CN106188281B - The preparation and application of anti-norovirus GII.4 type source of mouse monoclonal antibody - Google Patents
The preparation and application of anti-norovirus GII.4 type source of mouse monoclonal antibody Download PDFInfo
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Classifications
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- 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
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
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Abstract
The invention discloses the preparations and application of a kind of anti-norovirus GII.4 type source of mouse monoclonal antibody.The experimental results showed that the monoclonal antibody has the high neutralization activity to norovirus GII.4, and the cross reaction with GI.1 virus-like particle is not present in the antibody, being capable of specific recognition GII.4 virus-like particle.
Description
Technical field
The invention belongs to biomedicine fields, specifically, the present invention relates to anti-norovirus GII.4 type source of mouse monoclonals
The preparation and application of antibody.
Background technique
Norovirus (NoVs) be lead to the Sporadic cases of acute gastroenteritis and one of the main pathogen broken out greatly, and
And norovirus can infect the people of all age brackets.Although caused symptom is generally relatively milder after norovirus infection,
There is self limiting, the course of disease continues 1-3 days or so, but can still cause more in the people of child, old man and immunologic inadequacy
Serious symptom, or even cause death.According to the amino acid sequence of VP1 capsid protein, norovirus can be divided into 6 genomes
(G1-GVI), but only GI, GII and GIV can infect the mankind.The infection of people's norovirus is mainly drawn by norovirus GII
It rises, and big outbreak of epidemic is mostly caused by norovirus GII.4.Norovirus caused by the variant of GII.4 Strain
It infects and accounts for about 55%-85% in the enterogastritis case in the whole world, and those state of an illness need hospitalization very than more serious
To causing dead case also to be caused mostly by norovirus GII.4.
Norovirus has prevalence in developed country and developing country, brings serious economic loss, youngster to various countries
Virgin, old man health causes very big threat.Up to the present, the therapeutic medicine of the preventative vaccine and special efficacy that do not list
Object.Norovirus lacks cell culture model, and also without small animal model, this brings to the research of vaccine and antiviral drugs
Very big obstruction.
Therefore, those skilled in the art are dedicated to the anti-norovirus drug that exploitation has good potential applicability in clinical practice.
Summary of the invention
The purpose of the present invention is to provide the preparations and application of a kind of anti-norovirus GII.4 source of mouse monoclonal antibody.
The first aspect of the present invention, provides a kind of heavy chain variable region of antibody, and the heavy chain variable region includes following
Three complementary determining region CDR:
CDR1 shown in SEQ ID NO:8,
CDR2 shown in SEQ ID NO:9, and
CDR3 shown in SEQ ID NO:10；
Preferably, the heavy chain variable region has amino acid sequence shown in SEQ ID NO:6.
The second aspect of the present invention, provides a kind of heavy chain of antibody, and the heavy chain has such as first aspect present invention
The heavy chain variable region and heavy chain constant region.
In another preferred example, the heavy chain amino acid sequence of the antibody is as shown in SEQ ID NO.:3.
The third aspect of the present invention provides a kind of light chain variable region of antibody, and the light chain variable region, which has, to be selected from down
The complementary determining region CDR of group:
CDR1' shown in SEQ ID NO:14,
CDR2' shown in SEQ ID NO:15, and
CDR3' shown in SEQ ID NO:16；
Preferably, the light chain variable region has amino acid sequence shown in SEQ ID NO:7.
The fourth aspect of the present invention, provides a kind of light chain of antibody, and the light chain has such as third aspect present invention
The light chain variable region and constant region of light chain.
In another preferred example, the light-chain amino acid sequence of the antibody is as shown in SEQ ID NO.:5.
The fifth aspect of the present invention, provides a kind of antibody, and the antibody includes
(1) heavy chain variable region as described in the first aspect of the invention；And/or
(2) light chain variable region as described in the third aspect of the present invention；
Alternatively, the antibody includes
Heavy chain as described in respect of the second aspect of the invention；And/or light chain as described in the fourth aspect of the present invention.
The sixth aspect of the present invention, provides a kind of recombinant protein, and the recombinant protein includes
(i) as described in the first aspect of the invention heavy chain variable region, heavy chain as described in respect of the second aspect of the invention, such as this hair
Light chain variable region described in the bright third aspect, light chain as described in the fourth aspect of the present invention or as described in fifth aspect present invention
Antibody；And
(ii) sequence label of optional assistance expression and/or purifying.
The seventh aspect of the present invention provides a kind of polynucleotides, it encodes polypeptide selected from the group below:
(1) as described in the first aspect of the invention heavy chain variable region, heavy chain as described in respect of the second aspect of the invention, such as this hair
Light chain variable region described in the bright third aspect, light chain as described in the fourth aspect of the present invention or as described in fifth aspect present invention
Antibody；Or
(2) recombinant protein as described in sixth aspect present invention.
In another preferred example, the sequence of the polynucleotides has such as SEQ ID NO.:2 and/or SEQ ID NO.:4
Shown in polynucleotide sequence.
The eighth aspect of the present invention provides a kind of carrier, it contains multicore described in the 7th aspect of invention
Thuja acid.
The ninth aspect of the present invention provides a kind of genetically engineered host cell, which is characterized in that it contains this hair
Polynucleotides described in seven aspect of the present invention are integrated in carrier described in bright eighth aspect or genome.
The tenth aspect of the present invention provides a kind of kit, includes: in the kit
Antibody described in fifth aspect present invention.
In another preferred example, the kit is enzyme-linked immunologic detecting kit.
The eleventh aspect of the present invention provides a kind of immune conjugate, which contains:
(a) recombinant protein described in the antibody described in fifth aspect present invention or sixth aspect present invention；With
(b) coupling moiety selected from the group below: detectable marker, drug, toxin, cell factor, radionuclide or
Enzyme.
The twelveth aspect of the present invention, provides a kind of pharmaceutical composition, and the composition includes fifth aspect present invention
Immune conjugate described in recombinant protein described in the antibody, sixth aspect present invention or the tenth one side of the invention；With
And
Pharmaceutically acceptable carrier.
The thirteenth aspect of the present invention provides a kind of preparation method of recombinant polypeptide, and this method includes:
(a) under conditions suitable for the expression, host cell described in ninth aspect present invention is cultivated；
(b) recombinant polypeptide is isolated from culture, the recombinant polypeptide is antibody described in fifth aspect present invention
Or recombinant protein described in sixth aspect present invention.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 shows the anti-GII.4 monoclonal antibody of Polyacrylamide Gel Electrophoresis purifying.The antibody of 5 kinds of purifying passes through respectively
It is loaded in 12% polyacrylamide gel after sample-loading buffer processing containing reducing agent and carries out electrophoresis, and is bright with coomassie
Indigo plant dyeing display protein band.M, Protein Marker；1, D11 monoclonal antibody；2, G9 monoclonal antibodies；3,2D8 monoclonal antibodies；4,7D8 monoclonal antibodies；5,
8E1 monoclonal antibody
Fig. 2 shows the binding ability of enzyme-linked immunosorbent assay (Elisa) identification monoclonal antibody and not synantigen.In Elisa
Every hole is coated with 100ngGII.4 (A) or GI.1 (B) virus-like particle respectively on plate, and every hole adds the purifying of various concentration respectively
Monoclonal antibody is incubated for 2 hours at 37 DEG C, is then incubated for the anti-mouse secondary antibody that HRP is marked.Anti-hepatitis B surface antigen (HBsAg) monoclonal antibody
It is used to do unrelated control.Each point shows the OD450nm average and standard deviation of three repeat samples measurement in figure.
Fig. 3 shows that Westernblot is analyzed.After processing, the polyacrylamide 12% is solidifying for GII.4 virus-like particle
Electrophoresis is carried out in glue, is then transferred on pvdf membrane, is hybridized with the monoclonal antibody of purifying.M, Protein Marker；1, D11 is mono-
It is anti-；2, G9 monoclonal antibodies；3,2D8 monoclonal antibodies；4,7D8 monoclonal antibodies；5,8E1 monoclonal antibodies；6, the anti-GII.4 virus-like particle clonal antibody of mouse.
Fig. 4 shows sandwich Elisa detection GI.1 and GII.4 virus-like particle.Every hole is coated with respectively on Elisa plate
The diluted rabbit-anti GII.4 (A) of 50ul 1:5000 or rabbit-anti GI.1 (B), every hole add the GII.4 virus-like of various concentration respectively
Particle (A) and GI.1 virus-like particle (B) are incubated for 2 hours at 37 DEG C, and then the monoclonal antibody of the purifying of 10ng is added in every hole, finally uses
The anti-mouse secondary antibody of HRP label is incubated for.Anti-hepatitis B surface antigen (HBsAg) monoclonal antibody is used to do unrelated control.
Fig. 5 shows that monoclonal antibody inhibits GII.4 virus-like particle and PGMIII to act on to the detection of substitution neutralization test after purification
Activity.Every hole is coated with 50ul 10ug/ml PGMII on Elisa plate, by the monoclonal antibody of various concentration and 0.5ug/ml GII.4 disease
Malicious sample particle is added in Elisa plate after incubation at room temperature 1 hour, is subsequently added into rabbit-anti GII.4, the anti-rabbit finally marked with HRP
Secondary antibody is incubated for.Anti-hepatitis B surface antigen (HBsAg) monoclonal antibody is used to do unrelated control.
Fig. 6 shows the identification of the monoclonal antibody of DNA recombinant expression.Every hole is coated with respectively on Elisa plate
100ngGII.4 virus-like particle, every hole add the monoclonal antibody of the purifying of various concentration to be incubated for 2 hours at 37 DEG C respectively, then use HRP
The anti-mouse secondary antibody of label is incubated for.The culture supernatant of the cell of untransfected plasmid is as blank control.Histogram is shown in figure
The OD450nm average and standard deviation of three repeat samples measurement.
Specific embodiment
The present inventor obtains a kind of anti-norovirus GII.4 source of mouse monoclonal antibody by extensive and in-depth research, real
Test the result shows that, the monoclonal antibody has the high neutralization activity to norovirus GII.4, and the antibody is not present
It, being capable of specific recognition GII.4 virus with the cross reaction of GI.1 virus-like particle.It is anti-that the present invention also provides above-mentioned monoclonals
The purposes of body.
Specifically, this research is prepared for the Dan Ke of energy specific recognition GII.4 using GII.4 virus-like particle as immunogene
Grand antibody 8E1.The methods of Elisa and substitution neutralization experiment illustrate that the antibody can be used to Sensitive Detection and analyze GII.4, heavier
What is wanted is that it also has powerful neutralization activity.
Before describing the present invention, it should be understood that the present invention is not limited to the specific method and experiment conditions, because this
Class method and condition can change.It should also be understood that its purpose of the term as used herein is only that description specific embodiment, and
And it is not intended to be restrictive, the scope of the present invention will be limited only by the claims which follow.
Unless otherwise defined, otherwise whole technologies used herein and scientific term all have such as fields of the present invention
The normally understood identical meanings of those of ordinary skill.As used herein, in use, term in mentioning the numerical value specifically enumerated
" about " mean that the value can change not more than 1% from the value enumerated.For example, as used herein, statement " about 100 " includes 99 Hes
101 and between whole values (for example, 99.1,99.2,99.3,99.4 etc.).
Although can be used in implementation or test of the invention and heretofore described similar or of equal value any method
And material, place enumerates preferred method and material herein.
Norovirus GII.4
Norovirus GII.4 belongs to norovirus category GII type, is the main pathogens for causing norovirus outbreak of epidemic.
Norovirus has prevalence in developed country and developing country, bring serious economic loss to various countries, children, old man
Health causes very big threat.Up to the present, the curative drug of the preventative vaccine and special efficacy that do not list.
The present invention is prepared for GII.4 monoclonal antibody using recombination GII.4 virus-like particle as immunogene.System of the present invention
Standby antibody serves not only as laboratory testing tool, and be prepare therapeutic humanization monoclonal antibody reliable candidate and
Develop the useful reagent of diagnostic method.
Virus-like particle, amino acid sequence are prepared for using norovirus GII.4 VP1 in the present invention are as follows:
MASSDANPSDGSAANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIRNNFVQAPGGEFTVSPRN
APGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVILAGNAFTAGKVIFAAVPPNFPTEGLSPSQVTMFPHIVV
DVRQLEPVLIPLPDVRNNFYHYNQSNDPTIKLIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPTVES
RTKPFSVPVLTVEEMTNSRFPIPLEKLFTGPSSAFVVQPQNGRCTTDGVLLGTTQLSPVNICTFRGDVTHITGSRN
YTMNLASQNWNNYDPTEEIPAPLGTPDFVGKIQGVLTQTTRTDGSTRGHKATVYTGSADFAPKLGRVQFETDTDHD
FEANQNTKFTPVGVIQDGSTTHRNEPQQWVLPSYSGRNTPNVHLAPAVAPTFPGEQLLFFRSTMPGCSGYPNMDLD
CLLPQEWVQYFYQEAAPAQSDVALLRFVNPDTGRVLFECKLHKSGYVTVAHTGQHDLVIPPNGYFRFDSWVNQFYT
LAPMGNGTGRRRAL (SEQ ID NO.:1, GenBank ID:KC631827.1), 309 serines (Ser) sport asparagus fern
Amide (Asn).
Virus-like particle, amino acid sequence are prepared for using norovirus GI.1VP1 in the present invention are as follows:
MASKDATSSVDGASGAGQLVPEVNASDPLAMDPVAGSSTAVATAGQVNPIDPWIINNFVQAPQGEFTI
SPNNTPGDVLFDLSLGPHLNPFLLHLSQMYNGWVGNMRVRIMLAGNAFTAGKIIVSCIPPGFGSHNLTIAQATLFP
HVIADVRTLDPIEVPLEDVRNVLFHNNDRNQQTMRLVCMLYTPLRTGGGTGDSFVVAGRVMTCPSPDFNFLFLVPP
TVEQKTRPFTLPNLPLSSLSNSRAPLPISSMGISPDNVQSVQFQNGRCTLDGRLVGTTPVSLSHVAKIRGTSNGTV
INLTELDGTPFHPFEGPAPIGFPDLGGCDWHINMTQFGHSSQTQYDVDTTPDTFVPHLGSIQANGIGSGNYVGVLS
WISPPSHPSGSQVDLWKIPNYGSSITEATHLAPSVYPPGFGEVLVFFMSKMPGPGAYNLPCLLPQEYISHLASEQA
PTVGEAALLHYVDPDTGRNLGEFKAYPDGFLTCVPNGASSGPQQLPINGVFVFVSWVSRFYQLKPVGTASSARGRL
GLRR (SEQ ID NO.:20, NP_056821.2)
Antibody
As used herein, term " antibody " or " immunoglobulin " are about 150000 dalton for having identical structure feature
Different four glycan albumen is made of two identical light chains (L) and two identical heavy chains (H).Every light chain is total by one
Valence disulfide bond is connected with heavy chain, and the disulfide bond number between the heavy chain of different Immunoglobulin Isotypes is different.Each heavy chain and
The intrachain disulfide bond at light chain also regular interval.One end of each heavy chain has variable region (VH), is followed by multiple constant regions.Every
One end of light chain has variable region (VL), and the other end has constant region；The constant region of light chain is opposite with first constant region of heavy chain, gently
The variable region of chain and the variable region of heavy chain are opposite.Special amino acid residue forms boundary between the variable regions of the light chain and the heavy chain
Face.
As used herein, " variable " the certain parts for indicating variable region in antibody of term are different in sequence, its shape
Combination and specificity at various specific antibodies to its specific antigen.However, changeability and being unevenly distributed over entire anti-
In body variable region.It concentrates in light chain and heavy chain variable region three segments being known as in complementary determining region (CDR) or hypervariable region
In.More conservative part is known as framework region (FR) in variable region.Four FR are respectively contained in the variable region of native heavy and light chain
Area, they are generally in beta sheet configuration, are connected by three CDR of formation connection ring, can form part β folding in some cases
Stack structure.CDR in every chain is by the area FR firmly against the antigen for together forming antibody together and with the CDR of another chain
Binding site (referring to Kabat etc., NIH Publ.No.91-3242, rolls up I, 647-669 pages (1991)).Constant region is not joined directly
With the combination of antibody and antigen, but they show different effector functions, such as participate in antibody dependent on the thin of antibody
Cellular toxicity.
" light chain " of vertebrate antibodies (immunoglobulin) can be classified as obviously not according to the amino acid sequence of its constant region
One kind in same two classes (referred to as κ and λ).According to the amino acid sequence of its heavy chain constant region, immunoglobulin can be divided into not
Same type.Mainly there are 5 immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM, some of them can also be further separated into subclass
(isotype), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.Light chain constant corresponding to different immunoglobulin like protein is distinguished
It is also known as α, δ, ε, γ and μ.The subunit structure and 3-d modelling of different immunoglobulin like protein are known to those skilled in the art
's.
As used herein, term " monoclonal antibody (monoclonal antibody) " refers to the antibody obtained from a kind of substantially uniform group, i.e.,
The single antibody for including in the group be it is identical, in addition to a small number of mutation that may be present naturally occurred.Monoclonal antibody is high
Specifically it is directed to single antigen site.Moreover, (usually having for different determinants from conventional polyclonal antibody preparation
Different antibodies) it is different, each monoclonal antibody is for the single determinant on antigen.Other than their specificity, monoclonal
The benefit of antibody, which also resides in them, to be synthesized by hybridoma culture, will not be polluted by other immunoglobulins.Modifier
" monoclonal " illustrates the characteristic of antibody, is obtained from substantially uniform antibody population, this is not construed as needing to use appointing
What specific process produces antibody.
It is anti-that the invention also includes the monoclonals of the corresponding amino acid sequence with the anti-GII.4 viral monoclonal antibodies
Body, the monoclonal antibody with the anti-GII.4 viral monoclonal antibodies variable region chain, and other eggs with these chains
White matter or protein conjugate and fusion expressed product.Specifically, the present invention include have containing hypervariable region (complementary determining region,
CDR any protein or protein conjugate and fusion expressed product (the i.e. immune conjugate and fusion table of light chain and heavy chain)
Up to product), as long as the hypervariable region is identical as the hypervariable region of light chain of the invention and heavy chain or at least 90% homology, preferably extremely
Few 95% homology.
As it is known by the man skilled in the art, immune conjugate and fusion expressed product include: drug, toxin, cell factor
(cytokine), radionuclide, enzyme and other diagnosis or treatment molecule and the anti-GII.4 viral monoclonal antibodies or its
Segment in conjunction with and the conjugate of formation.The invention also includes with the anti-GII.4 viral monoclonal antibodies or its segment knot
The cell surface marker object or antigen of conjunction.
The present invention not only includes complete monoclonal antibody, further includes having immunocompetent antibody fragment, such as Fab or
(Fab')2Segment；Heavy chain of antibody；Antibody light chain.
As used herein, term " heavy chain variable region " and " VH" be used interchangeably.
As used herein, term " variable region " and " complementary determining region (complementarity determining
Region, CDR) " it is used interchangeably.
In a preferred embodiment of the invention, the heavy chain variable region of the antibody includes that following three complementations are determined
Determine area CDR:
CDR1, amino acid sequence are GYSFTDYYMH (SEQ ID NO:8), and coding nucleotide sequence is,
GGTTACTCATTCACTGACTACTACATGCAC(SEQ ID NO.:11)；
CDR2, amino acid sequence are YIDPFNGDTGYNQKFKV (SEQ ID NO.:9), coding nucleotide sequence
For TATATTGATCCTTTCAATGGTGATACTGGCTACAACCAGAAATTCAAGGTC (SEQ ID NO.:12)；
CDR3, amino acid sequence are DDWGSPFSY (SEQ ID NO.:10), and coding nucleotide sequence is,
GATGACTGGGGATCCCCGTTTTCTTAC(SEQ ID NO.:13)。
In another preferred example, the amino acid sequence of the heavy chain variable region are as follows:
EIQLQQSGPELMKPGASVKISCKASGYSFTDYYMHWVKQSHGKSLEWIGYIDPFNGDTGYNQKFKVRA
TLTVDESSTTAYMHLSSLTSDDSAVYYCARDDWGSPFSYWGQGTLVTVSA(SEQ ID NO.:6)。
In a preferred embodiment of the invention, the heavy chain of the antibody includes above-mentioned heavy chain variable region and heavy chain
Constant region, the heavy chain constant region can be source of mouse or source of people.
In another preferred example, the heavy chain amino acid sequence of the antibody are as follows:
MGWSWIFLFLLSGTAGVHSEIQLQQSGPELMKPGASVKISCKASGYSFTDYYMHWVKQSHGKSLEWIG
YIDPFNGDTGYNQKFKVRATLTVDESSTTAYMHLSSLTSDDSAVYYCARDDWGSPFSYWGQGTLVTVSAAKTTPPS
VYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCN
VAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDV
EVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQM
AKDKVSLTCMITDFFPEDITVEWQWNGQPAENCKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHN
HHTEKSLSHSPGK(SEQ ID NO.:3)
As used herein, term " light chain variable region " and " VL" be used interchangeably.
In a preferred embodiment of the invention, the light chain variable region of antibody according to the present invention has and is selected from
The complementary determining region CDR of the following group:
CDR1', amino acid sequence are RASHDIGSNLD (SEQ ID NO:14), and coding nucleotide sequence is CGG
GCAAGTCATGACATTGGTAGTAACTTAGAC(SEQ ID NO.:17)；
CDR2', amino acid sequence are ATSTLDS (SEQ ID NO:15), and coding nucleotide sequence is,
GCCACATCCACTTTAGATTCT(SEQ ID NO.:18)
CDR3', amino acid sequence are LQYASSPRT (SEQ ID NO:16), and coding nucleotide sequence is,
CTACAATATGCTAGTTCTCCTCGGACG(SEQ ID NO.:19)
In another preferred example, the amino acid sequence of the light chain variable region are as follows:
DIQMTQSPSSLSASLGESVSLTCRASHDIGSNLDWLQQEPDGTIKRLIYATSTLDSGVPKRFSGSRSG
SDYSLTISSLESEDFVEYYCLQYASSPRTFGGGTKLEIK(SEQ ID NO.:7)。
In a preferred embodiment of the invention, the light chain of the antibody includes above-mentioned light chain variable region and light chain
Constant region, the constant region of light chain can be source of mouse or source of people.
In another preferred example, the light-chain amino acid sequence of the antibody are as follows:
MRAAAQIFGFLLLLFPGTRCDIQMTQSPSSLSASLGESVSLTCRASHDIGSNLDWLQQEPDGTIKRLI
YATSTLDSGVPKRFSGSRSGSDYSLTISSLESEDFVEYYCLQYASSPRTFGGGTKLEIKRADAAPTVSIFPPSSEQ
LTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT
STSPIVKSFNRNEC(SEQ ID NO.:5)
In the present invention, term " antibody of the present invention ", " albumen of the present invention " or " polypeptide of the present invention " is used interchangeably, all
Refer to the antibody for specifically binding anti-GII.4 virus, such as with heavy chain (such as amino acid sequence of SEQ ID NO.:3) and/or gently
The albumen or polypeptide of chain (such as amino acid sequence of SEQ ID NO.:5).They can be with or without initial methionine.
In another preferred example, the antibody is mouse or the people's mouse chimeric mAb for resisting anti-GII.4 virus, it
Heavy chain constant region and/or constant region of light chain can be the heavy chain constant region or constant region of light chain of humanization.It is highly preferred that described
The heavy chain constant region or constant region of light chain of humanization are the heavy chain constant region or constant region of light chain of human IgG1, IgG2 etc..
The present invention also provides other protein or fusion expressed product with antibody of the present invention.Specifically, of the invention
It is (i.e. immune even including any protein or protein conjugate and fusion expressed product with heavy chain and light chain containing variable region
Join object and fusion expressed product), as long as the variable region is identical as the variable region of the heavy chain of antibody of the present invention and light chain or at least
90% homology, preferably at least 95% homology.
Generally, the antigenic binding property of antibody can be described by being located at 3 specific regions of heavy chain and light chain variable region,
The section is partitioned into 4 frame areas (FR) by referred to as Variable Area (CDR), and the amino acid sequence of 4 FR is relatively conservative,
Association reaction is not participated in directly.These CDR form cyclic structure, the β-pleated sheet formed by FR therebetween phase on space structure
Mutually close, the CDR on CDR and corresponding light chain on heavy chain constitutes the antigen binding site of antibody.It can be by comparing similar
The amino acid sequence of the antibody of type determines be which Amino acid profile FR or CDR region domain.
The heavy chain of antibody of the present invention and/or the variable region of light chain are particularly interesting, because at least partly relating in them
And combine antigen.Therefore, the present invention includes those molecules with the monoclonal antibody light chain with CDR and heavy chain variable region, only
Want its CDR and CDR that identifies herein have 90% or more (preferably 95% or more, most preferably 98% or more) homology.
The present invention not only includes complete monoclonal antibody, further include have immunocompetent antibody segment or antibody with
The fusion protein that other sequences are formed.Therefore, the invention also includes the segments of the antibody, derivative and analogue.
As used herein, term " segment ", " derivative " refer to that be kept substantially antibody of the present invention identical with " analog "
Biological function or active polypeptide.Polypeptide fragment of the invention, derivative or the like, which can be (i), one or more
Conservative or non-conservative amino acid residue (preferably conservative amino acid) substituted polypeptide, and such substituted amino
Sour residue, which can be, may not be by genetic code encoding, or (ii) has substitution in one or more amino acid residues
The polypeptide of group, or (iii) mature polypeptide and another compound (for example extend the compound of polypeptide half-life period, such as poly- second
Glycol) fusion is formed by polypeptide, or (iv) additional amino acid sequence is fused to this polypeptide sequence and the polypeptide that is formed is (as before
Lead sequence or secretion sequence or for purifying the sequence of this polypeptide or proprotein sequence, or merge egg with what 6His label was formed
It is white).According to the teaching of this article, these segments, derivative and analogue belong to scope known to those skilled in the art.
Antibody of the present invention, which refers to, combines polypeptide active, including above-mentioned CDR region with anti-GII.4 virus.The term also wraps
Include the variant form with polypeptide with antibody identical function of the present invention, comprising above-mentioned CDR region.These variant forms include
(but being not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10)
Missing, insertion and/or the substitution of amino acid, and C-terminal and/or N-terminal addition it is one or several (usually 20 with
It is interior, be more preferably within 5 within preferably 10) amino acid.For example, in the art, use is similar in performance
When amino acid is replaced, the function of protein is not usually changed.For another example, C-terminal and/or N-terminal add one or
Several amino acid will not generally also change the function of protein.The term further includes that the active fragment of antibody of the present invention and activity are spread out
Biology.
The variant form of the polypeptide includes: homologous sequence, conservative variant, allelic variant, natural mutation, induction
Mutant, the encoded albumen of DNA that can hybridize with the coding DNA of antibody of the present invention under the conditions of high or low stringency, with
And more peptide or proteins of the antiserum acquisition using anti-antibody of the present invention.
The present invention also provides other polypeptides, such as the fusion protein comprising human antibody or its segment.In addition to almost overall length
Outside polypeptide, the invention also includes the segments of antibody of the present invention.In general, the segment has at least about 50 companies of antibody of the present invention
Continue amino acid, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, most preferably at least about
100 continuous amino acids.
In the present invention, " conservative variant of antibody of the present invention " refers to compared with the amino acid sequence of antibody of the present invention,
There are at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acid are with similar or analogous properties
Amino acid is replaced and forms polypeptide.These conservative variation's polypeptides carry out amino acid substitution preferably based on Table A and generate.
Table A
The present invention also provides encoding such antibodies or the polynucleotide molecules of its segment or its fusion protein.Of the invention
Polynucleotides can be DNA form or rna form.DNA form includes cDNA, genomic DNA or artificial synthesized DNA.DNA can
To be single-stranded or double-strand.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be with
Coding region sequence shown in SEQ ID NO.:2 or 4 is identical or the variant of degeneracy.As used herein, " the variation of degeneracy
Body " refer in the present invention coding have amino acid sequence identical with polypeptide of the invention, but with SEQ ID NO.:3,5,6,
7, the differentiated nucleic acid sequence of coding region sequence shown in 8,9,10,14,15,16.
The polynucleotides for encoding mature polypeptide of the invention include: the coded sequence of an encoding mature polypeptide；Mature polypeptide
Coded sequence and various additional coding sequences；The coded sequence (and optional additional coding sequence) of mature polypeptide and non-volume
Code sequence.
The term polynucleotides of polypeptide " coding " can be the polynucleotides including encoding this polypeptide, be also possible to further include
The polynucleotides of additional code and/or non-coding sequence.
The invention further relates to hybridizing with above-mentioned sequence and having at least 50% between two sequences, preferably at least
70%, more preferably at least polynucleotides of the 80% phase same sex.The present invention is more particularly directed under strict conditions with it is of the present invention more
The interfertile polynucleotides of nucleotide.In the present invention, " stringent condition " refers to: (1) compared with low ionic strength and higher temperature
Under hybridization and elution, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；Or added with denaturant, such as 50% (v/v) formyl when (2) hybridization
Amine, 0.1% calf serum/0.1% Ficoll, 42 DEG C etc.；Or (3) only the phase same sex between two sequences at least 90%
More than, more preferably 95% or more when, just hybridizes.Further, the polypeptide of interfertile polynucleotide encoding and SEQ ID NO.:
Mature polypeptide shown in 12 and/or SEQ ID NO.:22 has identical biological function and activity.
The nucleotide full length sequence of antibody of the invention or its segment can usually use PCR amplification method, recombination method or artificial
Synthetic method obtains.A kind of feasible method is that manually synthetic method synthesizes related sequence, especially fragment length
When shorter.In general, being then attached the very long segment of available sequence again by first synthesizing multiple small fragments.In addition, may be used also
The coded sequence of heavy chain and expression label (such as 6His) are fused together, fusion protein is formed.
Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will
It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.
Biomolecule (nucleic acid, albumen etc.) according to the present invention includes existing biomolecule in a separate form.
At present, it is already possible to obtain encoding albumen of the present invention (or its segment or its derivative by chemical synthesis completely
Object) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or such as carrier) and
In cell.In addition, mutation can be also introduced into protein sequence of the present invention by chemical synthesis.
The invention further relates to the carriers comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence.This
A little carriers can be used for converting host cell appropriate, allow it to expression protein.
Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high
Equal eukaryocytes, such as mammalian cell.Representative example has: Escherichia coli, streptomyces；The bacterium of salmonella typhimurium
Cell；Fungal cell's such as yeast；The insect cell of drosophila S2 or Sf9；CHO, COS7, zooblast of 293 cells etc..
It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original
When core biology such as Escherichia coli, the competent cell that can absorb DNA can harvest after exponential phase of growth, use CaCl2Method processing, institute
With the step of it is generally well-known in the art.Another method is using MgCl2.If desired, conversion can also use the side of electroporation
Method carries out.When host is eucaryote, following DNA transfection method can be selected: calcium phosphate precipitation, conventional mechanical methods are such as
Microinjection, electroporation, liposome packaging etc..
The transformant of acquisition can use conventional method culture, express the polypeptide of coded by said gene of the invention.According to used
Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of being suitable for host cell growth
It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction)
Cell is further cultured for a period of time by the promoter for inducing selection.
Recombinant polypeptide in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.Such as
Fruit needs, and can be separated by various separation methods and purify the albumen of recombination using its physics, chemical and other characteristics.This
A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to: conventional renaturation process is used
Protein precipitant handles (salting-out method), centrifugation, permeates broken bacterium, super processing, ultracentrifugation, sieve chromatography (gel filtration), inhales
The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.
Antibody of the invention can be used alone, can also be with detectable marker (for diagnostic purpose), therapeutic agent, PK (egg
White kinases) combination of modified part or any the above substance combines or coupling.
Detectable marker for diagnostic purposes includes but is not limited to: fluorescence or luminous marker, radioactively labelled substance,
MRI (magnetic resonance imaging) or CT (x-ray tomography of electronic computer) contrast agent can generate detectable product
Enzyme.
The present invention also provides a kind of compositions.In preference, the composition is pharmaceutical composition, it contains
The antibody stated or its active fragment or its fusion protein and pharmaceutically acceptable carrier.In general, these substances can be prepared
In nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is usually about 5-8, and preferably pH is about
6-8, although pH value can be varied with the property and illness to be treated for being formulated substance.Prepared pharmaceutical composition
It can be administered by conventional route, including (but being not limited to): tumor is interior, peritonaeum is interior, intravenous or local administration.
Pharmaceutical composition of the invention can be directly used for combining GII.4 virus-like particle, thus can be used for preventing and treating
Norovirus (NoVs) is to lead to acute gastroenteritis.In addition, also can be used simultaneously other therapeutic agents.
Pharmaceutical composition of the invention contain safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more
Good ground 0.1-80wt%) the above-mentioned monoclonal antibody (or its conjugate) and pharmaceutically acceptable carrier or tax of the present invention
Shape agent.This kind of carrier include (but being not limited to): salt water, buffer, glucose, water, glycerol, ethyl alcohol, and combinations thereof.Drug system
Agent should match with administration mode.Pharmaceutical composition of the invention can be made into injection form, such as with physiological saline or contain
There are glucose and the aqueous solution of other adjuvants to be prepared by conventional method.Pharmaceutical composition such as injection, solution are preferably sterile
Under the conditions of manufacture.The dosage of active constituent is therapeutically effective amount, such as about 5 mg/kg of about 1 microgram/kg body weight-daily
Weight.In addition, polypeptide of the invention can be also used together with other therapeutic agents.
It is that the immune conjugate of safe and effective amount is applied to mammal when using pharmaceutical composition, the wherein safety
Effective quantity typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg weight, preferably
The ground dosage is about 1 mg/kg weight of about 10 micrograms/kg body weight-.Certainly, specific dosage is also contemplated that administration route, disease
The factors such as people's health status, within the scope of these are all skilled practitioners technical ability.
Hybridoma cell strain
The present invention also provides can produce the present invention for the hybridoma cell strain of anti-GII.4 viral monoclonal antibodies；It is excellent
Choosing, the present invention provides the hybridoma cell strains for anti-GII.4 viral monoclonal antibodies of high-titer.
After obtaining the hybridoma for producing anti-GII.4 viral monoclonal antibodies of the invention, those skilled in the art can
Easily to prepare antibody using the hybridoma cell strain.In addition, those skilled in the art can also easily know the present invention
Antibody structure (such as heavy chain variable region and light chain variable region of antibody), then can prepare the present invention by recombination method
Monoclonal antibody.
The preparation of monoclonal antibody
Antibody of the invention can be prepared by various technologies known to those skilled in the art.For example, this hair
Bright antigen can be applied to animal to induce the generation of monoclonal antibody.For monoclonal antibody, can using hybridoma technology come
Preparation is (see Kohler et al., Nature 256；495,1975；Kohler et al., Eur.J.Immunol.6:511,1976；
Kohler et al., Eur.J.Immunol.6:292,1976；Hammerling et al., In Monoclonal Antibodies
And T Cell Hybridomas, Elsevier, N.Y., 1981) or recombinant DNA method (U.S. Patent number 4,816,567) can be used
Preparation.
Representative myeloma cell is effective integration, the stabilization Gao Shui for supporting by the antibody produced cell of selection antibody
It shows no increases in output and gives birth to and to those of culture medium (HAT medium matrix) sensitivity myeloma cell, including myeloma cell strain, such as mouse
The myeloma cell strain of class (is purchased from Salk including the myeloma cell strain derived from MOPC-21 and MPC-11 mouse tumor
Institute Cell Distribution Center, Santiago, California, the U.S.) and SP-2, NZ0 or
X63-Ag8-653 cell (is purchased from American Type Culture Collection, Luo Keweier, Maryland, the U.S.).
Human myeloma and mouse-human heteromyeloma's cell strain be also described for generate human monoclonal antibodies [Kozbor,
J.Immunol., (1984) 133:3001；Brodeur etc., the production technology and application (Monoclonal of monoclonal antibody
Antibodies Production Techniques and Applications), 51-63 pages (Marcel Dekker,
Inc., New York, 1987)].
Growth of Hybridoma Cell is analyzed in culture medium therein to detect and have the monoclonal of required specificity anti-
The generation of body, e.g., by external binding analysis for example, Enzyme Linked Immunoadsorbent Assay (ELISA) or radiommunoassay (RIA).
The position for expressing the cell of antibody can be detected with FACS.Then, hybridoma clone can be formed by limiting dilution procedures
It is subcloned (subcloned), and grows (Goding, monoclonal antibody (Monoclonal by standard method
Antibodies): principle and practicing (Principles and Practice), Academic Press (1986) 59-103
Page).The suitable culture medium used to reach this purpose includes, for example, DMEM or RPMI-1640 culture medium.In addition,
Hybridoma can be grown as ascites tumor in animal body.
Pass through conventional immunoglobulin purification from culture medium, ascites or serum by the monoclonal antibody of subclone secretion
Technique is suitably separated, these purifying process are for example, Protein A-agarose method (protein A-Sepharose), hydroxyl
Base apatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
The present invention provides a kind of monoclonal antibodies for GII.4 virus.In of the invention one preferred scheme,
Monoclonal antibody is using culture hybridoma method preparation.The supernatant for taking Hybridoma Cell Culture is heavy through saturated ammonium sulfate
Shallow lake method slightly proposes IgG, then the antibody slightly mentioned is purified through affinity column (Protein G-Sephrose).
In the preferred scheme of of the invention one, monoclonal antibody is using Balb/C mouse ascites production monoclonal antibody
Method preparation.About hybridoma is inoculated into the mouse peritoneal of sensitization, visible abdomen obviously swells within 10 days or so.Extract abdomen
Water is pure through affinity column (Protein G-Sephrose) after saturated ammonium sulphate method slightly mentions, then by the antibody slightly mentioned
Change.
The immunoglobulin (antibody) of label
In a preference of the invention, the immunoglobulin has detectable marker.More preferably, the mark
Note object is selected from the group: colloid gold label object, horseradish peroxidase-labeled, colored labels or fluorescent marker.
Method known to those skilled in the art progress can be used in colloid gold label.In a preferred scheme of the invention
In, the monoclonal antibody colloid gold label of anti-GII.4 virus obtains the monoclonal antibody of colloid gold label.
Anti- GII.4 viral monoclonal antibodies of the invention have specificity well, very high potency.
Detection plate and its material
Detection plate material commonly used in the art can be used in detection plate of the invention, using conventional detection plate preparation method system
At.
The present invention detects the plate for detecting immunity of GII.4 virus, and the support plate including test-strips and support test-strips can such as adopt
With PVC polyester offset plate etc.；The test-strips are by filter sample paper, chromatographic material, nitrocellulose filter and blotting paper successively overlap joint group
At overlapping part can be fixedly connected using conventional method, such as adhesive tape；Wherein: the pre-coated colloid gold label of chromatographic material
Or the anti-GII.4 viral monoclonal antibodies or polyclonal antibody of coloured label, it is preferably single by the anti-GII.4 virus of colloid gold label
Clonal antibody adsorbs detection line and nature controlling line on nitrocellulose filter；
In a preferred scheme: the anti-GII.4 viral monoclonal antibodies of pre-coated colloid gold label on chromatographic material
It is that use concentration to carry out for the anti-GII.4 viral monoclonal antibodies solution of 0.5-1.5mg/ml colloid gold label pre-coated, packet
It is measured as 50 μ l/cm2；Preferred concentration is 0.5 or 1.5mg/ml, 50 μ l/cm2；
Detection method and result judgement
Detection plate is laid flat, by sample drop on filter sample paper, tomographic results are observed in sample about 120 μ l, 3~5min.According to
The fringe position of appearance carrys out judging result.
Negative: there is apparent colour band in quality control region, detection zone, are shown as negative；
It is positive: only obvious colour band occur in quality control region, and be shown as positive without colour band in detection zone；
Invalid: quality control region, detection zone are without any colour band or colour band do not occur in quality control region and colour band occur in detection zone, table
Bright detection method mistake or detection plate go bad or failure, and Ying Chongxin exchanges detection plate detection for.
Method and sample
The present invention relates in the method for cell and/or the pattern detection norovirus of histolysis.This method step
Suddenly approximately as: obtain cell and/or tissue samples；In the medium by sample dissolution；Detection is in the sample of the dissolution
The level of GII.4 virus.Sample used in the method for the present invention can be the appointing including cell being present in cell-preservation liquid
What sample, as used in liquid basal cell detection method.
Kit
The present invention also provides a kind of reagents for referring to and containing antibody (or its segment) or detection plate of the invention of the invention
Box, in a preference of the invention, the kit further includes container, operation instructions, buffer etc..
The present invention is further designed for the detection kit of detection GII.4 virus levels, which includes that identification is anti-
The antibody of GII.4 virus detects required common reagent and buffer, such as various bufferings for dissolving the cracking medium of sample
Liquid, detection label, detection substrate etc..The detection kit can be in-vitro diagnosis device.
The present invention is further designed and developed for the GII.4 virus infection correlation circumstance diagnostic assessment from solution sample
Kit, the kit can detecte be present in sample solution GII.4 virus, wherein save sample cell-preservation liquid
It can be the cell-preservation liquid in such as liquid basal cell detection method.
Anti- GII.4 viral monoclonal antibodies of the invention have many advantages, such as high-affinity, high specific, can be widely used in
The detection field of GII.4 virus, such as the preparation field of detection reagent or detection device are prepared, in specificity, sensitivity and inspection
Survey rate etc. has significant advantage compared with traditional detection method or detection reagent.
Main advantages of the present invention are:
(1) monoclonal antibody 8E1 of the invention is capable of the identification norovirus sample particle of specificity；
(2) monoclonal antibody 8E1 of the invention is capable of the combination norovirus GII.4 of specificity, with norovirus GI.1
No cross reaction, to realize the identification to norovirus GI.1 and norovirus GII.4.
(3) monoclonal antibody 8E1 of the invention has powerful neutralization activity to norovirus.
Combined with specific embodiments below, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip
Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and
Number is calculated by weight.Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel
?.
Material and method
The preparation of 1 antigen and mouse immune
Using baculovirus-insect expression system, virus-like is prepared for by expressing norovirus GII.4 VP1
Grain[1].6 weeks females of peritoneal immunity after the virus-like particle (50ul volume) of 10ug is mixed with isometric aluminium adjuvant (500ug)
Balb/c mouse, 0 week, 2 weeks, 4 weeks it is each immune primary.At the 6th week, mice serum is taken to detect dilution factor.7th week
When, the highest mouse of dilution factor passes through tail vein booster immunization 15ugGII.4 virus-like particle.After 3 days, mice spleen is taken
It is dirty to be used to prepare hybridoma.
The preparation and screening of 2 hybridoma cell strains
After mouse tail vein booster immunization 3 days, Mouse spleen cells is taken to melt with myeloma cell SP2/0 by PEG1500
It closes, prepares hybridoma.After 9 days, specific secretion is screened by enzyme-linked immunosorbent assay and is directed to GII.4 virus-like
The antibody of grain.In short, GII.4 virus-like particle be coated with 96 orifice plates, every hole 100ng, 4 DEG C coating overnight, with contain 5% degreasing
The PBST of milk is closed, and every hole adds 50ul hybridoma culture fluids to be incubated at 37 DEG C 2 hours, the secondary antibody then marked with HRP
(sigma) it is incubated for 1 hour, finally carries out chromogenic reaction, read the light absorption value of OD450.
The preparation of 3 ascites and antibody purification
Female Balb/c mouse peritoneal injects 500ul saxol, after two weeks, every mouse peritoneal inject 300,000 it is miscellaneous
Hand over oncocyte.After 7 days, No. 12 syringe needles collect ascites, and 10,000rpm centrifugation 10min remove upper layer grease and lower sediment, take
Clear ascites carries out antibody purification.According to specification, HiTrap HiTrapTM Protein G affinity column (GE is utilized
Health care) purifying ascites acquisition antibody.
4 enzyme-linked immunosorbent assays identify monoclonal antibody
It is coated with 96 hole Elisa plates overnight with every hole 100ngGI.1 or 4 DEG C of GII.4 virus-like particle, identifies the combination of monoclonal antibody
Ability.Elisa plate through the PBST containing 5% skim milk after 37 DEG C of 1 hours of closing, by every hole 50ul by various concentration
(5ug/ml, 2.5ug/ml, 1.25ug/ml and 0.625ug/ml) is added 37 DEG C of monoclonal antibody and is incubated for 2 hours, then with HRP label
Anti- mouse secondary antibody is incubated for, and light absorption value OD450 is last read.
5 polyacrylamide gel electrophoresises and western blot analysis
After protein sample is mixed with SDS-PAG sample-loading buffer, processing 10min is boiled, through 12% polyacrylamide gel
Protein isolate sample.Protein band is shown by coomassie brilliant blue staining or will be carried out on protein delivery to pvdf membrane
Western blot analysis.Monoclonal antibody is diluted in the PBST containing 1% skim milk by ultimate density 1ug/ml.Mouse is anti-
GII.4 polyclonal antibody 1:1000 dilution uses, and is then incubated for the mouse secondary antibody (sigma) that HPR is marked, finally uses LAS-
400 luminescence image analysis instrument are recorded.
The preparation of 6 rabbit-anti GI.1 virus-like particles or GII.4 virus-like particle polyclonal antibody
GI.1 virus-like particle or GII.4 virus-like particle mixed in equal volume with Freund's complete adjuvant it is fully emulsified, subcutaneously
Inject healthy rabbit 150ug/ only.By the GI.1 virus-like particle of 150ug or GII.4 virus-like particle and equivalent after 3 weeks and 6 weeks
The mixing of incomplete Freund's adjuvant adjuvant carries out booster immunization after fully emulsified.2 weeks collection serum after last time is immune, packing
It saves backup for -80 DEG C afterwards.
7 sandwich Elisa detect GI.1 and GII.4 virus-like particle
Respectively with the polyvalent antibody (preparation method is same as above) of rabbit-anti GI.1 virus-like particle and rabbit-anti GII.4 virus-like particle
4 DEG C of polyvalent antibody (preparation method is same as above) 1:5000 dilution (hole 50ul/) be coated with 96 hole Elisa plates overnight, Elisa plate warp
PBST containing 5% skim milk virus-like particle is added in Elisa plate, the hole 40ng/50ul/ after 37 DEG C of 2 hours of closing
Begin, 2 concentration of doubling dilution 12,37 DEG C of 2 hours of incubation, the then hole monoclonal antibody 10ng/50ul/ that virus-like particle is special
37 DEG C are incubated for 1 hour, are then incubated for the mouse secondary antibody that HPR is marked, last read light absorption value OD450.
8 external substitutions neutralize experiment
With porcine gastric mucin III (PGM) (Shanghai Yuan Mu Biotechnology Co., Ltd) (hole 50ul/) room temperature packet of 10ug/ml
By 96 hole Elisa plates, Elisa plate is spare after 4 DEG C of closings overnight through the PBST containing 5% skim milk.By GII.4 virus-like
Grain specific monoclonal antibody 8ug/ml begins, 2 doubling dilutions, is incubated at room temperature 1 with the GII.4 virus-like particle of isometric 0.5ug/ml
It is added on the 96 hole Elisa plates for being coated with PGM after hour, is incubated at room temperature 1 hour, rabbit-anti GII.4 virus-like is then added
37 DEG C of dilution of polyclonal antibody (preparation method is same as above) 1:1000 of grain are incubated for 1 hour, the rabbit secondary antibody then marked with HPR
(sigma) it is incubated for, last reads light absorption value OD450.
The gene order of 9 monoclonal antibodies expands and the building of expression vector
The cell of hybridoma cell strain Trizol reagent is first extracted into total serum IgE, then according to 5 ' RACE kit explanations
Book amplifies heavy chain and light chain full-length gene.It is introduced respectively using the method for PCR amplification at the 5 ' ends and 3 ' ends of heavy chain and light chain
HindIII and EcoRI restriction enzyme site, and the full gene of next heavy chain and light chain will be amplified and be cloned into pGEM-T respectively
(Promage) in, positive colony sequencing is filtered out, is then correctly cloned sequence with HindIII and EcoRI double digestion, warp
After agarose gel electrophoresis is purified into target fragment, connect with plasmid pcDNA3.1 (Promage) the T4DNA ligase of same digestion
It connects, is built into eukaryotic expression vector pcDNA3.1-(m8E1H) and pcDNA3.1- (m8E1L).
The recombinant expression of 10 monoclonal antibody genes is identified
Using liposome method cotransfection pcDNA3.1- (m8E1H) and pcDNA3.1- (m8E1L) arrive Chinese hamster ovary celI, 72
Culture supernatant is collected after hour to be analyzed, the expression of the antibody in culture supernatant is determined using ELISA: with GI.1 virus-like
Grain wrapper sheet is closed 1 hour with the PBST containing 5% milk in 37 DEG C, and 37 DEG C of culture supernatant to be measured that different dilutions are added are incubated for 2
Hour, it is then incubated for the anti-mouse IgG secondary antibody that HRP is marked, last reads light absorption value OD450.
Embodiment 1 secretes the screening of the hybridoma of GII.4 specific antibody
The spleen cell that GII.4 virus-like particle mouse has been immunized is used to prepare hybridoma.Pass through Elisa testing sieve
Hybridoma supernatant is selected, so that the hybridoma cell strain in conjunction with virus capable can be secreted by obtaining.Finally, have for five plants
The monoclonal antibody of excellent binding ability is screened out, they can combine GII.4 virus-like particle.Subtype identification shows, G9,
D11,7D8 and 8E1 belong to IgG1, and 2D8 belongs to IgG3.
Table 1. secretes the hybridoma cell strain identification of monoclonal antibody
| Hybridoma cell strain | Heavy chain | Light chain | With GII.4VLP binding ability * |
| G9 | IgG1 | kappa | +++ |
| D11 | IgG1 | kappa | +++ |
| 2D8 | IgG3 | kappa | +++ |
| 7D8 | IgG1 | kappa | +++ |
| 8E1 | IgG1 | kappa | +++ |
Sample for analysis is 50ul hybridoma culture cell.
* ,+: OD450nm > 0.15；++: OD450nm > 0.3；+++: OD450nm > 0.5.
The specificity analysis of the anti-GII.4 monoclonal antibody of embodiment 2
The purity and integrality of the GII.4 monoclonal antibody purified from ascites by SDS-PAGE identification first.Fig. 1 shows five
The heavy chain and light chain of kind monoclonal antibody are respectively 50KD and 25KD or so.Then, monoclonal antibody and not synantigen are had detected by Elisa method
Reactivity, including GI.1 virus-like particle and GII.4 virus-like particle.Fig. 2 shows that G9, D11,2D8 and 8E1 can be special
Property identification GII.4 virus-like particle, there is no the cross reactions with GI.1 virus-like particle, and some monoclonal antibodies (e.g., 7D8), together
When in conjunction with GI.1 virus-like particle and GII.4 virus-like particle, can not specific recognition GII.4 virus.
Finally, analyzing the combination situation of monoclonal antibody and GII.4 by Western blot, Fig. 3 is shown, in five kinds of antibody:
D11, G9 and 8E1 can identify the GII.4 virus-like particle of natural structure, the GII.4 virus-like after can not identifying denaturation
Grain, prompting the epitope of D11, G9 and 8E1 identification may be comformational epitope.
Sandwich Elisa of the embodiment 3 based on monoclonal antibody specifically can delicately detect GI.1 and GII.4 virus-like particle
Monoclonal antibody is measured by sandwich Elisa (as OD450nm > 0.15, to sentence the minimum detectability degree of virus-like particle
For the positive).Fig. 4 shows that G9, D11,7D8 and 8E1 monoclonal antibody specifically can delicately detect GII.4 virus-like particle, minimum
Limit of detection is respectively as follows: 0.3125ng, 0.3125ng, 0.3125ng, 0.625ng.
The potential neutralization activity of 4 monoclonal antibody of embodiment
Organizing blood group antigens (HBGA) is the carbohydrate being present on mucosal tissue and red blood cell, is needed for norovirus infection
Receptor.The combination of HBGA inhibits test to be widely used as the substitution neutralization test for antibody-mediated norovirus.Pig stomach is viscous
Contain HBGA in liquid element III (PGM), being verified, which can be used for, substitutes neutralization test[2].It is right respectively by substitution neutralization test
The potential neutralization activity of five kinds of monoclonal antibodies of G9, D11,2D8,7D8 and 8E1 is detected.Fig. 5 shows that D11, G9 and 8E1 are to GII.4
Show good neutralization activity, their EC50 of prevention virus-like particle in conjunction with PGM are respectively: 0.0979ug/ml,
0.6948ug/ml and 0.04357ug/ml.The above results show that 8E1 will be much better than other antibody to GII.4 neutralization activity
Strain, it is 16 times or so of G9 antibody strain that 8E1 monoclonal antibody, which is D11 antibody strain mostly twice to the neutralization activity of GII.4,.
The gene sequencing of 5 monoclonal antibody of embodiment
Heavy chain and the sequence of light chain for cloning the monoclonal antibody of the 8E1 come are following (wherein,Single underscorePart is signal peptide sequence
Column, italicized item is variable region sequences,For constant-region sequences):
8E1 monoclonal antibody heavy chain nucleotide sequence:
ATGGGATGGAGCTGGATCTTTCTCTTCCTTCTGTCAGGAACTGCAGGTGTCCACTCT (SEQ ID NO.:2)
8E1 monoclonal antibody heavy chain amino acid sequence:
MGWSWIFLFLLSGTAGVHS (SEQ ID NO.:3)
8E1 monoclonal antibody light chain nucleotide sequence:
ATGAGGGCTGCTGCACAGATTTTTGGCTTCTTGTTGCTCTTGTTTCCAGGTACCAGATGT (SEQ ID NO.:4)
8E1 monoclonal antibody light-chain amino acid sequence:
MRAAAQIFGFLLLLFPGTRC (SEQ ID NO.:5)
8E1 monoclonal antibody heavy chain variable region and light-chain variable sequence are further analyzed, 8E1 monoclonal antibody heavy chain variable amino acid is such as
Under (underscore mark be heavy chain CDR region):
EIQLQQSGPELMKPGASVKISCKASGYSFTDYYMHWVKQSHGKSLEWIGYIDPFNGDTGYNQKFKVRA
TLTVDESSTTAYMHLSSLTSDDSAVYYCARDDWGSPFSYWGQGTLVTVSA(SEQ ID NO.:6)
Above-mentioned heavy chain variable region belongs to IGHV1 subgroup.
8E1 monoclonal antibody chain variable region amino acid is following (underscore mark is heavy chain CDR region):
DIQMTQSPSSLSASLGESVSLTCRASHDIGSNLDWLQQEPDGTIKRLIYATSTLDSGVPKRFSGSRSG
SDYSLTISSLESEDFVEYYCLQYASSPRTFGGGTKLEIK(SEQ ID NO.:7)
Above-mentioned light chain variable region belongs to IGKV9 subgroup.
Each CDR region amino acid sequence and nucleotide sequence are summarized in table 2.
Table 2
The recombinant expression and identification of 6 monoclonal antibody gene of embodiment
In order to which whether the gene for verifying cloned 8E1 monoclonal antibody is correct, the coded sequence of heavy chain and light chain is inserted respectively
Enter into pcDNA3.1, construction of expression vector pcDNA3.1- (m8E1H) and pcDNA3.1- (m8E1L), then cotransfection CHO is thin
Born of the same parents, and whether detected in cell conditioned medium by ELISA with the presence of the antibody for specifically binding GII.4 virus-like particle.Fig. 6 is aobvious
Show, the cell conditioned medium of expression 8E1 monoclonal antibody sequence has very high binding signal, and related to the extension rate of supernatant；Without
The supernatant for transfecting the control cell of related plasmids is all not bound with signal in spite of dilution.The result illustrates of the invention
8E1 monoclonal antibody can be expressed successfully in host cell.
It discusses
Present invention obtains can specific bond GII.4 virion monoclonal antibody 8E1, with it is powerful it is potential in
And activity, therapeutic monoclonal antibodies drug can be used as after humanization or be used as detection reagent.It is examined by sandwich ELISA
It surveys, monoclonal antibody 8E1 is 0.625ng to the minimum detectability degree of GII.4 virus-like particle, this is by these monoclonal antibodies
It develops into norovirus detection kit and provides advantageous theoretical foundation.And the obtained Dan Ke for GII.4 of the present invention
EC50 of the grand antibody 8E1 to the interaction of GII.4 and PGM are as follows: 0.04357ug/ml is much better than other monoclonal antibodies.
Moreover, Western shows that the epitope of 8E1 monoclonal antibody identification is comformational epitope, this makes 8E1 that can only identify that space conformation is correctly sick
Malicious particle, and cannot identify depolymerization and denaturation virion, it is quantitative to can be used in reliably virion.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Bibliography
1.Huhti,L.,et al.,A comparison of methods for purification and
concentration of norovirus GII-4 capsid virus-like particles.Arch Virol,
2010.155(11):p.1855-8
2.Lindesmith,L.C.,et al.,Immunogenetic mechanisms driving norovirus
GII.4 antigenic variation.PLoS Pathog,2012.8(5):p.e1002705.
Claims (13)
1. a kind of antibody, which is characterized in that the antibody has heavy chain variable region and light chain variable region,
Wherein, the heavy chain variable region includes following three complementary determining region CDR:
CDR1 shown in SEQ ID NO:8,
CDR2 shown in SEQ ID NO:9, and
CDR3 shown in SEQ ID NO:10；
Also, the light chain variable region includes following three complementary determining region CDR:
CDR1' shown in SEQ ID NO:14,
CDR2' shown in SEQ ID NO:15, and
CDR3' shown in SEQ ID NO:16.
2. antibody as described in claim 1, which is characterized in that the amino acid sequence of the heavy chain variable region such as SEQ ID
Shown in NO:6.
3. antibody as described in claim 1, which is characterized in that the amino acid sequence of the heavy chain of the antibody such as SEQ ID NO:
Shown in 3.
4. antibody as described in claim 1, which is characterized in that the amino acid sequence of the light chain variable region such as SEQ ID
Shown in NO:7.
5. antibody as described in claim 1, which is characterized in that the amino acid sequence of the light chain of the antibody such as SEQ ID NO:
Shown in 5.
6. a kind of recombinant protein, which is characterized in that the recombinant protein includes
(i) antibody as described in any one in claim 1-5；And
(ii) sequence label of optional assistance expression and/or purifying.
7. a kind of polynucleotides, which is characterized in that it encodes polypeptide selected from the group below:
(1) antibody as described in any one in claim 1-5；Or
(2) recombinant protein as claimed in claim 6.
8. a kind of carrier, which is characterized in that it contains polynucleotides as claimed in claim 7.
9. a kind of genetically engineered host cell, which is characterized in that it contains in carrier or genome according to any one of claims 8
It is integrated with polynucleotides as claimed in claim 7.
10. a kind of kit, which is characterized in that include: in the kit
Antibody of any of claims 1-5.
11. a kind of immune conjugate, which is characterized in that the immune conjugate contains:
(a) antibody of any of claims 1-5 or recombinant protein as claimed in claim 6；With
(b) coupling moiety selected from the group below: detectable marker, drug, toxin, cell factor, radionuclide or enzyme.
12. a kind of pharmaceutical composition, which is characterized in that the composition includes of any of claims 1-5 anti-
Immune conjugate described in body, recombinant protein as claimed in claim 6 or claim 11；And
Pharmaceutically acceptable carrier.
13. a kind of preparation method of recombinant polypeptide, which is characterized in that this method includes:
(a) under conditions suitable for the expression, host cell as claimed in claim 9 is cultivated；
(b) recombinant polypeptide is isolated from culture, the recombinant polypeptide is of any of claims 1-5 anti-
Body or recombinant protein as claimed in claim 6.
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| CN201510216744.1A CN106188281B (en) | 2015-04-30 | 2015-04-30 | The preparation and application of anti-norovirus GII.4 type source of mouse monoclonal antibody |
| PCT/CN2016/080793 WO2016173558A1 (en) | 2015-04-30 | 2016-04-29 | Preparation and use of anti-norovirus gii.4 type murine monoclonal antibody |
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| CN108727488B (en) * | 2017-04-13 | 2022-07-22 | 中国科学院上海巴斯德研究所 | Preparation and application of anti-norovirus GII.17 monoclonal antibody |
| CN109081868B (en) * | 2017-06-14 | 2022-06-24 | 中国科学院上海巴斯德研究所 | Monoclonal antibody targeting Zika virus envelope protein conserved epitope and application thereof |
| CN109957014B (en) * | 2017-12-25 | 2022-03-29 | 中国科学院上海巴斯德研究所 | Preparation and application of anti-norovirus GII.3 murine monoclonal antibody |
| CN109265542B (en) * | 2018-09-27 | 2021-06-15 | 国药中生生物技术研究院有限公司 | Antibody specifically binding norovirus GII.4 genotype VP1 protein or VLP, and preparation method and application thereof |
| CN109734801A (en) * | 2019-03-15 | 2019-05-10 | 南方医科大学 | A kind of Preparation method and use of GII.4 type norovirus broad-spectrum monoclonal antibody |
| CN110221080B (en) * | 2019-06-25 | 2021-09-17 | 上海交通大学 | Human norovirus immune colloidal gold kit and cell strain |
| CN112159797B (en) * | 2020-09-18 | 2022-08-05 | 广东省微生物研究所（广东省微生物分析检测中心） | Hybridoma cell strain 3G 71B 10, anti-GII.4 type norovirus P protein monoclonal antibody and application |
| CN112175912B (en) * | 2020-09-18 | 2022-07-29 | 广东省微生物研究所（广东省微生物分析检测中心） | Hybridoma cell strain 3G41D6, anti-GII.4 type norovirus P protein monoclonal antibody and application |
| JP2024503274A (en) * | 2020-12-30 | 2024-01-25 | 遠大賽威信生命科学（南京）有限公司 | Norovirus virus-like particles, immune compositions or kits, and uses thereof |
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| WO2014126921A1 (en) * | 2013-02-12 | 2014-08-21 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Monoclonal antibodies that neutralize norovirus |
| CN104098698A (en) * | 2013-04-08 | 2014-10-15 | 中国人民解放军第二军医大学 | Antibody against CD3, and preparation method and application thereof |
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|---|---|---|---|---|
| WO2014126921A1 (en) * | 2013-02-12 | 2014-08-21 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Monoclonal antibodies that neutralize norovirus |
| CN104098698A (en) * | 2013-04-08 | 2014-10-15 | 中国人民解放军第二军医大学 | Antibody against CD3, and preparation method and application thereof |
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