CN108680744A - It is a kind of detection novel duck reovirus antibody indirect ELISA testing kit and application - Google Patents

Abstract

The invention discloses a kind of indirect ELISA testing kit of detection novel duck reovirus antibody, the kit is coated with the ELISA Plate of novel duck reovirus σ C protein recombinant antigens；The preparation method of the novel duck reovirus σ C protein recombinant antigens is as follows：With deposit number for CCTCC NO：The complete genome sequence of the duck reovirus of V201818 is template, obtains σ C genetic fragments using primer pair amplifies, builds recombinant expression carrier, pass through prokaryotic expression duck reovirus σ C protein recombinant antigens.The indirect ELISA testing kit of the present invention can fast 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

It is a kind of detection novel duck reovirus antibody indirect ELISA testing kit and Using

Technical field

The present invention relates to biological product technical fields, and in particular to a kind of to detect the indirect of novel duck reovirus antibody ELISA detection kit and application.

Background technology

Avianreovirus belongs to Hu Changgubing sections (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 2016, the outburst on a large scale of the ground such as China Shandong, Hebei, Henan and Jiangsu duck is with gambrel enlargement for main disease The communicable disease of shape, the disease mainly cause each age level duck gambrel swelling, gambrel often to contain a small amount of yellow or blood Sample exudate；Several cases have cheesy exudate in articular cavity, suffer from duck and different degrees of limping occur.The disease mainly results in The decline that kind duck is laid eggs with meat duck weight, feedstuff-meat ratio increase, and meat duck delivers qualification rate significant decrease for sale, to meat duck and kind duck cultivation Industry causes serious financial consequences.

Research confirms that this by the cause of disease of the high incidence duck infectious disease of cardinal symptom of arthritis is a kind of novel duck Reovirus belongs to emerging infectious disease, and step has still been in 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.

Invention content

For the above-mentioned prior art, the object of the present invention is to provide a kind of the indirect of detection novel duck reovirus antibody ELISA detection kit and application.It can be to newly breaking out using arthritis as the novel duck reovirus antibody of cardinal symptom It is fast and effectively detected, to monitor the popularity of novel duck reovirus in duck group.

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

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

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

With deposit number for CCTCC NO：The complete genome sequence of the duck reovirus of V201818 is template, utilizes primer σ C genetic fragments are obtained to amplification, recombinant expression carrier is built, passes through prokaryotic expression novel duck reovirus σ C Protein reconstitution antigen；

The primer pair includes：

Sense primer σ c-F:5’-ATGGACGGATTAACTCAACAGCAGCGA-3’；(SEQ ID NO.1)

Downstream primer σ C-R:5’-ACCTAAGGTGTGGCGCCGTACGGG-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 antigens of purifying are obtained.

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

Further, the indirect ELISA testing kit also includes：Enzyme labelled antibody, sample diluting liquid, cleaning solution, the moon 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 domain of invention；

The deposit number of the novel duck reovirus is CCTCC NO：V201818.

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

(1) it is coated with：With deposit number for CCTCC NO：The novel duck reovirus σ C recombinant protein conducts of V201818 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：5% skimmed milk power, 200 holes μ L/ are diluted using PBST to be closed, and are got rid of after being incubated 1h under the conditions of 37 DEG C It is dry, it is washed with PBST；

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

(4) enzyme labelled antibody is added：Enzyme mark goat-anti duck antibody PBST is pressed 1：500 dilutions add 100 μ l, 37 DEG C of items per hole It dries after acting on 1h under part, is washed with PBST；

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

(6) reaction is terminated：50 μ L terminate liquid color development stoppings are added to react per hole；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 OD₄₅₀0.071 or more, it is determined as the positive.

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

With deposit number for CCTCC NO：The complete genome sequence of the novel duck reovirus of V201818 is template, is utilized Primer pair amplifies obtain σ C genetic fragments, build recombinant expression carrier；Recombinant expression carrier is converted to BL21 (DE3) competence In cell, IPTG is added and carries out induced expression；By expression product by being denaturalized with after renaturation, the duck reovirus of purifying is obtained σ C recombinant proteins；

The primer pair includes：

Sense primer σ c-F:5’-ATGGACGGATTAACTCAACAGCAGCGA-3’；(SEQ ID NO.1)

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

Beneficial effects of the present invention：

For the newfound novel duck reovirus that can result in duckling gambrel enlargement, the present invention establishes detection The indirect ELISA detection method of the novel duck reovirus antibody, the detection method have easy, quick, stable, specificity By force, the characteristics such as sensitivity height, can to duck group in novel duck reovirus σ C specific antibodies be detected, and as according to According to come detect duck group in reovirus antibody level, to monitor duck group in novel duck reovirus popularity.

Description of the drawings

Fig. 1：Recombinant protein σ C induced expression and protein purification SDS-PAGE analysis charts in BL21 competence；Wherein, M swims Road 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, the 4 swimming lanes are to be precipitated after induction bacterium cracks.

Specific implementation mode

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 technology is introduced, from 2016, the ground such as China Shandong, Hebei, Henan and Jiangsu duck is quick-fried on a large scale Hair is using gambrel enlargement as the communicable disease of cardinal symptom.The symptom of disease duck reovirus infection different from the past, There is the symptom of gambrel enlargement in duck group for the first time, thus deduce, which may be to exhale the lonely disease of intestines by novel Caused by poison.It there is no the drug and method that can effectively control the novel duck reovirus infection at present.Based on this, this hair It is bright to propose a kind of indirect ELISA testing kit that detect novel duck reovirus antibody, to find to feel early Dye duck group takes it isolation or slaughters measure, is lost caused by novel duck reovirus infection with reducing.

It is known that the host range of Avianreovirus is wider, this virus of generally existing 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 larger variability, pathogenic, gene sequences Row and the coding of major protein all have differences.

Present inventor isolates one plant of duck reovirus N-DAV-SD16 from the tendon tissue of morbidity duck swelling, Genome sequencing carried out to the duck reovirus N-DAV-SD16 of above-mentioned new separation, and respectively by 10 genetic fragments with The Avianreovirus of existing report has carried out sequence alignment and homology analysis, as a result, it has been found that, new isolated strain N-DAV- L1, L2, L3, M1, M2, S3, S4 are respectively positioned in a relatively independent branch in 10 genetic fragments of SD16, and at present Through uploading, reovirus-originated duck homology is relatively low in Genbank, illustrates that the strain N-DAV-SD16 newly detached is different from it His Avianreovirus, it can be assumed that duck reovirus found in the present invention with it is existing it has been reported that duck exhale intestines lonely Virus exists compared with Big mutation rate, is 1 independent kind of Orthoreovirus, and the novel duck reovirus is preserved in China Type Tissue Collection, deposit number are CCTCC NO：V201818.Therefore, present invention novel duck to be detected exhales intestines There are variability for the duck reovirus of lonely virus and existing report, in the prior art still for the duck reovirus reported Without effective detection method, for its difficulty bigger of the detection of the novel duck reovirus.

The present invention carries out antibody test with serological method to novel duck reovirus for the first time.It can be to newly breaking out Fast and effeciently detected by the novel duck reovirus antibody of main feature of gambrel enlargement, be a kind of easy The indirect ELISA detection method used suitable for base.

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 institutes Show.According to the σ c protein gene orders of the novel duck reovirus obtained, two primers are designed：

Sense primer σ c-F:5’-ATGGACGGATTAACTCAACAGCAGCGA-3’

Downstream primer σ C-R:5’-ACCTAAGGTGTGGCGCCGTACGGG-3’.

The cDNA that reverse transcription obtains is amplified by the σ C genes that length is 1000bp 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 clone after purification is arrived In prokaryotic expression carrier PET-28a (+), recombinant prokaryotic expression vector PET28a- σ C are built；By recombinant prokaryotic expression vector PET- σ C are converted into BL21 (DE3) competent cell, express recombinant protein σ C through 1mM IPTG inducement efficients, the albumen is with packet The form for containing body exists, and inclusion body is by denaturation and renaturation, using the reovirus σ C proteins of purifying as envelope antigen.

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

1. being coated with：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 washings of 0.05% tween (mass concentration) are three times, every all over 4min；

2. closing：Closing dilutes 5% skimmed milk power (mass concentration) 200 holes μ L/ using PBST, and number is incubated 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：Appropriate serum to be checked is added per hole and dilutes mixed liquor, after being incubated 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, per 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 holes μ L/ of tmb substrate developing solution are protected from light effect 15min under the conditions of 37 DEG C；

6. terminating reaction：Add 50 μ L2M H per hole₂SO₄Terminate liquid color development stopping is reacted；

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 OD₄₅₀0.071 or more, it is determined as the positive.

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 exhales intestines according to the fowl delivered on GenBank during the test The major antigenic sites region difference of the code area of σ C proteins and the novel duck reovirus in the gene order of lonely virus Design primer pair, amplification obtain genetic fragment, build recombinant expression carrier；Recombinant expression carrier is converted to BL21 (DE3) and is felt In by state cell, IPTG is added and carries out induced expression；By expression product after denaturation and renaturation, purification of recombinant proteins is obtained, Using the recombinant protein of acquisition as envelope antigen, indirect ELISA testing kit is built.The blood made a definite diagnosis on applying clinical The specificity of this indirect ELISA testing kit prepared to different envelope antigens of final proof is investigated.The results show that with this hair The indirect ELISA testing kit that the reovirus σ C proteins of bright purifying are prepared as envelope antigen is to having made a definite diagnosis serum sample The rate of accuracy reached 100% of detection, the sensibility and specificity of detection, which is significantly better than using other recombinant proteins as coating, to be resisted Indirect ELISA testing kit prepared by original.

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 solution：1 × carbonate buffer solution (100mL, pH=9.6)：Na₂CO₃0.2756g, NaHCO₃ 0.6216g with distillation water dissolution 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, use distilled water Dissolve and be settled to 500mL, pH value 7.1-7.3.

PBST solution：Tween-20 0.5mL are added per 1L PBS, mix well.

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

TMB developing solutions：

buffer A：It weighs 66.5063g sodium citrates 800mL distillation water dissolution and uses dense HCl tune pH to 4.0, addition H₂O is settled to 1L, 4 DEG C of preservations；

buffer B：0.2956g tetramethyl benzidines (TMB) are weighed, 0.0633g tetrabutyl ammonium borohydrides (TBABH) are molten In 30ml dimethylacetylamides (DMA), 4 DEG C are kept in dark place；

In use, by buffer A and buffer B with volume 39:1 ratio mixing, matching while using.

2M H₂SO₄Terminate liquid：By dense H₂SO₄With volume ratio 1:5 ratio is added in distilled water, and 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 editors such as Luke, Science Press, 2002, Molecular Cloning:A Laboratory guide (third edition)；D.L. the chief editors such as Spector, science go 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： V201818) pair of primers is designed in complete genome sequence σ c proteins code area, and two sections of primer is respectively equipped with I digestion of BamH I and Sal Site, it is contemplated that the genetic fragment of 1000bp in the novel duck reovirus gene group can be amplified.

Sense primer σ c-F:5’-ATGGACGGATTAACTCAACAGCAGCGA-3’；

Downstream primer σ C-R:5’-AGTCGACTTAGGTATCGATGCCCGT-3’.

1.2PET28a- the structure of σ C prokaryotic expression carriers：

σ C genetic fragments are gone out by PCR amplification using specific upstream and downstream primer, segment after purification is carried with PMD18-T Body connects, and connection product is transformed into DH5 α competent cells, and picking positive bacterium colony shakes bacterium extraction T- σ recombinant plasmids.With limitation Property restriction endonuclease BamH I and Sal I (being purchased from Dalian treasured biotech firm) respectively to T- σ recombinant plasmids and PET-28a (+) carrier into Row double digestion, with both T4DNA ligases (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 results of picking show to expand the purpose band to 1000bp or so, and pre- The σ C genetic fragments of meter are in the same size.The single positive colony bacterium of picking extracts plasmid after dropping into row Zengjing Granule, with restricted interior Enzyme cutting BamH I and Sal I carries out digestion identification, and digestion band is correct.Positive colony sequencing result shows that σ C genes are inserted into position Set, direction of insertion and reading frame it is correct, the results showed that recombinant vector PET28a- σ C are built successfully.

1.3 recombinant protein σ C induced expressions and identification：

Successful recombinant vector PET28a- σ C plasmids will be built to be transformed into BL21 competent cells, picking single bacterium colony Zengjing Granule is carried out, next day presses 1:100 ratio is inoculated in containing 100 μ g/ml kan⁺2 × YT culture mediums in, 37 DEG C, 200r/ Min shake cultures are to OD₆₀₀1mL bacterium solutions are taken out between 0.6-1.0 as being compareed 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 mixings, boils Concussion cracks bacterium after handling 5min, and supernatant is taken to carry out SDS-PAGE after slightly centrifuging, 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 bacterials to limpid, 10,000 under the conditions of 4 DEG C × g/min centrifuges 5min, and supernatant precipitation suspension is taken to add 2 × sds gel sample loading buffer mixing respectively, boils processing 5min SDS-PAGE identifies protein expression mode afterwards.

1.4 protein purification：

A large amount of Fiber differentiations are carried out according to protein induced expression condition in 1.3.Take inclusion body with reference 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 expressions in BL21 competent cells, all with inclusion body Form exists, and molecular weight of albumen size is 50kDa, (referring to Fig. 1) in the same size with expected recombination σ C.

1.5Western Blotting analyses：According to method induction expression protein in 1.3, induction and non-induced is taken respectively Full bacterium SDS-PAGE, is operated with reference to routine Western Blotting methods, finally using enhancing HRP-DAB substrates colour developing Reagent develops the color.As a result it shows：Compared with SDS-PAGE, after PET28a- σ C conversion BL21 competent cells induction There is the band of specificity in Western Blotting corresponding positions, 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 do envelope antigen and establish indirect ELISA method, are groped using square formation method ELISA optimum proteins peridium concentration and serum diluted concentration and ELISA optimum reaction conditions.It is final to determine that condition is as follows：

2.1 coating：Antigen presses 1 with 1 × carbonate buffer solution (pH=9.6):After 1000 dilutions, 10 μ L coatings to 96 holes are taken In elisa plate, preservative film is wrapped, 37 DEG C of incubation 2h, three times using the PBST washings containing 0.05% tween, every all over 4min.Detection The hole holes σ C protein package amount 500ng/；

2.2 closing：Closing is used using being dried after being incubated 1h under the conditions of 200 37 DEG C of the holes μ L/ of PBST 5% skimmed milk powers of dilution 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 are added per hole Liquid is dried after being incubated 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 presses 1 with PBST：500 dilutions add 100 μ per hole L is dried under the conditions of 37 DEG C after effect 1h, is washed 4 times with PBST, each 4min；

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

2.6 terminate reaction：Add 50 μ L2M H per hole₂SO₄Terminate liquid color development stopping is reacted；

2.7 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 OD₄₅₀0.071 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, only (deposit number is CCTCC NO to this method with novel duck reovirus：V201818) positive serum reaction is in sun Property, illustrate that this method has specificity well.

The foregoing is merely the preferred embodiments of the application, are not intended to limit this application, for the skill of this field For art personnel, the application can have various modifications and variations.Within the spirit and principles of this application, any made by repair Change, equivalent replacement, improvement etc., should be included within the protection domain of the application.

SEQUENCE LISTING

<110>Shandong Agricultural University

<120>It is a kind of detection novel duck reovirus antibody indirect ELISA testing kit and application

<130> 2018

<160> 13

<170> PatentIn version 3.5

<210> 1

<211> 27

<212> DNA

<213>Artificial sequence

<400> 1

atggacggat taactcaaca gcagcga 27

<210> 2

<211> 24

<212> DNA

<213>Artificial sequence

<400> 2

acctaaggtg tggcgccgta cggg 24

<210> 3

<211> 3928

<212> DNA

<213>L1 genetic fragments

<400> 3

ctttttctcc gatctccgaa atgagttcgc gcaaagtggc tagacgtcgt cataaggatg 60

ctactgaatt taaatacact aagaacgtta acaagtctaa gccatcctct actgacgtta 120

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

tggactctcc ctcgtcgtcg cgtgaatgtg atcttcgcct cgccccacct acagagagta 1020

atgctcgatc attctcaatg ctgctcaata ctggtgcgac tccagaaggt actttcaacc 1080

ccaacaccct tcgtatgaat gtgctgcaga tgtgtcttca gtatgttctg gctaacctac 1140

acttgaatcg tagcactcag ttcaccatgg atttgactgc cgcagctccc aatctgtcgg 1200

cgtctcaact ccgtatcgtt ccagatgata aagatggtaa atggttccct gtcatgtatc 1260

catctcgcgt gaacatccca ctgttcaata agacagctga ttttgttaat cagtgcattc 1320

gtgacagaat tggccgatat gaccgcgccc agactttcgc tggcgcaccc tctgaatggg 1380

ctgacatgtg ggaaacagcg gacgcgttaa ctctctccgt ccgtgaaatg tggatgtcac 1440

gtatttccca aatgaatatc tctcctgctg atatcgctga cgctatctct cgatgttctc 1500

agtccctgct cactgttgcc gcgccaacag ctccttctgt agctcgtctg ttaccttggc 1560

gggttagttc cgatgagagg cagctcctcc aattgttgat gtacttgaac gttgggacta 1620

gtgccgacta cgtccaacct attctgtctg cgttcgctcg aactctatct cgcgtgtcac 1680

cattgcgtat taatcccact ttaatcgcta atgccatgtc gacaattgtc gagagcacta 1740

ctaataccca gagtcctgcg gcagctattt tgtcgaagct taaacctgtt gcctcggact 1800

tctccgattt taggttggcg tgtgccgctt ggttgtataa tggttgcgtc cagacatact 1860

tgtctgagga ttcatatcca agcagtggtg gatctgttac tagcattgat acattggttg 1920

atatgtttgt gtgcttgttg gctttgcctc ttgttactga tcctaatgct ccttgccaag 1980

cctttatggt tgtcgctaat gccatggttg gttacgagaa tctaccgatg gacgacccta 2040

attttactca gcaaagattg gctgcagcgt tcaacaatcc cacgacctgg cctcaatgtt 2100

tcctccaccc tcaaaacatc gatcgacgcc aatgtccgat tctctcatgg tgggctcagc 2160

agattcatcg taattggccc acaccgtctc aaattactta tggggcgcct gacatcattg 2220

ggtccgctaa cttgtttact cctcctgacg tgctgttgct tccattacag cataggccca 2280

tccgtattac taatcccacc ctgaacttcg ataatgagtt gacgacttgg cgtaacaccg 2340

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 genetic fragments

<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 genetic fragments

<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 genetic fragments

<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 genetic fragments

<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 genetic fragments

<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 genetic fragments

<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 genetic fragments

<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 genetic fragments

<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 genetic fragments

<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>σ C genes

<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 (10)

1. a kind of indirect ELISA testing kit of detection 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 antigens.

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

With deposit number for CCTCC NO：The complete genome sequence of the duck reovirus of V201818 is template, is expanded using primer pair Increasing obtains σ C genetic fragments, builds recombinant expression carrier, passes through prokaryotic expression novel duck reovirus σ C proteins 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 induced expression；By expression product by being denaturalized with after renaturation, obtain The novel duck reovirus σ C protein recombinant antigens 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 holes 500ng/.

5. according to claim 1-4 any one of them indirect ELISA testing kits, 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. claim 1-6 any one of them indirect ELISA testing kit is in the epidemiology of novel duck reovirus Application in investigation；

The deposit number of the novel duck reovirus is CCTCC NO：V201818.

8. a kind of indirect ELISA detection method of novel duck reovirus antibody, which is characterized in that include the following steps：

(1) it is coated with：With deposit number for CCTCC NO：The duck reovirus σ C recombinant proteins of V201818 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 containing 0.05% tween Washing；

(2) it closes：5% skimmed milk power, 200 holes μ L/ are diluted using PBST to be closed, and are dried, are used after being incubated 1h under the conditions of 37 DEG C PBST is washed；

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

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

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

(6) reaction is terminated：50 μ L terminate liquid color development stoppings are added to react per hole；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 OD₄₅₀0.071 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 proteins are prepared by the following method：

With deposit number for CCTCC NO：The complete genome sequence of the duck reovirus of V201818 is template, is expanded using primer pair Increasing obtains σ C genetic fragments, builds recombinant expression carrier；Recombinant expression carrier is converted into BL21 (DE3) competent cell, IPTG is added and carries out induced 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.

10. deposit number is CCTCC NO：The duck reovirus of V201818 leads to the duck of duck gambrel swelling in preparation detection Application in the indirect ELISA testing kit of reovirus.