Fusion protein lsdv090-133 and its use in detecting antibodies to bovine dermatophilus virus

Abstract

This invention belongs to the field of molecular biology and discloses the fusion protein LSDV090-133 and its application in detecting antibodies against bovine nodular dermatitis virus (LSDV) infection and differentiating antibody responses to vaccines. This invention utilizes a phage display library to screen for specific peptides against serum infected with LSDV and serum immunized with a goatpox attenuated live vaccine. Through bioinformatics analysis, three peptides specific to LSDV-infected serum were selected and tandemly linked to the pET28a vector via flexible peptide transfer. The resulting protein was synthesized and expressed in prokaryotes to obtain the LSDV090-133 fusion protein, with a molecular weight of approximately 20 kDa. Verification showed that this protein exhibits strong reactivity with LSDV-infected bovine positive serum, with a significantly higher grayscale value than serum immunized 60 days post-goatpox attenuated live vaccine immunization. It represents a potential new diagnostic target and can be used to differentiate antibody responses to LSDV infection and goatpox attenuated live vaccine immunization, providing a reference tool for the serological diagnosis and control of bovine nodular dermatitis.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to the artificially synthesized fusion protein LSDV090-133 and its application in identifying bovine nodular dermatitis virus antibodies. Background Technology

[0002] Lumpy skin disease (LSD) is an infectious disease of cattle caused by bovine lumpyskin disease virus (LSDV). The morbidity rate of LSD ranges from 5% to 45%, with a mortality rate of approximately 5%, which can reach as high as 20% in severe cases. LSDV is a member of the genus Capripoxvirus (CaPV) within the family Poxviridae. It is a double-stranded DNA virus with an envelope, a large genome containing 156 presumed genes, and a single serotype.

[0003] LSDV shares over 97% nucleotide sequence similarity with sheep and goat pox viruses of the same genus, and has high antigenic similarity. Therefore, my country uses intradermal injection of 5 times the dose of attenuated goat pox vaccine to prevent LSD.

[0004] Currently, goatpox attenuated live vaccine is used clinically to control bovine nodular dermatitis. However, serologically, it is difficult to distinguish between different members of the poxvirus genus. Therefore, there is an urgent need to develop serum antibody kits for differential diagnosis of wild-type virus infection or vaccine immunization, in order to promptly detect and cull infected cattle and control the epidemic as quickly as possible. Thus, developing a kit with high sensitivity and specificity to differentiate between bovine nodular dermatitis virus-infected serum and goatpox attenuated live immune serum is extremely important.

[0005] To address the aforementioned issues, the applicant disclosed a bovine nodular dermatitis virus-specific multi-fragment tandem fusion protein in CN 116751264 A for identifying serum from natural infection and vaccine immunization. In this invention, the applicant utilizes phage display library screening of specific peptides and proteins in infected serum, combined with Western blotting and mass spectrometry detection, to discover new fusion proteins that can be used for bovine nodular dermatitis virus antibody detection through multiple pathways, providing a new supplementary approach in this field of diagnostic reagents. Summary of the Invention

[0006] The purpose of this invention is to provide a fusion protein LSDV090-133 for detecting bovine nodular dermatitis virus antibodies, the fusion protein being shown in SEQ ID NO.2.

[0007] Another object of the present invention is to provide the application of the fusion protein LSDV090-133 in the detection of bovine nodular dermatitis virus antibodies.

[0008] Another objective of this invention is to provide the application of the fusion protein LSDV090-133 in distinguishing between bovine nodular dermatosis virus-infected serum and goatpox attenuated live vaccine-immunized serum.

[0009] To achieve the above objectives, the applicant has adopted the following technical measures:

[0010] Specific peptides for bovine nodular dermatitis were screened using phage display libraries for infected serum and serum immunized with goatpox attenuated live vaccine. Bioinformatics analysis identified peptides specific to bovine nodular dermatitis infected serum, including one specific peptide encoding the LSDV090 gene and two specific peptides encoding the LSDV133 gene, both obtained from phage display library screening. Simultaneously, the applicant incubated GTPV with infected serum and immunized serum from different time points (0 dpi, 15 dpi, 30 dpi, 45 dpi, and 60 dpi). AV41 viral particles were analyzed by Western blot analysis to identify differential bands. The gel was excised and sent to a company for mass spectrometry analysis to screen for new targets with immunogenicity or potential for differential diagnosis. A series of target protein sequences were obtained, among which two target protein sequences were highly homologous to the proteins encoded by the LSDV090 and LSDV133 genes. Therefore, the applicant identified one specific polypeptide encoded by the LSDV090 gene and two specific polypeptides encoded by the LSDV133 gene as the target specific polypeptides. The applicant used a flexible peptide to connect the above three polypeptides, and the constructed fusion protein is shown in SEQ ID NO.2.

[0011] The gene encoding the fusion protein LSDV090-133 is also within the scope of protection of this invention.

[0012] The method for expressing the above protein includes: cloning the codon-optimized polynucleotide encoding the fusion protein LSDV090-133 (shown in SEQ ID NO.1) into the pET28a vector to obtain the recombinant plasmid pET28a-LSDV090-133 linked with the target gene, and transforming the constructed pET28a-LSDV090-133 plasmid into Escherichia coli BL21(DE3) competent cells for protein expression.

[0013] The scope of protection of this invention also includes:

[0014] Application of fusion protein LSDV090-133 in the preparation of an antibody kit for detecting bovine nodular dermatitis virus.

[0015] Application of fusion protein LSDV090-133 in the preparation of a kit to differentiate between bovine nodular dermatosis virus-infected serum and goatpox attenuated live vaccine immunizing serum.

[0016] A kit for the diagnosis of bovine nodular dermatitis, the kit comprising the fusion protein LSDV090-133.

[0017] The kit described above is preferably an indirect ELISA diagnostic kit;

[0018] Preferably, in the ELISA diagnostic kit described above, the coating concentration of the fusion protein is 4 μg / mL, and the dilution factor of the serum sample is 1:800 (volume).

[0019] When the above kits are used in non-diagnostic testing, the results should be interpreted as follows:

[0020] If P / N > 2.1, the result is positive.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] This invention is the first to screen specific peptides in bovine nodular dermatitis (BND) infected serum and serum immunized with goatpox attenuated live vaccine using a phage display library. Through bioinformatics analysis, peptides specific to bovine nodular dermatitis infected serum were selected, and a fusion protein that can specifically distinguish between vaccine immunization and natural infection was obtained using flexible peptide tandem technology. The invention also verified that the protein has high reactivity with LSDV infected serum.

[0023] Based on the above-mentioned fusion protein, this invention assembles an indirect ELISA detection kit. The LSDV090-133 protein coating concentration in the kit is 4 μg / mL, and the serum dilution ratio is 1:800. The optimal protein coating conditions are 37℃ for 1 h. The determined enzyme-labeled antibody dilution is 1:8000. The optimal blocking solution is 1% (w / v) fish gelatin. The optimal blocking solution incubation time is 2 h. The determined color development time is 37℃ for 10 min. The diagnostic sensitivity is 100% (95% CI: 47.8%-100.0%), and the diagnostic specificity is 96.67% (95% CI: 83.33%-99.83%). Attached Figure Description

[0024] Figure 1 This is the PCR identification result of the recombinant plasmid pET28a-LSDV090-133;

[0025] In the figure: Lane 1: DL2000; Lanes 2-7: Bacterial culture samples; Lane 8: Negative control.

[0026] Figure 2 The results are SDS-PAGE (A) and Western Blot (B) analyses of the LSDV090-133 protein.

[0027] In the figure: Lane 1: Protein molecular weight standard; Lane 2: pET28a; Lane 3: pET28a induced at 37℃ for 5h; Lane 4: LSDV090-133 protein; Lane 5: LSDV090-133 protein induced at 37℃ for 5h.

[0028] Figure 3 These are the SDS-PAGE analysis results of purified LSDV090-133 protein;

[0029] In the diagram: Lane 1: Protein molecular weight standard; Lane 2: LSDV090-133 protein denaturation solution; Lane 3: LSDV090-133 protein refolding solution; Lane 4: Purified LSDV090-133 protein solution A; Lane 5: Purified LSDV090-133 protein solution B.

[0030] Figure 4 The results are SDS-PAGE (A), Western Blot (B and C), and serum incubation analysis for the differential diagnosis of LSDV090-133 protein.

[0031] In the figure: Lane 1: Protein molecular weight standard; Lane 2: LSDV090-133 protein; Lane 3: AXA19967.1 protein.

[0032] Figure 5 This is a scatter plot of samples from 5 bovine serum samples with high-titer antibodies and 30 bovine serum samples immunized with attenuated goatpox vaccine at 60 dpi, which are known to be used in this invention for diagnosing the disease. Detailed Implementation

[0033] The present invention will be described in detail below with reference to specific embodiments.

[0034] AXA19967.1 protein: Another protein developed by the applicant is a GTPV putative protein, approximately 23 kDa in size. This protein produces strong specific bands against both immune and infected sera and can serve as a universal diagnostic target for LSDV and GTPV in the goatpoxvirus genus. Its sequence is shown in SEQ ID NO.3.

[0035] Goatpox virus (AV41 strain) attenuated live vaccine: used to immunize bovine animals to improve their resistance to goatpox virus genus, purchased commercially from Jinyu Biotechnology Co., Ltd.

[0036] Other biological materials or reagents not mentioned in this article are all conventional materials in this field.

[0037] The present invention constructs the fusion protein LSDV090-133 by prokaryotic expression. Other conventional methods of protein synthesis in the field, such as eukaryotic expression and artificial synthesis, can also achieve the present invention.

[0038] Example 1: Preparation of LSDV090-133 protein

[0039] 1. Design and synthesize the recombinant plasmid pET28a-LSDV090-133.

[0040] The applicant used a phage display library to screen for specific peptides in bovine nodular dermatitis infection serum and serum immunized with goatpox attenuated live vaccine. Through bioinformatics analysis, the applicant finally obtained specific peptides that bind to bovine nodular dermatitis infection serum, namely one specific peptide of the LSDV090 gene and two specific peptides of the LSDV133 gene. Meanwhile, the applicant incubated GTPV AV41 virus particles with infected serum and immune serum at different time points (0 dpi, 15 dpi, 30 dpi, 45 dpi, and 60 dpi), analyzed the differential bands observed in Western blotting, excised the gels and sent them to the company for mass spectrometry analysis, and screened for new targets with immunogenicity or potential for differential diagnosis, obtaining a series of target protein sequences. Among them, two target protein sequences were highly homologous to the proteins encoded by the LSDV090 gene and the LSDV133 gene. Therefore, the applicant identified one specific polypeptide encoded by the LSDV090 gene and two specific polypeptides encoded by the LSDV133 gene as the target specific polypeptides. The applicant used a flexible peptide to connect the above three polypeptides, and constructed the fusion protein shown in SEQ ID NO.2.

[0041] The applicant tandem these three polypeptides using two "GGSSGGSS" flexible peptides and sent them to a company for codon optimization synthesis. The synthesized sequence was 467 bp in length, with the following structure: cc aagctt was added to the 5' end of SEQ ID NO.1, where cc ensures that the constructed recombinant protein gene sequence will not result in incorrect protein expression or no protein expression due to frameshift, and aagctt is a HindIII restriction site; a ctcgag XhoI restriction site was added to the 3' end of SEQ ID NO.1. This synthesized sequence was cloned into the pET28a vector to obtain a recombinant plasmid containing the target gene. The constructed pET28a-LSDV090-133 plasmid was transformed into E. coli BL21(DE3) competent cells for protein expression, with a protein size of approximately 20 kDa. The recombinant plasmid pET28a-LSDV090-133, containing the target gene, was obtained by cloning into the pET28a vector. The size of the band in the recombinant bacterial pET28a-LSDV090-133 was identified by PCR. Figure 1 The results showed that the size of the bacterial bands was correctly identified, and no base mutations or frameshifts were found when the bacterial culture was sent to the company for sequencing.

[0042] 2. Small-scale preparation of plasmid DNA

[0043] The method for preparing plasmid DNA in small quantities was performed according to the instructions of the Tiangen High Purity Plasmid Mini-Prep Kit. The specific steps are as follows:

[0044] (1) Take 5 mL of overnight cultured bacterial solution and add it to a centrifuge tube. Centrifuge at 8000 r / min for 3 min to collect the bacterial precipitate and discard the supernatant as much as possible.

[0045] (2) Add 250 μL of Buffer P1 to the centrifuge tube containing the bacterial precipitate. First check to ensure that RNase A has been added. Use a 1 mL pipette or vortex mixer to mix thoroughly and suspend the bacterial precipitate.

[0046] (3) Add 250 μL of Buffer P2 to the centrifuge tube and gently invert it 4-6 times to mix thoroughly to lyse the bacteria. At this point, the solution should become clear and viscous.

[0047] (4) Add 350 μL of Buffer P3 to the centrifuge tube, and immediately gently invert it 4-6 times to mix thoroughly. At this time, a white flocculent precipitate should appear. Centrifuge at 13000 r / min for 10 min.

[0048] (5) Transfer the supernatant obtained in step 4 to the adsorption column Spin Column CM with the collection tube already installed, centrifuge at 13000 r / min for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0049] (6) Add 750 μL Buffer PW to the adsorption column to check that anhydrous ethanol has been added, centrifuge at 13000 r / min for 1 min, and discard the waste liquid in the collection tube.

[0050] (7) Put the adsorption column back into the collection tube, centrifuge at 13000r / min for 2min, discard the waste liquid, and place the adsorption column at room temperature for several minutes to dry it completely.

[0051] (8) Place the adsorption column in a new centrifuge tube, add 50 μL of Buffer EB to the middle of the adsorption membrane, place at room temperature for 3 min, centrifuge at 13000 r / min for 1 min, collect in a centrifuge tube, and store the plasmid at -20℃.

[0052] 3. Plasmid was transformed into E. coli BL21(DE3) competent cells.

[0053] (1) Take 100 μL of competent cell suspension and add 1 μL of plasmid. Gently rotate to mix the contents and place on ice for 30 min (an empty vector control without plasmid DNA was set up in the experiment).

[0054] (2) Place the centrifuge tubes in a circulating water bath preheated to 42°C for 45 seconds for heat shock.

[0055] (3) Quickly transfer the centrifuge tube to ice to cool the cells for 2 minutes.

[0056] (4) Add 700 μL of LB liquid medium to each tube. Heat the medium to 37°C in a water bath, and then transfer the centrifuge tube to a 37°C shaker and incubate for 1 hour to allow the bacteria to recover (to achieve effective transformation, the rotation speed should not exceed 225 r / min during recovery).

[0057] (5) Take 100 μL of transformed competent cells and transfer them to an LB agar plate containing antibiotics (Kana resistant). Use a sterile bent glass rod to spread the transformed cells evenly on the surface of the agar plate, which is considered a low concentration. Centrifuge at 5000 r / min for 3 min, collect the bacteria, discard the culture medium, and keep 100 μL to resuspend the bacteria. Spread the resuspended bacteria evenly on the surface of the agar plate, which is considered a high concentration.

[0058] (6) Incubate the petri dish upright at 37°C until the liquid is absorbed, then invert the petri dish and incubate for 10-12 hours to observe the results.

[0059] 4. Low-level expression and confirmation of expression form of LSDV090-133 protein

[0060] (1) The plasmid with correct sequencing was transformed into the BL21 expression strain and cultured overnight on a plate containing Kana resistance.

[0061] (2) Pick a single clone of bacterial cell containing recombinant plasmid and add it to 5 mL LB liquid medium (50 mg / mL Kana resistance, added at a concentration of 0.1%, i.e., 5 μL added to 5 mL of medium) and incubate overnight at 37°C; at the same time, pick the corresponding bacterial cells transformed with empty vector as a late control.

[0062] (3) Transfer the inoculum at a ratio of 1:100. Generally, add 100 μL of recombinant plasmid bacterial culture and empty vector plasmid bacterial culture to two bacterial bottles containing 10 mL of liquid LB medium (ensure thorough shaking of the LB medium). Add Kana antibiotic and incubate at 37°C with shaking until OD. 600 The value reaches 0.6-0.8 (it takes about 3 hours; pay attention to the turbidity of the bacterial cells, i.e., the initial turbidity of the bacterial solution).

[0063] (4) Take 1 mL of liquid as the uninduced control, and add IPTG inducer to the remainder to make the final concentration reach 0.5 mM (IPTG: liquid culture medium = 1:1000, that is, 10 μL of 10 mL of culture medium is added). Induce protein expression by shaking at 37℃ for 5 h.

[0064] (5) After confirming that the protein could be stably expressed in E. coli BL21, 10 mL of expression bacteria were induced again. Batch collection of bacterial culture was performed in 2 mL centrifuge tubes. The culture was centrifuged at 12000 r / min for 2 min, washed with PBS, and the bacterial pellet was resuspended in 1 mL PBS. The bacterial cells were then disrupted using an ultrasonic homogenizer and centrifuged at 12000 r / min for 10 min at 4℃. The supernatant and pellet were processed separately to prepare protein electrophoresis samples for SDS-PAGE detection to determine the expression form of the prokaryotic recombinant protein. Western blotting was then used to determine whether the protein was expressed. Figure 2 The results showed that LSDV090-133 protein expression was confirmed by both SDS-PAGE and Western Blot results.

[0065] 5. Large-scale expression and purification of LSDV090-133 protein

[0066] (1) Dilute the seed bacteria at a ratio of 1:100. Generally, add 10 mL of recombinant plasmid bacterial solution to a culture flask containing 1 L of LB medium (use a large conical flask for shaking to ensure sufficient bacterial agitation), add Kana antibiotic, and incubate at 37°C with shaking until OD. 600 The value reached 0.6-0.8, and the time was about 3 hours.

[0067] (2) Add IPTG at a ratio of 1:1000 to achieve a final concentration of 0.5mM, and induce culture at 37℃ with shaking for 5h.

[0068] (3) Before high-pressure disruption, collect the bacterial culture in a 50 mL centrifuge tube, centrifuge at 12000 r / min for 10 min at 4℃ to collect the bacteria, and resuspend and wash twice with PBS. Then, high-pressure disruption is used to make the bacterial cells clear and transparent, and the liquid is collected.

[0069] (4) Pre-cool the high-speed centrifuge to 4°C, centrifuge at 12000r / min for 10min and collect the inclusion bodies.

[0070] (5) Wash the inclusion bodies with 1M and 2M urea to remove impurities and proteins, so that the clumps turn milky white.

[0071] (6) Resuspend the centrifuge tube in 20 mL of 6M urea and let it dissolve overnight at 4°C.

[0072] (7) The next day, centrifuge at 4°C and 12,000 r / min for 10 min to collect the supernatant.

[0073] (6) Equilibrate the His-labeled Ni-NTA packed column bed with 30 mL of 1% (v / v) imidazole.

[0074] (7) Filter the supernatant with a 0.45μm filter before passing through the column. Collect the liquid after passing through the column and consider it as solution A.

[0075] (8) Wash non-specifically bound proteins with 30 mL of 10% (v / v) imidazole and collect the solution as solution B.

[0076] (9) The eluted target protein liquid was concentrated by ultrafiltration using a 10 kDa ultrafiltration tube. The target protein was then displaced from the high concentration of imidazole using PBS solution. The mixture was centrifuged at 4000 r / min at 4℃. The ultrafiltration time depended on the filtration efficiency of the ultrafiltration tube. SDS-PAGE electrophoresis was performed to observe the purity of the LSDV090-133 fusion protein after ultrafiltration and to ensure its purity. Figure 3 ).

[0077] (10) The protein concentration was determined using the BCA kit, aliquoted, and stored at -80℃ for later use. The results showed that the final protein concentration determined by the BCA kit was 1.383 mg / mL.

[0078] Example 2:

[0079] SDS-PAGE and Western Blot were used to verify the differential diagnostic role of LSDV090-133 protein.

[0080] Take out two prepared SDS-polyacrylamide gels, and use SDS-PAGE to confirm that the protein loading amount is consistent. Use Western Blot to verify whether LSDV090-133 protein has a differential diagnostic function.

[0081] (1) Preparation of separating gel: Select an appropriate volume of separating gel, refer to the formula table, add the separating gel to the appropriate amount, add isopropanol and press the gel, after the separating gel solidifies, pour out the isopropanol, rinse lightly with pure water and then absorb with absorbent paper.

[0082] (2) Preparation of concentrated glue: Select an appropriate volume of concentrated glue, refer to the formula table, add concentrated glue until full, insert the comb, and after the concentrated glue solidifies, pull out the comb.

[0083] (3) Sample preparation: Mix the sample with SDS-PAGE loading buffer, treat at 100℃ for 10 min, immediately in an ice bath for 2 min, centrifuge at 10000 r / min for 30 s, and use 1×glycine buffer as electrophoresis buffer.

[0084] (4) Sample loading: Under normal circumstances, 10 μL of sample is loaded into each well. In this experiment, the concentrations of LSDV090-133 protein and AXA19967.1 protein were first determined using the BCA kit to adjust the specific sample loading volume. At the same time, 5 μL of marker was loaded (mixed with 5 μL of 1×Loading Buffer).

[0085] (5) Electrophoresis: Keep the constant voltage at 80V until the sample reaches the boundary between the stacking gel and the separating gel. Then adjust to 120V to ensure that the bromophenol blue is electrophoresed to the bottom position.

[0086] (6) After protein gel electrophoresis, directly place the protein in Coomassie brilliant blue staining solution for 4-6 hours, and then transfer it to destaining solution (10% glacial acetic acid, 5% ethanol) for destaining.

[0087] (7) If a Western blotting experiment is required, the size of the gel must be measured: the specific dimensions of its length and width. Based on the size of the gel, cut one PVDF membrane and three sheets of filter paper slightly larger than the gel. Soak the gel and paper in transfer buffer, and then soak the membrane in methanol for 5 minutes before transferring it to transfer buffer.

[0088] (8) Transfer: On the transfer plate, starting from the white layer, lay filter paper, membrane, gel, and filter paper in sequence. Cover each layer with sufficient transfer buffer and remove air bubbles. Insert into the transfer tank and set a constant voltage of 70V for 1 hour for transfer.

[0089] (9) Washing the membrane: Rinse the membrane 3 times with TBST buffer, every 5 min.

[0090] (10) Blocking: Incubate with 5% (w / v) skim milk powder blocking buffer at room temperature for 2-3 hours.

[0091] (11) Same as (9).

[0092] (12) Primary antibody incubation: 1×TBST diluted serum (the specific titer ratio of infected serum and immune serum was determined according to AXA19967.1 protein to ensure that the specific antibody titer in the serum remained consistent. Therefore, the dilution of infected serum was determined to be 1:800 and the dilution of immune serum was determined to be 1:200), and incubated overnight on a horizontal shaker at 4°C.

[0093] (13) Same as (9).

[0094] (14) Secondary antibody incubation: Dilute horseradish peroxidase-labeled rabbit anti-bovine IgG (H+L) with 1×TBST, with a dilution of 1:5000, and incubate at room temperature on a shaker for no more than 2 hours.

[0095] (15) Same as (9).

[0096] (16) Color development: Equal volumes of chemiluminescent color development solutions A and B were mixed on the membrane for development. The purified LSDV090-133 protein was approximately 20 kDa in size. Incubation with infected serum showed a clear target band for the protein. Figure 4 The grayscale value of the band incubated with immune serum was weaker than that of the band incubated with infected serum. Figure 4In the study (D), the positive control AXA19967.1 protein showed the corresponding target band at approximately 23 kDa. One polypeptide from the LSDV090 gene and two polypeptides from the LSDV133 gene were compared with the nucleotide sequences of the homologous genes AXA19987.1 and AXA19893.1 of the goatpox attenuated live vaccine (AV41 strain), respectively. The results showed only 6 and 4 base mismatches, respectively. Nucleotide sequence analysis indicated minimal differences in the amino acid epitopes that recognize serum antibodies between the two genes. Therefore, under the premise of a valid positive control, incubation of LSDV090-133 protein with immune serum still resulted in the appearance of the target band, although it was significantly weaker than that of LSDV090-133 protein incubated with infected serum. Therefore, the fusion protein LSDV090-133 prepared in this invention can not only be used to detect bovine nodular dermatitis virus antibodies but also to differentiate between bovine nodular dermatitis virus-infected serum and goatpox attenuated live vaccine immune serum.

[0097] Example 3:

[0098] Establishment of an indirect ELISA method to differentiate between bovine nodular dermatitis virus-infected serum and goatpox attenuated live vaccine-immunized serum:

[0099] 1. Optimized testing conditions

[0100] (1) Determination of protein coating concentration and serum dilution factor: LSDV090-133 protein was diluted in a gradient of 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL to coat the microplates, with each concentration coating two rows of eight wells. Negative and positive sera were serially diluted with PBS in PCR tubes at dilution factors of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, respectively. 100 μL of each dilution was then added to the corresponding protein gradient. OD was measured using a microplate reader following standard ELISA procedures. 450 Numerical analysis was conducted to compare the P / N values ​​corresponding to the same protein concentration, and the protein coating concentration and serum dilution factor corresponding to the largest P / N value were selected as the optimal conditions.

[0101] (2) Determining the optimal protein coating conditions: The coating conditions for LSDV090-133 protein were optimized according to the optimized protein coating concentration and serum dilution factor. With other experimental conditions fixed, LSDV090-133 protein was diluted with coating buffer and then transferred to 37℃ for 1 h, 37℃ for 2 h, 4℃ for 16 h, and 37℃ for 2 h followed by 4℃ for 14 h. Following standard iELISA procedures, three replicates were performed for each sample, and OD values ​​were read. 450The P / N values ​​of each group were compared, and the maximum P / N value was selected as the optimal coating condition for the protein.

[0102] (3) Determining the optimal blocking solution: The blocking solution was optimized according to the optimal protein coating concentration, coating conditions, and serum dilution factor. With other experimental conditions fixed, 5% (w / v) skim milk, 2% (w / v) BSA, and 1% (w / v) fish gelatin were tested. Three replicates were performed for each sample according to the standard iELISA procedure, and OD values ​​were read. 450 Numerical analysis was performed, comparing the P / N values ​​of each group, and the sealing liquid with the largest P / N value was selected as the optimal sealing liquid.

[0103] (4) Determining the optimal blocking time: The blocking time was optimized based on the optimized protein coating concentration, coating conditions, negative and positive serum dilution factors, and optimal blocking solution conditions. Blocking times of 37℃ for 1 h, 37℃ for 1.5 h, 37℃ for 2 h, 37℃ for 2.5 h, and 37℃ for 3 h were explored, with three replicates performed for each. The condition corresponding to the highest P / N value was selected as the optimal blocking time.

[0104] (5) Optimization of optimal enzyme-labeled antibody working concentration: Based on the above-determined optimal conditions, fix other experimental conditions, dilute the enzyme-labeled antibody at ratios of 1:4000, 1:6000, 1:8000, and 1:10000 respectively, and perform three replicates for each sample according to the standard ELISA operating procedure. Determine the optimal enzyme-labeled antibody working concentration based on the P / N value.

[0105] (6) Optimization of optimal substrate reaction time: With other experimental conditions fixed, the substrate was reacted for 5 min, 10 min, 15 min and 20 min respectively. The reaction time was repeated for three samples according to the standard ELISA procedure. The optimal substrate reaction time was determined based on the P / N value.

[0106] The detailed steps for finalizing the detection method are as follows:

[0107] (1) Coating: The purified protein was coated with carbonate buffer (CBS, pH 9.6) at a concentration of 4 μg / mL onto the microplate, 100 μL / well, and incubated at 37°C for 1 h.

[0108] (2) Washing: 300 μL of 0.1% (v / v) PBST washing solution per well, wash twice, 3 min each time.

[0109] (3) Blocking: 200 μL / well of 1% (w / v) fish gelatin blocking solution, incubated at 37°C for 2 h.

[0110] (4) Washing: 300 μL of washing solution per well, wash 3 times, 3 min each time.

[0111] (5) Add serum (infected serum, immune serum diluted at 1:800), 100 μL / well, and incubate at 37°C for 1 h.

[0112] (6) Same as (4).

[0113] (7) Enzyme-labeled antibody: diluted 1:8000, 100 μL / well, incubated at 37℃ for 1 h.

[0114] (8) Same as (4).

[0115] (9) Substrate: 100 μL / well of TMB single-component colorimetric solution, incubated at 37°C in the dark for 10 min.

[0116] (10) Termination solution: 50 μL / well.

[0117] (11) Reading: The microplate reader reads the OD value. 450 Numerical value.

[0118] Note: Positive control serum OD 450 / Negative control serum OD 450 >2.1, meaning P / N>2.1.

[0119] 2. Determine the criteria for judging ELISA test kits.

[0120] Positive control serum OD 450 OD in negative control serum 450 If the ratio is greater than 2.1, i.e., P / N > 2.1, then the test result is valid; the OD of the serum to be tested... 450 / Negative control serum OD 450 >2.1 is used as the positive criterion (PBST < 0.2 in the blank group, otherwise the test is invalid).

[0121] 3. ELISA diagnostic sensitivity and specificity test

[0122] Five bovine serum samples with high-titer antibodies and known background were tested, along with 30 bovine serum samples immunized with goatpox attenuated live vaccine at 60 dpi. Positive and negative controls were also included. OD values ​​were read. 450 Numerical values ​​were used to plot a scatter plot of the sample data using Graph Prism 8. Figure 5 Positive controls were sera infected with bovine nodular dermatosis virus (BND) in a known background, and negative controls were sera immunized with goatpox attenuated live vaccine in a known background at 60 days post-immunization (dpi). Both positive and negative control sera, as well as the sera to be tested, were subjected to [further testing / treatment]. The results of the bovine nodular dermatitis / sheep pox double antigen test kit were as expected.

[0123] The results showed that when P / N > 2.1 was used as the positive criterion, the diagnostic sensitivity was 100% (95% CI: 47.8%-100.0%) and the diagnostic specificity was 96.67% (95% CI: 83.33%-99.83%). There were extremely significant differences between immune sera detected by LSDV090-133 protein and infected sera with high antibody titers (p<0.0001).

[0124] 4. ELISA Specificity and Sensitivity Tests

[0125] To test the analytical specificity of the iELISA method based on LSDV090-133 protein, positive sera for bovine nodular dermatitis virus, bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, and bovine mycoplasma were diluted 1:800, and three replicates were performed for each sample. OD values ​​were read. 450 Numerical values. To test the analytical sensitivity of the iELISA method based on LSDV090-133 protein, infected serum and immune serum were diluted 1:100 to 1:12800, and OD values ​​were read. 450 Numerical value.

[0126] The results showed that, except for a specific binding reaction with positive sera of bovine nodular dermatitis virus, there was no cross-reactivity with positive sera of bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, or bovine mycoplasma, indicating that the iELISA kit has good specificity. Based on the LSDV090-133 protein, the limit of detection for positive sera using the iELISA kit was 1:800, according to the iELISA kit's criteria.

Claims

1. A synthetically produced fusion protein LSDV090-133, wherein the fusion protein is shown in SEQ ID NO.

2.

2. A gene encoding the fusion protein of claim 1.

3. The gene according to claim 2, which is shown in SEQ ID NO.

1.

4. A method for prokaryotic expression of the protein of claim 1, comprising: The gene shown in SEQ ID NO.1 was cloned into the pET28a vector to obtain a recombinant plasmid containing the target gene. pET28a-LSDV090-133 , will be constructed pET28a- LSDV090-133 The plasmid was transformed into E. coli BL21(DE3) competent cells for protein expression.

5. The use of the fusion protein LSDV090-133 according to claim 1 in the preparation of an antibody kit for detecting bovine nodular dermatitis virus infection.

6. The use of the fusion protein LSDV090-133 according to claim 1 in the preparation of a kit for distinguishing bovine nodular dermatosis virus-infected serum and goatpox attenuated vaccine immune serum.

7. A kit for the diagnosis of bovine nodular dermatitis, the kit comprising the fusion protein LSDV090-133.

8. The reagent kit according to claim 7, characterized in that, The kit described is an indirect ELISA diagnostic kit.

9. The kit according to claim 8, wherein the coating concentration of the fusion protein in the kit is 4 µg / mL, and the dilution factor of the serum sample is 1:800 (volume ratio).

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