An indirect ELISA kit for detecting Tembusu virus antibodies and application of Tembusu virus E protein domain III

Abstract

The present invention provides an indirect ELISA kit for detecting Tembusu virus antibodies and the application of Tembusu virus E protein domain III, belonging to the field of biological detection technology. The application of Tembusu virus E protein domain III in the preparation of a reagent or kit for detecting Tembusu virus antibodies. The present invention also provides an indirect ELISA kit for detecting Tembusu virus antibodies, comprising a detection plate coated with Tembusu virus E protein domain III and an enzyme-labeled secondary antibody. The kit has good detection specificity and provides technical support for epidemiological surveys, immune effect evaluation, and epidemic prevention and control of Tembusu virus.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to an indirect ELISA kit for detecting Tembusu virus antibodies and application of Tembusu virus E protein domain III. Background Art

[0002] Tembusu virus (TMUV) is a single-stranded positive-strand RNA virus of the genus Flaviviridae, with a genome size of about 11kb, only one open reading frame, and a polyprotein is translated. The polyprotein can be processed into 3 structural proteins (capsid protein C, outer membrane protein PrM / M and envelope protein E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) by the protease of the virus and the host. Wherein capsid protein C is related to replication, and the prior art reports that the replication ability of the strain is weakened by deleting the partial sequence of C protein, and the virulence is reduced (CN113234693A). Envelope protein E is not only the main structural protein of Tembusu virus, but also the main antigenic component, which plays an important role in virus adsorption, penetration, pathogenicity, tissue tropism, hemagglutination, serum specificity and inducing the immune response of the host. There are highly conserved regions and variable regions in the E protein sequence, and all cysteine ​​residues are highly conserved regions. The E protein forms three different domains in its three-dimensional structure: domain I, domain II, and domain III.

[0003] Detection of Tembusu virus antibodies can not only evaluate the vaccine immune effect, but also assist clinical diagnosis, which is a prerequisite for effectively controlling Tembusu virus infection. The ELISA method has the characteristics of easy operation, rapid, high sensitivity, easy to promote at the grassroots level, and can be used in large quantities for serological detection, and has become the most widely used serological detection method. For example, the patent of CN103063854A discloses a duck Tembusu virus antibody solid phase competition ELISA detection kit, which is coated on an ELISA plate with inactivated duck Tembusu virus, and also includes rabbit anti-duck Tembusu virus positive serum and duck anti-duck Tembusu virus positive control serum. Although the kit can eliminate the species source difference of serum samples, the preparation of viral antigens is obtained through cell passage culture, and there is a great risk of virus leakage or contamination. In addition, the patent with publication number CN109580941A discloses an indirect ELISA method for detecting duck Tembusu virus antibodies, which is coated with NS1 protein as an antigen, and the IFA compliance rate is high in the test results, and the test results have good repeatability, but the kit has some false positive results, which is difficult to meet accurate requirements. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a use of Tembusu virus E protein domain III in the preparation of a reagent or kit for detecting Tembusu virus antibodies, so that the prepared reagent or kit has no cross reaction and has good detection specificity.

[0005] The present invention provides the use of Tembusu virus E protein domain III in preparing a reagent or a kit for detecting Tembusu virus antibodies.

[0006] Preferably, the amino acid sequence of domain III of the Tembusu virus E protein is as shown in SEQ ID NO:1.

[0007] Preferably, the kit is prepared using the following immunoassay techniques: enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, immunochromatography, immunoblotting and immunoprecipitation.

[0008] The invention provides an indirect ELISA kit for detecting Tembusu virus antibodies, comprising a detection plate coated with Tembusu virus E protein domain III and an enzyme-labeled secondary antibody.

[0009] Preferably, the amino acid sequence of domain III of the Tembusu virus E protein is as shown in SEQ ID NO:1.

[0010] Preferably, the coating concentration of the Tembusu virus E protein domain III is 0.8-1.2 μg / ml.

[0011] Preferably, the coating condition of the Tembusu virus E protein domain III is 3-5° C. overnight.

[0012] Preferably, when preparing the test plate coated with Tembusu virus E protein domain III, the Tembusu virus E protein domain III is coated on the test plate and then blocked;

[0013] The blocking reagent is a 0.01M pH 7.4 PBS buffer solution containing BSA at a mass concentration of 0.8% to 1.2%.

[0014] Preferably, the sealing time is 110 to 130 minutes.

[0015] Preferably, the method further comprises at least one of the following reagents: a sample diluent, a washing solution, a stop solution and a color developing solution.

[0016] The present invention provides an application of Tembusu virus E protein domain III in the preparation of a reagent or kit for detecting Tembusu virus antibodies. The present invention prepares a recombinant protein of Tembusu virus E protein domain III by means of recombinant expression, coats the recombinant protein in a test plate, and uses the specific binding of antigen and antibody to detect Tembusu virus positive serum and positive serum of common viruses that infect poultry. The results show that the recombinant protein has no cross reaction with duck parvovirus, new duck reovirus, and duck plague virus positive serum, and only detects positive for Tembusu virus positive control serum. This shows that the Tembusu virus E protein domain III can be used for the detection of Tembusu virus antibodies, and the detection results are accurate and reliable.

[0017] The present invention provides an indirect ELISA kit for detecting antibodies to Tembusu virus, comprising a detection plate coated with domain III of the E protein of Tembusu virus and an enzyme-labeled secondary antibody. The present invention uses the indirect ELISA kit to detect the serum of ducks infected with Tembusu virus, with the indirect immunofluorescence detection method as a reference. The results show that the S / P values ​​of 22 sera detected by the kit are all >0.14, and the test results are positive, which is 100% consistent with the results of the indirect immunofluorescence test. It can be seen that the kit provided by the present invention provides technical support for epidemiological surveys, immune effect evaluation, and disease prevention and control of Tembusu virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the SDS-PAGA analysis chart of the Tembusu virus E protein domain III recombinant protein (TMUV-EIII protein); wherein, M: protein marker; 1: TMUV-EIII protein with a concentration of 1.353 mg / ml; 2: TMUV-EIII protein with a concentration of 1.589 mg / ml; 3: TMUV-EIII protein with a concentration of 1.316 mg / ml.

[0019] Biomaterial Deposit Information

[0020] Tembusu virus Goose / Guangdong / DT01 / 2020 strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on May 10, 2024. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the biological deposit number is CGMCC No.45979. DETAILED DESCRIPTION

[0021] The present invention provides the use of Tembusu virus E protein domain III in preparing a reagent or a kit for detecting Tembusu virus antibodies.

[0022] In the present invention, the amino acid sequence of the Tembusu virus E protein domain III is preferably as shown in SEQ ID NO: 1 (KGMTYPMCSNTFSLAKNPTDTGHGTVVVELSYAGTDGPCRVPISMSADLNDMTPVGRLITVNPYVSTSSTGAKIMVEVEPPFGDSFILVGSGKEQIRYQWHRSGSTIGK), with a length of 109 amino acids. The preparation method of the Tembusu virus E protein domain III is preferably obtained by expressing it in a prokaryotic expression system. In an embodiment of the present invention, recombinant expression is obtained using Escherichia coli as a host bacterium. The specific method preferably constructs a recombinant prokaryotic expression vector containing the Tembusu virus E protein domain III, transforms the recombinant prokaryotic expression vector into an Escherichia coli competent state, cultures and induces expression, separates the protein and purifies it to obtain the target recombinant protein. The nucleotide sequence of domain III of Tembusu virus E protein is preferably as shown in SEQ ID NO: 2 (AAAGGAATGACCTACCCGATGTGTAGCAACACATTTTCTCTAGCGAAGAATCCTACTGACACTGGGCATGGCACTGTTGTGGTGGAATTGTCTTATGCAGGTACTGATGGGCCCTGTAGAGTTCCCATATCCATGTCGGCAGATCTGAATGACATGACACCAGTTGGACGCTTGATAACAGTCAACCCATACGTGTCGACCTCCTCCACGGGTGCCAAGATAATGGTGGAAGTGGAACCTCCATTCGGGGATTCATTCATCTTAGTAGGAAGTGGAAAAGAACAGATCAGGTACCAGTGGCACAGAAGTGGGAGCACAATTGGAAAA). The method for constructing a recombinant prokaryotic expression vector comprising the E protein domain III of the Tembusu virus preferably reverse transcribes the viral RNA of the Tembusu virus Goose / Guangdong / DT01 / 2020 strain to obtain cDNA, and uses the cDNA as a template to perform PCR amplification using a primer pair, and the obtained PCR amplification product is cloned into a prokaryotic expression vector, and a recombinant prokaryotic expression vector is obtained after verification. The primer pair preferably includes a forward primer having a nucleotide sequence such as SEQ ID NO: 3 (ctcgagtgcggccgcaagcttATGAAAGGAATGACCTACCCGA, lowercase letters are homologous recombination arm sequences) and a reverse primer having a nucleotide sequence such as SEQ ID NO: 4 (tcgagctccgtcgacaagcttTTATTTTCCAATTGTGCTCCCA, lowercase letters are homologous recombination arm sequences).The reaction procedure of the PCR amplification is preferably: pre-denaturation at 92°C for 2min; in the third stage, PCR amplification at 94°C for 30s, 58°C for 30s, 72°C for 1min, for a total of 30 cycles, and finally extension at 72°C for 5min. After obtaining the PCR amplification product, it is preferably included to purify it. The purification method is preferably completed using a DNA fragment product purification kit. The backbone vector of the recombinant prokaryotic expression vector is preferably pET32a. The cloning method is preferably completed by homologous recombination method. The verification method is preferably to transform the homologous recombination reaction product into Escherichia coli competent state, amplify the colony PCR, and extract the plasmid from the colony where the target fragment is obtained. The method of the transformation of the present invention is preferably calcium chloride transformation method. The Escherichia coli competent state is preferably Escherichia coli BL21 (DE3) competent cells. The induced expression is preferably induced expression for 8h at a final concentration of 0.5mM IPTG. After the induction expression is completed, the bacteria are separated, broken, and the protein is separated. The purification method is preferably Ni-NTA affinity chromatography column purification. After SDS-PAGE detection, the purified protein was a protein of the expected target size, and sequencing results showed that the purified protein was a recombinant protein of the Tembusu virus E protein domain III.

[0023] In the present invention, the recombinant protein of the E protein domain III of the Tembusu virus is coated in an ELISA plate, and the positive serum of the Tembusu virus and the positive serum of pathogens such as duck parvovirus, new duck reovirus, and duck plague virus are detected by an indirect ELISA detection method. The results show that only the positive serum of the Tembusu virus is positive, while the positive serum of pathogens such as duck parvovirus, new duck reovirus, and duck plague virus is negative. It can be seen that the recombinant protein of the E protein domain III of the Tembusu virus only binds to the Tembusu virus antibody and has good specificity. Therefore, the kit is preferably prepared by the following immunoassay techniques: enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, immunochromatography, immunoblotting, and immunoprecipitation. The present invention does not place any special restrictions on the preparation method of the kit, and the preparation method known in the art can be used. The present invention does not place any special restrictions on the selection of other reagents of the kit, and the reagents involved in each detection technique known in the art can be used.

[0024] The invention provides an indirect ELISA kit for detecting Tembusu virus antibodies, comprising a detection plate coated with Tembusu virus E protein domain III and an enzyme-labeled secondary antibody.

[0025] In the present invention, the amino acid sequence of the Tembusu virus E protein domain III is preferably as shown in SEQ ID NO: 1. The preparation method of the Tembusu virus E protein domain III is the same as the above technical solution, which will not be described in detail here.

[0026] In the present invention, the coating concentration of the Tembusu virus E protein domain III is preferably 0.8-1.2 μg / ml, more preferably 1.0 μg / ml. The coating solution of the Tembusu virus E protein domain III is preferably 0.05M pH 9.6 carbonate buffer. In the embodiment of the present invention, a screening experiment of antigen coating concentration and serum dilution was carried out. The experiment showed that when the antigen coating concentration was 1.0 μg / ml and the antibody dilution was 1:1000, the OD of the positive serum was 450nm The value was close to 1.0, and the P / N value was the largest, while the detection effect was poor at the coating concentrations of 0.5 μg / ml, 2.0 μg / ml, and 4.0 μg / ml and the combinations of serum dilutions of 1:200, 1:500, and 1:2000.

[0027] In the present invention, the coating condition of the Tembusu virus E protein domain III is preferably 3-5°C overnight coating, more preferably 4°C overnight coating. In order to better improve the stability of the coated antigen, the optimization of the antigen coating conditions was also carried out, specifically, three coating conditions were set, and the results showed that the P / N value of the 4°C overnight coating condition was the highest, while the detection effect of the 4°C overnight coating condition after 37°C treatment for 1h and the 4°C overnight coating condition after 37°C coating for 5h was not good.

[0028] In the present invention, when preparing the test plate coated with the Tembusu virus E protein domain III, it is preferred to coat the Tembusu virus E protein domain III on the test plate and then block it. The blocking reagent is preferably a 0.01M pH 7.4 PBS buffer containing a mass concentration of 0.8% to 1.2% BSA, more preferably a 0.01M pH7.4 PBS buffer containing 1.0% BSA. The blocking temperature is preferably 36 to 37°C, more preferably 37°C. The blocking time is preferably 110 to 130min, more preferably 120min. In an embodiment of the present invention, in order to improve the detection sensitivity, the type and blocking conditions of the blocking solution are also optimized. Experiments show that compared with 3% gelatin solution, 5% FBS solution and 5% skim milk, when blocked with 1% BSA solution, the P / N value is the largest, so 1% BSA solution is selected as the best blocking solution. The blocking time is set to 30min, 60min, 90min and 120min, respectively, and the results show that the optimal blocking time is 120min.

[0029] In the present invention, the enzyme-labeled secondary antibody is preferably a goat anti-duck IgG (H+L) enzyme-labeled secondary antibody. In the embodiment of the present invention, the enzyme-labeled secondary antibody is purchased from KPL Company. The working concentration of the enzyme-labeled secondary antibody is preferably 1:2000.

[0030] In the present invention, the kit preferably further comprises at least one of the following reagents: a sample diluent, a washing solution, a stop solution and a color developing solution. The sample diluent is preferably a PBS buffer with a concentration of 0.01M pH 7.4. The sample diluent is used to dilute a sample or an antigen. The washing solution is preferably PBST, specifically a PBS buffer with a concentration of 0.01M and a pH of 7.2 to 7.4 containing 0.05% Tween. The stop solution is preferably a strong acid or strong base solution, such as a 2M sulfuric acid solution or a 2M sodium hydroxide solution. The color developing solution is a substrate for an enzyme, such as TMB, which is prepared by mixing equal volumes of substrate diluent A and substrate diluent B, and is specifically purchased from Wuhan Huamei Bioengineering Co., Ltd. The color developing solution is used to generate an enzymatic hydrolysis signal.

[0031] In the present invention, the detection method of the indirect ELISA kit for detecting Tembusu virus antibodies preferably comprises the following steps:

[0032] The diluted serum is added to a test plate coated with Tembusu virus E protein domain III, incubated, washed, an enzyme-labeled secondary antibody is added to the test plate, incubated, washed, a color developer is added to the washed test plate for color development, incubated, a stop solution is added, a signal is detected, a test result is obtained, and whether the sample is a positive sample is determined based on the test result.

[0033] In the present invention, the source of the serum is preferably poultry serum. The types of poultry preferably include Mallard, Cherry Valley Duck, Muscovy Duck, Muscovy Duck, etc. In the embodiment of the present invention, Mallard is used as a representative to illustrate the detection effect of the indirect ELISA kit. The dilution of the serum is preferably 1:800-1200, and the most preferred is 1:1000. The amount of serum added is preferably 100 μL / well. The incubation time of the serum is preferably 55-65 min, and the most preferred is 60 min. The number of washes is preferably 2-3 times. The incubation time of the enzyme-labeled secondary antibody is preferably 35-45 min, and the most preferred is 40 min. The color development time is preferably 18-22 min, and the most preferred is 20 min. The detection signal preferably detects OD 450nm The absorbance value.

[0034] In the present invention, the judgment method is to determine the critical value of the Tembusu virus indirect ELISA antibody detection method as 0.14, that is, S / P ≥ 0.14 is judged as positive, and S / P < 0.14 is judged as negative. The S / P value = (clinical sample OD 450nm Value - negative control serum OD 450nm Mean) / (positive control serum OD 450nm Mean - negative control serum OD 450nm mean).

[0035] In the present embodiment, the Tembusu virus DT01 strain was used as 10 6.0 TCID 50 Duck serum collected on the 3rd, 5th and 7th days after 14-day-old healthy Tembusu virus antibody-negative ducks were infected with 0.1 ml / 0.1 ml was used as the detection object, and the indirect ELISA kit constructed by the present invention was used for detection. At the same time, an indirect immunofluorescence detection kit was used as a control. The results showed that the S / P values ​​of the 22 sera detected by the kit were all 0.14, which was 100% consistent with the indirect immunofluorescence test results. This shows that the detection results of the indirect ELISA kit developed by the present invention are accurate and reliable. The indirect ELISA kit has good detection sensitivity, and the detection serum dilution is 1:12800.

[0036] The present invention provides an indirect ELISA kit for detecting Tembusu virus antibodies and the application of Tembusu virus E protein domain III provided by the present invention in detail in combination with the embodiments below, but they should not be understood as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] Prokaryotic expression and purification of domain III recombinant protein of Tembusu virus E protein

[0039] 1. Isolation and identification of Tembusu virus strain Goose / Guangdong / DT01 / 2020

[0040] The livers of sick geese from a farm in Guangdong Province were collected and ground. The ground liver slurry was inoculated into duck embryos through the allantoic cavity route. After the primary duck embryo allantoic fluid was harvested, a strain was isolated and named Goose / Guangdong / DT01 / 2020. Hemagglutination test was performed on it according to Appendix 3403 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China. PCR (references NY / T560-2018, SNT2744-2010, DB35 / T 1822-2019, DB 34 / T 3653-2020, DB34T3401-2019) and RT-PCR (references NY / T3233-2018, NYT772-2013, GBT16550-2020, DB37 / T 3821-2019, DB34 / T 3660-2020) was tested and preliminarily identified as Tembusu virus. The isolated virus was purified for 5 generations using duck embryos by limiting dilution method. The purified virus was tested for purity according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China, and the results confirmed that the virus was only Tembusu virus.

[0041] The Goose / Guangdong / DT01 / 2020 strain is deposited in a depository institution with the deposit number CGMCCNo.45979.

[0042] 2. Primer Design and Synthesis

[0043] According to the gene sequence information of the Tembusu virus Goose / Guangdong / DT01 / 2020 strain, primers for the pET32a-TMUV-EIII plasmid were designed and constructed, including the upstream primer TMUV-EIII-F: 5'-ctcgagtgcggccgcaagcttATGAAAGGAATGACCTACCCGA-3'; the downstream primer TMUV-EIII-R: 5'-tcgagctccgtcgacaagcttTTATTTTCCAATTGTGCTCCCA-3'; the italic part is the homologous recombination arm sequence. The primers were synthesized by Beijing Liuhe BGI Technology Co., Ltd.

[0044] 3. Amplification of EIII gene

[0045] The viral RNA of the Tembusu virus Goose / Guangdong / DT01 / 2020 strain (deposit number CGMCC No. 45979) was extracted according to the instructions of TaKaRa MiniBEST ViraL RNA / DNAExtractionKitVer.5.0 (TaKaRa, Code NO.9766), and the RT-PCR reaction was performed according to the instructions of the one-step RT-PCR kit PrimeScript TM One Step RT-PCRKitVer.2 (Dye PLus) (TaKara, Code No.RR057A). The RT-PCR reaction procedure is as follows: in the first stage, reverse transcription at 50°C for 30 min; in the second stage, pre-denaturation at 94°C for 2 min; in the third stage, PCR amplification at 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min, for a total of 30 cycles, and finally extension at 72°C for 5 min. The target fragment was recovered from agarose gel, purified using a DNA product purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number: DP204), and prepared for ligation with the pET32a plasmid.

[0046] 4. Construction, expression and purification of prokaryotic expression vector

[0047] The pET32a plasmid was linearized by HindIII endonuclease, and the purified target fragment was homologously recombined with the linearized pET32a plasmid using the CLonExpress ULtra One Step CLoning Kit (Novozyme Biotech Co., Ltd., Catalog No.: C115-01), and the expression vector pET32a-TMUV-EIII was successfully constructed. The expression vector pET32a-TMUV-EIII was transformed into Escherichia coli BL21 (DE3) competent cells to obtain the Escherichia coli BL-TMUV-EIII strain, which was induced by IPTG (0.5mM) for 8h, and the expression product was centrifuged at 4℃, 6000r / min for 10min to collect the cells, and the supernatant was discarded. After a series of pre-treatments such as resuspension with lysis buffer (containing 0.25mg / mL lysozyme), ultrasonic disruption, and urea washing, the purified TMUV-EIII protein was obtained by Ni-NTA affinity chromatography column purification, and the purified TMUV-EIII protein was packaged at -80℃ for use.

[0048] Example 2

[0049] Preparation of positive and negative control serum

[0050] 1. Preparation of duck positive control serum against Tembusu virus

[0051] The virus content was 10 6.0 TCID 50 / 0.1ml of Tembusu virus Goose / Guangdong / DT01 / 2020 strain (DT01 strain for short) was inactivated with 0.2% formaldehyde solution at 37°C for 24 hours, and then the oil phase (6 parts of Siben-80 were added to 94 parts of white oil for injection, mixed well, and sterilized under high pressure) and the water phase were mixed and emulsified in a volume ratio of 3:2 to prepare the Tembusu virus inactivated vaccine.

[0052] Ten 14-day-old healthy ducks with negative Tembusu virus antibodies were immunized by intramuscular injection, 1 ml per duck. 21 days later, the second immunization was performed in the same way as the first immunization. 42 days after the first immunization, a booster immunization was performed. 14 days after the booster immunization, the serum of the infected ducks was collected, and the serum that was detected as TMUV antibody positive by indirect immunofluorescence test was mixed, filtered with a filter membrane with a pore size of 0.22 μm, and then stored at -20°C as a positive control serum for ducks against Tembusu virus.

[0053] The specific method of indirect immunofluorescence test is as follows:

[0054] (1) Virus inoculation: Take the Tembusu virus DT01 strain, dilute it 1000 times with DMEM / F12 medium, and inoculate it into a 48-well cell plate filled with DF1 cells. Inoculate 0.1 mL (containing 103.0 TCID 50 ), and a blank cell control was set up without inoculation. After being cultured in a 37°C, 5% CO2 incubator for 72 hours, the cells were taken out for indirect immunofluorescence experiment.

[0055] (2) Indirect immunofluorescence test: Take out the cultured 48-well cell plate and discard the liquid, wash it once with PBS (pH 7.2-7.4, the same below), discard the liquid, add 300 μl of cold methanol to each well, let it stand at room temperature for 15 min, discard the liquid, dry it, wash it once with PBS, discard the liquid, add 100 μl of the original or appropriately diluted serum to be tested to each well, incubate it at 37°C for 1 h, remove and discard the liquid, wash it 3 times with PBS containing 0.05% Tween-20, then wash it 2 times with PBS, and gently shake and wash it for 1 min between each wash. After washing, add 100 μl of rabbit anti-duck IgY-FITC secondary antibody (Beijing Boerxi Technology Co., Ltd., catalog number: B230217) to each well, incubate it at 37°C for 1 h, remove and discard the liquid, wash it 3 times with PBS containing 0.05% Tween-20, then wash it 2 times with PBS, and gently shake and wash it for 1 min between each wash. After cleaning, place it under an inverted microscope for observation and photography.

[0056] 2. Preparation of Tembusu virus duck negative control serum Serum was collected from 70-day-old healthy Tembusu virus antibody-negative ducks, filtered through a filter with a pore size of 0.22 μm, and stored in aliquots at -20°C as Tembusu virus duck negative control serum.

[0057] Example 3

[0058] Establishment of TMUV-EIII-ELISA detection method

[0059] 1. Determination of the optimal working concentration of antigen and antibody

[0060] The purified TMUV-EIII recombinant protein was diluted with coating solution to concentrations of 0.5μg / ml, 1.0μg / ml, 2.0μg / ml, and 4.0μg / ml, and the diluted protein was added to the ELISA plate from left to right, 100μl / well, and incubated overnight at 4°C; the next day, it was washed three times with PBST; and blocked with PBS buffer containing 1% BSA at 37°C for 2 h, 200μl / well. The standard positive and negative serum was diluted in a gradient of 1:200, 1:500, 1:1000, and 1:2000, 100 μl / well, added to the ELISA plate from top to bottom, incubated at 37°C for 1 h, washed 3 times with PBST, and patted dry for the last time; 1:2000 diluted enzyme-labeled secondary antibody was added, incubated at 37°C for 40 min, washed 5 times with PBST, and patted dry for the last time; 100 μl TMB colorimetric solution was added to each well, incubated at 37°C in the dark for 20 min, 50 μl stop solution was added to terminate the reaction, and OD was immediately measured using an ELISA reader. 450nm Value. According to OD 450nm The optimal working concentration of antigen and antibody was determined by the P / N value.

[0061] The results showed that when the antigen coating concentration was 1.0 μg / ml and the antibody dilution was 1:1000, the OD of the positive serum was 450nm The value is close to 1.0, and the P / N value is the largest. Therefore, the optimal coating concentration of ELISA antigen is determined to be 1.0 μg / ml, and the optimal serum dilution is 1:1000. The results are shown in Table 1.

[0062] Table 1 Optimal working concentration test results of antigen and antibody (OD 450nm value)

[0063]

[0064]

[0065] 2. Determination of the optimal dilution of enzyme-labeled secondary antibody

[0066] The ELISA plate was coated with the antigen at the optimal concentration, the positive and negative serum was diluted at the optimal dilution, and the enzyme-labeled secondary antibody was diluted to 1:500, 1:1000, 1:2000, and 1:4000. Each dilution was repeated 3 times. The OD 450nm The optimal working concentration of the enzyme-labeled secondary antibody was determined by the PCR product value and P / N value.

[0067] The results showed that when the concentration of enzyme-labeled secondary antibody was 1:2000, the P / N value was the largest. The results are shown in Table 2.

[0068] Table 2 Optimal dilution test results of enzyme-labeled secondary antibody (OD 450nm value)

[0069]

[0070] 3. Determination of the optimal coating time

[0071] Coat the ELISA plate with the optimal concentration of antigen and set three coating conditions:

[0072] (1) Incubate at 37°C for 1 h and coat at 4°C overnight;

[0073] (2) Incubate at 37°C for 5 h and coat at 4°C overnight;

[0074] (3) Coating was performed overnight at 4°C. Each coating condition was repeated three times.

[0075] Dilute the positive and negative serum and enzyme-labeled secondary antibody at the optimal dilution and measure the OD value. 450nm The optimal coating time of the antigen was determined by the value and P / N value.

[0076] The results showed that the best coating condition was coating at 4°C overnight. The results are shown in Table 3.

[0077] Table 3 Optimal coating conditions test results (OD 450nm value)

[0078]

[0079] 4. Selection of the best blocking solution

[0080] The ELISA plate was coated with the optimal concentration of antigen at 4°C overnight. After washing, 3% gelatin in PBS, 5% FBS in PBS, 5% skim milk in PBS, and 1% BSA in PBS were used as blocking solutions. Three replicates were made for each condition. The negative and positive sera and enzyme-labeled secondary antibodies were diluted at the optimal dilution and the OD values ​​were calculated. 450nm The results showed that when 1% BSA was used for blocking, the P / N value was the largest, so 1% BSA in PBS was selected as the best blocking solution. The results are shown in Table 4.

[0081] Table 4 Determination of the best blocking solution (OD 450nm value)

[0082]

[0083] 5. Determination of closure time

[0084] The ELISA plate was coated with the optimal concentration of antigen at 4°C overnight, washed, and blocked with PBS buffer containing 1% BSA for 30 min, 60 min, 90 min, and 120 min, respectively. Each condition was repeated 3 times. 450nm The optimal blocking solution was determined by the value and P / N value.

[0085] The results showed that the optimal sealing time was 120 minutes. The results are shown in Table 5.

[0086] Table 5 Determination of closure time (OD 450nm value)

[0087]

[0088] 6. Determination of the optimal reaction time of serum and enzyme-labeled secondary antibody

[0089] Dilute the negative and positive serum and enzyme-labeled secondary antibody at the optimal dilution, and incubate for 30 min, 45 min, 60 min, and 90 min, respectively. Repeat 3 times for each condition to explore the optimal serum and enzyme-labeled secondary antibody incubation time. 450nm The optimal reaction time of serum and enzyme-labeled secondary antibody was determined by the value and P / N value. The results showed that the optimal serum incubation time was 60 minutes and the optimal secondary antibody incubation time was 40 minutes. The results are shown in Table 6.

[0090] Table 6 Determination of the optimal reaction time of serum and enzyme-labeled secondary antibody (OD 450nm value)

[0091]

[0092]

[0093] 7. Determination of color development time

[0094] Add colorimetric solution and incubate at 37°C in the dark for 15 min, 20 min, and 25 min, respectively. Repeat 3 times for each condition. 450nm value and P / N value to determine the optimal color development time.

[0095] The results showed that when the reaction time was 20 min, the P / N value was the largest, so the optimal color development time of the substrate was 20 min. The results are shown in Table 7.

[0096] Table 7 Determination of color development time (OD 450nm value)

[0097]

[0098] 8. Determination of the reaction procedure of the indirect ELISA kit

[0099] By optimizing the indirect ELISA reaction conditions for detecting Tembusu virus, the reaction procedure of the indirect ELISA kit was determined. The specific steps are as follows:

[0100] (1) Coating: The ELISA plate was coated with TMUV-EIII recombinant protein. The optimal coating concentration of TMUV-EIII recombinant protein was 1.0 μg / ml, 100 μl / well, incubated at 4°C overnight, washed three times with PBST buffer the next day, and patted dry for the last time;

[0101] (2) Blocking: Add PBS buffer containing 1% BSA for blocking, 200 μl / well, block at 37°C for 2 h, wash three times with PBST buffer, and pat dry for the last time;

[0102] (3) Serum incubation: add 1:1000 diluted serum to be tested, 100 μl / well, incubate at 37°C for 60 min, wash three times with PBST buffer, and pat dry for the last time;

[0103] (4) Secondary antibody incubation: add 1:2000 enzyme-labeled secondary antibody, 100 μl / well, incubate at 37°C for 40 min, wash 5 times, and pat dry for the last time;

[0104] (5) Color development: Add 100 μl / well of TMB color development solution and incubate at 37°C in the dark for 20 min;

[0105] (6) Stop: Add stop solution 2M sulfuric acid, 50 μl / well;

[0106] (7) Reading: Read OD using an ELISA reader 450nm value.

[0107] 9. Determination of critical value of indirect ELISA kit

[0108] The critical value of this kit is determined by using the established indirect ELISA method for detecting Tembusu virus antibodies to test the negative samples determined by the indirect immunofluorescence test, and the OD value of clinical negative serum samples is determined by the indirect ELISA method for detecting Tembusu virus antibodies to test the negative samples determined by the indirect immunofluorescence test. 450nm The S / P value of each sample (N01-N86 are Tembusu virus negative serum) was calculated based on the measurement results. S / P value = (Clinical sample OD 450nm Value - negative control serum OD 450nm Mean) / (positive control serum OD 450nm Mean - negative control serum OD 450nm The mean value (Table 8) was used to analyze the test results using the mean value + 3SD method to obtain the critical value. The calculated mean value was 0.050 and the SD was 0.029.

[0109] Table 8 Results of indirect ELISA detection method for clinical negative serum samples

[0110]

[0111]

[0112] Based on the above test results, the critical value of the indirect ELISA antibody detection method for Tembusu virus was determined to be 0.14, that is, S / P ≥ 0.14 was judged as positive, and S / P < 0.14 was judged as negative.

[0113] 10. Specificity of indirect ELISA kit

[0114] The established indirect ELISA method was used to detect the positive sera of known duck parvovirus, new duck reovirus, duck plague virus and other pathogens, and the specificity of this indirect ELISA kit was analyzed. The results showed (Table 9) that the S / P values ​​were all <0.14, which was judged as negative, indicating that the TMUV-EIII recombinant protein had no cross reaction with the positive sera of the above viruses, and the indirect ELISA kit had good specificity.

[0115] Table 9 Tembusu virus indirect ELISA kit specificity detection results

[0116]

[0117] 11. Indirect ELISA kit sensitivity

[0118] The established indirect ELISA method was used to detect 5 duck sera with a neutralization titer of 1:32 prepared in Example 2, and the sensitivity of the indirect ELISA kit was analyzed. The results showed (Table 10) that when the 5 sera were diluted to 1:12800, the S / P values ​​were still all>0.14 (the average value of the positive control was 1.602, and the average value of the negative control was 0.196), indicating that the indirect ELISA kit has good sensitivity.

[0119] Table 10 Sensitivity test results of indirect ELISA kit for detecting Tembusu virus antibodies

[0120]

[0121]

[0122] Example 4

[0123] Application of indirect ELISA kit for detecting antibodies against Tembusu virus

[0124] The test kit of the present invention is used to detect the pathogenicity of Tembusu virus to ducks. The tested serum is Tembusu virus Goose / Guangdong / DT01 / 2020 strain (deposit number CGMCC No.45979) with 10 6.0 TCID 50 / 0.1ml duck serum collected on the 3rd, 5th and 7th days after infection of 14-day-old healthy Tembusu virus antibody-negative ducks, a total of 22 samples. The 22 sera were verified as Tembusu virus antibody positive by indirect immunofluorescence test. The test results show (Table 11): The S / P values ​​of the 22 sera detected by the kit were all >0.14, which was 100% consistent with the results of the indirect immunofluorescence test.

[0125] Table 11 Results of serum samples tested for pathogenicity using the indirect ELISA kit for Tembusu virus

[0126]

[0127] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant protein of domain III of Tembusu virus E protein, characterized in that: The amino acid sequence is shown in SEQ ID NO:

1.

2. Use of the Tembusu virus E protein domain III recombinant protein according to claim 1 in the preparation of a reagent or kit for detecting Tembusu virus antibodies, wherein the Tembusu virus is the Tembusu virus Goose / Guangdong / DT01 / 2020 strain.

3. The application according to claim 2, characterized in that: The kit is prepared by using the following immunoassay techniques: enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, immunochromatography, immunoblotting and immunoprecipitation.

4. Use of the Tembusu virus E protein domain III recombinant protein according to claim 1 in the preparation of an indirect ELISA kit for detecting Tembusu virus antibodies, the indirect ELISA kit comprising a detection plate coated with the Tembusu virus E protein domain III recombinant protein and an enzyme-labeled secondary antibody.

5. An indirect ELISA kit for detecting Tembusu virus antibodies, characterized in that: It includes a detection plate coated with a recombinant protein of domain III of the E protein of Tembusu virus and an enzyme-labeled secondary antibody; The amino acid sequence of the Tembusu virus E protein domain III recombinant protein is shown in SEQ ID NO:

1.

6. The indirect ELISA kit according to claim 5, characterized in that: The coating concentration of the Tembusu virus E protein domain III recombinant protein is 0.8-1.2 μg / ml.

7. The indirect ELISA kit according to claim 5, characterized in that: The coating conditions of the Tembusu virus E protein domain III recombinant protein are 3-5°C overnight coating 。 8. The indirect ELISA kit according to claim 5, characterized in that: When preparing the test plate coated with the Tembusu virus E protein domain III recombinant protein, the Tembusu virus E protein domain III recombinant protein is coated on the test plate and then blocked; The blocking reagent is a 0.01 M pH 7.4 PBS buffer containing 0.8% to 1.2% BSA by mass concentration.

9. The indirect ELISA kit according to claim 8, characterized in that: The sealing time is 110 to 130 minutes.

10. The indirect ELISA kit according to any one of claims 5 to 9, characterized in that: The kit further comprises at least one of the following reagents: a sample diluent, a washing solution, a stop solution and a color developing solution.

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