Antibody or antigen binding fragment specifically binding to avian leukosis virus p27 protein and application thereof

Abstract

The invention relates to the technical field of antibodies, in particular to an antibody or an antigen binding fragment specifically binding to avian leukosis virus p27 protein and application of the antibody or the antigen binding fragment. The antibody or the antigen binding fragment contains a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown as SEQ.ID NO.1 or SEQ.ID NO.5, and the amino acid sequence of the light chain variable region is shown as SEQ.ID NO.3 or SEQ.ID NO.7. The antibody or the antigen binding fragment is positive in reaction with the avian leukosis virus, so that the antibody or the antigen binding fragment has good reaction characteristics with the avian leukosis virus.

Description

Technical Field

[0001] The present application relates to the field of antibody technology, and in particular to an antibody or antigen-binding fragment specifically binding to avian leukosis virus p27 protein and application thereof. Background Art

[0002] Avian leukosis virus (ALV) is a member of the Retroviridae family, which can cause leukosis and various tumor diseases in birds. There are two main transmission routes for exogenous ALV: vertical transmission, which is transmitted from parents to the next generation, and horizontal transmission, which is transmitted between individuals through the environment. Most birds are infected through direct contact with congenitally infected birds, and then spread to their offspring. Often, the offspring chicks of hens with viremia are also infected. Vertical transmission determines the continuity and persistence of infection, while horizontal transmission ensures that vertical transmission is maintained, making it difficult to completely eradicate the disease. Moreover, it can also cause immunosuppression in chickens, secondary infection with other bacteria and viruses, and cause serious economic losses. The homology of the gag gene sequence among the subgroups of exogenous ALV (A, B, C, D, J) is as high as 90%, and it mainly encodes viral nucleocapsid protein. The p27 protein encoded by it is a group-specific antigen of avian leukosis virus, a major component of nucleocapsid protein, highly conservative, accounting for more than 30% of the total protein, and is suitable for establishing clinical diagnostic methods for avian leukosis.

[0003] Therefore, the preparation of its monoclonal antibody can provide an important tool for ALV detection.

[0004] In view of this, this application is hereby filed. Summary of the invention

[0005] The purpose of the present application is to provide an antibody or antigen binding fragment that specifically binds to the p27 protein of avian leukosis virus, and the prepared ELISA antigen detection kit, so as to provide an effective means for the detection of avian leukosis virus.

[0006] In order to achieve the above purpose, this application adopts the following technical solutions:

[0007] An antibody or antigen-binding fragment that specifically binds to avian leukosis virus p27 protein, the antibody or antigen-binding fragment contains a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ.ID NO.1 or SEQ.ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.3 or SEQ.ID NO.7.

[0008] The N-termini of both the heavy and light chains of antibodies contain variable regions with greatly varying amino acid compositions and arrangements, which contain antigen binding sites that can complementarily bind to antigen epitopes. These sites determine the specificity of the antibody binding to the corresponding antigen epitope and are responsible for identifying and binding to antigens, thereby exerting an immune effect.

[0009]

[0010]

[0011]

[0012]

[0013] In some embodiments, the antibody or antigen binding fragment is a Fab, Fab', F(ab')2, scFv or Fv fragment. In some embodiments, the antibody or antigen binding fragment is an IgG1, IgG2, IgG3 or IgG4 antibody or a fragment thereof. In some embodiments, the antibody or antigen binding fragment is a monoclonal antibody or an antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment is a single chain antibody.

[0014] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment is shown in SEQ.ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.3. Further, the antibody is a monoclonal antibody 1G5, the amino acid sequence of the heavy chain variable region is shown in SEQ.ID NO.1, the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.3, the heavy chain subclass is IgG1, and the light chain subclass is kappa. Further, the antibody is a single-chain antibody 1G5, the amino acid sequence of the heavy chain variable region is shown in SEQ.ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.3.

[0015] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment is shown in SEQ.ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.7. Further, the antibody is a monoclonal antibody 2C4, the amino acid sequence of the heavy chain variable region is shown in SEQ.ID NO.5, the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.7, the heavy chain subclass is IgG1, and the light chain subclass is kappa. Further, the antibody is a single-chain antibody 2C4, the amino acid sequence of the heavy chain variable region is shown in SEQ.ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.7.

[0016] Antibodies are immunoglobulins (Igs) used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. Immunoglobulins are mainly divided into five types: IgA, IgD, IgE, IgG and IgM. IgG antibodies have different subtypes: IgG1, IgG2a, IgG2b, IgG3, IgG4, and the position and number of disulfide bonds between different subtypes are different. Immunoglobulin (Ig) light chains are divided into two subtypes: κ (kappa) and λ (lambda). After experimental detection and identification, the monoclonal antibodies 1G5 and 2C4 protected in this application are both IgG1 in heavy chain subclass and kappa in light chain subclass.

[0017] The present application provides a biological material related to the antibody or antigen-binding fragment, wherein the biological material is any of the following:

[0018] (a) A nucleic acid molecule comprising a sequence encoding the heavy chain variable region and / or light chain variable region of the antibody or antigen-binding fragment.

[0019] (b) An expression cassette comprising the nucleic acid molecule described in (a).

[0020] (c) A recombinant vector comprising the nucleic acid molecule described in (a) or the expression cassette described in (b).

[0021] (d) A recombinant eukaryotic cell containing the nucleic acid molecule in (a), the expression cassette in (b) or the recombinant vector in (c).

[0022] (e) A recombinant prokaryotic cell containing the nucleic acid molecule in (a), the expression cassette in (b) or the recombinant vector in (c).

[0023] The "nucleic acid molecule" can be DNA, such as cDNA or recombinant DNA, or RNA, such as mRNA or hnRNA.

[0024] An "expression cassette" comprises a polynucleotide sequence encoding a polypeptide (antibody) to be expressed and a sequence controlling its expression, such as a promoter, a signal peptide sequence, and optionally an enhancer sequence, including any combination of cis-acting transcriptional control units. Sequences that control gene expression (i.e., its transcription and translation of the transcription product) are often referred to as regulatory units. Most of the regulatory unit is located upstream of the coding sequence of the gene and is operably linked thereto. The expression cassette may also contain a downstream 3' untranslated region comprising a polyadenylation site.

[0025] The vector backbone of a "recombinant vector" can be a plasmid, a phage or a virus.

[0026] The host cell of "recombinant eukaryotic cell" can be yeast or mammalian cell, etc.

[0027] The host cell of the "recombinant prokaryotic cell" may be bacteria or algae, etc.

[0028] In some embodiments, the nucleotide sequence encoding the heavy chain variable region is shown as SEQ.ID NO.2 or SEQ.ID NO.6, and the nucleotide sequence encoding the light chain variable region is shown as SEQ.ID NO.4 or SEQ.ID NO.8.

[0029]

[0030]

[0031]

[0032]

[0033] In one embodiment, the nucleotide sequence encoding the heavy chain variable region is shown as SEQ.ID NO.2, and the nucleotide sequence encoding the light chain variable region is shown as SEQ.ID NO.4.

[0034] In one embodiment, the nucleotide sequence encoding the heavy chain variable region is shown as SEQ.ID NO.6, and the nucleotide sequence encoding the light chain variable region is shown as SEQ.ID NO.8.

[0035] The present application provides the use of the antibody or antigen-binding fragment or the biological material in detecting avian leukosis virus products.

[0036] In some embodiments, the product is a reagent or a kit. The heavy chain variable region and the light chain variable region provided in the present application can specifically bind to the avian leukosis virus. Therefore, this property and the commonly used detection means in the art can be used to detect the avian leukosis virus and prepare it into a product. A variety of different types of detection kits can be prepared according to specific needs. Depending on the specific detection method of the kit, the specific supporting reagents in the kit are also different, but all can be combined according to the preparation method of the known kit.

[0037] In a preferred embodiment, the kit is an avian leukosis virus double antibody sandwich ELISA antigen detection kit.

[0038] The present application relates to an avian leukosis virus double antibody sandwich ELISA antigen detection kit, which comprises: a supporting medium coated with a coating antibody or antigen-binding fragment, an enzyme-labeled enzyme-labeled antibody or antigen-binding fragment, and a detection reagent; wherein the coating antibody or antigen-binding fragment contains a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ.ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.3; the enzyme-labeled antibody or antigen-binding fragment contains a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ.ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.7.

[0039] In some embodiments, the coating antibody or antigen-binding fragment is monoclonal antibody 1G5; the enzyme-labeled antibody or antigen-binding fragment is monoclonal antibody 2C4.

[0040] In some embodiments, the coating concentration of the monoclonal antibody 1G5 is 40 ng / well to 60 ng / well, and the working concentration of the monoclonal antibody 2C4 is 1:30000 to 1:50000.

[0041] In some embodiments, the support medium is a microtiter plate.

[0042] In some embodiments, the enzyme labeled is horseradish peroxidase, alkaline phosphatase, or β-D-galactosidase.

[0043] In some embodiments, the coating concentration of the monoclonal antibody 1G5 is 50 ng / well, and the working concentration of the enzyme-labeled monoclonal antibody 2C4 is 1:40,000.

[0044] In some embodiments, the detection reagent includes a color development solution and a stop solution.

[0045] In some embodiments, the color developing solution includes color developing solution A and color developing solution B, color developing solution A contains 1.47% w / v disodium hydrogen phosphate, 0.93% w / v citric acid and 0.03% w / v urea peroxide, and color developing solution B contains 0.02% w / v tetramethylbenzenediamine and 10% v / v anhydrous ethanol.

[0046] In some embodiments, the stop solution is 0.3% HF (hydrofluoric acid) solution.

[0047] In some embodiments, the kit further comprises a positive control, a negative control, a support medium blocking solution, and a washing solution.

[0048] In some embodiments, the positive control is a PBS solution containing 10% v / v inactivated ALV J subgroup virus solution or ALVp27 protein and 0.05% v / v Proclin 300.

[0049] In some embodiments, the negative control is a PBS solution containing 10% v / v DF-1 cell culture supernatant and 0.05% v / v Proclin 300.

[0050] In some embodiments, the wash solution is phosphate buffered saline.

[0051] In some embodiments, the blocking solution is 0.01 M PBS containing 5% w / v sucrose, 20% v / v newborn calf serum, and 0.05% v / v Proclin 300 with a pH value of 7.4.

[0052] Compared with the prior art, the technical effects of this application are:

[0053] The antibody or antigen-binding fragment provided in the present application recognizes an antigenic epitope located on the p27 protein of avian leukosis virus and reacts positively with avian leukosis virus, indicating that it has good reaction characteristics with avian leukosis virus. The antibody can provide an important tool for ALV detection. DETAILED DESCRIPTION

[0054] Below, the specific implementation methods of the present application are described in detail.

[0055] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for possible spontaneous mutations that may occur in small amounts. Thus, the modifier "monoclonal" refers to the nature of the antibody as not being a mixture of discrete antibodies. Preferably, monoclonal antibodies include monovalent or single-chain antibodies, double-chain antibodies, chimeric antibodies, avian antibodies, and derivatives, functional equivalents, and homologs of the above antibodies, as well as antibody fragments and any polypeptides containing an antigen binding domain.

[0056] The term "antibody" has the same meaning as immunoglobulin, and is intended to encompass any specific binding factor having a binding domain with the desired specificity. Thus, this term encompasses antibody fragments, derivatives, avianized antibodies, and functional equivalents and homologs thereof, as well as any polypeptide containing an antigen binding domain, whether natural or synthetic. Examples of antibodies are immunoglobulin subtypes (such as IgG, IgE, IgM, IgD, and IgA) and their subtypes and subclasses; fragments containing an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. Chimeric molecules or equivalents containing an antigen binding domain fused to another polypeptide are also included. The cloning and expression of chimeric antibodies are described in EP.A.0120694 and EP.A.0125023. Antibodies can be modified in many ways, and DNA recombinant technology can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. This technique may include introducing DNA encoding the immunoglobulin variable region or complementarity determining regions (CDRs) of an antibody into the constant region or constant region plus framework region of a different immunoglobulin, see EP.A.184187, GB2188638A or EP.A.239400. Genetic mutations or other changes may also be made to hybridoma cells or other cells producing antibodies, which may or may not change the binding specificity of the produced antibody.

[0057] The "monoclonal antibody" used in the present application can also be prepared by the hybridoma method, because the DNA sequence encoding the antibody of the present application can be obtained by conventional means well known to those skilled in the art, such as artificially synthesizing a nucleotide sequence according to the amino acid sequence disclosed in the present application or amplifying it by PCR, and thus can also be obtained by recombinant DNA methods, and the sequence can be linked to a suitable expression vector by various methods well known in the art. Under conditions suitable for the expression of the antibody of the present application, the transformed host cells are cultured, and then the monoclonal antibody of the present application is purified by conventional separation and purification means well known to those skilled in the art. The antibody comprises a geometric body of polypeptide chains connected together by disulfide bridges, and the two polypeptide main chains called light chains and heavy chains constitute all the main structural categories (isotypes) of antibodies. Both the heavy chain and the light chain can be further divided into some subregions called variable regions and constant regions. The heavy chain includes a single variable region and three different constant regions, while the light chain includes 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 chain and the light chain are responsible for the binding specificity of the antibody.

[0058] The term "heavy chain variable region" refers to a polypeptide having a length of 110 to 125 amino acids, and its amino acid sequence corresponds to the heavy chain amino acid sequence of the monoclonal antibody of the present application starting from the heavy chain N-terminal amino acid. Similarly, the term "light chain variable region" refers to a polypeptide having a length of 95 to 115 amino acids, and its amino acid sequence corresponds to the light chain amino acid sequence of the monoclonal antibody of the present application starting from the light chain N-terminal amino acid.

[0059] The variable regions of each heavy chain / light chain pair (VH and VL) form an antigen binding site, respectively. Thus, for example, a complete IgG antibody has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are identical.

[0060] The variable regions of antibody heavy and light chains exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions (also called complementarity determining regions or CDRs). The term "variable" refers to the fact that certain sites of the variable domains differ extensively in sequence among antibodies and are involved in the binding and specificity of each particular antibody for its particular antigen. The variability is primarily located in the CDRs, which are separated by more highly conserved framework regions (FRs).

[0061] The term "antibody fragment" refers to a complete or full-length chain or portion of an antibody, typically the target binding region or variable region. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments. "Antigen binding fragment" is generally synonymous with "antibody fragment" and may refer to fragments that can prevent or substantially reduce the ability of African swine fever virus to bind to a receptor, such as Fv, Fab, F(ab')2, etc.

[0062] The present application is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0063] All chemical reagents used in the examples of this application were of analytical grade and purchased from Sinopharm Group.

[0064] The experimental methods described in this application are all conventional methods unless otherwise specified; the biological materials described can be obtained from commercial channels unless otherwise specified.

[0065] Example 1 Preparation and Identification of ALV p27 Protein

[0066] Referring to the genome sequence of ALV J subgroup ALV HLJ09MDJ-1 strain published by GenBank (accession number: JN624880), Shanghai Shenggong Biotechnology Co., Ltd. was commissioned to synthesize the codon-optimized p27 gene sequence. Upstream and downstream primers were designed, with BamH I restriction site added to the upstream primer and Xho I restriction site added to the downstream primer. The synthesized p27 gene sequence was used as a template for PCR amplification. After purification and recovery of the PCR product, it was digested with BamH I and Xho I restriction endonucleases at 37°C for 1h together with the pET-30a vector, and purified and recovered. The recovered product and the vector were connected by T4 DNA ligase at room temperature for 1h, and then transformed into DH5α competent cells. After picking a single colony to extract the plasmid, double enzyme digestion and sequencing were performed. The plasmid with correct enzyme digestion and sequencing was the recombinant plasmid pET-30a-p27. The recombinant plasmid pET-30a-p27 was transformed into BL21 (DE3) competent cells, and the p27 protein was expressed by IPTG induction. The protein was purified by nickel column affinity chromatography. The purified protein was detected by SDS-PAGE, and a clear protein band was seen at about 27KDa. The purity was determined to be 94% by spectral scanning. The protein concentration was determined to be 1.0 mg / ml by BCA protein concentration determination kit. The purified protein was packaged and stored below -70°C for later use.

[0067] Example 2 Preparation and identification of monoclonal antibodies against ALV p27 protein

[0068] 2.1 Screening of hybridoma cells

[0069] Five female BALB / c mice aged 4 to 6 weeks were immunized with ALVp27 protein at a dose of 100 μg / mouse (volume 200 μl) by subcutaneous multi-point immunization every 3 weeks. The first immunization was performed with an equal volume of Freund's complete adjuvant and ALVp27 protein emulsified, and the subsequent immunization was performed with an equal volume of Freund's incomplete adjuvant and ALVp27 protein emulsified, for a total of 3 immunizations. After the three immunizations, the serum of mice was collected and the serum titer was determined by the indirect ELISA method of ALVp27 protein: the protein was diluted to 0.5 μg / ml and then coated on the ELISA plate, 100 μl / well, and allowed to stand at 2-8°C for 16-24 hours; the liquid in the plate was discarded, and the blocking solution was added, 200 μl / well, and the plate was blocked at 2-8°C for 16-24 hours, and the plate was washed; the sample to be tested (hybridoma cell supernatant was diluted 1:100 and then diluted in multiples, mouse serum was diluted 1:1000 and then diluted in multiples) was added, 100 μl / well, and a negative plate with PBS (0.01 mol / L, pH 7.4) was set at the same time. Control, incubate at 37℃ for 60 minutes, wash the plate; add the secondary antibody diluted to the working concentration, 100μl / well, incubate at 37℃ for 60 minutes, wash the plate; add the colorimetric reagent A and B solution, 50μl / well, shake and mix, incubate at 37℃ in the dark for 15 minutes, add the stop solution, 50μl / well; set the wavelength of the microplate reader at 630nm, and detect the OD value of each well; when the negative control OD value is less than 0.1, the test is established, and the S / N (sample OD value / negative control OD value) ≥2.1 is judged as positive; S / N (sample OD value / negative control OD value) <2.1 is judged as negative. The highest sample dilution corresponding to the positive well is taken as the titer of the sample. The sera of 5 mice were tested using this method, and the results showed that the titer of the serum of 5# mouse was as high as 1:5.12 million. ALV p27 protein was intraperitoneally injected into 5# mice at a dose of 50 μg / mouse for shock immunization. Cell fusion was performed 3 days after immunization. The fused cells were subjected to multiple subclone screenings to obtain 18 positive hybridoma cells.

[0070] 2.2 Selection of paired monoclonal antibodies for ELISA

[0071] The titer of the supernatant of 18 hybridoma cells was determined by the indirect ELISA method of ALV p27 protein in 2.1. Among them, the three hybridoma cells with the highest ELISA titer were 1G5, 2C4, and 5H9. Ascites was prepared and purified by Protein G affinity chromatography. The purified monoclonal antibodies were enzyme-labeled in small batches according to Example 3.1. The three monoclonal antibodies were matched as coating antibodies and enzyme-labeled antibodies according to Table 1, respectively, with a coating concentration of 0.5 μg / ml and an enzyme-labeled antibody working concentration of 1:40000. The ALVp27 protein prepared in Example 1 and its different multiple dilutions, 10-fold diluted ALV J subgroup virus liquid, together with 5 ALV-negative cloacal swabs and 5 ALV-negative egg white samples, were detected by double antibody sandwich ELISA method. The results are shown in Table 1. The combination of coated monoclonal antibody 1G5 and enzyme-labeled monoclonal antibody 2C4 has the highest sensitivity in detecting ALV p27 protein. The 10-fold diluted ALV J subgroup virus fluid was positive, and the 10 clinical samples were all negative. Therefore, the pairing method was determined to be 1G5 as the coated monoclonal antibody and 2C4 as the enzyme-labeled monoclonal antibody for subsequent research.

[0072] Table 1 Results of the monoclonal antibody pairing test

[0073] 2.3 Identification of monoclonal antibodies

[0074] 2.3.1 Subclass identification

[0075] The subclasses of monoclonal antibodies 1G5 and 2C4 were identified using a monoclonal antibody subclass identification kit. The results showed that the heavy chain subclasses of 1G5 and 2C4 were both IgG1, and the light chain subclasses were both kappa.

[0076] 2.3.2 Western blot identification

[0077] First, the ALVp27 protein was subjected to SDS-PAGE electrophoresis, and then transferred to the membrane. The monoclonal antibody 1G5 and 2C4 dilutions were used as primary antibodies, and the HRP-labeled goat anti-mouse IgG dilution was used as the secondary antibody for Western blot detection. The results showed that both monoclonal antibodies reacted with the ALVp27 protein to produce specific bands, indicating that both monoclonal antibodies can recognize the ALVp27 protein.

[0078] 2.3.3 Indirect immunofluorescence identification

[0079] Avian leukosis virus of subgroup A, B, and J was inoculated into a 96-well cell culture plate of DF-1 cells that had grown into a good monolayer, and a healthy cell control was set up at the same time, and cultured in an incubator at 37°C and 5% CO2 for 7 days, fixed with 80% cold acetone, and stored below -20°C for later use after drying. When used, each antigen plate was taken back to temperature, washed once with PBS, and then added with monoclonal antibody 1G5 and 2C4 dilutions, and detected by conventional IFA method. The results showed that specific fluorescence was visible in the virus inoculation wells with monoclonal antibody 1G5 and 2C4, and no visible fluorescence was visible in the healthy cell control wells, indicating that monoclonal antibodies 1G5 and 2C4 can react with avian leukosis virus of subgroup A, B, and J.

[0080] 2.3.4 Specificity identification

[0081] The double antibody sandwich ELISA method was used to detect H5 subtype avian influenza hemagglutination inhibition test antigen, H7 subtype avian influenza hemagglutination inhibition test antigen, H9 subtype avian influenza hemagglutination inhibition test antigen, infectious bursal disease virus agar expansion test antigen, Newcastle disease virus hemagglutination inhibition test antigen, Marek's disease virus, Escherichia coli BL21 (DE3) culture supernatant, Escherichia coli BL21 (DE3) bacterial lysate, Escherichia coli BL21 (DE3)-pET-30a culture supernatant, and Escherichia coli BL21 (DE3)-pET-30a bacterial lysate, and all were negative.

[0082] Example 3 Establishment of ALV double antibody sandwich ELISA antigen detection kit

[0083] 3.1 Preparation and identification of enzyme-labeled antibodies

[0084] 3.1.1 Preparation

[0085] The monoclonal antibody 2C4 was labeled with horseradish peroxidase (HRP) using the modified sodium periodate method. Weigh 20 mg of horseradish peroxidase (HRP) and dissolve it in 1 ml of ultrapure water, add 1 ml of freshly prepared NaIO4 solution (30 mg of NaIO4 dissolved in 1 ml of ultrapure water, prepared immediately before use), mix well, and incubate at 2-8°C in the dark for 30 minutes; add 40 μl of ethylene glycol to the above solution and incubate at 2-8°C in the dark for 30 minutes; according to the ratio of 1 mg of purified monoclonal antibody to 100 μl of the above mixture, mix the two well, add them to the dialysis bag, mix well, and dialyze with CB buffer for 6 hours. The whole operation needs to be carried out in the dark; transfer the dialyzed mixture to a 1.5ml EP tube, add 10μl of freshly prepared NaBH4 solution (20mg NaBH4 dissolved in 1ml ultrapure water, prepared immediately before use), act at room temperature for 2 hours, and mix once every 30 minutes; add an equal volume of saturated ammonium sulfate, mix well, and act at 2-8℃ for 15 minutes. Centrifuge at 12000r / min for 10 minutes and discard the supernatant. Resuspend the precipitate with a mixture of PBS and glycerol (V:V=1:1) with an equal volume of the purified antibody.

[0086] 3.1.2 Identification

[0087] Appearance: At room temperature, it is a reddish brown liquid with no flocculent precipitation.

[0088] Quality evaluation: Dilute the enzyme-labeled antibody 10 times and use a UV spectrophotometer to detect the absorbance value A of the enzyme-labeled antibody at 403nm and 280nm. Calculate the corresponding enzyme parameters according to the formula:

[0089] Enzyme amount (mg / ml) = A 403nm ×0.4×dilution factor (10 times).

[0090] IgG amount (mg / ml) = (A 280nm -A 403nm ×0.3)×0.62×dilution factor (10 times).

[0091] Molecular ratio (E / P) = enzyme amount × 4 / IgG amount.

[0092] Marking rate = A 403nm / A 280nm .

[0093] After absorbance detection and calculation, the specific results are shown in Table 2:

[0094] Table 2 Quality evaluation results of enzyme-labeled antibodies

[0095] 3.2 Preparation of ALV double antibody sandwich ELISA antigen detection kit

[0096] Antigen coated plate: Dilute the 1G5 monoclonal antibody prepared in Example 2 to 0.5 μg / ml with carbonate buffer (0.05 mol / L, pH 9.6) for coating, 100 μl / well, let stand at 2-8°C for 16-24 hours, wash with detergent, add blocking solution (weigh 50 g sucrose, add 200 ml newborn calf serum, 0.5 ml Proclin300, add PBS (0.01 mol / L, pH 7.4) to make the volume 1000 ml), block at 2-8°C for 16-24 hours, discard the blocking solution and dry for 4 hours, package and store at 2-8°C for use.

[0097] Enzyme labeling reagent: dilute the enzyme-labeled monoclonal antibody 2C41 prepared in Example 3.1.1 at a ratio of 40000 with enzyme labeling diluent (measure 200 ml of newborn calf serum, 0.5 ml of Proclin 300, 0.5 ml of Tween 20, 0.04 g of AM dye, and add PBS (0.01 mol / L, pH 7.4) to make up to 1000 ml) as the enzyme labeling reagent and store at 2-8°C.

[0098] Positive control: Take 100 ml of inactivated ALV J subgroup virus liquid or ALV p27 protein, 0.5 ml of Proclin 3000, add PBS (0.01 mol / L, pH 7.4) to make up to 1000 ml, mix well and filter with a 0.22 μm filter, aseptically divide into quantitative portions as a positive control, and store at 2-8°C.

[0099] Negative control: Take 100 ml of DF-1 cell culture supernatant and 0.5 ml of Proclin 3000, add PBS buffer (0.01 mol / L, pH 7.4) to make up to 1000 ml, mix well and filter with a 0.22 μm filter, aseptically divide into quantitative portions as negative control, and store at 2-8°C.

[0100] 20× concentrated washing solution: Take 160g sodium chloride, 58g disodium hydrogen phosphate, 4.8g potassium dihydrogen phosphate, 4g potassium chloride, 800ml purified water, 10ml Tween 20, dissolve completely, make up to 1000ml with purified water, mix well, filter with 0.22μm filter membrane, and dispense aseptically. Dilute 20 times with purified water before use.

[0101] Color developing solution: Disodium hydrogen phosphate 14.7g, citric acid 9.3g, urea peroxide 0.3g, dissolved in purified water, dilute to 1000ml, mix well and aseptically dispense, color developing solution A. Tetramethylbenzyl diamine (TMB) 0.2g, anhydrous ethanol 10ml, dissolved in purified water, dilute to 1000ml, mix well and aseptically dispense, color developing solution B.

[0102] Stop solution: 0.3% HF (hydrofluoric acid) solution.

[0103] Assemble the above components into a test kit and store at 2-8°C.

[0104] 3.3 Establishment of detection method

[0105] The detection steps are as follows:

[0106] (1) Numbering: Number the corresponding microplates of samples in sequence. Each plate should have 2 negative control (NC) wells and 2 positive control (PC) wells.

[0107] (2) Sample addition: Add 100 μl of the sample to be tested and 100 μl of the negative and positive controls to the corresponding wells, and gently shake to mix. Seal the plate and incubate at 37°C for 60 minutes.

[0108] (3) Washing: Wash 4 times with detergent, and try to dry it as much as possible the last time.

[0109] (4) Add enzyme labeling reagent: Add 100 μl of enzyme labeling reagent to each well, seal the plate and incubate at 37°C for 60 min.

[0110] (5) Washing: Wash 4 times with detergent and try to dry it as much as possible the last time.

[0111] (6) Color development: Add 50 μl of color development solution A and 50 μl of color development solution B to each well, shake gently to mix, and incubate at 37°C in the dark for 15 min.

[0112] (7) Stopping: Add 50 μl of stop solution to each well, gently shake to mix, and measure the results using an ELISA reader within 10 minutes.

[0113] (8) Determination: Single wavelength determination, set the wavelength of the microplate reader to 630 nm and measure the A value of each well.

[0114] (9) Result determination: Calculate the S / P of the sample = (sample A value - average A value of negative control) / (average A value of positive control - average A value of negative control). When S / P ≥ 0.2, the sample is judged to be positive for avian leukosis virus p27 antigen; when S / P < 0.2, the sample is judged to be negative for avian leukosis virus p27 antigen.

[0115] Evaluation of 3.4ALV double antibody sandwich ELISA antigen detection kit

[0116] 3.4.1 Sensitivity

[0117] The test kit prepared in Example 3.2 was used to detect the gradient dilution of ALV p27 protein, and the results showed that the detection limit was 0.25 ng / ml. The ALV A, B, C, D, and J virus liquids were all positive.

[0118] The kit prepared in Example 3.2 was used to detect 30 ALV-positive egg white samples, positive cloacal swab samples, positive semen samples, and positive meconium samples collected from clinics, and the results showed that all of them were positive, indicating that the sensitivity of the kit was good.

[0119] 3.4.2 Specificity

[0120] The kit prepared in Example 3.2 was used to detect H5 subtype avian influenza hemagglutination inhibition test antigen, H7 subtype avian influenza hemagglutination inhibition test antigen, H9 subtype avian influenza hemagglutination inhibition test antigen, infectious bursal disease virus agar expansion test antigen, Newcastle disease virus hemagglutination inhibition test antigen, Marek's disease virus, Escherichia coli BL21 (DE3) culture supernatant, Escherichia coli BL21 (DE3) bacterial lysate, Escherichia coli BL21 (DE3)-pET-30a culture supernatant, Escherichia coli BL21 (DE3)-pET-30a bacterial lysate, and 30 clinically collected ALV negative egg white samples, negative cloacal swab samples, negative semen samples, and negative meconium samples. The results showed that all were negative, indicating that the specificity of this kit is good.

[0121] 3.4.3 Repeatability

[0122] According to Example 3.2, 3 batches of test kits were prepared, and 3 ALV-positive egg white samples were tested 5 times each. The intra-batch and inter-batch repeatability were calculated. The results showed that the intra-batch and inter-batch repeatability were both less than 10%, which was a good repeatability.

[0123] 3.4.4 Clinical application

[0124] According to the above results, 3 batches of test kits prepared in Example 3.2 were used for clinical application to detect 565 clinical samples (of which 102 were positive by PCR and 463 were negative). The results showed that the test kit detected 95 positive samples and 470 negative samples, with a positive compliance rate of 93.1%, a negative compliance rate of 100%, and a total compliance rate of 98.8%. This shows that the sensitivity and specificity of the test kit are good and can be used for the detection of clinical samples.

[0125] Example 4 Determination of variable region sequences of ALV p27 protein monoclonal antibodies 1G5 and 2C4

[0126] According to the sequence characteristics of mouse monoclonal antibodies, the 1G5 heavy chain variable region primer sequence was designed:

[0127] F1: 5'-ACTAGTCGACATGAACTTYGGG-3'

[0128] R1: 5'-CCAGGGRCCARKGGATARACN-3'

[0129] Design 1G5 light chain variable region primer sequence:

[0130] F2: 5'-ACTAGTCGACATGGAGWCAGACA-3'

[0131] R2: 5'-CCCAAGCTTACTGGATGGTGGG-3'

[0132] Design 2C4 heavy chain variable region primer sequence:

[0133] F3: 5'-GGGAATTCATGRAATGSASCTG-3'

[0134] R3: 5'-CCAGGGRCCARKGGATARACN-3'

[0135] Design 2C4 light chain variable region primer sequence:

[0136] F4: 5'-ACTAGTCGACATGAAGTTGCCTG-3'

[0137] R4: 5'-CCCAAGCTTACTGGATGGTGGG-3'

[0138] The hybridoma cells were collected, RNA was extracted and reverse transcribed as a template, the variable region sequences were amplified using the above primers, and the amplified products were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The results showed that the amino acid sequences of the heavy chain variable region and light chain variable region of monoclonal antibodies 1G5 and 2C4 were as shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, and SEQ ID No.7, respectively, and the gene sequences were as shown in SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, and SEQ ID No.8, respectively.

[0139] Example 5 Preparation and identification of single-chain antibodies 1G5 and 2C4

[0140] The heavy chain variable region (VH) gene and light chain variable region (VL) gene of the monoclonal antibody were amplified, and after the connecting peptide was transferred, they were connected to the prokaryotic expression vector pET-32a (+), and the recombinant plasmids were constructed respectively, and the BL21 (DE3) competent cells were transformed for expression to obtain the fusion protein. The corresponding single-chain antibodies 1G5 and 2C4 were prepared using the variable region sequences of the monoclonal antibodies 1G5 and 2C4 according to the method described in Example 5. The ELISA titers of 1G5 and 2C4 were determined according to the method of Example 2. As a result, the ELISA titers of the two single-chain antibodies were ≥1:512,000, which had good reactivity.

[0141] The above results show that the variable region sequences shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7 or SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8 can be used for the preparation of genetically engineered antibodies against avian leukosis virus.

[0142] The above description is only a preferred embodiment of the present application and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An antibody or antigen-binding fragment that specifically binds to the p27 protein of avian leukosis virus, characterized in that: The antibody or antigen-binding fragment contains a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ.ID NO.1 or SEQ.ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.3 or SEQ.ID NO.

7.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that The antibody or antigen-binding fragment is a Fab, Fab', F(ab')2, scFv or Fv fragment; Optionally, the antibody or antigen-binding fragment is an IgG1, IgG2, IgG3 or IgG4 antibody or a fragment thereof; Optionally, the antibody or antigen-binding fragment is a monoclonal antibody or an antigen-binding fragment thereof; Optionally, the antibody or antigen-binding fragment is a single-chain antibody; Optionally, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment is as shown in SEQ.ID NO.1, and the amino acid sequence of the light chain variable region is as shown in SEQ.ID NO.3; Optionally, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment is shown in SEQ.ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.

7.

3. The antibody or antigen-binding fragment according to claim 1, characterized in that The antibody is monoclonal antibody 1G5, the amino acid sequence of the heavy chain variable region is shown in SEQ.ID NO.1, the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.3, the heavy chain subclass is IgG1, and the light chain subclass is kappa; Optionally, the antibody is monoclonal antibody 2C4, the amino acid sequence of the heavy chain variable region is shown in SEQ.ID NO.5, the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.7, the heavy chain subclass is IgG1, and the light chain subclass is kappa; Optionally, the antibody is a single-chain antibody 1G5, the amino acid sequence of the heavy chain variable region is shown in SEQ.ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.3; Optionally, the antibody is a single-chain antibody 2C4, the amino acid sequence of the heavy chain variable region is shown in SEQ.ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.

7.

4. A biomaterial related to the antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that: The biological material is any of the following: (a) a nucleic acid molecule comprising a sequence encoding the heavy chain variable region and / or light chain variable region of the antibody or antigen-binding fragment; (b) an expression cassette comprising the nucleic acid molecule described in (a); (c) a recombinant vector comprising the nucleic acid molecule described in (a) or the expression cassette described in (b); (d) a recombinant eukaryotic cell containing the nucleic acid molecule in (a), the expression cassette in (b) or the recombinant vector in (c); (e) A recombinant prokaryotic cell containing the nucleic acid molecule in (a), the expression cassette in (b) or the recombinant vector in (c).

5. The biomaterial according to claim 4, characterized in that The nucleotide sequence encoding the heavy chain variable region is shown in SEQ.ID NO.2 or SEQ.ID NO.6, and the nucleotide sequence encoding the light chain variable region is shown in SEQ.ID NO.4 or SEQ.ID NO.

8.

6. Use of the antibody or antigen-binding fragment according to claims 1 to 3 or the biological material according to claims 4 to 5 in detecting avian leukosis virus products.

7. A double antibody sandwich ELISA antigen detection kit for avian leukosis virus, characterized in that: The kit comprises: a supporting medium coated with a coating antibody or antigen-binding fragment, an enzyme-labeled antibody or antigen-binding fragment, and a detection reagent; The coating antibody or antigen-binding fragment contains a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ.ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.3; The enzyme-labeled antibody or antigen-binding fragment contains a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ.ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ.ID NO.

7.

8. The kit according to claim 7, characterized in that The coating antibody or antigen-binding fragment is monoclonal antibody 1G5; The enzyme-labeled antibody or antigen-binding fragment is the monoclonal antibody 2C4.

9. The kit according to claim 8, characterized in that The coating concentration of the monoclonal antibody 1G5 is 40 ng / well to 60 ng / well, and the working concentration of the monoclonal antibody 2C4 is 1:30000 to 1:50000; Optionally, the support medium is a microtiter plate; Optionally, the enzyme labeled is horseradish peroxidase, alkaline phosphatase or β-D-galactosidase; Optionally, the coating concentration of the monoclonal antibody 1G5 is 50 ng / well, and the working concentration of the enzyme-labeled monoclonal antibody 2C4 is 1:40,000.

10. The kit according to claim 7, characterized in that The detection reagent includes a color developing solution and a stop solution; Optionally, the color developing solution includes color developing solution A and color developing solution B, color developing solution A contains 1.47% w / v disodium hydrogen phosphate, 0.93% w / v citric acid and 0.03% w / v urea peroxide, and color developing solution B contains 0.02% w / v tetramethylbenzenediamine and 10% v / v anhydrous ethanol; Optionally, the stop solution is 0.3% HF solution; Optionally, the kit further comprises a positive control, a negative control, a support medium blocking solution and a washing solution; Optionally, the positive control is a PBS solution containing 10% v / v inactivated ALV J subgroup virus solution or ALVp27 protein and 0.05% v / v Proclin 300; Alternatively, the negative control is a PBS solution containing 10% v / v DF-1 cell culture supernatant and 0.05% v / v Proclin 300; Optionally, the washing solution is phosphate buffered saline; Optionally, the blocking solution is 0.01 M PBS containing 5% w / v sucrose, 20% v / v newborn calf serum, and 0.05% v / v Proclin 300 with a pH value of 7.4.

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