CN107266566B - The anti-EV71 virus neutrality antibody E1 of source of people and its application - Google Patents
The anti-EV71 virus neutrality antibody E1 of source of people and its application Download PDFInfo
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- CN107266566B CN107266566B CN201710418696.3A CN201710418696A CN107266566B CN 107266566 B CN107266566 B CN 107266566B CN 201710418696 A CN201710418696 A CN 201710418696A CN 107266566 B CN107266566 B CN 107266566B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
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- C07K2317/55—Fab or Fab'
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- C07—ORGANIC CHEMISTRY
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C07—ORGANIC CHEMISTRY
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- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Communicable Diseases (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a kind of anti-EV71 virus neutrality antibody E1 of source of people obtained using display technique of bacteriophage screening, the amino acid sequence of light chain and heavy chain variable region is respectively as shown in SEQ ID No.1 and SEQ ID No.2.The antibody can specific recognition EV71 virion antigen, significant enzyme linked immunoassay can occur with EV71 virus, and have the function of the neutralization activity of anti-EV71 virus infection.In addition, antibody of the invention can be made to the specific antibodies medicine for preventing and treating hand-foot-and-mouth disease, thus for preventing and treating the hand-foot-and-mouth disease as caused by EV71 virus in clinic.
Description
Technical field
The present invention relates to genetic engineering and display technique of bacteriophage fields, specifically, being related to a kind of anti-EV71 disease of source of people
Malicious neutrality antibody E1 and its application.
Background technique
Hand-foot-and-mouth disease (Hand foot mouth disease, HFMD) is the infectious disease being caused by enterovirus, and is mostly occurred
The infant below in 5 years old, can cause fash, the ulcer at the positions such as fever and hand, foot, oral cavity, and few patients can cause cardiac muscle
The complication such as inflammation, pulmonary edema, aseptic meningoencephalitis.The enterovirus for causing hand-foot-and-mouth disease has more than 20 kinds, wherein Coxsack disease
Malicious (Cox Asckievirus) A16 type (Cox A16) and enterovirns type 71 (Enterovirus71.EV71) are most common.
Schmidt in 1974 et al. reports that breaks out (1969~1973 years) from California, USA shows as nervous system disease for the first time
The patient's body of shape disease is separated to EV71, and then, many countries report EV71 virus in different regions in succession in the world
Popularity, people gradually recognize that EV71 virus is the main pathogen of hand-foot-and-mouth disease.
Since hand-foot-and-mouth disease is currently without corresponding vaccine and specific drug, prepare that efficient, inexpensive, side reaction is small
Passive immunity preparation have become a hot topic of research.Using the source of people containing specific antibody or animal blood serum immunoglobulin come pre-
Anti- and treatment infectious disease is long-standing.The extracorporeal antivirus effect neutralization activity of monoclonal antibody and in vivo protection body are resisted virus and are attacked
It hits and has obtained many it is demonstrated experimentally that such as Hantaan virus, measles virus, RSV virus, rabies viruses neutralizing monoclonal antibody can
Protect animal from virus attack in vivo 100%.
Immunoglobulin (immunoglobulin, Ig) is exempted from as antibody component mainly from donor (convalescent)
Epidemic disease serum takes a long time from positive serum is obtained to by safety detection, and need to put into a large amount of manpower and financial resources, this just makes
Its a large amount of preparation is restricted, simultaneously because antibody is mainly derived from serum, therefore is easy to happen the sense of hematogenous infectious disease
Dye.And these defects can be then overcome using source of people gene engineering product substitution blood product, as human genetically engineered antibody is studied
Deepen continuously, to this field biological products development bring new hope and bright prospects.
(Phage Display Techniques, is shown in Barbas, C.F. to the display technique of bacteriophage that the beginning of the nineties rises
Equal, 1991) source of people or genetic engineering antibody and the development of whole gene engineered antibody technical field of research, have been greatly facilitated
Developmental research, and substantive application study and development phase are stepped by phase of basic research.Source of people Virus-resistant genetic engineering is anti-
The research success of body, especially source of people whole antibody, prevents and treats to the specificity of various viral infectious and opens new think of
Road, and a new class of antiviral agent is gradually developed in viral infection resisting biomedicine field.
Summary of the invention
The object of the present invention is to provide a kind of anti-EV71 virus neutrality antibody E1 of source of people and its applications.
In order to achieve the object of the present invention, the anti-EV71 virus neutrality antibody E1 of source of people of the present invention or its active fragment, it is described
The amino acid sequence of the light chain and heavy chain hypervariable region CDR1, CDR2 and CDR3 of antibody E1 or its active fragment is as shown in the table:
Antibody E1, i above-mentioned) its light chain variable region amino acid sequence is as shown in SEQ ID No.1 or the sequence is through replacing
Change, lack or add one or several amino acids formed amino acid sequences with same function.For example, by the 15th
Arg, which replaces with Lys, will not influence the function of albumen.
Ii) amino acid sequence of its heavy chain variable region as shown in SEQ ID No.2 or the sequence through replacement, lack or add
Add one or several amino acids formed amino acid sequences with same function.For example, the 8th Lys is replaced with Ala not
It will affect the function of albumen.
The present invention also provides the genes of encoding said antibody E1.Wherein, the core of coding light chain variable region and heavy chain variable region
Nucleotide sequence is respectively as shown in SEQ ID No.3 and SEQ ID No.4.
The present invention also provides expression cassette, expression vector or cloning vectors comprising the gene comprising encoding said antibody E1
The nucleic acid of sequence.
The present invention also provides the encoding gene containing the antibody E1 or the host cells of the expression cassette, carrier.
The present invention also provides the engineered obtained single-chain antibody ScFv of the antibody E1 or its active fragment or Fab antibody or
Whole antibody Immunoglobulin IgG.
The anti-EV71 virus neutrality antibody E1 of source of people of the present invention, can be prepared as follows: utilize phage display technology
Show technology, acquire multiple patient's EV71 convalescence peripheral blood lymphocytes, the anti-EV71 of source of people is constructed by gene engineering method
Virus gene engineering antibody library, and screen the genetic engineering antibody Fab segment for obtaining the anti-EV71 virus of specificity.
The antibody is hypervariable region (CDRs) specific gene sequence by being present in antibody light chain and heavy chain gene variable region
What column determined, and the functional antibodies of the specific binding EV71 virus of effective expression can be obtained in prokaryotic cell.It is special
Opposite sex identification EV71 virion antigen with EV71 virus there is apparent enzyme linked immunological (ELISA) to react and anti-EV71 be viral
The neutralization activity function of infection.
The light chain and heavy chain variable region gene of antibody E1 specificity are from EV71 antiviral antibody gene pool anti-to source of people
Specific rich product screening, the foundation of the antibody library derive from China EV71 virosis human peripheral lymphocyte gene.Its light chain
Framework sequence between the corresponding three CDR region combined sequences of heavy chain variable region and its CDR region constitutes the antibody variable
Region sequence feature, E1 belong to antibody light chain family VL1.Antibody protein function is by being present in antibody gene light chain and heavy chain variable region
Determinant complementary region CDR1, CDR2 and CDR3 in specific nucleotide sequences and its complementary series determined that 6 corresponding
CDR region amino acid sequence constitutes the antigen-specific binding region domain of antibody, determines the antigen binding characteristics of antibody of the present invention
And anti-EV71 viral function feature.
In addition, it is contemplated that the degeneracy of codon, such as can be in its code area, in the condition for not changing amino acid sequence
Under, the gene order for encoding above-mentioned Fab fragment antibody is transformed, the gene for encoding antibody with the same function is obtained.
Those skilled in the art can be according to the codon-bias of expression antibody host, artificial synthesized modifying gene, to improve antibody
Expression efficiency.
Further, the present invention recombinates the light chain variable region of above-mentioned Fab antibody and heavy chain variable region, obtains molecule
Measure smaller single-chain antibody (ScFv), the antibody equally can specific recognition EV71 viral surface antigen, have exempt from into the cell
The effect of epidemic disease.Single-chain antibody penetration power is strong, easily enters local organization and plays a role.
Can be by the gene of above-mentioned encoding Fab antibodies, ScFv gene cloning into expression vector, and then host is converted, pass through
Inducing expression obtains Fab antibody and single-chain antibody.
In addition, the light chain encoding gene of above-mentioned Fab antibody and heavy chain encoding gene can be cloned into complete anti-expression vector,
And import in host cell, obtain the full anti-immunoglobulin for expressing anti-EV71 virus.
Using ELISA, SDS-PAGE, the methods of experiment is neutralized to the E1 antibody progress Function Identification of acquisition, the results showed that
Human antibody E1 has specific binding for FUYANG-0805 plants of EV71 virions, is carried out using experiment is neutralized to Fab antibody
Function Identification, the results showed that E1 has preferable neutralization activity.
The present invention also provides the antibody E1 or its active fragment in preparation prevention or to treat the hand as caused by EV71 virus
Application in the drug of sufficient stomatosis.
The present invention also provides the antibody E1 or its active fragment in preparation EV71 virus antigen detection reagent or detection examination
Application in agent box.
The present invention further provides drug, detection reagent or detection reagents containing the antibody E1 or its active fragment
Box.
The present invention utilizes display technique of bacteriophage, and the source of people neutrality antibody that specificity is directed to EV71 virus has successfully been obtained
E1；Using the anti-EV71 virus gene engineering antibody variable region gene of the source of people neutrality of above-mentioned acquisition, Fab antibody gene and it is somebody's turn to do
Full-antibody gene under antibody gene feature, can in prokaryotic cell, yeast cells, eukaryocyte and any recombination system table
The antibody or improved any other gene containing the antibody gene based on this are reached and produced, obtains to have and neutralizes
The antibody products of EV71 virus infection are made clinically for preventing and treating the specific antibodies medicine of hand-foot-and-mouth disease.
Detailed description of the invention
Fig. 1 is the SDS-PAGE electrophoresis of E1 Antibody Fab fragment after purification in the embodiment of the present invention 1；Wherein, 1 is anti-for purifying
Body Fab E1, M are albumen Marker.
Fig. 2 is the ELISA testing result of anti-EV71 virus E1 Antibody Fab fragment in the embodiment of the present invention 1；Wherein, FabE1 is light
Chain, heavy chain variable region amino acid sequence respectively as shown in SEQ ID No.1,2, the mutant that FabE1 ' is FabE1 (will
15th Arg of FabE1 light chain variable region replaces with Lys, and the 8th Lys of FabE1 heavy chain variable region is replaced with Ala institute
The mutant of formation), positive control is anti-for business mouse, and negative control is Coxsack A4 antiviral antibody.
Fig. 3 is the SDS-PAGE electrophoresis in the embodiment of the present invention 3 after EV71 virus E1 antibody construction whole antibody IgG；Its
In, 1 is EV71 virus E1 whole antibody IgG, and M is albumen Marker.
Fig. 4 is the neutralization experimental result of anti-EV71 virus source of people E1 whole antibody IgG in the embodiment of the present invention 3；Wherein, negative
Control is Coxsack A4 antiviral antibody.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW,
Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
The building in 1 source of people of embodiment anti-EV71 antiviral antibody library and the screening of Fab antibody
1.1 materials and method
1.1.1 virus, cell and carrier source: FUYANG-0805 plants of EV71 virus document (Yang F, Jin Q,
He Y,Li L,Hou Y.2001.The complete genome of Enterovirus 71China strain.Sci
China C Life Sci and Wu Z, Yang F, Zhao R, Zhao L, Guo D, Jin are Q.2009.Identification
of small interfering RNAs which inhibit the replication of several
Enterovirus 71strains in China.J Virol Methods) in disclose, given birth to by Chinese Academy of Medical Sciences's cause of disease
Wu Xue research institute provides.Cell for neutralizing experiment outside EV71 virion is RD cell, is purchased from ATCC.Bacterial strain is XL1-Blue
(Stratagene, the U.S.), carrier pComb3H (are provided) by Scripps research institute, the U.S..
1.1.2 prepared by antigen
EV71 viral purification: culture supernatant after harvest FUYANG-0805 plants of infection RD cells of EV71 virus, through formalin-inactivated
After safety inspection, with 20% sucrose density gradient 35000g, 4 DEG C of centrifugation 3h purified virus particles (Beckman SW28).
1.1.3 the building of phage antibody library
Lymphocyte is separated from patient's EV71 convalescence anticoagulated blood with lymphocyte separation medium (Sigma, the U.S.), is used
RNeasy Mini Kit (QIAGEN, Germany) extracts total cell RNA, and using Oligo-dT as primer, the RNA of extraction adopts for template
With the first chain synthetic agent box (SuperScriptTMIIIFirst-Strand Synthesis of Invitrogen company
System for RT-PCR.Cat No.18080-051) reverse transcription generation cDNA.With one group of amplification human antibody IgG1 heavy chain
The primer of Fd and light chain Kappa and Lambda, the primer sequence used are as follows:
(1) area heavy chain Fd primer
The end 5'
VH1a 5'-CAG GTG CAG CTC GAG CAG TCT GGG-3'
VH1f 5'-CAG GTG CAG CTG CTC GAG TCT GGG-3'
VH2f 5'-CAG GTG CAG CTA CTC GAG TCG GG-3'
VH3a 5'-GAG GTG CAG CTC GAG GAG TCT GGG-3'
VH3f 5'-GAG GTG CAG CTG CTC GAG TCT GGG-3'
VH4f 5'-CAG GTG CAG CTG CTC GAG TCG GG-3'
VH6f 5'-CAG GTG CAG CTA CTA GAG TGG GG-3'
VH6a 5'-CAG GTA CAG CTC GAG CAG TCA GG-3'
The end 3'
CG1Z 5'-GCA TGT ACT AGT TTT GTC ACA AGA TTT GGG-3'
(2) light chain primer
The κ chain variable region end 5'-
VK1a 5'-GAC ATC GAG CTC ACC CAG TCT CCA-3'
VK2a 5'-GAT ATT GAG CTC ACT CAG TCT CCA-3'
VK3a 5'-GAA ATT GAG CTC ACG CAG TCT CCA-3'
The κ chain variable region end 3-
CK1d 5'-GCG CCG TCT AGA ATT AAC ACT CTC CCC TGT TGA AGC TCT
TTG TGA CGG GCG AAC TCA-3'
The λ chain variable region end 5-
VL1 5'-AAT TTT GAG CTC ACT CAG CCC CAC-3'
VL2 5'-TCT GCC GAG CTC CAG CCT GCC TCC GTG-3'
VL3 5'-TCT GTG GAG CTC CAG CCG CCC TCA GTG-3'
VL4 5'-TCT GAA GAG CTC CAG GAC CCT GTT GTG TCT GTG-3'
VL5 5'-CAG TCT GAG CTC ACG CAG CCG CCC-3'
VL6 5'-CAG ACT GAG CTC ACT CAG GAG CCC-3'
The λ chain variable region end 3-
CL2 5'-CGC CGT CTA GAA TTA TGA ACA TTC TGT AGG-3'
PCR amplification is carried out to source of people light chain and heavy chain Fab gene.PCR condition are as follows: 94 DEG C of 1min, 54 DEG C of 1min, 72 DEG C
2min, totally 35 recycle.Banking process substantially by document (Barbas, C.FIII., Kang, A.S., and larner,
R.A.Assembly of combinatorial antibody libraries on phage surface:the geneIII
site.Proc.Natl.Acad.Sci.USA.1991；88 (18): 7978-7982) it carries out.It is specific as follows:
All different heavy chains and light chain PCR product respective group is pressed respectively first to mix.It learns from else's experience XbaI and SacI enzyme
Digestion is cut, and pComb3H carrier DNA 1.5-2 μ g after purification by electrophoresis and light chain mixture 500-700ng, it is highly concentrated that 2 μ l are added
It spends ligase (NEB2000U/ μ l), connection buffer, l6 DEG C of connection overnight is added.100 μ l purified waters are added in next day, and 3M is added
16 μ l of NaAc, adds 2.5-3 times of dehydrated alcohol precipitating DNA, and centrifugation is added to competence after precipitating is resuspended with 20 μ l pure water
In bacterium XL1-Blue, voltage 2.5kv shocks by electricity 1 minute.2ml SOC culture solution is added after turning in electricity immediately, is transferred to bacterium immediately after
In incubator, 37 DEG C shaken cultivation 1 hour.Bacterium solution is all applied on LB plate with ampicillin, 37 DEG C of overnight incubations.
Next day, 10-15ml culture solution was added into plate, scraped bacterial plaque, is sub-packed in centrifuge tube, abandons supernatant after 12000rpm centrifugation, all
It mentions plasmid kit greatly with QIAGEN and extracts plasmid pComb3H-L, frozen after mixing stand-by in -20 DEG C.L chain will be cloned into
PComb3H-L and Fd chain purified pcr product carries out double enzyme digestion reaction, 37 DEG C of digestion 3-4h with XhoI and SpeI respectively.Electrophoresis returns
Corresponding band is received, recovery product after plasmid and Fd digestion is quantified.The 2 μ g of carrier DNA of recovery purifying after being digested is taken,
2 μ l high concentration ligases are added in heavy chain PCR products 600ng or so, corresponding connection buffer are added, 16 DEG C of connections are overnight.It is secondary
Day plus 100 μ l purified waters, add 16 μ l of 3M NaAc, add 2.5-3 times of dehydrated alcohol precipitating DNA, centrifugation, with 20 μ l pure water weights
It is outstanding to precipitate and be added in competence bacteria XL1-Blue, voltage 2.5kv, it shocks by electricity 1 minute.2ml SOC training is added in electricity immediately after turning
Nutrient solution is transferred in bacteriological incubator immediately after, 37 DEG C of 200rpm shaken cultivation 1h.Bacterium solution is transferred in a triangular flask, is added
Enter contain 20 μ g/ml ampicillins 10ml SB-A+ culture solution, 37 DEG C 200rpm shaken cultivation 1 hour.100ml SB is added
Culture solution (Tet of Amp and 20 μ g/ml containing 100 μ g/ml), shake culture 1 hour.It is added 1012Pfu helper phage
VCSM13,37 DEG C of standings infect 20min, and then the Kan of final concentration of 70 μ g/ml, 37 DEG C of overnight incubations are added in 37 DEG C of culture 2h.
To OD600When about 1,4 DEG C of 4000rpm of bacterium solution are centrifuged 15min, supernatant is shifted into sterile triangular flask, 4% (w/v) is added
PEG8000 and 3% (w/v) NaCl, is completely dissolved the rear above precipitating phage of ice bath 30min.4 DEG C of 9000rpm are centrifuged 20-
30min abandons supernatant and precipitating 2ml PBS is resuspended, and brief centrifugation, supernatant is Fab phage antibody library.
1.1.4 the inducing expression of the enrichment isolation of phage antibody library and Fab section antibody
It is screening antigen with FUYANG-0805 plants of inactivated virus particle of ultracentrifugation purifying.0.1M is used when use
NaHCO3(pH8.6) solution dilutes, and is coated with immune pipe, is added above-mentioned after 37 DEG C of closing 2h with the PBS liquid for containing 4% defatted milk
Phage antibody library, every pipe 1ml, 37 DEG C of incubation 2h are washed 20 times repeatedly with the TBS liquid containing 5%Tween-20, last per effective
The glycine-HCI elution of 1ml pH2.2, and neutralized with the Tris liquid of pH9.6.Bacteriophage after elution continues to infect
2ml fresh OD600For 1.0 or so XL1-Blu bacterium, infected through helper phage VCSM13 (Stratagene, the U.S.) laggard
The screening of row next round.It screens 3~4 times repeatedly.The inducing expression of specific enrichment screening method and Fab segment is substantially by document
(Barbas,C.FIII.,Kang,A.S.,and larner,R.A.Assembly of combinatorial antibody
libraries on phage surface:the geneIII site.Proc.Natl.Acad.Sci.USA.1991；88
(18): 7978-7982 it) carries out.
The enrichment of phage antibody library: 0.1M NaHCO is used3(pH8.6) solution presses the FUYANG-0805 strain of purifying
1:100 dilution is coated with 96 orifice plates respectively, and 4 DEG C overnight.Secondary daily PBS-T (0.05%Tween-20 is added in 20mM PBS) washes away not
The antigen of absorption, with 3% 37 DEG C of closing 1h of defatted milk.Confining liquid is abandoned, 60 μ l phage antibody libraries, 37 DEG C of incubations are added in every hole
2h.Discard in hole be not associated with bacteriophage, with TBS-T (50mM Tris-HCl, 150mM NaCl, 0.5%Tween-20,
PH7.5) washing lotion rinses each hole, and when flushing is blown and beaten repeatedly with pipettor, washes altogether 20 times, sufficiently to wash away unadsorbed bacteriophage.
Finally use ddH2O is washed twice, remaining liq in the hole that exhausts.The eluent of 50 μ l Glycine-HCl (pH2.2), room is added in every hole
Temperature is incubated for 10min, in the process, blows and beats (attention does not blow out bubble) repeatedly with suction pipette head.Eluent is incorporated in one
2M Tris is added according to the ratio of every 3 μ l of hole in a centrifuge tube, and making solution ph is about 7, to neutralize the bacteriophage under elution.It will
(OD in the fresh XL1-Blue bacterium solution of 2ml is added in the bacteriophage of elution immediately600=1), it is incubated at room temperature 20min.It is transferred to one
In 250ml triangular flask, it is added 10ml SB (the 20 μ g/ml containing 20 μ g/ml of ampicillin and tetracycline), 10 μ l is taken to be applied to immediately
Ammonia benzyl plate, for titrating bacteriophage.37 DEG C of shaken cultivation 1h of remaining liquid.100ml SB (100 μ containing ampicillin is added
20 μ g/ml of g/ml and tetracycline), 37 DEG C of shaken cultivation 2h.Then helper phage VCSM13 (9 × 10 is added12pfu/ml)
1ml, 37 DEG C of standings 20min, 37 DEG C of shaken cultivation 2h.It is added kanamycins (70 μ g/ml of final concentration), 37 DEG C of shaken cultivation mistakes
Night.OD is grown to bacterium600When about 1,4 DEG C of 6500rpm of bacterium solution are centrifuged 15min, transfer supernatant to sterile triangular flask is added
4% (w/v) PEG8000 and 3% (w/v) NaCl, is completely dissolved the rear abundant precipitating phage of ice bath 30min or more.4℃
9000rpm is centrifuged 20-30min, abandons supernatant.Precipitating 2ml PBS is resuspended, brief centrifugation, supernatant is first round enrichment sieve
Select Fab phage antibody library.
The expression of Fab positive colony: selecting 2000 single bacteriums after 3 enrichment isolations at random and fall in 96 deep-well plates,
Every 800 μ l culture medium of hole, 37 DEG C of overnight incubations.Next day is forwarded to containing 800 μ l SB culture mediums with the ratio of 1:20 (containing Amp
100 μ g/ml) 96 orifice plates in, 37 DEG C of cultures are to OD600When=0.2-0.3, the IPTG of final concentration of 1mM, 30 DEG C of inductions are added
Express 8-10hr.4 DEG C of 4000rpm are centrifuged 15min, and supernatant is for detecting.
1.1.5 the ELISA detection of the anti-EV71 virus Fab antibody of source of people
(1) expression of Fab is detected
With 0.1M NaHCO3(pH9.6) anti-human Fab antibody (is used, Sigma, the U.S.) packet by solution after 1:2000 dilution
By on ELISA Plate, 4 DEG C overnight；The Fab antibody of expression, 37 DEG C of 1h are added in the closing of 4% defatted milk, 37 DEG C of 1h；It is anti-that enzyme mark is added
Human Fab's secondary antibody (uses, Sigma, the U.S.) after 1:2000 dilution, 37 DEG C of 1h；Developing solution colour developing, 2M H2SO4Terminate reaction, enzyme mark
Instrument detects absorbance A value.
(2) it is active in conjunction with EV71 virus to detect Fab for Dot-ELISA
Use the inactivation EV71 virion of purifying as envelope antigen, remaining step is same as above.
1.1.6 the nucleic acid sequence analysis of source of people Fab antibody variable region gene
Nucleic acid sequence analysis is carried out with Qiagen Miniprep Kit (QIAGEN, Germany) preparation Plasmid DNA.Light and weight chain
Sequencing primer be respectively 5 '-AAACTAGCTAGTCGCC AAGGA-3 ' and 5 '-CCGCGGTGGCGGCCGCAAAT-3 '.It will survey
Sequence result is compared with IgG gene order in Internet V-Base gene pool.
1.1.7 the purifying of the anti-EV71 virus Fab antibody of source of people
2ml affinity column is prepared with the antibody (Sigma, the U.S.) of anti-human Fab, by the bacterium solution containing Fab antibody of filtration
It is added in affinity column, iterative cycles 30min to 1h.The pre- wash buffer of PBS of pH value 6.8 is added, washes away non-specific suction
Attached albumen.The Fab antibody albumen of the glycine HCI buffer elution of bound of pH value 2.7 is added, the Tris- of pH value 9.0 is added
HCl buffer neutralizes the solution eluted, then uses evaporating column centrifugal concentrating.Fab segment protein 10 after taking purifying to be concentrated
~20 μ g carry out SDS-PAGE electrophoresis.
1.1.8 detection is tested in the neutralization of the anti-EV71 virus Fab antibody of source of people
(1) experimental procedure
By 100TCID50The 25 μ l of antibody of EV71 25 μ l of virus and doubling dilution in 37 DEG C it is common be incubated for 2 hours, then plus
Entering concentration is 2 × 105The 50 μ l of cell culture fluid of a RD cell/ml.Observation 7 days records Cytopathic effect.
(2) result judgement
When there is 1 hole cytopathy occur in 2 holes of highest dilution antibody, there is not cytopathy in another 1 hole, the dilution
Inverse be the antibody neutralize antibody titers；When the complete lesion in 2 hole of high dilution, adjacent low 2 hole of dilution is not completely sick
Become, then the inverse of the two Average dilution is the neutralize antibody titers of the antibody；When two adjacent dilution antibody occur 1
There is not cytopathy in hole cytopathy, another 1 hole, then the inverse of the two Average dilution is the neutralizing antibody effect of the antibody
Valence.
1.2 result
1.2.1 the screening in source of people anti-EV71 antiviral antibody library
Enrichment isolation is carried out to phage antibody library with FUYANG-0805 plants of EV71 virion of purifying, after 3 wheel screenings
1000 clones of random picking.With anti-human Fab antibody (being used after 1:2000 dilution, Sigma, the U.S.) and EV71 virion
Sample to be tested supernatant is added in FUYANG-0805 plants of 96 orifice plates of antigen coat, (is made after 1:2000 dilution with the anti-human Fab secondary antibody of enzyme mark
It is detected with Sigma, the U.S.).It obtains 425 source of people Fab altogether as the result is shown and expresses positive colony, wherein 231 clones can
Specifically bind FUYANG-0805 plants of EV71 virion (table 1).
1FUYANG-0805 plants of table to phage antibody library enrichment isolation result
1.2.2 the sequence analysis of the anti-EV71 virus Fab antibody of source of people
Fab segment is analyzed and processed with DNASTAR sequence analysis software, compares Internet V-Base gene pool
In IgG sequence, above-mentioned 231 specifically bind EV71 virus source of people Fab monoclonal antibody in, have 11 Fab segments
Sequence it is different.Therefore the present invention successfully screens and clones 11 with different antibody weight chain variable region sequence and its group
The antibody of conjunction, heavy chain variable region principally fall into IgG VH3 and VH4 family, and light chain variable region principally falls into IgG VL1, VK1
With VK3 family.Wherein, E1 belongs to antibody light chain family VL1, the amino acid sequence difference of light chain variable region and heavy chain variable region
As shown in SEQ ID No.1 and SEQ ID No.2, the nucleotide sequence of coding light chain variable region and encoding heavy chain variable region is distinguished
As shown in SEQ ID No.3 and SEQ ID No.4.
1.2.3 the purifying of the anti-EV71 virus Fab antibody of source of people
2ml affinity column is prepared with the antibody (Sigma, the U.S.) of anti-human Fab, purifying Fab antibody is expressed supernatant, passed through
SDS-PAGE examines the expression and purifying situation of Fab antibody, as a result confirms to obtain compared with pure protein, after can obviously observing unwinding
Antibody light chain and Fd chain (Fig. 1).
1.2.4 the ELISA detection of the anti-EV71 virus Fab antibody of source of people
FUYANG-0805 plants of inactivation EV71 virion of purifying are used as envelope antigen, detects the combination activity of antibody,
As a result as shown in Figure 2.
The ELISA of antigen-binding activity is detected: using 0.1M NaHCO3The solution of (pH 9.6) is coated with the inactivation of purifying respectively
FUYANG-0805 plants of EV71 virion, 4 DEG C overnight, with PBS-T washing lotion board-washing 3 times, sufficiently removes liquid and 5% degreasing is added
The 50 μ l of antibody supernatant of prokaryotic expression is added in milk 100 μ l, 37 DEG C of incubation 1h, PBS-T washing lotion board-washing 3 times, sufficiently removal liquid,
50 μ l weak vibrations of 5%PBS- defatted milk are added into every hole again to mix, 37 DEG C of incubation 1h.PBS-T washing lotion board-washing 6 times, sufficiently
Liquid is removed, the anti-human Fab antibody of enzyme mark (Sigma company, 1:2000 dilution use) 100 μ l, 37 DEG C of incubations 1h, PBS- are added
Tween20 washing lotion board-washing 6 times, sufficiently removal liquid, are added developing solution A, B color development at room temperature 10min, 2M H2SO4Reaction is terminated,
OD450Reading.
1.2.5 the neutralization experiment of the anti-EV71 virus Fab antibody of source of people
FUYANG-0805 plants of EV71 virus are used as poison strain is attacked, the neutralization activity of antibody is detected in RD cell, as a result
As shown in table 2.
Experimental procedure: using Fuyang-0805 plants of EV71 virus as poison strain is attacked, detected in RD cell in antibody and
Activity.
By the 100TCID of 50 μ l50The antibody of EV71 virus (Fuyang-0805 plants) and 50 μ l doubling dilutions (make by embodiment 1
It is standby) 37 DEG C it is common be incubated for 2 hours after, density 2 × 10 is added5In the 100 μ l of cell suspension of a/ml.Observation 7 days records cell
Lesion result.
Wherein, (Fuyang-0805 plants) of used EV71 virus is by following processing: virus prepared by embodiment 1
Particle is 10 by 10 times of gradient dilutions-1To 10-10Virus liquid, each to be added in cell plates, every 50 μ l of hole, each dilution is added 4
Hole cell；Every hole adds 50 μ l of cell suspension, while setting cell controls (+50 μ l cell suspension of 50 μ l dilution), 37 DEG C of culture 7d,
Observe cytopathy；The TCID of isolated viral strain is calculated by Karber formula50；log TCID50=L-d (S-0.5).Wherein:
The log value of minimum dilution used in L=experiment；The log value of d=dilution gradient；The summation of positive part when S=sentences eventually
(there is the sum of ratio shared by the cell hole of CPE).
1.2.6 influence of the antibody after non-hypervariable region mutation to EV71 virus resistance
Using the method for site-directed point mutation, light chain VL sequence the 15th Arg of E1 is replaced with into Lys, heavy chain VH sequence
8th Lys of column replaces with Ala.By weight chain gene after light chain and heavy chain nucleic acid sequence encoding after being respectively synthesized mutation
It is cloned into pComb3H, obtains mutant E1 '.Immunology detection is carried out to the mutant, ELISA experiment shows that E1 ' can be special
Property combination EV71 virus.Neutralization reaction of the antibody in vitro with FUYANG-0805 plants is detected using experiment is neutralized, as the result is shown
Its property and E1 it is essentially identical (in table 2 data be antibody extension rate).
Table 2 antibody E1 and its mutant E1 ' is to FUYANG-0805 plants of neutralization activity
The application of the anti-EV71 virus neutrality antibody E1 of 2 source of people of embodiment
Vaccine and drug that hand-foot-and-mouth disease has no specificity are prevented and treated at present, clinically used immunoglobulin
The positive serum of the anti-EV71 of people is derived from, this prepares it largely to be restricted, simultaneously because deriving from serum so holding
The infection of hematogenous infectious disease easily occurs.The anti-EV71 virus gene engineering antibody replacement blood source of source of people obtained using the present invention
Property immunoglobulin, for hand-foot-and-mouth disease treatment provide newly by way of.
The method that embodiment 3 prepares whole antibody Immunoglobulin IgG using neutrality antibody E1
The expression and purifying of 1.1 whole antibody IgG
1.1.1 the building of whole antibody recombinant expression plasmid: primer (upstream primer 5'-cccAAGCTTGTTGCTCTG is first used
GATCTCTGGTGCCTACGGGgaaattgtgttgacccagtctcc-3', downstream primer 5'-
CtagTCTAGAATTAACACTCTCCCCTG-3') amplification obtains the light chain section of Fab antibody, with XbaI/HindIII digestion, gram
It is grand enter PIgG carrier (by Scripps research institute, the U.S. provide) (Christoph Rader, Mikhail Popkov, John
A.Neves,and Carlos F.Barbas III.Integrinαvβ3-targeted therapy for Kaposi.s
sarcoma with an in vitro-evolved antibody.The FASEB Journal.(October 18,2002)
10.1096/fj.02-0281fje.), obtain carrier PIgG-L.Primer (upstream primer 5'-gagGAGCTCACTCCgag is used again
Gtgcagctgttggagtctgggggaggcttggtac-3', downstream primer 5'-gagGGGCCCTTGGTGGAGGCTGAGGAG
ACGGT-3' the heavy chain section for) expanding Fab antibody, enters carrier PIgG-L using SacI/ApaI enzyme cutting clone, is built into whole antibody table
Up to carrier.
1.1.2 transfect: using the transfection reagent box for being purchased from U.S. Invitrogen company, operating method outlines as follows: by 5
After μ g recombinant plasmid dna is mixed with transfection reagent, the 293T cell that transfection stand density is 70%, 37 DEG C of 5%CO2Culture.
1.1.3 whole antibody IgG is purified: supernatant is collected after culture 3d, using the Protein-A parent purchased from Amersham company
Supernatant (Harlow E, Lane D.Antibodies:A Laboratory Manual [M] is expressed with chromatographic column direct purification
.New York:Cold Spring Harbor Laboratory Press,1988).Using ELISA and IFA to the pure of acquisition
The functional characteristic for changing IgG antibody is identified.
The SDS-PAGE electrophoresis result for constructing obtained whole antibody IgG is shown in Fig. 3.
In 1.2 whole antibody IgG and test
In order to further verify the neutralization activity of whole antibody IgG, use FUYANG-0805 plants of EV71 virus it is malicious as attacking
Strain detects the neutralization activity of antibody, as a result as shown in Figure 4 in RD cell.
Experimental procedure: using Fuyang-0805 plants of EV71 virus as poison strain is attacked, detected in RD cell in antibody and
Activity.
By the 100TCID of 50 μ l50The antibody of EV71 virus (Fuyang-0805 plants) and 50 μ l doubling dilutions (make by embodiment 3
It is standby) 37 DEG C it is common be incubated for 2 hours after, density 2 × 10 is added5In the 100 μ l of cell suspension of a/ml.Observation 7 days records cell
Lesion result.
Wherein, (Fuyang-0805 plants) of used EV71 virus is by following processing: virus prepared by embodiment 1
Particle is 10 by 10 times of gradient dilutions-1To 10-10Virus liquid, each to be added in cell plates, every 50 μ l of hole, each dilution is added 4
Hole cell；Every hole adds 50 μ l of cell suspension, while setting cell controls (+50 μ l cell suspension of 50 μ l dilution), 37 DEG C of culture 7d,
Observe cytopathy；The TCID of isolated viral strain is calculated by Karber formula50；log TCID50=L-d (S-0.5).Wherein:
The log value of minimum dilution used in L=experiment；The log value of d=dilution gradient；The summation of positive part when S=sentences eventually
(there is the sum of ratio shared by the cell hole of CPE).
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Institute of Pathogen Biology, Chinese Academy of Medical Sciences
<120>the anti-EV71 virus neutrality antibody E1 of source of people and its application
<130> KHP171113303.0
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 109
<212> PRT
<213>antibody E1 chain variable region amino acid sequence
<400> 1
Glu Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln Arg Val
1 5 10 15
Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp
20 25 30
Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asn Tyr Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly
50 55 60
Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln Ala
65 70 75 80
Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu Ser
85 90 95
Gly Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 2
<211> 116
<212> PRT
<213>antibody E1 heavy chain variable amino acid sequence
<400> 2
Leu Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val
1 5 10 15
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr Tyr Met His Trp
20 25 30
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Asn
35 40 45
Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val
50 55 60
Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser
65 70 75 80
Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Glu
85 90 95
Trp Gln Leu Asp Gly Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 3
<211> 327
<212> DNA
<213>antibody E1 light chain variable region nucleic acid sequence
<400> 3
gagctcacgc agccgccctc agtgtctggg gccccagggc agagggtcac catctcctgc 60
actgggagca gctccaacat cggggcaggt tatgatgtac actggtacca gcagcttcca 120
ggaacagccc ccagactcct catctataat tacagcaatc ggccctcagg ggtccctgac 180
cgattctctg gctccaagtc tggcacctca gcctccctgg ccatcactgg gctccaggct 240
gaggatgagg ctgattatta ctgccagtcc tatgacagca gcctgagtgg ttcggtattc 300
ggcggaggga ccaagctgac cgtccta 327
<210> 4
<211> 348
<212> DNA
<213>antibody E1 heavy chain variable region nucleic acid sequence
<400> 4
ctcgagtctg gggctgaggt gaagaagcct ggggcctcag tgaaggtctc ctgcaaggct 60
tctggataca ccttcaccgg ctactatatg cactgggtgc gacaggcccc tggacaaggg 120
cttgagtgga tgggatggat caaccctaac agtggtggca caaactatgc acagaagttt 180
cagggcaggg tcaccatgac cagggacacg tccatcagca cagcctacat ggagctgagc 240
aggctgagat ctgacgacac ggccgtgtat tactgtgcga gaggagaatg gcagttggac 300
gggtggttcg acccctgggg ccagggaacc ctggtcaccg tctcctca 348
Claims (11)
1. the anti-EV71 virus neutrality antibody E1 of source of people, which is characterized in that light chain hypervariable region CDR1, CDR2 of the antibody E1 and
The amino acid sequence of CDR3 are as follows: SSNIGAGYD, NYS and QSYDSSLSGSV, heavy chain hypervariable region CDR1, CDR2 of the antibody E1
With the amino acid sequence of CDR3 are as follows: GYTFTGYY, INPNSGGT and ARGEWQLDGWFDP.
2. antibody E1 according to claim 1, which is characterized in that the amino acid sequence of its light chain variable region such as SEQ ID
Shown in No.1, the amino acid sequence of heavy chain variable region is as shown in SEQ ID No.2；Alternatively,
15th Arg of sequence shown in SEQ ID No.1 replaces with Lys, and the 8th Lys of sequence shown in SEQ ID No.2 is replaced
It is changed to the antibody of Ala composition.
3. encoding the gene of antibody E1 described in claim 2.
4. gene according to claim 3, which is characterized in that the nucleotides sequence of coding light chain variable region and heavy chain variable region
Column are respectively as shown in SEQ ID No.3 and SEQ ID No.4.
5. expression cassette, expression vector or cloning vector comprising the nucleic acid comprising gene order as described in claim 3 or 4.
6. the host cell containing expression cassette described in the gene of claim 3 or 4 or claim 5, carrier.
7. the engineered obtained single-chain antibody ScFv of antibody E1 as claimed in claim 1 or 2 or Fab antibody or whole antibody immune globulin
White IgG.
8. in the drug of antibody E1 as claimed in claim 1 or 2 hand-foot-and-mouth disease as caused by EV71 virus in preparation prevention or treatment
Application.
9. antibody E1 as claimed in claim 1 or 2 is preparing the application in EV71 virus antigen detection reagent or detection kit.
10. containing the detection reagent or detection kit of antibody E1 as claimed in claim 1 or 2.
11. containing the drug of antibody E1 as claimed in claim 1 or 2.
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| CN102718865A (en) * | 2012-06-21 | 2012-10-10 | 中国医学科学院病原生物学研究所 | Humanized anti-EV71 virus neutralizing antibody EV71FabL11, preparation method and application thereof |
| CN102718864A (en) * | 2012-06-21 | 2012-10-10 | 中国医学科学院病原生物学研究所 | Human anti-EV71 (enterovirus 71) neutralizing antibody EV71FabK7 and preparation method and application thereof |
| WO2014070109A1 (en) * | 2012-10-29 | 2014-05-08 | Temasek Life Sciences Laboratory Limited | An enterovirus 71 specific antibody |
| CN105085626A (en) * | 2014-05-22 | 2015-11-25 | 厦门大学 | Broad-spectrum affinity epitope polypeptide and antibody for human enteroviruses and use thereof |
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| CN102718865A (en) * | 2012-06-21 | 2012-10-10 | 中国医学科学院病原生物学研究所 | Humanized anti-EV71 virus neutralizing antibody EV71FabL11, preparation method and application thereof |
| CN102718864A (en) * | 2012-06-21 | 2012-10-10 | 中国医学科学院病原生物学研究所 | Human anti-EV71 (enterovirus 71) neutralizing antibody EV71FabK7 and preparation method and application thereof |
| WO2014070109A1 (en) * | 2012-10-29 | 2014-05-08 | Temasek Life Sciences Laboratory Limited | An enterovirus 71 specific antibody |
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