Hybridoma cell line secreting monoclonal antibody against grass carp IL-4 / 13B, monoclonal antibody and application thereof

Abstract

The present invention provides a hybridoma cell line secreting monoclonal antibody against grass carp IL-4 / 13B, the monoclonal antibody and its application. The preservation number of the hybridoma cell line is CCTCC NO: C2022213. The monoclonal antibody against grass carp IL-4 / 13B is secreted by the hybridoma cell line, and the monoclonal antibody specifically recognizes the prokaryotic expression product, eukaryotic expression product and extracellular expression functional region protein of grass carp IL-4 / 13B. The preparation method of the grass carp IL-4 / 13B recombinant protein in the present invention is simple and highly expressed in animal cells. The expressed grass carp IL-4 / 13B protein can be used to find protein molecules that can activate or inhibit grass carp IL-4 / 13B, and can also be used to prepare monoclonal antibodies against grass carp IL-4 / 13B. The antibody can be used to prepare reagents and drugs for diagnosing, treating or preventing diseases related to the expression of grass carp IL-4 / 13B protein.

Description

Technical Field

[0001] The present invention belongs to the technical field of immune applications, and specifically relates to a hybridoma cell line secreting monoclonal antibodies against grass carp IL-4 / 13B, monoclonal antibodies, and applications, that is, a preparation method and applications of grass carp interleukin-4 / 13B (GCIL-4 / 13B) recombinant protein and monoclonal antibodies. The antibody is a monoclonal antibody produced by a hybridoma cell line secreting monoclonal antibodies against the extracellular region of grass carp IL-4 / 13B. Background Art

[0002] Grass carp (GC) is one of the most important economic fish species in China and also one of the freshwater fish species with the highest aquaculture production globally. In recent years, with the continuous expansion of the aquaculture scale and the continuous deterioration of the water ecological environment, diseases have become a key problem hindering the development of grass carp aquaculture. Therefore, the primary purpose is to solve the diseases that occur during the aquaculture process, and the immune system of the body plays an important role in resisting external pathogens and the like.

[0003] Th2 immunity is the main defense pathway of the host against metazoan pathogens. In mammals, two important cytokines involved in this immune response are interleukin-4 (IL-4) and interleukin-13 (IL-13). Both IL-4 and IL-13 are cytokines secreted by activated T cells. As important components that can regulate the immune system, they play a certain role in improving the body's resistance and can be used as an immune adjuvant to improve the immune protection effect of vaccines; in addition, IL-4 / IL-13 can also limit the expansion of the inflammatory response, thereby protecting the body from damage caused by excessive inflammatory responses and playing an important role in the immune response against the invasion of pathogenic microorganisms; furthermore, IL-4 / IL-13 can regulate the differentiation and proliferation of Th2 cells and inhibit the differentiation and proliferation of Th1 cells that can secrete pro-inflammatory cytokines. These results all indicate that IL-4 / IL-13 plays an important role in regulating innate and adaptive immune responses.

[0004] In teleost fish, IL-4 / 13 has not yet differentiated into two separate genes, and two gene subtypes, IL-4 / 13A and IL-4 / 13B, have been formed due to genome duplication events or local duplication events. In previous studies, researchers have carried out many studies on the origin and evolution of Th2 immune responses around the IL-4 and IL-13 genes in order to understand the important role played by this immune response in vertebrates. However, there are not many relevant studies on IL-4 / 13 in fish. Current research on fish mainly focuses on gene structure, transcriptome analysis, etc., and little is known about the structure at the protein level, mainly because of the lack of corresponding high-quality monoclonal antibodies. Therefore, there is an urgent need in this field to establish high-affinity and high-specificity IL-4 / 13 monoclonal antibodies and use the IL-4 / 13 monoclonal antibodies to detect the expression levels of IL-4 / 13 in grass carp tissues and sera. Understanding the significance of IL-4 / 13 cytokines in the proliferation and differentiation of Th2 cells in fish can not only provide clues for important evolutionary events in the formation of the immune system from aquatic organisms to terrestrial organisms, but also play a key role in finding new strategies to strengthen the fish immune system and has potential application value for the prevention and treatment of inflammatory diseases.

[0005] Previous studies have selected a polypeptide containing 14 - 20 amino acid residues for the preparation of polyclonal antibodies against grass carp IL-4 / 13B by analyzing the characteristics of the amino acid sequence of grass carp IL-4 / 13B. However, polyclonal antibodies have many defects in research, mainly because they can recognize multiple antigenic epitopes, and the target protein can bind more than one antibody molecule at multiple epitopes, resulting in their recognition of proteins with high homology to the immunogenic protein and the production of a large number of non-specific antibodies, which may sometimes generate background signals in some antibody applications. This will have an adverse impact on some experimental results and lead to inaccurate results. With the development of monoclonal antibody technology, the defects of polyclonal antibodies have been gradually overcome.

[0006] The monoclonal antibody hybridoma technology is a highly precise means of serological research. By in vitro inducing the fusion of antibody-secreting B cells and myeloma cells that can be cultured infinitely, hybridoma cells that can both secrete antibodies and be cultured and preserved in vitro for a long time are obtained. It has the advantages of high purity, strong specificity, good repeatability, etc. Existing monoclonal antibodies against human IL-4 and IL-13 cannot be used as effective detection antibodies for fish-related research because their amino acid sequences have low homology with grass carp IL-4 / 13 and cannot recognize antigenic epitopes. Therefore, there is an urgent need to prepare grass carp-derived IL-4 / 13 monoclonal antibodies with high affinity and high specificity to provide an effective means for monitoring the expression levels of IL-4 / 13 in grass carp tissues and sera and lay a foundation for studying the proliferation, differentiation, and functions of grass carp Th2 cells.

[0007] Monoclonal antibodies also have disadvantages. For example, there are certain requirements for technology, and monoclonal antibodies are more likely to lose epitopes than polyclonal antibodies after chemical treatment with antigens. Summary of the Invention

[0008] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a hybridoma cell line secreting monoclonal antibodies against grass carp IL-4 / 13B, the monoclonal antibodies, and their applications. During the preparation of monoclonal antibodies, several positive hybridoma cell lines can be screened after cell fusion, and the deficiencies in the prior art can be overcome by screening two or more monoclonal antibodies against the same antigen for compensation.

[0009] To achieve the above object, the solution of the present invention is as follows:

[0010] In the first aspect, the present invention provides a hybridoma cell line secreting monoclonal antibodies against grass carp IL-4 / 13B. The hybridoma cell line GC5IL-4 / 13B was deposited on August 17, 2022, at the China Center for Type Culture Collection, located on the campus of Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number CCTCC NO: C2022213.

[0011] In the second aspect, the present invention provides a monoclonal antibody against grass carp IL-4 / 13B secreted by the above hybridoma cell line, which specifically recognizes the prokaryotic expression product, eukaryotic expression product, and extracellular expression functional region protein of grass carp IL-4 / 13B.

[0012] Preferably, the subtype of the monoclonal antibody against grass carp IL-4 / 13B is IgG.

[0013] In the third aspect, the present invention provides the application of the above hybridoma cell line in the preparation of reagents for diagnosing, preventing, or treating diseases related to the expression of grass carp IL-4 / 13B protein.

[0014] In the fourth aspect, the present invention provides the application of the above monoclonal antibody against grass carp IL-4 / 13B in the preparation of reagents for diagnosing, preventing, or treating diseases related to the expression of grass carp IL-4 / 13B protein.

[0015] In the fifth aspect, the present invention provides the application of the above monoclonal antibody against grass carp IL-4 / 13B in the preparation of reagents, drugs, or test kits for detecting the expression of grass carp IL-4 / 13B protein.

[0016] In the sixth aspect, the present invention provides a reagent, drug, or test kit for detecting the expression of grass carp IL-4 / 13B protein, including the above hybridoma cell line and / or the monoclonal antibody against grass carp IL-4 / 13B.

[0017] Due to the above - mentioned solution, the beneficial effects of the present invention are as follows:

[0018] First, the preparation method of the grass carp IL - 4 / 13B recombinant protein of the present invention is simple and highly expressed in animal cells. The expressed grass carp IL - 4 / 13B protein can be used to find protein molecules that can activate or inhibit grass carp IL - 4 / 13B, and can also be used to prepare monoclonal antibodies against grass carp IL - 4 / 13B. The prepared monoclonal antibodies can highly specifically recognize the prokaryotic expression products, eukaryotic expression products and extracellular expression functional region proteins of grass carp IL - 4 / 13B. The antibody can be applied to the immunological detection of grass carp IL - 4 / 13B, including specifically detecting the concentration of IL - 4 / 13 in grass carp blood or body fluids by ELISA technology, and detecting the production of IL - 4 / 13 in tissues by laser confocal microscopy through frozen tissue sections, providing parameter indicators for diagnosing, treating or preventing diseases related to the expression of grass carp IL - 4 / 13B protein, and having potential application value for the prevention and treatment of grass carp inflammatory diseases.

[0019] Second, the preparation method of the GCIL - 4 / 13B - 5 monoclonal antibody in the present invention is simple, and the obtained monoclonal antibody has high specificity and strong stability, and can be used to prepare reagents and drugs for diagnosing, treating or preventing diseases related to the expression of grass carp IL - 4 / 13B protein.

[0020] Third, the present invention obtains the recombinant protein of grass carp IL - 4 / 13B through the prokaryotic expression method of Escherichia coli, purifies it and prepares its monoclonal antibody, and identifies and applies the antibody, laying a foundation for studying the proliferation, differentiation and function of grass carp Th2 cells, and providing technical support for developing new measures for fish disease prevention and control. Brief Description of the Drawings

[0021] Figure 1 It is a result diagram of the purification and SDS - PAGE analysis of the grass carp IL - 4 / 13B recombinant protein in Example 1 of the present invention (where M represents the protein molecular weight standard Marker, and 1 represents the grass carp IL - 4 / 13B recombinant protein).

[0022] Figure 2 It is a determination diagram of the antibody titer in the serum of immunized mice in Example 2 of the present invention.

[0023] Figure 3 It is a Western blotting detection and analysis diagram of the cell supernatant for the preparation of the grass carp IL - 4 / 13B protein antibody in Example 2 of the present invention.

[0024] Figure 4Purity analysis chart of the grass carp monoclonal antibody GCIL-4 / 13B-5 in Example 2 of the present invention (where 1: protein molecular weight standard; 2: GCIL-4 / 13B-5 monoclonal antibody).

[0025] Figure 5 Detection chart of the affinity constant of the grass carp monoclonal antibody GCIL-4 / 13B-5 in Example 2 of the present invention.

[0026] Figure 6 Verification result chart of eukaryotic protein of the GCIL-4 / 13B-5 purified strain monoclonal antibody in Example 3 of the present invention ((A) Using the Myc-tag antibody as the primary antibody, (B) The prepared GCIL-4 / 13B-5 monoclonal antibody as the primary antibody; where M is the protein molecular weight standard Marker; 1 is the cell lysate transfected with the pcDNA3.1-empty plasmid; 2 is the cell lysate transfected with the pcDNA3.1-GCIL-4 / 13B-Myc plasmid).

[0027] Figure 7 Specific immunofluorescence analysis chart of the GCIL-4 / 13B-5 purified strain monoclonal antibody in Example 3 of the present invention.

[0028] Figure 8 Cross-reaction verification result chart of the GCIL-4 / 13B-5 purified strain monoclonal antibody and zebrafish in Example 3 of the present invention ((A) Using the His-tag antibody as the primary antibody, (B) The prepared GCIL-4 / 13B-5 monoclonal antibody as the primary antibody; where M is the protein molecular weight standard Marker; 1 is the cell lysate transfected with the pcDNA3.1-empty plasmid; 2 is the cell lysate transfected with the zebrafish pcDNA3.1-zfIL-4 / 13A-His plasmid; 3 is the cell lysate transfected with the zebrafish pcDNA3.1-zfIL-4 / 13B-His plasmid; 4 is the cell lysate transfected with the pcDNA3.1-GCIL-4 / 13B-His plasmid).

[0029] The hybridoma cell line GC5IL-4 / 13B was deposited at the China Center for Type Culture Collection, Wuhan University on August 17, 2022, and its deposit number is: CCTCC NO: C2022213. Detailed implementation manners

[0030] The present invention provides a hybridoma cell line secreting a monoclonal antibody against grass carp IL-4 / 13B, the monoclonal antibody and its application.

[0031] First, the specific operations for the induced expression and purification of grass carp IL-4 / 13B protein are as follows: After optimizing the open reading frame sequence of grass carp IL-4 / 13B (removing the coding signal peptide sequence) according to the Escherichia coli codon preference table, it was respectively ligated to the expression vector pEHISTVb, and then transferred into the expression strains BL21(C43) and Rosetta competent cells. After induction with IPTG, the collected bacteria were cyclically disrupted by a high-pressure crusher for about 10 min to extract inclusion bodies. The inclusion bodies were renatured by the dilution renaturation method at 4 °C for 30 h. After concentration and replacement of the protein buffer solution, the renaturation solution was centrifuged at high speed (4 °C, 10,000 rpm, 5 min), purified using an AKTA protein purifier, and then analyzed and verified by SDS-PAGE.

[0032] Secondly, the monoclonal antibody against grass carp IL-4 / 13B protein and its preparation are as follows: The obtained recombinant grass carp IL-4 / 13B protein was used to immunize mice. Spleen cells were fused with sp2 / 0 cells, and several hybridoma cell lines capable of secreting monoclonal antibodies against GCIL-4 / 13B were screened. The antibody typing results all belonged to the IgG subclass (belonging to IgG1, IgG2a, and IgG2b), and two strains were selected from the screened positive hybridoma cells for purification and labeling.

[0033] The obtained monoclonal antibodies were verified and identified using laser confocal and Western Blot techniques. It was found that the prepared monoclonal antibodies could specifically recognize the recombinant protein expressed by grass carp IL-4 / 13B, with the characteristics of strong specificity and high stability. In addition, the recombinant protein can be used as a potential molecular immune adjuvant, and the ELISA method can be used to detect the concentrations of IL-4 / 13 in grass carp blood and body fluids, estimate the health status of grass carp, and provide parameter indicators for monitoring grass carp inflammation.

[0034] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0035] Example 1:

[0036] Preparation of grass carp IL-4 / 13B recombinant protein

[0037] (1) Induced expression of recombinant protein and extraction of inclusion bodies

[0038] The open reading frame sequence of grass carp IL-4 / 13B (NCBI accession number: MK799653.1) removing the coding signal peptide sequence (ATG AGGACTTTCTTGCTCTTGGCACTGACATTTGTAGCCGTTAATGGATCACAACCTGATCTTAGGAAGACTCTTTTAAAGGACATCATAGTGTTTGTGAACCAGACCCTACATAACCATTCAGAGAAGAATCTTAAACAATTTGTGAGAGACATATTTCAGAGCGTGAGATGCTCAGATGAAGCCCTTTGCCAAGCAGCAAAGGTCCTGAATGGGGTGCATCTAAACACAGATCCAAATAACATGATATATAGGAATCTGTTCACCTATGCAGATTTAACCGTGCACCGGAACTGCAGCGTTACAGCTTCAGAGGAACATCCAGTGAAGGATTTTCTCAAAAAAATCAAAGACTGCTGCCAATTACTTTACTCTAAACCTGTATCACGTCCAAAAAGAGTGAAA TGA , with the underscore in front being the promoter and the underscore at the back being the stop codon) (SEQ ID NO.1). Optimized according to the codon preference table of Escherichia coli, it was ligated to the expression vector pEHISTVb to construct the recombinant prokaryotic expression plasmid pEHISTVb-GCIL-4 / 13B, which was then transferred into the expression strains BL21(C43) and Rosetta competent cells. Positive monoclonal colonies were picked and cultured until the OD 600 value was between 0.6 - 0.8, and IPTG (isopropyl β-D-thiogalactoside, used at a concentration of 1 mmol / L) was added for induction expression. After inducing the recombinant bacteria for 10 h, the cells were collected by centrifugation (7500 rpm, 5 min), resuspended in sterile PBS, and lysed using an ultra-low temperature high-pressure continuous cell disruptor. After centrifugation, the supernatant was discarded, and the inclusion bodies were washed with Washing Buffer containing 1‰ DTT (dithiothreitol). After discarding the supernatant, the inclusion bodies were resuspended in Resuspension Buffer in a new centrifuge tube that had been weighed. 20 μL was taken for SDS-PAGE to detect the purity of the inclusion bodies. After centrifuging and discarding the supernatant, the inclusion bodies were dissolved in Dissolution Buffer to a final concentration of 30 mg / mL and stored at -20 °C for later use.

[0039] (2) Renaturation and purification of the recombinant protein

[0040] The prokaryotic protein expressed in the form of inclusion bodies was refolded by the dilution refolding method. After the refolding solution and the protein buffer were prepared, they were filtered through a 0.22 μm nitrocellulose filter membrane and placed in a clean beaker. 10 mL of the inclusion body solution in step (1) was added dropwise to 1 L of the prepared refolding solution, and in vitro refolding was carried out with the assistance of a magnetic stirrer (200 rpm) (refolding at 4 °C for 30 h). The diluted protein refolding solution was concentrated using a concentrator cup and a concentrator tube until the ideal loading volume of 3 - 5 mL was reached (filtered through a 0.22 μm filter membrane before loading). Finally, the protein was separated and purified by gel filtration chromatography using an AKTA protein purifier and a Superdex 200 column (GE Healthcare). According to the elution position of the separated and purified protein, the protein peak corresponding to the molecular weight was selected for concentration and SDS-PAGE analysis. The results showed that the purified GCIL-4 / 13B protein had a single band ( Figure 1 ).

[0041] Among them, the amino acid sequence of the grass carp IL-4 / 13B recombinant protein:

[0042] MRTFLLLALTFVAVNG SQPDLRKTLLKDIIVFVNQTLHNHSEKNLKQFVRDIFQSVRCSDEALCQAAKVLNGVHLNTDPNNMIYRNLFTYADLTVHRNCSVTASEEHPVKDFLKKIKDCCQLLYSKPVSRPKRVK (the underlined part is the signal peptide, and the rest is the mature peptide) (SEQ ID NO.2).

[0043] Example 2: Preparation of monoclonal antibody against grass carp IL-4 / 13B

[0044] (1) Mouse immunization

[0045] The purified grass carp IL-4 / 13B recombinant protein was provided to Beijing Genomics Institute at Shenzhen for the preparation of monoclonal antibodies. The specific steps were as follows: BALB / c female mice were immunized with the purified grass carp IL-4 / 13B recombinant protein, 30 - 60 μg per mouse, and intraperitoneally injected at two-week intervals. After four immunizations, blood was collected from the orbital cavity, and the serum antibody titer was measured by enzyme linked immunosorbent assay (ELISA). After reaching a certain value, the spleen cells of the immunized mice were taken for fusion ( Figure 2 ). Among them, the mice of boost #2 were selected for cell fusion experiments.

[0046] (2) Cell fusion and screening

[0047] 1) Gently pipette the healthy sp2 / 0 cells off the culture flask wall and aspirate them into a 50 mL centrifuge tube.

[0048] 2) Collect blood from the mouse by eye enucleation, then sacrifice the mouse by cervical dislocation and soak it in 75% alcohol for 5 min.

[0049] 3) Pour a small amount of serum-free IMDM medium into a petri dish and place the cell sieve and the syringe plunger in the dish. Use scissors and forceps to remove the mouse spleen and place it on the cell sieve. Gently crush the spleen thoroughly with the syringe plunger and aspirate the crushed cells into the centrifuge tube containing sp2 / 0 cells. Centrifuge at 1500 rpm for 5 min.

[0050] 4) Use scissors and forceps to remove the mouse thymus and crush it. Transfer the crushed thymus cells to a 15 mL centrifuge tube, then add 2 mL of HAT medium and 1 mL of HT medium and set aside in the incubator.

[0051] 5) For the centrifuged cells, discard the supernatant, gently and carefully resuspend the cells with serum-free IMDM medium, and centrifuge (1500 rpm, 5 min).

[0052] 6) Try to discard as much of the supernatant of the centrifuged cells as possible. Tap the bottom of the centrifuge tube to fully suspend the cells, place the centrifuge tube in warm water at 37°C, and slowly add 1 mL of PEG1450 (fusogen) within about 1 min. After adding, let it stand in the warm water for 1 min. Then slowly add 2 mL of serum-free IMDM medium within 2 min, and then slowly add 8 mL of serum-free IMDM medium within 2 min. Centrifuge (1000 rpm, 5 min).

[0053] 7) Discard the supernatant, add 10 mL of serum, carefully resuspend the cells, and pour in the previously prepared thymus cells. Then add sterilized semi-solid medium to make up to 50 mL, mix well, and evenly pour into 30 cell culture dishes. Place the cell culture dishes in a wet box and then place them in the incubator for culture.

[0054] (3) Screening and subtype identification of monoclonal antibodies

[0055] Transfer the cells after the fusion experiment to a semi-solid medium for culture. Pick the monoclonal colonies growing on the semi-solid medium into a 96-well culture plate for culture and subsequent screening. Replace the supernatant of the monoclonal cells in the 96-well culture plate for the first screening. Then coat the plate with grass carp IL-4 / 13B recombinant protein and use ELISA to screen for positive hybridoma cell lines twice successively. Identify the subtypes of the obtained positive cell strains, culture them in large quantities and cryopreserve them. Specific steps of the ELISA experiment: Dilute the coating antibody to 0.5 μg / mL with 100 mmol / L PBS buffer (pH 7.4), add 0.1 mL to each well, wash twice with PBS-T after overnight incubation at 4°C, add 200 μL of blocking solution to each well, and incubate at 37°C for 2 h. After washing 3 times with PBS-T, add 100 μL of hybridoma cell supernatant to each well and incubate at 37°C for 1 h. After washing 3 times with PBS-T, add 0.1 mL of HRP-labeled antibody (SBAClonotyping System-HRP, Southern Biotech) diluted 1:2000 with the blocking solution to each well and incubate at 37°C for 1 h. Wash 3 times with PBS-T; add 50 μL of substrate solution to each well and measure the absorbance at dual wavelengths (lambda;450, lambda;630) within 10 - 20 min.

[0056] The positive strains screened out were subjected to antibody subclass identification and Western Blotting analysis. Among them, GCIL-4 / 13B-36 and 45 were of IgG1 subtype, GCIL-4 / 13B-44 was of IgG2a subtype, and GCIL-4 / 13B-1, 4, 5, 13, 15, 33 were of IgG2b subtype (Table 1). Using the corresponding prokaryotic recombinant protein as the antigen, the positive cell supernatant as the primary antibody (v / v = 1:4), and HRP-goat anti-mouse IgG as the secondary antibody (v / v = 1:5000), finally, several monoclonal antibody cell supernatants with good specificity were screened out from GCIL-4 / 13B. Among them, the GCIL-4 / 13B recombinant protein had specific reactions with the monoclonal antibody cell supernatants of cell lines No. 4, 5, 13, 15, 33, 36, 44, and 45 of the immunized mice, and no specific reaction with cell line No. 1 Figure 3 ).

[0057] Table 1 shows the identification of the subtypes of monoclonal cell lines

[0058] Cell line number OD value against immunogen Subtype 1 1.091 G2b 4 1.069 G2b 5 1.206 G2b 13 1.211 G2b 15 1.206 G2b 33 1.092 G2b 36 0.784 G1 44 0.848 G2a 45 0.714 G1 Negative 0.048 Empty 0.041 Positive 1.339

[0059] (4) Purification and labeling of monoclonal antibodies

[0060] One antibody was selected from the positive monoclonal antibodies screened from GCIL-4 / 13B for purification and labeling. The monoclonal antibody was purified by affinity chromatography using a HiTrapr Protein A FF column, and the purity of the antibody was detected by SDS-PAGE. Then, the affinity constant of the antibody was detected by ELISA (affinity constant = 150,000 × A / antibody concentration, where A represents the antibody dilution factor corresponding to 1 / 2 of the maximum OD value, and the OD value was measured at dual wavelengths of 450 nm and 630 nm). The antibody numbered 5 was selected for purification and named GCIL-4 / 13B-5( Figure 4 ). The affinity constant of the purified antibody was detected and found to have a high titer( Figure 5 ). The monoclonal antibody was labeled with FITC (fluorescein isothiocyanate), and the antibody purification and labeling were completed by Beijing Genomics Institute at Shenzhen.

[0061] Example 3: Identification and Specificity Analysis of Grass Carp IL-4 / 13B Monoclonal Antibody

[0062] (1) Detection of Eukaryotic Protein Expression by Western Blotting

[0063] The grass carp pcDNA3.1-GCIL-4 / 13B-Myc plasmid was transfected into HEK293T cells, and cells transfected with the pcDNA3.1-empty plasmid were set as the control group. After 24 h of transfection, the cells were collected to extract proteins for Western Blotting experiments. Western Blotting used the electrotransfer method (25 v, 25 mA, 7 min) to transfer the protein samples onto a PVDF membrane. After blocking the PVDF membrane with 5% skim milk powder at 4 °C for 2 h, the prepared GCIL-4 / 13B-5 monoclonal antibody was incubated (overnight at 4 °C, monoclonal antibody diluted 1:200). HRP-labeled goat anti-mouse was used as the secondary antibody (diluted 1:10,000) and incubated at room temperature for 1 h, followed by washing 3 times with TBS-T buffer, 5 min each time. The PVDF membrane was placed on an Odyssey CLx Imaging System for photographing and analysis. The results showed that corresponding target bands appeared in the cell lysate samples transfected with pCDNA3.1-GCIL-4 / 13B-Myc, proving that GCIL-4 / 13B was well expressed in HEK293T cells. No bands appeared in the protein samples transfected with the empty plasmid, indicating that the screened monoclonal antibody could specifically recognize the grass carp IL-4 / 13B protein expressed in HEK293T cells. Among them, the band incubated with GCIL-4 / 13B-5 was slightly larger than the theoretical molecular mass, but the band was single. It was speculated that chemical modifications such as glycosylation and phosphorylation might occur during the expression of the GCIL-4 / 13B eukaryotic protein, resulting in the protein band being larger than the theoretical molecular mass.(Figure 6 )

[0064] (2) Direct immunofluorescence detection and analysis

[0065] Culture HEK293T cells in a 6-well plate with cell slides. When the cell confluence reaches about 70 - 80%, transfect the grass carp pcDNA3.1-GCIL-4 / 13B-Myc plasmid. At the same time, set the cells transfected with pcDNA3.1-empty plasmid as the control group. After 24 h of transfection, discard the culture supernatant, wash with PBS buffer solution, and then fix the cells with 4% paraformaldehyde for 15 min. Remove the liquid, wash with PBS buffer solution 3 times, 5 min each time. Permeabilize the cells with 0.5% Triton-X100 at room temperature for 10 min. Add an adequate amount of blocking solution (5% BSA) and incubate at room temperature for 1 h. Discard the blocking solution. Add the FITC-labeled GCIL-4 / 13B-5-FITC monoclonal antibody diluted with 5% BSA (antibody diluted at a ratio of 1:200) to the control group and the experimental group respectively, and incubate at room temperature for 2 h. Wash with PBS buffer solution 3 times, then add DAPI and stain at room temperature for 10 min. Wash with PBS buffer solution 3 times, 5 min each time. After adding the anti-fluorescence quencher, seal the slides and observe and photograph under a laser confocal microscope. The results show that GCIL-4 / 13B-5 can recognize the GCIL-4 / 13B protein located in the cytoplasm of HEK293T cells, while no specific fluorescence signal and positive cells are observed in the control group, indicating that the prepared GCIL-4 / 13B-5 monoclonal antibody can specifically bind to the GCIL-4 / 13B protein and can be used for the specific detection of cells or tissues secreting GCIL-4 / 13B( Figure 7 )

[0066] (3) Whether there is a cross-reaction between the grass carp IL-4 / 13B monoclonal antibody and zebrafish

[0067] The grass carp pcDNA3.1-GCIL-4 / 13B-His, zebrafish pcDNA3.1-zfIL-4 / 13A-His, and pcDNA3.1-zfIL-4 / 13B-His plasmids were transfected into HEK293T cells, and cells transfected with the pcDNA3.1-empty plasmid were set as the corresponding control group. After 24 h of transfection, the cells were collected to extract proteins for Western Blotting experiments. The experimental method was the same as described in (1). The results showed that by incubating with the His-tag antibody carried by the plasmid, no band appeared in the protein sample transfected with the empty plasmid, while corresponding bands appeared in the cell lysate samples transfected with pCDNA3.1-GCIL-4 / 13B-His, pcDNA3.1-zfIL-4 / 13A-His, and pcDNA3.1-zfIL-4 / 13B-His, proving that both GCIL-4 / 13B and zfIL-4 / 13B were well-expressed in HEK293T cells. The selected GCIL-4 / 13B-5 monoclonal antibody could specifically recognize the grass carp IL-4 / 13B protein expressed in HEK293T cells, and there was no cross-reaction with zebrafish IL-4 / 13A / B( Figure 8 ). It indicated that the prepared grass carp monoclonal antibody had the characteristic of strong specificity and could be used for the evaluation of the health and disease resistance of grass carp in fish farming.

[0068] The recombinant proteins and monoclonal antibodies obtained through the above examples have the following application prospects:

[0069] 1. Application of the purified grass carp IL-4 / 13B recombinant protein as a vaccine molecular adjuvant

[0070] Using adjuvants for antigen immunization has always been an effective strategy to enhance the immunogenicity of vaccines, which is also important for vaccines with IL-4 / 13B as an antigen. The grass carp IL-4 / 13B recombinant protein developed and prepared in the present invention has biological activity. To improve the protection efficiency of the vaccine, the purified grass carp IL-4 / 13B recombinant protein can be used alone or in combination. After immunizing grass carp, the IL-4 / 13B antibody level in the serum can be detected, and different bacteria or viruses can be used to infect the immunized grass carp to detect the cross-immunoprotection effect and evaluate the effect of related vaccine adjuvants. It is expected to increase the immunogenicity of the vaccine, improve the immune protection effect of the vaccine, thereby enhancing the antiviral ability of grass carp through immunization, and contributing to the research and application of new vaccines.

[0071] 2. Application of the monoclonal antibody in ELISA to detect the concentration of IL-4 / 13B in grass carp blood or body fluids. The specific experimental operation is as follows:

[0072] (1) Coating: Dilute the GCIL-4 / 13B-5 monoclonal antibody with coating buffer to a protein content of 1-10 μg / mL. Add 100 μL to each reaction well of the ELISA plate, incubate at 37°C for 2 h or overnight at 4°C.

[0073] (2) Washing the plate: Discard the liquid in the wells, spin dry, wash the plate 3 times with washing buffer, 3 min each time (abbreviated as washing, the same below). Spin dry (or gently tap to dry the liquid in the wells).

[0074] (3) Blocking: Use 10 mmol / L PBST (10 mmol / L PBS, 0.05% Tween-20) containing 1% BSA or 5% skim milk as the blocking solution, 350-400 μL / well, incubate at 37°C for 2 h.

[0075] (4) Washing the plate: The same as step (2).

[0076] (5) Adding samples: Add 100 μL of the diluted sample to be tested to the above-coated reaction wells, incubate at 37°C for 1 h. Then wash (set blank wells, negative control wells, positive control wells, standard wells, and test sample wells respectively).

[0077] (6) Adding enzyme-labeled antibody: Add 100 μL of freshly diluted enzyme-labeled antibody (dilution after titration) to each reaction well. Incubate at 37°C for 0.5-1 h, then wash.

[0078] (7) Adding substrate solution for color development: Add 100 μL of freshly prepared TMB substrate solution to each reaction well, incubate at 37°C for 10-30 min.

[0079] (8) Terminating the reaction: Add 50 μL of 2 mol / L sulfuric acid to each reaction well.

[0080] (9) Use an ELISA detector to measure the OD value of each well at 450 nm (read within 5 min after adding the termination solution).

[0081] (10) Result determination.

[0082] 1) The OD value of each standard and sample must be subtracted by the OD value of the zero well. If duplicate wells are set, take the average value;

[0083] 2) Use the concentration of the standard as the abscissa and the OD value as the ordinate to make a standard curve. According to the OD value of the sample, calculate the corresponding fitted concentration from the standard curve, and then multiply by the dilution factor to obtain the measured concentration of the sample.

[0084] 3. Application of monoclonal antibody in immunohistochemistry (laser confocal microscopy) to localize the distribution of IL-4 / 13B in various tissues of grass carp. The specific operation is as follows:

[0085] (1) Fixation and embedding of grass carp tissues: Collect fresh tissues of grass carp such as gills, head, kidney, spleen, liver and intestine, fix them with 4% paraformaldehyde at 4°C overnight. Put the fixed tissues into a small box made of aluminum foil, pour in the embedding agent OCT, arrange the tissues in place, and place them at -80°C until solidified for sectioning.

[0086] (2) Preparation of frozen sections: Quickly place the frozen embedded tissues in a cryostat pre-cooled to -20°C in advance, fix the tissue embedding block on the knife holder and use a single-sided blade for sectioning (adjust the thickness to 6μm for sectioning). Stretch out the cut tissue blocks with a brush, adhere the tissues to the glass slides and make marks.

[0087] (3) Immunofluorescence staining: Block the glass slides with 1% BSA at room temperature for 30 min, and wash them 3 times with PBS buffer. Incubate with the GCIL-4 / 13B-5 monoclonal antibody labeled with FITC or Cy5 at room temperature for 1 h. Wash the glass slides with PBS again 3 times, mount them with a fluorescence signal enhancement mounting medium, and collect photos with a TCS SP8 confocal microscope (Leica) for localization analysis.

[0088] The above description of the embodiments is for those of ordinary skill in the art to understand and use the present invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments. Modifications and improvements made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

[0089]

[0090]

[0091]

Claims

1. A hybridoma cell line secreting monoclonal antibody against grass carp IL-4 / 13B, characterized in that: The hybridoma cell line was deposited in the China Center for Type Culture Collection of Wuhan University on August 17, 2022, with the deposit number CCTCC NO: C2022213.

2. A monoclonal antibody against grass carp IL-4 / 13B secreted by the hybridoma cell line described in claim 1, characterized in that: It specifically recognizes the recombinant protein expressed by grass carp IL-4 / 13B.

3. The anti-grass carp IL-4 / 13B monoclonal antibody according to claim 2, characterized in that: The subtype of the monoclonal antibody against grass carp IL-4 / 13B is IgG.

4. Use of the anti-grass carp IL-4 / 13B monoclonal antibody according to claim 2, characterized in that: Use of the monoclonal antibody against grass carp IL-4 / 13B in the preparation of reagents or kits related to the detection of the expression of grass carp IL-4 / 13B protein.

5. A reagent or kit related to the detection of the expression of grass carp IL-4 / 13B protein, characterized in that: It includes the hybridoma cell line described in claim 1 and / or the monoclonal antibody against grass carp IL-4 / 13B described in claim 2.

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