Anti-swine SLFN11 protein monoclonal antibody and application thereof

Abstract

The invention discloses an anti-swine SLFN11 protein monoclonal antibody and application thereof, and relates to the technical field of biology. The anti-swine SLFN11 protein monoclonal antibody comprises a heavy chain variable region of which the amino acid sequence is shown as SEQ ID NO.1 and a light chain variable region of which the amino acid sequence is shown as SEQ ID NO.3. The invention further discloses a preparation method of the anti-swine SLFN11 protein monoclonal antibody. A heavy chain constant region of the monoclonal antibody is of an IgG1 type, a light chain constant region of the monoclonal antibody is of a Kappa type, and the monoclonal antibody has high affinity and stability and can be specifically combined with swine SLFN11 protein. The invention provides a good immunological tool for further exploring the antiviral function and mechanism of the SLFN11 protein, and lays a foundation for developing genetically engineered antibody drugs and novel vaccines for preventing or treating porcine viral diseases.

Description

Technical Field

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

[0002] The Schlafen (SLFN) gene family was originally discovered in humans and mice. It is an important gene involved in regulating biological functions in species such as humans and mice and has extensive homology. However, the functional scope of SLFN is still partially understood. Studies have shown that the SLFN protein encoded by the SLFN gene has multiple biological functions, which can regulate cell proliferation, differentiation of T cells and macrophages, inhibit the migration and invasion of cancer cells, inhibit the replication of viruses, etc.

[0003] SLFN family proteins are divided into three subgroups according to their size and structure: subgroup I (37-42kDa), subgroup II (58-68kDa) and subgroup III (100-104kDa). SLFN11 is a member of the SLFN gene family and belongs to subgroup III. It is an interferon-induced antiviral restriction factor that has the function of inhibiting the replication of multiple viruses and is closely related to the treatment of tumors. Li et al.'s research shows that SLFN11 can inhibit the synthesis of HIV viral proteins by binding to tRNA, offsetting the A / U preference of tRNA caused by the presence of HIV, and can be used as a new antiviral factor against HIV. In addition to HIV, SLFN11 has also been found to inhibit the replication activity of other viruses. Guo et al. showed that in addition to human SLFN11, bovine and monkey SLFN11 can also inhibit the replication of the prototype foamy virus. This inhibitory effect is achieved by disrupting the synthesis of the prototype foamy virus protein through the different codon usage between the virus and the host. In addition, they also found that the dephosphorylation of the SLFN11 protein, the activity of ATP synthase and helicase are also necessary for SLFN11 to exert its antiviral activity. However, there are no reports on the role of SLFN11 in major swine diseases.

[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, PRV, etc. The antiviral effect of SLFN may become a new target for anti-swine-related susceptible viruses. Therefore, the present invention constructs a porcine pET28a(+)-SLFN11-His recombinant expression plasmid and induces expression, purifies the recombinant protein, and prepares monoclonal antibodies against porcine SLFN11 recombinant protein after immunizing mice, in order to provide a good immunological tool for further studying the antiviral function and mechanism of porcine SLFN11. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-porcine SLFN11 protein monoclonal antibody and its application to solve the problems existing in the above-mentioned prior art. The heavy chain constant region of the monoclonal antibody is of IgG1 type and the light chain constant region is of Kappa type, which has strong affinity and stability and can specifically bind to the 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 the anti-porcine SLFN11 protein monoclonal antibody. At the same time, with the help of genetic engineering and protein engineering methods, the porcine SLFN11 protein monoclonal antibody can be modified in various ways, including adding, deleting or replacing one or more amino acids to obtain its active fragments or conservative variants, which provides the possibility of further improving the specificity and affinity of the antibody.

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

[0010] Further, the encoding gene includes 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 coding gene, gene expression cassette, recombinant expression vector or recombinant host cell in the preparation of the above-mentioned anti-swine SLFN11 protein monoclonal antibody.

[0017] The present invention also provides the use of the above-mentioned anti-swine SLFN11 protein monoclonal antibody in the preparation of a product for detecting the pig-derived 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] The present invention provides a monoclonal antibody against porcine SLFN11 protein, the heavy chain constant region of the monoclonal antibody is of IgG1 type, the light chain constant region is of Kappa type, it has strong affinity and stability, and can specifically bind to porcine SLFN11 protein. The present invention provides a good immunological tool for further exploring the antiviral function and mechanism of SLFN11 protein, and lays a foundation for the development of genetically engineered antibody drugs and new vaccines for preventing or treating 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

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

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

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

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

[0027] Figure 5 This is a statistical diagram 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 a result diagram of the specificity of the monoclonal antibody detected by Western Blot, where M represents Marker, 1 represents an irrelevant protein control, and 2 represents a 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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 fragment

[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 for expansion culture. 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. The culture was shaken at 37°C and 220 r / min. When the bacterial solution OD 600nm When the value reaches 0.6, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.1mmol / L. After 8 hours of culture, centrifuge and discard the supernatant. Add 15mL 1×PBS to resuspend the precipitate, and use an ultrasonic disruptor to break it on ice for 30 minutes, centrifuge and discard the supernatant. Resuspend the precipitate with 15mL 8M urea lysis buffer, shake it in a rotary mixer at 4℃ for 12 hours, and centrifuge to obtain the supernatant. Fill the purification column with Ni Sepharose6Fast Flow filler, purify the target protein and renature it.

[0041] Western Blot identification of SLFN11 protein: 12.5% ​​SDS-PAGE gel was selected and electrophoresis was performed at 120V. After electrophoresis, the protein was transferred to the PVDF membrane using the full wet transfer method, and the transfer time was 75min. After the transfer, it was blocked with 5% skim milk at room temperature for 2h. Subsequently, a 1:5000 diluted Anti-His mouse monoclonal antibody was used as the primary antibody and incubated at room temperature for 1h. After incubation, the membrane was washed 3 times with TBST, each for 10min. Then, a 1:5000 diluted horseradish peroxidase-labeled goat anti-mouse IgG (H+L) was used as the secondary antibody and incubated at room temperature for 1h. After incubation, the membrane was washed 3 times with TBST, each for 10min. Finally, the color developing solution was added under light-proof conditions, and after incubation for 15min, the membrane was developed and observed using an ultra-sensitive multifunctional imager.

[0042] The results of SDS-PAGE analysis of the prokaryotic expression of the 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 female BALB / c mice aged 6 to 8 weeks were selected, and the purified recombinant SLFN11 protein was renatured and uniformly emulsified with Freund's complete adjuvant at a ratio of 1:1. The mice were immunized by intraperitoneal injection (25 μg of recombinant SLFN11 protein was injected into each mouse). The interval between each immunization was two weeks. The second and subsequent immunizations were uniformly emulsified with incomplete Freund's adjuvant and recombinant SLFN11 protein at a ratio of 1:1. One week after the third immunization, the tail of the mice was bled. The mouse blood was graded 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. The protein concentration was 5 μg / mL, and the antibody titer was detected by indirect ELISA method (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 super-immunized 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 them in a 37°C, 5% CO2 incubator for culture. After the cells grow for 7-10 days, use the cell supernatant as the primary antibody and horseradish peroxidase-labeled goat anti-mouse IgG (H+L) as the secondary antibody, and use the indirect ELISA method to screen the positive wells.

[0049] 2. Preparation of monoclonal antibodies by ascites induction method

[0050] 1. Monoclonal Cell Subcloning and Ascites Preparation

[0051] Select indirect ELISA to detect OD 450nmThe wells with values ​​2.15 times higher than the negative control were transferred to 96-well plates, diluted according to the gradient, and observed under a microscope until there were about 90 to 100 cells in the wells. They were fully mixed with 1640 medium containing HAT, spread on 96-well plates, and expanded for culture; after the subclone cells were full, ELISA was performed, and the positive clone wells were selected for the second subclone; after the second subclone, ELISA was performed again until the positive rate of the cell supernatant of the monoclonal well was 100%. Inject Freund's incomplete adjuvant into the peritoneal cavity of BALB / c mice, 500μL per mouse, and hybridoma cells were injected into the peritoneal cavity 7 days later. After the abdomen of the mouse was obviously swollen, ascites was extracted, centrifuged at 12000r / min for 10min, and the supernatant was taken.

[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 resuspended solution in 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, that is, 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 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. IFA Identification of Monoclonal Antibodies

[0060] 293T cells were plated in a 24-well plate containing a cell slide. When the cells grew to 50% to 60%, the pET28a(+)-SLFN11-His eukaryotic plasmid and the empty plasmid were transfected into the 293T cells respectively. After the cells grew for 24 hours, the culture medium was discarded, and 5% paraformaldehyde solution was added to fix for 30 minutes, washed three times with PBS, and permeabilized with TritonX100 for 15 minutes. After permeabilization, it was washed three times and blocked with 1% BSA at room temperature for 2 hours. The positive control was SLFN11 mouse positive serum and Flag mouse monoclonal antibody, and the negative control was 293T cells transfected with an empty vector. The selected 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 the diluted antibody was added to the SLFN11 plasmid transfection group, and 250 μL of the serum diluted at the same ratio was added to the empty plasmid transfection group. Incubate at room temperature for 1.5 hours. 250 μL of FITC-labeled goat anti-mouse IgG diluted 1:1000 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 taken out and the slides were stained with Fluoroshield TM With DAPI staining and sealing, observe the fluorescence under a fluorescence microscope, the results are shown in 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 only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A monoclonal antibody against porcine SLFN11 protein, characterized in that: It comprises 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 anti-swine SLFN11 protein monoclonal antibody as claimed in claim 1.

3. The coding gene according to claim 2, characterized in that The encoding genes include a heavy chain variable region encoding gene and a light chain variable region encoding gene; 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 described in 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 coding 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 the preparation of the anti-porcine SLFN11 protein monoclonal antibody according to claim 1.

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

9. A product for detecting porcine SLFN11 protein, characterized in that: It 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.

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