Enzyme-linked immunoassay kit for detecting antibody of chicken infectious laryngotracheitis virus and fusion protein and biological material used thereby

Abstract

The application discloses an enzyme-linked immunoassay kit for detecting chicken infectious laryngotracheitis virus antibody and a fusion protein and a biological material used in the enzyme-linked immunoassay kit. The application provides a method for preparing a recombinant protein, wherein the recombinant protein is obtained by expressing a coding gene of the recombinant protein in cells; the recombinant protein has, in sequence from an N terminus to a C terminus, a gp67 signal peptide and an extracellular region of gD protein; and the extracellular region of gD protein is located at 85-406 of the gD protein. The application also protects the recombinant protein and the application of the recombinant protein as an antigen for specifically combining with chicken infectious laryngotracheitis virus antibody. The application also protects the application of the protein in preparing a kit for detecting chicken infectious laryngotracheitis virus antibody. The recombinant protein of the application has the advantages of high sensitivity and strong specificity when detecting ILTV antibody in serum of chicken groups, and is suitable for large-scale epidemiological monitoring and immune effect evaluation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of peptides in the field of biotechnology, and relates to an enzyme-linked immunoreagent kit for detecting antibodies of chicken infectious laryngotracheitis virus and a fusion protein and a biological material used by the enzyme-linked immunoreagent kit. BACKGROUND

[0002] Chicken infectious laryngotracheitis virus (ILTV) belongs to Herpesviridae, Alphaherpesvirinae and Iltovirus, and can cause an acute upper respiratory tract infectious disease of poultry, i.e., chicken infectious laryngotracheitis (ILT). ILTV mainly infects chicken flocks, and causes symptoms such as cough, panting, salivation, laryngeal hemorrhage and dyspnea, and can cause large-scale death in severe cases, thereby causing huge economic losses to the global poultry industry. In particular, under the current intensive breeding environment, the occasional outbreak of ILT poses a severe challenge to the prevention and control of poultry diseases.

[0003] Glycoprotein D (also referred to as gD protein) is an important structural protein of ILTV, belongs to type II transmembrane protein, widely exists on the outer membrane of the virus, participates in the combination of the virus and the host cell receptor and the membrane fusion process, and is one of key factors for the virus to infect the host. The gD protein has good immunogenicity, and can effectively induce the body to produce specific neutralizing antibodies. Therefore, the gD protein is not only an important candidate antigen for vaccine research and development, but also an ideal target in ILTV serological detection.

[0004] Traditional gD protein expression systems generally use Escherichia coli or mammalian cell expression systems. However, the gD protein itself belongs to a virus protein with complex glycosylation modification. The Escherichia coli expression system is easy to cause protein misfolding and inclusion body formation, thereby affecting the immunological activity and structural function thereof. Although the mammalian cell expression system can realize protein glycosylation modification, the culture cost thereof is high, the operation thereof is complex, and the yield thereof is low, thereby limiting the large-scale application thereof. SUMMARY

[0005] The purpose of the present application is to provide an enzyme-linked immunoreagent kit for detecting antibodies of chicken infectious laryngotracheitis virus and a fusion protein and a biological material used by the enzyme-linked immunoreagent kit.

[0006] The present application provides a method for preparing a recombinant protein, comprising the following steps: obtaining the recombinant protein by expressing a coding gene of the recombinant protein in a cell;

[0007] The recombinant protein is as follows (a1) or (a2) or (a3) or (a4):

[0008] (a1) a protein having, in order from the N terminus to the C terminus, a gp67 signal peptide and a gD protein extracellular region; the gD protein extracellular region is positions 85-406 of a gD protein;

[0009] (a2) a protein obtained by inserting a purification tag in (a1); the insertion position of the purification tag is downstream of the gD protein extracellular region or the insertion position of the purification tag is between the gp67 signal peptide and the gD protein extracellular region;

[0010] (a3) a protein having a gD protein extracellular region; the gD protein extracellular region is positions 85-406 of a gD protein;

[0011] (a4) a protein obtained by inserting a purification tag in (a3); the insertion position of the purification tag is upstream of the gD protein extracellular region or downstream of the gD protein extracellular region.

[0012] The gD protein is a gD protein derived from Infectious laryngotracheitis virus.

[0013] As an example, the gD protein is as shown in GenBank: WRQ97535.1 (17-JAN-2024).

[0014] As an example, positions 85-406 of the gD protein correspond to positions 42-363 of SEQ ID NO: 1.

[0015] In the recombinant protein, the gD protein extracellular region is as shown in positions 42-363 of SEQ ID NO: 1.

[0016] In the recombinant protein, the gD protein extracellular region is as shown in positions 41-363 of SEQ ID NO: 1.

[0017] As an example, the purification tag is a His tag, which can be a 6xHis tag or an 8xHis tag.

[0018] As an example, the gp67 signal peptide is as shown in positions 1-38 of SEQ ID NO: 1.

[0019] As an example, the recombinant protein is as shown in positions 42-363 of SEQ ID NO: 1.

[0020] As an example, the recombinant protein is as shown in positions 41-363 of SEQ ID NO: 1.

[0021] As an example, the recombinant protein is as shown in SEQ ID NO: 1 from the 39th to the 363rd.

[0022] As an example, the recombinant protein is as shown in SEQ ID NO: 1 from the 42nd to the 373rd.

[0023] As an example, the recombinant protein is as shown in SEQ ID NO: 1 from the 41st to the 373rd.

[0024] As an example, the recombinant protein is as shown in SEQ ID NO: 1 from the 39th to the 373rd.

[0025] As an example, the recombinant protein is as shown in SEQ ID NO: 1 from the 42nd to the 379th.

[0026] As an example, the recombinant protein is as shown in SEQ ID NO: 1 from the 41st to the 379th.

[0027] As an example, the recombinant protein is as shown in SEQ ID NO: 1 from the 39th to the 379th.

[0028] As an example, the recombinant protein is as shown in SEQ ID NO: 1 from the 1st to the 363rd.

[0029] As an example, the recombinant protein is as shown in SEQ ID NO: 1 from the 1st to the 373rd.

[0030] As an example, the recombinant protein is as shown in SEQ ID NO: 1 from the 1st to the 379th.

[0031] As an example, the recombinant protein is as shown in SEQ ID NO: 1.

[0032] As an example, the coding gene of the recombinant protein is as follows: the coding region of the extracellular region of the gD protein is as shown in SEQ ID NO: 2 from the 124th to the 1089th.

[0033] As an example, the coding gene of the recombinant protein is as follows: the coding region of the extracellular region of the gD protein is as shown in SEQ ID NO: 2 from the 121st to the 1089th.

[0034] As an example, the coding gene of the recombinant protein is as follows: any one of (c1) to (c13):

[0035] (c1) a DNA molecule with a coding region as shown in SEQ ID NO: 2 from the 124th to the 1089th.

[0036] (c2) a DNA molecule whose coding region is represented by positions 121-1089 of SEQ ID NO: 2;

[0037] (c3) a DNA molecule whose coding region is represented by positions 115-1089 of SEQ ID NO: 2;

[0038] (c4) a DNA molecule whose coding region is represented by positions 124-1119 of SEQ ID NO: 2;

[0039] (c5) a DNA molecule whose coding region is represented by positions 121-1119 of SEQ ID NO: 2;

[0040] (c6) a DNA molecule whose coding region is represented by positions 115-1119 of SEQ ID NO: 2;

[0041] (c7) a DNA molecule whose coding region is represented by positions 124-1137 of SEQ ID NO: 2;

[0042] (c8) a DNA molecule whose coding region is represented by positions 121-1137 of SEQ ID NO: 2;

[0043] (c9) a DNA molecule whose coding region is represented by positions 115-1137 of SEQ ID NO: 2;

[0044] (c10) a DNA molecule whose coding region is represented by positions 1-1089 of SEQ ID NO: 2;

[0045] (c11) a DNA molecule whose coding region is represented by positions 1-1119 of SEQ ID NO: 2;

[0046] (c12) a DNA molecule whose coding region is represented by positions 1-1137 of SEQ ID NO: 2;

[0047] (c13) a DNA molecule whose coding region is represented by SEQ ID NO: 2.

[0048] In the method, the coding gene of the recombinant protein is expressed in cells by using a baculovirus expression system. The baculovirus-insect cell expression system (BICS) has the advantages of large protein expression amount, complete post-expression translation modification, and short culture cycle. The system can efficiently express target proteins in insect cells (such as Sf9 cells or High Five cells), and has good protein folding ability and secretion mechanism, and is suitable for the preparation of vaccine antigens and diagnostic antigens. Therefore, developing a method for large-scale and soluble expression of target proteins based on the baculovirus expression system, and combining it to construct an ELISA detection system with simple operation, strong specificity and high sensitivity, becomes the key path to solve the bottleneck of the prior art. This not only helps to improve the immune prevention and control level of ILTV, but also provides strong technical support for the healthy development of poultry breeding industry.

[0049] As an example, the method for preparing a recombinant protein provided by the present application comprises the following steps:

[0050] (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 coding gene of the recombinant protein into a baculovirus transfer vector;

[0051] (2) culturing the recombinant Escherichia coli obtained in step (1) and extracting a plasmid, which is a recombinant Bacmid;

[0052] (3) transfecting the recombinant Bacmid obtained in step (2) into Sf9 cells and culturing, and collecting a culture supernatant, which is a P1 generation virus liquid;

[0053] (4) infecting Sf9 cells with the P1 generation virus liquid and culturing, and collecting a culture supernatant, which is a P2 generation virus liquid;

[0054] (5) infecting Sf9 cells with the P2 generation virus liquid and culturing, and collecting a culture supernatant, which is a P3 generation virus liquid;

[0055] (6) infecting High Five cells with the P3 generation virus liquid, collecting a culture supernatant, and purifying to obtain the recombinant protein.

[0056] Illustratively, the baculovirus transfer vector is a pFastBacI vector.

[0057] Specifically, the recombinant plasmid is a recombinant plasmid obtained by replacing a small fragment between the restriction enzyme recognition sequences of BamH I and Hind III in the pFastBacI vector with a double-stranded DNA molecule shown in SEQ ID NO: 2.

[0058] Specifically, the step (3) can be: transfecting the Sf9 cells with the recombinant Bacmid obtained in step (2), then culturing at 27°C for 72h, and harvesting the supernatant, which is the P1 generation virus liquid. The transfection is assisted by Cellfectin™ II reagent. 3μg of the recombinant Bacmid is used to transfect the Sf9 insect cells to obtain 2mL of the P1 generation virus liquid.

[0059] Specifically, the step (4) can 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 with 110rpm oscillation until more than 80% of the cells show pathological changes, and harvesting the supernatant, which is the P2 generation virus liquid.

[0060] Specifically, the step (5) can 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 with 110rpm oscillation until more than 80% of the cells show pathological changes, and harvesting the supernatant, which is the P3 generation virus liquid.

[0061] The preparation method of the cell suspension in step (4) and step (5) is: collecting the Sf9 cells in the logarithmic growth phase, resuspending with SIM SF Expression Medium to make the cell content 1.0×10 6 cells / mL.

[0062] 2mL of the P1 generation virus liquid is used for the amplification to obtain about 200mL of the P3 generation virus liquid.

[0063] Specifically, the step (6) can be: inoculating the P3 virus liquid into the cell suspension (the volume ratio of the virus liquid to the cell suspension is 1:100), culturing at 27°C with 120rpm oscillation for 48h, then centrifuging at 1500rpm for 15min, collecting the supernatant, and then purifying.

[0064] The preparation method of the cell suspension in step (6) is: taking the High Five cells, resuspending with SIM HF Expression Medium to make the cell content 1.5-2.0×10 6 cells / mL.

[0065] 200mL of the P3 generation virus liquid is used to prepare about 20L of the supernatant.

[0066] Exemplarily, the purification is affinity chromatography purification.

[0067] Exemplarily, the purification is nickel column purification.

[0068] The gp67 signal peptide is derived from a baculovirus natural secretion signal peptide, which can effectively guide the post-translational transport of the recombinant gD polypeptide chain, promote it to enter the endoplasmic reticulum of the host cell (in the endoplasmic reticulum, the signal peptide is self-cleaved by the cell), and be guided to the extracellular environment through the classical secretion pathway, thereby improving the secretion efficiency and correct folding of the exogenous protein. The gD protein is an important transmembrane glycoprotein on the surface of ILTV, and there are problems such as low expression amount and poor solubility in the process of heterologous expression of the full-length protein in vitro. Therefore, the extracellular region is cut from the full-length protein, and the codon optimization design is further carried out for the baculovirus expression system to ensure efficient expression in insect cells. Using 3 μg of recombinant Bacmid to transfect Sf9 cells, 2 mL of P1 generation recombinant baculovirus liquid can be successfully obtained. Subsequently, the P1 generation recombinant baculovirus liquid is inoculated into Sf9 cells at an infection amount of 10%, and the P2 generation recombinant baculovirus liquid is obtained. Subsequently, the P2 generation recombinant baculovirus liquid is inoculated into Sf9 cells at an infection amount of 10%, and about 200 mL of high-titer P3 generation recombinant baculovirus liquid is obtained. The P3 generation recombinant baculovirus liquid is used to infect High Five cells at an infection amount of 1%, and the expression peak of the gD protein can be reached after 48 hours of culture, and 20 L of culture supernatant can be harvested. Through detection, the expression amount of the target protein in the High Five cell culture liquid is as high as 6.67 mg / L, which meets the demand of large-scale protein preparation. Under the preferred conditions, the Sf9 cells and High Five cells used in the application are cultured in a suspension culture mode, and a serum-free culture medium system is used, which reduces the risk of exogenous protein pollution, is beneficial to simplify the downstream purification process, improves the batch consistency and controllability of product quality, and reduces the production cost. The nickel column affinity chromatography purification process is simple, and the protein purity is high. The recombinant protein prepared by the method of the application has a purity of not less than 90% detected by SDS-PAGE, and presents a single specific band detected by Western blot. It is suitable for subsequent ELISA antigen coating and application development, and has good operability and commercialization ability.

[0069] The application also protects a protein, which is any of the above-mentioned recombinant proteins.

[0070] Specifically, the protein is a recombinant protein prepared by the preparation method of any of the above-mentioned recombinant proteins.

[0071] The application also protects a biological material, which is any of the following (b1) to (b8):

[0072] (b1) a gene encoding the recombinant protein;

[0073] (b2) an expression cassette having (b1);

[0074] (b3) a recombinant vector having (b1);

[0075] (b4) a recombinant vector having (b2);

[0076] (b5) a recombinant cell having (b1);

[0077] (b6) a recombinant cell having (b2);

[0078] (b7) a recombinant cell having (b3);

[0079] (b8) a recombinant cell having (b4).

[0080] Specifically, the coding gene of the recombinant protein is any of the above.

[0081] Specifically, the recombinant vector is a recombinant plasmid obtained by inserting the coding gene of the recombinant protein into a pFastBacI vector. Specifically, the recombinant vector is a recombinant plasmid obtained by substituting a double-stranded DNA molecule shown in SEQ ID NO: 2 between positions 1-1119 with a small fragment between the restriction enzyme recognition sequences of BamH I and Hind III. Specifically, the recombinant vector is a recombinant Bacmid obtained by introducing the above-mentioned recombinant plasmid into E. coli DH10Bac and culturing.

[0082] The recombinant cell is a recombinant animal cell or a recombinant plant cell or a recombinant microorganism.

[0083] In certain embodiments, the animal cell line can be a non-reproductive material. In certain embodiments, the animal cell can be an ex vivo 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), Chinese hamster ovary subline 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 mammary tumor cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblast cells, bone marrow cell lines, T cells, or NK cells, etc. Avian cells include, but are not limited to, chicken cells, duck cells, or goose cells, etc. Amphibian cells include, but are not limited to, Xenopus laevis cells or Megalobatrachus knoepffli, etc. Fish cells include, but are not limited to, grass carp cells, carp cells, rainbow trout cells, or catfish cells, etc. Insect cells include, but are not limited to, Sf21 cells, Sf-9 cells, or High Five cells (Hi-5 cells), etc. In certain embodiments, the mammalian cell does not include animal reproductive cells, animal zygotes, and animal embryonic stem cells. In certain embodiments, the mammalian cell can be a somatic cell or a cell line.

[0084] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes, algae, etc. The bacteria can be from the genus Corynebacterium Corynebacterium sp. , such as Corynebacterium glutamicum, Corynebacterium pekinense, Corynebacterium crenatum, etc. The bacteria can be from the genus Brevibacterium Brevibacterium sp. , such as Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium ammoniagenes, etc. The bacteria can be from the genus Escherichia Escherichia sp. , such as Escherichia coli. The bacteria can be from the genus Erwinia Erwinia sp. . The bacteria can be from the genus Agrobacterium Agrobacterium sp. , such as Agrobacterium tumefaciens. The bacteria can be from the genus Flavobacterium (Flavobacterium sp. . The bacteria can be from the genus Alcaligenes Alcaligenes sp. . The bacteria can be from the genus Pseudomonas Pseudomonas sp. . The bacteria can be from the genus Bacillus Bacillus sp. , such as Bacillus sp., etc. Exemplarily, the Escherichia coli can be Escherichia coli DH10Bac. The viruses can include rotavirus, baculovirus, retrovirus (such as lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papovavirus (such as SV40), and herpesvirus (such as herpes simplex virus), etc. The fungi can be from the genus Saccharomyces Saccharomyces sp.), such as Saccharomyces cerevisiae, Candida, Pichia, etc. The fungus can be from Fusarium (Fusarium sp.) Fusarium sp. ). The fungus can be from Rhizoctonia (Rhizoctonia sp.) Rhizoctonia sp. ). The fungus can be from Verticillium (Verticillium sp.) Verticillium sp. ). The fungus can be from Penicillium (Penicillium sp.) Penicillium sp. ). The fungus can be from Aspergillus (Aspergillus sp.) Aspergillus sp. ). The fungus can be from Cephalosporium (Cephalosporium sp.) Cephalosporium sp. ). The actinomycete can be from Streptomyces (Streptomyces sp.) Streptomyces sp. ), such as Streptomyces. The algae can be from Cyanophyta (Cyanophyta sp.) Cyanophyta ), such as blue algae. The algae can be from Fucus (Fucus sp.) Fucus sp. ). The algae can be from Chlorella (Chlorella sp.) Achnanthes sp. ). The algae can be from Coccomyxa (Coccomyxa sp.) Amphiprora sp. ). The algae can be from Dimorphococcus (Dimorphococcus sp.) Amphora sp. ). The algae can be from Oedogonium (Oedogonium sp.) Ankistrodesmus sp. ). The algae can be from Platymonas (Platymonas sp.) Asteromonas sp. ). The algae can be from Chrysochromulina (Chrysochromulina sp.) Boekelovia sp. ).

[0085] Exemplarily, the recombinant cell is a recombinant cell into which the gene encoding the recombinant protein is introduced into E. coli DH10Bac. Exemplarily, the recombinant cell is a recombinant cell into which the recombinant vector is introduced into E. coli DH10Bac. Exemplarily, the recombinant cell is a recombinant cell into which the gene encoding the recombinant protein is introduced into Sf9 cell. Exemplarily, the recombinant cell is a recombinant cell into which the recombinant vector is introduced into Sf9 cell. Exemplarily, the recombinant cell is a recombinant cell into which the gene encoding the recombinant protein is introduced into High Five cell. Exemplarily, the recombinant cell is a recombinant cell into which the recombinant vector is introduced into High Five cell.

[0086] The present application also protects the use of the protein as an antigen; the antigen is an antigen specifically binding with the antibody of chicken infectious laryngotracheitis virus. Exemplarily, the antigen is a coating antigen.

[0087] The present application also protects the use of the protein or the biological material in the preparation of a kit; the kit is a kit for detecting the antibody of chicken infectious laryngotracheitis virus. In the kit, the protein serves as a coating antigen.

[0088] The present application also protects a kit, which is as follows (d1) or (d2):

[0089] (d1) a kit, which comprises the protein;

[0090] (d2) a kit comprising an enzyme labeled plate coated with the protein;

[0091] The kit is a kit for detecting antibodies of chicken infectious laryngotracheitis virus.

[0092] As an example, in the enzyme labeled plate coated with the protein, the coating concentration is 0.125 μg / mL.

[0093] The kit further comprises a secondary antibody working solution.

[0094] The secondary antibody working solution is obtained by diluting an enzyme labeled secondary antibody.

[0095] As an example, the secondary antibody working solution is obtained by diluting an enzyme labeled secondary antibody to 5000 times the volume.

[0096] As an example, the enzyme labeled secondary antibody is horseradish peroxidase labeled goat anti-chicken IgG.

[0097] As an example, the enzyme labeled secondary antibody is a reagent with the product number bs-0310G-HRP from Boosen Biotech Co., Ltd.

[0098] The present application establishes a method for detecting ILTV antibodies in serum samples of chicken flocks 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.

[0099] The method for detecting ILTV antibodies in serum samples of chicken flocks is an indirect ELISA detection method.

[0100] The indirect ELISA detection method comprises the following steps:

[0101] (1) dilute the recombinant protein to 0.125 μg / mL, coat an enzyme labeled plate, and incubate at 4°C;

[0102] (2) use 5% skim milk solution to block at 37°C for 60 min;

[0103] (3) add the serum diluent to be detected (the dilution multiple of the serum to be detected is 1:1600), and incubate at 37°C for 60 min;

[0104] (4) add the enzyme labeled secondary antibody working solution (the dilution multiple of the enzyme labeled secondary antibody is 1:5000), and incubate at 37°C for 45 min;

[0105] (5) add TMB color developing solution, terminate the reaction after 10 min, and measure the OD value at 450 nm wavelength;

[0106] (6) when the OD 450nmOD value ≥ 0.169 is determined as positive; OD value < 0.137 is determined as negative; and OD value ≥ 0.137 and < 0.169 is determined as a suspicious sample. 450nm OD value < 0.137 is determined as negative; OD value ≥ 0.137 and < 0.169 is determined as a suspicious sample. 450nm OD value ≥ 0.137 and < 0.169 is determined as a suspicious sample. BRIEF DESCRIPTION OF DRAWINGS

[0107] Figure 1 Figure 1 is a schematic diagram of elements of recombinant plasmid pFastBacl-gD.

[0108] Figure 2 Figure 2 is a schematic diagram of exemplary results of PCR identification of recombinant Bacmid.

[0109] Figure 3 Figure 3 is a schematic diagram of results of Western blot detection of P3 virus solution.

[0110] Figure 4 Figure 4 is a schematic diagram of SDS-PAGE identification results of purification of target protein using Ni-NTA affinity chromatography column.

[0111] Figure 5 Figure 5 is a schematic diagram of results of Western blot detection of gD-His solution.

[0112] Figure 6 Figure 6 is a schematic diagram of results of SDS-PAGE electrophoresis and Coomassie brilliant blue staining of gD-His solution.

[0113] Figure 7 Figure 7 is a schematic diagram of results of Western Blot detection in Comparative Example 1.

[0114] Figure 8 Figure 8 is a schematic diagram of results of Western Blot detection in Comparative Example 2. DETAILED DESCRIPTION

[0115] The present application will be further described in conjunction with the specific embodiments, and the examples given are only for the purpose of illustrating the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0116] The experimental methods in the following examples 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, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified. The quantitative tests in the following examples are all set up in triplicate, and the results are averaged, unless otherwise specified. pFastBacI vector (pFastBac1 vector): Yobo Biotech Co., Ltd., product number VT1166. DH10Bac competent cells: Beijing Bomeide 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. Avian infectious laryngotracheitis virus HB201806 strain, recorded in GenBank: PP062931.1 (17-JAN-2024). Skimmed milk powder: Biyun Tian Biotechnology Co., Ltd., item number P0216. ELISA coating solution (10x): Solabio Technology Co., Ltd., item number C1055. Dilute the ELISA coating solution (10x) with distilled water to 10 times the volume, i.e. 1x ELISA coating solution. 5% skimmed milk solution: PBST solution containing 5 g / 100 mL skimmed milk powder. Two-component TMB developing solution: Solabio Technology Co., Ltd., item number PR1210. ELISA stop solution: Solabio Technology Co., Ltd., item number C1058. Cellfectin™ II reagent: Thermo Fisher Scientific, product number 10362100, used according to the instructions. Horseradish peroxidase-labeled goat anti-chicken IgG (Goat Anti-Chicken IgG H&L, HRP conjugated): Boaosen Biotech Co., Ltd., bs-0310G-HRP, diluted with 5% skimmed milk solution when used.

[0117] Example 1, Preparation of Target Protein

[0118] I. Construction of Recombinant Plasmid pFastBacI-gD

[0119] The double-stranded DNA molecule shown in SEQ ID NO: 2 was used to replace the small fragment between the BamH I and Hind III enzyme cleavage sites in the pFastBacI vector to obtain the recombinant plasmid pFastBacI-gD. The recombinant plasmid pFastBacI-gD has been sequenced and verified, and the schematic diagram of its elements is shown in Figure 1The DNA shown in SEQ ID NO: 2 is in a recombinant plasmid.

[0120] The DNA shown in SEQ ID NO: 2 encodes the protein shown in SEQ ID NO: 1. In SEQ ID NO: 1, the first to 38th amino acid residues constitute a gp67 signal peptide, the 42nd to 363rd amino acid residues constitute an extracellular region of gD protein, and the 366th to 373rd amino acid residues constitute an 8xHis tag. The signal peptide is cleaved by the cell in the endoplasmic reticulum, forming a mature protein (the mature protein is shown in SEQ ID NO: 1 from the 39th to 379th positions). In SEQ ID NO: 2, the first to 114th nucleotides encode the gp67 signal peptide, the 124th to 1089th nucleotides encode the extracellular region of gD protein (which has been codon-optimized), and the 1096th to 1119th nucleotides encode the 8xHis tag.

[0121] II. Construction of a recombinant baculovirus expression vector

[0122] 1. Add 1-10 ng of the recombinant plasmid to 100 μL of DH10Bac competent cells, mix well, and then place the centrifuge tube in a 42°C metal bath for heat shock treatment for 45 s, immediately ice-bath for 2 min, add 900 μL of liquid LB medium, and incubate at 37°C, 220 rpm for 4 h.

[0123] 2. After step 1 is completed, take 100 μL of the bacterial solution and spread it on 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 at 37°C in the dark for 24-48 h. At this time, white single colonies can be observed.

[0124] 3. After step 2 is completed, pick a uniform white colony from the plate and inoculate it into 1 mL of liquid LB medium containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, and 10 μg / ml tetracycline, and incubate at 37°C, 220 rpm for 12 h.

[0125] 4. After step 3 is completed, take the bacterial solution 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 at 37°C for 24-48 h.

[0126] 5、After step 4, pick uniform white colonies from the plate and inoculate into 1 mL of liquid LB medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin and 10 μg / mL tetracycline, and incubate at 37°C, 220 rpm for 12 h.

[0127] 6、After step 5, sample the bacterial solution and perform PCR identification using a primer pair consisting of M13-F and M13-R. If a characteristic band of about 3300 bp is shown, it indicates that the recombinant Bacmid with the DNA shown in SEQ ID NO: 2 is obtained. An exemplary result is shown in Figure 2 , showing a characteristic band of about 3300 bp.

[0128] M13-F (SEQ ID NO: 5): 5'-GTTTTCCCAGTCACGAC-3';

[0129] M13-R (SEQ ID NO: 6): 5'-CAGGAAACAGCTATGAC-3'.

[0130] III. Preparation of recombinant baculovirus

[0131] 1. Take 2 mL of the bacterial solution with recombinant Bacmid obtained in step ii and inoculate into 200 mL of liquid LB medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin and 10 μg / mL tetracycline, and incubate at 37°C, 220 rpm for 14-16 h. Then perform plasmid extraction using a kit to obtain the recombinant Bacmid. The kit used for plasmid extraction is called NucleoBond Xtra Midi Plus kit for transfection-grade plasmid DNA, MACHEREY-NAGEL, product number 740412.50, and is operated according to the instructions.

[0132] 2. Transfect Sf9 cells with the recombinant Bacmid obtained in step 1 (transfection is assisted by Cellfectin™ II reagent), and then incubate at 27°C for 72 h (cytopathic effect can be observed), and harvest the supernatant, which is the P1 generation virus solution.

[0133] Use 3 μg of recombinant Bacmid to transfect Sf9 insect cells to obtain 2 mL of P1 generation virus solution.

[0134] IV. Subculture and amplification of recombinant baculovirus

[0135] 1. Inoculate the P1 generation virus solution obtained in step 3 into the cell suspension (virus solution to cell suspension volume ratio of 1:10), then culture at 27℃ and 110 rpm with shaking until more than 80% of the cells show cytopathic effects. Harvest the supernatant, which is the P2 generation virus solution. Cell suspension preparation method: Collect Sf9 cells in logarithmic growth phase and resuspend them in SIM SF Expression Medium to achieve a cell concentration of 1.0 × 10⁻⁶ cells / mL. 6 cells / mL. SIM SF Expression Medium (For SF9, SF21) (Serumfree): Sinocare, product number MSF1.

[0136] 2. Inoculate the P2 generation virus solution obtained in step 1 into the cell suspension (the volume ratio of virus solution to cell suspension is 1:10), and then culture at 27°C and 110 rpm with shaking until more than 80% of the cells show cytopathic effects. Harvest the supernatant, which is the P3 generation virus solution. The preparation method of the cell suspension is the same as that in step 1.

[0137] 3. Take the P3 generation virus solution obtained in step 2 and perform Western blot detection (primary antibody: Mouse anti-His-Tag mAb, iBiotech Biotechnology Co., Ltd., catalog number AE003; secondary antibody: Goat Anti-mouse IgG-HRP, Jinpulai Biotechnology Co., Ltd., catalog number P03S01L). See results below. Figure 3 . Figure 3 In the diagram, lane 1 was the negative control (Sf9 cells), and lane 2 was the P3 generation virus solution. The P3 generation virus solution showed the target band at a molecular weight of approximately 60 kDa.

[0138] Approximately 200 mL of P3 generation virus solution was obtained by passage and amplification of 2 mL of P1 generation virus solution.

[0139] V. Large-scale expression and purification of gD protein

[0140] 1. Inoculate the P3 virus solution obtained in step four into the cell suspension (virus solution to cell suspension volume ratio is 1:100), incubate at 27℃ and 120 rpm with shaking for 48 h, then centrifuge at 1500 rpm for 15 min and collect the supernatant. Cell suspension preparation method: Take High Five cells and resuspend them in SIM HF Expression Medium to achieve a cell concentration of 1.5-2.0 × 10⁶ cells / mL. 6SIM HF Expression Medium (For Hi5) (Serum free): Yikai Shenzhou Co., Ltd., product number MHF1. About 20 L supernatant was prepared from 200 mL P3 virus liquid.

[0141] 2. The supernatant obtained in step 1 was purified by using a Ni-NTA affinity column (Solebo Technology Co., Ltd.) to obtain the target protein with His tag. Elution process: the column-passed solution was collected after elution with eluent containing 25 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM and 500 mM imidazole, respectively. Composition of eluent: imidazole, 50 mM Tris and 300 mM NaCl, and the rest was H20.

[0142] 3. The column-passed solution was sampled and identified by SDS-PAGE. The results are shown in Figure 4 . Figure 4 Lane 1: supernatant obtained in step 1; Lane 2: sample flow-through; Lane 3: column-passed solution eluted with 25 mM imidazole eluent; Lane 4: column-passed solution eluted with 50 mM imidazole eluent; Lane 5: column-passed solution eluted with 100 mM imidazole eluent; Lane 6: column-passed solution eluted with 200 mM imidazole eluent; Lane 7: column-passed solution eluted with 300 mM imidazole eluent; Lane 8: column-passed solution eluted with 400 mM imidazole eluent.

[0143] 4. According to the electrophoresis results of step 3, the column-passed solutions eluted with 50 mM imidazole eluent, 100 mM imidazole eluent and 200 mM imidazole eluent were combined, and then centrifuged and concentrated in a Millipore ultrafiltration tube, and the system was replaced (the purpose of system replacement is to replace the system with PBS buffer) to obtain a solution containing the target protein, also known as gD-His solution. The target protein, i.e. the protein shown in SEQ ID NO: 1 from 39 to 379, is named gD-His protein.

[0144] Six, identification of gD-His protein

[0145] The gD-His solution was taken for Western blot detection (the primary antibody used was Mouse anti His-Tag mAb, Abways Biotech Co., Ltd., product number AE003). The results are shown in Figure 5 . A single band was shown, and the molecular weight was consistent with the prediction.

[0146] The gD-His solution was taken for SDS-PAGE electrophoresis detection. The photo after Coomassie brilliant blue staining is shown inFigure 6 Clear target protein bands were observed. The gel was scanned by a gel imaging system and analyzed by ImageJ software. The purity of the target protein reached 92%.

[0147] The gD-His solution was quantified using a BCA kit, and the protein concentration was 1 mg / mL. BCA Protein Assay Kit: Kangwei Century Co., Ltd., Catalog No. CW0014, according to the instructions. According to the quantitative results, the content of the target protein in the supernatant obtained in step 1 was 6.67 mg / L.

[0148] Example 2, preparation of standard serum

[0149] Standard positive serum (denoted as P): 3 four-week-old SPF white leihang chickens were infected with chicken infectious laryngotracheitis virus HB201806 strain by eye-pointing (dose of 10 4.0 EID 50 ), and blood was collected from the subclavian vein after 2 weeks, then centrifuged at 8000 rpm for 15 min to obtain 3 serum samples. The 3 sera were detected by Infectious Bursal Disease Virus Antibody test kit (BioChek Co., Ltd., Catalog No. CK124 ILT, according to the instructions), and all were positive. Then, the 3 serum samples were mixed in equal volume, which was the standard positive serum.

[0150] Standard negative serum (denoted as N): 3 six-week-old SPF white leihang chickens were collected from the subclavian vein, then centrifuged at 8000 rpm for 15 min to obtain 3 serum samples. The 3 sera were detected by Infectious Bursal Disease Virus Antibody test kit (BioChek Co., Ltd., Catalog No. CK124 ILT, according to the instructions), and all were negative. Then, the 3 serum samples were mixed in equal volume, which was the standard negative serum.

[0151] Example 3, establishment of indirect ELISA detection method

[0152] I. Optimization of antigen coating concentration and serum dilution

[0153] The optimal antigen coating concentration and serum dilution were determined by square array titration. The OD 450nm values of positive and negative sera under each combination were determined by cross combination. The combination with positive serum OD 450nm value close to 1.0 and negative serum OD 450nm value close to 0.0 and high P / N value was the optimal reaction condition.

[0154] 1. Take 96-well ELISA reaction plates, add 100 μL of protein coating solution to each well, and incubate at 4°C for 12-14 h.

[0155] Preparation of protein coating solution: The purified gD-His protein prepared in Example 1 was diluted with 1x 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.

[0156] 2. After completion of step 1, take the reaction plates, wash with PBST solution 3 times (300 μL per well each time, 3 min each time).

[0157] 3. After completion of step 2, add 300 μL of 5% skim milk solution to each well, and incubate at 37°C for 60 min (blocking).

[0158] 4. After completion of step 3, take the reaction plates, wash with PBST solution 3 times (300 μL per well each time, 3 min each time).

[0159] 5. After completion of step 4, take the reaction plates, add serum diluent (100 μL per well), and incubate at 37°C for 60 min.

[0160] The serum diluent was positive serum diluent or negative serum diluent. Each dilution of each serum was set up in triplicate. The positive serum diluent: the standard positive serum was gradient-diluted with 5% skim milk solution to 400-fold volume, 800-fold volume, 1600-fold volume, 3200-fold volume, or 6400-fold volume. The negative serum diluent: the negative serum (N) was gradient-diluted with 5% skim milk solution to 400-fold volume, 800-fold volume, 1600-fold volume, 3200-fold volume, or 6400-fold volume.

[0161] 6. After completion of step 5, take the reaction plates, wash with PBST solution 3 times (300 μL per well each time, 3 min each time).

[0162] 7. After completion of step 6, take the reaction plates, add 100 μL of enzyme-labeled secondary antibody working solution to each well, and incubate at 37°C for 60 min.

[0163] Enzyme-labeled secondary antibody working solution: horseradish peroxidase-labeled goat anti-chicken IgG was diluted to 10000-fold volume.

[0164] 8. After completion of step 7, take the reaction plates, wash with PBST solution 3 times (300 μL per well each time, 3 min each time).

[0165] 9. After step 8, take the reaction plate, add 100 μL TMB color developing solution to each well, and incubate at 37°C for 10 min in the dark.

[0166] 10. After step 9, take the reaction plate, add 50 μL ELISA termination solution to each well to terminate the reaction, and then use an enzyme label meter to measure the OD value of each well at 450 nm wavelength.

[0167] The results are shown in Table 1 (N=3, data are average values). When the antigen concentration was 0.125 μg / mL and the serum dilution factor was 1:1600, the OD value of the positive serum was 0.975 (close to 1), the OD value of the negative serum was 0.048 (close to 0), and the P / N value reached 20.455. Therefore, the final determination of the antigen coating concentration was 0.125 μg / mL, and the serum dilution factor was 1:1600. 450 450

[0168]

[0169] II. Determination of blocking time

[0170] 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 h.

[0171] Preparation method of protein coating solution: dilute the purified gD-His protein prepared in Example 1 with 1x ELISA coating solution to a protein concentration of 0.125 μg / mL.

[0172] 2. Step 2 of section I.

[0173] 3. After 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.

[0174] 4. Step 4 of section I.

[0175] 5. After step 4, take the reaction plate, add serum diluent (100 μL / well), and incubate at 37°C for 60 min.

[0176] The serum diluent is positive serum diluent (standard positive serum is diluted with 5% skim milk solution to 1600 times the volume) and negative serum diluent (standard negative serum is diluted with 5% skim milk solution to 1600 times the volume), and each serum diluent is set up in triplicate.

[0177] 6. Step 6 of section I.

[0178] 7. Step 7 of section I.

[0179] ​​8. The same as step 1, 8.

[0180] 9. The same as step 1, 9.

[0181] 10. The same as step 1, 10.

[0182] The results are shown in Table 2. The OD value of the positive serum was 1.008 and the OD value of the negative serum was 0.058 when the blocking was performed for 60 min. The P / N value was the highest. Therefore, the blocking time was determined to be 60 min. 450nm 450nm The results are shown in Table 2. The OD value of the positive serum was 1.008 and the OD value of the negative serum was 0.058 when the blocking was performed for 60 min. The P / N value was the highest. Therefore, the blocking time was determined to be 60 min.

[0183]

[0184] Three, determination of the incubation time of the secondary antibody serum

[0185] 1. The same as step 2, 1.

[0186] 2. The same as step 1, 2.

[0187] 3. The same as step 1, 3.

[0188] 4. The same as step 1, 4.

[0189] 5. After step 4 is completed, the reaction plate is taken out, serum diluent (100 μL / well) is added, and incubation is performed at 37°C for 30 min, 60 min or 90 min.

[0190] The serum diluent is positive serum diluent (standard positive serum is diluted to 1600 times by gradient dilution with 5% skim milk solution) and negative serum diluent (standard negative serum is diluted to 1600 times by gradient dilution with 5% skim milk solution), and three replicate wells are set for each serum diluent.

[0191] 6. The same as step 1, 6.

[0192] 7. The same as step 1, 7.

[0193] 8. The same as step 1, 8.

[0194] 9. The same as step 1, 9.

[0195] 10. The same as step 1, 10.

[0196] The results are shown in Table 3. The OD value of the positive serum was 1.014 and the OD value of the negative serum was 0.063 when the serum and the antigen were reacted for 60 min. Although the value of the positive serum increased to 1.455 when the incubation was performed for 90 min, the negative reaction did not increase significantly, indicating that the gD protein antigen has good specificity. Therefore, the serum incubation time was determined to be 60 min. 450nm 450nm The results are shown in Table 3. The OD value of the positive serum was 1.014 and the OD value of the negative serum was 0.063 when the serum and the antigen were reacted for 60 min. Although the value of the positive serum increased to 1.455 when the incubation was performed for 90 min, the negative reaction did not increase significantly, indicating that the gD protein antigen has good specificity. Therefore, the serum incubation time was determined to be 60 min.

[0197] ​​

[0198] Four, determination of dilution ratio and incubation time of enzyme-labeled secondary antibody

[0199] 1. 1 of step two in parallel.

[0200] 2. 2 of step one in parallel.

[0201] 3. 3 of step one in parallel.

[0202] 4. 4 of step one in parallel.

[0203] 5. 5 of step two in parallel.

[0204] 6. 6 of step one in parallel.

[0205] 7. After step 6 is completed, take the reaction plate and add 100 μL of enzyme-labeled secondary antibody working solution to each well, incubate at 37°C for 30 min, 45 min or 60 min.

[0206] The enzyme-labeled secondary antibody working solution is: dilute horseradish peroxidase-labeled goat anti-chicken IgG to a volume of 5000 times, 10000 times, 20000 times or 40000 times.

[0207] 8. 8 of step one in parallel.

[0208] 9. 9 of step one in parallel.

[0209] 10. 10 of step one in parallel.

[0210] The results are shown in Table 4. As the dilution ratio of the secondary antibody increases, the OD 450nm value of the positive serum shows a downward trend, while the background value of the negative serum remains at a low level. When the dilution ratio of the secondary antibody is 1:5000 and the incubation time is 45 min, the OD 450 value of the standard positive serum is 1.103, the OD 450nm value of the negative serum is 0.064, and the P / N value is 17.274. Under this condition, the signal-to-noise ratio is optimal, and both the positive signal intensity and the negative background value are within the ideal range. Therefore, the optimal dilution ratio of HRP-labeled goat anti-chicken IgG secondary antibody is determined to be 1:5000, and the incubation time is 45 min.

[0211]

[0212] Five, determination of the cut-off value

[0213] Take 42 SPF chicken sera (ILTV negative sera from white lai chicken provided by the Livestock and Poultry Disease Diagnosis Research Center of China Agricultural University) as test sera.

[0214] 1. Same as step 2, step 1.

[0215] 2. Same as step 2 in step one.

[0216] 3. Same as step 1, step 3.

[0217] 4. Same as step 4 in step one.

[0218] 5. After completing step 4, take the reaction plate, add serum diluent (100 μL / well), and incubate at 37°C for 60 min.

[0219] The serum diluent was obtained by serially diluting the test serum with 5% skim milk solution to a volume of 1600 times, and each serum diluent was prepared in 3 replicates.

[0220] 6. Same as step 6 in step one.

[0221] 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 min.

[0222] Enzyme-labeled secondary antibody working solution: Dilute horseradish peroxidase-labeled goat anti-chicken IgG to 5000 times volume.

[0223] 8. Same as step 1, step 8.

[0224] 9. Same as step 1, step 9.

[0225] 10. Same as step one, number 10.

[0226] Statistical analysis was performed to calculate the OD values ​​of 42 negative serum samples. 450nm The average value ( The OD and standard deviation (SD) of the sample are used as the cutoff criteria for negative and positive results in the ELISA method. 450nm Value ≥ +3SD indicates a positive ILTV result, when the serum OD 450nm Value < +2SD is considered negative. +2SD ≤OD 450nm Value < +3SD indicates a suspicious sample, and retesting is recommended. OD values ​​for 42 SPF chicken negative serum samples... 450nm Average value ( The value was 0.073, and the standard deviation (SD) was 0.032.

[0227] Therefore, the judgment criteria are established as follows:

[0228] When OD 450nm A value ≥ 0.169 is considered positive;

[0229] OD 450nm value < 0.137 is determined as negative;

[0230] OD 450nm value ≥ 0.137 and < 0.169 is determined as suspicious sample.

[0231] Example 4, Performance analysis of indirect ELISA method

[0232] I. Reproducibility test

[0233] 1. Intra-plate reproducibility test

[0234] The standard positive serum prepared in Example 2 and the standard negative serum prepared in Example 2 were used as test serum respectively. The operation was carried out according to the procedure of Example 3, step five. The reproducibility of the standard positive serum and the standard negative serum in the same reaction plate was evaluated. Specifically, 16 parallel holes were set for each of the positive serum and the negative serum, and the OD 450nm value of each hole was determined, and the arithmetic mean value (X ), standard deviation (SD) and coefficient of variation (CV) were calculated. The results are shown in Table 5. The average OD 450nm value of the standard positive serum was 1.062, and the coefficient of variation was 6.3%; the average OD 450nm value of the standard negative serum was 0.049, and the coefficient of variation was 8.0%. The coefficients of variation of the two groups of samples were both less than 10%, indicating that the ELISA system had good intra-plate reproducibility.

[0235]

[0236] 2. Inter-plate reproducibility test

[0237] The standard positive serum prepared in Example 2 and the standard negative serum prepared in Example 2 were used as test serum respectively. The operation was carried out according to the procedure of Example 3, step five. The reproducibility of the positive serum and the negative serum on different reaction plates was tested. 16 parallel holes were set for each serum sample, and the OD 450nm value of each plate was determined, and the average value (X ), standard deviation (SD) and coefficient of variation (CV) were calculated. The results are shown in Table 6. After the standard positive serum was repeated 16 times on different reaction plates, the average value was calculated to be 1.079, and the coefficient of variation was 5.3%; the average value of the repeated standard negative serum was 0.049, and the coefficient of variation was 8.5%. The coefficients of variation of the two standard sera were both less than 10%, indicating that the new method had good inter-plate reproducibility.

[0238]

[0239] III. Sensitivity test

[0240] 1. Same as 1 of Step 2 of Example 3.

[0241] 2. Same as 2 of Step 1 of Example 3.

[0242] 3. Same as 3 of Step 1 of Example 3.

[0243] 4. Same as 4 of Step 1 of Example 3.

[0244] 5. After Step 4 is completed, take the reaction plate and add serum diluent (100 μL / well), incubate at 37°C for 60 min.

[0245] Serum diluent: standard positive serum is diluted with 5% skim milk solution in gradient, respectively diluted to 200 times volume, 400 times volume, 800 times volume, 1600 times volume, 3200 times volume, 6400 times volume, 12800 times volume or 25600 times volume.

[0246] 6. Same as 6 of Step 1 of Example 3.

[0247] 7. Same as 7 of Step 5 of Example 3.

[0248] 8. Same as 8 of Step 1 of Example 3.

[0249] 9. Same as 9 of Step 1 of Example 3.

[0250] 10. Same as 10 of Step 1 of Example 3.

[0251] The results are shown in Table 7. The results show that when the positive serum is diluted to 1:12800, the OD value detected is still higher than the set positive-negative judgment critical value, and the judgment result is positive. When the dilution multiple reaches 1:25600, the detection signal decreases to below the critical value. In summary, the minimum detection limit of the ELISA method is 1:12800, which shows that it has high detection sensitivity and is suitable for the detection needs of low titer antibody samples. 450nm

[0252]

[0253] Four, specificity test

[0254] ​The test sera were as follows (all were chicken sera from Bai Laihang chicken, identified and provided by the Poultry Disease Diagnosis 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 for the test sera, respectively.

[0255] The procedure of Step Five in Example 3 was followed.

[0256] The results are shown in Table 8. The OD 450nm values of each test serum were significantly lower than the established positive and negative judgment critical values, and the judgment results were all negative. No obvious cross-reaction was observed. This indicates that the method has good specificity and can effectively distinguish ILTV antibodies from other common avian pathogen antibodies, and is suitable for specific detection of actual clinical samples.

[0257]

[0258] Comparative Example 1,

[0259] I. Construction of Control Plasmid 1

[0260] Control Plasmid 1 was constructed. Compared with recombinant plasmid pFastBac I-gD, the only difference of Control Plasmid 1 was that the segment shown in SEQ ID NO: 2 at positions 124-1089 was replaced with the DNA shown in SEQ ID NO: 3.

[0261] II. Comparison of the ability of recombinant plasmid pFastBac I-gD or Control Plasmid 1 to express the target protein

[0262] The test plasmids were recombinant plasmid pFastBac I-gD or Control Plasmid 1, respectively.

[0263] The test plasmid was taken and the method of step two of Example 1 was used. Then, 2 mL of the bacterial solution with the recombinant Bacmid was inoculated into 200 mL of liquid LB medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin and 10 μg / mL tetracycline, and cultured at 37°C, 220 rpm for 14-16 h, and then the plasmid was extracted using a kit to obtain the recombinant Bacmid. Then, 3 μg of the recombinant Bacmid was transfected into Sf9 cells (transfection was performed with Cellfectin™ II reagent), and then the cells were incubated at 27°C for 72 h, and then the supernatant was harvested and subjected to Western Blot (the primary antibody used was Mouse anti His-Tag mAb, Abmole Biotechnology Co., Ltd., item number AE003).

[0264] The results are shown in Figure 7 . Figure 7 Lane M is a protein molecular weight marker; lane 1 is a control of Sf9 cells not transfected with the recombinant Bacmid; lane 2 is the supernatant obtained using the control plasmid 1; and lane 3 is the supernatant obtained using the recombinant plasmid pFastBac I-gD. The target bands shown in the supernatant obtained using the recombinant plasmid pFastBac I-gD were subjected to gray scale scanning quantification. Figure 7 Compared with the target bands shown in the supernatant obtained using the control plasmid 1, the protein abundance represented by the target bands shown in the supernatant obtained using the recombinant plasmid pFastBac I-gD was 1.2 times.

[0265] Comparative Example 2,

[0266] I. Construction of Control Plasmid 2

[0267] The control plasmid 2 was constructed. Compared with the recombinant plasmid pFastBac I-gD, the difference of the control plasmid 2 was only that the segment shown in SEQ ID NO: 2 at positions 124-1089 was replaced with the DNA molecule shown in SEQ ID NO: 4.

[0268] II. Comparison of the ability of the recombinant plasmid pFastBac I-gD or the control plasmid 2 to express the target protein

[0269] The test plasmid was the recombinant plasmid pFastBac I-gD or the control plasmid 2.

[0270] The method was the same as step two of Comparative Example 1.

[0271] The results are shown in Figure 8 . Figure 8Lane M is protein molecular weight marker; lane 1 is Sf9 cell control without transfection of recombinant Bacmid; lane 2 is supernatant obtained by using recombinant plasmid pFastBacl-gD; lane 3 is supernatant obtained by using control plasmid 2. The results show that the control plasmid 2 fails to effectively express the target protein.

[0272] The present application has been described in detail. For those skilled in the art, without departing from the spirit and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range of equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In summary, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including the change made by the conventional technology known in the art, which is out of the range disclosed in this application. Some basic features can be applied according to the scope of the following attached claims.

Claims

1. Use of a recombinant protein in the preparation of a kit; the kit is a kit for detecting antibodies of chicken infectious laryngotracheitis virus; the recombinant protein is shown in SEQ ID NO: 1 at positions 39-379; the recombinant protein is prepared according to a method comprising the following steps: (1) introducing a recombinant plasmid into E. coli DH10Bac to obtain a recombinant E. coli; the recombinant plasmid is a recombinant plasmid obtained by replacing a small fragment between a BamHI and a HindIII enzyme digestion recognition sequence in a pFastBacI vector with a double-stranded DNA molecule shown in SEQ ID NO: 2 at positions 1-1119; (2) culturing the recombinant E. coli obtained in step (1) and extracting a plasmid to obtain a recombinant Bacmid; (3) transfecting Sf9 cells with the recombinant Bacmid obtained in step (2) and culturing, and collecting a culture supernatant to obtain P1 generation virus liquid; (4) infecting Sf9 cells with the P1 generation virus liquid and culturing, and collecting a culture supernatant to obtain P2 generation virus liquid; (5) infecting Sf9 cells with the P2 generation virus liquid and culturing, and collecting a culture supernatant to obtain P3 generation virus liquid; (6) infecting High Five cells with the P3 generation virus liquid, collecting a culture supernatant, and purifying to obtain the recombinant protein.

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