A monoclonal antibody against Penaeus chinensis hemocyanin and its application

Abstract

The present application relates to the technical field of antibodies, and in particular to a monoclonal antibody against Penaeus chinensis hemocyanin and its application. The monoclonal antibody against Penaeus chinensis hemocyanin is anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 or anti-SHC-6D6, which can be expressed by recombinant Expi293 cells containing a recombinant plasmid pcDNA3.1 containing a monoclonal antibody gene against Penaeus chinensis hemocyanin; the monoclonal antibody against Penaeus chinensis hemocyanin has good specificity, 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 detecting the content of the monoclonal antibody against Penaeus chinensis hemocyanin in animals immunized with Penaeus chinensis hemocyanin.

Description

Technical Field

[0001] The present application relates to the field of shrimp molecular immunology, and in particular to a monoclonal antibody against Chinese shrimp hemocyanin and its application. Background Art

[0002] 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.

[0003] 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.

[0004] 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.

[0005] 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

[0006] 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.

[0007] The technical solution of this application

[0008] 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-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 or anti-SHC-6D6;

[0009] The nucleotide sequence encoding the light chain of the anti-SHC-2B3 is shown in SEQ ID NO.1, and the nucleotide sequence of the light chain of the anti-SHC-2B3 contains a light chain variable region VL and a light chain constant region CL; the nucleotide sequence encoding the heavy chain of the anti-SHC-2B3 is shown in SEQ ID NO.2, and the nucleotide sequence of the heavy chain of the anti-SHC-2B3 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;

[0010] The nucleotide sequence encoding the light chain of anti-SHC-5C8 is shown in SEQ ID NO.5, and the nucleotide sequence of the light chain of anti-SHC-5C8 contains a light chain variable region VL and a light chain constant region CL; the nucleotide sequence encoding the heavy chain of anti-SHC-5C8 is shown in SEQ ID NO.6, and the nucleotide sequence of the heavy chain of anti-SHC-5C8 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.5 is shown in SEQ ID NO.7, and the amino acid sequence corresponding to SEQ ID NO.6 is shown in SEQ ID NO.8;

[0011] The nucleotide sequence encoding the light chain of the anti-SHC-1A1 is shown in SEQ ID NO.9, and the nucleotide sequence of the light chain of the anti-SHC-1A1 contains a light chain variable region VL and a light chain constant region CL; the nucleotide sequence encoding the heavy chain of the anti-SHC-1A1 is shown in SEQ ID NO.10, and the nucleotide sequence of the heavy chain of the anti-SHC-1A1 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.9 is shown in SEQ ID NO.11, and the amino acid sequence corresponding to SEQ ID NO.10 is shown in SEQ ID NO.12;

[0012] The nucleotide sequence encoding the light chain of the anti-SHC-14B3 is shown in SEQ ID NO.13, and the nucleotide sequence of the light chain of the anti-SHC-14B3 contains a light chain variable region VL and a light chain constant region CL; the nucleotide sequence encoding the heavy chain of the anti-SHC-14B3 is shown in SEQ ID NO.14, and the nucleotide sequence of the heavy chain of the anti-SHC-14B3 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.13 is shown in SEQ ID NO.15, and the amino acid sequence corresponding to SEQ ID NO.14 is shown in SEQ ID NO.16;

[0013] The nucleotide sequence encoding the light chain of anti-SHC-6D6 is shown in SEQ ID NO.17, and the nucleotide sequence of the light chain of anti-SHC-6D6 contains a light chain variable region VL and a light chain constant region CL; the nucleotide sequence encoding the heavy chain of anti-SHC-6D6 is shown in SEQ ID NO.18, and the nucleotide sequence of the heavy chain of anti-SHC-6D6 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.17 is shown in SEQ ID NO.19, and the amino acid sequence corresponding to SEQ ID NO.18 is shown in SEQ ID NO.20;

[0014] The amino acid sequence of the light chain variable region of the anti-SHC-2B3 described above includes CDR1 as shown in SEQ ID NO.21, CDR2 as shown in SEQ ID NO.22, and CDR3 as shown in SEQ ID NO.23; the amino acid sequence of the heavy chain variable region of the anti-SHC-2B3 includes CDR1 as shown in SEQ ID NO.24, CDR2 as shown in SEQ ID NO.25, and CDR3 as shown in SEQ ID NO.26;

[0015] The amino acid sequence of the light chain variable region of anti-SHC-5C8 includes CDR1 as shown in SEQ ID NO.27, CDR2 as shown in SEQ ID NO.28, and CDR3 as shown in SEQ ID NO.29; the amino acid sequence of the heavy chain variable region of anti-SHC-5C8 includes CDR1 as shown in SEQ ID NO.30, CDR2 as shown in SEQ ID NO.31, and CDR3 as shown in SEQ ID NO.32;

[0016] The amino acid sequence of the light chain variable region of anti-SHC-1A1 includes CDR1 as shown in SEQ ID NO.33, CDR2 as shown in SEQ ID NO.34, and CDR3 as shown in SEQ ID NO.35; the amino acid sequence of the heavy chain variable region of anti-SHC-1A1 includes CDR1 as shown in SEQ ID NO.36, CDR2 as shown in SEQ ID NO.37, and CDR3 as shown in SEQ ID NO.38;

[0017] The amino acid sequence of the light chain variable region of anti-SHC-14B3 includes CDR1 as shown in SEQ ID NO.39, CDR2 as shown in SEQ ID NO.40, and CDR3 as shown in SEQ ID NO.41; the amino acid sequence of the heavy chain variable region of anti-SHC-14B3 includes CDR1 as shown in SEQ ID NO.42, CDR2 as shown in SEQ ID NO.43, and CDR3 as shown in SEQ ID NO.44;

[0018] The CDR1 in the amino acid sequence of the light chain variable region of anti-SHC-6D6 is shown as SEQ ID NO.45, the CDR2 is shown as SEQ ID NO.46, and the CDR3 is shown as SEQ ID NO.47; the CDR1 in the amino acid sequence of the heavy chain variable region of anti-SHC-6D6 is shown as SEQ ID NO.48, the CDR2 is shown as SEQ ID NO.49, and the CDR3 is shown as SEQ ID NO.50.

[0019] By adopting the above technical scheme, the antigenic determinants of the monoclonal antibodies anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6 against Chinese shrimp hemocyanin are all located on two subunits of shrimp hemocyanin with molecular weights of 73 kDa and 75 kDa, and therefore all have good specificity.

[0020] In a second aspect, a hybridoma cell expressing monoclonal antibodies anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 or anti-SHC-6D6 against hemocyanin of Penaeus chinensis.

[0021] In a third aspect, the present application provides a recombinant plasmid pcDNA3.1 containing a gene encoding the above-mentioned monoclonal antibody anti-Chinese shrimp hemocyanin anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 or anti-SHC-6D6.

[0022] In a fourth aspect, the present application provides a recombinant Escherichia coli DH5α containing the recombinant plasmid pcDNA3.1.

[0023] In a fifth aspect, the present application provides a recombinant Expi293 cell containing the recombinant plasmid pcDNA3.1.

[0024] In a sixth aspect, the present application provides the above-mentioned monoclonal antibody against Penaeus chinensis hemocyanin as a positive internal reference, which is used to qualitatively determine 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.

[0025] Beneficial technical effects of this application

[0026] The monoclonal antibodies anti-Penaeus chinensis hemocyanin, anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6, of the present application can all be stably expressed by recombinant Expi293 cells, and the purity of the obtained monoclonal antibodies against Penaeus chinensis hemocyanin after purification is ≥90%.

[0027] Furthermore, the monoclonal antibodies anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6 against Penaeus chinensis hemocyanin of the present application all have good specificity, and the antigenic determinants specifically binding to the monoclonal antibodies against Penaeus chinensis hemocyanin are all located on the two subunits of Penaeus chinensis hemocyanin with molecular weights of 73 kDa and 75 kDa.

[0028] Furthermore, the monoclonal antibodies anti-Penaeus chinensis hemocyanin, anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6, of the present application can all be used as positive internal references to qualitatively determine whether the immunization of animals immunized with Penaeus chinensis hemocyanin is successful or not, or to quantitatively detect the content of monoclonal antibodies against Penaeus chinensis hemocyanin in animals immunized with Penaeus chinensis hemocyanin. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1, electrophoretograms of purity determination of concentrated solutions of monoclonal antibodies anti-Penaeus chinensis hemocyanin, anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6, respectively, using SDS-PAGE gel electrophoresis;

[0030] Figure 2 , The Western blot was used to detect the concentrated solutions of monoclonal antibodies anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6 against Chinese shrimp hemocyanin. DETAILED DESCRIPTION

[0031] 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.

[0032] 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;

[0033] 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

[0034] A hybridoma cell expressing monoclonal antibodies anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 or anti-SHC-6D6 against Penaeus chinensis hemocyanin is obtained by a method comprising the following steps:

[0035] 1. Preparation of Antigen

[0036] According to the method described in CN118005773A, Penaeus chinensis hemocyanin is extracted and purified to obtain Penaeus chinensis hemocyanin.

[0037] 2. Immunization of mice

[0038] (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;

[0039] (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;

[0040] The above-mentioned BALB / c mice were purchased from Southern Model Organisms Corporation;

[0041] 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.

[0042] The dosage of Penaeus chinensis hemocyanin injected into the above BALB / c mice was 100 μg / mouse;

[0043] 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.

[0044] The amount of Penaeus chinensis hemocyanin injected into the above BALB / c mice was 100 μg / mouse.

[0045] 3. Cell fusion

[0046] (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;

[0047] (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;

[0048] (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;

[0049] The SP2 / 0 cells are derived from the cell bank of the Chinese Academy of Sciences;

[0050] (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;

[0051] The amounts of PEG1450 and PBS buffer are calculated based on a ratio of PEG1450:PBS buffer of 10 g:100 mL;

[0052] (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;

[0053] (6) Add 0.5 mL of PEG1450 solution preheated to 37° C. to the mixed cell pellet of SP2 / 0 cells and spleen cells obtained above, mix well and incubate in a cell culture incubator at 37° C. and 5% CO2 volume percentage concentration for 1 min to obtain cell suspension I;

[0054] (7) Add antibiotics to the HAT medium and mix well to obtain a HAT medium containing antibiotics;

[0055] The antibiotics are penicillin and streptomycin mixed solution / double antibiotic (100×) (Shanghai Yamei Biopharmaceutical Technology Co., Ltd., catalog number: CB010);

[0056] 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;

[0057] (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;

[0058] (9) Add the cell suspension II obtained above to a culture dish, and then place the culture dish containing the cell suspension II in a cell culture incubator at 37° C. and a CO2 volume percentage concentration of 5% for 5 days, and regularly observe the cell growth in the culture dish until a cell cluster with a diameter of 100 μm is obtained, thereby obtaining a fusion cell clone;

[0059] (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).

[0060] 4. Identify monoclonal antibodies using Elisa

[0061] (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;

[0062] (2) Coating: Add 50 μL of coating solution to each well of a 96-well plate and coat at 4°C for 16 h.

[0063] (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.

[0064] (4) Blocking: Add blocking buffer at 100 μL / well and incubate at room temperature (25°C) for 1-2 h to prevent nonspecific binding;

[0065] 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;

[0066] (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.

[0067] (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.

[0068] (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.

[0069] (8) Secondary antibody incubation: Add the secondary antibody solution at 200 μL / well and incubate at room temperature (25°C) for 1 h.

[0070] 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.

[0071] (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.

[0072] (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;

[0073] (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;

[0074] (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;

[0075] 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.

[0076] 5. Single cell clone screening of hybridomas

[0077] (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;

[0078] The volume ratio of FBS to RPMI 1640 medium is 10:90, and FBS can be replaced by other serum supplements.

[0079] (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;

[0080] 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;

[0081] (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;

[0082] (4) Cultivate cells: Place the new well plate in a cell culture incubator at 37°C and 5% CO2 volume concentration for culturing;

[0083] (5) When the cell coverage rate is above 60%, the monoclonal antibody is tested by using the ELISA method in step 4 above;

[0084] Five hybridoma monoclonal cells with high specificity against Penaeus chinensis hemocyanin were obtained by the above method.

[0085] Example 2

[0086] A gene sequence of a monoclonal antibody anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 or anti-SHC-6D6 against Penaeus chinensis hemocyanin is obtained by a method comprising the following steps:

[0087] 1. Extraction of Total RNA

[0088] Total RNA was extracted from the five hybridoma monoclonal cells obtained above using a total RNA extraction reagent (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd., catalog number: YY101L), and named as total RNA I, total RNA II, total RNA III, total RNA IV and total RNA V, respectively.

[0089] 2. 5' RACE amplification

[0090] Total RNA I, total RNA II, total RNA III, total RNA IV and total RNA V were respectively subjected to 5'RACE amplification using SMARTer RACE 5' / 3'Kit (TAKARA, catalog number: 634859) to obtain solutions containing DNA I, DNA II, DNA III, DNA IV and DNA V, respectively.

[0091] 3. Gel electrophoresis and purification

[0092] The solutions containing DNA I, DNA II, DNA III, DNA IV and DNA V obtained above were subjected to agarose gel electrophoresis analysis, and the gels were cut to obtain the target fragments; then, the target fragments were purified using a DNA recovery and purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Catalog No.: DP214) to obtain purified antibody fragments I, antibody fragment II, antibody fragment III, antibody fragment IV and antibody fragment V corresponding to DNA I, DNA II, DNA III, DNA IV and DNA V.

[0093] 4. Cloning and sequencing

[0094] The purified antibody fragment I, antibody fragment II, antibody fragment III, antibody fragment IV and antibody fragment V were connected to the pMD19-T vector (GenScript Biotech Co., Ltd.) by a vector construction method (the vector construction method is a common vector construction method in the prior art), and then respectively transferred into Escherichia coli DH5α (Shanghai Weidi Biotechnology Co., Ltd.), and the positive clones corresponding to the antibody fragment I, antibody fragment II, antibody fragment III, antibody fragment IV and antibody fragment V were obtained by blue-white screening method;

[0095] The positive clones corresponding to antibody fragment I, antibody fragment II, antibody fragment III, antibody fragment IV and antibody fragment V were picked out respectively, and sequenced by Sanger method (completed by Shanghai Qingke Biotechnology Co., Ltd.) to obtain antibody gene I, antibody gene II, antibody gene III, antibody gene IV, antibody gene V corresponding to antibody fragment I, antibody fragment II, antibody fragment III, antibody fragment IV and antibody fragment V respectively;

[0096] The nucleotide sequence encoding the light chain of the antibody gene I is shown in SEQ ID NO.1, and the nucleotide sequence of the light chain of the antibody gene I 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 I is shown in SEQ ID NO.2, and the nucleotide sequence of the heavy chain of the antibody gene I 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;

[0097] The nucleotide sequence encoding the light chain of the antibody gene II is shown in SEQ ID NO.5, and the nucleotide sequence of the light chain of the antibody gene II 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 II is shown in SEQ ID NO.6, and the nucleotide sequence of the heavy chain of the antibody gene II 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.5 is shown in SEQ ID NO.7, and the amino acid sequence corresponding to SEQ ID NO.6 is shown in SEQ ID NO.8;

[0098] The nucleotide sequence encoding the light chain of the antibody gene III is shown in SEQ ID NO.9, and the nucleotide sequence of the light chain of the antibody gene III 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 III is shown in SEQ ID NO.10, and the nucleotide sequence of the heavy chain of the antibody gene III 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.9 is shown in SEQ ID NO.11, and the amino acid sequence corresponding to SEQ ID NO.10 is shown in SEQ ID NO.12;

[0099] The nucleotide sequence encoding the light chain of the antibody gene IV is shown in SEQ ID NO.13, and the nucleotide sequence of the light chain of the antibody gene IV 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 IV is shown in SEQ ID NO.14, and the nucleotide sequence of the heavy chain of the antibody gene IV 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.13 is shown in SEQ ID NO.15, and the amino acid sequence corresponding to SEQ ID NO.14 is shown in SEQ ID NO.16;

[0100] The nucleotide sequence encoding the light chain of the antibody gene V is shown in SEQ ID NO.17, and the nucleotide sequence of the light chain of the antibody gene V 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 V is shown in SEQ ID NO.18, and the nucleotide sequence of the heavy chain of the antibody gene V 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.17 is shown in SEQ ID NO.19, and the amino acid sequence corresponding to SEQ ID NO.18 is shown in SEQ ID NO.20;

[0101] Through analysis, it can be seen that CDR1 in the amino acid sequence of the light chain variable region of antibody gene I is shown as SEQ ID NO.21, CDR2 is shown as SEQ ID NO.22, and CDR3 is shown as SEQ ID NO.23; CDR1 in the amino acid sequence of the heavy chain variable region of antibody gene I is shown as SEQ ID NO.24, CDR2 is shown as SEQ ID NO.25, and CDR3 is shown as SEQ ID NO.26;

[0102] The amino acid sequence of the light chain variable region of antibody gene II includes CDR1 as shown in SEQ ID NO.27, CDR2 as shown in SEQ ID NO.28, and CDR3 as shown in SEQ ID NO.29; the amino acid sequence of the heavy chain variable region of antibody gene II includes CDR1 as shown in SEQ ID NO.30, CDR2 as shown in SEQ ID NO.31, and CDR3 as shown in SEQ ID NO.32;

[0103] The amino acid sequence of the light chain variable region of antibody gene III includes CDR1 as shown in SEQ ID NO.33, CDR2 as shown in SEQ ID NO.34, and CDR3 as shown in SEQ ID NO.35; the amino acid sequence of the heavy chain variable region of antibody gene III includes CDR1 as shown in SEQ ID NO.36, CDR2 as shown in SEQ ID NO.37, and CDR3 as shown in SEQ ID NO.38;

[0104] The amino acid sequence of the light chain variable region of antibody gene IV includes CDR1 as shown in SEQ ID NO.39, CDR2 as shown in SEQ ID NO.40, and CDR3 as shown in SEQ ID NO.41; the amino acid sequence of the heavy chain variable region of antibody gene IV includes CDR1 as shown in SEQ ID NO.42, CDR2 as shown in SEQ ID NO.43, and CDR3 as shown in SEQ ID NO.44;

[0105] The CDR1 in the amino acid sequence of the light chain variable region of the antibody gene V is shown as SEQ ID NO.45, the CDR2 is shown as SEQ ID NO.46, and the CDR3 is shown as SEQ ID NO.47; the CDR1 in the amino acid sequence of the heavy chain variable region of the antibody gene V is shown as SEQ ID NO.48, the CDR2 is shown as SEQ ID NO.49, and the CDR3 is shown as SEQ ID NO.50.

[0106] Example 3

[0107] A recombinant plasmid pcDNA3.1 containing the monoclonal antibody anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 or anti-SHC-6D6 gene against Penaeus chinensis hemocyanin is obtained by the following technical scheme:

[0108] 1. Double-digest the vector plasmid pcDNA3.1 with BamH I and EcoR1 to obtain linearized plasmid pcDNA3.1;

[0109] The above-mentioned vector plasmid pcDNA3.1 was sourced from GenScript Biotech Co., Ltd.;

[0110] The enzyme digestion system is shown in the following table:

[0111] Enzyme digestion system components Volume / Mass Vector plasmid pcDNA3.1 1 μg BamHI (10U / μL) 1μL EcoR1 (10U / μL) 1μL 10× Buffer 1μL <![CDATA[H2O]]> Make up to 10 μL

[0112] React at 37°C for 30 min.

[0113] 2. Perform PCR amplification on antibody fragment I, antibody fragment II, antibody fragment III, antibody fragment IV and antibody fragment V respectively

[0114] Respectively performing PCR amplification on antibody fragment I, antibody fragment II, antibody fragment III, antibody fragment IV and antibody fragment V to obtain PCR amplified antibody fragment I, antibody fragment II, antibody fragment III, antibody fragment IV and antibody fragment V solutions, respectively;

[0115] The specific components and dosage of the PCR amplification system are shown in the following table:

[0116] PCR amplification system components Volume / Mass Antibody fragment I, antibody fragment II, antibody fragment III, antibody fragment IV or antibody fragment V 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[ddH2O]]> Make up to 50 μL

[0117] The above-mentioned 2×Phanta Max Buffer, dNTP Mix (10 mM each), Phanta Max Super-Fidelity DNA Polymerase and ddH2O were all from Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novogene Biotech Co., Ltd., Cat. No.: P505-d1);

[0118] The upstream primer sequences are AGCTCGGATCC + 20 bp sequence of 5' of antibody fragment I, antibody fragment II, antibody fragment III, antibody fragment IV or antibody fragment V;

[0119] The aforementioned downstream primer sequences are GAATTCGG + 20 bp sequence of the 3' of antibody fragment I, antibody fragment II, antibody fragment III, antibody fragment IV or antibody fragment V;

[0120] 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).

[0121] 3. Ligation reaction

[0122] Using T4 DNA ligase, the antibody fragment I, antibody fragment II, antibody fragment III, antibody fragment IV and antibody fragment V amplified by PCR were connected to the linearized plasmid pcDNA3.1, respectively, to obtain corresponding solutions containing recombinant plasmid pcDNA3.1 I, recombinant plasmid pcDNA3.1 II, recombinant plasmid pcDNA3.1 III, recombinant plasmid pcDNA3.1 IV and recombinant plasmid pcDNA3.1 V, respectively;

[0123] The composition and dosage of the reactants, related reagents, etc. used in the above connection process are shown in the following table:

[0124] Reactants, related reagents, etc. Volume / Mass T4 DNA ligase (10U / μL) 1μL Antibody fragment I, antibody fragment II, antibody fragment III, antibody fragment IV or antibody fragment V after PCR amplification 200ng Linearized plasmid pcDNA3.1 500ng 10×T4 DNA ligase reaction buffer 2μL <![CDATA[H2O]]> Add to 20 μL

[0125] The reactants and related reagents used in the above connection process were mixed evenly and reacted at 16°C for 12 hours.

[0126] 4. Transformation of recombinant plasmid

[0127] Take 10 μL of the solution containing recombinant plasmid pcDNA3.1 I, recombinant plasmid pcDNA3.1 II, recombinant plasmid pcDNA3.1III, recombinant plasmid pcDNA3.1 IV or recombinant plasmid pcDNA3.1 V, 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, transfer it to a 42°C environment for 90 s, then let it stand on ice for 2 min, then transfer it to 5 mL LB liquid culture medium, incubate it at 37°C for 1 h, then take samples and evenly spread them on the pre-prepared LB agar plate containing 10 μg / mL ampicillin antibiotics, and invert and culture at 37°C for 16 h to obtain recombinant Escherichia coli DH5α I, recombinant Escherichia coli DH5α corresponding to recombinant plasmid pcDNA3.1 I, recombinant plasmid pcDNA3.1 II, recombinant plasmid pcDNA3.1 III, recombinant plasmid pcDNA3.1 IV and recombinant plasmid pcDNA3.1 V, respectively. II, recombinant E. coli DH5α III, recombinant E. coli DH5α IV, and recombinant E. coli DH5α V colonies;

[0128] Pick the above-mentioned single colonies of Escherichia coli DH5α corresponding to the recombinant plasmid pcDNA3.1 I, recombinant plasmid pcDNA3.1 II, recombinant plasmid pcDNA3.1 III, recombinant plasmid pcDNA3.1 IV and recombinant plasmid pcDNA3.1 V and inoculate them into 5 mL LB liquid culture medium for fermentation for 16 hours, and obtain the fermentation broths of recombinant Escherichia coli DH5α I, recombinant Escherichia coli DH5α II, recombinant Escherichia coli DH5α III, recombinant Escherichia coli DH5αIV and recombinant Escherichia coli DH5α V corresponding to the recombinant plasmid pcDNA3.1 I, recombinant plasmid pcDNA3.1 II, recombinant plasmid pcDNA3.1 III, recombinant plasmid pcDNA3.1 IV and recombinant plasmid pcDNA3.1 V respectively;

[0129] Sanger sequencing (completed by Shanghai Qingke Biotechnology Co., Ltd.) was used for verification, and the results showed that the recombinant Escherichia coli DH5α I, recombinant Escherichia coli DH5α II, recombinant Escherichia coli DH5α III, recombinant Escherichia coli DH5α IV, and recombinant Escherichia coli DH5α V were successfully obtained.

[0130] 5. Recombinant plasmid extraction

[0131] The fermentation broths of the recombinant E. coli DH5α I, recombinant E. coli DH5α II, recombinant E. coli DH5α III, recombinant E. coli DH5α IV, and recombinant E. coli DH5α V obtained above were used to extract recombinant plasmids using a plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number: DP103) to obtain recombinant plasmids pcDNA3.1 I, recombinant plasmids pcDNA3.1 II, recombinant plasmids pcDNA3.1 III, recombinant plasmids pcDNA3.1 IV, and recombinant plasmids pcDNA3.1V, respectively;

[0132] Sanger sequencing (completed by Shanghai Qingke Biotechnology Co., Ltd.) was used for verification, and the results showed that recombinant plasmid pcDNA3.1 I, recombinant plasmid pcDNA3.1 II, recombinant plasmid pcDNA3.1 III, recombinant plasmid pcDNA3.1 IV, and recombinant plasmid pcDNA3.1 V were successfully obtained.

[0133] Example 4

[0134] A recombinant Expi293 cell containing a recombinant plasmid pcDNA3.1 I, a recombinant plasmid pcDNA3.1 II, a recombinant plasmid pcDNA3.1 III, a recombinant plasmid pcDNA3.1 IV or a recombinant plasmid pcDNA3.1 V is obtained by the following technical scheme:

[0135] 1. Expi293 cell transfection

[0136] PEI transfection reagent (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: 5644123) and recombinant plasmid were mixed at a ratio of 0.1 mL PEI transfection reagent: 10 μg recombinant plasmid to obtain a transfection reagent / recombinant plasmid mixture, and allowed to stand at 30°C for 15 min;

[0137] The recombinant plasmids are respectively the above-mentioned recombinant plasmid pcDNA3.1 I, recombinant plasmid pcDNA3.1 II, recombinant plasmid pcDNA3.1 III, recombinant plasmid pcDNA3.1 IV and recombinant plasmid pcDNA3.1 V;

[0138] The transfection reagent / recombinant plasmid mixture obtained above was transfected into Expi293 cells, with 10 μg of recombinant plasmid corresponding to 10 6The ratio of Expi293 cells was calculated, and the recombinant Expi293 cells I, recombinant Expi293 cells II, recombinant Expi293 cells III, recombinant Expi293 cells IV and recombinant Expi293 cells V corresponding to the recombinant plasmid pcDNA3.1 I, recombinant plasmid pcDNA3.1 II, recombinant plasmid pcDNA3.1 III, recombinant plasmid pcDNA3.1 IV and recombinant plasmid pcDNA3.1 V were obtained respectively.

[0139] 2. Recombinant Expi293 Cell Culture

[0140] Recombinant Expi293 cells I, recombinant Expi293 cells II, recombinant Expi293 cells III, recombinant Expi293 cells IV, and recombinant Expi293 cells V were cultured in a cell culture incubator at 37° C. and a CO2 volume percentage concentration of 5% for one week, and recombinant Expi293 cell culture fluids containing monoclonal antibody I against Penaeus chinensis hemocyanin, monoclonal antibody II against Penaeus chinensis hemocyanin, monoclonal antibody III against Penaeus chinensis hemocyanin, monoclonal antibody IV against Penaeus chinensis hemocyanin, and monoclonal antibody V against Penaeus chinensis hemocyanin were obtained respectively;

[0141] The recombinant Expi293 cell culture fluid is centrifuged to collect the supernatant of the recombinant Expi293 cell culture fluid containing monoclonal antibody I against Penaeus chinensis hemocyanin, monoclonal antibody II against Penaeus chinensis hemocyanin, monoclonal antibody III against Penaeus chinensis hemocyanin, monoclonal antibody IV against Penaeus chinensis hemocyanin, and monoclonal antibody V against Penaeus chinensis hemocyanin respectively;

[0142] The Expi293 cells mentioned above were from Shanghai Duoning Biotechnology Co., Ltd.

[0143] 3. Antibody Purification

[0144] 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;

[0145] The Protein A affinity chromatography column was equilibrated with 3 column volumes of PBS buffer;

[0146] 1 L of the collected supernatant of the recombinant Expi293 cell culture medium containing monoclonal antibody I against Penaeus chinensis hemocyanin, monoclonal antibody II against Penaeus chinensis hemocyanin, monoclonal antibody III against Penaeus chinensis hemocyanin, monoclonal antibody IV against Penaeus chinensis hemocyanin, and monoclonal antibody V against Penaeus chinensis hemocyanin were loaded onto a Protein A affinity chromatography column equilibrated with PBS buffer, respectively, and the flow rate was controlled at 2 mL / min;

[0147] After the above-mentioned sample addition is completed, firstly, non-specifically bound proteins and impurities are eluted using PBS buffer to respectively retain the monoclonal antibody I against Penaeus chinensis hemocyanin, the monoclonal antibody II against Penaeus chinensis hemocyanin, the monoclonal antibody III against Penaeus chinensis hemocyanin, the monoclonal antibody IV against Penaeus chinensis hemocyanin, and the monoclonal antibody V against Penaeus chinensis hemocyanin bound to the Protein A affinity chromatography resin;

[0148] Then, the monoclonal antibodies I, II, III, IV, and V bound to the Protein A affinity chromatography resin were eluted using a 0.5 mM glycine elution buffer at pH 3.5, and adjusted to pH 7.2 using a 0.1 mM Tris-HCl aqueous solution at pH 10;

[0149] Then, ultrafiltration and concentration are performed to obtain corresponding concentrated solutions of monoclonal antibody I against hemocyanin of Penaeus chinensis, monoclonal antibody II against hemocyanin of Penaeus chinensis, monoclonal antibody III against hemocyanin of Penaeus chinensis, monoclonal antibody IV against hemocyanin of Penaeus chinensis, and monoclonal antibody V against hemocyanin of Penaeus chinensis;

[0150] The above-mentioned monoclonal antibody I against Penaeus chinensis hemocyanin, monoclonal antibody II against Penaeus chinensis hemocyanin, monoclonal antibody III against Penaeus chinensis hemocyanin, monoclonal antibody IV against Penaeus chinensis hemocyanin, and monoclonal antibody V against Penaeus chinensis hemocyanin are named anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6, respectively.

[0151] 4. SDS-PAGE gel electrophoresis

[0152] The purity of the concentrates of anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6 was determined by SDS-PAGE gel electrophoresis. The electrophoretic diagrams are shown in Figure 1 As shown, grayscale analysis showed that the purities of monoclonal antibodies anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6 in the corresponding concentrates were all ≥90%.

[0153] 5. Western blot

[0154] The monoclonal antibodies anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6 concentrates were analyzed by Western blot. The specific steps are as follows:

[0155] (1) Dilute the Penaeus chinensis hemocyanin with WB loading buffer to a concentration of 1 mg / mL and boil at 95°C for 5 min to obtain a pretreated Penaeus chinensis hemocyanin sample;

[0156] The WB loading buffer mentioned above was 100 mM DTT, 1% SDS, 20% glycerol, and 1% methyl blue;

[0157] (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;

[0158] (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;

[0159] The pore size of the above-mentioned nitrocellulose membrane is 0.22 μm;

[0160] (4) After the transfer, remove the nitrocellulose membrane, wash it once with water, 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;

[0161] 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;

[0162] (5) Wash the nitrocellulose membrane three times with PBST buffer, 5 min each time;

[0163] (6) The anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3, and anti-SHC-6D6 obtained above were added as specific primary antibodies to PBST buffer containing 5% skim milk powder to obtain primary antibody solutions with a concentration of 1 μg / mL. The nitrocellulose membranes were placed in the primary antibody solutions and incubated at room temperature (25°C) for 1 h.

[0164] (7) Wash the nitrocellulose membrane three times with PBST buffer, 5 min each time;

[0165] (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;

[0166] (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;

[0167] (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 determinants to which the monoclonal antibodies anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6 against Penaeus chinensis hemocyanin specifically bind are all located on the two subunits of the shrimp hemocyanin with molecular weights of 73 kDa and 75 kDa.

[0168] 6. Sequencing verification

[0169] The obtained monoclonal antibodies anti-Chinese shrimp hemocyanin anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6 were verified for amino acid sequences, and the results showed that the light chain amino acid sequence (light chain variable region VL + light chain constant region CL) of anti-SHC-2B3 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-2B3 was consistent with SEQ ID NO.4; the light chain amino acid sequence (light chain variable region VL + light chain constant region CL) of anti-SHC-5C8 was consistent with SEQ ID NO.7, and the heavy chain amino acid sequence (heavy chain variable region VH + heavy chain constant region CH1) of anti-SHC-5C8 was consistent with SEQ ID NO.8; the light chain amino acid sequence (light chain variable region VL + light chain constant region CL) of anti-SHC-1A1 was consistent with SEQ ID NO. NO.11, the heavy chain amino acid sequence (heavy chain variable region VH + heavy chain constant region CH1) of anti-SHC-1A1 is consistent with SEQ ID NO.12; the light chain amino acid sequence (light chain variable region VL + light chain constant region CL) of anti-SHC-14B3 is consistent with SEQ ID NO.15, and the heavy chain amino acid sequence (heavy chain variable region VH + heavy chain constant region CH1) of anti-SHC-14B3 is consistent with SEQ ID NO.16; the light chain amino acid sequence (light chain variable region VL + light chain constant region CL) of anti-SHC-6D6 is consistent with SEQ ID NO.19, and the heavy chain amino acid sequence (heavy chain variable region VH + heavy chain constant region CH1) of anti-SHC-6D6 is consistent with SEQ ID NO.20.

[0170] Application Examples

[0171] Application Example 1

[0172] 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:

[0173] According to the Elisa method of step 4 of Example 1, the anti-Penaeus chinensis hemocyanin monoclonal antibodies anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6 concentrated solutions obtained in Example 4 were used as the first antibody solution in step 4 (6) of Example 1, and the serum of mice immunized with Penaeus 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 average OD 450 The values ​​are shown in the following table:

[0174] Primary antibody sample <![CDATA[Average OD 450 value]]> anti-SHC-2B3 concentrate 2.11 anti-SHC-5C8 concentrate 2.24 anti-SHC-1A1 concentrate 2.14 anti-SHC-14B3 concentrate 2.35 anti-SHC-6D6 concentrate 2.21 Serum of mice immunized with Penaeus chinensis hemocyanin four times 2.09 Negative control 0.14

[0175] From the data analysis in the above table, we can see that the positive signals (average OD 450 The anti-Penaeus chinensis hemocyanin monoclonal antibodies anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6 concentrates also detected obvious positive signals (average OD 450 The values ​​were 2.11-2.35), indicating that the monoclonal antibodies anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6 against Penaeus chinensis hemocyanin can all be used as positive internal controls 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 is successful or not.

[0176] In summary, the monoclonal antibodies anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6 against Penaeus chinensis hemocyanin of the present application can all be stably expressed by recombinant Expi293 cells, and the obtained monoclonal antibodies anti-SHC-2B3, anti-SHC-5C8, anti-SHC-1A1, anti-SHC-14B3 and anti-SHC-6D6 against Penaeus chinensis hemocyanin can all be specifically bound to the two subunits of Penaeus chinensis hemocyanin with molecular weights of 73 kDa and 75 kDa, and can all be used as positive internal references for content detection of monoclonal antibodies against Penaeus chinensis hemocyanin.

[0177] 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-2B3; The light chain amino acid sequence of the anti-SHC-2B3 is shown in SEQ ID NO.3, and the light chain amino acid sequence of the anti-SHC-2B3 contains a light chain variable region VL and a light chain constant region CL; the heavy chain amino acid sequence of the anti-SHC-2B3 is shown in SEQ ID NO.4, and the heavy chain amino acid sequence of the anti-SHC-2B3 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-2B3 according to claim 1; the nucleotide sequence of the nucleic acid encoding the light chain amino acid sequence of anti-SHC-2B3 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-2B3 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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