CN108956985B - Indirect ELISA (enzyme-linked immunosorbent assay) detection kit for detecting novel goose astrovirus antibody and application - Google Patents

Abstract

The invention discloses an indirect ELISA detection kit for detecting a novel goose astrovirus antibody, which comprises an ELISA plate coated with a novel goose astrovirus ORF1b protein recombinant antigen; the preparation method of the novel goose star virus ORF1b protein recombinant antigen comprises the following steps: the preservation number is CCTCC NO: v201808, using the whole gene sequence of the goose astrovirus as a template, amplifying by using a primer pair to obtain an ORF1b gene fragment, constructing a recombinant expression vector, and expressing the goose astrovirus ORF1b protein recombinant antigen by a prokaryotic expression system. The indirect ELISA detection kit can be used for quickly and effectively detecting the novel goose astrovirus antibody which takes gout as a main symptom and is newly developed so as to monitor the epidemic situation of the novel goose astrovirus in goose flocks.

Description

Indirect ELISA (enzyme-linked immunosorbent assay) detection kit for detecting novel goose astrovirus antibody and application

Technical Field

The invention relates to the technical field of biological products, in particular to an indirect ELISA detection kit for detecting a novel goose astrovirus antibody and application thereof.

Background

Astrovirus (AStV) is a single-stranded positive-strand RNA virus which has no envelope, is spherical and has a diameter of 28-30nm, the genome of the virus has infectivity and a length of 6.4-7.9kb, and the infection of the virus has certain prevalence and can cause enteritis and diarrhea of human beings or animals, and some diseases are accompanied by vomit and abdominal pain. The astrovirus family can be divided into mammalian astrovirus and avian astrovirus according to different infection hosts, and the avian astrovirus can cause poultry to generate various diseases, and the clinical manifestations of the avian astrovirus are different due to different virus strains, virulence or infection hosts. Goose astrovirus (GAstV), which mainly attacks young goslings, has already occurred in a plurality of provinces, so that the death rate of the goslings is increased, and the healthy development of the Goose industry in China is greatly damaged.

In 2017, in 2-12 months, the gosling group in Shandong, Anhui, Jiangsu, Liaoning, Henan, Guangdong and the like in China outbreaks an infectious disease which is mainly characterized by gout. The disease mainly occurs to goslings of 5-20 days old, and the death rate can reach 50% at most. The body cavities and joints of the sick goslings are seriously deposited with urate, the goslings are tired in lying and difficult to take, the growth of the goslings is slow, the feed-meat ratio is increased, the slaughtering qualification rate of the meat geese is obviously reduced, and the serious economic loss is caused to the meat goose breeding industry.

Researches prove that the pathogeny of the goose infectious disease with high morbidity taking gout as a main symptom is a novel goose astrovirus, belongs to a new infectious disease, the researches on the biological characteristics, the pathogenic mechanism and the like of the virus are still in a starting stage, effective vaccine immunity and prevention control measures are still lacked at present, and a serological detection method for the novel virus is not reported.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide an indirect ELISA detection kit for detecting a novel goose astrovirus antibody and application thereof. The novel goose astrovirus antibody which takes gout as a main symptom and is newly developed can be quickly and effectively detected so as to monitor the epidemic situation of the novel goose astrovirus in goose groups.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an indirect ELISA detection kit for detecting a novel goose astrovirus antibody, which comprises an ELISA plate coated with a novel goose astrovirus ORF1b protein recombinant antigen;

the preparation method of the novel goose star virus ORF1b protein recombinant antigen comprises the following steps:

the preservation number is CCTCC NO: v201808, using the whole gene sequence of the goose astrovirus as a template, amplifying by using a primer pair to obtain an ORF1b gene fragment, constructing a recombinant expression vector, and expressing a novel goose astrovirus ORF1b protein recombinant antigen by a prokaryotic expression system;

the primer pair comprises:

an upstream primer ORF1b-F: 5'-ATGTTTGAAAAATTTTTCTATC-3'; (SEQ ID NO.1)

The downstream primer ORF1b-R: 5'-CCAGATTTGAGAAAAGAAGTC-3'. (SEQ ID NO.2)

Further, the recombinant expression vector is transformed into BL21(DE3) competent cells, and IPTG is added for induction expression; and (3) denaturing and renaturing the expression product to obtain a purified novel goose astrovirus ORF1b protein recombinant antigen.

Preferably, the coating amount of the novel goose star virus ORF1b protein recombinant antigen is 500 ng/hole.

Further, the indirect ELISA detection kit further comprises: enzyme-labeled antibody, sample diluent, washing liquid, negative control serum, positive control serum, substrate developing liquid A, B and stop solution.

Preferably, the enzyme-labeled antibody is an enzyme-labeled rabbit anti-goose antibody.

The application of the indirect ELISA detection kit in the epidemiological investigation of the novel goose astrovirus is also the protection scope of the invention;

the preservation number of the novel goose astrovirus is CCTCC NO: and V201808.

In a second aspect of the present invention, there is provided a novel indirect ELISA method for detecting an anseriavirus antibody, comprising the following steps:

(1) coating: the preservation number is CCTCC NO: v201808, taking the novel goose star virus ORF1b recombinant protein as a coating antigen, diluting the antigen, adding the diluted antigen into an enzyme-labeled plate hole, incubating overnight at 4 ℃ or incubating for 2-4h at 37 ℃, and washing by using PBST containing 0.05% Tween;

(2) and (3) sealing: diluting 5% skimmed milk powder with PBST (Poly-p-phenylene benzobisoxazole) 200 muL/hole, sealing, incubating at 37 ℃ for 1h, spin-drying, and washing with PBST;

(3) serum action conditions: adding diluted mixed solution of serum to be detected into each hole, incubating for 1h at 37 ℃, spin-drying, and washing with PBST;

(4) adding an enzyme-labeled antibody: enzyme-labeled rabbit anti-goose antibodies were expressed in PBST as 1: diluting with 500 μ l, adding 100 μ l into each well, drying at 37 deg.C for 1 hr, and washing with PBST;

(5) substrate color development: developing solution of TMB substrate at 100 μ L/hole, and keeping away from light at 37 deg.C for 15 min;

(6) and (3) terminating the reaction: adding 50 mu L of stop solution into each hole to stop the color reaction; reading data under the condition that the absorbance of a microplate reader is 450 nm;

(7) determination of negative and positive cut-off values: according to the formula: negative-positive cut-off value (negative sample OD)₄₅₀The average value plus the standard deviation of 3SD to obtain a positive and negative critical value; when OD is reached₄₅₀When the concentration is 0.065 or more, the specimen is judged to be positive.

In the step (1), the novel goose star virus ORF1b recombinant protein is prepared by the following method:

the preservation number is CCTCC NO: the whole gene sequence of the novel goose astrovirus of V201808 is used as a template, and an ORF1b gene fragment is obtained by amplification of a primer pair, so that a recombinant expression vector is constructed; transforming the recombinant expression vector into BL21(DE3) competent cells, and adding IPTG (isopropyl-beta-thiogalactoside) for induction expression; after the expression product is denatured and renatured, purified goose astrovirus ORF1b recombinant protein is obtained;

the primer pair comprises:

an upstream primer ORF1b-F: 5'-ATGTTTGAAAAATTTTTCTATC-3'; (SEQ ID NO.1)

The downstream primer ORF1b-R: 5'-CCAGATTTGAGAAAAGAAGTC-3'. (SEQ ID NO.2)

The invention has the beneficial effects that:

aiming at the newly discovered novel goose astrovirus capable of causing gosling gout, the invention establishes an indirect ELISA detection method for detecting the novel goose astrovirus antibody, the detection method has the characteristics of simplicity, convenience, rapidness, stability, strong specificity, high sensitivity and the like, can detect the specific antibody of the novel goose astrovirus ORF1b in the goose group, and detects the antibody level of the novel goose astrovirus in the goose group by taking the specific antibody as the basis, thereby monitoring the epidemic situation of the novel goose astrovirus in the goose group.

Drawings

FIG. 1: SDS-PAGE analysis picture of the induction expression and protein purification of recombinant protein ORF1b in BL21 competence; wherein, lane M is a protein standard; lane 1 is empty vector; lane 2 is the pellet after lysis of the inducer.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As introduced in the background art, the gosling group in Shandong, Anhui, Jiangsu, Liaoning, Henan, Guangdong and the like in China outbreaks an infectious disease which is mainly characterized by gout in 2017. The symptoms of the disease are different from the prior astrovirus infection, and the symptoms of gout appear in goose flocks for the first time, so that the infectious disease is probably caused by the novel astrovirus. There is currently no drug or method available to effectively control this new astrovirus infection. Based on the detection, the invention provides an indirect ELISA detection kit for detecting the novel goose astrovirus antibody, so that the infected goose groups can be found as soon as possible and isolation or killing measures can be taken on the infected goose groups, and the loss caused by the novel goose astrovirus infection can be reduced.

As the astrovirus is an RNA virus and has a plurality of segments, different strains have certain differences in antigen structure, pathogenicity, cell culture characteristics and host specificity, and are easy to mutate during genetic evolution. The astrovirus isolated from different hosts therefore has a great variability, differing in pathogenicity, genetic sequence and coding for major proteins.

The inventor of the application separates a novel virus N-AStV-SDPY from tissues such as the liver, the spleen, the kidney and the like of a gosling died from diseases, and identifies the novel virus N-AStV-SDPY as a goose astrovirus. The invention carries out whole genome sequencing on the strain N-AStV-SDPY, and carries out homology analysis and genetic evolution analysis with the astrovirus reported at present, and the result shows that the homology of each gene fragment of the strain N-AStV-SDPY is less than 70 percent; genetic evolution analysis shows that the strain N-AStV-SDPY is in a variation branch; the above results illustrate that: the strain N-AStV-SDPY is different from other astrovirus and is a new species of avian astrovirus. The novel goose astrovirus is preserved in China center for type culture Collection with the preservation number of CCTCC NO: and V201808. Therefore, the novel goose astrovirus to be detected by the invention and the existing reported astrovirus have variability, and the detection of the novel goose astrovirus is more difficult.

The invention firstly uses a serological method to detect the antibody of the novel goose astrovirus. The method can quickly and effectively detect the novel goose astrovirus antibody which is newly outbreaked and mainly characterized by gout, and is a simple and convenient indirect ELISA detection method suitable for being used in a basement layer.

In one embodiment of the invention, the novel goose astrovirus indirect ELISA detection method comprises the following steps:

(1) preparation of coating antigen:

obtaining a whole gene sequence of the novel goose astrovirus by a second-generation sequencing technology, wherein the whole gene sequence is shown as SEQ ID NO. 3; according to the obtained ORF1b protein gene of the novel goose star virus, two primers are designed:

an upstream primer ORF1b-F: 5'-ATGTTTGAAAAATTTTTCTATC-3';

the downstream primer ORF1b-R: 5'-CCAGATTTGAGAAAAGAAGTC-3'.

ORF1b gene sequence (shown as SEQ ID NO. 4) with the length of 1287bp is amplified by the primer through a common RT-PCR method, and is purified through gel electrophoresis and gel recovery, the purified product is cloned into a prokaryotic expression vector PET-28a (+), and a recombinant prokaryotic expression vector PET28a-ORF1b is constructed; the recombinant prokaryotic expression vector PET-ORF1b is transformed into BL21(DE3) competent cells, and the recombinant protein ORF1b is efficiently expressed by induction with 1mM IPTG, the protein exists in the form of inclusion bodies, and the inclusion bodies are denatured and renatured, and the purified astrovirus ORF1b protein is used as a coating antigen.

(2) And (3) establishing indirect ELISA by using the purified recombinant protein of the astrovirus ORF1b as a coating antigen:

coating: diluting the antigen with PBS (pH 7.2 or so) or 1 Xcarbonate buffer solution (pH 9.6) according to the required concentration, coating the antigen in a 96-well enzyme label plate (ELISA plate), coating a preservative film, incubating overnight at 4 ℃ or incubating for several hours at 37 ℃, and washing for three times and 4min each time by using PBST containing 0.05% Tween (mass concentration);

sealing: sealing and diluting 5% skimmed milk powder (mass concentration) with PBST (PBST) at 200 μ L/hole, incubating at 37 deg.C for several hours, spin-drying, washing with PBST for 3 times, each time for 4 min;

③ serum action conditions: adding a proper amount of diluted mixed solution of the serum to be detected into each hole, incubating for several hours at 37 ℃, then spin-drying, and washing for 3 times by PBST, wherein each time is 4 min;

fourthly, the action condition of the rabbit anti-goose enzyme-labeled secondary antibody is as follows: diluting rabbit anti-goose enzyme labeled secondary antibody with PBST, performing action for 1h at 37 ℃ after 100 mu L of the diluted secondary antibody is applied to each hole, spin-drying, and washing for 4 times with PBST, wherein each time is 4 min;

fifthly, substrate color development: developing solution of TMB substrate at 100 μ L/hole, and keeping away from light at 37 deg.C for 15 min;

sixthly, terminating the reaction: add 50. mu.L of 2M H per well₂SO₄Stopping the color development reaction by the stop solution;

and reading is carried out: and reading data by adopting a microplate reader under the condition that the absorbance is 450 nm.

(3) And (4) result judgment standard:

according to the formula: negative-positive cut-off value (negative sample OD)₄₅₀The average value plus the standard deviation of 3SD to obtain a positive and negative critical value; when OD is reached₄₅₀When the concentration is 0.065 or more, the specimen is judged to be positive.

For the indirect ELISA detection of the novel goose astrovirus, the selection of the coating antigen is very critical, and the specificity of the detection method is directly determined. In order to optimize the envelope antigen, primer pairs are respectively designed according to the coding region of ORF1b protein in the gene sequence of the avian astrovirus published on GenBank and the main antigen site region of the novel goose astrovirus in the test process, and gene fragments are obtained by amplification to construct a recombinant expression vector; transforming the recombinant expression vector into BL21(DE3) competent cells, and adding IPTG (isopropyl-beta-thiogalactoside) for induction expression; and (3) obtaining purified recombinant proteins after denaturation and renaturation of the expression products, and constructing an indirect ELISA detection kit by using the obtained recombinant proteins as envelope antigens respectively. The specificity of indirect ELISA detection kits prepared by different coated antigens is investigated by applying clinically confirmed serum samples. The result shows that the accuracy of the indirect ELISA detection kit prepared by using the purified goose astrovirus ORF1b protein as the coating antigen to the detection of the serum sample which is confirmed to be diagnosed reaches 100%, and the detection sensitivity and specificity are both obviously superior to those of the indirect ELISA detection kit prepared by using other recombinant proteins as the coating antigens.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available. Part of reagents and components used in the invention are as follows:

coating buffer solution: 1 × carbonate buffer (100mL, pH 9.6): na (Na)₂CO₃ 0.2756g，NaHCO₃0.6216g, dissolving with distilled water, fixing the volume to 100mL, keeping the pH value at 9.5-9.7, and storing at 4 ℃.

PBS solution: NaCl 4.25g, NaH₂PO₄·2H₂O 0.178g，Na₂HPO₄·12H₂And 1.386g of O, dissolving with distilled water and fixing the volume to 500mL, wherein the pH value is 7.1-7.3.

PBST solution: tween-200.5 mL was added to 1L of PBS, and the mixture was mixed well.

PBST blocking liquid: 5g of drymylk was dissolved in 100mL of PBST dilution and stored at 4 ℃ for a short period and-20 ℃ for a long period.

TMB color development liquid:

buffer A: 66.5063g of sodium citrate are weighed out and dissolved in 800mL of distilled water and the pH is adjusted to 4.0 with concentrated HCl, 314. mu. L H are added₂O is constant volume to 1L and stored at 4 ℃;

buffer B: 0.2956g of Tetramethylbenzidine (TMB) and 0.0633g of tetrabutylammonium borohydride (TBABH) were weighed out and dissolved in 30ml of Dimethylacetamide (DMA), and stored at 4 ℃ in the dark;

when in use, the buffer A and the buffer B are mixed according to the volume ratio of 39:1, and the mixture is prepared as it is.

2M H₂SO₄Stopping liquid: adding concentrated H₂SO₄Adding distilled water according to the volume ratio of 1:5, uniformly mixing and cooling to room temperature to obtain 2M H₂SO₄And (4) stopping the solution.

In the examples of the present invention, the specific experimental conditions and methods are not specified, and the conventional conditions such as J. SummBruker et al, science publishers, 2002, molecular cloning guidelines (third edition); master catalog of speekt et al, scientific press, 2001, cell experimental guidelines; or according to conditions recommended by the manufacturer.

Example 1: preparation of coating antigen

1.1 specific primer design and Synthesis: according to the whole gene sequence (shown as SEQ ID NO. 3) of the novel goose astrovirus (with the preservation number of CCTCC NO: V201808), a pair of primers is designed on the ORF1b protein gene sequence, the two segments of the primers are respectively provided with BamH I enzyme cutting sites and Sal I enzyme cutting sites, and a 1287bp gene fragment (shown as SEQ ID NO. 4) in the novel goose astrovirus genome can be predicted to be amplified.

An upstream primer ORF1b-F: 5'-ATGTTTGAAAAATTTTTCTATC-3';

the downstream primer ORF1b-R: 5'-CCAGATTTGAGAAAAGAAGTC-3'.

1.2 construction of prokaryotic expression vector PET28a-ORF1 b:

ORF1b gene fragments are amplified by PCR through specific upstream and downstream primers, the purified fragments are connected with a PMD18-T vector, a connecting product is transformed into DH5 alpha competent cells, and positive bacterial colonies are selected and shaken to extract T-ORF recombinant plasmids. The T-ORF recombinant plasmid and the PET-28a (+) vector were each double-digested with restriction enzymes BamHI and SalI (available from Dalibao Bio Inc.), and the digested products were ligated with T4DNA ligase (available from Dalibao Bio Inc.). The ligation product is transformed into DH5 alpha competent cells, positive clones are identified by a PCR method and enzyme digestion, and the positive clones are sent to Qingdao Stricture biotechnology limited company for sequencing.

The PCR identification result of a colony of a single positive clone is selected, and the colony can be amplified to a target band of about 1287bp and is consistent with the size of an expected ORF1b gene fragment. And (3) selecting a single positive clone colony, performing enrichment culture, extracting plasmids, and performing enzyme digestion identification by using restriction enzymes BamH I and Sal I to obtain a correct enzyme digestion band. The positive clone sequencing result shows that the ORF1b gene insertion position, insertion direction and reading frame are correct, and the result shows that the recombinant vector PET28a-ORF1b is successfully constructed.

1.3 recombinant protein ORF1b induced expression and identification:

transforming the successfully constructed recombinant vector PET28a-ORF1b plasmid into BL21 competent cells, selecting a single colony for enrichment culture, and inoculating the single colony to a strain containing 100 mu g/mL kan according to the ratio of 1:100 the next day⁺In 2 XYT medium, cultured at 37 ℃ with shaking at 200r/min to OD₆₀₀Taking out 1mL of bacterial liquid between 0.6-1.0 as a control before induction, adding IPTG into the rest culture till the final concentration is 1mM, continuing to culture at 37 ℃ for 6h under the condition of 200r/min oscillation, and finishing the induction. Respectively taking induction and non-induction2mL of bacterial solution, collecting bacterial precipitates after centrifugation, and resuspending the bacteria by 50 mu of LPBS. And adding 50 mu L of 2 xSDS gel sample adding buffer solution into the sample, uniformly mixing, boiling for 5min, shaking to crack bacteria, slightly centrifuging, taking supernatant, performing SDS-PAGE, and identifying whether the protein is expressed or not. Centrifuging induced culture bacteria liquid, removing supernatant, collecting bacteria precipitate, suspending bacteria with PBS, ultrasonically cracking to clear, centrifuging at 4 deg.C for 5min at 10,000 Xg/min, respectively taking supernatant and precipitate suspension, adding 2 XSDS gel sample-adding buffer solution, mixing, boiling for 5min, and SDS-PAGE identifying protein expression mode.

1.4 protein purification:

and (3) carrying out mass induction culture according to the protein induction expression conditions in the step 1.3. And (3) taking the inclusion body to purify the protein according to the instruction of the kit, recovering a protein sample, and performing SDS-PAGE to identify the purity of the protein.

SDS-PAGE analysis showed that recombinant protein ORF1b was successfully expressed in BL21 competent cells, all in the form of inclusion bodies, with a protein molecular weight size consistent with the expected recombinant ORF1b size (see FIG. 1).

1.5 Western Blotting analysis: protein is induced and expressed according to the method in 1.3, induced and non-induced whole bacteria SDS-PAGE are respectively taken, the operation is carried out according to the conventional Western Blotting method, and finally, an enhanced HRP-DAB substrate chromogenic reagent is adopted for developing. The results show that: compared with SDS-PAGE, PET28a-ORF1b transformed BL21 competent cells showed specific bands at the corresponding positions of Western Blotting after induction, while the control non-induced bacteria showed no bands. The result shows that the recombinant protein ORF1b can react with the antibody in the positive serum to generate specific antigen-antibody reaction.

Example 2: indirect ELISA detection of novel goose astrovirus antibody

The purified recombinant protein ORF1b in example 1 is taken as a coating antigen to establish an indirect ELISA method, and the ELISA optimal protein coating concentration, serum dilution concentration and ELISA optimal reaction conditions are searched by a square matrix method. The final determination conditions were as follows:

2.1 coating: the antigen was diluted 1:1000 in 1 Xcarbonate buffer (pH 9.6), 10. mu.L was coated onto a 96-well ELISA plate, coated with plastic wrap, incubated at 37 ℃ for 2h, and washed three times with 0.05% Tween in PBST for 4min each time. Detecting the coating amount of ORF1b protein of the hole by 500 ng/hole;

2.2 sealing: sealing, diluting 5% skimmed milk powder with PBST, incubating at 37 deg.C for 1h, spin-drying, washing with PBST for 3 times (4 min each time);

2.3 serum action conditions: adding 10 μ L of mixed solution of blood serum to be detected and 5% skimmed milk powder diluted by 90 μ L of BST into each hole, incubating at 37 deg.C for 1h, spin-drying, washing with PBST for 3 times, and each time for 4 min;

2.4 the action conditions of the enzyme-labeled rabbit anti-goose antibody are as follows: enzyme-labeled rabbit anti-goose antibodies were expressed in PBST as 1: diluting with 500, adding 100ul per well, drying at 37 deg.C for 1 hr, washing with PBST for 4 times, each time for 4 min;

2.5 substrate color development: developing solution of TMB substrate at 100 μ L/hole, and keeping away from light at 37 deg.C for 15 min;

2.6 termination of the reaction: add 50. mu.L of 2M H to each well₂SO₄Stopping the color development reaction by the stop solution;

2.7 reading: reading data under the condition that the absorbance of a microplate reader is 450 nm;

3. and (4) result judgment standard:

according to the formula: negative-positive cut-off value (negative sample OD)₄₅₀The average value plus the standard deviation of 3SD to obtain a positive and negative critical value; when OD is reached₄₅₀When the concentration is 0.065 or more, the specimen is judged to be positive.

4. Cross-reactivity: the method established by the invention is used for detecting the positive serum and the negative serum of goose parvovirus, goose tembusu virus, goose adenovirus, goose influenza virus, goose astrovirus GsFJ2017 strain (the accession number of GenBank is MF576430) and goose astrovirus CastV-8 strain, and the method only reacts positively with the positive serum of novel goose astrovirus (the preservation number is CCTCC NO: V201808), thereby showing that the method has good specificity.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shandong university of agriculture

<120> indirect ELISA detection kit for detecting novel goose astrovirus antibody and application

<130> 2018

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 22

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<213> Artificial sequence

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atgtttgaaa aatttttcta tc 22

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<212> DNA

<213> Artificial sequence

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ccagatttga gaaaagaagt c 21

<210> 3

<211> 7252

<212> DNA

<213> Goose astrovirus (Goose astrovirus)

<400> 3

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ccaagatgaa aggcttttat ggtcgcactc cagcctggag tgctctcatg aagtgtaatg 300

ccatctatct taaagattgg aaaacagcta taggggttga caatgggagg tatggtctct 360

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cgctgaaacg atgtggagct cttgtccaac aagttcttga gaagagcaaa caagcaaagg 540

aacaaatgca aaaagccgag gaattacaga agaagattga tcaaattaat gaaagcaaca 600

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tgactttcct agcatggatg tgtgttgcga tcattgcttt cagcttgatt caattggcgg 840

atgctagtga gattaaggat aacagaacag aaaattgttt cccaacatct ggctgtgttc 900

tctcagacgt tccttggact aagtcgtaca cctttatgga gctaatggac aagtgtgtga 960

ccggtactgc catcatacct aaaaacacat ttaatggtga agggcttgag ctggagtgta 1020

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atacaatagc ggttataaag ctgccccccg aattacagaa tttgaaaccc ataaaactcg 1980

caaaacgtgt tgagtccaca tacttaacat tgacagcttt tgatcctaat tttcaaaatg 2040

ttgtgtcata cactggctgg tgtattattg atggaaattg gatcagtaat gcatttaata 2100

ctcaatttgg aaacagtggt ggcccatacg ttgatgcaga gggtaaattg gttgggattc 2160

atcttggaac tcagggagtt atgtcccaag gtattgttgt ggtggatgcc cttaaaacgc 2220

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ttgcaggaac aaagacatca catgaagcaa ttctcaaagg tattgaggaa ttgcgagagg 2340

acgtcgacac gctcaagaac aagtgcaaga attatgatga gtactggctt gttcaaacta 2400

tttttggcca gaagaaaaag ggcaaaacaa agaaaacagc ccgggggaat aaacatcttg 2460

taacaaagag attcctgagt aaaggccact tcatgaaact caaaatgctc accgatgagg 2520

aatataatca tatgattgag caaggtttca ccgctgatga gatacgtgag gcagtcaatg 2580

agctgagaga acaagcatgg ctcaactact gcattgataa tgatattgat gaagaaggtg 2640

cggaagagtg gtatgaagat atgattgaag atgaccaaat taatgaaaag attgatgaag 2700

ccattgagaa agcaatggaa gaaaggggcg agttttatca agtcaaaaaa aggaaaacat 2760

ttgcacaaca agcattatta cacctcatcc gtgtcagtaa agccaggagc caaactgcaa 2820

agcaagaggt tcagaaagaa aacgcagatc agctttacga gatgttcaaa tctgccgtaa 2880

aggatgaaca agttgatgaa ggcacaacct cttacgcact cttcgccaat ggtgatcaga 2940

ttgagatggt cgaaggaaag gaacttgatt ttgaaaaggt aaagcttatc cacgttgatg 3000

gtgaacaaaa aaatgagacc attccgggga cgaaagtcac tgagatttct actggccctg 3060

agaataagaa gaacattttg cgcaagaagg acactcaaat taatccagaa caagctcaac 3120

ctcttgagca gcgcaagagg atctgcaagt ggtgtgggtc gagccaaaag catgactttc 3180

gtgattgcaa gttccagcgt gagaaaaggt tttgtgtctt ctgtgcaaag atgcactcat 3240

tgtttgatgg tcatcaacgg gacgtcttgt gcgacgtttg tgggaagaag ttcaagagtg 3300

tagaggagct tgaacagcac gtaactcgtg aaacgtgtga aaaaaactag ataaggggga 3360

cgatgccatc tgggtccccc gcataccacc tgatgattct cacatttttt cttggcagga 3420

tgatattatt gagtgtgata agaaaatcac tttgcccgac tatatagata taattggttt 3480

tatacctgta gataaattag ttgtcagagc aaagaccatt catgacccct tattaaactt 3540

ggttgagcat tggcaacaag atgaatatgc tcctactaaa tgggactata aagcctatac 3600

caagatgttt gaaaaatttt tctatcatga acctcaggat tttgtaaaca attttccaga 3660

gctggtcatc ttaagtgata gtgttgtgtt agaagaacat tcttatatgg ccaattccaa 3720

tttaacaccc ataatggcaa ctaagaagaa tgtcgatagt acccctggtt atccaaaatt 3780

taagtacttt gacacagaga aggactatct tgaacaatgt ggctggggtg agtacttgaa 3840

ggctgtatca gatattgatg agactgtgca aaataggccc ctctggtggt gttttctcaa 3900

aaatgaggtc ctcaaaaaga agaaaatcat ggataatgat attcgtatga tcttgtgtgc 3960

tgatccagta tatacaagga ttggagccat gtttgagcag gaccagaatg agaaaatgaa 4020

gcaacagaca gaacggcggg ctgcacaagt tggttggaca cccttttttg gtggaataca 4080

tcagcgagta tctaggctca ttggtggtgg tgacaggttt tttgtagaga cggactggac 4140

gcgttatgat ggtacgttgc ccaagcctct cttctggcgg atacgacaga tgcgttactt 4200

tttcctgtcc aaccatcata agacaccaca gcttaagaaa ctctatgatt ggtatgtcaa 4260

aaacttagtt gaaaaaatta tattattacc aactggggag gtttgtacag ttaagaaggg 4320

aaatccaagt ggccaatatt caacaacagt ggacaacaat atgtgtaatg tctggctcac 4380

ccattttgag atagcttatc tttattggaa acagcatggg tcattgccga cgctcagatt 4440

actcagggat aatgtcacca tgatttgcta tggcgatgat aggcttgtgt caataaggaa 4500

gggttttatt aattacgaca cgaatgtcgt catagatatg tataaaaata tttttggtat 4560

gtgggttaag ccagaaaatg tcaaggtctc tgatgatatt gagggtatga ctttctgtgg 4620

tcttacgttg gttaaaagga gtgatggtag atttgttgga gttcccaatg ttgataagat 4680

attgtcaaca cttgaaaatc ctgttagaaa attacccaac ttagaatcac tctggggtaa 4740

attggtttct ttaaggatct tgtgcgaaaa tgcacccaat agtgttagag agtttcttga 4800

taaacaaatc aacacggttg aggagcacgc tgccagtgaa aatattaact tacctgaggt 4860

cgggcccgac ttcttttctc aaatctggtg agtggcggac cgaaataaaa tggcagacag 4920

ggcggtggcc ccgcgcgaga aggtgaccaa gaaggttaca aaagtagtca ccgttaagaa 4980

aaaacaccca aaaaagaaac caaagcagaa agtatataag ccccaaaaat tacccatgaa 5040

ggccgagagg aagcttgaga aagaagtgaa aggtttgaag aaaagagtag ctggaccacc 5100

cgttaatgac aaaatgacta ccacgataac acttggtcag atcactggga attcaacaga 5160

cacactcgac cggaagcata aatacttcac aaatccactc atgatgaaaa accaggaaaa 5220

tgggcaaaca gcaactcctc taagtataag ggcctcacaa tataacttgt ggaggatcag 5280

aaagctgcat atccgccttg ttccacttgc tggtagggcc aatattcttg gctcggttgt 5340

cttcctagat atagaacagg aagctaacac agctgggcca gagtccatag ataccataaa 5400

agctcggccg catcttgaac tcccgattgg ctcaaaacat ctttggaggg ttcaacccag 5460

gctgatgcag ggaccccggc aaggatggtg gaatgtagac cccggggatt cacctactga 5520

ctcacttggt ccagcaatca atatgtggac atatttgaaa acagtaaatg cactttcaac 5580

acgggcgcag gcacaacaag ttccctatac ttctgccctc ttcctggttg aagcaacggt 5640

cacttatgag ttctcgaact atggtccaaa gccagggctc tcactcatga ccagtgaaac 5700

actttcagca tcagggaaaa cggcaaccct tgtaaatacc caggatggtg cacttgcttt 5760

aacagttagt ggcgcattgc agagattcct cgatgagaag gagcaacaca ggcgcgtttc 5820

aaatgcgcag acctctggtg tcggtgaggt gttttgggct gtttccacag aggtggtcga 5880

gaccgtagcg agtgcacttg gaggctgggg ttggctcttg aaaggaggat ggttcgtgat 5940

cagaaaattg tttggtgctg cttcaaattc tggttccact tatctgatct attcctcagt 6000

ttcagacgcc cagatagaca gcaggattta tcagacagtt ccaccgaaca cgccactaca 6060

gctggctgca aatacagtta agcttgtaca gctaacacag ccaaatgtga atacaactgg 6120

acaaggtacc actgtccttt cacgggatgc cgattacctg cctctgccag tggcaccaat 6180

gcaggttact ccctcgcttg tgtacaactt ccagggtgaa aggcagagca ctacagaatc 6240

gtgctcattc ctggtgtttg gaataccaca ggcagaatcc aggtcaagat acaatgcaaa 6300

tataaccttc aatgttggct atcgtggaag gacttcaaca tcatttacac ttggaacaca 6360

caattggtgg gctgttatga cactttcaca aacaggagta atttttgcac caccggctgt 6420

gggcacaggg gtctgtaata cattggctac agccatacaa cacttaaacc ctgagcttga 6480

aacagcagtc ctgcgtgtga ataccagtac aacatctact ggtgggctaa taacggaact 6540

caggaatcgg ctcaacatcg ctgatgggga ctatgtgatt tcaatgggtg atccgcaagg 6600

aaataggtca gcactgtact ttaggaattc agaccagaaa tgggtgtggc tctgggctgg 6660

cgactctaac cctggtgaaa ctttccaaag ttttaagatg ccagtcctaa ttaattggtc 6720

agtgtcagat tcccaggaac aatataatgc tagagtcagg atggtacagt atgctaatgc 6780

acaacagcag actttgacag accctgagga agatgatgat cccctctctg atgtcacttc 6840

gctttttgat ccaacagcgg aagatgagac tgacttccac ctagcagtct cactcaagac 6900

ctctgactac ttaaaagaag aggctgagta ctggaaagcg aaggcgcagg ccttgcttat 6960

ggagaaggca ctaagtgcac cacaagcagg gacagtccgc tttgagaagg gcggacatga 7020

gtgatctctt cactagcagc cgcggccacg ccgagtagga tcgagggtac agctgcacct 7080

tctcattagg cttgagggag aatcggccct ctctgcctta aattaacccg gtgacagtca 7140

ggtggtcacc cacacaaatc gcgtgcgggt acccctgacc gggtttttgt ttaaagaatc 7200

aaatgtgatt tttaagcatt taaaatcttt aaaaaaaaaa aaaaaaaaaa aa 7252

<210> 4

<211> 1287

<212> DNA

<213> ORF1b gene

<400> 4

atgtttgaaa aatttttcta tcatgaacct caggattttg taaacaattt tccagagctg 60

gtcatcttaa gtgatagtgt tgtgttagaa gaacattctt atatggccaa ttccaattta 120

acacccataa tggcaactaa gaagaatgtc gatagtaccc ctggttatcc aaaatttaag 180

tactttgaca cagagaagga ctatcttgaa caatgtggct ggggtgagta cttgaaggct 240

gtatcagata ttgatgagac tgtgcaaaat aggcccctct ggtggtgttt tctcaaaaat 300

gaggtcctca aaaagaagaa aatcatggat aatgatattc gtatgatctt gtgtgctgat 360

ccagtatata caaggattgg agccatgttt gagcaggacc agaatgagaa aatgaagcaa 420

cagacagaac ggcgggctgc acaagttggt tggacaccct tttttggtgg aatacatcag 480

cgagtatcta ggctcattgg tggtggtgac aggttttttg tagagacgga ctggacgcgt 540

tatgatggta cgttgcccaa gcctctcttc tggcggatac gacagatgcg ttactttttc 600

ctgtccaacc atcataagac accacagctt aagaaactct atgattggta tgtcaaaaac 660

ttagttgaaa aaattatatt attaccaact ggggaggttt gtacagttaa gaagggaaat 720

ccaagtggcc aatattcaac aacagtggac aacaatatgt gtaatgtctg gctcacccat 780

tttgagatag cttatcttta ttggaaacag catgggtcat tgccgacgct cagattactc 840

agggataatg tcaccatgat ttgctatggc gatgataggc ttgtgtcaat aaggaagggt 900

tttattaatt acgacacgaa tgtcgtcata gatatgtata aaaatatttt tggtatgtgg 960

gttaagccag aaaatgtcaa ggtctctgat gatattgagg gtatgacttt ctgtggtctt 1020

acgttggtta aaaggagtga tggtagattt gttggagttc ccaatgttga taagatattg 1080

tcaacacttg aaaatcctgt tagaaaatta cccaacttag aatcactctg gggtaaattg 1140

gtttctttaa ggatcttgtg cgaaaatgca cccaatagtg ttagagagtt tcttgataaa 1200

caaatcaaca cggttgagga gcacgctgcc agtgaaaata ttaacttacc tgaggtcggg 1260

cccgacttct tttctcaaat ctggtga 1287

Claims (4)

1. An indirect ELISA detection kit for detecting a novel goose astrovirus antibody is characterized by comprising an ELISA plate coated with a novel goose astrovirus ORF1b protein recombinant antigen;

the preservation number of the novel goose astrovirus is CCTCC NO: v201808;

the preparation method of the novel goose star virus ORF1b protein recombinant antigen comprises the following steps:

the preservation number is CCTCC NO: v201808, using the whole gene sequence of the novel goose astrovirus as a template, obtaining an ORF1b gene segment by utilizing primer pair amplification, constructing a recombinant expression vector, and expressing a novel goose astrovirus ORF1b protein recombinant antigen through a prokaryotic expression system;

the sequences of the primer pairs are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;

transforming the recombinant expression vector into BL21 competent cells, and adding IPTG (isopropyl-beta-thiogalactoside) for induction expression; after the expression product is denatured and renatured, a purified novel goose astrovirus ORF1b protein recombinant antigen is obtained;

the coating amount of the novel goose astrovirus ORF1b protein recombinant antigen is 500 ng/hole.

2. The indirect ELISA detection kit of claim 1, further comprising: enzyme-labeled antibody, sample diluent, washing liquid, negative control serum, positive control serum, substrate developing liquid A, substrate developing liquid B and stop solution.

3. The indirect ELISA detection kit of claim 2 wherein the enzyme-labeled antibody is an enzyme-labeled rabbit anti-goose antibody.

4. An indirect ELISA detection method of a novel goose astrovirus antibody is characterized by comprising the following steps:

(1) coating: the preservation number is CCTCC NO: v201808, taking the novel goose star virus ORF1b recombinant protein as a coating antigen, diluting the antigen, adding the diluted antigen into an enzyme-labeled plate hole, incubating overnight at 4 ℃ or incubating for 2-4h at 37 ℃, and washing by using PBST containing 0.05% Tween;

(2) and (3) sealing: diluting 5% skimmed milk powder with PBST (Poly-p-phenylene benzobisoxazole) 200 muL/hole, sealing, incubating at 37 ℃ for 1h, spin-drying, and washing with PBST;

(3) serum action conditions: adding diluted mixed solution of serum to be detected into each hole, incubating for 1h at 37 ℃, spin-drying, and washing with PBST;

(4) adding an enzyme-labeled antibody: enzyme-labeled rabbit anti-goose antibodies were expressed in PBST as 1: diluting with 500 μ l, adding 100 μ l into each well, drying at 37 deg.C for 1 hr, and washing with PBST;

(5) substrate color development: developing solution of TMB substrate at 100 μ L/hole, and keeping away from light at 37 deg.C for 15 min;

(6) and (3) terminating the reaction: adding 50 mu L of stop solution into each hole to stop the color reaction; reading data under the condition that the absorbance of a microplate reader is 450 nm;

(7) determination of negative and positive cut-off values: according to the formula: positive-negative cutoff = negative sample OD₄₅₀The average value plus the standard deviation of 3SD to obtain a positive and negative critical value; when OD is reached₄₅₀Positive when the concentration is more than 0.065;

in the step (1), the novel goose star virus ORF1b recombinant protein is prepared by the following method:

the preservation number is CCTCC NO: the whole gene sequence of the novel goose astrovirus of V201808 is used as a template, and an ORF1b gene fragment is obtained by amplification of a primer pair, so that a recombinant expression vector is constructed; transforming the recombinant expression vector into BL21 competent cells, and adding IPTG (isopropyl-beta-thiogalactoside) for induction expression; after the expression product is denatured and renatured, purified novel goose astrovirus ORF1b recombinant protein is obtained;

the sequences of the primer pairs are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.

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