CN110078821A - The sequence and its application of 68 type VP1 monoclonal antibody of enterovirus D group - Google Patents
The sequence and its application of 68 type VP1 monoclonal antibody of enterovirus D group Download PDFInfo
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- CN110078821A CN110078821A CN201910211598.1A CN201910211598A CN110078821A CN 110078821 A CN110078821 A CN 110078821A CN 201910211598 A CN201910211598 A CN 201910211598A CN 110078821 A CN110078821 A CN 110078821A
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses the sequences and its application of a kind of 68 type VP1 monoclonal antibody of enterovirus D group, utilize extraction EV-D68 virus genome RNA, the cDNA of VP1 gene is obtained by reverse transcription and PCR amplification, it is cloned into cloning vector, it is transferred to expression vector again and obtains recombinant expression plasmid, it converts host strain and is expressed with IPTG inducible protein, expression product obtains can be used as the VP1 fusion protein of antidiastole antigen by the extraction, denaturation, purifying of inclusion body.Using recombination VP1 albumen as immunogene, immune BALb/c mouse takes immune mouse spleen and merges it with myeloma cell SP2/0, obtain the hybridoma cell strain that can express VP1 antibody, this cell can stably excreting monoclonal antibody, the immunogenicity of antibody is detected by flow cytometry.The present invention obtains the H chain, L chain variable region nucleotide and amino acid sequence of VP1 monoclonal antibody simultaneously.
Description
Technical field: the invention belongs to field of biotechnology, are related to a kind of specific monoclonal for being directed to EVD68-VP1 of energy
Antibody, and in particular to the variable region sequences and flow cytometer detection method of the antibody.
Background technique: 68 type of enterovirus D group (Enterovirus D68, EV-D68) belongs to Picornaviridae,
It most separated and obtains in the throat swab sample that the U.S. adds the lower respiratory tract infection in the state Li Fuliya to be hospitalized people earlier than 1962.EV-
Diverse clinical manifestations caused by D68 virus infection, from the infection of the upper respiratory tract that symptoms are mild to serious adult and children's meninx
Inflammation shape.Studies have shown that EV-D68 can cause ACUte flaccid paralysis and cranial nerve function sexual dysfunction in children.In recent years,
EV-D68 virus has prevalence in China and world wide.From -2010 years 2008, EV-D68 virus was in Asia, Europe, beauty
The ground such as state have small range popular.In December, 2014,47, the whole America state and Colombia area, have 1152 people be diagnosed as by
Acute respiratory infection caused by EV-D68 virus.In China, 5 years old young girl of in August, 2014, BJ Children's Hospital is examined
Break as EV-D68 virus-positive, then development is serious pulmonary infection.In China, Xiang et al. passes through in August, 2006-
6942 of in April, 2010 are related with acute respiratory infection (Acute respiratory tract infection, ARTI)
The nose of adult patient (age >=15 year old) wipe, swallow and wipe pattern detection discovery, wherein 130 samples are enterovirus infection sample
Originally, for the enterovirus infected based on CA21 and EV-D68, EV-D68 infection proportion is 10%, however, this report lacks youngster
The data of virgin ARTI.LU et al. between -2012 years 2009 41 children's enterovirus infection case pattern detections find,
EV-D68 cases of infection are 7, and ratio is up to 17.1%.Another research of Xiao et al. is to the Chinese Chongqing 2012-2014
1876 respiratory tract infection in area (the nose swab sample of Respiratory tract infections, RTD children is examined
It surveys, 19 EV-D68 infection samples is found, wherein 13 samples are to detect in September, 2014 to October, and divide from sample
From 13 strain virus are obtained, phylogenetic analysis finds that this 13 strain virus and U.S.'s Major Epidemic strain in 2014 have very high homology
Property.Currently, there is no the effective antiviral object and preventative vaccine for EV-D68, so the blood of EV-D68 antibody anti-to crowd
Clear detection, and the effective EV-D68 curative drug of research and development, preventative vaccine are control EV-D68 virus infection and prevalence
Key.Enterovirus D68 type VP1 gene acts not only as the foundation that different serotypes are classified in enterovirus genus, and
It can also classify according to not belonged in VP1 gene pairs Picornaviridae.VP1 is the dominant capsid of principal immune of EV-D68
Albumen, VP1 full length gene 927bp contain virulent Main Antigenic.
Summary of the invention
It is an object of the invention to deficiencies according to prior art, prepare a kind of monoclonal antibody for EVD68-VP1,
The variable region nucleotide sequence and amino acid sequence of monoclonal antibody are provided.And antibody is detected with flow cytometry.
The technical solution of the present invention is as follows:
The variable region sequences of 68 type VP1 monoclonal antibody of enterovirus D group, the antibody includes heavy-chain variable domains
VH and light variable domains VL；Wherein, the nucleotides sequence of heavy-chain variable domains VH is classified as SEQ ID NO:1, amino acid sequence
It is classified as SEQ ID NO:2；Heavy-chain variable domains VH successively include hypervariable region FRH1, CDRH1, FRH2, CDRH2, FRH3,
CDRH3 and FRH4, the nucleotide sequence are followed successively by SEQ ID NO:3,5,7,9,11,13,15, the amino acid sequence according to
Secondary is SEQ ID:4,6,8,10,12,14,16；The nucleotides sequence of light variable domains VL is classified as SEQ ID NO:17, amino
Acid sequence is SEQ ID NO:18；Light variable domains VL successively include hypervariable region FRL1, CDRL1, FRL2, CDRL2,
FRL3, CDRL3 and FRL4, the nucleotide sequence are followed successively by the amino of SEQ ID NO:19,21,23,25,27,29,31
Acid sequence is followed successively by SEQ ID:20,22,24,26,28,30,32.
A kind of antibody or antigen-binding fragment or polypeptide, can be in conjunction with EVD68-VP1, and it includes 68 types of enterovirus D group
Any amino acid sequence described in the variable region sequences of VP1 monoclonal antibody.
A kind of antibody or antigen-binding fragment or polypeptide, can be in conjunction with EVD68-VP1, sequence and the heavy chain and light chain
The sequence similarity of sequence at least 91% and 64%.
A kind of genetic engineering antibody, the heavy chain and light-chain variable sequence that it is included are consistent with the variable region sequences
Or at least 64% similitude.
A kind of nucleic acid, comprising above-mentioned nucleotide sequence, including SEQ ID:1,3,5,7,9,11,13,15,17,19,21,
23、25、27、29、31。
A kind of antibody or antigen or nucleic acid are when studying EVD68 with the application in Flow cytometry immunogenicity.
Beneficial effects of the present invention: the method that the present invention devises a kind of recombined human EV-D68VP1 of high yield, and generate
A kind of anti-VP1 monoclonal antibody of high-affinity, obtains the variable region nucleotide sequence and amino acid sequence of the antibody, has
There are the potentiality of excellent basis studies and clinical application.
Detailed description of the invention:
Fig. 1 .EVD68-VP1 protein gene pcr amplification product electrophoretogram；Wherein M:DNA marker；1:PCR product；
Fig. 2 .EVD68-VP1 protein gene pcr amplification product and recombinant expression plasmid pGEX6p-1 double digestion glue figure
(BamH1/XhoI)；Wherein M:DNA marker；1:PCR product；2: carrier；
Fig. 3 .EVD68-VP1 recombinant protein, which is taken temperature, to be reached；Wherein M: protein marker；1: before induction；2: after induction；
The Western blot of Fig. 4 .EVD68-VP1 recombinant protein purification product is analyzed；Wherein M: protein marker；1,
2: fusion protein；
The specific binding situation of Fig. 5 Flow cytometry antibody.
Specific embodiment:
The present invention is described in further detail below in conjunction with embodiment.It should be appreciated, however, that these drawings and examples
It has been intended merely to illustration, and has been not for limiting the present invention.It is single to present invention preparation under concept thereof of the invention
The simple transformation of clonal antibody and preparation method thereof, belongs to the scope of protection of present invention.
Using enterovirus D group Fermon plants of Structural protein VP1 genes of 68 type as template, design primer amplifies target fragment
VP1 is connected on coli expression carrier pGEX6p-1.Recombinant expression carrier pGEX6p-1-VP1 is converted into large intestine bar
Bacterium expresses bacterial strain Rosetta (DE3), carries out inducing expression with IPTG to the engineering bacteria.After cellular lysate with SDS-PAGE electrophoresis,
Western-blot identifies fusion protein；Expression thallus be denaturalized after high pressure is broken with 8M urea, albuminate into
Row affinitive layer purification.By the way that BALB/c mouse is repeatedly immunized using full-length proteins as immunogene, reach to its serum titer
It is required to fusion, takes its splenocyte merge with myeloma cell and prepare anti-VP1 monoclonal antibody, carry out on a molecular scale
Heavy chain of antibody, light-chain variable region gene transfer and with flow cytometry identify antibody specificity.
Embodiment 1:
The clone of VP1 protein gene:
Expanded respectively from the plasmid containing VP1 gene previously constructed using polymerase chain reaction (PCR) and archaeal dna polymerase
Increase VP1 DNA fragmentation: 95 DEG C 2 minutes, 30 circulations (95 DEG C 20 seconds, 57 DEG C 20 seconds and 72 DEG C 20 seconds), finally in 72 DEG C of extensions
10 minutes.Primer 1CGGGATCCTTAAACCACTTACATGGAGC and primer 2 CCGCTCGAGTTAGTGATGATGATGATGAT
GATGATGATGATGGGTGGTTACTATGTTGTGAGG is used for the amplification of VP1.Pcr amplification product successively uses 1% agarose solidifying
Glue (Biowest) electrophoretic analysis (as shown in Figure 1), with BamH1/XhoI restriction enzyme (NEB) double digested, (verifying is as schemed after digestion
Shown in 2), it is finally cloned into pGEX6p-1 (Novagen).Final construct is named as pGEX6p-1-VP1, by its turn
Change into coli strain DH5 α competent cell (Life technologies), is coated on respectively containing 50 μ g/mL
On (LB) agar of ampicillin and at 37 DEG C overnight.Second day, contained in culture tube using single colonie inoculation 5mL LB
In the culture medium of 50 μ g/mL chlorampenicol resistants, is stayed overnight at 37 DEG C with 220rpm shaken cultivation, be then sequenced.
Embodiment 2: the expression and purification of albumen
By pEGX6p-1-VP1 expression plasmid convert Escherichia coli Rosseta (DE3) competent cell, by colony inoculation in
In LB culture medium containing 50 μ g/ml ampicillins and 34 μ g/ml chloramphenicol.At 37 DEG C, overnight incubation under 220rpm.When
When the OD600 of culture reaches 0.4-0.6, IPTG to final concentration of 0.6mM is added.At 16 DEG C after incubation 16-18 hours, with
4000rpm is centrifuged 20 minutes harvest bacterial precipitations, with the high pressure destroyer smudge cells in lysis buffer.(lysis buffer:
50mM Tris, 300mM NaCl, 10mM imidazoles, 1.4mM beta -mercaptoethanol, PH7.8).40 points are centrifuged at 4 DEG C with 18000rpm
Zhong Hou collects clear supernatant and is denaturalized inclusion body protein with 8M urea (PH7.8).It is then centrifuged for, collects supernatant
For purifying.According to His gene fusion system handbook, Ni-NTA agarose (GE Healthcare, USA) purifying protein is used
Matter.With 50mL washing buffer (washing buffer: 50mM Tris, 300mM NaCl, 20mM imidazoles, 1.4mM beta -mercaptoethanol,
8M urea, pH7.4) continuous washing more than ten times, 10mL elution buffer eluted (elution buffer: 50mM Tris,
300mM NaCl, 500mM imidazoles, 1.4mM beta -mercaptoethanol, 8M urea, pH7.4).Using SDS-PAGE (such as Fig. 3, shown in 4:
Respectively take temperature up to and protein purification glue figure) and Nano Drop measure lipidated protein and concentration.
The preparation of embodiment 3:EVD68-VP1 monoclonal antibody
1. animal immune
1.1 choose Balb/c female mice 4, can purchase 4 week old mouse, injections of antigens in raising 1-2 weeks；
1.2 animal immunes:
Just exempt from: the 1st day, 200ug antigen/mouse+same volume not formula Freund's complete adjuvant, the mode mutually injected with syringe
Form Water-In-Oil compound, subcutaneous multi-point injection；
Two exempt from: the 14th day, 200ug antigen/mouse+same volume not formula Freund's incomplete adjuvant was mutually injected with syringe
Mode forms Water-In-Oil compound, subcutaneous multi-point injection；
Three exempt from: the 28th day, 200ug antigen/mouse+same volume not formula Freund's incomplete adjuvant was mutually injected with syringe
Mode forms Water-In-Oil compound, subcutaneous multi-point injection；
Tail blood examination is surveyed: the 35-36 days, tail vein acquired about 20ul tail blood, after room temperature 30min, 5000rpm centrifugation 5min,
Haemocyte precipitating is abandoned, serum is drawn, detects potency for ELISA；
Four exempt from: the 42nd day, 200ug antigen/mouse+same volume not formula Freund's incomplete adjuvant was mutually injected with syringe
Mode forms Water-In-Oil compound, subcutaneous multi-point injection；
1.3 bioactivities: enzyme linked immunosorbent assay (ELISA)
Coating: 10ug antigen+10ml coating buffer (1.6gNaCO3+2.92gNaHCO3+100ml deionized water) mixes,
The hole 100ul/ spreads elisa plate, 37 ° of incubation 2h；
Closing: the plank being coated with, which is inhaled, abandons antigen coat liquid, is directly added into confining liquid (5% skimmed milk power+coating buffer)
The hole 300ul/, board-washing 3 times after 37 ° of incubation 2h；
Detection: serum and 1XPBS are prepared with 1:1000, are mixed, and inhale 150ul dilution and first hole is added, the hole 2-8 is equal
100ul 1XPBS is added, inhales 50ul liquid from first hole and the second hole is added, suction is drawn 50ul to third hole, inhaled after blowing 10 times
50ul is inhaled after blowing 10 times to the 4th hole, is successively inhaled and is drawn 50ul discarding after blowing to the 7th hole, octal is as negative control；37 ° incubate
It educates deionization after 1h to wash 3 times, adds the hole 100ul/ secondary antibody (sheep anti-mouse igg-HRP 1:1000 1XPBS), after 37 ° of incubation 30min
Deionization is washed 3 times, is added developing solution (according to kit specification use), the hole 100ul/, 5-10min, and color change is observed.
2. cell fusion
2.1 cell fusions: choosing 1 mouse and reinforce eventually, carries out cell fusion with PEG routine fusion method.6 pieces are spread altogether
Plate, progress cell conditioned medium detection in the 8th day；
2.2 fusion screenings: liquid processing was changed in fused 3rd, 6 day, the 7-14 days screening fused cells after fusion: is adopted
Fused cell supernatant is screened with indirect ELISA method, carries out monoclonal by screening final confirmation positive cell hole twice.
3. being subcloned and building strain
It is subcloned using positive cell strain of the limited dilution method to all screening confirmations, is grown to cell colony
Supernatant is drawn when field of microscope 1/3 to be screened；When reaching 100% wait be subcloned positive rate, strain is formally built, every plant of cell freezes
Deposit 2 or more.
Embodiment 4: the potency of antibody is detected by ELISA
1. coating: known antigens being diluted to 1~10 μ g/ml with coating buffer, every hole adds 0.1ml, and 4 DEG C overnight.It is secondary
Day, solution in hole is discarded, is washed 3 times, every time 3 minutes with washing buffer.(referred to as washing, similarly hereinafter).
2. sample-adding: adding certain diluted antibody 0.1ml in the above-mentioned reacting hole being coated with, set 37 DEG C and be incubated for 1 hour, wash
It washs.(while doing blank, feminine gender and positive hole control) is in reacting hole.
3. washing: be added 0.1ml diluted fresh enzyme mark secondary antibody, 37 DEG C incubations 30-60 minutes, wash, last
All over being washed with DDW.
4. adding substrate solution colour developing: 0.1ml tmb substrate solution being added in each reacting hole, 37 DEG C are reacted 10 minutes.
5. terminating reaction: 0.05ml 2M sulfuric acid being added in each reacting hole.
6. result judgement: can be in the result that in white background, directly detects by an unaided eye: color be deeper in reacting hole, positive journey
Degree is stronger, and to be colourless or extremely shallow, the depth of the be in color of foundation is indicated negative reaction with "+", "-" number.OD value can also be surveyed:
On ELISA detector, at 450nm, each hole OD value is surveyed after returning to zero with blank control wells, if more than defined negative control
2.1 times of OD value, it is as positive.
Embodiment 5: the immunogenicity of Flow cytometry antibody
1, the collection of cell
RD cell discards culture medium, is cleaned twice with PBS, and 0.25% pancreas enzyme -EDTA of 0.5ml is added and places cell culture
1min is digested in case and is terminated with DMEM (10%FBS) and is digested, and 2x10 is collected in cell count6A cell.At this point, two parts need to be taken thin
Born of the same parents, a part are used as blank control, and another part is measurement sample.Every part 1x106A cell.
2, cell cleans
Cell will be collected, 1000rpm centrifugation 5min is centrifuged by centrifuge, upper layer culture medium is abandoned in suction, and (note: this process is best
Most of liquid first is drawn with 1ml pipette tips, then with the careful sucked away liquid of 200ul pipette tips, is prevented cell in operating process
Siphon away), 2ml PBS is added, cell (gently blowing and beating cell cleaning) is resuspended, is centrifuged 5min again, inhales and abandon supernatant fluid, repeat
Cleaning is primary.
3, cell surface is closed
Closing is to cause false positive phenomenon in order to which antibody discord cell carries out non-specific binding.Confining liquid, using PBS
(2%FBS).0.5ml confining liquid is added, 4 degree of refrigerators are closed 30 minutes.Supernatant is abandoned in centrifugation, is added 100ulPBS (2%FBS),
This process still uses the PBS of confining liquid system effectively false positive can be prevented to generate, while 20ul monoclonal antibody (10 μ are added
G/mL it) is incubated with 30 minutes with irrelevant antibody (SFTSV-4H1,10 μ g/mL), irrelevant antibody is as negative control.It is washed with PBS
After washing twice, sheep anti-mouse antibodies (the SUNGENE BIOTECH of FITC conjugation is added；Dilution 1:100), and cell is existed
It is incubated for again in dark 30 minutes.
4, it cleans again
After incubation process, 1000rpm is centrifuged 5min, inhales and abandons supernatant, 2ml PBS is added, piping and druming cell is gently resuspended,
Cleaning 2 times as described in step 2 process.Ultimately join machine on 300ul PBS.
We, can be with it will be clear that monoclonal antibody detects the high positive rate of enterovirus infection from Fig. 5
Clearly distinguish infection cell and non-infected cells.
Embodiment 6: the gene sequencing and analysis of anti-EVD68-VP1 monoclonal antibody weight light variable domains
The RNA that hybridoma sample is extracted with Trizol method, obtains cDNA after reverse transcription, expands and obtain heavy chain and light
Amplified production is carried out library construction and carries out quality Q C by chain variable region, carries out high-flux sequence using Miseq 2X300PE,
By bioinformatic analysis, it is compared with database.Sequencing result is shown, successfully obtains hybridoma cell strain antibody variable
Domain gene.Analysis the result shows that: the nucleotides sequence of hybridoma cell strain heavy chain of antibody variable domains VH is classified as SEQ ID
NO:1, amino acid sequence are SEQ ID NO:2；Heavy-chain variable domains VH successively include hypervariable region FRH1, CDRH1, FRH2,
CDRH2, FRH3, CDRH3 and FRH4, the nucleotide sequence is followed successively by SEQ ID NO:3,5,7,9,11,13,15, described
Amino acid sequence is followed successively by SEQ ID NO:4,6,8,10,12,14,16；The nucleotides sequence of light variable domains V L is classified as
SEQ ID NO:17, amino acid sequence are SEQ ID NO:18；Light variable domains V L successively include hypervariable region FRL1,
CDRL1, FRL2, CDRL2, FRL3, CDRL3 and FRL4, the nucleotide sequence be followed successively by SEQ ID NO:19,21,23,
25,27,29,31 amino acid sequences are followed successively by SEQ ID:20,22,24,26,28,30,32.
<110>University Of Tianjin
<120>sequence and its application of 68 type VP1 monoclonal antibody of enterovirus D group
<130>
<160> 32
<170>
<210> 1
<211> 354
<212> DNA
<213>artificial sequence
<400> 1
gaagtgaagc ttgaggagtc tggaggaggc ttggtgcaac ctggaggatc catgaaactc 60
tcctgtgttg cctctggatt cactttcaat aactactgga tgaactgggt ccgccagtct 120
ccagagaagg ggcttgaatg gattgctgaa attagattga aatctgataa ttatgcaaca 180
cattatgcgg agtctgtgaa agggaggttc accatctcaa gagatgattc caaaagtagt 240
gtctacctgc aaatgaacaa cttaagagct gaagacactg gcatttatta ctgtaccagg 300
cactgggacg tccttgacta ctggggccaa ggcaccactc tcacagtctc ctca 354
<210> 2
<211> 118
<212> PRT
<213>artificial sequence
<400> 2
Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asn Asn Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile
35 40 45
Ala Glu Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His Tyr Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr
85 90 95
Tyr Cys Thr Arg His Trp Asp Val Leu Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Leu Thr Val Ser Ser
115
<210> 3
<211> 75
<212> DNA
<213>artificial sequence
<400> 3
gaagtgaagc ttgaggagtc tggaggaggc ttggtgcaac ctggaggatc catgaaactc 60
tcctgtgttg cctct 75
<210> 4
<211> 25
<212> PRT
<213>artificial sequence
<400> 4
Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Met Lys Leu Ser Cys Val Ala Ser
20 25
<210> 5
<211> 24
<212> DNA
<213>artificial sequence
<400> 5
ggattcactt tcaataacta ctgg 24
<210> 6
<211> 8
<212> PRT
<213>artificial sequence
<400> 6
Gly Phe Thr Phe Asn Asn Tyr Trp
1 5
<210> 7
<211> 51
<212> DNA
<213>artificial sequence
<400> 7
atgaactggg tccgccagtc tccagagaag gggcttgaat ggattgctga a 51
<210> 8
<211> 17
<212> PRT
<213>artificial sequence
<400> 8
Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Ala
1 5 10 15
Glu
<210> 9
<211> 30
<212> DNA
<213>artificial sequence
<400> 9
attagattga aatctgataa ttatgcaaca 30
<210> 10
<211> 10
<212> PRT
<213>artificial sequence
<400> 10
Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr
1 5 10
<210> 11
<211> 111
<212> DNA
<213>artificial sequence
<400> 11
cattatgcgg agtctgtgaa agggaggttc accatctcaa gagatgattc caaaagtagt 60
gtctacctgc aaatgaacaa cttaagagct gaagacactg gcatttatta c 111
<210> 12
<211> 37
<212> PRT
<213>artificial sequence
<400> 12
His Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
1 5 10 15
Ser Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp
20 25 30
Thr Gly Ile Tyr Tyr
35
<210> 13
<211> 33
<212> DNA
<213>artificial sequence
<400> 13
tgtaccaggc actgggacgt ccttgactac tgg 33
<210> 14
<211> 11
<212> PRT
<213>artificial sequence
<400> 14
Cys Thr Arg His Trp Asp Val Leu Asp Tyr Trp
1 5 10
<210> 15
<211> 30
<212> DNA
<213>artificial sequence
<400> 15
ggccaaggca ccactctcac agtctcctca 30
<210> 16
<211> 10
<212> PRT
<213>artificial sequence
<400> 16
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
1 5 10
<210> 17
<211> 339
<212> DNA
<213>artificial sequence
<400> 17
gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gagaattact 60
atgagttgta agtccagtca gagcctttta tatcgtaaca atcaaaagaa ctacttggtc 120
tggtaccaac agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 180
gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240
atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatacctat 300
ccgctcacgt tcggtgctgg gaccaagctg gagctgaaa 339
<210> 18
<211> 113
<212> PRT
<213>artificial sequence
<400> 18
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Arg Ile Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Arg
20 25 30
Asn Asn Gln Lys Asn Tyr Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Thr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Glu
100 105 110
Lys
<210> 19
<211> 78
<212> DNA
<213>artificial sequence
<400> 19
gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gagaattact 60
atgagttgta agtccagt 78
<210> 20
<211> 26
<212> PRT
<213>artificial sequence
<400> 20
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Arg Ile Thr Met Ser Cys Lys Ser Ser
20 25
<210> 21
<211> 36
<212> DNA
<213>artificial sequence
<400> 21
cagagccttt tatatcgtaa caatcaaaag aactac 36
<210> 22
<211> 12
<212> PRT
<213>artificial sequence
<400> 22
Gln Ser Leu Leu Tyr Arg Asn Asn Gln Lys Asn Tyr
1 5 10
<210> 23
<211> 51
<212> DNA
<213>artificial sequence
<400> 23
ttggtctggt accaacagaa accagggcag tctcctaaac tgctgattta c 51
<210> 24
<211> 17
<212> PRT
<213>artificial sequence
<400> 24
Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
1 5 10 15
Tyr
<210> 25
<211> 9
<212> DNA
<213>artificial sequence
<400> 25
tgggcatcc 9
<210> 26
<211> 3
<212> PRT
<213>artificial sequence
<400> 26
Trp Ala Ser
<210> 27
<211> 105
<212> DNA
<213>artificial sequence
<400> 27
actagggaat ctggggtccc tgatcgcttc acaggcagtg gatctgggac agatttcact 60
ctcaccatca gcagtgtgaa ggctgaagac ctggcagttt attac 105
<210> 28
<211> 35
<212> PRT
<213>artificial sequence
<400> 28
Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly
1 5 10 15
Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala
20 25 30
Val Tyr Tyr
35
<210> 29
<211> 33
<212> DNA
<213>artificial sequence
<400> 29
tgtcagcaat attataccta tccgctcacg ttc 33
<210> 30
<211> 11
<212> PRT
<213>artificial sequence
<400> 30
Cys Gln Gln Tyr Tyr Thr Tyr Pro Leu Thr Phe
1 5 10
<210> 31
<211> 27
<212> DNA
<213>artificial sequence
<400> 31
ggtgctggga ccaagctgga gctgaaa 27
<210> 32
<211> 9
<212> PRT
<213>artificial sequence
<400> 32
Gly Ala Gly Thr Lys Leu Glu Glu Lys
1 5
Claims (6)
1. the variable region sequences of 68 type VP1 monoclonal antibody of enterovirus D group, it is characterised in that the antibody includes that heavy chain can
Structure changes domain VH and light variable domains VL；Wherein, the nucleotides sequence of heavy-chain variable domains VH is classified as SEQ ID NO:1,
Amino acid sequence is SEQ ID NO:2；Heavy-chain variable domains VH successively include hypervariable region FRH1, CDRH1, FRH2, CDRH2,
FRH3, CDRH3 and FRH4, the nucleotide sequence are followed successively by SEQ ID NO:3,5,7,9,11,13,15, the amino acid
Sequence is followed successively by SEQ ID:4,6,8,10,12,14,16；The nucleotides sequence of light variable domains VL is classified as SEQ ID NO:
17, amino acid sequence is SEQ ID NO:18；Light variable domains VL successively include hypervariable region FRL1, CDRL1, FRL2,
CDRL2, FRL3, CDRL3 and FRL4, the nucleotide sequence are followed successively by the institute of SEQ ID NO:19,21,23,25,27,29,31
It states amino acid sequence and is followed successively by SEQ ID:20,22,24,26,28,30,32.
2. a kind of antibody or antigen-binding fragment or polypeptide, can be in conjunction with EVD68-VP1, it is characterised in that it includes claims
Any amino acid sequence described in 1.
3. a kind of antibody or antigen-binding fragment or polypeptide, can be in conjunction with EVD68-VP1, it is characterised in that its sequence and right 2
The sequence similarity of the heavy chain and sequence of light chain at least 91% and 64%.
4. a kind of genetic engineering antibody, the heavy chain and light-chain variable sequence and variable region described in claim 1 that it is included
Sequence is consistent or similitude at least 64%.
5. a kind of nucleic acid, comprising the nucleotide sequence described in claim 1, including SEQ ID:1,3,5,7,9,11,13,15,
17、19、21、23、25、27、29、31。
6. any antibody or antigen or nucleic acid are when studying EVD68 with Flow cytometry immunogene in claim 1-5
Application in property.
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