Monoclonal antibody, preparation method and application thereof
阅读说明：本技术 一种单克隆抗体、其制备方法及用途 (Monoclonal antibody, preparation method and application thereof ) 是由 李玲 董春娜 吴冬荀 张艳宾 张蕾 张彤 李静 刘新月 肖进 齐鹏 于 2020-07-29 设计创作，主要内容包括：本发明公开了一种抗猪瘟病毒E2单克隆抗体、其制备方法及用途。所述单克隆抗体包括重链可变区和轻链可变区,重链可变区的CDR1的氨基酸序列如序列1所示；重链可变区的CDR2的氨基酸序列如序列2所示；重链可变区的CDR3的氨基酸序列如序列3所示；轻链可变区的CDR1的氨基酸序列如序列4所示；轻链可变区的CDR2的氨基酸序列如序列5所示；轻链可变区的CDR3的氨基酸序列如序列6所示。本发明所述的单克隆抗体能特异性与猪瘟病毒E2蛋白结合,具有很好的抗原结合活性和一定的病毒中和活性,既可用于猪瘟的检测诊断又可以用于预防治疗,在开发猪瘟病毒新型诊断试剂和治疗药物中具有较好的生产应用前景。(The invention discloses a swine fever virus resisting E2 monoclonal antibody, a preparation method and application thereof. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of CDR1 of the heavy chain variable region is shown as a sequence 1; the amino acid sequence of CDR2 of the heavy chain variable region is shown as sequence 2; the amino acid sequence of CDR3 of the heavy chain variable region is shown as sequence 3; the amino acid sequence of CDR1 of the light chain variable region is shown as sequence 4; the amino acid sequence of CDR2 of the light chain variable region is shown as sequence 5; the amino acid sequence of CDR3 of the variable region of the light chain is shown in sequence 6. The monoclonal antibody disclosed by the invention can be specifically combined with classical swine fever virus E2 protein, has good antigen binding activity and certain virus neutralizing activity, can be used for detection and diagnosis of classical swine fever virus and prevention and treatment, and has good production and application prospects in development of novel diagnostic reagents and treatment medicines of classical swine fever virus.)
1. An anti-classical swine fever virus E2 monoclonal antibody or an antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, both of which are composed of a determinant complementary region and a framework region, both of the determinant complementary regions of the heavy chain variable region and the light chain variable region are composed of CDR1, CDR2 and CDR3, characterized in that: the amino acid sequence of CDR1 of the heavy chain variable region is shown as sequence 1 or has at least 80% homology with the amino acid sequence; the amino acid sequence of CDR2 of the heavy chain variable region is shown as sequence 2 or has at least 80% homology with the amino acid sequence; the amino acid sequence of CDR3 of the heavy chain variable region is shown as sequence 3 or has at least 80% homology with the amino acid sequence; the amino acid sequence of CDR1 of the light chain variable region is shown as sequence 4 or has at least 80% homology with the amino acid sequence; the amino acid sequence of CDR2 of the light chain variable region is shown as sequence 5 or has at least 80% homology with the amino acid sequence; the amino acid sequence of CDR3 of the light chain variable region is shown as sequence 6 or has at least 80% homology with the amino acid sequence;
the antigen binding fragment is selected from Fab, Fab ', F (ab') 2, Fv, Fab/c, single chain antibody or diabody.
2. The anti-classical swine fever virus E2 monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein said anti-classical swine fever virus E2 monoclonal antibody comprises:
1) a heavy chain variable region consisting of the sequence:
an amino acid sequence shown in sequence 7, or a sequence having from 93% or more to less than 100% sequence identity to a sequence shown in sequence 7;
2) a light chain variable region consisting of the sequence:
an amino acid sequence shown in sequence 8, or a sequence having greater than or equal to 93% to less than 100% sequence identity to a sequence shown in sequence 8.
3. A nucleic acid molecule encoding the anti-classical swine fever virus E2 monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2;
preferably, the nucleic acid molecule sequence encoding CDR1 of the heavy chain variable region is shown in seq id No. 11, the nucleic acid molecule sequence encoding CDR2 of the heavy chain variable region is shown in seq id No. 12, the nucleic acid molecule sequence encoding CDR3 of the heavy chain variable region is shown in seq id No. 13, the nucleic acid molecule sequence encoding CDR1 of the light chain variable region is shown in seq id No. 14, the nucleic acid molecule sequence encoding CDR2 of the light chain variable region is shown in seq id No. 15, and the nucleic acid molecule sequence encoding CDR3 of the light chain variable region is shown in seq id No. 16;
more preferably, the nucleic acid molecule encoded by the heavy chain variable region of the anti-classical swine fever virus E2 monoclonal antibody or antigen binding fragment thereof is represented by SEQ ID No. 9, and the nucleic acid molecule encoded by the light chain variable region is represented by SEQ ID No. 10.
4. A recombinant vector comprising the nucleic acid molecule of claim 3.
5. A recombinant cell into which the nucleic acid molecule of claim 3 has been introduced or into which the recombinant vector of claim 4 has been transfected.
6. A method of producing the anti-classical swine fever virus E2 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising the steps of:
1) cloning the nucleic acid molecule sequence of claim 3 into an expression vector to obtain a recombinant expression vector;
2) transfecting the recombinant expression vector into a host cell;
3) harvesting the transfected cell culture supernatant of step 2) multiple times;
and/or obtaining recombinant cells through the step 2), culturing the recombinant cells in a target culture medium to obtain cell strains capable of expressing the antibody, gradually amplifying the cell strains obtained by culture, and harvesting culture supernatants;
4) purifying the culture supernatant obtained in step 3) to obtain the anti-classical swine fever virus E2 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-2;
preferably, the host cell is a prokaryotic cell, a eukaryotic cell, an insect cell or a fibroblast; the prokaryotic cell is escherichia coli or bacillus subtilis, the fungal cell is a yeast cell or aspergillus, the insect cell is an S2 drosophila cell or an Sf9 cell, and the fibroblast is a CHO cell, a COS cell, a BHK cell or an HEK293 cell.
7. A method for detecting the expression of classical swine fever virus E2 protein for non-diagnostic purposes, said method comprising the steps of:
1) preparing or extracting a sample containing classical swine fever virus E2 protein;
2) contacting or incubating the sample obtained in step 1) with the specific classical swine fever virus E2 monoclonal antibody or antigen binding fragment thereof as described above;
3) detecting an immune reaction of the sample with the antibody or antigen-binding fragment thereof.
8. A kit for testing the titer of a live virus of a swine fever virus and/or the efficacy of a live vaccine, comprising: the anti-classical swine fever virus E2 monoclonal antibody or the FITC-labeled anti-classical swine fever virus E2 monoclonal antibody of any one of claims 1-2.
9. A blocking ELISA kit for detecting classical swine fever virus antibodies, comprising: an HRP-labeled anti-classical swine fever virus E2 monoclonal antibody according to any one of claims 1-2 or anti-classical swine fever virus E2 monoclonal antibody according to any one of claims 1-2.
10. Use of the anti-CSFV E2 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-2 for the preparation of CSFV diagnostic, therapeutic or CSFV antibody detection reagents.
Technical Field
The invention belongs to the fields of immunology and molecular biology, and relates to a swine fever virus E2-resistant monoclonal antibody, and a preparation method and application thereof.
Background
Classical Swine Fever (CSF) is an acute, febrile and highly contagious viral infectious disease caused by infection of domestic pigs and wild pigs with CSF virus (CSFV), and is characterized by acute onset, high fever retention and fine vascular degeneration, causing generalized punctate bleeding throughout the body, and spleen infarction. Pigs of different breeds, sexes and ages are susceptible, and swine fever is a serious infectious disease seriously harming the pig industry. The pathogen of hog cholera, hog cholera virus, belongs to the flaviviridae family and pestivirus genus, and the members of the same genus also include bovine viral diarrhea virus, sheep border disease virus, etc. The classical swine fever virus genome is a single-stranded positive-stranded RNA of about 12.3kb in length, comprising both untranslated regions (UTRs) and an intermediate large Open Reading Frame (ORF) encoding a large polyprotein that is processed by host cell and virus-encoded proteolytic enzymes to yield functional mature proteins, including 4 structural proteins (Core, Erns, E1, and E2) and 8 nonstructural proteins (Npro, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS 5B). The structural protein E2 is the most important immunogenic protein, induces an organism to generate a neutralizing antibody, can protect pigs against the attack of virulent strains of swine fever virus, and is also an important target protein for the development of swine fever genetic engineering vaccines.
China pays great attention to the prevention and control of swine fever, 21 months at 2017, Chinese & ltnational swine fever prevention and control guidance opinions (2017 and 2020) are issued by the department of agricultural rural areas, clear requirements are put forward on the purification of the swine fever in the swine farm, all the swine farms and partial areas in China reach the purification standard of the swine fever by the end of 2020, and the range of the purification area of the swine fever is further expanded. The immunization is a main measure for preventing and controlling the occurrence and prevalence of the swine fever, and the quality of the swine fever vaccine directly influences the immune effect of the swine fever. Currently, there are three main types of marketed swine fever vaccines: the vaccine is a traditional hog cholera lapinized virus live vaccine; the second is a subunit vaccine expressed by an insect cell-baculovirus eukaryotic system based on a main protective antigen E2 of the classical swine fever virus, and comprises a domestic classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine (Rb-03 strain) of Xinjiang Tiankang, and foreign countriesCSF E2 and
Based on the type and production process of the existing commercial swine fever vaccine, the antigen content of the live virus and/or the live vaccine is quantified, namely the efficacy test method of the swine fever vaccine-half of the Fluorescent Antibody Infectious Dose (FAID)50) The method is a classical method for measuring the titer of live viruses in the field of virology, has the characteristics of accurate measurement result, stable method, simple operation and the like, and can be used for measuring the content of the live viruses of the classical swine fever viruses by using an indirect or direct immunofluorescence method through the monoclonal antibody of the classical swine fever virus E2. Serological differential diagnosis of matched classical swine fever E2 subunit vaccine and recombinant live virus vector vaccine CP7_ E2alf requires simultaneous anti-swine fever E2 subunit vaccine and recombinant live virus vector vaccineHog cholera virus ErnsAnd E2 monoclonal antibody to distinguish vaccine immunized animals and eliminate wild virus infected animals. In addition, the classical swine fever virus E2 monoclonal antibody based on high specificity and affinity is central to the development of blocking ELISA kits. Meanwhile, the treatment of wild virus infected animals has higher requirements on the neutralizing activity of the monoclonal antibody against classical swine fever virus E2.
Disclosure of Invention
One of the purposes of the invention is to provide a monoclonal antibody which specifically binds to classical swine fever virus E2 and has the characteristics of high affinity and better specificity.
It is a further object of the present invention to provide a heavy chain, a light chain or a fragment thereof of the above antibody.
It is a further object of the present invention to provide nucleic acid molecules or fragments thereof encoding the above monoclonal antibodies or antigen binding fragments thereof, and recombinant vectors and recombinant cells for recombinant expression of the above antibodies inserted into these nucleic acid molecules.
The fourth purpose of the invention is to provide a preparation method and application of the monoclonal antibody.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a monoclonal antibody or an antigen binding fragment thereof specifically binding to classical swine fever virus E2, wherein the monoclonal antibody or the antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, both the heavy chain variable region and the light chain variable region consist of a determinant complementary region and a framework region, and both the determinant complementary regions of the heavy chain variable region and the light chain variable region consist of CDR1, CDR2 and CDR3, namely, heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR 3;
the heavy chain CDR1 has the amino acid sequence shown in
the heavy chain CDR2 has the amino acid sequence shown in sequence 2 or an amino acid sequence with at least 80% homology with the amino acid sequence;
the heavy chain CDR3 has the amino acid sequence shown in sequence 3 or an amino acid sequence at least 80% homologous thereto;
light chain CDR1 has the amino acid sequence set forth in sequence No. 4 or an amino acid sequence at least 80% homologous thereto;
light chain CDR2 has the amino acid sequence set forth in sequence 5 or an amino acid sequence at least 80% homologous thereto;
the light chain CDR3 has the amino acid sequence shown in sequence 6 or an amino acid sequence at least 80% homologous thereto;
preferably, heavy chain CDR1 has the amino acid sequence shown in
Further, the heavy chain variable region of the monoclonal antibody has an amino acid sequence shown in sequence 7 or an amino acid sequence having at least 80% homology thereto, and the light chain variable region of the monoclonal antibody has an amino acid sequence shown in
Preferably, the heavy chain variable region of the monoclonal antibody has an amino acid sequence shown in sequence 7; the variable region of the light chain of the monoclonal antibody has an amino acid sequence shown in a
Antibodies comprising conservative sequence variants of the amino acid sequences of preferred antibodies are also included within the scope of the invention. Conservative amino acid sequence variants include modifications in the amino acid sequence that do not significantly alter the binding properties of the monoclonal antibodies of the invention, such as variants resulting from similar amino acid substitutions, amino acid deletions, additions well known in the art.
The monoclonal antibody of the invention also comprises porcine and non-porcine antibodies and all antibodies with the same functions or modification and optimization as the monoclonal antibody.
Further, the antigen-binding fragment of the monoclonal antibody includes Fab, Fab ', F (ab') 2, Fv, or single chain antibody.
Fab refers to the portion of an antibody molecule that contains one light chain variable and constant region and one heavy chain variable and constant region that are disulfide bonded.
Fab' refers to a Fab fragment that contains part of the hinge region.
F (ab ') 2 refers to a dimer of Fab'.
Fv refers to the smallest antibody fragment containing the variable regions of the heavy and light chains of an antibody and having all antigen binding sites.
The single-chain antibody is an engineered antibody formed by directly connecting a light chain variable region and a heavy chain variable region or connecting the light chain variable region and the heavy chain variable region through a peptide chain.
The monoclonal antibodies disclosed herein may comprise one or more glycosylation sites in the heavy and light chain variable regions, as is well known in the art, the presence of one or more glycosylation sites in the variable regions may result in enhanced immunogenicity of the antibody.
Monoclonal antibodies of the invention can be designed to include modifications in the Fc region, typically to alter 1 or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In addition, the antibodies of the invention may be chemically modified (e.g., one or more chemical groups may be attached to the antibody), or modified to alter glycosylation thereof, thereby altering one or more functional properties of the antibody.
The invention also provides a nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof as described above, comprising a nucleotide sequence encoding a heavy chain variable region of a monoclonal antibody, a nucleotide sequence encoding a light chain variable region of a monoclonal antibody, a nucleotide sequence encoding a heavy chain of a monoclonal antibody, or a nucleotide sequence encoding a light chain of a monoclonal antibody.
The nucleic acid molecules of the present invention encoding the aforementioned monoclonal antibodies or antigen-binding fragments thereof include nucleic acid molecules having conservative nucleotide sequence variants of preferred nucleotide sequences. So-called conservative nucleotide sequence variants arise from degenerate and/or silent variants of the genetic code, and substitutions, deletions and insertions of nucleotides are also included.
Specifically, the nucleic acid molecule sequence encoding the heavy chain CDR1 is shown in sequence 11, the nucleic acid molecule sequence encoding the heavy chain CDR2 is shown in sequence 12, the nucleic acid molecule sequence encoding the heavy chain CDR3 is shown in sequence 13, and the nucleic acid molecule sequence encoding the heavy chain variable region is shown in sequence 9; the nucleic acid molecule sequence encoding the light chain CDR1 is shown in SEQ ID No. 14, the nucleic acid molecule sequence encoding the light chain CDR2 is shown in SEQ ID No. 15, the nucleic acid molecule sequence encoding the light chain CDR3 is shown in SEQ ID No. 16, and the nucleic acid molecule sequence encoding the light chain variable region is shown in SEQ ID No. 10.
The present invention also provides a recombinant vector comprising the nucleic acid molecule as described above, and further comprising an expression control sequence operably linked to the sequence of the nucleic acid molecule.
"vector" in the present invention means, in turn, a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell so that the genetic material elements it carries are expressed in the host cell. By way of example, the carrier includes: plasmids, phages, cosmids, artificial chromosomes, phages, animal viruses, and the like. The animal virus species used as vectors are lentivirus, adenovirus, herpesvirus, poxvirus, baculovirus, papilloma virus and the like. A vector may contain a variety of expression control elements including promoter sequences, reverse transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site. The vector may also include components which assist its entry into the cell, such as viral particles, liposomes or protein coats, but not exclusively.
In a specific embodiment of the present invention, the recombinant vector is constructed by inserting the nucleic acid molecule shown above into a pCAGGS vector (Nova life, Singapore).
The present invention also provides a method for constructing the recombinant vector described above, which comprises inserting the nucleic acid molecule described above into a pCAGGS vector.
Specifically, the method for constructing the recombinant vector of the present invention is as follows:
respectively merging signal peptide (coding sequence such as sequence 17), antibody variable region and corresponding constant region sequence published by NCBI, adding termination codon TAA to gene sequence C end, artificially synthesizing complete gene sequence for expressing heavy chain and light chain, respectively cloning to expression vector pCAGGS by using molecular cloning method, and respectively constructing to obtain recombinant expression plasmid.
Specifically, the recombinant expression vector is obtained by inserting a DNA fragment shown in a sequence 20 in a sequence table or a fragment shown in a sequence 21 in the sequence table between EcoRI and BglII enzyme recognition sites of an expression vector pCAGGS, and respectively constructing a recombinant expression plasmid pC-HC for expressing a heavy chain or a recombinant expression vector pC-LC for expressing a light chain.
The present invention also provides a recombinant cell into which the nucleic acid molecule or the recombinant vector as described above has been introduced.
The term "recombinant cell" as used herein refers to a cell into which a nucleic acid molecule or vector has been introduced, and includes a number of cell types, such as prokaryotic cells, e.g., E.coli or Bacillus subtilis, fungal cells, e.g., yeast cells or Aspergillus, insect cells, e.g., S2 Drosophila Sf9, or fibroblast, CHO cells, COS cells, BHK cells, HEK293 cells, etc.
In a specific embodiment of the invention, the recombinant cell is a HEK293 cell.
The present invention also provides a method of producing an antibody or antigen-binding fragment using a recombinant cell as hereinbefore described, the method comprising culturing a recombinant cell as hereinbefore described under suitable conditions and recovering the antibody, the method comprising the steps of: transfecting a host cell or the recombinant cell with a recombinant expression vector, and harvesting cell culture supernatants after transfection for multiple times, and/or culturing the recombinant cell in a target culture medium, harvesting the culture supernatants and purifying to obtain the anti-CSFV E2 monoclonal antibody or the antigen binding part thereof.
The invention also provides an antibody or antigen-binding fragment produced by the above method.
The invention also provides a method for detecting the expression of classical swine fever virus E2 protein for non-diagnostic purposes, which is characterized by comprising the following steps:
1) preparing or extracting a sample containing classical swine fever virus E2 protein;
2) contacting or incubating the sample obtained in step 1) with the specific classical swine fever virus E2 monoclonal antibody or antigen binding fragment thereof as described above;
3) detecting an immune reaction of the sample with the antibody or antigen-binding fragment thereof.
The detection product containing the anti-classical swine fever virus E2 monoclonal antibody is also within the protection scope of the invention. The detection product includes, but is not limited to, a detection reagent, a kit, a chip or a test paper. All products capable of detecting the expression of classical swine fever virus E2 are included in the scope of the present invention.
Specifically, the invention provides a kit for testing the titer of live viruses and/or the efficacy of live vaccines of classical swine fever viruses, which is characterized by comprising: the anti-CSFV E2 monoclonal antibody or FITC marked anti-CSFV E2 monoclonal antibody.
Specifically, the kit for testing the titer and/or the efficacy of the live vaccine of the classical swine fever virus comprises the following 1) or 2):
1) the anti-classical swine fever virus E2 monoclonal antibody and the FITC labeled anti-antibody;
2) FITC labels the anti-CSFV E2 monoclonal antibody and the anti-antibody.
The FITC-labeled anti-antibody is preferably a FITC-labeled goat anti-mouse IgG secondary antibody; the anti-antibody is preferably a goat anti-mouse IgG secondary antibody.
The kit also comprises a cell culture plate, PK-15 cells, cell culture solution and other reagents required by an indirect immunofluorescence method, such as fixing solution, PBS and the like.
The invention also provides a method for testing the efficacy of the live virus and/or live vaccine of the classical swine fever virus by using the kit, which is characterized by comprising the following steps: the anti-CSFV E2 monoclonal antibody is used as a primary antibody, or FITC marked anti-CSFV E2 monoclonal antibody.
The method for testing the efficacy of the live virus and/or the live vaccine of the classical swine fever virus comprises the following steps:
1) digesting and dispersing the full monolayer PK-15 cells by using EDTA-pancreatin, and paving the prepared passage cell suspension on a 96-hole cell culture plate;
2) within 24h, when the cell density in a 96-hole cell culture plate is 80-95%, washing the cells once by using PBS for later use;
3) re-dissolving the freeze-dried classical swine fever live vaccine to be detected or the live classical swine fever virus by using a DMEM medium containing 2% FBS, diluting according to a gradient of 10 times, adding the diluted solution into the cell culture plate obtained in the step 2) at a concentration of 100 mu l/hole respectively, and placing the cell culture plate at a temperature of 37 ℃ and 5% CO2Culturing in an incubator, and simultaneously setting a non-virus-inoculated normal cell control;
4) after 72h of culture, the expression of the viral protein E2 in the cells of step 3) was detected by indirect immunofluorescence
Taking out the cell culture plate from the incubator, discarding the liquid in the culture plate hole, and washing for 2 times by PBS;
② discarding PBS, adsorbing the residual liquid in the cell culture plate with absorbent paper, adding fixing liquid (methanol: acetone in volume ratio of 1:1), and fixing at-20 deg.C for 30 min;
thirdly, removing the fixing liquid, and washing for 2 times by PBS;
adding the anti-classical swine fever virus E2 monoclonal antibody diluted by PBS, and incubating for 90min at 37 ℃;
fifthly, primary antibody is discarded, PBS is used for washing for 2 times, FITC labeled goat anti-mouse IgG secondary antibody diluted by PBS is added, and incubation is carried out for 60min at 37 ℃;
sixthly, discarding the secondary antibody, washing for 2 times by PBS, and observing the fluorescence condition of each hole in the cell culture plate under a fluorescence microscope;
5) using viral titres FAID according to the Indirect immunofluorescence protocol50The formula for the calculation of/0.1 ml is:
LogFAID50the log of the highest dilution above 50% positive porosity + distance ratio x difference in log of dilution;
the difference between the log of dilutions-log of the highest dilution below 50% positive porosity-log of the highest dilution above 50% positive porosity;
distance ratio (percentage of more than 50% positive porosity-50)/(percentage of more than 50% positive porosity-percentage of less than 50% positive porosity);
the invention also provides a blocking ELISA kit for detecting the antibody of the classical swine fever virus, which is characterized by comprising: HRP-labeled anti-CSFV E2 monoclonal antibody or anti-CSFV E2 monoclonal antibody.
Specifically, the blocking ELISA kit for detecting the antibody of the classical swine fever virus comprises: and (3) labeling the swine fever virus E2-resistant monoclonal antibody and the swine fever virus E2 protein by HRP to form an enzyme-linked reaction plate.
Wherein the sequence of the classical swine fever virus E2 protein is an amino acid residue sequence from 41-379 sites of an amino terminal of a sequence 18 in a sequence table; for convenience of expression and purification, the amino acid of the protein is connected with gp67 signal peptide, the C end of the protein is connected with 6 XHis tag, and the gp67 signal peptide sequence is amino acid residue sequence from 1-41 of amino end of sequence 18 in the sequence table.
The kit can also comprise positive control serum and negative control serum, wherein the positive control serum is swine serum collected after immunization of swine fever live vaccine, and the negative control serum is swine serum without Specific Pathogen (SPF).
The kit can also comprise: sample diluent, 20-fold concentrated washing solution, substrate solution A, substrate solution B and stop solution.
The sample dilution contained 5mg/ml casein in 0.01M phosphate buffer (pH 7.4).
The substrate solution A was a citrate phosphate buffer solution containing 0.6mg/ml urea hydrogen peroxide.
The substrate solution B was a Tetramethylbenzidine (TMB) solution of 0.2 mg/ml.
The 20-fold concentrated washing solution is 0.01M phosphate buffer solution with pH value of 7.4 and contains 0.8-1.2% (ml/ml) of Tween-20.
The stop solution is a 2M sulfuric acid solution.
If necessary, a sample dilution plate (2, 96 wells/block) may be included in the kit for sample dilution.
The invention also provides the use of a monoclonal antibody or antigen-binding fragment thereof as hereinbefore described, which comprises any one of:
1) the blocking ELISA kit for preparing the hog cholera virus antibody detection is characterized by comprising the following components: the HRP-labeled anti-classical swine fever virus E2 monoclonal antibody;
2) the application in the preparation of diagnostic preparations of hog cholera virus;
3) application in preparing preparation for treating swine fever is provided.
The swine fever virus resisting E2 monoclonal antibody can specifically and efficiently combine with a swine fever virus E2 protein, does not generate cross reaction with exogenous viruses such as bovine viral diarrhea virus, porcine circovirus type 2, porcine pseudorabies and the like, has good antigen combination activity and certain virus neutralization activity, can be used for detection and diagnosis of swine fever, can be used for prevention and treatment, and has good production and application prospects in development of novel diagnostic reagents and therapeutic drugs of swine fever viruses.
Drawings
FIG. 1 is a graph showing the results of indirect immunofluorescence screening of classical swine fever virus E2 monoclonal antibody;
FIG. 2 is a graph showing the results of comparing the anti-CSFV E2 monoclonal antibody of the present invention with a commercial product using an indirect immunofluorescence assay;
FIG. 3 is a graph showing the results of specific fluorescence reactions of the anti-CSFV E2 monoclonal antibody on different cells;
FIG. 4 is a SDS-PAGE electrophoresis result of affinity chromatography purification of anti-CSFV E2 monoclonal antibody;
FIG. 5 is a graph showing the Western blotting result of classical swine fever virus E2 protein using monoclonal antibody against classical swine fever virus E2;
FIG. 6 is a cross-reaction of the anti-CSFV E2 monoclonal antibody with BVDV, PCV2 and PRV;
FIG. 7 is an indirect immunofluorescence assay showing neutralization of classical swine fever virus vaccine C strain with an anti-classical swine fever virus E2 monoclonal antibody.
FIG. 8 is a schematic view of the enzyme linked immunosorbent assay plate of the kit.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions.