CN109306007A - Anti- porcine reproductive and respiratory syndrome virus nsp4 protein gene engineered antibody and application - Google Patents
Anti- porcine reproductive and respiratory syndrome virus nsp4 protein gene engineered antibody and application Download PDFInfo
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- CN109306007A CN109306007A CN201811121302.9A CN201811121302A CN109306007A CN 109306007 A CN109306007 A CN 109306007A CN 201811121302 A CN201811121302 A CN 201811121302A CN 109306007 A CN109306007 A CN 109306007A
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
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Abstract
The invention belongs to antibody applied technical fields, more particularly to anti-porcine reproductive and respiratory syndrome virus nsp4 protein gene engineered antibody and application, the present invention provides a kind of genetic engineering antibodies, for shown in SEQ ID NO.6 and SEQ ID NO.8, " Y " font antibody is formed after the mixing of both albumen, can and porcine reproductive and respiratory syndrome virus nsp4 albumen into specificity combination, only one epitope of the antibody, compatibility is good, it is specific high, it can be prepared into the ELISA reagent of porcine reproductive and respiratory syndrome virus, Western-Blot detection reagent, IFA detection reagent etc..It is provided by the invention to be obtained by artificial synthesized method, therefore the anti-porcine reproductive and respiratory syndrome virus nsp4 genetic engineering antibody of simple and direct acquisition can be more convenient, animal welfare issues when effectively solving hybridoma and save the disadvantages of at high cost, phenotype is easy to be lost, while having evaded preparation ascites.
Description
Technical field
The invention belongs to antibody applied technical fields, and in particular to anti-porcine reproductive and respiratory syndrome virus nsp4 albumen base
Because of engineered antibody and application.
Background technique
Porcine reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome
Virus, PRRSV), belong to shell type virales Arteriviridae Arterivirus, is to cause porcine reproductive and respiratory syndrome
Cause of disease, can lead to pregnant sow late abortion, premature labor and produce stillborn foetus, piglet and fattening porcine respiratory disease.From in June, 2006
Since just, occur in succession in more than ten of province such as China Hubei, Hunan, Jiangxi, Anhui using highly pathogenic PRRSV as main pathogen
Pig " nameless high-fever syndrome ", cause serious economic loss to the pig breeding industry in China.
Porcine reproductive and respiratory syndrome virus is the single strand plus RNA virus for having cyst membrane, and genome length is about 15kb,
With 5 ' end cap and 3 ' end Poly (A) structures.Full-length genome at least contain 10 open reading frame (ORF1a, ORF1b,
ORF2a, ORF2b, ORF3, ORF4, ORF5a, ORF5b, ORF6, ORF7), it overlaps between adjacent reading frame.Coding
ORF2~ORF7 of virus structural protein is located at 3 ' ends of porcine reproductive and respiratory syndrome virus genome.ORF2~ORF4 is compiled
Code virus secondary structure albumen, ORF5, ORF6 and ORF7 coding virus major structural protein, be respectively envelope protein GP5,
Cyst membrane stromatin (M) and nucleocapsid protein (N).And the ORF1a and ORF1b that genome 5 ' is held account for about the five of full-length genome
/ tetra-, it can produce 4 kinds of polyproteins, respectively pp1a, pp1a-nsp2TF, pp1a-nsp2N and pp1ab.These polies
Albumen can at least translate 16 non-structural proteins relevant to virus replication.It can be encoded at least after wherein pp1a is hydrolyzed
10 non-structural proteins (nsp1~8), the non-structural protein including 4 with hydrolase of proteolysis, respectively nsp1 α,
Nsp1 β, nsp2 and nsp4.
Wherein nsp4 has 3C sample serine protease, and function is cracking porcine reproductive and respiratory syndrome virus coding
Precursor polyprotein, to generate other non-structural proteins, these albumen collectively constitute virus replicase and transcriptase it is multiple
Zoarium come complete virus duplication and proliferation.During long-term evolution, nsp4 as protease cracking polyprotein in addition to producing
Non-structural protein needed for raw virus replication, additionally it is possible to target Partial joints molecule or kinases in innate immunity signal path and come
Escape host immune response.
Antibody is the globulin that can be specifically bound with corresponding antigens, has immune function, therefore be widely used in immunology
The fields such as analysis, radio-immuno-image and immune target therapy.Kohler and Milstein utilizes Cell culture invitro within 1975
Monoclonal antibody is successfully prepared with screening technique, becomes the milestone of antibody research field.As what is deepened continuously to antibody grinds
Studying carefully, has evolved to third generation genetic engineering antibody at present, assembling antibody molecule can be transformed in people again at the genetic level,
Genetic engineering antibody not only retains or increases the main physiological activity of natural antibody, also overcome monoclonal antibody preparation and
Certain defects of application aspect illustrate strong momentum in field of biotechnology.
Summary of the invention
In view of the above-mentioned problems, the present invention provides a kind of anti-porcine reproductive and respiratory syndrome virus nsp4 protein gene engineerings
Antibody, the genetic engineering antibody are protein peptide fragment shown in SEQ ID NO.6 and SEQ ID NO.8.
It is another object of the present invention to provide anti-porcine reproductive and respiratory syndrome virus nsp4 protein gene engineerings
The application of antibody.Using the genetic engineering antibody, the detection kit of porcine reproductive and respiratory syndrome virus can be prepared into.
In order to achieve the above object, the present invention takes following technical measures:
Applicant is directed to porcine reproductive and respiratory syndrome virus nsp4 albumen, is prepared for a kind of anti-pig breeding and integrates with breathing
Virus nsp4 protein gene engineered antibody is levied, the antibody includes protein shown in SEQ ID NO.6 and SEQ ID NO.8
Peptide fragment forms " Y " font antibody after the mixing of both albumen, can be with porcine reproductive and respiratory syndrome virus nsp4 albumen into special
Property combination, only one epitope of the antibody, compatibility is good, and specificity is high, the property with monoclonal antibody specificity having the same
Matter.
Protection scope of the present invention further includes encoding protein peptide fragment shown in SEQ ID NO.6 and SEQ ID NO.8
Polynucleotides, the polynucleotides may be selected to be SEQ ID NO.5 and SEQ ID NO.7.
Protection scope of the present invention further includes SEQ ID NO.2 and the SEQ ID for including in said gene engineered antibody
Variable region amino acid sequence shown in NO.4.
Protection scope of the present invention further includes encoding variable domain polypeptide shown in SEQ ID NO.2 and SEQ ID NO.4
Polynucleotides, the polynucleotides may be selected to be SEQ ID NO.1 and SEQ ID NO.3.
Anti- porcine reproductive and respiratory syndrome virus nsp4 protein gene engineered antibody is integrated in the breeding of preparation pig with breathing
The application in the detection kit of virus is levied, including the use of protein peptide fragment shown in SEQ ID NO.6 and SEQ ID NO.8,
It is prepared into the ELISA reagent of porcine reproductive and respiratory syndrome virus, Western-Blot detection reagent, IFA detection reagent etc..
Compared with prior art, the invention has the following advantages that
The present invention provides a kind of anti-porcine reproductive and respiratory syndrome virus nsp4 protein gene engineered antibody for the first time, this is anti-
Body affinity is strong, can be used in the experiment such as Western-Blot, IFA, is porcine reproductive and respiratory syndrome virus basic research, stream
Row disease learns investigation and clinical diagnosis and the foundation of identification method provides material and support.
The nucleotide sequence of antibody of the present invention can be obtained by artificial synthesized method, therefore can be more convenient simple and direct obtain
Anti- porcine reproductive and respiratory syndrome virus nsp4 genetic engineering antibody is obtained, hybridoma is effectively solved and saves at high cost, phenotype
The disadvantages of easy to be lost, while having evaded the animal welfare issues being related to when preparing ascites.
Detailed description of the invention
Fig. 1 is building plasmid schematic diagram；
Fig. 2 is that Western-Blot determines antibody-secreting Best Times qualification figure；
Fig. 3 is that Western-Blot identifies secretory antibody schematic diagram.
Fig. 4 is that IFA identifies secretory antibody schematic diagram.
Fig. 5 is genetic engineering antibody prepared by the present invention and the monoclonal antibody conduct of porcine reproductive and respiratory syndrome virus nsp4 albumen
Primary antibody carries out Western-Blot experimental result schematic diagram.
Fig. 6 is IFA detection schematic diagram of the genetic engineering antibody prepared by the present invention as primary antibody.
Specific embodiment
Illustrate that embodiments of the present invention, technological means used in the present invention are below by way of specific specific example
Method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, to be not intended to limit the present invention model
It encloses, the spirit and scope of the invention are limited only by the claims that follow.Reagent or material of the present invention, if not otherwise specified,
Derive from commercial channel.
Embodiment 1:
The preparation of the genetic engineering antibody of anti-porcine reproductive and respiratory syndrome virus nsp4 albumen:
The genetic engineering antibody of anti-porcine reproductive and respiratory syndrome virus nsp4 albumen can be according to SEQ ID NO.6 and SEQ
Amino acid sequence shown in ID NO.8 is combined to obtain by industry；Or obtained by conventional protein expression mode, this
Following manner is taken in invention:
1) using sequence shown in SEQ ID NO.5 as template, design specific primer IgG1-HV-F1, IgG1-HC-R amplification
Heavy chain overall length polynucleotides containing leader sequence correspond to the heavy chain region AH1 of genetic engineering antibody；With SEQ ID NO.7 institute
Showing that sequence is template, design specific primer κ-LV-F1, κ-LC-R1 expand the light chain overall length polynucleotides containing leader sequence,
The light chain region AL1 of corresponding genetic engineering antibody.
Among the segment EcoRI that is separately connected in pABL carrier and BamHI the two restriction enzyme sites that amplification is obtained,
Successfully 2 plasmids (H1 and L1) of building, plasmid construct such as Fig. 1 are identified in sequencing.
2) by plasmid H1 and L1 cotransfection HEK-293T cell, according to Thermo Fisher2000 turns
The specification of transfection reagent is operated, concrete operations are as follows: is taken heavy chain and each 0.5ug of light chain plasmids (by taking 24 orifice plates as an example), is added
Into 50 μ l Opti-MEM, 2.5 μ l of transfection reagent is added, mixed liquor is added in cell after room temperature effect 15min, 4-6h
After be changed toFreeStyleTM293Expression Medium。
HEK-293T cell is transfected by above-mentioned pairing transfection method, changes liquid after 6h, and respectively at 48h, 72h, 96h and 120h
Collect cell conditioned medium, be added 5 × SDS-PAGE Loading Buffer, 100 DEG C of boiling water baths 10min, 4 DEG C of 12000r/min from
Heart 10min draws supernatant and carries out SDS-PAGE electrophoresis, with glue and the step of falling glue according to BIO-RAD protein electrophoresis instrument operation instruction
Book is operated.Albumen is gone into pvdf membrane later.Sheep anti-mouse igg-HRP is added after closing pvdf membrane, is incubated at room temperature 1h, TBST
It washes film and Super Signal Western Pico chemiluminescent substrate is added afterwards three times, observed in chemiluminescence imaging system
And preservation of taking pictures is as a result, experimental result such as Fig. 2.
The result shows that antibody-secreting amount is best after plasmid H1 and L1 cotransfection cells 96h.
Using the above method, the genetic engineering antibody (AH1 and AL1) of HEK-293T cell expression is largely prepared, is made
It is 500 μ g/mL using mouse immune globulin G (IgG) ELISA kit detection antibody protein concentration, for following for primary antibody
Embodiment.
Embodiment 2:
The specificity identification of anti-porcine reproductive and respiratory syndrome virus nsp4 genetic engineering antibody:
Specificity identification (ELISA detection):
1) by the porcine reproductive and respiratory syndrome virus nsp4 albumen of purifying (by plasmid pCAGGS-nsp4 (with reference to text
It offers: Tao R, Fang L, Bai D, Ke W, Zhou Y, Wang D, Xiao S.Porcine Reproductive and
Respiratory Syndrome Virus Nonstructural Protein 4Cleaves Porcine DCP1a To
Attenuate Its Antiviral Activity.J Immunol.2018Aug 29.pii:ji1701773.doi:
10.4049/jimmunol.1701773.), transfection cell progress eukaryotic expression obtains) coated elisa plate, peridium concentration is 1 μ g/
ML, 4 DEG C are coated with overnight.It is washed three times with PBST, the genetic engineering antibody that embodiment 1 is obtained does not do transfection processing as primary antibody
HEK-293T cell conditioned medium as control, every 100 μ L of hole, which is placed in 37 DEG C of incubator, is incubated for 1h, and PBST washing three is taken second place
Diluted sheep anti-mouse igg-HRP the ELIAS secondary antibody of 1:4000 of 100 μ L is added in every hole afterwards, and use after 1h is incubated in 37 DEG C of incubators
PBST is washed three times, and substrate is added and is protected from light colour developing 10min, result observation and analysis are carried out after colour developing.HEK- as the result is shown
Antibody in 293T cell conditioned medium can interact with porcine reproductive and respiratory syndrome virus nsp4 albumen, and gene
The concentration of engineered antibody is higher, and OD630nm value still has 2.662+0.067 when 96h, therefore OD630nm value does not show the time
Variation, is all larger than 2.
Then, genetic engineering antibody prepared by doubling dilution embodiment 1 carries out ELISA experiment, experimental result such as Fig. 3 institute
Show.As the result is shown when the dilution of genetic engineering antibody is greater than 1:2560, OD630nm value is just less than 1.
Specificity identification (Western-Blot):
(1) identification for 4 albumen of porcine reproductive and respiratory syndrome virus nsp: plasmid pCAGGS-nsp4 (ginseng is taken respectively
Examine document: Tao R, Fang L, Bai D, Ke W, Zhou Y, Wang D, Xiao S.Porcine Reproductive and
Respiratory Syndrome Virus Nonstructural Protein 4Cleaves Porcine DCP1a To
Attenuate Its Antiviral Activity.J Immunol.2018Aug 29.pii:ji1701773.doi:
10.4049/jimmunol.1701773.) 6ug, 3ug, 1.5ug, 0.75ug and 0ug transfection HEK-293T cell (3.5cm training
Support ware) eukaryotic expression is carried out, and blank control is set up, sample is received after 36h.The genetic engineering antibody prepared using embodiment 1 is as one
Anti- progress Western-Blot experiment, as a result as shown in Figure 4.
The result shows that genetic engineering antibody provided by the invention can be the same with monoclonal antibody, specific band is generated, band is clear
Clear and single, not extra miscellaneous band illustrates that genetic engineering antibody provided by the invention can be integrated with pig breeding is crossed with breathing
It levies virus nsp4 protein-specific to combine, and has the characteristics of high specificity of monoclonal antibody.
(2) for the identification of porcine reproductive and respiratory syndrome virus: MARC-145 cell being connected to 3.5cm culture dish, carefully
Born of the same parents it is long to single layer when be inoculated with WUH3 the plant of porcine reproductive and respiratory syndrome virus of 1MOI, distinguish after inoculation 12h, for 24 hours, 36h and
48h receives sample, and the sample of acquisition is as antigen.Genetic engineering antibody, porcine reproductive and respiratory syndrome virus prepared by embodiment 1
Nsp4 albumen monoclonal antibody (being prepared according to the method that the myeloma cell of this field routine is merged with splenocyte) as primary antibody into
Row Western-Blot experiment, experimental result are as shown in Figure 5.
The result shows that genetic engineering antibody provided by the invention can occur instead with porcine reproductive and respiratory syndrome virus
It answers, and the band of genetic engineering antibody is equally clear and single with the band of monoclonal antibody control group as seen from the figure, without other miscellaneous
Band illustrates that genetic engineering antibody provided by the invention has high specificity identical with monoclonal antibody and specificity.
Specificity identification (IFA detection)
Be put into cell climbing sheet in 24 orifice plates, then access MARC-145 cell, cell it is long to single layer when be inoculated with the pig of 1MOI
WUH3 plants of Reproductive and respiratory syndrome virus, respectively after inoculation 12h, for 24 hours, 36h and 48h receive sample, while set up do not connect it is malicious thin
Born of the same parents' control.After cell sample prepares, cell culture medium is discarded, with PBS (1X, pH=7.4, similarly hereinafter) wash 3 times, every time
Every hole adds 1mL PBS, each 5min.When being added and discarding liquid, movement slowly will avoid blowing afloat in cell.Hole is added
The paraformaldehyde of 400 μ L 4%, fixed cell 15min.Methanol after -20 DEG C of pre-coolings are added, the hole 1mL/, permeabilization cell
10min, PBS are washed 3 times.400 hole μ L/ of confining liquid is added, closes 45min~60min.Embodiment 1 is added and prepares genetic engineering
1h is made in antibody, 250 holes μ L/, 37 DEG C of senses, and PBS is washed 3 times.The sheep anti-mouse igg of fluorescein isothiocynate (FITC) label is added
(doing 100 times of dilutions with PBS), 250 holes μ L/, 37 DEG C of senses abandon fluorescence secondary antibody after making 1h, and PBS is washed 3 times, paid attention to being protected from light operation.Most
Mounting, upper sem observation afterwards.Experimental result such as Fig. 6.
The result shows that genetic engineering antibody provided by the invention can identify porcine reproductive and respiratory syndrome virus nsp4 egg
It is white, and extension at any time, specificity fluorescent increase, and genetic engineering antibody can have bright with porcine reproductive and respiratory syndrome virus
Aobvious fluorescence reaction.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should be covered by the claims of the present invention.
Sequence table
<110>Hua Zhong Agriculture University
<120>porcine reproductive and respiratory syndrome virus nsp4 protein gene engineered antibody and application
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cctggacatg gccttgagtg gattggagag atttcacctg gaagtggtaa tactaattat 180
aatgacaagt ttaagggcaa ggccacattc actgcagaaa catcctccaa cacagcctac 240
atgcaactca gcagcctgac atctgacgac tctgccgtct atttctgtgc atccatttat 300
tactacggta gtagcatctt cgatgtctgg ggcgccggga cctcagtcac cgtctcctca 360
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tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180
tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240
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ctcacgttcg gtgctgggac caagctggag ctgaaacgg 339
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<212> PRT
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Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
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Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Leu Val His Ser
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Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
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atgggatgca gctgtgtaat gctcttcctc ctgtcagtaa ctgcaggtgt ccactcccag 60
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tgtaaggcta ctggctacac attcagtagt tactggatag agtggataaa gcagaggcct 180
ggacatggcc ttgagtggat tggagagatt tcacctggaa gtggtaatac taattataat 240
gacaagttta agggcaaggc cacattcact gcagaaacat cctccaacac agcctacatg 300
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aaaacgacac ccccatctgt ctatccactg gcccctggat ctgctgccca aactaactcc 480
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aactctggat ccctgtccag cggtgtgcac accttcccag ctgtcctgca gtctgacctc 600
tacactctga gcagctcagt gactgtcccc tccagcacct ggcccagcga gaccgtcacc 660
tgcaacgttg cccacccggc cagcagcacc aaggtggaca agaaaattgt gcccagggat 720
tgtggttgta agccttgcat atgtacagtc ccagaagtat catctgtctt catcttcccc 780
ccaaagccca aggatgtgct caccattact ctgactccta aggtcacgtg tgttgtggta 840
gacatcagca aggatgatcc cgaggtccag ttcagctggt ttgtagatga tgtggaggtg 900
cacacagctc agacgcaacc ccgggaggag cagttcaaca gcactttccg ctcagtcagt 960
gaacttccca tcatgcacca ggactggctc aatggcaagg agttcaaatg cagggtcaac 1020
agtgcagctt tccctgcccc catcgagaaa accatctcca aaaccaaagg cagaccgaag 1080
gctccgcagg tgtacaccat tccacctccc aaggagcaga tggccaagga taaagtcagt 1140
ctgacctgca tgataacaga cttcttccct gaagacatta ctgtggagtg gcagtggaat 1200
gggcagccag cggagaacta caagaacact cagcccatca tggacacaga tggctcttac 1260
ttcgtctaca gcaagctcaa tgtgcagaag agcaactggg aggcaggaaa tactttcacc 1320
tgctctgtgt tacatgaggg cctgcacaac caccatactg agaagagcct ctcccactct 1380
cctggtaaat ga 1392
<210> 6
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<212> PRT
<213>artificial sequence (Artificial Sequence)
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Met Gly Cys Ser Cys Val Met Leu Phe Leu Leu Ser Val Thr Ala Gly
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Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys
20 25 30
Pro Gly Ala Ser Val Arg Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe
35 40 45
Ser Ser Tyr Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His Gly Leu
50 55 60
Glu Trp Ile Gly Glu Ile Ser Pro Gly Ser Gly Asn Thr Asn Tyr Asn
65 70 75 80
Asp Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala Glu Thr Ser Ser Asn
85 90 95
Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val
100 105 110
Tyr Phe Cys Ala Ser Ile Tyr Tyr Tyr Gly Ser Ser Ile Phe Asp Val
115 120 125
Trp Gly Ala Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro
130 135 140
Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser
145 150 155 160
Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val
165 170 175
Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe
180 185 190
Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr
195 200 205
Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala
210 215 220
His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp
225 230 235 240
Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val
245 250 255
Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr
260 265 270
Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu
275 280 285
Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln
290 295 300
Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser
305 310 315 320
Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys
325 330 335
Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile
340 345 350
Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro
355 360 365
Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met
370 375 380
Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn
385 390 395 400
Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr
405 410 415
Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn
420 425 430
Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu
435 440 445
His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
450 455 460
<210> 7
<211> 717
<212> DNA
<213>artificial sequence (Artificial Sequence)
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atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat 60
gttgtgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120
tcttgcagat ctagtcagac ccttgtacac agtaatggaa acacctattt acattggtac 180
ctgcagaagc caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct 240
ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300
agagtggagg ctgaggatct gggagtttat ttctgctctc aaagtacaca tgttcctctc 360
acgttcggtg ctgggaccaa cctggagctg aaacgggctg atgctgcacc aactgtatcc 420
atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 480
aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 540
aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 600
agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 660
actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgttag 717
<210> 8
<211> 238
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 8
Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala
1 5 10 15
Ser Ser Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val
20 25 30
Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Leu
35 40 45
Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro
50 55 60
Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
65 70 75 80
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys
100 105 110
Ser Gln Ser Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Asn Leu
115 120 125
Glu Leu Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro
130 135 140
Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu
145 150 155 160
Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly
165 170 175
Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser
180 185 190
Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp
195 200 205
Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr
210 215 220
Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
225 230 235
<210> 9
<211> 34
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
aggaattcgc caccatggga tgcagctgtg taat 34
<210> 10
<211> 34
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
aggaattcgc caccatggga tgcagctgtg taat 34
<210> 11
<211> 34
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
aggaattcgc caccatgaag ttgcctgtta ggct 34
<210> 12
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
cgaggatccc taacactcat tcctgttgaa g 31
Claims (7)
1. a kind of anti-porcine reproductive and respiratory syndrome virus nsp4 protein gene engineered antibody, the antibody includes SEQ ID
Protein peptide fragment shown in NO.6 and SEQ ID NO.8.
2. encoding the polynucleotides of protein peptide fragment shown in SEQ ID NO.6 and SEQ ID NO.8.
3. polynucleotides according to claim 2 are shown in SEQ ID NO.5 and SEQ ID NO.7.
4. the variable region of genetic engineering antibody described in claim 1 is shown in SEQ ID NO.2 and SEQ ID NO.4.
5. encoding the polynucleotides of variable region shown in claim 4.
6. polynucleotides according to claim 5 are shown in SEQ ID NO.1 and SEQ ID NO.3.
7. sequence shown in claims 1 or 2 or 4 or 5 is in the detection kit for preparing porcine reproductive and respiratory syndrome virus
Application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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