A bovine viral diarrhea virus ns2 truncated protein and use in bvdv antibody detection

Abstract

The application belongs to the technical field of biology and particularly relates to a bovine viral diarrhea virus NS2 truncated protein and application thereof in BVDV antibody detection. The application provides a truncated protein of BVDV NS2 protein. The truncated protein is soluble expressed after being fused with glutathione S transferase (GST) and horseradish peroxidase (HRP) respectively, has good reactivity with BVDV antibody positive serum, does not react with BVDV antibody negative serum, and can be used for BVDV antibody detection. Furthermore, the application constructs a double antigen sandwich ELISA method for BVDV antibody detection by taking the fusion protein of GST and the truncated protein as a coating antigen and taking the fusion protein of HRP and the truncated protein as a detection antigen. The method has the technical advantages of high sensitivity, good specificity, simple operation, easy popularization, low cost and the like, and can be applied to screening of BVDV antibodies in a cattle herd.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and particularly relates to a bovine viral diarrhea virus NS2 truncated protein and its application in BVDV antibody detection. Background Art

[0002] Bovine viral diarrhea-mucosal disease (BVD-MD) is an infectious disease of cattle caused by infection with the bovine viral diarrhea virus (BVDV). It is listed as a notifiable infectious disease of cattle by the World Organization for Animal Health (OIE) and as a Category III animal disease in the Ministry of Agriculture and Rural Affairs of my country's "List of Category I, II, and III Animal Diseases." BVD-MD has a broad host spectrum, encompassing domestic animals such as cattle (dairy cows, yaks, and buffaloes), deer, camels, sheep, and pigs, as well as some wild cloven-hoofed animals. However, the clinical symptoms of BVD-MD are most typical in cattle, which can present with a wide range of symptoms, including diarrhea, respiratory symptoms, hemorrhagic syndrome, chronic mucosal disease, immune tolerance, immunosuppression, persistent infection, and reproductive failure.

[0003] Currently, in Nordic countries such as Norway and Sweden, BVD purification is achieved through monitoring and elimination of persistently infected cattle. my country's current "Health Standards for Breeding Animals" also lists the absence of bovine viral diarrhea as a health standard for breeding cattle.

[0004] Currently, the indirect ELISA method used in the detection of bovine viral diarrhea virus antibodies has low specificity. Although the competitive ELISA established with auxiliary monoclonal antibodies can improve the specificity of the detection, it significantly increases the cost of the detection reagents. Summary of the Invention

[0005] To address the problems of the prior art, the present invention proposes a truncated NS2 protein of bovine viral diarrhea virus and its application in BVDV antibody detection. Specifically, the present invention includes the following contents:

[0006] In the first aspect, the present invention provides a bovine viral diarrhea virus NS2 truncated protein NS2 aa 345-373 , the NS2 aa345-373 The amino acid sequence is shown in SEQ ID NO.1.

[0007] In a second aspect, the present invention provides a method for encoding the NS2 aa 345-373 The nucleic acid sequence is shown in SEQ ID NO.2.

[0008] In a third aspect, the present invention provides the bovine viral diarrhea virus NS2 truncated protein NS2 described in the first aspect. aa 345-373 Application in the preparation of bovine viral diarrhea virus detection reagents.

[0009] In a fourth aspect, the present invention provides a fusion protein of a truncated protein of bovine viral diarrhea virus NS2, wherein the fusion protein is a fusion protein GST-NS2 of glutathione S transferase (GST) and the truncated protein of bovine viral diarrhea virus NS2 described in the first aspect. aa 345-373 or horseradish peroxidase (HRP) and the fusion protein HRP-NS2 of the bovine viral diarrhea virus NS2 truncated protein described in the first aspect above aa 345-373 .

[0010] Preferably, the fusion protein GST-NS2 aa 345-373 The amino acid sequence is shown in SEQ ID NO.3; the fusion protein HRP-NS2 aa 345-373 The amino acid sequence is shown in SEQ ID NO.5.

[0011] In a fifth aspect, the present invention provides a nucleic acid encoding the fusion protein described in the fourth aspect, wherein the nucleic acid encoding the fusion protein GST-NS2 aa 345-373 The nucleic acid sequence is shown in SEQ ID NO.4; encoding the fusion protein HRP-NS2 aa 345-373 The nucleic acid sequence is shown in SEQ ID NO.6.

[0012] In a sixth aspect, the present invention provides the use of the fusion protein described in the fourth aspect in preparing a bovine viral diarrhea virus detection reagent.

[0013] In a seventh aspect, the present invention provides a bovine viral diarrhea virus protein complex, the complex comprising the fusion protein GST-NS2 described in the fourth aspect above. aa 345-373 and fusion protein HRP-NS2 aa 345-373 .

[0014] In an eighth aspect, the present invention provides a kit for detecting antibodies to bovine viral diarrhea virus, the kit comprising the fusion protein GST-NS2 described in the fourth aspect. aa 345-373 and fusion protein HRP-NS2 aa 345-373.

[0015] In a ninth aspect, the present invention provides the use of the kit described in the eighth aspect in the detection of bovine viral diarrhea virus for purposes other than disease diagnosis.

[0016] Preferably, the method for using the kit comprises the following steps:

[0017] (1) Fusion protein GST-NS2 aa 345-373 Conduct antigen coating for coating antigen;

[0018] (2) Blocking, washing the plate, and adding the serum sample to be tested for incubation;

[0019] (3) After washing the plate, add the fusion protein HRP-NS2 aa 345-373 incubation;

[0020] (4) After washing the plate, add TMB colorimetric solution for color development;

[0021] (5) Determination of OD 450 The absorbance value of the sample is used to calculate the S / P value, where S / P = (sample OD 450nm - Average negative control OD 450nm ) / (Average positive control OD 450nm - Average negative control OD 450nm );

[0022] (6) Result determination: When the S / P value is ≥ 0.1825, it is determined to be positive; when the S / P value of the test sample is < 0.1825, it is determined to be negative.

[0023] The beneficial effects of the present invention are:

[0024] The present invention provides a bovine viral diarrhea virus NS2 truncated protein NS2 aa 345-373 , compared to the truncated protein NS2 with a higher antigenic index aa 296-319 , the truncated protein NS2 described in this application aa 345-373 On the basis of having good reactivity with bovine viral diarrhea virus positive serum, it can induce the expected protein expression; and the truncated protein NS2 aa 296-319 It can be fused with glutathione S-transferase (GST) or horseradish peroxidase (HRP) to obtain the fusion protein GST-NS2 aa 345-373 Can be used to capture bovine viral diarrhea virus antibodies, and the fusion protein HRP-NS2 aa 345-373The application can detect bovine viral diarrhea virus antibody, and can be used for constructing a double-antigen sandwich ELISA method for detecting bovine viral diarrhea virus antibody, which has the technical advantages of high sensitivity, good specificity, simple operation, easy popularization, low cost, etc., and can be applied to the screening of BVDV antibody in a cattle herd.

[0025] HRP and NS2 aa 296-319 In E. coli, the expression is in the form of a fusion protein. Since E. coli naturally has no HRP activity, it does not react with the substrate, thereby avoiding the purity requirement of NS2 aa 296-319 , reducing the background noise caused by E. coli protein pollution, avoiding the complex process of coupling HRP to NS2 protein in vitro, the stability of activity, and the non-specificity caused by coupling with E. coli protein, and improving the specificity and convenience of detection. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 GST, GST-NS2 aa 345-373 , GST-NS2 aa 296-319 Protein expression results; wherein lanes 1, 4, and 7 are protein markers, from top to bottom, 130kD, 95kD, 65kD, 45kD, 35kD, 26kD, 20kD, and 14kD; lane 2 is the bacterial protein before induction of the GST recombinant bacteria; lane 3 is the induction product of the GST recombinant bacteria; lane 5 is the bacterial protein before induction of the GST-NS2 aa 345-373 recombinant bacteria; lane 6 is the induction product of the GST-NS2 aa 345-373 recombinant bacteria; lane 8 is the induction product of the GST-NS2 aa 296-319 recombinant bacteria; lane 9 is the induction product of the GST-NS2 aa 345-373 recombinant bacteria.

[0027] Figure 2 GST, GST-NS2 aa 345-373 , GST-NS2 aa 296-319 Protein solubility analysis and purification results; wherein lanes 4, 8, and 12 are protein markers, from top to bottom, 130kD, 95kD, 65kD, 45kD, 35kD, 26kD, 20kD, and 14kD; lanes 1, 2, and 3 are the supernatant, precipitate, and purified product of the induction product of the GST recombinant bacteria after ultrasonic treatment; lanes 5, 6, and 7 are the induction product of the GST-NS2aa 296-319 Supernatant, precipitate, and purified product of the recombinant bacteria induced product after ultrasonication; lanes 9, 10, and 11 are GST-NS2 aa 345-373 After ultrasonication, the supernatant of the recombinant bacteria-induced product was precipitated and purified.

[0028] Figure 3 GST, GST-NS2 aa 345-373 Protein, GST-NS2 aa 296-319 Results of reactivity analysis with BVDV-positive sera.

[0029] Figure 4 HRP-NS2 aa 296-319 Induction expression results of recombinant bacteria; Lane 1 is the protein marker, from top to bottom are 180kD, 130kD, 100kD, 70kD, 55kD, 40kD, 35kD, 25kD, 15kD, 10kD; Lane 1 is the recombinant bacteria pre-induction bacterial protein, Lane 2 is HRP-NS2 aa 296-319 After induction of the recombinant bacteria, there was no obvious expression of the target protein.

[0030] Figure 5 HRP-NS2 aa 345-373 The induced expression results of the recombinant bacteria; among them, lane 1 is the bacterial protein of the recombinant bacteria before induction, lane 2 is the bacterial protein of the recombinant bacteria after induction, and about 39kD protein expression can be seen. Lane 3 is the protein marker, which are 180kD, 130kD, 100kD, 70kD, 55kD, 40kD, 35kD, 25kD, 15kD, and 10kD from top to bottom.

[0031] Figure 6 HRP-NS2 aa 345-373 Western blot of the supernatant and precipitate of the induced expression product of the recombinant bacteria after sonication; Lane 1 is the protein marker, which is 130kD, 95kD, 65kD, 45kD, 35kD, 26kD, 20kD, and 14kD from top to bottom; Lane 2 is HRP-NS2 aa 345-373 Supernatant sample after sonication of recombinant bacteria, lane 3 is HRP-NS2 aa 345-373 The recombinant bacteria were sonicated and the samples were precipitated.

[0032] Figure 7MedCalcv 20.0.10 for interactive dot plot analysis results.

[0033] Figure 8 Sample test results. DETAILED DESCRIPTION

[0034] The embodiments of the present application will be described in detail below with examples. Those skilled in the art will understand that the following examples are only preferred embodiments of the present application, in order to better understand the present application, and thus should not be regarded as limiting the scope of the present application. For those skilled in the art, the present application can have various modifications and changes, any modification, equivalent replacement or improvement within the spirit and principles of the present application should be included in the protection scope of the present application.

[0035] The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used are all purchased from conventional biochemical reagent manufacturers unless otherwise specified.

[0036] The present application finds that the fusion protein (GST-NS2 aa 296-319 ) of GST and NS2 truncated protein NS2 aa 345-373 is used as a coating antigen for antibody capture, and the fusion protein (HRP-NS2 aa 296-319 ) of HRP and NS2 truncated protein NS2 aa 345-373 is used for detection, which improves the specificity and convenience of detection, and can be used for antibody detection of BVDV infected cattle, and specifically includes the following contents:

[0037] Example 1 Preparation of NS2 truncated protein and fusion protein

[0038] According to the bovine viral diarrhea virus genome sequence (GenBank accession number KC695814) determined by the laboratory in the early stage, the nucleotide sequence (474bp) of the coding region of NS2 aa 296-453 is selected, as shown in SEQ ID NO. 7, and the recombinant vector Puc-NS2.474 is synthesized by Beijing Qikexin Biotechnology Co., Ltd. Xi'an Branch.

[0039] Primers P1: 5'-gatctggttccgcgtggatccTCTGGGCGGTTGAGGGCC-3' (shown in SEQ ID NO.9) and P2: 5'-gtcacgatgcggccgctcgagtcatcatcttaagatccatcctaggtgtt-3' (shown in SEQ ID NO.10) were designed to amplify NS2 using Puc-NS2.474 as a template. aa 345-373 The target fragment was recovered from the coding region, and 10 ng of the fragment was taken and combined with the pGEX-4T-1 vector (100 ng) treated with BamHI and XhoI double enzymes for recombination reaction using a homologous recombination cloning kit (NEB). The recombinant product was transformed into BL21 (DE3) competent cells, and positive clones were screened using agar medium containing 60 μg / mL ampicillin. After verification by colony PCR and sequencing, the recombinant bacteria were cultured and fermented. 600 When the concentration of IPTG was 0.6, IPTG (final concentration was 0.8 mM) was added and cultured at 37 °C for 6-8 h. The fusion protein of GST and NS2 truncated protein (GST-NS2) was purified using a GST tag protein purification kit. aa 345-373 ).

[0040] At the same time, targeting the truncated protein NS2 with a higher antigenicity index aa 296-319 (shown in SEQ ID NO.8), primers P3: 5'-gatctggttccgcgtggatccAAGTTTTTTATACTGTCT-3' (shown in SEQ ID NO.11) and P4: 5'-gt cacgatgcggccgctcgagtcaTGCTACAGTCTGGTTCCTAA-3' (shown in SEQ ID NO.12) were set, and GST-NS2 was obtained by amplification and recombination using the same method as above. aa 296-319 BL21 (DE3) expression bacteria were used to express GST-NS2. aa 296-319 .

[0041] The pGEX-4T-1 vector was recombined into BL21 (DE3) bacteria and cultured and fermented under the same conditions, and the GST protein control was purified. aa 345-373 Protein, GST-NS2 aa 296-319The proteins were coated on enzyme-labeled plates and conventional indirect ELISA was performed to detect the positive and negative serum of bovine BVDV. The serum was diluted at a ratio of 1:10 and the HRP-labeled rabbit anti-bovine IgG (Sigma) was diluted at a ratio of 1:10000. The results were used to determine the GST-NS2 aa 345-373 Protein, GST-NS2 aa 296-319 Reactivity of the protein with bovine BVDV-positive sera.

[0042] The results are as follows Figure 1-3 As shown, GST recombinant bacteria, GST-NS2 aa 345-373 Recombinant bacteria, GST-NS2 aa 296-319 After induction, the recombinant bacteria can express proteins of about 26kD, 27kD, and 27kD, respectively, but GST-NS2 aa 296-319 The expression of recombinant bacteria is low (e.g. Figure 1 The supernatant after sonication contained the target protein expression, and after purification, a high-purity protein could be obtained, but GST-NS2 aa 296-319 The yield is low (e.g. Figure 2 As shown). It shows that compared with NS2 protein, the truncated protein NS2 described in this application aa 345-373 Able to be expressed soluble and with a higher antigenic index than the truncated protein NS2 aa 296-319 , the truncated protein NS2 described in this application aa 345-373 The soluble expression level of GST was significantly increased.

[0043] ELISA was performed using duplicate positive and negative sera. The three proteins did not react with BVDV negative serum. The fusion protein GST-NS2 aa 345-373 Protein, GST-NS2 aa 296-319 It has good reactivity with bovine viral diarrhea virus positive serum (such as Figure 3 shown).

[0044] The designed primers P5: 5'-tatcggaattaattcggatccgtatccatatgatgttccagattatgct-3' (shown in SEQ ID NO.13) and P6: 5'-gagagttgctgttgaccactctgc-3' (shown in SEQ ID NO.14) were used to amplify the HRP coding region using pCAG HRP-TM plasmid (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) as a template, and the target DNA frag1 was recovered. The synthesized primers P7: 5'-agtggtcaacagcaactctccgcggggcggtggcggtagcaacaagcactgcataatatgcaca-3' (shown in SEQ ID NO.15) and P8: 5'-gtggtggtggtggtgctcgaggggcttcccgtagcgacc-3' (shown in SEQ ID NO.16) were used to amplify NS2 using Puc-NS2.474 as a template. aa 345-373 At the same time, primers P9: 5'-agtggtcaacagcaactctccgcggggcggtggcggtagcAAGTTTTTTATACTGTCT-3' (shown in SEQ ID NO.17) and P10: 5'-gtggtggtggtggtgctcgagTGCTACAGTCTGGTTCCTAA-3' (shown in SEQ ID NO.18) were used to amplify NS2 aa 296-319 , the target DNA Frag3 was recovered, 10ng frag2 DNA and 10ng frag3 DNA were paired with 20ng frag1 DNA respectively, and the recombination reaction was carried out with the pET-22b vector treated with BamHI and XhoI double enzymes using a homologous recombination cloning kit (NEB Company), and BL21 (DE3) competent cells were transformed. Positive clones were screened using agar medium containing 60μg / mL ampicillin. After verification by colony PCR and sequencing, HRP-NS2 aa 345-373、 HRP-NS2 aa 296-319 The recombinant bacteria were cultured and fermented, and the OD 600 When the pH value was 0.6, IPTG (final concentration was 0.1 mM) was added, and the cells were induced at 37°C for 10 h. Protein electrophoresis was performed to determine the expression of the target protein, and Western Blot analysis was performed on the target protein in the supernatant and precipitate after induction of the recombinant bacteria. The antibody used was an anti-His monoclonal antibody diluted 1:1000.

[0045] The results are as follows Figure 4-6 As shown, HRP-NS2 aa 296-319 The recombinant bacteria did not express the expected protein after induction (such as Figure 4 as shown), HRP-NS2 aa 345-373 The recombinant bacteria were induced to express the expected target protein, about 39kD (such as Figure 5 Soluble HRP-NS2 was present in the supernatant after sonication. aa 345-373 The target protein (such as Figure 6 As shown). This shows that compared with the truncated protein NS2 with a higher antigenic index aa 296-319 , the protein NS2 described in this application aa 345-373 It can be expressed soluble with HRP and can be used for the detection of bovine viral diarrhea antibodies.

[0046] Therefore, HRP-NS2 was selected aa 345-373 Recombinant bacteria for protein fusion protein HRP-NS2 aa 345-373 Preparation: After the induction expression is completed, the bacteria are collected and resuspended in PBS. After ultrasonic treatment, centrifugation is carried out at 10000g for 10 min at 4°C. The ultrasonic treatment supernatant containing soluble substances is collected and PC-300 liquid biological preservative (Beijing Solebow Technology Co., Ltd.) is added at a final concentration of 0.1% for later use.

[0047] Example 2 Establishment of a Double Antigen Sandwich ELISA Detection Method for Bovine Viral Diarrhea Antibodies

[0048] Based on the fusion protein prepared in Example 1, a method for detecting bovine viral diarrhea virus antibodies was further established. The method specifically comprises the following steps:

[0049] 1. Coating ELISA plate: The fusion protein GST-NS2 prepared in Example 1 aa 345-373 Dilute with carbonate-bicarbonate buffer (capsules purchased from Sigma-Aldrich, each capsule is dissolved in 100 mL of deionized water) to concentrations of 1.0 μg / mL, 0.5 μg / mL, and 0.25 μg / mL, mix equally, and take 100 μL to coat a 96-well ELISA plate at 4°C overnight.

[0050] 2. Establishment of detection method:

[0051] (1) Discard the antigen solution in the coated ELISA plate, wash the plate three times with PBST solution, block with 5% skim milk powder at 37°C for 60 min, and wash the plate three times with PBST solution.

[0052] (2) Dilute the positive serum and negative sample serum to 1:10, 1:20, and 1:40 respectively with serum diluent, add to the ELISA plate, gently shake to mix, seal with sealing film, and incubate at 37°C for 30 min;

[0053] (3) Add 300 μL of washing solution (1×) to each well, wash three times, and pat dry;

[0054] (4) Add HRP-NS2 aa 345-373 The supernatant was ultrasonically treated, 100 μL was added to each well, sealed with sealing film, and incubated at 37°C for 30 min;

[0055] (5) Remove the ELISA plate, open the sealing film, add 300 μL of washing solution (1×) to each well, wash three times, and pat dry;

[0056] (6) Add 100 μL of substrate to each well, seal with sealing film, and incubate at 37°C in the dark for 10 min;

[0057] (7) Add 100 μL of stop solution to each well and read the absorbance at 450 nm (OD450 nm value) using a microplate reader;

[0058] (8) P / N value (positive control serum OD 450 Mean / negative control serum OD 450 The protein coating concentration at the maximum mean value and the optimal serum dilution factor were used as the optimal detection parameters, and the fusion protein GST-NS2 was finally determined. aa 345-373 The coating concentration was 0.5 μg / mL, and the detection dilution of the serum to be tested was 1:20.

[0059] Example 3 Kit Preparation

[0060] The kit components include: ELISA plate, sealing film, positive control, negative control, serum diluent, color development solution, concentrated washing solution, and stop solution.

[0061] Coated ELISA plate: fusion protein GST-NS2 aa 345-373 Use carbonate-bicarbonate buffer (0.5 μg / mL) and take 100 μL to coat a 96-well ELISA plate at 4°C overnight. The next day, discard the antigen solution, wash the plate three times with PBST solution, block with 5% skim milk powder at 37°C for 60 min, wash the plate three times with PBST solution, air-dry, vacuum-pack, and store at 4°C.

[0062] Detection method: (1) Take out the ELISA plate, balance it to room temperature, add 95 μL serum diluent to each well, then add 5 μL of the sample to be tested, 15 negative bovine sera with clear background, 20 positive bovine sera with clear background, 2 wells of positive control serum, and 2 wells of negative control serum, gently shake to mix, seal with sealing film, and incubate at 37°C for 30 minutes; (2) Add 300 μL washing solution (1×) to each well, wash 3 times, and pat dry; (3) Add fusion protein HRP-NS2 to each well aa 345-373 Ultrasonic treatment of 100 μL of supernatant, sealing with sealing film, incubation at 37 ° C for 30 min; (4) Remove the ELISA plate, add 300 μL of washing solution (1×) to each well, wash 3 times, and pat dry; (5) Add 100 μL of substrate to each well, seal with sealing film, and incubate at 37 ° C in the dark for 10 min; (6) Add 100 μL of stop solution to each well, and read the absorbance value (OD) at 450 nm using an ELISA reader. 450nm value); (7) using the formula S / P = (sample OD 450nm - Average negative control OD 450nm ) / (Average positive control OD 450nm - Average negative control OD450nm ), and MedCalcv20.0.10 was used for interactive dot plot analysis to determine the critical value.

[0063] The results are as follows Figure 7 As shown in the figure, when the S / P value is 0.1825, it is the critical value, and the detection can have high sensitivity and specificity; when the S / P value is ≥0.1825, it is judged as positive; when the S / P value of the test sample is <0.1825, it is judged as negative.

[0064] Sealing film: purchased from Beijing Solebow Technology Co., Ltd.

[0065] Serum diluent: purchased from Jinan Biotech Co., Ltd.

[0066] Concentrated washing solution: 10x PBST: purchased from Beijing Solebow Technology Co., Ltd., stored in aliquots.

[0067] Color development solution: Single-component TMB color development solution, purchased from Beijing Solebow Technology Co., Ltd.

[0068] Stop solution: 2M H2SO4: Take 108.7mL of 98% concentrated sulfuric acid and 891.3mL of deionized water and store in aliquots.

[0069] Positive control: serum from bovine naturally infected with bovine BVDV screened by virus neutralization test, stored at -20°C after aliquoting.

[0070] Negative control: Aseptically collect whole blood from cattle with no history of vaccination or BVDV infection, separate the serum, and store it at -20°C after aliquoting.

[0071] Example 4 Sample Detection

[0072] The prepared kit was used to test sera immune to common bovine viral diseases or vaccines, including standard positive sera for foot-and-mouth disease type O and type A, to verify the specificity of the test. The positive sera were diluted at 1:20, 1:40, 1:80, and 1:160, respectively, to evaluate the sensitivity of the detection method. The kit of the present invention was used in conjunction with a commercially available kit to test 50 clinical serum samples to verify the kit's detection accuracy.

[0073] The results are as follows Figure 8 As shown, the test results for O-type foot-and-mouth disease standard-positive serum and A-type foot-and-mouth disease standard-positive serum were negative, while BV DV-positive serum was still positive at a 1:80 dilution, demonstrating the kit's excellent specificity and sensitivity. In compliance testing, 7 samples that tested negative with commercially available kits were negative with this kit, and 38 of 43 samples that tested positive with commercially available kits were positive with this kit, for an overall compliance rate of 90%.

[0074] The basic principles, main features and advantages of the present invention are shown and described above. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.

Claims

1. A bovine viral diarrhea virus NS2 truncated protein NS2 aa345-373 , the NS2 aa345-373 The amino acid sequence is shown in SEQ ID NO.

1.

2. Encoding NS2 according to claim 1 aa345-373 A nucleic acid characterized in that The nucleic acid sequence is shown as SEQ ID NO.

2.

3. A fusion protein of a truncated protein of NS2 of bovine viral diarrhea virus, characterized in that: The fusion protein is a fusion protein GST-NS2 of glutathione S transferase (GST) and the bovine viral diarrhea virus NS2 truncated protein according to claim 1 aa345-373 , or a fusion protein HRP-NS2 of horseradish peroxidase (HRP) and the bovine viral diarrhea virus NS2 truncated protein according to claim 1 aa345-373 ; The fusion protein GST-NS2 aa345-373 The amino acid sequence is shown in SEQ ID NO.3; the fusion protein HRP-NS2 aa345-373 The amino acid sequence is shown in SEQ ID NO.

5.

4. The nucleic acid encoding the fusion protein according to claim 3, characterized in that Encoding the fusion protein GST-NS2 aa345-373 The nucleic acid sequence is shown in SEQ ID NO.4; encoding the fusion protein HRP-NS2 aa345-373 The nucleic acid sequence is shown in SEQ ID NO.

6.

5. Use of the bovine viral diarrhea virus NS2 truncated protein according to claim 1 or the fusion protein according to claim 3 in the preparation of a bovine viral diarrhea virus antibody detection reagent.

6. A kit for detecting bovine viral diarrhea virus antibodies, characterized in that: The kit comprises the fusion protein GST-NS2 described in claim 3 aa345-373 and fusion protein HRP-NS2 aa345-373 .

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