CN105542002B - A kind of monoclonal antibody Q206 and application - Google Patents
A kind of monoclonal antibody Q206 and application Download PDFInfo
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- CN105542002B CN105542002B CN201610069699.6A CN201610069699A CN105542002B CN 105542002 B CN105542002 B CN 105542002B CN 201610069699 A CN201610069699 A CN 201610069699A CN 105542002 B CN105542002 B CN 105542002B
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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Abstract
The invention discloses a kind of monoclonal antibody Q206 and applications.The monoclonal antibody Q206 that the present invention protects is a kind of IgG antibody, is made of light chain and heavy chain；CDR1, CDR2 and CDR3 in heavy chain variable region in the heavy chain is followed successively by the sequence 3 of sequence table from N-terminal 45-52 amino acids residue, 70-79 amino acids residue and 118-128 amino acids residue；CDR1, CDR2 and CDR3 in light chain variable region in the light chain is followed successively by the sequence 5 of sequence table from N-terminal 45-53 amino acids residue, 71-73 amino acids residue and 110-123 amino acids residue.The present invention has great application value for the prevention and control of Ebola virus Zaire hypotype.
Description
Technical field
The present invention relates to a kind of monoclonal antibody Q206 and applications.
Background technique
Ebola virus is that most one of fatal infection venereal disease poison known to the mankind, average case fatality rate are up to 50% so far
Left and right.Due to the high lethality rate of Ebola hemorrhagic fever, and there has been no any preventions or treatment method at present can be clinically wide
General use.Ebola virus is listed in the bio-safety fourth stage (Biosafety Level 4) virus, while being considered to be biology
One of tool of terrorism.
2014 spring year start to have attracted worldwide attention in Ebola's epidemic situation that West Africa various countries are broken out.Cut-off is extremely
On 2 27th, 2015, Ebola virus, which is made a definite diagnosis, had reached 23825 people with suspected case, and death toll reaches 9660 people, the U.S.
There is the propagation of Introduced cases with some European countries, and has had death.Therefore, prevention and control Ebola virus is global host country
The cardinal task of family.However enable owner's anxiety: up to the present there are no an approved research of Ebola vaccine
It is listed with drug.By the emergency therapeutic agent ZMapp that America & Canada is developed jointly be it is currently the only in model animal and
Small-scale patients clinical verifies effective drug.China's emergency drug still not similar at present, once there is Chinese citizen's infection
Ebola virus, consequence are hardly imaginable.Urgent Ebola virus antibody of developing is for China or even global Ebola virus epidemic situation
Prevention and control have great importance, while to society stabilization harmony have far-reaching influence.
Summary of the invention
The object of the present invention is to provide a kind of monoclonal antibody Q206 and applications.
The monoclonal antibody Q206 that the present invention protects is a kind of IgG antibody, is made of light chain and heavy chain；In the heavy chain
Heavy chain variable region in CDR1, CDR2 and CDR3 be followed successively by the sequence 3 of sequence table from N-terminal 45-52 amino acids residue,
70-79 amino acids residue and 118-128 amino acids residue；The CDR1 in light chain variable region in the light chain,
CDR2 and CDR3 be followed successively by the sequence 5 of sequence table from N-terminal 45-53 amino acids residue, 71-73 amino acids residue and
110-123 amino acids residue.
The heavy chain can for following (a) or (b): (a) sequence 3 of sequence table is from N-terminal 20-467 amino acids residue
The protein of composition；(b) protein shown in the sequence 3 of sequence table.
The light chain can for following (c) or (d): the sequence 5 of (c) sequence table is from N-terminal 20-237 amino acids residue
The protein of composition；(d) protein shown in the sequence 5 of sequence table.
The present invention also protects the gene for encoding the IgG antibody, it is characterised in that:
The gene for encoding the heavy chain is following (1) or (2) or (3):
(1) sequence 4 of sequence table DNA molecular shown in the nucleotide of 5 ' end 889-2292；
(2) sequence 4 of sequence table DNA molecular shown in the nucleotide of 5 ' end 946-2292；
(3) DNA molecular shown in the sequence 4 of sequence table；
The gene for encoding the light chain is following (4) or (5) or (6):
(4) sequence 6 of sequence table DNA molecular shown in the nucleotide of 5 ' end 889-1602；
(5) sequence 6 of sequence table DNA molecular shown in the nucleotide of 5 ' end 946-1602；
(6) DNA molecular shown in the sequence 6 of sequence table.
The present invention also protects the IgG antibody preparing answering in the drug for inhibiting Ebola virus Zaire hypotype
With.
The present invention also protects a kind of for inhibiting the drug of Ebola virus Zaire hypotype, and active constituent is described
IgG antibody.
The present invention also protects the IgG antibody answering in preparation is used for and in the drug of Ebola virus Zaire hypotype
With.
The present invention also protects a kind of for neutralizing the drug of Ebola virus Zaire hypotype, and active constituent is described
IgG antibody.
The present invention also protects the IgG antibody to draw in preparation for preventing and/or treating Ebola virus Zaire hypotype
Application in the drug of the Ebola hemorrhagic fever risen.
The present invention also protects a kind of for preventing and/or treating Ebola's bleeding caused by Ebola virus Zaire hypotype
The drug of heat, active constituent are the IgG antibody.
Ebola virus Zaire hypotype is also known as Zaire Ebola virus, english abbreviation EBOV.
The present invention is screened anti-using the GP △ m albumen of EBOV as bait from the peripheral blood mononuclear cells of immune monkey
The memory B cell that body generates, obtaining can be the same as the monoclonal antibody of GP △ m albumen specific bond.It is infected by EBOV pseudotype virus
Model, screening have obtained with neutralization activity while having had the monoclonal antibody of good specificity.
The present invention has great application value for the prevention and control of Zaire Ebola virus, and the society for generating far-reaching is anticipated
Justice.
Detailed description of the invention
Fig. 1 is result of the antibody to the neutralization activity of EBOV.
Fig. 2 is result of the antibody to the neutralization activity of VSV.
Fig. 3 is result of the antibody to the neutralization activity of HIV.
Fig. 4 is result of the antibody to the neutralization activity of SUDV.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Plasmid pcDNA3.1 (+): Invitrogen company, catalog number V790-20.293T cell: Gaede, CRL-
11268.Rhesus macaque: blue island biology.PMD18T carrier: Takara, catalog number D101A.VeroE6 cell: vircell is public
Department, catalog number VCV6.
Plasmid pNL4-3R-E-luciferase (skeleton plasmid): bibliography: He J, Choe S, Walker R, Di
Marzio P,Morgan DO,Landau NR.J Virol 69:6705–6711,1995.。
The discovery of embodiment 1, antibody
One, the preparation of GP △ m albumen
1, construction recombination plasmid
(1) double chain DNA molecule shown in the sequence 2 of composition sequence table.
Protein shown in the sequence 1 of double chain DNA molecule polynucleotide shown in the sequence 2 of sequence table, wherein opening
Reading frame is sequence 2 from the nucleotide of 5 ' end 18-1451.In the sequence 1 of sequence table, from the 1st to 19 amino acids of N-terminal
Residue forms signal peptide, and the 472nd to 477 amino acids residue forms His6Label.Protein shown in the sequence 1 of sequence table
It is expected that molecular weight is 200kDa.
(2) double chain DNA molecule obtained with restriction enzyme BamHI and NotI double digestion step 1, recycling digestion produce
Object.
(3) restriction enzyme BamHI and NotI double digestion plasmid pcDNA3.1 (+) is used, the carrier of about 5400bp is recycled
Skeleton.
(4) digestion products that step 2 recycles are connected with the carrier framework that step 3 recycles, obtains recombinant plasmid
pcDNA3.1-GP△m.According to sequencing result, structure is carried out to recombinant plasmid pcDNA3.1-GP △ m and is described as follows: in plasmid
The sequence 2 of sequence table is inserted between BamHI the and NotI restriction enzyme site of pcDNA3.1 (+) from the core of 5 ' end the 11st to 1453
Double chain DNA molecule shown in thuja acid.
2, recombinant cell is constructed
The Transfected Recombinant Plasmid 293T cell that step 1 is obtained, obtains recombinant cell.
3, GP △ m albumen is prepared
(1) recombinant cell for taking step 2 to obtain, in the DMEM culture medium culture 72h for containing 2% fetal calf serum, then
4000rpm is centrifuged 30min, collects supernatant.
(2) affinity chromatography
The chromatographic column specification of affinity chromatography: length 3cm, internal diameter 1cm；
The column packing of affinity chromatography: nickel column beads (is purchased from Qiagen company, catalog number 30230)；
Operating procedure: being 1. splined on affinity column for the supernatant that 300mL step (1) obtains, and 4 DEG C are incubated for 3 hours；②
Pillar is washed with the sample-loading buffer of 100mL imidazoles containing 20mM；3. eluting purpose with the elution buffer of 30mL imidazoles containing 500mM
Albumen collected solution after column.Sample-loading buffer, that is, HEPEs buffer.
(3) solution after crossing column that step (2) obtains is taken, (is purchased from Merck company, catalog number is with 30kD concentration tube
UFC800396 it) is concentrated, obtains the concentrate that volume is 1ml.
(4) gel permeation chromatography
The chromatographic column specification of gel permeation chromatography: length 24cm, internal diameter 2cm；
The column packing of gel permeation chromatography: superdex200increase 10/300GL is (public purchased from GEHealthcare
Department, catalog number 28-9909-44)；
Operating procedure: the concentrate that loading 0.5ml step (3) obtains, the PBS buffer solution for being 0.5ml/min with flow velocity
Peak corresponding solution, as GP △ m protein solution after crossing column that retention volume is 11ml are collected in (pH7.2,10mM) elution.
Two, the separation antibody variable region gene from the menory B cell of GP △ m albumen specific bond
1, it using GP △ m albumen as immunogen immune rhesus macaque, then takes peripheral blood and separates mononuclearcell (PBMC).
2, it is capable of B cell (the CD3 of specific recognition GP △ m albumen using selected by flow cytometry apoptosis-CD16-CD235a-
CD19+CD27+CD38-IgG+GP△m BCR+)。
3, the B cell for taking step 2 to obtain, extracts total serum IgE and reverse transcription is cDNA.
4, the cDNA obtained using step 3 is carried out nest-type PRC, pcr amplification product is sequenced as template.
Obtain the nucleotide sequence of heavy chain variable region and the nucleotide sequence of light chain variable region.
Three, antibody gene is obtained
By heavy chain variable region upstream and CMV segment, the constant region in heavy chain variable region downstream and human IgG1 and ployA piece
Section carries out overlapping PCR, obtains the DNA fragmentation that can express entire heavy chain.
By the constant region of antibody's light chain variable region upstream and CMV segment, antibody's light chain variable region downstream and light chain κ/λ and
PloyA segment carries out overlapping PCR, obtains the DNA fragmentation that can express Whole light chains.
The amino acid sequence of Q206 total length heavy chain is as shown in the sequence 3 of sequence table, the sequence 4 of encoding gene such as sequence table
It is shown.In the sequence 3 of sequence table, the 1st to 19 amino acids residue forms signal peptide (pilot protein is secreted into extracellularly), and the 20th
Heavy chain variable region is formed to 137 amino acids residues, the 138th to 467 amino acids residue forms heavy chain constant region.Sequence table
In sequence 4, the 1st to 888 nucleotide forms CMV promoter, 3 institute of sequence of the 889th to 2292 nucleotide coding sequence table
The total length heavy chain shown, the 2293rd to 2490 nucleotide are ployA segment.
The amino acid sequence of Q206 full-length light chains is as shown in the sequence 5 of sequence table, the sequence 6 of encoding gene such as sequence table
It is shown.In the sequence 5 of sequence table, the 1st to 19 amino acids residue forms signal peptide (pilot protein is secreted into extracellularly), and the 20th
Light chain variable region is formed to 131 amino acids residues, the 132nd to 237 amino acids residues forms constant region of light chain.Sequence table
Sequence 6 in, the 1st to 888 nucleotide forms CMV promoter, the sequence 5 of the 889th to 1602 nucleotide coding sequence table
Shown in full-length light chains, the 1603rd to 1750 nucleotide is ployA segment.
The preparation of embodiment 2, antibody
One, the building of recombinant plasmid
Double chain DNA molecule shown in sequence 4 by sequence table is inserted into PMD18T carrier, obtains heavy chain expression vector.
Double chain DNA molecule shown in sequence 6 by sequence table is inserted into PMD18T carrier, obtains light chain expression vector.
Two, the building of recombinant cell
By heavy chain expression vector and light chain expression vector cotransfection 293T cell, recombinant cell is obtained.
Three, the preparation of antibody
1, the recombinant cell for taking step 2 to obtain, in the DMEM culture medium culture 72h containing 2% fetal calf serum, then 4 DEG C,
4000rpm is centrifuged 30min, collects supernatant.
2, affinity chromatography
The chromatographic column specification of affinity chromatography: length 3cm, internal diameter 1cm；
The column packing of affinity chromatography: protein A beads (Thermo, catalog number 10006D)；
Operating procedure: being 1. splined on affinity column for the supernatant that 300mL step (1) obtains, and 4 DEG C are incubated for 16 hours；
2. washing pillar with 60ml combination buffer；3. eluting destination protein with 30mL elution buffer, solution after column was collected.
Combination buffer: taking glycine 112.6g, sodium chloride 175.2g, is dissolved in water and is settled to 1L with water, uses hydroxide
Sodium tune pH to 8.0.
Elution buffer: taking glycine 7.5g, is dissolved in water and is settled to 500ml with water, with hydrochloric acid tune pH to 3.0.
3, the solution after crossing column that step 2 obtains is taken, is concentrated with ultrafiltration concentration pipe and system is replaced into PBS buffer solution
(pH7.2,10mM) obtains the antibody-solutions that 1ml protein concentration is 2mg/ml.
Embodiment 3, the effect of antibody
One, the preparation of EBOV pseudovirus
1, double chain DNA molecule shown in the sequence 8 by artificial synthesized sequence table is inserted into plasmid pcDNA3.1 (+)
Between BamHI and NotI restriction enzyme site, recombinant plasmid pcDNA3.1-GP △ muc is obtained.Double-strand shown in the sequence 8 of sequence table
Protein shown in the sequence 7 of DNA molecular polynucleotide (the GP △ muc albumen of EBOV).
2, by recombinant plasmid pcDNA3.1-GP △ muc and plasmid pNL4-3R-E-luciferase cotransfection 293T cell,
Obtain recombinant cell.
Containing the virus genomic full gene of HIV-1 (with wild HIV-1 disease in plasmid pNL4-3R-E-luciferase
The difference of virus gene group is only that frameshit has occurred in envelope gene).Recombinant plasmid pcDNA3.1-GP △ muc and plasmid
PNL4-3R-E-luciferase cotransfection host cell can express EBOV pseudovirus.EBOV pseudovirus only has virion
The memebrane protein on surface is GP △ muc albumen, other are the ingredient of HIV-1 virus, can only replicate base in infected cell
Because of group and expressing luciferase reporter gene, the virion of infectious cannot be generated again.The GP of GP △ muc albumen and EBOV
Albumen is compared, and difference, which is only that, has lacked one section of region mucin-like domain, and pseudovirus can be significantly improved by lacking this region
Into the ability of permissive cell.
3, the recombinant cell for obtaining step 2, in the DMEM culture medium culture 60h for containing 10% fetal calf serum.
4, after completing step 3, culture supernatant, the as virus liquid (abbreviation EBOV virus liquid) of EBOV pseudovirus are taken.
5, the virus titer in the ELISA kit detection EBOV virus liquid of p24 quantitative detection is utilized.
Two, the neutralization activity detection of monoclonal antibody
1, antibody-solutions prepared by Example 2, carry out doubling dilution with PBS buffer solution (pH7.2,10mM), are resisted
Body dilution.
2,96 porocyte culture plates are taken, 100 microlitres of antibody diluents and 50 microlitres of EBOV virus liquids are added in every hole, so that mixed
Antibody concentration in zoarium system is 50 μ g/ml, 16.67 μ g/ml, 5.56 μ g/ml, 1.85 μ g/ml, 0.62 μ g/ml, 0.21 μ g/
Ml, 0.07 μ g/ml or 0.02 μ g/ml (in terms of protein concentration), 37 DEG C stationary incubation 1 hour.With isometric PBS buffer solution
(pH7.2,10mM) replaces antibody diluent, as virus control.With isometric DMEM culture medium generation for containing 10% fetal calf serum
For EBOV virus liquid, as cell controls.
3, after completing step 2, the tissue culture plate is taken, every hole is inoculated with 100 microlitres of VeroE6 cell suspensions and (is used to prepare
The solvent of cell suspension is the DMEM culture medium containing 10% fetal calf serum, and the VeroE6 cell concentration in cell suspension is 2 × 105
A cell/ml), 37 DEG C stationary incubation 64 hours.
4, after completing step 3, the tissue culture plate is taken, inhales and abandons supernatant, 150 microlitres of lysates are added in every hole, and (micro- lattice are drawn
This biotechnology, article No. T003, by specification operation), 37 DEG C stationary incubation 5 minutes.
5, after completing step 4, the tissue culture plate is taken, detects uciferase activity.
Fluorescence intensity results are shown in Table 1, and (three multiple holes, secondary series, third column and the 4th column difference of table 1 is arranged in each processing
List three respective values of multiple holes).
The result of 1 fluorescence intensity of table
| Virus control | 453716 | 416989 | 515392 |
| Cell controls | 955 | 1081 | 1105 |
| Antibody concentration is 0.02 μ g/ml | 419562 | 478111 | 458209 |
| Antibody concentration is 0.07 μ g/ml | 407524 | 421839 | 311511 |
| Antibody concentration is 0.21 μ g/ml | 299560 | 348442 | 312615 |
| Antibody concentration is 0.62 μ g/ml | 151200 | 133032 | 126311 |
| Antibody concentration is 1.85 μ g/ml | 50524 | 44045 | 31867 |
| Antibody concentration is 5.56 μ g/ml | 37881 | 35521 | 27259 |
| Antibody concentration is 16.67 μ g/ml | 32032 | 38608 | 26517 |
| Antibody concentration is 50 μ g/ml | 23149 | 29638 | 21385 |
Neutralization activity (%)=[1- (fluorescence intensity-cell controls fluorescence intensity of test group)/(virus control it is glimmering
Luminous intensity-cell controls fluorescence intensity)] × 100%.
The result is shown in Figure 1 of neutralization activity.
Antibody concentration when neutralization activity is 50%, the i.e. IC of antibody are calculated using 5 software of Prism50Value, antibody
IC50Value is 0.36 μ g/ml.
Embodiment 4, the specificity of antibody
One, the preparation (VSV, that is, vesicular stomatitis virus) of VSV pseudovirus
1, double chain DNA molecule shown in the sequence 9 by artificial synthesized sequence table is inserted into plasmid pcDNA3.1 (+)
Between BamHI and NotI restriction enzyme site, recombinant plasmid is obtained.Double chain DNA molecule shown in the sequence 9 of sequence table encodes VSV's
Shell G glycoprotein.
2, the recombinant plasmid and plasmid pNL4-3R-E-luciferase cotransfection 293T cell obtained step 1, obtains
Recombinant cell.
3, the recombinant cell for obtaining step 2, in the DMEM culture medium culture 60h for containing 10% fetal calf serum.
4, after completing step 3, culture supernatant, the as virus liquid (abbreviation VSV virus liquid) of VSV pseudovirus are taken.
5, the virus titer in the ELISA kit detection VSV virus liquid of p24 quantitative detection is utilized.
Two, the preparation of HIV pseudovirus
1, double chain DNA molecule shown in the sequence 10 by artificial synthesized sequence table is inserted into plasmid pcDNA3.1 (+)
Between BamHI and NotI restriction enzyme site, recombinant plasmid is obtained.Double chain DNA molecule shown in the sequence 10 of sequence table encodes HIV-
The GP albumen of 1CNE30.
2, the recombinant plasmid and plasmid pNL4-3R-E-luciferase cotransfection 293T cell obtained step 1, obtains
Recombinant cell.
3, the recombinant cell for obtaining step 2, in the DMEM culture medium culture 60h for containing 10% fetal calf serum.
4, after completing step 3, culture supernatant, the as virus liquid (abbreviation inhibition of HIV liquid) of HIV pseudovirus are taken.
5, the virus titer in the ELISA kit detection inhibition of HIV liquid of p24 quantitative detection is utilized.
Three, the preparation (SUDV, that is, Ebola virus the Sudan hypotype) of SUDV pseudovirus
1, double chain DNA molecule shown in the sequence 11 by artificial synthesized sequence table is inserted into plasmid pcDNA3.1 (+)
Between BamHI and NotI restriction enzyme site, recombinant plasmid is obtained.Double chain DNA molecule shown in the sequence 11 of sequence table encodes SUDV
GP △ muc albumen.
2, the recombinant plasmid and plasmid pNL4-3R-E-luciferase cotransfection 293T cell obtained step 1, obtains
Recombinant cell.
3, the recombinant cell for obtaining step 2, in the DMEM culture medium culture 60h for containing 10% fetal calf serum.
4, after completing step 3, culture supernatant, the as virus liquid (abbreviation SUDV virus liquid) of SUDV pseudovirus are taken.
5, the virus titer in the ELISA kit detection SUDV virus liquid of p24 quantitative detection is utilized.
Four, the neutralization activity detection of monoclonal antibody
EBOV virus liquid is replaced with VSV virus liquid, inhibition of HIV liquid or SUDV virus liquid, the step of the other the same as in Example 3
Two.
Antibody is shown in Fig. 2 to the result of the neutralization activity of VSV virus.
Antibody is shown in Fig. 3 to the result of the neutralization activity of inhibition of HIV.
Antibody is shown in Fig. 4 to the result of the neutralization activity of SUDV virus.
The result shows that antibody-solutions prepared by embodiment two are to VSV virus, the neutralization activity of inhibition of HIV and SUDV virus
It is 20% hereinafter, for negative findings.
Claims (9)
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| CN111574621B (en) * | 2020-04-30 | 2022-03-29 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Monoclonal antibody for neutralizing EB virus and application thereof |
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