CN114426974B - Antibodies or antibody fragments specifically binding to the CD2v protein of African swine fever virus - Google Patents

Antibodies or antibody fragments specifically binding to the CD2v protein of African swine fever virus Download PDF

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CN114426974B
CN114426974B CN202011186928.5A CN202011186928A CN114426974B CN 114426974 B CN114426974 B CN 114426974B CN 202011186928 A CN202011186928 A CN 202011186928A CN 114426974 B CN114426974 B CN 114426974B
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swine fever
african swine
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田克恭
郝丽影
张超林
邓均华
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Luoyang Pu Tai Biotechnology Co ltd
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Abstract

The invention relates to an African swine fever virus CD2v protein, and a kit and an antibody prepared from the same. The gene sequence of the African swine fever virus CD2v protein provided by the invention comprises a nucleotide sequence shown as SEQ ID No.1 or a nucleotide sequence which is at least 90% homologous with the sequence of SEQ ID No. 1. According to the invention, codon optimization is carried out on the gene sequence of the African swine fever virus CD2v protein according to a preferred codon of a CHO expression system, and then fusion proteins mFc-CD2v and fusion proteins gD-CD2v are prepared through the CHO expression system. ELISA antibody detection kit or monoclonal antibody prepared by fusion protein mFc-CD2v can be used for detecting African swine fever virus antibody, and has the advantages of rapid and sensitive detection and good specificity.

Description

Antibodies or antibody fragments specifically binding to the CD2v protein of African swine fever virus
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a CHO expressed African swine fever virus CD2v protein, an ELISA antibody detection kit prepared by using the protein, a preparation method and application thereof, and a monoclonal antibody prepared by using the protein and application thereof.
Background
African swine fever (African swine fever, ASF) is an acute, febrile, highly contagious infectious disease of pigs caused by African Swine Fever Virus (ASFV), and is clinically characterized by high fever, cyanosis of skin, extensive bleeding of lymph nodes and viscera, short course of disease and high mortality rate of 100%. Clinical symptoms and pathological changes are similar to those of acute swine fever, and are easy to misdiagnose during diagnosis, and high fever, skin congestion, cyanosis, abortion, edema and visceral hemorrhage are manifested. The disease is classified as a type of animal epidemic disease in China, which belongs to the legal report animal epidemic disease of the world animal health Organization (OIE). The disease has been developed in 1921 in various countries such as kenya and spanish in europe, in caucasian and russia, and in 2018 in 8 months in northeast China and rapidly developed in various provinces (regions). The number of live pigs in China is 50% of the world, and ASFV is spread in China, which seriously threatens the world farming industry.
ASFV CD2v is a envelope glycoprotein encoded by the EP402R gene, whose extracellular immunoglobulin-like domain has an amino acid sequence similar to that of the host CD2 protein, and can be expressed in T cells and NK cells; the intracellular portion has no obvious similarity to the host CD2 protein. The protein can make virus-infected cells and extracellular virus particles adsorb erythrocytes, and promote the diffusion and transmission of viruses in a host body. Meanwhile, researchers classify viruses into serotypes based on the division of original ASFV strains into gene types I/II according to HAI characteristics of CD2 v. It follows that research based on CD2v protein is significant. The preparation of the monoclonal antibody can provide an important tool for ASFV detection and structural analysis of CD2v protein thereof.
At present, animal biological product enterprises actively develop African swine fever vaccines (see https:// www.pig66.com/zbfz/feizhouzhuwen/2019/0905/18190547. HtmL), such as attenuated live vaccines prepared by ASFV CD2v and MGF360-505R combined deletion strain developed by Harbin veterinary research institute of China academy of agriculture (see China patent CN 110093324A), attenuated live vaccines prepared by ASFV CD2v and MGF combined deletion strain developed by military medical institute of military science, and development of ASFV CD2v subunit vaccines developed by Qingdao Yibang bioengineering Co (see China patent CN110078801A, CN 110157737A). However, the current african swine fever detection is mainly focused on antigen detection, and the effect evaluation after vaccine immunization of pigs is still blank.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides an African swine fever virus CD2v protein gene sequence, a fusion protein containing the African swine fever virus CD2v protein gene sequence is prepared by utilizing a molecular biological means, and a kit and a monoclonal antibody are prepared by using the fusion protein.
In a first aspect the invention provides a gene sequence of the CD2v protein of african swine fever virus, the gene sequence comprising the nucleotide sequence shown as SEQ ID No.1 or a nucleotide sequence which is at least 90% homologous to the sequence of SEQ ID No. 1.
According to some embodiments of the invention, the gene sequence is codon optimized according to CHO expression system preference codons. The invention adopts the CHO expression system to favor codons to carry out codon optimization to obtain the gene sequence, thereby facilitating the preparation of fusion proteins through the CHO expression system in the later stage.
According to some embodiments of the invention, the gene sequence comprises a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence of SEQ ID No. 1.
In a second aspect the invention provides a fusion protein comprising a gene sequence according to the first aspect.
According to some embodiments of the invention, the fusion protein comprises the fusion protein mFc-CD2v and/or the fusion protein gD-CD2v, the gene sequence of the fusion protein mFc-CD2v comprising the nucleotide sequence shown in SEQ ID No.2, and the gene sequence of the fusion protein gD-CD2v comprising the nucleotide sequence shown in SEQ ID No. 3.
According to some embodiments of the invention, the gene sequence of the fusion protein mFc-CD2v comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to SEQ id No. 2.
According to some embodiments of the invention, the gene sequence of the fusion protein gD-CD2v comprises a nucleotide sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to SEQ id No. 3.
In a third aspect, the present invention provides a method for preparing a fusion protein according to the second aspect, comprising the steps of:
s1: the gene sequences of the first aspect are respectively connected with the gene sequences of the mouse Fc fragment and/or the gene sequences of the porcine pseudorabies virus gD protein through fusion to respectively construct expression plasmids;
S2: respectively transfecting the expression plasmids constructed in the step S1 into CHO cells to express fusion proteins mFc-CD2v and/or fusion proteins gD-CD2v;
s3: harvesting said fusion protein mFc-CD2v and/or said fusion protein gD-CD2v.
According to a fourth aspect of the present invention, there is provided a fusion protein according to the second aspect or a fusion protein obtained by the method of preparation according to the third aspect, which can be used as an immunogen for monoclonal antibodies to the CD2v protein of african swine fever virus, an antigen for screening for a coated antigen of an ELISA antibody detection kit or monoclonal antibodies to the CD2v protein of african swine fever virus.
In a fifth aspect, the invention provides an ELISA antibody detection kit for African swine fever virus, which comprises a support medium coated with the fusion protein mFc-CD2v in the fusion protein according to the second aspect, an enzyme-labeled reagent, a positive control, a negative control and a detection reagent.
According to some embodiments of the invention, the fusion protein mFc-CD2v has a coating concentration of 0.5-0.8 μg/mL.
According to some embodiments of the invention, the coating concentration may be 0.5 μg/mL, 0.51 μg/mL, 0.52 μg/mL, 0.53 μg/mL, 0.54 μg/mL, 0.55 μg/mL, 0.56 μg/mL, 0.57 μg/mL, 0.58 μg/mL, 0.59 μg/mL, 0.6 μg/mL, 0.61 μg/mL, 0.62 μg/mL, 0.63 μg/mL, 0.64 μg/mL, 0.65 μg/mL, 0.66 μg/mL, 0.67 μg/mL, 0.68 μg/mL, 0.69 μg/mL, 0.7 μg/mL, 0.71 μg/mL, 0.72 μg/mL, 0.73 μg/mL, 0.74 μg/mL, 0.75 μg/mL, 0.76 μg/mL, 0.77 μg/mL, 0.78 μg/mL, 0.79 μg/mL.
According to one embodiment of the invention, the coating concentration of the fusion protein mFc-CD2v in the kit is 0.6. Mu.g/mL.
According to some embodiments of the invention, the support medium is selected from a microtiter plate.
According to some embodiments of the invention, the enzyme-labeled reagent is selected from a solution of enzyme-labeled goat anti-pig IgG or enzyme-labeled rabbit anti-pig IgG diluted with an enzyme-labeled diluent.
According to some embodiments of the invention, the enzyme-labeled enzyme is selected from horseradish peroxidase, alkaline phosphatase, or β -D-galacto-glycase.
According to some embodiments of the invention, the positive control is selected from the group consisting of positive serum of the fusion protein mFc-CD2v immunized pig.
According to some embodiments of the invention, the negative control is selected from serum that is negative for african swine fever virus antigen antibodies.
According to some embodiments of the invention, the detection reagent comprises a chromogenic liquid.
According to some embodiments of the invention, the color developing solution comprises color developing solution a comprising 1.47% w/v disodium hydrogen phosphate, 0.93% w/v citric acid, and 0.03% w/v carbamide peroxide, and color developing solution B comprising 0.02% w/v tetramethylbiphenyl diamine and 10% v/v absolute ethanol.
According to some embodiments of the invention, the stop solution is 2M H 2 SO 4 A solution.
According to some embodiments of the invention, the kit further comprises a wash solution and/or a sample diluent.
According to some embodiments of the invention, the washing solution is a phosphate buffer.
According to some embodiments of the invention, the sample diluent comprises a PBS solution of 20% V/V neonatal bovine serum and 0.01% V/VProclin 300.
The kit disclosed by the invention can be used for detecting the African swine fever virus antibody, and has the advantages of rapid and sensitive detection and good specificity.
In a sixth aspect, the invention provides an antibody or antibody fragment which specifically binds to the CD2v protein of african swine fever virus, wherein the antibody or antibody fragment comprises the heavy chain variable region of the amino acid sequence shown in seq id No.4 or a conservative variant thereof and the light chain variable region of the amino acid sequence shown in seq id No.6 or a conservative variant thereof.
According to the invention, the conservative variants may be obtained by adding, deleting, replacing or modifying conservative mutations of one or more amino acids.
According to some embodiments of the invention, the amino acid sequence of the heavy chain variable region is encoded by the base sequence shown in SEQ ID No.5 or a degenerate sequence thereof.
According to some embodiments of the invention, the amino acid sequence of the light chain variable region is encoded by the base sequence shown in SEQ ID No.7 or a degenerate sequence thereof.
According to some embodiments of the invention, the antibody is a monoclonal antibody or a genetically engineered antibody.
According to some embodiments of the invention, the genetically engineered antibody comprises one or more selected from the group consisting of a single chain antibody or fragment thereof, a chimeric monoclonal antibody or fragment thereof, a reshaped monoclonal antibody or fragment thereof.
According to the invention, the antibody or fragment thereof retains the ability to specifically bind to african swine fever virus.
According to some embodiments of the invention, the antibodies or antibody fragments of the invention are prepared from fusion protein mFc-CD2v as immunogen and fusion protein gD-CD2v as screening antigen, which is composed of the basic units of Ig monomers comprising four heterologous polypeptide chains, wherein the two heavy chains with the larger molecular weight are light chains, the two light chains with the smaller molecular weight are identical in amino acid composition between the two heavy chains in the same Ig molecule, and the two light chains in the same Ig molecule are identical in amino acid composition, wherein each heavy chain has a heavy chain variable region on the N-terminus, the amino acid sequence of which is shown in SEQ ID No.4, and each light chain variable region on the N-terminus, the amino acid sequence of which is shown in SEQ ID No. 5.
The heavy chain variable region and the light chain variable region of the antibody or the antibody fragment have good binding capacity to African swine fever virus, and recognize CD2v protein of the African swine fever virus, and the ELISA titer is 1: 1280000.
According to some embodiments of the invention, the antibody is IgA, igD, igE, igG or IgM.
As a preferred embodiment of the present invention, the antibody or fragment thereof is monoclonal antibody 2D1, the heavy chain variable region of monoclonal antibody 2D1 is shown as SEQ ID No.4, and the light chain variable region is shown as SEQ ID No. 5. The antigen epitope recognized by the monoclonal antibody 2D1 is positioned on the CD2v protein of the African swine fever virus.
According to some embodiments of the invention, the ELISA titer of the monoclonal antibody 2D1 to the African swine fever virus CD2v is 1: 1280000, and the monoclonal antibody is positive in reaction with the African swine fever virus, can be used for immunohistochemical detection of a plurality of tissues with high titer, and shows that the monoclonal antibody has good reaction characteristics with the African swine fever virus.
According to some embodiments of the invention, the method for preparing an antibody or antibody fragment comprises preparing the fusion protein mFc-CD2v of the fusion protein according to the second aspect as an immunogen and the fusion protein gD-CD2v as a screening antigen.
In a seventh aspect the invention provides a hybridoma cell producing an antibody or antibody fragment according to the sixth aspect.
According to some embodiments of the invention, the hybridoma cells are prepared from the fusion protein mFc-CD2v as an immunogen and the fusion protein gD-CD2v as a screening antigen.
According to some embodiments of the invention, the hybridoma cell line 2D1 secretes the monoclonal antibody 2D1.
In an eighth aspect, the invention provides the use of a kit according to the fifth aspect or an antibody or antibody fragment according to the sixth aspect in the detection of an african swine fever virus antibody.
The beneficial technical effects of the invention are as follows:
according to the invention, codon optimization is carried out on the gene sequence of the African swine fever virus CD2v protein according to a preferred codon of a CHO expression system, and then fusion proteins mFc-CD2v and fusion proteins gD-CD2v are prepared through the CHO expression system.
The ELISA antibody detection kit for the African swine fever virus CD2v protein prepared by using the fusion protein mFc-CD2v as a coating antigen can be used for detecting the African swine fever virus antibody, and has the advantages of rapid and sensitive detection and good specificity.
The monoclonal antibody 2D1 prepared by taking fusion protein mFc-CD2v as an immunogen and fusion protein gD-CD2v as a screening antigen has the identified epitope located on the African swine fever virus CD2v protein, has ELISA titer of 1: 1280000 on the African swine fever virus CD2v, is positive in reaction with the African swine fever virus, can be used for immunohistochemical detection of a plurality of tissues with high titer, and shows that the monoclonal antibody has good reaction characteristics with the African swine fever virus.
Detailed Description
Hereinafter, embodiments of the present invention will be described.
The term African swine fever virus (African swine fever virus, ASFV) is a large virus of nucleoplasmic double-stranded DNA, which is in an icosahedral structure and is coated with a capsule, the genome sizes of different strains are slightly different, the length is between 170 and 190kb, the virus comprises 1 middle conservation region and 2 variable regions distributed at two ends, 150 main open reading frames are provided, and about 200 proteins are encoded, wherein a plurality of proteins related to virus replication, immune escape, virus transmission and the like are provided.
The term "African swine fever virus CD2v protein" is abbreviated as "ASFV CD2v", also called ASFV EP402R, gene full length 1083bp, encoded protein size 41kDa, and designated CD2v because the amino acid sequence of the extracellular region immunoglobulin-like domain of encoded protein is very similar to that of the CD2 of the host cell; is a envelope glycoprotein coded by EP402R gene, can help ASFV infected cells and extracellular virus particles adsorb erythrocytes, promote the diffusion and transmission of viruses in a host body, and can inhibit lymphocyte proliferation caused by mitogen stimulation so as to inhibit lymphocyte function.
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 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 will be apparent to those 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 antibodies specifically disclosed herein, one or more amino acid additions, deletions, substitutions, etc. may be modified by conventional genetic engineering and protein engineering methods to obtain conservative variants, while still maintaining specific binding to feline panleukopenia 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 advantages and features of the present invention will become more apparent from the following description of the embodiments. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
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.
In order that the invention may be more readily understood, the invention will be further described with reference to the following examples. It should be understood that these examples are only for the purpose of the present invention and are not intended to limit the scope of the present invention. The experimental methods provided by the invention are conventional methods unless specified; the biological material, unless otherwise specified, is commercially available.
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 expression of fusion protein MFc-CD2v primer sequences for plasmid construction
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.
And carrying out fusion PCR by taking the CD2v fragment and the mFc fragment as templates to obtain the MFc-CD2v fragment. Reaction system 50 μl: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 fusion protein MFc-CD2v fragment 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-MFc-CD2v. And the bacterial liquid of the plasmid is largely shaken, and then the plasmid is extracted by Endo-free PlasmidMidi 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.
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.
gD-CD2v fragment was obtained by fusion PCR using CD2v fragment and gD fragment as templates. Reaction system 50 μl:HSDNA Polymerase 0.5. Mu.L, 5 XPrimeSTARbuffer 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.
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 MFc-CD2v 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 L ExpiFectamineTM 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 Westernblotting identification of fusion proteins MFc-CD2v and fusion proteins 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, performing Westernblotting detection on the fusion protein MFc-CD2V and the fusion protein gD-CD2V by using a PRVGD monoclonal antibody and a goat anti-mouse antibody. Results: the purified fusion proteins MFc-CD2V and the fusion proteins gD-CD2V have protein molecular masses of about 113kDa and 92kDa, respectively, and the protein sizes are both 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 MFc-CD2V and the fusion proteins gD-CD2V were 1.2mg/mL and 2.3mg/mL, respectively.
Example 2 preparation and application of ASFV CD2v ELISA antibody detection kit
2.1 preparation of the kit
Antigen coated plate: the fusion protein mFc-CD2v prepared in example 1 was diluted to 0.5-0.8. Mu.g/mL with carbonate buffer (pH 9.6,0.05 mol/L), coated, 100. Mu.L/well, left to stand for 16-24 hours at 2-8℃and washed with washing solution, and then a blocking solution (weighing 50g of sucrose, adding 200mL of neonatal bovine serum, 0.5mL of Proclin300, adding PBS (0.01 mol/L, pH 7.4) to a constant volume of 1000 mL), blocking for 16-24 hours at 2-8℃and then drying, sealing and storing at 2-8℃for later use.
Enzyme-labeled reagent: the commercial enzyme-labeled goat anti-pig IgG or enzyme-labeled rabbit anti-pig is diluted 50000 times by enzyme-labeled diluent (200 mL of new born calf serum, 0.5mL Proclin300, 0.5mL of Tween 20 and 0.04g of AM dye are measured, PBS (0.01 mol/L, pH value is 7.4) is added for volume fixation to 1000 mL) to be used as an enzyme-labeled reagent, and the reagent is stored at 2-8 ℃.
Positive control: taking 10mL of positive serum of a fusion protein mFc-CD2v immunized pig, 200mL of new born calf serum and 0.5mL Proclin300, adding PBS (0.01 mol/L, pH value of 7.4) to 1000mL, uniformly mixing, filtering by a 0.22 mu m filter, and carrying out aseptic quantitative split charging to serve as a positive control, and preserving at 2-8 ℃.
Negative control: 200mL of new born calf serum and 0.5mL of Proclin300 are measured, PBS buffer solution (0.01 mol/L, pH value is 7.4) is added to 1000mL, the mixture is uniformly mixed, a 0.22 mu m filter is used for filtration, and sterile quantitative split charging is used as negative control, and the mixture is stored at the temperature of 2-8 ℃.
Sample dilution: 8g of sodium chloride, 2.9g of disodium hydrogen phosphate, 0.24g of potassium dihydrogen phosphate, 0.2g of potassium chloride, 600mL of purified water, 3001mL of Proclin and 200mL of newborn calf serum are taken, after complete dissolution, the volume is fixed to 1000mL by the purified water, after uniform mixing, 0.22 mu m filtration, sterile split charging and 2-8 ℃ storage are carried out.
20 x concentrated wash: 160g of sodium chloride, 58g of disodium hydrogen phosphate, 4.8g of potassium dihydrogen phosphate, 4g of potassium chloride, 800mL of ultrapure water and 10mL of tween 20 are taken, after complete dissolution, the volume is fixed to 1000mL by using the ultrapure water, and the filtration is carried out by using a 0.22 mu m filter membrane, and the sterile split charging is carried out. The product is diluted 20 times with distilled water.
Color development liquid: 14.7g of disodium hydrogen phosphate, 9.3g of citric acid and 0.3g of carbamide peroxide are taken and dissolved in purified water to reach 1000mL of constant volume, and the color development liquid A is obtained by aseptic split charging after uniform mixing and filtration. 0.2g of tetramethyl biphenyl diamine (TMB) and 10mL of absolute ethyl alcohol are taken and dissolved in purified water to reach a constant volume of 1000mL, and the color development liquid B is obtained by mixing, filtering and sterile split charging.
Stop solution: 2M H 2 SO 4 A solution.
The components are assembled into a kit, and the kit A (coating concentration 0.5 mug/mL), the kit B (coating concentration 0.6 mug/mL), the kit C (coating concentration 0.7 mug/mL) and the kit D (coating concentration 0.8 mug/mL) are marked in sequence according to the size of the coating concentration.
2.2 establishment of the detection method
The detection steps are as follows:
(1) Sample adding: the sample to be detected is diluted by 100 times by the sample diluent respectively, 100 mu L of the diluted sample is added into the corresponding hole respectively, meanwhile, a positive control hole 2, a negative control hole 2 and a blank control hole 1 are arranged (blank control can not be arranged in dual-wavelength detection), and the plate is put at 37 ℃ for 30 minutes for incubation.
(2) Washing: washing with washing liquid for 5 times, and finally buckling.
(3) And (3) adding an enzyme-labeled reagent: except for blank control wells, 100 μl of enzyme-labeled reagent diluent was added to each well, and the plates were incubated at 37deg.C for 30 minutes.
(4) Washing: washing with washing liquid for 5 times, and finally buckling.
(5) Color development: 50 μl of each of the color development liquid A and the color development liquid B is added into each hole, and the mixture is uniformly mixed and developed for 15 minutes at 37 ℃ in a dark place.
(6) Terminating and detecting: each well was subjected to 50. Mu.l of stop solution and OD450nm values were measured by an enzyme-labeled instrument within 10 minutes, and the measurement was carried out according to the following results.
When the dual wavelength is measured, the dual wavelength is set to 450nm/600-650nm, and the value A of each hole is measured. In single wavelength measurement, the wavelength of the enzyme label instrument is set at 450nm, and the A value of each well is measured after the blank control well is zeroed. The test is established when the A value of the negative control hole is less than or equal to 0.1 and the A value of the positive control hole is more than or equal to 0.3, otherwise, the test is invalid. Calculation method, S/P value = sample a value/positive control a value mean. And (3) result judgment:
The S/P value is more than or equal to 0.5, the ASFV CD2v protein antibody is positive,
the S/P value < 0.5 is negative for ASFV CD2v protein antibody.
2.3 evaluation of the kit
2.3.1 specificity
7 parts of porcine common virus positive serum (comprising porcine pseudorabies virus positive serum, porcine reproductive and respiratory syndrome virus positive serum, swine fever virus positive serum, porcine circovirus type 2 positive serum, porcine epidemic diarrhea virus positive serum, porcine transmissible gastroenteritis virus 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-kit D prepared in example 2.1, and the result is that: the S/P values are all less than 0.5, and are all negative, which indicates that the specificity of the kit A-kit D is good.
2.3.2 sensitivity
Positive serum-fold dilution of ASFV CD2v protein immunized pigs was tested using kit a-kit D prepared in example 2.1, as follows: the kit A and the kit C and the kit D detect positive serum 1:125 times of diluent of ASFV CD2v protein immune pigs, and detect 1:250 times of diluent as negative; only the kit B detects that the S/P value of the 1:250-fold dilution of positive serum of ASFV CD2v protein immunized pigs is more than or equal to 0.5, and still is positive.
Positive serum from ASFV CD2v protein immunized pigs was tested for 20 parts using kit B prepared in example 2.1, results: the S/P values are all more than or equal to 0.5, and are positive.
2.3.3 clinical application
According to the above results, the kit B prepared in example 2.1 was used for clinical application to detect 20 parts of ASFV positive porcine serum and 100 parts of negative porcine serum collected before 2018, resulting in: the S/P values of 20 ASFV positive pig serum detected are all more than or equal to 0.5, and are positive; the S/P value of 100 parts of pig serum collected before 2018 is less than 0.5, and the pig serum is negative.
EXAMPLE 3ASFV CD2v protein monoclonal antibody preparation and identification
3.1 preparation of monoclonal antibodies
5 female BALB/c mice of 4-6 weeks old were immunized with 100 μg/mouse (400 μl volume) of the fusion protein mFc-CD2v 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 ELISA.
Wherein, kit B was an indirect ELISA method constructed with fusion protein gD-CD2v as coating antigen, prepared as in example 2.1, with a coating amount of 0.5. Mu.g/mL.
The detection method comprises the following steps: 1) Sample adding: the serum of the mice to be detected is diluted by PBS and then subjected to multiple dilution, 100 mu L of the diluted serum is added into the corresponding holes respectively, PBS is used as negative control, and the plates are sealed and incubated for 60 minutes at 37 ℃.
2) Washing: washing with washing liquid for 5 times, and finally buckling.
3) Adding a secondary antibody: 100 μl of HRP-goat anti-mouse IgG 1:10000 dilution was added to each well, and incubated at 37deg.C for 45 minutes after sealing.
4) Washing: washing with washing liquid for 5 times, and finally buckling.
5) Color development: 50 μl of each of the color development liquid A and the color development liquid B is added into each hole, and the mixture is uniformly mixed and developed for 15 minutes at 37 ℃ in a dark place.
6) Terminating and detecting: each well was subjected to 50. Mu.l of stop solution and OD450nm values were measured by an enzyme-labeled instrument within 10 minutes, and the measurement was carried out according to the following results.
ELISA antibody titers of sera of 5 mice were determined with kit B, as a result: only 1 mouse (3#) had a serum titer as high as 1: 256000. Mice # 3 were immunized with the fusion protein gD-CD2v by intraperitoneal injection and cell fusion was performed 3 days after the immunization. The fused cells are subjected to multiple subcloning screening, positive hybridoma cells are finally obtained, 5 strains (1A 1, 2D1, 3E5, 4F1 and 5C 2) are preferably selected after cell supernatant is evaluated, ascites is prepared by a mouse ascites method, ELISA antibody titers of monoclonal antibodies are measured by using a kit B, and the results (see Table 3) are shown as follows: the yields of the monoclonal antibodies 1A1 and 3E5 ascites are not ideal and are discarded; the monoclonal antibody 4F1 ELISA antibody has lower titer and is discarded; the monoclonal antibodies 2D1 and 5C2 have ideal results for subsequent evaluation.
Table 3 5 summary of evaluation information of ASFV CD2v monoclonal antibodies
Name of the name The ascites is generated ELISA antibody titers Application case
1A1 No ascites Unmeasured test Discarding
2D1 The yield of ascites is 15 mL/unit, and the yield is higher 1︰1280000 Reservation of
3E5 The ascites collection amount is only 1 mL/unit, and the yield is low 1︰80000 Discarding
4F1 The ascites yield is 6 mL/unit, and the yield is moderate 1︰40000 Discarding
5C2 The yield of ascites is 8 mL/unit, and the yield is moderate 1︰320000 Reservation of
3.2 evaluation of reactivity of monoclonal antibodies 2D1, 5C2 with ASFV
Monoclonal antibodies 2D1, 5C2 were tested by IFA method and were purchased from European African swine fever reference laboratory (Centro de)en Sanidad Animal (CISA-INIA), madrid, spain) African swine fever virus antigen plate is warmed, washed 1 time with PBS, diluted solutions of monoclonal antibodies 2D1 and 5C2 are respectively added, negative control with PBS is arranged, and the mixture is placed in a 37 ℃ incubator to act for 1 hour; washing 3 times with PBS, adding FITC-labeled rabbit anti-mouse IgG secondary antibody diluent, and reacting at 37 ℃ for 50 minutes; after washing 3 times with PBS, the cells were observed under a fluorescence microscope. Results: obvious yellow-green fluorescence is visible in the cell wells reacted by the monoclonal antibody 2D1, no yellow-green fluorescence is visible in the cell wells reacted by the monoclonal antibody 5C2, and no yellow-green fluorescence is visible in the negative control wells, which indicates that only the monoclonal antibody 2D1 can react with African swine fever virus. Monoclonal antibody 2D1 was used for subsequent identification.
3.3 identification of monoclonal antibody 2D1
3.3.1 subclass identification
The subclass of monoclonal antibody 2D1 was identified using the monoclonal antibody subclass identification kit, resulting in: the heavy chain subclass is IgG2a and the light chain subclass is kappa.
3.3.2Westernblot identification
SDS-PAGE electrophoresis is carried out on the gD protein expressed by CHO and the fusion protein gD-CD2v, and after transfer, westernblot detection is carried out by taking monoclonal antibody 2D1 diluent as primary antibody and goat anti-mouse IgG diluent marked by HRP as secondary antibody. Results: monoclonal antibodies can only react with the fusion protein gD-CD2v to generate specific bands. The monoclonal antibody 2D1 was shown to recognize only african swine fever virus CD2v protein.
3.3.3 specific identification
Swine fever virus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine circovirus type 2 and porcine parvovirus are respectively prepared into IFA antigen plates, the IFA antigen plates are fixed by 80% cold acetone, the temperature is returned, PBS is used for washing for 1 time, then monoclonal antibody 2D1 is added, and IFA detection is carried out according to the example 3.2, and the result is that: no yellow-green fluorescence is seen in the cell holes of the monoclonal antibody 2D1 reaction, which indicates that the monoclonal antibody 2D1 does not react with other viruses of porcine origin, and the specificity is good.
3.3.4 immunohistochemical detection
Tissue samples of tonsils, lungs, lymph nodes, spleen, kidneys, livers and the like of positive pigs infected by ASFV are collected and sampled by PCR, and are rapidly placed in formalin for fixation, and paraffin sections are prepared according to a conventional method. After fixing, washing with flowing water, dewatering, transparentizing, immersing in wax, embedding, slicing, spreading and mounting on treated slide, baking. Dewaxing the slide to distilled water, and dripping 3%H 2 O 2 Standing to inhibit endogenous enzymes. The baked slide is treated with antigen thermal remediation such as autoclaving, boiling thermal remediation, microwave thermal remediation, or enzymatic digestion to repair the antigen. Washing with phosphate buffer for 3 times, and dripping blocking solution such as horse serum or bovine serum albumin BSA for blocking. After removing the blocking solution, diluted primary antibody (monoclonal antibody 2D1 dilution, comparison with ASFV positive serum) was added, incubated at room temperature for 1 hr or at 37℃for 45-60Incubation overnight at min or 2-8deg.C; washing 3 times with phosphate buffer solution, adding HPR marked goat anti-mouse IgG as enzyme-labeled secondary antibody (ASFV positive serum uses HRP marked goat anti-pig IgG as enzyme-labeled secondary antibody), and incubating at room temperature for 1 hr or 37 deg.C for 45-60 min. Washing 3 times with phosphate buffer solution, adding AEC or DAB color development solution for color development, and stopping with phosphate buffer solution or distilled water according to the dyeing condition; hematoxylin is added to line the cell nucleus for counterstaining, dehydration, transparency, sealing and microscopic examination are carried out if necessary. Result determination criteria: the negative control background is clear, the background is not subjected to nonspecific staining, cells of the positive control tissue are stained in red, and the test is established; the tissue cells to be detected are stained in red, namely ASFV antigen positive, otherwise negative. Results (see table 4): compared with ASFV positive serum, the monoclonal antibody 2D1 can be used for immunohistochemical detection of a plurality of tissues with high titer, and the background is clean, and particularly, the monoclonal antibody 2D1 can be diluted by 1:800-1:1600 and can also be used for detection of a plurality of tissues.
TABLE 4 immunohistochemical application comparison results
3.3.5 determination of variable region sequences
According to the sequence characteristics of the murine monoclonal antibody, the heavy chain variable region primer sequence is designed:
F：5’-GGGAATTCATGRAATGSASCTGG-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 2D1 are shown as SEQ ID No.4 and SEQ ID No.6 respectively, and the gene sequences are shown as SEQ ID No.5 and SEQ ID No.7 respectively.
Example 4 preparation and identification of Single chain antibody 2D1
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 2D1 was prepared as described in example 3 using the variable region sequence of monoclonal antibody 2D1. ELISA antibody titers were determined for single chain antibody 2D1 as described in example 2, resulting in: ELISA antibody titer of single-chain antibody 2D1 to ASFVCD2v protein is more than 1:80000, which shows that single-chain antibody 2D1 and CD2v protein have good reaction characteristics. IFA assays were performed using ASFV antigen plates from african swine fever reference laboratory (Centro de Investigaci pulp n en Sanidad Animal (CISA-INIA), madrids, spain) and were positive, indicating that single chain antibodies recognized ASFV.
The results show that the variable region sequences shown in 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 genetically engineered antibody.
It should be noted that the above description is only a preferred embodiment of the present invention, and is not intended to limit the invention, but rather the above description is intended to limit the invention to the particular embodiment disclosed, and any and all simple modifications, equivalent changes and modifications made to the above embodiments according to the technical principles of the present invention will fall within the scope of the technical proposal of the present invention, as long as the equivalent embodiments can be changed or modified to equivalent changes without departing from the scope of the technical proposal of the present invention.
<110> Luoyang Putai Biotechnology Co., ltd
<120> African swine fever virus CD2v protein, kit and antibody prepared from the same
<160> 7
<170> PatentIn version3.5
<210>1
<211>573
<212>DNA
<213> Gene sequence of African swine fever Virus CD2v protein (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> nucleotide sequence of fusion protein CD2v-Fc (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> nucleotide sequence of fusion protein gD-CD2v
<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> amino acid sequence of heavy chain variable region (Artificial sequence)
<400>4
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Ile Ser Cys Gln Thr Ser Gly Tyr Thr Phe Thr Glu His
20 25 30
Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser His Glu Trp Ile
35 40 45
Gly Val Ile Asp Pro Asp Ser Gly Gly Ile 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 Asp Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Tyr Ala Lys Gly Val Ala Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Ser Val Thr Val Ser Ser
115
<210>5
<211>354
<212>DNA
<213> nucleotide sequence of heavy chain variable region (Artificial sequence)
<400>5
gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctgggtcttc agtgaagata 60
tcctgccaga cttctggata cacatttact gaacacacca tgcactgggt gaagcagagc 120
catggaaaga gccatgagtg gattggagtt attgatcctg acagtggtgg tataacctat 180
aaccagaaat tcaagggcaa ggccacattg actgtagaca agtcctccag cacagcctac 240
atggacttcc gcagcctgac atctgaggat tctgcagtct attattgttc aagatatgct 300
aaaggggttg ctatggacta ctggggtcaa ggaacctcag tcaccgtctc ctca 354
<210>6
<211>112
<212>PRT
<213> amino acid sequence of light chain variable region (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 Ser 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 Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95
Thr Leu Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210>7
<211>336
<212>DNA
<213> nucleotide sequence of light chain variable region (Artificial sequence)
<400>7
gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60
atctcttgca gatctagtca gagccttgta cacaggaatg gaaacagtta tttacattgg 120
tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtctc caaccgattt 180
tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240
agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac actttttccg 300
tggacgttcg gtggaggcac caagctggaa atcaaa 336

Claims (4)

1. An antibody or antibody fragment that specifically binds to the CD2v protein of african swine fever virus, wherein the antibody or antibody fragment comprises a heavy chain variable region of the amino acid sequence shown in seq id No.4 and a light chain variable region of the amino acid sequence shown in seq id No. 6.
2. The antibody or antibody fragment of claim 1, wherein the amino acid sequence of the heavy chain variable region is encoded by the base sequence shown in SEQ ID No.5 or a degenerate sequence thereof; and/or the amino acid sequence of the light chain variable region is encoded by the base sequence shown in SEQ ID No.7 or a degenerate sequence thereof.
3. The antibody or antibody fragment according to claim 1 or 2, wherein the antibody is a monoclonal antibody or a genetically engineered antibody.
4. The antibody or antibody fragment according to claim 3, wherein the genetically engineered antibody comprises one or more selected from the group consisting of a single chain antibody or fragment thereof, a chimeric monoclonal antibody or fragment thereof.
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CN115073591B (en) * 2022-06-30 2023-01-20 中国人民解放军军事科学院军事医学研究院 Monoclonal antibody capable of identifying ASFV outer membrane glycosylation modified protein and application thereof
CN115232206B (en) * 2022-06-30 2023-04-11 中国人民解放军军事科学院军事医学研究院 anti-CD 2v protein monoclonal antibody and application thereof
CN116643050B (en) * 2023-04-17 2024-05-24 新希望六和股份有限公司 IgA antibody ELISA detection kit for resisting porcine group A rotavirus and detection method

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102741279A (en) * 2009-08-31 2012-10-17 艾普利穆恩公司 B7-h4 fusion proteins and methods of use thereof
CN105658664A (en) * 2013-07-31 2016-06-08 美国安进公司 Stabilization of Fc-containing polypeptides
CN110078801A (en) * 2019-05-22 2019-08-02 青岛易邦生物工程有限公司 A kind of Chinese hamster ovary celI strain of high efficient expression African swine fever CD2V albumen
CN110759973A (en) * 2019-10-30 2020-02-07 广州伯尼兹生物科技有限公司 Cell strain for expressing African swine fever virus CD2v protein and application thereof
CN111393531A (en) * 2019-01-03 2020-07-10 浙江海隆生物科技有限公司 Subunit fusion protein CD2V-Fc and preparation method and application thereof
CN111471089A (en) * 2019-01-24 2020-07-31 浙江海隆生物科技有限公司 Recombinant African swine fever virus CD2V subunit protein and preparation method and application thereof
CN111518174A (en) * 2020-05-12 2020-08-11 浙江鼎持生物制品有限公司 Optimized African swine fever CD2v protein and high-efficiency expression method and application thereof
CN111793128A (en) * 2020-07-30 2020-10-20 北京世纪元亨动物防疫技术有限公司 Hybridoma cell line and monoclonal antibody for resisting African swine fever virus CD2v protein

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102741279A (en) * 2009-08-31 2012-10-17 艾普利穆恩公司 B7-h4 fusion proteins and methods of use thereof
CN105658664A (en) * 2013-07-31 2016-06-08 美国安进公司 Stabilization of Fc-containing polypeptides
CN111393531A (en) * 2019-01-03 2020-07-10 浙江海隆生物科技有限公司 Subunit fusion protein CD2V-Fc and preparation method and application thereof
CN111471089A (en) * 2019-01-24 2020-07-31 浙江海隆生物科技有限公司 Recombinant African swine fever virus CD2V subunit protein and preparation method and application thereof
CN110078801A (en) * 2019-05-22 2019-08-02 青岛易邦生物工程有限公司 A kind of Chinese hamster ovary celI strain of high efficient expression African swine fever CD2V albumen
CN110759973A (en) * 2019-10-30 2020-02-07 广州伯尼兹生物科技有限公司 Cell strain for expressing African swine fever virus CD2v protein and application thereof
CN111518174A (en) * 2020-05-12 2020-08-11 浙江鼎持生物制品有限公司 Optimized African swine fever CD2v protein and high-efficiency expression method and application thereof
CN111793128A (en) * 2020-07-30 2020-10-20 北京世纪元亨动物防疫技术有限公司 Hybridoma cell line and monoclonal antibody for resisting African swine fever virus CD2v protein

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