Bovine viral diarrhea virus NS2 truncated protein and application thereof in BVDV antibody detection

Abstract

The invention 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 invention firstly provides a truncated protein of a BVDV NS2 protein, the truncated protein is subjected to soluble expression after being fused with glutathione S transferase (GST) and horse radish peroxidase (HRP) respectively, has better reactogenicity with positive serum of a BVDV antibody, does not react with negative serum of the BVDV antibody, and can be used for detecting the BVDV antibody; secondly, a fusion protein of GST and the truncated protein is taken as a coating antigen, a fusion protein of HRP and the truncated protein is taken as a detection antigen, a double-antigen sandwich ELISA method for BVDV antibody detection is constructed, and the method has the technical advantages of high sensitivity, good specificity, simplicity and convenience in operation, easiness in popularization, low cost and the like, and can be applied to screening of BVDV antibodies in cattle.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a truncated NS2 protein of bovine viral diarrhea virus and its application in BVDV antibody detection. Background Technology

[0002] Bovine Viral Diarrhea-Mucosal Disease (BVD-MD) is an infectious disease of cattle caused by Bovine viral diarrhea virus (BVDV). It is listed as a notifiable infectious disease of cattle by the World Organisation for Animal Health (OIE), and classified as a Class III animal disease in my country's Ministry of Agriculture and Rural Affairs' "List of Class I, II, and III Animal Diseases". BVDV has a broad host spectrum, including livestock such as cattle (dairy cows, yaks, and buffalo), deer, camels, sheep, and pigs, as well as some wild cloven-hoofed animals. However, cattle show the most typical clinical symptoms after infection, which can manifest as a wide range of symptoms, including diarrhea, respiratory symptoms, hemorrhagic syndrome, chronic mucosal disease, immune tolerance, immunosuppression, persistent infection, and reproductive disorders.

[0003] Currently, Nordic countries such as Norway and Sweden are achieving BVD eradication by monitoring and culling cattle with persistent infections. my country's current "Standards for the Health of Breeding Animals" also lists the absence of bovine viral diarrhea as a health standard for breeding cattle.

[0004] Currently, the indirect ELISA method used for detecting bovine viral diarrhea virus antibodies has low specificity. While competitive ELISA methods using auxiliary monoclonal antibodies can improve the specificity of detection, they significantly increase the cost of detection reagents. Summary of the Invention

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

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

[0007] Secondly, the present invention provides encoding the NS2 described in the first aspect above. aa 345-373 The nucleic acid, the sequence of which is shown in SEQ ID NO.2.

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

[0009] Fourthly, the present invention provides a fusion protein of bovine viral diarrhea virus NS2 truncated protein, wherein the fusion protein is a fusion protein GST-NS2 of glutathione S-transferase (GST) and the bovine viral diarrhea virus NS2 truncated protein described in the first aspect. aa 345-373 ; or horseradish peroxidase (HRP) and the truncated bovine viral diarrhea virus NS2 protein HRP-NS2 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] Fifthly, the present invention provides a nucleic acid encoding the fusion protein described in the fourth aspect above, wherein the fusion protein GST-NS2 is encoded. 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 application of the fusion protein described in the fourth aspect above in the preparation of 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] Eighthly, the present invention provides a kit for detecting bovine viral diarrhea virus antibodies, the kit comprising the fusion protein GST-NS2 described in the fourth aspect above. aa 345-373 and fusion protein HRP-NS2 aa 345-373 .

[0015] In a ninth aspect, the present invention provides the application of the kit described in the eighth aspect above in the detection of bovine viral diarrhea virus for non-disease diagnosis purposes.

[0016] Preferably, the method of using the kit includes the following steps:

[0017] (1) Using the fusion protein GST-NS2 aa 345-373 Antigen coating is performed for the coating antigen;

[0018] (2) After sealing and washing the plate, add the serum sample to be tested and incubate.

[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 Calculate the S / P value from the absorbance value of the sample, where S / P = (sample OD) / (sample OD). 450nm - Average negative control OD 450nm ) / (mean 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 sample is <0.1825, it is determined to be negative.

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

[0024] This invention provides a truncated protein NS2 of bovine viral diarrhea virus NS2. aa 345-373 Compared to the truncated protein NS2, which has a higher antigenic index, aa 296-319 The truncated protein NS2 described in this application aa 345-373 Based on its good reactivity with bovine viral diarrhea virus positive serum, it can induce the expression of the expected protein; and the truncated protein NS2 aa 296-319 The fusion protein GST-NS2 can be fused with glutathione S-transferase (GST) or horseradish peroxidase (HRP) for expression. aa 345-373 It can be used to capture bovine viral diarrhea virus antibodies, and the fusion protein HRP-NS2 aa 345-373 This method can detect bovine viral diarrhea virus (BVDV) antibodies and can be used to construct a double-antigen sandwich ELISA method for detecting BVDV antibodies. The method has technical advantages such as high sensitivity, good specificity, simple operation, easy promotion, and low cost, and can be applied to the screening of BVDV antibodies in cattle herds.

[0025] Moreover, HRP and NS2 aa 296-319 It is expressed in E. coli as a fusion protein. Because the native protein in E. coli has no HRP activity and does not react with the substrate, NS2 is avoided. aa 296-319The purity requirements are met, and the background of E. coli protein contamination is reduced, which has a noise reduction effect. It also avoids the complex process of HRP coupling to NS2 protein in vitro, the problems of activity stability and nonspecificity caused by coupling with E. coli protein, thus improving the specificity and convenience of detection. Attached Figure Description

[0026] Figure 1 GST, GST-NS2 aa 345-373 GST-NS2 aa 296-319 Protein expression results; lanes 1, 4, and 7 are protein markers, with values ​​of 130kD, 95kD, 65kD, 45kD, 35kD, 26kD, 20kD, and 14kD from top to bottom, respectively; lane 2 contains pre-inducible somatic protein of GST recombinant bacteria; lane 3 contains GST recombinant bacteria induction product; lane 5 contains GST-NS2. aa 345-373 Recombinant bacterial induction of procell protein; lane 6 is GST-NS2 aa 345-373 Recombinant bacterial induced product; lane 8 is GST-NS2 aa 296-319 Pre-inducible bacterial protein; supernatant after sonication; lane 9 is GST-NS2. aa 345-373 Recombinant bacterial induced products.

[0027] Figure 2 GST, GST-NS2 aa 345-373 GST-NS2 aa 296-319 The results of protein fusibility analysis and purification are shown below. Lanes 4, 8, and 12 are protein markers, with values ​​of 130 kD, 95 kD, 65 kD, 45 kD, 35 kD, 26 kD, 20 kD, and 14 kD from top to bottom, respectively. Lanes 1, 2, and 3 are the supernatant, precipitate, and purified product of the GST recombinant bacterial induction product after sonication, respectively. Lanes 5, 6, and 7 are GST-NS2... aa 296-319 The supernatant, precipitate, and purified product of the recombinant bacterial induction product were obtained after sonication; lanes 9, 10, and 11 contained GST-NS2. aa 345-373 The supernatant, precipitate, and purified product were obtained after sonication of the recombinant bacterial induced product.

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

[0029] Figure 4 HRP-NS2 aa 296-319The results of recombinant bacterial induction expression; lane 1 is the protein marker, with values ​​from top to bottom of 180kD, 130kD, 100kD, 70kD, 55kD, 40kD, 35kD, 25kD, 15kD, and 10kD; lane 1 also shows the recombinant bacterial pre-induction somatic protein, and lane 2 shows HRP-NS2. aa 296-319 After induction, the recombinant bacteria did not show significant expression of the target protein in their cell proteins.

[0030] Figure 5 HRP-NS2 aa 345-373 The results of recombinant bacterial induction expression are shown. Lane 1 shows the pre-induction bacterial somatic protein, lane 2 shows the post-induction bacterial somatic protein, with approximately 39kD of protein expression. Lane 3 shows the protein markers, with values ​​of 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 images of the supernatant and precipitate of the recombinant bacterial induction expression product after sonication; lane 1 is the protein marker, with values ​​from top to bottom of 130kD, 95kD, 65kD, 45kD, 35kD, 26kD, 20kD, and 14kD; lane 2 is the HRP-NS2 marker. aa 345-373 The supernatant sample after sonication of the recombinant bacteria, lane 3 contains HRP-NS2. aa 345-373 The recombinant bacteria were precipitated after sonication.

[0032] Figure 7 Interactive dot plot analysis results were performed using MedCalc v20.0.10.

[0033] Figure 8 Sample test results. Detailed Implementation

[0034] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are merely preferred embodiments of the present invention to facilitate a better understanding of the invention, and therefore should not be considered as limiting the scope of the invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the experimental materials used, unless otherwise specified, were purchased from conventional biochemical reagent manufacturers.

[0036] This invention discovers that GST and NS2 can be used to shorten the NS2 protein. aa 296-319 fusion protein (GST-NS2) aa 345-373 HRP is used as the coating antigen to capture antibodies, and HRP is used to truncate the NS2 protein NS2. aa 296-319 The fusion protein (HRP-NS2) aa 345-373 This method improves the specificity and convenience of testing and can be used for antibody detection in cattle infected with BVDV. Specifically, it includes the following:

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

[0038] Based on the bovine viral diarrhea virus genome sequence previously determined by our laboratory (GenBank accession number KC695814), NS2 was selected. aa 296-453 The coding region nucleotide sequence (474bp), as shown in SEQ ID NO.7, was used to synthesize the recombinant vector Puc-NS2.474 by Beijing Qingke Biotechnology Co., Ltd. Xi'an Branch.

[0039] Primers P1: 5'-gatctggttccgcgtggatccTCTGGGCGGTTGAGGGCC-3' (SEQ ID NO. 9) and P2: 5'-gtcacgatgcggccgctcgagtcatcatcttaagatccatcctaggtgtt-3' (SEQ ID NO. 10) were designed to amplify NS2 using Puc-NS2.474 as a template. aa 345-373 The coding region was extracted, the target fragment was recovered, and 10 ng of it was mixed with 100 ng of pGEX-4T-1 vector (double-digested with BamHI and XhoI). Recombination was performed using a homologous recombination cloning kit (NEB). The recombinant product was transformed into BL21(DE3) competent cells. 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 at OD200. 600 IPTG (final concentration 0.8 mM) was added at a concentration of 0.6, and the mixture was induced and cultured at 37°C for 6-8 h. The fusion protein of GST and NS2 truncated protein (GST-NS2) was obtained by purification using a GST-tagged protein purification kit. aa 345-373 ).

[0040] Meanwhile, targeting the truncated protein NS2, which has a high antigenicity index... aa 296-319(As shown in SEQ ID NO. 8), primers were set: P3: 5'-gatctggttccgcgtggatccAAGTTTTTTATACTGTCT-3' (as shown in SEQ ID NO. 11), P4: 5'-gtcacgatgcggccgctcgagtcaTGCTACAGTCTGGTTCCTAA-3' (as shown in SEQ ID NO. 12). Amplification and recombination were performed using the same method as described above to obtain GST-NS2. aa 296-319 GST-NS2 was obtained by purifying BL21(DE3)-expressing bacteria after induction of expression. aa 296-319 .

[0041] Recombinant BL21(DE3) bacteria using the pGEX-4T-1 vector were cultured and fermented under the same conditions, and the GST protein control was purified. Using 0.5 μg / mL GST protein and GST-NS2... aa 345-373 Protein, GST-NS2 aa 296-319 The proteins were coated onto ELISA plates, and bovine BVDV positive and negative sera were detected by routine indirect ELISA. The serum dilution ratio was 1:10, and the HRP-labeled rabbit anti-bovine IgG (Sigma) was diluted 1:10000. GST-NS2 was determined based on the results. aa 345-373 Protein, GST-NS2 aa 296-319 Reactivity of protein with bovine BVDV-positive serum.

[0042] The results are as follows Figure 1-3 As shown, this indicates that GST recombinant bacteria and GST-NS2 aa 345-373 Recombinant bacteria, GST-NS2 aa 296-319 The recombinant bacteria could express proteins of approximately 26kD, 27kD, and 27kD after induction, respectively, but GST-NS2... aa 296-319 The recombinant bacteria had lower surface area (e.g. Figure 1 (As shown); the target protein was expressed in the supernatant after sonication, and high-purity protein could be obtained after purification, but GST-NS2... aa 296-319 The harvest was low (e.g.) Figure 2 (As shown). This indicates that, compared to the NS2 protein, the truncated protein NS2 described in this application... aa 345-373 It can be expressed in a soluble manner and has 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 content of GST was significantly increased.

[0043] ELISA was performed using paired positive and negative sera. The three proteins did not react with BVDV-negative serum, indicating the presence of the fusion protein GST-NS2. aa 345-373 Protein, GST-NS2aa 296-319 It has good reactivity with bovine viral diarrhea virus positive serum (e.g. Figure 3 (As shown).

[0044] Using designed primers P5: 5'-tatcggaattaattcggatccgtatccatatgatgttccagattatgct-3' (SEQ ID NO.13) and P6: 5'-gagagttgctgttgaccactctgc-3' (SEQ ID NO.14), with pCAG HRP-TM plasmid (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) as a template, the HRP coding region was amplified, and the target DNA frag1 was recovered. Primers P7: 5'-agtggtcaacagcaactctccgcggggcggtggcggtagcaacaagcactgcataatatgcaca-3' (SEQ ID NO.15) and P8: 5'-gtggtggtggtggtgctcgaggggcttcccgtagcgacc-3' (SEQ ID NO.16), with Puc-NS2.474 as a template, NS2 was amplified. aa 345-373 The coding region was extracted, and the target DNA Frag2 was recovered. Simultaneously, NS2 was amplified using primers P9: 5'-agtggtcaacagcaactctccgcggggcggtggcggtagcAAGTTTTTTATACTGTCT-3' (SEQ ID NO.17) and P10: 5'-gtggtggtggtggtgctcgagTGCTACAGTCTGGTTCCTAA-3' (SEQ ID NO.18). aa 296-319 The target DNA Frag3 was recovered. 10 ng of frag2 DNA and 10 ng of frag3 DNA were paired with 20 ng of frag1 DNA, respectively. These were then used to perform recombination reactions with the pET-22b vector, which had been double-digested with BamHI and XhoI, using a homologous recombination cloning kit (NEB). The resulting cells were transformed into BL21(DE3) competent cells. Positive clones were screened using agar medium containing 60 μg / mL ampicillin. After verification by colony PCR and sequencing, HRP-NS2 was used to... aa 345-373、 HRP-NS2 aa 296-319 The recombinant bacteria were cultured and fermented at OD. 600IPTG (final concentration 0.1 mM) was added when the concentration was 0.6, and the mixture was induced and cultured at 37℃ 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 recombinant bacteria. The antibody used was a 1:1000 diluted anti-His monoclonal antibody.

[0045] The results are as follows Figure 4-6 As shown, this indicates that HRP-NS2 aa 296-319 The recombinant bacteria did not show obvious expression of the expected protein after induction (e.g. Figure 4 As shown), HRP-NS2 aa 345-373 The recombinant bacteria induced significant expression of the intended target protein, approximately 39 kDa (e.g., ...). Figure 5 As shown in the figure, soluble HRP-NS2 was found in the supernatant after ultrasound. aa 345-373 Target protein (e.g.) Figure 6 (As shown). This indicates that, compared to 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 in soluble form with HRP and can be used for the detection of antibodies against bovine viral diarrhea.

[0046] Therefore, HRP-NS2 was selected. aa 345-373 Recombinant bacteria fusion protein HRP-NS2 aa 345-373 In preparation, after the induction of expression was completed, the bacterial cells were collected and resuspended in PBS. After sonication, the cells were centrifuged at 10000g for 10 min at 4℃. The soluble sonicated supernatant was collected and PC-300 liquid biological preservative (Beijing Solarbio Science & Technology Co., Ltd.) with a final concentration of 0.1% was added for later use.

[0047] Example 2: Establishment of a double-antigen sandwich ELISA method for detecting bovine viral diarrhea antibodies.

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

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

[0050] 2. Establishment of detection methods:

[0051] (1) Discard the antigen solution in the coated ELISA plate, wash the plate 3 times with PBST solution, block with 5% skim milk powder at 37℃ for 60 min, and wash the plate 3 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 them 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 3 times, and pat dry;

[0054] (4) Add HRP-NS2 aa 345-373 Ultrasonic treatment was performed on the supernatant, 100 μL per well, and the wells were sealed with sealing film and incubated at 37°C for 30 min.

[0055] (5) Remove the microplate, open the sealing film, add 300 μL of washing buffer (1×) to each well, wash 3 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 an ELISA reader;

[0058] (8) Using the P / N value (positive control serum OD) 450 Mean / Negative control serum OD 450 The optimal detection parameters were the protein coating concentration at which the mean value was maximized and the optimal serum dilution factor. The fusion protein GST-NS2 was ultimately determined to be the optimal protein for detection. 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: Reagent Kit Preparation

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

[0061] Coating the ELISA plate: The fusion protein GST-NS2 was coated onto the plate. aa 345-373 Use 0.5 μg / mL carbonate-bicarbonate buffer to coat 100 μL of a 96-well microplate and incubate overnight at 4°C. 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, equilibrate to room temperature, add 95 μL of serum diluent to each well, and then add 5 μL of the sample to be tested, namely 15 negative bovine serum samples with clear background, 20 positive bovine serum samples with clear background, 2 positive control serum samples, and 2 negative control serum samples. Gently shake to mix, seal with sealing film, and incubate at 37℃ for 30 min; (2) Add 300 μL of washing buffer (1×) to each well, wash 3 times, and pat dry; (3) Add fusion protein HRP-NS2 to each well. aa 345-373 (3) Sonicate 100 μL of supernatant, seal with sealing film, and incubate at 37°C for 30 min; (4) Remove the microplate, add 300 μL of washing buffer (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 at 450 nm (OD) using a microplate reader. 450nm (7) Using the formula S / P = (sample OD) 450nm - Average negative control OD 450nm ) / (mean positive control OD) 450nm - Average negative control OD450nm Interactive dot plot analysis was performed using MedCalc v20.0.10 to determine the critical values.

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

[0064] Sealing film: Purchased from Beijing Solarbio Technology Co., Ltd.

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

[0066] Concentrated washing solution: 10x PBST: purchased from Beijing Solarbio Technology Co., Ltd., and stored in smaller containers.

[0067] Colorimetric solution: Single-component TMB colorimetric solution, purchased from Beijing Solarbio Technology Co., Ltd.

[0068] Termination solution: 2M H2SO4: Take 108.7mL of 98% concentrated sulfuric acid and 891.3mL of deionized water, and store them separately.

[0069] Positive control: Bovine serum naturally infected with bovine BVDV, screened using a virus neutralization test, aliquoted and stored at -20°C.

[0070] Negative control: Whole blood was aseptically collected from bovines with no history of vaccination or BVDV infection. Serum was separated, aliquoted, and stored at -20°C.

[0071] Example 4 Sample Detection

[0072] The prepared kit was used to detect common bovine viral diseases or vaccine-immunized sera, including O-type foot-and-mouth disease standard positive sera and A-type foot-and-mouth disease standard positive sera, to verify the specificity of the detection. Positive sera were diluted at 1:20, 1:40, 1:80, and 1:160 to evaluate the sensitivity of the detection method. The kit of this invention was compared with commercially available kits using 50 clinical serum samples to verify the detection concordance rate of the kit.

[0073] The results are as follows Figure 8 As shown, the test results for O-type and A-type foot-and-mouth disease standard positive sera were negative, and BVDV positive sera were still positive at a 1:80 dilution, indicating that the kit has good specificity and sensitivity. In the concordance test, 7 negative samples tested with commercially available kits were negative with this kit, and 38 positive samples tested with this kit were positive out of 43 commercially available kits, resulting in an overall concordance 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 a 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 truncated protein of bovine viral diarrhea virus NS2 according to claim 1 aa345-373 .

4. The fusion protein according to claim 3, characterized in that 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.

5. The nucleic acid encoding the fusion protein according to claim 4, 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.

6. Use of the truncated protein of bovine viral diarrhea virus NS2 according to claim 1, or the fusion protein according to any one of claims 3 to 4 in the preparation of a bovine viral diarrhea virus detection reagent.

7. A bovine viral diarrhea virus protein complex, characterized in that: The complex comprises the fusion protein GST-NS2 described in claim 3 or 4 aa345-373 and fusion protein HRP-NS2 aa345-373 .

8. A kit for detecting bovine viral diarrhea virus antibodies, characterized in that: The kit comprises the fusion protein GST-NS2 described in claim 3 or 4 aa345-373 and fusion protein HRP-NS2 aa345-373 .

9. Use of the kit as claimed in claim 8 in the detection of bovine viral diarrhea virus antibodies.

10. The use according to claim 9, characterized in that The method for using the kit comprises the following steps: (1) Fusion protein GST-NS2 aa345-373 Conducting antigen coating for coating antigen; (2) Blocking, washing the plate, and adding the serum sample to be tested for incubation; (3) After washing, add fusion protein HRP-NS2 aa345-373 Incubation; (4) After washing the plate, add TMB color developing solution for color development; (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 - Mean negative control OD 450nm ) / (average positive control OD 450nm - Mean negative control OD 450nm ); (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.

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