CN108017715A - A kind of Zaire types Ebola virus detection antibody and preparation method and application - Google Patents
A kind of Zaire types Ebola virus detection antibody and preparation method and application Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
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Abstract
The present invention provides Zaire types Ebola virus detection antibody and preparation method and application.The Zaire types Ebola virus detection antibody, it is characterised in that be human monoclonal antibody；The amino acid sequence of its light chain hypervariable region CDR1, CDR2 and CDR3 are respectively SEQ ID No.3, DDS, and the amino acid sequence of SEQ ID No.5, heavy chain hypervariable region CDR1, CDR2 and CDR3 are respectively SEQ ID No.6, SEQ ID No.7, and SEQ ID No.8.Antibody of the present invention can be good at detecting Zaire type Ebola virus envelope protein GP, have important economy and social effect.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of Zaire types Ebola virus detection antibody, its preparation side
Method and application.
Background technology
Ebola virus (Ebolavirus) finds for 1976 in the Sudan and Democratic Republic of the Congo respectively.At present,
Send out 5 kinds of hypotypes：Zaire's type (Zaire), the Sudan's type (Sudan), Ben Dibujiao types (Bundibugyo), Cote d'lvoire's type
(Cote d ' Ivoire), and Christopher Eccleston type (Reston), wherein preceding 4 hypotypes to the mankind have it is pathogenic.The last angstrom
The rich viral infection event of drawing, which is broken out, to be started from 2 months 2014, and the death rate may be up to 90%, and the cause of death is mainly apoplexy, cardiac muscle stalk
Plug, hypovolemic shock or organ failure.
Ebola virus not yet determines that evidence suggests the virus is animal sources venereal disease in the origin of nature and host
Poison, bat are probably its potential natural reservoir (of bird flu viruses).Ebola virus has a very strong infectiousness, the blood of infected patient, sperm,
Sweat, saliva and secretion are respectively provided with infectivity, can infect Close contacts by approach such as skin, conjunctiva or respiratory tracts.Angstrom
Rich to draw virus infected cell to be mediated by envelope protein GP, GP is processed by furin or furin-like protease, formed GP1 with
GP2 Liang Ge subunits, GP1 are connected by a pair of of disulfide bond with GP2 and are formed heterodimer, form the trimeric form of maturation.GP1
Subunit is responsible for virus being anchored to target cell, and GP2 subunits are responsible for merging for peplos and cell membrane, and then by viral genome
Host cell is discharged into, this process needs endogenous cysteine protease to participate in completing.
GP plays a significant role at the pathogenic aspect of Ebola virus, optionally reduces cell surface and is glued with cell
The expression of the relevant macromolecular of immune function is echoed, so as to cause the death that comes off of cell.In addition, sGP is bound to by CD16b
Neutrophil leucocyte, interacts with host immune system, changes physical and functional mutual between Fc γ RIIIB and CR3
Effect, and then suppress removing of the Neutrophil to virus, so as to cause immunologic escape.
At present, in the market only has Taibei Abnova mouse sources related to two companies of Yi Qiao Divine Land, Beijing producing and selling and rabbit
Source is single (more) anti-.Common antibody screening method has phage displaying antibody library screening, single B cell sequencing technologies, B cell
Immortalization and the screening of humanization mouse hybridoma cell etc., we are obtained using phage displaying antibody library screening methods
A kind of Zaire types Ebola virus detects antibody.The present invention utilizes the phage displaying antibody text constructed by the PBMC of normal person
Storehouse, obtains the monoclonal antibody gene sequence with affinity by the way of affine screening, simplifies the screening process of antibody,
Also the research and development for antibody drug provide a shortcut.
The content of the invention
Deficiency and actual demand for existing market supply, the object of the present invention is to provide Zaire type ebola diseases
Poison detection antibody, its preparation method and application.Zaire types Ebola virus provided by the invention detects antibody, is ebola disease
The detection of malicious Infect And Diagnose and Ebola virus antigens GP provides new selection, has important economy and social effect.
In order to achieve the above object, the present invention provides a kind of Zaire types Ebola virus to detect antibody, its feature exists
In being human monoclonal antibody；The amino acid sequence of its light chain hypervariable region CDR1, CDR2 and CDR3 are respectively SEQ ID No.3
Or the sequence one or more amino acids formed has the function of equal amino acid sequence, DDS through replacing, lacking or add
(E-1C1 CDR-L2, Asp Asp Ser) or the sequence one or more amino acids formed have through replacing, lacking or add
The amino acid sequence of equal function, and SEQ ID No.5 or the sequence are through replacing, lacking or adding one or more amino acid shapes
Into there is equal amino acid sequence, the amino acid sequence of heavy chain hypervariable region CDR1, CDR2 and CDR3 are respectively SEQ
ID No.6 or the sequence one or more amino acids formed have the function of equal amino acid sequence through replacing, lacking or add
One or more is amino acids formed has the function of equal ammonia through replacing, lacking or add for row, SEQ ID No.7 or the sequence
Base acid sequence, and SEQ ID No.8 or the sequence through replace, lack or add it is one or more it is amino acids formed have it is equal
The amino acid sequence of function.
Preferably, the amino acid sequence of the light chain variable region of Zaire types Ebola virus detection antibody is SEQ
ID No.1 or the sequence one or more amino acids formed have the function of equal amino acid sequence through replacing, lacking or add
Row, the amino acid sequence of heavy chain variable region are SEQ ID No.2 or the sequence through replacing, lacking or add one or more amino
What acid was formed has the function of equal amino acid sequence.
Preferably, the amino of light chain framework region FR1, FR2 and FR3 of Zaire types Ebola virus detection antibody
Acid sequence is respectively that SEQ ID No.9 or the sequence are one or more amino acids formed with same through replacing, lacking or add
Amino acid sequence, SEQ ID No.10 or the sequence etc. function are formed through replacing, lacking or adding one or more amino acid
There is equal amino acid sequence and SEQ ID No.11 or the sequence through replacing, lacking or adding one or more
It is amino acids formed that there is equal amino acid sequence；The amino acid sequence of heavy chain framework regions FR1, FR2 and FR3 is respectively
SEQ ID No.12 or the sequence one or more amino acids formed have the function of equal amino through replacing, lacking or add
Acid sequence, SEQ ID No.13 or the sequence one or more amino acids formed have equal work(through replacing, lacking or add
The amino acid sequence and SEQ ID No.14 of energy or the sequence are one or more amino acids formed through replacing, lacking or add
There is equal amino acid sequence.
Preferably, the coding light chain variable region of described Zaire types Ebola virus detection antibody and heavy chain variable region
Nucleotide sequence is respectively the tool that SEQ ID No.15 or the sequence are formed through replacing, lacking or adding one or more nucleotide
Have equal function nucleotide sequence and SEQ ID No.16 or the sequence through replacing, lacking or adding one or more nucleosides
What acid was formed has the function of equal nucleotide sequence.
Present invention also offers a kind of expression vector, it is characterised in that the expression vector includes SEQ ID No.15
Or the sequence through replace, lack or add one or more nucleotide are formed have the function of equal nucleotide sequence and
One or more nucleotide are formed has the function of equal nucleosides through replacing, lacking or add for SEQ ID No.16 or the sequence
It is at least one in acid sequence.
Present invention also offers a kind of host cell, it is characterised in that the host cell includes above-mentioned expression vector.
Present invention also offers the preparation method of above-mentioned Zaire types Ebola virus detection antibody, it is characterised in that bag
Include following steps：By SEQ ID No.15-SEQ ID No.20 and above-mentioned sequence through replacing, lacking or adding one or more cores
What thuja acid was formed have the function of in equal nucleotide sequence it is at least one be connected with expression vector, amplification, it is thin to transfect 293T
Born of the same parents, collect 293T cell conditioned mediums, hang proA columns, elution, obtains Zaire types Ebola virus detection antibody.
Preferably, the expression vector is mammalian expression vector.
It is highly preferred that the mammalian expression vector is pFuse-Fc and pCAGGS mammalian expression vectors.
Present invention also offers above-mentioned Zaire types Ebola virus detection antibody to prepare to Ebola virus GP antigens
With affinity, to the application in the detection of Ebola virus.
In the first aspect of the present invention, the present invention provides a kind of Zaire types Ebola virus to detect antibody, the antibody
For human monoclonal antibody.According to an embodiment of the invention, the amino acid sequence of light chain hypervariable region CDR1, CDR2 and CDR3 of antibody
Row are respectively as shown in SEQ ID No.3, DDS (E-1C1CDR-L2, Asp Asp Ser), SEQ ID No.5；Its heavy chain hypervariable region
The amino acid sequence of CDR1, CDR2 and CDR3 are respectively as shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8.
The amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID No.1, or the sequence is through replacing, lacking
Or addition is one or more amino acids formed has the function of equal amino acid sequence；The ammonia of the heavy chain variable region of the antibody
Base acid sequence is as shown in SEQ ID No.2, or the sequence one or more amino acids formed has through replacing, lacking or add
The amino acid sequence of equal function.
Sequence can include the equivalent amino acid of some biological functions or " conservative replacement ", and other sequences can include
The aniso- amino acid of function or " non-conservation replacement ", it is transformed through genetic engineering to improve the spy of CDR or the antibody containing CDR
Property.
Foregoing amino acid sequence is as follows：
SEQ ID No.1(E-1C1 L)：
YVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVE
AGDEADYYCQVWDSSSDHWVFGGGTKLTVLG。
SEQ ID No.2(E-1C1 H)：
MAQVQLVQSGAEVKKPGASVKASCKASGYTFTSYYISWVRQAPGQGLEWMGIINPGDAGTTYAQKFQGRVTMTRDTS
TSTVYMELSSLRSEDTGVYYCARGRSGWYGMDVWGQGTTVTVSS。
SEQ ID No.3(E-1C1 CDR-L1)：NIGSKS.
SEQ ID No.5(E-1C1 CDR-L3)：QVWDSSSDHWV.
SEQ ID No.6(E-1C1 CDR-H1)：GYTFTSYY.
SEQ ID No.7(E-1C1 CDR-H2)：INPGDAGT.
SEQ ID No.8(E-1C1 CDR-H3)：ARGRSGWYGMDV.
The amino acid sequence of light chain framework region FR1, FR2 and FR3 of the antibody such as SEQ ID No.9, SEQ ID
Shown in No.10, SEQ ID No.11；The amino acid sequence of its heavy chain framework regions FR1, FR2 and FR3 such as SEQ ID No.12, SEQ
Shown in ID No.13, SEQ ID No.14.
According to an embodiment of the invention, foregoing amino acid sequence is as follows：
SEQ ID No.9(E-1C1 FR-L1)：YVLTQPPSVSVAPGQTARITCGGN.
SEQ ID No.10(E-1C1 FR-L2)：VHWYQQKPGQAPVLVVY.
SEQ ID No.11(E-1C1FR-L3)：DRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYC.
SEQ ID No.12(E-1C1FR-H1)：MAQVQLVQSGAEVKKPGASVKASCKAS.
SEQ ID No.13(E-1C1 FR-H2)：ISWVRQAPGQGLEWMGI.
SEQ ID No.14(E-1C1FR-H3)：TYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTGVYYC.
The antibody is human monoclonal antibody.
The antibody is single-chain antibody, double-chain antibody, chimeric antibody or derivatives thereof.
The antibody or its antigen-binding fragment of the present invention, can be good at combining Ebola virus surface sole antigenic-capsule
Memebrane protein GP.
In the second aspect of the present invention, the present invention provides two kinds of DNA fragmentations.According to an embodiment of the invention, these DNA
The antibody of fragment coding as described in relation to the first aspect.
According to an embodiment of the invention, DNA fragmentation described above, can comprising heavy chain variable region coded sequence and light chain
Become area's coded sequence；The nucleotide sequence of coding light chain variable region and heavy chain variable region is respectively such as SEQ ID No.15, SEQ ID
Shown in No.16.
According to an embodiment of the invention, foregoing nucleotide sequence is as follows：
SEQ ID No.15(E-1C1 L chain)：
TATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTGTGGGGGAAACAACAT
TGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGATAGCGACC
GGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAA
GCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAGTGATCATTGGGTGTTCGGCGGAGGGACCAA
GCTGACCGTCCTAGGT。
SEQ ID No.16(E-1C1 H chain)：
ATGGCACAGGTCCAGCTTGTGCAGTCTGGGGCTGAAGTCAAGAAGCCTGGGGCCTCAGTGAAGGCTTCCTGCAAGGC
ATCTGGATATACCTTCACCAGCTACTATATCTCCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAA
TAATCAATCCTGGTGATGCTGGCACAACCTACGCACAGAAGTTTCAGGGCAGAGTCACCATGACCAGGGACACGTCC
ACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGGCGTGTATTACTGTGCGCGGGGTAGGAG
TGGCTGGTACGGTATGGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA。
By using the polynucleotides of the present invention, Zaire types Ebola virus detection antibody or anti-can be effectively synthesized
Former binding fragment.
In the third aspect of the present invention, present invention also offers two kinds of expression vectors, the expression vector includes at least one
The DNA fragmentation as described in second aspect of a copy.
In the fourth aspect of the present invention, the present invention provides a kind of host cell, the host cell includes such as third party
Expression vector described in face.
By using the method according to the invention, can effectively be synthesized according to this hair using foregoing polynucleotides
The Zaire types Ebola virus detection antibody or antigen-binding fragment of bright embodiment.It is anti-on the detection of Zaire types Ebola virus
Body or antigen-binding fragment, the equally applicable polynucleotides of feature and advantage described above, details are not described herein.
In the fifth aspect of the present invention, the present invention provides the detection antibodies Antibodies of Ebola virus as described in relation to the first aspect
Preparation method, includes the following steps：
(1) using affine screening technique screen from phage displaying antibody library can specific bond Zaire types angstrom win
The monoclonal of viral envelope proteins GP is drawn, the hypervariable region consecutive nucleotides sequence that sequencing obtains its heavy chain and light chain is carried out to it
Column information；
(2) antibody expression vector is built by PCR amplification, digestion, connection method.
According to an embodiment of the invention, the preparation method of the antibody can use technology well known to those skilled in the art
Carry out, do not do herein specifically limited.
According to an embodiment of the invention, the carrier described in step (2) is mammalian expression vector, is preferably pFuse-Fc
With pCAGGS mammalian expression vectors.
In view of the degeneracy of codon, such as can be in its code area, under conditions of amino acid sequence is not changed, to compiling
The gene order of the above-mentioned antibody of code is transformed, and obtains the gene of antibody of the coding with identical function.Those skilled in the art
Can be according to the codon-bias of expression antibody host, artificial synthesized modifying gene, to improve the expression efficiency of antibody.
Further, the present invention is recombinated the light chain variable region and heavy chain variable region of antibody noted earlier, can be obtained
The single-chain antibody (ScFv) of molecular weight smaller, the antibody can equally identify Zaire type Ebola virus antigens GP.Single-chain antibody
Penetration power is strong, easily enters local organization and plays a role.The gene of above-mentioned encoding antibody, ScFv gene clonings can be carried to expression
In body, and then conversion or transfection host cell, obtain the antibody and single-chain antibody.
In addition, the light chain variable region encoding gene and heavy chain variable region gene of antibody noted earlier can be cloned into complete anti-table
Up in carrier, and import in host cell, obtain the full anti-immunoglobulin of detection of expression Ebola virus.
Compared with prior art, the present invention has the advantages that：
Antibody sources of the present invention can preferably embody virus and the interaction of human immune system in human body,
Antibody (ScFv-Fc forms) and the affinity KD of GP albumen are 3.93nM.Antibody of the present invention can be good at detecting Zaire
Type Ebola virus envelope protein GP, has important economy and social effect.
Brief description of the drawings
Fig. 1 is that two kinds of antibody formations of ScFv-Fc and IgG of E-1C1 prepared by the present invention and Ebola GP are protein bound
ELISA results；
The ScFv-Fc antibody that Fig. 2 is antibody E-1C1 of the present invention is protein bound in the GP of 293T cell surfaces with expression
IFC results.
Fig. 3 is that the IgG antibody Western Blot methods of antibody E-1C1 of the present invention detect the result of Ebola's GP albumen.
Embodiment
With reference to specific embodiment, the present invention is further explained.It is to be understood that these embodiments are merely to illustrate the present invention
Rather than limit the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, people in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Scope.
The phage displaying antibody library used in following embodiments is provided by Richard Lerner, and GAO ' s Lib, are pressed
According to Zhang H, Wilson IA, Lerner RA：Selection of antibodies that regulate phenotype
from intracellular combinatorial antibody libraries.Proc Natl Acad Sci
USA2012,109 (39)：Construction method structure described in 15728-15733..
Zaire type Ebola virus envelope protein GP (the NCBI GenBank used in following embodiments：
AHX24658.2, build to insect expression plasmid to express obtain) according to being documented in Wang H, Shi Y, Song J, Qi J, Lu
G, Yan J, Gao GF：Ebola Viral Glycoprotein Bound to Its Endosomal
ReceptorNiemann-Pick C1.Cell 2016,164 (1-2)：258-268. method prepare.
sGP(NCBI GenBank：U23187.1, build to insect expression plasmid to express obtain) according to being documented in Mohan
GS, Li W, Ye L, Compans RW, Yang C：Antigenic Subversion：A Novel Mechanism of Host
Immune Evasion by Ebola Virus.PLoS Pathogens 2012,8 (12)：Preparation method in e1003065
Prepare.
ssGP(NCBI GenBank：NP_066248, build to insect expression plasmid to express obtain) according to being documented in
Radoshitzky SR, Warfield KL, Chi X, Dong L, Kota K, Bradfute SB, Gearhart JD,
Retterer C, Kranzusch PJ, Misasi JN et al：Ebolavirus delta-peptide
immunoadhesins inhibit marburgvirus and ebolavirus cell entry.Journal of
Virology 2011,85 (17)：Construction method structure in 8502-8513.
The preparation of 1 antibody of embodiment
Zaire types Ebola virus GP protein detects the preparation method of antibody, includes the following steps：
1.1 using affine screening techniques screen from phage displaying antibody library can specific bond Zaire types angstrom win
Draw the monoclonal of viral envelope proteins GP：
1.1.1 using NHS-PEG4-Biotin (Thermo Fisher, 21955) to containing primaquine in antigen (GP) molecule
The amino acid of base carries out biotinylation, obtains the antigen Bio-GP after biotinylation：
1.1.1.1 NHS- is made using the mole of NHS-PEG4-Biotin according to needed for being calculated antigen GP moles
50 times of GP moles of mole of PEG4-Biotin, computational methods are as follows：
mL protein*(mg protein/mL protein)*(mmol protein/mg protein)*(50mmol
Biotin/mmol protein)=mmol Biotin；
The NHS-PEG4-Biotin and the molar ratio of antigen molecule that 50=recommends；
1.1.1.2. the volume that the concentration according to needed for being calculated 1.1.1.1 is 20mMNHS-PEG4-Biotin, computational methods are such as
Under：
* 170 μ L/2.0mg of mmol Biotin* (589mg/mmol Biotin))=μ L Biotin Solution
589=NHS-PEG4-Biotin molecular weight；
170=dissolving 2.0mg NHS-PEG4-Biotin are the solvent volume needed for molar concentration 20mM；
1.1.1.3 200 μ g GP are dissolved into 700 μ L PBS；
1.1.1.4 NHS-PEG4-Biotin is dissolved as 20mM using the solvent in 170 μ L kits；
1.1.1.5 the NHS-PEG4-Biotin of 4 μ L is added in GP, mixes, is incubated 2h on ice；
1.1.1.6 the protein sample being incubated is added into desalting column (Thermo Fisher, 89882) medium center, makes egg
It is white to infiltrate to desalting column；
1.1.1.7 1000g, 2min are centrifuged, and the stream river liquid obtained is the Bio-GP after biotinylation；
1.1.2 screened using paramagnetic particle method：
1.1.2.1 first day：
1.5ml centrifuge tubes are closed using 5%BSA (AMRESCO, 0332)-PBST, are incubated at room temperature 1h；
Magnetic bead (the every samples of 200 μ L, Thermo Fisher, 11206D) is washed using PBS 4 times；
5%BSA-PBST closes library (200 μ L), is incubated at room temperature 30min；
Above-mentioned library is added in the magnetic bead that PBS washes (1/4 volume magnetic bead), is incubated at room temperature 30min；
Step supernatant adds the antigen Bio-GP after 5 μ g biotinylations upwards, is incubated at room temperature 2h；
Slightly centrifuge, will be added to obtained by upper step in the remaining 3/4 volume magnetic bead of back, be incubated at room temperature 20min；
Magnetic bead is recycled, PBST is washed 4 times, and PBS is washed 2 times；
Magnetic bead is recycled, adds 300 μ L pH 2.2Glycine-HCl, reacts at room temperature 10min；
To new centrifuge tube, the 115 μ L of Tris for adding pH 8.0 immediately are neutralized upper step supernatant；
Gained supernatant is added into the XL1-Blue (Agilent, 200228) that 9mL OD600 are 0.5-0.7, is mixed, 37 DEG C,
30min；
Titration：
Output OutPut：1 μ L of step are taken into 200 μ L LB, then carrying out 100 times of concentration gradients using LB dilutes, and applies ammonia
Benzyl tablet；
Put into Input：Take in 1 μ L to 1mL LB of library, carry out 1000 times of gradient dilutions using LB, add to 200 μ L XL1-
In Blue, ammonia benzyl tablet is applied；
By obtained by upper step, 3000RPM, 4 DEG C, 15min, abandons supernatant, adds 200 μ L LB, mixes, and applies the big plates of Amp；
Titer plate is placed in 37 DEG C overnight, and big plate is placed in 30 DEG C overnight；
1.1.2.2 second day
Big plate is scraped using spreading rod, 25mL LB are scraped, and 5mL LB are washed；
Survey OD values；
The scraper plate liquid of suitable volumes is diluted with LB (LB contains 10%Glucose, 100 μ g/mL Amp, 5 μ g/mL Tet) extremely
100mL, OD600 are 0.1 or so, 37 DEG C of 2h, to OD600 0.5 or so；
Add 1.5mL Helper phage (Thermo Fisher, 18311019), 37 DEG C, 0.5h, one is rocked per 10min
It is secondary；
37 DEG C, 200RPM, 1.5h, OD600 is surveyed, is diluted to OD6000.8；
4 DEG C, 3000RPM, 15min, 50mL LB resuspensions, then centrifuge once；
50mLLB is resuspended, and takes 40mL (LB contains 100 μ g/mL Amp, 5 μ g/mL Tet, 35 μ g/mL Kan) in new bottle；
30 DEG C, overnight；
1.1.2.3 the 3rd day
Take and shook bacterium yesterday, 4 DEG C, 5000RPM, 15min；
Supernatant adds PEG-NaCl (1: 4), on ice 1h into new centrifugal barrel；
4 DEG C, 14000g, 30min, abandon supernatant；
4 DEG C, 14000g, 5min, abandon supernatant；
2mL 1%BSA-PBS are resuspended, 14000g, 5min, 2 times, take 200 μ L to be screened for next round, this is complete for a wheel
Screening；
Repeat last round of screening step；
1.1.2.4 the 4th day
Repeat new round screening；
1.1.2.5 the 5th day
Repeat new round screening；
1.1.2.6 the 6th day
Repeat new round screening, complete within six days four-wheel screening.
Output devoting rate is calculated, it is as a result as shown in the table：
1. ratio for input and output example of table
The variable region sequences nucleotide sequence letter of sequencing its heavy chain of acquisition and light chain must be carried out to clone's phasmid of gained
The nucleotide sequence of breath, coding light chain variable region and heavy chain variable region is respectively SEQ ID No.15 and SEQ ID No.16, E-
The sequencing result of 1C1 is as follows：
B09_S148806_S625854a_EGP-1-1-C1_PGMF, (SEQ ID NO：18)：
TGCATTAGGAGGATTTAAATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCC
CAGCCGGCCATGGCACAGGTCCAGCTTGTGCAGTCTGGGGCTGAAGTCAAGAAGCCTGGGGCCTCAGTGAAGGCTTC
CTGCAAGGCATCTGGATATACCTTCACCAGCTACTATATCTCCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGT
GGATGGGAATAATCAATCCTGGTGATGCTGGCACAACCTACGCACAGAAGTTTCAGGGCAGAGTCACCATGACCAGG
GACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGGCGTGTATTACTGTGCGCG
GGGTAGGAGTGGCTGGTACGGTATGGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGCGGCGGCGGCT
CTGGCGGAGGTGGCAGCGGCGGTGGCGGATCCTCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGA
CAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCA
GGCCCCTGTGCTGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTG
GGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGT
AGTAGTGATCATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTGGCCTCGGGGGCCTGGTCGACTACAA
AGATGACGATGACAAATAGACTAGTGGCCAGGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTG
AGGGAGGCGGTTCCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAGATGGCAACGCTAATAAGGGGCTAT
GACCGAA
1.2a uses the combination angstrom that restriction endonuclease SfiI (Thermo Fisher, FD1824,1 μ L/ reactions) digestion is screened
The rich clone's phasmid (digestion obtains VH-VL, SEQ.ID No.17) and destination carrier plasmid pFuse- for drawing virus GP protein
Fc (InvivoGen, pfuse-hg1fc2), 37 DEG C, 30min；
1.2b respectively with the phasmid (SEQ.ID No.18) that is sieved to for template, conventionally to expressed sequence into
Row gene chemical synthesis (GENEray) (both sides introduce EcoRI, XhoI restriction enzyme sites), obtains heavy chain variable region-heavy chain constant region (VH-
CH, SEQ.ID No.19), light chain variable region-constant region of light chain (VL-CL, SEQ.ID No.20), uses restriction endonuclease EcoRI
(Thermo Fisher, FD0274,1 μ L/ reactions), XhoI (Thermo Fisher, FD0694,1 μ L/ reactions) digestion VH-CH,
VL-CL and target plasmid pCAGGS (Addgene, 12445), 37 DEG C, 30min；
1.3 carry out DNA gel electrophoresis, by purpose antibody fragment (ScFv forms VH-VL, SEQ.ID No.17 and IgG shapes
Formula heavy chain total length VH-CH, SEQ.ID No.19, light chain total length VL-CL, SEQ.ID No.20) and destination carrier plasmid
(pFuse-Fc and pCAGGS, wherein, pFuse-Fc is used to be connected with VH-VL, and pCAGGS is used to be connected with VH-CH and VL-CL)
Cut glue and recycled purpose antibody fragment and carrier segments using gel reclaims kit (CWBIO, CW2302)；
SEQ ID No.17 (E-1C1 ScFv, VH-VL)：
ATGGCACAGGTCCAGCTTGTGCAGTCTGGGGCTGAAGTCAAGAAGCCTGGGGCCTCAGTGAAGGCTTCCTGCAAGGC
ATCTGGATATACCTTCACCAGCTACTATATCTCCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAA
TAATCAATCCTGGTGATGCTGGCACAACCTACGCACAGAAGTTTCAGGGCAGAGTCACCATGACCAGGGACACGTCC
ACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGGCGTGTATTACTGTGCGCGGGGTAGGAG
TGGCTGGTACGGTATGGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGCGGCGGCGGCTCTGGCGGAG
GTGGCAGCGGCGGTGGCGGATCCTCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCC
AGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGT
GCTGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGG
CCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAGTGAT
CATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT。
SEQ.ID No.19(E-1C1VH-CH)：
GAATTCGCCACCatggagttcggcctgagctgggtgttcctggtggccatcatcaagggcgtgcaatgccagATGGC
ACAGGTCCAGCTTGTGCAGTCTGGGGCTGAAGTCAAGAAGCCTGGGGCCTCAGTGAAGGCTTCCTGCAAGGCATCTG
GATATACCTTCACCAGCTACTATATCTCCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATC
AATCCTGGTGATGCTGGCACAACCTACGCACAGAAGTTTCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAG
CACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGGCGTGTATTACTGTGCGCGGGGTAGGAGTGGCT
GGTACGGTATGGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAgcgagcaccaaaggcccgagcgtgttt
ccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccgga
accggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcg
gcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaac
cataaaccgagcaacaccaaagtggataaacgcgtgGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTG
CCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCT
CACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAG
CTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG
CAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCA
AGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC
CACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGACTCGAG。
SEQ.ID No.20(E-1C1 VL-CL)：
GAATTCGCCACCatggcotgggctctgctattcctcaccctcctcactcagggcacagggtcctgggccTATGTGCT
GACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTA
AAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGATAGCGACCGGCCCTCA
GGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGA
TGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAGTGATCATTGGGTGTTCGGCGGAGGGACCAAGCTGACCG
TCCTAGGTGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAG
GCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGT
CAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGA
CGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTG
GCCCCTACAGAATGTTCAcaccaccaceaccaccacTAACTCGAG。
1.4 are carried out purpose antibody fragment and carrier segments using connection kits (Thermo Fisher, 15224041)
Connection, 22 DEG C, 60min；
Gained connection product is converted DH5 α competence (CWBIO, CW0808,100 μ L) by 1.5, places 30min on ice；
1.6 42 DEG C of heat shock 1min；
1.7 place 1min on ice；
1.8 add LB culture mediums to after 1mL, and 37 DEG C, 200RPM, shakes bacterium 60min；
1.9 take 200 μ L to apply LB tablets (ammonia benzyl resistance), 37 DEG C, stay overnight；
1.10 picking tablet yesterday monoclonals add glycerine and carry out monoclonal guarantor bacterium (final glycerol concentration 10%) and sequencing, and
Monoclonal sequence is compared, obtains the correct monoclonal of sequence (SEQ.ID NO.17, SEQ.ID NO.19 and SEQ.ID
NO.20)；
1.11, which use the glycerol stock correctly cloned to be inoculated into according to 1: 1000 volume ratio in LB culture mediums, shakes bacterium, 37 DEG C, mistake
Night；
1.12 carry plasmid kit (TIANGEN, DP117) extraction plasmid using big；
1.13 use PEI (Sigma-Aldrich, P3143, mass ratio PEI: plasmid, 3: 1) transfect 293T cells and (be purchased fromCRL-3216)；
Collect 293T cell conditioned mediums after 1.14 4 days, after 0.22 μm of membrane filtration, hang proA columns (GE Healthcare,
17-0403-01), 2mL/min；
1.15 use AKTA pure (GE Healthcare, 17-0403-01) antibody elution, then with 1M Tris
(pH8.0) neutralized；
1.16 by gained protein concentration, and it is PBS solvents to change liquid；
1.17SDS-PAGE electrophoresis detections obtain whether albumen (IgG forms and ScFv-Fc forms) is antibody protein.
This plant of monoclonal antibody is named as E-1C1 (SEQ.ID NO.17, SEQ.ID NO.19 and SEQ.ID NO.20), is
Human monoclonal antibody；The amino acid sequence of its light chain hypervariable region CDR1, CDR2 and CDR3 are respectively SEQ ID No.3, DDS
(E-1C1CDR-L2, AspAsp Ser), and SEQ ID No.5, the amino acid sequence of heavy chain hypervariable region CDR1, CDR2 and CDR3
Respectively SEQ ID No.6, SEQ ID No.7, and SEQ ID No.8；The amino acid sequence of light chain variable region is SEQ ID
No.1, the amino acid sequence of heavy chain variable region is SEQ ID No.2；The amino acid sequence of light chain framework region FR1, FR2 and FR3 point
Wei not SEQ D No.9, SEQ ID No.10 and SEQ ID No.11；The amino acid sequence of heavy chain framework regions FR1, FR2 and FR3
Respectively SEQ ID No.12, SEQ ID No.13 and SEQ ID No.14.
1.18ELISA
The antigen protein in 200ng/ holes is coated with 96 hole elisa plates, 100 μ L/ holes, 4 DEG C overnight；200 μ L/ hole PBS wash 3
Time；200 μ L/ holes, 5% skimmed milk power-PBST room temperatures closing 1h；Add 10 times of concentration gradient diluted E-1C1 (ScFv-Fc forms
And IgG forms), 100 μ L/ holes (100 μ g/ml of initial concentration), is incubated at room temperature 1h；200 μ L/ hole PBST are washed 3 times；Add with
100 μ L/ holes of HRP- goat-anti people secondary antibodies (ZSGB-BIO, ZB-2304), are incubated at room temperature 1h；200 μ L/ hole PBST are washed 3 times；Add TMB
50 μ L/ holes of nitrite ion (Beyotime, P0209), 2min；Add 2M H250 μ L/ holes of SO4 terminate reaction；Read OD450 numbers
Value；Data analysis is carried out using Graphpad Prism 5, calculates EC50.
As a result as illustrated by fig. 1, two kinds of forms of the antibody can combine GP albumen well, while the antibody
IgG forms can also combine secreting type GP albumen (sGP, NCBI GenBank：U23187.1, and ssGP, NCBI GenBank：NP_
066248, build to insect expression plasmid and express acquisition), table 2 shows ScFv-Fc the and IgG forms and ebola disease of E-1C1
The half effective concentration EC50 of malicious GP.
Table 2
2 antigen-antibody affinity of embodiment is tested
Using OCTET technologies, by the ScFv-Fc forms (4.125 μ g/mL) of the E-1C1 obtained in embodiment 1 by affine
Effect is fixed on Protein A probes (PALL ForteBio, 18-5010), and fixed 500s, makes response reach 1-2nm,
Liquid phase antigen protein Ebola virus GP presses 2 doubling dilutions (concentration range 3.125nM-200nM).Carry out kinetic parameter survey
It is fixed, binding time 500s, Dissociation time 500s.Then, utilizeSoftware calculates affinity, and table 3 shows E-1C1
ScFv-Fc forms and Ebola virus GP affinity measurement result, affinity is respectively 3.93nM.
Table 3
3 antibody of embodiment and the IFC of cell surface GP albumen are tested
293T cells (CRL-3216 GP protein expression vector pEGFP-N1-GP (pEGFP- are transiently transfected in)
N1, BD Biosciences, #6085-1) (GENEray companies synthesize GP sequences, NCBI GenBank：AHX24658.2 both ends
Introduce XhoI, BamHI restriction enzyme sites, digestion insertion pEGFP-N1, SEQ.ID NO.4), 24 it is small when after use embodiment 1 in
The ScFv-Fc forms of the E-1C1 arrived are incubated altogether as primary antibody (10 μ g/mL, 200 μ L) with the 293T cells of transfection expression GP albumen
Educate, then using Goat anti Human-PE fluorescence antibodies (eBioscienceTM, 12-4998-82,1: 200,200 μ L) into
One step dyes, and observation shooting is carried out using fluorescence microscope, determines that screened antibody and the in situ of GP combine activity, as a result
As shown in Figure 1, the GP albumen of E-1C1 and cell surface expression, which has, combines activity.
SEQ.ID NO.4(EGFP-N1-GP)：
ctcgagaccaccATGGGCGTTACAGGAATATTGCAGTTACCTCGTGATCGATTCAAGAGGACATCATTCTTTCTTTG
GGTAATTATCCTTTTCCAAAGAACATTTTCCATCCCACTTGGAGTCATCCACAATAGCACATTACAGGTTAGTGATG
TCGACAAACTAGTTTGTCGTGACAAACTGTCATCCACAAATCAATTGAGATCAGTTGGACTGAATCTCGAAGGGAAT
GGAGTGGCAACTGACGTGCCATCTGCAACTAAAAGATGGGGCTTCAGGTCCGGTGTCCCACCAAAGGTGGTCAATTA
TGAAGCTGGTGAATGGGCTGAAAACTGCTACAATCTTGAAATCAAAAAACCTGACGGGAGTGAGTGTCTACCAGCAG
CGCCAGACGGGATTCGGGGCTTCCCCCGGTGCCGGTATGTGCACAAAGTATCAGGAACGGGACCGTGTGCCGGAGAC
TTTGCCTTCCATAAAGAGGGTGCTTTCTTCCTGTATGATCGACTTGCTTCCACAGTTATCTACCGAGGAACGACTTT
CGCTGAAGGTGTCGTTGCATTTCTGATACTGCCCCAAGCTAAGAAGGACTTCTTCAGCTCACACCCCTTGAGAGAGC
CGGTCAATGCAACGGAGGACCCGTCTAGTGGCTACTATTCTACCACAATTAGATATCAGGCTACCGGTTTTGGAACC
AATGAGACAGAGTACTTGTTCGAGGTTGACAATTTGACCTACGTCCAACTTGAATCAAGATTCACACCACAGTTTCT
GCTCCAGCTGAATGAGACAATATATACAAGTGGGAAAAGGAGCAATACCACGGGAAAACTAATTTGGAAGGTCAACC
CCGAAATTGATACAACAATCGGGGAGTGGGCCTTCTGGGAAACTAAAAAAAACCTCACTAGAAAAATTCGCAGTGAA
GAGTTGTCTTTCACAGTTGTATCAAACGGAGCCAAAAACATCAGTGGTCAGAGTCCGGCGCGAACTTCTTCCGACCC
AGGGACCAACACAACAACTGAAGACCACAAAATCATGGCTTCAGAAAATTCCTCTGCAATGGTTCAAGTGCACAGTC
AAGGAAGGGAAGCTGCAGTGTCGCATCTAACAACCCTTGCCACAATCTCCACGAGTCCCCAATCCCTCACAACCAAA
CCAGGTCCGGACAACAGCACCCATAATACACCCGTGTATAAACTTGACATCTCTGAGGCAACTCAAGTTGAACAACA
TCACCGCAGAACAGACAACGACAGCACAGCCTCCGACACTCCCTCTGCCACGACCGCAGCCGGACCCCCAAAAGCAG
AGAACACCAACACGAGCAAGAGCACTGACTTCCTGGACCCCGCCACCACAACAAGTCCCCAAAACCACAGCGAGACC
GCTGGCAACAACAACACTCATCACCAAGATACCGGAGAAGAGAGTGCCAGCAGCGGGAAGCTAGGCTTAATTACCAA
IACTATTGCTGGAGTCGCAGGACTGATCACAGGCGGGAGAAGAACTCGAAGAGAAGCAATTGTCAATGCTCAACCCA
AATGCAACCCTAATTTACATTACTGGACTACTCAGGATGAAGGTGCTGCAATCGGACTGGCCTGGATACCATATTTC
GGGCCAGCAGCCGAGGGAATTTACATAGAGGGGCTAATGCACAATCAAGATGGTTTAATCTGTGGGTTGAGACAGCT
GGCCAACGAGACGACTCAAGCTCTTCAACTGTTCCTGAGAGCCACAACTGAGCTACGCACCTTTTCAATCCTCAACC
GTAAGGCAATTGATTTCTTGCTGCAGCGATGGGGCGGCACATGCCACATTCTGGGACCGGACTGCTGTATCGAACCA
CATGATTGGACCAAGAACATAACAGACAAAATTGATCAGATTATTCATGATTTTGTTGATAAAACCCTTCCGGACCA
GGGGGACAATGACAATTGGTGGACAGGATGGAGACAATGGATACCGGCAGGTATTGGAGTTACAGGCGTTATAATTG
CAGTTATCGCTTTATTCTGTATATGCAAATTTGTCTTTggggatccACCGGTCGCCACCATGGTG。
The Western Blot experiments of 4 antibody test GP albumen of embodiment
(volume ratio 4: 1 and 5*Loading buffer (Beyotime, P0015) are mixed the sample preparation of GP albumen, boiling water bath
10min), SDS-PAGE glue (5 μ g/mL, 10 μ L) is run, after transferring film (nitrocellulose filter, Thermo Fisher, IB301001),
The IgG forms (10 μ g/mL, 1mL) of the E-1C1 obtained using embodiment 1 are incubated as primary antibody, room temperature 1h, then using Goat
Anti Human-HRP (ZSGB-BIO, ZDR-5301,1: 2000,1mL) are incubated, room temperature 1h, finally using CN/ as secondary antibody
DAB Substrate Kit are developed the color (Thermo Fisher, 34000,2mL), and the results are shown in Figure 3, used E-
1C1 can detect GP albumen in Western Blot levels.
It can be seen from above-described embodiment that antibody sources of the present invention are in normal pbmc, and ELISA,
Ebola virus envelope protein GP can be combined well in OCTET, IFC and Western Blot experiments, therefore is had
Certain economy and social effect.
Applicant states that the present invention illustrates the detailed construction feature of the present invention by above-described embodiment, but the present invention is simultaneously
Above-mentioned detailed construction feature is not limited to, that is, does not mean that the present invention has to rely on above-mentioned detailed construction feature and could implement.Institute
Belong to those skilled in the art it will be clearly understood that any improvement in the present invention, to the equivalence replacement of component selected by the present invention
And the increase of accessory, selection of concrete mode etc., all fall within protection scope of the present invention and the open scope.
The preferred embodiment of the present invention described in detail above, still, during present invention is not limited to the embodiments described above
Detail, in the range of the technology design of the present invention, a variety of simple variants can be carried out to technical scheme, this
A little simple variants belong to protection scope of the present invention.
It is further to note that each particular technique feature described in above-mentioned embodiment, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
Sequence table
<110>Shanghai Science and Technology Univ.
<120>A kind of Zaire types Ebola virus detection antibody and preparation method and application
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 108
<212> PRT
<213>People (Homo sapiens)
<400> 1
Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr
1 5 10 15
Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His
20 25 30
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp
35 40 45
Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn
50 55 60
Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp
65 70 75 80
Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His Trp
85 90 95
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105
<210> 2
<211> 121
<212> PRT
<213>People (Homo sapiens)
<400> 2
Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
1 5 10 15
Gly Ala Ser Val Lys Ala Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30
Ser Tyr Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
35 40 45
Trp Met Gly Ile Ile Asn Pro Gly Asp Ala Gly Thr Thr Tyr Ala Gln
50 55 60
Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr
65 70 75 80
Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Gly Val Tyr
85 90 95
Tyr Cys Ala Arg Gly Arg Ser Gly Trp Tyr Gly Met Asp Val Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 3
<211> 6
<212> PRT
<213>People (Homo sapiens)
<400> 3
Asn Ile Gly Ser Lys Ser
1 5
<210> 4
<211> 2067
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ctcgagacca ccatgggcgt tacaggaata ttgcagttac ctcgtgatcg attcaagagg 60
acatcattct ttctttgggt aattatcctt ttccaaagaa cattttccat cccacttgga 120
gtcatccaca atagcacatt acaggttagt gatgtcgaca aactagtttg tcgtgacaaa 180
ctgtcatcca caaatcaatt gagatcagtt ggactgaatc tcgaagggaa tggagtggca 240
actgacgtgc catctgcaac taaaagatgg ggcttcaggt ccggtgtccc accaaaggtg 300
gtcaattatg aagctggtga atgggctgaa aactgctaca atcttgaaat caaaaaacct 360
gacgggagtg agtgtctacc agcagcgcca gacgggattc ggggcttccc ccggtgccgg 420
tatgtgcaca aagtatcagg aacgggaccg tgtgccggag actttgcctt ccataaagag 480
ggtgctttct tcctgtatga tcgacttgct tccacagtta tctaccgagg aacgactttc 540
gctgaaggtg tcgttgcatt tctgatactg ccccaagcta agaaggactt cttcagctca 600
caccccttga gagagccggt caatgcaacg gaggacccgt ctagtggcta ctattctacc 660
acaattagat atcaggctac cggttttgga accaatgaga cagagtactt gttcgaggtt 720
gacaatttga cctacgtcca acttgaatca agattcacac cacagtttct gctccagctg 780
aatgagacaa tatatacaag tgggaaaagg agcaatacca cgggaaaact aatttggaag 840
gtcaaccccg aaattgatac aacaatcggg gagtgggcct tctgggaaac taaaaaaaac 900
ctcactagaa aaattcgcag tgaagagttg tctttcacag ttgtatcaaa cggagccaaa 960
aacatcagtg gtcagagtcc ggcgcgaact tcttccgacc cagggaccaa cacaacaact 1020
gaagaccaca aaatcatggc ttcagaaaat tcctctgcaa tggttcaagt gcacagtcaa 1080
ggaagggaag ctgcagtgtc gcatctaaca acccttgcca caatctccac gagtccccaa 1140
tccctcacaa ccaaaccagg tccggacaac agcacccata atacacccgt gtataaactt 1200
gacatctctg aggcaactca agttgaacaa catcaccgca gaacagacaa cgacagcaca 1260
gcctccgaca ctccctctgc cacgaccgca gccggacccc caaaagcaga gaacaccaac 1320
acgagcaaga gcactgactt cctggacccc gccaccacaa caagtcccca aaaccacagc 1380
gagaccgctg gcaacaacaa cactcatcac caagataccg gagaagagag tgccagcagc 1440
gggaagctag gcttaattac caatactatt gctggagtcg caggactgat cacaggcggg 1500
agaagaactc gaagagaagc aattgtcaat gctcaaccca aatgcaaccc taatttacat 1560
tactggacta ctcaggatga aggtgctgca atcggactgg cctggatacc atatttcggg 1620
ccagcagccg agggaattta catagagggg ctaatgcaca atcaagatgg tttaatctgt 1680
gggttgagac agctggccaa cgagacgact caagctcttc aactgttcct gagagccaca 1740
actgagctac gcaccttttc aatcctcaac cgtaaggcaa ttgatttctt gctgcagcga 1800
tggggcggca catgccacat tctgggaccg gactgctgta tcgaaccaca tgattggacc 1860
aagaacataa cagacaaaat tgatcagatt attcatgatt ttgttgataa aacccttccg 1920
gaccaggggg acaatgacaa ttggtggaca ggatggagac aatggatacc ggcaggtatt 1980
ggagttacag gcgttataat tgcagttatc gctttattct gtatatgcaa atttgtcttt 2040
ggggatccac cggtcgccac catggtg 2067
<210> 5
<211> 11
<212> PRT
<213>People (Homo sapiens)
<400> 5
Gln Val Trp Asp Ser Ser Ser Asp His Trp Val
1 5 10
<210> 6
<211> 8
<212> PRT
<213>People (Homo sapiens)
<400> 6
Gly Tyr Thr Phe Thr Ser Tyr Tyr
1 5
<210> 7
<211> 8
<212> PRT
<213>People (Homo sapiens)
<400> 7
Ile Asn Pro Gly Asp Ala Gly Thr
1 5
<210> 8
<211> 12
<212> PRT
<213>People (ARGRSGWYGMDV)
<400> 8
Ala Arg Gly Arg Ser Gly Trp Tyr Gly Met Asp Val
1 5 10
<210> 9
<211> 24
<212> PRT
<213>People (Homo sapiens)
<400> 9
Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr
1 5 10 15
Ala Arg Ile Thr Cys Gly Gly Asn
20
<210> 10
<211> 17
<212> PRT
<213>People (Homo sapiens)
<400> 10
Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val
1 5 10 15
Tyr
<210> 11
<211> 36
<212> PRT
<213>People (Homo sapiens)
<400> 11
Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly
1 5 10 15
Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala
20 25 30
Asp Tyr Tyr Cys
35
<210> 12
<211> 27
<212> PRT
<213>People (Homo sapiens)
<400> 12
Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
1 5 10 15
Gly Ala Ser Val Lys Ala Ser Cys Lys Ala Ser
20 25
<210> 13
<211> 17
<212> PRT
<213>People (Homo sapiens)
<400> 13
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
1 5 10 15
Ile
<210> 14
<211> 38
<212> PRT
<213>People (Homo sapiens)
<400> 14
Thr Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr
1 5 10 15
Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
20 25 30
Thr Gly Val Tyr Tyr Cys
35
<210> 15
<211> 324
<212> DNA
<213>People (Homo sapiens)
<400> 15
tatgtgctga ctcagccacc ctcggtgtca gtggccccag gacagacggc caggattacc 60
tgtgggggaa acaacattgg aagtaaaagt gtgcactggt accagcagaa gccaggccag 120
gcccctgtgc tggtcgtcta tgatgatagc gaccggccct cagggatccc tgagcgattc 180
tctggctcca actctgggaa cacggccacc ctgaccatca gcagggtcga agccggggat 240
gaggccgact attactgtca ggtgtgggat agtagtagtg atcattgggt gttcggcgga 300
gggaccaagc tgaccgtcct aggt 324
<210> 16
<211> 363
<212> DNA
<213>People (Homo sapiens)
<400> 16
atggcacagg tccagcttgt gcagtctggg gctgaagtca agaagcctgg ggcctcagtg 60
aaggcttcct gcaaggcatc tggatatacc ttcaccagct actatatctc ctgggtgcga 120
caggcccctg gacaagggct tgagtggatg ggaataatca atcctggtga tgctggcaca 180
acctacgcac agaagtttca gggcagagtc accatgacca gggacacgtc cacgagcaca 240
gtctacatgg agctgagcag cctgagatct gaagacacgg gcgtgtatta ctgtgcgcgg 300
ggtaggagtg gctggtacgg tatggatgtc tggggccaag ggaccacggt caccgtctcc 360
tca 363
<210> 17
<211> 735
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
atggcacagg tccagcttgt gcagtctggg gctgaagtca agaagcctgg ggcctcagtg 60
aaggcttcct gcaaggcatc tggatatacc ttcaccagct actatatctc ctgggtgcga 120
caggcccctg gacaagggct tgagtggatg ggaataatca atcctggtga tgctggcaca 180
acctacgcac agaagtttca gggcagagtc accatgacca gggacacgtc cacgagcaca 240
gtctacatgg agctgagcag cctgagatct gaagacacgg gcgtgtatta ctgtgcgcgg 300
ggtaggagtg gctggtacgg tatggatgtc tggggccaag ggaccacggt caccgtctcc 360
tcaggcggcg gcggctctgg cggaggtggc agcggcggtg gcggatcctc ctatgtgctg 420
actcagccac cctcggtgtc agtggcccca ggacagacgg ccaggattac ctgtggggga 480
aacaacattg gaagtaaaag tgtgcactgg taccagcaga agccaggcca ggcccctgtg 540
ctggtcgtct atgatgatag cgaccggccc tcagggatcc ctgagcgatt ctctggctcc 600
aactctggga acacggccac cctgaccatc agcagggtcg aagccgggga tgaggccgac 660
tattactgtc aggtgtggga tagtagtagt gatcattggg tgttcggcgg agggaccaag 720
ctgaccgtcc taggt 735
<210> 18
<211> 1000
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
tgcattagga ggatttaaat gaaataccta ttgcctacgg cagccgctgg attgttatta 60
ctcgcggccc agccggccat ggcacaggtc cagcttgtgc agtctggggc tgaagtcaag 120
aagcctgggg cctcagtgaa ggcttcctgc aaggcatctg gatatacctt caccagctac 180
tatatctcct gggtgcgaca ggcccctgga caagggcttg agtggatggg aataatcaat 240
cctggtgatg ctggcacaac ctacgcacag aagtttcagg gcagagtcac catgaccagg 300
gacacgtcca cgagcacagt ctacatggag ctgagcagcc tgagatctga agacacgggc 360
gtgtattact gtgcgcgggg taggagtggc tggtacggta tggatgtctg gggccaaggg 420
accacggtca ccgtctcctc aggcggcggc ggctctggcg gaggtggcag cggcggtggc 480
ggatcctcct atgtgctgac tcagccaccc tcggtgtcag tggccccagg acagacggcc 540
aggattacct gtgggggaaa caacattgga agtaaaagtg tgcactggta ccagcagaag 600
ccaggccagg cccctgtgct ggtcgtctat gatgatagcg accggccctc agggatccct 660
gagcgattct ctggctccaa ctctgggaac acggccaccc tgaccatcag cagggtcgaa 720
gccggggatg aggccgacta ttactgtcag gtgtgggata gtagtagtga tcattgggtg 780
ttcggcggag ggaccaagct gaccgtccta ggtggcctcg ggggcctggt cgactacaaa 840
gatgacgatg acaaatagac tagtggccag gagggtggtg gctctgaggg tggcggttct 900
gagggtggcg gctctgaggg aggcggttcc cggtggtggc tctggttccg gtgattttga 960
ttatgaaaga tggcaacgct aataaggggc tatgaccgaa 1000
<210> 19
<211> 1434
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
gaattcgcca ccatggagtt cggcctgagc tgggtgttcc tggtggccat catcaagggc 60
gtgcaatgcc agatggcaca ggtccagctt gtgcagtctg gggctgaagt caagaagcct 120
ggggcctcag tgaaggcttc ctgcaaggca tctggatata ccttcaccag ctactatatc 180
tcctgggtgc gacaggcccc tggacaaggg cttgagtgga tgggaataat caatcctggt 240
gatgctggca caacctacgc acagaagttt cagggcagag tcaccatgac cagggacacg 300
tccacgagca cagtctacat ggagctgagc agcctgagat ctgaagacac gggcgtgtat 360
tactgtgcgc ggggtaggag tggctggtac ggtatggatg tctggggcca agggaccacg 420
gtcaccgtct cctcagcgag caccaaaggc ccgagcgtgt ttccgctggc gccgagcagc 480
aaaagcacca gcggcggcac cgcggcgctg ggctgcctgg tgaaagatta ttttccggaa 540
ccggtgaccg tgagctggaa cagcggcgcg ctgaccagcg gcgtgcatac ctttccggcg 600
gtgctgcaga gcagcggcct gtatagcctg agcagcgtgg tgaccgtgcc gagcagcagc 660
ctgggcaccc agacctatat ttgcaacgtg aaccataaac cgagcaacac caaagtggat 720
aaacgcgtgg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 780
gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 840
atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 900
gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 960
gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1020
tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1080
gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1140
ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1200
tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1260
accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1320
gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1380
cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaatgact cgag 1434
<210> 20
<211> 738
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
gaattcgcca ccatggcctg ggctctgcta ttcctcaccc tcctcactca gggcacaggg 60
tcctgggcct atgtgctgac tcagccaccc tcggtgtcag tggccccagg acagacggcc 120
aggattacct gtgggggaaa caacattgga agtaaaagtg tgcactggta ccagcagaag 180
ccaggccagg cccctgtgct ggtcgtctat gatgatagcg accggccctc agggatccct 240
gagcgattct ctggctccaa ctctgggaac acggccaccc tgaccatcag cagggtcgaa 300
gccggggatg aggccgacta ttactgtcag gtgtgggata gtagtagtga tcattgggtg 360
ttcggcggag ggaccaagct gaccgtccta ggtggtcagc ccaaggctgc cccctcggtc 420
actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 480
ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 540
aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 600
agctacctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 660
acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc acaccaccac 720
caccaccact aactcgag 738
Claims (10)
1. a kind of Zaire types Ebola virus detects antibody, it is characterised in that is human monoclonal antibody；Its light chain hypervariable region
The amino acid sequence of CDR1, CDR2 and CDR3 are respectively SEQ ID No.3 or the sequence through replacing, lacking or adding one or more
A amino acids formed have the function of equal amino acid sequence, DDS or the sequence through replacing, lacking or adding one or more
It is amino acids formed that there is equal amino acid sequence, and SEQ ID No.5 or the sequence through replacing, lacking or adding one
It is a or multiple amino acids formed there is equal amino acid sequence, the amino of heavy chain hypervariable region CDR1, CDR2 and CDR3
Acid sequence is respectively that SEQ ID No.6 or the sequence are one or more amino acids formed with same through replacing, lacking or add
Amino acid sequence, SEQ ID No.7 or the sequence etc. function is one or more amino acids formed through replacing, lacking or add
There is equal amino acid sequence, and SEQ ID No.8 or the sequence through replacing, lacking or adding one or more amino
What acid was formed has the function of equal amino acid sequence.
2. Zaire types Ebola virus as claimed in claim 1 detects antibody, it is characterised in that the Zaire types angstrom are rich
The amino acid sequence for drawing the light chain variable region of viral diagnosis antibody is SEQ ID No.1 or the sequence through replacing, lacking or add
One or more is amino acids formed to have the function of equal amino acid sequence, and the amino acid sequence of heavy chain variable region is SEQ ID
No.2 or the sequence one or more amino acids formed have the function of equal amino acid sequence through replacing, lacking or add.
3. Zaire types Ebola virus as claimed in claim 1 detects antibody, it is characterised in that the Zaire types angstrom are rich
The amino acid sequence for drawing light chain framework region FR1, FR2 and FR3 of viral diagnosis antibody is respectively SEQ ID No.9 or sequence warp
Replace, missing or addition it is one or more it is amino acids formed have the function of equal amino acid sequence, SEQ ID No.10 or
The sequence one or more amino acids formed has the function of equal amino acid sequence and SEQ through replacing, lacking or add
ID No.11 or the sequence one or more amino acids formed have the function of equal amino acid sequence through replacing, lacking or add
Row；The amino acid sequence of heavy chain framework regions FR1, FR2 and FR3 be respectively SEQ ID No.12 or the sequence through replacing, lack or
Addition one or more is amino acids formed to have the function of equal amino acid sequence, SEQ ID No.13 or the sequence through replacing
Change, lack or add it is one or more it is amino acids formed have the function of equal amino acid sequence and SEQ ID No.14 or
The sequence one or more amino acids formed has the function of equal amino acid sequence through replacing, lacking or add.
4. Zaire types Ebola virus as claimed in claim 1 detects antibody, it is characterised in that the Zaire types angstrom are rich
The coding light chain variable region of viral diagnosis antibody and the nucleotide sequence of heavy chain variable region is drawn to be respectively SEQ ID No.15 or be somebody's turn to do
Through replacing, lacking or add, one or more nucleotide are formed sequence has the function of equal nucleotide sequence and SEQ ID
One or more nucleotide are formed has the function of equal nucleotide sequence through replacing, lacking or add for No.16 or the sequence.
5. a kind of expression vector, it is characterised in that the expression vector includes SEQ ID No.15 or the sequence through replacing, lacking
Lose or what the one or more nucleotide of addition were formed has the function of equal nucleotide sequence and SEQ ID No.16 or the sequence
Row are through replacing, lacking or adding have the function of in equal nucleotide sequence at least the one of one or more nucleotide formation
It is a.
6. a kind of host cell, it is characterised in that the host cell includes the above-mentioned expression vector described in claim 5.
7. the preparation method of the Zaire types Ebola virus detection antibody described in claim 1-4, it is characterised in that including such as
Lower step：By SEQ ID No.15-SEQ ID No.20 and above-mentioned sequence through replacing, lacking or adding one or more nucleotide
Formed have the function of in equal nucleotide sequence it is at least one be connected with expression vector, amplification, transfection 293T cells, receipts
293T cell conditioned mediums are taken, hang proA columns, elution, obtains Zaire types Ebola virus detection antibody.
8. the preparation method of Zaire types Ebola virus detection antibody as claimed in claim 7, it is characterised in that described
Expression vector is mammalian expression vector.
9. the preparation method of Zaire types Ebola virus detection antibody as claimed in claim 8, it is characterised in that described
Mammalian expression vector is pFuse-Fc and pCAGGS mammalian expression vectors.
10. the Zaire types Ebola virus detection antibody described in claim 1-4 has Ebola virus GP antigens in preparation
Affinity, to the application in the detection of Ebola virus.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001016183A1 (en) * | 1999-08-30 | 2001-03-08 | U.S. Army Medical Research Institute Of Infectious Diseases | Monoclonal antibodies and vaccines against epitopes on the ebola virus glycoprotein |
| CN105112375A (en) * | 2015-08-21 | 2015-12-02 | 浙江大学医学院附属第一医院 | Hybridoma cell strain ZJED0-02, anti-Ebola virus GP protein monoclonal antibody, and preparation and application thereof |
-
2017
- 2017-12-29 CN CN201711497938.9A patent/CN108017715B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001016183A1 (en) * | 1999-08-30 | 2001-03-08 | U.S. Army Medical Research Institute Of Infectious Diseases | Monoclonal antibodies and vaccines against epitopes on the ebola virus glycoprotein |
| CN105112375A (en) * | 2015-08-21 | 2015-12-02 | 浙江大学医学院附属第一医院 | Hybridoma cell strain ZJED0-02, anti-Ebola virus GP protein monoclonal antibody, and preparation and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| A. L. MOEKOTTE等: "Monoclonal antibodies for the treatment of Ebola virus disease", 《EXPERT OPINION ON INVESTIGATIONAL DRUGS》 * |
| 周荣苹等: "抗埃博拉病毒核蛋白ＮＰ人－ 鼠嵌合抗体的研究", 《病毒学报》 * |
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