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-14B3, 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 with an application date of October 30, 2024, application number 2024115287370, and invention name “A monoclonal antibody against Chinese shrimp hemocyanin and its application”. Technical Field

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

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

[0004] Shrimp hemocyanin (SHC) is an important component of crustacean hemolymph and has multiple physiological functions. In addition to carrying oxygen, hemocyanin also has phenoloxidase activity, antibacterial and antiviral activity, metal ion transport, protein storage, osmotic pressure regulation, molting hormone transport, and participation in the solidification of the epidermis. Therefore, the study of crustacean hemocyanin has become a hot area 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 hemocyanin in Chinese shrimp can easily and quickly detect and monitor the fluctuation of hemocyanin concentration in Chinese shrimp.

[0006] The monoclonal antibodies against Penaeus chinensis hemocyanin reported in the literature are mainly expressed through hybridoma cells, but hybridoma cell expression has the technical problem of unstable expression. Summary of the invention

[0007] The purpose of the present application is to provide a monoclonal antibody against Penaeus chinensis hemocyanin in order to solve the above-mentioned problem of unstable expression of hybridoma cells. The monoclonal antibody against Penaeus chinensis 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 Penaeus chinensis hemocyanin.

[0008] The technical solution of this application In a first aspect, the present application provides a monoclonal antibody against Penaeus chinensis hemocyanin, wherein the monoclonal antibody against Penaeus chinensis hemocyanin is anti-SHC-14B3; The light chain amino acid sequence of the anti-SHC-14B3 is shown in SEQ ID NO.3, and the light chain amino acid sequence of the anti-SHC-14B3 contains a light chain variable region VL and a light chain constant region CL; the heavy chain amino acid sequence of the anti-SHC-14B3 is shown in SEQ ID NO.4, and the heavy chain amino acid sequence of the anti-SHC-14B3 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-mentioned anti-SHC-14B3 is shown as SEQ ID NO.5, CDR2 is shown as SEQ ID NO.6, and CDR3 is shown as SEQ ID NO.7; the CDR1 in the amino acid sequence of the heavy chain variable region of anti-SHC-14B3 is shown as SEQ ID NO.8, CDR2 is shown as SEQ ID NO.9, and CDR3 is shown as SEQ ID NO.10.

[0009] In a second aspect, the present application provides a nucleic acid, which encodes a monoclonal antibody anti-SHC-14B3 against Chinese shrimp hemocyanin, the nucleotide sequence of the nucleic acid encoding the light chain amino acid sequence of anti-SHC-14B3 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-14B3 is shown in SEQ ID NO.2.

[0010] By adopting the above technical scheme, the antigenic determinant cluster of the obtained monoclonal antibody anti-SHC-14B3 against Chinese shrimp hemocyanin is located on two subunits of shrimp hemocyanin with molecular weights of 73kDa and 75kDa, and has good specificity.

[0011] In a third aspect, the present application provides a hybridoma cell expressing the monoclonal antibody anti-SHC-14B3 against Penaeus chinensis hemocyanin.

[0012] In a fourth aspect, the present application provides a recombinant plasmid pcDNA3.1 containing the nucleic acid encoding the above-mentioned nucleic acid.

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

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

[0015] In a seventh aspect, the present application provides a monoclonal antibody against Penaeus chinensis hemocyanin as a positive internal reference for qualitatively judging whether the immunization of an animal immunized with Penaeus chinensis hemocyanin is successful or not, or quantitatively detecting the content of the monoclonal antibody against Penaeus chinensis hemocyanin in an animal immunized with Penaeus chinensis hemocyanin.

[0016] Beneficial technical effects of this application The monoclonal antibody anti-SHC-14B3 against Penaeus chinensis hemocyanin of the present application can be stably expressed by recombinant Expi293 cells, and the obtained monoclonal antibody against Penaeus chinensis hemocyanin has a purity of ≥90% after purification.

[0017] Furthermore, the monoclonal antibody anti-SHC-14B3 against Chinese shrimp hemocyanin of the present application has good specificity, and the antigenic determinant specifically bound to the monoclonal antibody against Chinese shrimp hemocyanin is located on the two subunits of shrimp hemocyanin with molecular weights of 73 kDa and 75 kDa.

[0018] Furthermore, the monoclonal antibody anti-SHC-14B3 against Penaeus chinensis hemocyanin of the present application can be used as a positive internal reference to qualitatively determine whether the immunization of animals immunized with Penaeus chinensis hemocyanin is successful or not, or to quantitatively detect the content of the monoclonal antibody against Penaeus chinensis hemocyanin in animals immunized with Penaeus chinensis hemocyanin. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 , an electrophoretic diagram showing the purity of the concentrated solution of the monoclonal antibody anti-SHC-14B3 against Penaeus chinensis hemocyanin determined by SDS-PAGE gel electrophoresis; Figure 2 , The Western blot was used to detect the concentrated solution of anti-SHC-14B3 monoclonal antibody against Chinese shrimp hemocyanin. DETAILED DESCRIPTION

[0020] The following is a specific embodiment and combined with the attached Figure 1-2 The present application is further described in detail, but the present application is not limited thereto.

[0021] In each embodiment of the present application, the PBS buffer used is a dilution obtained by diluting 10×PBS buffer (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd., catalog number: PS110S) 10 times with deionized water; The PBST buffer used was a dilution obtained by diluting 10×PBS / Tween buffer (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd., catalog number: PS102S) 10 times with deionized water. Example Example 1

[0022] A hybridoma cell expressing the monoclonal antibody anti-SHC-14B3 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 spleens of the BALB / c mice are removed by aseptic operation, and the adherent adipose 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 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 8 / mL of SP2 / 0 cell suspension; The SP2 / 0 cells are derived from the cell bank of the Chinese Academy of Sciences; (4) Dissolve PEG1450 (Sigma-Aldrich, catalog number: P7181) in PBS buffer, and then filter with a 0.45 μm polyvinylidene fluoride membrane (PVDF membrane) to obtain a PEG1450 solution; The amounts of PEG1450 and PBS buffer are calculated based on a ratio of PEG1450:PBS buffer of 10 g:100 mL; (5) The SP2 / 0 cell suspension obtained above was mixed with the spleen cell suspension, and the number of SP2 / 0 cells in the mixture was controlled to be 1×10 7 The number of spleen cells is 1×10 8 The cells were mixed evenly and then centrifuged at a speed of 2000 r / min for 5 min, and the supernatant was discarded to obtain a mixed cell pellet containing SP2 / 0 cells and spleen cells; (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 place at 37°C, CO 2 Incubate in a cell culture incubator with a volume percentage concentration of 5% for 1 min to obtain cell suspension I; (7) Add antibiotics to the HAT medium and mix well to obtain a HAT medium containing antibiotics; The antibiotics are penicillin and streptomycin mixed solution / double antibiotic (100×) (Shanghai Yamei Biopharmaceutical Technology Co., Ltd., catalog number: CB010); 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; (8) Add 8-10 mL of HAT medium containing antibiotics preheated to 37° C. to the cell suspension I obtained above and mix well to obtain cell suspension II; (9) Add the cell suspension II obtained above into a culture dish, and then place the culture dish containing the cell suspension II at 37°C and CO 2 The cells were cultured in a cell culture incubator at a volume percentage concentration of 5% for 5 days, and the cell growth in the culture dish was observed regularly until a cell cluster with a diameter of 100 μm was obtained, and a fusion cell clone was obtained; (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 second antibody solution is prepared by mixing horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd., catalog number: LF101) and PBS buffer in a volume ratio of 1:1000. (9) Washing: discard the liquid in the wells and wash with PBST buffer at 200 μL / well. Gently shake the plate, discard the liquid in the wells, and repeat washing 2-4 times to remove unbound secondary antibody. (10) Color development: Add the substrate 3,3',5,5'-tetramethylbenzidine (TMB) to the wells at a dosage of 50 μL / well and incubate at room temperature (25°C) for 15 min to allow it to react with the HRP on the secondary antibody; (11) Termination: Add 200 μL of 0.1 mol / L hydrochloric acid aqueous solution to each well to terminate the reaction and obtain a terminated reaction solution; (12) Determination of OD 450 Value: At a working wavelength of 450 nm, measure the OD of the termination reaction solution in each well after the termination reaction. 450 Calculate the average OD of the first antibody solution, positive control, and negative control. 450 value; Evaluation criteria: Quality control qualified (negative control average OD 450 Value < 0.2, average OD of positive control 450 After the value is greater than 1.5), select the positive wells (average OD 450 The samples with a PCR value > 1.5 were used for single cell clone screening of hybridomas.

[0026] 5. Single cell clone screening of hybridomas (1) Preparation of culture medium for single cell clone screening of hybridomas: Add fetal bovine serum (FBS) to RPMI 1640 culture medium to obtain serum-containing RPMI 1640 culture medium; The amount of FBS added is 10:90 in the volume ratio of FBS to RPMI 1640 medium, wherein FBS can be replaced by other serum supplements; (2) Dilute the sample: select the positive well sample in (12) in the above step 4 for identifying the monoclonal antibody using the Elisa method and perform limiting 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; For example, the concentration of sample hybridoma cells is 1×10 6 / mL, take out 0.1mL of sample from the sample hybridoma cells and add it to 0.9mL of PBS buffer to obtain dilution I, so that the total volume of dilution I is 1mL; then, take out 0.1mL from dilution I and add it to 0.9mL of PBS buffer to obtain dilution II; and so on to obtain the final dilution, and the final 1mL of final dilution contains 10 cells; (3) Add 0.9 mL of serum-containing RPMI 1640 medium to each well of the new plate, then add 0.1 mL of the final dilution obtained in step (2) above to each well and mix by pipetting; (4) Cultivate cells: Place the new well plate in a 37°C, CO 2 Cultured in a cell culture incubator at a volume percentage concentration of 5%; (5) When the cell coverage rate is above 60%, the monoclonal antibody is tested by using the ELISA method in step 4 above; A hybridoma monoclonal cell strain with high specificity against Penaeus chinensis hemocyanin was obtained by the above method. Example 2

[0027] A gene sequence of a monoclonal antibody anti-SHC-14B3 against Penaeus chinensis hemocyanin is obtained by a method comprising the following steps: 1. Extraction of Total RNA Total RNA was extracted from the hybridoma monoclonal cells obtained above using a total RNA extraction reagent (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd., catalog number: YY101L).

[0028] 2. 5' RACE amplification The total RNA was amplified by 5'RACE using SMARTer RACE 5' / 3'Kit (TAKARA, Cat. No. 634859) to obtain a solution containing DNA.

[0029] 3. Gel electrophoresis and purification The DNA-containing solution obtained above was subjected to agarose gel electrophoresis analysis, and the gel was cut to obtain the target fragment; then, the target fragment was purified using a DNA recovery and purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number: DP214) to obtain a purified antibody fragment corresponding to the DNA.

[0030] 4. Cloning and sequencing The purified antibody fragment was connected to 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 prior art), and then transferred into Escherichia coli DH5α (Shanghai Weidi Biotech Co., Ltd.), and the positive clones of the corresponding antibody fragments were obtained by blue-white screening method; The positive clones corresponding to the antibody fragments were selected and sequenced by the Sanger method (completed by Shanghai Qingke Biotechnology Co., Ltd.) to obtain the antibody genes corresponding to the antibody fragments; 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 the above 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; Through analysis, it can be seen that CDR1 in the amino acid sequence of the light chain variable region of the antibody gene is shown as SEQ ID NO.5, CDR2 is shown as SEQ ID NO.6, and CDR3 is shown as SEQ ID NO.7; CDR1 in the amino acid sequence of the heavy chain variable region of the antibody gene is shown as SEQ ID NO.8, CDR2 is shown as SEQ ID NO.9, and CDR3 is shown as SEQ ID NO.10. Example 3

[0031] A recombinant plasmid pcDNA3.1 containing the monoclonal antibody anti-SHC-14B3 gene against Penaeus chinensis hemocyanin is obtained by the following technical scheme: 1. Double-digest the vector plasmid pcDNA3.1 with BamH I and EcoR1 to obtain linearized plasmid pcDNA3.1; The above-mentioned vector plasmid pcDNA3.1 was sourced from GenScript Biotech Co., Ltd.; The enzyme digestion system is shown in Table 1 below: Table 1 Enzyme digestion system React at 37°C for 30 min.

[0032] 2. PCR amplification of antibody fragments Performing PCR amplification on the antibody fragment to obtain a PCR amplified antibody fragment solution; The specific components and dosage of the PCR amplification system are shown in Table 2 below: Table 2 PCR amplification system PCR amplification system components Volume / Mass Antibody fragments 1ng Upstream primer (10 μM) 2μL Downstream primer (10 μM) 2μL 2×Phanta Max Buffer 25μL dNTP Mix (10 mM each) 1μL Phanta Max Super-Fidelity DNA Polymerase 1μL <![CDATA[ddH 2 The]]> Make up to 50 μL The above 2×Phanta Max Buffer, dNTP Mix (10mM each), Phanta Max Super-Fidelity DNA Polymerase and ddH 2 O was from Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novozyme Biotechnology Co., Ltd., catalog number: P505-d1); The upstream primer sequence is AGCTCGGATCC + 20 bp sequence of the 5' of the antibody fragment; The aforementioned downstream primer sequence is GAATTCGG + 20 bp sequence of the 3' of the antibody fragment; The program settings of the PCR amplification system were: pre-denaturation (95°C, 5 min), cyclic amplification [30 cycles in total, 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 The antibody fragments amplified by PCR were connected to the linearized plasmid pcDNA3.1 using T4 DNA ligase to obtain the corresponding solution containing the recombinant plasmid pcDNA3.1; The composition and dosage of the reactants, related reagents, etc. used in the above connection process are shown in Table 3 below: Table 3 Ligation reaction system Reactants, related reagents, etc. Volume / Mass T4 DNA ligase (10U / μL) 1μL PCR amplified antibody fragments 200ng Linearized plasmid pcDNA3.1 500ng 10×T4 DNA ligase reaction buffer 2μL <![CDATA[H 2 The]]> Add to 20 μL The reactants and related reagents used in the above connection process were mixed evenly and reacted at 16°C for 12 hours.

[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 minutes, transfer it to a 42°C environment for 90 seconds, then let it stand on ice for 2 minutes, then transfer it to 5 mL of LB liquid culture medium, incubate it at 37°C for 1 hour, then take a sample and evenly spread it on a pre-prepared LB agar plate containing 10 μg / mL ampicillin antibiotics, and invert it at 37°C for 16 hours to obtain the recombinant Escherichia coli DH5α colonies corresponding to the recombinant plasmid pcDNA3.1; A single colony of the Escherichia coli DH5α corresponding to the recombinant plasmid pcDNA3.1 was selected and inoculated into 5 mL of LB liquid medium for fermentation for 16 h to obtain a fermentation broth of the recombinant Escherichia coli DH5α corresponding to the recombinant plasmid pcDNA3.1; The results of the Sanger sequencing (completed by Shanghai Qingke Biotechnology Co., Ltd.) showed that the recombinant Escherichia coli DH5α was successfully obtained.

[0035] 5. Recombinant plasmid extraction The recombinant plasmid was extracted from the fermentation broth of the recombinant Escherichia coli DH5α obtained above using a plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number: DP103) to obtain the recombinant plasmid pcDNA3.1; The results of Sanger sequencing (completed by Shanghai Qingke Biotechnology Co., Ltd.) showed that the recombinant plasmid pcDNA3.1 was successfully obtained. Example 4

[0036] A recombinant Expi293 cell containing a recombinant plasmid pcDNA3.1 is obtained by the following technical scheme: 1. Expi293 cell transfection 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 PEI transfection reagent: 10 μg recombinant plasmid pcDNA3.1 to obtain a transfection reagent / recombinant plasmid pcDNA3.1 mixture, and allowed to stand at 30°C for 15 min; The mixture of transfection reagent / recombinant plasmid pcDNA3.1 obtained above was transfected into Expi293 cells, with 10 μg of recombinant plasmid corresponding to 10 6 The ratio of Expi293 cells to recombinant Expi293 cells was calculated to obtain the recombinant Expi293 cells corresponding to the recombinant plasmid pcDNA3.1.

[0037] 2. Recombinant Expi293 Cell Culture The recombinant Expi293 cells were cultured at 37°C and CO 2 After culturing in a cell culture incubator with a volume percentage concentration of 5% for one week, a recombinant Expi293 cell culture fluid containing a monoclonal antibody against Penaeus chinensis hemocyanin was obtained; Centrifuging the above-mentioned recombinant Expi293 cell culture fluid, collecting the supernatant corresponding to the recombinant Expi293 cell culture fluid containing the monoclonal antibody against Penaeus chinensis hemocyanin; The Expi293 cells mentioned above were from Shanghai Duoning Biotechnology Co., Ltd.

[0038] 3. Antibody Purification The Protein A affinity chromatography resin (Suzhou Nanovita Technology Co., Ltd., Catalog No.: 17013-090100) was loaded into the chromatography column to form a Protein A affinity chromatography column; The Protein A affinity chromatography column was equilibrated with 3 column volumes of PBS buffer; The collected 1L supernatant of the recombinant Expi293 cell culture medium containing the monoclonal antibody against Penaeus chinensis hemocyanin was loaded into a Protein A affinity chromatography column equilibrated with PBS buffer, and the flow rate was controlled at 2mL / min; After the above-mentioned sample loading is completed, non-specifically bound proteins and impurities are first eluted using PBS buffer to retain the monoclonal antibody against Penaeus chinensis hemocyanin bound to the Protein A affinity chromatography resin; Then, the monoclonal antibody against Penaeus chinensis hemocyanin bound to the Protein A affinity chromatography resin was eluted using a 0.5 mM glycine elution buffer at pH 3.5, and the pH was adjusted to 7.2 using a 0.1 mM Tris-HCl aqueous solution at pH 10; Then ultrafiltration and concentration are performed to obtain a concentrated solution of the corresponding monoclonal antibody against Penaeus chinensis hemocyanin; The monoclonal antibody against Penaeus chinensis hemocyanin is named anti-SHC-14B3.

[0039] 4. SDS-PAGE gel electrophoresis The purity of the anti-SHC-14B3 concentrate was determined by SDS-PAGE gel electrophoresis. The electrophoresis diagram is shown in Figure 1 As shown, the purity of the monoclonal antibody anti-SHC-14B3 in the corresponding concentrated solution can be obtained by grayscale analysis ≥ 90%.

[0040] 5. Western blot The monoclonal antibody anti-SHC-14B3 concentrate was analyzed by Western blot. The specific steps are as follows: (1) Dilute the hemocyanin of Penaeus chinensis with WB loading buffer to a concentration of 1 mg / mL and boil at 95°C for 5 min to obtain a pretreated hemocyanin sample of Penaeus chinensis; The WB loading buffer mentioned above was 100 mM DTT, 1% SDS, 20% glycerol, and 1% methyl blue; (2) Add the pretreated Penaeus chinensis hemocyanin sample into the sample wells of the precast gel, add 100 ng of the pretreated Penaeus chinensis hemocyanin sample to each well, perform electrophoresis for 1 h at a constant voltage of 120 V, and remove the gel; (3) Cut a piece of nitrocellulose membrane of the same size as the gel, soak the nitrocellulose membrane and filter paper with rapid transfer buffer (Shanghai Yazyme Biotechnology Co., Ltd., catalog number: PS117), and then place them in a wet transfer apparatus in a "sandwich" manner. The order is: electrode (-) - sponge - filter paper - gel - nitrocellulose membrane - filter paper - sponge - electrode (+), constant current 300mA, power on for 1h; The pore size of the above-mentioned nitrocellulose membrane is 0.22 μm; (4) After the transfer, remove the nitrocellulose membrane, wash it with water once, and then place it in PBST buffer containing 5% skimmed milk powder and block it at 30°C for 10 minutes to prevent nonspecific binding; The above-mentioned PBST buffer containing 5% skimmed milk powder has a mass volume ratio of skimmed milk powder to PBST buffer of 5 g:100 mL; (5) Wash the nitrocellulose membrane three times with PBST buffer, 5 min each time; (6) Add the anti-SHC-14B3 obtained above as a specific primary antibody into PBST buffer 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 three times with PBST buffer, 5 min each time; (8) Add horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd., catalog number: LF101) as the secondary antibody into PBST buffer 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 three times with PBST buffer for 5 min each time to remove non-specifically bound secondary antibodies and other impurities; (10) Use ECL color developing solution to develop the final band until the color is clear. The resulting blot is as follows: Figure 2 As shown, from Figure 2 It can be seen that the antigenic determinant cluster to which the monoclonal antibody anti-SHC-14B3 against Chinese shrimp hemocyanin specifically binds is located on the two subunits of shrimp hemocyanin with molecular weights of 73kDa and 75kDa.

[0041] 6. Sequencing verification The amino acid sequence of the obtained monoclonal antibody anti-Chinese shrimp hemocyanin anti-SHC-14B3 was verified, and the results showed that the light chain amino acid sequence (light chain variable region VL + light chain constant region CL) of anti-SHC-14B3 was consistent with SEQ ID NO.3, and the heavy chain amino acid sequence (heavy chain variable region VH + heavy chain constant region CH1) of anti-SHC-14B3 was consistent with SEQ ID NO.4. Application Examples Application Example 1

[0042] A monoclonal antibody against Penaeus chinensis hemocyanin is used as a positive internal reference in the detection of the content of anti-Penaeus chinensis hemocyanin, and is obtained by the following technical scheme: According to the Elisa method of step 4 of Example 1, the anti-SHC-14B3 monoclonal antibody concentrate against Penaeus chinensis hemocyanin obtained in Example 4 was used as the first antibody solution in (6) of step 4 of Example 1, and the serum of mice immunized with Penaeus chinensis hemocyanin four times in step 2 of Example 1 was used as the control of the first antibody solution. The rest of the experimental operations were the same as those in step 4 of Example 1. The average OD 450 The values ​​are shown in Table 4 below: Table 4 Test data of application examples From the data analysis in the above table, we can see that the positive signals (average OD 450 The anti-SHC-14B3 concentrate, a monoclonal antibody against Penaeus chinensis hemocyanin, also detected a significant positive signal (mean OD 450 The value was 2.35), which showed that the monoclonal antibody anti-SHC-14B3 against Penaeus chinensis hemocyanin could be used as a positive internal reference to quantitatively detect the content of antibodies against Penaeus chinensis hemocyanin in animals immunized with Penaeus chinensis hemocyanin or to qualitatively determine whether the immunization of animals immunized with Penaeus chinensis hemocyanin was successful or not.

[0043] In summary, the anti-Penaeus chinensis hemocyanin monoclonal antibody anti-SHC-14B3 of the present application can be stably expressed by recombinant Expi293 cells, and the obtained anti-Penaeus chinensis hemocyanin monoclonal antibody anti-SHC-14B3 can specifically bind to two subunits of Penaeus chinensis hemocyanin with molecular weights of 73 kDa and 75 kDa, and can be used as a positive internal reference for content detection of the anti-Penaeus chinensis hemocyanin monoclonal antibody.

[0044] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, any equivalent changes made according to the structure, shape, and principle of the present application should be included in 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-14B3; The light chain amino acid sequence of the anti-SHC-14B3 is shown in SEQ ID NO.3, and the light chain amino acid sequence of the anti-SHC-14B3 contains a light chain variable region VL and a light chain constant region CL; the heavy chain amino acid sequence of the anti-SHC-14B3 is shown in SEQ ID NO.4, and the heavy chain amino acid sequence of the anti-SHC-14B3 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-Chinese shrimp hemocyanin anti-SHC-14B3 according to claim 1, the nucleotide sequence of the nucleic acid encoding the light chain amino acid sequence of anti-SHC-14B3 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-14B3 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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