CN107033242A

CN107033242A - A kind of monoclonal antibody of anti-Ebola virus envelope glycoprotein in people source and application

Info

Publication number: CN107033242A
Application number: CN201710246616.0A
Authority: CN
Inventors: 陈薇; 于长明; 迟象阳; 范鹏飞; 侯利华; 李建民; 徐俊杰; 吴诗坡; 刘威岑; 宋小红; 陈旖; 张晓鹏; 房婷; 付玲
Original assignee: Institute of Bioengineering Chinese Academy of Military Medical Sciences
Current assignee: Institute of Bioengineering Chinese Academy of Military Medical Sciences
Priority date: 2017-04-16
Filing date: 2017-04-16
Publication date: 2017-08-11
Anticipated expiration: 2037-04-16
Also published as: CN107033242B

Abstract

The invention discloses a kind of anti-Ebola virus envelope glycoprotein GP in people source monoclonal antibody, the antibody has unique CDR region, and with unique action site, computer simulation analysis and biological experiment result confirm that the antibody had both acted on GP NPC1 receptor binding domains, GP glycan cap region is also acted on simultaneously, by playing unique neutralization mechanism with the collective effect of two domains of GP, play a part of anti-Ebola virus infection.The antibody shows higher antigen-binding activity and neutralization activity; 50% cell by virus attack can just be protected by only needing about 0.3 μ g/ml antibody; when antibody concentration is 100 μ g/ml; protective rate reaches 100%; therefore, the antibody that the present invention is provided is with a wide range of applications in ebola disease viral disease medicine is prepared.

Description

A kind of monoclonal antibody of anti-Ebola virus envelope glycoprotein in people source and application

Technical field

The invention discloses a kind of monoclonal antibody of anti-Ebola virus envelope glycoprotein and application, belong to microorganism and Field of immunology.

Background technology

Ebola virus (Ebola virus, EBOV) can trigger acute severe haemorrhage in the mankind and non-human primates body Pyreticosis, is one of fatal rate highest virus for finding so far, fatal rate is up to 90%, can have directly by contact transmission Extremely strong infectiousness and fatal rate.Ebola virus is by U.S. NIH (National Institutes of Health) and CDC (Centers for Disease Control) is classified as A classes biological agent/bio-terrorism agent (Biological agents), by it One of pathogen for being classified as there is major hidden danger and risk to national security and public health, at present still without granted prevention or Treat the medicine of Ebola virus.After Ebola virus is found within 1967,12 fairly large propagation are experienced altogether.2014 Maximum-norm, Ebola's epidemic situation most rambunctious in history have been broken out in year West Africa, are identified as Zaire type Ebola virus, this Secondary epidemic situation causes 10,000 many cases dead and the infection of 2.5 ten thousand many cases is (according to WHO [World Health Organization] 2016 Ebola's report of infectious disease of issue in 17 days 2 months), epidemic situation is traveled to beyond the African continent first, worldwide causes pole Big fear.The highest attention of the public and authorities has quickly propelled the research of Ebola's vaccine and antiviral drugs.

Several promising Ebola's vaccine clinical experiments are carried out rapidly both at home and abroad：Gorilla adenovirus type III carrier epidemic disease Seedling, Adenovirus Type 5 carrier bacterin, vesicular stomatitis virus carrier bacterin etc..The effective prevention class medicine of development is undeniably It is an important target, and precautionary measures are believed to save substantial amounts of life, but vaccine is difficult in some cases To play a role, such as：1) host individual there may be difference (such as immunocompetence of older, Jr. and immunocompromised It is weak)；2) immune effect gradually successively decreases after vaccine is immune for a long time；3) vaccine excites the immune of host can not resist high dose Ebola virus exposes；4) most patients only just seek medicine help after there is Ebola virus infection signal, and vaccine exists It is difficult to excite the effective immune response of host in of short duration treatment window phase.Therefore, Ebola controls in the process of vaccine development The research and development for treating medicine equally can not be ignored.Although currently without Ebola's medicine of approval, thering are some test-types to resist Among ebola disease cytotoxic drug is being studied, including siRNA, antisense oligonu-cleotides medicine, nucleotide analog, antibody Medicine etc..Wherein, the antibody drug " Zmapp " that is mixed by three plants of monoclonal antibodies and its optimization strain " MIL77 " are angstrom rich During drawing Epidemic outbreak of disease, the life of many people is successfully saved, its effect and treatment window phase length are far more than other kinds of resist Virus drugs, substantially increase drug safety and patient's cure rate, have given people greatly to inspire.Treated using antibody drug Filamentous virus causes worldwide concern, the focus as Ebola's drug research field.

GP on Ebola virus envelope surface has almost mediated all links of cell entry cell, is one important Virucidin's action target spot.Ebola virus GP genes are processed to two kinds of albumen, and a kind of is the non-structural type GP of secretion (secreted glycoprotein, sGP)；Another is structural type GP.Ebola GP is synthesized as a polypeptide first, Then GP1 (1-501 amino acids) and GP2 (502-676 amino acids) subunit are turned into by the digestion of Furin enzymes, two subunits lead to Cross disulfide bond to be connected, the transmembrane region of the tripolymer of formation inside GP2 is secured within envelope membrane surface, GP1 includes mucin (Mucin), the domain such as glycan cap (Glycan cap), head (Head), base portion (Base).However, simple Furin digestions Process is still not enough to excite the generation of Ebola's GP film fusion process.

After Ebola virus enters in vivo, combined with cell membrane surface receptors, but these host cell membrane surfaces is viscous The attached factor, is not that the acceptor that film fusion enters host cell occurs for real Ebola virus.Virus is incorporated into cell surface After adhesion factor, endocytosis and pinocytosis have been mediated by clathrin, the transport of primary and secondary endosome is there occurs.GP is in endosome It is middle by histone enzyme B and histone enzyme L digestions, eliminate on GP1 include mucin (Mucin) and glycan cap (Glycan cap) 60% amino acid inside, forms activated state GP (primed GP, GPcl), now have activated conclusive film fusion process Generation.GPcl combines generation film with interior body film albumen Niemann-Pick C1 (NPC1) and merged, so that viral RNA enters The viral genome that kytoplasm completes is replicated and transcribed, and new virus albumen post-assembly is into progeny virion, and from host cell Sprout on surface.

Three plants of people mouse that Zmapp is expressed by Tobacco System are fitted together to the cocktail that monoclonal antibody (c2G4, c4G7, c13C6) is constituted Therapeutic strategy, wherein c2G4 (epitope is C511, N550, G553, C556) and c4G7 (epitope is C511, D552, C556) are combined In the GP2 subunits of the glycoprotein GP on Ebola virus envelope surface, epitope exists overlapping；C13C6 (epitope is T270, K272) Glycan cap region is incorporated into approximately perpendicular angle.MIL77 is to optimize the Antibody Combination in Zmapp, MIL77-1/-2/-3 points The not other variable region containing c2G4, c4G7 and c13C6, its skeleton area is expressed in the engineered CHO of glycosyl after humanization modified Cell.The cocktail thereof therapy of MIL77-1 and the strain antibodies of ML77-3 two can rise after infecting 72 hours to non-human primates To 100% protection activity.

The multiple complementary effect of the monoclonal antibody of the Zmapp anti-Ebola virus envelope glycoprotein of successful application prompting Site is the key factor for treating Ebola virus infection, and obtaining the antibody with this unique effect site turns into prior art One technical need urgently to be resolved hurrily, it is with unique CDR region, simultaneously it is therefore an object of the present invention to provide a kind of full people source Experiments verify that with unique effect site and neutralizing mechanism, excellent antigen-binding activity, angstrom of neutralization activity are thus shown It is rich to draw monoclonal antibody, provide a kind of more efficiently candidate therapeutic means for Ebola virus infection.

The content of the invention

Based on foregoing invention purpose, present invention firstly provides a kind of list of the complete anti-Ebola virus envelope glycoprotein in people source Clonal antibody, the amino acid sequence such as SEQ ID NO in CDR1, CDR2 and CDR3 area of the weight chain variable district of the antibody：1 Shown in 26-33,51-58,97-116 amino acids sequence, CDR1, CDR2 and CDR3 area such as SEQ ID of the light chain variable district NO：Shown in 5 27-32,50-52,89-99 amino acids sequences.

In a preferred technical scheme, the amino acid sequence such as SEQ ID NO of the weight chain variable district of the antibody：1 It is shown, the amino acid sequence such as SEQ ID NO of the light chain variable district：Shown in 5.

In a technical scheme being more highly preferred to, the amino acid sequence such as SEQ ID of the heavy chain constant region of the antibody NO：Shown in 3, the amino acid sequence such as SEQ ID NO of the constant region of light chain：Shown in 7 or 9.

Second, present invention also offers a kind of alkali yl coding sequence for encoding said monoclonal antibody heavy chain and light chain, institute The alkali yl coding sequence of weight chain variable district of antibody is stated by SEQ ID NO：Shown in 2, the base of the light chain variable district of the antibody Coded sequence is by SEQ ID NO：Shown in 6.

In a preferred technical scheme, the alkali yl coding sequence of the heavy chain constant region of the antibody is by SEQ ID NO： Shown in 4, the alkali yl coding sequence of the constant region of light chain of the antibody is by SEQ ID NO：Shown in 8 or 10.

3rd, it can express the nucleosides of above-mentioned coding monoclonal antibody heavy and/or light chain present invention also offers a kind of The function element of coding sequences, this function element can be traditional expression vector.

In a preferred technical scheme, the function element is linear expression cassette.

4th, present invention also offers a kind of host cell containing above-mentioned linear expression cassette.

In a preferred technical scheme, the cell is 293T cells.

Finally, the application present invention also offers said monoclonal antibody in ebola disease viral disease medicine is prepared.

The monoclonal antibody for the anti-Ebola virus envelope glycoprotein in people source that the present invention is provided has unique CDR region, and With unique action site, computer simulation analysis and biological experiment result confirm that the antibody had both acted on GP NPC1 Receptor binding domain, while GP glycan cap region is also acted on, by playing uniqueness with the collective effect of two domains of GP Neutralization mechanism, plays a part of anti-Ebola virus infection.Based on this unique neutralization mechanism, the antibody that the present invention is provided shows Shown excellent antigen-binding activity and neutralization activity, it is only necessary to the μ g/ml of offer 0.3 antibody can just protect 50% by virus The cell of attack, when antibody concentration is 100 μ g/ml, protective rate reaches 100%.Therefore the result of the present invention is shown, described anti- Body has the wide variety of prospect in ebola disease viral disease medicine is prepared.

Brief description of the drawings

Fig. 1 flow sorted cells instrument sorts cell flow and sorting collection of illustrative plates；

The linear expression cassette schematic diagrames of Fig. 2；

Fig. 3 .ELISA detect monoclonal antibody and GP binding specificity schematic diagrames；

With the neutralization activity curve map of experiment detection antibody in Fig. 4 pseudovirus；

Fig. 5 .ELISA verify monoclonal antibody and GP and its truncate the combination schematic diagram of antigen；

The optimal mode that Fig. 6 computer simulation analysis 1B3 and GP are combined；

Fig. 7 A.Western Blot analyze the combination schematic diagram of 1B3 and GP mutant；

The combination schematic diagram of Fig. 7 B. flow cytometer showed 1B3 and GP mutant；

Fig. 8 competition binding elisa assay 1B3's acts on glycan cap area schematic

Embodiment

The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and It is apparent.But these embodiments are only exemplary, do not constitute any limitation to protection scope of the present invention.

The screening and preparation of the monoclonal antibody of the anti-Ebola virus envelope glycoprotein in the people source of embodiment 1

1. blood sample is gathered

After informed consent form is obtained, blood after collection restructuring Ebola vaccine clinical experiment subject is immune 14 days for the first time Liquid sample 5mL, for subsequent experimental.

2. airflow classification is unicellular

The blood sample of collection is separated into PBMC using Ficoll density-gradient centrifugation methods, process is as follows：

1) fresh anticoagulated whole blood, EDTA anti-freezings are taken.With isometric PBS or 0.09%NaCl dilution whole bloods.

2) separating liquid of certain volume is added in centrifuge tube, separating liquid ullage is arrived into the blood sample tiling after dilution, Keep two liquid level interfaces clear.Separating liquid, the not diluted whole blood of anti-freezing, PBS (or physiological saline) volume are 1:1:1.

3) trim, room temperature, horizontal rotor 700-800g (2000-2500rpm), 3~4acc of acceleration centrifuge 20- 30min。

4) after centrifugation terminates, ttom of pipe is red blood cell, and intermediate layer is separating liquid, and the superiors are blood plasma/tissue homogenate layers, blood plasma Layer is one layer of thin and finer and close tunica albuginea with separating between liquid layer, i.e.,：Mononuclearcell (including lymphocyte and monocyte) Layer.Careful tunica albuginea layer of drawing is into another centrifuge tube.

5) certain volume is diluted to PBS/1640, overturns and mix.Room temperature, horizontal rotor 250-400g (1000- 1500rpm), 5-10min is centrifuged, supernatant is abandoned.Repeated washing 1-2 times.

6) lymphocyte is resuspended with PBS or suitable culture mediums standby.

PBMC is used into streaming antibody staining：anti-CD3-FITC,anti-CD20-FITC,anti-CD19-PE- Alexa 610, anti-CD27-PE, anti-CD38-PE-Cy5, every 5 × 10⁶Individual PBMC cells respectively add the 5 above-mentioned antibody of μ L, After being incubated 30 minutes, PBS repeated washings 2-3 times containing 2%FBS are used.Utilize MoFlo XDP flow sorted cells instrument, selection Special cell surface marker thing (CD3neg/CD20low/CD19high/CD27high/CD38high) the sorting slurry of thick liquid cell is thin Born of the same parents (as shown in Figure 1), directly sort single thick liquid cell into 96 orifice plates, in 96 orifice plates per hole contain 5U RNase inhibitors and 19.8 μ L are gone in the water of RNase, -80 DEG C of preservations.

3. singe-cell PCR expands full Human monoclonal antibody gene

1) reverse transcription PCR

With specific reference to specification (QIAGEN, 210212), program is simply described below：

By airflow classification 2112 it is unicellular, therefore have 2112 reverse transcription reaction systems.To each reaction system In add simultaneously and following whole be directed to heavy chain (H), κ light chains, the special primer of each hypotype of lambda light chain (primer sequence is shown in Table 1).

Primer：

H：5 ' L-VH 1, L-VH 3, the L-VH 5 of L-VH 4/6,5 ', HuIgG-const-anti, 3 ' C μ CH1

κ：5′L Vκ1/2、5′L Vκ3、5′L Vκ4、3′Cκ543–566

λ：5′L Vλ1、5′L Vλ2、5′L Vλ3、5′L Vλ4/5、5′L Vλ6、5′L Vλ7、5′L Vλ8、3′Cλ

The reverse transcription PCR primer of table 1

Included in PCR reaction systems：The μ L of 5 × buffer solution 6, dNTP 1.2 μ L, the μ L of reverse transcriptase 1.2, primer as above, template For unicellular, water polishing to 30 μ L.

PCR reaction conditions are：50 DEG C of reverse transcriptions 30min, 95 DEG C of pre-degeneration 15min, then 95 DEG C of 40s, 55 DEG C of 30s, 72 DEG C 1min, 40 circulations, last 72 DEG C of extensions 10min.

2) nest-type PRC

Using reverse transcription product as template, 3 nest-type PRCs reaction amplification H, κ, λ are carried out respectively, and detailed process is as follows：

Primer：

H：VH3a-sense、VH3b-sense、MuD、PW-Cgamma

κ：5′Pan Vκ、3′Cκ494–516

λ：5 ' AgeI V λ 1,5 ' AgeI V λ 2,5 ' AgeI V λ 3,5 ' AgeI V λ 4/5,5 ' AgeI V λ 6,5 ' AgeI V λ 7/8、3′XhoI Cλ

The nest-type PRC primer of table 2

Included in PCR reaction systems：The μ L of 10 × buffer solution 2.5, the μ L of 10mM dNTP 0.5, the μ L of archaeal dna polymerase 0.25, draw Thing as above, template be the μ L of reverse transcription product 1, water polishing to 25 μ L.

PCR reaction conditions are：94 DEG C of pre-degeneration 4min, then 94 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 45min, 40 circulations, Last 72 DEG C of extensions 10min.

3) agarose gel electrophoresis

One unicellular middle heavy chain and light chain gene expand successful clone, it is considered to be the clone of successful matching.Take 5 μ L nested PCR amplifications products take the positive colony of pairing to be sequenced after 1% agarose gel electrophoresis, and it is anti-that sequencing is obtained Body variable region sequences are analyzed with Vector NTI softwares and IMGT websites, and carry out antibody protein expression and functional verification.

4. linear expression cassette expresses antibody

By above-mentioned reverse transcription reaction, the antibody sequence of 462 pairings is obtained in single cell clone, if using tradition Cloning expression method waste time and energy, antibody can quickly be expressed by the method for building linear expression cassette.This method it is basic Principle is by promoter sequence (GENBANK registration numbers directly by Overlap extension PCR：X03922.1), the volume of antibody leader peptide Code sequence is (by SEQ ID NO:Shown in 11), antibody variable region (being obtained from unicellular middle amplification), (raw work is biological for antibody constant region Synthesis, heavy chain constant region sequence is by SEQ ID NO:Shown in 3, DNA encoding sequence is by SEQ ID NO:Shown in 4, Kappa type light chains Constant-region sequences are by SEQ ID NO:Shown in 7, DNA encoding sequence is by SEQ ID NO:Shown in 8, Lamda type constant region of light chain sequences Row are by SEQ ID NO:Shown in 9, DNA encoding sequence is by SEQ ID NO:Shown in 10), poly A tract (GENBANK registration numbers： X03896.1) connect, the DNA transfections of the linear forms are entered in cell and carry out antibody expression.

Detailed process is as follows：

1) with pSec Tag2 (Invitrogen) for template, amplification promoter-leader fragment, poly A tract fragment.

The PCR reaction systems of amplification promoter-leader fragment include：Template plasmid pSec Tag2 (Invitrogen) 1ng, the μ L of 10 × buffer solution 5, the μ L of 10mM dNTP 1, the μ L of archaeal dna polymerase 0.5, primer 5'CMV-FORWARD (CGATGTACGGGCCAGATATACGCGTTG), primer 3'leader-sequence (GTCACCAGTGGAACCTGGAACCCA), water polishing is to 50 μ L.

The PCR reaction systems of amplification poly A tract fragment include：Template plasmid pSec Tag2 (Invitrogen) 1ng, The μ L of 10 × buffer solution 5, the μ L of 10mM dNTP 1, the μ L of archaeal dna polymerase 0.5, primer 5'POLY (A) (GCCTCGACTGTGCCTTCTAGTTGC), primer 3'POLY (A) (TCCCCAGCATGCCTGCTATTGTCT), water polishing to 50 μL。

PCR reaction conditions are：94 DEG C of pre-degeneration 4min, then 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 30 circulations, most 72 DEG C extend 10min afterwards.

2) antibody constant region is expanded.

Included in H chain constant region PCR systems：Heavy chain constant region template 10ng, the μ L of 10 × buffer solution 5, the μ of 10mM dNTP 1 L, the μ L of archaeal dna polymerase 0.5, primer 5'CH (ACCAAGGGCCCATCGGTCTTCCCC), primer 3'CH (GCAACTAGAAGGCACAGTCGAGGCTTTACCCGGAGACAGGGA), water polishing is to 50 μ L.

Included in κ chain constant region PCR systems：κ chain constant region templates 10ng, the μ L of 10 × buffer solution 5, the μ L of 10mM dNTP 1, The μ L of archaeal dna polymerase 0.5, primer 5'C κ (ACTGTGGCTGCACCATCTGTCTTC), primer 3'C κ (GCAACTAGAAGGCACAGTCGAGGCACACTCTCCCCTGTTGAAGCT), water polishing is to 50 μ L.

Included in λ chain constant region PCR systems：λ chain constant region templates 10ng, the μ L of 10 × buffer solution 5, the μ L of 10mM dNTP 1, The μ L of archaeal dna polymerase 0.5, primer 5'C λ (GAGGAGCTTCAAGCCAACAAGGCCACA), primer 3'C λ (GCAACTAGAAGGCACAGTCGAGGCTGAACATTCTGTAGGGGCCAC), water polishing is to 50 μ L.

PCR reaction conditions are：94 DEG C of pre-degeneration 4min, then 94 DEG C of 30s, 60 DEG C of 60s, 72 DEG C of 3min, 30 circulations, most 72 DEG C extend 10min afterwards.

3) antibody variable region is expanded.

Included in PCR system：Template is reverse transcription PCR product 10ng, the μ L of 10 × buffer solution 5,10mM dNTP 1 μ L, DNA The μ L of polymerase 0.5, primer (after mixing heavy chain and light chain primer respectively in addition system), water polishing to 50 μ as shown in table 3 L。

PCR reaction conditions are：94 DEG C of pre-degeneration 4min, then 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 3min, 30 circulations, most 72 DEG C extend 10min afterwards.

The linear list of table 3. reaches frame construction PCR primer

4) PCR primer recovery purifying：By above PCR primer after 1% agarose gel electrophoresis, cut glue and use OMEGA companies QIAquick Gel Extraction Kit is reclaimed.

5) the linear expression cassette of heavy chain and light chain is expanded respectively.

The splicing sequential schematic of linear expression cassette is as shown in Figure 2.In Fig. 2, A is the linear expression cassette of H chains, and B is that κ chains are linear Expression cassette, C is the linear expression cassette of λ chains.

PCR reaction systems include：

Template：Promoter after purification-leader fragment 10ng, heavy chain/light chain variable district fragment 10ng, heavy chain/light Chain constant segments 10ng, poly A tract fragment 10ng, the μ L of 10 × buffer solution 2.5, the μ L of 10mM dNTP 0.5, archaeal dna polymerase 0.25 μ L, primer 5'CMV-FORWARD (CGATGTACGGGCCAGATATACGCGTTG) and 3'POLY (A) (TCCCCAGCATGCCTGCTATTGTCT), water polishing is to 25 μ L.

6) PCR primer recovery purifying：PCR reaction products are directly reclaimed with OMEGA companies QIAquick Gel Extraction Kit.

7) DNA is quantitative：PCR recovery products are quantified with Nano (GE Healthcare).

8) cell is inoculated with：By 293T cells with 2 × 10⁵/ mL is inoculated in 24 porocyte culture plates, is containing 5%CO₂'s In cell incubator, 37 DEG C of overnight incubations.

9) cell cotransfection：Next day, into the MEM culture mediums of 200 μ L serum-frees, add the heavy chain and light chain successfully constructed Linear expression cassette each 1 μ g of PCR primer, added after mixing 4 μ L transfection reagents Turbofect (Thermo Scientific, R0531), added to dropwise in the 293T cell culture wells of incubated overnight after common incubation 15-20min.Containing 5%CO₂It is thin It is standby that cells and supernatant is received in born of the same parents' incubator, after 37 DEG C of culture 48h.

Antibody of the 5.ELISA screenings with binding activity

1) the hole elisa plate of the previous day 96 is tested, lectin and Goat anti-human IgG-Fc antibody is coated with respectively.With Lectin and Goat anti-human IgG-Fc antibody is diluted to final concentration of 10 μ g/mL by ELISA coating buffers respectively, per hole 100 μ L are coated with.And other antigens such as PA, LF, EF are coated with, coated elisa plate is put into wet box, 4 DEG C are overnight.

2) experimental day, which is got rid of, abandons liquid in hole, is washed with ELISA cleaning solutions 2 times.

3) 100 μ L confining liquids are added per hole, are placed 1 hour at room temperature.

4) get rid of and abandon liquid in hole, washed with cleaning solution 2 times.

5) GP antigens are caught, by GP protein expressions supernatant and sample diluting liquid 1:1 dilution, is added to being coated with lectin's In hole, room temperature is placed 1 hour.

6) get rid of and abandon liquid in hole, washed with cleaning solution 6 times.

7) 100 μ L monoclonal antibody dilution is added, using MIL77-2 as positive control, the moon is used as using other nonspecific antibodies Property control.

8) it is stored at room temperature 1 hour.

9) get rid of and abandon liquid in hole, washed with cleaning solution 6 times.

10) the goat anti-human igg's secondary antibody for marking HPR is with 1:20000 are diluted with dilution, are added to per the μ L of hole 100 In elisa plate corresponding aperture, it is incubated at room temperature 1 hour；

11) get rid of and abandon liquid in hole, washed 6 times, discarded for the last time after cleaning solution, by residual liquid in blotting paper with cleaning solution It is upper softly to pat dry as far as possible；

12) 100 μ L TMB one-component nitrite ions are added per hole, are developed the color 5 minutes, room temperature lucifuge adds 50 μ L per hole afterwards Terminate liquid terminating reaction；

13) with the OD values detected on ELIASA at 450-630nm, keeping records initial data；

As a result：462 plants of monoclonal antibodies are screened, and specificity identification is carried out to Ebola GP binding antibody.Such as Fig. 3, In 24 strain antibodies with binding activity, have 17 plants with Ebola GP can specific bond, including 1B3,1D8,2G10,4C4, 4F2,5C3,5D2,5G1,10B7,17B5,17C2,17F3,19A8,19G9,20A10,20E8,20H7, and other several strain antibodies Then there is non-specific binding with PA or other antigens.

6. there is the antibody of neutralization activity in pseudovirus with experiment screening

1) pseudovirus is pressed 1:6 are diluted with 1640 culture mediums, with pseudovirus diluted monoclonal antibody, and are carried out twice successively Dilution, the hole to add MIL77-2 antibody is not added with pseudovirus and only adds the hole of culture medium as complete living pair as positive control According to being added without the hole of antibody only to add pseudovirus and be used as complete dead control.

2) pseudovirus and mixtures of antibodies are placed into 37 DEG C of incubators to be incubated 1 hour.

3) one piece of 96 orifice plate separately is taken, 2 × 10 will be spread after 293 cell counts per hole⁴Cell, per the μ L of hole 100.

4) the antibody virus solution in 96 orifice plates of incubation is transferred in the hole of correspondence cell plates, 100 μ L is shifted per hole； Full control wells living add 100 μ L culture medium, and complete dead control wells add 100 μ L pseudovirus solution.

5) 96 porocyte culture plates are put into after being cultivated 36~48 hours in incubator, take out Tissue Culture Plate, it is careful to suction out Nutrient solution is discarded.

6) each hole adds 350rpm concussions 15min on 100 μ L cell pyrolysis liquids, oscillator.Afterwards, 3000rpm is centrifuged 10min。

7) by Luciferase Assay Substrate (lyophilized, detection substrate is freeze-dried) and Luciferase Assay Buffer (detection buffer solution) mixing blow it is even after, fill whole signal piping.

8) 20 μ L of supernatant readings are drawn, fluorescent value, protective rate of the calculating antibody to cell is read.

As a result：In such as Fig. 4, the monoclonal antibody of 17 plants of Ebola's GP specific bonds, there is one plant of monoclonal antibody 1B3 with neutralization activity, 50% cell can just be protected by only needing about 0.3 μ g/ml antibody, and when antibody concentration is 100 μ g/ml, protective rate reaches 100%.And cell with extremely compareing indifference entirely in hole where other monoclonal antibodies, such as 4F2, hole where also a kind of monoclonal antibody, such as 20E8 Middle cell survival rate is less than complete dead control.Pass through sequence analysis, the amino acid sequence such as SEQ ID of monoclonal antibody 1B3 weight chain variable district NO：Shown in 1, the amino acid sequence such as SEQ ID NO of light chain variable district：Shown in 3, by further analyzing, the heavy chain of antibody can Become the amino acid sequence such as SEQ ID NO in CDR1, CDR2 and CDR3 area in area：1 26-33,51-58,97-116 amino acids Shown in sequence, CDR1, CDR2 and CDR3 area such as SEQ ID NO of the light chain variable district：3 27-32,50-52,89-99 Shown in amino acid sequence.

Embodiment 2：The Characterization of antigenic epitopes that neutralization monoclonal antibody 1B3 is combined with GP

1.1B3 and GP and its combination analysis for truncating antigen

ELISA detects monoclonal antibody and truncates the combination of antigen.Three kinds of GP truncation antigen GP1 (33~500aa) of structure, GPdmucin (33~310aa, 463~632aa), sGP (33~294aa) expression plasmid, with Expi293 mammalian cell systems Above-mentioned three kinds of truncations antigen is expressed, GP total lengths is caught respectively with the lectin for being coated in elisa plate and truncates antigen, identify 17 plants The binding activity of specific antibody and GP and its truncation antigen.With Zmapp optimization strain MIL77-1/2/3 and the special monoclonal antibody of anthrax 8A7 is respectively as positive and negative control.

As a result：Can combine such as Fig. 5, MIL77-3 and the GP of four kinds of forms, MIL77-1/2 be only capable of with GP total lengths and GPdmucin is combined, and the combination epitope region of document report is consistent, negative control antibody 8A7 then with four kinds of form antigens It can not combine.In 17 plants of specific antibodies, it can be combined comprising 16 strain antibodies including 1B3 and the GP of four kinds of forms, illustrate 1B3 The epitope that monoclonal antibody is combined with GP is located at 33~294aa regions on GP1.

2.1B3 and GP action sites computer simulation analysis

Acted on using Discovery Studio softwares simulation 1B3 with GP albumen, input 1B3 variable region nucleic acid sequences, led to Cross Macromolecules modules and three-dimensional modeling is carried out to 1B3Fab first.The Ebola's GP crystal structures downloaded from PDB storehouses File 3CXY.1B3 and GP are docked using ZDock modules, the combination Pose of antigen-antibody is given a mark, each ammonia is calculated The comprehensive grading of base acid.The high amino acid of selection marking is screened, and is obtained antigen of the combination interface comprising key amino acid and is resisted Body combination Pose gathers, and therefrom analysis obtains optimal combination Pose.

As a result：Optimal mode such as Fig. 6 that the 1B3 and GP of computer simulation analysis are combined, 1B3 be incorporated into GP 33~ Between 294aa, the region is located at GP1 subunits, and analog result is consistent with above-mentioned ELISA results.On GP1 subunits, GP and 1B3 is tied It is 172,112,117,119,120,118,114,58,90,54 successively to close 10 amino acid sites before RCF algorithms marking highest Site, wherein the critical sites that GPcl and NPC1 that 112,114 and 118 sites are document reports are combined.Illustrate that 1B3 is very possible It is incorporated into GP NPC1 receptor binding domains.

The identification of 3.1B3 and GP1 NPC1 receptor binding domains combination

Western Blot and flow cytometer showed 1B3 and GP mutant combination.Select the pass of computer simulation epitope analysis Key amino acid：170th, 171,172,112,114,117,118,119 and 120 are mutated into alanine respectively, are cloned into surface exhibition Show in plasmid pDisplay (Invitrogen).Transfect after 293T cells, be used for Western in after 48h, taking a little cell Blot, remaining cell is used for streaming staining analysis.The HA labels for being located at Insert Fragment c-terminus in pDisplay plasmids are selected to make To weigh the basis for estimation of GP expression quantity.

As a result：Western Blot results show, in the case of identical GP applied sample amounts are ensured, 117,118 and 171 ammonia GP and 1B3 combination is significantly reduced (Fig. 7 A) after base acid is mutated respectively.Flow cytometer showed result shows 118 and 172 amino acids The GP and 1B3 of site mutation binding activity lose (Fig. 7 B).The above results show that 117,118,171 and 172 amino acids are The key amino acid that GP is combined with 1B3, the adjacent amino acid of these two pair is located at the two ends of GP acceptor NPC1 lands.Therefore 1B3 May be by these sites being incorporated into the way of on receptor binding domain, and then by influenceing GP and NPC1 combination to play guarantor Shield activity.

The identification of 4.1B3 and GP1 glycan cap area combination

Using competition binding elisa assay 1B3 antigenic action epitope.With the MIL77-1/2/3 of known combination epitope and Remaining two plants are incorporated into the specific antibody in sGP regions as control antibodies, and these antibody and GP knot are investigated using competitive ELISA Epitope is closed with the presence or absence of overlapping.Keep the amount of detection antibody constant in experiment, add the biotin labeling of various concentrations gradient Antibody is competed, the competition antibody whether investigation detection antibody and GP combination can be incubated together is blocked.

As a result：Such as Fig. 8,1B3 antibody exists with MIL77-3,2G10,5C3 to be competed, and illustrates these antibody binding epitope spaces Approach or exist overlapping.MIL77-3 is the monoclonal antibody of one plant of 270 and 272 amino acids for acting on GP1 glycan caps area, illustrates 1B3 GP NPC1 receptor binding domains are not only incorporated into, while acting on close to the glycan cap region of MIL77-3 epitopes in GP1, and are somebody's turn to do The ebola antibody of the mode of action has no report at present.In view of 1B3 and MIL77-3 has close epitope, it is in having preferably again With the full Human monoclonal antibody of activity, 1B3 very likely substitutes MIL77-3 and MIL77-1 and constitutes better cocktail treatments plan Slightly.

Sequence table

<110>Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A

<120>A kind of monoclonal antibody of anti-Ebola virus envelope glycoprotein in people source and application

<160> 11

<170> PatentIn version 3.3

<210> 1

<211> 127

<212> PRT

<213> Homo sapiens

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Glu Ile Trp Phe Gly Glu Leu Leu Tyr Thr Ser Tyr

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 2

<211> 381

<212> DNA

<213> Homo sapiens

<400> 2

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt agctatagca tgaactgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtttcatac attagtagta gtagtagtac catatactac 180

gcagactctg tgaagggccg attcaccatc tccagagaca atgccaagaa ctcactgtat 240

ctgcaaatga acagcctgag agacgaggac acggctgtgt attactgtgc gagagatcgg 300

gaaatatggt tcggggagtt attatatacg agctacggta tggacgtctg gggccaaggg 360

accacggtca ccgtctcctc a 381

<210> 3

<211> 330

<212> PRT

<213> Homo sapiens

<400> 3

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 4

<211> 990

<212> DNA

<213> Homo sapiens

<400> 4

gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcag ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 960

cagaagagcc tctccctgtc tccgggtaaa 990

<210> 5

<211> 109

<212> PRT

<213> Homo sapiens

<400> 5

Asp Ile Val Met Thr Gln Thr Pro Val Ile Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

Gly Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 6

<211> 328

<212> DNA

<213> Homo sapiens

<400> 6

gatattgtga tgacccagac tccagtcatc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccctccggg ctacactttt 300

ggccagggga ccaaggtgga aatcaaac 328

<210> 7

<211> 106

<212> PRT

<213> Homo sapiens

<400> 7

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 8

<211> 318

<212> DNA

<213> Homo sapiens

<400> 8

actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 60

actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 120

aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 180

aaggacagca cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa 240

cacaaagtct acgcctgcga agtcacccat cagggcctga gttcgcccgt cacaaagagc 300

ttcaacaggg gagagtgt 318

<210> 9

<211> 106

<212> PRT

<213> Homo sapiens

<400> 9

Arg Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

35 40 45

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 10

<211> 318

<212> DNA

<213> Homo sapiens

<400> 10

cgtcagccca aggctgcccc ctcggtcact ctgttcccac cctcgagtga ggagcttcaa 60

gccaacaagg ccacactggt gtgtctcata agtgacttct acccgggagc cgtgacagtg 120

gcctggaagg cagatagcag ccccgtcaag gcgggagtgg agaccaccac accctccaaa 180

caaagcaaca acaagtacgc ggccagcagc tacctgagcc tgacgcctga gcagtggaag 240

tcccacaaaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga gaagacagtg 300

gcccctacag aatgttca 318

<210> 11

<211> 60

<212> DNA

<213> Homo sapiens

<400> 11

atggattcac aggcccaggt tcttatgtta ctgctgctat gggtatctgg tacctgtggg 60

Claims

1. a kind of monoclonal antibody of the anti-Ebola virus envelope glycoprotein in people source, it is characterised in that the heavy chain of the antibody can Become the amino acid sequence such as SEQ ID NO in CDR1, CDR2 and CDR3 area in area：1 26-33,51-58,97-116 amino acids Shown in sequence, CDR1, CDR2 and CDR3 area such as SEQ ID NO of the light chain variable district：5 27-32,50-52,89-99 Shown in amino acid sequence.

2. antibody according to claim 1, it is characterised in that the amino acid sequence such as SEQ of the weight chain variable district of the antibody ID NO：Shown in 1, the amino acid sequence such as SEQ ID NO of the light chain variable district：Shown in 5.

3. antibody according to claim 2, it is characterised in that the amino acid sequence such as SEQ of the heavy chain constant region of the antibody ID NO：Shown in 3, the amino acid sequence such as SEQ ID NO of the constant region of light chain：Shown in 7 or 9.

4. a kind of alkali yl coding sequence of coding claim the 1-3 any monoclonal antibody heavy and light chain, its feature exists In the alkali yl coding sequence of the weight chain variable district of the antibody is by SEQ ID NO：Shown in 2, the light chain variable district of the antibody Alkali yl coding sequence is by SEQ ID NO：Shown in 6.

5. sequence according to claim 4, it is characterised in that the alkali yl coding sequence of the heavy chain constant region of the antibody by SEQ ID NO：Shown in 4, the alkali yl coding sequence of the constant region of light chain of the antibody is by SEQ ID NO：Shown in 8 or 10.

6. a kind of can express the nucleotide coding sequence of coding monoclonal antibody heavy and/or light chain described in claim 5 Function element.

7. the function element according to claim requirement 6, it is characterised in that the function element is linear expression cassette.

8. a kind of host cell containing linear expression cassette described in claim 7.

9. the host cell according to claim requirement 8, it is characterised in that the cell is 293T cells.

10. application of any described monoclonal antibodies of claim 1-3 in ebola disease viral disease medicine is prepared.