Antibodies or antigen-binding fragments specifically binding to avian leukosis virus p27 protein and uses thereof

Abstract

The application relates to the technical field of antibodies, in particular to an antibody or antigen binding fragment specifically binding to p27 protein of avian leukosis virus and application thereof. 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 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 antigen binding fragment is positive to avian leukosis virus, which indicates that the antibody or antigen binding fragment has good reaction characteristics with the avian leukosis virus.

Description

TECHNICAL FIELD

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

[0002] Avian leukosis virus (ALV) is a member of retrovirus family and retrovirus genus, which can cause avian leukosis and various tumor diseases. There are two main transmission routes of exogenous ALV. One is vertical transmission, which is the main transmission route, and the other is horizontal transmission, which is transmitted between individuals through the environment. Most birds are infected by directly contacting with birds with congenital infection, and then transmitted to offspring. The offspring of hens with viremia also carry the virus. Vertical transmission determines the continuity and persistence of infection, and horizontal transmission ensures the maintenance of vertical transmission, making it difficult to completely eradicate the disease. Moreover, it can also cause immune suppression in chickens, secondary infection of other bacteria and viruses, and cause serious economic losses. gag gene sequence has high homology of up to 90% among exogenous ALV (A, B, C, D, J) subgroups, which mainly encodes viral nucleocapsid protein. The p27 protein encoded by gag gene is a group-specific antigen of avian leukosis virus and the main component of nucleocapsid protein, which is highly conserved and accounts for more than 30% of total protein, and is suitable for the establishment of clinical diagnosis method for avian leukosis.

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

[0004] Therefore, the present application is proposed. SUMMARY

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

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

[0007] An antibody or antigen binding fragment specifically binding to p27 protein of avian leukosis virus, which 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 variable region of the heavy chain and the light chain of the antibody contains a variable region with great variation in amino acid composition and arrangement at the N-terminus, which contains an antigen binding site capable of complementary binding with an epitope of an antigen, determines the specificity of the antibody in binding with the corresponding antigen epitope, is responsible for recognizing and binding with the antigen, and thus plays 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 fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a single chain antibody.

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

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

[0016] Antibodies are immunoglobulins (Igs) used by the immune system to identify and neutralize foreign invaders such as bacteria, viruses, and the like. Immunoglobulins are mainly divided into five types: IgA, IgD, IgE, IgG, and IgM. IgG antibodies have different subtypes: IgGl, IgG2a, IgG2b, IgG3, IgG4, and the positions and numbers of disulfide bonds are different between different subtypes. Immunoglobulin (Ig) light chains are divided into two subtypes: kappa (kappa) and lambda (lambda). Through experimental detection and identification, the heavy chain subclass of the monoclonal antibodies 1G5 and 2C4 protected by the present application is IgGl, and the light chain subclass is kappa.

[0017] The present application provides biological materials related to the antibody or antigen-binding fragment, and the biological materials are any one of the following:

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

[0019] (b) an expression cassette comprising the nucleic acid molecule of (a).

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

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

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

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

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

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

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

[0027] The host cell of the "recombinant prokaryotic cell" can be a bacterium or an alga, etc.

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

[0029]

[0030]

[0031]

[0032]

[0033]

[0034] In one embodiment, the nucleotide sequence encoding the heavy chain variable region is set forth in SEQ. ID NO. 2, and the nucleotide sequence encoding the light chain variable region is set forth in SEQ. ID NO. 4.

[0035] In one embodiment, the nucleotide sequence encoding the heavy chain variable region is set forth in SEQ. ID NO. 6, and the nucleotide sequence encoding the light chain variable region is set forth in SEQ. ID NO. 8.

[0036] The present application provides use of the antibody or antigen binding fragment or the biological material in detecting avian leukosis virus products.

[0037] 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 avian leukosis virus, so that the detection of avian leukosis virus can be realized by using the property and the detection means commonly used in the art, and the product can be prepared. A variety of different types of detection kits can be prepared according to specific needs. According to the specific detection method of the kit, the specific matching reagents in the kit are also different, but they can be combined according to the preparation method of the known kit.

[0038] In preferred embodiments, the kit is an avian leukosis virus double antibody sandwich ELISA antigen detection kit.

[0039] The present application relates to an avian leukosis virus double antibody sandwich ELISA antigen detection kit, which comprises a support 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 as SEQ. ID NO. 1, and the amino acid sequence of the light chain variable region is shown as 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 as SEQ. ID NO. 5, and the amino acid sequence of the light chain variable region is shown as SEQ. ID NO. 7.

[0040] In some embodiments, the coating antibody or antigen binding fragment is a monoclonal antibody 1G5; and the enzyme-labeled antibody or antigen binding fragment is a monoclonal antibody 2C4.

[0041] 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.

[0042] In some embodiments, the support medium is a microtiter plate.

[0043] In some embodiments, the enzyme-labeled enzyme is horseradish peroxidase, alkaline phosphatase or β-D-galactosidase.

[0044] 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 1:40000.

[0045] In some embodiments, the detection reagent comprises a chromogenic solution and a termination solution.

[0046] In some embodiments, the developing solution comprises developing solution A and developing solution B, developing solution A comprises 1.47% w / v disodium hydrogen phosphate, 0.93% w / v citric acid and 0.03% w / v urea peroxide, and developing solution B comprises 0.02% w / v tetramethylbenzidine and 10% v / v absolute ethanol.

[0047] In some embodiments, the stopping solution is 0.3% HF (hydrofluoric acid) solution.

[0048] In some embodiments, the kit further comprises a positive control, a negative control, a blocking solution for support medium and a washing solution.

[0049] In some embodiments, the positive control is a PBS solution containing 10% v / v inactivated ALV J subgroup virus solution or ALV p27 protein and 0.05% v / v Proclin 300.

[0050] 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.

[0051] In some embodiments, the washing solution is phosphate buffer.

[0052] In some embodiments, the blocking solution is 0.01M PBS with pH value of 7.4 containing 5% w / v sucrose, 20% v / v newborn calf serum and 0.05% v / v Proclin 300.

[0053] Compared with the prior art, the technical effects of the present application are:

[0054] The antibody or antigen binding fragment provided by the present application recognizes an antigen epitope located in the avian leukosis virus p27 protein, and is positive in reaction with the avian leukosis virus, indicating that it has good reaction characteristics with the avian leukosis virus. The antibody can provide an important tool for ALV detection. DETAILED DESCRIPTION

[0055] Hereinafter, the specific embodiments of the present application will be described in detail.

[0056] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present. Thus, the modifier "monoclonal" indicates the character of the antibody as not being an admixture of discrete antibody species. Preferably, the monoclonal antibodies include monovalent or single-chain antibodies, diabodies, chimeric antibodies, avianized antibodies, and derivatives, functional equivalents and homologues of the above antibodies, and also include antibody fragments and any polypeptide containing an antigen binding domain.

[0057] The term "antibody" is synonymous with immunoglobulin and encompasses any specific binding member having a binding domain of the desired specificity, and thus the term encompasses antibody fragments, derivatives, avianized antibodies and functional equivalents and homologues of antibodies which are homologous thereto, as well as any polypeptide comprising an antigen binding domain, whether naturally occurring or synthetically produced. Examples of antibodies are immunoglobulin subtypes (such as IgG, IgE, IgM, IgD and IgA) and subtypes thereof; also fragments comprising an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. Chimeric molecules comprising an antigen binding domain fused to another polypeptide or equivalent are also included. Cloning and expression of chimeric antibodies is described in EP.A. 0 120 694 and EP.A. 0 125 023. Antibodies can be modified in a number of ways, and other antibodies or chimeric molecules retaining the specificity of the original antibody can be produced using DNA recombination techniques. Such techniques can involve introducing DNA encoding the 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. 0 184 187, GB 2188638A or EP.A. 0 239 400. Hybridoma cells or other cells producing antibodies can also be subjected to genetic mutation or other alteration, which can or can not alter the binding specificity of the antibody produced.

[0058] "Monoclonal antibodies" for use in the application can also be produced using hybridoma methods, since DNA sequences encoding antibodies of the application can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibodies, or fragments thereof), and the DNA sequences can be artificially synthesized based on the amino acid sequences disclosed herein. Once isolated, the DNA sequences can be placed into expression vectors, which are then transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulins. These host cells are then cultured in tissue culture media under conditions suitable for the expression of the DNA sequences. The monoclonal antibodies produced by the host cells can then be recovered from the culture media. Antibodies comprise a geometric arrangement of polypeptide chains, two polypeptide backbones, called light and heavy chains, which make up all the major classes (isotypes) of antibodies. Both the heavy and light chains are further divisible into regions called variable and constant regions. The heavy chain includes a single variable region and three constant regions, and the light chain includes a single variable region (different from that of the heavy chain) and a single constant region (different from that of the heavy chain). The variable regions of the heavy and light chains are responsible for the binding specificity of the antibody.

[0059] The term "heavy chain variable region" refers to a polypeptide of 110 to 125 amino acids in length which corresponds to the sequence of amino acids in the heavy chain of a monoclonal antibody of the present application beginning with the N-terminal amino acid of the heavy chain. Similarly, the term "light chain variable region" refers to a polypeptide of 95 to 115 amino acids in length which corresponds to the sequence of amino acids in the light chain of a monoclonal antibody of the present application beginning with the N-terminal amino acid of the light chain.

[0060] The variable regions of each heavy / light chain pair (VH and VL) form an antigen binding site, respectively. Thus, for example, an intact IgG antibody has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are identical.

[0061] The variable regions of the heavy and light chains of an antibody exhibit the same general structure. Each heavy and light chain variable region is composed of a single structural unit, which consists of four FRs connected by three CDRs. From N- to C-terminus, the CDRs are denoted as CDR1, CDR2 and CDR3, respectively. The CDRs are primarily responsible for binding to an antigen, while the FRs are more highly conserved and serve to maintain a proper conformation of the binding site. Individual CDRs (i.e., CDR1, CDR2 and CDR3) can be further subdivided into regions of even higher specificity, denoted as CDR1-1, CDR1-2, CDR2-1, CDR2-2, CDR3-1 and CDR3-2, respectively.

[0062] The term "antibody fragment" refers to a portion of an intact or full-length chain or antibody, typically the target binding or variable region. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments. The term "antigen binding fragment" is generally synonymous with "antibody fragment" and can refer to a fragment, such as Fv, Fab, F(ab')2, etc., that is capable of preventing or substantially reducing the ability of the African swine fever virus to bind to a receptor.

[0063] The present application is further illustrated by the following examples. Unless otherwise indicated, the materials in the examples were prepared according to known procedures or purchased directly from the market.

[0064] The chemical reagents used in the examples of the present application are all of analytical purity, purchased from the National Pharmaceutical Group.

[0065] The experimental methods described in the present application are all routine methods unless otherwise specified; the biological materials described, if not specifically stated, can be obtained from commercial channels.

[0066] Example 1 Preparation and identification of ALV p27 protein

[0067] The p27 gene sequence was synthesized by Shanghai Shengong Bioengineering Co., Ltd. according to the ALV J subgroup ALV HLJ09MDJ-1 strain genome sequence published in GenBank (accession number: JN624880). The upstream and downstream primers were designed, and the upstream primer was added with a BamH I enzyme cutting site, and the downstream primer was added with a Xho I enzyme cutting site. The synthesized p27 gene sequence was used as a template for PCR amplification, and the PCR product was purified and recovered. The pET-30a vector was cut by BamH I and Xho I restriction endonucleases at 37℃ for 1h, and then 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 identification were performed. The plasmid with correct enzyme digestion and sequencing identification was the recombinant plasmid pET-30a-p27. The recombinant plasmid pET-30a-p27 was transformed into BL21 (DE3) competent cells, and p27 protein was expressed by IPTG induction. The protein was purified by nickel column affinity chromatography, and the purified protein was detected by SDS-PAGE. A clear protein band was observed at about 27KDa, and the purity was determined to be 94% by spectral scanning method. The protein concentration was 1.0mg / ml determined by BCA protein concentration determination kit. The purified protein was divided and stored at below -70℃ for standby.

[0068] Example 2 Preparation and identification of ALV p27 protein monoclonal antibody

[0069] 2.1 Screening of hybridoma cells

[0070] Take 4-6 weeks old female BALB / c mice 5, with subcutaneous multi-point immunization method every 3 weeks according to 100 μg / one (volume 200 μl) ALVp27 protein immunization. First immunization with Freund's complete adjuvant and ALVp27 protein emulsified with equal volume, followed by Freund's incomplete adjuvant and ALVp27 protein emulsified with equal volume, a total of 3 times. Three immunization of mice serum with ALVp27 protein indirect ELISA method to determine the serum titer: the protein was diluted to 0.5 μg / ml after coating the enzyme-labeled plate, 100 μl / well, 2-8℃ for 16-24 hours; discard the plate, add blocking solution, 200 μl / well, 2-8℃ blocking 16-24 hours, washing plate; add the sample (hybridoma cell supernatant 1:100 dilution and then dilution, mouse serum 1:1000 dilution and then dilution), 100 μl / well, while setting up the negative control with PBS (0.01 mol / L, pH 7.4), 37℃ incubation for 60 minutes, washing plate; adding dilution to the working concentration of the second antibody, 100 μl / well, 37℃ incubation for 60 minutes, washing plate; adding color reagent A, B liquid, 50 μl / well, shaking mixed, 37℃ avoid light incubation for 15 minutes, add stop solution, 50 μl / well; set the wavelength of the enzyme-labeled instrument at 630 nm, detection of each well OD value; when the negative control OD value <0.1, the test is valid, S / N (sample OD value / negative control OD value) ≥2.1, judged as positive; S / N (sample OD value / negative control OD value) <2.1, judged as negative. The highest sample dilution corresponding to the positive well as the titer of the sample. The method was used to detect 5 mice serum, the results showed that the serum titer of 5 mice was up to 1:512 million. ALV p27 protein was injected intraperitoneally 50 μg / one 5# mice for impact immunization, and 3 days after immunization, the cells were fused, and the fused cells were subcloned several times to obtain 18 positive hybridoma cells.

[0071] 2.2 ELISA with the selection of paired monoclonal antibodies

[0072] The titers of the supernatants of 18 hybridoma cells were determined by the indirect ELISA method of ALV p27 protein in 2.1, and the three hybridoma cells with the highest ELISA titers were 1G5, 2C4 and 5H9. The ascites were prepared, and the ascites were purified by Protein G affinity chromatography. The purified monoclonal antibodies were subjected to small-scale enzyme labeling according to Example 3.1. The three monoclonal antibodies were matched as coating antibodies and enzyme-labeled antibodies according to Table 1, the coating concentration was 0.5 μg / ml, and the working concentration of the enzyme-labeled antibody was 1:40,000. The ALV p27 protein prepared in Example 1 and its different dilutions, 10-fold diluted ALV J subgroup virus liquid, together with 5 ALV negative cloaca swabs and 5 ALV negative egg white samples, were detected by double antibody sandwich ELISA method. The results are shown in Table 1. The coating monoclonal antibody 1G5 and the enzyme-labeled monoclonal antibody 2C4 were matched to detect ALV p27 protein with the highest sensitivity, and the 10-fold diluted ALV J subgroup virus liquid was positive, and the detection of 10 clinical samples was negative. Therefore, the pairing mode was determined as 1G5 as the coating monoclonal antibody and 2C4 as the enzyme-labeled monoclonal antibody for subsequent research.

[0073] Table 1 Results of monoclonal antibody pairing test

[0074]

[0075] 2.3 Identification of monoclonal antibodies

[0076] 2.3.1 Subclass identification

[0077] The subclasses of monoclonal antibodies 1G5 and 2C4 were identified by a monoclonal antibody subclass identification kit. The results showed that the heavy chain subclasses of 1G5 and 2C4 were IgG1, and the light chain subclasses were kappa.

[0078] 2.3.2 Western blot identification

[0079] ALV p27 protein was first subjected to polyacrylamide gel electrophoresis (SDS-PAGE), and then monoclonal antibodies 1G5 and 2C4 dilutions were used as primary antibodies, and HRP-labeled goat anti-mouse IgG dilutions were used as secondary antibodies for Western blot detection. The results showed that the two monoclonal antibodies reacted with ALV p27 protein to produce specific bands, indicating that the two monoclonal antibodies could recognize ALV p27 protein.

[0080] 2.3.3 Indirect immunofluorescence identification

[0081] The A, B, J subgroup avian leukemia virus was inoculated into the DF-1 cells which grew well in the 96-hole cell culture plate, and healthy cell control was set up, and the plate was cultured in a 37℃, 5% CO2 incubator for 7 days, and then was fixed with 80% cold acetone, and then was dried and stored in -20℃ for later use. When used, the plate was taken out, washed with PBS once, and then the monoclonal antibody 1G5 and 2C4 diluent was added, and the conventional IFA method was used for detection. The results showed that specific fluorescence was observed in the virus inoculation holes added with the monoclonal antibody 1G5 and 2C4, and no fluorescence was observed in the healthy cell control holes, indicating that the monoclonal antibody 1G5 and 2C4 could react with the A, B, and J subgroup avian leukemia virus.

[0082] 2.3.4 Specificity identification

[0083] The H5 subtype avian influenza hemagglutination inhibition test antigen, H7 subtype avian influenza hemagglutination inhibition test antigen, H9 subtype avian influenza hemagglutination inhibition test antigen, chicken infectious bursal disease virus antigen, chicken Newcastle disease virus hemagglutination inhibition test antigen, chicken Marek's disease virus, E. coli BL21(DE3) culture supernatant, E. coli BL21(DE3) bacterial lysis solution, E. coli BL21(DE3)-pET-30a culture supernatant, and E. coli BL21(DE3)-pET-30a bacterial lysis solution were all negative.

[0084] Example 3 Establishment of ALV double antibody sandwich ELISA antigen detection kit

[0085] 3.1 Preparation and identification of enzyme-labeled antibody

[0086] 3.1.1 Preparation

[0087] The monoclonal antibody 2C4 was labeled with horseradish peroxidase (HRP) by using the improved sodium periodate method. 20 mg of horseradish peroxidase (HRP) was dissolved in 1 ml of ultrapure water, 1 ml of freshly prepared NaIO4 solution (30 mg of NaIO4 was dissolved in 1 ml of ultrapure water, and it was prepared immediately before use) was added, and it was mixed well, and it was kept in the dark at 2-8℃ for 30 minutes; 40 μl of ethylene glycol was added to the above solution, and it was kept in the dark at 2-8℃ for 30 minutes; according to the proportion of 1 mg of purified monoclonal antibody plus 100 μl of the above mixture, the two were mixed well, then added to the dialysis bag, mixed well, and dialyzed with CB buffer for 6 hours. The whole operation needs to be carried out in the dark; the dialyzed mixture was transferred to a 1.5 ml EP tube, 10 μl of freshly prepared NaBH4 solution (20 mg of NaBH4 was dissolved in 1 ml of ultrapure water, and it was prepared immediately before use) was added, and it was kept at room temperature for 2 hours, and it was mixed well every 30 minutes; an equal volume of saturated ammonium sulfate was added, mixed well, and kept at 2-8℃ for 15 minutes. Centrifuged at 12000 r / min for 10 minutes, and the supernatant was discarded. The precipitate was suspended with an equal volume of PBS and glycerol mixture (V:V=1:1) as the purified antibody.

[0088] 3.1.2 Identification

[0089] Appearance: at room temperature, it is a red-brown liquid, and no flocculent precipitate is observed.

[0090] Quality evaluation: after 10-fold dilution of the enzyme-labeled antibody, the absorbance A of the enzyme-labeled antibody at 403 nm and 280 nm was detected by ultraviolet spectrophotometer. The corresponding enzyme parameters were calculated according to the formula:

[0091] Enzyme amount (mg / ml) = A 403nm × 0.4 × dilution factor (10 times).

[0092] IgG amount (mg / ml) = (A 280nm -A 403nm × 0.3) × 0.62 × dilution factor (10 times).

[0093] Molar ratio (E / P) = enzyme amount × 4 / IgG amount.

[0094] Labeling rate = A 403nm / A 280nm .

[0095] After the absorbance detection and calculation, the specific results are shown in Table 2:

[0096] Table 2 Quality evaluation results of enzyme-labeled antibody

[0097]

[0098] 3.2 Preparation of ALV double antibody sandwich ELISA antigen detection kit

[0099] Antigen-coated plate: the 1G5 monoclonal antibody prepared in Example 2 was diluted to 0.5 μg / ml with carbonate buffer (0.05 mol / L, pH 9.6) for coating, 100 μl / well, 2-8°C for 16-24 hours, washed with washing solution, 200 μl / well of blocking solution (sucrose 50 g, 200 ml of newborn calf serum, 0.5 ml of Proclin 300, supplemented with PBS (0.01 mol / L, pH 7.4) to 1000 ml) was added, 2-8°C for 16-24 hours, the blocking solution was discarded and dried for 4 hours, and then stored at 2-8°C after packaging.

[0100] Enzyme-labeled reagent: the enzyme-labeled monoclonal antibody 2C41 prepared in Example 3.1.1 was diluted 1:40000 with enzyme-labeled diluent (200 ml of newborn calf serum, 0.5 ml of Proclin 300, 0.5 ml of Tween 20, 0.04 g of AM dye, supplemented with PBS (0.01 mol / L, pH 7.4) to 1000 ml) as the enzyme-labeled reagent, and stored at 2-8°C.

[0101] Positive control: 100 ml of inactivated ALV J subgroup virus liquid or ALV p27 protein, 0.5 ml of Proclin 300, supplemented with PBS (0.01 mol / L, pH 7.4) to 1000 ml, mixed and filtered with a 0.22 μm filter, sterilely aliquoted and stored at 2-8°C as the positive control.

[0102] Negative control: 100 ml of DF-1 cell culture supernatant, 0.5 ml of Proclin 300, supplemented with PBS buffer (0.01 mol / L, pH 7.4) to 1000 ml, mixed and filtered with a 0.22 μm filter, sterilely aliquoted and stored at 2-8°C as the negative control.

[0103] 20x concentrated washing solution: sodium chloride 160 g, disodium hydrogen phosphate 58 g, potassium dihydrogen phosphate 4.8 g, potassium chloride 4 g, purified water 800 ml, Tween 20 10 ml, completely dissolved, made up to 1000 ml with purified water, mixed and filtered with a 0.22 μm filter, sterilely aliquoted. When used, dilute 20 times with purified water.

[0104] Color developing solution: disodium hydrogen phosphate 14.7 g, citric acid 9.3 g, urea peroxide 0.3 g, dissolved in purified water, made up to 1000 ml, mixed and sterilely aliquoted as color developing solution A. Tetramethylbenzidine (TMB) 0.2 g, anhydrous ethanol 10 ml, dissolved in purified water, made up to 1000 ml, mixed and sterilely aliquoted as color developing solution B.

[0105] Stop solution: 0.3% HF (hydrofluoric acid) solution.

[0106] The above components are assembled into a kit, which is stored at 2-8°C.

[0107] 3.3 Establishment of detection method

[0108] The detection steps are as follows:

[0109] (1) Numbering The sample corresponding microwell plate is numbered in order, and 2 wells of negative control (NC) and 2 wells of positive control (PC) should be set for each plate.

[0110] (2) Sample addition: 100 μl of each sample to be tested, 100 μl of negative and positive controls are added to the corresponding wells respectively, and gently shaken to mix. After sealing the plate, incubate at 37°C for 60 minutes.

[0111] (3) Washing: wash with washing solution for 4 times, and try to dry at the last time.

[0112] (4) Addition of enzyme-labeled reagent: add 100 μl of enzyme-labeled reagent to each well, and seal the plate after incubation at 37°C for 60 minutes.

[0113] (5) Washing: wash with washing solution for 4 times, and try to dry at the last time.

[0114] (6) Color development: add 50 μl of color developing solution A and color developing solution B to each well in turn, gently shake to mix, and incubate at 37°C for 15 minutes in the dark.

[0115] (7) Termination: add 50 μl of termination solution to each well, gently shake to mix, and measure the results within 10 minutes using an enzyme-labeled instrument.

[0116] (8) Measurement: single wavelength measurement, set the wavelength of the enzyme-labeled instrument at 630 nm, and measure the A value of each well.

[0117] (9) Result determination: calculate the S / P of the sample = (sample A value - average value of negative control A value) / (average value of positive control A value - average value of negative control A value), when S / P≥0.2, the sample is determined to be positive for avian leukosis virus p27 antigen; when S / P<0.2, the sample is determined to be negative for avian leukosis virus p27 antigen.

[0118] 3.4 Evaluation of ALV double antibody sandwich ELISA antigen detection kit

[0119] 3.4.1 Sensitivity

[0120] The kit prepared in Example 3.2 was used to detect ALV p27 protein gradient dilutions, and the results showed that the detection lower limit was 0.25 ng / ml. The detection of ALV A, B, C, D, and J group virus liquids were all positive.

[0121] The reagent kit prepared in Example 3.2 was used to detect 30 clinically collected ALV positive egg white samples, positive cloaca swab samples, positive semen samples, and positive meconium samples, and the results showed that all were positive. This indicates that the sensitivity of the reagent kit is good.

[0122] 3.4.2 Specificity

[0123] The reagent 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, chicken infectious bursal disease virus ELISA test antigen, chicken Newcastle disease virus hemagglutination inhibition test antigen, chicken Marek's disease virus, E. coli BL21(DE3) culture supernatant, E. coli BL21(DE3) cell lysate, E. coli BL21(DE3)-pET-30a culture supernatant, E. coli BL21(DE3)-pET-30a cell lysate, and 30 clinically collected ALV negative egg white samples, negative cloaca swab samples, negative semen samples, and negative meconium samples, and the results showed that all were negative, indicating that the specificity of the reagent kit is good.

[0124] 3.4.3 Reproducibility

[0125] Three batches of reagent kits were prepared according to Example 3.2, and each of the three batches was used to detect 3 ALV positive egg white samples for 5 times, and the intra-batch and inter-batch reproducibility was calculated. The results showed that the intra-batch and inter-batch reproducibility was not higher than 10%, and the reproducibility was good.

[0126] 3.4.4 Clinical application

[0127] According to the above results, three batches of reagent kits prepared according to Example 3.2 were used for clinical application, and 565 clinical samples were detected (of which 102 were positive and 463 were negative by PCR). The results showed that 95 were positive and 470 were negative by the reagent kit, the positive coincidence rate was 93.1%, the negative coincidence rate was 100%, and the total coincidence rate was 98.8%. This indicates that the sensitivity and specificity of the reagent kit are good, and it can be used for the detection of clinical samples.

[0128] Example 4 Determination of ALV p27 protein monoclonal antibody 1G5 and 2C4 variable region sequences

[0129] According to the sequence characteristics of the mouse-derived monoclonal antibody, the 1G5 heavy chain variable region primer sequence was designed as follows:

[0130] F1: 5'-ACTAGTCGACATGAACTTYGGG-3'

[0131] R1: 5'-CCAGGGRCCARKGGATARACN-3'

[0132] Design of 1G5 light chain variable region primer sequence:

[0133] F2: 5'-ACTAGTCGACATGGAGWCAGACA-3'

[0134] R2: 5'-CCCAAGCTTACTGGATGGTGGG-3'

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

[0136] F3: 5'-GGGAATTCATGRAATGSASCTG-3'

[0137] R3: 5'-CCAGGGRCCARKGGATARACN-3'

[0138] Design of 2C4 light chain variable region primer sequence:

[0139] F4: 5'-ACTAGTCGACATGAAGTTGCCTG-3'

[0140] R4: 5'-CCCAAGCTTACTGGATGGTGGG-3'

[0141] The hybridoma cells were collected, and after extraction of RNA, reverse transcription was performed as a template, and the variable region sequence was amplified using the above primers. The amplification product was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The results showed that the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibodies 1G5 and 2C4 were respectively as shown in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, and the gene sequences were respectively as shown in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8.

[0142] Example 5 Preparation and identification of single-chain antibodies 1G5 and 2C4

[0143] The heavy chain variable region (VH) gene and the light chain variable region (VL) gene of the monoclonal antibody were amplified, and after being moved into a connecting peptide, they were connected to the prokaryotic expression vector pET-32a(+). Recombinant plasmids were constructed, and BL21 (DE3) competent cells were transformed for expression to obtain fusion proteins. The variable region sequences of the monoclonal antibodies 1G5 and 2C4 were used to prepare the corresponding single-chain 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, and the results showed that the ELISA titers of the two single-chain antibodies were both ≥1: 51.2 million, and they had good reactivity.

[0144] The above results show that the variable region sequence 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.

[0145] The above merely provides the preferred embodiment of the present application, and not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall fall within the protection scope of the present application.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the p27 protein of avian leukosis virus, characterized in that, The antibody or its 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; Alternatively, 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.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment is in the form of Fab, Fab', F(ab')2, scFv or Fv fragment.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment is an IgG1, IgG2, IgG3, or IgG4 antibody.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment.

5. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment is a single-chain antibody.

6. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a monoclonal 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. The heavy chain subclass is IgG1, and the light chain subclass is kappa.

7. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a monoclonal 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. The heavy chain subclass is IgG1, and the light chain subclass is kappa.

8. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a single-chain antibody 1G5, with the amino acid sequence of the heavy chain variable region as shown in SEQ. ID NO.1 and the amino acid sequence of the light chain variable region as shown in SEQ. ID NO.

3.

9. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a single-chain antibody 2C4, with the amino acid sequence of the heavy chain variable region as shown in SEQ. ID NO.5 and the amino acid sequence of the light chain variable region as shown in SEQ. ID NO.

7.

10. A biomaterial relating to the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 9, characterized in that, The biomaterial is any one of the following: (a) A nucleic acid molecule containing sequences encoding a heavy chain variable region and a light chain variable region encoding the antibody or an antigen-binding fragment thereof; (b) Expression cassette containing the nucleic acid molecules in (a); (c) A recombinant vector containing the nucleic acid molecule in (a) or the expression cassette in (b); (d) Recombinant eukaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c); (e) Recombinant prokaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c).

11. The biomaterial according to claim 10, characterized in that, The nucleotide sequences encoding the heavy chain variable region are shown in SEQ. ID NO.2 and the nucleotide sequences encoding the light chain variable region are shown in SEQ. ID NO.4; Alternatively, the nucleotide sequence encoding the variable region of the heavy chain is shown in SEQ. ID NO.6 and the nucleotide sequence encoding the variable region of the light chain is shown in SEQ. ID NO.

8.

12. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 9 in the preparation of a product for detecting avian leukosis virus.

13. A double-antibody sandwich ELISA antigen detection kit for avian leukosis virus, characterized in that, The kit includes: a support medium coated with a coating antibody or its antigen-binding fragment, an enzyme-labeled antibody or its antigen-binding fragment, and a detection reagent; The coating antibody or its 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 its 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.

14. The kit according to claim 13, characterized in that, The coating antibody or its antigen-binding fragment is a monoclonal antibody 1G5; The enzyme-labeled antibody or its antigen-binding fragment is a monoclonal antibody 2C4.

15. The kit according to claim 14, 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.

16. The reagent kit according to claim 15, characterized in that, The supporting medium is a microtiter plate.

17. The kit according to claim 15, characterized in that, The enzyme labeled is horseradish peroxidase, alkaline phosphatase, or β-D-galactosidase.

18. The reagent kit according to claim 15, characterized in that, The monoclonal antibody 1G5 coating concentration is 50 ng / well, and the enzyme-labeled monoclonal antibody 2C4 working concentration is 1:40000.

19. The reagent kit according to claim 13, characterized in that, The detection reagents include a colorimetric solution and a stop solution.

20. The kit according to claim 19, characterized in that, The colorimetric solution includes colorimetric solution A and colorimetric solution B. Colorimetric solution A contains 1.47% w / v disodium hydrogen phosphate, 0.93% w / v citric acid and 0.03% w / v urea peroxide, and colorimetric solution B contains 0.02% w / v tetramethylbenzidine and 10% v / v anhydrous ethanol.

21. The reagent kit according to claim 19, characterized in that, The stop solution is a 0.3% HF solution.

22. The reagent kit according to claim 19, characterized in that, The kit also includes a positive control, a negative control, a supporting blocking solution, and a washing solution.

23. The reagent kit according to claim 22, characterized in that, Positive controls were PBS solutions containing 10% v / v inactivated ALV J subset virus or ALVp27 protein and 0.05% v / v Proclin 300.

24. The kit according to claim 22, characterized in that, The negative control was a PBS solution containing 10% v / v DF-1 cell culture supernatant and 0.05% v / v Proclin 300.

25. The reagent kit according to claim 22, characterized in that, The washing solution was phosphate buffer.

26. The reagent kit according to claim 22, characterized in that, The blocking solution was 0.01M PBS at pH 7.4 containing 5% w / v sucrose, 20% v / v newborn calf serum, and 0.05% v / v Proclin 300.

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