Enzyme linked immunosorbent assay kit for detecting avian infectious laryngotracheitis virus antibody and fusion protein and biological material used by enzyme linked immunosorbent assay kit

Abstract

The invention discloses an enzyme linked immunosorbent assay kit for detecting an avian infectious laryngotracheitis virus antibody, and a fusion protein and a biological material used by the enzyme linked immunosorbent assay kit. The invention provides a method for preparing a recombinant protein. The method comprises the following steps: expressing a coding gene of the recombinant protein in a cell to obtain the recombinant protein; a gp67 signal peptide and a gD protein extracellular domain are sequentially arranged from the N end to the C end of the recombinant protein; and the gD protein extracellular region is the 85th to 406th sites of the gD protein. The invention also protects the recombinant protein and application of the recombinant protein as an antigen specifically bound with the avian infectious laryngotracheitis virus antibody. The invention also protects application of the protein in preparation of a kit for detecting the avian infectious laryngotracheitis virus antibody. The recombinant protein provided by the invention is used for detecting the ILTV antibody in chicken flock serum, has the advantages of high sensitivity and strong specificity, and is suitable for large-scale epidemiological monitoring and immune effect evaluation.

Description

Technical Field

[0001] The invention belongs to the peptide field in the field of biotechnology, and relates to an enzyme-linked immunosorbent assay kit for detecting antibodies to infectious laryngotracheitis virus and a fusion protein and biological material used therein. Background Art

[0002] Infectious laryngotracheitis virus (ILTV) belongs to the genus Iltovirus, subfamily Alphaherpesvirinae, family Herpesviridae, and causes infectious laryngotracheitis (ILT), an acute upper respiratory tract infection in poultry. ILTV primarily infects chickens, causing symptoms such as coughing, wheezing, salivation, laryngeal bleeding, and respiratory distress. In severe cases, it can cause mass mortality and significant economic losses to the global poultry industry. Particularly in today's intensive farming environment, recurring outbreaks of ILT pose a significant challenge to disease prevention and control in the poultry industry.

[0003] Glycoprotein D (also known as gD protein) is a key structural protein of ILTV. A type II transmembrane protein, it is widely present on the viral outer membrane and participates in the binding and membrane fusion processes between the virus and host cell receptors. It is a key factor in viral infection. The gD protein has excellent immunogenicity and can effectively induce the production of specific neutralizing antibodies. Therefore, the gD protein is not only an important candidate antigen for vaccine development, but also an ideal target for ILTV serological testing.

[0004] Traditional gD protein expression systems typically utilize Escherichia coli or mammalian cell expression systems. However, gD is a viral protein that undergoes complex glycosylation modifications. E. coli expression systems are prone to protein misfolding and inclusion body formation, which can affect its immunological activity and structural function. While mammalian cell expression systems can achieve protein glycosylation modification, their high culture costs, complex procedures, and low yields limit their large-scale application. Summary of the Invention

[0005] The purpose of the present invention is to provide an enzyme-linked immunosorbent assay kit for detecting antibodies to infectious laryngotracheitis virus and a fusion protein and biological materials used therein.

[0006] The present invention provides a method for preparing a recombinant protein, comprising the following steps: obtaining the recombinant protein by expressing a gene encoding the recombinant protein in a cell; The recombinant protein is as follows (a1) or (a2) or (a3) or (a4): (a1) a protein having a gp67 signal peptide and a gD protein extracellular region from its N-terminus to its C-terminus, wherein the gD protein extracellular region is positions 85-406 of the gD protein; (a2) a protein obtained by inserting a purification tag into (a1); the purification tag is inserted downstream of the gD protein extracellular region or the purification tag is inserted between the gp67 signal peptide and the gD protein extracellular region; (a3) a protein comprising a gD protein extracellular region; the gD protein extracellular region is from position 85 to position 406 of the gD protein; (a4) A protein obtained by inserting a purification tag into (a3); the purification tag is inserted upstream of the extracellular region of the gD protein or downstream of the extracellular region of the gD protein.

[0007] The gD protein is derived from the gD protein of Infectious laryngotracheitis virus.

[0008] As an example, the gD protein is shown in GenBank: WRQ97535.1 (17-JAN-2024).

[0009] As an example, positions 85-406 of the gD protein correspond to positions 42-363 of SEQ ID NO: 1.

[0010] In the recombinant protein, the extracellular region of the gD protein is shown in positions 42-363 of SEQ ID NO: 1.

[0011] In the recombinant protein, the extracellular region of the gD protein is shown as positions 41-363 in SEQ ID NO: 1.

[0012] As an example, the purification tag is a His tag, for example, a 6×His tag or an 8×His tag.

[0013] As an example, the gp67 signal peptide is shown in positions 1 to 38 of SEQ ID NO: 1.

[0014] As an example, the recombinant protein is shown in positions 42-363 of SEQ ID NO: 1.

[0015] As an example, the recombinant protein is shown in positions 41-363 of SEQ ID NO: 1.

[0016] As an example, the recombinant protein is shown in positions 39-363 of SEQ ID NO: 1.

[0017] As an example, the recombinant protein is shown in positions 42-373 of SEQ ID NO: 1.

[0018] As an example, the recombinant protein is shown in positions 41-373 of SEQ ID NO: 1.

[0019] As an example, the recombinant protein is shown in positions 39-373 of SEQ ID NO: 1.

[0020] As an example, the recombinant protein is shown in positions 42-379 of SEQ ID NO: 1.

[0021] As an example, the recombinant protein is shown in positions 41-379 of SEQ ID NO: 1.

[0022] As an example, the recombinant protein is shown in positions 39-379 of SEQ ID NO: 1.

[0023] As an example, the recombinant protein is shown as positions 1-363 in SEQ ID NO: 1.

[0024] As an example, the recombinant protein is shown as positions 1-373 in SEQ ID NO: 1.

[0025] As an example, the recombinant protein is shown as positions 1-379 in SEQ ID NO: 1.

[0026] As an example, the recombinant protein is shown in SEQ ID NO: 1.

[0027] As an example, in the gene encoding the recombinant protein, the coding region of the gD protein extracellular region is shown in positions 124-1089 of SEQ ID NO: 2.

[0028] As an example, in the gene encoding the recombinant protein, the coding region of the gD protein extracellular region is shown in positions 121-1089 of SEQ ID NO: 2.

[0029] As an example, the gene encoding the recombinant protein is any one of the following (c1) to (c13): (c1) a DNA molecule having a coding region as shown in positions 124-1089 of SEQ ID NO: 2; (c2) a DNA molecule having a coding region as shown in positions 121-1089 of SEQ ID NO: 2; (c3) a DNA molecule having a coding region as shown in positions 115-1089 of SEQ ID NO: 2; (c4) a DNA molecule having a coding region as shown in positions 124 to 1119 of SEQ ID NO: 2; (c5) a DNA molecule having a coding region as shown in positions 121 to 1119 of SEQ ID NO: 2; (c6) a DNA molecule having a coding region as shown in positions 115 to 1119 of SEQ ID NO: 2; (c7) a DNA molecule having a coding region as shown in positions 124-1137 of SEQ ID NO: 2; (c8) a DNA molecule having a coding region as shown in positions 121 to 1137 of SEQ ID NO: 2; (c9) a DNA molecule having a coding region as shown in positions 115 to 1137 of SEQ ID NO: 2; (c10) a DNA molecule having a coding region as shown in positions 1 to 1089 of SEQ ID NO: 2; (c11) a DNA molecule having a coding region as shown in positions 1 to 1119 of SEQ ID NO: 2; (c12) a DNA molecule having a coding region as shown in positions 1 to 1137 of SEQ ID NO: 2; (c13) A DNA molecule whose coding region is shown in SEQ ID NO: 2.

[0030] In this method, the gene encoding the recombinant protein is expressed in cells using a baculovirus expression system. The Baculovirus-Insect Cell Expression System (BICS) offers advantages such as high protein expression, complete post-translational modification, and a short culture cycle. This system can efficiently express target proteins in insect cells (such as Sf9 or High Five cells) and exhibits excellent protein folding and secretion mechanisms, making it suitable for the preparation of vaccine and diagnostic antigens. Therefore, developing a baculovirus-based method for large-scale, soluble expression of target proteins, combined with the construction of an ELISA detection system that is simple, specific, and highly sensitive, is a key approach to overcoming existing technical bottlenecks. This approach will not only help improve ILTV immune control but also provide strong technical support for the healthy development of the poultry industry.

[0031] As an example, the method for preparing a recombinant protein provided by the present invention comprises the following steps: (1) introducing a recombinant plasmid into Escherichia coli DH10Bac to obtain a recombinant Escherichia coli; the recombinant plasmid is a recombinant plasmid obtained by inserting a DNA molecule having a gene encoding the recombinant protein into a baculovirus transfer vector; (2) culturing the recombinant E. coli obtained in step (1) and extracting the plasmid, which is the recombinant Bacmid; (3) Transfecting Sf9 cells with the recombinant Bacmid obtained in step (2) and culturing the cells, collecting the culture supernatant, which is the P1 generation virus solution; (4) Infecting Sf9 cells with the P1 virus solution and culturing them, and collecting the culture supernatant, which is the P2 virus solution; (5) Infecting Sf9 cells with the P2 virus solution and culturing them, collecting the culture supernatant, which is the P3 virus solution; (6) Infecting High Five cells with the P3 generation virus solution, collecting the culture supernatant, and purifying to obtain the recombinant protein.

[0032] Exemplarily, the baculovirus transfer vector is a pFastBacI vector.

[0033] Specifically, the recombinant plasmid is a double-stranded DNA molecule represented by positions 1 to 1119 in SEQ ID NO: 2 that replaces the BamH I and Hind The recombinant plasmid was obtained by enzyme III digesting the small fragment between the recognition sequences.

[0034] Specifically, step (3) can be as follows: transfecting Sf9 cells with the recombinant Bacmid obtained in step (2), then culturing at 27°C for 72 hours, and harvesting the supernatant to obtain the P1 generation virus solution. Transfection is performed using Cellfectin™ II reagent. 3 μg of recombinant Bacmid is used to transfect Sf9 insect cells to obtain 2 mL of P1 generation virus solution.

[0035] Specifically, step (4) may be: inoculating the P1 generation virus liquid into the cell suspension (the volume ratio of the virus liquid to the cell suspension is 1:10), then culturing at 27°C and 110 rpm with shaking until more than 80% of the cells show pathological changes, and harvesting the supernatant, which is the P2 generation virus liquid.

[0036] Specifically, step (5) may be: inoculating the P2 generation virus liquid into the cell suspension (the volume ratio of the virus liquid to the cell suspension is 1:10), then culturing at 27°C and 110 rpm with shaking until more than 80% of the cells show pathological changes, and harvesting the supernatant, which is the P3 generation virus liquid.

[0037] Preparation of cell suspension in step (4) and step (5): Collect Sf9 cells in the logarithmic growth phase and resuspend them in SIMSF Expression Medium to a cell content of 1.0×10 6 cells / mL.

[0038] 2 mL of P1 virus solution was passaged and amplified to obtain approximately 200 mL of P3 virus solution.

[0039] Specifically, step (6) may be: inoculating the P3 virus solution into the cell suspension (the volume ratio of the virus solution to the cell suspension is 1:100), culturing at 27°C and 120 rpm for 48 hours, then centrifuging at 1500 rpm for 15 minutes, collecting the supernatant, and then purifying.

[0040] Preparation of cell suspension in step (6): Take High Five cells and resuspend them in SIM HF Expression Medium to a cell content of 1.5-2.0×10 6 cells / mL.

[0041] 200 mL of P3 generation virus solution was prepared to obtain approximately 20 L of supernatant.

[0042] Exemplarily, the purification is affinity chromatography purification.

[0043] Exemplarily, the purification is nickel column purification.

[0044] The gp67 signal peptide, derived from the natural baculovirus secretion signal peptide, effectively directs the post-translational transport of the recombinant gD polypeptide chain, promoting its entry into the host cell's endoplasmic reticulum (where the signal peptide is excised by the cell itself) and export to the extracellular environment via the classical secretory pathway, thereby improving the secretion efficiency and correct folding of the exogenous protein. The gD protein is a key transmembrane glycoprotein on the surface of ILTV. Heterologous expression of the full-length protein in vitro suffers from low expression levels and poor solubility. Therefore, the present invention truncated the extracellular region from the full-length protein and further performed codon optimization for the baculovirus expression system to ensure efficient expression in insect cells. Transfection of Sf9 cells with 3 μg of recombinant bacmid successfully yielded 2 mL of P1 recombinant baculovirus. Subsequently, the P1 recombinant baculovirus solution was inoculated into Sf9 cells at a 10% infection rate to yield P2 recombinant baculovirus. Subsequently, the P2 recombinant baculovirus solution was inoculated into Sf9 cells at a 10% infection dose to obtain approximately 200 mL of high-titer P3 recombinant baculovirus solution. The P3 recombinant baculovirus solution was infected with High Five cells at a 1% infection dose, and the gD protein expression peak was reached after 48 hours of culture, and 20 L of culture supernatant could be harvested. After testing, the expression level of the target protein in the High Five cell culture medium was as high as 6.67 mg / L, meeting the needs of large-scale protein production. Under optimal conditions, the Sf9 cells and High Five cells used in the present invention were both cultured in suspension and serum-free medium systems, which reduced the risk of exogenous protein contamination, helped simplify the downstream purification process, improved the batch consistency and controllability of product quality, and reduced production costs. Nickel column affinity chromatography purification has a simple purification process and high protein purity. The recombinant protein prepared by the method of the present invention was detected to have a purity of not less than 90% by SDS-PAGE, and a single specific band was detected by Western blot. It is suitable for subsequent ELISA antigen coating and application development, and has good operability and commercialization capabilities.

[0045] The present invention also protects a protein, which is any of the above-mentioned recombinant proteins.

[0046] Specifically, the protein is a recombinant protein prepared by any of the above methods for preparing recombinant proteins.

[0047] The present invention also protects biological materials as described in any one of the following (b1) to (b8): (b1) a gene encoding the recombinant protein; (b2) having the expression cassette of (b1); (b3) a recombinant vector comprising (b1); (b4) a recombinant vector comprising (b2); (b5) recombinant cell having (b1); (b6) recombinant cells having (b2); (b7) recombinant cells having (b3); (b8) Recombinant cells with (b4).

[0048] Specifically, the gene encoding the recombinant protein is any one of the above.

[0049] Specifically, the recombinant vector is a recombinant plasmid obtained by inserting the coding gene of the recombinant protein into the pFastBacI vector. Specifically, the recombinant vector is a recombinant plasmid obtained by replacing the double-stranded DNA molecule shown at positions 1-1119 in SEQ ID NO: 2 with the double-stranded DNA molecule shown at positions 1-1119 in SEQ ID NO: 2. BamH I and Hind Specifically, the recombinant vector is a recombinant Bacmid obtained by introducing the above-mentioned recombinant plasmid into Escherichia coli DH10Bac and culturing it.

[0050] The recombinant cell is a recombinant animal cell, a recombinant plant cell or a recombinant microorganism.

[0051] In certain embodiments, the animal cell line may be non-reproductive material. In certain embodiments, the animal cell may be an isolated mammalian cell. In certain embodiments, the animal cell is a mammalian cell, an avian cell, an amphibian cell, a fish cell, or an insect cell. Mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO cells), a substrain of Chinese hamster ovary cells (CHO-K1 cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), baby hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells, or natural killer (NK) cells. Avian cells include, but are not limited to, chicken cells, duck cells, or goose cells. Amphibian cells include, but are not limited to, African clawed frog cells or giant salamander cells. Fish cells include, but are not limited to, grass carp cells, common carp cells, rainbow trout cells, or catfish cells. Insect cells include, but are not limited to, Sf21 cells, Sf-9 cells, or High Five cells (Hi-5 cells). In certain embodiments, the mammalian cells do not include animal germ cells, animal fertilized eggs, or animal embryonic stem cells. In certain embodiments, the mammalian cells may be somatic cells or cell lines.

[0052] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes, algae, etc. The bacteria may be from the genus Corynebacterium ( Corynebacterium sp.), such as Corynebacterium glutamicum, Corynebacterium pekinensis, Corynebacterium crenulate, etc. The bacteria may be from the genus Brevibacterium ( Brevibacterium sp. ), such as Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium ammoniaphagoides, etc. The bacteria may be from the genus Escherichia ( Escherichia sp. ), such as Escherichia coli. The bacteria may be from the genus Erwinia ( Erwinia sp. The bacteria may be from the genus Agrobacterium ( Agrobacterium sp. ), such as Agrobacterium tumefaciens. The bacteria may be from the genus Flavobacterium (Flavobacterium sp. The bacteria may be from the genus Alcaligenes ( Alcaligenes sp. The bacteria may be from the genus Pseudomonas ( Pseudomonas sp. The bacteria may be from the genus Bacillus ( Bacillus sp. ), such as Bacillus, etc. For example, the Escherichia coli can be Escherichia coli DH10Bac. The virus can include rotavirus, baculovirus, retrovirus (such as lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papillomavirus (such as SV40) and herpes virus (such as herpes simplex virus). The fungus can be from the genus Saccharomyces ( Saccharomyces sp. ), such as Saccharomyces cerevisiae, Candida, Methanol yeast, Pichia pastoris, etc. The fungus may be from the genus Fusarium ( Fusarium sp. The fungus may be from the genus Rhizoctonia ( Rhizoctonia sp. The fungus may be from the genus Verticillium ( Verticillium sp. The fungus may be from the genus Penicillium ( Penicillium sp. The fungus may be from the genus Aspergillus ( Aspergillus sp. The fungus may be from the genus Cephalosporium ( Cephalosporium sp. The actinomycetes may be from the genus Streptomyces ( Streptomyces sp. ), such as Streptomyces. The algae may be from the phylum Cyanobacteria ( Cyanophyta ), such as cyanobacteria. The algae may be from the genus Fucus ( Fucus sp. The algae may be from the genus Aspergillus ( Achnanthes sp. The algae may be from the genus Cocos algae ( Amphiprora sp. The algae may be from the genus Diplophyta ( Amphora sp. The algae may be from the genus Cellulose ( Ankistrodesmus sp. The algae may be from the genus Astrophytum ( Asteromonas sp. The algae may be from the genus Chromophytes ( Boekelovia sp. ).

[0053] Exemplarily, the recombinant cell is a recombinant cell obtained by introducing the gene encoding the recombinant protein into Escherichia coli DH10Bac. Exemplarily, the recombinant cell is a recombinant cell obtained by introducing the recombinant vector into Escherichia coli DH10Bac. Exemplarily, the recombinant cell is a recombinant cell obtained by introducing the gene encoding the recombinant protein into Sf9 cells. Exemplarily, the recombinant cell is a recombinant cell obtained by introducing the recombinant vector into Sf9 cells. Exemplarily, the recombinant cell is a recombinant cell obtained by introducing the gene encoding the recombinant protein into High Five cells. Exemplarily, the recombinant cell is a recombinant cell obtained by introducing the recombinant vector into High Five cells.

[0054] The present invention also protects the use of the protein as an antigen; the antigen is an antigen that specifically binds to an antibody against infectious laryngotracheitis virus of avian influenza virus. Exemplarily, the antigen is a coating antigen.

[0055] The present invention also protects the use of the protein or the biological material in preparing a kit; the kit is a kit for detecting antibodies to infectious laryngotracheitis virus of chickens. In the kit, the protein serves as a coating source.

[0056] The present invention also protects a kit, which is as follows (d1) or (d2): (d1) a kit comprising the protein; (d2) a kit comprising an ELISA plate coated with the protein; The kit is a kit for detecting antibodies to infectious laryngotracheitis virus of chickens.

[0057] As an example, the coating concentration of the ELISA plate coated with the protein is 0.125 μg / mL.

[0058] The kit also includes a secondary antibody working solution.

[0059] The secondary antibody working solution is obtained by diluting the enzyme-labeled secondary antibody.

[0060] As an example, the secondary antibody working solution is obtained by diluting the enzyme-labeled secondary antibody to 5000 times the volume.

[0061] As an example, the enzyme-labeled secondary antibody is goat anti-chicken IgG labeled with horseradish peroxidase.

[0062] As an example, the enzyme-labeled secondary antibody is a reagent with the product number bs-0310G-HRP from Biosun Biotechnology Co., Ltd.

[0063] The present invention establishes a method for detecting ILTV antibodies in chicken serum samples based on the recombinant protein, which has the advantages of high sensitivity and strong specificity and is suitable for large-scale epidemiological monitoring and immune effect evaluation.

[0064] The method for detecting ILTV antibodies in chicken serum samples was an indirect ELISA test method.

[0065] The indirect ELISA test method includes the following steps: (1) Dilute the recombinant protein to 0.125 μg / mL, coat it on an ELISA plate, and incubate at 4°C; (2) Block with 5% skim milk solution at 37°C for 60 minutes; (3) Add the serum diluent to be tested (the dilution ratio of the serum to be tested is 1:1600) and incubate at 37°C for 60 min; (4) Add enzyme-labeled secondary antibody working solution (the dilution ratio of enzyme-labeled secondary antibody is 1:5000) and incubate at 37°C for 45 minutes; (5) Add TMB colorimetric solution, terminate the reaction after 10 min, and measure the OD value at a wavelength of 450 nm; (6) When OD 450nm When the value is ≥0.169, it is considered positive; when OD 450nm When the OD value is less than 0.137, it is considered negative; 450nm When the value is ≥0.137 and <0.169, it is judged as a suspicious sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 Schematic diagram of the elements of the recombinant plasmid pFastBacⅠ-gD.

[0067] Figure 2 This is an exemplary result diagram of PCR identification of recombinant bacmid.

[0068] Figure 3 This is the result of Western blot detection of P3 virus liquid.

[0069] Figure 4 This is the SDS-PAGE identification result of the target protein purified using Ni-NTA affinity chromatography column.

[0070] Figure 5 This is the result of Western blot detection of gD-His solution.

[0071] Figure 6 This is the result of SDS-PAGE electrophoresis of gD-His solution and Coomassie Brilliant Blue staining.

[0072] Figure 7This is the result of Western Blot detection in Comparative Example 1.

[0073] Figure 8 This is the result of Western Blot detection in Comparative Example 2. DETAILED DESCRIPTION

[0074] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0075] Unless otherwise noted, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in literature in the field or according to product specifications. Materials and reagents used in the following examples are commercially available unless otherwise noted. Unless otherwise noted, quantitative assays in the following examples were performed in triplicate, and the results were averaged. pFastBacI vector (pFastBac1 vector): U-Bio Biotechnology, product number VT1166. DH10Bac competent cells: Beijing Biomed Gene Technology Co., Ltd., product number BC112-01. Sf9 cells (Sf9 cells in Sf-900™ II SFM): Thermo Fisher Scientific, product number 11496015. High Five cells (High Five™ cells in Express Five™ medium): Thermo Fisher Scientific, product number B85502. Infectious laryngotracheitis virus (ILTV) strain HB201806, registered in GenBank: PP062931.1 (JAN 17, 2024). Skim milk powder: BioSky Biotechnology Co., Ltd., catalog number P0216. ELISA coating buffer (10×): Solebio Technology Co., Ltd., catalog number C1055. Dilute the 10× ELISA coating buffer to 10 times its volume with distilled water to create a 1× ELISA coating buffer. 5% skim milk solution: PBST containing 5g / 100mL skim milk powder. Two-component TMB colorimetric solution: Solebio Technology Co., Ltd., catalog number PR1210. ELISA stop buffer: Solebio Technology Co., Ltd., catalog number C1058. Cellfectin™ II reagent: Thermo Fisher Scientific, catalog number 10362100. All procedures were performed according to the manufacturer's instructions. Goat Anti-Chicken IgG H&L, HRP conjugated: Biosun Biotechnology Co., Ltd., bs-0310G-HRP, diluted with 5% skim milk solution.

[0076] Example 1: Preparation of target protein 1. Construction of recombinant plasmid pFastBacⅠ-gD The double-stranded DNA molecule shown at positions 1 to 1119 in SEQ ID NO: 2 was used to replace the DNA in the pFastBacI vector. BamH I and Hind The recombinant plasmid pFastBacⅠ-gD was obtained by sequencing. The schematic diagram of its elements is shown in Figure 1 The recombinant plasmid contains the DNA shown in SEQ ID NO: 2.

[0077] The DNA set forth in SEQ ID NO:2 encodes the protein set forth in SEQ ID NO:1. In SEQ ID NO:1, amino acid residues 1-38 constitute the gp67 signal peptide, amino acid residues 42-363 constitute the gD protein extracellular region, and amino acid residues 366-373 constitute the 8×His tag. The signal peptide is autologously cleaved off in the endoplasmic reticulum, forming the mature protein (represented as positions 39-379 in SEQ ID NO:1). In SEQ ID NO:2, nucleotides 1-114 encode the gp67 signal peptide, nucleotides 124-1089 encode the gD protein extracellular region (codon-optimized), and nucleotides 1096-1119 encode the 8×His tag.

[0078] 2. Construction of recombinant baculovirus expression vector 1. Add 1-10 ng of recombinant plasmid to 100 μL of DH10Bac competent cells, mix well and place on ice for 30 minutes. Then place the centrifuge tube in a 42°C metal bath for heat shock treatment for 45 seconds. Immediately place on ice for 2 minutes, add 900 μL of liquid LB medium, and culture at 37°C, 220 rpm, with shaking for 4 hours.

[0079] 2. After completing step 1, sample 100 μL of bacterial solution and spread it onto a solid LB medium plate containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline, 100 μg / ml X-gal and 40 μg / ml IPTG. Incubate inverted at 37°C in the dark for 24-48 hours. At this time, white monoclonal colonies can be observed.

[0080] 3. After completing step 2, pick uniform white colonies from the plate and inoculate them into 1 mL of liquid LB medium containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, and 10 μg / ml tetracycline. Incubate at 37°C and 220 rpm for 12 h.

[0081] 4. After completing step 3, take a sample of the bacterial liquid and streak it onto a solid LB medium plate containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline, 100 μg / ml X-gal and 40 μg / ml IPTG, and incubate it in an inverted static culture at 37°C for 24-48 hours.

[0082] 5. After completing step 4, pick uniform white colonies from the plate and inoculate them into 1 mL of liquid LB medium containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, and 10 μg / ml tetracycline. Incubate at 37°C and 220 rpm with shaking for 12 h.

[0083] 6. After completing step 5, sample the bacterial solution and perform PCR identification using the primer pair consisting of M13-F and M13-R. If a characteristic band of about 3300 bp is displayed, it means that a recombinant bacmid with the DNA shown in SEQ ID NO: 2 has been obtained. Figure 2 , showing a characteristic band of about 3300bp.

[0084] M13-F (SEQ ID NO: 5): 5'-GTTTTCCCAGTCACGAC-3'; M13-R (SEQ ID NO: 6): 5'-CAGGAAACAGCTATGAC-3'.

[0085] 3. Preparation of recombinant baculovirus 1. Take 2 mL of the bacterial suspension containing the recombinant bacmid obtained in step 2 and inoculate it into 200 mL of liquid LB medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline. Incubate at 37°C with shaking at 220 rpm for 14-16 hours. Then, use a kit to extract the recombinant bacmid using the NucleoBondXtra Midi Plus kit for transfection-grade plasmid DNA, available from Macherey-Nagel, product number 740412.50. Follow the instructions.

[0086] 2. Transfect Sf9 cells with the recombinant Bacmid obtained in step 1 (using Cellfectin™ II reagent), then incubate at 27°C for 72 hours (cytopathic effects can be observed), and harvest the supernatant, which is the P1 virus solution.

[0087] 3 μg of recombinant Bacmid was used to transfect Sf9 insect cells to obtain 2 mL of P1 virus solution.

[0088] 4. Passaging and Amplification of Recombinant Baculovirus 1. Inoculate the P1 virus solution obtained in step 3 into the cell suspension (the volume ratio of virus solution to cell suspension is 1:10), then culture at 27°C and 110 rpm with shaking until more than 80% of the cells show pathological changes. Harvest the supernatant, which is the P2 virus solution. Preparation of cell suspension: Collect Sf9 cells in the logarithmic growth phase and resuspend them in SIM SF Expression Medium to a cell content of 1.0×10 6 cells / mL. SIM SF Expression Medium (For SF9, SF21) (Serumfree): Sino Biological, product number MSF1.

[0089] 2. Inoculate the P2 virus solution obtained in step 1 into the cell suspension (volume ratio of virus solution to cell suspension is 1:10). Then, culture at 27°C and 110 rpm with shaking until more than 80% of the cells show pathological changes. Harvest the supernatant, which is the P3 virus solution. The cell suspension preparation method is the same as that in step 1.

[0090] 3. Take the P3 generation virus solution obtained in step 2 and perform Western blot detection (the primary antibody is Mouse anti-His-Tag mAb, Abotek Biotechnology Co., Ltd., catalog number AE003; the secondary antibody is Goat Anti-mouse IgG-HRP, Jinpulai Biotechnology Co., Ltd., catalog number P03S01L). The results are shown in Figure 3 . Figure 3 In the figure, lane 1 is the negative control (Sf9 cells), and lane 2 is the P3 virus solution. The P3 virus solution shows the target band at a molecular weight of approximately 60 kDa.

[0091] 2 mL of P1 virus solution was passaged and amplified to obtain approximately 200 mL of P3 virus solution.

[0092] 5. Large-Scale Expression and Purification of gD Protein 1. Inoculate the P3 virus solution obtained in step 4 into the cell suspension (the volume ratio of virus solution to cell suspension is 1:100), shake and culture at 27°C and 120 rpm for 48 hours, then centrifuge at 1500 rpm for 15 minutes and collect the supernatant. Preparation of cell suspension: Take High Five cells and resuspend them in SIM HF Expression Medium to a cell content of 1.5-2.0×10 6cells / mL. SIM HF Expression Medium (For Hi5) (Serum-free): Sino Biological, product number MHF1. 200 mL of P3 generation virus solution was used to prepare approximately 20 L of supernatant.

[0093] 2. The supernatant from step 1 was purified using a Ni-NTA affinity chromatography column (Solaibao Technology Co., Ltd.) with the His-tagged target protein. Elution was performed using eluents containing 25 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM imidazole, respectively. The post-column fractions were collected separately. The eluent composition consisted of imidazole, 50 mM Tris, and 300 mM NaCl, with the balance being HO.

[0094] 3. After passing through the column, sample the solution and perform SDS-PAGE identification. Figure 4 . Figure 4 Middle: Lane 1, supernatant obtained in step 1; Lane 2: load flow-through; Lane 3: post-column solution eluted with 25 mM imidazole eluent; Lane 4: post-column solution eluted with 50 mM imidazole eluent; Lane 5: post-column solution eluted with 100 mM imidazole eluent; Lane 6: post-column solution eluted with 200 mM imidazole eluent; Lane 7: post-column solution eluted with 300 mM imidazole eluent; Lane 8: post-column solution eluted with 400 mM imidazole eluent.

[0095] 4. Based on the electrophoresis results from step 3, the post-column solution eluted with 50 mM imidazole, the post-column solution eluted with 100 mM imidazole, and the post-column solution eluted with 200 mM imidazole were combined and placed in a Millipore ultrafiltration tube for centrifugal concentration and system exchange (the purpose of the system exchange is to replace the system with PBS buffer) to obtain a solution containing the target protein, also referred to as gD-His solution. The target protein is the protein represented by positions 39-379 in SEQ ID NO: 1, and is designated as gD-His protein.

[0096] VI. Identification of gD-His Protein Take the gD-His solution and perform Western blot detection (the primary antibody used is Mouse anti-His-Tag mAb, Abotec Biotechnology Co., Ltd., catalog number AE003). Figure 5 A single band was shown, and the molecular weight was consistent with the prediction.

[0097] Take gD-His solution and perform SDS-PAGE electrophoresis. See the photo after Coomassie Brilliant Blue staining. Figure 6Clear target protein bands were observed. Scanning the gel with an imager and analyzing it with ImageJ software revealed a purity of 92%.

[0098] The gD-His solution was quantified using a BCA assay kit at a protein concentration of 1 mg / mL. The BCA Protein Assay Kit (Cat. No. CW0014, available from Kangwei Century Co., Ltd.) was used according to the manufacturer's instructions. Based on the quantification results, the target protein content in the supernatant obtained in step 1 was calculated to be 6.67 mg / L.

[0099] Example 2: Preparation of standard serum Standard positive serum (indicated by P): Three 4-week-old SPF white Leghorn chickens were infected with HB201806 strain of avian infectious laryngotracheitis virus by eye drop (the dose was 10 4.0 EID 50 Two weeks later, blood was collected from the subwing vein and centrifuged at 8000 rpm for 15 minutes, with the supernatant aspirated to obtain three serum samples. Each serum sample was tested using the Infectious Bursal Disease Virus Antibody test kit (BioChek, Cat. No. CK124 ILT, following the manufacturer's instructions) and all were positive. Equal volumes of the three serum samples were then mixed to create the standard positive serum.

[0100] Standard negative serum (indicated by N): Blood was collected from the subwing vein of three 6-week-old SPF White Leghorn chickens. The blood was then centrifuged at 8000 rpm for 15 minutes, and the supernatant was aspirated. Three serum samples were obtained. Each serum sample was tested using the Infectious Bursal Disease Virus Antibody test kit (BioChek, Catalog No. CK124 ILT, following the manufacturer's instructions) and all were negative. Equal volumes of the three serum samples were then combined to create the standard negative serum.

[0101] Example 3. Establishment of indirect ELISA detection method 1. Optimization of antigen coating concentration and serum dilution multiple The optimal antigen coating concentration and serum dilution multiple were determined by matrix titration. The OD values of positive serum and negative serum under each combination were determined by cross-combination. 450nm Positive serum OD 450nm Values close to 1.0 and negative serum OD 450nm The combination with a value close to 0.0 and a higher P / N value was regarded as the optimal reaction condition.

[0102] 1. Take a 96-well ELISA reaction plate, add 100 μL of protein coating solution to each well, and incubate at 4°C for 12-14 hours.

[0103] Preparation of protein coating solution: The purified gD-His protein prepared in Example 1 was serially diluted with 1× ELISA coating solution to obtain protein concentrations of 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, or 0.125 μg / mL, i.e., protein coating solutions of different dilutions.

[0104] 2. After completing step 1, take the reaction plate and wash it three times with PBST solution (300 μL per well each time, 3 minutes each time).

[0105] 3. After completing step 2, add 300 μL of 5% skim milk solution to each well and incubate at 37°C for 60 minutes (blocking).

[0106] 4. After completing step 3, take the reaction plate and wash it three times with PBST solution (300 μL per well each time, 3 minutes each time).

[0107] 5. After completing step 4, take the reaction plate, add serum diluent (100 μL / well), and incubate at 37°C for 60 minutes.

[0108] Serum diluents were either positive serum diluent or negative serum diluent, with three replicates per well for each serum dilution. Positive serum diluent: Standard positive serum was serially diluted with 5% skim milk solution to 400-fold, 800-fold, 1600-fold, 3200-fold, or 6400-fold volume, respectively. Negative serum diluent: Negative serum (N) was serially diluted with 5% skim milk solution to 400-fold, 800-fold, 1600-fold, 3200-fold, or 6400-fold volume, respectively.

[0109] 6. After completing step 5, take the reaction plate and wash it three times with PBST solution (300 μL per well each time, 3 minutes each time).

[0110] 7. After completing step 6, take the reaction plate, add 100 μL of enzyme-labeled secondary antibody working solution to each well, and incubate at 37°C for 60 minutes.

[0111] Enzyme-labeled secondary antibody working solution: dilute horseradish peroxidase-labeled goat anti-chicken IgG to 10,000 times the volume.

[0112] 8. After completing step 7, take the reaction plate and wash it three times with PBST solution (300 μL per well each time, 3 minutes each time).

[0113] 9. After completing step 8, take the reaction plate, add 100 μL of TMB colorimetric solution to each well, and incubate at 37°C in the dark for 10 minutes.

[0114] 10. After completing step 9, take the reaction plate, add 50 μL of ELISA stop solution to each well to stop the reaction, and then use a microplate reader to measure the OD value of each well at a wavelength of 450 nm.

[0115] The results are shown in Table 1 (N=3, data are average values). When the antigen concentration was 0.125μg / mL and the serum dilution ratio was 1:1600, the positive serum OD 450 The value is 0.975 (close to 1), and the negative serum OD 450 The value was 0.048 (close to 0), and the P / N value reached 20.455. Therefore, the final determination of the antigen coating concentration for this method was 0.125 μg / mL and the serum dilution factor was 1:1600.

[0116]

[0117] 2. Determination of Closure Time 1. Take a 96-well ELISA reaction plate, add 100 μL of protein coating solution to each well, and incubate at 4°C for 12-14 hours.

[0118] Preparation of protein coating solution: The purified gD-His protein prepared in Example 1 was diluted with 1× ELISA coating solution to a protein concentration of 0.125 μg / mL.

[0119] 2. Same as step 2 in step 1.

[0120] 3. After completing step 2, add 300 μL of 5% skim milk solution to each well and incubate at 37°C for 30 min, 60 min, or 90 min.

[0121] 4. Same as step 1, step 4.

[0122] 5. After completing step 4, take the reaction plate, add serum diluent (100 μL / well), and incubate at 37°C for 60 minutes.

[0123] The serum diluents were positive serum diluent (the standard positive serum was gradiently diluted to 1600 times the volume with 5% skim milk solution) and negative serum diluent (the standard negative serum was gradiently diluted to 1600 times the volume with 5% skim milk solution), and 3 replicate wells were set for each serum diluent.

[0124] 6. Same as step 6 in step 1.

[0125] 7. Same as step 7 in step 1.

[0126] 8. Same as step 8 in step 1.

[0127] 9. Same as step 9 in step 1.

[0128] 10. Same as step 10.

[0129] The results are shown in Table 2. OD of positive serum at 60 min after blocking 450nm The value was 1.008, negative serum OD 450nm The value is 0.058, which is the highest P / N value. Therefore, the sealing time is determined to be 60 minutes.

[0130]

[0131] 3. Determination of the primary antibody serum incubation time 1. Same as step 1 in step 2.

[0132] 2. Same as step 2 in step 1.

[0133] 3. Same as step 3 in step 1.

[0134] 4. Same as step 4 in step 1.

[0135] 5. After completing step 4, take the reaction plate, add serum diluent (100 μL / well), and incubate at 37°C for 30 min, 60 min, or 90 min.

[0136] The serum diluents were positive serum diluent (the standard positive serum was gradiently diluted to 1600 times the volume with 5% skim milk solution) and negative serum diluent (the standard negative serum was gradiently diluted to 1600 times the volume with 5% skim milk solution), and 3 replicate wells were set for each serum diluent.

[0137] 6. Same as step 6 in step 1.

[0138] 7. Same as step 7 in step 1.

[0139] 8. Same as step 8 in step 1.

[0140] 9. Same as step 9 in step 1.

[0141] 10. Same as step 10 in step 1.

[0142] The results are shown in Table 3. When the serum reacted with the antigen for 60 minutes, the OD of the positive serum was 450nm The value reached 1.014, negative serum OD 450nm The value was 0.063. Although the positive serum value increased to 1.455 at 90 minutes of incubation, the negative reaction still did not increase significantly, indicating that the gD protein antigen has good specificity. Therefore, the serum incubation time was determined to be 60 minutes.

[0143]

[0144] 4. Determination of enzyme-labeled secondary antibody dilution multiple and action time 1. Same as step 1 in step 2.

[0145] 2. Same as step 2 in step 1.

[0146] 3. Same as step 3 in step 1.

[0147] 4. Same as step 4 in step 1.

[0148] 5. Same as step 5 in step 2.

[0149] 6. Same as step 6 in step 1.

[0150] 7. After completing step 6, take the reaction plate, add 100 μL of enzyme-labeled secondary antibody working solution to each well, and incubate at 37°C for 30 min, 45 min, or 60 min.

[0151] The enzyme-labeled secondary antibody working solutions were: horseradish peroxidase-labeled goat anti-chicken IgG diluted to 5000 times volume, 10000 times volume, 20000 times volume or 40000 times volume.

[0152] 8. Same as step 8 in step 1.

[0153] 9. Same as step 9 in step 1.

[0154] 10. Same as step 10 in step 1.

[0155] The results are shown in Table 4. As the dilution factor of the secondary antibody increases, the OD 450nm The OD value of the standard positive serum showed a downward trend, while the background value of the negative serum remained at a low level. When the dilution ratio of the secondary antibody was 1:5000 and the incubation time was 45 minutes, the OD value of the standard positive serum 450 The value was 1.103, and the OD of negative serum 450nm The value was 0.064, and the P / N value was 17.274. Under these conditions, the detection signal-to-noise ratio was optimal, and the positive signal intensity and negative background value were both within the ideal range. Therefore, the optimal dilution of the HRP-labeled goat anti-chicken IgG secondary antibody was determined to be 1:5000, and the incubation time was 45 minutes.

[0156]

[0157] 5. Determination of critical value 42 SPF chicken sera (ILTV-negative sera from white Leghorn chickens provided by the Animal Disease Diagnosis and Research Center of China Agricultural University) were used as test sera.

[0158] 1. Same as step 1 in step 2.

[0159] 2. Same as step 2 in step 1.

[0160] 3. Same as step 3 in step 1.

[0161] 4. Same as step 1, step 4.

[0162] 5. After completing step 4, take the reaction plate, add serum diluent (100 μL / well), and incubate at 37°C for 60 minutes.

[0163] Serum diluent was prepared by gradiently diluting the test serum to 1600 times the volume with 5% skim milk solution, and 3 replicate wells were set for each serum dilution.

[0164] 6. Same as step 6 in step 1.

[0165] 7. After completing step 6, take the reaction plate, add 100 μL of enzyme-labeled secondary antibody working solution to each well, and incubate at 37°C for 45 minutes.

[0166] Enzyme-labeled secondary antibody working solution: dilute horseradish peroxidase-labeled goat anti-chicken IgG to 5000 times the volume.

[0167] 8. Same as step 8 in step 1.

[0168] 9. Same as step 9 in step 1.

[0169] 10. Same as step 10 in step 1.

[0170] Statistical analysis was performed to calculate the OD of 42 negative serum samples. 450nm The average value of the value ( ) and standard deviation (SD), which are used as the critical standard for negative and positive judgment of ELISA method. 450nm Value ≥ +3SD, it is determined to be ILTV positive. 450nm Value< +2SD was considered negative. +2SD ≤OD 450nm Value< If the sample is +3SD, it is considered suspicious and retesting is recommended. 450nm Average value ( ) was 0.073, and the standard deviation (SD) was 0.032.

[0171] Therefore, the judgment criteria are established as follows: When OD 450nm A value ≥0.169 was considered positive; When OD 450nm A value < 0.137 was considered negative; When OD 450nmWhen the value is ≥0.137 and <0.169, it is judged as a suspicious sample.

[0172] Example 4. Performance analysis of indirect ELISA method 1. Repeatability test 1. Intra-plate repeatability test The standard positive serum prepared in Example 2 and the standard negative serum prepared in Example 2 were used as test serum. The operation was carried out according to step 5 of Example 3. The repeatability of the standard positive serum and the standard negative serum in the same reaction plate was evaluated. The specific method was as follows: 16 parallel wells were set for each of the positive serum and the negative serum, and the OD of each well was measured. 450nm values, calculate their arithmetic mean ( ), standard deviation (SD) and coefficient of variation (CV). The results are shown in Table 5. The OD of the standard positive serum 450nm The average value was 1.062, and the coefficient of variation was 6.3%; the standard negative serum OD 450nm The average value was 0.049, and the coefficient of variation was 8.0%. The coefficient of variation for both groups of samples was less than 10%, indicating that the ELISA system has good intra-plate reproducibility.

[0173]

[0174] 2. Inter-plate repeatability test The standard positive serum prepared in Example 2 and the standard negative serum prepared in Example 2 were used as test serum. The operation was carried out according to step 5 of Example 3. The positive serum and negative serum were tested on different reaction plates for reproducibility. 16 parallel wells were set for each serum sample, and the OD values of each plate were measured. 450nm values, calculate the average value ( ), standard deviation (SD), and coefficient of variation (CV). The results are shown in Table 6. Sixteen replicates of the standard positive serum on different reaction plates yielded an average value of 1.079 and a coefficient of variation of 5.3%. The average value of replicates of the standard negative serum was 0.049 and a coefficient of variation of 8.5%. The coefficients of variation for both standard sera were less than 10%, demonstrating that the new method has good inter-plate reproducibility.

[0175]

[0176] 3. Sensitivity Test 1. Same as step 1 of step 2 in Example 3.

[0177] 2. Same as step 2 of step 1 in Example 3.

[0178] 3. Same as step 3 of step 1 in Example 3.

[0179] 4. Same as step 4 of step 1 in Example 3.

[0180] 5. After completing step 4, take the reaction plate, add serum diluent (100 μL / well), and incubate at 37°C for 60 minutes.

[0181] Serum diluent: dilute the standard positive serum with 5% skim milk solution in a gradient manner to 200-fold, 400-fold, 800-fold, 1600-fold, 3200-fold, 6400-fold, 12800-fold or 25600-fold volume, respectively.

[0182] 6. Same as step 6 of step 1 in Example 3.

[0183] 7. Same as step 7 of step 5 in Example 3.

[0184] 8. Same as step 8 of step 1 in Example 3.

[0185] 9. Same as step 9 of step 1 in Example 3.

[0186] 10. Same as 10 in step 1 of Example 3.

[0187] The results are shown in Table 7. The results showed that when the positive serum was diluted to 1:12800, the OD 450nm The value was still above the set negative / positive threshold, and the result was determined to be positive. When the dilution ratio reached 1:25,600, the detection signal dropped below the critical value. In summary, the minimum detection limit of this ELISA method is 1:12,800, indicating that it has high detection sensitivity and is suitable for the detection needs of low-titer antibody samples.

[0188]

[0189] 4. Specificity Test The test sera were as follows (all chicken sera from White Leghorn chickens, identified and provided by the Animal and Poultry Disease Diagnostic Research Center of China Agricultural University): H5 subtype avian influenza virus (AIV-H5)-positive serum, H7 subtype avian influenza virus (AIV-H7)-positive serum, infectious bronchitis virus (IBV)-positive serum, Newcastle disease virus (NDV)-positive serum, and fowl adenovirus type 4 (FAdV-4)-positive serum. The standard positive serum prepared in Example 2 and the standard negative serum prepared in Example 2 were used as positive and negative controls, respectively, for the test sera.

[0190] Proceed according to step 5 of Example 3.

[0191] The results are shown in Table 8. The OD of each serum to be tested 450nmThe values were significantly lower than the established cutoff values for positive / negative determination, indicating that all results were negative, with no significant cross-reactivity observed. This demonstrates that this method has good specificity and can effectively distinguish ILTV antibodies from antibodies against other common avian pathogens, making it suitable for specific detection in actual clinical samples.

[0192]

[0193] Comparative Example 1 1. Construction of control plasmid 1 Construction of control plasmid 1. Compared with the recombinant plasmid pFastBacI-gD, the control plasmid 1 differs only in that the segment represented by positions 124 to 1089 in SEQ ID NO: 2 is replaced by the DNA represented by SEQ ID NO: 3.

[0194] 2. Comparison of the ability of the recombinant plasmid pFastBacⅠ-gD or the control plasmid 1 to express the target protein The test plasmids were recombinant plasmid pFastBacⅠ-gD or control plasmid 1.

[0195] Take the test plasmid and follow the procedure of step 2 in Example 1. Then, take 2 mL of the bacterial solution containing the recombinant bacmid and inoculate it into 200 mL of liquid LB medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline. Incubate with shaking at 37°C and 220 rpm for 14-16 hours. Then, use a kit to extract the plasmid to obtain the recombinant bacmid. Then, transfect 3 μg of the recombinant bacmid into Sf9 cells (using Cellfectin™ II reagent), incubate at 27°C for 72 hours, and harvest the supernatant for Western blotting (the primary antibody used was a mouse anti-His-Tag mAb, available from Abotek Biotechnology Co., Ltd., catalog number AE003).

[0196] See the results Figure 7 . Figure 7 In the figure, lane M is a protein molecular weight standard; lane 1 is a control of Sf9 cells that were not transfected with recombinant Bacmid; lane 2 is the supernatant obtained by using control plasmid 1; lane 3 is the supernatant obtained by using recombinant plasmid pFastBacⅠ-gD. Figure 7 Grayscale scanning was performed for quantification. Compared with the target band shown in the above step using control plasmid 1, the protein represented by the target band shown in the above step using recombinant plasmid pFastBacⅠ-gD was 1.2 times more abundant.

[0197] Comparative Example 2 1. Construction of control plasmid 2 Construction of control plasmid 2. Compared with the recombinant plasmid pFastBacI-gD, the only difference of control plasmid 2 is that the segment represented by positions 124 to 1089 in SEQ ID NO: 2 is replaced by the DNA molecule represented by SEQ ID NO: 4.

[0198] 2. Comparison of the ability of the recombinant plasmid pFastBacⅠ-gD or the control plasmid 2 to express the target protein The test plasmids were recombinant plasmid pFastBacⅠ-gD or control plasmid 2.

[0199] The method is the same as step 2 of Comparative Example 1.

[0200] See the results Figure 8 . Figure 8 In the figure, lane M is a protein molecular weight standard; lane 1 is a control of Sf9 cells not transfected with recombinant Bacmid; lane 2 is the supernatant obtained by using the recombinant plasmid pFastBacⅠ-gD; and lane 3 is the supernatant obtained by using control plasmid 2. The results show that control plasmid 2 failed to effectively express the target protein.

[0201] 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 practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A method for preparing a recombinant protein, comprising the steps of: obtaining the recombinant protein by expressing a gene encoding the recombinant protein in a cell; The recombinant protein is as follows (a1) or (a2) or (a3) or (a4): (a1) a protein having a gp67 signal peptide and a gD protein extracellular region from its N-terminus to its C-terminus, wherein the gD protein extracellular region is positions 85-406 of the gD protein; (a2) a protein obtained by inserting a purification tag into (a1); the purification tag is inserted downstream of the gD protein extracellular region or the purification tag is inserted between the gp67 signal peptide and the gD protein extracellular region; (a3) a protein comprising a gD protein extracellular region; the gD protein extracellular region is from position 85 to position 406 of the gD protein; (a4) A protein obtained by inserting a purification tag into (a3); the purification tag is inserted upstream of the extracellular region of the gD protein or downstream of the extracellular region of the gD protein.

2. The method according to claim 1, wherein: In the recombinant protein, the extracellular region of the gD protein is as shown in positions 42 to 363 of SEQ ID NO: 1 or as shown in positions 41 to 363 of SEQ ID NO:

1.

3. The method according to claim 2, wherein: In the recombinant protein encoding gene, the coding region of the gD protein extracellular region is shown as positions 124-1089 in SEQ ID NO: 2 or as positions 121-1089 in SEQ ID NO:

2.

4. The method according to any one of claims 1 to 3, wherein: In the method, a baculovirus expression system is used to express the gene encoding the recombinant protein in cells.

5. The method according to claim 4, wherein: The method comprises the following steps: (1) introducing a recombinant plasmid into Escherichia coli DH10Bac to obtain a recombinant Escherichia coli; the recombinant plasmid is a recombinant plasmid obtained by inserting a DNA molecule having a gene encoding the recombinant protein into a baculovirus transfer vector; (2) culturing the recombinant E. coli obtained in step (1) and extracting the plasmid, which is the recombinant Bacmid; (3) Transfecting Sf9 cells with the recombinant Bacmid obtained in step (2) and culturing the cells, collecting the culture supernatant, which is the P1 generation virus solution; (4) Infecting Sf9 cells with the P1 virus solution and culturing them, and collecting the culture supernatant, which is the P2 virus solution; (5) Infecting Sf9 cells with the P2 virus solution and culturing them, collecting the culture supernatant, which is the P3 virus solution; (6) Infecting High Five cells with the P3 generation virus solution, collecting the culture supernatant, and purifying to obtain the recombinant protein.

6. Protein, which is (a1) or (a2) or (a3) or (a4) as follows: (a1) a protein having a gp67 signal peptide and a gD protein extracellular region from its N-terminus to its C-terminus, wherein the gD protein extracellular region is positions 85-406 of the gD protein; (a2) a protein obtained by inserting a purification tag into (a1); the purification tag is inserted downstream of the gD protein extracellular region or the purification tag is inserted between the gp67 signal peptide and the gD protein extracellular region; (a3) a protein comprising a gD protein extracellular region; the gD protein extracellular region is from position 85 to position 406 of the gD protein; (a4) A protein obtained by inserting a purification tag into (a3); the purification tag is inserted upstream of the extracellular region of the gD protein or downstream of the extracellular region of the gD protein.

7. Biological material, any of the following (b1) to (b8): (b1) a gene encoding the protein according to claim 6; (b2) having the expression cassette of (b1); (b3) a recombinant vector comprising (b1); (b4) a recombinant vector comprising (b2); (b5) recombinant cell having (b1); (b6) recombinant cells having (b2); (b7) recombinant cells having (b3); (b8) Recombinant cells with (b4).

8. Use of the protein according to claim 6 as an antigen; the antigen is an antigen that specifically binds to antibodies against infectious laryngotracheitis virus of avian influenza virus.

9. Use of the protein according to claim 6 or the biological material according to claim 7 in preparing a kit; the kit is a kit for detecting antibodies against infectious laryngotracheitis virus.

10. A kit comprising the following (d1) or (d2): (d1) a kit comprising the protein according to claim 6; (d2) a kit comprising an ELISA plate coated with the protein of claim 6; The kit is a kit for detecting antibodies to infectious laryngotracheitis virus of chickens.

Images