Recombinant protein, kit and ELISA method for detecting serotype 4 avian adenovirus

Abstract

The present invention discloses a recombinant protein, a kit and an ELISA detection method for detecting serotype 4 avian adenovirus. Specifically disclosed are a recombinant protein 100K (672-1053) having an amino acid sequence of SEQ ID No. 1 and its use as a detection antigen in the preparation of a product for detecting serotype 4 avian adenovirus. The present invention also discloses a FAdV-4 antibody detection kit and an ELISA detection method. The recombinant protein of the present invention specifically binds to FAdV-4 positive serum and has good reactivity. The detection kit and method established using the recombinant protein have strong specificity, no cross-reaction, high sensitivity, and good repeatability. At the same time, it can distinguish between animals immunized with inactivated vaccines and animals naturally infected to produce antibodies, and can identify FAdV-4 infection, provide technical support for the purification of FAdV-4, and provide reliable and effective means for the prevention and control of HHS diseases.

Description

Technical Field

[0001] The invention relates to a recombinant protein, a kit and an ELISA detection method for detecting serum type 4 avian adenovirus, and belongs to the field of biomedicine. Background Art

[0002] Hydropericardium hepatitis syndrome (HHS) is a new poultry disease with high infectiousness and high mortality caused mainly by fowl adenovirus serotype 4 (FAdV-4). Poultry infected with fowl adenovirus serotype 4 (FAdV-4) suffer from acute death, with the peak of death concentrated within 1 week. Dead poultry show typical symptoms of pericardial effusion and inclusion body hepatitis. In addition, FAdV-4 infection can lead to host immunosuppression, and the resulting immune failure, secondary infection, and mixed infection further aggravate the harm of hydropericardium hepatitis syndrome (HHS). FAdV-4 can infect chickens, ducks, ostriches, etc., mainly chickens, especially 3-7 week old broilers. Clinically, chickens with the disease show symptoms such as lethargy, ruffled feathers, and yellow-green loose feces. Autopsy shows typical lesions such as yellowish effusion in the pericardial cavity and swollen and yellow liver (Luan Yongjiao, Xie Zhixun, Wang Sheng, et al. Pathological observation of artificial infection of SPF chickens with serotype 4 avian adenovirus isolate from Guangxi [J]. Chinese Journal of Veterinary Science, 2020, 50(09): 1183-1192.). It has a high incidence rate, rapid mortality, and strong infectivity, causing huge economic losses to poultry production. At present, the detection methods for FAdV-4 include restriction endonuclease analysis (REA), DNA probe in situ hybridization, polymerase chain reaction (PCR), real-time fluorescence quantitative PCR and high-resolution melting curve (High-resolution melting, HRM), agar diffusion test, and enzyme-linked immunosorbent assay (ELISA) detection methods. However, the current detection methods still cannot meet the needs of clinical detection, and their sensitivity and specificity need to be further improved. The ELISA detection method is simple to operate, has good specificity and low cost, is widely used in clinical practice, and is suitable for large-scale testing of clinical samples. However, the conventional ELISA detection method mainly detects antibodies to the structural proteins of FAdV-4, which can be obtained through immunization or caused by natural infection. Therefore, it cannot distinguish between animals immunized with inactivated vaccines and naturally infected animals, and cannot detect and eliminate naturally infected animals in a timely manner.

[0003] 100K protein is one of the main non-structural proteins of FAdV-4. It does not constitute the structure of the virus particles, but plays an important role in the infection and replication of the virus. 100K protein is expressed in large quantities in the late stage of infection, which will stimulate the animal body to produce a large number of structural protein and non-structural protein antibodies. However, most of the non-structural proteins are removed in the inactivated vaccine. Therefore, the inactivated vaccine immunized animals do not produce or rarely produce non-structural protein antibodies. There are differences in the levels of non-structural protein antibodies in infected animals and inactivated vaccine immunized animals. The detection of non-structural protein antibodies can be used to identify naturally infected animals, providing a reliable and effective means for the prevention and control of HHS diseases. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to detect fowl adenovirus serotype 4 (FAdV-4) more specifically, sensitively, reliably and / or accurately, and / or how to distinguish animals naturally infected with fowl adenovirus serotype 4 and animals immunized with inactivated vaccines. The technical problem to be solved is not limited to the technical subject matter described, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0005] In order to solve the above technical problems, the present invention first provides a recombinant protein named 100K (672-1053), and the recombinant protein 100K (672-1053) can be any of the following:

[0006] A1) a protein whose amino acid sequence is SEQ ID No. 1;

[0007] A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No.1;

[0008] A3) A fusion protein having the same function is obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of A1) or A2).

[0009] The recombinant protein 100K (672-1053) is a truncated 100K protein (NCBI: ANV21399.1), and compared with the 100K protein, 671 amino acids at the N-terminus are truncated.

[0010] Proteins of different families and proteins with the same functions from different sources homologous to the protein 100K (672-1053) are all within the protection scope of the present invention.

[0011] As used herein, the terms “recombinant protein 100K (672-1053)”, “100K (672-1053) recombinant protein”, “100K (672-1053) protein”, “protein 100K (672-1053)”, “100K (672-1053)”, and “100K (672-1053) antigen” have the same meaning and can be used interchangeably.

[0012] In order to facilitate purification or detection of the protein in A1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing.

[0013] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0014] A person skilled in the art can easily mutate the nucleotide sequence encoding the recombinant protein 100K (672-1053) of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the recombinant protein 100K (672-1053) isolated from the present invention are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the recombinant protein 100K (672-1053) and have the function of the recombinant protein 100K (672-1053).

[0015] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0016] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

[0017] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0018] The present invention also provides a nucleic acid molecule, which may be any of the following:

[0019] B1) a nucleic acid molecule encoding the recombinant protein 100K (672-1053);

[0020] B2) the coding sequence is the DNA molecule shown in SEQ ID No. 2;

[0021] B3) The nucleotide sequence is the DNA molecule shown in SEQ ID No.2.

[0022] The amino acid sequence encoded by the DNA molecule shown in SEQ ID No. 2 is the recombinant protein 100K (672-1053) of SEQ ID No. 1.

[0023] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA.

[0024] The present invention also provides a biomaterial, which may be any one of the following C1) to C4):

[0025] C1) an expression cassette containing the nucleic acid molecule;

[0026] C2) a recombinant vector containing the nucleic acid molecule, or a recombinant vector containing the expression cassette described in C1);

[0027] C3) a recombinant microorganism containing the nucleic acid molecule, or a recombinant microorganism containing the expression cassette described in C1), or a recombinant microorganism containing the recombinant vector described in C2);

[0028] C4) a recombinant cell containing the nucleic acid molecule, or a recombinant cell containing the expression cassette of C1), or a recombinant cell containing the recombinant vector of C2).

[0029] The vectors described herein are well known to those skilled in the art, including but not limited to: plasmids, bacteriophages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 artificial chromosomes (PAC) or Ti plasmid artificial chromosomes (TAC), etc.), viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses or herpes viruses (such as herpes simplex virus), etc.), and the vectors can be specifically 20-T Vecter and / or pET-32a(+) vector.

[0030] Microorganisms described herein can be yeast, bacteria, algae or fungi. Wherein, bacteria can be from Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, etc.

[0031] The cells described herein refer to cells that can be used to introduce vectors, including but not limited to eukaryotic cells (such as yeast cells, Aspergillus), animal cells (such as mammalian cells, insect cells) or prokaryotic cells (such as Escherichia coli or Bacillus subtilis). The cells can specifically be Trans5α Chemically Competent Cells and / or BL(21)DE3 competent cells.

[0032] The recombinant vector can be specifically a recombinant vector pET-32a-100K (672-1053). The recombinant vector pET-32a-100K (672-1053) is a recombinant expression vector obtained by replacing the fragment (small fragment) between the EcoR I and Hind III recognition sites of the pET-32a (+) vector with a DNA fragment whose nucleotide sequence is SEQ ID No. 2 in the sequence list, while keeping the other nucleotide sequences of the pET-32a (+) vector unchanged. The recombinant vector pET-32a-100K (672-1053) expresses the recombinant protein 100K (672-1053) whose amino acid sequence is shown in SEQ ID No. 1.

[0033] The recombinant cell may specifically be a recombinant cell BL(21)DE3 / pET-32a-100K(672-1053).

[0034] The recombinant cell BL(21)DE3 / pET-32a-100K(672-1053) is a recombinant cell expressing the recombinant protein 100K(672-1053) obtained by introducing the recombinant vector pET-32a-100K(672-1053) into BL(21)DE3 competent cells. The recombinant cell BL(21)DE3 / pET-32a-100K(672-1053) contains the gene of the recombinant protein 100K(672-1053) shown in SEQ ID No.2.

[0035] The present invention also provides any of the following applications of the recombinant protein 100K (672-1053), and / or the nucleic acid molecule, and / or the biomaterial:

[0036] D1) Use in detecting serotype 4 avian adenovirus or in preparing a product for detecting serotype 4 avian adenovirus;

[0037] D2) Use in the diagnosis or auxiliary diagnosis of diseases caused by serotype 4 avian adenovirus infection or in the preparation of products for the diagnosis or auxiliary diagnosis of diseases caused by serotype 4 avian adenovirus infection;

[0038] D3) Use in screening or assisting screening for diseases caused by serotype 4 avian adenovirus infection or in preparing products for screening or assisting screening for diseases caused by serotype 4 avian adenovirus infection;

[0039] D4) Use in distinguishing animals naturally infected with serotype 4 avian adenovirus and animals immunized with inactivated vaccines, or in preparing products for distinguishing animals naturally infected with serotype 4 avian adenovirus and animals immunized with inactivated vaccines;

[0040] D5) Use in the preparation of a detection antigen for detecting serotype 4 avian adenovirus;

[0041] D6) Use in the preparation of serotype 4 avian adenovirus antibodies;

[0042] D7) in the prevention and control of diseases caused by serotype 4 avian adenovirus infection.

[0043] Furthermore, the disease caused by the infection with Fowl adenovirus serotype 4 (FAdV-4) may be Hydropericardium hepatitis syndrome (HHS).

[0044] Furthermore, the product may be a reagent or a kit.

[0045] The present invention also provides a kit for detecting serum type 4 avian adenovirus, the kit comprising the recombinant protein 100K (672-1053).

[0046] Furthermore, the kit may be an enzyme-linked immunosorbent assay kit.

[0047] The present invention also provides a method for preparing the kit, which comprises the steps of preparing the recombinant protein 100K (672-1053) into a 6 μg / mL solution and coating the solution on a solid phase carrier to obtain an antigen-coated solid phase carrier.

[0048] In the above method, the conditions for coating the solid phase carrier may be placing at 37° C. for 2 hours and then placing at 4° C. for 14 hours.

[0049] The coating means that the antigen is bound to the solid phase carrier through physical adsorption.

[0050] Furthermore, the solid phase carrier may be an ELISA plate, a membrane carrier, a microsphere, a biochip or a magnetic bead but is not limited thereto.

[0051] The material of the solid phase carrier may be polystyrene, cellulose, cross-linked dextran, polyacrylamide, polyethylene, polypropylene, polyvinyl chloride, cross-linked dextran, glass, silicone rubber or agarose gel, but is not limited thereto.

[0052] The membrane carrier may be a nitrocellulose membrane, a glass cellulose membrane or a nylon membrane but is not limited thereto.

[0053] The name of the above-mentioned kit can be an indirect ELISA detection kit for avian adenovirus serum type 4 or a FAdV-4 ELISA detection kit.

[0054] Furthermore, the kit also includes a coating solution, a standard positive serum, a standard negative serum and an enzyme-labeled secondary antibody.

[0055] The enzyme-labeled secondary antibody refers to a second antibody that is labeled with an enzyme and can specifically recognize and bind to the antibody to be tested. The enzyme used for labeling can be horseradish peroxidase (HRP), alkaline phosphatase (AKP), β-galactosidase (β-Galactosidase), glucose oxidase (GOD), acid phosphatase but is not limited thereto.

[0056] Furthermore, the kit also includes a sample diluent, an enzyme-labeled antibody diluent, a substrate color developing solution, a stop solution and a washing solution.

[0057] Furthermore, the enzyme-labeled secondary antibody in the kit can be an HRP-labeled goat anti-chicken IgG antibody.

[0058] Furthermore, the kit also includes a readable carrier recording the method described herein for detecting antibodies to serotype 4 avian adenovirus. The readable carrier may be a kit instruction sheet (e.g., a printed instruction sheet) for practicing the method of the present invention or a computer-readable medium (e.g., a floppy disk, a CD, etc.) on which information has been recorded.

[0059] The preparation method of the ELISA plate coated with the antigen of the present invention (recombinant protein 100K (672-1053)) is as follows: the antigen (purified recombinant protein 100K (672-1053)) is diluted with ELISA coating solution and then coated with the ELISA plate (100 μL per well), the antigen coating concentration is 6 μg / mL, and the coating conditions are: standing at 37°C for 2 hours and then coating at 4°C for 14 hours; after washing the plate with PBST, 200 μL of blocking solution (5% skim milk) is added to each well, and blocked at 37°C for 60 minutes.

[0060] The standard positive serum of the present invention is the positive serum of chicken infected with FADV-4, the standard negative serum is the negative serum of SPF chicken, the sample diluent is a 1% BSA solution, the enzyme-labeled antibody diluent is a 1% BSA solution, the substrate color developing solution is a TMB color developing solution, the stop solution is 2 mol / L H2SO4, and the washing solution is PBST (trade name 1×PBST buffer, company Beijing Solebow Technology Co., Ltd., item number P1031).

[0061] The invention also provides application of the kit in detecting serum type 4 avian adenovirus antibodies.

[0062] The present invention also provides a primer pair for amplifying the gene of the recombinant protein 100K (672-1053), wherein the primer pair can be composed of primer 100K (672-1053) -F and primer 100K (672-1053) -R, wherein the primer 100K (672-1053) -F can be a single-stranded DNA molecule shown in SEQ ID No. 3; and the primer 100K (672-1053) -R can be a single-stranded DNA molecule shown in SEQ ID No. 4.

[0063] The present invention also provides a method for detecting serum type 4 avian adenovirus antibodies, wherein the method uses the recombinant protein 100K (672-1053) as an antigen and adopts an ELISA indirect method to detect serum type 4 avian adenovirus antibodies in a sample to be tested.

[0064] In the above method, the method may include the following steps:

[0065] E1) adding a sample to be tested to any of the kits described herein;

[0066] E2) adding the sample to be tested and incubating for 30 min;

[0067] E3) adding enzyme-labeled secondary antibody and incubating for 30 min, wherein the enzyme-labeled secondary antibody is HRP-labeled goat anti-chicken IgG antibody;

[0068] E4) Add enzyme reaction substrate to develop color.

[0069] In the above method, the sample to be tested may be serum.

[0070] Furthermore, in one embodiment of the present invention, the method of the present invention comprises the following steps:

[0071] 1. Antigen-coated ELISA plate: Dilute the antigen (purified recombinant protein 100K (672-1053)) with ELISA coating solution (Beijing Solebow Technology Co., Ltd., product number C1050) and coat the ELISA plate (100 μL per well). The antigen coating concentration is 6 μg / mL. The coating conditions are: incubate at 37°C for 2 hours and then coat at 4°C for 14 hours.

[0072] 2. Washing: discard the coated antigen in the ELISA plate, pat dry on absorbent paper, fill the well with washing solution (PBST), let it stand for 2-3 minutes, pat dry on absorbent paper, and wash 3 times;

[0073] 3. Blocking: Add 200 μL of blocking solution (5% skim milk) to each well and block for 60 min at 37°C;

[0074] 4. Wash the plate: same as step 2;

[0075] 5. Add the sample to be tested: dilute the sample serum (primary antiserum) to be tested with diluent (1% BSA) at a ratio of 1:100, add 100 μL to each well of the ELISA plate, and set up positive serum control, negative serum control and blank control, and incubate at 37°C for 30 minutes;

[0076] 6. Wash the plate: same as step 2;

[0077] 7. Add enzyme-labeled secondary antibody: dilute the enzyme-labeled secondary antibody (HRP-labeled goat anti-chicken IgG antibody) at a ratio of 1:5000 with diluent (1% BSA), add it to the ELISA plate, 100 μL per well, and incubate at 37°C for 30 minutes;

[0078] 8. Wash the plate: same as step 2;

[0079] 9. Add substrate for color development: add 100 μL of TMB color development solution (Beijing Solebow Technology Co., Ltd., product number PR1201) to each well and incubate at 37°C for 11 min;

[0080] 10. Stop the reaction: Add 50 μL of stop solution (2 mol / L H2SO4) to each well to stop the reaction, and finally detect with an ELISA reader.

[0081] Result judgment: negative and positive cut-off value (cut-off) is: 0.346, that is, when D 450nm When the value is greater than or equal to 0.346, it is judged as positive (indicating that the sample to be tested contains FAdV-4 antibodies); D 450nm When the value is less than 0.346, it is judged as negative (indicating that the sample to be tested does not contain FAdV-4 antibodies).

[0082] The purposes of the above-mentioned applications and methods may be disease diagnosis, disease prognosis and / or disease treatment, and their purposes may also be non-disease diagnosis, non-disease prognosis and non-disease treatment purposes; their direct purpose may be to obtain information on intermediate results of disease diagnosis results, disease prognosis results and / or disease treatment results, and their direct purpose may be non-disease diagnosis, non-disease prognosis and / or non-disease treatment purposes.

[0083] The sample to be tested in the above application may be a sample from a non-living human or animal body, such as an environmental sample (such as air) or a food (such as frozen food or fresh food).

[0084] Herein, the term enzyme linked immunosorbent assay (ELISA) refers to adsorbing an antigen or antibody onto a solid phase carrier, and in the detection process, the antibody (or antigen) to be tested and the enzyme marker are sequentially added to the reaction system in a sequential order, so that they react with the antigen (or antibody) on the solid phase carrier to form an antigen-antibody complex, and then the unbound free enzyme marker and free antigen (or antibody) are washed away, and the enzyme activity of the bound enzyme marker is measured to determine the content of the antibody (or antigen) to be tested in the sample. ELISA includes direct method, indirect method, double antibody sandwich method, competitive method and anti-enzyme antibody method. Although the embodiment provided by the present invention adopts the ELISA indirect method to detect serum type 4 avian adenovirus antibodies, the present invention is not limited to this specific method. Those skilled in the art may adopt other methods, as long as the method of combining the immune reaction of the antigen (100K (672-1053)) of the present invention with the antibody and the efficient catalytic action of the enzyme does not depart from the scope of the present invention, and the present invention should include these alternative methods.

[0085] In order to establish an ELISA detection method for identifying serotype 4 fowl adenovirus (FAdV-4) infection, the present invention designs and screens a pair of specific primers based on the FAdV-4 non-structural protein 100K gene sequence on GenBank, connects the PCR amplification product to the pET-32a (+) expression vector, successfully constructs the pET-32a-100K (672-1053) recombinant plasmid, transfers it into BL (21) DE3 competent cells, and obtains efficient expression through IPTG induction to obtain the recombinant protein 100K (672-1053). Western-blot identification results show that the 100K (672-1053) recombinant protein specifically binds to FAdV-4 positive serum and has good reactivity. The FAdV-4 non-structural protein 100K (672-1053) recombinant protein is used as an ELISA coating antigen, the reaction conditions are optimized, and an indirect ELISA detection method (referred to as 100K-ELISA method) for detecting FAdV-4 antibodies is established. This method specifically binds to positive serum infected with FAdV-4, has no cross-reaction with other positive serums of poultry diseases, and has good specificity; positive serum diluted 1280 times can still be detected by 100K-ELISA method, with high sensitivity; the maximum coefficient of variation within and between batches of 100K-ELISA method is less than 10%, with good repeatability. 50 negative serum samples of SPF chickens artificially infected with FAdV-4 were detected by 100K-ELISA method, and the results were all positive; while when 50 negative serum samples of SPF chickens immunized with FAdV-4 inactivated vaccine were detected, the results were all negative, indicating that the 100K-ELISA method can distinguish between chickens immunized with inactivated vaccine and chickens that produce antibodies naturally, and can identify FAdV-4 infection, providing technical support for the purification of FAdV-4, and providing a reliable and effective means for the prevention and control of HHS diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 This is the physicochemical property analysis curve of 100K (672-1053) protein.

[0087] Figure 2 The PCR amplification gel imaging system observation results of the 100K (672-1053) protein gene, where M represents DL2000 Marker, and 1-4 represents the amplified products of the 100K (672-1053) protein gene.

[0088] Figure 3 The double enzyme digestion identification result of the recombinant plasmid (pET-32a-100K (672-1053)), wherein M represents DNA Marker, and 1 represents the double enzyme digestion product of pET-32a-100K (672-1053).

[0089] Figure 4The SDS-PAGE electrophoresis results of the induced expression of the recombinant protein 100K (672-1053). Figure 4 A in the middle represents the expression products at different induction times. M represents protein marker; 1 represents empty bacteria; 2-7 represent the expression products at induction times of 2, 4, 6, 8, 10, and 12 h, respectively. Figure 4 B in the figure indicates the expression products at different IPTG concentrations. M indicates protein marker; 1 indicates empty bacteria; 2-7 indicate the expression products at IPTG concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mmol / L, respectively.

[0090] Figure 5 It is the solubility identification of the recombinant protein 100K (672-1053). M represents protein marker, 1 represents whole bacteria, 2 represents supernatant of induced bacterial solution, and 3 represents precipitate of induced bacterial solution.

[0091] Figure 6 The purification results of the recombinant protein 100K (672-1053) are shown in Figure 1. M represents protein marker, 1 represents empty bacterial cell control, 2 represents protein before purification, and 3-4 represents protein after purification.

[0092] Figure 7 This is the Western-blot identification of the recombinant protein 100K (672-1053). M represents the protein molecular weight standard, 1 represents the recombinant protein 100K (672-1053), and 2 represents the negative control.

[0093] Figure 8 This is the specificity test result of the FAdV-4 ELISA detection method in Example 4.

[0094] Fig. 9 This is the sensitivity test result of the FAdV-4 ELISA detection method in Example 4.

[0095] Fig.10 This is the sample identification test result of the FAdV-4 ELISA detection method in Example 4. DETAILED DESCRIPTION

[0096] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0097] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0098] The FAdV-4 isolate GX2019-010 in the following examples is recorded in the following document: Luan Yongjiao, Xie Zhixun, Wang Sheng, Luo Sisi, Zhang Lei, Xie Liji, Xie Zhiqin, Deng Xianwen, Zhang Minxiu, Zhang Yanfang, Zeng Tingting, Fan Qing. Whole genome sequencing and analysis of serotype 4 avian adenovirus Guangxi isolate GX2019-010 [J / OL]. Chinese Journal of Animal Husbandry and Veterinary Medicine: 1-12 [2022-06-13]. http: / / kns.cnki.net / kcms / detail / 11.4843.S.20200806.1044.002.html. The public can obtain the biological material from the applicant, which is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0099] The positive serum of FAdV-4 infected chicken (FAdV-4 positive serum for short), serum sample of chicken immunized with FAdV-4 inactivated vaccine and negative serum of SPF chicken in the following embodiments are collected and stored by the laboratory where the inventor of the present application is located; the positive serum of Newcastle disease virus (NDV), avian reovirus (ARV), H5 subtype avian influenza virus (AIV-H5), H9 subtype avian influenza virus (AIV-H9), infectious bursal disease virus (IBDV) and infectious bronchitis virus (IBV) are stored by the laboratory where the inventor of the present application is located; the public can obtain the above-mentioned biological materials from the applicant, which are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0100] The clinical samples in the following examples are 96 serum samples of chickens immunized with FAdV-4 inactivated vaccine and 217 serum samples of non-immunized chickens collected from Guangxi Fufeng Chicken Farm. The public can obtain the above biological materials from the applicant, which are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0101] In the following examples, 2× -T PCR SuperMix was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0102] The carrier in the following examples 20-T vector was purchased from Takara Company, and pET-32a(+) vector was purchased from Beijing Solebow Technology Co., Ltd.

[0103] The Trans5α Chemically Competent Cell and BL(21)DE3 competent cells in the following examples were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0104] Example 1. Preparation of recombinant protein antigen for detecting serotype 4 avian adenovirus

[0105] In this embodiment, the recombinant protein antigen used to detect serotype 4 fowl adenovirus (FAdV-4) is a truncated protein of the non-structural protein 100K of serotype 4 fowl adenovirus (FAdV-4), named 100K (672-1053), which is expressed by the Escherichia coli prokaryotic expression system.

[0106] The recombinant protein 100K (672-1053) is a truncated 100K protein (NCBI: ANV21399.1), which has 671 amino acids at the N-terminus truncated compared with the 100K protein.

[0107] The amino acid sequence of the recombinant protein 100K (672-1053) is SEQ ID No. 1, and the coding sequence (CDS) of the recombinant protein 100K (672-1053) is SEQ ID No. 2.

[0108] 1. Cloning of FAdV-4 100K (672-1053) protein gene

[0109] 1-1. Primer design

[0110] Referring to the gene sequence of FAdV-4 nonstructural protein 100K published in GenBank, the amino acid sequence of 100K protein was analyzed using Protean software in DNAStar7.1. The potential epitope regions were analyzed by comprehensively considering antigenicity, hydrophilicity, hydrophobicity and surface accessibility ( Figure 1 ). Specific primers were designed using Primer 5.0, and appropriate restriction sites were designed at the 5′ end according to the multiple cloning site of the expression vector. The primers were synthesized by BGI Biotechnology Co., Ltd. The specific information of the primers is shown in Table 1.

[0111] Table 1. Specific primer sequences for amplifying FAdV-4 100K (672-1053) protein gene

[0112]

[0113] 1-2-1. Viral DNA extraction

[0114] According to the instructions of the DNA / RNA co-extraction kit (product of Beijing Quanshijin Biotechnology Co., Ltd.), the DNA (FAdV-4DNA) of the FAdV-4 Guangxi isolate (GX2019-010) was extracted and placed in a -35°C refrigerator for use.

[0115] 1-2-2. PCR amplification of 100K (672-1053) protein gene

[0116] Using the FAdV-4DNA extracted in step 1-2-1 as a template, amplify the 100K (672-1053) protein gene. The PCR reaction system and reaction procedure are shown in Tables 2 and 3. Four systems are prepared for gene amplification of each sample, i.e., 200 μL (50 μL for each system). After the PCR reaction is completed, the reaction product is electrophoresed on a 1% agarose gel at 80V, 60min, and DL2000 Marker is used as a control. The results observed by the gel imaging system are as follows Figure 2 As shown, the results showed that the size of the 100K (672-1053) protein gene amplification band was 1149 bp, which was consistent with expectations.

[0117] Table 2. PCR amplification system of 100K (672-1053) protein gene

[0118]

[0119] Table 3. PCR reaction procedure of 100K (672-1053) protein gene

[0120]

[0121] 1-2-3. Recovery of PCR products

[0122] The PCR product was recovered using a gel recovery kit (product of OMEGA). The specific steps are as follows:

[0123] (1) Cut the target band completely, place it in a 2 mL EP tube, weigh it, and add binding buffer at a ratio of 1 g / mL. Place it in a 55560℃ water bath for nearly 7 minutes, shaking it every 253 minutes until the gel is completely melted.

[0124] (2) Place the centrifuge column on the collection tube, add 650 μL of gel mixture to the centrifuge tube, centrifuge at 10,000 × g for 1 min, and discard the liquid in the collection tube until all the liquid has filtered through the centrifuge column.

[0125] (3) Add 300 μL binding buffer, centrifuge at 12,000 × g for 1 min, and discard the liquid in the collection tube.

[0126] (4) Add 700 μL SPW Wash Buffer, centrifuge at 12,000 × g for 1 min, discard the collected solution, and repeat this step twice.

[0127] (5) Centrifuge the empty centrifuge tube at 12,000 × g for 2 min to dry the centrifuge column. Open the centrifuge tube lid and let it stand for 2 min to fully evaporate the ethanol.

[0128] (6) Transfer the centrifuge column to a clean EP tube, add 30 μL of deionized water to the center of the column membrane, let it stand for 2 min, centrifuge at maximum speed for 1 min, and store the eluted DNA (i.e., the recovered product: 100K (672-1053) protein gene DNA) at -35°C for later use.

[0129] 1-2-4. PCR products and cloning vectors 20-T Vecter Connection

[0130] Take 4 μL of the recovered product into an EP tube and add 1 μL 20-T Vecter, then add 5μL T4 ligation Mix (Takara), mix gently, and connect at 16℃ overnight (14h). Add all the ligation products to 100μL Trans5α Chemically Competent Cell, ice bath for 30min, heat shock in 42℃ water bath for 45s, ice bath for 2min, add 500μL sterile LB medium, mix well, place in 37℃ shaker, culture at 200rpm for 1h to recover the bacteria. Pipette 200μL of transformed competent cells and add them to LB plate containing ampicillin (Amp), 5-bromo-4-chloro-3-indole-β-D-galactoside (X-gal), isopropyl-β-D-thiogalactoside (IPTG), spread evenly, place the plate in a 37℃ incubator, invert the plate, and culture overnight. White colonies were picked by blue-white colony screening, inoculated into 1 mL of LB medium containing Amp, and cultured at 37°C, 200 rpm for 4 h. After PCR detection, the positive bacterial solution was sent to Bio-Tech for sequencing to verify whether the target gene was successfully inserted into the vector.

[0131] 1-2-5. Extraction of recombinant plasmid

[0132] The correctly sequenced bacterial solution was diluted 1:100 and inoculated into LB medium containing Amp. The culture was incubated overnight at 37°C and 200 rpm. The plasmid was extracted using a plasmid extraction kit (product of OMEGA) as follows:

[0133] (1) Centrifuge the overnight culture at 4000 × g for 10 min and discard the supernatant.

[0134] (2) Add 500 μL of Solution I, vortex to mix thoroughly, and transfer the suspension to a new 2 ml centrifuge tube.

[0135] (3) Add 500 μL Solution II and gently invert the tube several times to mix to obtain a clear lysate.

[0136] (4) Add 700 μL of Solution III and immediately invert to mix until white flocs are produced.

[0137] (5) Centrifuge at 12,000 × g for 10 min at room temperature.

[0138] (6) Transfer the supernatant to a centrifuge column and centrifuge at maximum speed for 1 min. Discard the flow-through and repeat this step until all the supernatant has passed through the column.

[0139] (7) Add 500 μL HBC buffer, centrifuge at 12,000 × g for 1 min, discard the filtrate, and reuse the collection tube.

[0140] (8) Add 700 μL DNA washing buffer, centrifuge at 12,000 × g for 1 min, discard the solution, and repeat twice.

[0141] (9) Centrifuge the empty tube for 2 minutes to dry the centrifuge column.

[0142] (10) Transfer the centrifuge column to a new EP tube, add 100 μL of sterile deionized water, centrifuge at maximum speed for 1 min, measure the plasmid concentration, and store at -35°C.

[0143] The extracted recombinant plasmid was named 20-T-100K (672-1053), which contained the 100K (672-1053) protein gene with the nucleotide sequence of SEQ ID No. 2.

[0144] 2. Construction of prokaryotic expression vector of FAdV-4 100K (672-1053) protein gene

[0145] According to the restriction sites (EcoR I and Hind III) selected in Table 1, refer to the Takara endonuclease instructions, the recombinant plasmid 20-T-100K (672-1053) and pET-32a (+) vector were double-digested, 50 μL system, 37 ° C for 3h. After the digestion products were separated by 0.8% agarose gel electrophoresis, the target fragment (100K (672-1053) protein gene) and the vector backbone were recovered, and the target fragment and the vector backbone were connected at a ratio of 10:1 at 16 ° C overnight. The ligation product was transferred to BL (21) DE3 competent cells, plated and cultured overnight, and the colonies were picked for PCR identification. The positive colonies were selected for double digestion identification (identification results are shown in Figure 3The size of the enzyme-cut fragment was consistent with the size of the target gene) and sent to Sangon Biotechnology for sequencing. The sequencing results were compared and analyzed using the Clustal W method in the MegAlign software to verify whether the fragment was correctly connected to the expression vector. The sequencing results showed that the target fragment was successfully inserted into the pET-32a(+) vector and the recombinant vector was successfully constructed. After sequencing identification, the positive recombinant vector was screened and named pET-32a-100K(672-1053).

[0146] The recombinant vector pET-32a-100K (672-1053) is a recombinant expression vector obtained by replacing the fragment (small fragment) between the EcoR I and Hind III recognition sites of the pET-32a (+) vector with a DNA fragment having a nucleotide sequence of SEQ ID No. 2 in the sequence table, while keeping the other nucleotide sequences of the pET-32a (+) vector unchanged. The recombinant vector pET-32a-100K (672-1053) expresses the recombinant protein 100K (672-1053) having an amino acid sequence as shown in SEQ ID No. 1.

[0147] The recombinant vector pET-32a-100K(672-1053) was introduced into BL(21)DE3 competent cells to obtain a recombinant cell expressing the recombinant protein 100K(672-1053), which was named BL(21)DE3 / pET-32a-100K(672-1053). The recombinant cell BL(21)DE3 / pET-32a-100K(672-1053) contained the gene of the recombinant protein 100K(672-1053) shown in SEQ ID No.2.

[0148] 3. Induced expression and purification of recombinant proteins

[0149] 3-1. Induced expression of recombinant protein

[0150] The positive bacterial solution with correct sequencing (i.e., the bacterial solution obtained by culturing the positive colonies with correct sequencing in step 2 in LB liquid medium) was inoculated into 10 mL of LB liquid medium containing Amp at a ratio of 1%, and cultured at 37°C, 200 rpm for 14 h as seed night. The seed solution was inoculated into 12 mL of LB liquid medium at a volume ratio of 1:100 for expansion culture, and divided into 6 test tubes (2 ml in each test tube), and cultured at 37°C, 200 rpm for 4 h until D 600nmWhen the concentration was between 0.65 and 0.8, IPTG was added to the final concentration of 0.2mmol / L, 0.4mmol / L, 0.6mmol / L, 0.8mmol / L, and 1mmol / L, respectively, and induced for 6 hours. 1mL of bacterial solution was taken, 12000×g, 2min, and the supernatant was discarded. 100μL of high-efficiency RIPA lysis solution (Beijing Solebow Technology Co., Ltd.) was added to the precipitate, mixed and placed on ice for 20min, 30Hz ultrasound for 3min, boiled for 10min, ice bathed for 10min, centrifuged, and the supernatant was taken for SDS-PAGE electrophoresis, 65V, 35min, and then 110V, 30min. The electrophoresis bands were analyzed with BandScan 4.3 software to select the best IPTG concentration. Repeat the above operation, induce with the best IPTG concentration for 2h, 4h, 6h, 8h, 10h, and 12h, and determine the best induction time by SDS-PAGE electrophoresis analysis.

[0151] After IPTG induction, the recombinant protein was well expressed, and the recombinant protein 100K (672-1053) (67kDa) was consistent with the expected size. The results of SDS-PAGE electrophoresis analyzed by BandScan 4.3 software showed that the recombinant protein 100K (672-1053) was expressed at the best level after 4h of induction at an IPTG concentration of 0.4mmol / L ( Figure 4 ).in Figure 4 A in the middle represents the expression products at different induction times. M represents protein marker; 1 represents empty bacteria; 2-7 represent the expression products at induction times of 2, 4, 6, 8, 10, and 12 h, respectively. Figure 4 B in the figure indicates the expression products at different IPTG concentrations. M indicates protein marker; 1 indicates empty bacteria; 2-7 indicate the expression products at IPTG concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mmol / L, respectively. Figure 4 Lane 3 of A shows that the expression level of the recombinant protein 100K (672-1053) is optimal when the induction time is 4 h; Figure 4 Lane 4 in B shows that the expression level of the recombinant protein 100K (672-1053) is optimal when the IPTG concentration is 0.4 mmol / L.

[0152] 3-2. Identification of the solubility of recombinant protein

[0153] After induction under the optimal induction conditions (IPTG concentration of 0.4mmol / L, induction time of 4h), 2mL of bacterial solution was centrifuged, the bacterial sludge was washed twice with PBS, 200μL PBS was added to resuspend the precipitate, lysozyme (final concentration 100μg / mL) was added, and the mixture was repeatedly frozen and thawed three times between -80℃ and 37℃, and ultrasonically lysed on ice until the bacterial solution was clear, 12000×g, 2min, the supernatant and the precipitate were separated, the precipitate was dissolved with inclusion body dissolution solution, loading buffer was added, 30μL / well was loaded for SDS-PAGE electrophoresis, and the expression form of the recombinant protein was analyzed. If the expression product is in the supernatant, it is soluble expression, and if the expression product is in the precipitate, it is inclusion body expression. The results are as follows Figure 5 As shown, after ultrasonication, the recombinant protein 100K (672-1053) was separated from the precipitate supernatant and analyzed by SDS-PAGE electrophoresis. The results showed that the expression product was mainly in the precipitate, which was inclusion body expression.

[0154] 3-3. Purification of recombinant protein

[0155] Preparation of inclusion body protein purification buffer: Prepare Binding Buffer with 20mmol / L Tris-HCl (pH 7.9), 5mmol / LImidazole, 0.5mol / L NaCl, and 8mol / L urea; increase the imidazole concentration based on Binding Buffer to prepare Elution Buffer with imidazole concentrations of 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, and 500mmol / L respectively.

[0156] Assembly of the chromatography column: Add 3 mL of Ni-Agarose Resin filler to the chromatography column, let it stand to separate the layers, open the bottom outlet, rinse the ethanol in the filler with 15 mL of deionized water after the ethanol flows out, and equilibrate the column with 24 mL of Binding Buffer.

[0157] Treatment and purification of bacterial solution: 600mL bacterial solution was induced under the optimal induction conditions, centrifuged at 10000×g for 10min, bacterial mud was collected, 20mL bacterial lysis solution was added, mixed and placed in ice water for lysis for 30min, and the bacteria were lysed by ultrasonication in an ice bath, 50Hz, 5s intervals for each ultrasonication, and ultrasonication until the liquid was no longer viscous. Centrifuged at 4℃, 10000×g for 15min, the precipitate was collected, and 200μL of the supernatant was reserved for testing. The precipitate was resuspended with 10mL Binding Buffer and shaken at room temperature for 20min to fully dissolve the inclusion bodies. Centrifuged at 10000×g for 15min, the supernatant was collected and loaded on the column, mixed thoroughly with the filler, placed on ice and mixed on a rocking platform for 20min to fully combine the protein with the filler. Let stand for stratification and collect the flow-through. Use 15 times the volume of the filler to wash away the impurities. Each gradient was eluted with 1 column volume of Elution Buffer, and the eluate was collected in separate tubes. The purity of the target protein was analyzed by SDS-PAGE electrophoresis ( Figure 6 ). The column was washed with Binding Buffer and deionized water, then sealed with 20% ethanol and stored at 2-8°C.

[0158] Renaturation of inclusion body protein: Select the gradient elution protein with higher purity and put it into the treated dialysis bag. Place the dialysis bag in the renaturation buffer (1×PBS, 1mmol / L ethylenediaminetetraacetic acid (EDTA), urea, pH 8.0), place it on a magnetic stirrer, and slowly dialyze at 4°C. The urea concentration of the renaturation buffer is 4mol / L, 3mol / L, 2mol / L, 1mol / L, and 0mol / L, respectively. The solution is changed every 10512h. After the dialysis, the concentration of the purified protein is determined using the BCA protein concentration determination kit, and the purified protein is stored in aliquots. Finally, the purified recombinant protein 100K (672-1053) is obtained. The results are as follows Figure 6 As shown, after the recombinant protein 100K (672-1053) was purified by nickel column affinity chromatography, the purification effect was detected by SDS-PAGE electrophoresis, and a single specific band was obtained. The purification effect was good, and the concentration was determined by BCA protein concentration determination kit to be 192 mg / L.

[0159] 3-4. Western-blot identification of recombinant protein

[0160] 3-4-1. Reagent preparation

[0161] (1) Transfer buffer (100 mL): weigh 0.29 g of glycine and 0.58 g of Tris, add 80 mL of deionized water, and then add 20 mL of methanol, mix well and set aside.

[0162] (2) Blocking buffer: Weigh 0.5 g skim milk powder and add it to 10 mL of 1× TBST solution (Beijing Solebow Technology Co., Ltd., catalog number T1085), and stir thoroughly to dissolve.

[0163] 3-4-2. Transfer

[0164] After SDS-PAGE electrophoresis, remove the gel, cut off the excess part, and soak it in transfer buffer. Prepare absorbent paper and polyvinylidene fluoride (PVDF) membrane, soak in transfer buffer for 10 minutes. Make a "sandwich" transfer block from bottom to top with "paper-membrane-gel-paper", flatten it, remove all bubbles, put it in the electrotransfer tank, cover it, and transfer the membrane with 0.5mA current for 30 minutes. After the transfer, remove the membrane and seal it with 5% skim milk for 3 hours.

[0165] 3-4-3. Immunohistochemical hybridization and color development

[0166] After blocking, put it in TBST solution, wash 3 times, 10 minutes each time, and then wash it once with Tris buffered saline solution (TBS, Beijing Solebow Technology Co., Ltd.) for 10 minutes, with an oscillation frequency of 80 times / min. The subsequent membrane washing is the same. Dilute FAdV-4 positive serum (primary antibody) with 0.5% skim milk (prepared with TBST) at a ratio of 1:200, block with disposable plastic film, and incubate at 4°C overnight. Discard the primary antibody and wash away the unbound antibody. Add the diluted HRP-labeled goat anti-chicken IgG antibody as the secondary antibody (1:2000, Sangon Biotech (Shanghai) Co., Ltd.), and incubate at 37°C for 1h. Wash away the unbound secondary antibody. After washing the membrane, take it out, lay the membrane flat with the protein side facing up in the color development box, add ECL working diluent (component in the ECL ultrasensitive chemiluminescence kit, Suzhou Xinsaimei Biotechnology Co., Ltd.) to cover the membrane, and incubate in the dark for 155 minutes. Exposure imaging was performed using the BIO-RAD imaging system.

[0167] Western-blot identification results of recombinant protein Figure 7 As shown, the purified recombinant protein was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, and Western-blot detection was performed using FAdV-4 positive serum as the primary antibody. The results showed that there was a target band at 67 KDa, indicating that the recombinant protein 100K (672-1053) had a specific reaction with FAdV-4 positive serum, indicating that the recombinant protein had good reactivity (antigenicity).

[0168] Example 2, Optimization of FAdV-4 ELISA detection conditions

[0169] The ELISA detection of the present invention is an indirect ELISA method, the principle of which is to adsorb a known antigen onto a solid phase carrier, add a sample to be tested (such as animal serum containing an antibody to be tested), allow the antibody to be tested to bind to the antigen, wash the unbound antibody, and then add an enzyme-labeled secondary antibody (a second antibody labeled with an enzyme that can specifically recognize and bind to the antibody to be tested) to form an antigen-antibody to be tested-enzyme-labeled secondary antibody complex. When a reaction substrate of the enzyme is further added to the reaction system, the amount of colored product formed is proportional to the amount of the antibody to be tested.

[0170] This example optimizes the detection conditions of the ELISA indirect method (also referred to herein as the 100K-ELISA method) based on the antigen protein (recombinant protein 100K (672-1053)) prepared in Example 1.

[0171] 1. Determination of the optimal antigen coating concentration and the optimal dilution of primary antibody serum

[0172] The purified 100K (672-1053) recombinant protein was diluted to 48, 24, 12, 6, and 3 μg / mL with coating solution (ELISA coating solution (10×), a product of Beijing Solebow Technology Co., Ltd.), and 100 μL / well was coated on the ELISA plate, with two columns coated at each concentration; the primary antibody serum was diluted 50, 100, 200, and 400 times in sequence, with one row for each gradient, and ELISA detection was performed according to the conventional ELISA procedure. Read D on the ELISA reader 450nm Value, select D 450nm The value is close to 1, the P / N value (positive serum D 450nm / negative serum D 450nm ) The largest well is the best reaction well, the coating concentration corresponding to the best reaction well is the best antigen coating concentration, and the corresponding primary antiserum dilution is the best dilution of the primary antiserum. The results are shown in Table 4. After the square array test, the best antigen coating concentration of the 100K-ELISA method is 6 μg / mL, and the best dilution of the primary antiserum is 1:100.

[0173] Table 4. Determination of the optimal coating concentration of 100K (672-1053) recombinant protein antigen and the optimal serum dilution

[0174]

[0175] Note: “+” indicates positive serum; “-” indicates negative serum.

[0176] 2. Determination of the optimal coating conditions for antigens

[0177] The antigen (purified 100K (672-1053) recombinant protein) was diluted to the optimal coating concentration and coated on the ELISA plate. The coating conditions were 4°C for 14h, 25°C for 14h, 37°C for 1h and then 4°C for 14h, 37°C for 2h and then 4°C for 14h, 37°C for 3h and then 4°C for 14h. The primary antiserum was diluted to the optimal dilution for ELISA detection. The other reaction conditions remained unchanged as above. The coating condition corresponding to the best reaction well was selected as the optimal coating condition. The results are shown in Table 5. Under the conditions of standing at 37°C for 2h and then coating at 4°C for 14h, the positive serum D 450nm The value is close to 1 and the P / N value is the largest, so the optimal coating condition of the 100K-ELISA method is determined to be 37°C for 2 hours and then 4°C for 14 hours.

[0178] Table 5. Determination of optimal antigen coating conditions

[0179]

[0180] Note: “+” indicates positive serum; “-” indicates negative serum.

[0181] 3. Determination of the optimal closure time

[0182] After coating according to the optimal coating concentration of antigen in step 1 and the optimal coating conditions in step 2, the blocking solution was added for 30min, 60min, 90min, and 120min respectively, and the primary antibody serum was diluted with the optimal primary antibody dilution (1:100) for ELISA reaction to determine the optimal blocking time. The results are shown in Table 6. When the blocking time is 60min, the positive serum D 450nm The value is close to 1, and the P / N value is the largest. The optimal blocking time of the 100K-ELISA method is determined to be 60 minutes.

[0183] Table 6. Determination of the optimal closure time

[0184]

[0185] Note: “+” indicates positive serum; “-” indicates negative serum.

[0186] 4. Determination of the optimal incubation time of primary antibody serum

[0187] The ELISA plate was coated and blocked according to the optimal conditions determined in steps 1, 2, and 3, and the primary antibody serum was added for 30 min, 60 min, 90 min, and 120 min, respectively. The other procedures remained unchanged, and the ELISA test was performed. The incubation time corresponding to the reaction well with the largest P / N value was selected as the optimal incubation time for the primary antibody serum. The results are shown in Table 7. When the primary antibody serum was incubated for 30 min, the positive serum D 450nm The value is close to 1 and the P / N value is the largest, so the optimal incubation time of the primary antibody serum in the 100K-ELISA method is determined to be 30 minutes.

[0188] Table 7. Determination of the optimal incubation time of primary antibody serum

[0189]

[0190]

[0191] Note: “+” indicates positive serum; “-” indicates negative serum.

[0192] 5. Determination of the optimal dilution and incubation time of enzyme-labeled secondary antibody

[0193] The optimal coating concentration of antigen and the optimal dilution of serum were used for ELISA detection, and the HRP-labeled goat anti-chicken IgG antibody was diluted at 1:2500, 1:5000, 1:7500, 1:10000, 1:15000, and 1:20000, respectively, to determine the optimal dilution of enzyme-labeled secondary antibody.

[0194] According to the optimized best reaction conditions, after adding the enzyme-labeled secondary antibody, the reaction time was 30min, 60min, 90min, and 120min respectively. Other conditions remained unchanged and ELISA determination was performed. Three replicates were set for each time period, and the time corresponding to the well with the largest P / N value was selected as the optimal incubation time for the enzyme-labeled secondary antibody.

[0195] The results are shown in Tables 8 and 9. When the enzyme-labeled secondary antibody was diluted 1:5000, the positive serum D 450nm The value is close to 1, and the P / N value is the largest. The optimal dilution of the enzyme-labeled secondary antibody in the 100K-ELISA method is determined to be 1:5000 (Table 8). When the enzyme-labeled secondary antibody is incubated for 30 minutes, the P / N value is the largest, and the optimal incubation time of the enzyme-labeled secondary antibody in the 100K-ELISA method is determined to be 30 minutes (Table 9).

[0196] Table 8. Determination of the optimal dilution of enzyme-labeled secondary antibody

[0197]

[0198] Note: “+” indicates positive serum; “-” indicates negative serum.

[0199] Table 9. Determination of the optimal incubation time for enzyme-labeled secondary antibodies

[0200]

[0201] Note: “+” indicates positive serum; “-” indicates negative serum.

[0202] 6. Determination of the best color development time

[0203] After the ELISA test was performed according to the optimal reaction conditions optimized according to steps 1-5, the color was developed for 3, 5, 7, 9, 11, 13, and 15 minutes respectively to determine the optimal color development time. The results are shown in Table 10. When the color was developed for 11 minutes, the P / N value of the 100K-ELISA method was the largest, and the optimal color development time of the 100K-ELISA method was determined to be 11 minutes.

[0204] Table 10. Determination of the optimal color development time

[0205]

[0206] Note: “+” indicates positive serum; “-” indicates negative serum.

[0207] 7. Determination of cut-off

[0208] 100 SPF chicken negative serum samples were tested using the optimized ELISA reaction procedure in steps 1-6 to determine D 450nm The value of x+3s was calculated as the critical value of the established method (100K-ELISA method). 450nm A value greater than or equal to the critical value was considered positive, D 450nm The value is less than the critical value and is considered negative. According to the above determination method, the negative and positive critical value (cut-off) is: 0.346, that is, when D 450nm When the value is greater than or equal to 0.346, it is judged as positive (indicating that the sample to be tested contains FAdV-4 antibodies); D 450nm When the value is less than 0.346, it is judged as negative (indicating that the sample to be tested does not contain FAdV-4 antibodies).

[0209] Example 3, FAdV-4 ELISA detection kit and detection method

[0210] 1. FAdV-4 ELISA detection method:

[0211] According to the optimization of Example 2, the following 100K-ELISA method was established:

[0212] ① Antigen-coated ELISA plate: The antigen prepared in Example 1 (purified recombinant protein 100K (672-1053)) was diluted with ELISA coating solution (Beijing Solebow Technology Co., Ltd., product number C1050) and then coated on the ELISA plate (100 μL per well). The antigen coating concentration was 6 μg / mL. The coating conditions were: standing at 37°C for 2 h and then coating at 4°C for 14 h.

[0213] ② Washing: discard the coated antigen in the ELISA plate, pat dry on absorbent paper, fill the well with washing solution (PBST), let it stand for 2-3 minutes, pat dry on absorbent paper, and wash 3 times;

[0214] ③ Blocking: Add 200 μL of blocking solution (5% skim milk) to each well and block for 60 min at 37°C;

[0215] ④Wash the plate: same as step ②;

[0216] ⑤ Add the sample to be tested: dilute the sample serum (primary antiserum) to be tested with diluent (1% BSA) at a ratio of 1:100, add 100 μL to each well of the ELISA plate, and set up positive serum control, negative serum control and blank control, and incubate at 37°C for 30 minutes;

[0217] ⑥Wash the plate: same as step ②;

[0218] ⑦ Add enzyme-labeled secondary antibody: dilute the enzyme-labeled secondary antibody (HRP-labeled goat anti-chicken IgG antibody) at a ratio of 1:5000 with diluent (1% BSA), add 100 μL to each well of the ELISA plate, and incubate at 37°C for 30 minutes;

[0219] ⑧Wash the plate: same as step ②;

[0220] ⑨ Add substrate for color development: add 100 μL of TMB color development solution (Beijing Solebow Technology Co., Ltd., product number PR1201) to each well and incubate at 37°C for 11 min;

[0221] ⑩Terminate the reaction: Add 50 μL of stop solution (2 mol / L H2SO4) to each well to terminate the reaction, and finally detect with an ELISA reader.

[0222] Result judgment: negative and positive cut-off value (cut-off) is: 0.346, that is, when D 450nm When the value is greater than or equal to 0.346, it is judged as positive (indicating that the sample to be tested contains FAdV-4 antibodies); D 450nm When the value is less than 0.346, it is judged as negative (indicating that the sample to be tested does not contain FAdV-4 antibodies).

[0223] 2. FAdV-4 ELISA detection kit

[0224] The FAdV-4 ELISA detection kit of the present invention comprises: an enzyme-labeled plate coated with the antigen of the present invention (recombinant protein 100K (672-1053)); standard positive serum; standard negative serum; HRP-labeled goat anti-chicken IgG antibody; sample diluent; enzyme-labeled antibody diluent; substrate color developing solution; stop solution; and washing solution.

[0225] The preparation method of the ELISA plate coated with the antigen of the present invention (recombinant protein 100K (672-1053)) is as follows: the antigen prepared in Example 1 (purified recombinant protein 100K (672-1053)) is diluted with ELISA coating solution (Solabo) and then coated with the ELISA plate (100 μL per well), the antigen coating concentration is 6 μg / mL, and the coating conditions are: standing at 37°C for 2 hours and then coating at 4°C for 14 hours; after washing the plate with PBST, 200 μL of blocking solution (5% skim milk) is added to each well, and blocked at 37°C for 60 minutes.

[0226] The standard positive serum is the positive serum of chicken infected with FADV-4.

[0227] The standard negative serum is SPF chicken negative serum.

[0228] The sample diluent was 1% BSA solution.

[0229] The enzyme-labeled antibody diluent was 1% BSA solution.

[0230] The substrate color developing solution is TMB color developing solution.

[0231] The stop solution is 2mol / L H2SO4.

[0232] The washing solution was PBST (trade name 1×PBST buffer, company Beijing Solebow Technology Co., Ltd., product number P1031).

[0233] Example 4: Validation of FAdV-4 ELISA Detection Method

[0234] 1. Specificity test

[0235] The 100K-ELISA method established in Example 3 was used to detect positive sera against NDV, IBDV, IBV, ARV, AIV-H5, AIV-H9, FAdV-4 inactivated vaccine-immunized chicken sera, and FAdV-4-infected chicken positive sera. SPF chicken negative serum samples were used as negative controls to evaluate the specificity of the ELISA method.

[0236] The results are as follows Figure 8 As shown, the results showed that in the 100K-ELISA method, the recombinant protein 100K (672-1053) only specifically bound to the positive serum of FAdV-4 infected chickens, and did not react with the positive serum of other avian viruses, nor with the immune serum of FAdV-4 inactivated vaccine, with good specificity.

[0237] 2. Sensitivity test

[0238] Three FAdV-4 infected chicken positive sera were randomly selected and diluted at 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560 and 1:5120, respectively, and the ELISA best reaction procedure (100K-ELISA method established in Example 3) was performed to evaluate the sensitivity of the ELISA method.

[0239] The results are as follows Fig. 9 As shown, when the positive serum was diluted 1:1280, the 100K-ELISA method was used to detect D 450 nm The value is still greater than its critical value, which is positive; the minimum detection limit of the 100K-ELISA method is 1:1280, indicating that the ELISA detection method established by the present invention has good sensitivity.

[0240] 3. Repeatability test

[0241] Randomly take 5 copies of FAdV-4 infected chicken positive serum and SPF chicken negative serum, use the same batch of antigen coated plates, detect at three different times, set 3 replicates each time, and conduct intra-batch repeatability test; on three different batches of antigen coated plates, the samples are tested at the same time, and the inter-batch repeatability test is conducted. The results are shown in Table 11. The same batch of coated ELISA plates are used to detect 5 copies of positive and negative samples at three different times. The coefficient of variation (coefficient of variation = variance / mean × 100%) of the 100K-ELISA method is between 2% and 54.5%, which is less than 10%, indicating that the intra-batch repeatability of the method is good. The ELISA plates coated with three different batches are used to detect 10 random samples at the same time. The results show that the coefficient of variation is less than 5%, indicating that the inter-batch repeatability of the method is good.

[0242] Table 11. 100K-ELISA method repeatability test results

[0243]

[0244] 4. Verification of identification tests

[0245] The 100K-ELISA method established by the present invention was used to detect 50 positive serum samples of FAdV-4 infected chickens and 50 serum samples of FAdV-4 inactivated vaccine-immunized chickens, as well as 100 negative serum samples of SPF chickens, to evaluate whether the established method can distinguish between serum antibodies of infected chickens and serum antibodies of inactivated vaccine-immunized chickens. The results are as follows Fig.10As shown, 50 positive serum samples from FAdV-4 infected chickens and 50 serum samples from chickens immunized with FAdV-4 inactivated vaccine and 100 negative serum samples from SPF chickens were detected by the 100K-ELISA method. The results showed that all 50 positive sera from FAdV-4 infected chickens were positive, while all 50 sera from chickens immunized with FAdV-4 inactivated vaccine and 100 negative sera from SPF chickens were negative, indicating that the established method can distinguish between infected serum antibodies and inactivated vaccine immune antibodies.

[0246] 5. Clinical sample test results

[0247] The 100K-ELISA method established by the present invention was used to detect 96 serum samples of chickens immunized with FAdV-4 inactivated vaccine and 217 serum samples of non-immunized chickens collected from Guangxi Fufeng Chicken Farm. At the same time, a commercial kit for group I fowl adenovirus (FAdV-I) (fowl adenovirus group I (FADV-I) antibody ELISA kit, referred to as FAdV-I ELISA kit, a product of BioChek, Netherlands, article number CK132) was used for comparative detection. The results are shown in Table 12. The samples (96 samples) immunized with inactivated vaccine were detected by the FAdV-I ELISA kit, and all of them were positive. The positive rate of the samples (96 samples) immunized with inactivated vaccine detected by the 100K-ELISA method was 3.1%. The positive rates of the non-immunized serum samples (217 samples) detected by the FAdV-I ELISA kit and the 100K-ELISA method were 44.7% and 30.9%, respectively.

[0248] Table 12. Clinical sample test results

[0249]

[0250] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims. SEQUENCE LISTING <110> Guangxi Zhuang Autonomous Region Veterinary Research Institute <120> Recombinant protein, kit and ELISA method for detecting serotype 4 avian adenovirus <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 382 <212> PRT <213> Artificial sequence <400> 1 Met Glu Arg Ser Asn Ile Pro Thr Thr Ala Ala Pro Phe Leu Pro Ser 1 5 10 15 Asp Phe Val Pro Leu Ser Phe Arg Gln Ala Gln Pro Leu Leu Trp Asp 20 25 30 Gln Val Tyr Leu Leu Gln Thr Ala Phe Phe Leu Cys Asn His Gly Gly 35 40 45 Tyr Leu Trp Glu Pro Glu Glu Thr Glu Asn Pro Asn Pro Arg Asp Arg 50 55 60 Thr Tyr Cys Pro Cys Asn Leu Cys Ser Pro His Arg Met Pro Gln His 65 70 75 80 Asn Val Pro Leu His Asn Glu Leu Leu Ala Ile Asn Thr Phe Glu Ile 85 90 95 Arg Thr Asp Asp Gly Lys Thr Phe Lys Leu Thr Pro Glu Leu Trp Ala 100 105 110 Asn Ala Tyr Leu Asp Lys Phe Glu Pro Lys Asp Tyr His Pro Phe Glu 115 120 125 Val Val His Phe Pro Gln His Glu Glu Ala Phe Ser Arg Asp Leu Thr 130 135 140 Ala Cys Val Thr Lys Ser Pro Glu Ile Leu Ser Leu Ile Arg Gln Ile 145 150 155 160 Gln Ala Ser Arg Glu Glu Phe Leu Leu Thr Arg Gly Lys Gly Val Tyr 165 170 175 Lys Asp Pro Asp Thr Gly Glu Val Leu Thr Pro Gln Pro Asp Leu Gln 180 185 190 Ala Gly Ala Ala Arg Arg Gln Ala Leu Pro Thr Ala Tyr Ala Asp His 195 200 205 Ala Arg Gly Ala Ala Thr Ser Ala Glu Pro Ser Arg Ala Leu Arg Pro 210 215 220 Thr Ser Val Ala Thr Ala Ala Gly Glu Thr Glu His Gly Gly Ala Leu 225 230 235 240 Gln Arg Ala Ile Gly Ser Val Gln Pro Ser Val Ala Gly Ala Thr Pro 245 250 255 His Gly Pro Glu Asn Gly Arg Pro Glu Gly Gln Gly Leu Gly Thr Ser 260 265 270 Gly Ala Arg Asn Leu Gln Ser Arg Gly Gly Asp Arg Val Arg Arg Arg 275 280 285 Asn Ser Arg Gln Arg Gly Tyr Arg Tyr Gly Arg Gly Pro Asp Glu His 290 295 300 Asp Leu Arg Arg Gly Gly Gly Gly Gly Arg Arg Gly Val Phe Arg Gly 305 310 315 320 Ser Gly Trp Gly Arg Gln Gly Glu Gln Pro Pro Tyr Asp Ser Pro Gln 325 330 335 Thr Gln Pro Lys Arg Ile Leu Arg Arg Pro Val Pro Gly Pro Asp Glu 340 345 350 Thr Ser Pro Ala Tyr Gln Gln His Arg Gln His Glu Gln His Arg Gln 355 360 365 Glu Asp Pro Ser Ala Ala Pro Thr Arg Pro Ser Thr Pro Arg 370 375 380 <210> 2 <211> 1149 <212> DNA <213> Artificial sequence <400> 2 atggagcgca gtaacattcc caccacggcc gcccccttcc taccctccga ctttgtgccg 60 ctctccttcc gacaagccca gcctctgctc tgggaccagg tgtacctcct ccaaaccgcc 120 tttttcctct gcaaccacgg aggatacctg tgggagcccg aggaaaccga gaatcccaac 180 cctcgcgatc gcacctactg tccgtgcaac ttgtgcagtc cgcaccggat gccccaacac 240 aacgtgcctc tgcacaacga actgctcgcc atcaacacgt ttgaaatccg cacggacgac 300 ggcaagacct tcaaattgac tcccgaactg tgggccaacg cctacctaga caaattcgaa 360 cccaaagact accacccttt cgaagtggtg cacttccctc aacacgagga agcgttctct 420 agagacctca cggcctgcgt caccaaaagc cccgaaatcc tcagtctgat tcgtcaaatt 480 caggcttcga gggaggagtt cctcctcacg cggggtaagg gcgtatacaa agaccccgac 540 accggcgagg tcctcactcc gcagccagat ctccaagctg gagcagcccg gcgacaagct 600 ctaccaaccg cttacgccga tcacgccaga ggagctgcga cgtcggcaga gccttctcga 660 gctctacggc ctaccagcgt cgcaaccgcc gccggcgaaa ccgaacacgg gggtgctctt 720 cagcgcgcta tcggctcggt ccaaccctcc gtcgcaggag caactcctca tggcccagag 780 aatggtcgac ctgaaggcca gggcctcgga acctccggag cccgaaatct acaatcccga 840 ggaggcgacc gagtccgacg gcgaaactct aggcagcgag gataccgata cggaagagga 900 ccagatgagc acgatctccg aagaggagga ggaggaggaa gacgaggcgt attccgcgga 960 tctggctggg gaagacaagg agaacagccc ccctacgatt ccccccaaac gcagccgaaa 1020 cgcatcctcc gtcgccccgt cccaggccct gacgagacct cccctgcgta ccaacaacac 1080 cgccaacacg agcagcaccg ccaggaggat ccgtccgcag cgcctacccg accgagcacc 1140 ccgaggtaa 1149 <210> 3 <211> 28 <212> DNA <213> Artificial sequence <400> 3 ccggaattca tggagcgcag taacattc 28 <210> 4 <211> 27 <212> DNA <213> Artificial sequence <400> 4 cccaagcttt tacctcgggg tgctcgg 27

Claims

1. A recombinant protein, characterized in that The amino acid sequence of the recombinant protein is shown in SEQ ID No.

1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule is any of the following: B1) The coding sequence is the DNA molecule shown in SEQ ID No. 2; B2) The nucleotide sequence is the DNA molecule shown in SEQ ID No.

2.

3. Biomaterial, characterized in that The biological material is any one of the following C1) to C4): C1) an expression cassette containing the nucleic acid molecule according to claim 2; C2) a recombinant vector containing the nucleic acid molecule of claim 2, or a recombinant vector containing the expression cassette of C1); C3) a recombinant microorganism containing the nucleic acid molecule of claim 2, or a recombinant microorganism containing the expression cassette of C1), or a recombinant microorganism containing the recombinant vector of C2); C4) A recombinant cell containing the nucleic acid molecule of claim 2, or a recombinant cell containing the expression cassette of C1), or a recombinant cell containing the recombinant vector of C2).

4. Any of the following uses of the recombinant protein according to claim 1, and / or the nucleic acid molecule according to claim 2, and / or the biomaterial according to claim 3: D1) Use in the preparation of products for the detection of serotype 4 avian adenovirus; D2) Use in the preparation of products for diagnosis or auxiliary diagnosis of serotype 4 avian adenovirus infection; D3) Use in the preparation of products for screening or assisting in screening for serotype 4 avian adenovirus infection; D4) Use in the preparation of products for identifying animals naturally infected with serotype 4 avian adenovirus and animals immunized with inactivated vaccines; D5) Use in the preparation of detection antigens for detecting serotype 4 avian adenovirus; D6) Use in the preparation of antibodies against serotype 4 avian adenovirus.

5. A kit for detecting serotype 4 avian adenovirus, characterized in that: The kit comprises the recombinant protein according to claim 1.

6. A method for preparing the kit according to claim 5, characterized in that: The method comprises the steps of preparing the recombinant protein according to claim 1 into a 6 μg / mL solution and coating the solution on a solid phase carrier to obtain an antigen-coated solid phase carrier.

7. The method according to claim 6, characterized in that The conditions for coating the solid phase carrier are: placing at 37° C. for 2 hours and then placing at 4° C. for 14 hours.

8. A primer pair for amplifying the gene of the recombinant protein according to claim 1, characterized in that: The primer pair consists of primer 100K (672-1053) -F and primer 100K (672-1053) -R, wherein the primer 100K (672-1053) -F is a single-stranded DNA molecule shown in SEQ ID No. 3; and the primer 100K (672-1053) -R is a single-stranded DNA molecule shown in SEQ ID No.

4.

9. A method for detecting antibodies to serotype 4 avian adenovirus for non-disease diagnosis purposes, characterized in that: The method uses the recombinant protein of claim 1 as an antigen and adopts an ELISA indirect method to detect serum type 4 avian adenovirus antibodies in the sample to be tested.

10. The method according to claim 9, characterized in that The method comprises the following steps: E1) adding a sample to be tested to the kit according to any one of claims 5 to 7; E2) Add the sample to be tested and incubate for 30 min; E3) adding enzyme-labeled secondary antibody and incubating for 30 min, wherein the enzyme-labeled secondary antibody is HRP-labeled goat anti-chicken IgG antibody; E4) Add enzyme reaction substrate to develop color.

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