A monoclonal antibody against porcine SLFN11 protein and its application

Abstract

The present invention discloses a monoclonal antibody against porcine SLFN11 protein and its application, relating to the field of biotechnology. The monoclonal antibody against porcine SLFN11 protein comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO.1 and a light chain variable region with the amino acid sequence shown in SEQ ID NO.3. The monoclonal antibody has an IgG1 heavy chain constant region and a kappa light chain constant region, exhibiting strong affinity and stability and capable of specifically binding to porcine SLFN11 protein. This invention provides an excellent immunological tool for further exploring the antiviral function and mechanism of the SLFN11 protein, laying the foundation for the development of genetically engineered antibody drugs and novel vaccines for the prevention or treatment of porcine viral diseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an anti-porcine SLFN11 protein monoclonal antibody and an application thereof. Background Art

[0002] The Schlafen (SLFN) gene family, originally discovered in humans and mice, is a key gene involved in regulating biological functions in humans and mice, sharing extensive homology. However, the scope of SLFN's functions remains partially understood. Research has shown that the SLFN protein encoded by the SLFN gene has diverse biological functions, including regulating cell proliferation, T cell and macrophage differentiation, inhibiting cancer cell migration and invasion, and suppressing viral replication.

[0003] SLFN family proteins are divided into three subgroups based on their size and structure: subgroup I (37-42 kDa), subgroup II (58-68 kDa), and subgroup III (100-104 kDa). SLFN11, a member of the SLFN gene family and belonging to subgroup III, is an interferon-induced antiviral restriction factor that inhibits the replication of various viruses and is closely related to tumor treatment. Research by Li et al. has shown that SLFN11 can bind to tRNA, counteracting the A / U bias of tRNA caused by the presence of HIV, inhibiting the synthesis of HIV viral proteins and potentially serving as a new antiviral factor against HIV. In addition to HIV, SLFN11 has also been found to inhibit the replication of other viruses. Guo et al.'s research results showed that in addition to human SLFN11, bovine and monkey SLFN11 could also inhibit the replication of the prototype foamy virus. This inhibitory effect was achieved by disrupting the synthesis of the prototype foamy virus protein through different codon usage between the virus and the host. In addition, they also found that dephosphorylation of the SLFN11 protein and the activities of ATP synthase and helicase are also required for SLFN11 to exert its antiviral activity. However, the role of SLFN11 in major swine diseases has not been reported.

[0004] Pigs are one of the most important economic animals for humans to obtain animal protein, but the pig farming industry is plagued by various diseases, such as ASFV, PRRSV, PEDV, and PRV. The antiviral effect of SLFN may become a new target for combating porcine-related susceptible viruses. Therefore, the present invention constructs a porcine pET28a(+)-SLFN11-His recombinant expression plasmid and induces expression. The recombinant protein is purified and then immunized in mice to prepare monoclonal antibodies against the porcine SLFN11 recombinant protein. This provides a good immunological tool for further studying the antiviral function and mechanism of porcine SLFN11. Summary of the Invention

[0005] The present invention aims to provide a monoclonal antibody against porcine SLFN11 protein and its application to address the above-mentioned problems in the prior art. The monoclonal antibody has an IgG1 heavy chain constant region and a kappa light chain constant region, and has strong affinity and stability, and can specifically bind to porcine SLFN11 protein.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an anti-porcine SLFN11 protein monoclonal antibody, comprising a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.3.

[0008] Genetic engineering can be used to generate monoclonal antibodies against porcine SLFN11 protein. Furthermore, genetic and protein engineering methods can be used to modify the porcine SLFN11 protein monoclonal antibody in various ways, including by adding, deleting, or replacing one or more amino acids to obtain active fragments or conservative variants, thereby further enhancing the antibody's specificity and affinity.

[0009] The present invention also provides a gene encoding the above-mentioned anti-porcine SLFN11 protein monoclonal antibody.

[0010] Furthermore, the encoding genes include a heavy chain variable region encoding gene and a light chain variable region encoding gene;

[0011] The nucleotide sequence of the heavy chain variable region encoding gene is shown in SEQ ID NO.2;

[0012] The nucleotide sequence of the light chain variable region encoding gene is shown in SEQ ID NO.4.

[0013] The present invention also provides a gene expression cassette comprising the above-mentioned coding gene.

[0014] The present invention also provides a recombinant expression vector comprising the above gene expression cassette.

[0015] The present invention also provides a recombinant host cell, comprising the above-mentioned recombinant expression vector.

[0016] The present invention also provides the use of the above-mentioned encoding gene, gene expression cassette, recombinant expression vector or recombinant host cell in preparing the above-mentioned anti-porcine SLFN11 protein monoclonal antibody.

[0017] The present invention also provides the use of the above-mentioned anti-porcine SLFN11 protein monoclonal antibody in the preparation of a product for detecting porcine SLFN11 protein.

[0018] The present invention also provides a product for detecting porcine SLFN11 protein, comprising the above-mentioned anti-porcine SLFN11 protein monoclonal antibody.

[0019] The product can be an ELISA detection kit.

[0020] The present invention discloses the following technical effects:

[0021] This invention provides a monoclonal antibody against the porcine SLFN11 protein. The heavy chain constant region of this monoclonal antibody is IgG1, and the light chain constant region is kappa. This monoclonal antibody has strong affinity and stability, and specifically binds to the porcine SLFN11 protein. This invention provides an excellent immunological tool for further exploring the antiviral function and mechanism of the SLFN11 protein, and lays a foundation for the development of genetically engineered antibody drugs and novel vaccines for the prevention or treatment of porcine viral diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is the identification result of the recombinant expression plasmid pET28a(+)-SLFN11-His; 1 represents the plasmid double enzyme digestion product, 2 represents the empty plasmid control, and M represents the marker;

[0024] Figure 2 The figure shows the results of SDS-PAGE analysis of the prokaryotic expression of recombinant SLFN11 protein; wherein, M represents Marker, 1 represents uninduced bacterial solution, 2 represents the supernatant after the uninduced bacterial solution is disrupted, 3 represents the precipitate after the uninduced bacterial solution is disrupted, 4 represents the induced bacterial solution, 5 represents the supernatant after the induced bacterial solution is disrupted, and 6 represents the precipitate after the induced bacterial solution is disrupted;

[0025] Figure 3 The results of SDS-PAGE and Western Blot detection of purified recombinant SLFN11 protein; where M represents Marker, 1 represents unpurified recombinant SLFN11 protein; 2 and 3 represent purified recombinant SLFN11 protein;

[0026] Figure 4 is a statistical graph of mouse serum titer;

[0027] Figure 5 This is a statistical chart of the titer of the purified monoclonal antibody;

[0028] Figure 6 This is a diagram showing the results of isotype identification of the monoclonal antibody SLFN11-1B4H4G8;

[0029] Figure 7 This is the result of Western Blot detection of the specificity of the monoclonal antibody, where M represents Marker, 1 represents an irrelevant protein control, and 2 represents the recombinant porcine SLFN11 protein;

[0030] Figure 8 This is the IFA identification result of the monoclonal antibody SLFN11-1B4H4G8. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Example 1 Preparation of porcine recombinant SLFN11 protein fragments

[0037] 1. Construction of recombinant expression plasmid pET28a(+)-SLFN11-His

[0038] The pET28a(+)-SLFN11-His recombinant expression plasmid was prepared by Nanjing GenScript Biotechnology Co., Ltd. The pET28a(+)-SLFN11-His recombinant plasmid was double-digested with restriction endonucleases BamH I and Xho I, and then identified by nucleic acid gel electrophoresis. The results are shown in Figure 1 The results showed that the recombinant plasmid was successfully constructed.

[0039] 2. Inducible expression of SLFN11 protein

[0040] The porcine pET28a(+)-SLFN11-His recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells and screened overnight with solid LB medium containing kanamycin. Single colonies with good growth were picked and expanded. 8 mL of activated bacterial solution was added to 300 mL of LB liquid medium without antibiotics at a ratio of 1:100, and then kanamycin was added at a ratio of 1:1000 to the bacterial solution and cultured in a shaking table at 37°C and 220 r / min. When the bacterial solution OD 600nm When the value reaches 0.6, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mmol / L. After 8 hours of incubation, centrifuge and discard the supernatant. Add 15 mL of 1× PBS to resuspend the pellet and use an ultrasonic disruptor on ice for 30 minutes. Centrifuge and discard the supernatant. Resuspend the pellet in 15 mL of 8 M urea lysis buffer and shake on a rotary mixer at 4°C for 12 hours. Centrifuge and collect the supernatant. Fill the purification column with Ni Sepharose 6 Fast Flow filler, purify the target protein, and then renature it.

[0041] Western blot analysis of SLFN11 protein: Electrophoresis was performed on a 12.5% ​​SDS-PAGE gel at 120 V. After electrophoresis, the protein was transferred to a PVDF membrane using a wet transfer method for 75 minutes. After transfer, the membrane was blocked with 5% skim milk for 2 hours at room temperature. Subsequently, an anti-His mouse monoclonal antibody (anti-His) was used as the primary antibody at a dilution of 1:5000 and incubated for 1 hour at room temperature. After incubation, the membrane was washed three times with TBST for 10 minutes each. Next, a horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) secondary antibody was used at a dilution of 1:5000 and incubated for 1 hour at room temperature. After incubation, the membrane was washed three times with TBST for 10 minutes each. Finally, a colorimetric solution was added, and the membrane was incubated for 15 minutes in the dark before visualization using an ultrasensitive multifunctional imager.

[0042] The results of SDS-PAGE analysis of prokaryotic expression of recombinant SLFN11 protein are shown in Figure 2 The results showed that the recombinant SLFN11 protein was expressed normally. The results of SDS-PAGE and Western Blot detection of the purified recombinant SLFN11 protein are shown in Figure 3 The results showed that the purified recombinant SLFN11 protein was prepared in this example.

[0043] Example 2 Preparation of monoclonal antibodies

[0044] 1. Establishment of hybridoma cell lines

[0045] 1. Mouse Immunization

[0046] Five 6-8 week old female BALB / c mice were taken, and the purified recombinant SLFN11 protein was renatured and emulsified with Freund's complete adjuvant in a 1:1 ratio. The mice were immunized by intraperitoneal injection (25 μg recombinant SLFN11 protein per mouse). Each immunization was separated by two weeks. The second and subsequent immunizations used incomplete Freund's adjuvant and recombinant SLFN11 protein in a 1:1 ratio. One week after the third immunization, the mice were bled from the tail. The blood was serially diluted as the primary antibody, and horseradish peroxidase-labeled goat anti-mouse IgG (H+L) was used as the secondary antibody. The renatured recombinant SLFN11 protein was used to coat the ELISA plate at a protein concentration of 5 μg / mL. The antibody titer was detected by indirect ELISA (see the results). Figure 4 ), and the mice with the highest antibody titer were selected for superimmunization.

[0047] 2. Fusion of spleen cells and myeloma cells

[0048] Select superimmunized mice, remove the spleen, grind to prepare a single cell suspension, and mix it with SP2 / 0 cells at a ratio of 10:1. Use PEG1500 for cell fusion, then use preheated 1640 medium to terminate the fusion and use HAT medium to evenly spread the plates. Place the cells in a 37°C, 5% CO2 incubator and culture them after 7-10 days of cell growth. Use the cell supernatant as the primary antibody and horseradish peroxidase-labeled goat anti-mouse IgG (H+L) as the secondary antibody to screen the positive wells using the indirect ELISA method.

[0049] 2. Preparation of Monoclonal Antibodies by Ascites Induction

[0050] 1. Monoclonal Cell Subcloning and Ascites Preparation

[0051] Select indirect ELISA to detect OD 450nmWells with a value 2.15 times higher than the negative control were transferred to a 96-well plate and serially diluted. The cells were observed under a microscope until approximately 90 to 100 cells were present in the well. The cells were then thoroughly mixed with 1640 medium containing HAT and plated onto a 96-well plate for expansion. After the subcloned cells had grown confluently, ELISA was performed, and positive clones were selected for a second subcloning. After the second subcloning, ELISA was performed again until the supernatant of the monoclonal wells reached a 100% positive rate. BALB / c mice were intraperitoneally injected with 500 μL of incomplete Freund's adjuvant per mouse. Seven days later, hybridoma cells were intraperitoneally injected. Ascites was aspirated after the abdomen became noticeably enlarged, and the supernatant was collected by centrifugation at 12,000 rpm for 10 minutes.

[0052] 2. Monoclonal Antibody Purification

[0053] Extract the supernatant of the ascites after centrifugation, dilute the supernatant with the same volume of PBS, mix well, add saturated ammonium sulfate solution dropwise, stirring while adding, until the final concentration of ammonium sulfate is 50%, place at 4°C for 4h, centrifuge at 12000r / min for 10min, discard the supernatant, retain the precipitate, resuspend the precipitate with an equal amount of PBS as the ascites, put the resuspension into a dialysis bag, dialyze at 4°C overnight, and dialyze to remove the saturated ammonium sulfate. The dialyzed ascites was loaded into a Protein G column for purification to prepare a monoclonal antibody named SLFN11-1B4H4G8. The titer of the purified monoclonal antibody is shown in Figure 5 .

[0054] 3. Characterization of Monoclonal Antibodies

[0055] 1. Monoclonal Antibody Subtype Identification

[0056] In order to determine the subtype of the monoclonal antibody SLFN11-1B4H4G8, the commercial kit Mouse Monoclonal Antibody Isotyping Elisa Kit was used to identify the subtype of the monoclonal antibody. The culture supernatant of the hybridoma cell line was reacted with anti-IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgM heavy chain antibodies and anti-light chain Kappa (κ chain) and Lambda (λ chain) antibodies, and the OD was measured after color development. 450nm Value, see the result Figure 6 The results showed that the heavy chain constant region of the monoclonal antibody SLFN11-1B4H4G8 was of IgG1 type, and the light chain constant region was of Kappa type.

[0057] 2. Monoclonal antibody specificity and application effect

[0058] The purified monoclonal antibody SLFN11-1B4H4G8 was used as the primary antibody for Western Blotting, and horseradish peroxidase-labeled goat anti-mouse IgG (H+L) was used as the secondary antibody to detect the specificity of the SLFN11 protein. The results are shown in Figure 7 The results showed that this monoclonal antibody reacted with the recombinant SLFN11 protein to produce specific bands, indicating that this monoclonal antibody can specifically recognize the recombinant SLFN11 protein.

[0059] 4. Monoclonal Antibody IFA Identification

[0060] 293T cells were plated into 24-well plates containing cell slides. When the cells reached 50% to 60% confluence, the pET28a(+)-SLFN11-His eukaryotic plasmid and an empty vector plasmid were transfected into the 293T cells. After 24 hours of growth, the culture medium was discarded and the cells were fixed with 5% paraformaldehyde solution for 30 minutes. The cells were washed three times with PBS and permeabilized with TritonX100 for 15 minutes. After permeabilization, the cells were washed three times and blocked with 1% BSA for 2 hours at room temperature. Positive controls included SLFN11 mouse positive serum and Flag mouse monoclonal antibody, and negative controls included 293T cells transfected with an empty vector. The screened monoclonal antibody, SLFN11 mouse serum, and Flag mouse monoclonal antibody were used as primary antibodies and diluted at a dilution ratio of 1:500. 250 μL of diluted antibody was added to the SLFN11 plasmid transfection group, and 250 μL of serum diluted at the same ratio was added to the empty plasmid transfection group. The cells were incubated at room temperature for 1.5 hours. 250 μL of 1:1000 diluted FITC-labeled goat anti-mouse IgG was added to each transfection group as a secondary antibody and incubated in the dark for 1.5 hours. After 1.5 hours of light protection, the slides were removed and stained with Fluoroshield. TM DAPI staining and sealing were performed, and fluorescence was observed under a fluorescence microscope. Figure 8 .

[0061] 5. Sequencing of variable regions of hybridoma monoclonal antibody genes

[0062] The variable region nucleic acid and amino acid sequences of the monoclonal antibody were obtained by molecular cloning technology and sent to Nanjing Detai Biotechnology Co., Ltd. for sequencing. The sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody SLFN11-1B4H4G8 were determined, and the results are as follows:

[0063] (1) Amino acid sequence of the heavy chain variable region (SEQ ID NO. 1):

[0064] EVMLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVATISSGGTYTFYPDSVKGRFTISRDHAKS TLYLQMGSLKSEDTAIYYCTRRDGDSGYFDVWGPGTTVTVSS.

[0065] (2) Nucleotide sequence of the gene encoding the heavy chain variable region (SEQ ID NO.2):

[0066] GAAGTGATGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTAAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTACTTACACCTTCTA TCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACCATGCCAAGAGCACCCTGTACCTACAAATGGGCAGTCTGAAGTCTGAGGACACGGCCATTTATTACTGTACAAGACGGGATGGTGACTCCGGGTACTTCGATGTCTGGGGCCCAGGGACCACGGTCACCGTCTCCTCA.

[0067] (3) Amino acid sequence of light chain variable region (SEQ ID NO.3):

[0068] DIQMTQTTSSLSASLGDRVTISCRTSQDITNYLNWYQQKPDGTINLLIYYTSRLHSGVPSRFSGSGSGTDYSLTINN LEQEDIATYFCQQGRTLPLTFGGGTKLELR.

[0069] (4) Nucleotide sequence of the gene encoding the light chain variable region (SEQ ID NO.4):

[0070] GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTC ACCATCAGTTGCAGGACAAGTCAGGACATTACCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTATTAATCTCCTGATCTACTACACATCACGATTACACTCAGGAGTCCCTTCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAACAACCTGGAACAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTCGTACGCTCCCGCTCACGTTCGGTGGTGGGACCAA GCTGGAGCTGAGA.

[0071] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A monoclonal antibody against porcine SLFN11 protein, characterized in that: It includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.

3.

2. A gene encoding the monoclonal antibody against porcine SLFN11 protein as claimed in claim 1.

3. The coding gene according to claim 2, characterized in that The encoding genes include heavy chain variable region encoding genes and light chain variable region encoding genes; The nucleotide sequence of the heavy chain variable region encoding gene is shown in SEQ ID NO.2; The nucleotide sequence of the light chain variable region encoding gene is shown in SEQ ID NO.

4.

4. A gene expression cassette, characterized in that Comprising the coding gene according to claim 2 or 3.

5. A recombinant expression vector, characterized in that: Comprising the gene expression cassette of claim 4.

6. A recombinant host cell, characterized in that Comprising the recombinant expression vector according to claim 5.

7. Use of the encoding gene according to claim 2 or 3, the gene expression cassette according to claim 4, the recombinant expression vector according to claim 5, or the recombinant host cell according to claim 6 in preparing the anti-porcine SLFN11 protein monoclonal antibody according to claim 1.

8. Use of the anti-porcine SLFN11 protein monoclonal antibody according to claim 1 in the preparation of a product for detecting porcine SLFN11 protein.

9. A product for detecting porcine SLFN11 protein, characterized in that: The invention comprises the anti-porcine SLFN11 protein monoclonal antibody according to claim 1.

10. The product according to claim 9, characterized in that The product is an ELISA detection kit.

Images