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-1A1, 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 title "A Monoclonal Antibody Against Shrimp 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 shrimp hemocyanin of Chinese shrimp and its application. Background Art

[0003] Chinese shrimp is an important economic animal for mariculture 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 farming industry in China. Therefore, it has become particularly urgent and necessary to study the immune mechanism of Chinese shrimp.

[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 phenol oxidase 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 monoclonal antibody against shrimp hemocyanin of Chinese shrimp can be used to conveniently and quickly detect and monitor the fluctuation law of the hemocyanin concentration in Chinese shrimp.

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

[0007] The purpose of this application is to provide a monoclonal antibody against shrimp hemocyanin of Chinese shrimp to solve the above-mentioned problem of unstable expression of hybridoma cells. This monoclonal antibody against shrimp hemocyanin of Chinese shrimp 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 of Chinese shrimp.

[0008] Technical Solution of This Application In the first aspect, this application provides a monoclonal antibody against shrimp hemocyanin of Chinese shrimp, and the monoclonal antibody against shrimp hemocyanin of Chinese shrimp is anti-SHC-1A1; The light chain amino acid sequence of the anti-SHC-1A1 is as shown in SEQ ID NO.3, and the light chain amino acid sequence of the anti-SHC-1A1 contains a light chain variable region VL and a light chain constant region CL; the heavy chain amino acid sequence of the anti-SHC-1A1 is as shown in SEQ ID NO.4, and the heavy chain amino acid sequence of the anti-SHC-1A1 contains a heavy chain variable region VH and a heavy chain constant region CH1; The CDR1 in the amino acid sequence of the light chain variable region of the above anti-SHC-1A1 is as shown in SEQ ID NO.5, the CDR2 is as shown in SEQ ID NO.6, and the CDR3 is as shown in SEQ ID NO.7; the CDR1 in the amino acid sequence of the heavy chain variable region of the anti-SHC-1A1 is as shown in SEQ ID NO.8, the CDR2 is as shown in SEQ ID NO.9, and the CDR3 is as shown in SEQ ID NO.10.

[0009] In a second aspect, the present application provides a nucleic acid that encodes a monoclonal antibody anti-SHC-1A1 against the hemocyanin of Fenneropenaeus chinensis. The nucleotide sequence of the nucleic acid encoding the light chain amino acid sequence of anti-SHC-1A1 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-1A1 is as shown in SEQ ID NO.2.

[0010] By adopting the above technical solution, the antigenic determinants of the obtained monoclonal antibody anti-SHC-1A1 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 it has good specificity.

[0011] In a third aspect, the present application provides a hybridoma cell that expresses a monoclonal antibody anti-SHC-1A1 against the hemocyanin of Fenneropenaeus chinensis.

[0012] In a fourth aspect, the present application provides a recombinant plasmid pcDNA3.1 that contains the encoding nucleic acid as described above.

[0013] In a fifth aspect, the present application provides a recombinant Escherichia coli DH5α that contains the recombinant plasmid pcDNA3.1.

[0014] In a sixth aspect, the present application provides a recombinant Expi293 cell that contains the recombinant plasmid pcDNA3.1.

[0015] 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 has been successfully immunized or for quantitatively detecting the content of the monoclonal antibody against shrimp hemocyanin in an animal immunized with shrimp hemocyanin.

[0016] Advantageous technical effects of the present application A monoclonal antibody anti-SHC-1A1 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.

[0017] Furthermore, a monoclonal antibody anti-SHC-1A1 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.

[0018] Furthermore, a monoclonal antibody anti-SHC-1A1 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 has been 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

[0019] Figure 1 Electrophoresis diagram of determining the purity of the concentrated solution of the monoclonal antibody anti-SHC-1A1 against shrimp hemocyanin by SDS-PAGE gel electrophoresis; Figure 2 Blotting diagram of detecting the concentrated solution of the monoclonal antibody anti-SHC-1A1 against shrimp hemocyanin by Western blot. Detailed implementation manners

[0020] The following further describes the present application in detail through specific examples in combination with the attached Figure 1-2 drawings, but does not limit the present application.

[0021] In each embodiment 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; The used PBST buffer solution is a dilution obtained by diluting a 10×PBS / Tween buffer solution (Shanghai Yaen Biopharmaceutical Technology Co., Ltd., product number: PS102S) 10 times with deionized water. Examples Example 1

[0022] A hybridoma cell expressing the monoclonal antibody anti-SHC-1A1 against Penaeus chinensis hemocyanin is obtained by a method comprising the following steps: 1. Preparation of Antigen According to the method described in CN118005773A, Penaeus chinensis hemocyanin is extracted and purified to obtain Penaeus chinensis hemocyanin.

[0023] 2. Immunization of mice (1) Dissolving the Penaeus chinensis hemocyanin obtained in step 1 in PBS buffer at a concentration of 1 mg / mL to obtain a Penaeus chinensis hemocyanin solution as an immunogen; (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 initial immunization, and the next three times were booster immunizations. Each immunization was separated by two weeks to obtain fully immunized BALB / c mice; The above-mentioned BALB / c mice were purchased from Southern Model Organisms Corporation; Primary immunization: BALB / c mice were subcutaneously injected with a Chinese shrimp hemocyanin solution supplemented with complete Freund's adjuvant (Sigma-Aldrich, Cat. No. F5881) at multiple points. The volume ratio of Chinese shrimp hemocyanin solution to complete Freund's adjuvant was 1:1, and the mixture was fully emulsified before injection. The dosage of Penaeus chinensis hemocyanin injected into the above BALB / c mice was 100 μg / mouse; Enhanced immunization: BALB / c mice were subcutaneously injected with a solution of Penaeus chinensis hemocyanin supplemented with incomplete Freund's adjuvant (Sigma-Aldrich, Cat. No. F5506) at multiple points. Penaeus chinensis hemocyanin and incomplete Freund's adjuvant were mixed at a volume ratio of 1:1 and injected after being fully emulsified. The amount of Penaeus chinensis hemocyanin injected into the above BALB / c mice was 100 μg / mouse.

[0024] 3. Cell fusion (1) The BALB / c mice that have completed the above immunization are killed by cervical dislocation, and the spleen of the BALB / c mice is removed by aseptic operation, and the adherent fat tissue and connective tissue are removed; (2) Rinse the 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 cell concentration of 1×10 8 / mL spleen cell suspension; (3) Take SP2 / 0 cells in the logarithmic growth phase, centrifuge them at 2000 r / min for 5 min, discard the supernatant, and then resuspend the SP2 / 0 cell pellet with incomplete DMEM medium to obtain an SP2 / 0 cell suspension with a cell concentration of 1×10 8 cells / mL; The SP2 / 0 cells are from the Cell Bank of the Chinese Academy of Sciences; (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; The dosages of the above PEG1450 and PBS buffer are calculated according to the ratio of PEG1450:PBS buffer of 10 g:100 mL; (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; (6) Drop 0.5 mL of the PEG1450 solution preheated to 37 °C into the mixed cell pellet of SP2 / 0 cells and spleen cells obtained above. After mixing evenly, place it in a cell culture incubator at 37 °C and 5% (v / v) CO 2 ₂ for incubation for 1 min to obtain cell suspension I; (7) Add antibiotics to HAT medium and mix evenly to obtain HAT medium containing antibiotics; The antibiotic is a mixture of penicillin and streptomycin / dual antibiotics (100×) (Shanghai Yaen Biopharmaceutical Technology Co., Ltd., catalog number: CB010); 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; (8) Add 8 - 10 mL of the HAT medium containing antibiotics preheated to 37 °C to the above-obtained cell suspension I, mix well to obtain cell suspension II; (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 culture incubator at 37 °C and 5% (v / v) CO 2 ₂ for culturing for 5 days, and regularly observe the cell growth in the culture dish until cell clusters with a diameter of 100 μm are obtained to obtain fused cell clones; (10) The fusion cell clones obtained in (9) were gradually expanded using DMEM complete medium, and the supernatant of the hybridoma cells was used for subsequent Elisa experiments (the monoclonal antibody against Penaeus chinensis hemocyanin is an extracellular product of the hybridoma cells).

[0025] 4. Identify monoclonal antibodies using Elisa (1) Preparation of coating solution: dissolve the Penaeus chinensis 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; (2) Coating: Add 50 μL of coating solution to each well of a 96-well plate and coat at 4°C for 16 h. (3) Washing: discard the liquid in the wells and wash with PBST buffer at 200 μL / well. Gently shake the well plate, discard the liquid in the wells, and repeat the PBST buffer wash 2-4 times to remove unbound Chinese shrimp hemocyanin. (4) Blocking: Add blocking buffer at 100 μL / well and incubate at room temperature (25°C) for 1-2 h to prevent nonspecific binding; The blocking buffer is bovine serum albumin (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd., catalog number: PS113) dissolved in PBS buffer, and the mass volume ratio of the bovine serum albumin to the PBS buffer is 5g:100mL; (5) Washing: discard the liquid in the wells and wash with PBST buffer at 200 μL / well. Gently shake the well plate, discard the liquid in the wells, and repeat the PBST buffer wash 2-4 times to remove the blocking solution to obtain a coated 96-well plate. (6) Primary antibody incubation: The supernatant of hybridoma cells was added as the primary antibody solution to the coated 96-well plate. The amount of the primary antibody solution was 200 μL / well. The serum of mice immunized with Penaeus chinensis hemocyanin was used as the positive control, and the serum of mice not immunized with Penaeus chinensis hemocyanin was used as the negative control. Two parallel samples were set for the primary antibody solution, positive control, and negative control, respectively. The plates were incubated at 37°C for 1 h. (7) Washing: discard the liquid in the wells and wash with PBST buffer at 200 μL / well. Gently shake the well plate, pour out the liquid, and repeat the washing 2-4 times to remove the unbound primary antibody. (8) Secondary antibody incubation: Add the secondary antibody solution at 200 μL / well and incubate at room temperature (25°C) for 1 h. The described second antibody solution is prepared by mixing goat anti-mouse IgG labeled with horseradish peroxidase (HRP) (Shanghai Yaen Biopharmaceutical Technology Co., Ltd., product number: LF101) and PBS buffer in a volume ratio. The ratio of goat anti-mouse IgG labeled with horseradish peroxidase (HRP) to PBS buffer is 1:1000; (9), Washing: Discard the liquid in the wells, wash with PBST buffer, with a dosage of 200 μL / well of PBST buffer. Gently shake the microplate, discard the liquid in the wells, and repeat the washing 2 - 4 times to remove the unbound second antibody; (10), Color development: Add the substrate 3,3’,5,5’-tetramethylbenzidine (TMB) to the wells, with a dosage of 50 μL / well. Incubate at room temperature (25 °C) for 15 min to allow it to react with the HRP on the second antibody; (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; (12), Measuring 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 two parallel samples of the first antibody solution, positive control, and negative control; Judgment criterion: After the quality control is qualified (negative control average OD 450 value < 0.2, positive control average OD 450 value > 1.5), select the positive wells (average OD 450 value > 1.5) of the samples for single-cell cloning screening of hybridomas.

[0026] 5. Single-cell cloning screening of hybridomas (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; The added amount of the above FBS, the volume ratio of FBS to RPMI 1640 medium is 10:90, and the FBS can be replaced with other serum supplements; (2), Diluting the sample: Select the positive well samples in (12) of the above step 4 for identifying monoclonal antibodies by the Elisa method 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 dilute the positive well samples to the required number of cells using PBS buffer; For example, the concentration of the sample hybridoma cells is 1×10 6cells / 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 Diluent I, making the total volume of Diluent I 1 mL; then, take 0.1 mL from Diluent I and add it to 0.9 mL of PBS buffer to obtain Diluent II; and so on to obtain the final diluent. The final 1 mL of the final diluent contains 10 cells; (3) Add 0.9 mL of RPMI 1640 medium containing serum to each well of a new well plate, and then add 0.1 mL of the final diluent obtained in (2) above to each well, and pipette and mix well; (4) Cultivate the cells: Place the new well plate in a cell culture incubator at 37 °C with a CO 2 volume percentage concentration of 5% and conduct the cultivation; (5) After the cell coverage rate is above 60%, detect according to the method of identifying the monoclonal antibody by the Elisa method in step 4 above; One hybridoma monoclonal cell with high specificity against the hemocyanin of Chinese shrimp is obtained by the above method. Example 2

[0027] The gene sequence of a monoclonal antibody anti-SHC-1A1 against the hemocyanin of Chinese shrimp is obtained by a method including the following steps: 1. Extraction of total RNA Extract total RNA from the above-obtained hybridoma monoclonal cells using a total RNA extraction reagent (Shanghai Yaen Biopharmaceutical Technology Co., Ltd., product number: YY101L).

[0028] 2. 5’RACE amplification Use SMARTer RACE 5’ / 3’Kit (TAKARA, product number: 634859) to perform 5’RACE amplification on the total RNA to obtain a solution containing DNA.

[0029] 3. Gel electrophoresis and purification Perform agarose gel electrophoresis analysis on the solution containing DNA obtained above, cut the gel to obtain the target fragment; then, use a DNA recovery and purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., product number: DP214) to purify the target fragment to obtain a purified antibody fragment corresponding to the DNA.

[0030] 4. Cloning and sequencing The purified antibody fragment was ligated to the pMD19-T vector (GenScript Biotech Corporation) using a vector construction method (a common vector construction method in the prior art), and then transferred into Escherichia coli DH5α (Shanghai Vidy Biotechnology Co., Ltd.). Positive clones corresponding to the antibody fragment were obtained by blue-white screening; The positive clones corresponding to the antibody fragment were picked out and sequenced by the Sanger method (completed by Shanghai Qingke Biotechnology Co., Ltd.) to obtain the antibody gene corresponding to the antibody fragment; 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 the light chain variable region VL and the 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 the heavy chain variable region VH and the heavy chain constant region CH1; the amino acid sequence corresponding to SEQ ID NO.1 above 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; Through analysis, CDR1 in the amino acid sequence of the light chain variable region of the antibody gene is shown in SEQ ID NO.5, CDR2 is shown in SEQ ID NO.6, and CDR3 is shown in SEQ ID NO.7; CDR1 in the amino acid sequence of the heavy chain variable region of the antibody gene is shown in SEQ ID NO.8, CDR2 is shown in SEQ ID NO.9, and CDR3 is shown in SEQ ID NO.10. Example 3

[0031] A recombinant plasmid pcDNA3.1 containing the monoclonal antibody anti-SHC-1A1 gene against the hemocyanin of Fenneropenaeus chinensis was obtained by the following technical scheme: 1. The vector plasmid pcDNA3.1 was double digested with BamH I and EcoR1 to obtain a linearized plasmid pcDNA3.1; The above vector plasmid pcDNA3.1 is from GenScript Biotech Corporation; The enzyme digestion system is shown in Table 1 below: Table 1 Enzyme digestion system Components of Restriction Enzyme System Volume / Mass Vector Plasmid pcDNA3.1 1 μg BamH I (10 U / μL) 1 μL EcoR1 (10 U / μL) 1 μL 10× Buffer 1 μL <![CDATA[H 2 O]]> Make up to 10 μL React at 37°C for 30 min.

[0032] 2. PCR amplification of the antibody fragment The antibody fragment was subjected to PCR amplification to obtain a solution of the antibody fragment after PCR amplification; The specific components and dosages of the PCR amplification system are shown in Table 2 below: Table 2 PCR Amplification System Components of PCR Amplification System 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 <![CDATA[ddH 2 O]]> Make up to 50 μL The above 2×Phanta Max Buffer, dNTP Mix (10 mM each), Phanta Max Super-Fidelity DNA Polymerase, and ddH 2 O all come from Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novoprotein Scientific Inc., Catalog No.: P505-d1); The above upstream primer sequence is AGCTCGGATCC + the 20 bp sequence at the 5' end of the antibody fragment; The above downstream primer sequence is GAATTCGG + the 20 bp sequence at the 3' end of the antibody fragment; 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).

[0033] 3. Ligation Reaction Using T4 DNA ligase, the antibody fragment after PCR amplification is ligated to the linearized plasmid pcDNA3.1 to obtain a corresponding solution containing the recombinant plasmid pcDNA3.1; The composition and dosages of the reactants, related reagents, etc. used in the above ligation process are shown in Table 3 below: Table 3 Ligation Reaction System 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[H 2 O]]> Make up to 20 μL Mix the reactants, related reagents, etc. used in the above ligation process evenly and control the reaction at 16°C for 12 h.

[0034] 4. Transformation of Recombinant Plasmid 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, 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, then take a sample and evenly coat 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 the recombinant Escherichia coli DH5α colonies corresponding to the recombinant plasmid pcDNA3.1; Pick a single colony of the above-mentioned Escherichia coli DH5α containing the recombinant plasmid pcDNA3.1 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; Verify by Sanger sequencing (completed by Shanghai Qingke Biotechnology Co., Ltd.), and the results show that recombinant Escherichia coli DH5α with successful recombination is obtained.

[0035] 5. Recombinant plasmid extraction 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-mentioned recombinant Escherichia coli DH5α to obtain the recombinant plasmid pcDNA3.1; Verify by Sanger sequencing (completed by Shanghai Qingke Biotechnology Co., Ltd.), and the results show that the recombinant plasmid pcDNA3.1 is successfully obtained. Example 4

[0036] A recombinant Expi293 cell containing the recombinant plasmid pcDNA3.1 is obtained by the following technical scheme: 1. Expi293 cell transfection Mix the PEI transfection reagent (Shanghai Aladdin Biochemical Technology Co., Ltd., product number: 5644123) and the recombinant plasmid pcDNA3.1 in a 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; Transfect the above-obtained mixture of transfection reagent / recombinant plasmid pcDNA3.1 into Expi293 cells, and according to the ratio of 10 μg of recombinant plasmid corresponding to 10 6 Expi293 cells, to obtain recombinant Expi293 cells corresponding to the recombinant plasmid pcDNA3.1.

[0037] 2. Culture of recombinant Expi293 cells Culture the recombinant Expi293 cells in a cell culture incubator at 37 °C and a CO 2 volume percentage concentration of 5% for 1 week to obtain the culture broth of recombinant Expi293 cells corresponding to the monoclonal antibody against Fenneropenaeus chinensis hemocyanin; Centrifuge the above-mentioned culture broth of recombinant Expi293 cells and collect the supernatant of the culture broth of recombinant Expi293 cells corresponding to the monoclonal antibody against Fenneropenaeus chinensis hemocyanin; The above-mentioned Expi293 cells are from Shanghai Duoning Biotechnology Co., Ltd.

[0038] 3. Purification of Antibody Load Protein A affinity chromatography resin (Suzhou NanoMicro Technologies Co., Ltd., product number: 17013 - 090100) into a chromatography column to form a Protein A affinity chromatography column; Equilibrate the Protein A affinity chromatography column with 3 column volumes of PBS buffer; Load the supernatant of 1 L of recombinant Expi293 cell culture medium containing monoclonal antibody against Fenneropenaeus chinensis hemocyanin into the Protein A affinity chromatography column that has been equilibrated with PBS buffer, and control the flow rate at 2 mL / min; After the above sample loading is completed, first use PBS buffer to elute non - specifically bound proteins and impurities to retain the monoclonal antibody against Fenneropenaeus chinensis hemocyanin bound to the Protein A affinity chromatography resin; Then use 0.5 mM glycine elution buffer with pH = 3.5 to elute the monoclonal antibody against Fenneropenaeus chinensis hemocyanin bound to the Protein A affinity chromatography resin, and adjust it to pH = 7.2 with 0.1 mM Tris - HCl aqueous solution with pH = 10; Then perform ultrafiltration concentration to obtain a concentrated solution of the corresponding monoclonal antibody against Fenneropenaeus chinensis hemocyanin; The above - mentioned monoclonal antibody against Fenneropenaeus chinensis hemocyanin is named anti - SHC - 1A1.

[0039] 4. SDS - PAGE Gel Electrophoresis Use SDS - PAGE gel electrophoresis to determine the purity of the concentrated solution of anti - SHC - 1A1. The obtained electrophoresis pattern is as Figure 1 shown. Through gray - scale analysis, the purity of the monoclonal antibody anti - SHC - 1A1 in the corresponding concentrated solution is ≥90%.

[0040] 5. Western blot Use Western blot to analyze the concentrated solution of the monoclonal antibody anti - SHC - 1A1. The specific steps are as follows: (1). Dilute Fenneropenaeus chinensis hemocyanin with WB loading buffer to a concentration of 1 mg / mL and boil it at 95°C for 5 min to obtain a pretreated Fenneropenaeus chinensis hemocyanin sample; The above - mentioned WB loading buffer is 100 mM DTT, 1% SDS, 20% glycerol and 1% methylene blue; (2) Add the pre-treated Chinese shrimp hemocyanin sample into the loading wells of the precast gel. Add 100 ng of the pre-treated Chinese shrimp hemocyanin sample to each well, and perform electrophoresis for 1 h under the condition of a constant voltage of 120 V. Then take out the gel. (3) Cut a piece of nitrocellulose membrane with the same size as the gel, soak the nitrocellulose membrane and filter paper with the rapid transfer buffer (Shanghai Yamei Biotechnology Co., Ltd., product number: PS117), and then place them in a wet transfer apparatus in the "sandwich" sandwich method in the following order: electrode (-) - sponge - filter paper - gel - nitrocellulose membrane - filter paper - sponge - electrode (+), with a constant current of 300 mA and electrify for 1 h. The pore size of the above-mentioned nitrocellulose membrane is 0.22 μm. (4) After the transfer is completed, take out the nitrocellulose membrane, wash it once with water, and then place it in a PBST buffer solution containing 5% skim milk powder and block it at 30 °C for 10 min to prevent non-specific binding. For the above-mentioned PBST buffer solution containing 5% skim milk powder, the dosages of skim milk powder and PBST buffer solution are in a mass-volume ratio of skim milk powder: PBST buffer solution of 5 g: 100 mL. (5) Wash the nitrocellulose membrane 3 times with PBST buffer solution, 5 min each time. (6) Add the obtained anti-SHC-1A1 as a specific primary antibody into the PBST buffer solution containing 5% skim 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. (7) Wash the nitrocellulose membrane 3 times with PBST buffer solution, 5 min each time. (8) Add horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Shanghai Yamei Biopharmaceutical Technology Co., Ltd., product number: LF101) as a secondary antibody into the PBST buffer solution containing 5% skim milk powder to obtain a secondary antibody solution with a concentration of 1 μg / mL, and incubate at room temperature (25 °C) for 1 h. (9) Wash the nitrocellulose membrane 3 times with PBST buffer solution, 5 min each time, to remove non-specifically bound secondary antibody and other impurities. (10) Use the ECL developing solution to develop the final bands until the color is clear. The obtained blot is as Figure 2 shown. It can be seen from Figure 2 that the antigenic determinants where the monoclonal antibody anti-SHC-1A1 against Chinese shrimp hemocyanin specifically binds are located on two subunits of shrimp hemocyanin with molecular weights of 73 kDa and 75 kDa.

[0041] 6. Sequencing verification The obtained monoclonal antibody anti-SHC-1A1 against Fenneropenaeus chinensis hemocyanin was verified for its amino acid sequence. The results showed that the amino acid sequence of the light chain of anti-SHC-1A1 (variable region of light chain VL + constant region of light chain CL) was consistent with SEQ ID NO.3, and the amino acid sequence of the heavy chain of anti-SHC-1A1 (variable region of heavy chain VH + constant region of heavy chain CH1) was consistent with SEQ ID NO.4. Application Example Application Example 1

[0042] An application of a monoclonal antibody against Fenneropenaeus chinensis hemocyanin as a positive internal reference in the detection of the content of anti-Fenneropenaeus chinensis hemocyanin was obtained by the following technical scheme: According to the Elisa method in step 4 of Example 1, the concentrated solution of the monoclonal antibody anti-SHC-1A1 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 mouse immunized with Fenneropenaeus chinensis hemocyanin four times in step 2 of Example 1 was used as a control for the first antibody solution. The rest of the experimental operations were the same as those in step 4 of Example 1. The final average OD 450 value is shown in Table 4 below: Table 4 Detection data of application example First Antibody Sample <![CDATA[Average OD 450 value]]> anti-SHC-1A1 Concentrate 2.14 Serum of Mice Immunized with Chinese Shrimp Hemocyanin Four Times 2.09 Negative Control 0.14 From the data analysis in the above table, it can be seen that the positive signal (average OD 450 value is 2.09) that can be detected by the serum of the mouse immunized with Fenneropenaeus chinensis hemocyanin four times, and the concentrated solution of the monoclonal antibody anti-SHC-1A1 against Fenneropenaeus chinensis hemocyanin can also detect an obvious positive signal (average OD 450 value is 2.14). Thus, it shows that the monoclonal antibody anti-SHC-1A1 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 immunization of animals immunized with Fenneropenaeus chinensis hemocyanin is successful.

[0043] In summary, the monoclonal antibody anti-SHC-1A1 against Fenneropenaeus chinensis hemocyanin of the present application can be stably expressed by recombinant Expi293 cells. The obtained monoclonal antibody anti-SHC-1A1 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.

[0044] 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 shall be covered within the protection scope of the present application.

Claims

1. A monoclonal antibody against Penaeus chinensis hemocyanin, characterized in that: The monoclonal antibody against Penaeus chinensis hemocyanin is anti-SHC-1A1; The light chain amino acid sequence of the anti-SHC-1A1 is shown in SEQ ID NO.3, and the light chain amino acid sequence of the anti-SHC-1A1 contains a light chain variable region VL and a light chain constant region CL; the heavy chain amino acid sequence of the anti-SHC-1A1 is shown in SEQ ID NO.4, and the heavy chain amino acid sequence of the 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 Chinese shrimp hemocyanin according to claim 1, the nucleotide sequence of the nucleic acid encoding the light chain amino acid sequence of anti-SHC-1A1 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-1A1 is shown in SEQ ID NO.

2.

3. A recombinant plasmid pcDNA3.1 containing the nucleic acid encoding the nucleic acid 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. The monoclonal antibody against Penaeus chinensis hemocyanin according to claim 1 is used as a positive internal reference to qualitatively judge whether the immunization of an animal immunized with Penaeus chinensis hemocyanin is successful or not, or to quantitatively detect the content of the monoclonal antibody against Penaeus chinensis hemocyanin in an animal immunized with Penaeus chinensis hemocyanin.

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