CN107238702A - Detect the enzyme linked immunological kit of PPR virus antibody - Google Patents

Abstract

The invention discloses the enzyme linked immunological kit of detection PPR virus antibody.The enzyme linked immunological kit of the detection PPR virus antibody, including envelope antigen, the envelope antigen are restructuring PPR virus H F fusion proteins, and the restructuring PPR virus H F fusion proteins are protein a) or b)：A) protein being made up of SEQ ID No.2 amino acid sequence；B) soluble protein that substitution and/or missing and/or addition by the amino acid sequence shown in SEQ ID No.2 by one or several amino acid residues is obtained.The enzyme linked immunological kit of the detection PPR virus antibody cannot be only used for the diagnosis of PPR, and evaluable immune effect of vaccine, in addition, it can also fast and accurately detect PPR, be conducive to the monitoring clinically to PPR, so that the propagation of PPR is preferably controlled for China will produce positive role.

Description

Detect the enzyme linked immunological kit of PPR virus antibody

Technical field

The present invention relates to the enzyme linked immunological kit that PPR virus antibody is detected in enzyme linked immunosorbent detection field.

Background technology

PPR (PPR) is the infectious disease of OIE statutory reports, is also classified as a class zoonosis by the Chinese government, The characteristics of it has high incidence and high mortality, the generation of the epidemic situation will not only be brought seriously to the Animal husbandry production for occurring state Economic loss, international trade can also be produced serious influence, therefore the disease causes many national great attentions.It is small to ruminate Epizootic disease (Peste des Petits Ruminants, PPR) is by PPR virus (Peste des Petits Ruminants Virus, PPRV) caused by a kind of acute, hot, contagious disease, with high incidence and high mortality The characteristics of.The control and elimination of epidemic disease, be unable to do without effective vaccine and quick sensitive detection means as technical support, therefore The R＆D intensity of quick effective, cheap diagnosis detection reagent is increased, accelerates to promote Related product listing, can be China National PPR eliminate the implementation of plan strong technical support be provided.

At present, it there is no effective ways to treat PPR, vaccine inoculation, epidemic situation can only be taken to slaughter and strengthen after occurring The method of regular sero monitoring controls the disease.Both at home and abroad it is most common report be with PPRV N after purification, H protein (total length) or Part restructuring N, H protein fragment comprising N protein Main Antigenic also have the PPRV totivirus with concentration as envelope antigen Indirect ELISA method detection PPR serum antibodies (Qiu Wenying, 2011 are set up as envelope antigen；Balamurugan et al., 2007) there is not yet the report of indirect ELISA method is set up, but by envelope antigen of H-F fused in tandem albumen.

The content of the invention

A technical problem to be solved by this invention is how to detect PPR virus antibody.

In order to solve the above technical problems, the invention provides the ELISA reagent of detection PPR virus antibody Box.

The enzyme linked immunological kit of detection PPR virus antibody provided by the present invention, including coating antigen, it is described Coating antigen is restructuring PPR virus H-F fusion proteins, the restructuring PPR virus H-F fusion proteins for a) or B) protein：

A) protein being made up of SEQ ID No.2 amino acid sequence；

B) by the amino acid sequence shown in SEQ ID No.2 is by the substitution of one or several amino acid residues and/or lacks Lose and/or add obtained soluble protein.

In above-mentioned enzyme linked immunological kit, protein name a) is referred to as rmHF1-Y (also known as rmHF1), is restructuring series connection PPRV H-F albumen.SEQ ID No.2 are made up of 1184 amino acid residues.

In above-mentioned enzyme linked immunological kit, the restructuring PPR virus H-F fusion proteins can be according to including as follows It is prepared by the method for step：The encoding gene of the restructuring PPR virus H-F fusion proteins is set to be expressed in biology Obtain the protein；It is described biological for microorganism, plant or non-human animal.

In above-mentioned enzyme linked immunological kit, the encoding gene of the restructuring PPR virus H-F fusion proteins is set to exist Expression is carried out in biology to be included the encoding gene of the restructuring PPR virus H-F fusion proteins importing the micro- life of acceptor Thing, obtains expressing the recombinant microorganism of the restructuring PPR virus H-F fusion proteins, cultivates the recombinant microorganism, Expression obtains the restructuring PPR virus H-F fusion proteins.

Any of in above-mentioned enzyme linked immunological kit, the recipient microorganism can be C1)-C4):

C1) prokaryotic micro-organisms；

C2) gramnegative bacterium；

C3) Escherichia bacteria；

C4) e. coli bl21 (DE3).

In above-mentioned enzyme linked immunological kit, the encoding gene of the restructuring PPR virus H-F fusion proteins is such as It is lower 1) or 2) shown in DNA molecular：

1) coded sequence is the DNA molecular shown in SEQ ID No.1 4-3558；

2) DNA molecular of the DNA molecular with more than 90% homogeneity and coding rmHF1-Y with 1) limiting.

Wherein, SEQ ID No.1 are made up of 3564 nucleotides, and SEQ ID No.1 4-3558 are rmHF1-Y genes Nucleotide sequence, the rmHF1-Y gene coding amino acid sequences shown in SEQ ID No.1 4-3558 are SEQ ID No.2 protein rmHF1-Y.

In above-mentioned enzyme linked immunological kit, 2) concretely nucleotide sequence and 1) have at least 91%, 92%, 95%, 96%th, the DNA molecular of 98% or 99% homogeneity.

In above-mentioned enzyme linked immunological kit, the recombinant microorganism can be that pET30a-rmHF1-Y is imported into Escherichia coli The express amino acid sequence that BL21 (DE3) is obtained is the recombinant microorganism of SEQ ID No.2 protein, the recombinant microorganism BL21 (DE3)/pET30a-rmHF1-Y is named as, the pET30a-rmHF1-Y is replaced with the DNA shown in SEQ ID No.1 Fragment between pET30a (+) Nde I and XhoI recognition sites is (including Nde I recognition sites and XhoI recognition sites Small fragment), keep pET30a (+) other sequences constant, obtained rmHF1-Y gene recombinant vectors.

Described to be expressed as induced expression in above-mentioned enzyme linked immunological kit, the induced expression is the IPTG with 0.75mM Induced 13-16 hours or 13-24 hours or 13 hours or 16 hours at 16 DEG C.

The restructuring PPR virus H-F fusion proteins are preparing the enzyme of detection detection PPR virus antibody Application in linked immunoassay reagent kit falls within protection scope of the present invention.

The enzyme linked immunological kit of above-mentioned detection PPR virus antibody is preparing PPR diagnostic kit In application fall within protection scope of the present invention.

The enzyme linked immunological kit of above-mentioned detection PPR virus antibody is preparing PPR monitoring reagent box In application fall within protection scope of the present invention.

It is demonstrated experimentally that using the indirect of the enzyme linked immunological kit for detecting PPR virus antibody foundation of the invention The method of ELISA detection PPR virus antibody has higher specificity, sensitiveness and good accuracy, and can be fast Speed, easy operation, are conducive to the monitoring clinically to PPR.Use the present invention's anti-using rmHF1-Y as coating The indirect ELISA detection PPR virus that the enzyme linked immunological kit of former detection PPR virus antibody is set up resists The method of body is 94.5% with France ID-VET PPR antibody assay kit coincidence rate, and to recombinate PPRV H eggs The method for the indirect ELISA detection PPR virus antibody that white rmH is set up as envelope antigen is small with France ID-VET's The coincidence rate for ruminating epizootic disease antibody assay kit only has 83.3%, is set up using recombinating PPRV F proteins rmF as envelope antigen Indirect ELISA detection PPR virus antibody method and France ID-VET PPR antibody assay kit Coincidence rate there was only 72.6%.Illustrate to detect PPR virus using rmHF1-Y as the indirect ELISA that envelope antigen is set up The method of antibody and the coincidence rate of France ID-VET PPR antibody assay kit are significantly higher than to recombinate PPRV H The indirect ELISA that albumen rmH is set up as envelope antigen detects the method for PPR virus antibody and to recombinate PPRV F The method that the indirect ELISA that albumen rmF is set up as envelope antigen detects PPR virus antibody.Using rmHF1-Y as The sensitiveness of the method for the indirect ELISA detection PPR virus antibody that envelope antigen is set up is significantly higher than to recombinate PPRV The indirect ELISA that H protein rmH is set up as envelope antigen detects the method for PPR virus antibody and to recombinate PPRV F The method that the indirect ELISA that albumen rmF is set up as envelope antigen detects PPR virus antibody.The detection of the present invention is small The enzyme linked immunological kit for ruminating epizootic disease antiviral antibody cannot be only used for the diagnosis of PPR, and evaluable vaccine immunity Effect, in addition, can also fast and accurately detect PPR, is conducive to the monitoring clinically to PPR, so as to be China preferably controls the propagation of PPR to produce positive role.

Brief description of the drawings

Fig. 1 composes for the SDS-PAGE of each bacterial strain expressing protein.

In figure, M is Marker, from top to bottom respectively 180kDa, 150kDa, 130kDa, 92kDa, 62kDa, 43kDa, 26kDa、10kDa；1st, the recipient bacterium whole bacterial protein liquid of induced expression, 2, BL21 (DE3)/pET30a- of induced expression RmHF1-Y contains albumen supernatant, 3, BL21 (DE3)/pET30a-rmHF1-Y of induced expression contain albumen precipitation, 4, induced expression BL21 (DE3)/pET30a-rmHF1-Y whole bacterial protein liquid, 5, the supernatant soluble protein after ni-sepharose purification, 6, molecule Sieve supernatant soluble protein after purification, 7, BL21 (DE3)/pET30a-rmHF1-W whole bacterial protein liquid of induced expression, 8, The BL21 (DE3) of induced expression/pET30a-rmHF2-Y whole bacterial protein liquid.

Molecular sieve purification identifications and Structural Identification of the Fig. 2 for recombinant protein rmHF1-Y.The signified purpose for purifying of arrow Protein peak.

Embodiment

The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining The bright present invention, the scope being not intended to be limiting of the invention.Experimental method in following embodiments, unless otherwise specified, be Conventional method.Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.

PET30a (+) is Novagen Products.PET28a (+) is Novagen Products.

Embodiment 1, solubility expression rmHF1

1st, synthetic gene

The application devises 3 kinds of PPR virus H and F protein fusion, respectively SEQ ID No.1 4- The rmHF2-Y genes and SEQ ID No.5 shown in rmHF1-Y genes, SEQ ID No.3 1-3684 shown in 3558 RmHF1-W genes shown in 4-3558.RmHF1-Y genes and rmHF2-Y genes differing only on nucleotide sequence 5' ends are different, and SEQ ID No.1 24-3564 are identical with SEQ ID No.3 150-3690 nucleotides.

RmHF1-Y genes and rmHF1-W genes encode protein rmHF1, the rmHF2-Y base shown in SEQ ID No.2 Because of the protein rmHF2 shown in coding SEQ ID No.3.RmHF1 and rmHF2 differ only in amino on amino acid sequence End is different, and SEQ ID No.2 8-1184 are identical with SEQ ID No.3 51-1227 amino acids residues.

2nd, the structure of recombinant expression carrier and recombinant bacterium

With shown in SEQ ID No.1 DNA replace pET30a (+) Nde I and XhoI recognition sites between fragment (including Small fragment including Nde I recognition sites and XhoI recognition sites), keep pET30a (+) other sequences constant, obtain RmHF1-Y gene recombinant vectors, are named as pET30a-rmHF1-Y.PET30a-rmHF1-Y contains rmHF1-Y genes, The nucleotide sequence of rmHF1-Y genes is SEQ ID No.1 4-3558 nucleotides, rmHF1-Y gene code SEQ ID Protein rmHF1 shown in No.2.

With shown in SEQ ID No.5 DNA replace pET30a (+) Nde I and XhoI recognition sites between fragment (including Small fragment including Nde I recognition sites and XhoI recognition sites), keep pET30a (+) other sequences constant, obtain RmHF1-W gene recombinant vectors, are named as pET30a-rmHF1-W.PET30a-rmHF1-W contains rmHF1-W genes, The nucleotide sequence of rmHF1-W genes is SEQ ID No.5 4-3558 nucleotides, rmHF1-W gene code SEQ ID Protein rmHF1 shown in No.2.

PET30a (+) BamHI and XhoI recognition sites are replaced with the DNA shown in SEQ ID No.3 145-3690 Between fragment (small fragment including BamHI recognition sites and XhoI recognition sites), keep pET30a (+) other sequences It is constant, rmHF2-Y gene recombinant vectors are obtained, pET30a-rmHF2-Y is named as.PET30a-rmHF2-Y contains RmHF2-Y genes, the nucleotide sequence of rmHF2-Y genes is SEQ ID No.3 1-3684 nucleotides, rmHF2-Y bases Because of the protein rmHF2 shown in coding SEQ ID No.4.

PET30a-rmHF1-Y, pET30a-rmHF1-W and pET30a-rmHF2-Y are individually converted into Escherichia coli BL21 (DE3) competent cell.It is spread evenly across on the LB flat boards containing kanamycins, 37 DEG C are cultivated 16 hours.Single bacterium colony Shaken cultivation is stayed overnight, and is extracted plasmid and is sequenced, sequencing result is shown into the recombination bacillus coli containing pET30a-rmHF1-Y BL21 (DE3)/pET30a-rmHF1-Y is named as, sequencing result is shown into the recombination bacillus coli containing pET30a-rmHF1-W BL21 (DE3)/pET30a-rmHF1-W is named as, sequencing result is shown into the recombination bacillus coli containing pET30a-rmHF2-Y It is named as BL21 (DE3)/pET30a-rmHF2-Y.

3rd, the analysis and identification of protein expression form

By BL21 (DE3)/pET30a-rmHF1-Y, BL21 (DE3)/pET30a-rmHF1-W, BL21 (DE3)/pET30a- RmHF2-Y and e. coli bl21 (DE3) (abbreviation recipient bacterium) this four bacterial strains are individually inoculated in containing 50 μ g/ml cards that is mould (concentration that kanamycins to kanamycins are added in LB fluid nutrient mediums is what 50 μ g/ml were obtained to the LB fluid nutrient mediums of element Culture medium) in, 37 DEG C, using the complete warm oscillator 200rpm shaken cultivations of Thermo MaxQ6000 types to 0D₆₀₀Value is (with containing 50 μ The LB fluid nutrient mediums of g/ml kanamycins are blank control) when reaching 0.6, add isopropylthio-β-D-galactoside (IPTG) induced expression is carried out.The induced expression of above-mentioned four plants of bacterium is to induce (be somebody's turn to do at 16 DEG C within 16 hours with 0.75mM IPTG Induced expression condition is by optimizing obtained highly-soluble induced expression to conditions such as temperature, time, IPTG concentration Condition).

Induced expression zymotic fluid is taken to be used for protein expression form analysis.Concretely comprise the following steps, take 1m L zymotic fluids to be placed in 1.5m In L centrifuge tubes, 8500rpm/min centrifugation 45min under the conditions of mark, 4 DEG C are carried out, supernatant is discarded, collects bacterial sediment.Add Precipitation is resuspended in 1m L PBS, and 8000rpm/min centrifugation 5min discard supernatant.200 μ L are added into the bacterial sediment washed PBS, high pressure crushes thalline, and cracking is no longer sticky to bacterium solution, obtains whole bacterial protein liquid.By whole bacterial protein liquid in 4 DEG C of centrifugations 18000rpm/min centrifuges 45min in machine, and supernatant (being named as containing albumen supernatant) is collected respectively and precipitation (is named as containing egg White precipitation), 50 μ L PBS are added in containing albumen precipitation washing precipitation is resuspended.To whole bacterial protein liquid, containing albumen supernatant and Containing 10 μ L 5 × SDS-PAGE loading Buffer are added in albumen precipitation, after fully mixing, put in boiling water bath and boil 5min, after sample cooling after, with hand held centrifuge wink from.6 μ L are taken to be used for SDS-PAGE electrophoretic analysis, and associated proteins gray scale Analysis software initial analysis protein content.Gel after electrophoresis is needed on NC films, using the sheep anti-mouse antibody of anti-His labels as knot Antibody DAB colour developings are closed, Western-blot identifications are carried out.Filtered by above-mentioned whole bacterial protein liquid and containing albumen supernatant with 0.22 μm Loading is to solution 1, (solute and its concentration are as follows in advance after membrane filtration：20mM Tris, 150mM NaCl, solvent are water, PH8.0 solution) the nickel post that has balanced.Nickel post is accessed on AKTA machines, respectively with the solution 1 of 10 column volumes and 10 posts (solute and its concentration are as follows for the solution 2 of volume：20mM Tris, 150mM NaCl, 50mM imidazoles, solvent are water, and pH8.0's is molten Liquid) impurity protein in cleaning nickel post, and monitor protein peak on AKTA machines.With solution 3, (solute and its concentration are as follows： 20mM Tris, 150mM NaCl, 300mM imidazoles, solvent are water, pH8.0 solution) destination protein hung on nickel post is rinsed, And the elution samples for destination protein peak occur are collected using AKTA, the sample is referred to as ni-sepharose purification destination protein sample.

The Superdex200 gel columns that the destination protein sample of ni-sepharose purification is produced with GE companies enter one by molecular sieve Step purifying.Mobile phase uses solution 1.By that can remove a large amount of imidazoles contained in sample after molecular sieve purification, collection is washed De- peak, obtains the destination protein sample of molecular sieve purification, right using NanoDrop2000 ultramicrospectrophotometers (ND2000) The content of albumen (i.e. soluble destination protein) carries out quantitative analysis in the destination protein sample of obtained molecular sieve purification.It is used in combination NanoDrop2000 ultramicrospectrophotometers (ND2000) determine the protein content in whole bacterial protein liquid, obtain thalline total Protein content.It will contain after albumen precipitation urea dissolves, be determined with NanoDrop2000 ultramicrospectrophotometers (ND2000) Content containing the protein in albumen precipitation.

As a result BL21 (DE3)/pET30a-rmHF1-Y of induced expression whole bacterial protein liquid is shown, containing albumen supernatant With containing in albumen precipitation containing size be 131kDa destination protein rmHF1；BL21 (DE3)/pET30a- of induced expression Destination protein rmHF1 in rmHF1-Y whole bacterial protein liquid accounts for the 45% of bacterial protein (full bacterium total protein), induced expression BL21 (DE3)/pET30a-rmHF1-Y the BL21 that induced expression is accounted for containing the destination protein rmHF1 in albumen supernatant (DE3) the 63% of destination protein HF-his in/pET30a-rmHF1-Y whole bacterial protein liquid, 63% destination protein RmHF1 is soluble protein；BL21 (DE3)/pET30a-rmHF1-Y of induced expression containing the destination protein in albumen precipitation RmHF1 accounts for 37% of destination protein rmHF1 in BL21 (DE3)/pET30a-rmHF1-Y of induced expression whole bacterial protein liquid, The 37% destination protein rmHF1 is insoluble inclusion body albumen；Illustrate BL21 (DE3)/pET30a-rmHF1- of induced expression Y destination protein rmHF1 accounts for the destination protein of 45%, BL21 (DE3)/pET30a-rmHF1-Y expression of bacterial protein 63% is soluble protein in rmHF1, and 37% is insoluble inclusion body albumen.BL21 (DE3)/pET30a- of induced expression The destination protein rmHF1 that size is 131kD is not contained in rmHF1-W whole bacterial protein liquid, illustrates BL21 (DE3)/pET30a- RmHF1-W does not have express express target protein rmHF1.BL21 (DE3)/pET30a-rmHF2-Y of induced expression whole bacterial protein liquid In do not contain size be 131kD destination protein rmHF2, illustrate that BL21 (DE3)/pET30a-rmHF2-Y does not express purpose egg White rmHF2.The whole bacterial protein liquid of the e. coli bl21 (DE3) of induced expression does not contain the destination protein that size is 131kD The rmHF1 and destination protein rmHF2 that size is 131kD；Illustrate that e. coli bl21 (DE3) does not have express express target protein rmHF1 With rmHF2 (Fig. 1).It can be seen that, although use same expression vector pET30a (+) and same Host Strains e. coli bl21 (DE3), the expression of different external source target gene is differed greatly, and rmHF1-Y genes are imported into large intestine by pET30a (+) Bacillus BL21 (DE3) can obtain the solution expression with high efficiency of rmHF1-Y genes, and rmHF1-W genes are imported by pET30a (+) In e. coli bl21 (DE3), rmHF1-W genes are not expressed but, and rmHF2-Y genes are imported into large intestine by pET30a (+) In bacillus BL21 (DE3), rmHF2-Y genes are not also expressed.

In addition, according to the method described above, the sequence between pET28a (+) restriction enzyme NheI and NotI sites is replaced The rmHF1-Y genes shown in SEQ ID No.1 4-3558 are changed to, keeps pET28a (+) other sequences constant, is contained There is the recombinant expression carrier of rmHF1-Y genes, the recombinant expression carrier is named as pET28a-rmHF1-Y.By pET28a- RmHF1-Y is transferred to e. coli bl21 (DE3) competent cell, by obtained recombination bacillus coli be named as BL21 (DE3)/ pET28a-rmHF1-Y.BL21 (DE3)/pET28a-rmHF1-Y is inoculated in the LB Liquid Cultures containing 50 μ g/ml kanamycins In base (concentration that kanamycins to kanamycins are added in LB fluid nutrient mediums is the culture medium that 50 μ g/ml are obtained), 37 DEG C, Using the complete warm oscillator 200rpm shaken cultivations of Thermo MaxQ6000 types to 0D₆₀₀Value is (with the LB containing 50 μ g/ml kanamycins Fluid nutrient medium is blank control) when reaching 0.6, add isopropylthio-β-D-galactoside (IPTG) and carry out induced expression. The induced expression is induced 16 hours at 16 DEG C with 0.75mM IPTG.The zymotic fluid of induced expression is taken to carry out according to the method described above Protein expression form analysis.As a result show do not have in BL21 (DE3)/pET28a-rmHF1-Y whole bacterial protein liquid of induced expression The expression of purposeful albumen.It can be seen that, although it is big using same external source target gene (rmHF1-Y genes) and same Host Strains Enterobacteria BL21 (DE3), in different BL21 (DE3) expression vector-pET28a (+) and pET30a (+), external source target gene Expression differ greatly, by rmHF1-Y genes by pET30a (+) import e. coli bl21 (DE3) can obtain The solution expression with high efficiency of rmHF1-Y genes, e. coli bl21 (DE3) is imported by rmHF1-Y genes by pET28a (+) In, rmHF1-Y genes are not expressed but.

4th, rmHF1 solubility expression and purifying

BL21 (DE3)/pET30a-rmHF1-Y is inoculated in the LB fluid nutrient mediums containing 50 μ g/ml kanamycins (in LB The concentration that kanamycins to kanamycins are added in fluid nutrient medium is the culture medium that 50 μ g/ml are obtained) in, 37 DEG C, use The complete warm oscillator 200rpm shaken cultivations of Thermo MaxQ6000 types are to 0D₆₀₀Value is (with the LB liquid containing 50 μ g/ml kanamycins Culture medium is blank control) when reaching 0.6, add IPTG and carry out induced expression.The induced expression is with 0.75mM IPTG 16 DEG C induction 16h.The zymotic fluid after IPTG induced expressions 16h is taken to collect bacterial sediment.Add PBS and precipitation, 8000rpm/min is resuspended 5min is centrifuged, supernatant is discarded.PBS is added into the bacterial sediment washed, high pressure crushes thalline, and cracking to bacterium solution is no longer glued Thick, 16000rpm/min centrifuges 30min in 4 DEG C of centrifuges, collects supernatant (being named as containing albumen supernatant), abandons precipitation. Albumen supernatant will be contained, and with loading after 0.22 μm of membrane filtration to advance solution 1, (solute and its concentration are as follows：20mM Tris、 150mM NaCl, solvent is water, pH8.0 solution) the nickel post that has balanced.Nickel post is accessed on AKTA machines, respectively with 10 (solute and its concentration are as follows with the solution 2 of 10 column volumes for the solution 1 of column volume：20mM Tris, 150mM NaCl, 50mM miaows Azoles, solvent is water, pH8.0 solution) impurity protein in cleaning nickel post, and monitor protein peak on AKTA machines.With solution 3 (solute and its concentration are as follows：20mM Tris, 150mM NaCl, 300mM imidazoles, solvent are water, pH8.0 solution) rinse nickel Post hangs over the destination protein on nickel post, and the elution samples for destination protein peak occur are collected using AKTA, and the sample is referred to as into nickel The rmHF1 albumen (ni-sepharose purification destination protein sample) of post purifying.

The Superdex200 gel columns that the rmHF1 albumen of ni-sepharose purification is produced with GE companies are further by molecular sieve Purifying, obtains the rmHF1 albumen of molecular sieve purification.Mobile phase in the molecular sieve purification uses above-mentioned solution 1.Pass through molecular sieve The a large amount of imidazoles contained in sample can be removed after purification, and the structure of rmHF1 albumen is monomer structure (Fig. 2).Collect monomer The eluting peak of structure, obtains the rmHF1 albumen (molecular sieve purification destination protein sample) of molecular sieve purification, uses The purity of albumen of the NanoDrop2000 ultramicrospectrophotometers (ND2000) to obtaining carries out quantitative analysis.

By its amino acid sequence of the rmHF1 albumen progress mass spectral analysis of molecular sieve purification, as a result show rmHF1 amino acid Sequence is as shown in SEQ ID No.2.

Embodiment 2, the detection PPR virus antibody by envelope antigen indirect ELISA method of rmHF1 albumen

Related solution in the present embodiment is as follows：

Concentration is 0.01M, and pH value is the preparation of 7.4 PBS：8.5g NaCl、0.2g KCl、2.9g Na₂HPO₄·12H₂O、0.59g NaH₂PO₄·2H₂O, 1L deionized water.

It is coated with buffer solution：0.05mol/L sodium carbonate-bicarbonate buffer solution (pH9.6), solvent is water, solute and its Concentration is as follows：Na₂CO₃1.59g/L and NaHCO₃ 2.93g/L。

Cleaning solution:0.5% tween cleaning solution.0.5% tween cleaning solution is prepared as follows：It is 0.01M in concentration, The content of addition polysorbas20 and sodium azide to sodium azide is the content of 5g/L, polysorbas20 in the PBS that pH value is 7.4 For 5mL/L, 0.5% tween cleaning solution is obtained.

Confining liquid：1%BSA confining liquids.1%BSA confining liquids are prepared as follows：It is 0.01M in concentration, pH value is The BSA solution for Jia 10% in 7.4 PBS, is diluted to 1% (volumn concentration), obtains 1%BSA confining liquids.

Secondary antibody dilution：Addition BSA to BSA content is in concentration is 0.01M, the PBS that pH value is 7.4 1% (volumn concentration), obtains secondary antibody dilution.

1st, the foundation and optimization of indirect ELISA reaction condition

RmHF1 albumen (the hereinafter referred to as rmHF1 eggs of molecular sieve purification in embodiment 1 are determined using chessboard square formation titration Most suitable coating concentration and optimal serum dilution in vain), optimal confining liquid concentration is determined with the BSA closing ELISA Plates of various concentrations, Serum and ELIAS secondary antibody best effort time are determined, optimizes ELIAS secondary antibody working concentration, criterion is：Positive serum and feminine gender The OD of serum₄₅₀Reaction condition corresponding to the maximum hole of ratio (P/N) is the optimum reaction condition of ELISA method.

By coated elisa plate after the rmHF1 protein ladders dilution of molecular sieve purification in embodiment 1, titrated using chessboard square formation Method determines the package amount and serum dilution of rmHF1 albumen；And it is excellent on the basis of antigen is most preferably coated with concentration and serum dilution Change ELISA testing conditions.As a result as shown in table 1, the mass concentration of rmHF1 albumen is 1.0 μ g/mL, test serum extension rate For 1:When 20, P/N values are maximum, it is thus determined that the optimal coating concentration of antigen (rmHF1 albumen) is 1.0 μ g/mL, test serum is dilute It is 1 to release multiple:20.This experiment simultaneously determines that the rabbit-anti goat IgG of HRP marks presses 1:20000 dilution proportion, 37 DEG C of effects 0.5h, adds OD values after TMB nitrite ions 10min optimal.

The reaction condition optimization result of table 1, ELISA detection method

	Coated antigen	Sealing condition	Test serum	Secondary antibody IgG-HRP
					Optimize dilution factor	1.0μg/mL	1%BSA	1:20	1:20000
Reaction condition	4℃/16h	37℃/2h	37℃/1h	37℃/0.5h

What the step was determined detects PPR virus antibody by envelope antigen indirect ELISA method of rmHF1 albumen Optimal Experimental method (hereinafter referred to as recombinate rmHF1 optimization indirect ELISA method) it is as follows：

1.1 coating：With rmHF1 albumen (the hereinafter referred to as rmHF1 of molecular sieve purification in coating buffer solution dilution embodiment 1 Albumen) to rmHF1 albumen concentration be 1.0 μ g/ml, obtain be coated with original solution, with the coating original solution be coated with experimental port, 100 μ L/ holes add to ELISA Plate, 4 DEG C of incubation 16h.

1.2 washing：Incline hole endoperidium original solution, is washed 5 times with 0.5% tween cleaning solution, each 3min；Pat dry.

1.3 closing：Add 1%BSA confining liquids, 100ul/ holes, 37 DEG C of incubation 2h.

1.4 sample-adding：

1.4.1 sample well

PPRV positive serums are diluted 20 times with coating buffer solution, test serum is obtained.100 μ L test serums are added to enzyme On target, 37 DEG C are reacted liquid in 1h, hole of inclining, and are then washed with cleaning solution 5 times, each 3min.Wherein, positive serum is method State ID-VET PPR antibody assay kit (IDPPR Competition PPR antibody Detection kit, production code member is PPRC-4P) it is detected as the sheep blood serum of PPRV antibody positives.

1.4.2 blank control wells

Test serum is replaced with to isometric high purity water with differing only in for 1.4.1, other steps are constant.

1.5 add ELIAS secondary antibody：Addition carries out 1 with secondary antibody dilution:The HRP mark rabbit-anti goat IgGs of 20000 dilutions, 100ul/ holes, 37 DEG C of 30min.

1.6 colour developing：TMB is added, 10min is reacted in 100ul/ holes.

1.7 terminate：Add 0.2mol/L H₂SO₄Solution terminating reaction, 100ul/ holes.

1.8 determine：Each hole OD is read with ELIASA_450nmNumerical value.

2nd, the determination of ELISA yin and yang attributes critical value

1000 parts that China Animal Disease Control And Prevention Center plant-eating animal and infecting both domestic animals and human ward are preserved are through French ID- VET PPR antibody assay kit (IDThe antibody test of PPR Competition PPRs is tried Agent box, production code member is PPRC-4P) to be detected as the negative sheep blood serums of PPRV indirect using the restructuring rmHF1 optimizations of step 1 PPRV positive serums in 1.4.1 (are replaced with 1000 parts of PPRV negative serums, other operations are identical) by ELISA method respectively Indirect ELISA detection is carried out, the average value (X) and standard deviation (SD) of 1000 parts of PPRV negative serums is calculated.It is judged to the positive；It is judged to feminine gender.

As a result show, 1000 parts of PPRV negative serum average valuesIt is 0.053 for 0.162, SD, therefore yin and yang attribute is critical ValueFor 0.321.

3rd, specific test

Using step 1 restructuring rmHF1 optimization indirect ELISA method to 10 parts of sheep johne's diseases, sheep cloth disease, tuberculosis, C.perfringens disease and aftosa positive serum are detected that observation has no cross reaction with Other diseases.Wherein, will 1.4.1 the PPRV positive serums in replace with above-mentioned serum respectively, and other operations are identical.As a result show that restructuring rmHF1 optimizes Cause of disease (sheep johne's disease, sheep cloth disease, tuberculosis, C.perfringens disease and aftosa of the indirect ELISA method to several Yang Yuan Positive serum) positive serum detected, its OD₄₅₀Value is respectively：0.169th, 0.171,0.122,0.194,0.226, it is respectively less than Critical value 0.321, shows rmHF1 albumen and sheep johne's disease, sheep cloth disease, tuberculosis, C.perfringens disease and aftosa sun Property the equal no cross reaction of serum, step 1 restructuring rmHF1 optimization indirect ELISA method there is good specificity.

4th, sensitivity tests

RmHF1 in the optimization indirect ELISA method of step 1 is replaced with into restructuring H protein rmH, other operations are constant, build Vertical restructuring H protein rmH optimization indirect ELISA methods.

RmHF1 in the optimization indirect ELISA method of step 1 is replaced with into restructuring F protein rmF, other operations are constant, build Vertical restructuring F protein rmF optimization indirect ELISA methods.

Wherein, above-mentioned restructuring H protein rmH according to rmHF1 in the step 4 of embodiment 1 solubility expression and purification process system It is standby, differ only in will carry out the recombination bacillus coli of soluble induced expression by recombination bacillus coli BL21 (DE3)/ PET30a-rmHF1-Y replaces with recombination bacillus coli BL21 (DE3)/pET30a-rmH-Y.Recombination bacillus coli BL21 (DE3)/ PET30a-rmH-Y is that pET30a-rmH-Y is imported into the recombinant bacterium that e. coli bl21 (DE3) is obtained.PET30a-rmH-Y is With rmH-Y replace pET30a (+) Nde I and XhoI recognition sites between fragment (including Nde I recognition sites and XhoI identification Small fragment including site), keep pET30a (+) other sequences constant, obtained rmH-Y gene recombinant vectors, life Entitled pET30a-rmH-Y.

PET30a-rmH-Y contains rmH-Y genes, and rmH-Y genes are by SEQ ID 1852-3555 nucleotides of No.1 Missing, the DNA that other invariant nucleotides are obtained, rmH-Y DNA encoding the protein rmH.RmH is by the of SEQ ID No.3 The constant obtained protein of the other amino acid residues of 617-1184 amino acids residue deletions.

Above-mentioned restructuring F protein rmF is prepared according to the solubility expression and purification process of rmHF1 in the step 4 of embodiment 1, area It is not only that the recombination bacillus coli by soluble induced expression is carried out by recombination bacillus coli BL21 (DE3)/pET30a- RmHF1-Y replaces with recombination bacillus coli BL21 (DE3)/pET30a-rmF-Y.Recombination bacillus coli BL21 (DE3)/pET30a- RmF-Y is that pET30a-rmF-Y is imported into the recombinant bacterium that e. coli bl21 (DE3) is obtained.PET30a-rmF-Y is to use rmF-Y (including Nde I recognition sites and XhoI recognition sites exist the fragment for replacing between pET30a (+) Nde I and XhoI recognition sites Interior small fragment), holding pET30a (+) other sequences are constant, and obtained rmF-Y gene recombinant vectors are named as pET30a-rmF-Y.PET30a-rmF-Y contains rmF-Y genes, and rmF-Y genes are by SEQ ID 25-1896 cores of No.1 Thuja acid is lacked, the DNA that other invariant nucleotides are obtained, rmF-Y DNA encoding the protein rmF.RmF is by SEQ ID No.3 The constant obtained protein of the other amino acid residues of 8-631 amino acids residue deletions.

By the PPR antibody assay kit (ID through French ID-VET PPR Competition PPR antibody assay kit, production code member is PPRC-4P) being detected as the positive sheep blood serums of PPRV, to carry out multiple proportions dilute Release, detected using the restructuring rmHF1 optimization indirect ELISA methods of step 1, while indirect using the rmH optimizations of restructuring H protein The PPR antibody test reagent of ELISA method, restructuring F protein rmF optimization indirect ELISA methods and the French ID-VET Box is compared, and draws greatest dilution during positive critical value.

As a result show sheep blood serum positive PPRV from 1：25 proceed by doubling dilution, using the restructuring rmHF1 of step 1 Optimization indirect ELISA method is detected that the dilution factor of sheep blood serum positive PPRV is still positive when being 1: 1600；Using restructuring H protein rmH optimization indirect ELISA methods are detected that the greatest dilution of sheep blood serum positive PPRV is 1:800；Using weight Group F protein rmF optimization indirect ELISA methods are detected that the greatest dilution of sheep blood serum positive PPRV is 1:800；Show The restructuring rmHF1 optimizations indirect ELISA method sensitiveness of step 1 is preferable, hence it is evident that higher than restructuring H protein rmH and restructuring F protein RmF as detection method during envelope antigen sensitiveness.

5th, replica test

Indirect ELISA method is optimized to 6 parts of sheep blood serums in same batch plate and different batches using the restructuring rmHF1 of step 1 Detected respectively on plate, parallel determination 5 times, calculate batch interior, interassay coefficient of variation (CV).As a result show, repeat to make a variation in batch Coefficient repeats the coefficient of variation between 2%~8%, between batch and is less than 9% (table 2).As a result show, the restructuring rmHF1 optimizations of step 1 Indirect ELISA method has good repeatability.

The restructuring rmHF1 optimization indirect ELISA method replica tests of the step 1 of table 2

6th, accordance is tested

Detected using the restructuring rmHF1 optimization indirect ELISA methods of step 1, while using the restructuring H eggs of step 4 White rmH optimizes the small anti-of indirect ELISA method, the restructuring F protein rmF optimization indirect ELISA methods of step 4 and France ID-VET Hay epizootic disease antibody assay kit (IDPPR Competition PPR antibody assay kits, product Numbering is PPRC-4P) to China Animal Disease Control And Prevention Center plant-eating animal and 500 parts of sheep blood of infecting both domestic animals and human ward preservation Detected clearly, calculate former three and the coincidence rate of France ID-VET PPR antibody assay kit.

As a result show to optimize the positive that indirect ELISA method detects 500 parts of sheep blood serums using the restructuring rmHF1 of step 1 Rate is 44.19%；French ID-VET PPR antibody assay kit Positive rate is 42.25%, the weight of step 1 Group rmHF1 optimization indirect ELISA methods and France ID-VET PPR antibody assay kit coincidence rate are 94.5%. And the PPR antibody assay kit of the restructuring H protein rmH optimization indirect ELISA methods and France ID-VET of step 4 Coincidence rate there was only 83.3%, the restructuring F protein rmF optimization indirect ELISA methods of step 4 ruminate beast with France's the small of ID-VET The coincidence rate of epidemic disease antibody assay kit only has 72.6%.Illustrate the indirect ELISA inspection for setting up rmHF1-Y as envelope antigen The method and the coincidence rate of France ID-VET PPR antibody assay kit for surveying PPR virus antibody are notable Higher than the side that the indirect ELISA set up using recombinating PPRV H proteins rmH as envelope antigen detects PPR virus antibody Method and the indirect ELISA set up using recombinating PPRV F proteins rmF as envelope antigen detect the side of PPR virus antibody Method.

<110>China Animal Disease Control And Prevention Center

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 3564

<212> DNA

<213>Artificial sequence

<220>

<221> CDS

<222> (4)..(3558)

<223>

<400> 1

catatgcacc atcaccacca tcacatgagc gcacaacgcg aacgcatcaa cgcgttctac 60

aaagacaacc tgcacaacaa aacccatcgc gttatcctgg atcgcgaacg tctgaccatt 120

gaacgtccgt atatcctgct gggggttctg ctggttatgt ttctgagcct gatcggtctg 180

ctggcaattg ctggtattcg tctgcatcgc gcaaccgttg gtaccgcaga aattcaaagc 240

cgtctgaaca ccaacatcga actgaccgaa agcatcgacc atcagaccaa agacgttctg 300

accccgctgt ttaaaatcat cggcgacgaa gtcggcattc gtattccgca gaaattcagc 360

gacctggtca aattcatcag cgacaaaatc aaattcctga acccggaccg cgaatacgat 420

tttcgcgatc tgcgttggtg tatgaatccg ccggaacgcg tcaaaatcaa cttcgaccag 480

ttctgcgaat acaaagcggc ggttaaaagc gtcgaacaca tcttcgaaag cagcctgaac 540

cgttctgaac gtctgcgtct gctgaccctg ggtccgggta ccggttgtct gggtcgtacc 600

gttacccgtg cacaatttag cgaactgacc ctgaccctga tggatctgga cctggagatg 660

aaacataacg tcagcagcgt ctttaccgtt gtggaagaag gtctgtttgg ccgtacctat 720

accgtttggc gttctgatac cggtaaaccg agtaccagtc cgggtattgg tcattttctg 780

cgcgtcttcg aaattggtct ggttcgcgat ctggaactgg gcgctccgat ttttcacatg 840

accaactacc tgaccgtcaa catgagcgac gattaccgtt cttgtctgct ggcagttggc 900

gaactgaaac tgaccgcact gtgtaccccg tctgaaaccg ttaccctgtc tgaatctggc 960

gttccgaaac gcgaaccgct ggttgtcgtt attctgaatc tggcaggtcc gaccctgggc 1020

ggcgaactgt attctgttct gccgaccacc gatccgaccg ttgaaaaact gtatctgagc 1080

agccatcgcg gtatcatcaa agacaacgaa gcgaattggg ttgttccgtc taccgacgtt 1140

cgtgatctgc aaaacaaagg cgagtgcctg gttgaagctt gtaaaacccg tccgccgagc 1200

ttttgtaacg gtaccggtat tggtccgtgg tctgaaggtc gtattccggc ttacggcgtt 1260

attcgcgttt ctctggatct ggcatctgat ccgggcgtag ttattaccag cgtttttggt 1320

ccgctgattc cgcatctgtc tggcatggac ctgtataata atccgtttag ccgcgcggct 1380

tggctggcag ttccgccgta cgaacagtct tttctgggca tgatcaacac cattggtttt 1440

ccggatcgcg ctgaagttat gccgcatatt ctgaccaccg aaattcgcgg tccgcgcggt 1500

cgttgtcatg ttccgattga actgagcagc cgtatcgacg acgacatcaa aatcggcagc 1560

aacatggttg tcctgccgac caaagatctg cgttatatca ccgcgaccta cgacgtttct 1620

cgtagcgaac acgcgatcgt ctactacatc tacgataccg gccgtagcag cagctatttt 1680

tacccggttc gtctgaactt tcgcggtaat ccgctgtctc tgcgtattga gtgctttccg 1740

tggtaccaca aagtctggtg ttaccacgac tgcctgatct acaacaccat caccaacgaa 1800

gaagtgcata cccgcggtct gaccggtatt gaagtcacct gcaacccggt tggtggcggt 1860

ggaatcggag gtggtggaag cggaggaggt ggaagcatgc accatcacca ccatcacatg 1920

acccgcgttg cgaccctggt ctttctgttt ctgttcccga acaccgttac ctgtcagatt 1980

cattggggca acctgagcaa aatcggcatt gttggtaccg gtagcgcttc ctacaaagtt 2040

atgacccgtc cgagtcatca gaccctggtt atcaaactga tgccgaacat caccgcgatt 2100

gataactgca ccaaaagcga gatcagcgag tacaaacgtc tgctgatcac cgttctgaaa 2160

ccggttgaag acgcactgag cgtcatcacc aaaaacgtcc gtccgattca aaccctgacc 2220

ccgggtcgtc gtacccgtcg ttttgtaggc gctgttctgg caggtgttgc actgggcgtt 2280

gcaaccgcag cacaaattac cgcaggcgtt gcactgcatc agtctctgat gaacagccag 2340

gcgattgaaa gcctgaaaac cagcctggag aaaagcaacc aggcaattga agaaatccgt 2400

ctggcgaaca aagaaaccat cctggcagtt cagggcgtcc aggattacat caacaacgag 2460

ctggtcccgt ctgttcatcg tatgagctgc gaactggtcg gtcataaact gagcctgaaa 2520

ctgctgcgct actacaccga gatcctgagc atctttggtc cgagtctgcg cgatccgatt 2580

gcagcggaaa ttagcattca ggcgctgagt tacgcgctgg gcggcgatat taacaaaatc 2640

ctggacaaac tgggctattc tggcggcgat tttctggcga ttctggaatc caaaggcatc 2700

aaagcgcgcg ttacctacgt tgatacccgc gattatttca tcatcctgag cattgcgtat 2760

ccgaccctga gcgaaatcaa aggcgttatc gtccacaaaa tcgaggcgat cagctacaac 2820

atcggcgctc aagagtggta taccacgatt ccgcgttacg ttgcgaccca gggttatctg 2880

attagcaact tcgacgagac cagctgcgtt tttaccccgg aaggtaccgt ttgtagccaa 2940

aacgctctgt atccgatgag tccgctgctg caagagtgtt ttcgcggtag caccaaaagt 3000

tgcgcacgta ccctggtttc tggtaccacc agtaatcgct tcatcctgag caaaggcaac 3060

ctgatcgcaa attgcgcctc tgttctgtgc aaatgctaca ccaccgaaac cgtcatcaac 3120

caggatccgg ataaactgct gaccgttatt gcctctgata aatgcccggt agttgaagtt 3180

gacggcgtca ccattcaggt tggtagccgc gaatatccgg atagcgtcta tctgcacgag 3240

attgatctgg gtccggcaat ttctctggag aaactggatg tcggtaccaa tctgggtaac 3300

gcagttacgc gtctggaaaa cgcaaaagaa ctgctggacg cgagcgatca gattctgaaa 3360

accgttaaag gcgttccgtt cagcggtaac atctacattg ccctggcagc ttgtattggc 3420

gttagtctgg gtctggttac cctgatttgc tgttgcaaag gccgttgtcg caacaaagag 3480

attccggcaa gcaaaattaa tccgggcctg aaaccggatc tgaccggtac cagcaaaagc 3540

tacgttcgta gcctgtaact cgag 3564

<210> 2

<211> 1184

<212> PRT

<213>Artificial sequence

<220>

<223>

<400> 2

Met His His His His His His Met Ser Ala Gln Arg Glu Arg Ile Asn

1 5 10 15

Ala Phe Tyr Lys Asp Asn Leu His Asn Lys Thr His Arg Val Ile Leu

20 25 30

Asp Arg Glu Arg Leu Thr Ile Glu Arg Pro Tyr Ile Leu Leu Gly Val

35 40 45

Leu Leu Val Met Phe Leu Ser Leu Ile Gly Leu Leu Ala Ile Ala Gly

50 55 60

Ile Arg Leu His Arg Ala Thr Val Gly Thr Ala Glu Ile Gln Ser Arg

65 70 75 80

Leu Asn Thr Asn Ile Glu Leu Thr Glu Ser Ile Asp His Gln Thr Lys

85 90 95

Asp Val Leu Thr Pro Leu Phe Lys Ile Ile Gly Asp Glu Val Gly Ile

100 105 110

Arg Ile Pro Gln Lys Phe Ser Asp Leu Val Lys Phe Ile Ser Asp Lys

115 120 125

Ile Lys Phe Leu Asn Pro Asp Arg Glu Tyr Asp Phe Arg Asp Leu Arg

130 135 140

Trp Cys Met Asn Pro Pro Glu Arg Val Lys Ile Asn Phe Asp Gln Phe

145 150 155 160

Cys Glu Tyr Lys Ala Ala Val Lys Ser Val Glu His Ile Phe Glu Ser

165 170 175

Ser Leu Asn Arg Ser Glu Arg Leu Arg Leu Leu Thr Leu Gly Pro Gly

180 185 190

Thr Gly Cys Leu Gly Arg Thr Val Thr Arg Ala Gln Phe Ser Glu Leu

195 200 205

Thr Leu Thr Leu Met Asp Leu Asp Leu Glu Met Lys His Asn Val Ser

210 215 220

Ser Val Phe Thr Val Val Glu Glu Gly Leu Phe Gly Arg Thr Tyr Thr

225 230 235 240

Val Trp Arg Ser Asp Thr Gly Lys Pro Ser Thr Ser Pro Gly Ile Gly

245 250 255

His Phe Leu Arg Val Phe Glu Ile Gly Leu Val Arg Asp Leu Glu Leu

260 265 270

Gly Ala Pro Ile Phe His Met Thr Asn Tyr Leu Thr Val Asn Met Ser

275 280 285

Asp Asp Tyr Arg Ser Cys Leu Leu Ala Val Gly Glu Leu Lys Leu Thr

290 295 300

Ala Leu Cys Thr Pro Ser Glu Thr Val Thr Leu Ser Glu Ser Gly Val

305 310 315 320

Pro Lys Arg Glu Pro Leu Val Val Val Ile Leu Asn Leu Ala Gly Pro

325 330 335

Thr Leu Gly Gly Glu Leu Tyr Ser Val Leu Pro Thr Thr Asp Pro Thr

340 345 350

Val Glu Lys Leu Tyr Leu Ser Ser His Arg Gly Ile Ile Lys Asp Asn

355 360 365

Glu Ala Asn Trp Val Val Pro Ser Thr Asp Val Arg Asp Leu Gln Asn

370 375 380

Lys Gly Glu Cys Leu Val Glu Ala Cys Lys Thr Arg Pro Pro Ser Phe

385 390 395 400

Cys Asn Gly Thr Gly Ile Gly Pro Trp Ser Glu Gly Arg Ile Pro Ala

405 410 415

Tyr Gly Val Ile Arg Val Ser Leu Asp Leu Ala Ser Asp Pro Gly Val

420 425 430

Val Ile Thr Ser Val Phe Gly Pro Leu Ile Pro His Leu Ser Gly Met

435 440 445

Asp Leu Tyr Asn Asn Pro Phe Ser Arg Ala Ala Trp Leu Ala Val Pro

450 455 460

Pro Tyr Glu Gln Ser Phe Leu Gly Met Ile Asn Thr Ile Gly Phe Pro

465 470 475 480

Asp Arg Ala Glu Val Met Pro His Ile Leu Thr Thr Glu Ile Arg Gly

485 490 495

Pro Arg Gly Arg Cys His Val Pro Ile Glu Leu Ser Ser Arg Ile Asp

500 505 510

Asp Asp Ile Lys Ile Gly Ser Asn Met Val Val Leu Pro Thr Lys Asp

515 520 525

Leu Arg Tyr Ile Thr Ala Thr Tyr Asp Val Ser Arg Ser Glu His Ala

530 535 540

Ile Val Tyr Tyr Ile Tyr Asp Thr Gly Arg Ser Ser Ser Tyr Phe Tyr

545 550 555 560

Pro Val Arg Leu Asn Phe Arg Gly Asn Pro Leu Ser Leu Arg Ile Glu

565 570 575

Cys Phe Pro Trp Tyr His Lys Val Trp Cys Tyr His Asp Cys Leu Ile

580 585 590

Tyr Asn Thr Ile Thr Asn Glu Glu Val His Thr Arg Gly Leu Thr Gly

595 600 605

Ile Glu Val Thr Cys Asn Pro Val Gly Gly Gly Gly Ile Gly Gly Gly

610 615 620

Gly Ser Gly Gly Gly Gly Ser Met His His His His His His Met Thr

625 630 635 640

Arg Val Ala Thr Leu Val Phe Leu Phe Leu Phe Pro Asn Thr Val Thr

645 650 655

Cys Gln Ile His Trp Gly Asn Leu Ser Lys Ile Gly Ile Val Gly Thr

660 665 670

Gly Ser Ala Ser Tyr Lys Val Met Thr Arg Pro Ser His Gln Thr Leu

675 680 685

Val Ile Lys Leu Met Pro Asn Ile Thr Ala Ile Asp Asn Cys Thr Lys

690 695 700

Ser Glu Ile Ser Glu Tyr Lys Arg Leu Leu Ile Thr Val Leu Lys Pro

705 710 715 720

Val Glu Asp Ala Leu Ser Val Ile Thr Lys Asn Val Arg Pro Ile Gln

725 730 735

Thr Leu Thr Pro Gly Arg Arg Thr Arg Arg Phe Val Gly Ala Val Leu

740 745 750

Ala Gly Val Ala Leu Gly Val Ala Thr Ala Ala Gln Ile Thr Ala Gly

755 760 765

Val Ala Leu His Gln Ser Leu Met Asn Ser Gln Ala Ile Glu Ser Leu

770 775 780

Lys Thr Ser Leu Glu Lys Ser Asn Gln Ala Ile Glu Glu Ile Arg Leu

785 790 795 800

Ala Asn Lys Glu Thr Ile Leu Ala Val Gln Gly Val Gln Asp Tyr Ile

805 810 815

Asn Asn Glu Leu Val Pro Ser Val His Arg Met Ser Cys Glu Leu Val

820 825 830

Gly His Lys Leu Ser Leu Lys Leu Leu Arg Tyr Tyr Thr Glu Ile Leu

835 840 845

Ser Ile Phe Gly Pro Ser Leu Arg Asp Pro Ile Ala Ala Glu Ile Ser

850 855 860

Ile Gln Ala Leu Ser Tyr Ala Leu Gly Gly Asp Ile Asn Lys Ile Leu

865 870 875 880

Asp Lys Leu Gly Tyr Ser Gly Gly Asp Phe Leu Ala Ile Leu Glu Ser

885 890 895

Lys Gly Ile Lys Ala Arg Val Thr Tyr Val Asp Thr Arg Asp Tyr Phe

900 905 910

Ile Ile Leu Ser Ile Ala Tyr Pro Thr Leu Ser Glu Ile Lys Gly Val

915 920 925

Ile Val His Lys Ile Glu Ala Ile Ser Tyr Asn Ile Gly Ala Gln Glu

930 935 940

Trp Tyr Thr Thr Ile Pro Arg Tyr Val Ala Thr Gln Gly Tyr Leu Ile

945 950 955 960

Ser Asn Phe Asp Glu Thr Ser Cys Val Phe Thr Pro Glu Gly Thr Val

965 970 975

Cys Ser Gln Asn Ala Leu Tyr Pro Met Ser Pro Leu Leu Gln Glu Cys

980 985 990

Phe Arg Gly Ser Thr Lys Ser Cys Ala Arg Thr Leu Val Ser Gly Thr

995 1000 1005

Thr Ser Asn Arg Phe Ile Leu Ser Lys Gly Asn Leu Ile Ala Asn

1010 1015 1020

Cys Ala Ser Val Leu Cys Lys Cys Tyr Thr Thr Glu Thr Val Ile

1025 1030 1035

Asn Gln Asp Pro Asp Lys Leu Leu Thr Val Ile Ala Ser Asp Lys

1040 1045 1050

Cys Pro Val Val Glu Val Asp Gly Val Thr Ile Gln Val Gly Ser

1055 1060 1065

Arg Glu Tyr Pro Asp Ser Val Tyr Leu His Glu Ile Asp Leu Gly

1070 1075 1080

Pro Ala Ile Ser Leu Glu Lys Leu Asp Val Gly Thr Asn Leu Gly

1085 1090 1095

Asn Ala Val Thr Arg Leu Glu Asn Ala Lys Glu Leu Leu Asp Ala

1100 1105 1110

Ser Asp Gln Ile Leu Lys Thr Val Lys Gly Val Pro Phe Ser Gly

1115 1120 1125

Asn Ile Tyr Ile Ala Leu Ala Ala Cys Ile Gly Val Ser Leu Gly

1130 1135 1140

Leu Val Thr Leu Ile Cys Cys Cys Lys Gly Arg Cys Arg Asn Lys

1145 1150 1155

Glu Ile Pro Ala Ser Lys Ile Asn Pro Gly Leu Lys Pro Asp Leu

1160 1165 1170

Thr Gly Thr Ser Lys Ser Tyr Val Arg Ser Leu

　　1175 1180

<210> 3

<211> 3690

<212> DNA

<213>Artificial sequence

<220>

<221> CDS

<222> (1)..(3684)

<223>

<400> 3

atgcaccatc atcatcatca ttcttctggt ctggtgccac gcggttctgg tatgaaagaa 60

accgctgctg ctaaattcga acgccagcac atggacagcc cagatctggg taccgacgac 120

gacgacaagg ccatggctga tatcggatcc atgagcgcac aacgcgaacg catcaacgcg 180

ttctacaaag acaacctgca caacaaaacc catcgcgtta tcctggatcg cgaacgtctg 240

accattgaac gtccgtatat cctgctgggg gttctgctgg ttatgtttct gagcctgatc 300

ggtctgctgg caattgctgg tattcgtctg catcgcgcaa ccgttggtac cgcagaaatt 360

caaagccgtc tgaacaccaa catcgaactg accgaaagca tcgaccatca gaccaaagac 420

gttctgaccc cgctgtttaa aatcatcggc gacgaagtcg gcattcgtat tccgcagaaa 480

ttcagcgacc tggtcaaatt catcagcgac aaaatcaaat tcctgaaccc ggaccgcgaa 540

tacgattttc gcgatctgcg ttggtgtatg aatccgccgg aacgcgtcaa aatcaacttc 600

gaccagttct gcgaatacaa agcggcggtt aaaagcgtcg aacacatctt cgaaagcagc 660

ctgaaccgtt ctgaacgtct gcgtctgctg accctgggtc cgggtaccgg ttgtctgggt 720

cgtaccgtta cccgtgcaca atttagcgaa ctgaccctga ccctgatgga tctggacctg 780

gagatgaaac ataacgtcag cagcgtcttt accgttgtgg aagaaggtct gtttggccgt 840

acctataccg tttggcgttc tgataccggt aaaccgagta ccagtccggg tattggtcat 900

tttctgcgcg tcttcgaaat tggtctggtt cgcgatctgg aactgggcgc tccgattttt 960

cacatgacca actacctgac cgtcaacatg agcgacgatt accgttcttg tctgctggca 1020

gttggcgaac tgaaactgac cgcactgtgt accccgtctg aaaccgttac cctgtctgaa 1080

tctggcgttc cgaaacgcga accgctggtt gtcgttattc tgaatctggc aggtccgacc 1140

ctgggcggcg aactgtattc tgttctgccg accaccgatc cgaccgttga aaaactgtat 1200

ctgagcagcc atcgcggtat catcaaagac aacgaagcga attgggttgt tccgtctacc 1260

gacgttcgtg atctgcaaaa caaaggcgag tgcctggttg aagcttgtaa aacccgtccg 1320

ccgagctttt gtaacggtac cggtattggt ccgtggtctg aaggtcgtat tccggcttac 1380

ggcgttattc gcgtttctct ggatctggca tctgatccgg gcgtagttat taccagcgtt 1440

tttggtccgc tgattccgca tctgtctggc atggacctgt ataataatcc gtttagccgc 1500

gcggcttggc tggcagttcc gccgtacgaa cagtcttttc tgggcatgat caacaccatt 1560

ggttttccgg atcgcgctga agttatgccg catattctga ccaccgaaat tcgcggtccg 1620

cgcggtcgtt gtcatgttcc gattgaactg agcagccgta tcgacgacga catcaaaatc 1680

ggcagcaaca tggttgtcct gccgaccaaa gatctgcgtt atatcaccgc gacctacgac 1740

gtttctcgta gcgaacacgc gatcgtctac tacatctacg ataccggccg tagcagcagc 1800

tatttttacc cggttcgtct gaactttcgc ggtaatccgc tgtctctgcg tattgagtgc 1860

tttccgtggt accacaaagt ctggtgttac cacgactgcc tgatctacaa caccatcacc 1920

aacgaagaag tgcatacccg cggtctgacc ggtattgaag tcacctgcaa cccggttggt 1980

ggcggtggaa tcggaggtgg tggaagcgga ggaggtggaa gcatgcacca tcaccaccat 2040

cacatgaccc gcgttgcgac cctggtcttt ctgtttctgt tcccgaacac cgttacctgt 2100

cagattcatt ggggcaacct gagcaaaatc ggcattgttg gtaccggtag cgcttcctac 2160

aaagttatga cccgtccgag tcatcagacc ctggttatca aactgatgcc gaacatcacc 2220

gcgattgata actgcaccaa aagcgagatc agcgagtaca aacgtctgct gatcaccgtt 2280

ctgaaaccgg ttgaagacgc actgagcgtc atcaccaaaa acgtccgtcc gattcaaacc 2340

ctgaccccgg gtcgtcgtac ccgtcgtttt gtaggcgctg ttctggcagg tgttgcactg 2400

ggcgttgcaa ccgcagcaca aattaccgca ggcgttgcac tgcatcagtc tctgatgaac 2460

agccaggcga ttgaaagcct gaaaaccagc ctggagaaaa gcaaccaggc aattgaagaa 2520

atccgtctgg cgaacaaaga aaccatcctg gcagttcagg gcgtccagga ttacatcaac 2580

aacgagctgg tcccgtctgt tcatcgtatg agctgcgaac tggtcggtca taaactgagc 2640

ctgaaactgc tgcgctacta caccgagatc ctgagcatct ttggtccgag tctgcgcgat 2700

ccgattgcag cggaaattag cattcaggcg ctgagttacg cgctgggcgg cgatattaac 2760

aaaatcctgg acaaactggg ctattctggc ggcgattttc tggcgattct ggaatccaaa 2820

ggcatcaaag cgcgcgttac ctacgttgat acccgcgatt atttcatcat cctgagcatt 2880

gcgtatccga ccctgagcga aatcaaaggc gttatcgtcc acaaaatcga ggcgatcagc 2940

tacaacatcg gcgctcaaga gtggtatacc acgattccgc gttacgttgc gacccagggt 3000

tatctgatta gcaacttcga cgagaccagc tgcgttttta ccccggaagg taccgtttgt 3060

agccaaaacg ctctgtatcc gatgagtccg ctgctgcaag agtgttttcg cggtagcacc 3120

aaaagttgcg cacgtaccct ggtttctggt accaccagta atcgcttcat cctgagcaaa 3180

ggcaacctga tcgcaaattg cgcctctgtt ctgtgcaaat gctacaccac cgaaaccgtc 3240

atcaaccagg atccggataa actgctgacc gttattgcct ctgataaatg cccggtagtt 3300

gaagttgacg gcgtcaccat tcaggttggt agccgcgaat atccggatag cgtctatctg 3360

cacgagattg atctgggtcc ggcaatttct ctggagaaac tggatgtcgg taccaatctg 3420

ggtaacgcag ttacgcgtct ggaaaacgca aaagaactgc tggacgcgag cgatcagatt 3480

ctgaaaaccg ttaaaggcgt tccgttcagc ggtaacatct acattgccct ggcagcttgt 3540

attggcgtta gtctgggtct ggttaccctg atttgctgtt gcaaaggccg ttgtcgcaac 3600

aaagagattc cggcaagcaa aattaatccg ggcctgaaac cggatctgac cggtaccagc 3660

aaaagctacg ttcgtagcct gtaactcgag 3690

<210> 4

<211> 1227

<212> PRT

<213>Artificial sequence

<220>

<223>

<400> 4

Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser

1 5 10 15

Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp

20 25 30

Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile

35 40 45

Gly Ser Met Ser Ala Gln Arg Glu Arg Ile Asn Ala Phe Tyr Lys Asp

50 55 60

Asn Leu His Asn Lys Thr His Arg Val Ile Leu Asp Arg Glu Arg Leu

65 70 75 80

Thr Ile Glu Arg Pro Tyr Ile Leu Leu Gly Val Leu Leu Val Met Phe

85 90 95

Leu Ser Leu Ile Gly Leu Leu Ala Ile Ala Gly Ile Arg Leu His Arg

100 105 110

Ala Thr Val Gly Thr Ala Glu Ile Gln Ser Arg Leu Asn Thr Asn Ile

115 120 125

Glu Leu Thr Glu Ser Ile Asp His Gln Thr Lys Asp Val Leu Thr Pro

130 135 140

Leu Phe Lys Ile Ile Gly Asp Glu Val Gly Ile Arg Ile Pro Gln Lys

145 150 155 160

Phe Ser Asp Leu Val Lys Phe Ile Ser Asp Lys Ile Lys Phe Leu Asn

165 170 175

Pro Asp Arg Glu Tyr Asp Phe Arg Asp Leu Arg Trp Cys Met Asn Pro

180 185 190

Pro Glu Arg Val Lys Ile Asn Phe Asp Gln Phe Cys Glu Tyr Lys Ala

195 200 205

Ala Val Lys Ser Val Glu His Ile Phe Glu Ser Ser Leu Asn Arg Ser

210 215 220

Glu Arg Leu Arg Leu Leu Thr Leu Gly Pro Gly Thr Gly Cys Leu Gly

225 230 235 240

Arg Thr Val Thr Arg Ala Gln Phe Ser Glu Leu Thr Leu Thr Leu Met

245 250 255

Asp Leu Asp Leu Glu Met Lys His Asn Val Ser Ser Val Phe Thr Val

260 265 270

Val Glu Glu Gly Leu Phe Gly Arg Thr Tyr Thr Val Trp Arg Ser Asp

275 280 285

Thr Gly Lys Pro Ser Thr Ser Pro Gly Ile Gly His Phe Leu Arg Val

290 295 300

Phe Glu Ile Gly Leu Val Arg Asp Leu Glu Leu Gly Ala Pro Ile Phe

305 310 315 320

His Met Thr Asn Tyr Leu Thr Val Asn Met Ser Asp Asp Tyr Arg Ser

325 330 335

Cys Leu Leu Ala Val Gly Glu Leu Lys Leu Thr Ala Leu Cys Thr Pro

340 345 350

Ser Glu Thr Val Thr Leu Ser Glu Ser Gly Val Pro Lys Arg Glu Pro

355 360 365

Leu Val Val Val Ile Leu Asn Leu Ala Gly Pro Thr Leu Gly Gly Glu

370 375 380

Leu Tyr Ser Val Leu Pro Thr Thr Asp Pro Thr Val Glu Lys Leu Tyr

385 390 395 400

Leu Ser Ser His Arg Gly Ile Ile Lys Asp Asn Glu Ala Asn Trp Val

405 410 415

Val Pro Ser Thr Asp Val Arg Asp Leu Gln Asn Lys Gly Glu Cys Leu

420 425 430

Val Glu Ala Cys Lys Thr Arg Pro Pro Ser Phe Cys Asn Gly Thr Gly

435 440 445

Ile Gly Pro Trp Ser Glu Gly Arg Ile Pro Ala Tyr Gly Val Ile Arg

450 455 460

Val Ser Leu Asp Leu Ala Ser Asp Pro Gly Val Val Ile Thr Ser Val

465 470 475 480

Phe Gly Pro Leu Ile Pro His Leu Ser Gly Met Asp Leu Tyr Asn Asn

485 490 495

Pro Phe Ser Arg Ala Ala Trp Leu Ala Val Pro Pro Tyr Glu Gln Ser

500 505 510

Phe Leu Gly Met Ile Asn Thr Ile Gly Phe Pro Asp Arg Ala Glu Val

515 520 525

Met Pro His Ile Leu Thr Thr Glu Ile Arg Gly Pro Arg Gly Arg Cys

530 535 540

His Val Pro Ile Glu Leu Ser Ser Arg Ile Asp Asp Asp Ile Lys Ile

545 550 555 560

Gly Ser Asn Met Val Val Leu Pro Thr Lys Asp Leu Arg Tyr Ile Thr

565 570 575

Ala Thr Tyr Asp Val Ser Arg Ser Glu His Ala Ile Val Tyr Tyr Ile

580 585 590

Tyr Asp Thr Gly Arg Ser Ser Ser Tyr Phe Tyr Pro Val Arg Leu Asn

595 600 605

Phe Arg Gly Asn Pro Leu Ser Leu Arg Ile Glu Cys Phe Pro Trp Tyr

610 615 620

His Lys Val Trp Cys Tyr His Asp Cys Leu Ile Tyr Asn Thr Ile Thr

625 630 635 640

Asn Glu Glu Val His Thr Arg Gly Leu Thr Gly Ile Glu Val Thr Cys

645 650 655

Asn Pro Val Gly Gly Gly Gly Ile Gly Gly Gly Gly Ser Gly Gly Gly

660 665 670

Gly Ser Met His His His His His His Met Thr Arg Val Ala Thr Leu

675 680 685

Val Phe Leu Phe Leu Phe Pro Asn Thr Val Thr Cys Gln Ile His Trp

690 695 700

Gly Asn Leu Ser Lys Ile Gly Ile Val Gly Thr Gly Ser Ala Ser Tyr

705 710 715 720

Lys Val Met Thr Arg Pro Ser His Gln Thr Leu Val Ile Lys Leu Met

725 730 735

Pro Asn Ile Thr Ala Ile Asp Asn Cys Thr Lys Ser Glu Ile Ser Glu

740 745 750

Tyr Lys Arg Leu Leu Ile Thr Val Leu Lys Pro Val Glu Asp Ala Leu

755 760 765

Ser Val Ile Thr Lys Asn Val Arg Pro Ile Gln Thr Leu Thr Pro Gly

770 775 780

Arg Arg Thr Arg Arg Phe Val Gly Ala Val Leu Ala Gly Val Ala Leu

785 790 795 800

Gly Val Ala Thr Ala Ala Gln Ile Thr Ala Gly Val Ala Leu His Gln

805 810 815

Ser Leu Met Asn Ser Gln Ala Ile Glu Ser Leu Lys Thr Ser Leu Glu

820 825 830

Lys Ser Asn Gln Ala Ile Glu Glu Ile Arg Leu Ala Asn Lys Glu Thr

835 840 845

Ile Leu Ala Val Gln Gly Val Gln Asp Tyr Ile Asn Asn Glu Leu Val

850 855 860

Pro Ser Val His Arg Met Ser Cys Glu Leu Val Gly His Lys Leu Ser

865 870 875 880

Leu Lys Leu Leu Arg Tyr Tyr Thr Glu Ile Leu Ser Ile Phe Gly Pro

885 890 895

Ser Leu Arg Asp Pro Ile Ala Ala Glu Ile Ser Ile Gln Ala Leu Ser

900 905 910

Tyr Ala Leu Gly Gly Asp Ile Asn Lys Ile Leu Asp Lys Leu Gly Tyr

915 920 925

Ser Gly Gly Asp Phe Leu Ala Ile Leu Glu Ser Lys Gly Ile Lys Ala

930 935 940

Arg Val Thr Tyr Val Asp Thr Arg Asp Tyr Phe Ile Ile Leu Ser Ile

945 950 955 960

Ala Tyr Pro Thr Leu Ser Glu Ile Lys Gly Val Ile Val His Lys Ile

965 970 975

Glu Ala Ile Ser Tyr Asn Ile Gly Ala Gln Glu Trp Tyr Thr Thr Ile

980 985 990

Pro Arg Tyr Val Ala Thr Gln Gly Tyr Leu Ile Ser Asn Phe Asp Glu

995 1000 1005

Thr Ser Cys Val Phe Thr Pro Glu Gly Thr Val Cys Ser Gln Asn

1010 1015 1020

Ala Leu Tyr Pro Met Ser Pro Leu Leu Gln Glu Cys Phe Arg Gly

1025 1030 1035

Ser Thr Lys Ser Cys Ala Arg Thr Leu Val Ser Gly Thr Thr Ser

1040 1045 1050

Asn Arg Phe Ile Leu Ser Lys Gly Asn Leu Ile Ala Asn Cys Ala

1055 1060 1065

Ser Val Leu Cys Lys Cys Tyr Thr Thr Glu Thr Val Ile Asn Gln

1070 1075 1080

Asp Pro Asp Lys Leu Leu Thr Val Ile Ala Ser Asp Lys Cys Pro

1085 1090 1095

Val Val Glu Val Asp Gly Val Thr Ile Gln Val Gly Ser Arg Glu

1100 1105 1110

Tyr Pro Asp Ser Val Tyr Leu His Glu Ile Asp Leu Gly Pro Ala

1115 1120 1125

Ile Ser Leu Glu Lys Leu Asp Val Gly Thr Asn Leu Gly Asn Ala

1130 1135 1140

Val Thr Arg Leu Glu Asn Ala Lys Glu Leu Leu Asp Ala Ser Asp

1145 1150 1155

Gln Ile Leu Lys Thr Val Lys Gly Val Pro Phe Ser Gly Asn Ile

1160 1165 1170

Tyr Ile Ala Leu Ala Ala Cys Ile Gly Val Ser Leu Gly Leu Val

1175 1180 1185

Thr Leu Ile Cys Cys Cys Lys Gly Arg Cys Arg Asn Lys Glu Ile

1190 1195 1200

Pro Ala Ser Lys Ile Asn Pro Gly Leu Lys Pro Asp Leu Thr Gly

1205 1210 1215

Thr Ser Lys Ser Tyr Val Arg Ser Leu

1220 1225

<210> 5

<211> 3564

<212> PRT

<213>Artificial sequence

<220>

<223>

<400> 5

catatgcatc atcaccatca ccatatgtcc gcacaaaggg aaaggatcaa tgccttctac 60

aaagacaatc ttcataataa gacccatagg gtaatcctgg atagggaacg cttaactatt 120

gaaagaccct acatcttact tggagtccta ctggtaatgt ttctgagtct aatcgggctg 180

ctggccattg cagggatcag gcttcaccga gccaccgttg ggactgcgga gatccagagt 240

cggctgaata ccaacattga gttgaccgaa tccattgatc atcaaactaa ggatgtctta 300

actcccctgt ttaaaatcat tggtgatgaa gtcggcatca gaattccaca gaagttcagt 360

gatcttgtca agttcatctc cgataagatt aagttcctca accctgacag agaatatgat 420

tttagggatc tccggtggtg tatgaacccc cctgagagag tcaaaattaa ctttgaccag 480

ttttgtgaat acaaagccgc agtcaagtca gttgaacata tatttgagtc atcactcaac 540

aggtcagaaa ggttgcgatt attgactctt gggcccggaa caggctgtct cggcaggaca 600

gtaacaagag ctcagttctc agagcttacg ctgaccctga tggacctgga tctcgagatg 660

aagcacaacg tgtcctcagt gtttaccgta gtcgaagagg gattattcgg aagaacatat 720

actgtctgga gatccgacac cggaaaaccg agcaccagtc caggtattgg ccatttttta 780

agagtcttcg agatcgggct ggtgagagat ctcgagctgg gtgcccctat tttccatatg 840

accaactacc tcacagtaaa catgagtgat gactatcgga gctgtctttt agcagtaggg 900

gagttgaagc tgacagccct atgcacccca tctgagactg tgactctgag tgagagtgga 960

gttccaaaga gagagcctct tgtggttgtg atactcaacc tagctgggcc tactctaggg 1020

ggcgaactat acagtgtatt gcctaccact gaccccacgg tggagaaact ctatttatcc 1080

tcacataggg ggattatcaa agataacgag gccaattggg tagtaccgtc gaccgatgtt 1140

cgtgatcttc aaaacaaagg agaatgtctg gtggaagcat gcaagactcg acctccttca 1200

ttttgcaatg gcacaggaat aggcccatgg tcagagggga gaatccctgc ctacggggtg 1260

atcagggtca gtcttgactt agctagtgac ccgggtgtag ttatcacttc agtgtttggc 1320

ccattgatac ctcacctatc cggtatggat ctttacaaca atccgttttc aagagctgca 1380

tggttggctg taccacctta tgagcagtca tttctaggaa tgataaatac aattggcttc 1440

ccggacagag cagaggttat gccgcacatt ttgaccacag agatcagagg gcctcgaggt 1500

cgttgtcatg ttcctataga gttgtccagc aggattgatg atgatatcaa gatcgggtcc 1560

aacatggttg tattgccgac gaaggacctg aggtacataa cagccactta tgatgtttcc 1620

aggagcgagc atgcaatcgt gtactatatc tatgacacgg gtcgctcatc atcttacttc 1680

tacccagttc gattgaattt caggggcaat cctctctctc tgaggataga gtgttttccc 1740

tggtatcata aggtgtggtg ctaccatgat tgtcttatat acaacaccat aacaaacgaa 1800

gaagtccaca cgagagggct gaccggtata gaggtaacat gtaatccagt cggtggcggt 1860

ggaatcggag gtggtggaag cggaggaggt ggaagcatgc atcatcacca tcaccatatg 1920

acacgggtcg caaccttagt atttctgttt cttttcccaa acactgtcac gtgccagatt 1980

cactggggca atctatccaa gatcgggatt gtaggaacgg ggagtgccag ctacaaggtg 2040

atgactaggc caagccacca aactctggtc ataaagttga tgccaaatat aacagccatc 2100

gacaattgta cgaaatcaga gatttcagag tacaaaagat tgctgatcac agtgttaaag 2160

cctgtagagg atgccctgtc agtgataacc aagaatgtaa gaccaattca aactctaaca 2220

cctgggcgca ggacccgccg ttttgtcggg gctgttctgg ccggagtagc acttggagtc 2280

gcgacagccg ctcaaataac tgccggagtc gcactccatc agtcattgat gaattcccaa 2340

gcaattgaaa gtttaaaaac cagtcttgag aagtcgaatc aggcaataga agaaatcaga 2400

cttgcaaata aggagaccat actggcggta cagggcgtcc aagactatat caacaatgag 2460

cttgtcccct ctgttcatag aatgtcatgt gagcttgtag gtcacaaact cagtctcaag 2520

ctccttaggt attataccga gatcctgtct atattcgggc ctagccttcg agacccgata 2580

gctgctgaaa tatcaatcca ggcactcagc tatgcattag gcggagacat caataaaatt 2640

ctggacaagc ttgggtatag cggcggggat ttccttgcta tcctagaaag caaggggata 2700

aaggcccggg tcacatatgt ggacacaaga gattacttta taattcttag catagcctac 2760

ccaaccttat ctgagatcaa gggggtgata gttcataaga tagaagctat atcctacaat 2820

attggggcac aggaatggta tactactatc cctagatatg tagccactca gggatatctg 2880

atatcgaatt tcgatgagac gtcatgcgtc ttcactccag aggggacagt ctgcagccag 2940

aatgcgttgt atccaatgag cccattgctt caggaatgct tcagggggtc gacaaaatcg 3000

tgcgccagaa ccctagtttc agggaccaca agtaatagat ttatcctatc aaaagggaac 3060

ttgattgcaa attgtgcgtc agttttgtgc aagtgttaca caacggagac agttatcaac 3120

caagatcctg ataaactact aactgttata gcctccgata agtgtcccgt agtcgaggtg 3180

gatggagtga caatacaggt cggcagtcga gagtacccag attctgtata cctacatgaa 3240

atagacttag gcccagccat ctccctggag aaactggatg taggcaccaa tttaggcaat 3300

gcagtcacaa gactggagaa tgcaaaggag ctactagatg catcagacca gatactgaag 3360

actgttaaag gggtaccttt cagtggcaat atatacatag cactggcagc ttgcattggg 3420

gtatccctag ggcttgtcac attaatatgc tgctgtaagg ggagatgtag gaacaaggag 3480

attcctgcct ccaaaatcaa cccagggctc aaacccgacc taaccgggac ttcaaagtcg 3540

tacgtgagat cactgtagct cgag 3564

Claims (10)

1. the enzyme linked immunological kit of PPR virus antibody is detected, including envelope antigen, the envelope antigen is restructuring PPR virus H-F fusion proteins, the restructuring PPR virus H-F fusion proteins are protein a) or b)：

A) protein being made up of SEQ ID No.2 amino acid sequence；

B) by the amino acid sequence shown in SEQ ID No.2 by one or several amino acid residues substitution and/or missing and/ Or the soluble protein that addition is obtained.

2. enzyme linked immunological kit according to claim 1, it is characterised in that：The restructuring PPR virus H-F Fusion protein is prepared according to the method comprised the following steps：Make the coding of the restructuring PPR virus H-F fusion proteins Gene in biology express obtaining the protein；It is described biological for microorganism, plant or non-human animal.

3. enzyme linked immunological kit according to claim 2, it is characterised in that：Make the restructuring PPR virus H- The encoding gene of F fusion proteins carries out expression in biology to be included the restructuring PPR virus described in claim 1 The encoding gene of H-F fusion proteins imports recipient microorganism, obtains recombinating PPR virus described in expression claim 1 The recombinant microorganism of H-F fusion proteins, cultivates the recombinant microorganism, and expression obtains recombinating small ruminate described in claim 1 Epizootic disease virus H-F fusion proteins.

4. enzyme linked immunological kit according to claim 3, it is characterised in that：The recipient microorganism is C1)-C4) in It is any:

C1) prokaryotic micro-organisms；

C2) gramnegative bacterium；

C3) Escherichia bacteria；

C4) e. coli bl21 (DE3).

5. according to any described enzyme linked immunological kit in claim 2-4, it is characterised in that：The restructuring PPR The encoding gene of viral H-F fusion proteins for it is following 1) or 2) shown in DNA molecular：

1) coded sequence is the DNA molecular shown in SEQ ID No.1 4-3558；

2) have with the DNA molecular that 1) limits more than 90% homogeneity and coding claim 1 described in restructuring small ruminate beast The DNA molecular of epidemic disease poison H-F fusion proteins.

6. according to any described enzyme linked immunological kit in claim 3-5, it is characterised in that：The recombinant microorganism is will The express amino acid sequence that pET30a-rmHF1-Y importing e. coli bl21s (DE3) are obtained is SEQ ID No.2 protein Recombinant microorganism, the recombinant microorganism is named as BL21 (DE3)/pET30a-rmHF1-Y, the pET30a-rmHF1-Y It is the fragment between Nde I and the XhoI recognition sites that pET30a (+) is replaced with the DNA shown in SEQ ID No.1, keeps pET30a Other sequences of (+) are constant, obtained recombinant expression carrier.

7. according to any described enzyme linked immunological kit in claim 2-6, it is characterised in that：It is described to be expressed as induction table Reach, the induced expression is 13-16 hours or 13-24 hours or 13 hours to be induced at 16 DEG C or 16 small with 0.75mM IPTG When.

8. PPR virus H-F fusion proteins are recombinated described in claim 1 is preparing detection detection PPR disease Application in the enzyme linked immunological kit of malicious antibody.

9. the enzyme linked immunological kit of the detection PPR virus antibody described in claim 1 is examined preparing PPR Application in disconnected kit.

10. the enzyme linked immunological kit of the detection PPR virus antibody described in claim 1 is preparing PPR Application in monitoring reagent box.