CN110092829A - Preparation method, product and its application of the anti-enteric virus71 type monoclonal antibody of source of people - Google Patents
Preparation method, product and its application of the anti-enteric virus71 type monoclonal antibody of source of people Download PDFInfo
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- CN110092829A CN110092829A CN201810092819.3A CN201810092819A CN110092829A CN 110092829 A CN110092829 A CN 110092829A CN 201810092819 A CN201810092819 A CN 201810092819A CN 110092829 A CN110092829 A CN 110092829A
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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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/14—Reoviridae, e.g. rotavirus, bluetongue virus, Colorado tick fever virus
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- Food Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention is a kind of preparation method of the anti-enteric virus71 type monoclonal antibody of source of people, product and its application, through acquisition by the human patients periphery blood of enteric virus71 type natural infection, screening induces the enterovirus 71 type specific immunoglobulin G secretion B cell of generation because of infection, then the immunoglobulin G region of variability gene of B cell is grown in choosing, and it is transfected into human cell's strain and shows, to obtain the anti-enteric virus71 type monoclonal antibody of source of people, the enteric virus71 type of several genes hypotype can be neutralized single-minded and potently using the anti-enteric virus71 type monoclonal antibody of above-mentioned source of people, especially those enteric virus71 type gene hypotype B4 in recent years prevailing in Taiwan and China, C4 and B5.
Description
Technical field
The present invention is a kind of monoclonal antibody, the preparation method of espespecially a kind of anti-enteric virus71 type monoclonal antibody of source of people, product
And its application.
Background technique
Effective antibody level of protection be fight the important immune key of enteric virus71 type, however, at present for the mankind in
The understanding that the antibody specificity and functional characteristic that generate are induced after natural infection enteric virus71 type is fairly limited.
Enteric virus71 type (EV71) is the virus that a kind of no outer embrane single-stranded RNA is inhereditary material, and cause Europe and
Children's hand stomatopod disease of sub- Pacific Province and the main pathogen of herpangina.When breaking out enteric virus71 type epidemic situation, it may go out
Now invade nervous centralis aseptic meningitis, like polio nervous disorders, encephalitis with cardiopulmonary failure, even death
Case.2011, serious epidemic situation was broken out in Vietnam, led to 174,67 infection cases and 200 deaths.2012 in China
Epidemic situation, have more than 2,000,000 cases and hundreds of Lethal cases.2016, Hispanic epidemic situation had 109 to make a definite diagnosis sternly
Grave illness example.Although the epidemic situation of enteric virus71 type is so seriously, in the majority state that epidemic situation still occurs, all there are no in approval
The vaccine in city and specific treatment method can be used to fight enteric virus71 type.
Enteric virus71 type is every about 2-3 in Prevalent district meeting Outbreak, the gene hypotype meeting occurred in the past sometimes
Occur once again, has new gene hypotype sometimes and occur, replace gene hypotype popular in the past.By past enteric virus71 type epidemic disease
The Strain of feelings can find, high mutation rate and Recombination Fraction are one of the characteristics of enteric virus71 type, once multiple discovery enterovirus
71 types have gene recombination, and between homogenic type and allogene type gene swapping can occur for this recombination.
The appearance of enteric virus71 type novel strain or new gene type, it may be possible under the pressure of host immune, mutation
Virus hides the attack of host immune system because being able to due to has survival advantage, and therefore infects more people.Taiwan was in 1998
When breaking out the infection of enteric virus71 type gene hypotype C2, there are about 120,000 or more infected cases, and result in 78 dead diseases
Example.By group's serological analysis, the results show that lacking protection antibody is the high infection rate of children group and high severe risk
One of the main reasons.In the prevalence analysis of Thailand outburst enteric virus71 type gene hypotype B5 in 2012, also there is similar research knot
Fruit.
In addition, researches show that in the child that some enteric virus71 types infect, although its antibody response is shown across gene
The neutralization titer of hypotype, but it is very low to certain gene hypotypes its neutralization titers or generated antibody is to certain gene hypotypes
Especially there is deviation.Past, many researchs were also shown, and the neutralizing antibody of host has promoted the evolution of virus.Nearest research
It shows, after mouse infection enteric virus71 type in generated anti-enteric virus71 type monoclonal antibody group, there is also to different diseases
Strain or different genes parting strain identification difference degree.However, our enteric virus71 type neutralizing antibodies anti-for the mankind at present
Antigen identification content it is still unclear, which has vaccine design and the following policy for utilizing Vaccine Control epidemic situation
Epochmaking relevance.More and more evidences show that it is all that the enteric virus71 type vaccine of existing exploitation is still unable to cross protection
Genotyping and Strain prevailing.
The protein coat of enteric virus71 type is as composed by its 4 gene product VP1-4.VP1, VP2 and VP3 table
The outside of present virus coat, and VP4 is then buried in inside.Using mouse monoclonal antibody, several enteric virus71s can be calibrated
In the Linear antigenic of type and determine position (linear neutralizing epitope), such as the circuit the GH (215- in VP1
220 Amino acid sequences) on, on the circuit EF (141-146 Amino acid sequence) of VP2 and in the region Knob of VP3.Research
The antibody decision position of specific enteric virus71 type Strain can be recognized (positioned at the 145th of VP1 the by also showing mouse monoclonal antibody
A Amino acid).Anyway, the enteric virus71 type antigen identification content of mouse monoclonal antibody can not represent human child's body
The antigen of interior potent confrontation enteric virus71 type antibody recognizes content.At least insofar, acute enteropathy is come from there has been no any
Malicious 71 types infect monoclonal antibody caused by the lower mankind, and (neutralization) epitope recognized with the antibody is detailed
Report.
Generally in children's patient of enteric virus71 type acute infection, at first week of infection, enteric virus71 type was special
Anisotropic immunoglobulin G secretion B cell (plasmablasts) largely can be induced, antibody secreting cell reaction
Peak period be clinical symptom appearance after the 4-7 days.In addition, research is it has also been found that metainfective seroreaction content has
The neutralization titer of intersection confrontation enteric virus71 type genotype B and C, however serum after some infection, there is relatively low anti-intestines
The antibody titer of 71 type genotype C of virus.
Prior art system monoclonal antibody is derived from BALB/c mouse, via the enteric virus71 type gene hypotype C4 that deactivates
After vaccine injection, then through monoclonal antibody caused by hybridoma cell strain integration technology, and hybridoma cell strain integration technology
Generated mouse monoclonal antibody is just for enteric virus71 type gene hypotype C4, and its antigen only has single target, does not wrap
Containing other enteric virus71 type the gene hypotype B4s and B5 popular in Taiwan and China.
At present also have technology choosing grow anti-enteric virus71 type antibody performance gene in yeast vector, and show and it is pure
Change in saccharomycete monoclonal antibody produced, or by synthesis enteric virus71 type VP1 victory peptide come after mouse is immunized, choosing
The antibody mediated immunity gene of wherein VP1 specificity is grown, and is found expression in humanizing cells, the monoclonal for producing this gene product is anti-
Body, but the heavy chain of the monoclonal antibody and light chain region of variability part are still source of mouse, in addition, source of mouse antibody is in the intracorporal effect of people
And it is bad, source of mouse monoclonal antibody is also without clearly indicating its area epitope for acting on enteric virus71 type (epitope)
Domain.
Summary of the invention
The present invention provides preparation method, product and its application of a kind of anti-enteric virus71 type monoclonal antibody of source of people, the anti-intestines of source of people
Viral 71 type monoclonal antibody systems are transformed as follows: (1) from the human sample blood for having infected enteric virus71 type
In, isolate the immunoglobulin G secretion B cell of enterovirus 71 type specific；(2) above-mentioned immunoglobulin G secretion B is grown in choosing
The immunoglobulin variable area gene of cell carries out recombinant clone, then the immunoglobulin G gene of above-mentioned recombinant clone is transfected
Into HEK293T human cell's strain, expresses immunoglobulin G and purify, it is anti-to can be obtained the anti-enteric virus71 type monoclonal of source of people
Body.
The present invention has the advantages that
(1) the anti-enteric virus71 type monoclonal antibody of source of people of the invention is human antibody, after the infection of enteric virus71 type
The raw monoclonal antibody in the human B cell institute source induced, compared to existing source of mouse monoclonal antibody, the strong 10- of neutralising capacity
100 times.
(2) the anti-enteric virus71 type monoclonal antibody of source of people manufactured by the present invention can fight single-minded and potently difference
The enteric virus71 type of gene hypotype, such as in Strain such as gene hypotype B4, B5, C4 of China and Taiwan Major Epidemic.
(3) present invention escapes the technology of mutant strain (escape mutant selection) using screening antibodies, to this
Epitope of the anti-enteric virus71 type monoclonal antibody action of 12 source of people of invention on enteric virus71 type shell mechanism is
It is analyzed.The anti-enteric virus71 type monoclonal antibody of source of people has the ability for neutralizing enteric virus71 type originally, and escapes to this
Strain loses the phenomenon that neutralising capacity, illustrates that this is escaped Strain and attacks by the anti-enteric virus71 type monoclonal antibody of source of people
It is mutated on the shell specific region hit, is compared through sequencing and with maternal plant virus, can define and escape outside virus
The crucial catastrophe point of shell gene.It is single-minded that the anti-enteric virus71 type monoclonal antibody institute of 12 source of people can be clearly indicated by this method
The specific region of sexual assault (neutralization) virus.
(4) the virus coat antigen that the anti-enteric virus71 type monoclonal antibody of source of people is recognized can be drawn through the present invention to determine
Position map, including valley Northern Margin area (canyon northern rim), sole area (canyon floor), gorge
Paddy south marginal zone (canyon southern rim), 3 times of axis uplift plateaus (3-fold plateau) and 2 times of axis plateaus
The five regions such as area (2-fold plateau).What the anti-enteric virus71 type monoclonal antibody of 12 plants of source of people of the invention was recognized
Epitope, it is quite conservative between different genes type, show that the anti-enteric virus71 type monoclonal of 12 plants of source of people of the invention is anti-
Body has the popularity and cross complementary of identification enteric virus71 type；The animal of poison infection enteric virus71 type is attacked in mouse brain
Model shows that the anti-enteric virus71 type monoclonal antibody 16-2-9D of source of people (identification sole area) and 17-2-2B (recognize valley
Marginal zone) potent can prevent neuropathy caused after enteric virus71 type gene hypotype B5 and C4 brain inner infection mouse with
It is slow to grow up.
(5) gene of the anti-enteric virus71 type monoclonal antibody of source of people of the invention it is optional grow to the carrier of Escherichia coli or
In other host cells, reach the analysis, transformation and persistence of facilitating gene sequence.
(6) the anti-enteric virus71 type monoclonal antibody of source of people of the invention can according to enteric virus71 type gene hypotype B4, B5 or
C4 carries out the manufacture of specific aim kit, reagent or medicament, and mentioned reagent box, reagent or medicament can neutralize enterovirus enteropathy
71 type gene hypotype B4, B5 or C4 of poison.
(7) the anti-enteric virus71 type monoclonal antibody of source of people of the invention can be made into a kind of detection kit, wherein the inspection
Test agent box is that infection enterovirus is verified whether by enzyme linked immunosorbent assay (ELISA) analytic approach, immuno-precipitation or Western blot
71 types.
(8) the anti-enteric virus71 type monoclonal antibody of the made source of people confrontation intestines of specificity protection in vivo be can verify that
The ability of viral 71 types, the method is as follows: plural mouse is attacked poison infection enteric virus71 type with intracerebral by (a)；It (b) will be above-mentioned small
Mouse dimidiation, is respectively designated as control group and experimental group, and the anti-enteric virus71 type monoclonal antibody of experimental group injection source of people is right
According to the buffer for organizing then injection same volume；(c) after 2~5 days, control group can obviously generate serious fortune compared to experimental group
The neurological symptoms result of dynamic disability, therefore the anti-enteric virus71 type monoclonal antibody of susceptible of proof injection source of people protects mouse with can dramatically
Movement disability occurred is infected from enteric virus71 type.
Detailed description of the invention
Fig. 1: for the process block diagram of the present invention manufacture anti-enteric virus71 type monoclonal antibody of source of people.
Fig. 2: for the present invention with regard to the anti-enteric virus71 type monoclonal antibody of source of people to the enteric virus71 from 1998 to 2016 years
The neutralization reaction of type clinical strain；* the anti-enteric virus71 type monoclonal antibody of source of people (representative mAb) complete neutralization disease
The minimum concentration symbol of poison is as follows: ++++: < 100ng/ml；+++: 0.1-1 μ g/ml；++: 1-10 μ g/ml；+: 10-50 μ
g/ml；: without neutralising capacity；Control group #: using no neutralising capacity the anti-enteric virus71 type monoclonal antibody of source of people, 17-1-
Human serum is as control group after 10B and 16-2-1A, with the infection of enteric virus71 type.
Fig. 3: for the anti-enteric virus71 type monoclonal antibody immunity precipitate virus coat protein of source of people of the invention；Do not feel
The RD cell culture supernatant of dye is as antigen control group (mock), and source of people resisiting influenza virus monoclonal antibody 2-12C is as anti-
Body control group.
Fig. 4: the virus coat key amino acid position recognized by the anti-enteric virus71 type monoclonal antibody of source of people of the invention
Point.It is guarded between each genotype (including A, B1, B2, B3, B4, B5, C1, C2, C3, C4 and C5) enteric virus71 type in the site
In the presence of.
Fig. 5 A: the virus coat key amino acid recognized by the anti-enteric virus71 type monoclonal antibody of source of people of the invention
Site, the antigen that the enteric virus71 type shell mechanism according to RCSB Protein Data Bank 3ZFF and 3VBS is drawn are determined
Determine base map.The figure system uses PyMOL, draws out virus coat side cartoon figure.Coat protein VP1: black；VP2: ash
Color；VP3: white.It is respectively 5 meanings as 5 times of axis that number, which represents meaning,；3 meanings are three times axis；2 meanings are 2 times of axis.
Fig. 5 B: the virus coat key amino acid recognized by the anti-enteric virus71 type monoclonal antibody of source of people of the invention
Site, the antigen that the enteric virus71 type shell mechanism according to RCSB Protein Data Bank 3ZFF and 3VBS is drawn are determined
Determine base map.The figure system uses PyMOL, draws out 5 times of apex regions as the pentamer surface view at center.Coat protein
VP1: black；VP2: grey；VP3: white.It is respectively 5 meanings as 5 times of axis that number, which represents meaning,；3 meanings are three times axis；2 meanings
Think to be 2 times of axis.
Fig. 6 A: for the in vivo protective capability of the anti-enteric virus71 type monoclonal antibody of source of people of the invention.Mouse is infecting
Time after enteric virus71 type in 0 to 14 day according to instruction measures movement disability score (the motor deficit of mouse
Scores), the performance for moving disability can then divide into 7 grades according to severity.Since the disability of the first order, every increase level-one,
The severity for representing its disability increases level-one.Therefore, a higher number, the then degree for representing its movement disability are more serious.
Asterisk, which is shown in, to be given and is not given to the enteric virus71 type of the anti-enteric virus71 type monoclonal antibody of source of people and infect, and is had
Significant difference.Upright triangle, handstand triangle represent: [enteric virus71 type 12-96015+ buffer PBS] v.s. [enteropathy
The malicious 71 anti-enteric virus71 type monoclonal antibodies of type 12-96015+ source of people].Gray squares, gray circular represent: [enteric virus71
Type 11-96023+ buffer PBS] v.s. [the anti-enteric virus71 type monoclonal antibody of enteric virus71 type 11-96023+ source of people].*:P
≤0.05；* *:P≤0.01；***:P≤0.001).The d.p.i. of abbreviation；days post-infection.
Fig. 6 B: for the in vivo protective capability of the anti-enteric virus71 type monoclonal antibody of source of people of the invention.Mouse is infecting
After enteric virus71 type, according to the time of instruction in 0 to 14 day, the weight of mouse is measured.Error line represents the standard error of mean value.
Asterisk is shown in be infectd with the enteric virus71 type without the anti-enteric virus71 type monoclonal antibody of source of people, has conspicuousness poor
It is different.Upright triangle, handstand triangle represent: [enteric virus71 type 12-96015+ buffer PBS] v.s. [enteric virus71 type
The anti-enteric virus71 type monoclonal antibody of 12-96015+ source of people].Gray squares, gray circular represent: [enteric virus71 type 11-
96023+ buffer PBS] v.s. [the enteric virus71 type 11-96023+anti-enteric virus71 type monoclonal antibody of source of people].*:P≤
0.05；**:P≤0.01；***:P≤ 0.001).The d.p.i. of abbreviation；days post-infection.
Specific embodiment
Refering to fig. 1, system of the present invention discloses a kind of preparation method of anti-enteric virus71 type monoclonal antibody of source of people, according to such as lower section
Method is transformed:
(1) from the patient infected through Laboratory Diagnosed for enteric virus71 type, the peripheral blood mononuclear cells of patient is harvested
(Peripheral blood mononuclear cells, PBMCs).PBMCs (cell number: 1 × 10 is marked first6) in
Cell surface marker antibody can be used to mark in Plasmablast B cell, including PB anti-CD3 (5 μ g/ml, UCHT1
Clone, Becton, Dickinson and Company, the U.S.), (1:10 dilution, it is real to be used in 100 μ L to FITC anti-CD19
Test in sample, HIB19 clone, Becton, Dickinson and Company, the U.S.), (1:20 is dilute by PE-Cy7anti-CD27
Release, be used in 100 μ L experiment samples, M-T271 clone, Becton, Dickinson and Company, the U.S.), APC-
H7anti-CD20 (5 μ g/ml, L27 clones, Becton, Dickinson and Company, the U.S.) and PE-Cy5anti-
(1:10 dilution, is used in 100 μ L experiment samples CD38, HIT2 clone, Becton, Dickinson and Company, beauty
State), then by flow cytometer (flow cytometry) method, confirmation and screening Plasmablast B cell and collect list
One cell is to the hole 96- micro porous disk.
(2) after, respectively by the Variable Area of the immunoglobulin G heavy chain of single Plasmablast B cell and light chain
Gene choosing is grown in immunoglobulin G heavy chain expression vector (IgG-AbVec display carriers [FJ475055]) and kappa or lambda
Immunoglobulin free light chain expression vector (Ig κ-AbVec display carriers [FJ475056] or Ig λ-AbVec display carriers
[FJ517647]), and the selected heavy chain grown and light chain gene carrier cloning transfection (transfection) are arrived into 293T cell strain
In (ATCC number: ATCC CRL-3216), free serum culture system expression monoclonal antibody is recycled.Culture 5 days after, with from
The method (400g [gravity], room temperature, 10 minutes) of the heart removes cell debris, collects supernatant liquor, can be obtained containing gene table
Up to it is rear it is secreted arrive the anti-enteric virus71 type monoclonal antibody of extracellular recombination human source.
The Plasmablast B cell provided by the patient infected by enteric virus71 type, is obtained altogether in this experiment
Obtained 191 plants of the anti-enteric virus71 type monoclonal antibody of source of people.By enzyme linked immunosorbent assay (ELISA) analytic approach (enzyme-
Linked immunosorbent assay, ELISA) method, it is made with the human immunoglobulin of serial dilution known concentration
The standard curve being made can estimate the relative concentration of the anti-enteric virus71 type monoclonal antibody of source of people in supernatant liquor.This source of people is anti-
The antigenic specificity identification capability (or potency) of enteric virus71 type monoclonal antibody by antigen binding test analysis and will neutralize
The methods of test measures.In experiment of the invention, the anti-enteric virus71 type monoclonal antibody energy specificity of 84 plants of source of people is shared
Virus coat is recognized, the anti-enteric virus71 type monoclonal antibody of these source of people being capable of immunoprecipitation virus coat as can be seen from Figure 2
Protein, but the protein of denaturation cannot be recognized.These results indicate that the anti-enteric virus71 type monoclonal of source of people of the invention is anti-
Body, main system recognize the structure antigenic structure of virus coat in specific manner, will make introductions all round respectively below.
Virus used in present invention experiment:
For testing the anti-enteric virus71 type monoclonal antibody combination of source of people or the experiment of virus neutralizing cpaacity: from 1998
Enteric virus71 type clinical strains separated to 2016 (including 98-2086,98-4215,99-1691,99-3351,00-
2278、01-1437、02-2792、03-70576、04-72232、05-1956、07-72043、 08-96016、10-96018、
11-96023,12-96015,14-51389,15-921,16-50444 and 16-50555).
Virus needed for experiment control group: other types of enterovirus, including 12-50891 (Coxsackie virus A2),
14-2060 (coxsackie virus A 16), 15-50909 (coxsackie virus A 16) and 14-2795 (enterovirus D68).
Operation: the culture of enteric virus71 type or other enteroviruses uses general common enterovirus growth medium
(the 100 μ g/ml of fetal calf serum/penicillin and strepto- of Dulbecco's Modified Eagles Medium [DMEM]/2%
100 μ g/ml of element) (ATCC is numbered: ATCC CCL-136), cell will generate cytopathy because of viral mass propagation with RD cell
Become (cytopathic effect, CPE), freezed repeatedly again at this time and thaws respectively three times after, with the method for centrifugation (400g,
Room temperature, 10 minutes) removal cell debris, and collect virus there are supernatant.On the other hand, for 12-96015 and 11-
96023 the two enteric virus71 types are used herein as RD cell and produce a large amount of purified virus liquid.In order to collect in supernatant liquor
Virion, NaCl (sodium chloride, 250mM) and 6000 (10%polyethylene of PEG is added
Glycol,The U.S.) after, it is handled 12 to 16 hours in 4 DEG C, then with supercentrifugal process (100,000g, 5 hours), sink
Shallow lake virion.(0.01% Triton X-100 and 10%protease inhibitor mixture is included with PBS
[Roche, Switzerland]) sediment of the back dissolving containing virion.Later, be added RNase-free DNase (0.05mg/ml, RQ1,
Promega, the U.S.) it is handled 30 minutes in 37 DEG C, to remove remaining DNA；EDTA is added again
(ethylenediaminetetraacetic acid, 10mM) stops reacting.It is then to utilize in order to which virus is further purified
10-35% potassium tartrate gradient (potassium tartrate gradient [10-35%, w/v]) is dissolved in 250mM HEPES
In (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, 250mM NaCl [pH 7.5]), with
Ultracentrifugation method (159,382g, 4 DEG C, 2 hours) purifying.Finally, virus concentration is then with BCA protein assay reagents
(The Thermo Scientific PierceTMBCA Protein Assay Kit, Thermo Fisher
Scientific, the U.S.) it estimates.It is thin with RD using Reed-Muench method for vial supernatant and purified virus liquid
Born of the same parents measurement can cause 50% cultured tissue infect virus titer (50%tissue culture infective dose,
TCID50)。
All enteric virus71 types in testing, detect the base of its virus capsid protein matter VP1, VP2, VP3 and VP4
Because of sequence (P1 gene region), that is, specific introduction is used, the area enteric virus71 Xing P1 is amplified with polymerase chain chain reaction
Domain, and the sequencing region.
For the enteric virus71 type Strain of genotype B:
VP1_F:GCGGCGGCCCAGAAGAA, VP1_R:GAGGTTTGCCCAGTCGTTA；
VP2_F:ACAGAGCCTCAAACAAGAC, VP2_R:GAAATTTGGCAGAATGGGTGC；
VP3_F:TTTGACCAAGGGGCAACCC, VP3_R:GTCCTGTGGGTGCCGGCAG；
VP4_F:ATCCGGTGTGCAATAGAGC, VP4_R:CATTCACCATAACCGACTATG.
For the enteric virus71 type Strain of genotype C:
VP1_F:GCGGCAGCCCAAAAGAA, VP1_R:AAGATTTGCCCAATCATTGTG；
VP2_F:ACAGAGTCTCAAGCAGGAT, VP2_R:GAAGTTTGGTAGAATAGGTGC；
VP3_F:TACGACCAAGGAGCGACGC, VP3_R:CTGTGGGTGCTGGTAGAGC；
VP4_F:ATCCGGTGTGCAACAGAGC, VP4_R:CACTCACCATAACCGACTATG.As a result: in this experiment
84 plants can with specificity recognize enteric virus71 type the anti-enteric virus71 type monoclonal antibody of source of people in, have 38 plants have it is potent
The ability of ground neutralization enteric virus71 type.And these viruses neutralize the anti-enteric virus71 type monoclonal antibodies of source of people, according to its heavy chain and
The protein coding sequence of light chain variable region (VDJ and VJ) is analyzed, then can divide into 12 groups.Therefore, this is further analyzed
12 representative viruses neutralize the characteristic of the anti-enteric virus71 type monoclonal antibody of source of people.The anti-enteric virus71 type Dan Ke of source of people
Grand antibody is that [(1-354 nucleotide are human monoclonal antibody 16-3-10B to heavy chain SEQ ID NO:1 to 16-3-10B respectively
The heavy chain region of variability of Immunoglobulin IgG.It is according to implementation method that the choosing of the heavy chain region of variability of human immunoglobulin IgG, which is grown,
" production of human monoclonal antibody (monoclonal antibody) " unit described in method complete.This artificial sequence
Gene pool is had been stored in, number is KY354551.), (1-330 nucleotide are mankind Dan Ke to light chain SEQ ID NO:13
The light chain region of variability of grand antibody 16-3-10B Immunoglobulin IgG.The choosing of the light chain region of variability of human immunoglobulin IgG is grown
It is complete according to method described in implementation method " production of human monoclonal antibody (monoclonal antibody) " unit
At.This artificial sequence has been stored in gene pool, and number is KY354565.)], 16-2-8C [heavy chain SEQ ID NO:2 (1-
402 nucleotide are the heavy chain region of variability of human monoclonal antibody's 16-2-8C Immunoglobulin IgG.Human immunoglobulin
It is according to implementation method " human monoclonal antibody (monoclonal antibody) that the choosing of the heavy chain region of variability of IgG, which is grown,
" method described in unit is completed for production.This artificial sequence has been stored in gene pool, and number is KY354552.), light chain
(1-330 nucleotide are the light chain region of variability of human monoclonal antibody's 16-2-8C Immunoglobulin IgG to SEQ ID NO:14.
It is according to implementation method " human monoclonal antibody that the choosing of the light chain region of variability of human immunoglobulin IgG, which is grown,
" method described in unit is completed for the production of (monoclonal antibody).This artificial sequence has been stored in gene pool,
Number is KY354566.)], [(1-402 nucleotide are human monoclonal antibody 16- to heavy chain SEQ ID NO:3 to 16-2-9D
The heavy chain region of variability of 2-9D Immunoglobulin IgG.It is according to implementation that the choosing of the heavy chain region of variability of human immunoglobulin IgG, which is grown,
Method described in " production of human monoclonal antibody (monoclonal antibody) " unit of method is completed.This person
Process column have been stored in gene pool, and number is KY354553.), (1-330 nucleotide, are people to light chain SEQ ID NO:15
The light chain region of variability of class monoclonal antibody 16-2-9D Immunoglobulin IgG.The light chain region of variability of human immunoglobulin IgG
It is according to described in implementation method " production of human monoclonal antibody (monoclonal antibody) " unit that choosing, which is grown,
Method is completed.This artificial sequence has been stored in gene pool, and number is KY354567.)], 16-2-11B [heavy chain SEQ ID
(1-372 nucleotide are the heavy chain region of variability of human monoclonal antibody's 16-2-11B Immunoglobulin IgG to NO:4.The mankind exempt from
It is according to implementation method " human monoclonal antibody (monoclonal that the choosing of the heavy chain region of variability of epidemic disease globulin IgG, which is grown,
Antibody " method described in unit is completed for production).This artificial sequence has been stored in gene pool, and number is
KY354554.), (1-330 nucleotide are human monoclonal antibody's 16-2-11B immunoglobulins to light chain SEQ ID NO:16
The light chain region of variability of IgG.It is according to implementation method " source of people Dan Ke that the choosing of the light chain region of variability of human immunoglobulin IgG, which is grown,
" method described in unit is completed for the production of grand antibody (monoclonal antibody).This artificial sequence has been stored in base
Yin Ku, number is KY354568.)], [(1-369 nucleotide, are human monoclonals to heavy chain SEQ ID NO:5 to 17-2-2B
The heavy chain region of variability of antibody 17-2-2B Immunoglobulin IgG.The choosing of the heavy chain region of variability of human immunoglobulin IgG grow be according to
Factually method described in " production of human monoclonal antibody (monoclonal antibody) " unit of applying method is complete
At.This artificial sequence has been stored in gene pool, and number is KY354555.), light chain SEQ ID NO:17 (1-330 nucleosides
Acid is the light chain region of variability of human monoclonal antibody's 17-2-2B Immunoglobulin IgG.The light chain of human immunoglobulin IgG
It is according to implementation method " production of human monoclonal antibody (monoclonal antibody) " unit that the choosing of region of variability, which is grown,
Described in method complete.This artificial sequence has been stored in gene pool, and number is KY354569.)], 16-2-12D [heavy chain
(1-402 nucleotide are the heavy chain region of variability of human monoclonal antibody's 16-2-12D Immunoglobulin IgG to SEQ ID NO:6.
It is according to implementation method " human monoclonal antibody that the choosing of the heavy chain region of variability of human immunoglobulin IgG, which is grown,
" method described in unit is completed for the production of (monoclonal antibody).This artificial sequence has been stored in gene pool,
It compiles
In Fig. 2, in order to analyze the anti-enteric virus71 type monoclonal antibody identification different genes hypotype enterovirus of representative source of people
The range of 71 types, the different genes hypotype enteric virus71 type clinical strain separated from 1998 to 2016 years are included into neutralization examination
Test examination.It was found that 10 plants of (16-3-10B, 16-2-8C, 16-2-11B, 17-2-2B, 16-2-9D, 16-2-12D, 16- therein
3-3C, 16-2-2D, 34-1-6D and 17-1-12A) the enteric virus71 type of several genes hypotype can be neutralized extensive and potently,
Including genotype B (containing B4 and B5) and C (containing C1, C4 and C5).But the above-mentioned anti-enteric virus71 type monoclonal antibody of source of people fights base
Because subtype C 2 enteric virus71 type when, effect is poor, even if still can not effectively neutralize virus at concentrations up to 50 μ g/ml.It is worth
It is noted that have the anti-enteric virus71 type monoclonal antibody of six plants of source of people (16-3-10B, 16-2-8C, 16-2-11B, 17-2-2B,
16-2-9D and 16-2-12D) at low concentration (< 1 μ g/ml), still have to enteric virus71 type gene hypotype B4, B5 and C4 very strong
Neutralization；Especially 16-3-10B and 16-2-8C still has superpower neutralization at lower concentrations (< 100ng/ml), changes
Yan Zhi, 16-3-10B and 16-2-8C have superior compared to other anti-enteric virus71 type monoclonal antibodies of 10 kinds of source of people
Protective capability.In addition, it has been found that the anti-enteric virus71 type monoclonal antibody 16-3-4D and 17-2-12A of source of people is typically only capable to fight
The enteric virus71 type of genotype B, and higher concentration (> 10 μ g/ml) is needed, just there is the effect for neutralizing virus.
Present invention experiment uses the binding test analytic approach based on flow cytometer (flow cytometry):
Present invention experiment tests the anti-enteric virus71 type monoclonal antibody of source of people of the invention and enterovirus with flow velocity cell instrument
The analysis method of 71 type antigen bindings is as follows:
First day, the enteric virus71 type of appropriate amount is infected into RD cell.
It second day, collects, cleaning and back dissolving these cells.It is solid with 2% formaldehyde (formaldehyde) of Fresh again
Determine cell (10 minutes, 37 DEG C) and with 0.1%Triton X-100 (15 minutes, 37 DEG C) permeabilized (permeabilizing)
Cell.Then with -3% bovine serum albumin(BSA) of saponin(e (sapoinin) (bovine serum albumin, BSA) by background area
Covering.These cell samples are acted on again with Primary antibodies and secondary antibody.Wherein Primary antibodies include the anti-intestines of source of people of the invention
(5 μ g/ml, are diluted in BD Perm/Wash to viral 71 type monoclonal antibodiesTMBuffer buffer solution [Thermo Fisher
Scientific, the U.S.]) or enteric virus71 type infection after human serum (1:125 is diluted in BD Perm/WashTMbuffer
Buffer solution [Thermo Fisher Scientific, the U.S.]) and anti-enteric virus71 type 3C monoclonal antibody (1 μ of source of mouse
G/ml, anti-Enterovirus 71 3C antibody, GeneTex, the U.S.).Secondary antibody is then containing fluorescent
(fluorescence) the goat source anti-human immunoglobulin g antibody (2.5 μ g/ml) of conjugation calibration or the anti-mouse in goat source
Immunoglobulin like protein G antibody (1.5 μ g/ml, Thermo Fisher Scientific, the U.S.).Utilize BD
FACSCantoTMII stream type cell analyzer analyzes these cells acted on by Primary antibodies and secondary antibody, that is, analyzes 10,
In the infection of enteric virus71 type (enteric virus71 type 3C is positive) cell event of 000 collection, the anti-enteric virus71 type monoclonal of source of people
The cell percentages that antibody is recognized.When implementing the binding test analytic approach, not infected RD cell is as antigen control
Group, in addition, the source of people resisiting influenza virus monoclonal antibody 2-12C and anti-enteric virus71 type VP2 monoclonal antibody MAB979 of source of mouse
(EMD Millipore, Germany) is as the antibody control group tested every time.
Present invention experiment uses enzyme linked immunosorbent assay (ELISA) analytic approach (Enzyme-linked immunosorbent
Assay):
The enteric virus71 type of purifying is first adsorbed on the hole 96- micro porous disk by present invention experiment.To avoid non-specific knot
The reaction of conjunction is covered background area with 3% bovine serum albumin(BSA).Primary antibodies, including the anti-enteric virus71 of source of people are added
Type monoclonal antibody supernatant (5 μ g/ml), the purifying anti-enteric virus71 type monoclonal antibody of source of people (5 μ g/ml) or enteric virus71
Human serum (1:125 dilution) after type infection, and the anti-enteric virus71 type monoclonal of source of people that these are combined with the virus of absorption
Antibody can then connect with horseradish peroxidase (horseradish peroxidase, HRP, Invitrogen, the U.S.) conjugation
The anti-immunoglobulin G antibody (0.5 μ g/ml, Rockland, the U.S.) of knot is detected as secondary antibody, the second level in connection
Antibody is then with 3', 3', 5', 5' ,-tetramethyl benzidine colour reagent (TMB substrate reagent, BD
Biosciences, the U.S.) it reacts, it is terminated react with 2M sulfuric acid later, and the multiple holes for obtaining each experiment sample are inhaled
Light (OD450 subtracts OD570) is averaged.PBS, source of people resisiting influenza virus monoclonal antibody 2-12C and the anti-enterovirus of source of mouse
71 type VP2 monoclonal antibody MAB979 (EMD Millipore, Germany) are as the Primary antibodies control group tested every time.
Before determining result, the extinction average value of all samples and positive controls MAB979 must be corrected, school
Positive light absorption value is equal to sample and positive controls MAB979 subtracts negative control group 2-12C, and difference is greater than or equal to 0.30
Person determines that the anti-enteric virus71 type monoclonal antibody sample of source of people is the positive to the binding ability of the enteric virus71 type.
Present invention experiment uses immunoprecipitation (immunoprecipitation) and Western (Western
Blotting) analytic approach:
Immunoprecipitation analysis be using magnetic bead conjugation connection protein G (Thermo Fisher
Scientific, the U.S.) antibody of immunoglobulin class g can firmly be caught, this is taken while precipitating magnetic bead with benefit
The invention institute anti-enteric virus71 type monoclonal antibody of source of people to be tested, and the anti-enteric virus71 type monoclonal antibody of this source of people can
To combine institute's enteric virus71 type to be tested in specific manner.Foundation laterStandard method dissociate (eluate)
The anti-enteric virus71 type monoclonal antibody of the source of people combined on magnetic bead out and the anti-enteric virus71 type monoclonal antibody institute of source of people are single-minded
In conjunction with the compound washings of enteric virus71 type antigen.Sodium dodecyl sulfate polyacrylamide gel electrophoresis method is penetrated again
(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) analysis washes out
Protein contained by object, and with silver staining (Pierce Silver Stain Kit, Thermo Fisher Scientific,
The U.S.) dye the protein belt on colloid.And it is detected contained in colloid with Western blot by the anti-enteric virus71 type of source of people
The enteric virus71 type antigen that monoclonal antibody specificity combines.Fig. 3 system utilizes the anti-enteric virus71 type VP2 monoclonal antibody of source of mouse
MAB979 (1:1000 dilution, EMD Millipore, Germany) and the anti-enteric virus71 type VP1 monoclonal antibody MAB1255- of source of mouse
M05 (1:1000 dilution, Abnova, Taiwan) as Primary antibodies, exempt from by the anti-muroid in goat source followed by HRP conjugation connection
Epidemic disease Lysozyme antibody (1:5000 dilution, Thermo Fisher Scientific, the U.S.) is as secondary antibody.Later, with
By matter chemical luminescence method (Pierce enhanced chemiluminescence western blotting substrate,
Thermo Fisher Scientific, the U.S.) detect the coat protein of enteric virus71 type.Enteric virus71 type 12-
Collected vial supernatant is as antigen after 96025 infection RD cells, for the anti-enteric virus71 type list of source of people of the invention
Clonal antibody reaction, to carry out immunoprecipitation.The RD cell culture supernatant being wherein uninfected by is as antigen control group
(mock), source of people resisiting influenza virus monoclonal antibody 2-12C is as antibody control group.
The present invention tests the neutralization test (Neutralization assay) used:
Principle: the anti-enteric virus71 type monoclonal antibody supernatant of source of people, the anti-enteric virus71 type list of source of people purified are used
Clonal antibody and control group serum equal samples carry out neutralization test, to confirm the anti-enteric virus71 type monoclonal of source of people of the invention
Whether antibody can neutralize enteric virus71 type clinical strain or enteric virus71 type escapes strain infection RD cell.
Serum sample can be handled 30 minutes at 56 DEG C in advance.Then, the anti-enteric virus71 type list of source of people is prepared in 96 porose discs
Virus (the 100TCID of clonal antibody (50 μ l) and equivalent50, 50 μ l) mixed liquor (every hole totally 100 μ l), and make in 37 DEG C
With 2 hours.Then, 100 μ l are added and include 8 × 104The suspension of a RD cell is cultivated 5 days at 37 DEG C.It tests each time
Setting, includes serum control group and viral serial dilution group after cell control group, infection, and each sample standard deviation is equipped with three and repeats.
At the end of infection reaction, then 5% glutaraldehyde (glutaraldehyde) fixed cell and 0.1% crystal violet stained cells are utilized.
Observation can completely inhibit the anti-enteric virus71 type monoclonal antibody minimum concentration of cytopathogenetic source of people, and repeat sequence in three
Reach consistent results person under column dilution, determines minimum neutralization concentration.
Enteric virus71 type virus in present invention experiment escapes the generation of strain:
The anti-enteric virus71 type monoclonal antibody institute's specificity identification of source of people is neutralized outside enteric virus71 type in order to analyze
Epitope on glutelin, the gene hypotype B5 in experiment using two plants of enteric virus71 types 12-96015 is (in gene pool
The serial number registered in [https: //www.ncbi.nlm.nih.gov/genbank/]: KX267854) and 11-96023
Gene hypotype C4 (serial number registered in gene pool: KX267855) escapes the maternal plant virus of virus mutation strain as screening,
The anti-enteric virus71 type monoclonal antibody action of source of people 1 hour that it is generated with the present invention respectively, it is thin to be then seeded to single layer RD
Born of the same parents.Inoculated cell after 37 DEG C are cultivated 4 days, with freeze and thaw repeatedly respectively three times after, with the method for centrifugation (400g,
Room temperature, 10 minutes) removal cell debris, and after collecting supernatant, it will be seeded to single layer RD cell again.Later, cell is 37
If DEG C culture generated cytopathy after 4 days, can with freeze and thaw repeatedly respectively three times after, with the method for centrifugation (400g,
Room temperature, 10 minutes) removal cell debris, and collect vial supernatant；But if cytopathy is not generated, by collected supernatant
Liquid is seeded to single layer RD cell again.
When virus is escaped plant screening experiment and carried out, it need to set up simultaneously without the anti-enteric virus71 type monoclonal antibody of source of people
Control group, it is however generally that, the viral generation needs two for escaping strain to repeated inoculation three times.And collected virus is escaped
De- strain supernatant need to carry out the verifying that virus escapes strain, the people with the anti-enteric virus71 type monoclonal antibody of the source of people acted on
The anti-enteric virus71 type monoclonal antibody in source escapes combination and the neutralising capacity of strain by losing or significantly reducing to the virus.Into one
Step analyzes this and escapes viral P1 gene order and compared with the P1 gene order of control group maternal plant 12-96015 or 11-96023
Compared with the point mutation position that acquisition virus capsid protein matter encodes.
If Fig. 4 is shown, the 11-96023 of the 12-96015 and gene hypotype C4 of enteric virus71 type gene hypotype B5 are by this
What the representative anti-enteric virus71 type monoclonal antibody of source of people of 12 of invention was filtered out escapes virus mutation strain (Escape
Mutant, E), filtering out 35 plants altogether can dash forward to avoid the virus of escaping neutralized by the anti-enteric virus71 type monoclonal antibody of source of people
Mutant finds that their overwhelming majority have single shell Amino acid catastrophe point after sequence is analyzed；However, there is two plants to escape
Virus-free mutant strain is then found respectively have the catastrophe point at two (such as the anti-enteric virus71 type monoclonal antibody 17-2-12A institute of source of people
What is filtered out escapes Δ 12-96015 enteric virus71 type [with VP3 K144E and VP3T148A] and the anti-enteric virus71 type list of source of people
What clonal antibody 16-3-10B was filtered out escapes Δ 11-96023 enteric virus71 type [with VP2T141M and VP1S283F]).
After the sequence for analyzing the Amino acid mutational site that this 35 plants are escaped virus, the displacement of a total of 25 kinds of Amino acids is found, occur
On coat protein Amino acid at 19, this at 19 amino acid be in the anti-enteric virus71 type monoclonal antibody of source of people and enteric virus71
The vital identification site of type, these sites are located on coat protein VP1 to VP3, and add collimation mark with font
Show, is guarded at 17 wherein having on various genotype enteric virus71 type coat protein sequences.In addition, due to the anti-enterovirus of source of people
71 type monoclonal antibody 16-3-4D and 17-2-12A can not neutralize enteric virus71 type genotype C, thus not further directed to
Its screening enteric virus71 type 11-96023's escapes Strain.
Referring next to Fig. 5 A, Fig. 5 B, 19 defined in the present invention at epitope, be to be located at enteric virus71 type shell
(key amino acid replacement site at the 19 of VP1 to VP3), we depict the anti-enteric virus71 type Dan Ke of these source of people to protein
Five regions position (including valley Northern Margin area (canyon in grand antibody and on the be located at shell of viral proteins in site
Northern rim), sole area (canyon floor), valley south marginal zone (canyon southern rim), 3
The five regions such as times axis uplift plateau (3-fold plateau) and 2 times of axis uplift plateaus (2-fold plateau)).And 12 plants of generations
The anti-enteric virus71 type monoclonal antibody of table source of people can according to the five regions position on the shell of viral proteins recognized
It is located at valley Northern Margin area to be divided into for 16-2-11B (heavy chain SEQ ID NO:4, light chain SEQ ID NO:16), 16-
3-3C (heavy chain SEQ ID NO:7, light chain SEQ ID NO:19) and 16-2-2D (heavy chain SEQ ID NO:8, light chain SEQ ID
NO:20), it is located at sole area for 16-2-8C (heavy chain SEQ ID NO:2, light chain SEQ ID NO:14), 16-2-9D (weight
Chain SEQ ID NO:3, light chain SEQ ID NO:15) and 16-2-12D (heavy chain SEQ ID NO:6, light chain SEQ ID NO:18),
It is 16-3-10B (heavy chain SEQ ID NO:1, light chain SEQ ID NO:13) and 17-2-2B (heavy chain positioned at valley Northern Margin area
SEQ ID NO:5, light chain SEQ ID NO:17), being located at three times axis uplift plateau is 34-1-6D (heavy chain SEQ ID NO:10, light chain
SEQ ID NO:22) and 16-3-4D (heavy chain SEQ ID NO:11, light chain SEQ ID NO:23), it is positioned at 2 times of axis uplift plateaus
17-1-12A (heavy chain SEQ ID NO:9, light chain SEQ ID NO:21) and 17-2-12A (heavy chain SEQ ID NO:12, light chain
SEQ ID NO:24).
The present invention tests the statistical analysis used:
The TCID of virus infection RD cell50The statistical analysis of value is using the calculating side Reed-Muench in SPSS software
Method.The difference of concentration is neutralized between two groups to be analyzed in the Mann-Whitney method of GraphPad Prism software.Work as p
Value is considered to have statistical significance less than 0.05.Figure is all with Microsoft Microsoft Excel and GraphPad
Made by the softwares such as Prism.
Wherein the anti-enteric virus71 type monoclonal antibody of the source of people can be used for defining recognized enteric virus71 type shell knot
The epitope in structure region, the method is as follows: (1) female using the anti-enteric virus71 type monoclonal antibody of source of people and enteric virus71 type
Strain effect, filters out the enteric virus71 type that can not be neutralized, is named as and escapes Strain；(2) by it is above-mentioned escape Strain with
Original enteric virus71 type maternal plant carries out sequencing, can define the envelope amino acid replacement site for escaping Strain key, should
Amino acid is in the anti-enteric virus71 type monoclonal antibody of source of people and the vital identification site of enteric virus71 type, that is, enteropathy
The epitope of malicious 71 type shells.
The protecting effect of present invention experiment anti-enteric virus71 type monoclonal antibody of source of people in Mice Body:
Refering to Fig. 6 A and Fig. 6 B, in vivo protected to determine whether the anti-enteric virus71 type monoclonal antibody of source of people has
Mouse fights the ability of enteric virus71 type, we establish the animal model of mouse brain inner infection enteric virus71 type.It is two weeks big
HSCARB2 (human scavenger receptor class B member 2) transgenic mice (C57BL/6 strain it is small
Mouse) it is used to build up the animal model of mouse brain inner infection enteric virus71 type, and the virus of infection is then using enteric virus71
Type clinical strains (the Strain 11-96023 of the Strain 12-96015 or gene hypotype C4 of gene hypotype B5).Then, I
Select the anti-enteric virus71 type monoclonal antibody 17-2-2B of the source of people for acting on virus coat valley south marginal zone
(500 μ g/ml) and another act on the anti-enteric virus71 type monoclonal antibody 16-2- of source of people in virus coat sole area
9D (500 μ g/ml) carries out the test of protection mouse infection.In antibody assay group, while the anti-enteric virus71 type list of source of people is added
The enteric virus71 type (the Strain 11-96023 of the Strain 12-96015 or gene hypotype C4 of gene hypotype B5) of clonal antibody
It is delivered to brain inner infection；It is then that the enteric virus71 type of same volume PBS is added to be delivered to brain inner infection in buffer control group；
In addition, thering is one group to have no the infection of enteric virus71 type but only bestowing PBS (every group n=4 to 8 of same total volume in mouse intracerebral
Mouse).With 4 × 106TCID50The brain of the enteric virus71 type infecting mouse of dosage, after infection 2 to 5 days, mouse can start
There is the neurological symptoms result of severe motion disability, and continues to after about infecting 14 days (shown in Fig. 6 A and Fig. 6 B), severe motion
The symptom of disability can be measured using being seven grades of measurement points-scoring systems, and score is higher, and the symptom that represents is more serious, the results show that people
The anti-enteric virus71 type monoclonal antibody in source and while enteric virus71 type injection really can be significant avoid mouse because of enteric virus71
Type infects growth retardation occurred, and reaches the effect of severe motion disability and growth delay occur after preventing brain inner infection, phase
For the control group in not adding at the anti-enteric virus71 type monoclonal antibody of source of people, there is significant difference (Fig. 6 A and Fig. 6 B institute
Show).
Claims (10)
1. a kind of preparation method of the anti-enteric virus71 type monoclonal antibody of source of people, is transformed as follows:
(1) from the human sample blood for having infected enteric virus71 type, enterovirus 71 type specific immunoglobulin G point is isolated
Secrete B cell；
(2) the immunoglobulin G region of variability gene that above-mentioned B cell is grown in choosing carries out recombinant clone, then above-mentioned recombinant clone is exempted from
Epidemic disease Lysozyme gene, which is transfected into human cell's strain, expresses immunoglobulin G and purifies, and can be obtained the anti-enteric virus71 type of source of people
Monoclonal antibody.
2. the preparation method of the anti-enteric virus71 type monoclonal antibody of source of people as described in claim 1, which is characterized in that the human cell
Strain uses HEK293T human cell strain.
3. the preparation method of the anti-enteric virus71 type monoclonal antibody of source of people as described in claim 1, which is characterized in that the source of people is anti-
Enteric virus71 type monoclonal antibody can be used for defining the epitope in recognized enteric virus71 type shell mechanism region, method
It is as follows:
(1) it is acted on using the anti-enteric virus71 type monoclonal antibody of source of people and enteric virus71 type maternal plant, and filters out and can not neutralize
Enteric virus71 type antibody escapes strain, is named as and escapes Strain；
(2) by the above-mentioned shell mechanism gene for escaping Strain and enteric virus71 type maternal plant carry out sequencing and compared with, that is, can define
The viral antigen that the anti-enteric virus71 type monoclonal antibody of source of people is acted on out determines position.
4. the preparation method of the anti-enteric virus71 type monoclonal antibody of source of people as claimed in claim 2, which is characterized in that the antigen is determined
Positioning system is plotted in the virus coat structure of enteric virus71 type maternal plant, including valley Northern Margin area, sole area, valley
Southern marginal zone, 2 times of axis uplift plateaus and 3 times of axis uplift plateaus, being located at valley Northern Margin area is 16-2-11B (heavy chain SEQ ID
NO:4, light chain SEQ ID NO:16), 16-3-3C (heavy chain SEQ ID NO:7, light chain SEQ ID NO:19) and 16-2-2D (weight
Chain SEQ ID NO:8, light chain SEQ ID NO:20), being located at sole area is 16-2-8C (heavy chain SEQ ID NO:2, light chain
SEQ ID NO:14), 16-2-9D (heavy chain SEQ ID NO:3, light chain SEQ ID NO:15) and 16-2-12D (heavy chain SEQ ID
NO:6, light chain SEQ ID NO:18), being located at valley Northern Margin area is 16-3-10B (heavy chain SEQ ID NO:1, light chain SEQ
ID NO:13) and 17-2-2B (heavy chain SEQ ID NO:5, light chain SEQ ID NO:17), being located at three times axis uplift plateau is 34-1-
6D (heavy chain SEQ ID NO:10, light chain SEQ ID NO:22) and 16-3-4D (heavy chain SEQ ID NO:11, light chain SEQ ID
NO:23), being located at 2 times of axis uplift plateaus is 17-1-12A (heavy chain SEQ ID NO:9, light chain SEQ ID NO:21) and 17-2-12A
(heavy chain SEQ ID NO:12, light chain SEQ ID NO:24).
5. the preparation method of the anti-enteric virus71 type monoclonal antibody of source of people as described in claim 1, wherein verifying made source of people
The method of the ability of the anti-enteric virus71 type monoclonal antibody confrontation enteric virus71 type of specificity protection in vivo is as follows:
(1) plural mouse is attacked into poison infection enteric virus71 type with intracerebral；
(2) by above-mentioned mouse dimidiation, control group and experimental group, the anti-enteric virus71 of experimental group injection source of people are respectively designated as
Type monoclonal antibody, the buffer of control group then injection same volume；
(3) after 2~5 days, control group can obviously generate the neurological symptoms result of severe motion disability compared to experimental group, therefore
The anti-enteric virus71 type monoclonal antibody of susceptible of proof injection source of people protects mice against the infection of enteric virus71 type and is occurred in which can dramatically
Movement disability.
6. the anti-enteropathy of source of people made by a kind of preparation method of the anti-enteric virus71 type monoclonal antibody of source of people as described in claim 1
Malicious 71 type monoclonal antibodies.
7. the anti-enteric virus71 type monoclonal antibody of source of people as claimed in claim 6, wherein the anti-enteric virus71 type list of the source of people
The mutant gene heavy chain of clonal antibody and corresponding light chain expression sequence are 16-3-10B (heavy chain SEQ ID NO:1, light chain SEQ
ID NO:13), 16-2-8C (heavy chain SEQ ID NO:2, light chain SEQ ID NO:14), 16-2-9D (heavy chain SEQ ID NO:3,
Light chain SEQ ID NO:15), 16-2-11B (heavy chain SEQ ID NO:4, light chain SEQ ID NO:16), 17-2-2B (heavy chain SEQ
ID NO:5, light chain SEQ ID NO:17), 16-2-12D (heavy chain SEQ ID NO:6, light chain SEQ ID NO:18), 16-3-3C
(heavy chain SEQ ID NO:7, light chain SEQ ID NO:19), 16-2-2D (heavy chain SEQ ID NO:8, light chain SEQ ID NO:20),
17-1-12A (heavy chain SEQ ID NO:9, light chain SEQ ID NO:21), 34-1-6D (heavy chain SEQ ID NO:10, light chain SEQ
ID NO:22), 16-3-4D (heavy chain SEQ ID NO:11, light chain SEQ ID NO:23) or 17-2-12A (heavy chain SEQ ID NO:
12, light chain SEQ ID NO:24).
8. the anti-enteric virus71 type monoclonal antibody of source of people as claimed in claim 6, wherein the anti-enteric virus71 type list of the source of people
The gene of clonal antibody is optional to be grown to the carrier of Escherichia coli or host cell.
9. it is a kind of for detecting the detection kit of enteric virus71 type, it include the anti-enteric virus71 type of source of people as claimed in claim 7
Monoclonal antibody, wherein the detection kit is by enzyme linked immunosorbent assay (ELISA) analytic approach, immuno-precipitation or Western
Method verifies whether infection enteric virus71 type.
10. it is a kind of for fighting the kit, reagent or medicament of enteric virus71 type, it include the anti-intestines of source of people as claimed in claim 7
Viral 71 type monoclonal antibodies, wherein the enteric virus71 type system that the kit, reagent or medicament neutralize is selected from enteric virus71 type
Gene hypotype B4, B5 or C4.
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| WO2017159996A1 (en) * | 2016-03-18 | 2017-09-21 | 한국생명공학연구원 | Antibody against drug-resistant influenza virus |
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2018
- 2018-01-31 CN CN201810092819.3A patent/CN110092829A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017159996A1 (en) * | 2016-03-18 | 2017-09-21 | 한국생명공학연구원 | Antibody against drug-resistant influenza virus |
| CN107188964A (en) * | 2016-12-15 | 2017-09-22 | 上海科医联创生物科技有限公司 | The anti-CD19 monoclonal antibodies of Quan Renyuan and its production method |
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| HUANG,K.-Y.A. ET AL.: "Homo sapiens clone 16-3-10B anti-EV71 immunoglobulin heavy chain variable region mRNA, partial cds", 《GENBANK ACCESSION：KY354551.1》 * |
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Application publication date: 20190806 |