CN107098979A - PPR virus H F fusion proteins and its relevant biological material and application - Google Patents

PPR virus H F fusion proteins and its relevant biological material and application Download PDF

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CN107098979A
CN107098979A CN201710483527.8A CN201710483527A CN107098979A CN 107098979 A CN107098979 A CN 107098979A CN 201710483527 A CN201710483527 A CN 201710483527A CN 107098979 A CN107098979 A CN 107098979A
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leu
rmhf1
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孙雨
宋晓晖
杨林
王传彬
董浩
杨天意
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CHINA ANIMAL BLIGHT PREVENTION AND CONTROL CENTER
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Abstract

The invention discloses PPR virus H F fusion proteins and its relevant biological material and application.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 method for the indirect ELISA detection PPR virus antibody that PPR virus H F fusion proteins are set up as envelope antigen has higher specificity, sensitiveness and good accuracy, and quick, the easy operation of energy, be conducive to the monitoring clinically to PPR.

Description

PPR virus H-F fusion proteins and its relevant biological material and application
Technical field
The present invention relates to PPR virus H-F fusion proteins in biological technical field and its relevant biological material with answering With.
Background technology
PPR (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 the characteristics of high mortality.At present, it there is no effective ways to treat PPR, can only take after vaccine inoculation, epidemic situation generation The method for slaughtering and strengthening regular sero monitoring controls the disease.Most common report is with PPRV N, H eggs after purification both at home and abroad (total length) or the part comprising N protein Main Antigenic recombinate N, H protein fragment as envelope antigen in vain, also have with concentration PPRV totivirus sets up indirect ELISA method detection PPR serum antibodies (Qiu Wenying, 2011 as envelope antigen； Balamurugan et al., 2007), but there is not yet set up indirect ELISA side using H-F fused in tandem albumen as envelope antigen The report of method.
PPRV genomes encode 6 kinds of structural proteins, i.e. nucleoprotein (N), phosphoprotein (P), polymerase large protein (L), base altogether Matter albumen (M), fusion protein (F) and hemagglutinin (H).Wherein, H gene total length 1852bp, by 609 amino acid encodings, Molecular weight is 68kDa.F full length gene 2321bp, containing an ORF, F protein contains 546 amino acid, and molecular weight is about 59kDa, it is highly conserved in numb epidemic disease Tobamovirus.F full length gene 2321bp, containing an ORF, F protein contains 546 amino Acid, molecular weight is about 59kDa, highly conserved in numb epidemic disease Tobamovirus.The antigenicity of H and F protein is stable, in virus infection Antibody in animal blood serum for H, F fusion protein is occupied an leading position, and is two good target genes as diagnostic antigen.But It is, PPR H, the usual inclusion bodies with insoluble monovalent albumen of F protein expression product of majority research expression In the presence of with soluble H-F fusion-activities protein expression, there is not been reported.The expression of the inclusion body protein of unit price is due to existing Expression quantity is not enough and space structure has false folding, it is impossible to forms high protein structure and natural space conformation epitope, leads It, which is caused, as diagnostic antigen can not form immune predicting space epitope well.Meanwhile, the expression product in monovalent inclusion body does not have There is biological activity, thus need to be denatured and renaturation process.The denaturation of albumen and renaturation are an extremely complex processes, The denaturing conditions of different albumen are different, and renaturation yield is difficult often to improve.This is the main restricting factor for limiting its application.
The content of the invention
A technical problem to be solved by this invention be how clinical quick detection PPR.
In order to solve the above technical problems, the invention provides the restructuring PPR virus H-F of solubility expression fusions Albumen and its relevant biological material.
In order to solve the above technical problems, the invention provides restructuring PPR virus H-F fusion proteins 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 is by the substitution of one or several amino acid residues and/or lacks Lose and/or add obtained soluble protein.
In above-mentioned protein, protein name a) is referred to as rmHF1-Y (also known as rmHF1), is the PPRV H-F of restructuring series connection Albumen.SEQ ID No.2 are made up of 1184 amino acid residues.
In above-mentioned protein, the protein can be prepared according to the method comprised the following steps：Make the volume of the protein Code gene in biology express obtaining the protein；It is described biological for microorganism, plant or non-human animal.
In above-mentioned protein, the encoding gene of the protein is carried out expression in biology is included described protein Encoding gene import recipient microorganism, obtain expressing the recombinant microorganism of the protein, cultivate the recombinant microorganism, table Reach the protein.
Any of in above-mentioned protein, 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 protein, the encoding gene of the protein 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) 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 protein, 2) concretely nucleotide sequence and 1) have at least 91%, 92%, 95%, 96%, 98% Or the DNA molecular of 99% homogeneity.
In above-mentioned protein, the recombinant microorganism is that pET30a-rmHF1-Y is imported into e. coli bl21 (DE3) to obtain To express amino acid sequence be SEQ ID No.2 protein recombinant microorganism, the recombinant microorganism is named as BL21 (DE3)/pET30a-rmHF1-Y, the pET30a-rmHF1-Y are to replace pET30a (+) with the DNA shown in SEQ ID No.1 Nde I and XhoI recognition sites between fragment (small fragment including Nde I recognition sites and XhoI recognition sites), protect The other sequences for holding pET30a (+) are constant, obtained rmHF1-Y gene recombinant vectors.
Described to be expressed as induced expression in above-mentioned protein, the induced expression is to be lured with 0.75mM IPTG at 16 DEG C Lead 13-16 hours or 13-24 hours or 13 hours or 16 hours.
Protection scope of the present invention is fallen within the biomaterial of the albumen qualitative correlation, the biomaterial can be following Any of B1) to B16)：
B1) the nucleic acid molecules of code for said proteins；
B2 B1) is contained) expression cassettes of the nucleic acid molecules；
B3 B1) is contained) recombinant vectors of the nucleic acid molecules；
B4 B2) is contained) recombinant vector of the expression cassette；
B5 B1) is contained) recombinant microorganisms of the nucleic acid molecules；
B6 B2) is contained) recombinant microorganism of the expression cassette；
B7 B3) is contained) recombinant microorganism of the recombinant vector；
B8 B4) is contained) recombinant microorganism of the recombinant vector；
B9 B1) is contained) the transgenetic animal cell systems of the nucleic acid molecules；
B10 B2) is contained) the transgenetic animal cell system of the expression cassette；
B11 B3) is contained) the transgenetic animal cell system of the recombinant vector；
B12 B4) is contained) the transgenetic animal cell system of the recombinant vector；
B13 B1) is contained) the transgenic plant cells systems of the nucleic acid molecules；
B14 B2) is contained) the transgenic plant cells system of the expression cassette；
B15 B3) is contained) the transgenic plant cells system of the recombinant vector；
B16 B4) is contained) the transgenic plant cells system of the recombinant vector.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules also may be used To be RNA, such as mRNA or hnRNA.
Herein, term " homogeneity " refers to the sequence similarity with native sequence nucleic acid.Homogeneity can with the naked eye or meter Calculation machine software is evaluated.Using computer software, the homogeneity between two or more sequences can use percentage (%) table Show, it can be for the homogeneity between evaluation correlated series.
In above-mentioned biomaterial, the nucleic acid molecules can 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) 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 biomaterial, 2) concretely nucleotide sequence and 1) have at least 91%, 92%, 95%, 96%, The DNA molecular of 98% or 99% homogeneity.
In above-mentioned biomaterial, the recombinant vector can be the pET30a-rmHF1-Y；
The recombinant microorganism can be E1) or E2)：
E1) recombinant microorganism is that the encoding gene of described protein is imported into recipient microorganism, obtains expressing institute Any of the recombinant microorganism of protein is stated, the recipient microorganism is C1)-C4):
C1) prokaryotic micro-organisms；
C2) gramnegative bacterium；
C3) Escherichia bacteria；
C4) e. coli bl21 (DE3).
E2) recombinant microorganism is that the pET30a-rmHF1-Y is imported into the table that e. coli bl21 (DE3) is obtained It is the recombinant microorganism of SEQ ID No.2 protein up to amino acid sequence.
In above-mentioned biomaterial, B9) to B16) it may include that propagating materials also may not include propagating materials.
D1 application) or D2) falls within protection scope of the present invention：
D1) application in PPR diagnostic antigen is prepared of the protein or the biomaterial, preparing inspection The application or the application in PPR diagnostic kit is prepared surveyed in the kit of PPR virus antibody；
D2) application of the protein in monoclonal antibody is prepared.
The present invention carries out PPR virus H and F protein gene to merge after series connection in large intestine bar after codon optimization Soluble fusion overexpression protection original rmHF1-Y (restructuring PPR virus H-F fusion proteins) is obtained in bacterium：The present invention is used DNA shown in SEQ ID No.1 replaces fragment (including the Nde I identifications between pET30a (+) Nde I and XhoI recognition sites Small fragment including site and XhoI recognition sites), keep pET30a (+) other sequences constant, obtained rmHF1-Y genes Recombinant expression carrier pET30a-rmHF1-Y, will
PET30a-rmHF1-Y imports the destination protein rmHF1 that e. coli bl21 (DE3) obtains solubility.With 0.75mM IPTG is induced 16 hours at 16 DEG C, and rmHF1 content reaches the 45% of bacterial protein, the rmHF1 63% of expression It is solvable.
It is demonstrated experimentally that detecting PPR virus antibody using rmHF1-Y as the indirect ELISA that envelope antigen is set up Method has higher specificity, sensitiveness and good accuracy, and quick, the easy operation of energy, and it is clinically right to be conducive to The monitoring of PPR.Using the rmHF1-Y indirect ELISA detection PPR virus antibody set up as envelope antigen Method and France ID-VET PPR antibody assay kit coincidence rate are 94.5%, and to recombinate PPRV H proteins The method for the indirect ELISA detection PPR virus antibody that rmH is set up as envelope antigen is small anti-with France ID-VET's The coincidence rate of hay epizootic disease antibody assay kit only has 83.3%, is set up as envelope antigen using to recombinate PPRV F proteins rmF The method and France ID-VET PPR antibody assay kit of indirect ELISA detection PPR virus antibody Coincidence rate only has 72.6%.Illustrate to resist rmHF1-Y as the indirect ELISA detection PPR virus that envelope antigen is set up The method of body and the coincidence rate of France ID-VET PPR antibody assay kit are significantly higher than to recombinate PPRV H eggs The indirect ELISA that white rmH is set up as envelope antigen detects the method for PPR virus antibody and to recombinate PPRV F eggs The method that the indirect ELISA that white rmF is set up as envelope antigen detects PPR virus antibody.It regard rmHF1-Y as bag The sensitiveness of the method for the indirect ELISA detection PPR virus antibody set up by antigen is 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 method for the indirect ELISA detection PPR virus antibody that envelope antigen is set up cannot be only used for examining for PPR It is disconnected, and evaluable immune effect of vaccine, in addition, this method can fast and accurately detect PPR, so that for China more The propagation of good control PPR will 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 It is thick, 16000 in 4 DEG C of centrifuges
Rpm/min centrifuges 30min, collects supernatant (being named as containing albumen supernatant), abandons precipitation.Albumen supernatant will be contained With loading after 0.22 μm of membrane filtration to advance solution 1, (solute and its concentration are as follows：20mM Tris, 150mM NaCl, it is molten Agent is 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 (solute and its concentration are as follows with the solution 2 of 10 column volumes：20mM Tris, 150mM NaCl, 50mM imidazoles, solvent is water, PH8.0 solution) impurity protein in cleaning nickel post, and monitor protein peak on AKTA machines.With (the solute and its dense of solution 3 Degree is as follows：20mM Tris, 150mM NaCl, 300mM imidazoles, solvent are water, pH8.0 solution) rinse nickel post hang on nickel post Destination protein, and collected using AKTA and the elution samples at destination protein peak occur, the sample is referred to as to the rmHF1 of ni-sepharose purification Albumen (ni-sepharose purification destination protein sample).
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 PPRs antibody is examined Test agent box, 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 ELIASA450nmNumerical 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 (IDPPR Competition PPR antibody test reagents Box, production code member is PPRC-4P) it is detected as restructuring rmHF1 optimization indirect ELISA of the negative sheep blood serums of PPRV using step 1 Method (the PPRV positive serums in 1.4.1 being replaced with into 1000 parts of PPRV negative serums respectively, other operations are identical) is carried out Indirect ELISA is detected, calculates the average value (X) and standard deviation (SD) of 1000 parts of PPRV negative serums. 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-VETPPR Competition are small Epizootic disease antibody assay kit is ruminated, production code member is PPRC-4P) the positive sheep blood serum progress doubling dilutions of PPRV are detected as, 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 is compiled Number it is PPRC-4P) 500 parts of sheep blood serums being preserved to China Animal Disease Control And Prevention Center plant-eating animal and infecting both domestic animals and human ward Detected, 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. Protein 1.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. 2. protein according to claim 1, it is characterised in that：The protein is according to the method system comprised the following steps It is standby：The encoding gene of the protein is set in biology express obtaining the protein；It is described biological for microorganism, plant Or non-human animal.
  3. 3. protein according to claim 2, it is characterised in that：The encoding gene of the protein is set to be carried out in biology Expression includes the encoding gene of the protein described in claim 1 importing recipient microorganism, obtains described in expression claim 1 The recombinant microorganism of protein, cultivates the recombinant microorganism, and expression obtains protein described in claim 1.
  4. 4. protein according to claim 3, it is characterised in that：Any of the recipient microorganism is C1)-C4):
    C1) prokaryotic micro-organisms；
    C2) gramnegative bacterium；
    C3) Escherichia bacteria；
    C4) e. coli bl21 (DE3).
  5. 5. according to any described protein in claim 2-4, it is characterised in that：The encoding gene of the protein is as follows Or 2) 1) DNA molecular shown in：
    1) coded sequence is the DNA molecular shown in SEQ ID No.1 4-3558；
    2) DNA molecular with 1) restriction is with protein DNA point described in more than 90% homogeneity and coding claim 1 Son.
  6. 6. according to any described protein in claim 3-5, it is characterised in that：The recombinant microorganism is by pET30a- The express amino acid sequence that rmHF1-Y importing e. coli bl21s (DE3) are obtained is that the restructuring of SEQ ID No.2 protein is micro- Biology, the recombinant microorganism is named as BL21 (DE3)/pET30a-rmHF1-Y, and the pET30a-rmHF1-Y is to use SEQ DNA shown in ID No.1 replaces the fragment between pET30a (+) Nde I and XhoI recognition sites, keeps pET30a (+) its Its sequence is constant, obtained recombinant expression carrier.
  7. 7. the biomaterial with the albumen qualitative correlation described in claim 1, the biomaterial is following B1) to B16) in appoint It is a kind of：
    B1) the nucleic acid molecules of protein described in coding claim 1；
    B2 B1) is contained) expression cassettes of the nucleic acid molecules；
    B3 B1) is contained) recombinant vectors of the nucleic acid molecules；
    B4 B2) is contained) recombinant vector of the expression cassette；
    B5 B1) is contained) recombinant microorganisms of the nucleic acid molecules；
    B6 B2) is contained) recombinant microorganism of the expression cassette；
    B7 B3) is contained) recombinant microorganism of the recombinant vector；
    B8 B4) is contained) recombinant microorganism of the recombinant vector；
    B9 B1) is contained) the transgenetic animal cell systems of the nucleic acid molecules；
    B10 B2) is contained) the transgenetic animal cell system of the expression cassette；
    B11 B3) is contained) the transgenetic animal cell system of the recombinant vector；
    B12 B4) is contained) the transgenetic animal cell system of the recombinant vector；
    B13 B1) is contained) the transgenic plant cells systems of the nucleic acid molecules；
    B14 B2) is contained) the transgenic plant cells system of the expression cassette；
    B15 B3) is contained) the transgenic plant cells system of the recombinant vector；
    B16 B4) is contained) the transgenic plant cells system of the recombinant vector.
  8. 8. biomaterial according to claim 7, it is characterised in that：The nucleic acid molecules 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) DNA molecular with 1) restriction is with protein DNA point described in more than 90% homogeneity and coding claim 1 Son.
  9. 9. biomaterial according to claim 8 or claim 9, it is characterised in that：The recombinant vector is described in claim 6 pET30a-rmHF1-Y；
    The recombinant microorganism is E1) or E2)：
    E1) recombinant microorganism is that the encoding gene of the protein described in claim 1 is imported into recipient microorganism, obtains table Any of up to the recombinant microorganism of protein described in claim 1, the recipient microorganism is C1)-C4):
    C1) prokaryotic micro-organisms；
    C2) gramnegative bacterium；
    C3) Escherichia bacteria；
    C4) e. coli bl21 (DE3).
    E2) recombinant microorganism is that pET30a-rmHF1-Y described in claim 6 is imported into e. coli bl21 (DE3) to obtain To express amino acid sequence be SEQ ID No.2 protein recombinant microorganism.
  10. 10.D1) or D2) application：
    D1 any described biomaterial detects small ruminate in preparation in protein or claim 7-9) described in claim 1 Application in the kit of epizootic disease antiviral antibody, the application in PPR diagnostic antigen is prepared are preparing small ruminate Application in epizootic disease diagnostic kit；
    D2) application of the protein described in claim 1 in monoclonal antibody is prepared.
CN201710483527.8A 2017-06-22 2017-06-22 Peste des petits ruminants virus H-F fusion protein and related biological material and application thereof Expired - Fee Related CN107098979B (en)

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CN110684782A (en) * 2019-10-29 2020-01-14 新疆畜牧科学院兽医研究所（新疆畜牧科学院动物临床医学研究中心） Codon-optimized peste des petits ruminants virus F gene nucleic acid vaccine
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