CN108680744B - Indirect ELISA detection kit for detecting novel duck reovirus antibody and application thereof - Google Patents

Abstract

The invention discloses an indirect ELISA detection kit for detecting a novel duck reovirus antibody, which comprises an ELISA plate coated with a novel duck reovirus sigma C protein recombinant antigen; the preparation method of the novel duck reovirus sigma C protein recombinant antigen comprises the following steps: the preservation number is CCTCC NO: the whole gene sequence of the duck reovirus V201818 is used as a template, a primer pair is used for amplification to obtain a sigma C gene segment, a recombinant expression vector is constructed, and a prokaryotic expression system is used for expressing the duck reovirus sigma C protein recombinant antigen. The indirect ELISA detection kit can be used for quickly and effectively detecting the novel duck reovirus antibody with the main symptom of tarsal joint enlargement in the new outbreak so as to monitor the epidemic situation of the novel reovirus in the duck group.

Description

Indirect ELISA detection kit for detecting novel duck reovirus antibody and application thereof

Technical Field

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

Background

Avian reovirus belongs to reoviridae (reoviridae) Orthoreovirus (Orthoreovirus) and can cause various diseases of poultry, and clinical manifestations of the avian reovirus vary according to virus strains, virulence or infected hosts.

In 2016, the ducks in Shandong, Hebei, Henan and Jiangsu provinces in China have wide-range outbreaks of infectious diseases with tarsal joint swelling as a main symptom, the disease mainly causes tarsal joint swelling of the ducks at various ages, and the tarsal joints often contain a small amount of yellow or blood sample exudates; in severe cases, there is a cheese-like exudate in the joint cavity, with varying degrees of lameness occurring in the affected ducks. The disease mainly causes the reduction of the egg laying of breeding ducks and the weight of meat ducks, the feed-meat ratio is increased, the slaughtering qualification rate of the meat ducks is obviously reduced, and the serious economic loss is caused to the breeding industry of the meat ducks and the breeding ducks.

Research proves that the pathogen of the duck infectious disease with high incidence rate taking arthritis as main symptom is a novel duck reovirus, belongs to new infectious diseases, the research on the biological characteristics, pathogenic mechanism and the like of the virus is still in the initial stage, effective vaccine immunization and prevention control measures are still lacked at present, and a serological detection method for the novel virus is not reported at present.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide an indirect ELISA detection kit for detecting a novel duck reovirus antibody and application thereof. The novel duck reovirus antibody with arthritis as a main symptom in a new outbreak can be quickly and effectively detected to monitor the epidemic situation of the novel duck reovirus in a duck group.

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 duck reovirus antibody, which comprises an ELISA plate coated with a novel duck reovirus sigma C protein recombinant antigen;

the preparation method of the novel duck reovirus sigma C protein recombinant antigen comprises the following steps:

the preservation number is CCTCC NO: v201818, using the whole gene sequence of the duck reovirus as a template, amplifying by using a primer pair to obtain a sigma C gene segment, constructing a recombinant expression vector, and expressing a novel duck reovirus sigma C protein recombinant antigen through a prokaryotic expression system;

the primer pair comprises:

5'-ATGGACGGATTAACTCAACAGCAGCGA-3' as upstream primer sigma c-F; (SEQ ID NO.1)

The downstream primer sigma C-R is 5'-ACCTAAGGTGTGGCGCCGTACGGG-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 the purified novel duck reovirus sigma C protein recombinant antigen.

Preferably, the coating amount of the novel duck reovirus sigma C 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 goat anti-duck antibody.

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

the preservation number of the novel duck reovirus is CCTCC NO: v201818.

In a second aspect of the invention, a novel indirect ELISA detection method for duck reovirus antibodies is provided, which comprises the following steps:

(1) coating: the preservation number is CCTCC NO: v201818, 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% of 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: the enzyme-labeled goat anti-duck antibody is prepared by using PBST according to the proportion of 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 number is 0.071 or more, the test piece is judged to be positive.

In the step (1), the novel duck reovirus sigma C recombinant protein is prepared by the following method:

the preservation number is CCTCC NO: v201818, using the whole gene sequence of the novel duck reovirus as a template, amplifying by using a primer pair to obtain a sigma C gene segment, and constructing a recombinant expression vector; transforming the recombinant expression vector into BL21(DE3) competent cells, and adding IPTG (isopropyl-beta-thiogalactoside) for induction expression; obtaining purified duck reovirus sigma C recombinant protein after denaturation and renaturation of the expression product;

the primer pair comprises:

5'-ATGGACGGATTAACTCAACAGCAGCGA-3' as upstream primer sigma c-F; (SEQ ID NO.1)

The downstream primer sigma C-R is 5'-ACCTAAGGTGTGGCGCCGTACGGG-3'. (SEQ ID NO.2)

The invention has the beneficial effects that:

aiming at the newly found novel duck reovirus capable of causing tarsal joint swelling of ducklings, the invention establishes an indirect ELISA detection method for detecting the novel duck reovirus antibody, the detection method has the characteristics of simplicity, convenience, rapidness, stability, strong specificity, high sensitivity and the like, can detect the sigma C specific antibody of the novel duck reovirus in a duck group, and detects the antibody level of the reovirus in the duck group by taking the sigma C specific antibody as the basis, thereby monitoring the epidemic situation of the novel duck reovirus in the duck group.

Drawings

FIG. 1: SDS-PAGE analysis picture of the induction expression and protein purification of the recombinant protein sigma C in BL21 competence; wherein, lane M is a protein standard; lane 1 is empty, lane 2 is uninduced whole bacteria, lane 3 is supernatant after lysis of the induced bacteria, and lane 4 is precipitate after lysis of the induced bacteria.

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 described in the background art, in 2016, the ducks in Shandong, Hebei, Henan and Jiangsu in China have wide-range outbreaks of infectious diseases with tarsal joint swelling as the main symptom. The symptoms of the disease are different from the prior duck reovirus infection, and the symptoms of tarsal joint swelling appear in the duck group for the first time, so that the infectious disease is probably caused by the novel reovirus. At present, no medicine and method capable of effectively controlling the novel duck reovirus infection exist. Based on the detection, the invention provides an indirect ELISA detection kit capable of detecting the novel duck reovirus antibody, so that infected ducks can be found as soon as possible and isolation or killing measures can be taken on the infected ducks, and the loss caused by the novel duck reovirus infection can be reduced.

It is known that the host range of avian reovirus is wide, the virus is generally existed in various poultry, and the reovirus has been isolated from chicken, goose, pigeon, ostrich, duck, turkey and other pheasant and other sick or healthy birds since 1954, but the reovirus isolated from different hosts has large variability, and the pathogenicity, gene sequence and main protein coding of the reovirus are different.

The inventor of the application separates a strain of duck reovirus N-DAV-SD16 from the swollen tendon tissue of diseased duck, carries out whole genome sequencing on the newly separated duck reovirus N-DAV-SD16, and respectively carries out sequence comparison and homology analysis on 10 gene segments and the existing reported avian reovirus, and as a result, the L1, the L2, the L3, the M1, the M2, the S3 and the S4 in the 10 gene segments of the newly separated strain N-DAV-SD16 are all located in a relatively independent branch, and have lower homology with the duck source reovirus which is uploaded in Genbank at present, which indicates that the newly separated strain N-DAV-SD16 is different from other avian reoviruses, and can determine that the duck reovirus found in the invention has larger variation with the existing reported duck reovirus and is 1 single species of orthoreovirus, the novel duck reovirus is preserved in China center for type culture Collection with the preservation number of CCTCC NO: v201818. Therefore, the novel duck reovirus to be detected by the invention and the duck reovirus reported in the prior art have variability, no effective detection method for the reported duck reovirus exists in the prior art, and the difficulty in detecting the novel duck reovirus is higher.

The invention firstly uses a serological method to detect the antibody of the novel duck reovirus. Can quickly and effectively detect the novel duck reovirus antibody which is newly outbreaked and mainly characterized by tarsal joint swelling, and is a simple indirect ELISA detection method suitable for basic use.

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

(1) preparation of coating antigen:

the whole gene sequence of the novel duck reovirus is obtained by a second-generation sequencing technology, and the sequences of 10 gene segments of L1, L2, L3, M1, M2, M3, S1, S2, S3 and S4 in the whole genome sequence are respectively shown as SEQ ID NO.3-SEQ ID NO. 12. According to the sigma c protein gene sequence of the obtained novel duck reovirus, two primers are designed:

upstream primer sigma c-F: 5'-ATGGACGGATTAACTCAACAGCAGCGA-3'

The downstream primer sigma C-R is 5'-ACCTAAGGTGTGGCGCCGTACGGG-3'.

Amplifying the cDNA obtained by reverse transcription by using the primer through a common PCR method to obtain a sigma C gene sequence (shown as SEQ ID NO. 13) with the length of 1000bp, purifying the cDNA by gel electrophoresis and gel recovery, cloning the purified cDNA into a prokaryotic expression vector PET-28a (+), and constructing a recombinant prokaryotic expression vector PET28 a-sigma C; the recombinant prokaryotic expression vector PET-sigma C is transformed into BL21(DE3) competent cells, the recombinant protein sigma C is efficiently expressed by induction of 1mM IPTG, the protein exists in the form of inclusion bodies, and the inclusion bodies are denatured and renatured, and the purified reovirus sigma C protein is used as envelope antigen.

(2) And (3) establishing indirect ELISA by taking the purified reosigma C recombinant protein 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 goat duck resistant enzyme-labeled secondary antibody is as follows: diluting goat anti-duck enzyme-labeled secondary antibody with PBST, drying after reacting at 37 deg.C for 1h with 100 μ L per well, washing 4 times with PBST, each time for 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 to each 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 number is 0.071 or more, the test piece is judged to be positive.

For the indirect ELISA detection of the novel duck reovirus, the selection of the coating antigen is very critical, and the specificity of the detection method is directly determined. In order to optimally select the envelope antigen, primer pairs are respectively designed according to the coding region of sigma C protein in the gene sequence of the avian reovirus published on GenBank and the main antigen site region of the novel duck reovirus in the test process, and are amplified to obtain gene segments so as 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 reovirus sigma C protein as the envelope antigen to the detection of the serum sample which is confirmed reaches 100 percent, and the detection sensitivity and specificity are obviously superior to those of the indirect ELISA detection kit prepared by using other recombinant proteins as the envelope antigen.

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, H is 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: a pair of primers is designed according to the sigma c protein coding region of the whole gene sequence of the novel duck reovirus (the preservation number is CCTCC NO: V201818), the two sections of the primers are respectively provided with BamHI enzyme cutting sites and SalI enzyme cutting sites, and a gene segment of 1000bp in the genome of the novel duck reovirus can be expected to be amplified.

5'-ATGGACGGATTAACTCAACAGCAGCGA-3' as upstream primer sigma c-F;

the downstream primer sigma C-R is 5'-AGTCGACTTAGGTATCGATGCCCGT-3'.

1.2 construction of prokaryotic expression vector PET28 a-sigma C:

amplifying a sigma C gene fragment by adopting specific upstream and downstream primers through PCR, connecting the purified fragment with a PMD18-T vector, transforming a connecting product into DH5 alpha competent cells, and selecting positive bacterial colony shake bacteria to extract a T-sigma recombinant plasmid. The T-sigma recombinant plasmid and the PET-28a (+) vector were double-digested with restriction enzymes BamHI and SalI (available from Dalibao Bio Inc.), respectively, and the two 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 single positive clone colony is selected, and the result shows that the target band about 1000bp can be amplified and is consistent with the size of the predicted sigma C 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 sigma C gene insertion position, the insertion direction and the reading frame are correct, and the result shows that the recombinant vector PET28 a-sigma C is successfully constructed.

1.3 recombinant protein sigma C induced expression and identification:

transforming the successfully constructed recombinant vector PET28 a-sigma C plasmid into BL21 competent cells, selecting a single colony for enrichment culture, and inoculating the single colony in a ratio of 1:100 in the next day100μg/ml kan⁺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. 2mL of induced and non-induced bacteria liquid are respectively taken, and after centrifugation, bacterial precipitates are collected and are resuspended by 50 mu 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 the recombinant protein σ C was successfully expressed in BL21 competent cells, all in the form of inclusion bodies, with a protein molecular weight of 50kDa, consistent with the expected size of recombinant σ C (see fig. 1).

1.5Western 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: in contrast to SDS-PAGE, PET28a- σ C-transformed BL21 competent cells induced a specific band at the position corresponding to Western Blotting, whereas the control non-induced bacteria did not. The result shows that the recombinant protein sigmaC can generate specific antigen-antibody reaction with the antibody in the positive serum.

Example 2: indirect ELISA detection of novel duck reovirus antibody

The purified recombinant protein sigma C 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 sigma C protein coating amount of the wells to be 500 ng/well;

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 90 μ L of 5% skimmed milk powder diluted by PBST into each well, 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 goat anti-duck antibody are as follows: enzyme-labeled goat anti-duck antibody was prepared using PBST according to the following formula 1: diluting 500, adding 100 μ L of the solution into each well, performing action at 37 deg.C for 1h, spin-drying, 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 number is 0.071 or more, the test piece 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 duck parvovirus, duck tembusu virus, duck adenovirus, duck influenza virus, duck reovirus NDRV-JM85 strain and duck reovirus NDRV-TH11 strain, and the method only reacts with the positive serum of novel duck reovirus (CCTCC NO: V201818) to show 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 duck reovirus antibody and application

<130> 2018

<160> 13

<170> PatentIn version 3.5

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acctaaggtg tggcgccgta cggg 24

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aagaacctgc ggatagtgcc acagataaga aagtcactgt tccatcacca gacaatccag 180

ccgcttctac tccctcttcc actgatggag cttctcaaac atccgttgct aagcagacga 240

atgataatga cgcctcagtt aaggaatcag ctcccaagcc taccgtatct agcgatggga 300

aagacggaat gcatggtgct gtgaagttgc aagacgctaa ggccactgca gctgtggata 360

gtaataagga tagagatgtg gtatttggtg gcgcaggctc tggtgacaaa aatgctatta 420

cgaagactgg ttccgttgac aatgatgggg gcgttaaagt cgttccagcc aaggatgcta 480

cgatatcttc ggccaaagcc atgatggaac agaagcagtt agttgctggt cttccgaagc 540

aaccgaagtc tgctaatcat ttgtgcaccg tttgtatggc acaattcgcg tctgctgacg 600

ccctaactat ccatcagact acgcactcca ttggctccaa tgctgctctg acgagcttct 660

cgatttctac tgctgttgaa gaattcattc aatcatgggc tgctgctacg tccactgcca 720

ataccaagac ggccttaact gtgtctgacg tggactcgct gatgatgact gaaggaatac 780

gtctcataac ttgggattct gggttatgta catcttttga gcttgtcccg attgtccatt 840

caaacactgt tcaagatgta atttcgtact catggttcac atcaagttat aatatcacaa 900

ctcccttccc acaggcgcct gtcgtgcgaa ttgttttacg tactaattgg gctgccaaat 960

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attttactca gcaaagattg gctgcagcgt tcaacaatcc cacgacctgg cctcaatgtt 2100

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tggttgatct ggttctgcgc atcatcgaca gtggtcggta ccagcccaat tggaatcagt 2400

ccatccgtgc ctctatgcgg aatgcgatga caaatttcag aattattaag tcctatactc 2460

ctgcttacat agcagaactg ctacctgtgg aactggcagc tatcgctcca actctaccct 2520

tccagccttt tcaggtgccg tttgcccgct tagatcgcga cgctatcgtc actcacgtca 2580

atgtatctag acaagctccc aacaatcttg ctcaacctgc attgaacatg tccatgacgt 2640

accagcgcac aggggttcca atctctctta gtgcccgtcc cttggcagtc gctcttctgt 2700

caggccagta ccctactgat ccccctcttc agactaatgt ttggtacgta aacactctca 2760

cgcctctata ttccaatgat ggtctcttta ataacgtcca gcacgctatg gttgcttctg 2820

aagcttacgc taccttgatc accatgctgg ctcagtgcac tgacatgcag taccctgtgg 2880

atcggccatt gaattggctt cgtcagatta atttggctgc taatgaggcg acgattttcg 2940

gtcgatcaat taattcactt ttccagactg ctttcgacct ttcaccttcc actgtattgc 3000

ttcaaccgtt tttggaatct gatccacgtg cgacacagct agccatttct tacgttcgtt 3060

ataatggtga cagtgaaacc ttcgtgccaa cagtgcgtcc atctatgatt tcagaagcga 3120

cattgctcgt tgagcgtact ctctcgcacg aatacaacct cttcggttta tgccgtggtg 3180

acatcatcct gggacagcac atgactccaa ctgcgttcaa tcctttggct ccgcctcctt 3240

ccgtcgtttt taacaggggt gatactgatg tctatgagtt tggctctcgt agcttcgcca 3300

acttcggcat gaatggggag gagatcttgg tcatggatgc gaacggcgtg cgtcgtccat 3360

tactcggccg gtgggttatg ccactgcagc ttttgatggt taatattggc gtatttccta 3420

agctgttgct ggatcgtatc ctgaaggggc gcttgtatat ccgacttgaa gttggcgcgt 3480

atccatatac ggtgcagtat taccagggac gtgagtttac ggatggcttc actctgcttg 3540

agcaatggat gtctaaggtg tcgcccatgg gtatccctcc cgtccctttc ctcatgccgc 3600

agtccgaagg acacaacatc acttcaggca tggttactca ttacatctgg tccactgaat 3660

ataatgatgg gtccctcttc gccacgaaca ccgacctacc agttactgtg ttcggccctg 3720

accgcaccat cccaatcgag cgttatcggg cactcgtgga tccaggtgct cttcccgcta 3780

ctaaccaact gccgcacacc attgatcttt actgctcact gagacggtac tacttggaga 3840

cacctcccat cactgcgacc gtcaccactt atggcgatgg actccccgcg ctgaaccatt 3900

agagcggcga ggctagacgc gagctgat 3928

<210> 4

<211> 3830

<212> DNA

<213> L2 Gene fragment

<400> 4

gctttttcct caccatgcat gtcaacgggt ttgatgatgc tactctctcc tacgcacagt 60

ccatatcggg ggttatccca atgacaaata agttatttga gcaagcatct acctcaatac 120

gtgccctacc acgctcacat gtttatgcta tattagataa tgttaatttt tcagtttcat 180

gtgtcatccc gaatcgtatt ttccatcatc ctgatcactc tgagtacttt tacattgatg 240

cggttaacag agttagacgg aaacaagtta tcgatcctga tgatgtattt gtgccaaact 300

gcaatttgca gggtcttatt actccaatgg agagattacc aaattatggt caattgtctg 360

agactatttc gtcgaacgct cgggatggct tgccatctgc acgcgtagca gctacttttt 420

ataacacttc ggtgtcacag gctcgccaag ttaaagctcc acttgaatcc tttttgttgc 480

ctttgctatt atctgagact tgctcattat cagacgatcc ttgcggactt gacaccgcag 540

cttctcctcc gatccatacc aatctggcgt tatgggtgtt acgcgaaatt agtcgaacta 600

tttgtggatc ttcgaatgac cgttcacctt ggttactgct cgattcaggg gttgcgtggt 660

tcatgtctcc gttaatgtca tcggccattc cacctcttat ggctgactta acgaatctag 720

cgatctacaa acaaatttgt tctgtgtctg atgagcttca ttcccttgca gttcaagtgg 780

ttttgcaggc cgcggcgtca caatcgtatg gacactacat attgcagacg aagtcagtat 840

tcccccaaaa tacgttacat aacatgtttc gtacgctcac tgatggtatt gttccggtca 900

tagattggtt agaaccgcgt tcaaactatc gcttcatgct tcaaggtgcg cgtaaagtga 960

cttcagatga tgcgaatcag gctccggata acacggaagc cgcggagcaa ctcggtcgca 1020

agatggggtg cctcgatgtc gtacgctcct tacgtaagat gtcctcgtcc atcactgtcc 1080

attcacacga tgccatgacc ttcgtacgtg atgctatgtc gtgcactagt ggcatcttta 1140

tcacgcgtca acctactgag accgttttaa aagagtacac tcaagcgccg accattgaag 1200

tccctattcc tcaatcggac tggtcaccgc ctattggatc tctgcgatat ctctctgatg 1260

cctgttccct ccccgctgtg tatctggcta gggcttggcg cagagctgcc tctgctgtag 1320

tagataatcc gcacacttgg gaccctttat atcaggccat ccttcgctct caatatgtga 1380

cgtcacgcgg tgggtccggt gccgcgttaa gagatgcttt gaaggctgcg gaagttgagc 1440

ttcctcagta tcctggggtc agtgttaaag tggcgaccaa gatttatcaa gcggctcaga 1500

ctgctgacgt gcctttcgat aaattatctc gtgctgttct agctccattg tcgatgggct 1560

tacgtaacca agttcagcga cgtccaagaa ccatcatgcc catgaatgtg gttcaacagc 1620

agatttcagc ggctcatact ctctccgctg actacattaa ttatcacatg aacttgtcga 1680

cgacctcggg tagcgctgtg attgagaagg tggttccatt aggtatgtat gcatcctgtc 1740

ctcccgctca agcggttaat attgatatta aggcttgtga cgcgtccatc acgtaccagt 1800

attttctttc tgttatcgtc ggtgccattc atgagggtgc agcaggccgt cgtgtctcgt 1860

cttcattcat gggcgttcca ccaagcgtgc tgtccgttgt cgatgctagc ggagtgactt 1920

catccatgcc catctcaggt tttcaagtca tgtgtcaatg gttggctaaa ctttaccagc 1980

gaggttttga gtatcaagtg acggatacat tctcacctgg caatattttc acgcatcaca 2040

ctactacttt tccctctggt tcaacagcga cgtctacaga acatactgcc aataatagca 2100

cgatgatgga tggcttcctg cgcgcttgga ttccttcctc cggtgcgtcc gacgtactga 2160

agaagttctg caaatccatc tcaatacaac ggaattacgt ttgtcagggt gacgatggtc 2220

taatggtcgt tgatgggcta tcgacaggta aattatcagg cgagataatc gatgaatttg 2280

ttaaggaatt gagagcctat ggtaaatcat ttgggtggaa ctatgatata gagtttaccg 2340

gaaatgcgga gtatctaaag ttgtatttcc taaacggttg ccgtatacct aatgtttctc 2400

gacatccgat ctgtggcaaa gagcgcgctt caggggacaa gttagaaatg tggccgtcca 2460

ccattgacat cttcaatggc atatttgtga atggtgtgca tgatggtttg ccgtggcgca 2520

gatggttacg ttattgttgg gctcttgctc ttatgtattc tggaaagacc gtgcgtcatg 2580

atgattctga ggtgttgatc caatatccta tgtggtcttt tgtgtattgg ggtttgcctc 2640

ccgtgagcgc gtttgggtct gatccatgga tcttttctcc atatatgccc actggtgatc 2700

atggtttcta ttcaatgttg actttagtgc gccctctgat cactaacttg tccccatcgt 2760

cagacgcttc gggattattt ggtcaatgcg atcacaacgt tttgttcaat tctgagctag 2820

tttatcaggg ctattacatg gctcaatgcc cacggcaacc ttctcgctcg aaccgtagag 2880

atgatcccga ctctgtacag cgcttcgtca aggccttgga gtcttacctt tacatttccc 2940

ctgagctaaa atcgcgagtg cgccttggtc gtgaccgctg gcagaagttg gttgggtata 3000

cggaaaaatc tcccccgtcg cttgatgacg tggcgttcaa atggttccgt agtgcacagg 3060

aagctgatct cccaaccgct acagagattc aaagcatgga tctggccttg ctggcagcca 3120

gacgtaggac atatcagggc ttctccaagt tgttgaatac ctatttaagg gtaacctggg 3180

atttatctga tcctgttgaa cacgctgtag atccccgcgt acccttgtgt gccggtgtct 3240

ctccatcaaa tagcgagccg ttcctcaaat tgtactccgt aggtcctatg atgcaatcca 3300

cgcgtaaata ctttagcaat acgctattta tccatcggac tgtgtctggt cttgacgtcg 3360

atgtcgtcga tcgtgcgctc cttaggttgc gtgcccttaa tgcgcctgat gacgtggttg 3420

tagctcaact tttgatggta gggttgtctg aagccgaagc cgctacatta gcagcgaaaa 3480

tacggacgat ggatatcaat gccgtgcaat tggccagagt tgtcaactta tccatccctg 3540

actcatggat gaccatggat tttgatcgct tgatacgaga tatcgtgtct gtcactcctc 3600

tgaccgttcg atccctaacc accgatctac cctctggcgt accgtgggct cgcgcgatct 3660

tacagttctt aggtgcgggt gtcgccatga cggccgtcgg acccttgcgt cgtccctact 3720

tacactcagt tgccggaggc atgtcttcat tcattaagca gttccgccgg tggatgcgtg 3780

ccgaaacgag gtagcgtccg tgcccggcat ggctcgagga attactcatc 3830

<210> 5

<211> 3876

<212> DNA

<213> L3 Gene fragment

<400> 5

gcttttccac ccatggctca gattcgaggc cttcggttgt ctacgacgct ctcagctcca 60

cctccacgca agattataac atctcacact tatgatgagc tgatctccgc tctgaagtta 120

gcaaccaagc cttggcgccc tttgaagtcg cgaaataatg attctgtcac ggcagtgcag 180

ctcctttttc cccttaatgg ttatattgaa cccatgttca tgttggaaaa ggatatgacc 240

cttagtgatt ttgaggcctg gttgacgcct cttctatctg cactcgctga ccagttgctt 300

agacactacc ctatcgccgc ctatcacggg cgtttgatta atccgctgct atctaatgca 360

attgttgccg ctttcttgtc taacgtgcct tatgcgcatg cattggatca tctcttcctt 420

gttagaggaa acgtcgagga tattatggat gcggggatcg caattcagaa tcacttgtgg 480

ttcgatcgcg gtgcactagt gacccctgct ggacagaaat ttgttcagct gactggctat 540

aacttctcct ctaatgatcc gtgtctattt tctaagcaat tgcgttgtta tggcctcgtt 600

tactactttc tcgacatggc cgaatgtctg gcgtattgtt ggcgtcatct atccaattca 660

actccactga tacactttga ccgtccgtcc aatggagttc attgtttggt gccctctgaa 720

tccacgacgc ctatcgctgg ttcgttacca gtgtcagcac tcagctctat tttgttggaa 780

tcctgtcttc agcaatctac aattaatgcc cttactccca ctggttcgcc cgtcattaga 840

caaatagaag cattgttgcc tatatcatca ccgtttttcg aacgacggaa cactctggaa 900

tattctctct tcgcactgtc aaatgctctg gtaaatggtt atcagcttgt agacttgcgt 960

tccggccacc ctgattgcgc tactgttgct gccatcctag ctagattgat tgatttctct 1020

aaggatatca ccgttattca accgcgccct gctctctttg ctatcaacag cgacagtccg 1080

cttacgtata gtggagaaaa tgctaatttt atttcgcgct tgacttgtgc gtccggtaga 1140

cctattggtc cggtcgttgt cggtaaatcc gttgatcatg ccgttggttg gatgccccag 1200

tttgaccccg ccacgtccta caaccctgat ctctcgatgg actcacttgc tcgtgccacg 1260

acactgcctc tccgtgctaa gtattcgact ttctggtcag gcccagcatt gttttctttc 1320

gcttcatgta ataggcacaa tggtgtatat gacatacagt tcatggctca atttcctcct 1380

acttacttta gtgatgatga cgccttttct agatcacgat tctcttctta tcgtgcagtt 1440

agggaccggt cattgttgaa ggataccgct aatttgatgt acatctcgaa tttgtccagc 1500

tctcacgacc atcgtcttgt cccagattct aaaactatga tttatgtggg ttcctctggt 1560

actcatgtag ataatcaacc ttctatcatt aaacctctct tagctggaac tcttccgggt 1620

gtttttcgcc ccctttctat aaaacaggtt ggttgggagg tcactaatgg aacgatttgt 1680

gatgttgagc ttcctttagc cactggtacg ttcttcttcg tgtacagtga tgtggatcaa 1740

gtgcaatcag gtgattctga tttggacgcc tcctcgcgtc gcttttgctc ccaattggac 1800

atgctaatga agttgacgtt tactggtgga tcgcttgtcg ttaaatgcaa ttttccgact 1860

agtctagtgt ggcgtcacat cttctccact gtttctcctt atttctcggc tattcattta 1920

atgaagcctc tcgtgtcaaa taatttagaa ctgtatctat tgtttgcgga gcgtttgccc 1980

gttcctgacg tcgcgttccg tccttcagcg gacgttgtcg ttttctggcg atcacagcta 2040

caacgctatc gagtgttgcg tgattccttt tctaatgtgc cctccatcgg gtccactctc 2100

actttagatg aacctttgac tgtctctatt ctcaattttg ttgacgtcac ctccctttct 2160

tctcttgagg atcaacgagc cttatctgct ttttcagttt taacttctct agggtcacag 2220

aaactctcgc ttcatcctta ctttgatagc taccgcacgc agctcactgg aataattact 2280

ccacattcac gtaatcttct agatagactg gcgtacgtcc cgcgcgtttt tccttcgacg 2340

attgatgtgc aacatcgtgt catggcctct tcagatccag aaatttttgg ttttcgttct 2400

aattcatgga ctcaactgtc cttcttctac gacgcgacgt taacttctat tgattttact 2460

gatgtaaagc actggttaga tttagggacc gggcctgagg cgcgaccgtt gtcttttctg 2520

ccttccgatc ttcccgtcac gttatgtgac actcgtccat tcatcttccc ttccggctgc 2580

tgggctactt tcactgattt cttaagttat gactaccttg tcacgaatgt cgtgctctca 2640

actggtgccg atgtcgtatc ctgtgttctt tctctgggtg cggcctgtgc tgatgccaac 2700

ataactttac atgaaggcgt gcggcagctg atttcccaat gcgtggatgc caatgtcaag 2760

acattgttcc tgcagcttaa ttgtcctctc ccatctgcgg gtgatgtatc tcgggagatt 2820

cttgagatgg ttcagactaa ttcaacttac gtgtttcata ccttgggtcg tattgaaccg 2880

ttcattccat actccgctct ttcagagata gttgaggact tgtgtcccgg catcgtcatt 2940

gaaattaaga ctatggatcc ctctctctca tggcttgatt acgctgttca atccaatgcg 3000

tcagtgacgt cggatgatat cgtattggca atgcgtctgt ctcacttctg tccacttttc 3060

gtgtttcatt ttgaccgtca gtctgctcaa tttccggatg atgcgcgtgt tgggactcct 3120

tttactgtca cgctgttaga ttatgaagat actcgttcat acgaggtgac gttagataat 3180

gtcactatcg ccaccgttac cgcaggtgct ttggtgggtt tctcatctgg tgtcagcgtc 3240

agttcaacca acaatcagct tattttaact atcgattccg caagtccagg aattctctcg 3300

gtcattcaga ttcttcccgc tcgtatctct ttaggcagtt gtgtgataga agcaccggat 3360

ccatctctct ccttgatctt ccccgccacg ttagatacct ctttgtcggg aaccgatttg 3420

gagctgtatt tgtctgactg gtacgatgtt gcattatttt acgtcgatga aatccactcc 3480

cgcttgctgc cagtgtccga taccaagtat gaaatatatc gcaaggatca ggcgccgaac 3540

agccgggtga tcaactatat tttcgatcgg tctgatgtgt ttttcaagct agtgttatgt 3600

gacgtatctc cctcaggagt aggccgtttc atctaccgtg agttgccaga attaagttca 3660

cctgtttggc cagacaacgc gcgcactttc ttgtccatac cgttcgagtc acccatggtg 3720

attgtctcgc cggacggacc tgttaattac gatggcgcaa actttactcc tccaaactca 3780

tggttgacgg ttgatggcag tacctgcgtt gtagatggcc gtccttcgtt ctacgtgccc 3840

cctggccgat atggtctggt gagagtctaa acgacc 3876

<210> 6

<211> 2199

<212> DNA

<213> M1 Gene fragment

<400> 6

atggcctatc tagccacacc tgtgctagga gtcggttctc gcattaccgc tttagatcgt 60

actattgatg ctatcacgtt gaaacctcga atcgacttac aagatgtata cacaattgat 120

cccactttga ctctgcgtca gatagagtta atctcttcgg gaacttcaat ggacgatatc 180

gctcgtggac tgttgcaccg agactggcgt cgtcaatcca tcatcgtttt gcttccctcg 240

cgtcgctctc tccttgagta tctattgtct aacccttctg tctgtccaga cggtttagat 300

cgttctcgac ttaaaggatt tcagaagcgt ccaaatgatt ttcgtgttca agatttcttc 360

tctccactga tcacggactc gacgtcaatt gctacatact ctcgatggct taatgcccac 420

cctgttgtgt actcaactac tcataaggtc gctggtgctc gggtgcgtct ctttggacct 480

gccaaattat acattctgtc acctgacgtt cttcgcgaat tatccatttt gagatccacg 540

gatcgtgtcc tcgttgtacc tacagcacgt gtatatgttg gttgctttcc tagcgcttcc 600

actagtaatt gtgtgctcac tgcacgcgac cgctggaatg ctcctgacgt tcatcccgtt 660

gtcaaggcaa tccaattagc atatgaccat caatatcgtg tcaccgctcg ctatctttcg 720

gatccccttg tctccgccct tcttgttggg aatcggtcgg tgaagacctt gaaggtacag 780

ccagtagagg ccagagcagc acgatcagtc ggcattcgcg ttcaagcgat gacgccccct 840

cgtggtatca acacctctat catccaagtc gttgatctca ggctgcaatg tcgacattct 900

ctcattccca ccgaaaggcc cttcccgctg acatttatcg gcctcccatc ctgtttgctc 960

cagcatttgg atttgacgct atctgacgat tgggtgccta ttcgtgatcc cacgggcatg 1020

tttgaaatgt ggttcatgat tcttacgctc acttgtgata agattcttga tggacggggc 1080

aacgctgttt ttctcatccc cagttctact aatgcattgt cgattaatta tgtacagctt 1140

acatcgaccg cgtctcaacg ccctcagtca ttagcggcaa atgcatctgg acggatagat 1200

tctatcggac tatgtatgcc taaggggtct tttaagtcaa ctatgattaa atttctcact 1260

ggcttggaga tttgcggcac acgagtgatg tactcggacg tcgtgatgga cagtgatgac 1320

gtgggtgacg ctttggatcc tacttttgaa acggctttgt atgatgcact ggtagcactt 1380

gatccgcctt ttgacgttga taagttggct agccccactg atctagttaa tcaggagtac 1440

gttgcgtctc atatgtaccc gacattctta cggcttgtca atgagctgct gactcctaag 1500

gcttcagagt tgtactctga gcgtagcgtt gaattccgat ctcttactta cgcgcacgct 1560

gattctgaat ttcttaactc atgctggacc gctcgcttaa tgcgttgctt tatcaactat 1620

catgaagagc agaatatctt acttcgtcct ggacgcgttg gtggggtgtt atttcaagtc 1680

gcgttgagcc gttgctataa gatgttcgct acttccactc ctgcttcccc tctgtcattg 1740

ttcctcaagt cgttgttcgt tccttggatt gagtctgccc cactgttagc gaatctaacg 1800

ccaaatgagt cttctcgtgt gttagcatgg tatattcctt cctcgtactg gagcgacaat 1860

ggttggtgcg tttgtgacac tcatcgtcac gtcaccttct ccttcatccg cggtcttccc 1920

gccgacctgt cggtgttaga tctgtttgat tggtctcgat tccgcgcgac tataaacgtg 1980

gacacgtctc tcgtggagct aggcgcagac attcgtgcgg tcaaagtatc agtccattgg 2040

acatctcaga agcccactgt ggacgttttt gacaatcgtg cgcttttcac ccccttccag 2100

cactaccatt tgagtctcca ctgtaattgc gcacctggtc gacctttctt cgcgaagaac 2160

atgaagctat atttgtcgac ggtaggaggc gagcactga 2199

<210> 7

<211> 2208

<212> DNA

<213> M2 Gene fragment

<400> 7

accaccgaga tctacactct ttccctacac gacgctcttc cgatctcttt ctcacaagat 60

gggtaacgcg acgtctgttg tgcagaactt caatatccaa ggtgatggta atcattttgc 120

tccatctgct gagactactt catccgccgt accgtcatta tctttgaatc ccggactgtt 180

aaatccaggt ggtaaggcgt gggtcctgat tgatccatct ctaaatgctt ccgatccttc 240

atcactacgt ctgatgactt cggctgatct atcaacactt cctcgatctg ctactagtaa 300

ctctaccggg tttctcccca cttctggcat gtatgccatt gctactaagg agacgttgag 360

tgtaattact gagcacgcga tttcccagtt tgataagtta cagatggctt gtgagttgga 420

ccgcgattat ctggatgcta gaggtgtttc tcctgagtct gtgaatattc atagttatat 480

agcctacgtt gattgcttcg tgggtgtatc tgcaaggcag gctgcgtcaa attttaagcg 540

gcatgtgcca gttatcacca aatctcgtat gacacaattt atgacatccg cgcagaatat 600

gttgcaagtg cttgggccct gggaacgtga tgttcgtgag ttactcacta ttcttcctac 660

ttccactacc gctggtaaaa ttacgtgcga catgaagtct gttgtcgctt tcattgatga 720

tcagctctct gataccagtt tgtgtcgtct gtaccccgac tgtgctgctg cggcggtggc 780

tagacgtaat ggtggcattc gatggaagac acctgatact gacgaggctc cttcacttgc 840

aactaacgat attgctgctt caactatggg tacgcttgcg aatactacac cactggctga 900

gaagtcgaac tcgggcgagg agtcgatgcg cttggttagt gatgttggcg tggacatcgt 960

ttgttctcgt ggccccatca gttcttcagt ttggtcccgc acggttgaac ccaaatcgta 1020

caatattaga acccttcgtg tagaagaagc gctttggcta cgcgagtgcc aagcgactac 1080

tggttttgat gtacagtaca cgctgcccga ccagactaca cataagcatt tctggcttca 1140

gaaggggtca gtcgtcataa atcttgagca aacgggtagt atgatgttcg atgtgaacat 1200

agcgggtaaa gattacaaga agggcacctt taatcctgat aatcataaat tggtcctctt 1260

ggttatgcag tcaaagatcc ctttcgagtc ttggaccgtc gcttctcaaa ttactggtat 1320

cgctcaagtg gctgaggtca ctgtgcatgc tgctgatagt tcgactccta accaaaagat 1380

aatcggtgaa acttcgctgt cttatttatt tgagagggag acggtgacca catccaatac 1440

tgaagtcaat acatatctgt tgtgcacttg gcagcttgac gacgcgcaga gcaatgacgc 1500

aaacgcctgg ccagatgctt gggacgggat cacaacattg accccactta cgtccggtac 1560

tgtaaccatc aaggggactt cggtggattc tgtcgtaccg tctgatttag ttggtgctta 1620

tacacctgag gctttggctg ccgcgcttcc taacgacgct gggttaattt tggctaataa 1680

ggcaactaaa ttggctgacg ccatcaataa ggaggatgat tctgtgattg atgagtcttc 1740

tccctttagc acccccattc aaggagttct ggctgttcaa caacttgata ccgtggggac 1800

acgcggtaca cgtgcactcc agcctccatc cattctgaaa cgcatcgcct cacgagctct 1860

tcacatgttt cttggtgatc caaagtctat tctaaaacag gcgacgcccg tattgaggga 1920

ccctgacgtt tggaccggct ttgttcaagg tgttagagac ggcatccgga ctaagtcgct 1980

atccgctgga gtacggtccg tgtataataa cgttaccgcc acacagtctg tacaaacgtg 2040

gaaacagggg ttcctgacga aaatacagac gttgttcaag ccatcgtgag gtgctaaggc 2100

ctctctctgc ggcgggtcgg tgggcacgtc gtagtgacgc tgaatgcacg gggaggtgac 2160

gctccctgga ttggcaagat cggaagagcg tcgtgtaggg aaagagtg 2208

<210> 8

<211> 1996

<212> DNA

<213> M3 Gene fragment

<400> 8

gctttttgag tcctagcgtg gatcatggcg tcaaccaagt ggggagacaa gccgatgtcg 60

ctctcaatgt ctcacgatgg atcatctatc cgcagtgccg cctcacagtt tctgtcggtt 120

cccctgtctc actcaacgcc aatcccacct caacggaaga ccgtattgct gaaattcatg 180

atcggtgatg acctggttac cgtgcagggc gcgctcgctc cttttgatga atactggtac 240

gataaccaac ctctattgtc tcaagctgtt gagctgctcg cctctgagga tcgtctgcgt 300

caattcgagc attatgagaa gtttctactt aagaagggcc accaaattgc tgagatcatg 360

aataggctac gtcttttctt caccgacgtt ctcaaagtga agatggaagc tgatgctctt 420

ccttctctag ctcaatacct gatggctggt actttggatg ctgtctccac cgttcacgaa 480

cctgatgctt gtgttccagt cacttcaaaa atcatagcta agcagcagac tgtgtccaag 540

tcccctggac gtcttgctga agaggagtat aatgttatta gatcacgttt cctcactcat 600

gagatctttg acttaacgtc tgacttgccc ggtgttcaac cattcatgga tatgtactat 660

gccaccgttc ctcgtgccga ttccaccggt tggtgtgtct accgcagaaa aggtctattg 720

attcatgccc ctgatgagca atactcggat ctgactattt tcaccacccg tcttacggca 780

gcgcgtgagt tacagcttgt ggctggggag gtcgttgtgg cttgcttcga tcttatggat 840

atctctgata ttgctccatc tcatcatgca tcggttcaag aggaacgtac tctcggcact 900

agcaagtatt ccaacgttac agctaatgag catccgttgg tattcttttc acccaatgca 960

ttacgctggg caatagatca tgcctgtact gattccttga tttccactag gaatattcgg 1020

gtctgcgtcg gtattgaccc cttagtgacc agatggactc gcgatggcgt gcaggaggct 1080

gcaattctta tggatgacaa gctaccctca gcaggacgtg ctcggatggc tctacgaacc 1140

ttgcttctag cgcgtcgctc accaatgacg tccttcttac taggtgctct caagcagtcc 1200

ggtggtcagc taatggaaca ttatcgatgt gatgcggcta ataggtatgg atctcccacc 1260

attccagttt ctcaccctcc accgtgtcca aaatgtcctg agctgaaaga acagatcacc 1320

aaactttcgt cagctcctgc gcctaaagtt gactcgtccg ctggtcctgc cgtgctgttg 1380

tcgaagatcg ctgagctcca acgtgctaac cgagaactgt ccttgaagtt agtggacgtt 1440

cagccagccc gagaagacca ccttctagct tacctcaacg agcacgtatg tgttaacgct 1500

aaagatcacg agaaaggtct actagcccgt tgtaatgtct cgagtgattc aatcgccgct 1560

atccttggtc aacgtttgaa aaatcgagaa cggtttgaaa cgagactacg gcacgaggct 1620

ggtgcggagt gggagccacg agtggaagcg ttgaatcaag agttggctaa ggcgcgtatt 1680

gagcaacaag atatgatgac tcagtcctta cagtacctga atgaacgtga tgaactgctg 1740

cgcgaggtgg atgagctcaa acgcgaactg gctaccctac ggtttgctaa tgtgagacta 1800

aatgccgata accaccgcat gagtcgtgcg acccgtgttg gagatgtatt cgtcagtgat 1860

gttgatccct taccacccgg tcttcctggt gaatcgaaac catccattga agaactggta 1920

gatgatctgt gagctttgcc ttgtgactcg acttctctct gattccatgt acccacggcg 1980

gactcggcta ttcatc 1996

<210> 9

<211> 1644

<212> DNA

<213> S1 Gene fragment

<400> 9

gctttttcag tctcttgtat cgatgttccg tatgtcttcc ggttcatgca acggcgcgac 60

gtcaatattt ggtaacgtgc attgtcaagc ggcccaaaat accgcaggcg gagatcttca 120

agctacctcg tcattaattg cttattggcc ttatctcgct gctggtggtg gtctcattat 180

aattttaatt attgttatag gtataatctg ttgttgtaag gccaaagtta aggctgacgc 240

taccagaagt gtgttctatc gggagttgct tgctctgaac tcgggcaagt gtaatgcagc 300

acctccgtca tacgacgttt gatgtgcggc ggtttgagtt ttctccgacg gtgtttgaag 360

agtgtttgac tccatctttt accgctgtga ctgacactga ccctgtgagg tactttaata 420

ttgagcttcc gtcaactcac cgtctcctcc cttggcttcc cgttcttctg ttccaatcct 480

gtaaagtgca tgtttcttta gtacgtagat tctctttatg ctcgacttta tctgatattt 540

gtgagtacga ttgcaaattg cttccgtcta ttaacgctat tgtgtcgaat ccagtgtcga 600

gcgcggtttc atctatcgtc gttcactggg atggacggat taactcaaca gcagcgaaga 660

gaagtcgtgg ggttgatact gtcgttgact tcgagcgtga gtataagttc tggcgatttg 720

acgcaaattc gtgagcgtct ttccgctttg gaatctgcga ctgcgtcgct gaacgaatcc 780

gttaatacag ctttgtctag gttagtggat ttgtctgcat cgcttgataa cgtggcggcc 840

tcgttagcgg agacgaaagt ggaaatgaac tcactagttt ctgacgttca gggtttgcga 900

gcttcccttg actcttctgc ttcagagctg gcttctctat cttcattggt gcgtgatcac 960

ggctcttcga ttgctggcct acagagagaa gtaagtgcct tatcgagtga ggtaggcaac 1020

cttaaaacct cggtatcatc gcagggcctt cctatcacta gccttgagaa acgagtgcaa 1080

gctttagaag gtggttctag tacgactctg tcatttgctg atcctcttaa gttagaggct 1140

gggaccgtgt cactcgaggt agatccgtat ttctgctctg tgaatcgtaa tctgacgtcg 1200

tattctgctg atgctcagtt gatgcaattt cagtggtctg tgaaagggga agatggcgcg 1260

gccaactcta ttgatatgga cgtgaacgct cactctcatg gttcacgcac tgattatctg 1320

atgtcaacca agcaatcatt gactgttaca acgtctcccg ctactcttgt ctttgaactg 1380

gataggatta ttgctcttcc ctccgacctt tctcgcctaa ttccatgtta tggttttcag 1440

caagccactt ttcccgttga tatctccttc cagcgagatg gcgtttcgca tacgtatcaa 1500

gtctatggga agtacacatc ttctcgcgtc ttcaagacta cgttctcgcc tggctcctca 1560

ggtcccgcag tgattaagtt tttgaccgtg cgtacgggca tcgataccta aggtgtggcg 1620

ccgtacgggg gctggttatt catc 1644

<210> 10

<211> 1322

<212> DNA

<213> S2 Gene fragment

<400> 10

gggcgcgtgc cgtgtacgac ttcttttcta cgcctttcgg gaatcgtggt ctagcgacga 60

atcgtactca actatcatca ctactatcaa gctcgaattc cccatggcaa cgttttctat 120

catcaatgac tccattgaca gcgccgggca tcgtttcgac acctgaagca ccctatccag 180

gttcgttaat gtatcaagag tctatgctcc acagtgctac cgtccctgga gtacttggca 240

atcgcgacgc ttggcgtacg ttcaatgtct tcggactttc atggactgac gaaggactgt 300

caggactagt ggctacccaa gatcctcctc ccgccgcccc gtatcagcca gcctctgctc 360

agtggtcgga tcttctcaac taccccagat gggcaaacag acgtcgtgag ctgcaatcta 420

agtacccgct tctgcttcgc tccactctgc tctctgccat gcgagctggt cctgttctat 480

atgttgagac gtggccgaat atgatttctg gacgattagc tgattggttt atgtcccaat 540

atggcaataa tttcgttgac atgtgtgcta ggttgaccca gtcttgttcg aacatgcctg 600

ttgaacctga tgggaattat gatcaacaga tgcgtgcttt aattagtttg tggcttctgt 660

catacattgg ggtaatcaac caaaccaaca ccatcagcgg tttctacttc tcctcaaaga 720

ctcggggtca agcgttggac agttggactt tgttctatac cacgaatact aatcgtgtcc 780

aaattacgca gagacatttt gcttatgtgt gcgcccgatc tcctgattgg aacgtggaca 840

aatcatggat cgctgctgcg aacttaaccg ccattgttat ggcttgccgt caaccgccga 900

tgtttgctaa tcaaggcgtc attaatcagg cgcagaaccg acccggattc tccatgaatg 960

gggggacgcc cgtccacgag ctcaacttac ttactactgc gcaagagtgc atcaggcagt 1020

gggtggtagc aggcttggtg tcggcagcaa aggggcaagc actaacgcag gaagctaatg 1080

acttctcaaa cctcatccag gcggatctag gccagatcaa ggcgcaggac gacgctttgt 1140

acaatcagca gccgggatac gcgaggagaa taaaaccttt cgttaatggt gactggacac 1200

caggtatgac cgctcaagct ctggccgttc tagccacttt taccgcctag gcgtagggtc 1260

gtacgctgcc cgagtccagc cctccggcag cccgtggaga tcggacgagc gtcgtgtagg 1320

ga 1322

<210> 11

<211> 1202

<212> DNA

<213> S3 Gene fragment

<400> 11

gctttttgag tccttagcgt gcaagccgca atggaggtac gtgtgccaaa ctttcactcg 60

ttcgttgaag gaataacatc tagctacttg aagactcctg cttgctggaa tgcacaaaca 120

gcttgggata ctgtgacctt tcacgtccct gatgtaatta gagttggtaa cgcctactgt 180

tgttctcaat gttgtggtgt actctactac gggactctgc cctcggacgg taattatttc 240

cctcatcaca agtgtcatca gcaacagttt aggactgata ctccactgct tcgatacgtg 300

cgcattggta gaaccactga gcatctgatg gatcaatatg ctgtcgctct ggagtccatt 360

gctgaacact atgacgagat tagtcaacgt atggtcgatg agccagagaa tgacgaggtt 420

acacctcttg atatcgttac gcgtaccgaa tctatcagga gtgacaaggc agtcgaccca 480

gacttttgga catacccact tgagcggcgt tctgatgatt ctcgtagaga catcgcctca 540

gcatgctgga aaatgattga cgcgtcggcg cgtagtctca ctcttccaaa ttgcctcgtc 600

tccccctctt tgcactctcg ctccgtcttt ggtcagatgc aaacgaccac cactatatac 660

gatgttgcgg catcgggaaa ggccgttaag ttttcaccaa tggttgctac actagcgcaa 720

cgtgatgctg gccctgtaat gcttgcgaat gctgacccgg cggaaggcgt gtactctttc 780

tggacgtcgc acttcgcttt ctcaccgctc atcggcggag ttggaattac gggacagtac 840

gctcgtgagt cgtaccatca agtgggtcat ccagtgattg ggagtggtaa gaaggcatcg 900

cattacagga atctgttcat ggaagcgtgg cgcgggtggt cgaagtcagc tttcgcatgt 960

gctactggaa tggagccagc tgaatgtgaa tctcgtctga gaggacacgc tcgtactatg 1020

ctcggacgct ctctgccgcc cgtttgtgac gatgatgttg ctcagcagtc tggcgcggta 1080

ctgacttcgc tgcagaaaac gaacaagttc accgttgtgg agtgtggttg gtaagtacct 1140

ccgggtcaaa atgcacatag gctcccacct atgtgacggt tagcgggact cgcctattca 1200

tc 1202

<210> 12

<211> 1192

<212> DNA

<213> S4 Gene fragment

<400> 12

gctttttgag tccttgcgca gccatggaca acaccgtgcg tgttggagtt tcccgcaaca 60

catccggcgc agctggtcag actgttttta gaaactttta cttactacga tgcaatatct 120

cagctgacgg tcgtaatgca acaaaagctg tgcaatccca ttttccattc ctttctcgtg 180

ctgtccgttg cctatctcct ctagccgctc attgtgctga taggactctt cgtcgtgaca 240

atgtgaaaca aattcttact cgtgagctgc catttccatc ggatttaatc aattacgcac 300

atcatgtgaa ctcatcctcc cttactactt ctcagggtgt cgaggcggca cgtctagtgg 360

cccaagtcta tggagaacag ctatcgtttg atcacattta tcccactggt tccgcaactt 420

actgccctgg agcgattgct aatgcgattt cccgtatcat ggctggtttt gtgccccacg 480

aaggtgacaa ctttaccccg gacggttcta ttgactatct cgccgccgac ctggtcgcgt 540

ataagttcgt gctcccttac atgctagata ttgtggacgg acgtccgcag attgttcttc 600

catcacacac tgttgaggag atgctgtcca acacgagttt gcttaattcg attgacgctt 660

catttggtat tgaatcgaag agcgatcaac gcatgacccg tgacgcggct gaaatgagtt 720

ctcgctcact taatgagctt gaggatcatg agcagagggg tcgaatgcct tggaaaatca 780

tgacggcaat gttcgcggcg caattgaagg tggagttgga cgccctagct gatgagcgcg 840

ttgaatctca ggctaacgct catgtgacat cttttgggtc tcgtctgttc aaccaaatgt 900

ctgcttttgt cccaattgat cgtgagttga tggagctggc tctactcatc aaagagcagg 960

gtttcgcaat gaatccaggg caagtcgcat ctaaatggtc gctgatacga cgatctggcc 1020

ccactcgccc gctatcaggc gcacgccttg agatcaggaa tggcaactgg acaattcgtg 1080

aaggtgacca gacgcttctg tctgtctccc cagctaggat ggcgtaaacg ggacccatgg 1140

tgcgggtgag gggccgccac accctctgcc gcgacctgga ctcttattca tc 1192

<210> 13

<211> 1000

<212> DNA

<213> sigma C Gene

<400> 13

atggacggat taactcaaca gcagcgaaga gaagtcgtgg ggttgatact gtcgttgact 60

tcgagcgtga gtataagttc tggcgatttg acgcaaattc gtgagcgtct ttccgctttg 120

gaatctgcga ctgcgtcgct gaacgaatcc gttaatacag ctttgtctag gttagtggat 180

ttgtctgcat cgcttgataa cgtggcggcc tcgttagcgg agacgaaagt ggaaatgaac 240

tcactagttt ctgacgttca gggtttgcga gcttcccttg actcttctgc ttcagagctg 300

gcttctctat cttcattggt gcgtgatcac ggctcttcga ttgctggcct acagagagaa 360

gtaagtgcct tatcgagtga ggtaggcaac cttaaaacct cggtatcatc gcagggcctt 420

cctatcacta gccttgagaa acgagtgcaa gctttagaag gtggttctag tacgactctg 480

tcatttgctg atcctcttaa gttagaggct gggaccgtgt cactcgaggt agatccgtat 540

ttctgctctg tgaatcgtaa tctgacgtcg tattctgctg atgctcagtt gatgcaattt 600

cagtggtctg tgaaagggga agatggcgcg gccaactcta ttgatatgga cgtgaacgct 660

cactctcatg gttcacgcac tgattatctg atgtcaacca agcaatcatt gactgttaca 720

acgtctcccg ctactcttgt ctttgaactg gataggatta ttgctcttcc ctccgacctt 780

tctcgcctaa ttccatgtta tggttttcag caagccactt ttcccgttga tatctccttc 840

cagcgagatg gcgtttcgca tacgtatcaa gtctatggga agtacacatc ttctcgcgtc 900

ttcaagacta cgttctcgcc tggctcctca ggtcccgcag tgattaagtt tttgaccgtg 960

cgtacgggca tcgataccta aggtgtggcg ccgtacgggg 1000

Claims (6)

1. An indirect ELISA detection kit for detecting a novel duck reovirus antibody is characterized by comprising an ELISA plate coated with a novel duck reovirus sigma C protein recombinant antigen;

the preparation method of the novel duck reovirus sigma C protein recombinant antigen comprises the following steps:

the preservation number is CCTCC NO: v201818, using the whole gene sequence of the duck reovirus as a template, amplifying by using a primer pair to obtain a sigma C gene segment, constructing a recombinant expression vector, and expressing a novel duck reovirus sigma C 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.

2. The indirect ELISA detection kit of claim 1, wherein the recombinant expression vector is transformed into BL21 competent cells, and IPTG is added for induction expression; and (3) denaturing and renaturing the expression product to obtain the purified novel duck reovirus sigma C protein recombinant antigen.

3. The indirect ELISA detection kit of claim 1, wherein the novel recombinant antigen of duck reovirus σ C protein is coated in an amount of 500 ng/well.

4. The indirect ELISA detection kit of any one of claims 1 to 3, wherein said 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.

5. The indirect ELISA detection kit of claim 4 wherein the enzyme-labeled antibody is an enzyme-labeled goat anti-duck antibody.

6. A novel indirect ELISA detection method for duck reovirus antibody is characterized by comprising the following steps:

(1) coating: the preservation number is CCTCC NO: v201818, 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% of 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: the enzyme-labeled goat anti-duck antibody is prepared by using PBST according to the proportion of 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 reached₄₅₀Above 0.071, judged positive;

in the step (1), the novel duck reovirus sigma C recombinant protein is prepared by the following method:

the preservation number is CCTCC NO: the whole gene sequence of the duck reovirus of V201818 is used as a template, a primer pair is used for amplification to obtain a sigma C gene segment, and a recombinant expression vector is constructed; transforming the recombinant expression vector into BL21 competent cells, and adding IPTG (isopropyl-beta-thiogalactoside) for induction expression; obtaining purified duck reovirus sigma C recombinant protein after denaturation and renaturation of the expression product;

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

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