Monoclonal antibody for resisting haemocyanin of Chinese prawns and application of monoclonal antibody

Abstract

The invention relates to the technical field of molecular immunology of prawns, in particular to a monoclonal antibody for resisting haemocyanin of Chinese prawns and application, the monoclonal antibody for resisting haemocyanin of Chinese prawns is anti-SHC-5C8, and the monoclonal antibody for resisting haemocyanin of Chinese prawns can be expressed through recombinant Expi293 cells of recombinant plasmids pcDNA3.1 containing genes of the monoclonal antibody for resisting haemocyanin of Chinese prawns; the monoclonal antibody for resisting the fenneropenaeus chinensis haemocyanin is good in specificity, can be specifically combined to two subunits with the molecular weights of 73kDa and 75kDa of the fenneropenaeus chinensis haemocyanin, and can be used as a positive internal reference for detecting the content of the monoclonal antibody for resisting the fenneropenaeus chinensis haemocyanin in animals immunized by the fenneropenaeus chinensis haemocyanin.

Description

[0001] This application is a divisional application of the application with the application date of October 30, 2024, application number 2024115287370, and invention name "A Monoclonal Antibody Against Shrimp Hemocyanin 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 shrimp hemocyanin and its application. Background Art

[0003] Fenneropenaeus chinensis is an important marine aquaculture economic animal in China. Since the outbreak of white spot syndrome virus (WSSV) in 1993, this disease has been a serious restricting factor for the development of the shrimp aquaculture industry in China. Therefore, it has become particularly urgent and necessary to study the immune mechanism of Fenneropenaeus chinensis.

[0004] Shrimp Hemocyanin (SHC) is an important component of the hemolymph of crustaceans and has various physiological functions. In addition to carrying oxygen, hemocyanin also has phenoloxidase activity, antibacterial and antiviral activities, metal ion transport, protein storage, osmotic pressure regulation, transport of ecdysteroids, and participation in the solidification of the cuticle. Therefore, the research on crustacean hemocyanin has become a hot field of concern for scholars at home and abroad.

[0005] Crustaceans perform various physiological functions by regulating the concentration and properties of hemocyanin. Therefore, the use of monoclonal antibodies against shrimp hemocyanin can conveniently and quickly detect and monitor the fluctuation law of hemocyanin concentration in shrimp.

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

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

[0008] Technical Solution of this Application

[0009] In the first aspect, this application provides a monoclonal antibody against shrimp hemocyanin, and the monoclonal antibody against shrimp hemocyanin is anti-SHC-5C8;

[0010] The light chain amino acid sequence of the anti-SHC-5C8 is as shown in SEQ ID NO.3, and the light chain amino acid sequence of the anti-SHC-5C8 contains a light chain variable region VL and a light chain constant region CL; the heavy chain amino acid sequence of the anti-SHC-5C8 is as shown in SEQ ID NO.4, and the heavy chain amino acid sequence of the anti-SHC-5C8 contains a heavy chain variable region VH and a heavy chain constant region CH1;

[0011] CDR1 in the amino acid sequence of the light chain variable region of the above anti-SHC-5C8 is as shown in SEQ ID NO.5, CDR2 is as shown in SEQ ID NO.6, and CDR3 is as shown in SEQ ID NO.7; CDR1 in the amino acid sequence of the heavy chain variable region of the anti-SHC-5C8 is as shown in SEQ ID NO.8, CDR2 is as shown in SEQ ID NO.9, and CDR3 is as shown in SEQ ID NO.10.

[0012] In a second aspect, the present application provides a nucleic acid that encodes a monoclonal antibody anti-SHC-5C8 against the hemocyanin of Fenneropenaeus chinensis. The nucleotide sequence of the nucleic acid encoding the light chain amino acid sequence of anti-SHC-5C8 is as shown in SEQ ID NO.1, and the nucleotide sequence of the nucleic acid encoding the heavy chain amino acid sequence of anti-SHC-5C8 is as shown in SEQ ID NO.2.

[0013] By adopting the above technical solution, the antigenic determinants of the monoclonal antibody anti-SHC-5C8 against the hemocyanin of Fenneropenaeus chinensis are located on two subunits of the hemocyanin of Fenneropenaeus chinensis with molecular weights of 73 kDa and 75 kDa, and have good specificity.

[0014] In a third aspect, the present application provides a hybridoma cell that expresses a monoclonal antibody anti-SHC-5C8 against the hemocyanin of Fenneropenaeus chinensis.

[0015] In a fourth aspect, the present application provides a recombinant plasmid pcDNA3.1 containing the encoding nucleic acid as described above.

[0016] In a fifth aspect, the present application provides a recombinant Escherichia coli DH5α containing the recombinant plasmid pcDNA3.1.

[0017] In a sixth aspect, the present application provides a recombinant Expi293 cell containing the recombinant plasmid pcDNA3.1.

[0018] In a seventh aspect, the present application provides a monoclonal antibody against shrimp hemocyanin as a positive internal reference for qualitatively determining whether an animal immunized with shrimp hemocyanin is successfully immunized or for quantitatively detecting the content of the monoclonal antibody against shrimp hemocyanin in an animal immunized with shrimp hemocyanin.

[0019] Advantageous technical effects of the present application

[0020] A monoclonal antibody anti-SHC-5C8 against shrimp hemocyanin of the present application can be stably expressed by recombinant Expi293 cells, and the obtained monoclonal antibody against shrimp hemocyanin has a purity of ≥90% after purification.

[0021] Furthermore, a monoclonal antibody anti-SHC-5C8 against shrimp hemocyanin of the present application has good specificity, and the antigenic determinants that specifically bind to the monoclonal antibody against shrimp hemocyanin are located on two subunits of shrimp hemocyanin with molecular weights of 73 kDa and 75 kDa.

[0022] Furthermore, a monoclonal antibody anti-SHC-5C8 against shrimp hemocyanin of the present application can be used as a positive internal reference for qualitatively determining whether an animal immunized with shrimp hemocyanin is successfully immunized or for quantitatively detecting the content of the monoclonal antibody against shrimp hemocyanin in an animal immunized with shrimp hemocyanin. Description of the drawings

[0023] Figure 1 Electrophoretogram of determining the purity of the concentrated solution of the monoclonal antibody anti-SHC-5C8 against shrimp hemocyanin by SDS-PAGE gel electrophoresis;

[0024] Figure 2 Blotting diagram of detecting the concentrated solution of the monoclonal antibody anti-SHC-5C8 against shrimp hemocyanin by Western blot. Detailed implementation manners

[0025] The following further elaborates on the present application through specific examples in combination with the attached Figure 1-2 and does not limit the present application.

[0026] In each example of the present application, the used PBS buffer solution is a dilution obtained by diluting a 10×PBS buffer solution (Shanghai Yaen Biopharmaceutical Technology Co., Ltd., product number: PS110S) 10 times with deionized water;

[0027] The PBST buffer used was a dilution obtained by diluting a 10×PBS / Tween buffer (Shanghai Yaen Biopharmaceutical Technology Co., Ltd., product number: PS102S) 10-fold with deionized water.

[0028] Example

[0029] Example 1

[0030] A hybridoma cell expressing the monoclonal antibody anti-SHC-5C8 against the hemocyanin of Chinese shrimp was obtained by a method comprising the following steps:

[0031] 1. Preparation of antigen

[0032] According to the method described in CN118005773A, the hemocyanin of Chinese shrimp was extracted and purified to obtain the hemocyanin of Chinese shrimp.

[0033] 2. Immunization of mice

[0034] (1). The hemocyanin of Chinese shrimp obtained in step 1 was dissolved in PBS buffer at a concentration of 1 mg / mL to obtain a hemocyanin solution of Chinese shrimp as the immunogen;

[0035] (2). Three healthy BALB / c mice weighing 20 - 25 g were selected for immunization. The immunization was divided into four times. The first time was the primary immunization, and the last three times were booster immunizations. The interval between each immunization was two weeks, and BALB / c mice with completed immunization were obtained;

[0036] The above-mentioned BALB / c mice were purchased from Southern Model Organisms Co., Ltd.;

[0037] Primary immunization: The BALB / c mice were subcutaneously injected with the hemocyanin solution of Chinese shrimp at multiple points, supplemented with complete Freund's adjuvant (Sigma-Aldrich, product number: F5881). The dosage of the hemocyanin solution of Chinese shrimp and complete Freund's adjuvant was in a volume ratio of 1:1, and they were fully emulsified before injection;

[0038] The dosage of the hemocyanin of Chinese shrimp injected into the above-mentioned BALB / c mice was 100 μg per mouse;

[0039] Booster immunization: The BALB / c mice were subcutaneously injected with the hemocyanin solution of Chinese shrimp at multiple points, supplemented with incomplete Freund's adjuvant (Sigma-Aldrich, product number: F5506). The hemocyanin of Chinese shrimp and incomplete Freund's adjuvant were mixed in a volume ratio of 1:1 and fully emulsified before injection;

[0040] The dosage of the hemocyanin of Chinese shrimp injected into the above-mentioned BALB / c mice was 100 μg per mouse.

[0041] 3. Cell fusion

[0042] (1) Sacrifice the immunized BALB / c mice by cervical dislocation. Remove the spleens of the BALB / c mice through aseptic operation, and remove the adherent adipose tissue and connective tissue;

[0043] (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 with DMEM incomplete medium to obtain a spleen cell suspension with a cell concentration of 1×10 8 cells / mL;

[0044] (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 with DMEM incomplete medium to obtain an SP2 / 0 cell suspension with a cell concentration of 1×10 8 cells / mL;

[0045] The above-mentioned SP2 / 0 cells are derived from the Cell Bank of the Chinese Academy of Sciences;

[0046] (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 a PEG1450 solution;

[0047] The dosages of the above-mentioned PEG1450 and PBS buffer are calculated according to the ratio of PEG1450:PBS buffer of 10 g:100 mL;

[0048] (5) Mix the above-obtained SP2 / 0 cell suspension and spleen cell suspension, and control the number of SP2 / 0 cells in the mixed solution to be 1×10 7 cells and the number of spleen cells to be 1×10 8 cells. Mix evenly and then centrifuge at 2000 r / min for 5 min, discard the supernatant, and obtain a mixed cell pellet containing SP2 / 0 cells and spleen cells;

[0049] (6) Drop 0.5 mL of the preheated PEG1450 solution at 37°C into the mixed cell pellet of SP2 / 0 cells and spleen cells obtained above. After mixing evenly, place it in a cell incubator at 37°C and a CO2 volume percentage concentration of 5% for incubation for 1 min to obtain cell suspension I;

[0050] (7) Add antibiotics to the HAT medium and mix evenly to obtain a HAT medium containing antibiotics;

[0051] The antibiotic mentioned above is a mixture of penicillin and streptomycin / double antibody (100×) (Shanghai YaMei Biotechnology Co., Ltd., product number: CB010);

[0052] In the above HAT medium containing antibiotics, the working concentration of penicillin is 100 U / mL, and the working concentration of streptomycin is 100 μg / mL;

[0053] (8) Add 8 - 10 mL of preheated (to 37 °C) HAT medium containing antibiotics to the above - obtained cell suspension I, mix well to obtain cell suspension II;

[0054] (9) Add the above - obtained cell suspension II into a culture dish, and then place the culture dish containing cell suspension II in a cell incubator at 37 °C with a CO₂ volume percentage concentration of 5% for 5 days. Regularly observe the cell growth in the culture dish until cell clusters with a diameter of 100 μm are obtained to get fused cell clones;

[0055] (10) Gradually expand the fused cell clones obtained in (9) using DMEM complete medium, and take the supernatant of the hybridoma cells for subsequent Elisa experiments (the monoclonal antibody against shrimp hemocyanin is an extracellular product of hybridoma cells).

[0056] 4. Identify the monoclonal antibody by the Elisa method

[0057] (1) Preparation of the coating solution: Dissolve the shrimp hemocyanin obtained in the preparation of the antigen in step 1 above in PBS buffer to prepare a coating solution with a concentration of 5 μg / mL;

[0058] (2) Coating: Add 50 μL of the coating solution to each well of a 96 - well plate and coat at 4 °C for 16 h;

[0059] (3) Washing: Discard the liquid in the wells, wash with PBST buffer. The amount of PBST buffer used is 200 μL / well. Gently shake the well plate, discard the liquid in the wells, and repeat the PBST buffer washing 2 - 4 times to remove the unbound shrimp hemocyanin;

[0060] (4) Blocking: Add 100 μL / well of blocking buffer and incubate at room temperature (25 °C) for 1 - 2 h to prevent non - specific binding;

[0061] The above blocking buffer is bovine serum albumin (Shanghai YaMei Biotechnology Co., Ltd., product number: PS113) dissolved in PBS buffer, and the mass - to - volume ratio of the bovine serum albumin to PBS buffer is 5 g:100 mL;

[0062] (5), Washing: Discard the liquid in the wells, wash with PBST buffer solution, the amount of PBST buffer solution is 200 μL / well, gently shake the microplate, discard the liquid in the wells, and repeat the PBST buffer solution washing 2 - 4 times to remove the blocking solution, obtaining the coated 96-well microplate;

[0063] (6), Primary antibody incubation: Add the supernatant of hybridoma cells as the first antibody solution to the coated 96-well microplate, the amount of the first antibody solution is 200 μL / well, use the serum of mice immunized with shrimp hemocyanin as the positive control, and use the serum of mice not immunized with shrimp hemocyanin as the negative control. Set 2 parallel samples for the first antibody solution, positive control, and negative control respectively, and incubate at 37 °C for 1 h;

[0064] (7), Washing: Discard the liquid in the wells, wash with PBST buffer solution, the amount of PBST buffer solution is 200 μL / well, gently shake the microplate, pour out the liquid, and repeat the washing 2 - 4 times to remove the unbound primary antibody;

[0065] (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 °C) for 1 h;

[0066] The described secondary antibody solution is composed of goat anti-mouse IgG labeled with horseradish peroxidase (HRP) (Shanghai Yaen Biopharmaceutical Technology Co., Ltd., product number: LF101) and PBS buffer solution calculated by volume ratio. The ratio of goat anti-mouse IgG labeled with horseradish peroxidase (HRP) to PBS buffer solution is 1:1000;

[0067] (9), Washing: Discard the liquid in the wells, wash with PBST buffer solution, the amount of PBST buffer solution is 200 μL / well, gently shake the microplate, discard the liquid in the wells, and repeat the washing 2 - 4 times to remove the unbound secondary antibody;

[0068] (10), Color development: Add the substrate 3,3’,5,5’-tetramethylbenzidine (TMB) to the wells, the amount of TMB is 50 μL / well, and incubate at room temperature (25 °C) for 15 min to react with the HRP on the secondary antibody;

[0069] (11), Termination: Add 200 μL of 0.1 mol / L hydrochloric acid aqueous solution to each well to terminate the reaction, obtaining the reaction-terminated solution;

[0070] (12), Measure OD 450 value: At a working wavelength of 450 nm, measure the OD 450 value of the reaction-terminated solution in each well after the reaction is terminated, and calculate the average OD 450 value of the 2 parallel samples of the first antibody solution, positive control, and negative control;

[0071] Judgment criteria: After the quality control is qualified (the average OD value of the negative control < 0.2, and the average OD value of the positive control > 1.5), select the positive wells (average OD value > 1.5) of the samples for the single-cell cloning screening of hybridomas. 450 value < 0.2, and the average OD 450 value > 1.5), and then select the positive well (average OD 450 value > 1.5) samples for the single-cell cloning screening of hybridomas.

[0072] 5. Single-cell cloning screening of hybridomas

[0073] (1) Preparation of the medium for single-cell cloning screening of hybridomas: Add fetal bovine serum (FBS) to RPMI 1640 medium to obtain RPMI 1640 medium containing serum;

[0074] For the addition amount of the above FBS, the volume ratio of FBS to RPMI 1640 medium is 10:90, and the FBS can be replaced by other serum supplements;

[0075] (2) Dilute the sample: Select the positive well samples in (12) of the identification of monoclonal antibodies by the Elisa method in step 4 above for limited dilution to obtain monoclonal cells; According to the concentration and volume of the positive well samples, calculate the expected number of cells in each positive well, and use PBS buffer to dilute the positive well samples to the required number of cells;

[0076] For example, the concentration of the sample hybridoma cells is 1×10 6 cells / mL. Take 0.1 mL of the sample from the sample hybridoma cells and add it to 0.9 mL of PBS buffer to obtain dilution I, so that the total volume of dilution I is 1 mL; Then, take 0.1 mL from dilution I and add it to 0.9 mL of PBS buffer to obtain dilution II; And so on, to obtain the final dilution. The final 1 mL of the final dilution contains 10 cells;

[0077] (3) Add 0.9 mL of RPMI 1640 medium containing serum to each well in a new well plate, and then add 0.1 mL of the final dilution obtained in (2) above to each well, and pipette and mix well;

[0078] (4) Culture the cells: Place the new well plate in a cell culture incubator at 37 °C and a CO2 volume percentage concentration of 5% for culture;

[0079] (5) After the cell coverage rate is above 60%, detect according to the method of identifying monoclonal antibodies by the Elisa method in step 4 above;

[0080] Through the above method, 1 hybridoma monoclonal cell with high specificity against the hemocyanin of Chinese shrimp is obtained.

[0081] Example 2

[0082] The gene sequence of a monoclonal antibody anti-SHC-5C8 against the hemocyanin of Chinese shrimp was obtained by a method including the following steps:

[0083] 1. Extraction of total RNA

[0084] Total RNA was extracted from the above-obtained hybridoma monoclonal cells using a total RNA extraction reagent (Shanghai Yamei Biopharmaceutical Technology Co., Ltd., product number: YY101L).

[0085] 2. 5’RACE amplification

[0086] The total RNA was subjected to 5’RACE amplification using SMARTer RACE 5’ / 3’Kit (TAKARA, product number: 634859) to obtain a solution containing DNA.

[0087] 3. Gel electrophoresis and purification

[0088] The solution containing DNA obtained above was analyzed by agarose gel electrophoresis, and the target fragment was obtained by cutting the gel; then, the target fragment was purified using a DNA recovery and purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., product number: DP214) to obtain a purified antibody fragment corresponding to the DNA.

[0089] 4. Cloning and sequencing

[0090] The purified antibody fragment was ligated to the pMD19-T vector (GenScript Biotech Corporation) using a vector construction method (the vector construction method is a common vector construction method in the prior art), and then transferred into Escherichia coli DH5α (Shanghai Vidy Biotechnology Co., Ltd.). The positive clone corresponding to the antibody fragment was obtained by the blue-white screening method;

[0091] The positive clone corresponding to the antibody fragment was picked out and sequenced by the Sanger method (completed by Shanghai Qingke Biotechnology Co., Ltd.) to obtain the antibody gene of the corresponding antibody fragment;

[0092] The nucleotide sequence encoding the light chain of the antibody gene is as 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 as 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 the above SEQ ID NO.1 is as shown in SEQ ID NO.3, and the amino acid sequence corresponding to SEQ ID NO.2 is as shown in SEQ ID NO.4;

[0093] Through analysis, it can be known that CDR1 in the amino acid sequence of the light chain variable region of the antibody gene is as shown in SEQ ID NO.5, CDR2 is as shown in SEQ ID NO.6, and CDR3 is as shown in SEQ ID NO.7; CDR1 in the amino acid sequence of the heavy chain variable region of the antibody gene is as shown in SEQ ID NO.8, CDR2 is as shown in SEQ ID NO.9, and CDR3 is as shown in SEQ ID NO.10.

[0094] Example 3

[0095] A recombinant plasmid pcDNA3.1 containing the monoclonal antibody anti-SHC-5C8 gene against the hemocyanin of Chinese shrimp is obtained by the following technical scheme:

[0096] 1. Use BamH I and EcoR1 to double digest the vector plasmid pcDNA3.1 to obtain the linearized plasmid pcDNA3.1;

[0097] The above vector plasmid pcDNA3.1 is derived from GenScript Biotech Corporation;

[0098] The enzyme digestion system is shown in Table 1 below:

[0099] Table 1 Enzyme digestion system

[0100] Components of the digestion system Volume / Mass Vector plasmid pcDNA3.1 1 μg BamHI (10 U / μL) 1 μL EcoR1 (10 U / μL) 1 μL 10× Buffer 1 μL <![CDATA[H2O]]> Make up to 10 μL

[0101] React at 37 °C for 30 min.

[0102] 2. Perform PCR amplification on the antibody fragment

[0103] Perform PCR amplification on the antibody fragment to obtain the antibody fragment solution after PCR amplification;

[0104] The specific components and dosages of the PCR amplification system are shown in Table 2 below:

[0105] Table 2 PCR amplification system

[0106] Components of the PCR amplification system Volume / Mass Antibody fragment 1 ng Forward primer (10 μM) 2 μL Reverse primer (10 μM) 2 μL 2× PhantaMax Buffer 25 μL dNTP Mix (10 mM each) 1 μL PhantaMax Super-Fidelity DNA Polymerase 1 μL <![CDATA[ddH2O]]> Make up to 50 μL

[0107] The above-mentioned 2×Phanta Max Buffer, dNTP Mix (10 mM each), Phanta Max Super-Fidelity DNA Polymerase, and ddH2O are all from Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novoprotein Scientific Inc., Catalog No.: P505-d1).

[0108] The above-mentioned upstream primer sequence is AGCTCGGATCC + the 20 bp sequence at the 5' end of the antibody fragment.

[0109] The above-mentioned downstream primer sequence is GAATTCGG + the 20 bp sequence at the 3' end of the antibody fragment.

[0110] The program settings for the PCR amplification system are: pre-denaturation (95°C, 5 min), cyclic amplification [a total of 30 cycles, each cycle including denaturation (95°C, 30 s), annealing (65°C, 30 s), extension (72°C, 10 s)], and final extension (72°C, 5 min).

[0111] 3. Ligation reaction

[0112] Using T4 DNA ligase, the antibody fragment after PCR amplification was ligated to the linearized plasmid pcDNA3.1 to obtain a corresponding solution containing the recombinant plasmid pcDNA3.1.

[0113] The composition and dosage of the reactants, related reagents, etc. used in the above ligation process are shown in Table 3 below.

[0114] Table 3 Ligation reaction system

[0115] Reactants, related reagents, etc. Volume / Mass T4 DNA ligase (10 U / μL) 1 μL Antibody fragment after PCR amplification 200 ng Linearized plasmid pcDNA3.1 500 ng 10× T4 DNA ligase reaction buffer 2 μL <![CDATA[H2O]]> Make up to 20 μL

[0116] Mix the reactants, related reagents, etc. used in the above ligation process evenly, and control the reaction at 16°C for 12 h.

[0117] 4. Transformation of recombinant plasmid

[0118] Take 10 μL of the solution containing the recombinant plasmid pcDNA3.1 and add it to 50 μL of competent Escherichia coli DH5α (Shanghai Weidi Biotechnology Co., Ltd.). Mix well, let it stand on ice for 30 min, then transfer it to an environment at 42 °C for 90 s, and then place it on ice for 2 min. Then transfer it to 5 mL of LB liquid medium, incubate at 37 °C for 1 h, take a sample and evenly spread it on a pre-prepared LB agar plate containing 10 μg / mL ampicillin antibiotic, and incubate it upside down at 37 °C for 16 h to obtain recombinant Escherichia coli DH5α colonies corresponding to the recombinant plasmid pcDNA3.1;

[0119] Pick a single colony of Escherichia coli DH5α containing the recombinant plasmid pcDNA3.1 described above and inoculate it into 5 mL of LB liquid medium for fermentation for 16 h to obtain the fermentation broth of recombinant Escherichia coli DH5α corresponding to the recombinant plasmid pcDNA3.1;

[0120] Verify by Sanger sequencing (completed by Shanghai Qingke Biotechnology Co., Ltd.). The results show that recombinant Escherichia coli DH5α with successful recombination is obtained.

[0121] 5. Recombinant plasmid extraction

[0122] Use a plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., product number: DP103) to extract the recombinant plasmid from the fermentation broth of the above-obtained recombinant Escherichia coli DH5α to obtain the recombinant plasmid pcDNA3.1;

[0123] Verify by Sanger sequencing (completed by Shanghai Qingke Biotechnology Co., Ltd.). The results show that the recombinant plasmid pcDNA3.1 is successfully obtained.

[0124] Example 4

[0125] A recombinant Expi293 cell containing the recombinant plasmid pcDNA3.1 is obtained by the following technical scheme:

[0126] 1. Expi293 cell transfection

[0127] Mix the PEI transfection reagent (Shanghai Aladdin Biochemical Technology Co., Ltd., product number: 5644123) with the recombinant plasmid pcDNA3.1 according to the ratio of PEI transfection reagent: recombinant plasmid pcDNA3.1 of 0.1 mL: 10 μg to obtain a mixture of transfection reagent / recombinant plasmid pcDNA3.1, and let it stand at 30 °C for 15 min;

[0128] Transfect the above-obtained mixture of transfection reagent / recombinant plasmid pcDNA3.1 into Expi293 cells, according to 10 μg of recombinant plasmid corresponding to 10 6The proportion of Expi293 cells was obtained to get recombinant Expi293 cells corresponding to the recombinant plasmid pcDNA3.1.

[0129] 2. Culture of recombinant Expi293 cells

[0130] The recombinant Expi293 cells were cultured in a cell incubator at 37 °C with a CO2 volume percentage concentration of 5% for 1 week to obtain a recombinant Expi293 cell culture solution corresponding to the monoclonal antibody against the hemocyanin of Chinese shrimp.

[0131] The above-mentioned recombinant Expi293 cell culture solution was centrifuged, and the supernatant of the recombinant Expi293 cell culture solution corresponding to the monoclonal antibody against the hemocyanin of Chinese shrimp was collected.

[0132] The above-mentioned Expi293 cells were from Shanghai Duoning Biotechnology Co., Ltd.

[0133] 3. Purification of the antibody

[0134] Protein A affinity chromatography resin (Suzhou NanoMicro Technologies Co., Ltd., product number: 17013-090100) was packed into a chromatography column to form a Protein A affinity chromatography column.

[0135] The Protein A affinity chromatography column was equilibrated with 3 column volumes of PBS buffer.

[0136] The supernatant of 1 L of the recombinant Expi293 cell culture solution containing the monoclonal antibody against the hemocyanin of Chinese shrimp collected was loaded onto the Protein A affinity chromatography column equilibrated with PBS buffer, and the flow rate was controlled at 2 mL / min.

[0137] After the above sample loading was completed, PBS buffer was first used to elute non-specifically bound proteins and impurities to retain the monoclonal antibody against the hemocyanin of Chinese shrimp bound to the Protein A affinity chromatography resin.

[0138] Then, a 0.5 mM glycine elution buffer with pH = 3.5 was used to elute the monoclonal antibody against the hemocyanin of Chinese shrimp bound to the Protein A affinity chromatography resin, and it was adjusted to pH = 7.2 with a 0.1 mM Tris-HCl aqueous solution with pH = 10.

[0139] Then ultrafiltration concentration was carried out to obtain a concentrated solution of the corresponding monoclonal antibody against the hemocyanin of Chinese shrimp.

[0140] The above-mentioned monoclonal antibody against the hemocyanin of Chinese shrimp was named anti-SHC-5C8.

[0141] 4. SDS-PAGE Gel Electrophoresis

[0142] The concentrated solution of anti-SHC-5C8 was assayed for purity by SDS-PAGE gel electrophoresis, and the obtained electrophoresis pattern is as Figure 1 shown. By gray-scale analysis, the purity of the monoclonal antibody anti-SHC-5C8 in the corresponding concentrated solution was ≥90%.

[0143] 5. Western Blot

[0144] The concentrated solution of the monoclonal antibody anti-SHC-5C8 was analyzed by Western blot. The specific steps are as follows:

[0145] (1). The hemocyanin of Chinese shrimp was diluted to a concentration of 1 mg / mL with WB loading buffer and boiled at 95 °C for 5 min to obtain a pretreated hemocyanin sample of Chinese shrimp;

[0146] The above WB loading buffer was 100 mM DTT, 1% SDS, 20% glycerol and 1% methylene blue;

[0147] (2). The above pretreated hemocyanin sample of Chinese shrimp was added into the loading wells of the precast gel. 100 ng of the pretreated hemocyanin sample of Chinese shrimp was added to each well. Under the condition of a constant voltage of 120 V, electrophoresis was carried out for 1 h, and then the gel was taken out;

[0148] (3). A piece of nitrocellulose membrane of the same size as the gel was cut. The nitrocellulose membrane and filter paper were soaked with a rapid transfer membrane buffer (Shanghai Yamei Biotechnology Co., Ltd., product number: PS117), and then placed in a wet transfer membrane apparatus in the form of a "sandwich". The order was: electrode (-)-sponge-filter paper-gel-nitrocellulose membrane-filter paper-sponge-electrode (+). A constant current of 300 mA was applied for 1 h;

[0149] The pore size of the above nitrocellulose membrane was 0.22 μm;

[0150] (4). After the transfer membrane was completed, the nitrocellulose membrane was taken out, washed once with water, and then placed in a PBST buffer containing 5% skim milk powder and blocked at 30 °C for 10 min to prevent non-specific binding;

[0151] For the above PBST buffer containing 5% skim milk powder, the dosages of skim milk powder and PBST buffer were in a mass-to-volume ratio of skim milk powder:PBST buffer of 5 g:100 mL;

[0152] (5). The nitrocellulose membrane was washed 3 times with PBST buffer, 5 min each time;

[0153] (6) Add the obtained anti-SHC-5C8 as a specific primary antibody to the PBST buffer containing 5% non-fat milk powder to obtain a primary antibody solution with a concentration of 1 μg / mL. Place the nitrocellulose membrane in the primary antibody solution and incubate at room temperature (25 °C) for 1 h.

[0154] (7) Wash the nitrocellulose membrane 3 times with PBST buffer, 5 min each time.

[0155] (8) Add goat anti-mouse IgG labeled with horseradish peroxidase (HRP) (Shanghai Yaen Biopharmaceutical Technology Co., Ltd., product number: LF101) as the secondary antibody to the PBST buffer containing 5% non-fat milk powder to obtain a secondary antibody solution with a concentration of 1 μg / mL. Incubate at room temperature (25 °C) for 1 h.

[0156] (9) Wash the nitrocellulose membrane 3 times with PBST buffer, 5 min each time, to remove non-specifically bound secondary antibody and other impurities.

[0157] (10) Use ECL chromogenic solution to develop the final bands until the color is clear. The obtained blot is as shown in Figure 2 shown. It can be seen from Figure 2 that the antigenic determinants where the monoclonal antibody anti-SHC-5C8 against the hemocyanin of Chinese shrimp binds specifically are located on two subunits of the hemocyanin of shrimp with molecular weights of 73 kDa and 75 kDa.

[0158] 6. Sequencing verification

[0159] Verify the amino acid sequence of the obtained monoclonal antibody anti-SHC-5C8 against the hemocyanin of Chinese shrimp. The results show that the light chain amino acid sequence (variable region of light chain VL + constant region of light chain CL) of anti-SHC-5C8 is consistent with SEQ ID NO.3, and the heavy chain amino acid sequence (variable region of heavy chain VH + constant region of heavy chain CH1) of anti-SHC-5C8 is consistent with SEQ ID NO.4.

[0160] Application examples

[0161] Application example 1

[0162] An application of a monoclonal antibody against the hemocyanin of Chinese shrimp as a positive internal reference in the detection of the content of the hemocyanin of Chinese shrimp is obtained by the following technical scheme:

[0163] According to the Elisa method in step 4 of Example 1, the concentrated monoclonal antibody anti-SHC-5C8 against Fenneropenaeus chinensis hemocyanin obtained in Example 4 was used as the first antibody solution in (6) of step 4 in Example 1. At the same time, the serum of the mice immunized with Fenneropenaeus chinensis hemocyanin four times in step 2 of Example 1 was used as a control for the first antibody solution. The remaining experimental operations were the same as those in step 4 of Example 1, and finally the average OD 450 value was obtained.

[0164] Through data analysis, it can be seen that the positive signal that can be detected by the serum of the mice immunized with Fenneropenaeus chinensis hemocyanin four times (the average OD 450 value is 2.09), and the concentrated monoclonal antibody anti-SHC-5C8 against Fenneropenaeus chinensis hemocyanin can also detect obvious positive signals (the average OD 450 value is 2.24). This shows that the monoclonal antibody anti-SHC-5C8 against Fenneropenaeus chinensis hemocyanin can be used as a positive internal reference for quantitatively detecting the content of antibodies against Fenneropenaeus chinensis hemocyanin in animals immunized with Fenneropenaeus chinensis hemocyanin or qualitatively judging whether the animals immunized with Fenneropenaeus chinensis hemocyanin are successfully immunized.

[0165] In summary, a monoclonal antibody anti-SHC-5C8 against Fenneropenaeus chinensis hemocyanin of the present application can be stably expressed by recombinant Expi293 cells. The obtained monoclonal antibody anti-SHC-5C8 against Fenneropenaeus chinensis hemocyanin can specifically bind to two subunits with molecular weights of 73 kDa and 75 kDa of Fenneropenaeus chinensis hemocyanin, and can be used as a positive internal reference for the content detection of monoclonal antibodies against Fenneropenaeus chinensis hemocyanin.

[0166] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereby. Therefore, all equivalent changes made according to the structure, shape, and principle of the present application should be covered within the protection scope of the present application.

Claims

1. A monoclonal antibody against the hemocyanin of Fenneropenaeus chinensis, characterized in that, The monoclonal antibody against Fenneropenaeus chinensis hemocyanin is anti-SHC-5C8; The light chain amino acid sequence of the anti-SHC-5C8 is shown in SEQ ID NO.3, and the light chain amino acid sequence of the anti-SHC-5C8 contains a light chain variable region VL and a light chain constant region CL; the heavy chain amino acid sequence of the anti-SHC-5C8 is shown in SEQ ID NO.4, and the heavy chain amino acid sequence of the anti-SHC-5C8 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-5C8 against Fenneropenaeus chinensis hemocyanin as claimed in claim 1. The nucleotide sequence of the nucleic acid encoding the light chain amino acid sequence of anti-SHC-5C8 is shown in SEQ ID NO.1, and the nucleotide sequence of the nucleic acid encoding the heavy chain amino acid sequence of anti-SHC-5C8 is shown in SEQ ID NO.

2.

3. A recombinant plasmid pcDNA3.1 containing the nucleic acid encoding as claimed in claim 2.

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

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

6. A monoclonal antibody against Fenneropenaeus chinensis hemocyanin as claimed in claim 1 is used as a positive internal reference for qualitatively judging whether an animal immunized with Fenneropenaeus chinensis hemocyanin is successfully immunized or for quantitatively detecting the content of the monoclonal antibody against Fenneropenaeus chinensis hemocyanin in an animal immunized with Fenneropenaeus chinensis hemocyanin.

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