Pig slpi monoclonal antibody and application thereof

Abstract

The application discloses a pig SLPI monoclonal antibody and application thereof, and belongs to the field of biotechnology and cell engineering. The application immunizes mice with a His-SLPI recombinant protein, and carries out cell fusion when the serum titer reaches 1:10000 or above. On the seventh day of the fusion, indirect ELISA is carried out on cell supernatant, and positive wells with higher OD values are screened out. The purified recombinant GST-SLPI protein is used as a coating antigen, the wells are expanded and cultured, and the cells are made into single cells in single wells by using an effective dilution method. After continuous screening to obtain 100% positive wells, the supernatant of the positive cells is used for specific identification of the pig-derived SLPI protein by Western blot and IFA. One hybridoma strain B with a higher binding titer is obtained by identification, the heavy chain amino acid sequence of the SLPI monoclonal antibody produced by the hybridoma strain B is shown as SEQ ID NO. 1, and the light chain amino acid sequence is shown as SEQ ID NO. 2.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and cell engineering, and particularly relates to a porcine SLPI monoclonal antibody and its application. Background Technology

[0002] Secretory leukocyte protease inhibitor (SLPI) plays a crucial regulatory role in both innate and adaptive immunity and is an important component of tissue regeneration. SLPI is a cationic inhibitor of elastase, a non-glycosylated protein molecule secreted by mucosal epithelial cells, consisting of a single-chain polypeptide of 107 amino acids with a relative molecular mass of 11.7 kDa. SLPI is a member of the whey acidic protein family and plays a key role in host anti-protease selection. SLPI has been confirmed to be mainly present in epithelial cells of the respiratory and digestive tracts, and is also distributed in cells of the parotid gland (e.g., in secreted saliva). SLPI consists of two highly homologous N-terminal and C-terminal regions. The C-terminal region broadly inhibits the activity of mast cell and leukocyte serine proteases, while the N-terminal region promotes the binding of the inhibitor to serine proteases by mediating interactions with heparin. As a natural protease inhibitor, SLPI can inhibit neutrophil elastase, cathepsin G, mast cell chymotrypsin-like proteins, trypsin, and chymotrypsin, maintaining a local balance between protease inhibitors and proteases. SLPI is known to attenuate the endotoxin response of monocytes / macrophages by inhibiting the transcription factor NF-κB. SLPI can antagonize the production of cytokines by monocytes and other leukocytes, and effectively prevent the cascade reaction of inflammatory cytokines, thus regulating anti-inflammatory effects. SLPI is effective against Gram-positive and Gram-negative bacteria because its cationic nature disrupts the stability and integrity of bacterial cell membranes, thereby killing cells. SLPI is also effective against viral infections such as herpes simplex virus (HSV), human papillomavirus (HPV), and Epstein-Barr virus (EBV). Furthermore, SLPI is expressed during skin wound healing, participates in tissue repair, and plays a role in cell proliferation, differentiation, and apoptosis.

[0003] SLPIs, as anti-inflammatory mediators, can counteract the body's inflammatory response. Secreted by intestinal mucosal epithelial cells, SLPIs can defend against the invasion of pathogens and harmful microorganisms in the intestine, enhancing their protective and anti-inflammatory effects and indicating their research value in the gut. PCV2 is highly pathogenic to pigs, seriously threatening the pig industry. Enteritis is one of the PCV2-related diseases, causing intestinal mucosal damage. Secretory immunoglobulin A (SlgA) participates in immune regulation in the intestinal mucosal immune system; disordered SlgA secretion affects the immune system, leading to diarrhea in piglets. Chemokines (CCL28) can induce widespread mucosal migration of IgA antibody-secreting cells. SIgA secretion depends on polymeric immunoglobulin receptor (pIgR). PCV2 infection downregulates the expression of both PIGR and CCL28, resulting in decreased SIgA secretion. SLPIs can prevent the production of pro-inflammatory cytokines and indirectly weaken adaptive inflammatory immune responses, maintaining the balance of immune responses in mucosal barrier tissues. SLPI can also inhibit SIgA secretion by suppressing NF-κB protein degradation and downregulating pIgR expression through the NF-κB signaling pathway. Experiments have shown that PCV2 can upregulate SLPI secretion from porcine intestinal epithelial cells. Therefore, the preparation of SLPI monoclonal antibodies is significant for studying the role of PCV2 infection in IPECs during SIgA secretion and for exploring the immunopathogenic mechanism of PCV2. Currently, there are no porcine SLPI monoclonal antibody products available. Therefore, based on previously obtained high-purity SLPI protein through prokaryotic expression and purification, monoclonal antibodies were prepared by immunizing mice using hybridoma cell technology. Western blotting, IFA, and ELISA were used for detection and specificity identification. The prepared monoclonal antibody provides a technical platform for establishing a detection method for porcine SLPI protein and exploring its role in intestinal immunity. Furthermore, the development of inexpensive, active, high-purity SLPI protein and its high-performance porcine SLPI monoclonal antibodies has important application value in detection and treatment. Summary of the Invention

[0004] This invention utilizes pET28a(+) as the expression vector in E. coli BL21(DE3) to fuse two tandem SLPI genes, controlled by the T7 promoter, with six His tags, BamHI, and XhoI restriction enzyme sites at their N-terminus, thereby achieving SLPI fusion protein inclusion body expression. After denaturation and renaturation, the inclusion bodies are purified using a Ni column to obtain a high-purity recombinant SLPI protein, which is used for antibody preparation. Meanwhile, pGEX-6P-1-2SLPI expression yields recombinant SLPI protein, which is used for antibody titer detection.

[0005] The present invention provides an SLPI monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the SLPI monoclonal antibody is shown in SEQ ID NO.1 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0006] The present invention also provides nucleotide sequences encoding the heavy and light chains of the SLPI monoclonal antibody.

[0007] The present invention also provides a kit for detecting SLPI antigen, the kit containing the above-mentioned SLPI monoclonal antibody.

[0008] The present invention also provides the application of the SLPI monoclonal antibody in the preparation of products for detecting SLPI antigens.

[0009] Furthermore, the SLPI antigen is a porcine antigen.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The monoclonal antibody produced by the hybridoma cell line of this invention has a very high binding titer. Attached Figure Description

[0012] Figure 1 This is a flowchart of the experimental process of the present invention.

[0013] Figure 2 The image shows the SDS-PAGE analysis results of the purified recombinant His-SLPI protein in Example 1, where M: protein marker; 1: recombinant His-SLPI protein; 2-7: wash buffer collected during purification; 8-9: elution buffer (purified recombinant His-SLPI protein).

[0014] Figure 3 The results of Western blot identification of the three monoclonal antibodies in Example 1 are shown, where M: Marker; 1: recombinant His-SLPI protein; 2: pET-28a empty vector; A: 9D10; B: 9G11; C: 10E2.

[0015] Figure 4 The results of IFA identification of the three monoclonal antibodies in Example 1 are shown; (+) positive serum control, (-) negative serum control, A: 9D10; B: 9G11; C: 10E2. Detailed Implementation

[0016] Example 1

[0017] The SLPI (NM_213870.1) gene sequence was found from NCBI. After optimizing the gene according to the codons of E. coli, the two SLPI gene sequences were tandemly linked and ligated into the pGEX-6P-1 and pET-28a vectors respectively to construct the pGEX-6P-1-2SLPI and pET-28a-2SLPI recombinant plasmids, which were then sent to the company for synthesis.

[0018] Remove E. coli Trans10 and BL21 competent cells and place them on crushed ice. Add 10 μL each of pGEX-6P-1-2SLPI and empty vector pGEX-6P-1 to E. coli Trans10 competent cells and label them. Add 10 μL each of pET-28a-2SLPI and empty vector pET-28a to BL21 competent cells, following the instructions for use of competent cells. Perform double enzyme digestion identification the next day.

[0019] 100 μL of each of the following bacterial strains—pGEX-6P-1-2SLPI, empty vector pGEX-6P-1, pET-28a-2SLPI, and empty vector pET-28a—were added to 10 mL of Amp+ / Kan+ (1:1000) liquid culture medium, respectively. The cultures were incubated at 37°C and 200 rpm for approximately 3 hours, and the OD600 nm value was read using a microplate reader. When the OD600 nm value reached approximately 0.6, 0.75 mM IPTG was added, and the culture was continued for 6 hours in a shaker to induce the expression of recombinant SLPI protein. The bacterial cells were collected, resuspended in 3 mL of PBS, and subjected to SDS-PAGE electrophoresis. The remaining cells were sonicated (on ice), and the supernatant and precipitated proteins were collected by low-temperature centrifugation and subjected to SDS-PAGE electrophoresis.

[0020] After identifying the soluble forms of the two recombinant proteins, large-scale bacterial cell preparation began. The recombinant His-SLPI protein was purified according to the instructions of the Kangwei Century His-tagged protein purification kit (inclusion body protein). The recombinant GST-SLPI protein expressed by the recombinant bacteria pGEX-6P-1-2SLPI was purified according to the instructions of the Beyotime GST-tagged protein purification kit, and the eluent was collected for SDS-PAGE protein purity assessment.

[0021] Cut the dialysis bags to the appropriate length as needed, and activate them according to the instructions. Weigh urea in advance and serially dilute it to 2 mol / L. Place the dialysis bags in an 8 mol / L urea beaker at low temperature, changing the medium every 12 hours, avoiding protein precipitation during this process. Concentrate the renatured protein by centrifuging at 4500 rpm for 20 minutes in an ultrafiltration tube. Immunize mice using the His-SLPI recombinant protein according to the immunization schedule. To exclude non-specific antibodies, use the purified recombinant GST-SLPI protein as the coating antigen to establish an indirect ELISA for determining the serum titer of immunized mice. When the titer reaches 1:10000 or higher, cell fusion begins. On day 7 of fusion, the cell supernatant is analyzed using an indirect ELISA, selecting positive wells with higher OD values ​​to screen for positive hybridoma cells. Positive hybridoma cells were cultured in expanded quantities, and the cells were diluted to single-cell wells using an effective dilution method. After continuous subcloning until 100% positive wells were obtained, the supernatant of the positive cells was used to specifically identify porcine SLPI protein using Western blot and IFA, respectively. A total of three hybridoma cell lines (A, B, and C) were identified. The identification results are shown below. Figure 3 and Figure 4 The results showed that the porcine SLPI monoclonal antibody produced by the cell tumor hybrid B had the strongest ability to specifically recognize both exogenous and endogenous porcine SLPI proteins.

[0022] The amino acid sequence of the heavy chain variable region of the monoclonal antibody produced by hybridoma cell line B is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0023] SEQ ID NO.1:Glu Val Leu Glu Ser Gly Gly Leu Val Gln Pro Gly Gly SerMet Arg Leu Cys Val Ala Ser Phe AspThr Phe Ser Asp Trp Gly Asp Val Gln ProGlu Lys Leu Glu Val Glu Ly Arg SerAlaAsn Asn His Pro Tyr Thr Serg With Tyr Gln Met AsnLeuArg Thr Asp Thr Gly With TyrCys ThrAsp Ser Ala Thr Trp Gly Gly Thr Val Thr Ser Ser

[0024] SEQ ID NO.2:Ser Ile Val Gln Thr Pro Ser Leu Val Ala Ser Ala Asp ArgVal Ile Thr Lys Ser Gln Ser Val Asn Asp Val Ala Tyr Gln Gln Phe Lys Pro GlySer Lys Leu Ile Phe TyrAla Leu Tyr Cys Gln Asp Tyr Ser Pro Pro Thr Phe GlySer Gly Lys Leu Glu Lys AlaAlaAla(SIVQTPSLVASADRVITKSQSVN DVAYQQFKPGSKLIFYASRNGVDRTSYGDFISQGAE LYCQDYSPPTFGSGKLEKAAA)。

[0025] NCBI GenBank SLPI database:

[0026] SEQ ID NO.3:

[0027] ggcaggggcctcttgcccttcgtgcttcttgccctgggaatccatggcaccttgggccgtggaaggtgctgaaaatgctttgaaagggggagc

[0028] ctgccctcctagaaaaattgtccagtgccttagatatgagaaacccaagtgcacaagtgactggcagtgtccagacaagaagaaatgttgccg

[0029] agatacttgcgcaatcaaatgcctgaaccctgttgctatcacgaacccagttaaggtgaagcctgggaagtgtccagtggtctatggccagtgta

[0030] tgatgctcaacccccccaatcactgcaagacagacagccagtgcctgggtgacttaaaatgctgcaagagcatgtgcgggaaagtctgcctca

[0031] cccctgtgaaagcctgattcctgccatttagaagaggctctggattcctactttgtgctgtctgggaattccatttccactcccagacgtggatccc

[0032] tggggcaggccgtgataaagacttggttctaccaccaccatctactttgcagaacatctcagcaccctgtaggcttccaggacatgcctgttgattggtgcatcaataaataagcatatttctctctgt。

[0033] Optimized tandem porcine SLPI nucleotide sequence:

[0034] SEQ ID NO.4:

[0035] ggatccATGGCACCGTGGGCGGTTGAAGGCGCGGAAAACGCACTGAAAGGTGGTGCGTGCCCGCCGCG

[0036] TAAAATTGTGCAGTGTCTGCGTTATGAAAAACCGAAATGTACCAGCGATTGGCAGTGTCCGGATAAAA

[0037] AGAAATGTTGTCGTGATACATGTGCCATCAAATGTCTGAATCCGGTTGCAATTACCAATCCAGTTAAAG

[0038] TTAAACCAGGTAAATGTCCGGTTGTTTATGGTCAGTGTATGATGCTGAATCCGCCGAATCATTGTAAAA

[0039] CGGATAGCCAGTGCCTGGGTGATCTGAAATGTTGCAAATCTATGTGCGGTAAAGTTTGTCTGACCCCG

[0040] GTTAAAGCCATGGCACCGTGGGCAGTTGAAGGTGCAGAAAATGCGCTGAAAGGTGGCGCATGTCCTC

[0041] CGCGTAAAATCGTTCAGTGCCTGCGTTATGAAAAACCTAAATGCACCTCTGATTGGCAGTGCCCGGAT

[0042] AAAAAGAAATGCTGTCGTGATAACCTGTGCCATCAAATGCCTGAATCCGGTGGCTATCACCAATCCGGT

[0043] TAAAGTTAAACCGGGTAAATGCCCGGTTGTTTATGGCCAGTGTATGATGCTGAACCCGCCGAATCATTG

[0044] CAAAACGGATTCCCAGTGTCTGGGTGATCTGAAATGCTGCAAATCTATGTGTGGTAAAGTTTGCCTGACGCCTGTTAAAGCCTAActcgag.

[0045] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An SLPI monoclonal antibody, characterized in that, The amino acid sequence of the heavy chain variable region of the SLPI monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

2. The nucleotide sequences encoding the heavy chain variable region and light chain variable region of the SLPI monoclonal antibody of claim 1.

3. A kit for detecting SLPI antigen, characterized in that, The kit contains the SLPI monoclonal antibody as described in claim 1.

4. The use of the SLPI monoclonal antibody according to claim 1 in the preparation of products for detecting SLPI antigen.

5. The application according to claim 4, characterized in that, The SLPI antigen is a porcine antigen.

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