Preparation method of monoclonal antibody of coccidian oocyst protein

Abstract

The invention provides a preparation method of a monoclonal antibody of coccidian oocyst protein, and belongs to the technical field of antibodies. The method comprises the following steps: carrying out flow sorting on single antigen-specific B lymphocytes, carrying out stimulated differentiation culture in a specific culture medium by taking EL4B5 as feeder layer cells, cracking obtained plasma cells, carrying out PCR (Polymerase Chain Reaction) amplification to obtain a variable region sequence of a monoclonal antibody, and carrying out recombinant expression to obtain the coccidian oocyst protein monoclonal antibody. The monoclonal antibody of the coccidian oocyst protein provided by the invention comprises at least one of coccidian monoclonal antibodies 6B10, 5C8 and 5D5. According to the present invention, the antigen affinity verification is performed on the monoclonal antibodies obtained through the recombinant expression, and the results show that the three monoclonal antibodies have good antigen affinity and good detection specificity, such that the effective tool is provided for the subsequent diagnosis and detection of the porcine coccidiosis and the porcine coccidiosis infection diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of antibodies, and particularly relates to a method for preparing a monoclonal antibody against coccidian oocyst protein. Background Art

[0002] Rabbits are a widely used host species for the production of polyclonal and monoclonal antibodies. Rabbit monoclonal antibodies exhibit exceptionally high specificity and sensitivity, recognize a wider range of epitopes, and possess excellent affinity, making them commonly used as diagnostic reagents in veterinary medicine and the life sciences.

[0003] Due to the lack of suitable fusion partner cells, initial attempts to develop rabbit hybridomas involved fusing rabbit spleen cells with mouse SP2 / 0 cells. However, the fusion rate of rabbit-mouse fusions was low, and the chromosomes of the fused cells were highly unstable, resulting in a significant loss of positive rates. Previous rabbit monoclonal antibody development was primarily based on monoclonal antibodies derived from rabbit hybridomas at Epitomics, which has strong rabbit monoclonal antibody development capabilities and whose rabbit myeloma cell-derived rabbit monoclonal antibody technology is still under patent protection.

[0004] With the advancement of molecular biology, immunology and flow cytometry sorting technology, it has become possible to sort out single antibody-secreting cells (plasma cells or B lymphocytes) from animals. The sorted cells are then cultured in vitro or subjected to single-cell omics sequencing to obtain the antibody-encoding gene sequence. The monoclonal antibodies obtained by in vitro recombinant expression have been widely used in animals such as mice, horses, pigs, cattle, sheep and rabbits. This can circumvent the patent technology barriers of Epitomics and prepare a new generation of monoclonal antibodies.

[0005] The widespread application of single-cell specific B cell in vitro culture and 10×genomics sequencing technology has made it possible to further screen for cells with high-affinity, high-expression antibodies and reduce false positive rates. This is particularly true for single-cell specific B cell in vitro culture, which utilizes a feeder layer of cells to support B cell proliferation and expansion. This feeder layer also incorporates relevant cytokines to simulate T cell stimulation of B cells.

[0006] Feeder layer matrix coverage facilitates cell attachment, growth, and differentiation. Some difficult-to-culture cells, especially those that are more difficult to culture at low cell densities, require living cells to maintain them (e.g., mouse embryonic fibroblasts). This may be due to leakage of metabolites from fibroblasts (actually primitive mesenchymal cells, not fibroblast metabolites) or supplementation of the culture medium with growth factors secreted by fibroblasts, or it may be due to the optimization of the cell attachment surface by cell products. After the monolayer culture reaches confluence, subsequent proliferation causes cells to detach from the artificial attachment surface and migrate to the surface of the monolayer. Cell morphology will also change, such as poor cell spreading, darker staining, and a high degree of differentiation. This is obviously not surprising, because the interaction between cells and their underlying layer is different from the interaction between cells and the synthetic matrix attachment surface, which causes cell morphology changes and reduces the cell's proliferation potential. This undoubtedly affects the preparation of single antibody-secreting cells.

[0007] The development of monoclonal antibodies against coccidian oocyst proteins is the basis for the diagnosis of porcine coccidiosis. Unfortunately, there are currently no reports of monoclonal antibodies with high affinity and specificity for Cryptosporidium parvum. Summary of the Invention

[0008] In view of this, the object of the present invention is to provide a method for preparing monoclonal antibodies against coccidian oocyst proteins, and to develop monoclonal antibodies with high affinity and specificity based on the screening and cultivation of single B lymphocytes.

[0009] The present invention provides a method for screening monoclonal antibodies to coccidian oocyst proteins, comprising the following steps:

[0010] Rabbits were immunized with coccidian oocyst protein and peripheral blood or spleen was collected;

[0011] isolating mononuclear cells from the peripheral blood of the rabbit and / or isolating spleen cells from the spleen;

[0012] flow cytometry-sorting antigen-specific B lymphocytes from the mononuclear cells and / or spleen cells;

[0013] The antigen-specific B lymphocytes are cultured in a culture medium containing EL4B5 feeder cells to obtain plasma cells;

[0014] The culture medium contains 10 ng / mL interleukin-1β, 10 ng / mL tumor necrosis factor α, 10 ng / mL IL-21, 10 ng / mL IL-2, 10 ng / mL IL-10, 10 ng / mL TNF-α, 10 ng / mL IL-6, and 10 ng / mL Staphylococcus aureus Cowans strain secretion SAC;

[0015] The cultured plasma cells were lysed, and the resulting cell lysate was used as a template to amplify the variable region of a rabbit monoclonal antibody.

[0016] Preferably, the cell density of the EL4B5 feeder cells is 1×10 7 ~1×10 9 / ml.

[0017] Preferably, the cell density of the antigen-specific B lymphocytes is 1×10 7 ~1×10 9 / ml.

[0018] Preferably, the amino acid sequence of the coccidian oocyst protein is shown in SEQ ID NO: 7.

[0019] Preferably, the primers for amplifying the variable region of the rabbit monoclonal antibody include nucleotide sequences as shown in SEQ ID NO: 8 to SEQ ID NO: 40.

[0020] The present invention provides a monoclonal antibody to coccidia oocyst protein obtained by screening using the screening method, comprising at least one of the following: coccidia monoclonal antibody 6B10, coccidia monoclonal antibody 5C8, and coccidia monoclonal antibody 5D5;

[0021] The amino acid sequence of the heavy chain variable region of the coccidia monoclonal antibody 6B10 is shown in SEQ ID NO: 1, and the amino acid sequence of the corresponding light chain variable region is shown in SEQ ID NO: 2;

[0022] The amino acid sequence of the heavy chain variable region of the coccidia monoclonal antibody 5C8 is shown in SEQ ID NO: 3, and the amino acid sequence of the corresponding light chain variable region is shown in SEQ ID NO: 4;

[0023] The amino acid sequence of the heavy chain variable region of the coccidia monoclonal antibody 5D5 is shown in SEQ ID NO: 5, and the amino acid sequence of the corresponding light chain variable region is shown in SEQ ID NO: 6.

[0024] The present invention provides application of the monoclonal antibody in preparing a product for detecting porcine coccidiosis.

[0025] Preferably, the product includes a diagnostic kit and a detection reagent.

[0026] Preferably, the product is prepared based on the following immunoassay technology:

[0027] Enzyme-linked immunosorbent assay, immunohistochemistry, in vitro chromatography, flow cytometry, hemagglutination, and immunoblotting.

[0028] The present invention provides a porcine coccidiosis detection kit based on immunoassay technology, comprising the monoclonal antibody and a detection antigen;

[0029] The detection antigen includes coccidian oocyst protein.

[0030] The present invention provides a method for screening monoclonal antibodies against coccidian oocyst proteins. Mononuclear cells isolated from the peripheral blood of an immunized animal or spleen cells isolated from the spleen are flow-sorted. The resulting B lymphocytes capable of producing specific IgG antibodies are stimulated and differentiated in a specific culture medium using EL4B5 cells as feeder cells, enabling them to differentiate into plasma cells that secrete specific IgG antibodies. The plasma cells are lysed, and the resulting lysate contains DNA fragments encoding the specific IgG antibodies. This is then amplified by PCR to obtain the variable region sequence of the monoclonal antibody. Thus, the screening method provided by the present invention promotes the proliferation and differentiation of single B lymphocytes by optimizing the feeder cells and culture medium, maintaining the proliferation and differentiation potential of single B lymphocytes, and thereby ensuring in vitro screening of monoclonal antibodies using single specific B cells.

[0031] The present invention provides monoclonal antibodies against coccidian oocyst proteins, including at least one of the following: coccidian monoclonal antibody 6B10, coccidian monoclonal antibody 5C8, and coccidian monoclonal antibody 5D5. Antigen affinity validation of the recombinantly expressed monoclonal antibodies was performed, and the results demonstrated that all three monoclonal antibodies exhibited good antigen affinity and specificity, providing an effective tool for the subsequent diagnosis and detection of porcine coccidian infections and their associated infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The amino acid sequence of the coccidian oocyst protein and the corresponding coding gene map;

[0033] Figure 2 The results of SDS-PAGE electrophoresis detection of recombinant monoclonal antibodies;

[0034] Figure 3 Results of flow cytometry sorting of single antigen-specific B lymphocytes; A is the lymphocytes circled by gate A, B is the single cell image circled by gate B, and C is the 7-AAD circled by negative screening of gate C. - Cell population, D is the D gate negative screening circle FITC cell population, E is the E gate positive screening circle APC + Antigen-specific cell populations, F is the distribution result diagram of each cell population in the loss cell analysis sample;

[0035] Figure 4 Principle of antibody affinity determination Figure 1 ;

[0036] Figure 5 Principle of antibody affinity determinationFigure 2 ;

[0037] Figure 6 Affinity detection results of monoclonal antibody 6B10;

[0038] Figure 7 Affinity detection results of monoclonal antibody 5C8;

[0039] Figure 8 Affinity detection results of monoclonal antibody 5D5. DETAILED DESCRIPTION

[0040] The present application provides a screening method of a monoclonal antibody of coccidian oocyst protein, comprising the following steps:

[0041] immunizing a rabbit with coccidian oocyst protein, and collecting peripheral blood or spleen of the rabbit;

[0042] isolating single nuclear cells from the peripheral blood of the rabbit and / or spleen cells from the spleen;

[0043] flow sorting antigen-specific B lymphocytes from the single nuclear cells and / or spleen cells;

[0044] culturing the antigen-specific B lymphocytes in a culture medium containing EL4B5 feeder cells to obtain plasma cells;

[0045] the culture medium contains 10 ng / mL interleukin-1β, 10 ng / mL tumor necrosis factor α, 10 ng / mL IL-21, 10 ng / mL IL-2, 10 ng / mL IL-10, 10 ng / mL TNF-α, 10 ng / mL IL-6 and 10 ng / mL Staphylococcus aureus Cowans strain exudate SAC;

[0046] lysis of the cultured plasma cells, and amplifying the variable region of the rabbit monoclonal antibody by taking the obtained cell lysate as a template.

[0047] The present application immunizes a rabbit with coccidian oocyst protein, and collects peripheral blood or spleen of the rabbit.

[0048] In the present invention, the amino acid sequence of the coccidian oocyst protein is PPVPSCPPGFTLEPRGCV RARQVPPVIRCPKKSVASGNECVTKDFAAGIEVCPEGFMEKNRKCRKVISVQPELGCKKGFALQAGGDCVRAGEDDVITRCPKHSKQTSKGCITVQKVDVEYNCPEGLELRKNSCVGTQNIDAVPSCPNGFLLESDMCVRKTATPPRVSCPKGYKAQDNGCVLIEKFDLDVSCREGEYDGKKHCRKTALEPPAPHCPPGALLKDKECVR (SEQ ID NO: 7). The coccidian oocyst protein is preferably obtained by recombinant expression.The nucleotide sequence of the gene encoding the coccidian oocyst protein is shown in SEQ ID NO: 41 (CCGCCTGTGCCATCA). The recombinant expression method preferably involves cloning the gene encoding the coccidian oocyst protein into a vector, transforming the resulting recombinant vector into a competent Escherichia coli prokaryotic expression system, culturing and inducing expression, and isolating and purifying the recombinant protein to obtain the coccidian oocyst protein.

[0049] In the present invention, the coccidian oocyst protein is prepared into a vaccine and then rabbits are immunized. The rabbits are preferably New Zealand white rabbits. The immunization method is preferably a multi-point immunization injection on the abdomen and back of the rabbit, with a total of 3 immunizations, 3 weeks between two adjacent injections, and a dose of 200 μg of antigen for each immunization. During the first immunization, the vaccine used is a mixture of the coccidian oocyst protein and Freund's complete adjuvant in a volume ratio of 1:1 and emulsified. During the second and third immunizations, the vaccine used is a mixture of the coccidian oocyst protein and Freund's incomplete adjuvant in a volume ratio of 1:1 and emulsified. After the three immunizations, the titer of antibodies against porcine coccidian oocyst wall antigens in the serum is preferably determined, and rabbits with higher titers are selected for a booster immunization. The antigen immunization dose for the booster immunization is preferably 200 μg. Three days after the booster immunization, the spleen or peripheral blood is collected.

[0050] After obtaining the peripheral blood or spleen of the immunized rabbit, the present invention separates mononuclear cells from the peripheral blood of the rabbit and / or separates spleen cells from the spleen.

[0051] The present invention has no particular limitation on the method for isolating mononuclear cells or spleen cells, and any separation method known in the art may be used, such as density gradient centrifugation to separate mononuclear cells.

[0052] After obtaining mononuclear cells and / or spleen cells, the present invention performs flow cytometry sorting of antigen-specific B lymphocytes from the mononuclear cells and / or spleen cells.

[0053] In the present invention, the principle of sorting antigen-specific B lymphocytes is to utilize the differences in markers on the surfaces of different lymphocytes, and to use negative screening of antibodies against cell surface markers to remove T, monocytes, and other lymphocytes, thereby increasing the abundance of B lymphocytes. B lymphocytes with the ability to secrete IgG antibodies are then enriched by removing B lymphocytes with IgM antibodies through negative screening, and then antigen-specific screening is performed using a pre-labeled screening antigen. Since the pre-screening labeling antigen formed by incubating the biotin-labeled immunogen with the avidin-labeled fluorescent dye is an "antigen-biotin-avidin-fluorescent dye" complex, the characteristic of this complex is that it can utilize the amplification effect of biotin-avidin to improve the efficiency of B lymphocyte screening. In an embodiment of the present invention, the method for sorting antigen-specific B lymphocytes can refer to the method for efficiently isolating single antigen-specific B lymphocytes from spleen cells disclosed in the patent publication number CN 110016462A.

[0054] After flow cytometry sorting, the present invention cultured the antigen-specific B lymphocytes in a culture medium containing EL4B5 feeder cells to obtain plasma cells.

[0055] In the present invention, the cell density of the EL4B5 feeder cells is preferably 1×107 ~1×10 9 / ml, more preferably 1×10 8 / ml. The cell density of the antigen-specific B lymphocytes is preferably 1×10 7 ~1×10 9 / ml, more preferably 1×10 8 / ml. The culture medium contains 10ng / mL interleukin-1β, 10ng / mL tumor necrosis factor α, 10ng / mL IL-21, 10ng / mL IL-2, 10ng / mL IL-10, 10ng / mL TNF-α, 10ng / mL IL-6 and Staphylococcus aureus Cowans strain secretion SAC. The dilution ratio of Staphylococcus aureus Cowans strain secretion SAC is preferably 1:75,000. The culture medium also preferably includes 10ng / mL BAFF (B cell activating factor of the tumor necrosis factor family). The culture medium and EL4B5 feeder cells are used to culture antigen-specific B lymphocytes, which is beneficial to simulate the stimulation of cytokines in vivo, promote cell proliferation and differentiation, and obtain a large number of plasma cells capable of secreting antibodies.

[0056] After obtaining plasma cells, the present invention lyses the cultured plasma cells and uses the obtained cell lysate as a template to amplify the variable region of the rabbit monoclonal antibody.

[0057] The present invention has no particular limitation on the method for lysing cells, and any cell lysing method known in the art, such as repeated freezing and thawing, may be used.

[0058] In the present invention, after cell lysis, the cell lysate is collected, and an antigen-coated ELISA detection kit is preferably used to identify positive clones. The single-well cell lysate of the positive clone is selected as a template, and after reverse transcription and nested PCR amplification, the obtained PCR product is a naturally paired rabbit monoclonal antibody light and heavy chain variable region gene (VH and VL) fragment. The primers for amplifying the variable region of the rabbit monoclonal antibody include nucleotide sequences as shown in SEQ ID NO: 8 to SEQ ID NO: 40. The PCR product is recombinantly expressed in a prokaryotic expression system as a rabbit monoclonal antibody, and then the antigen-antibody specific affinity is determined to obtain a monoclonal antibody against coccidian oocyst protein.

[0059] The present invention provides a monoclonal antibody to coccidia oocyst protein obtained by screening using the screening method, comprising at least one of the following: coccidia monoclonal antibody 6B10, coccidia monoclonal antibody 5C8, and coccidia monoclonal antibody 5D5.

[0060] In the present invention, the coccidian monoclonal antibody 6B10 specifically binds to coccidian oocyst protein, has a heavy chain variable region (VH) defined according to the Kabat numbering system, and the heavy chain variable region (VH) has a single complementarity determining region (CDRs). The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1 (METGLRWLLLVAVFKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLDTYHI SWVRQAPGKGLEWIGAVTRDDVICFANWAKGRFTISKTSTTVDLKINSPTTEDTATYFCVRDDDFSSRNLWGPGTLVTVSS), and comprises a light chain variable region (VL) defined according to the Kabat numbering system, and the light chain variable region (VL) has three complementarity determining regions (CDRs). The corresponding amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2 (MDTRAPTQLLGLLLLWLPGARCAYDMTQTPASVEVAVGDTVTIKCQASQN VNIRLSWVQQKSGQPPKQLIYAASNLASGVPSRFEGSGSGTEFTLTIRDLECAD AAIYYCQQTASYHDVDDAFGGGTEVVVK).

[0061] In the present invention, the coccidia monoclonal antibody 5C8 specifically binds to coccidia oocyst protein. The antibody includes a heavy chain variable region (VH) and a light chain variable region (VL). The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 3 (METGLRWLLLVAVLKGVRCQSMEESGGRLVTPGTPLTLTCTVSGFT ISDDTINWVRQAPGKGLEWIGSIYAATGGTYYASWARGRFTISRTSTTVDLKM TSLTTEDTATYFCARYLALWGPGTLVTVSS), and the corresponding light chain variable region amino acid sequence is shown in SEQ ID NO: 4 (MDTRAPTQLLGLLLLWLPGATFAIVMTQTPASVSVPV GDTVTINCQASESVYRKNWLAWFQQKPGQPPKLLIYQASTLASGVPSRFRGS GSGTQFTLTISDVVCDDAATYYCAGYKGALTDGTAFGGGTEVVVK).

[0062] In the present invention, the coccidia monoclonal antibody 5D5 specifically binds to coccidia oocyst protein, and the antibody includes a heavy chain variable region (VH) and a light chain variable region (VL). The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5 (METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFDL SRESMGWVRQAPGKGLESIGMIGSSGSTYYASWAKGRFTISKTSSTTVDLKM NSLTTEDTATYFCARAWDLWGPGILVTVSS), and the corresponding light chain variable region amino acid sequence is shown in SEQ ID NO: 6 (MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAV GGTVTINCQSSQSVYGNNWLAWYQQKPGQPPKRLIYLASTLASGVPSRFKGS GSGTQFTLTISDVQCDDAATYYCQGGYSTQIDPFGGGTEVVVK).

[0063] In the present invention, the affinity of the obtained antibodies was accurately measured using the GatorPlus Biolayer Interferometry (BLI) system. The results showed that all three monoclonal antibodies had good binding specificity to coccidian oocyst protein.

[0064] Based on the good specific affinity of the monoclonal antibody to porcine coccidia antigens, the present invention provides the use of the monoclonal antibody in preparing a product for detecting porcine coccidiosis.

[0065] The product preferably comprises a diagnostic kit and / or a detection reagent. The product is preferably prepared based on the following immunoassay techniques: enzyme-linked immunosorbent assay, immunohistochemistry, in vitro chromatography, flow cytometry, hemagglutination, and immunoblotting.

[0066] The present invention provides a pig coccidiosis detection kit based on immune detection technology, comprising the monoclonal antibody and a detection antigen; the detection antigen preferably comprises coccidian oocyst protein.

[0067] In the present invention, the detection kit is preferably a colloidal gold immunochromatographic test strip or card. Based on competitive detection, the colloidal gold immunochromatographic test strip or card preferably includes a conjugate pad carrying the monoclonal antibody labeled with colloidal gold, a detection area coated with coccidian oocyst protein to form a detection line, and a quality control line formed by mouse anti-rabbit IgG antibody. The present invention does not particularly limit the preparation method of the detection kit; any test strip known in the art can be used.

[0068] In order to make the purpose, technical solutions and advantages of this application more clear, the present application is further described in detail below with reference to the examples. The specific implementation cases described in this description are only used to illustrate this application and are not intended to limit this application. Reagents not described in detail in this application are conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are conventional experimental methods and can be obtained from the prior art.

[0069] Explanation of terms

[0070] In this application, the term "antibody" is explained in detail. Antibodies are immunoglobulins produced by B cells under the stimulation of antigenic substances and can specifically bind to corresponding antigens. Globulins with antibody activity and similar structures to antibodies are collectively referred to as immunoglobulins (Ig). Antibodies are mainly distributed in the blood and also exist in other parts of the body, including: (1) plasma and interstitial fluid where antibody-secreting B cells gather; (2) in the cytoplasm, such as membrane-bound organelles such as the endoplasmic reticulum and Golgi apparatus, and on the surface of B lymphocytes that can synthesize antibody molecules; (3) on the surface of certain immune effector cells, such as natural killer cells (NK) and mononuclear phagocytes, which, although they cannot synthesize antibodies, can connect to specific receptors of antibody molecules; (4) in certain secretions, such as milk and mucus.

[0071] The "composition of an antibody molecule" is explained in this application.

[0072] The basic structure of an antibody molecule is a symmetrical structure consisting of four peptide chains connected by disulfide bonds. Two long chains are called heavy chains, and two short chains are called light chains. The four chains form a Y shape through the disulfide bonds between the chains.

[0073] Light chains (L chains) consist of approximately 214 amino acids and are of two types: kappa (κ) and lambda (λ). While the L chain types on the same Ig are identical, the same individual can harbor both types of light chains.

[0074] The heavy chain (H chain) consists of 450-550 amino acid residues. Different heavy chains have varying antigenicity due to differences in the order of amino acid residues, the number and location of disulfide bonds, and the type and quantity of sugars they contain. Based on their antigenicity, H chains can be classified as μ, γ, α, and δ chains. The two heavy chains in each IgG molecule are identical.

[0075] Variable and constant regions of antibodies:

[0076] The free amino or carboxyl groups at each end of the four peptide chains in an Ig monomer are oriented in the same direction, referred to as the amino terminus (N-terminus) and carboxyl terminus (C-terminus), respectively. At the N-terminus of the Ig monomer molecule, the composition and order of amino acids in half of the light chain and a quarter of the heavy chain vary depending on the antibody's specificity. This region is called the variable region (V region), which includes the light chain variable region (VL) and the heavy chain variable region (VH). The L chain variable region has three CDR regions. According to the Kabat numbering system, the CDR1 region is located at amino acids 24-34, the CDR2 region is located at amino acids 50-56, and the CDR3 region is located at amino acids 89-97. The H chain also has three CDR regions, located at amino acids 31-35, 50-65, and 95-102, respectively. The V region confers specificity to the antibody's antigen binding. At the C-terminus of the Ig polypeptide chain, the remaining half of the light chain and three-quarters of the heavy chain have relatively stable amino acid numbers, types, order, and sugar content. This region is known as the constant region (C region), which also includes the light chain constant region (CL) and the heavy chain constant region (CH). The C region forms the framework of the Ig, mediates secondary reactions, and determines the antigenicity of the Ig.

[0077] The following is a detailed description of the method for preparing a monoclonal antibody against a coccidian oocyst protein provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0078] Example 1

[0079] A method for screening monoclonal antibodies against coccidian oocyst protein

[0080] New Zealand white rabbits are immunized with porcine coccidian oocyst wall antigens as immunogens; B lymphocytes from the rabbit spleen are then isolated and cultured; ELISA tests are then performed to obtain well plate cells with high ELISA test results, and RNA from the B lymphocytes is then extracted and reverse transcribed into cDNA; the cDNA is amplified by PCR to obtain naturally paired rabbit monoclonal antibodies; and the heavy chain variable region (VH) and light chain variable region (VL) genes of the paired rabbit monoclonal antibodies are then expressed. Specifically, the preparation process of rabbit monoclonal antibodies to porcine coccidian oocyst wall antigens includes:

[0081] 1. Preparation of Immunogen

[0082] According to previous literature mining and sequence comparison, the protein has a high homology with the oocyst wall protein rTgOWP1-f protein used for diagnosis of Toxoplasma gondii), and the 56-282aa region of Isospora suis CSUI_006207 has good specificity and antigenic epitope prediction. It was constructed into the BamHI / HindIII multiple cloning site of the pET-28a vector, and the constructed recombinant vector was transformed into Escherichia coli for conventional culture and induction expression of the recombinant protein ( Figure 1 ), the recombinant protein was purified and tested by PAGE-SDS electrophoresis. Figure 2 .

[0083] 2. Animal immunization

[0084] New Zealand white rabbits were immunized with the recombinant protein prepared above as an immunogen, with 200 μg per rabbit. For the first immunization, the protein was mixed and emulsified with Freund's complete adjuvant in a 1:1 ratio and injected at multiple sites on the rabbit's abdomen and back. Subsequently, 200 μg of the antigen was mixed with Freund's incomplete adjuvant in a 1:1 ratio and emulsified at three-week intervals for two more immunizations. After the three immunizations, rabbit serum samples were collected and their titers against porcine coccidian oocyst wall antigens were determined using ELISA. Rabbits with high serum titers were boosted with 200 μg of the immunogen injected subcutaneously at multiple sites. Spleens were harvested three days later.

[0085] The reagents and detection methods of ELISA are as follows:

[0086] A. Reagents and configuration specifications

[0087] 1) ELISA plate, 100 μl tip, 1 ml Eppendorf tube, wet box.

[0088] 2) Coating diluent: 0.05 mol / L sodium carbonate-sodium bicarbonate buffer, pH 9.6;

[0089] To a mixture of 0.15 g of sodium carbonate, 0.29 g of sodium bicarbonate, and 0.02 g of sodium azide, add double-distilled water to 100 ml and adjust the pH to 9.6.

[0090] 3) Blocking solution: 5% calf serum in PBS; including 5 ml of calf serum and 95 ml of 1×PBS (pH 7.4).

[0091] 4) Washing solution: PBST, pH 7.4;

[0092] NaCl 0.8g, KH2PO4 0.02g, Na2HPO4.12H2O 0.29g, KCl 0.02g, Tween 200.05ml, sodium azide 0.01g, add double distilled water to 100ml, and adjust to pH 7.4.

[0093] 5) Sample diluent: PBS, pH 7.4;

[0094] NaCl 0.8g, KH2PO4 0.02g, Na2HPO4.12H2O 0.29g, KCl 0.02g, sodium azide 0.01g, add double distilled water to 100ml, and adjust to pH 7.4.

[0095] 6) The dilution range of the enzyme-labeled secondary antibody (goat anti-rabbit) is 1:5,000 to 1:100,000.

[0096] 7) Substrate solution: TMB-hydrogen peroxide urea solution;

[0097] ① Substrate solution A: TMB 20 mg, anhydrous ethanol 10 ml, add double distilled water to 100 ml.

[0098] ② Substrate solution B: 0.1 mol / L citric acid-0.2 mol / L sodium dihydrogen phosphate buffer, pH 5.0-5.4; preparation method: Na2HPO4 1.46g, citric acid 0.933g, 0.75% urea hydrogen peroxide 0.64ml, add triple-distilled water to 100ml, and adjust to pH 5.0-5.4.

[0099] ③ Mix substrates A and B in a ratio of 1:1 to form TMB-hydrogen peroxide urea solution.

[0100] 8) Stop solution: 2 mol / L H2SO4 solution;

[0101] 200ml of double-distilled water and 34ml of concentrated sulfuric acid are slowly added dropwise while stirring continuously, and double-distilled water is added to 300ml.

[0102] 9) 0.9% normal saline.

[0103] B. Operation steps

[0104] 1) Coating process (note the setting of blank control and negative control):

[0105] Dilute the antigen to be used with coating diluent to an appropriate concentration (generally, the required antigen coating amount is 20-200 μg per well), add 100 μl of antigen to each well, and incubate at 37°C for 4 hours; discard the liquid in the well (to avoid evaporation, the plate should be covered or placed flat in a metal wet box with wet gauze on the bottom).

[0106] 2) Seal the enzyme-labeled reaction wells:

[0107] Block with 5% calf serum at 37°C for 40 minutes. Fill each well with blocking solution and remove any bubbles. Wash the wells three times with washing solution, each time for 3 minutes.

[0108] Washing method: drain the reaction solution in the wells, fill the wells with washing solution, place for 2 minutes, shake slightly, drain the solution in the wells, pour out the liquid and pat dry on absorbent paper. Washing times: 3 times

[0109] 3) Add the sample to be tested (establish a suitable concentration gradient):

[0110] A dilution of 1:50-1:400 is generally used during testing, and a larger dilution volume should be used to ensure that the sample aspirate volume is generally >20μl.

[0111] Add the diluted sample to the enzyme-labeled reaction wells, add at least two wells for each sample, 100 μl per well, and incubate at 37°C for 60 minutes. Wash the wells three times with washing solution, each time for 3 minutes.

[0112] 4) Add enzyme-labeled antibody:

[0113] Enzyme-labeled antibody: Follow the reference working dilution provided by the enzyme conjugate provider. Incubate at 37°C for 30-60 minutes. Results are often unstable if the time is shorter than 30 minutes. Add 100 μl to each well and wash as before.

[0114] 5) Add substrate solution (prepare immediately before use): TMB-hydrogen peroxide urea solution is preferred, followed by OPD-hydrogen peroxide substrate solution system.

[0115] Substrate addition amount: 100 μl per well, place at 37°C in the dark for 3-5 minutes, add stop solution for color development.

[0116] 6) Termination of reaction: Add 50 μl of stop solution to each well to terminate the reaction and measure the experimental results within 20 minutes.

[0117] 7) Result Interpretation: OPD development is performed at a wavelength of 492 nm, while TMB reaction product detection requires a wavelength of 450 nm. Always zero the blank wells during testing. The ratio of the absorbance of the assay well to the average absorbance of a group of negative specimen wells (P / N) is used to represent the antibody titer. A P / N greater than 2 is considered the antibody titer (the value depends on the specific test requirements).

[0118] 3. Isolation of specific B lymphocytes

[0119] Collect fresh whole blood from immune rabbits and dilute it with serum-free 1640 medium or PBS at a volume ratio of 1:1. Then, obtain B cells by density gradient centrifugation. The specific steps are as follows:

[0120] Add 5 mL of separation reagent to a 15 mL centrifuge tube, then add 5 mL of the dilution obtained in the previous step on top, and then centrifuge at 1500 g for 30 minutes at room temperature (swinging rotor); then aspirate the middle buffy coat layer (mononuclear cells) and add it to a new centrifuge tube, then resuspend it in serum-free medium, centrifuge at 800 g for 10 minutes, obtain the precipitate, and finally resuspend the cells in 1× PBS).

[0121] Screening of single antigen-specific B lymphocytes: Biotin-pET28a-4xoocystwallprotein was used to biotinylate the porcine coccidian oocyst wall antigen to obtain biotin-labeled antigen. Biotin-labeled antigen was added to the resuspended cells obtained above and incubated at room temperature for 30 minutes. Then, FITC-labeled lymphocyte markers (7AAD, CD4, CD8) antibodies and IgM antibodies were used in sequence to incubate the antigen at room temperature for 30 minutes. The cells were then washed several times with PBS and then subjected to flow cytometry sorting ( Figure 3 ), single B cells were obtained, and single antigen-specific B lymphocytes were obtained by live cell sorting. For specific operation steps, please refer to patent publication number CN 110016462A (Method for efficiently isolating single antigen-specific B lymphocytes from spleen cells).

[0122] 4. Cloning of the Gene Encoding Rabbit Monoclonal Antibody

[0123] After single B cells are sorted in vitro, they need to be cultured in specific feeder cells and stimulated to differentiate in a specific culture medium to differentiate into plasma cells that can secrete antibodies. After that, the antibodies in the culture supernatant are detected and the positive wells are selected for subsequent antibody gene amplification. The cells used are EL4B5 feeder cells. In the co-culture, interleukin-1β, tumor necrosis factor α, IL-21, IL-2, IL-10, TNF-α, IL-6, BAFF (B cell activating factor of the tumor necrosis factor family) and SAC (Staphylococcus aureus Cowans strain) are used. The above cytokine concentration is 10 ng / mL and the dilution factor of SAC is 1:75,000. The cultured B cell supernatant is identified by antigen-coated ELISA (steps as described in step 2) to identify positive clones.

[0124] Single-well cells from positive clones were collected and lysed. The resulting lysate was used as a template for reverse transcription to obtain cDNA. Nested PCR was used to amplify the naturally paired light and heavy chain variable region genes (VH and VL) of rabbit monoclonal antibodies from the cDNA of the corresponding positive clones. The nested PCR reaction procedure and required primers are shown in Tables 1 and 2, and primer sequences are shown in Table 3. After amplification, the PCR products were sequenced to obtain their specific sequences.

[0125] Table 1 The first round (1 st )PCR reaction system

[0126] cDNA template 2 μl <![CDATA[1 st F Primer]]> 1 μl <![CDATA[1 st R Primers]]> 1 μl 2.5 mM dNTP 4 μl 5 x PrimeSTAR GXL buffer 10 μl PrimeSTAR GXL polymerase 2 μl <![CDATA[ddH2O]]> 30 μl

[0127] 1 st The PCR program settings were:

[0128] 95°C for 3 min; then 98°C for 10 s, 55°C for 30 s, and 72°C for 30 s for 35 cycles; 72°C for 5 min, and then stored at 4°C.

[0129] Table 2 Second round 2 nd PCR system

[0130] <![CDATA[1 st PCR products]]> 2 μl <![CDATA[2 nd F Primer]]> 1 μl <![CDATA[2 nd R Primers]]> 1 μl 2.5 mM dNTP 8 μl 5 x PrimeSTAR GXL buffer 10 μl PrimeSTAR GXL polymerase 2 μl <![CDATA[ddH2O]]> 26 μl

[0131] 2 nd The PCR setup procedure is as follows:

[0132] 95°C for 3 min; then 98°C for 10 s, 55°C for 30 s, and 72°C for 30 s for 35 cycles; 72°C for 5 min, and then stored at 4°C.

[0133] Table 3 Primer sequence list

[0134]

[0135]

[0136] Note: R stands for A+G; Y stands for C+T; M stands for A+C; K stands for G+T; S stands for C+G; W stands for A+T; B stands for C+G+T; V stands for A+C+G.

[0137] Example 2

[0138] Method for preparing monoclonal antibody against coccidian oocyst protein

[0139] The sequences of the PCR products amplified in Example 1 were loaded onto the pDNA3.4(+) plasmid, and the vectors were transfected into host cells (competent Escherichia coli). The host cells were cultured, and the desired monoclonal antibodies were isolated and purified from the culture medium of the host cells. The specific method is as follows:

[0140] Expression and purification of monoclonal antibodies

[0141] One day before transfection, 1.3 × 10 6 cells / mL to separate host cells; adjust the cell density to 2.6×10 6cells / mL, dilute the recombinant light and heavy chain expression plasmids to be transfected in OptiMEM in a sterile tube, and dilute PEI in OptiMEM in a sterile tube. Mix and let stand for 20 minutes, then add the light and heavy chain expression plasmid / PEI mixture to the host cells. Harvest the supernatant 96-120 hours after transfection and incubate it with Protein A agarose. Centrifuge and collect the antibody-adsorbed Protein A agarose, wash it, and elute the antibody-adsorbed Protein A agarose. Dialyze the eluate into 1× PBS to obtain the antibody. After the antibody is verified to be qualified, aliquot it and store it at -20°C until use.

[0142] result:

[0143] In this example, three monoclonal antibodies that can recognize oocyst proteins of porcine coccidia were screened and obtained. The heavy chain of monoclonal antibody 6B10 consists of the amino acid sequence set forth in SEQ ID NO: 1; it comprises a light chain variable region (VL) defined according to the Kabat numbering system, the light chain variable region (VL) having three complementarity determining regions (CDRs), and consisting of the amino acid sequence set forth in SEQ ID NO: 2. The heavy chain of monoclonal antibody 5C8 consists of the amino acid sequence set forth in SEQ ID NO: 3; it comprises a light chain variable region (VL) defined according to the Kabat numbering system, the light chain variable region (VL) having three complementarity determining regions (CDRs), and consisting of the amino acid sequence set forth in SEQ ID NO: 4. The heavy chain of monoclonal antibody 5D5 consists of the amino acid sequence set forth in SEQ ID NO: 5; it comprises a light chain variable region (VL) defined according to the Kabat numbering system, the light chain variable region (VL) having three complementarity determining regions (CDRs), and consisting of the amino acid sequence set forth in SEQ ID NO: 6.

[0144] Example 3

[0145] Detection of the affinity of a monoclonal antibody against coccidian oocyst protein

[0146] Affinity testing of affinity purified antibodies:

[0147] The affinity of the obtained antibodies was accurately measured using the GatorPlus Biolayer Interferometry (BLI) system biomolecular interaction analyzer. The BLI system is a common method for molecular interaction analysis based on the optical surface plasmon resonance (SPR) principle. SPR uses the principles of physical optics to detect changes in the angle caused by changes in the dielectric constant due to molecular binding. Changes in the angle will lead to changes in the response value. Its working principle is as follows Figure 4 and Figure 5 shown.

[0148] BLI experiments involve immobilization and interaction analysis. Immobilization involves the reversible or irreversible binding of immobilized molecules to the sensor surface. Interaction analysis involves the binding of the analyte to the sensor surface during the binding phase and the dissociation phase, during which the analyte dissociates from the sensor surface. The specific steps include baseline 1, sample loading, baseline 2, binding, and dissociation.

[0149] Baseline: 1000 ppm Protein A probe was immersed in Kbuffer; the probe Protein A was purchased from GatorBio.

[0150] Sample loading was then performed: a Protein A probe (GatorBio) was immersed in a diluted antibody solution to allow the probe to bind to the antibody. A baseline procedure was then repeated, specifically by immersing the Protein A probe in K buffer to wash away any residual antibody. Binding was then performed, with the Protein A probe immersed in diluted antigen, allowing the bound antibody to bind to different concentrations of antigen (50 nM and 100 nM) and measuring the antigen-antibody binding curve. Dissociation was then performed, with the Protein A probe immersed in K buffer to monitor the antigen-antibody dissociation curve. The curve was then fitted, background was subtracted, and the Kd was calculated. The Kd value was used to assess the high affinity of the obtained antibody for the recombinant oocyst antigen.

[0151] Kd value results are shown in Figure 6 to Figure 8 Monoclonal antibody 6B10, monoclonal antibody 5C8, and monoclonal antibody 5D5 have good affinity for the antigen.

[0152] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for screening monoclonal antibodies against coccidian oocyst proteins, characterized in that: The following steps are involved: Rabbits were immunized with coccidian oocyst protein and peripheral blood or spleen was collected; isolating mononuclear cells from the peripheral blood of the rabbit and / or isolating spleen cells from the spleen; flow cytometry-sorting antigen-specific B lymphocytes from the mononuclear cells and / or spleen cells; The antigen-specific B lymphocytes were cultured in a culture medium containing EL4B5 feeder cells, wherein the culture medium contained 10 ng / mL interleukin-1β, 10 ng / mL tumor necrosis factor α, 10 ng / mL IL-21, 10 ng / mL IL-2, 10 ng / mL IL-10, 10 ng / mL TNF-α, 10 ng / mL IL-6 and 10 ng / mL Staphylococcus aureus Cowans strain secretion SAC; Cultured antigen-specific B lymphocytes were lysed, and the resulting cell lysate was used as a template to amplify the variable region of a rabbit monoclonal antibody.

2. The screening method according to claim 1, characterized in that The cell density of the EL4B5 feeder cells was 1×10 7 ~1×10 9 / ml.

3. The screening method according to claim 1, characterized in that The cell density of the antigen-specific B lymphocytes was 1×10 7 ~1×10 9 / ml.

4. The screening method according to claim 1, characterized in that The amino acid sequence of the coccidian oocyst protein is shown in SEQ ID NO:

7.

5. The screening method according to claim 1, characterized in that The primers for amplifying the variable region of the rabbit monoclonal antibody include nucleotide sequences as shown in SEQ ID NO: 8 to SEQ ID NO:

40.

6. A monoclonal antibody against coccidian oocyst protein obtained by screening according to any one of claims 1 to 5, characterized in that: Including at least one of the following: coccidia monoclonal antibody 6B10, coccidia monoclonal antibody 5C8, coccidia monoclonal antibody 5D5; The amino acid sequence of the heavy chain variable region of the coccidia monoclonal antibody 6B10 is shown in SEQ ID NO: 1, and the amino acid sequence of the corresponding light chain variable region is shown in SEQ ID NO: 2; The amino acid sequence of the heavy chain variable region of the coccidia monoclonal antibody 5C8 is shown in SEQ ID NO: 3, and the amino acid sequence of the corresponding light chain variable region is shown in SEQ ID NO: 4; The amino acid sequence of the heavy chain variable region of the coccidia monoclonal antibody 5D5 is shown in SEQ ID NO: 5, and the amino acid sequence of the corresponding light chain variable region is shown in SEQ ID NO:

6.

7. Use of the monoclonal antibody according to claim 6 in the preparation of a product for detecting porcine coccidiosis.

8. The application according to claim 7, characterized in that: The products include diagnostic kits and detection reagents.

9. The application according to claim 8, characterized in that: The product is prepared based on the following immunoassay technology: Enzyme-linked immunosorbent assay, immunohistochemistry, in vitro chromatography, flow cytometry, hemagglutination, and immunoblotting.

10. A porcine coccidiosis detection kit based on immunoassay technology, characterized in that: comprising the monoclonal antibody according to claim 6 and a detection antigen; The detection antigen includes coccidian oocyst protein.

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