CN117007808A - African swine fever virus CD2v protein blocking biochip antibody detection kit - Google Patents

African swine fever virus CD2v protein blocking biochip antibody detection kit Download PDF

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CN117007808A
CN117007808A CN202210454844.8A CN202210454844A CN117007808A CN 117007808 A CN117007808 A CN 117007808A CN 202210454844 A CN202210454844 A CN 202210454844A CN 117007808 A CN117007808 A CN 117007808A
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antibody
cd2v
solution
protein
kit
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田克恭
郝丽影
李玉芳
彭伍平
邓均华
张许科
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Luoyang Zhongke Biochip Technology Co ltd
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Luoyang Zhongke Biochip Technology Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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Abstract

The invention relates to the field of biotechnology, and particularly provides an antibody detection kit for an African swine fever virus CD2v protein blocking biochip. The antibody adopted by the enzyme-labeled reagent in the kit contains a heavy chain variable region with an amino acid sequence shown as SEQ ID NO.4 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 6. The kit has high detection sensitivity, good specificity, rapidness and simple operation.

Description

African swine fever virus CD2v protein blocking biochip antibody detection kit
Technical Field
The invention relates to the field of biotechnology, in particular to an antibody detection kit of an African swine fever virus CD2v protein blocking biochip.
Background
African swine fever (African swine fever, ASF) is a high-mortality infectious disease caused by infection of domestic pigs and wild pigs with African swine fever virus (African swine fever virus, ASFV) and the like. ASFV is a double-stranded large nuclear DNA virus (NCLDV), the only member of the African swine fever virus family. ASFV is composed of more than 3 thousands of protein subunits, presents a regular icosahedral structure, has a multi-layer material structure, and is composed of an outer envelope, capsid protein, double-layer inner membrane, core protein capsid and double-stranded DNA genome, respectively, to constitute spherical virus particles with radius of about 130 nm. The major components of ASFV viral capsids are pp220, pp62, p72, p54, p30, CD2v, etc.
CD2v is a protein encoded by the EP402R gene, and is named CD2v because of extremely high similarity to the T lymphocyte surface adhesion receptor leukocyte differentiation antigen 2 (CD 2). The CD2v protein can help ASFV adsorb to the cell membrane of erythrocytes and susceptible cells, the former facilitating viral transmission in the host, and the latter facilitating viral infection of cells. CD2v also plays an important role in ASFV escape host immune system monitoring. In addition, CD2v helps ASFV adsorb to the erythrocyte membrane, allowing the virus to express the c-lectin encoded by ORF EP153R on erythrocytes, and allowing the originally abnormally unstable and fragile ASFV adsorbed erythrocyte membrane to stabilize. Meanwhile, ASFV strains may be typed according to the hemagglutination inhibition reaction of CD2v for a total of 8 serotypes, in addition to the genotyping system initially based on p 72. It can be seen that CD2v plays a critical role in ASFV.
CD2v is a transmembrane glycoprotein assembled from a signal peptide, two immunoglobulin-like domains, a transmembrane region, an acidic domain and a leucine rich domain, but the true structure of CD2v has not been completely analyzed. The extracellular domain of CD2v protein has a remarkable similarity to that of CD2 protein in T cells, but its cytoplasmic domain is completely different from that of CD2 protein, and the function of this unique cytoplasmic domain is unknown.
Based on the above, developing antibodies against CD2v proteins can provide an important tool for ASFV detection and structural analysis of CD2v proteins.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims at providing an African swine fever virus CD2v protein blocking biochip antibody detection kit.
The second purpose of the invention is to provide the application of the antibody specifically binding with the African swine fever virus CD2v protein in preparing an African swine fever virus CD2v protein blocking biochip antibody detection kit.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
the African swine fever virus CD2v protein blocking biochip antibody detection kit comprises an African swine fever virus CD2v protein antibody detection chip and an enzyme-labeled reagent; the enzyme-labeled reagent is an antibody which specifically binds with the African swine fever virus CD2v protein, and the antibody contains a heavy chain variable region with an amino acid sequence shown as SEQ ID NO.4 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 6.
YDHDYAKLGTELGSTSNGRQCAGIALTSRMKLWLNWVKQSHGKSLEWIGVIDPNNGGTTYNQKFKGKATLTVDKSSSTAYMEVRSLTSEYSAVYYCARYGKGIGLDYWGQGTSVTVSS(SEQ ID NO.4)。
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAVDLGVYFCSQSTHVPWTFGGGTKLEIK(SEQ ID NO.6)。
In a preferred embodiment, the african swine fever virus CD2v protein antibody detection chip is provided with a spotting point, the spotting point comprises a quality control product point, a detection point and a blank control point, the quality control product point is coated with a quality control product, the quality control product is goat anti-mouse IgG, and the coating amount is 3-5 ng/point, preferably 4 ng/point.
In a preferred embodiment, the detection spot is coated with the fusion protein CD2v-mFc in an amount of 1-64 ng/spot, preferably 8-32 ng/spot, more preferably 16 ng/spot.
In a preferred embodiment, the nucleotide sequence encoding the fusion protein CD2v-mFc is shown in SEQ ID NO. 2.
GCCACCATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCGGCCGCCACAGGCGCGCACTCCCACCACCATCACCATCATATCGATTATTGGGTGAGCTTTAACAAGACTATCATTCTGGACAGCAATATTACTAACGACAATAACGACATCAACGGCGTGAGCTGGAACTTCTTTAACAACAGCTTCAATACTCTCGCCACATGTGGCAAGGCCGGCAACTTCTGCGAGTGTAGCAACTATAGCACAAGCATCTACAACATTACTAACAACTGCTCTCTGACTATCTTCCCACATAACGACGTCTTCGACACTACATACCAAGTGGTCTGGAATCAGATCATTAACTACACAATCAAACTGCTGACTCCAGCCACTCCACCTAACATCACATACAACTGCACAAATTTTCTGATTACTTGTAAAAAAAATAATGGCACAAATACAAATATCTATCTGAACATCAACGATACATTCGTCAAGTACACAAACGAGTCCATTCTGGAGTACAACTGGAACAACTCCAACATCAATAATTTCACTGCCACTTGCATCATTAATAACACAATCTCCACTAGCAATGAGACTACACTGATCAACTGCACATATCTGACTCTGAGCTCCAACTACTTTTATACTTTCTTCAAACTGTACGAGAATTTGTACTTTCAAGGCGAGCCAAGGGGACCTACAATCGAGCCAAGGGGACCTACAATCAAGCCTTGCCCACCATGCAAGTGCCCAGCTCCTAATCTGCTGGGCGGACCATCCGTGTTCATCTTCCCACCTAAAATCAAAGATGTGCTCATGATCTCCCTCTCCCCTATCGTGACTTGCGTGGTGGTGGATGTGAGCGAGGACGACCCAGATGTCCAGATCAGCTGGTTCGTGAACAACGTGGAGGTGCATACTGCTCAGACACAGACTCATAGGGAGGACTACAACAGCACACTGAGAGTCGTGTCCGCTCTGCCAATCCAGCATCAAGATTGGATGAGCGGCAAGGAGTTTAAGTGCAAGGTCAACAACAAGGATCTGCCAGCCCCTATCGAGAGGACAATCAGCAAGCCAAAAGGCAGCGTGAGGGCTCCTCAAGTGTACGTGCTCCCTCCTCCAGAGGAGGAGATGACTAAAAAGCAAGTGACTCTCACTTGCATGGTGACAGACTTCATGCCAGAGGACATCTACGTGGAGTGGACTAACAACGGCAAGACTGAACTGAATTACAAAAACACAGAGCCAGTGCTGGACTCCGACGGAAGCTACTTCATGTACAGCAAGCTGAGGGTCGAGAAGAAGAACTGGGTCGAGAGGAATTCCTACAGCTGTTCCGTGGTGCACGAAGGACTGCACAACCACCACACTACTAAGTCCTTTTCTAGGACACCCGGCAAGTAA(SEQ ID NO.2)。
In a preferred embodiment, the blank spots are coated with a blank.
In the invention, the number of quality control product points is 1-4, preferably 3. The buffer solution of the goat anti-mouse IgG, the buffer solution of the fusion protein CD2v-mFc and the blank control are respectively sample application liquid, and the sample application liquid is prepared by uniformly mixing 5w/v% glycerol solution, 5w/v% sorbitol solution, 0.05v/v% triton solution, DMSO solution and pH6.8PBS solution according to the volume ratio of 10:15:0.1:50:100.
In a preferred embodiment, the edge distance between any two points among the detection point, the quality control point and the blank control point is equal to or more than 700 μm, and the edge distance between any one point and the edge of the chip is equal to or more than 5mm, preferably equal to or more than 8mm.
In a more preferred embodiment, the sample application mode of each chip hole of the detection chip can be shown as a-E in fig. 1, points (1) - (3) are quality control product points, point (4) is blank control point, point (5) is detection point, quality control product point (1), quality control product point (2), quality control product point (3) and blank control sample application point (4) are respectively positioned at the left upper corner, the left lower corner, the right lower corner and the right upper corner of the detection chip, detection point (5) can be applied to other positions of the detection chip, and the distance between the points of points (1) to (5) and the point edge is more than or equal to 700 μm; the distances from the quality control product point (1), the quality control product point (2), the quality control product point (3) and the blank control point (4) to the upper edge and the lower edge of the detection chip subunit are more than or equal to 8mm, and the distances from the left edge and the right edge of the detection chip subunit are more than or equal to 5mm.
In a preferred embodiment, the antibody is a monoclonal antibody or a genetically engineered antibody, wherein the genetically engineered antibody comprises a chimeric antibody, a modified antibody or a small molecule antibody, which may be, for example, fab (consisting of an intact light chain and Fd), fv (consisting of VH and VL) or ScFv (single chain antibody, with VH and VL linked by a linker peptide). Preferably monoclonal antibodies or single chain antibodies.
In a preferred embodiment, the antibody is IgA, igD, igE, igG or IgM, preferably IgG, further preferably, the antibody is monoclonal antibody 5G8 selected in the examples of the invention, the heavy chain subclass is IgG2a, the light chain subclass is kappa, or the antibody is single chain antibody 5G8 constructed in the invention.
In a preferred embodiment, the nucleotide sequence encoding the heavy chain variable region of the antibody is shown in SEQ ID NO. 5; preferably, the nucleotide sequence encoding the light chain variable region of the antibody is shown in SEQ ID NO. 7. The enzyme includes horseradish peroxidase, alkaline phosphatase or beta-D-galactosidase.
TATGACCATGATTACGCCAAGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTGCCCTTACTAGTCGCATGAAGTTGTGGTTGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGTTATTGATCCTAACAATGGTGGTACTACCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTATATGGAGGTCCGCAGCCTGACATCTGAATATTCTGCAGTCTATTACTGTGCAAGATATGGTAAAGGGATTGGTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO.5)。
GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGAAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAACTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGTGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SEQ ID NO.7)。
In a preferred embodiment, the kit further comprises a wash solution, a sample dilution solution, and a substrate solution. The washing solution is preferably a PBS solution of 1v/v% Tween 20; the sample diluent is preferably 10v/v% fetal bovine serum, 0.05v/v% to 0.2v/v% Triton-100, 0.1v/v% Proclin300 in PBS; the substrate solution is preferably a TMB solution.
The invention also provides application of the antibody specifically binding with the African swine fever virus CD2v protein in preparing the African swine fever virus CD2v protein blocking biochip antibody detection kit, wherein the antibody contains a heavy chain variable region with an amino acid sequence shown as SEQ ID NO.4 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 6.
The invention also provides a preparation method of the kit, which comprises the following steps:
the method comprises the following steps of (1) preparing African swine fever virus CD2v-mFc protein, respectively diluting with spotting liquid together with goat anti-mouse IgG, and spotting on a membrane to prepare an antibody detection chip of the African swine fever virus CD2v protein;
the step (2) comprises independent detection holes by using the bottom end, the upper grating and the antibody detection chips arranged in the step (1) therebetween, wherein the antibody detection chips are arranged between the bottom end and the upper grating and clamped by the bottom end and the upper grating, one detection hole corresponds to one detection chip subunit, and when the antibody joint detection kit comprises a plurality of antibody detection chip subunits of African swine fever virus, each independent detection chip subunit is integrated into a microarray chip;
respectively labeling an African swine fever virus CD2v protein monoclonal antibody 5G8 by using enzymes, diluting the monoclonal antibody with enzyme-labeled diluent according to the final concentration of 75ng/ml, and uniformly mixing the monoclonal antibody with enzyme-labeled diluent which is PBS solution containing 20v/v% of fetal bovine serum as an enzyme-labeled reagent;
Preparing a sample diluent, a washing liquid and a substrate liquid;
and (5) assembling the subunit of the African swine fever virus antibody detection chip prepared in the step (2) or the subunit microarray chip, the enzyme-labeled reagent prepared in the step (3), the sample diluent, the washing liquid and the substrate liquid prepared in the step (4) into a kit.
Compared with the prior art, the invention has the technical effects that:
the invention provides an African swine fever virus CD2v protein blocking biochip antibody detection kit, wherein an antibody adopted by an enzyme-labeled reagent is an antibody specifically binding with the African swine fever virus CD2v protein, the antibody is prepared by using a fusion protein CD2v-mFc as an immunogen and a fusion protein gD-CD2v as a screening antigen, and the identified epitope is positioned on the African swine fever virus CD2v protein, and the blocking rate of the antibody to African swine fever virus positive serum is 91%. The African swine fever virus CD2v protein blocking biochip antibody detection kit which is used as a competitive antibody for detection has the advantages of high detection sensitivity, good specificity, rapidness and simple operation.
In addition, compared with the traditional ELISA kit which is provided with negative control, positive control or standard substance as the quality control substance in the kit, the kit provided by the invention is provided with the quality control substance on the African swine fever virus CD2v protein antibody detection chip, and the quality control substance is commercial goat anti-mouse IgG, and the negative control and positive control are not required to be arranged in the kit, namely, complicated preparation steps of immunization or virus attack, serum collection and the like and labor and animal test site cost are not required to be screened in advance, and the operation convenience and accuracy of the kit are greatly improved. According to the invention, the sheep anti-mouse IgG with specific content can be used as a quality control product without negative control and positive control, and the sheep anti-mouse IgG has the function of being used as a reaction control when no antibody is blocked in a single detection hole, is used for evaluating the effectiveness of a test, and reflects the reaction degree of an enzyme-labeled reagent when no blocking exists. The goat anti-mouse IgG can be purchased commercially or prepared by a conventional method, and is simple to operate.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of sample application modes of each chip hole on an African swine fever virus CD2v protein antibody detection chip provided by the invention, wherein A, B, C, D, E in FIG. 1 respectively shows different sample application modes;
FIG. 2 is a partial enlarged view of three kits having different numbers of the antibody detection chip subunits of the African swine fever virus CD2v protein, showing one detection chip of the antibody detection chip subunits of the African swine fever virus CD2v protein, wherein O, P, Q is a kit having one, three and a plurality of the antibody detection chip subunits, respectively.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The description will be given of some of the matters related to the present invention.
The term "CHO cell" is a chinese hamster ovary cell (Chinese Hamster Ovary Cell, CHO cell for short), an epithelial cell line derived from chinese hamster ovary, and is commonly used in biological and medical research and commercial production of therapeutic proteins. The term "CHO expression system" also called "CHO cell expression system" means an expression system in which a gene of a protein or a fragment thereof is transferred into CHO cell culture by molecular biological means to obtain a secreted target protein or a fragment thereof.
The term "codon optimization" refers to the fact that a gene can be synthesized using codons that are favored and avoid low availability or rare codons, such redesign of the gene is called codon optimization.
The term "antibody" is an immunoglobulin with immune function which is synthesized and secreted by B cells after the B cells differentiate and mature into plasma cells after the immune cells of the organism are activated by antigens and can be specifically combined with the corresponding antigens.
The term "Ig", immunoglobulin (immunolobulin), refers to a globulin having antibody (Ab) activity or chemical structure, similar to an antibody molecule. Immunoglobulin is a tetrapeptide chain structure formed by two identical light chains and two identical heavy chains joined by interchain disulfide bonds.
The term "IgA", immunoglobulin A (abbreviated IgA), is present in serum in amounts of 10-20% of serum immunoglobulins, next to IgG, in mucosal tissues such as the digestive tract, respiratory tract and genitourinary system. Mucosal tissue, with mucosal layer lymphoid tissue, produces IgA to avoid invasion by pathogens, and is also found in saliva, tears and milk, especially colostrum, where IgA content is quite high. In humans, the structure of IgA exists mainly in the form of monomers and dimers. According to the distribution of IgA in the body, it can be divided into serotypes and secretes. Serotype IgA is monomeric and has weaker immune effects. Secretory IgA has two and three bodies, is the main component of the body mucosa defense system, and is widely distributed in milk, saliva, gastrointestinal tract, respiratory tract and genitourinary tract mucosa secretion.
The term "IgD", immunoglobulin D (IgD) is present in serum in very low amounts, about 1% of total Ig, and in large individual amounts, and can be present on the surface of B cells as a membrane receptor.
The term "IgE", igE, is an antibody that mediates type i responses, and thus detection of serum total IgE and specific IgE is valuable for diagnosis of type i responses and determination of allergens.
The term "IgG", immunoglobulin G (IgG) is synthesized in spleen and lymph node, and has the highest content in human serum (75% of Ig content), and is mainly distributed in serum and tissue fluid, and is a main component of antibacterial, antitoxin and antiviral antibodies, and is also an important material basis in the process of anti-infectious immunity of the organism.
The term "IgM", immunoglobulin M (IgM) is the largest molecular weight immunoglobulin secreted and synthesized mainly by plasma cells in the spleen and lymph nodes, and is divided into two subtypes, igML and IgM 2. Mainly distributed in serum, exists in a pentamer form and accounts for 5% -10% of the total Ig in the serum. IgM has powerful bactericidal, complement activating, immunoregulatory and agglutinating effects and is also involved in the pathological processes of certain autoimmune diseases and hypersensitivity reactions.
The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical, except that there may be a small number of possible spontaneous mutations. Thus, the modifier "monoclonal" refers to a mixture of antibodies whose properties are not discrete. Preferably, the monoclonal antibodies include monovalent or single chain antibodies, diabodies, chimeric antibodies, canine, feline, mink-humanized antibodies, as well as derivatives, functional equivalents, and homologs of the above antibodies, as well as antibody fragments and any polypeptide comprising an antigen binding domain. Antibodies are any specific binding factor that encompasses a binding domain having the desired specificity, and thus this term encompasses antibody fragments, derivatives, caninized antibodies, and functional equivalents and homologs of antibodies that are homologous thereto, as well as any polypeptide, whether naturally or synthetically produced, that comprises an antigen binding domain. Examples of antibodies are immunoglobulin subtypes (e.g., igG, igE, igM, igD and IgA) and subtype subclasses thereof; fragments comprising an antigen binding domain such as Fab, scFv, fv, dAb, fd; and diabodies (diabodies). Chimeric molecules or equivalents comprising an antigen binding domain fused to another polypeptide are also included. Cloning and expression of chimeric antibodies is described in ep.a.0126694 and ep.a.012623. Antibodies can be modified in a number of ways and DNA recombination techniques can be used to produce other antibodies or chimeric molecules that retain the original antibody specificity. Such techniques may involve introducing DNA encoding the immunoglobulin variable or Complementarity Determining Regions (CDRs) of an antibody into the constant or constant region plus framework regions of different immunoglobulins, see ep.a.184387, GB2188638A or ep.a.239400. The hybridoma cells or other antibody-producing cells may also be subjected to genetic mutations or other alterations, which may or may not alter the binding specificity of the produced antibody. The "monoclonal antibodies" used in the present invention may also be prepared by hybridoma methods, as DNA sequences encoding the murine antibodies of the present invention may be obtained by conventional means well known to those skilled in the art, such as by artificially synthesizing nucleotide sequences from the amino acid sequences disclosed herein or amplifying them by PCR, and thus may also be obtained by recombinant DNA methods, and the sequences may be ligated into suitable expression vectors by various methods well known in the art. Finally, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention, and then purified by conventional isolation and purification means well known to those skilled in the art to obtain the monoclonal antibodies of the invention. Antibodies comprise a geometry of polypeptide chains linked together by disulfide bridges, two polypeptide backbones, termed the light and heavy chains, constituting all major structural classes (isotypes) of antibodies. Both heavy and light chains can be further divided into several sub-regions called variable and constant regions. Heavy chains comprise a single variable region and three different constant regions, while light chains comprise a single variable region (different from the variable region of the heavy chain) and a single constant region (different from the constant region of the heavy chain). The variable regions of the heavy and light chains are responsible for the binding specificity of the antibody.
The term "heavy chain variable region" refers to a polypeptide which is 110 to 125 amino acids in length and whose amino acid sequence corresponds to the heavy chain amino acid sequence of a monoclonal antibody of the invention starting from the N-terminal amino acid of the heavy chain. Similarly, the term "light chain variable region" refers to a polypeptide that is 95 to 115 amino acids in length and whose amino acid sequence corresponds to the amino acid sequence of the light chain of the monoclonal antibody of the invention starting from the N-terminal amino acid of the light chain. It is obvious to one of ordinary skill in the art that, based on the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody specifically disclosed in the present invention, one or more amino acids may be added, deleted, substituted, etc. modified by conventional genetic engineering and protein engineering methods to obtain conservative variants, while still being able to maintain specific binding to african swine fever virus. Monoclonal antibodies of the invention also include active fragments or conservative variants thereof.
The term "conservative variant" refers to a variant that substantially retains the properties of its parent, such as the basic immunological biological, structural, regulatory, or biochemical properties. Generally, the amino acid sequence of a conservative variant of a polypeptide differs from that of the parent polypeptide, but the differences are limited so that the sequence of the parent polypeptide is generally very similar to the conservative variant and is identical in many regions. The difference in amino acid sequence between the conservative variant and the parent polypeptide may be, for example: substitutions, additions and deletions of one or more amino acid residues, and any combination thereof. The amino acid residues that are replaced or inserted may or may not be encoded by the genetic code. Conservative variants of a polypeptide may occur naturally, or it may be non-naturally occurring variants. Non-naturally occurring conservative variants of a polypeptide may be produced by mutagenesis techniques or by direct synthesis.
The term "enzyme" includes, but is not limited to horseradish peroxidase, alkaline phosphatase, beta-D-galactosidase. Wherein, the substrate used by horseradish peroxidase is o-phenylenediamine (OPD) or tetramethyl benzidine (TMB), preferably tetramethyl benzidine (TMB); the substrate used for alkaline phosphatase is p-nitrophenyl phosphate (p-NPP); the substrate used for the beta-D-galactosidase was 4-methylumbelliferone-beta-D-galactoside (4 MUG).
The term "microarray chip" refers to a micro device capable of parallel processing and analyzing biological or chemical information in a sample, which is set on a substrate in an array manner, wherein the dot diameter of the array is within 500 μm, and the minimum distance between the centers of two adjacent dots is based on that no signal crossing occurs (see GB/T279990-2011, basic terminology of biochip).
The invention is further illustrated by the following examples. The materials in the examples were prepared according to the existing methods or were directly commercially available unless otherwise specified.
The phosphate buffer used in the examples of the present invention was PBS buffer (pH 7.4), 1L of which was formulated by volume: 8.0g NaCl, 0.2g KCl and Na 2 HPO 4 12H 2 O 2.9g、KH 2 PO 4 0.24g, but this embodiment does not constitute a limitation of the present invention in any way.
The chemical reagents used in the invention are all analytically pure and purchased from the national drug group.
EXAMPLE 1 preparation of ASFV CD2v fusion protein
1.1 construction of recombinant plasmid for fusion expression of CD2v Gene and murine Fc fragment
1.1.1 optimization and Synthesis of the CD2v Gene and murine Fc fragment
The sequence of the gene encoding CD2v in the ASFV SY18 genome (accession number MH 766894.1) registered in GenBank was referenced, analyzed by TMHMM software, and the extracellular region was selected for fusion with the murine Fc fragment (i.e., mFc). CD2v was codon optimized according to CHO expression system preference codons (see SEQ ID No.1 for details) and pUC57-CD2v plasmid, pCDNA3.4-mFc plasmid was synthesized by Suzhou Jin Weizhi Biotechnology Co.
1.1.2 primer design
The best signal peptide SP (italic sequence in Table 1) selected for screening was selected for expression of the protein of interest. The restriction sites XbaI and HindIII (underlined sequences in Table 1) were added upstream and downstream of the target gene, respectively, his tags (underlined sequences in Table 1) were added before the target gene to facilitate protein purification and detection, and TEV restriction sites (lower-case sequences in Table 1) were added between the fusion proteins, and the primer synthesis was as shown in the following table.
TABLE 1 primer sequences for construction of CD2v-mFc plasmid expressing fusion proteins
1.1.3 construction and identification of recombinant plasmids
The primers shown in Table 1 were used to amplify the CD2v and mFc gene fragments, respectively, using the pUC57-CD2v and pCDNA3.4-mFc plasmids thus synthesized as templates, and the fragments were identified by 1.0% agarose gel electrophoresis, as a result: the amplified CD2v fragment was about 680bp and the mFc fragment was about 750bp.
Fusion PCR was performed using the CD2v fragment and the mFc fragment as templates to obtain a CD2v-mFc fragment. Reaction system 50 μl:HS DNA Polymerase 0.5. Mu.L, 5 XPrimeSTAR buffer 10. Mu.L, dNTP mix (2.5 mM) 4. Mu.L, CD2v fragment amplification upstream primer, mFc fragment amplification downstream primer 1. Mu.L each, template 1. Mu.L each, ddH 2 O was added to 50. Mu.L. PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 98℃for 10 seconds, annealing at 55℃for 30 seconds, elongation at 72℃for 1 minute for 30 seconds, 30 cycles total; extension was carried out at 72℃for 10 minutes. The PCR products were identified by 1.0% agarose gel electrophoresis, and the results: the CD2v-mFc fragment of the fusion protein was about 1391bp. Purifying the target fragment by using an OMEGA gel recovery kit.
The obtained target fragment and pCDNA3.4 vector were digested with XbaI and HindIII, respectively, the target fragment and vector were ligated with T4 DNA ligase at 22℃for 2 hours, and the ligation product was transformed into TOP10 competent cells to obtain an expression plasmid, which was identified by double digestion to obtain a target fragment of about 1391bp and a pCDNA3.4 vector fragment of about 5970bp, which was identified by sequencing (see SEQ ID No.2 for details) as expected, and the expression plasmid was designated pCDNA3.4-CD2v-mFc. And the bacterial liquid of the plasmid is largely shaken, and then the plasmid is extracted by Endo-free Plasmid Midi Kit of endotoxin-free plasmid extraction kit, the concentration is 814 ng/. Mu.L, and the plasmid is preserved at-20 ℃ for standby.
1.2 construction of recombinant plasmid for fusion expression of PRVGD Gene and ASFV CD2v Gene
1.2.1 optimization and Synthesis of PRVGD Gene and CD2v Gene
The PRV HN1201 gD gene was codon optimized according to the CHO expression system preferred codons and pUC57-gD plasmid was synthesized by Suzhou Jin Weizhi Biotechnology Co.
1.2.2 primer design
The signal peptide HT2 (italic sequence in Table 2) screened in the laboratory was selected for expression of the protein of interest. The restriction sites XbaI and HindIII (underlined sequences in Table 2) were added upstream and downstream of the target gene, respectively, his tags (underlined sequences in Table 2) were added before the termination of the codes to facilitate protein purification and detection, and EK restriction sites (lower-case sequences in Table 2) were added between the fusion proteins, and the primer synthesis was as shown in the following table.
TABLE 2 primer sequences for construction of gD-CD2v plasmid expressing fusion protein
1.2.3 construction and identification of recombinant plasmids
The primers in Table 2 were used to amplify the CD2v and gD gene fragments, respectively, using the pUC57-CD2v and pUC57-gD plasmids thus synthesized as templates, and the fragments were identified by 1.0% agarose gel electrophoresis, as a result: the amplified CD2v fragment is about 640bp, and the gD fragment is about 1121bp.
The CD2v fragment and the gD fragment are used as templates to obtain the gD-CD2v fragment through fusion PCR, and the sequence is shown as SEQ ID NO. 3. Reaction system 50 μl: HS DNA Polymerase 0.5. Mu.L, 5 XPrimeSTAR buffer 10. Mu.L, dNTP mix (2.5 mM) 4. Mu.L, gD gene upstream primer, CD2v gene downstream primer 1. Mu.L each, template 1. Mu.L, ddH 2 O was added to 50. Mu.L. PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 98℃for 10 seconds, annealing at 55℃for 30 seconds, elongation at 72℃for 90 seconds, 30 cycles total; extension was carried out at 72℃for 10 minutes. The PCR products were identified by 1.0% agarose gel electrophoresis, and the results: the fragment of the fusion protein gD-CD2v was about 1629bp. Purifying the target fragment by using an OMEGA gel recovery kit.
GCCACCATGAACCTGCTGCTGATCCTGACCTTTGTGGCCGCCGCCGTGGCCGCTGATGTGGATGCCGTGCCCGCTCCCACCTTTCCTCCTCCTGCCTACCCCTACACCGAGAGCTGGCAGCTGACACTGACCACAGTGCCTTCCCCCTTCGTGGGCCCTGCCGATGTGTACCACACCAGGCCCCTGGAGGATCCTTGCGGAGTGGTGGCTCTCATCAGCGACCCTCAGGTCGACAGGCTGCTGAACGAGGCTGTGGCCCACAGGAGGCCTACATACAGGGCCCACGTGGCCTGGTACAGGATCGCCGACGGCTGTGCCCACCTGCTGTACTTTATCGAGTACGCTGACTGCGACCCCAGGCAGATTTTCGGCAGGTGCCGGAGGAGGACCACCCCTATGTGGTGGACCCCCTCCGCCGACTACATGTTCCCCACCGAGGACGAGCTGGGCCTGCTGATGGTGGCCCCTGGCAGGTTCAATGAGGGCCAGTACAGGAGGCTGGTGTCCGTGGACGGCGTGAACATCCTCACCGACTTCATGGTGGCCCTGCCTGAGGGCCAGGAATGTCCTTTCGCCCGGGTCGACCAGCACCGGACCTACAAGTTCGGCGCCTGCTGGTCCGACGACTCCTTCAAGAGGGGCGTGGACGTGATGAGGTTCCTGACCCCCTTCTATCAGCAGCCCCCCCACAGGGAGGTGGTGAACTACTGGTACAGGAAGAACGGCAGGACACTGCCCCGGGCTTATGCTGCCGCCACACCTTACGCCATCGACCCCGCTAGGCCCAGCGCTGGATCCCCCAGGCCCCGTCCCCGTCCCCGTCCTCGGCCCCGTCCTAAACCTGAGCCTGCCCCTGCTACACCTGCTCCCCCTGGAAGGCTGCCTGAACCTGCTACCCGGGATCACGCTGCTGGCGGAAGGCCTACACCCAGGCCTCCCCGTCCTGAGACCCCTCATAGGCCTTTCGCTCCCCCTGCTGTCGTCCCTTCCGGATGGCCTCAGCCTGCCGAGCCTTTTCCCCCCAGGACAACCGCCGCTCCTGGAGTCTCCAGGCATAGGgatctgtacgacgatgacgataagATCGATTATTGGGTGAGCTTTAACAAGACTATCATTCTGGACAGCAATATTACTAACGACAATAACGACATCAACGGCGTGAGCTGGAACTTCTTTAACAACAGCTTCAATACTCTCGCCACATGTGGCAAGGCCGGCAACTTCTGCGAGTGTAGCAACTATAGCACAAGCATCTACAACATTACTAACAACTGCTCTCTGACTATCTTCCCACATAACGACGTCTTCGACACTACATACCAAGTGGTCTGGAATCAGATCATTAACTACACAATCAAACTGCTGACTCCAGCCACTCCACCTAACATCACATACAACTGCACAAATTTTCTGATTACTTGTAAAAAAAATAATGGCACAAATACAAATATCTATCTGAACATCAACGATACATTCGTCAAGTACACAAACGAGTCCATTCTGGAGTACAACTGGAACAACTCCAACATCAATAATTTCACTGCCACTTGCATCATTAATAACACAATCTCCACTAGCAATGAGACTACACTGATCAACTGCACATATCTGACTCTGAGCTCCAACTACTTTTATACTTTCTTCAAACTGTACCACCACCATCACCATCATTAA(SEQ ID NO.3)。
The obtained target fragment and pCDNA3.4 vector were digested with XbaI and HindIII, respectively, the target fragment and vector were ligated with T4 DNA ligase at 22℃for 2 hours, and the ligation product was transformed into TOP10 competent cells to obtain an expression plasmid, which was identified by double digestion to obtain a target fragment of about 1629bp and a pCDNA3.4 vector fragment of about 5970bp, which was identified by sequencing (see SEQ ID No.3 for details) as expected, and the expression plasmid was designated pCDNA3.4-gD-CD2v. And the bacterial liquid of the plasmid is largely shaken, and then Endo-free Plasmid Midi Kit is used for extracting the plasmid, the concentration is measured to be 600 ng/. Mu.L, and the plasmid is preserved at-20 ℃ for standby.
1.3 expression of fusion proteins CD2v-mFc and fusion proteins gD-CD2v
The ExpiCHO cells were resuscitated and passaged as per the instructions of the ExpiCHO-S expression system. Cell count before transfection, and dilution of cells to 6X 10 based on the count result 6 Each viable cell/mL, the volume used for transfection was 25 mL/serving of plasmid. Adding 20 μg recombinant plasmid into 1mL OptiPROTM SFM for dilution, adding 80 μ LExpiFectamineTM CHO reagent into 1mL OptiPROTM SFM for dilution, adding ExpiFectamineTM CHO reagent into diluted recombinant plasmid, mixing, incubating at room temperature for 4-5 min, slowly transferring the mixed solution into diluted cells, shaking culture flask while adding, shaking, labeling, and placing at 37deg.C and 8% CO 2 Orbital shaker culture at 125 rpm. 150. Mu.L of the ExpiCHOTM enhancer and 6mL of the ExpiCHOTM adjuvant were added 18-22 hours after transfection. And 7-9 days after transfection, when the cell viability is reduced to about 80%, harvesting and detecting culture supernatant after centrifugation at 5000 rpm for 20 minutes.
1.4 purification and Western blotting identification of fusion proteins CD2v-mFc and fusion protein gD-CD2v
The culture supernatant was collected, filtered through a 0.45 μm filter, and then purified by a His Gravitrap protein purification column, the protein was washed off with a equilibration solution, and the eluate at the time of 250mmol/L imidazole was collected. Purified proteins were transferred to NC membrane after SDS-PAGE electrophoresis, blocked with 5% skim milk for 1 hour, added with ASFV positive serum (100-fold dilution), washed 3 times with PBST at 37 ℃ and incubated with HRP-labeled goat anti-swine IgG (2000-fold dilution), washed 3 times with PBST at 37 ℃ and developed using DAB kit. And simultaneously, carrying out Western blotting detection on the fusion protein CD2V-mFc and the fusion protein gD-CD2V by using a PRVGD monoclonal antibody and a goat anti-mouse antibody. Results: the purified fusion proteins CD2V-mFc and gD-CD2V had protein molecular masses of about 113kDa and 92kDa, respectively, with both protein sizes being higher than expected. Glycosylation sites are present by predictive analysis of glycosylation of protein sequences.
Purified proteins were quantified using BCA protein quantification kit. As a result, the concentrations of the fusion proteins CD2V-mFc and gD-CD2V were 1.4mg/mL and 2.5mg/mL, respectively.
EXAMPLE 2ASFV CD2v protein monoclonal antibody preparation and identification
2.1 preparation of monoclonal antibodies
5 female BALB/c mice of 4-6 weeks old were immunized with CD2v-mFc protein at a dose of 100. Mu.g/dose (400. Mu.L in volume) every 3 weeks by subcutaneous multipoint immunization. Immunization is carried out after isovolumetric emulsification of Freund's complete adjuvant and 100 mu g of protein in the first immunization, and immunization is carried out after isovolumetric emulsification of Freund's incomplete adjuvant and 100 mu g of protein in the subsequent immunization for 3 times. Mouse serum was collected after three-phase and serum titers were determined by fusion protein gD-CD2v protein indirect ELISA method: diluting the protein to 0.5 mug/ml, coating an ELISA plate, 100 mug/hole, and standing for 16-24 hours at 2-8 ℃; discarding the liquid in the plate, adding a sealing liquid, 200 μl/hole, sealing at 2-8deg.C for 16-24 hours, and washing the plate; adding a sample to be tested (diluted 1:1000 of mouse serum and diluted in a doubling ratio), 100 μl/well, setting negative control added with PBS (0.01M, pH value 7.4), incubating at 37deg.C for 60 min, and washing the plate; adding the secondary antibody diluted to the working concentration, 100 μl/hole, incubating at 37deg.C for 45 min, and washing the plate; sequentially adding a developer A, B solution, 50 μl/hole, shaking and mixing, incubating at 37deg.C in dark for 15 min, and adding a stop solution, 50 μl/hole; setting the wavelength of an enzyme label instrument at 450nm, and detecting the OD value of each hole; when the OD value of the negative control is less than 0.2, the test is established, and the S/N (sample OD value/negative control OD value) is more than or equal to 2.1, and the test is positive; otherwise, judging as negative. The highest sample dilution corresponding to the positive well was taken as the titer of the sample. Serum from 5 mice was tested by this method, as a result: only 1 mouse (5 #) had a serum titer as high as 1: 512000. And immunizing a 5# mouse by intraperitoneal injection of a CD2v-mFc protein, carrying out cell fusion 3 days after the immunization, and carrying out multiple subcloning screening on the fused cells to obtain 18 positive hybridoma cells. Positive serum blocking test is carried out on the supernatants of 18 hybridoma cells, and the operation steps are as follows: detecting 100 μl of cell supernatant, setting the hole with sample diluent as negative control, incubating at 37deg.C for 60 min, and washing the plate; adding ASF positive serum diluted 1:800 times, 100 μl/well, incubating at 37deg.C for 60 min, and washing the plate; adding diluted HRP-marked anti-pig IgG,100 μl/well, incubating at 37deg.C for 30 min, and washing the plate; sequentially adding a developer A, B solution, 50 μl/hole, shaking and mixing, incubating at 37deg.C in dark for 15 min, and adding a stop solution, 50 μl/hole; setting the wavelength of an enzyme label instrument at 450nm, and detecting the OD value of each hole; sample blocking rate = [ (negative control well OD value-sample well OD value)/negative control well OD value ] ×100%. Preferably, the 4 hybridoma cells (3A 3, 4C12, 5G1, 5G 8) with the highest blocking rate were used for ascites preparation, and after 100-fold dilution of the ascites, the positive serum blocking test was used to evaluate the blocking rate of the ascites, and the results (see Table 3): the blocking rate of the monoclonal antibodies 3A3 and 4C12 is not higher than 50 percent, and the monoclonal antibodies are discarded; the blocking rate of the monoclonal antibodies 5G1 and 5G8 is more than 80%, and the results are ideal and are used for subsequent evaluation.
Table 3 4 results of ASFV CD2v monoclonal antibody positive serum blocking test
Name of the name Blocking rate
3A3 44％
4C12 51％
5G1 87％
5G8 91％
2.2 subclass identification
The subclasses of monoclonal antibodies 5G1, 5G8 were identified using the monoclonal antibody subclass identification kit, resulting in: the heavy chain subclasses of the clone antibodies 5G1 and 5G8 were IgG2a, and the light chain subclasses were kappa.
2.3Western blot identification
Carrying out SDS-PAGE electrophoresis on fusion protein gD-CD2V, separately expressed gD protein and CHO cell supernatant, and carrying out Western blot detection by taking monoclonal antibody 5G1 and 5G8 dilutions as primary antibodies and HRP-marked goat anti-mouse IgG dilutions as secondary antibodies respectively after transferring. Results: the 2 monoclonal antibodies react with the fusion protein gD-CD2v to generate specific bands, and do not react with gD protein and CHO cell supernatant. It was shown that all 2 monoclonal antibodies recognize CD2v protein.
2.4 specificity identification
The method comprises the steps of respectively preparing IFA antigen plates from swine fever virus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine circovirus type 2, porcine parvovirus and porcine epidemic diarrhea virus, fixing with 80% cold acetone, returning to temperature, washing for 1 time with PBS, adding monoclonal antibody 5G1 or 5G8, and detecting by adopting a conventional IFA method, wherein the result is that: no yellow-green fluorescence is seen in the cell holes inoculated with different viruses added with the monoclonal antibodies 5G1 or 5G8, which indicates that the monoclonal antibodies 5G1 and 5G8 do not react with other viruses of porcine origin, and the specificity is good.
Example 3 establishment of ASFV CD2v protein blocking biochip antibody detection kit
3.1 preparation of spotting solution
Preparation of 5% glycerol solution: precisely weighing 5.00g of glycerol, placing into a 100ml volumetric flask, adding a small amount of purified water, slightly rotating to dissolve thoroughly, avoiding excessive bubbles, adding purified water, placing scale marks, and turning upside down and shaking for 10 times for standby;
preparation of 5% sorbitol solution: precisely weighing 5.00g sorbitol, placing in a 250ml beaker, adding a proper amount of purified water, stirring to dissolve completely, transferring to a 100ml volumetric flask completely, adding a scale mark of the purified water, and turning upside down and shaking for 10 times for standby;
preparation of 0.05% triton solution: measuring 50 μl of triton by a pipette, putting into a 100ml volumetric flask, adding a proper amount of purified water to dissolve completely, adding a scale mark of the purified water, and turning upside down and shaking for 10 times for standby;
DMSO solution: directly adopting DMSO reagent;
PBS (pH 6.8) solution: firstly preparing 0.2mol/L disodium hydrogen phosphate solution and 0.3mol/L sodium dihydrogen phosphate solution, and then mixing the two solutions according to the volume ratio of 49.5:51 to obtain phosphate buffer solution with the pH value of 6.8;
the solution is mixed evenly according to the volume ratio of 10:15:0.1:50:100 to be used as the sample application liquid.
3.2 preparation of enzyme-labeled antibody
3.2.1 preparation
The monoclonal antibodies 5G1 and 5G8 of the CD2v protein of the African swine fever virus are respectively marked by horseradish peroxidase (HRP) by adopting a modified sodium periodate method.
20mg of horseradish peroxidase (HRP) was weighed into 1ml of ultrapure water, and 1ml of freshly prepared NaIO was added 4 Solution (30 mg NaIO) 4 Dissolving in 1ml of ultrapure water), mixing, and performing light-shielding action for 30 minutes at 4 ℃; adding 40 μl of ethylene glycol into the above solution, and keeping out of light at 4deg.C for 30 min; the purified monoclonal antibody was added to 100. Mu.l of the above mixture in a ratio of 1mg, and the mixture was added to a dialysis bag, and the mixture was dialyzed against CB buffer for 6 hours. The whole operation needs to be carried out in a dark place; the dialyzed mixture was transferred to a 1.5ml EP tube and 10. Mu.l of freshly prepared NaBH was added 4 Solution (20 mg NaBH) 4 Dissolving in 1ml of ultrapure water), allowing the mixture to act at room temperature for 2 hours, and uniformly mixing the mixture once every 30 minutes; an equal volume of saturated ammonium sulfate was added and after mixing, the mixture was allowed to act at 4℃for 15 minutes. Centrifuge at 12000 rpm for 10 min, discard supernatant. The pellet was suspended by blowing with an equal volume of PBS and glycerol mixture (V: V=1:1) to purify the antibody.
3.2.2 identification
Appearance: at room temperature, as a reddish brown liquid, no floc precipitate was seen.
And (3) quality evaluation: the absorbance A of the enzyme-labeled antibody at 403nm and 280nm was measured by an ultraviolet spectrophotometer. The corresponding enzyme parameters were calculated according to the formula:
enzyme amount (mg/ml) =a403 nm×0.4×dilution.
IgG amount (mg/ml) = (a280 nm ≡a403nm×0.3) ×0.62×dilution.
Molar ratio (E/P) =enzyme amount×4/IgG amount.
Labeling rate = a403nm/a280nm.
Specific results of detection and calculation of absorbance are shown in Table 4.
TABLE 4 quality assessment of different enzyme-labeled antibodies
3.3 preparation of kit
The CD2v-mFc protein prepared in example 1 was diluted to 800. Mu.g/ml with spotting solution as spotting solution at the detection point; diluting goat anti-mouse IgG to 200 mug/ml by using a sample application liquid, and respectively applying sample application liquid as a quality control point sample application liquid to points (1), (2) and (3) in the figure 1 so as to carry out data statistical analysis according to a quality control point average value; the spotting fluid was spotted as a blank spotting fluid at spot (4). Starting the spotting instrument, setting programs and spotting parameters, and spotting the detection point spotting liquid at the point (5), the quality control point spotting liquid at the point (1), (2), (3) in fig. 1, and the blank control point spotting liquid at the point (4) in fig. 1 according to the volume spotting of 20 nL/point. Taking out the spotted film, placing the spotted film in the middle of a bottom plate of the chip, pressing the middle of the bottom plate by a cover plate, fixing edge strips on two edge cards, assembling the ASFV CD2v blocking method chip, packaging, and preserving at 2-8 ℃. Besides the points (1), (2), (3) and (4) are respectively fixed at the left upper corner, the left lower corner, the right lower corner and the right upper corner of the detection chip, the point (5) can be spotted at any other position of the detection chip, and the distance between the points and the edge of other points is not less than 700 mu m, and the shortest distance between the points is not less than 700 mu m. The distances from the quality control product point (1), the quality control product point (2), the quality control product point (3) and the blank control sample application point (4) to the upper edge and the lower edge of the detection chip subunit are more than or equal to 8mm, and the distances from the left edge and the right edge of the detection chip subunit are more than or equal to 5mm.
The antibody detection kit comprises one or more African swine fever virus CD2v protein antibody detection chip subunits, wherein the antibody detection chip subunits consist of a bottom end, an upper grille and detection chips clamped by the bottom end and the upper grille and are arranged between the bottom end and the upper grille to form independent detection holes, one detection hole corresponds to one detection chip subunit, and when the antibody joint detection kit comprises a plurality of African swine fever virus CD2v protein antibody detection chip subunits, each independent detection chip subunit is integrated into a microarray chip. FIG. 2 shows the kit with different numbers of the antibody detection chip subunits of the African swine fever virus CD2v protein, and O, P, Q in FIG. 2 is the kit with one, three or more antibody detection chip subunits, respectively, and the partial enlarged view of the kit shows the spotting point on the detection chip in one of the antibody detection chip subunits of the African swine fever virus CD2v protein.
Enzyme-labeled reagent: 200ml of PBS solution and fetal calf serum are taken and mixed uniformly, and PBS is added to be 1L in volume to be used as enzyme-labeled diluent. The ELISA monoclonal antibodies 5G1 and 5G8 prepared in example 2 were diluted and used as ELISA reagents, filtered at 0.22 μm, and packaged aseptically.
Sample dilution: PBS solution containing 10% V/V fetal calf serum, 0.1% V/V Tween 20, 1% W/V BSA, 0.05% -0.5% W/V Casein, 1% W/V Proclin300, 0.22 μm filtration, and sterile packaging.
Washing liquid: the PBS solution containing 1%V/V Tween 20 was filtered through 0.22 μm and aseptically packaged. The sample was diluted 20-fold with purified water.
Substrate solution: TMB (3, 3', 5' -tetramethylbenzidine) solution, commercial product, and sterile packaging.
The above components are assembled into a kit, and labeled as a kit A (ELISA monoclonal antibody 5G 1) and a kit B (ELISA monoclonal antibody 5G 8).
3.4 detection method establishment
The operation steps are as follows:
(1) Numbering, the chip wells are numbered according to the sample sequence.
(2) Soaking, adding 300 mu L/hole of washing liquid, soaking for 3 minutes, discarding liquid, and beating to dry.
(3) Adding a sample, adding 50 mu L/hole of a sample diluent, adding 50 mu L/hole of a sample to be detected, and placing a constant-temperature oscillating incubator at 37 ℃ for 1000 revolutions/minute for 20 minutes; discarding the solution, adding 300 mu L/hole of washing solution, soaking for 30-60 seconds, and discarding the solution. Repeatedly washing for 5 times, and finally beating to dry.
(4) Adding enzyme, adding 100 μl/hole of enzyme-labeled reagent, placing in a constant temperature shaking incubator at 37deg.C for 20 min at 1000 rpm; discarding the solution, adding 300 mu L/hole of washing solution, soaking for 30-60 seconds, and discarding the solution. Repeatedly washing for 5 times, and finally beating to dry.
(5) Color development, adding substrate solution 100. Mu.L/well, placing at 37 ℃, 1000 rpm for 15 minutes.
(6) Measuring, vertically discarding liquid, removing the upper cover of the chip, reversely buckling on dust-free paper, lightly pressing to dryness, and measuring the result by using a micropore disc chip imager within 10 minutes to obtain PI (PI= (1-sample S value/quality control point N value) ×100%).
Test effectiveness judgment: the quality control point S value is equal to or more than 6000, the blank contrast S value is equal to or less than 3000, otherwise, the test is invalid, and the judgment is automatically completed through an internal data processing analysis system. The result judgment (the judgment is also completed by the internal one-key intelligent data processing analysis system without the need of a technician to perform data operation and statistical analysis) is as follows:
calculation of PI: pi= (1-sample S value/quality control point N value) ×100%
Positive ASFV CD2v antibody is positive when PI is more than or equal to 40%;
when the PI is less than or equal to 30%, the ASFV CD2v antibody is negative;
when the suspicious PI is more than 30% and less than 40%, the ASFV CD2v antibody is suspicious, sampling again is needed for detection, and if the detection is still suspicious, the detection is negative.
3.4 evaluation of ASFV CD2v protein blocking biochip antibody detection kit
3.4.1 specificity
7 parts of porcine common virus positive serum (comprising porcine pseudorabies positive serum, porcine reproductive and respiratory syndrome positive serum, swine fever positive serum, porcine circovirus type 2 positive serum, porcine epidemic diarrhea positive serum, porcine transmissible gastroenteritis positive serum, porcine rotavirus positive serum), 20 parts of SPF porcine serum and 50 parts of ASFV antigen negative porcine serum were detected by using the kit A and the kit B prepared in the example 3.2, and the result is that: PI is less than or equal to 30%, and both are negative, which indicates that the specificity of the kit A and the kit B is good.
3.4.2 sensitivity
ASF standard positive serum-fold ratio dilutions were tested using kit a, kit B prepared in example 3.2, results: the kit A detects that the 1:32 times of diluent of standard positive serum is positive, and the 1:64 times of diluent is negative; the kit B detects that the 1:64 times of diluent of ASF standard positive serum is positive and the 1:128 times of diluent is negative. The reagent kit B is shown to have higher sensitivity.
Positive serum from CD2v protein immunized pigs was tested for 20 parts using kit B prepared in example 3.2, results: all positive.
3.4.3 clinical application
According to the above results, the kit B prepared in example 3.2 was used for clinical application to detect 20 ASF positive pig serum and 1000 negative pig serum collected before 2018, resulting in: PI of 20 ASF positive pig serum detected is equal to or more than 40%, and the pig serum is positive; and detecting that 1000 parts of pig serum collected before 2018 is negative and has PI less than or equal to 30 percent.
EXAMPLE 4ASFV CD2v protein monoclonal antibody 5G8 variable region sequence determination
According to the sequence characteristics of the murine monoclonal antibody, the heavy chain variable region primer sequence is designed:
F：5’-ACTAGTCGACATGAAGWTGTGGBTRA-3’；
R：5’-CCAGGGRCCARKGGATARACN-3’；
designing a light chain variable region primer sequence:
F：5’-ACTAGTCGACATGAAGTTGCCTGTTA-3’；
R：5’-CCCAAGCTTACTGGATGGTGGG-3’；
hybridoma cells were collected, RNA was extracted and reverse transcribed as a template, and the variable region sequence was amplified using the above primers, and the amplified product was sent to Suzhou Jin Weizhi Biotechnology Co., ltd for sequencing. Results: the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 5G8 are respectively shown as SEQ ID No.4 and SEQ ID No.6, and the gene sequences are respectively shown as SEQ ID No.5 and SEQ ID No. 7.
EXAMPLE 5 preparation and identification of Single chain antibody 5G8
Amplifying heavy chain variable region (VH) gene and light chain variable region (VL) gene of monoclonal antibody, transferring into connecting peptide, connecting to prokaryotic expression vector pET-32a (+), respectively constructing recombinant plasmid, and transforming BL21 competent cells for expression to obtain fusion protein. The corresponding single chain antibody 5G8 was prepared as described in example 4 using the variable region sequence of monoclonal antibody 5G8. Positive serum blocking assays were performed on single chain antibody 5G8 as described in example 2, resulting in: the blocking rate of the single-chain antibody 5G8 is more than 80%, and the result is ideal, which shows that the prepared single-chain antibody has good activity.
The results show that the variable region sequences shown by SEQ ID No.4 and SEQ ID No.6 or SEQ ID No.5 and SEQ ID No.7 can be used for preparing the African swine fever virus CD2v protein genetic engineering antibody.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
SEQUENCE LISTING
<110> Luoyang midke biochip technologies Co., ltd
<120> African swine fever virus CD2v protein blocking biochip antibody detection kit
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 573
<212> DNA
<213> artificial sequence
<400> 1
atcgattatt gggtgagctt taacaagact atcattctgg acagcaatat tactaacgac 60
aataacgaca tcaacggcgt gagctggaac ttctttaaca acagcttcaa tactctcgcc 120
acatgtggca aggccggcaa cttctgcgag tgtagcaact atagcacaag catctacaac 180
attactaaca actgctctct gactatcttc ccacataacg acgtcttcga cactacatac 240
caagtggtct ggaatcagat cattaactac acaatcaaac tgctgactcc agccactcca 300
cctaacatca catacaactg cacaaatttt ctgattactt gtaaaaaaaa taatggcaca 360
aatacaaata tctatctgaa catcaacgat acattcgtca agtacacaaa cgagtccatt 420
ctggagtaca actggaacaa ctccaacatc aataatttca ctgccacttg catcattaat 480
aacacaatct ccactagcaa tgagactaca ctgatcaact gcacatatct gactctgagc 540
tccaactact tttatacttt cttcaaactg tac 573
<210> 2
<211> 1398
<212> DNA
<213> artificial sequence
<400> 2
gccaccatgg actggacctg gaggatcctc ttcttggtgg cggccgccac aggcgcgcac 60
tcccaccacc atcaccatca tatcgattat tgggtgagct ttaacaagac tatcattctg 120
gacagcaata ttactaacga caataacgac atcaacggcg tgagctggaa cttctttaac 180
aacagcttca atactctcgc cacatgtggc aaggccggca acttctgcga gtgtagcaac 240
tatagcacaa gcatctacaa cattactaac aactgctctc tgactatctt cccacataac 300
gacgtcttcg acactacata ccaagtggtc tggaatcaga tcattaacta cacaatcaaa 360
ctgctgactc cagccactcc acctaacatc acatacaact gcacaaattt tctgattact 420
tgtaaaaaaa ataatggcac aaatacaaat atctatctga acatcaacga tacattcgtc 480
aagtacacaa acgagtccat tctggagtac aactggaaca actccaacat caataatttc 540
actgccactt gcatcattaa taacacaatc tccactagca atgagactac actgatcaac 600
tgcacatatc tgactctgag ctccaactac ttttatactt tcttcaaact gtacgagaat 660
ttgtactttc aaggcgagcc aaggggacct acaatcgagc caaggggacc tacaatcaag 720
ccttgcccac catgcaagtg cccagctcct aatctgctgg gcggaccatc cgtgttcatc 780
ttcccaccta aaatcaaaga tgtgctcatg atctccctct cccctatcgt gacttgcgtg 840
gtggtggatg tgagcgagga cgacccagat gtccagatca gctggttcgt gaacaacgtg 900
gaggtgcata ctgctcagac acagactcat agggaggact acaacagcac actgagagtc 960
gtgtccgctc tgccaatcca gcatcaagat tggatgagcg gcaaggagtt taagtgcaag 1020
gtcaacaaca aggatctgcc agcccctatc gagaggacaa tcagcaagcc aaaaggcagc 1080
gtgagggctc ctcaagtgta cgtgctccct cctccagagg aggagatgac taaaaagcaa 1140
gtgactctca cttgcatggt gacagacttc atgccagagg acatctacgt ggagtggact 1200
aacaacggca agactgaact gaattacaaa aacacagagc cagtgctgga ctccgacgga 1260
agctacttca tgtacagcaa gctgagggtc gagaagaaga actgggtcga gaggaattcc 1320
tacagctgtt ccgtggtgca cgaaggactg cacaaccacc acactactaa gtccttttct 1380
aggacacccg gcaagtaa 1398
<210> 3
<211> 1677
<212> DNA
<213> artificial sequence
<400> 3
gccaccatga acctgctgct gatcctgacc tttgtggccg ccgccgtggc cgctgatgtg 60
gatgccgtgc ccgctcccac ctttcctcct cctgcctacc cctacaccga gagctggcag 120
ctgacactga ccacagtgcc ttcccccttc gtgggccctg ccgatgtgta ccacaccagg 180
cccctggagg atccttgcgg agtggtggct ctcatcagcg accctcaggt cgacaggctg 240
ctgaacgagg ctgtggccca caggaggcct acatacaggg cccacgtggc ctggtacagg 300
atcgccgacg gctgtgccca cctgctgtac tttatcgagt acgctgactg cgaccccagg 360
cagattttcg gcaggtgccg gaggaggacc acccctatgt ggtggacccc ctccgccgac 420
tacatgttcc ccaccgagga cgagctgggc ctgctgatgg tggcccctgg caggttcaat 480
gagggccagt acaggaggct ggtgtccgtg gacggcgtga acatcctcac cgacttcatg 540
gtggccctgc ctgagggcca ggaatgtcct ttcgcccggg tcgaccagca ccggacctac 600
aagttcggcg cctgctggtc cgacgactcc ttcaagaggg gcgtggacgt gatgaggttc 660
ctgaccccct tctatcagca gcccccccac agggaggtgg tgaactactg gtacaggaag 720
aacggcagga cactgccccg ggcttatgct gccgccacac cttacgccat cgaccccgct 780
aggcccagcg ctggatcccc caggccccgt ccccgtcccc gtcctcggcc ccgtcctaaa 840
cctgagcctg cccctgctac acctgctccc cctggaaggc tgcctgaacc tgctacccgg 900
gatcacgctg ctggcggaag gcctacaccc aggcctcccc gtcctgagac ccctcatagg 960
cctttcgctc cccctgctgt cgtcccttcc ggatggcctc agcctgccga gccttttccc 1020
cccaggacaa ccgccgctcc tggagtctcc aggcataggg atctgtacga cgatgacgat 1080
aagatcgatt attgggtgag ctttaacaag actatcattc tggacagcaa tattactaac 1140
gacaataacg acatcaacgg cgtgagctgg aacttcttta acaacagctt caatactctc 1200
gccacatgtg gcaaggccgg caacttctgc gagtgtagca actatagcac aagcatctac 1260
aacattacta acaactgctc tctgactatc ttcccacata acgacgtctt cgacactaca 1320
taccaagtgg tctggaatca gatcattaac tacacaatca aactgctgac tccagccact 1380
ccacctaaca tcacatacaa ctgcacaaat tttctgatta cttgtaaaaa aaataatggc 1440
acaaatacaa atatctatct gaacatcaac gatacattcg tcaagtacac aaacgagtcc 1500
attctggagt acaactggaa caactccaac atcaataatt tcactgccac ttgcatcatt 1560
aataacacaa tctccactag caatgagact acactgatca actgcacata tctgactctg 1620
agctccaact acttttatac tttcttcaaa ctgtaccacc accatcacca tcattaa 1677
<210> 4
<211> 118
<212> PRT
<213> artificial sequence
<400> 4
Tyr Asp His Asp Tyr Ala Lys Leu Gly Thr Glu Leu Gly Ser Thr Ser
1 5 10 15
Asn Gly Arg Gln Cys Ala Gly Ile Ala Leu Thr Ser Arg Met Lys Leu
20 25 30
Trp Leu Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Val Ile Asp Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Val Arg Ser Leu Thr Ser Glu Tyr Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Gly Lys Gly Ile Gly Leu Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
<210> 5
<211> 354
<212> DNA
<213> artificial sequence
<400> 5
tatgaccatg attacgccaa gcttggtacc gagctcggat ccactagtaa cggccgccag 60
tgtgctggaa ttgcccttac tagtcgcatg aagttgtggt tgaactgggt gaagcagagc 120
catggaaaga gccttgagtg gattggagtt attgatccta acaatggtgg tactacctac 180
aaccagaagt tcaagggcaa ggccacattg actgtagaca agtcctccag cacagcctat 240
atggaggtcc gcagcctgac atctgaatat tctgcagtct attactgtgc aagatatggt 300
aaagggattg gtttggacta ctggggtcaa ggaacctcag tcaccgtctc ctca 354
<210> 6
<211> 112
<212> PRT
<213> artificial sequence
<400> 6
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Val Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 7
<211> 336
<212> DNA
<213> artificial sequence
<400> 7
gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60
atctcttgca gatctagtca gagccttgta cacagaaatg gaaacaccta tttacattgg 120
tacctgcaga agccaggcca gtctccaaaa ctcctgatct acaaagtttc caaccgattt 180
tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240
agcagagtgg aggctgtgga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300
tggacgttcg gtggaggcac caagctggaa atcaaac 336
<210> 8
<211> 107
<212> DNA
<213> artificial sequence
<400> 8
tctagagcca ccatggactg gacctggagg atcctcttct tggtggcggc cgccacaggc 60
gcgcactccc accaccatca ccatcatatc gattattggg tgagctt 107
<210> 9
<211> 67
<212> DNA
<213> artificial sequence
<400> 9
gattgtaggt ccccttggct cgccttgaaa gtacaaattc tcgtacagtt tgaagaaagt 60
ataaaag 67
<210> 10
<211> 67
<212> DNA
<213> artificial sequence
<400> 10
cttttatact ttcttcaaac tgtacgagaa tttgtacttt caaggcgagc caaggggacc 60
tacaatc 67
<210> 11
<211> 32
<212> DNA
<213> artificial sequence
<400> 11
aagcttttac ttgccgggtg tcctagaaaa gg 32
<210> 12
<211> 76
<212> DNA
<213> artificial sequence
<400> 12
tctagaatga acctgctgct gatcctgacc tttgtggccg ccgccgtggc cgctgatgtg 60
gatgccgtgc ccgctc 76
<210> 13
<211> 65
<212> DNA
<213> artificial sequence
<400> 13
aagctcaccc aataatcgat cttatcgtca tcgtcgtaca gatccctatg cctggagact 60
ccagg 65
<210> 14
<211> 65
<212> DNA
<213> artificial sequence
<400> 14
cctggagtct ccaggcatag ggatctgtac gacgatgacg ataagatcga ttattgggtg 60
agctt 65
<210> 15
<211> 47
<212> DNA
<213> artificial sequence
<400> 15
aagcttttaa tgatggtgat ggtggtggta cagtttgaag aaagtat 47

Claims (10)

1. The African swine fever virus CD2v protein blocking biochip antibody detection kit is characterized by comprising an African swine fever virus CD2v protein antibody detection chip and an enzyme-labeled reagent;
the enzyme-labeled reagent is an antibody which specifically binds with the African swine fever virus CD2v protein, and the antibody contains a heavy chain variable region with an amino acid sequence shown as SEQ ID NO.4 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 6.
2. The kit according to claim 1, wherein the african swine fever virus CD2v protein antibody detection chip is provided with a spotting spot, the spotting spot comprises a quality control spot, a detection spot and a blank control spot, the quality control spot is coated with a quality control substance, the quality control substance is goat anti-mouse IgG, and the coating amount is 3-5 ng/spot, preferably 4 ng/spot;
Preferably, the detection spot is coated with the fusion protein CD2v-mFc in an amount of 1-64 ng/spot, preferably 8-32 ng/spot, further preferably 16 ng/spot;
preferably, the nucleotide sequence encoding the fusion protein CD2v-mFc is shown in SEQ ID NO. 2;
preferably, the blank spots are coated with a blank.
3. The kit according to claim 2, wherein the number of quality control spots is 1-4, preferably 3.
4. The kit according to claim 2, wherein the buffer solution of goat anti-mouse IgG, the buffer solution of the fusion protein CD2v-mFc and the blank control are respectively spotting solutions, and the spotting solutions are prepared by uniformly mixing 5w/v% glycerol solution, 5w/v% sorbitol solution, 0.05v/v% triton solution, DMSO solution and ph6.8 PBS solution according to a volume ratio of 10:15:0.1:50:100.
5. The kit according to claim 2, wherein the edge distance between any two points among the detection point, the quality control point and the blank control point is equal to or more than 700 μm, and the edge distance between any point and the chip is equal to or more than 5mm, preferably equal to or more than 8mm.
6. Kit according to claim 1, wherein the antibody is a monoclonal antibody or a genetically engineered antibody, wherein the genetically engineered antibody comprises a chimeric, a diabody or a small molecule antibody, preferably the antibody is a monoclonal or a single chain antibody;
Preferably, the antibody is IgA, igD, igE, igG or IgM;
preferably, the nucleotide sequence encoding the heavy chain variable region of the antibody is shown in SEQ ID NO. 5;
preferably, the nucleotide sequence encoding the light chain variable region of the antibody is shown in SEQ ID NO. 7.
7. The kit of claim 1, wherein the enzyme comprises horseradish peroxidase, alkaline phosphatase, or beta-D-galactosidase.
8. The kit of claim 1, further comprising a wash solution, a sample diluent, and a substrate solution.
9. The kit of claim 8, wherein the wash solution is a 1v/v% tween 20 in PBS;
preferably, the sample diluent is a PBS solution of 10v/v% fetal bovine serum, 0.05v/v% to 0.2v/v% Triton-100, 0.1v/v% Proclin 300;
preferably, the substrate solution is a TMB solution.
10. Use of an antibody specifically binding to african swine fever virus CD2v protein in the preparation of an african swine fever virus CD2v protein blocking biochip antibody detection kit according to any one of claims 1 to 9, wherein the antibody comprises a heavy chain variable region having an amino acid sequence shown as SEQ ID No.4 and a light chain variable region having an amino acid sequence shown as SEQ ID No. 6.
CN202210454844.8A 2022-04-27 2022-04-27 African swine fever virus CD2v protein blocking biochip antibody detection kit Pending CN117007808A (en)

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