Middle mountain disease virus truncated protein VP3s as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a truncated protein VP3s of a Zhongshan disease virus as well as a preparation method and application of the truncated protein VP3s. The Zhongshan disease virus antibody detection method has the technical advantages of being high in sensitivity, good in specificity, easy and convenient to operate, easy to popularize, low in cost and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a truncated protein VP3s of Zhongshan disease virus and a preparation method and application thereof. Background Art

[0002] Chuzan disease, also known as bovine abnormal birthing disease, is an insect-borne disease caused by the Chuzan virus (CHUV) transmitted by Culicoides midges from cattle. Cattle are susceptible to the disease, which primarily affects the central nervous system of calves and causes hydrocephalus and cerebellar hypoplasia, a syndrome characterized by anencephaly or brainstem hypoplasia. Adult cattle are latently infected and do not show clinical symptoms. However, infection in pregnant cows can lead to abnormal births, primarily miscarriage, premature birth, stillbirth, or birth of malformed fetuses. The disease is particularly harmful during epidemics. In 2016, Zhang Yishuang et al. conducted a serological survey of CHUV in cattle and sheep in nine provinces and regions in my country and found widespread positive sera in eight of them, with the disease being more severe in southern China. These reports indicate that CHUV is widespread in my country, posing a significant risk of developing the disease.

[0003] CHUV, a subgroup of the Palyam virus in the genus Orbivirus within the family Reoviridae, is a double-stranded RNA virus widely distributed in tropical, subtropical, and temperate regions, infecting a wide range of ruminant species. Its genome consists of 10 double-stranded RNA segments (Seg-1 to Seg-10) encoding seven structural viral proteins (VP1-VP7) and four nonstructural proteins (NS1-NS4). VP2 and VP5 exhibit high variability in their genome sequences, which correlates with viral serotype specificity. Overall, with the exception of VP2, structural proteins are more conserved than nonstructural proteins, with VP3 showing the highest sequence conservation.

[0004] Because adult cattle often harbor latent infections and exhibit no clinical symptoms, detecting antibodies to CHUV has become the best method for investigating the prevalence of the disease. Several methods for detecting CHUV antibodies have been developed. Yang Zhenxing et al. directly coated the CHUV virus to establish the C-ELISA method, which they used to successfully conduct epidemiological surveys of Zhongshan disease in cattle and sheep in multiple provinces and cities in my country between 2016 and 2018. However, this method requires the preparation of highly purified CHUV virus, making it relatively cumbersome. Zhang Yishuang et al. also developed an ELISA method for detecting viral antibody titers by recombinantly expressing the VP7 protein. However, the poor conservation of the VP7 protein may affect the reliability of the test results. Other CHUV antibody detection methods, such as serum neutralization tests and agar immunodiffusion, have been phased out in practice due to common drawbacks such as complex operation and low sensitivity.

[0005] The structural protein VP3 is an ideal antigen for detecting CHUV-specific antibodies. However, because VP3 is a hydrophobic protein, its soluble expression is challenging, and therefore its application in CHUV antibody detection has been limited. This paper proposes a truncated Zhongshan disease virus VP3 protein and its preparation method, and applies it to CHUV antibody detection, establishing a rapid, sensitive, and specific indirect ELISA method. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a truncated protein VP3s of Zhongshan disease virus and its nucleotide sequence to solve the technical problem of the lack of an indirect ELISA detection method for detecting Zhongshan disease virus in the prior art.

[0007] The second object of the present invention is to provide a method for preparing the above-mentioned truncated protein VP3s of Zhongshan disease virus.

[0008] A third object of the present invention is to provide a method for detecting antibodies to Zhongshan disease virus for non-diagnostic purposes.

[0009] A fourth object of the present invention is to provide an indirect ELISA kit for detecting antibodies to Zhongshan disease virus.

[0010] The fifth object of the present invention is to provide the use of the above-mentioned Zhongshan disease virus truncated protein VP3s in the preparation of Zhongshan disease virus vaccine, the preparation of Zhongshan disease virus antibodies, the preparation of Zhongshan disease virus diagnostic antigens, the preparation of reagents and / or kits for detecting Zhongshan disease virus, and the preparation of reagents and / or kits for detecting Zhongshan disease virus antibodies.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0012] In a first aspect, the present invention provides a truncated protein VP3s of Zhongshan disease virus, the nucleotide sequence of which is shown in SEQ ID No.4.

[0013] In a second aspect, the present invention provides a method for preparing the truncated protein VP3s of the Zhongshan disease virus.

[0014] Furthermore, the preparation method includes:

[0015] (1) The gene encoding the truncated protein VP3s was constructed into the pET-28a(+) expression vector;

[0016] (2) Transform the expression vector identified correctly in step (1) into BL21 (DE3) competent cells for culture and expression;

[0017] (3) Purify the target protein expressed in step (2) by immobilized metal affinity chromatography and remove the tag.

[0018] In a third aspect, the present invention provides a method for detecting antibodies to Zhongshan disease virus for non-diagnostic purposes, which uses the truncated protein VP3s to detect the sample to be tested.

[0019] Furthermore, the non-diagnostic method for detecting antibodies to Zhongshan disease virus comprises the following specific steps:

[0020] S1, antigen coating using truncated protein VP3s as coating antigen;

[0021] S2: Block, wash the plate, and then add the diluted serum sample to be tested for incubation;

[0022] S3, after washing the plate, add enzyme-labeled secondary antibody for incubation;

[0023] S4, after washing the plate, add TMB color developing solution for color development;

[0024] S5. After color development, add the stop solution and measure the absorbance value at OD450nm;

[0025] S6. Result judgment: When OD 450 When OD ≥ 0.196, it was determined to be positive; 450 <0.168, it is judged as negative, and when 0.168≤OD 450 When the value is less than 0.196, it is judged as suspicious.

[0026] Furthermore, in the above steps, the coating solution is a 0.05 M carbonate buffer solution with a pH value of 9.6, the blocking solution is a PBS solution containing 1% BSA (w / v), the washing solution is PBST containing 0.25% v / v Tween-20, the diluent is a 1% BSA (w / v) PBS solution, the enzyme-labeled secondary antibody is rabbit anti-bovine IgG-HRP, and the stop solution is a commercial stop solution.

[0027] In a fourth aspect, the present invention provides an indirect ELISA kit for detecting antibodies to Zhongshan disease virus.

[0028] Furthermore, the indirect ELISA kit includes an enzyme-labeled plate coated with the Zhongshan disease virus truncated protein VP3s, an enzyme-labeled secondary antibody, a washing solution, a diluent, a coating solution, a blocking solution, a color developing solution, a stop solution, a negative control, a positive control, and a result judgment standard.

[0029] Furthermore, the steps for preparing the ELISA plate coated with the truncated protein VP3s of the Zhongshan disease virus are as follows:

[0030] (1) Coat the truncated protein VP3s of Zhongshan disease virus onto an ELISA plate at 4°C overnight;

[0031] (2) Add 1% BSA blocking solution and block at 37°C for 2 h;

[0032] (3) Add PBST washing solution and wash the plate three times;

[0033] (4) Add diluted Zhongshan disease virus-positive serum and incubate at 37°C for 30 min;

[0034] (5) After taking out, shake out the liquid in the wells, add PBST washing solution and wash the plate three times, pat dry the droplets in the wells, and air-dry in a 37°C incubator to obtain an ELISA plate coated with the truncated protein VP3s of Zhongshan disease virus.

[0035] Furthermore, in the preparation step (1) of the above-mentioned ELISA plate, the concentration of the truncated protein VP3s of the Zhongshan disease virus coated on each well was 1 μg / mL.

[0036] Furthermore, the positive serum diluted in the above-mentioned ELISA plate preparation step (4) is diluted 1:200.

[0037] In the fifth aspect, the present invention provides the use of the above-mentioned truncated protein VP3s of Zhongshan disease virus in the preparation of Zhongshan disease virus vaccine, the preparation of Zhongshan disease virus antibody, the preparation of Zhongshan disease virus diagnostic antigen, the preparation of reagents and / or kits for detecting Zhongshan disease virus, and the preparation of reagents and / or kits for detecting Zhongshan disease virus antibodies.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The truncated protein of Zhongshan disease virus VP3 provided by the present invention has good solubility, simpler operation steps during protein expression, and more stable protein structure, thereby improving protein expression efficiency.

[0040] (2) The method for detecting antibodies to Zhongshan disease virus provided by the present invention can efficiently detect the antibody level of CHUV in bovine serum. The method has the characteristics of good repeatability, high specificity and sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 The hydrophobicity analysis of the VP3 amino acid sequence shows that the red box represents the most hydrophilic region, i.e., amino acids 410-517.

[0043] Figure 2 This is the VP3s protein after recombinant, expression and purification in Example 2. DETAILED DESCRIPTION

[0044] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms is non-limiting.

[0045] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0046] In one aspect, the present invention provides a truncated protein VP3s of Zhongshan disease virus, the nucleotide sequence of which is shown in SEQ ID No.4.

[0047] According to another aspect of the present invention, a method for preparing the truncated protein VP3s of Zhongshan disease virus is also provided.

[0048] In some specific embodiments, the preparation method comprises:

[0049] (1) The gene encoding the truncated protein VP3s was constructed into the pET-28a(+) expression vector;

[0050] (2) Transform the expression vector identified correctly in step (1) into BL21 (DE3) competent cells for culture and expression;

[0051] (3) Purify the target protein expressed in step (2) by immobilized metal affinity chromatography and remove the tag.

[0052] According to another aspect of the present invention, a method for detecting antibodies to Zhongshan disease virus for non-diagnostic purposes is provided, which uses the truncated protein VP3s to detect a sample to be tested.

[0053] In some specific embodiments, the non-diagnostic method for detecting antibodies to Zhongshan disease virus comprises the following steps:

[0054] S1, antigen coating using truncated protein VP3s as coating antigen;

[0055] S2: Block, wash the plate, and then add the diluted serum sample to be tested for incubation;

[0056] S3, after washing the plate, add enzyme-labeled secondary antibody for incubation;

[0057] S4, after washing the plate, add TMB color developing solution for color development;

[0058] S5. After color development, add the stop solution and measure the absorbance value at OD450nm;

[0059] S6. Result judgment: When OD 450 When OD ≥ 0.196, it was determined to be positive; 450 <0.168, it is judged as negative, and when 0.168≤OD 450 When the value is less than 0.196, it is judged as suspicious.

[0060] In some specific embodiments, in the above steps, the coating solution is a 0.05 M carbonate buffer solution with a pH value of 9.6, the blocking solution is a PBS solution containing 1% BSA (w / v), the washing solution is PBST containing 0.25% v / v Tween-20, the diluent is a 1% BSA (w / v) PBS solution, the enzyme-labeled secondary antibody is rabbit anti-bovine IgG-HRP, and the stop solution is a commercial stop solution.

[0061] According to another aspect of the present invention, an indirect ELISA kit for detecting antibodies to Zhongshan disease virus is also provided.

[0062] The indirect ELISA kit includes an enzyme-labeled plate coated with the Zhongshan disease virus truncated protein VP3s, an enzyme-labeled secondary antibody, a washing solution, a diluent, a coating solution, a blocking solution, a color developing solution, a stopping solution, a negative control, a positive control, and a result judgment standard.

[0063] In some specific embodiments, the steps for preparing the ELISA plate coated with the truncated protein VP3s of the Zhongshan disease virus are as follows:

[0064] (1) Coat the truncated protein VP3s of Zhongshan disease virus onto an ELISA plate at 4°C overnight;

[0065] (2) Add 1% BSA blocking solution and block at 37°C for 2 h;

[0066] (3) Add PBST washing solution and wash the plate three times;

[0067] (4) Add diluted Zhongshan disease virus-positive serum and incubate at 37°C for 30 min;

[0068] (5) After taking out, shake out the liquid in the wells, add PBST washing solution and wash the plate three times, pat dry the droplets in the wells, and air-dry in a 37°C incubator to obtain the enzyme-labeled plate for the truncated protein VP3s of Zhongshan disease virus.

[0069] In some specific embodiments, the concentration of the truncated protein VP3s of the Zhongshan disease virus coated on each well in the preparation step (1) of the above-mentioned ELISA plate is 1 μg / mL.

[0070] In some specific embodiments, the positive serum diluted in step (4) of preparing the ELISA plate is diluted 1:200.

[0071] According to another aspect of the present invention, the use of the above-mentioned truncated protein VP3s of Zhongshan disease virus in preparing Zhongshan disease virus vaccine, preparing Zhongshan disease virus antibody, preparing Zhongshan disease virus diagnostic antigen, preparing reagents and / or kits for detecting Zhongshan disease virus, and preparing reagents and / or kits for detecting Zhongshan disease virus antibodies is also provided.

[0072] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0073] The main reagents and their sources used in the following examples are shown in Table 1.

[0074] Table 1 Reagents and their manufacturers

[0075]

[0076] Example 1 Screening and Optimization of Zhongshan Disease Virus VP3 Truncated Sequences

[0077] The amino acid sequence of Zhongshan disease virus VP3 is based on the data published by NCBI (Accession No.: BAA34936), with a total of 904 amino acids, and the specific sequence information is SEQ ID No. 1. The corresponding nucleotide sequence is AB014728, with a total of 2774bp, and the specific sequence information is SEQ ID No. 2. Analysis of the VP3 amino acid sequence shows that the overall hydrophobicity of the VP3 protein is very high, and it is difficult to achieve full-length soluble expression and the probability of successful expression is low. However, a region with high hydrophilicity was obtained after screening (see Figure 1 ), whose amino acid sequence is SEQ ID No. 3, consisting of 108 amino acids and a molecular weight of approximately 12.5 kDa, and designated VP3s. Further redesign and optimization of the nucleotide sequence resulted in the nucleotide sequence shown in SEQ ID No. 4. This nucleotide sequence was synthesized at Sangon Biotech (Shanghai) Co., Ltd. and used for subsequent cloning and recombinant expression.

[0078] Example 2 Expression, purification and activity testing of the truncated VP3s of the Zhongshan disease virus VP3 protein

[0079] The synthesized sequence SEQ ID No. 4 was constructed into the pET28a(+) expression vector and transformed into BL21(DE3) competent cells after identification. The prokaryotic expression of the truncated protein was attempted to obtain the soluble expressed target protein. After the protein was purified by Ni-NTA affinity chromatography, the recombinantly expressed Zhongshan disease virus truncated VP3s protein was finally obtained. The electrophoresis results of VP3s protein are as follows Figure 2 As shown, the amount of protein is very small, with a target band at approximately 15 kD, and both purity and expression level are high.

[0080] The purified recombinant VP3s protein was quickly verified for antigenic activity using the ELISA method. The purified VP3s protein was coated at 1 μg / mL, 100 μL / well, coated overnight at 4°C, blocked with 1% BSA at 37°C for 2 h, washed three times with 250 μL / well PBST solution (0.1%), and 100 μL / well of 100-fold diluted CHUV clinical positive serum (or clinical negative serum) was added. Each serum was repeated three times, incubated at 37°C for 30 min, washed three times with 250 μL / well PBST solution (0.1%), patted dry, added with 100 μL / well of rabbit anti-bovine IgG-HRP secondary antibody (diluted 1:10000 in PBS), reacted at 37°C for 30 min, washed again three times, patted dry, added with 100 μL / well of TMB color development solution, developed at 25°C for 10 min, and finally added with 50 μL / well of stop solution to terminate the reaction. The OD450 nm value was measured using a microplate reader. The ratio of the OD values ​​of positive and negative serum is shown in Table 2. The larger the ratio, the more obvious the antigen positivity. The expressed truncated protein VP3s of Zhongshan disease virus reacted with clinical positive serum but had no significant reaction with clinical negative serum, thus having good antigenic activity.

[0081] Table 2 Antigen activity verification

[0082]

[0083] Note: P: positive serum OD value; N: negative serum OD value; P / N: ratio of positive serum OD value to negative serum OD value.

[0084] Example 3 Establishment of indirect ELISA detection method

[0085] 1. Determination of the optimal antigen coating concentration and serum dilution

[0086] Using the checkerboard method, the coating antigen (truncated VP3s) was diluted to 2, 1.5, 1, 0.75, 0.5, and 0.25 μg / mL using coating buffer (0.05 M bicarbonate, pH 9.6). The plate was then coated onto an ELISA plate at 100 μL per well and incubated at 4°C for 16 hours. After blocking with 1% BSA (w / v), Zhongshan disease virus-positive and -negative sera were diluted 1:50, 1:100, 1:200, and 1:400, respectively. Rabbit anti-bovine IgG-HRP was diluted 1:10,000 and added to each well at 100 μL. TMB single-component colorimetric solution was then added to each well, and color was developed at 25°C for 10 minutes. The reaction was terminated by adding 50 μL of stop solution to each well. The absorbance at 450 nm in each well was measured using a microplate reader, and the P / N ratio was calculated. Thus, the optimal antigen coating concentration and serum dilution were determined.

[0087] The results showed that the optimal coating concentration of the antigen was 1 μg / mL and the optimal serum dilution concentration was 1:200. The results are shown in Table 3.

[0088] Table 3 Optimal antigen coating concentration and serum dilution concentration

[0089]

[0090]

[0091] 2. Determination of critical value

[0092] According to the optimal reaction conditions measured above, 40 negative sera were tested, and the absorbance at a wavelength of 450 nm in each well was detected using an enzyme-labeled instrument. According to the principle of statistical analysis, the positive value was determined to be OD 450 ≥ Mean value + 3 × standard deviation, calculate the critical value of this detection method.

[0093] According to the measured data, the average value is 0.154 and the standard deviation is 0.014. 450 When OD ≥ 0.196, it was determined to be positive; 450 <0.168, it is judged as negative, and when 0.168≤OD 450 When the value is less than 0.196, it is judged as suspicious.

[0094] Example 4 Clinical Validation of the Indirect ELISA Detection Kit for Zhongshan Disease Virus Antibodies

[0095] 1. Specificity test

[0096] The Zhongshan Disease Virus Antibody Level Detection Kit was used to test serum positive for standard antibodies to bovine viral diarrhea virus (BVDV), bovine Brucella (Bru), and bovine rhinotracheitis virus (IBR), as well as serum positive for CHUV virus antibodies. The results are shown in Table 4. With the exception of the CHUV-positive serum, which had a P / N value greater than 3, the P / N values ​​for all other sera were less than 2. This met the criteria for negative sera, demonstrating the good specificity of this method.

[0097] Table 4 Specific serum detection

[0098]

[0099] 2. Sensitivity test

[0100] Three CHUV virus antibody-positive sera and three CHUV virus antibody-negative sera were diluted at various dilutions, ranging from 1:200 to 1:6400. The sensitivity of the indirect ELISA assay was verified. Results showed that positive results were still detected after a dilution of 1:3200, as shown in Table 5. This demonstrates the good sensitivity of this assay.

[0101] Table 5 Sensitivity of indirect ELISA method

[0102]

[0103] 3. Intra-batch and inter-batch repeatability testing

[0104] Under the same reaction conditions as above, three CHUV virus antibody positive sera (P) and three negative sera (N) were randomly selected for detection on the same ELISA coated plate, and three replicates were performed for each serum to perform inter-batch reproducibility test; similarly, three positive sera and three negative sera were randomly selected for ELISA detection on ELISA enzyme-labeled plates coated at three different time points, and the test results were analyzed and the OD was calculated. 450nm The mean value, standard deviation and coefficient of variation were used to verify the repeatability of the method. The results are shown in Table 6. It can be seen that the coefficient of variation is within 8%, indicating that the detection method has good repeatability.

[0105] Table 6 Intra-batch and inter-batch repeatability tests

[0106]

[0107] Comparative Example 1 Comparison between the method of the present invention and the C-ELISA method

[0108] 43 clinical samples were randomly selected, and the detection results were compared using the indirect ELISA method established by the present invention and the C-ELISA method.

[0109] The C-ELISA method uses purified virus as the antigen and coats the ELISA plate at a concentration of 4.12 μg / mL, with 50 μL / well. A rabbit anti-CHUV polyclonal antibody is used as the competing antibody, with an optimal dilution ratio of 1:12,000. When using the competitive ELISA method to detect samples, an inhibition rate ≥50% is considered positive; an inhibition rate between 40% and 50% is considered suspect; and an inhibition rate <40% is considered negative.

[0110] The results are shown in Table 7. It can be seen that the positive coincidence rate of the two methods is 120%, and the positive detection rate of this method is higher. The negative coincidence rate of the two methods is 100%. The overall coincidence rate is 97.7%.

[0111] Table 7 Comparison of the compliance rate with C-ELISA method

[0112]

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A truncated protein VP3s of Zhongshan disease virus, characterized in that Its nucleotide sequence is shown in SEQ ID No.

4.

2. The method for preparing the truncated protein VP3s of Zhongshan disease virus according to claim 1, characterized in that: The steps include: (1) The gene encoding the truncated protein VP3s was constructed into the pET-28a(+) expression vector; (2) Transform the expression vector identified correctly in step (1) into BL21 (DE3) competent cells for culture and expression; (3) Purify the target protein expressed in step (2) by immobilized metal affinity chromatography and remove the tag.

3. A method for detecting antibodies to Zhongshan disease virus for non-diagnostic purposes, characterized in that: The sample to be tested is detected using the truncated protein VP3s described in any one of claims 1-2.

4. The method for detecting antibodies to Zhongshan disease virus for non-diagnostic purposes according to claim 3, characterized in that: The specific steps are: S1, antigen coating using truncated protein VP3s as coating antigen; S2: Block, wash the plate, and then add the diluted serum sample to be tested for incubation; S3, after washing the plate, add enzyme-labeled secondary antibody for incubation; S4, after washing the plate, add TMB color developing solution for color development; S5. After color development, add stop solution and measure OD 450nm The absorbance value of S6. Result judgment: When OD 450 When OD ≥ 0.196, it was determined to be positive; 450 <0.168, it is judged as negative, and when 0.168≤OD 450 When the value is less than 0.196, it is judged as suspicious.

5. The method for detecting antibodies to Zhongshan disease virus for non-diagnostic purposes according to claim 4, characterized in that: In the above steps, the coating solution is 0.05M carbonate buffer, the blocking solution is a PBS solution containing 1% BSA (w / v), the washing solution is PBST, the diluent is a 1% BSA (w / v) PBS solution, the enzyme-labeled secondary antibody is rabbit anti-bovine IgG-HRP, and the stop solution is a commercial stop solution; preferably, the washing solution contains 0.25% v / v Tween-20; and the pH of the coating solution is 9.

6.

6. An indirect ELISA kit for detecting antibodies to Zhongshan disease virus, characterized in that: The invention comprises an enzyme-labeled plate coated with the truncated protein VP3s according to any one of claims 1 to 2, an enzyme-labeled secondary antibody, a washing solution, a diluent, a coating solution, a blocking solution, a color developing solution, a stop solution, a negative control, a positive control and a result judgment standard.

7. The indirect ELISA kit for detecting Zhongshan disease virus antibodies according to claim 6, characterized in that: The steps for preparing the ELISA plate coated with the truncated protein VP3s of the Zhongshan disease virus are as follows: A1. Coat the truncated protein VP3s of Zhongshan disease virus onto the ELISA plate at 4°C overnight. A2. Add 1% BSA blocking solution and block at 37°C for 2 hours; A3. Add PBST washing solution and wash the plate three times; A4. Add diluted Zhongshan disease virus-positive serum and incubate at 37°C for 30 minutes; A5. After taking out, shake out the liquid in the wells, add PBST washing solution and wash the plate three times, pat dry the droplets in the wells, and air-dry in a 37°C incubator to obtain the ELISA plate coated with the truncated protein VP3s of Zhongshan disease virus.

8. The preparation method according to claim 7, characterized in that The concentration of the truncated protein VP3s of Zhongshan disease virus coated in each well in step A1 is 1 μg / mL.

9. The preparation method according to claim 7, characterized in that The positive serum diluted in step A4 is 1:

200.

10. Use of the truncated protein VP3s of Zhongshan disease virus according to claim 1 or the truncated protein VP3s of Zhongshan disease virus obtained by the preparation method according to claim 2 in the following B1 to B5: B1. Preparation of Zhongshan disease virus vaccine; B2. Preparation of antibodies against Zhongshan disease virus; B3. Preparation of diagnostic antigen for Zhongshan disease virus; B4. Preparing reagents and / or kits for detecting Zhongshan disease virus; B5. Prepare reagents and / or kits for detecting antibodies to Zhongshan disease virus.

Images