A monoclonal antibody cell line 6F6 targeting camel immunoglobulin G and its application

Abstract

The present invention discloses a monoclonal antibody cell strain 6F6 for camel immunoglobulin G and its application. The 6F6 strain has been deposited in the General Microbiology Center CGMCC of the China Microbiological Culture Collection Administration Committee, and its address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named as a monoclonal antibody cell strain of camel immunoglobulin G (IgG), with a deposit date of August 9, 2023, and a deposit number of CGMCC No.45650. Through artificial expression, purification, immunization and screening of camel IgG protein, a monoclonal antibody cell strain 6F6 that can stably and efficiently express camel IgG was obtained. It was identified as the IgG1 subtype, and the recognition epitope was KEPPSMRLKARPG. It has been successfully applied to the antibody detection of camel brucellosis, and the established method has good sensitivity and specificity.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a monoclonal antibody cell line 6F6 targeting camel immunoglobulin G (IgG) and applications thereof. Background Art

[0002] With the development of my country's economy and the improvement of awareness of food quality, camels, as economic animals with specific uses, are generating more and more value in industries such as textiles, food and tourism. According to relevant data from the National Bureau of Statistics, the number of camels raised has been steadily increasing in the past decade. However, compared with other domestic animals, there is a very scarce variety of biological products such as antibodies, vaccines and diagnostic products for camels, which makes it difficult to prevent and control camel diseases.

[0003] Immunoglobulin G (IgG) is the most important immunoglobulin produced by the body and is the main component of immunoglobulins, accounting for about 75% of the total immunoglobulin content in serum. IgG is the most important antibody in the body, with antiviral, virus neutralization, antibacterial and immune regulation functions. Changes in IgG often indicate changes in the occurrence and development of diseases. However, there are currently no monoclonal antibodies and products targeting camel IgG, which restricts the diagnosis of camel diseases and the development of related products.

[0004] Camels are one of the important natural hosts of Brucella. At present, the diagnosis of camel brucellosis at home and abroad is still based on cattle, sheep and other animals, and the traditional agglutination tests such as tiger red plate agglutination test and test tube agglutination test are still the main references. However, the sensitivity and specificity of the above methods are poor, which is prone to missed diagnosis or mistaken killing. There has been a blank in the diagnosis method at home and abroad, so it is urgent to create and optimize it. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a monoclonal antibody cell line 6F6 against camel immunoglobulin G (IgG) and its application, so as to achieve efficient and stable expression of camel IgG antibodies, which can be used in the research of camel-related biology and epidemic diseases.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0007] In the first aspect, a monoclonal antibody cell line 6F6 against camel immunoglobulin G is provided. The 6F6 strain has been deposited in the General Microbiology Center of the China Culture Collection Administration CGMCC, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named as the monoclonal antibody cell line of camel immunoglobulin G (IgG), with a deposit date of August 9, 2023 and a deposit number of CGMCC No.45650.

[0008] In a second aspect, a monoclonal antibody of camel immunoglobulin G is provided, which is secreted by the 6F6 strain.

[0009] In a third aspect, a composition for detecting camel immunoglobulin G is provided, wherein the composition contains 6F6 strain and / or monoclonal antibody.

[0010] In a fourth aspect, a kit for detecting camel immunoglobulin G is provided, the kit containing one or more of the 6F6 strain, a monoclonal antibody and a composition.

[0011] In a fifth aspect, a kit comprising a 6F6 strain, a monoclonal antibody and a combination is provided for use in detecting camel immunoglobulin G.

[0012] The beneficial effects of the present invention are:

[0013] 1. The present invention develops a monoclonal antibody against camel immunoglobulin G, which makes up for the plight of no monoclonal antibody specifically against camel IgG at home and abroad.

[0014] 2. The monoclonal antibody developed by the present invention has a good reaction effect on camel IgG, and the effective antibody titer is above 1:64000.

[0015] 3. The monoclonal antibodies developed by the present invention have no cross-reactivity with immunoglobulins of other animals.

[0016] 4. The present invention can be applied to the research of camel-related biology and epidemic diseases.

[0017] 5. The monoclonal antibody developed by the present invention is used in the detection of camel brucellosis antibodies, and the sensitivity and specificity of the established kit are both above 95%. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the SDS-PAGE image of the purified monoclonal antibody;

[0019] Figure 2 This is the result diagram of the optimal working concentration of HRP-labeled anti-camel IgG monoclonal antibody;

[0020] Figure 3 This is the result of indirect ELISA test of known positive and negative serum;

[0021] Figure 4 This is the ROC curve of the camel brucellosis indirect ELISA method based on HRP-labeled anti-camel IgG monoclonal antibody. DETAILED DESCRIPTION

[0022] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0023] Example 1

[0024] ——Expression and purification of camel IgG protein

[0025] (1) Design upstream and downstream primers based on the gene sequence of camel IgG from NCBI (gene number: NM_001303570.1), insert restriction site sequences into the upstream and downstream primers, and design primers. Primer sequence F: ctcgaggcagggcctgct, R: ggatcctcagcatctttggaaag.

[0026] (2) Camel peripheral blood leukocyte RNA was used as a template to amplify the target fragment after reverse transcription. The target fragment was 720 bp.

[0027] (3) The PCR product and the PET-28a empty vector were digested with enzymes respectively, and the digested products were ligated overnight and transformed into BL21 competent cells.

[0028] (4) After culturing at 37°C for 16 to 18 hours, single colonies were picked for identification. Positive clones were inoculated into LB medium and cultured at 37°C with shaking. When the absorbance reached 0.4 to 0.8, IPTG at a final concentration of 1 mM was added for induction for 3 to 6 hours.

[0029] (5) The culture medium was centrifuged to remove the supernatant, the bacterial cells were collected, and washed 2-3 times with 0.01 M phosphate buffer. After removing the culture medium, the precipitate was resuspended and disrupted by ultrasonication.

[0030] (6) The product after sonication is centrifuged to remove the precipitate and then purified using a nickel column to obtain the target protein.

[0031] Example 2

[0032] ——Screening, preparation and identification of camel IgG monoclonal antibodies

[0033] (1) The expressed and purified camel IgG protein was diluted to 0.1 mg / ml with physiological saline, emulsified with an equal volume of Freund's complete adjuvant, and then subcutaneously injected into 8-week-old BALB / c mice. Fourteen days after immunization, Freund's incomplete adjuvant was used for another immunization at the same dose, and the immunization was repeated three times in succession.

[0034] (2) After the last immunization, collect the mouse serum and measure the IgG and IgM content of each mouse serum against the immunogen. If the serum is mainly IgG, fusion can be performed. Use the same dose of antigen without adding adjuvant for booster immunization 2 to 3 days before fusion. Otherwise, re-immunization should be performed according to the original method until IgG is mainly present in the serum.

[0035] (3) The spleen cells of the mice were fused with SP2 / 0 myeloma cells according to standard procedures.

[0036] (4) 7 to 10 days after fusion, the hybridoma cell supernatant was collected and an indirect enzyme-linked immunosorbent assay was performed using a 96-well ELISA plate coated with LPS to screen for hybridoma cell lines that strongly reacted with LPS.

[0037] (5) Subcloning was performed three times according to the limiting dilution method to obtain a monoclonal antibody cell line. After expansion culture, the cell line was cryopreserved in liquid nitrogen.

[0038] (6) Experimental mice were intraperitoneally injected with 0.4 mL of liquid paraffin or Freund's incomplete adjuvant 7 to 10 days before ascites preparation, with 0.2 mL in each abdominal cavity.

[0039] (7) The revived and expanded monoclonal antibody cells were gently washed three times with serum-free DMEM, blown off and counted. 5 The cells were injected intraperitoneally into mice, and the ascites of the mice were collected 10 days after the injection.

[0040] (8) The collected mouse ascites was centrifuged at 8000-10000 rpm / min at 4°C for 10 minutes, the supernatant was collected, the titer was determined, and then stored at -80°C for later use.

[0041] Example 3

[0042] ——Determination of monoclonal antibody titer

[0043] (1) Add 100 μl (1 μg / mL) of target protein (camel IgG protein) to each well of the ELISA plate and coat overnight.

[0044] (2) After washing, add 100 μl of PBS containing 5% skim milk to each well, block at 37°C for 2 hours, and tap the cleaned plate to use as an ELISA plate.

[0045] (3) The prepared ascites was diluted 1:1000 with PBS solution, and then diluted serially to 1:128000.

[0046] (4) Add 100 μl of diluted monoclonal antibody to each well and vortex to mix for 5 minutes.

[0047] (5) After incubation at 37°C for 30 minutes, remove the reaction plate, discard the reaction solution, add 300 μl of 1× washing solution to each well, wash three times, and spin dry.

[0048] (6) Dilute HRP-labeled goat anti-mouse IgG with PBS at a ratio of 1:10,000 and add 100 μl to each well. Incubate at 37°C for 30 minutes, wash three times, and spin dry.

[0049] (7) Immediately add the substrate color development solution to the ELISA reaction plate, 100 μl / well, and color development at room temperature in the dark for 15 minutes. Then add 50 μl of stop solution to each well to terminate the reaction. After the reaction is terminated, measure the OD value with an ELISA reader within 20 minutes. 450nm Value. 50nm The maximum dilution ≥0.2 was taken as the final titer of the mAb.

[0050] (8) The OD value of the monoclonal antibody used in the present invention when diluted 64,000 times 450nm The reading is 0.239, so the antibody titer is 64,000.

[0051] Example 4

[0052] ——Purification and identification of monoclonal antibodies

[0053] (1) Take 1 portion of pretreated ascites and add 2 portions of 0.06 mol / L pH 5.0 acetate buffer, and adjust the pH to 4.8 with 1 mol / L HCl.

[0054] (2) Add octanoic acid dropwise at a ratio of 11 μl octanoic acid per ml of diluted ascites under stirring at room temperature. Add the octanoic acid within 30 min. Let stand at 4°C for 2 h. Centrifuge at 15,000 g for 30 min and discard the precipitate.

[0055] (3) The supernatant was filtered through a nylon mesh (125 μm), 1 / 10 volume of 0.01 mol / L PBS was added, and the pH was adjusted to 7.2 with 1 mol / L NaOH; saturated ammonium sulfate was added to 45% saturation at 4°C, and the mixture was allowed to react for 30 minutes and allowed to stand for 1 hour.

[0056] (4) Centrifuge at 10,000 g for 30 minutes and discard the supernatant.

[0057] (5) The precipitate was dissolved in an appropriate amount of PBS (containing 137 mmol / L NaCl, 2.6 mol / L KCl, and 0.2 mmol / L EDTA) and dialyzed against 50 to 100 times the volume of PBS at 4°C overnight, during which the water was changed more than 3 times.

[0058] (6) Take out the sample and centrifuge at 10,000 g for 30 minutes to remove the insoluble sediment. After determining the protein content, the sample is packaged. The purification effect of the monoclonal antibody is identified by SDS-PAGE ( Figure 1 ).

[0059] (7) Take the purified monoclonal antibody and add it to the target protein coated ELISA plate, 100 μL / well, incubate at 37°C for 30 min, wash the plate three times with PBST, add 6 secondary antibodies labeled with HRP, including goat anti-mouse IgA, IgG1, IgG2a, IgG2b, IgG3, and IgM, 100 μL / well, add 2 wells for each enzyme-labeled secondary antibody, incubate at 37°C for 30 min, wash the plate three times with PBST, add 100 μL of substrate color development solution to each well for 20 min, add 50 μL of stop solution to stop the reaction, and read the OD 450nm OD value of the wells with HRP-labeled goat anti-mouse IgG1 secondary antibody only 450nm The average value is greater than 0.8 (1.9925), and the OD of other reaction wells 450nm The average values ​​were all less than 0.1, and were identified as IgG1 subtype (the results are shown in Table 1).

[0060] Table 1 Results of antibody subtype identification of monoclonal antibody 6F6 strain

[0061] Antibody subtype IgA IgG1 IgG2a Ig2b Ig3 IgM <![CDATA[Monoclonal antibody 6F6 OD 450nm Average value]]> 0.093 1.9925 0.0875 0.0375 0.0443 0.086

[0062] Example 5

[0063] ——Identification of the reactivity of anti-camel IgG monoclonal antibody 6F6 with IgG from other animals

[0064] (1) Add 100 μl (1 μg / mL) of commercial bovine IgG and horse IgG, which are similar to camel species, to each well of the ELISA plate and coat overnight.

[0065] (2) After washing, add 100 μl of PBS containing 5% skim milk to each well, block at 37°C for 2 hours, and tap the cleaned plate to use as an ELISA plate.

[0066] (3) The prepared ascites was diluted 1:30,000 with PBS solution and added to ELISA plates coated with different proteins, 100 μl per well, and shaken for 5 minutes.

[0067] (5) After incubation at 37°C for 30 minutes, remove the reaction plate, discard the reaction solution, add 300 μl of 1× washing solution to each well, wash three times, and spin dry.

[0068] (6) Dilute HRP-labeled goat anti-mouse IgG with PBS at a ratio of 1:10,000 and add 100 μl to each well. Incubate at 37°C for 30 minutes, wash three times, and spin dry.

[0069] (7) Immediately add the substrate color development solution to the ELISA reaction plate, 100 μl / well, and color development at room temperature in the dark for 15 minutes. Then add 50 μl of stop solution to each well to terminate the reaction. After the reaction is terminated, measure the OD value with an ELISA reader within 20 minutes.450nm value.

[0070] (8) The study found that the anti-camel IgG monoclonal antibody invented and developed had no reactivity to bovine and horse IgG (OD 450nm value<0.2).

[0071] Table 2 Reactivity results of anti-camel IgG monoclonal antibodies to bovine and horse IgG

[0072] Animal Type camel ox horse <![CDATA[Monoclonal antibody 6F6 OD 450nm Average value]]> 2.118 0.116 0.181

[0073] Example 6

[0074] ——Identification of the specific antigen epitope recognized by anti-camel IgG monoclonal antibody 6F6

[0075] (1) Take 70 μL of the expressed and purified IgG protein after ultrasonic lysis and mix it with an equal amount of loading buffer (containing 1% DTT and 1% amphoteric electrolytes). Take out the IPG precast strip from the refrigerator and place it at room temperature for 10 minutes. Continuously and linearly add samples to the focusing disk. Remove the protective layer of the IPG precast strip. Place the strip correctly into the focusing disk. Cover the back of the strip evenly and continuously with mineral oil and perform isoelectric focusing electrophoresis.

[0076] (2) Prepare SDS-PAGE gel, take out the IPG strip that has completed the isoelectric focusing procedure, rinse with ultrapure water, dry with filter paper, and place the plastic plate side of the strip close to the long glass plate of the SDS gel, with the short glass plate facing up. Place a small piece of filter paper stained with a protein marker on the acidic side of the IPG strip. Quickly pour the hot-melt low-melting point agarose sealing liquid into the glass plate interlayer, immerse and seal the IPG strip and marker filter paper, remove all bubbles, and perform electrophoresis and Coomassie Brilliant Blue staining.

[0077] (3) Take a PVDF membrane that is the same size as the gel, soak it in methanol for 15 seconds, then transfer it to double distilled water and soak it for 2 minutes, and finally soak it in electrotransfer solution for 5 minutes. Then perform electrotransfer. After the electrotransfer is completed, take out the PVDF membrane and put it in TBST solution containing 5% skim milk powder, and block it at room temperature for 2 hours. After blocking, wash the membrane in TBST for 3 times. Put the washed membrane in a 1:2000 diluted purified anti-camel IgG 6F6 solution and react it at room temperature for 1 hour. After the reaction is completed, wash the membrane in TBST for 3 times. Put the reaction membrane in a 1:2000 diluted goat anti-mouse IgG solution and react it at room temperature for 1 hour. Wash the reacted membrane in TBST for 3 times. Under light-proof conditions, DAB substrate luminescent developer was added to the washed membrane, incubated at room temperature for 3 minutes, and then the developer was dried. Finally, the membrane was placed in a chemiluminescent imager. The results of the 2D-Western blot test showed that there were positive reaction points in the range of 35kDa and pH=3-5, and no obvious specific reaction was observed in other areas of the membrane. Protein spots were extracted for mass spectrometry identification, and the anti-camel IgG specific recognition antigen epitope KEPPSMRLKARPG was obtained, which is the precursor epitope of the large subunit p51 of the camel IgG receptor FcRn terminal.

[0078] Example 7

[0079] ——Horseradish peroxidase (HRP) labeling

[0080] (1) Dissolve 5 mg of HRP in 0.3 mol / L pH 8.1 NaHCO3 solution, add 0.1 ml of 1% dinitrofluorobenzene anhydrous ethanol solution, and stir at room temperature for 1 hour to block the α and ε amino groups on the HRP molecule.

[0081] (2) Add 1 ml of 0.06 mol / L sodium periodate solution and stir gently for 30 minutes at room temperature in the dark until the solution turns yellow-green. Then add 1 ml of 0.06 mol / L ethylene glycol and stir gently for 1 hour to terminate the oxidation reaction.

[0082] (3) Transfer the sample to a dialysis bag and dialyze it against 1 L of 0.01 mol / L pH 9.5 carbonate buffer at 4°C overnight, changing the buffer three times. Protect from light.

[0083] (4) Take 3 ml of the above-formylated HRP solution, add 1 ml of carbonate buffer containing 5 mg of purified monoclonal antibody, stir gently at room temperature for 2-3 hours, and protect from light; add 5 mg of NaBH4 and leave at 4°C for 3 hours or overnight, or use 0.2 ml of ethanolamine (2 mol / L pH 9.5) and let it act for 7 hours.

[0084] (5) The sample was transferred to a dialysis bag and dialyzed in 0.02 mol / L pH 7.4 PBS for 24 h, with the buffer changed three times.

[0085] (6) Centrifuge at 3000 r / min for 30 minutes to remove the precipitate. The supernatant is then salted out three times with half-saturated ammonium sulfate, and the precipitate is dissolved with a small amount of PBS. After desalting by dialysis or chromatography, the HRP-labeled anti-camel IgG monoclonal antibody is obtained.

[0086] Example 8

[0087] ——Establishment of indirect ELISA antibody detection method for Brucella camelid

[0088] (1) Determination of the optimal coating concentration: The purified Brucella lipopolysaccharide (LPS) was coated on the ELISA plate at 20 μg / ml, 10 μg / ml, 5 μg / ml, and 2.5 μg / ml, and the one with the highest positive control / negative control (P / N value) was selected as the optimal coating concentration. The study found that the coating effect was best when the coating concentration was 10 μg / ml.

[0089] Table 3 Comparison of the effects of different coating concentrations

[0090]

[0091] (2) Determination of the optimal working concentration of HRP-labeled anti-camel IgG monoclonal antibody: HRP-labeled anti-camel IgG monoclonal antibody was diluted from 1:10,000 to 1:10,000 to 40,000 times, and the OD values ​​of positive and negative serum samples were selected. 450nm The dilution with the highest ratio is taken as the optimal working concentration ( Figure 2 ). When HRP-labeled anti-camel IgG monoclonal antibody was diluted 30,000 times, the OD values ​​of positive and negative sera were 450nm Therefore, the working concentration of HRP-labeled anti-camel IgG monoclonal antibody was determined to be 30,000 times diluted.

[0092] (3) Determination of critical value: 127 camel sera that were positive in the bengal plate agglutination test, tube agglutination test, and complement fixation test and 142 camel sera that were negative were collected. OD was determined according to the established indirect ELISA method. 450nm value( Figure 3 ), and the test results were analyzed using SPSS17.0 software. The ROC curve was constructed using the non-parametric method, and the cut-off point with the maximum Youden index was used as the critical point for positive and negative judgment, and the sensitivity and specificity of the kit were determined at the same time. According to SPSS statistical software, the P% value was determined to be 30%, and the sensitivity corresponding to this critical value was 98.4% and the specificity was 96.5% ( Figure 4 ).

[0093] In the implementation process of the present invention, a monoclonal antibody cell line 6F6 that can stably and efficiently express camel IgG was obtained by artificially expressing, purifying, immunizing and screening camel IgG protein. It was identified as IgG1 subtype, and the recognition epitope was KEPPSMRLKARPG. The HRP-labeled purified monoclonal antibody was applied to the camel Brucella indirect ELISA antibody detection kit. The kit can effectively and accurately detect camel brucellosis, and its sensitivity and specificity are both above 95%.

[0094] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0095] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A monoclonal antibody cell line 6F6 against camel immunoglobulin G, characterized in that: The 6F6 strain has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named as a monoclonal antibody cell strain of camel immunoglobulin G (IgG). The deposit date is August 9, 2023, and the deposit number is CGMCC No.45650.

2. A monoclonal antibody of camel immunoglobulin G, characterized in that It is secreted by the 6F6 strain described in claim 1, is of IgG1 subtype, and recognizes the epitope KEPPSMRLKARPG.

3. A composition for detecting camel immunoglobulin G, characterized in that The composition contains the 6F6 strain according to claim 1 and / or the monoclonal antibody according to claim 2.

4. A kit for detecting camel immunoglobulin G, characterized in that: The kit contains one or more of the 6F6 strain according to claim 1 , the monoclonal antibody according to claim 2 , and the composition according to claim 3 .

5. The kit according to claim 4, characterized in that It is used for detection by ELISA method.

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