application has excellent specificity. The antigenic determinants that specifically bind to the monoclonal antibody against hemocyanin of Chinese shrimp are located on two subunits of hemocyanin with molecular weights of 73kDa and 75kDa.

[0022] Furthermore, the monoclonal antibody anti-SHC-1A1 against Chinese shrimp hemocyanin of this application can be used as a positive internal control to qualitatively determine whether the immunization of animals immunized with Chinese shrimp hemocyanin was successful, or to quantitatively detect the content of monoclonal antibody against Chinese shrimp hemocyanin in animals immunized with Chinese shrimp hemocyanin. Attached Figure Description

[0023] Figure 1. Electrophoresis diagram of the purity determination of the concentrated anti-SHC-1A1 monoclonal antibody against hemocyanin of Chinese shrimp using SDS-PAGE gel electrophoresis.

[0024] Figure 2. Western blot diagram of the concentrated anti-SHC-1A1 monoclonal antibody against hemocyanin of Chinese shrimp. Detailed Implementation

[0025] The present application will be further described in detail below with reference to specific embodiments and Figures 1-2, but this does not limit the present application.

[0026] In the various embodiments of this application, the PBS buffer used is a diluted solution obtained by diluting 10×PBS buffer (Shanghai Yamei Biomedical Technology Co., Ltd., catalog number: PS110S) 10 times with deionized water;

[0027] The PBST buffer used was a diluted solution obtained by diluting 10×PBS / Tween buffer (Shanghai Yamei Biomedical Technology Co., Ltd., catalog number: PS102S) 10 times with deionized water. Example 1

[0028] A hybridoma cell expressing a monoclonal antibody anti-SHC-1A1 against hemocyanin in Chinese shrimp was obtained by a method comprising the following steps:

[0029] 1. Preparation of antigens

[0030] According to the method described in CN118005773A, hemocyanin of Chinese shrimp was extracted and purified to obtain hemocyanin of Chinese shrimp.

[0031] 2. Immunity in mice

[0032] (1) Dissolve the Chinese shrimp hemocyanin obtained in step 1 in PBS buffer at a concentration of 1 mg / mL to obtain Chinese shrimp hemocyanin solution, which is used as an immunogen;

[0033] (2) Three healthy BALB / c mice weighing 20-25g were selected for immunization. The immunization was divided into four times. The first time was the primary immunization, and the next three times were booster immunizations. Each immunization was two weeks apart, and BALB / c mice that had completed immunization were obtained.

[0034] The BALB / c mice mentioned above were purchased from Southern Model Biotechnology Co., Ltd.

[0035] Primary immunization: BALB / c mice were injected subcutaneously at multiple sites with Chinese shrimp hemocyanin solution, supplemented with complete Freund's adjuvant (Sigma-Aldrich, catalog number: F5881). The volume ratio of Chinese shrimp hemocyanin solution to complete Freund's adjuvant was 1:1, and the mixture was fully emulsified before injection.

[0036] The dosage of Chinese shrimp hemocyanin injected into the above BALB / c mice was 100 μg / mouse;

[0037] Enhanced immunization: BALB / c mice were injected subcutaneously at multiple sites with Chinese shrimp hemocyanin solution, supplemented with incomplete Freund's adjuvant (Sigma-Aldrich, catalog number: F5506). Chinese shrimp hemocyanin and incomplete Freund's adjuvant were mixed at a volume ratio of 1:1, fully emulsified, and then injected.

[0038] The dosage of Chinese shrimp hemocyanin injected into the BALB / c mice was 100 μg / mouse.

[0039] 3. Cell fusion

[0040] (1) The BALB / c mice that have completed the above immunization were euthanized by cervical dislocation. The spleen of the BALB / c mice was removed by aseptic operation, and the adhering adipose tissue and connective tissue were removed.

[0041] (2) After rinsing the removed spleen with DMEM incomplete medium, centrifuge at 2000 r / min for 5 min, discard the supernatant, and then resuspend the spleen cell pellet in DMEM incomplete medium to obtain a cell concentration of 1×10⁻⁶. 8 Spleen cell suspension of 1 cell / mL;

[0042] (3) Take SP2 / 0 cells in the logarithmic growth phase, centrifuge at 2000 r / min for 5 min, discard the supernatant, and then resuspend the SP2 / 0 cell pellet in DMEM incomplete medium to obtain a cell concentration of 1×10⁻⁶. 8 SP2 / 0 cell suspension per mL;

[0043] The SP2 / 0 cells mentioned above were obtained from the Cell Bank of the Chinese Academy of Sciences;

[0044] (4) Dissolve PEG1450 (Sigma-Aldrich, catalog number: P7181) in PBS buffer, and then filter it through a 0.45 μm polyvinylidene fluoride membrane (PVDF membrane) to obtain PEG1450 solution.

[0045] The amounts of PEG1450 and PBS buffer mentioned above are calculated based on a ratio of PEG1450:PBS buffer of 10g:100mL.

[0046] (5) Mix the SP2 / 0 cell suspension and spleen cell suspension obtained above, and control the number of SP2 / 0 cells in the mixture to be 1×10⁻⁶. 7 The number of spleen cells is 1×10. 8 Mix the cells thoroughly and centrifuge at 2000 r / min for 5 min. Discard the supernatant to obtain a mixed cell pellet containing SP2 / 0 cells and spleen cells.

[0047] (6) Add 0.5 mL of PEG1450 solution preheated to 37°C to the mixed cell pellet of SP2 / 0 cells and spleen cells obtained above, mix well and incubate in a cell culture incubator at 37°C and CO2 volume percentage concentration of 5% for 1 min to obtain cell suspension I.

[0048] (7) Add antibiotics to HAT medium and mix well to obtain HAT medium containing antibiotics.

[0049] The antibiotic in question is a mixture of penicillin and streptomycin / double antibiotic (100×) (Shanghai Yamei Biomedical Technology Co., Ltd., catalog number: CB010);

[0050] The working concentration of penicillin in the above-mentioned HAT medium containing antibiotics is 100 U / mL, and the working concentration of streptomycin is 100 μg / mL.

[0051] (8) Add 8-10 mL of HAT medium containing antibiotics preheated to 37°C to the cell suspension I obtained above, mix well, and obtain cell suspension II;

[0052] (9) Add the cell suspension II obtained above to a culture dish, and then place the culture dish containing cell suspension II in a cell culture incubator at 37°C and CO2 volume percentage concentration of 5% for 5 days. Observe the cell growth in the culture dish regularly until a cell cluster with a diameter of 100 μm is obtained, and a fusion cell clone is obtained.

[0053] (10) The fusion cell clones obtained in (9) were expanded stepwise using DMEM complete medium. The supernatant of the hybridoma cells was used for subsequent ELISA experiments (the monoclonal antibody against Chinese shrimp hemocyanin is an extracellular product of hybridoma cells).

[0054] 4. Identification of monoclonal antibodies using ELISA.

[0055] (1) Preparation of coating solution: Dissolve the Chinese shrimp hemocyanin obtained in the preparation of antigen in step 1 above in PBS buffer to prepare a coating solution with a concentration of 5 μg / mL.

[0056] (2) Coating: Add 50 μL of coating solution to each well of the 96-well plate and coat at 4°C for 16 h;

[0057] (3) Washing: Discard the liquid in the wells, wash with PBST buffer (200 μL / well), gently shake the plate, discard the liquid in the wells, and repeat the PBST buffer washing 2-4 times to remove unbound Chinese shrimp hemocyanin.

[0058] (4) Blocking: Add 100 μL of blocking buffer to each well and incubate at room temperature (25°C) for 1-2 hours to prevent nonspecific binding;

[0059] The above blocking buffer is bovine serum albumin (Shanghai Yamei Biomedical Technology Co., Ltd., catalog number: PS113) dissolved in PBS buffer, and the mass-volume ratio of bovine serum albumin to PBS buffer is 5g:100mL;

[0060] (5) Washing: Discard the liquid in the wells, wash with PBST buffer (200 μL / well), gently shake the plate, discard the liquid in the wells, and repeat the washing with PBST buffer 2-4 times to remove the blocking solution and obtain the coated 96-well plate.

[0061] (6) Primary antibody incubation: The supernatant of hybridoma cells was added to the coated 96-well plate as the primary antibody solution. The amount of primary antibody solution was 200 μL / well. The serum of mice immunized with Chinese shrimp hemocyanin was used as the positive control, and the serum of mice not immunized with Chinese shrimp hemocyanin was used as the negative control. Two parallel samples were set up for the primary antibody solution, positive control and negative control respectively. The samples were incubated at 37°C for 1 h.

[0062] (7) Washing: Discard the liquid in the wells, wash with PBST buffer (200 μL / well), gently shake the plate, pour out the liquid, and repeat the washing 2-4 times to remove unbound primary antibody.

[0063] (8) Secondary antibody incubation: Add the secondary antibody solution, the amount of the secondary antibody solution is 200 μL / well, and incubate at room temperature (25℃) for 1 h;

[0064] The second antibody solution is prepared by mixing horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Shanghai Yamei Biomedical Technology Co., Ltd., catalog number: LF101) and PBS buffer at a volume ratio of 1:1000.

[0065] (9) Washing: Discard the liquid in the wells, wash with PBST buffer (200 μL / well), gently shake the plate, discard the liquid in the wells, and repeat the washing 2-4 times to remove unbound secondary antibodies.

[0066] (10) Color development: Add substrate 3,3',5,5'-tetramethylbenzidine (TMB) to the wells, with a TMB volume of 50 μL / well, and incubate at room temperature (25°C) for 15 min to allow it to react with HRP on the second antibody;

[0067] (11) Termination: Add 200 μL of 0.1 mol / L hydrochloric acid aqueous solution to each well to terminate the reaction and obtain the termination reaction solution;

[0068] (12) Determination of OD 450 Value: The OD value of the termination reaction solution in each well was measured at a working wavelength of 450 nm after the reaction was terminated. 450 Calculate the average OD values ​​for the first antibody solution, positive control, and negative control in two parallel samples. 450 value;

[0069] Evaluation criteria: Quality control passed (average OD of negative control) 450 Value <0.2, average OD of positive control 450 After the value > 1.5, select the positive wells (average OD). 450Samples with a value > 1.5 were used for single-cell clone screening of hybridomas.

[0070] 5. Single-cell clone screening of hybridomas

[0071] (1) Preparation of culture medium for single-cell clone screening of hybridoma: Fetal bovine serum (FBS) was added to RPMI 1640 medium to obtain serum-containing RPMI 1640 medium;

[0072] The above-mentioned amount of FBS added, the volume ratio of FBS to RPMI 1640 medium is 10:90, where FBS can be replaced by other serum supplements;

[0073] (2) Diluting the sample: Select the positive well sample from (12) in step 4 above, which uses ELISA to identify monoclonal antibodies, and perform limited dilution to obtain monoclonal cells; calculate the expected number of cells in each positive well based on the concentration and volume of the positive well sample, and use PBS buffer to dilute the positive well sample to the required number of cells.

[0074] For example, the concentration of hybridoma cells in the sample is 1×10⁻⁶. 6 The sample was taken from the hybridoma cells and 0.1 mL was added to 0.9 mL of PBS buffer to obtain dilution I, making the total volume of dilution I 1 mL. Then, 0.1 mL of dilution I was taken and added to 0.9 mL of PBS buffer to obtain dilution II. This process was repeated to obtain the final dilution. The final 1 mL of the final dilution contained 10 cells.

[0075] (3) Add 0.9 mL of serum-containing RPMI 1640 medium to each well of the new plate, and then add 0.1 mL of the final dilution obtained in (2) above to each well and mix by pipetting.

[0076] (4) Cell culture: Place the new well plate in a cell culture incubator at 37°C and a CO2 volume percentage concentration of 5% for culture;

[0077] (5) When the cell coverage is above 60%, the monoclonal antibody is identified by ELISA as described in step 4 above.

[0078] A hybridoma monoclonal cell line with high specificity against hemocyanin in Chinese shrimp was obtained using the above methods. Example 2

[0079] The gene sequence of a monoclonal antibody against hemocyanin in Chinese shrimp, anti-SHC-1A1, was obtained by a method comprising the following steps:

[0080] 1. Extraction of total RNA

[0081] Total RNA was extracted from the hybridoma monoclonal cells obtained above using a total RNA extraction reagent (Shanghai Yamei Biomedical Technology Co., Ltd., catalog number: YY101L).

[0082] 2. 5' RACE amplification

[0083] Total RNA was amplified by 5' RACE using the SMARTer RACE 5' / 3' Kit (TAKARA, catalog number: 634859) to obtain a solution containing DNA.

[0084] 3. Gel electrophoresis and purification

[0085] The DNA-containing solution obtained above was analyzed by agarose gel electrophoresis, and the target fragment was obtained by gel cutting. Then, the target fragment was purified using a DNA recovery and purification kit (Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP214) to obtain the purified antibody fragment corresponding to the DNA.

[0086] 4. Cloning and sequencing

[0087] The purified antibody fragments were ligated into the pMD19-T vector (GenScript Biotech Co., Ltd.) using a vector construction method (the vector construction method is a common vector construction method in the existing technology), and then transformed into Escherichia coli DH5α (Shanghai Weidi Biotechnology Co., Ltd.). Positive clones of the corresponding antibody fragments were obtained by blue-white screening method.

[0088] Positive clones corresponding to the antibody fragments were selected and sequenced using the Sanger method (completed by Shanghai Qingke Biotechnology Co., Ltd.) to obtain the antibody genes corresponding to the antibody fragments;

[0089] The nucleotide sequence encoding the light chain of the antibody gene is shown in SEQ ID NO.1, and the nucleotide sequence of the light chain of the antibody gene contains a light chain variable region VL and a light chain constant region CL; the nucleotide sequence encoding the heavy chain of the antibody gene is shown in SEQ ID NO.2, and the nucleotide sequence of the heavy chain of the antibody gene contains a heavy chain variable region VH and a heavy chain constant region CH1; the amino acid sequence corresponding to SEQ ID NO.1 is shown in SEQ ID NO.3, and the amino acid sequence corresponding to SEQ ID NO.2 is shown in SEQ ID NO.4;

[0090] Analysis revealed that the amino acid sequences of the light chain variable region of the antibody gene, CDR1 as shown in SEQ ID NO.5, CDR2 as shown in SEQ ID NO.6, and CDR3 as shown in SEQ ID NO.7, and the amino acid sequences of the heavy chain variable region of the antibody gene, CDR1 as shown in SEQ ID NO.8, CDR2 as shown in SEQ ID NO.9, and CDR3 as shown in SEQ ID NO.10. Example 3

[0091] A recombinant plasmid pcDNA3.1 containing the gene for the monoclonal antibody anti-SHC-1A1, which targets hemocyanin in Chinese shrimp, was obtained using the following technical method:

[0092] 1. The vector plasmid pcDNA3.1 was double-digested with BamHI and EcoR1 to obtain the linearized plasmid pcDNA3.1;

[0093] The aforementioned vector plasmid pcDNA3.1 was derived from Genscript Biotech Inc.

[0094] The enzyme digestion system is shown in Table 1 below:

[0095] Table 1 Enzyme digestion system

[0096] Enzyme digestion system components (volume / mass): Vector, plasmid, pcDNA 3.11 μg, BamHI (10 U / μL) 1 μL, EcoR1 (10 U / μL) 1 μL, 10× buffer 1 μL, H2O to 10 μL surface

[0097] React at 37℃ for 30 minutes.

[0098] 2. Perform PCR amplification on the antibody fragment.

[0099] The antibody fragment was amplified by PCR to obtain a PCR-amplified antibody fragment solution.

[0100] The specific components and amounts of the PCR amplification system are shown in Table 2 below:

[0101] Table 2 PCR amplification system

[0102] PCR amplification system components (volume / mass): Antibody fragment 1 ng, Forward primer (10 μM) 2 μL, Reverse primer (10 μM) 2 μL, 2×Phanta Max Buffer 25 μL, dNTP Mix (10 mM each) 1 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL, ddH2O to 50 μL. surface

[0103] The above-mentioned 2×Phanta Max Buffer, dNTP Mix (10mM each), Phanta Max Super-Fidelity DNA Polymerase and ddH2O are all from Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novizan Biotechnology Co., Ltd., catalog number: P505-d1).

[0104] The upstream primer sequence mentioned above is the 20bp sequence of the 5' end of the AGCTCGGATCC+ antibody fragment;

[0105] The downstream primer sequence described above is the 20bp sequence of the 3' of the GAATTCGG+ antibody fragment;

[0106] The PCR amplification system was programmed as follows: pre-denaturation (95℃, 5 min), cyclic amplification [30 cycles in total, each cycle including denaturation (95℃, 30 s), annealing (65℃, 30 s), extension (72℃, 10 s)] and final extension (72℃, 5 min).

[0107] 3. Connection reaction

[0108] Using T4 DNA ligase, the antibody fragment amplified by PCR was ligated into the linearized plasmid pcDNA3.1 to obtain the corresponding solution containing the recombinant plasmid pcDNA3.1;

[0109] The composition and amount of reactants and related reagents used in the above connection process are shown in Table 3 below:

[0110] Table 3 Connection Reaction System

[0111] Reactants and related reagents, in equal volumes / masses: T4 DNA ligase (10 U / μL) 1 μL; PCR-amplified antibody fragment 200 ng; linearized plasmid pcDNA 3.1; 1500 ng; 10×T4 DNA ligase reaction buffer 2 μL; H2O to bring the total to 20 μL. surface

[0112] Mix the reactants and related reagents used in the above connection process thoroughly and react at 16°C for 12 hours.

[0113] 4. Transformation of recombinant plasmids

[0114] Take 10 μL of the solution containing recombinant plasmid pcDNA3.1 and add it to 50 μL of competent Escherichia coli DH5α (Shanghai Weidi Biotechnology Co., Ltd.). Mix well, incubate on ice for 30 min, then transfer to a 42℃ environment for 90 s, then incubate on ice for 2 min, then transfer to 5 mL of LB liquid medium and incubate at 37℃ for 1 h. Then take a sample and spread it evenly on a pre-prepared LB agar plate containing 10 μg / mL ampicillin antibiotic, and incubate upside down at 37℃ for 16 h to obtain recombinant Escherichia coli DH5α colonies with the corresponding recombinant plasmid pcDNA3.1.

[0115] A single colony of Escherichia coli DH5α containing the recombinant plasmid pcDNA3.1 was picked and inoculated into 5 mL of LB liquid medium for fermentation for 16 h to obtain the fermentation broth of recombinant Escherichia coli DH5α containing the recombinant plasmid pcDNA3.1.

[0116] The results, obtained using Sanger sequencing (performed by Shanghai Qingke Biotechnology Co., Ltd.), confirmed the successful recombinant Escherichia coli DH5α.

[0117] 5. Recombinant plasmid extraction

[0118] The recombinant plasmid pcDNA3.1 was obtained by extracting the recombinant Escherichia coli DH5α fermentation broth obtained above using a plasmid extraction kit (Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP103).

[0119] Verification was performed using Sanger sequencing (conducted by Shanghai Qingke Biotechnology Co., Ltd.), and the results showed that the recombinant plasmid pcDNA3.1 was successfully obtained. Example 4

[0120] A recombinant Expi293 cell containing the recombinant plasmid pcDNA3.1 was obtained using the following technical method:

[0121] 1. Expi293 cell transfection

[0122] PEI transfection reagent (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: 5644123) and recombinant plasmid pcDNA3.1 were mixed at a ratio of 0.1 mL: 10 μg to obtain a mixture of transfection reagent and recombinant plasmid pcDNA3.1. The mixture was then incubated at 30°C for 15 min.

[0123] The above-obtained mixture of transfection reagent / recombinant plasmid pcDNA3.1 was transfected into Expi293 cells at a ratio of 10 μg recombinant plasmid to 10 μg of recombinant plasmid. 6The proportion of each Expi293 cell was used to obtain recombinant Expi293 cells corresponding to the recombinant plasmid pcDNA3.1.

[0124] 2. Recombinant Expi293 cell culture

[0125] After culturing recombinant Expi293 cells in a cell culture incubator at 37°C and 5% CO2 for 1 week, a culture medium containing a monoclonal antibody against hemocyanin of Chinese shrimp was obtained.

[0126] Centrifuge the above-mentioned recombinant Expi293 cell culture medium and collect the supernatant corresponding to the recombinant Expi293 cell culture medium containing monoclonal antibodies against hemocyanin of Chinese shrimp.

[0127] The Expi293 cells mentioned above were obtained from Shanghai Duoning Biotechnology Co., Ltd.

[0128] 3. Antibody purification

[0129] Protein A affinity chromatography resin (Suzhou Nanomicro Technology Co., Ltd., product number: 17013-090100) is packed into a chromatography column to form a Protein A affinity chromatography column;

[0130] The Protein A affinity chromatography column was equilibrated using 3 column volumes of PBS buffer.

[0131] The supernatant of 1 L of recombinant Expi293 cell culture medium containing monoclonal antibodies against hemocyanin of Chinese shrimp was loaded into a Protein A affinity chromatography column that had been equilibrated with PBS buffer, and the flow rate was controlled at 2 mL / min.

[0132] After the above sample addition is completed, non-specifically bound proteins and impurities are first washed away with PBS buffer to retain the monoclonal antibody against Chinese shrimp hemocyanin bound to Protein A affinity chromatography resin.

[0133] The monoclonal antibody against Chinese shrimp hemocyanin bound to Protein A affinity chromatography resin was then eluted using 0.5 mM glycine elution buffer at pH 3.5, and the pH was adjusted to 7.2 using 0.1 mM Tris-HCl aqueous solution at pH 10.

[0134] Then, ultrafiltration concentration was performed to obtain a concentrated solution of the corresponding monoclonal antibody against hemocyanin of Chinese shrimp.

[0135] The aforementioned monoclonal antibody against hemocyanin in Chinese shrimp is named anti-SHC-1A1.

[0136] 4. SDS-PAGE gel electrophoresis

[0137] The purity of the concentrated anti-SHC-1A1 solution was determined by SDS-PAGE gel electrophoresis. The resulting electrophoresis pattern is shown in Figure 1. Gray-scale analysis showed that the purity of the monoclonal antibody anti-SHC-1A1 in the concentrated solution was ≥90%.

[0138] 5. Western blot

[0139] The concentrated anti-SHC-1A1 monoclonal antibody was analyzed by Western blot, and the specific steps are as follows:

[0140] (1) Dilute Chinese shrimp hemocyanin with WB loading buffer to a concentration of 1 mg / mL and boil at 95℃ for 5 min to obtain pretreated Chinese shrimp hemocyanin sample;

[0141] The above-mentioned WB loading buffer consisted of 100 mM DTT, 1% SDS, 20% glycerol, and 1% methylene blue.

[0142] (2) Add the pretreated Chinese shrimp hemocyanin sample to the sample well of the precast gel, add 100 ng of the pretreated Chinese shrimp hemocyanin sample to each well, and electrophores for 1 h under constant voltage of 120 V, and then take out the gel.

[0143] (3) Cut a piece of nitrocellulose membrane the same size as the gel, and soak the nitrocellulose membrane and filter paper in rapid transfer buffer (Shanghai Yamei Biotechnology Co., Ltd., product number: PS117). Then, place it in a wet transfer apparatus in a "sandwich" manner, in the following order: electrode (-) - sponge - filter paper - gel - nitrocellulose membrane - filter paper - sponge - electrode (+), constant current 300mA, energized for 1h;

[0144] The nitrocellulose membrane described above has a pore size of 0.22 μm;

[0145] (4) After the transfer is complete, take out the nitrocellulose membrane, wash it once with water, and then place it in PBST buffer containing 5% skim milk powder and block it at 30°C for 10 min to prevent non-specific binding.

[0146] The above-mentioned PBST buffer containing 5% skim milk powder is prepared by using skim milk powder and PBST buffer in a mass-volume ratio of 5g:100mL.

[0147] (5) Wash the nitrocellulose membrane three times with PBST buffer, 5 min each time;

[0148] (6) The anti-SHC-1A1 obtained above was added as a specific primary antibody to PBST buffer containing 5% skim milk powder to obtain a primary antibody solution with a concentration of 1 μg / mL. The nitrocellulose membrane was placed in the primary antibody solution and incubated at room temperature (25℃) for 1 h.

[0149] (7) Wash the nitrocellulose membrane three times with PBST buffer, 5 min each time;

[0150] (8) Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Shanghai Yamei Biomedical Technology Co., Ltd., catalog number: LF101) was added as a secondary antibody to PBST buffer containing 5% skim milk powder to obtain a secondary antibody solution with a concentration of 1 μg / mL, and incubated at room temperature (25℃) for 1 h.

[0151] (9) Wash the nitrocellulose membrane three times with PBST buffer for 5 minutes each time to remove non-specifically bound secondary antibodies and other impurities;

[0152] (10) Use ECL staining solution to develop the final band until the color is clear. The resulting blot is shown in Figure 2. As can be seen from Figure 2, the antigenic determinant of the monoclonal antibody anti-SHC-1A1 against Chinese shrimp hemocyanin is located on the two subunits of shrimp hemocyanin with molecular weights of 73kDa and 75kDa.

[0153] 6. Sequencing verification

[0154] The obtained monoclonal antibody against hemocyanin of Chinese shrimp, anti-SHC-1A1, was verified by amino acid sequence verification. The results showed that the light chain amino acid sequence of anti-SHC-1A1 (light chain variable region VL + light chain constant region CL) was consistent with SEQ ID NO.3, and the heavy chain amino acid sequence of anti-SHC-1A1 (heavy chain variable region VH + heavy chain constant region CH1) was consistent with SEQ ID NO.4.

[0155] Application Examples

[0156] Application Example 1

[0157] A monoclonal antibody against hemocyanin in Chinese shrimp was used as a positive internal control in the detection of hemocyanin content in Chinese shrimp, and was obtained using the following technical solution:

[0158] Following the ELISA method in step 4 of Example 1, the concentrated anti-SHC-1A1 monoclonal antibody against Chinese shrimp hemocyanin obtained in Example 4 was used as the first antibody solution in step 4(6) of Example 1. Simultaneously, serum from mice immunized four times with Chinese shrimp hemocyanin in step 2 of Example 1 was used as a control for the first antibody solution. All other experimental procedures were the same as in step 4 of Example 1. The final average OD... 450 The values ​​are shown in Table 4 below:

[0159] Table 4 Detection data from application examples

[0160] The average OD of the first antibody sample 450 The serum concentration of anti-SHC-1A1 concentrate was 2.14 g / mL; the serum concentration of mice immunized four times with Chinese shrimp hemocyanin was 2.09 g / mL; and the negative control concentration was 0.14 g / mL. surface

[0161] Analysis of the data in the table above shows that the serum of mice immunized with Chinese shrimp hemocyanin four times could detect a positive signal (average OD). 450 The value was 2.09), and a significant positive signal (mean OD) could also be detected in the concentrated anti-SHC-1A1 monoclonal antibody against hemocyanin in Chinese shrimp (mean OD value was 2.09). 450 The value was 2.14, indicating that the monoclonal antibody anti-SHC-1A1 against Chinese shrimp hemocyanin can be used as a positive internal control for quantitative detection of the antibody content against Chinese shrimp hemocyanin in animals immunized with Chinese shrimp hemocyanin or for qualitative judgment of whether the immunization of animals with Chinese shrimp hemocyanin was successful.

[0162] In summary, the monoclonal antibody anti-SHC-1A1 against hemocyanin of Chinese shrimp proposed in this application can be stably expressed in recombinant Expi293 cells. The resulting monoclonal antibody anti-SHC-1A1 can specifically bind to two subunits of hemocyanin of Chinese shrimp with molecular weights of 73kDa and 75kDa, and can be used as a positive internal control for detecting the content of monoclonal antibody against hemocyanin of Chinese shrimp.

[0163] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A monoclonal antibody against hemocyanin in Chinese shrimp, characterized in that, The monoclonal antibody against hemocyanin in Chinese shrimp is anti-SHC-1A1; the light chain amino acid sequence of anti-SHC-1A1 is shown in SEQ ID NO.3, and the light chain amino acid sequence of anti-SHC-1A1 contains a light chain variable region VL and a light chain constant region CL; the heavy chain amino acid sequence of anti-SHC-1A1 is shown in SEQ ID NO.4, and the heavy chain amino acid sequence of anti-SHC-1A1 contains a heavy chain variable region VH and a heavy chain constant region CH1.

2. A nucleic acid, characterized in that, The nucleic acid encodes the monoclonal antibody anti-SHC-1A1 against hemocyanin of Chinese shrimp as described in claim 1. The nucleotide sequence of the light chain amino acid sequence of the nucleic acid encoding anti-SHC-1A1 is shown in SEQ ID NO.1, and the nucleotide sequence of the heavy chain amino acid sequence of the nucleic acid encoding anti-SHC-1A1 is shown in SEQ ID NO.

2. 3.

1. A recombinant plasmid pcDNA containing the nucleic acid as described in claim 2.

4. A recombinant Escherichia coli DH5α containing the recombinant plasmid pcDNA3.1 as described in claim 3.

5. A recombinant Expi293 cell containing the recombinant plasmid pcDNA3.1 as described in claim 3.

6. The monoclonal antibody against Chinese shrimp hemocyanin as described in claim 1 can be used as a positive internal control to qualitatively determine whether the immunization of animals immunized with Chinese shrimp hemocyanin was successful, or to quantitatively detect the content of monoclonal antibody against Chinese shrimp hemocyanin in animals immunized with Chinese shrimp hemocyanin.

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