CN108982847A - A kind of indirect ELISA detection method for the duck reovirus leading to duck spleen necrosis - Google Patents

Abstract

The invention discloses a kind of indirect ELISA testing kit for detecting novel duck reovirus antibody, the kit is coated with the ELISA Plate of novel duck reovirus σ C protein recombinant antigen；The novel duck reovirus σ C protein recombinant antigen the preparation method is as follows: using the complete genome sequence for the duck reovirus that deposit number is CCTCC NO:V201843 as template, σ C genetic fragment is obtained using primer pair amplifies, recombinant expression carrier is constructed, prokaryotic expression duck reovirus σ C protein recombinant antigen is passed through.Indirect ELISA testing kit of the invention can quickly and effectively be detected to what is newly broken out by the novel duck reovirus antibody of cardinal symptom of gambrel enlargement, to monitor the popularity of novel reovirus in duck group.

Description

A kind of indirect ELISA detection method for the duck reovirus leading to duck spleen necrosis

Technical field

The present invention relates to biological product technical fields, and in particular to a kind of duck reovirus for leading to duck spleen necrosis Indirect ELISA detection method.

Background technique

Avianreovirus belongs to Hu Changgubing section (Reovirdae) Orthoreovirus (Orthoreovirus), can To cause birds that a variety of diseases occur, clinical manifestation is different and variant because virus stain, virulence or infection host's.

From 2017, it is main that the ground such as China Shandong, Hebei, Henan and Jiangsu, Anhui duck, which is broken out on a large scale with spleen necrosis, The communicable disease of symptom, the disease mainly cause each age level duck spleen enlargement, bleeding and necrosis, cause kind of a duck lay eggs and There is the different necrosis region of several degree in meat duck appetite stimulator, syntexis, retarded growth, spleen, and enlargement, bleeding, edge increase It is raw.Duck feedstuff-meat ratio increases, and meat duck delivers qualification rate significant decrease for sale, causes serious financial consequences to meat duck and kind duck aquaculture.

Research confirms, this using spleen necrosis as the cause of disease of the high incidence duck infectious disease of cardinal symptom is a kind of novel Duck reovirus, belongs to emerging infectious disease, has still been in step to the research of biological characteristics, the pathogenic mechanism of the virus etc. Section, still lacks effective vaccine immunity and Control Measure at present, current for the Serology test of the new virus Also it not yet has been reported that.

Summary of the invention

For the above-mentioned prior art, the object of the present invention is to provide a kind of duck reoviruses for leading to duck spleen necrosis Indirect ELISA detection method.It can be to newly breaking out using spleen necrosis as the antibody of the novel duck reovirus of cardinal symptom It is quickly and effectively detected, to monitor the popularity of novel duck reovirus in duck group.

The deposit number of the novel duck reovirus is CCTCC NO:V201843, and depositary institution is Chinese Typical Representative culture Object collection, the deposit date is on July 18th, 2018, preservation address was Wuhan, China, and Wuhan University, classification naming is novel N-DRV-XT18 plants of duck reovirus.

To achieve the above object, the present invention adopts the following technical scheme:

The first aspect of the present invention provides a kind of indirect ELISA detection reagent for detecting novel duck reovirus antibody Box, the indirect ELISA testing kit are coated with the ELISA Plate of novel duck reovirus σ C protein recombinant antigen；

The novel duck reovirus σ C protein recombinant antigen the preparation method is as follows:

Using the complete genome sequence for the duck reovirus that deposit number is CCTCC NO:V201843 as template, primer is utilized σ C genetic fragment is obtained to amplification, recombinant expression carrier is constructed, passes through prokaryotic expression novel duck reovirus σ C Protein reconstitution antigen；

The primer pair includes:

Upstream primer σ c-F:5 ,-ATACGCCTGATACTTTCCCT-3 '；(SEQ ID NO.1)

Downstream primer σ C-R:5 '-GCGCAATGAGAAGAGCTATTCATC-3 '.(SEQ ID NO.2)

Further, the recombinant expression carrier is converted into BL21 (DE3) competent cell, IPTG is added and is lured Lead expression；By expression product by being denaturalized with after renaturation, the novel duck reovirus σ C protein recombinant antigen of purifying is obtained.

Preferably, the package amount of the novel duck reovirus σ C protein recombinant antigen is the hole 500ng/.

Further, the indirect ELISA testing kit also includes: enzyme labelled antibody, sample diluting liquid, cleaning solution, yin Property control serum, positive control serum, substrate developing solution A, B and terminate liquid.

Preferably, the enzyme labelled antibody is enzyme mark goat-anti duck antibody.

Application of the above-mentioned indirect ELISA testing kit in the epidemiological survey of novel duck reovirus is also this The protection scope of invention；

The deposit number of the novel duck reovirus is CCTCC NO:V201843.

The second aspect of the present invention provides a kind of indirect ELISA detection method of novel duck reovirus antibody, including Following steps:

(1) be coated with: using deposit number be CCTCC NO:V201843 novel duck reovirus σ C recombinant protein as Envelope antigen will be added in ELISA Plate hole after antigen diluent, 4 DEG C of overnight incubations or 37 DEG C of incubation 2-4h, using containing 0.05% The PBST of tween is washed；

(2) it closes: being closed using PBST dilution 5% skimmed milk power, 200 hole μ L/, got rid of after being incubated for 1h under the conditions of 37 DEG C It is dry, it is washed with PBST；

(3) serum action condition: every hole is added serum to be checked and dilutes mixed liquor, dries, uses after being incubated for 1h under the conditions of 37 DEG C PBST washing；

(4) it adds enzyme labelled antibody: enzyme mark goat-anti duck antibody being diluted with PBST by 1:500, every hole adds 100 μ l, 37 DEG C of items It dries after acting on 1h under part, is washed with PBST；

(5) substrate develops the color: 100 hole μ L/ of tmb substrate developing solution, and effect 15min is protected from light under the conditions of 37 DEG C；

(6) terminate reaction: every hole adds 50 μ L terminate liquid color development stoppings to react；Using being read under microplate reader absorbance 450nm Data；

(7) determination of yin and yang attribute critical value: according to formula: yin and yang attribute critical value=negative sample OD₄₅₀Average value+standard Deviation 3SD obtains yin and yang attribute critical value；Work as OD₄₅₀0.072 or more, it is determined as the positive.

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

Using the complete genome sequence for the novel duck reovirus that deposit number is CCTCC NO:V201843 as template, utilize Primer pair amplifies obtain σ C genetic fragment, construct recombinant expression carrier；Recombinant expression carrier is converted to BL21 (DE3) and is experienced In state cell, IPTG is added and carries out inducing expression；Expression product is exhaled into the lonely disease of intestines by the duck for being denaturalized with after renaturation, obtaining purifying Malicious σ C recombinant protein；

The primer pair includes:

Upstream primer σ c-F:5 ,-ATACGCCTGATACTTTCCCT-3 '；(SEQ ID NO.1)

Downstream primer σ C-R:5 '-GCGCAATGAGAAGAGCTATTCATC-3 '.(SEQ ID NO.2)

Beneficial effects of the present invention:

For the newfound novel duck reovirus that can result in duck spleen necrosis, the present invention establishes detection, and this is new The indirect ELISA detection method of type duck reovirus antibody, the detection method have it is easy, quickly, stabilization, high specificity, The characteristics such as sensitivity height can detect novel duck reovirus σ C specific antibody in duck group, and on this basis Come detect duck group in reovirus antibody level, thus monitor duck group in novel duck reovirus popularity.

Detailed description of the invention

Fig. 1: recombinant protein σ C in BL21 competence inducing expression and protein purification SDS-PAGE analysis chart；Wherein, M Swimming lane is protein standards；1st swimming lane is zero load, and the 2nd swimming lane is not induce full bacterium, and the 3rd swimming lane is supernatant after induction bacterium cracking, 4th swimming lane is to precipitate after induction bacterium cracks.

Specific embodiment

It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field The identical meanings of understanding.

As background technique is introduced, from 2017, the big model of the ground such as China Shandong, Hebei, Henan and Jiangsu, Anhui duck Outburst is enclosed using spleen necrosis as the communicable disease of cardinal symptom.It has been investigated that the communicable disease is by novel reovirus Caused.It there is no the drug and method that can effectively control the novel duck reovirus infection at present.It is of the invention based on this A kind of indirect ELISA testing kit that can detect novel duck reovirus antibody is proposed, it is bad with spleen to what is newly broken out The antibody of the dead novel duck reovirus for cardinal symptom is quickly and effectively detected, and is exhaled with monitoring novel duck in duck group The popularity of the lonely virus of intestines, to find that infected duck group takes it isolation or slaughters measure early, to reduce novel duck Loss brought by reovirus infection.

It is known that the host range of Avianreovirus is wider, generally existing this virus in various birds, from 1954 It has been separated in the birds of a variety of morbidities such as chicken, goose, pigeon, ostrich, duck, turkey and other pheasants or health since year Reovirus, and the reovirus being separated to from different hosts its there are biggish variability, pathogenic, gene sequences Column and the coding of major protein all have differences.

Present inventor isolates one plant of duck reovirus N-DRV-XT18 from morbidity duck spleen tissue, to above-mentioned The duck reovirus N-DRV-XT18 newly separated has carried out genome sequencing, and respectively by 10 genetic fragments and existing report The Avianreovirus in road has carried out sequence alignment and homology analysis, as a result, it has been found that, the 10 of new isolated strain N-DRV-XT18 L1, L2, L3, M1, M2, S3, S4 are respectively positioned in a relatively independent branch in a genetic fragment, and with uploaded at present Reovirus-originated duck sequence variability is larger in Genbank, illustrates that the strain N-DRV-XT18 newly separated is different from it really His Avianreovirus, it can be assumed that duck reovirus found in the present invention and it is existing it has been reported that duck exhale intestines lonely Virus exists compared with Big mutation rate, is 1 new independent kind of Orthoreovirus.Therefore, which is exhaled intestines lonely by the present invention For preservation in China typical culture collection center, deposit number is CCTCC NO:V201843.

The present invention carries out antibody test to novel duck reovirus with serological method for the first time.It can be to new outburst Fast and effeciently detected by the novel duck reovirus antibody of cardinal symptom of spleen necrosis, be a kind of easy to be suitable In the indirect ELISA detection method that base uses.

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

(1) preparation of envelope antigen:

The complete genome sequence that novel duck reovirus is obtained by two generation sequencing technologies, L1, L2 in whole genome sequence, The sequence of this 10 genetic fragments of L3, M1, M2, M3, S1, S2, S3, S4 is respectively such as SEQ ID NO.3-SEQ ID NO.12 institute Show.According to the σ c protein gene order of novel duck reovirus obtained, two primers are designed:

Upstream primer σ c-F:5 ,-ATACGCCTGATACTTTCCCT-3 '

Downstream primer σ C-R:5 '-GCGCAATGAGAAGAGCTATTCATC-3 '.

The cDNA that reverse transcription obtains is amplified by the σ C gene that length is 980bp by regular-PCR method with above-mentioned primer Sequence (shown in SEQ ID NO.13), and purified by way of gel electrophoresis and glue recycling, cDNA after purification is cloned into In prokaryotic expression carrier PET-28a (+), recombinant prokaryotic expression vector PET28a- σ C is constructed；By recombinant prokaryotic expression vector PET- σ C is converted into BL21 (DE3) competent cell, expresses recombinant protein σ C through 1mM IPTG inducement efficient, the albumen is with packet The form for containing body exists, and inclusion body is by denaturation and renaturation, using the reovirus σ C albumen of purifying as envelope antigen.

(2) with after purification exhale intestines orphan's σ C recombinant protein to do envelope antigen to establish indirect ELISA:

1. coating: antigen is dilute with PBS (pH=7.2 or so) or with 1 × carbonate buffer solution (pH=9.6) by required concentration Coating in 96 hole elisa Plates (elisa plate), wrap by preservative film after releasing, and 4 DEG C are incubated overnight or 37 DEG C of incubation a few hours, using containing The PBST washing of 0.05% tween (mass concentration) is three times, every all over 4min；

2. closing: closing dilutes 5% skimmed milk power (mass concentration) 200 hole μ L/ using PBST, is incubated for number under the conditions of 37 DEG C It dries after hour, is washed 3 times with PBST, it is every all over 4min；

3. serum action condition: every hole is added appropriate serum to be checked and dilutes mixed liquor, after being incubated for a few hours under the conditions of 37 DEG C Drying, is washed 3 times with PBST, every all over 4min；

4. goat-anti duck ELIAS secondary antibody action condition: after goat-anti duck ELIAS secondary antibody does appropriate dilution with PBST, every 100 μ L of hole, It dries, is washed 4 times with PBST, each 4min after acting on 1h under the conditions of 37 DEG C；

5. substrate develops the color: 100 hole μ L/ of tmb substrate developing solution is protected from light effect 15min under the conditions of 37 DEG C；

6. terminating reaction: every hole adds 50 μ L2M H₂SO₄The reaction of terminate liquid color development stopping；

7. reading: using reading data under microplate reader absorbance 450nm.

(3) result judgement standard:

According to formula: yin and yang attribute critical value=negative sample OD₄₅₀Average value+standard deviation 3SD, it is critical to obtain yin and yang attribute Value；Work as OD₄₅₀0.072 or more, it is determined as the positive.

Since Avianreovirus is RNA virus, there are multiple segmentations, in antigenic structure, pathogenic, cell between different strains Cultural character and host specificity etc. have a certain difference, and variation is easy to happen in genetic evolution.Therefore, different The coding of the antigenic, pathogenic of reovirus, gene order and major protein can all have differences.Using indirect ELISA Reovirus antibody is detected, above all the sequence of hard objectives gene, designs synthetic antigen egg according to target gene sequence It is white to be detected.But the present invention duck reovirus to be detected for leading to duck spleen necrosis is the strain newly separated, Gene composition it is unknown, and with it is existing it has been reported that duck reovirus exist compared with Big mutation rate, also can not be according to existing duck The genome sequence of reovirus designs synthetic antigen albumen.

For novel duck reovirus indirect ELISA detection, the selection of envelope antigen is very crucial, directly certainly Determine the specificity of detection method.For preferred envelope antigen, the present invention is during the test according to the novel duck reovirus Major antigenic sites region separately designs primer pair, and amplification obtains genetic fragment, constructs recombinant expression carrier；Recombinant expression is carried Body is converted into BL21 (DE3) competent cell, and IPTG is added and carries out inducing expression；By expression product by denaturation and renaturation Afterwards, purification of recombinant proteins is obtained, using the recombinant protein of acquisition as envelope antigen, constructs indirect ELISA testing kit. The specificity for the indirect ELISA testing kit that the serum sample made a definite diagnosis on applying clinical prepares different envelope antigens carries out It investigates.The results show that the indirect ELISA detection examination prepared using the reovirus σ C protein that the present invention purifies as envelope antigen To the accuracy rate of serum sample detection has been made a definite diagnosis up to 100%, the sensibility and specificity of detection is significantly better than with it agent box The indirect ELISA testing kit that his recombinant protein is prepared as envelope antigen.

In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool The technical solution of the application is described in detail in the embodiment of body.

The test material that test material is this field routine is not specifically described used in the embodiment of the present invention, It can be commercially available by commercial channel.Used portion of reagent and component are as follows in the present invention:

It is coated with buffer: 1 × carbonate buffer solution (100mL, pH=9.6): Na₂CO₃0.2756g, NaHCO₃ 0.6216g dissolves with distilled water and is settled to 100mL, pH value 9.5-9.7,4 DEG C of preservations.

PBS solution: NaCl 4.25g, NaH₂PO₄·2H₂O 0.178g, Na₂HPO₄·12H₂O 1.386g, uses distilled water Dissolve and be settled to 500mL, pH value 7.1-7.3.

PBST solution: Tween-20 0.5mL is added in every 1L PBS, mixes well.

PBST confining liquid: 5g drymilk is dissolved in 100mL PBST dilution, and short-term preservation is long-term to protect in 4 DEG C It is stored in -20 DEG C.

TMB developing solution:

Buffer A: weighing 66.5063g sodium citrate 800mL distilled water and dissolve and use dense HCl tune pH to 4.0, is added H₂O is settled to 1L, 4 DEG C of preservations；

Buffer B: weighing 0.2956g tetramethyl benzidine (TMB), and 0.0633g tetrabutyl ammonium borohydride (TBABH) is molten In 30ml dimethyl acetamide (DMA), 4 DEG C are kept in dark place；

In use, buffer A and buffer B are mixed with the ratio of volume 39:1, matching while using.

2M H₂SO₄Terminate liquid: by dense H₂SO₄It is added in distilled water with the ratio of volume ratio 1:5, mixing is cooled to room temperature i.e. For 2M H₂SO₄Terminate liquid.

Specific experiment condition and method are not specified in the embodiment of the present invention, usually according to normal condition, such as J. Pehanorm cloth The chief editor such as Luke, Science Press, 2002, Molecular Cloning:A Laboratory guide (third edition)；D.L. Spector etc. is edited, and science goes out Version society, 2001, cell experiment guide；Or the condition suggested according to manufacturer.

Embodiment 1: the preparation of envelope antigen

1.1 specific primer designs and synthesis: according to the novel duck reovirus (deposit number is CCTCC NO: V201843) pair of primers is designed in complete genome sequence σ c protein code area, and two sections of primer are respectively equipped with the I digestion position BamH I and Sal Point, it is contemplated that the genetic fragment of 980bp in the novel duck reovirus gene group can be amplified.

Upstream primer σ c-F:5 ,-ATACGCCTGATACTTTCCCT-3 '；

Downstream primer σ C-R:5 '-GCGCAATGAGAAGAGCTATTCATC-3 '.

1.2PET28a- the building of σ C prokaryotic expression carrier:

σ C genetic fragment is gone out by PCR amplification using specific upstream and downstream primer, by after purification segment and PMD18-T Carrier connection, connection product are transformed into DH5 α competent cell, and picking positive bacterium colony shakes bacterium and extracts T- σ recombinant plasmid.With limit Property restriction endonuclease BamH I and Sal I processed (being purchased from Dalian treasured biotech firm) respectively to T- σ recombinant plasmid and PET-28a (+) carrier into Row double digestion, with both T4DNA ligase (being purchased from Dalian treasured biotech firm) connections digestion products.Connection product is transformed into DH5 α In competent cell, identify that positive colony, positive colony send to Qingdao and hold up the limited public affairs of section's biotechnology using PCR method and digestion Department's sequencing.

The single positive colony bacterium colony PCR qualification result of picking shows to expand the purpose band to 980bp or so, and estimated σ C genetic fragment it is in the same size.The single positive colony bacterium of picking extracts plasmid after dropping into row Zengjing Granule, uses restriction enzyme Enzyme BamH I and Sal I carries out digestion identification, and digestion band is correct.Positive colony sequencing as the result is shown σ C gene insertion position, insert Enter direction and reading frame is correct, the results showed that recombinant vector PET28a- σ C is constructed successfully.

1.3 recombinant protein σ C inducing expressions and identification:

Successful recombinant vector PET28a- σ C plasmid will be constructed to be transformed into BL21 competent cell, picking single bacterium colony Zengjing Granule is carried out, next day is inoculated in the ratio of 1:100 containing 100 μ g/ml kan⁺2 × YT culture medium in, 37 DEG C, 200r/ Min shake culture is to OD₆₀₀1mL bacterium solution is taken out between 0.6-1.0 as compareing before induction, and IPTG is added in remaining culture To final concentration of 1mM, continue 37 DEG C of 200r/min shaken cultivation 6h, terminates induction.Induction and non-induced bacterium solution 2mL are taken respectively, Bacterial precipitation is collected after centrifugation, bacterium is resuspended with 50 μ LPBS.Sample adds 2 × sds gel sample loading buffer, 50 μ L to mix, and boils Concussion cracks bacterium after handling 5min, and supernatant is taken to carry out SDS-PAGE after being slightly centrifuged, and whether identification albumen expresses.Take Fiber differentiation Supernatant is abandoned after bacterium solution centrifugation and collects bacterial precipitation, with ultrasonic treatment after PBS suspended bacterial to limpid, 10,000 under the conditions of 4 DEG C × g/min is centrifuged 5min, takes supernatant precipitation suspension that 2 × sds gel sample loading buffer is added to mix respectively, boils processing 5min SDS-PAGE identifies protein expression mode afterwards.

1.4 protein purifications:

A large amount of Fiber differentiations are carried out according to expression condition protein induced in 1.3.Take inclusion body referring to kit specification into Row protein purification carries out SDS-PAGE and identifies purity of protein after going recycling protein example processing.

SDS-PAGE analysis shows that, recombinant protein σ C successful expression in BL21 competent cell, all with inclusion body Form exists, and molecular weight of albumen size is 40kDa, (referring to Fig. 1) in the same size with expected recombination σ C.

1.5Western Blotting analysis: according to method induction expression protein in 1.3, induction and non-induced is taken respectively Full bacterium SDS-PAGE, is operated referring to routine Western Blotting method, finally aobvious using enhancing HRP-DAB substrate Color reagent develops the color.As the result is shown: with SDS-PAGE compare, PET28a- σ C convert BL21 competent cell induction after There is the band of specificity in Western Blotting corresponding position, and compare non-induction bacterium then without.The result shows that recombinant protein With antibody in positive serum specific antigen-antibody reaction can occur for sigmaC.

Embodiment 2: indirect ELISA detects novel duck reovirus antibody

1 purification of recombinant proteins σ C of Example does envelope antigen and establishes indirect ELISA method, is groped using square matrix method ELISA optimum protein peridium concentration and serum diluted concentration and ELISA optimum reaction condition.It is final to determine that condition is as follows:

2.1 coatings: after antigen is diluted with 1 × carbonate buffer solution (pH=9.6) by 1:1000, take 10 μ L coating to 96 In the elisa plate of hole, preservative film is wrapped, 37 DEG C of incubation 2h, three times using the PBST washing containing 0.05% tween, every all over 4min.Inspection The hole gaging hole σ C protein package amount 500ng/；

2.2 closings: closing is used using drying after being incubated for 1h under the conditions of PBST dilution 5% skimmed milk power, 200 37 DEG C of the hole μ L/ PBST is washed 3 times, every all over 4min；

2.3 serum action conditions: 10 μ L serum to be checked and the diluted 5% skimmed milk power mixing of 90 μ L PBST is added in every hole Liquid is dried after being incubated for 1h under the conditions of 37 DEG C, wash 3 times with PBST, every time 4min；

2.4 enzyme mark goat-anti duck antibody action conditions: enzyme mark goat-anti duck antibody is diluted with PBST by 1:500, and every hole adds 100 μ L is dried after acting on 1h under the conditions of 37 DEG C, is washed 4 times with PBST, each 4min；

2.5 substrates develop the color: 100 hole μ L/ of tmb substrate developing solution, and effect 15min is protected from light under the conditions of 37 DEG C；

2.6 terminate reaction: every hole adds 50 μ L2M H₂SO₄The reaction of terminate liquid color development stopping；

2.7 readings: using reading data under microplate reader absorbance 450nm；

3, result judgement standard:

According to formula: yin and yang attribute critical value=negative sample OD₄₅₀Average value+standard deviation 3SD, it is critical to obtain yin and yang attribute Value；Work as OD₄₅₀0.072 or more, it is determined as the positive.

4, cross reactivity: with the method that the present invention establishes to Duck parvovirus, duck tembusu virus, Ana 1 aviadenovirus, duck Influenza virus, NDRV-JM85 plants of duck reovirus, NDRV-TH11 plants of positive serums of duck reovirus and negative serum into Row detection, this method are only reacted with novel duck reovirus (deposit number is CCTCC NO:V201843) positive serum in sun Property, illustrate that this method has specificity well.

The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.

SEQUENCE LISTING

<110>Shandong Agricultural University

<120>a kind of indirect ELISA detection method for the duck reovirus for leading to duck spleen necrosis

<130> 2018

<160> 13

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213>artificial sequence

<400> 1

atacgcctga tactttccct 20

<210> 2

<211> 24

<212> DNA

<213>artificial sequence

<400> 2

gcgcaatgag aagagctatt catc 24

<210> 3

<211> 3959

<212> DNA

<213>the L1 gene of strain N-DAV-XT18

<400> 3

gctttttctc cgaacgccga aatgagttcg cgcaaagtgg ctagacgtcg tcataaggat 60

gctaccgaca ctaaagattc taagtacact gataaagata agtcctcagc cacaactact 120

gaaacgaagt ccgtcgactc cactaacaag agtgtaagcg ctccaaaatc tgatactcca 180

gcagcatcaa caccatcctc tacagatggc gcttctcagg ttgcagtcgc taaacaaacc 240

aatgataatg acgcttcttc taaagattct tcccctaaac caactgtcac tcccgacgga 300

aaggatggaa tgcatggtgc ggttaaatct caagacgcta aagctaccat tgctgttgat 360

gataacaagg acagggacgt ggtcttcggt ggcaagggtt caggtgataa aaatgctatt 420

acaaagactg ggtccgtgga taatgatggt ggtgttaaaa cgataccggc taaggatact 480

actatcgcat ccgctaaagc catgatggaa cagaaacagt tagttgcggg tcttccgaag 540

cagccaaaac ctgccagcca tctatgcact gtttgcatgg ctcaattcgc ttcagctgat 600

gcattgacta tacaccaaac aactcattcg attggatcca acgctgcact gactagcttt 660

tcgatctcta ccgccgttga ggagttcatt caatcttggg cctcggctac ttccactgct 720

aatactaaaa cagcactaac tgtggcggac gttgattctt taatgatgac tgaagggata 780

cgtttaatca cttgggattc cggactctgt acctcctttg agcttgtgcc gatagtgcag 840

tctaacactg tccaagacgt tatctcatac tcttggttca catctagtta caatatcgct 900

actcctttcc ctcaagcgac tgtggtgcgt attgtgttac gtacgaactg ggctgctaga 960

ttggattccc catcttcctc tcgtgaatgc gatcttcggc ttgctcctcc gacagaaagt 1020

aatgcacgtt cttttgctat gctcctcaac actggtgcta ctcctgaagg tacatttaat 1080

cctaacactc tccgtatgaa cgtcttgcag atgtgcctcc agtatgtgtt agctaacctc 1140

catttaaaca ggagtactca atttacgatg gacctgaccg ctgccgctcc caatttatcc 1200

gcttctcaac ttcgaatcgt gcccgaagac aaggatggaa aatggttccc ggtcatgtac 1260

ccttctcgtg taaacatccc cctctttaat aaaacagccg atttcgttaa ccaatgtatt 1320

cgtgacagaa tcggtcgcta cgatcgcgct caaactttcg ctggtgctcc ttctgaatgg 1380

gctgatatgt gggagactgc agactcatta acactctcgg ttcgtgaaat gtggatgtct 1440

cggatctcac aaatgaacat ctctcccgcc gatatcgccg acgcaatctc gcgttgttcg 1500

cagtcgctcc tgacggtagc cgcacctact gctccctccg tagctcgtct cctgccgtgg 1560

cgtgttagtt ctgatgagcg ccaattatta caactgctga tgtatcttaa cgtaggtact 1620

agcgccgatt acatccagcc aattctatct gcgttcgctc gcactctctc gcgtgtttca 1680

cctttgcgta ttaaccccac tctcatcgct aacgcaatgt ctactattgt tgaaagcact 1740

actaacaccc agagtccagc cgctgccatc ttgtcaaagt tgaagccagt cgcatccgac 1800

ttctcagact tccgtctcgc atgtgctgct tggctgtata atggttgtgt ccagacgtac 1860

ttatctgaag actcttaccc cagtagcggc ggatctgtca ctagtattga cactctggtt 1920

gatatgtttg tctgtctctt ggctctcccg cttgtcactg atcctaatgc gccttgtcaa 1980

gctttcatgg tcgtggctaa tgctatggtc ggctatgaga atctgccgat ggatgatcct 2040

aactttaccc aacagagact ggctgccgcg tttaataacc ctaccacctg gccgcagtgc 2100

tttctacacc ctcagaacat cgatcgccgc caatgtccga tcctatcctg gtgggcacaa 2160

cagattcacc gcaactggcc tactccatca cagatcacct atggcgcgcc tgatattatc 2220

ggttcagcca acctattcac tccccctgat gttctgctgc tcccgctcca gcatcgcccc 2280

attcgcatta cgaatccaac tttgaacttc gacaacgagt taacaacctg gcgtaacacg 2340

gtagtagatt tgatcctacg catcattgac agtggcagat accaaccgaa ctggaatcag 2400

tccatccgcg cttcaatgcg aaacgctatg acgaacttta ggattattaa atcgtacact 2460

cccgcttaca tcgctgaatt gttacccgtt gagttggccg ctatcgctcc cacccttcct 2520

ttccagcctt tccaggttcc cttcgctcgt ctagaccgtg atgctattgt cacccatgtc 2580

aacgtgtcgc gtcaggcccc gaacgtgctt gctcagccag cgttaaacat gtccatgacg 2640

tatcaacgca ctggcgtgcc aatatcgttg agcgcccgtc ctcttgcagt tgctcttctg 2700

tccggccaat atcctactga gccacctctg cagacgaatg tgtggtatgt gaatacgctt 2760

acccctctgt attcaaatga tggattattt aataacgtcc agcatgctat ggtggccgct 2820

gaagcttacg ccactcttat cactatgtta gcacagtgta cggacatgca gtatcccgtg 2880

gaccgtccct taaactggtt acgtcaaatc aacctagcgg cgaatgaggc taccatcttt 2940

gggcgctcca tcaattctct cttccagacc gcctttgatc tttccccttc tactgttctc 3000

ctgcaaccgt ttttggagtc cgaccctcgc tctacacaac tcgccatctc ctacgtccgc 3060

tataatggtg atagcgaaac cttcgtgcct accgtccgcc cgtctatgat agctgaggct 3120

actctactcg tcgagcgcgt tctgtcacat gagtataact tatttggact ctgtcgcggt 3180

gacatcattt taggacagca catgacccct tccgcattta acccgttagc ccctcctcct 3240

tccgtcgtct ttagtcgcgg tgaccccgag gtctacgagt tcggccagcg tagtttcgcc 3300

aactttggta tgaatggtga agagatcctc gttatggacg cgaatggcgt acgtcgtccc 3360

ctgcttggta gatgggtgat gcccctacag ctactgatgg tgaacattgg cgtgttccct 3420

aagctcctgc tggaccggat tctgaaaggg cgtttgtaca ttaggctaga ggtcggtgcc 3480

tatccttaca ctgttcagta ctatcagggt cgagagttca ctgatggatt tacgctgttg 3540

gagcagtgga tgtcgaaagt gtcgcccatg ggcataccac ccgttccgtt cctgatgcca 3600

caatctgaag gtcataacat tacttctggc atggtgaccc actacatttg gtccactgag 3660

tataacgatg gctccctttt cgccacaaac acagacttgc ctgttactgt cttcggtcca 3720

gaccgcacta tccctattga gagataccgt gctttggtcg atccgggcgc tctgccagcc 3780

acaaaccaac tcccccacac tatcgatctc tactgttcgt tgagacgcta ctacttggaa 3840

acacctccga ttacagcgac ggtgacaact tatggtgatg gtctcccagc gttgaaccat 3900

tagagcggcg aggctagacg cgagctgatc gcgtcgactc tcgttggagg ttactcatc 3959

<210> 4

<211> 3830

<212> DNA

<213>the L2 gene of strain N-DAV-XT18

<400> 4

gctttttcct caccatgcat gtcaatgggt ttgatgatgc tactctcgca tacgcacgct 60

ccatatcggg tcttacttca atgtcaaatg atttgtttga gagagcttca ttgtctatgc 120

gctcattccc acgttctcat gcttatgaca tattagataa agtagaattt tctgtctctt 180

gtgtcattcc taatgccata tttcatcacc cggaccacgt tgagtacttt tatatcgatg 240

ctgtcaatag ggtcaggcgt aaacatgtca ttgattcgga tgatgtattt gtaccaaact 300

gtaattttca gggtctctta actcctatgg ataagttacc aaactatggt caactatctg 360

aggtcatctc ctcgaatgct cgcgacgggt tagcttcggc acgaatcgcc aatactttct 420

ataacattgc tgtctcacaa gctaggcaag tcaaagcgcc tcttgaatcg tttctccttc 480

ccctattact ttctgaaacc tgccctttat ctgatgaccc atgtggattg gatacttccc 540

cgtctcctcc gatcagtaga aatctggcgt tatgggtact gcgcgagtta agccgcacta 600

tttgtggatc gtcaggcgat cggtcaccat ggttgttgtt agattcaggt gtagcatggt 660

tcctttcacc actgatgtct tcagctatcc ctccccttat ggctgattta acgaatctgg 720

ctatctataa acaggtatgc tccgtctccg atgatatgca ctccctcgcc gttcaaatgg 780

tgttgcaagc agctgcctcg caatcctacg gacattatgt gctgcaaact aaggccgttt 840

ttccgcaaaa cactttgcat aacatgttta gaaccatcac tgacggtctg gtccctatga 900

tagactggct ggagcccagg tctaactacc gcttcatgct gcagggtgcg cgtaaggtga 960

catcagatga tgcgaatcaa tctccagata acacggaggc tgctgagaaa gttggccata 1020

agatgggttg tttagatgtt gttagatcct tacggaagat gtcttctgcc attacagtgc 1080

attcgcatga tgcgatgacg tttgttcggg acgccatgtc ttgtaccagt ggtattttca 1140

ttactcgcca acctactgaa actgtgttga aggaatatac gcaagctccg acgattgaag 1200

ttcccattcc tcaaactgat tggtctccac caattggatc ccttcgctac ttatctgatg 1260

cctgtgctct acccgctgtg tacttggctc gttcttggcg tcgagctgcc tcagcagtcg 1320

tagataatcc tcacacttgg gatccactat atcaagctat tctccgctcc caatacgtca 1380

catcacgcgg tggttcgggc gctgcgttac gagacgcgtt aaaagctgct gaagtggagc 1440

ttccacagta tcctggtgtc agtattaagg tcgctacaaa gatataccag gcagctcaaa 1500

ctgctgatgt gccattcgat aagctctccc gtgccgtcct tgctcccttg tcgatggggc 1560

tgcgtaatca ggtccagcgt cgtcctcgca ccatcatgcc tatgaacgtc gtgcaacaac 1620

aggtctcagc agctcatact ctatctgccg actatatcaa ctatcacatg aacttatcca 1680

ccacatcggg tagtgccgtt atcgagaagg tcgttccgtt gggcatgtac gcctcctgtc 1740

cgcctgctca ggcagtcaat attgatatta aagcgtgtga cgcgtctatt acgtatcagt 1800

acttcctctc cgtcattgtt ggtgctattc atgaaggtgc tgctggacgt cgtgtatcct 1860

cctcgtttat gggtgttcct cccagcgtat tgtcggtcgt tgattccagt ggagtcacat 1920

cttcaatgcc catttccggt ttccaggtta tgtgtcaatg gttagcgaaa ctctatcagc 1980

gtggttttga gtatcaggtc acggacacat tttcacccgg caatatcttt acgcaccaca 2040

cgacgacttt tccttccgga tctaccgcca cttccaccga gcacactgcc aataatagca 2100

cgatgatgga tggatttcta agatcttggg tcccctcctc tgatgcatca gatattttga 2160

agaagttctg tcgctctatt tccatccagc gaaattatgt ctgtcagggt gacgatggcc 2220

taatgattgt cgatggtctc tcaacgggta agttatcggg cgaggtgatt gctgagtttg 2280

tcaaggagtt gcgagcgtac ggtaagtctt ttggatggaa ctatgacatc gagtttactg 2340

ggaacgctga gtatcttaag ttgtattttc tgaacggttg ccgggtacct aatgtgtcgc 2400

gacaccctat atgtggtaaa gagcgagctt ccggagataa gttagagatg tggccttcta 2460

ccatcgatat ttttaatggc atctttgtca acggtgtgca tgatggtctg ccttggcgtc 2520

gttggcttcg ctactgctgg gccttggcca cgttatattc tggaaagcgt gtgcgtcatg 2580

agaatgttga agtgtcaatc caatatccta tgtggtcatt tatctactgg ggcctgcctc 2640

ctgtcagcgt ctttggttcc gatccgtgga tattctctcc ctacatgccc actggcgatc 2700

acggattcta ttccatgcta actcttgtta gacccctaat taccgccctc tctccttcat 2760

cttcaatgga tggacttttt ggtcagtgcg atcacaacgc tctctttaac tccgagatgg 2820

tgtaccaggg ttactatatg gcgcagtgtc cgcgtcagcc atcgagatcc aaccgtcgtg 2880

atgacccgga gtctgtccaa cgatttgtta gggcgctcga atcgtactta tacatatccc 2940

cggagttgaa agcacgagtc aggcttggtc gtgaccgctg gcagaaatta gttgggtata 3000

ctgaaaaatc tccgccctct ctcgatgacg tcgctcataa atggttccgt agtgcgcagg 3060

aagccgactt acctaccacc tcggaaattc aggccatgga cttagcgtta atcgcggccc 3120

gccgtaaaac ataccaaggc ttctctaagc tacttaacac gtaccttcga gtaacttggg 3180

acctgaccga gccaatcgag catgccgtcg atccccgggt accactctgt gctggcattt 3240

ctccctcgaa tagtgagccc tttttgaagc tgtactcgat cggtcctatg atgcagtcga 3300

ctcggaaata tttcagtaac accttgttca ttcatcgtac cgtatctggc cttgacgtcg 3360

acgttgtaga tcgcgctctt ctacgtctga gagcccttaa tgctcctgat gaggtggtca 3420

ttgctcaact cctcatggtc ggcctgtccg aagcagaagc ggcgactttg gcagctaaga 3480

ttaggactat ggacatcaat gccgttcagt tggctcgagt ggtgaatctg tcaatacccg 3540

actcttggat gacgatggat ttcgatcgtt taatacgtga tattgtgtct actacccctt 3600

taactattcg ttctttgacg actgacctgc cctctggtgt cccatgggtt cgtgccatat 3660

tgcagttcct cggtgctgga gtcgccatga ccgccgttgg tcccgttaga cgcccatacc 3720

ttcactcagt cgctggtggc atgtcgtcat tcattaaaca gtttcgccgg tggatgcggg 3780

ctgagacgag gtagcgtccg tgcccagcat ggctcgagga attattcatc 3830

<210> 5

<211> 3906

<212> DNA

<213>the L3 gene of strain N-DAV-XT18

<400> 5

gcttttcacc catggctcag attagaggcc ttcgattgtc tacgacactc tcagcaccgc 60

cttcacgtcg atcgtacaca cctcagacat atgatgacct catttcagcg ctgaaattaa 120

ctgtcaagcc ctggcgaccg ttgcgatctg gcgctcaaga tgttatcacg gccgtgcaac 180

tgttctttcc actaaatggg tacgttgaac cactgttcat gctcgaaaag gaggtgagct 240

atgaagattt tgaagcatgg ttatcgccaa ttctatctgc cttggctgac cagttcttgc 300

gtcggtatcc catcgcctca taccatggcc gtttagtgaa tcctctgcta gcgaacgcca 360

ttgtagctgc gtttttgtca aatgcgccat atgcgcacgc tattgaacat ctcttcttag 420

tgcgtggagt gatagaagac attgtcgacg ctggtttatc cattcaaaat cacctgtggt 480

tcgatcgtgg cgctctcgtg tcacccgctg gtcagaaatt cattcagctt gacaagtact 540

ctttctcgtc tcgtgaccca tgtctcttcg ctaagcagtc tcgctactat ggcctggttt 600

actatttcct caacatatcg gactgcttgt cctattgcta tcgtcatctc tcaaacttca 660

cacctctgct gcactttgat cgaccatcca acggcgtgca ttgtctagta ccgtcggaat 720

ccacgactcc catagccggg tctttacctg tgtcctccct gagtgccatc ttattagaat 780

cttgcattca gcagtctatt ctgaacactc ggactccgac gggcgcacca tcgacgagac 840

agatagaggc tcttttgccc gtgtcctctc ccttctttga acgtcccacc actttggaat 900

actctttgtt ttctctttct aacgctttag taggtggtta ccagctatta gatctacctt 960

ctggtcaccc tgattgttct accgttgctg ctttgttggc tcgattaacg gatttttcca 1020

aggagatcac agtcattcaa cctattccgt cgcgttttac tttgtacgct gacagtccat 1080

tgaattacag tggtgagaac gccatgtttc taagccgttt gccatgtgaa tctggtaaat 1140

ccgtcggtcc tgtctttact ggtaaacctg ttaataggag tattggttgg atgcctcagt 1200

tcgatcctgc tacgtcatat gatcctgaaa tggcagctga gtccctagcc aaagcgacca 1260

ctctcccatt gcgtgcaaaa ttttcatcat tctggtccgg acccgcactt ttctctgccg 1320

cctcatgtga cagacacaat ggtgtgtatg acctgcgctt tatgccacct tttccatcca 1380

catactttga tgaggatgac gtgttttctc gctctcggtt ttcatcttat cgtgctgtcc 1440

gggaccgttc gcttttgaag gataccgcca atctcatgta tgtctccaat ttgtcgaatt 1500

cacatgacca gcgtcttctt cctgaatcta aaactatggt gtatgtgggc gcttctggta 1560

ctcacactga taatcagcca tcaattatca agcccttgtt agacggcact cttcctggcg 1620

tcttcaagcc cctttcggtt aaacaggttg gttgggaggt taccaacggt actatctgtg 1680

atatcgagtt acccttggct actggaacat tcttcttcgt atacagtgat gtcgatcagg 1740

tacagtcggg cgattccgat ctcgtctcct cttctcgacg tttctgtctt caattagaca 1800

tgctgatgaa gctgacgtat acctctggtt ctgtgatcgt gaagtgcaat ttccctactg 1860

acttagtgtg gcgtcacatt ttctcaacta tctctccata tttcatgtcg atccatctta 1920

tgaagccgct tgtctccaat aacttagaac tgtacattct atttgctgaa aagttatccg 1980

tccctgacgc gcctttcaat ccctccgctg acgtggtgac attctggcgc tctcaacttc 2040

agcgctacag agttctccgg gattcttttt cctccgtccc tcagattgac tcagctctta 2100

cgctggacga cccactaacc gtgtcggtct taaatttcgt agatgtcaca tcactttctt 2160

cgctggatga tcagcgtgca ctggctgcct tctctgttct aacatctttg gggtcacaaa 2220

agttgtctat tcatccatat tttgacagtt accgcacaca gctgaccggc atcgtgacac 2280

ctcattctcg caacctcgtt gatcgcttag cgtacgtgcc gcgcgtgttc ccttctacca 2340

ttgacgtcca acatcgcact atcgctcctt ctgatcctga gatattcggt tttcgttcca 2400

acgcgtggac gcagctctct ttcttctacg atcacgcact cacgttgatg gattttactg 2460

atgttaagca ttggctcgac ctcggcacag gtcctgaagc gcgtccctta tctttcttgc 2520

cttccgacct tccagtgact ctttgtgacg tacgcccctt cgtcctgccc accggatgtt 2580

ggtccacatt taccgatatg ctgaattatg attatttaac aactaatgtg gtgttgtcca 2640

ctggtgcgga tgttgtctcg tgtgttctct ctctgggtgc ggcttgcgct gatgcaaaca 2700

tgtcccttca tgatggtgta cgccagttag tctctcagtg tgttgaagct aatgtccgca 2760

cacttttcct ccaacttaac tgtcctctcc cttccgcggg cgatgtatct cgtgatgttt 2820

tggagttggt ccagtctaac tcaacttatg tcttccattc ccttggacgc gtcgagccct 2880

tccttcctta ctcagctttg cttgaattga ttgaagatat ctgccctggc gttgtggttg 2940

agattaaaac catggatcct tccctagcat ggctaagtta cgcggtgcaa gccaacgctt 3000

cggtaacatc cgatgacatg gcattagcga tgcggctgtc ccacttctgc ccgttatttg 3060

ttttccgttt cgatcaaaga tctgcccaat ttccggacga cgctcgcgtc ggcgttccgt 3120

tctccgtgat tgtgtctaac tatgatgcta cacactctta tgaggtcact cttgacaacg 3180

ttaccatcgc gaccgtcact gctggttcct taataggctt ctcttctggt gtgactgttt 3240

cagttcaagg tactctactg actctctcta ttaatccgtc tagccctggt gttctctccg 3300

tagttcagac attgccagtt cgcatctctc ttggcagttg cgttatagac gctccggatc 3360

catcgctgtc tcttgtgttc ccttcagtcc tagatacttc tttgtcgggg acgaacttag 3420

agttgtactt gtccgactgg tacgatgttg cactcttcta catcgacgag gtcaactctc 3480

gactgattcc tctatccgat tcaaaatatg aaatctttcg acgtgatcaa gcccctaata 3540

gccgcaccct caactatatc ttcgaccgat cggatgtttt ctttaaaatc gtgttgtgtg 3600

acgtctcatc ctctggtgta ggccgattca tctatcgtga acttcctgag ctgagttctc 3660

cggtatggcc tgacaacatg cgcaccttct tgtcattgcc ttttgatgct cctatggtga 3720

ttatctcccc ggatggtccc gtgaattacg atggtgctgt aatcacccct cctacttcat 3780

ggctgacggt ggatggtagt acctgcgtca ttgatggacg tccatcattt tacgttcctc 3840

ctggtcgata cggtctggtg agagtctaaa cgatcgcggg cctccagtag aagggtgtta 3900

ctcatc 3906

<210> 6

<211> 2264

<212> DNA

<213>the M1 gene of strain N-DAV-XT18

<400> 6

atctagccac accggtgcta ggagttggtt ctcgtattac cgcattggat cgcactattg 60

acgcacttac tttgaaacca cgtaccgaac tgcacgatgt ctacactctt gatccatctt 120

taactctacg ccagattgag ttgatttcat ctggtacttc aatggacgat atcgctcgcg 180

gtttgcttca tcgggattgg cgtcgtcaaa ctgtcattac tctgcttcct tctcgtcgga 240

ctttactcga ctatctactc tctaatcctt cgttgtgtcc tgatggttta gatcgttcca 300

ggctcaaagg gttcaagaag cgtcccaatg acttccgcat cagtgacttc ttttctccgt 360

taatcactga gtccacttct attagcacat actcacgctg gctcaatgcc catcctgtta 420

tattctccat cactcacaag gtagtcggcg ctcgcttacg cctattcggt ccgtctaaat 480

tatatacatt atctcccgac gttcttcgag agctatcgat tttgaagtca actgatcgtg 540

ttcttgtcac acccactgcg cgtgtgtacg tgagttgttt ccctagtgct tctacgagta 600

actgcgtcct ctctgcccgc gaacgttgga acgctcccga tgtccatccc gtcgttaaag 660

ccatacaact ggtctacgat caccagtacc gcgtcaccgc gcgttatctc tccgatccct 720

tagcttcggc tttattgctt gggactcagt cggtacgctc tctgaaggtt ccccccatcg 780

aggctagagc tgctcgttca gttggcgttc gtgtacaggc aatgacccct cctcgtggta 840

tcaacacctc catcatacaa gtcatcgatc tgcgtttgca gtgccgtcac tcacttatcc 900

ctgttgagag accatttcca ttaactctcg taggcttgcc gtcctgcttg cttcagcatt 960

tggagttgac tttatccgat agctggacgc ctattcgaga ctcaactggt atgtttgaga 1020

tgtggttcat ggtactcact cttacgtgcg acaagattgt ggatggacgt ggtaatgctg 1080

tctttctaac ccctggttct actgcctctt cttcaattaa ttatgttcag ctcgtgtcta 1140

cttcttctcc tcggccgcaa tcgctggcat ctaacgcgtc tggtcgtatt gactccgtag 1200

gtttgtgtat gccaaagggt tcattcaaat cgactatgat taagttcctg actggcttag 1260

aaatatgtgg cacgcgcgta atgtactctg atgtcgtcat ggacagcgac gatgttggtg 1320

attcattgga ccctaccttt gagactactc tctttgatga gttgatggcg ttggatcctc 1380

ctttcgactt agataagtta gccagttcaa ctgaccttgt cgatcagtct tacatcgcct 1440

ctcacatgta tcccaccttt ctgaggcttg ttaacgaact gttgactcct cgagcttctg 1500

agctatactc agaacacagt gctgagttta ggtctttgac ttacgcccat gcagactctg 1560

aatttcttaa cgcttgttgg acagcacgat tgatgcgctg ctttattaac tactatgagg 1620

agcagaacat cctgctacgt cctgggagag ttggaggtgt attgtttcaa gttgcgctca 1680

gtcgttgcta taagatgttc gcgacatcca ctccttcagt tccattgtca ttattcctaa 1740

aatcactgtt cgtcccttgg attgaatcgg caccccttct cgccccactg actctcaacg 1800

aatcctcacg cgtactcgct tggtatatac ccgagtcaca atggacggaa aatggatggt 1860

gcatgtgtga taagcatcgt catgtgacct tttcttttat tcgtggtttg cctgctgacc 1920

tgtccgtcct tgatctgttc gactggtcgc gattccgcgc aactatcgga gtagacacaa 1980

ctctcgtgga gctgggtacc tccattcgtg ccgttcgcgt ctccgttttc tggacttccc 2040

aaaaacccac tgtggacgtg tttgataacc gcgctctctt caccccattc aaacactatc 2100

atcttagtct tcactgtcgg tgcgcccttg gtcgaccatt caccgcgaag aacatgaaat 2160

tgtatttgtc cacggtagga aacgagaact gacgggccgt ggggcggtga cacccaggga 2220

gggtatgctg gtaaccctgg gttagtcgtc ttgagatact catc 2264

<210> 7

<211> 2158

<212> DNA

<213>the M2 gene of strain N-DAV-XT18

<220>

<221> misc_feature

<222> (1)..(4)

<223> n is a, c, g, or t

<400> 7

nnnntttgag tgctaacctt tctcacacga tgggtaacgc gacgtctgtg gttcagaact 60

tcaacataca aggtgatggt aaccattttt ctccttctgc cgagactacc tcctccgctg 120

taccgtcgct atcattaaat ccaggtctgc ttaatcctgg cggtaaagca tggaccctta 180

tcaacccaac gctcaatgca tccgatccct cgtcgttacg attgatgaca tccgctgact 240

tgtccacctt gtcccaatcc gcgattggta actccactgg ccttctccca acgtcaggca 300

tgtataccgt ggctaataaa gagacgttga gtgtggtgac gaatcatgca ctgtctcaat 360

ttgagaagct tcaaatggcc tgcgaactgg atcgtgacta tctggatgct cgaggcgtgt 420

caccagaatc cgtggatatc cacaattaca tagtctacgt ggactgcttt gttggtgtgt 480

ccgctcgaca agcggcttct aattttcagc agcacgttcc agtcatcacc aagtcaagaa 540

tgacacagtt catgacgtcc gcgcagaata tgcttcaggt ccttggccca tgggaacatg 600

acgttcgtga gcttctcacg atattgccga cttctaccac tgcaggcaag atcacttgtg 660

acatgaaatc cgtggttaac tttgtcaacg accagctctc tgacacaaat ttatgccgtc 720

tctatcctga atgtgctgct tcgtctgtag ctaaacgtaa tggcgggata cgatggaagc 780

aacctgacac tgacgaggct ccctcccttg caactaacga tatcgccgct tccacgatgg 840

gtgcgcttgc taacaccacc cccctggctg agaagtctaa ttctggtgaa gagtctatgc 900

gtttagtcaa cgacgtaggt gtggatatca tctgttctag agcgccagtc agctcctcag 960

tgtggtcacg tactgttgag ccaaaatcgt ataacatcag gacactccgc gtggaagaag 1020

cgctttggtt gcgggaatgt cagacatcta atggttttga cgttcagtac actctgccag 1080

atcagacaac tcataagcat ttctggcttc aacaaggttc cacagtgatt aacttggagc 1140

agacgggcgg catgatgttt gaggtgaata tctctggtaa agactacaag aagggcactt 1200

ttgatccaga taaccagaag ttggtgctcc tagttatgca atccaaaata ccttttgagt 1260

catggacttc cgcagcacag attactggta tagctcaagt ggctgaagtc actgtacacg 1320

ccgccgacag ctcgacacct ggtcggaaaa tcattggtga gacatcgtta tcgtatctgt 1380

ttgaaagaga gaccgttact acagctaaca ccgaggtgaa cacctacctc ttttgcacgt 1440

ggcaacttga cgatgctcag agtaatggcg acaatgcttg gcaggatgcg tgggatggta 1500

ttaccacttt gaccccactc acttcaggca ccgtgacggt taagggtacg tctgtggact 1560

ctgtcgtgcc gactgactta gtgggctctt atactcctga agctctggct gctgcccttc 1620

caaacgacgc cggacgtatt ctcgccaata aggctattaa gctggctgat gctatcaaga 1680

aagaggatga ttctgtcatt gacgagtctt ctccattcag cacccccatt cagggagtcc 1740

tcgccgtcca acagctagat accgttggga cacgtggtgt gcgtatcatc caacctccct 1800

cctttctgaa acgtgtcgct tcacgtgctc ttcacatgtt cctcggcgac cctcagtcca 1860

tcttgaagca ggcgacaccc gttcttcgag acccggacgt ttggactggc ttcatccaag 1920

gagttcgtga tgggatccgc accaagtctc tttccgctgg agttagatct gtctacaaca 1980

acgtcaccgc aactcaatca gtgcagacct ggaagcaggg atttctgact aagatacaga 2040

cgttgttcag accatagtga ggtgctaagg cctctctgcg cggcgggtcg gtgggcacgt 2100

cgcgttgatg ctgaatgcac ggggaggtga cgctctctgg gttagcacgt tactcatc 2158

<210> 8

<211> 1996

<212> DNA

<213>the M3 gene of strain N-DAV-XT18

<220>

<221> misc_feature

<222> (1)..(4)

<223> n is a, c, g, or t

<400> 8

nnnntttgag tcctagcgtg gatcatgtcg tcaaccaagt ggggagacaa accgatgtcg 60

ctctcaatgt ctcatgatgg gtcgtccatc cgttctgctg catcacaatt tttgtcggtt 120

ccattgtccc actccactcc tattccacct caacggaaga ctgtgcttct gaagttcatg 180

ctcggcgaag atctggttac cgttcagggt actctggctc cgtttgatga ctactggttc 240

gataatcaac ccctgctttc ccaggctgtg gaactgttgg catctgagga ccgtctacgt 300

aattttgagc attatgagaa gtttcttctc aagaagggtc atcagctacc ggagattatg 360

aacagactgc gtttattctt cactgatgtg ttgaaggtga aaatggaagc tgacaatcta 420

ccgtcccttg ctcaatattt gatggctgga accatggatg ccgtttctac tggtcaccct 480

cccggcgcct ctgtgccaga cgtctctaaa gtggtagcta agcagcaaac gatctctaaa 540

tcgcccggtc gtttggatga ggaagagtat aacgtaatac gttcacgctt cttaactcac 600

gaggtatttg acctgtcatc cgatctccca ggtgtccaac cctttctgga catgtattat 660

gctaccgttc cacgtgctga tgctactggc tggtgcgctt cacgacgcaa agggttactt 720

gttcacgctc ctggcgaacg tttcgaggat ctaaccatat ttgccactga tacctctcta 780

ccaaatgagc tcatattaac ggcaggtgat gtcactgtag cgcgctttga ccttcttgac 840

gtgtccggga tagcccctca acatcacgct tcagtccaag aagagcgtac tgttggaacg 900

agtcggtatt cagccattac tgcaaacgat caccctttgg tgttcttctc tccctcagcc 960

attcgttggg cgattgatca ttcgtgcact gattcgctca tttctcctcg gaatatcaga 1020

gtgtgcgttg gcatcgaccc gttgattacc cgttggacgc gcgatggtgt gcaagaagct 1080

gctattctga tggatgacaa gctcccgtct gtcggtcgcg ctcgcatggc tctcaggaca 1140

ttgactctcg ctcgtcggtc gccgatgatt tctttcatga ttggagcgtt gaagcactca 1200

ggtggtcaac tcatggaaca ctatcgatgt gacgcagcta atcgatacgg ttcgcccacg 1260

gtgccagcct ctcaccctcc accctgcgct aagtgcccgg agcttagaga acaaatcacg 1320

aggctatctt ctcaacccgt cgccaattca caacctctgg ccggtccagc tgctctgctg 1380

tcgaagatct ctgaattgca acgcctcaac cgggaattgt ctctgaagtt ggcagatgtt 1440

caaccggcac gtgaagacca tctcctagcg tatttgaacg agcatgtgtg cgtgaacgcc 1500

aaggatcatg agaaggacct ccttgcccgt tgcaatgtgt ctggtgactc agtcaatacc 1560

gtcgtcgccc agagagctaa gaatcgtgac aggtttgagg cccgcttgcg tcaggaagcc 1620

aacgctgaat gggagccccg catggctgca ctaaatcaag agctcacaca atcacgcgct 1680

gaacagcagg agatgatgac gcaggcgctg caatacctca acgagcgtga cgaattggct 1740

caagagttgg atgagttgaa acgggagatc actactctga gatctgcgaa cgtgaggttg 1800

aatgctgaga accaccgaat gagtcgagcg accagagtgg gtgattcttt tgtcagtgat 1860

gtcctctcga cgccttccga cgttccacga acttctgcac cttccatgga tgacctggtg 1920

gacgacctgt gagctttgac ctgtgactcg gtttctctct gactccatga ccccacggcg 1980

gactcggtta tccatc 1996

<210> 9

<211> 1568

<212> DNA

<213>the S1 gene of strain N-DAV-XT18

<400> 9

gcttttttct tctctgccca tggctgacgg tgcatgcaat cacgctactt ctatttttgg 60

agccgtatat tgtcaaatat cacagaatat tgcacacgga aatattgatt cttacacctc 120

ttggacttct tatttacctc ctattctagg cggtggtttt ggtcttattg ttcttctcgt 180

gcttgtggtt ttgatcgtgt attgttgtaa gcattcaaaa attctttctg ccgttaaggc 240

gactgattct gccgtgacaa ctttgcttcg tgatgtcact cccgccaacc ccgatcctgt 300

tcaagtcgtt taaagtccac tcgtggcgtc ttttgtccca atccccgttc cacgttcagt 360

tctgcgacgc aggactccaa tcctacgacg tctattcacg tttcccttct gtgtgtgacc 420

tttcgctctg ttattatctt aatacacctt tcgagtttgg aattgctgct atagaggggc 480

gtcccggtga ctattatttg ctgttcgctg gcaagtccag tgactctaac tcacgagtat 540

ctttgtacgc tacgcggcaa gccggtgacg atggatcgca acgaggtgat acgcctgata 600

ctttccctcc tcccctacca gtcaagcgac gtcgatcatt tgacgacaca gatcaaatcc 660

ctccaaagcg ccgtcgactc actgaaagaa tcacaagtgg tagtgttgag acgcctgact 720

acgattacgt cgacggtggc ggatctacaa tcaacaactg aattgttgac ctcacaggtg 780

gcaggactta gttcccgtgt ggcttcagtg actgatgagg tagtccgtgt aaattcagtg 840

attggaacta cgatcactaa tcttgacaat gtccggtccg agctatcctc tctctcctcc 900

caagtctcgt cgcagacgtc cactctaacg aatcttacat caaccgtttc atcccagtct 960

cttgcgattt ctgatctcca gcgacgagtt acggccttag aacgatcggg tggtgcgccg 1020

acacaatttg aagctccctt gcacctacaa aacggagtcg tctcactcca agcatctccc 1080

tctttctgtt ctttgtctcc gatcctctcc ggacctgctg atgctgctgt tttcaaggtt 1140

ggtgagtggc tgggaactgt catatctggt caaagtcagt catctgcaat catgaacgtg 1200

cggattcatt catttgggca gcggaccatg ttgcttatgt cttcgcaaaa tgtattcact 1260

attccgccag gttcgggtgc gtctttgcag ctagatgtga atcgtataac gacccctgcc 1320

attgacgctg ctatggtaac tccttccgct gcttttgctt ctgcttcctt tatggctgac 1380

atagctttca aagactctaa gacaggagaa gtccatgctt tacacactac tggctctttt 1440

cgatcacctt ctttctctat cgtttgggtc ccggttgctt cggaaactcg taattatcaa 1500

ataatggcgt tacgcttcac cgtcgccacg ggctaggctg tggcgcgcaa tgagaagagc 1560

tattcatc 1568

<210> 10

<211> 1324

<212> DNA

<213>the S2 gene of strain N-DAV-XT18

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1323)..(1324)

<223> n is a, c, g, or t

<400> 10

nctttttctt ccacgatggc gcgtgccgtg tacgactttt tatctacgcc tttcggtaac 60

cgtggtctag caactaaccg tactcaactg tcgtcactac tgtcaagttc aaattcgcca 120

tggcaacgct ttttgtccgc cttaactcca ctgaccgctc caggcattgt ttcaacccct 180

gaggcaccct acccgggttc atcgttatac caggagtcca tgcttcacag cgctactatc 240

cctgggattc taggtaatag agacgcgtgg cgcaatttca acgtattcgg cttttcatgg 300

acagatgaag gtttgtcagg acttgtcgct gctcaggacc ctcctccagc tccaccgtac 360

caaccagctt cgggacagtg gtctgatctg cttcagtatc ctcgatgggc taatcgtcaa 420

cgtgagttgc agtctaaata tcccattctg ttgagatcta ctttgctttc agctatgcgc 480

gctggaccgg tgttgtacgt tgagacttgg cccaacatga tttctggtcg acttgctgac 540

tggttcatgt cgcagtatgg aaacaacttc gttgacatgt gtgcacgatt aactcagtcc 600

tgcatgaaca tgccagttga gccggatggt aattatgacc agcagatgcg agcattaatt 660

agtctgtggc tcctctcgta tattggcgtt gtgaatcaat ctaacactat taacggcttc 720

tactttgctt ccaagacgcg tgggcaggct atggataact ggacgctgtt ctatgctacc 780

aacaccaatc gggtgcagat tactcagcgc cactttgcct acgtttgcgc tcgctccccc 840

gactggaatg tcgataagtc ctggatcagc gctgccaatt tgactgccat tatcatggct 900

tgccgccagc cgccagcctt tgccaaccag ggggtaatca accaggccca gaatcgaccc 960

ggattctcga tgaatggtgg aacgcctgtg cacgagctga acctgctgac taccgctcaa 1020

gcttgtatcc agcagtgggt cgtagctggt ttgatctcag cagctaaggg tcagtccatg 1080

acgcaggagg cgaatgactt ctcgaacctc atccaagccg atcttgggcg gatcaaagcg 1140

caagatgacg cgttgtacaa ccaacaacct ggttacgctc gtcgcattaa gccgtttgcc 1200

aacggagatt ggacgcctgg aatgactgcg caagcattag ctctactagc cacttttacc 1260

gtctaggcgt agggtcgtac gctgcctgag tccagccctc cggcagcacg tggacgtact 1320

cann 1324

<210> 11

<211> 1202

<212> DNA

<213>the S3 gene of strain N-DAV-XT18

<400> 11

gctttttgag tccttagcgt gcaagccgca atggaggtgc gtgtgccaaa ctttcactcc 60

tttatcgaag gtattactac tagttacttg tgttctcctg cgtgctggaa ttcgaagacg 120

ttatgggata ttgaagaatt tcacacacct gacgttatca gggtcggcaa cgcttattgt 180

tgcactcagt gctgtggtgt tctgtactat ggtgcccctc cctctgatgg aaactgtttt 240

ccacatcaca agtgtcatca acagcaatat cgtactgaga ctccgctcat gagatatatt 300

aaggtgggtc gcactacaga gcaactgctt gatcaatatg ccattgctct gcatgtcatt 360

gcagattact atgatgaggc gagtaagcaa cctcatgata ttgctgaagc tgagtcaatc 420

gcaccatttg atatcgtaac caggactgaa tctattcgca gtgaccgtgc cgttgacccg 480

gaattctgga cttatccgtt agagaggcga ggatacgacg cgcgacatga gattgctaga 540

gcgggttgga agatgatcga tgcttcatcg cgaagtcaca ctcttcctga atgtctggtg 600

tcaaatatgc tacatactag gcatgtcttc agccaaatgt tgaccacgac aaccatctat 660

gatgtcgctg tcacgggtaa ggccgttaaa ttcagtccga tggtagcaac catgccaact 720

cgaggagatg gtgctgtggc tctgtcaaga ggtaacttgg atcatgatgt cgaggactgt 780

tggatggatg gttttgcatt ctcccccctc atcggcggtg ttggcatcac tggtcaattt 840

gagcgtggtt cctgccataa ttttgggcac cccatgattg ggagcggtaa gaaagcttct 900

cactaccgca atttgttcat ggaatcctgg cgtggatggt caaagtcgtg ctttacatgt 960

gctgcaggga tggagcccgc ggagtgcgaa tctaggctgc gaggccacgc cagaactatg 1020

ttcggacgtt ctcttccgga tatctgtgac ttcgaggaga ctacccacgt tggccagtcg 1080

tccgcgccat taaagaaggc cacgaaattg tccttcctgg agtgtaggtg gtaagcacct 1140

ctgggtcaaa atgcacatag gctcccacct atgtgacggt tagcgggact cacctattca 1200

tc 1202

<210> 12

<211> 1191

<212> DNA

<213>the S4 gene of strain N-DAV-XT18

<220>

<221> misc_feature

<222> (1)..(4)

<223> n is a, c, g, or t

<400> 12

nnnntttgag tccttgttca gccatggaca acaccgtgcg tgttggagtt tcccgcaaca 60

catccgtcgc cgctggtcag actgttttca agaacttcta cttactccga tgcaacattt 120

cagctgacgg tcgaaatgcc acgaaagcgg tccagtctca cttcccatac ttgtctcgcg 180

ctgtccgttg cttgtccccg cttgctgctc actgtgctga tagaactctt cgtcgtgata 240

acgtcaagaa cattttgacg cgtgatctgc cctttccatc cgatctcatt aactacgctc 300

accacgtgaa ctcgtcctcc ctcaccactt cccaaggcgt tgaagccgct cgtctcgtgt 360

cccaggttta cggggagcaa gttcctttcg accacgttta tccatctggt tcagcaacct 420

actgtcccgg tgccgtcgcc aacgctatct ctcggcttat ggcaggtttt gtgcctcagg 480

aaggtgacga tttcatgccg actggtccaa tcgattatct cgcagctgat cttgtggcgt 540

acaagtttgt tttaccgtat atgttagata tggtggatgg acgtccccaa attgtgctac 600

cctctcacac cgttgaggaa atgttgactg acactggcct gctgaatgct attgatgcct 660

catttggcat tgagtctaaa agtgatcaac gcatgacgcg tgatgctgct gagatgagct 720

ctcgttcgct caatgagtta gagaaccatg aacatcgcgg gcgcatgccg tggaagatta 780

tgcttgctat gatggccgcg caactgaagc ttgaactgga cgcattagca gacgaacgca 840

ctgagtctca ggctaacgct catgttacat ccttcggctc acgtctattc aatcagatgt 900

cagcctttgt gcctatcgat cgtgagttga tggagcttgc tctcctaatc aaagaacaag 960

gctttgccat gaatcctggt caggttgcgg ccaaatggtc gtctattagg aggtccagtg 1020

cagtacgttc cctggcgagt gcgcgtcttg agattcgaaa tgggaactgg atgatccgcg 1080

aaggcgacca gacgctgctg tctgtctctc cagctaggat ggcgtagacg ggacccatgg 1140

tgtgggtgag gggggccacg acctctgcca cgacttggac tcttattcat c 1191

<210> 13

<211> 980

<212> DNA

<213>σ C gene

<400> 13

tacgcctgat actttccctt ggatcgcaac gaggtgatac gcctgatact ttccctcctc 60

ccctaccagt caagcgacgt cgatcatttg acgacacaga tcaaatccct ccaaagcgcc 120

gtcgactcac tgaaagaatc acaagtggta gtgttgagac gcctgactac gattacgtcg 180

acggtggcgg atctacaatc aacaactgaa ttgttgacct cacaggtggc aggacttagt 240

tcccgtgtgg cttcagtgac tgatgaggta gtccgtgtaa attcagtgat tggaactacg 300

atcactaatc ttgacaatgt ccggtccgag ctatcctctc tctcctccca agtctcgtcg 360

cagacgtcca ctctaacgaa tcttacatca accgtttcat cccagtctct tgcgatttct 420

gatctccagc gacgagttac ggccttagaa cgatcgggtg gtgcgccgac acaatttgaa 480

gctcccttgc acctacaaaa cggagtcgtc tcactccaag catctccctc tttctgttct 540

ttgtctccga tcctctccgg acctgctgat gctgctgttt tcaaggttgg tgagtggctg 600

ggaactgtca tatctatgaa cgtgcggatt cattcatttg ggcagcggac catgttgctt 660

atgtcttcgc aaaatgtatt cactattccg ccaggttcgg gtgcgtcttt gcagctagat 720

gtgaatcgta taacgacccc tgccattgac gctgctatgg taactccttc cgctgctttt 780

gcttctgctt cctttatggc tgacatagct ttcaaagact ctaagacagg agaagtccat 840

gctttacaca ctactggctc ttttcgatca ccttctttct ctatcgtttg ggtcccggtt 900

gcttcggaaa ctcgtaatta tcaaataatg gcgttacgct tcaccgtcgc cacggggcgc 960

aatgagaaga gctattcatc 980

Claims (9)

1. a kind of indirect ELISA testing kit for detecting novel duck reovirus antibody, which is characterized in that described indirect ELISA detection kit is coated with the ELISA Plate of novel duck reovirus σ C protein recombinant antigen；The novel duck is exhaled The deposit number of the lonely virus of intestines is CCTCC NO:V201843.

2. indirect ELISA testing kit according to claim 1, which is characterized in that the novel duck reovirus σ C protein recombinant antigen the preparation method is as follows:

Using the complete genome sequence for the duck reovirus that deposit number is CCTCC NO:V201843 as template, expanded using primer pair Increasing obtains σ C genetic fragment, constructs recombinant expression carrier, passes through prokaryotic expression novel duck reovirus σ C protein Recombinant antigen；

The sequence of the primer pair is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.

3. indirect ELISA testing kit according to claim 2, which is characterized in that turn the recombinant expression carrier Change into BL21 (DE3) competent cell, IPTG is added and carries out inducing expression；By expression product by being denaturalized with after renaturation, obtain The novel duck reovirus σ C protein recombinant antigen that must be purified.

4. indirect ELISA testing kit according to claim 1, which is characterized in that the novel duck reovirus σ The package amount of C protein recombinant antigen is the hole 500ng/.

5. indirect ELISA testing kit according to claim 1-4, which is characterized in that the indirect ELISA Detection kit also includes: enzyme labelled antibody, sample diluting liquid, cleaning solution, negative control sera, positive control serum, substrate are aobvious Color liquid A, B and terminate liquid.

6. indirect ELISA testing kit according to claim 5, which is characterized in that the enzyme labelled antibody is enzyme mark sheep Anti- duck antibody.

7. indirect ELISA testing kit described in any one of claims 1-6 is in the epidemiology of novel duck reovirus Application in investigation；

The deposit number of the novel duck reovirus is CCTCC NO:V201843.

8. a kind of indirect ELISA detection method of novel duck reovirus antibody, which comprises the following steps:

(1) it is coated with: using duck reovirus σ C recombinant protein that deposit number is CCTCC NO:V201843 as envelope antigen, It will be added to after antigen diluent in ELISA Plate hole, 4 DEG C of overnight incubations or 37 DEG C of incubation 2-4h, using the PBST for containing 0.05% tween Washing；

(2) it closes: being closed using PBST dilution 5% skimmed milk power, 200 hole μ L/, dry, use after being incubated for 1h under the conditions of 37 DEG C PBST washing；

(3) serum action condition: every hole is added serum to be checked and dilutes mixed liquor, dries after being incubated for 1h under the conditions of 37 DEG C, uses PBST Washing；

(4) add enzyme labelled antibody: by enzyme mark goat-anti duck antibody with PBST by 1:500 dilution, every hole adds 100 μ l, under the conditions of 37 DEG C It dries after effect 1h, is washed with PBST；

(5) substrate develops the color: 100 hole μ L/ of tmb substrate developing solution, and effect 15min is protected from light under the conditions of 37 DEG C；

(6) terminate reaction: every hole adds 50 μ L terminate liquid color development stoppings to react；Using reading data under microplate reader absorbance 450nm；

(7) determination of yin and yang attribute critical value: according to formula: yin and yang attribute critical value=negative sample OD₄₅₀Average value+standard deviation 3SD obtains yin and yang attribute critical value；Work as OD₄₅₀0.072 or more, it is determined as the positive.

9. indirect ELISA detection method according to claim 8, which is characterized in that in step (1), the novel duck is exhaled Intestines orphan's virus σ C recombinant protein is prepared by the following method:

Using the complete genome sequence for the duck reovirus that deposit number is CCTCC NO:V201843 as template, expanded using primer pair Increasing obtains σ C genetic fragment, constructs recombinant expression carrier；Recombinant expression carrier is converted into BL21 (DE3) competent cell, IPTG is added and carries out inducing expression；Expression product is recombinated by the duck reovirus σ C for being denaturalized with after renaturation, obtaining purifying Albumen；

The sequence of the primer pair is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.