CN106188281A - The preparation of anti-norovirus GII.4 type Mus resource monoclonal antibody and application - Google Patents
The preparation of anti-norovirus GII.4 type Mus resource monoclonal antibody and application Download PDFInfo
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- CN106188281A CN106188281A CN201510216744.1A CN201510216744A CN106188281A CN 106188281 A CN106188281 A CN 106188281A CN 201510216744 A CN201510216744 A CN 201510216744A CN 106188281 A CN106188281 A CN 106188281A
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- 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 preparation and the application of a kind of anti-norovirus GII.4 type Mus resource monoclonal antibody.Test result indicate that, described monoclonal antibody has the high neutralization activity to norovirus GII.4, and this antibody does not exist and the cross reaction of GI.1 virus-like particle, it is possible to specific recognition GII.4 virus-like particle.
Description
Technical field
The invention belongs to biomedicine field, specifically, the present invention relates to anti-norovirus GII.4 type Mus
The preparation of resource monoclonal antibody and application.
Background technology
Norovirus (NoVs) be the Sporadic cases causing acute gastroenteritis and the main pathogen broken out greatly it
One, and, norovirus can infect the people of all age brackets.Although, the symptom that norovirus causes after infecting
General the gentleest, there is self limiting, the course of disease continues about 1-3 days, but in child, old man and immunologic function
The most full people still can cause the most serious symptom, even cause death.Aminoacid according to VP1 capsid protein
Sequence, 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 mainly by caused by norovirus GII, and big outbreak of epidemic many by promise as sick
Poison GII.4 causes.The gastroenteritis disease infected in the whole world of the norovirus that the variant of GII.4 Strain is caused
Example account for about 55%-85%, and those state of an illness are than the more serious case needing hospitalization to even result in death
Also mostly caused by norovirus GII.4.
Norovirus all has popular in developed country and developing country, brings serious economic loss to various countries,
Child, the health of old man cause the biggest threat.Up to the present, there is no preventative vaccine and the specially good effect of listing
Curative drug.Norovirus lacks cell culture model, does not also have small animal model, and this gives vaccine and disease-resistant
The research of cytotoxic drug brings the biggest obstruction.
Therefore, those skilled in the art are devoted to develop the anti-norovirus medicine with good potential applicability in clinical practice.
Summary of the invention
It is an object of the invention to provide the preparation of a kind of anti-norovirus GII.4 Mus resource monoclonal antibody and answer
With.
A first aspect of the present invention, it is provided that the variable region of heavy chain of a kind of antibody, described variable region of heavy chain bag
Include three below 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, described variable region of heavy chain has the aminoacid sequence shown in SEQ ID NO:6.
A second aspect of the present invention, it is provided that the heavy chain of a kind of antibody, described heavy chain has such as the present invention
Variable region of heavy chain described in Yi Fangmian and CH.
In another preference, the heavy chain amino acid sequence of described antibody is as shown in SEQ ID NO.:3.
A third aspect of the present invention, it is provided that the variable region of light chain of a kind of antibody, described variable region of light chain has
Complementary determining region CDR selected from lower group:
CDR1' shown in SEQ ID NO:14,
CDR2' shown in SEQ ID NO:15, and
CDR3' shown in SEQ ID NO:16；
Preferably, described variable region of light chain has the aminoacid sequence shown in SEQ ID NO:7.
A fourth aspect of the present invention, it is provided that the light chain of a kind of antibody, described light chain has such as the present invention
Variable region of light chain described in three aspects and constant region of light chain.
In another preference, the light-chain amino acid sequence of described antibody is as shown in SEQ ID NO.:5.
A fifth aspect of the present invention, it is provided that a kind of antibody, described antibody has:
(1) variable region of heavy chain as described in the first aspect of the invention；And/or
(2) variable region of light chain as described in third aspect present invention；
Or, described antibody has:
Heavy chain as described in respect of the second aspect of the invention；And/or the light chain as described in fourth aspect present invention.
A sixth aspect of the present invention, it is provided that a kind of recombiant protein, described recombiant protein has:
(i) variable region of heavy chain as described in the first aspect of the invention, heavy chain as described in respect of the second aspect of the invention,
Variable region of light chain as described in third aspect present invention, the light chain as described in fourth aspect present invention or as this
Invent the antibody described in the 5th aspect；And
(ii) optional assistance is expressed and/or the sequence label of purification.
A seventh aspect of the present invention, it is provided that a kind of polynucleotide, its coding is selected from the polypeptide of lower group:
(1) variable region of heavy chain as described in the first aspect of the invention, heavy chain as described in respect of the second aspect of the invention,
Variable region of light chain as described in third aspect present invention, the light chain as described in fourth aspect present invention or as this
Invent the antibody described in the 5th aspect；Or
(2) recombiant protein as described in sixth aspect present invention.
In another preference, the sequence of described polynucleotide has such as SEQ ID NO.:2 and/or SEQ ID
Polynucleotide sequence shown in NO.:4.
A eighth aspect of the present invention, it is provided that a kind of carrier, it contains described in invention the 7th aspect
Polynucleotide.
A ninth aspect of the present invention, it is provided that a kind of genetically engineered host cell, it is characterised in that it
The multinuclear described in the present invention seven aspect is had containing the carrier described in eighth aspect present invention or genome are integrated
Thuja acid.
A tenth aspect of the present invention, it is provided that a kind of test kit, described test kit includes:
Antibody described in fifth aspect present invention.
In another preference, described test kit is enzyme-linked immunologic detecting kit.
A eleventh aspect of the present invention, it is provided that a kind of immune conjugate, this immune conjugate contains:
Antibody described in (a) fifth aspect present invention or the recombiant protein described in sixth aspect present invention；With
B () is selected from the coupling moiety of lower group: detectable, medicine, toxin, cytokine, radiation
Property nucleic or enzyme.
A twelveth aspect of the present invention, it is provided that a kind of pharmaceutical composition, described compositions comprises the present invention the
Antibody described in five aspects, the recombiant protein described in sixth aspect present invention or the present invention the 11st aspect institute
The immune conjugate stated；And
Pharmaceutically acceptable carrier.
A thirteenth aspect of the present invention, it is provided that the preparation method of a kind of recombinant polypeptide, the method comprises:
A () under conditions suitable for the expression, cultivates the host cell described in ninth aspect present invention；
B () isolates recombinant polypeptide from culture, described recombinant polypeptide is described in fifth aspect present invention
Antibody or sixth aspect present invention described in recombiant protein.
In should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and below (as implemented
Example) in can be combined with each other between each technical characteristic of specifically describing, thus constitute new or preferred skill
Art scheme.As space is limited, the most tired at this state.
Accompanying drawing explanation
Fig. 1 shows the anti-GII.4 monoclonal antibody of Polyacrylamide Gel Electrophoresis purification.Resisting of 5 kinds of purification
Body is loaded in the polyacrylamide gel of 12% carry out respectively after the sample-loading buffer containing reducing agent processes
Electrophoresis, and show protein band with coomassie brilliant blue staining.M, Protein Marker；1, D11 monoclonal antibody；
2, G9 monoclonal antibodies；3,2D8 monoclonal antibodies；4,7D8 monoclonal antibodies；5,8E1 monoclonal antibodies
Fig. 2 shows that enzyme-linked immunosorbent assay (Elisa) identifies the binding ability of monoclonal antibody and not synantigen.
On Elisa plate, every hole is coated 100ngGII.4 (A) or GI.1 (B) virus-like particle, every hole respectively
The monoclonal antibody of the purification adding variable concentrations respectively is hatched 2 hours at 37 DEG C, then resists with the anti-Mus two of HRP labelling
Hatch.Anti-hepatitis B surface antigen (HBsAg) monoclonal antibody is used to do unrelated control.In figure, each point shows
OD450nm meansigma methods and the standard deviation of three repeat samples mensuration are shown.
Fig. 3 shows that Westernblot analyzes.GII.4 virus-like particle after treatment, 12% poly-
Acrylamide gel carries out electrophoresis, is then transferred on pvdf membrane, hybridize with the monoclonal antibody of purification.
M, Protein Marker；1, D11 monoclonal antibody；2, G9 monoclonal antibodies；3,2D8 monoclonal antibodies；4,7D8 monoclonal antibodies；5,
8E1 monoclonal antibody；6, mouse-anti GII.4 virus-like particle clonal antibody.
Fig. 4 shows that sandwich Elisa detects GI.1 and GII.4 virus-like particle.On Elisa plate often
Hole is coated the anti-GII.4 of rabbit (A) or the anti-GI.1 of the rabbit (B) of 50ul 1:5000 dilution respectively, and every hole is divided
The GII.4 virus-like particle (A) and the GI.1 virus-like particle (B) that do not add variable concentrations are hatched at 37 DEG C
2 hours, the then monoclonal antibody of the purification of every hole addition 10ng, finally incubate with the anti-Mus two of HRP labelling is anti-
Educate.Anti-hepatitis B surface antigen (HBsAg) monoclonal antibody is used to do unrelated control.
Fig. 5 shows that replacement neutralization test detects monoclonal antibody suppression GII.4 virus-like particle and PGMIII after purification
The activity of effect.On Elisa plate, every hole is coated 50ul 10ug/ml PGMII, by the monoclonal antibody of variable concentrations
Add in Elisa plate after incubated at room 1 hour with 0.5ug/ml GII.4 virus-like particle, then add
Enter the anti-GII.4 of rabbit, finally hatch with the anti-rabbit two of HRP labelling is anti-.Anti-hepatitis B surface antigen (HBsAg)
Monoclonal antibody is used to do unrelated control.
Fig. 6 shows the qualification of the monoclonal antibody of DNA recombinant expression.On Elisa plate, every hole is wrapped respectively
By 100ngGII.4 virus-like particle, every hole adds the monoclonal antibody of the purification of variable concentrations respectively and hatches 2 at 37 DEG C
Hour, then hatch with the anti-Mus two of HRP labelling is anti-.The culture supernatant of the cell of untransfected plasmid is made
For blank.In figure, block diagram shows OD450nm meansigma methods and the standard deviation of three repeat samples mensuration.
Detailed description of the invention
The present inventor is by extensively in-depth study, it is thus achieved that a kind of anti-norovirus GII.4 Mus resource monoclonal
Antibody, test result indicate that, described monoclonal antibody has the high neutralization to norovirus GII.4 and lives
Property, and this antibody does not exist and the cross reaction of GI.1 virus-like particle, it is possible to specific recognition GII.4
Virus.Present invention also offers the purposes of said monoclonal antibody.
Specifically, this research is prepared for energy specific recognition GII.4 using GII.4 virus-like particle as immunogen
Monoclonal antibody 8E1.Elisa and substituting neutralize the method such as experiment illustrate this antibody can be used to Sensitive Detection and
Analyze GII.4, it is often more important that it also has powerful neutralization activity.
Before describing the present invention, it should be understood that the invention is not restricted to described concrete grammar and experiment condition,
Because this kind of method and condition can change.It should also be understood that its purpose of term used herein is only that description
Specific embodiments, and it is not intended to be restrictive, the scope of the present invention is by only by appended claim
Book limits.
Unless otherwise defined, whole technology the most used herein and scientific terminology are respectively provided with such as institute of the present invention
The identical meanings that the those of ordinary skill in genus field is generally understood that.As used herein, specifically enumerate mentioning
When using in numerical value, term " about " means that this value can be not more than 1% from the value variation enumerated.Such as,
As used herein, statement " about 100 " include 99 and 101 and between whole values (such as, 99.1,99.2,
99.3,99.4 etc.).
Although implementing or can use in test and heretofore described similar or of equal value appointing in the present invention
Where method and material, place enumerates preferred method and material herein.
Norovirus GII.4
Norovirus GII.4 belongs to norovirus and belongs to GII type, is cause norovirus outbreak of epidemic main
Pathogen.Norovirus all has popular in developed country and developing country, brings serious warp to various countries
Ji loss, child, the health of old man cause the biggest threat.Up to the present, there is no the prevention of listing
Property vaccine and the curative drug of specially good effect.
The present invention utilizes restructuring GII.4 virus-like particle to be prepared for GII.4 monoclonal antibody as immunogen.
Antibody prepared by the present invention serves not only as test in laboratory instrument, and is to prepare therapeutic humanization
The reliable candidate of monoclonal antibody and the useful reagent of exploitation diagnostic method.
Using norovirus GII.4 VP1 to be prepared for virus-like particle in the present invention, its aminoacid sequence is:
MASSDANPSDGSAANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIRNNFVQAPGG
EFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVILAGNAFTAGKVIFA
AVPPNFPTEGLSPSQVTMFPHIVVDVRQLEPVLIPLPDVRNNFYHYNQSNDPTIKLIAML
YTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPTVESRTKPFSVPVLTVEEMTNSRF
PIPLEKLFTGPSSAFVVQPQNGRCTTDGVLLGTTQLSPVNICTFRGDVTHITGSRNYTMN
LASQNWNNYDPTEEIPAPLGTPDFVGKIQGVLTQTTRTDGSTRGHKATVYTGSADFAPKL
GRVQFETDTDHDFEANQNTKFTPVGVIQDGSTTHRNEPQQWVLPSYSGRNTPNVHLAPAV
APTFPGEQLLFFRSTMPGCSGYPNMDLDCLLPQEWVQYFYQEAAPAQSDVALLRFVNPDT
GRVLFECKLHKSGYVTVAHTGQHDLVIPPNGYFRFDSWVNQFYTLAPMGNGTGRRRAL
(SEQ ID NO.:1, GenBank ID:KC631827.1), 309 serines (Ser) sport sky
Winter amide (Asn).
Using norovirus GI.1VP1 to be prepared for virus-like particle in the present invention, its aminoacid sequence is:
MASKDATSSVDGASGAGQLVPEVNASDPLAMDPVAGSSTAVATAGQVNPIDPWIINNFVQ
APQGEFTISPNNTPGDVLFDLSLGPHLNPFLLHLSQMYNGWVGNMRVRIMLAGNAFTAGK
IIVSCIPPGFGSHNLTIAQATLFPHVIADVRTLDPIEVPLEDVRNVLFHNNDRNQQTMRL
VCMLYTPLRTGGGTGDSFVVAGRVMTCPSPDFNFLFLVPPTVEQKTRPFTLPNLPLSSLS
NSRAPLPISSMGISPDNVQSVQFQNGRCTLDGRLVGTTPVSLSHVAKIRGTSNGTVINLT
ELDGTPFHPFEGPAPIGFPDLGGCDWHINMTQFGHSSQTQYDVDTTPDTFVPHLGSIQAN
GIGSGNYVGVLSWISPPSHPSGSQVDLWKIPNYGSSITEATHLAPSVYPPGFGEVLVFFM
SKMPGPGAYNLPCLLPQEYISHLASEQAPTVGEAALLHYVDPDTGRNLGEFKAYPDGFLT
CVPNGASSGPQQLPINGVFVFVSWVSRFYQLKPVGTASSARGRLGLRR
(SEQ ID NO.:20, NP_056821.2)
Antibody
As used herein, term " antibody " or " immunoglobulin " are have identical architectural feature about 150000
Daltonian different four polysaccharide albumen, it is made up of the heavy chain (H) that two identical light chains (L) are identical with two.
Every light chain is connected with heavy chain by a covalent disulfide bonds, and between the heavy chain of different Immunoglobulin Isotype
Disulfide bond number different.Every heavy chain and the intrachain disulfide bond at the most regular interval of light chain.Every heavy chain
There is variable region (VH) one end, is followed by multiple constant region.There is variable region (VL) one end of every light chain, another
End has constant region；The constant region of light chain is relative with the first of heavy chain constant region, the variable region of light chain and heavy chain
Variable region relative.Special amino acid residue forms interface between light chain and the variable region of heavy chain.
As used herein, term " variable " represent in antibody some part of variable region in sequence the most not
With, it defines various specific antibodies to the combination of its specific antigen and specificity.But, transmutability is not
It is evenly distributed in whole antibody variable region.It concentrates on and is referred to as complementation decision in light chain and variable region of heavy chain
In three fragments in district (CDR) or hypervariable region.Part more conservative in variable region is referred to as framework region (FR).
Each self-contained four FR districts in the variable region of native heavy and light chain, they are generally in beta sheet configuration,
It is connected by forming three CDR connecting ring, in some cases can forming part β-pleated sheet structure.Every chain
In CDR by FR district firmly against together and together form the antigen of antibody with the CDR of another chain
Binding site (sees Kabat etc., NIH Publ.No.91-3242, roll up I, 647-669 page (1991)).
Constant region the most directly participates in the combination of antibody and antigen, but they show different effector functions, such as
Participate in the cytotoxicity depending on antibody of antibody.
" light chain " of vertebrate antibodies (immunoglobulin) can be classified as according to the aminoacid sequence of its constant region
A class in visibly different two classes (referred to as κ and λ).According to the aminoacid sequence of its CH, immunity
Globulin can be divided into different kinds.Mainly have 5 immunoglobulin like protein: IgA, IgD, IgE, IgG and
IgM, some of them also can be further separated into subclass (isotype), such as IgG1, IgG2, IgG3, IgG4, IgA
And IgA2.CH corresponding to inhomogeneity immunoglobulin is called α, δ, ε, γ and μ.
The subunit structure of inhomogeneity immunoglobulin and 3-d modelling are known to those skilled in the art.
As used herein, term " monoclonal antibody (monoclonal antibody) " refers to obtain from the colony that a class is substantially uniform
The single antibody comprised in antibody, i.e. this colony is identical, except dashing forward of minority natural generation that may be present
Outside change.Monoclonal antibody is with high specificity for single antigen site.And, with conventional polyclonal antibody system
Agent (being typically have the different antibodies for different determinants) is different, and each monoclonal antibody is on antigen
Single determinant.In addition to their specificity, it is by miscellaneous that the benefit of monoclonal antibody also resides in them
Hand over tumor cultivation to synthesize, will not be polluted by other immunoglobulin.Modifier " monoclonal " illustrates anti-
The characteristic of body, is to obtain from substantially uniform antibody population, and this is not construed as needing with any special
Method produces antibody.
Present invention additionally comprises the list of the corresponding aminoacid sequence with described anti-GII.4 viral monoclonal antibodies
Clonal antibody, there is the monoclonal antibody of described anti-GII.4 viral monoclonal antibodies variable region chain, and
There is other protein of these chains or protein conjugate and fusion expressed product.Specifically, bag of the present invention
Include and there is the light chain containing hypervariable region (complementary determining region, CDR) and any protein of heavy chain or protein molecule
Thing and fusion expressed product (i.e. immune conjugate and fusion expressed product), as long as this hypervariable region is with the present invention's
Light chain is identical with the hypervariable region of heavy chain or at least 90% homology, preferably at least 95% homology.
As it is known by the man skilled in the art, immune conjugate and fusion expressed product include: medicine, toxin,
Cytokine (cytokine), radionuclide, enzyme and other diagnosis or treatment molecule and described anti-GII.4
That viral monoclonal antibodies or its fragment combine and the conjugate that formed.Present invention additionally comprises and resist with described
Cell surface marker thing that GII.4 viral monoclonal antibodies or its fragment combine or antigen.
The present invention not only includes complete monoclonal antibody, also includes having immunocompetent antibody fragment, as
Fab or (Fab')2Fragment；Heavy chain of antibody；Light chain of antibody.
As used herein, term " variable region of heavy chain " and " VH" be used interchangeably.
As used herein, term " variable region " and " complementary determining region (complementarity determining
Region, CDR) " it is used interchangeably.
One of the present invention preferred embodiment in, the variable region of heavy chain of described antibody includes three below
Complementary determining region CDR:
CDR1, its aminoacid sequence is GYSFTDYYMH (SEQ ID NO:8), its coding nucleotide sequence
It is classified as, GGTTACTCATTCACTGACTACTACATGCAC (SEQ ID NO.:11)；
CDR2, its aminoacid sequence is YIDPFNGDTGYNQKFKV (SEQ ID NO.:9), its encoding nucleoside
Acid sequence is, TATATTGATCCTTTCAATGGTGATACTGGCTACAACCAGAAATTCAAGGTC (SEQ ID
NO.:12)；
CDR3, its aminoacid sequence is DDWGSPFSY (SEQ ID NO.:10), its coding nucleotide
Sequence is, GATGACTGGGGATCCCCGTTTTCTTAC (SEQ ID NO.:13).
In another preference, the aminoacid sequence of described variable region of heavy chain is:
EIQLQQSGPELMKPGASVKISCKASGYSFTDYYMHWVKQSHGKSLEWIGYIDPFNGDTGYNQK
FKVRATLTVDESSTTAYMHLSSLTSDDSAVYYCARDDWGSPFSYWGQGTLVTVSA(SEQ ID
NO.:6)。
One of the present invention preferred embodiment in, the heavy chain of described antibody includes above-mentioned variable region of heavy chain
And CH, described CH can be Mus source or people source.
In another preference, the heavy chain amino acid sequence of described antibody is:
MGWSWIFLFLLSGTAGVHSEIQLQQSGPELMKPGASVKISCKASGYSFTDYYMHWVKQSHGKS
LEWIGYIDPFNGDTGYNQKFKVRATLTVDESSTTAYMHLSSLTSDDSAVYYCARDDWGSPFSYWGQG
TLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSD
LYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDV
LTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGK
EFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWN
GQPAENCKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ
ID NO.:3)
As used herein, term " variable region of light chain " and " VL" be used interchangeably.
One of the present invention preferred embodiment in, according to the variable region of light chain of the antibody of the present invention, tool
There is a complementary determining region CDR selected from lower group:
CDR1', its aminoacid sequence is RASHDIGSNLD (SEQ ID NO:14), its coding nucleotide
Sequence is, CGGGCAAGTCATGACATTGGTAGTAACTTAGAC (SEQ ID NO.:17)；
CDR2', its aminoacid sequence is ATSTLDS (SEQ ID NO:15), and its coding nucleotide sequence is,
GCCACATCCACTTTAGATTCT(SEQ ID NO.:18)
CDR3', its aminoacid sequence is LQYASSPRT (SEQ ID NO:16), its coding nucleotide sequence
It is classified as, CTACAATATGCTAGTTCTCCTCGGACG (SEQ ID NO.:19)
In another preference, the aminoacid sequence of described variable region of light chain is:
DIQMTQSPSSLSASLGESVSLTCRASHDIGSNLDWLQQEPDGTIKRLIYATSTLDSGVPKRFS
GSRSGSDYSLTISSLESEDFVEYYCLQYASSPRTFGGGTKLEIK(SEQ ID NO.:7)。
One of the present invention preferred embodiment in, the light chain of described antibody includes above-mentioned variable region of light chain
And constant region of light chain, described constant region of light chain can be Mus source or people source.
In another preference, the light-chain amino acid sequence of described antibody is:
MRAAAQIFGFLLLLFPGTRCDIQMTQSPSSLSASLGESVSLTCRASHDIGSNLDWLQQEPDGT
IKRLIYATSTLDSGVPKRFSGSRSGSDYSLTISSLESEDFVEYYCLQYASSPRTFGGGTKLEIKRAD
AAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSS
TLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(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 "
It is used interchangeably, all refers to the antibody of specific binding anti-GII.4 virus, such as, there is heavy chain (such as SEQ ID
The aminoacid sequence of NO.:3) and/or the albumen or many of light chain (such as the aminoacid sequence of SEQ ID NO.:5)
Peptide.They can be with or without initial methionine.
In another preference, described antibody be anti-GII.4 virus Mus or people's Mus chimeric monoclonal resist
Body, its CH and/or constant region of light chain can be humanized CH or chain constant
District.It is highly preferred that described humanized CH or constant region of light chain are human IgG1, IgG2 etc.
CH or constant region of light chain.
Present invention also offers other protein or the fusion expressed product with antibody of the present invention.Specifically,
Any protein that the present invention includes having heavy chain and light chain containing variable region or protein conjugate and fusion
Expression product (i.e. immune conjugate and fusion expressed product), if this variable region and the heavy chain of antibody of the present invention
Or at least 90% homology, preferably at least 95% homology identical with the variable region of light chain.
Typically, the antigenic binding property of antibody can be by 3 the specific regions being positioned at heavy chain and variable region of light chain
Describe, referred to as Variable Area (CDR), this intersegmental is divided into 4 frame areas (FR), the ammonia of 4 FR
Base acid sequence is the most conservative, the most directly participates in association reaction.These CDR form circulus, pass through
The β-pleated sheet that FR therebetween is formed is close to each other on space structure, on CDR on heavy chain and corresponding light chain
CDR constitute the antigen binding site of antibody.Can be by comparing the aminoacid sequence of the antibody of same type
Determine be which Amino acid profile FR or CDR region territory.
The heavy chain of antibody of the present invention and/or the variable region of light chain are particularly interesting, because in them at least
Part relates to conjugated antigen.Therefore, the present invention includes that those have the monoclonal antibody light chain of band CDR and weight
The molecule of chain variable region, if its CDR and the CDR herein identified have more than 90% (preferably more than 95%,
Most preferably more than 98%) homology.
The present invention not only includes complete monoclonal antibody, the fragment that also includes there is immunocompetent antibody or
The fusion protein that antibody is formed with other sequences.Therefore, present invention additionally comprises the fragment of described antibody, derive
Thing and analog.
As used herein, term " fragment ", " derivant " and " analog " refers to be kept substantially this
Biological function that invention antibody is identical or the polypeptide of activity.The polypeptide fragment of the present invention, derivant or similar
Thing can be that (i) has one or more conservative or non-conservative amino acid residue (preferably conservative amino acid is residual
Base) polypeptide that is replaced, and such substituted amino acid residue can be to may not be by genetic code
Encode, or (ii) has the polypeptide of substituted radical in one or more amino acid residues, or (iii) becomes
Ripe polypeptide merges institute with another compound (such as extending the compound of polypeptide half-life, such as Polyethylene Glycol)
The polypeptide formed, or the polypeptide that (iv) additional aminoacid sequence is fused to this peptide sequence and is formed is (as front
Lead sequence or secretion sequence or for the sequence of this polypeptide of purification or proprotein sequence, or with 6His tag-shaped
The fusion protein become).According to teaching herein, it is ripe that these fragments, derivant and analog belong to this area
Practice scope known to technical staff.
Antibody of the present invention refers to have polypeptide that anti-GII.4 virus combines activity, that include above-mentioned CDR region.Should
Term also includes having and antibody identical function of the present invention, the polypeptide that comprises above-mentioned CDR region variant form.
These variant forms include (but being not limited to): one or more (usually 1-50, preferably 1-30
Individual, more preferably 1-20, most preferably 1-10) amino acid whose disappearance, insert and/or replace, Yi Ji
C-terminal and/or N-terminal add one or several (usually within 20, within preferably 10,
Within being more preferably 5) aminoacid.Such as, in the art, with similar nature or similar aminoacid
When replacing, generally will not change the function of protein.The most such as, add at C-terminal and/or N-terminal
Add one or several aminoacid generally also will not change the function of protein.This term also includes antibody of the present invention
Active fragment and reactive derivative.
The variant form of this polypeptide includes: homologous sequence, conservative variant, allelic variant, natural prominent
Variant, induced mutants, can hybridize with the coding DNA of antibody of the present invention under the conditions of high or low stringency
The albumen coded by DNA and utilize the polypeptide or albumen that the antiserum of anti-antibody of the present invention obtains.
Present invention also offers other polypeptide, as comprised the fusion protein of people's antibody or its fragment.Except almost
Outside the polypeptide of total length, present invention includes the fragment of antibody of the present invention.Generally, this fragment has the present invention
At least about 50 continuous amino acids of antibody, the most at least about 50 continuous amino acids, the most at least
About 80 continuous amino acids, the most at least about 100 continuous amino acids.
In the present invention, " conservative variant of antibody of the present invention " refers to the aminoacid sequence with antibody of the present invention
Row are compared, and have at most 10, the most at most 8, the most at most 5, the most at most 3
Aminoacid is replaced by the aminoacid that character is similar or close and is formed polypeptide.These conservative variation's polypeptide are
Carry out aminoacid replacement according to Table A and produce well.
Table A
Present invention also offers encoding such antibodies or its fragment or the polynucleotide molecule of its fusion protein.This
The polynucleotide of invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA
Or the DNA of synthetic.DNA can be strand or double-strand.DNA can be coding strand or non-coding
Chain.The coding region sequence of encoding mature polypeptide can be with the coding region sequence shown in SEQ ID NO.:2 or 4
Identical or the variant of degeneracy.As used herein, " variant of degeneracy " refers to compile in the present invention
Code has an aminoacid sequence identical with the polypeptide of the present invention, but with SEQ ID NO.:3,5,6,7,8,
9, the differentiated nucleotide sequence of coding region sequence shown in 10,14,15,16.
The polynucleotide of the mature polypeptide of code book invention include: the coded sequence of an encoding mature polypeptide；Become
The coded sequence of ripe polypeptide and various additional coding sequence；The coded sequence of mature polypeptide (adds with optional
Coded sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide including encoding such peptides, it is also possible to
It is the polynucleotide also including additional code and/or non-coding sequence.
The invention still further relates to have at least 50%, the most extremely between above-mentioned sequence hybridization and two sequences
Few 70%, the polynucleotide of more preferably at least 80% homogeny.The present invention be more particularly directed under strict conditions with
The interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " refers to: (1)
Hybridization under relatively low ionic strength and higher temperature and eluting, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；
Or added with denaturant during (2) hybridization, such as 50% (v/v) Methanamide, 0.1% calf serum/0.1% Ficoll,
42 DEG C etc.；Or (3) only homogeny between two sequences is at least more than 90%, when more preferably more than 95%
Just hybridize.Further, the polypeptide of interfertile polynucleotide encoding and SEQ ID NO.:12 and/or SEQ
Mature polypeptide shown in ID NO.:22 has identical biological function and activity.
The nucleotide full length sequence of the antibody of the present invention or its fragment generally can use PCR TRAP, recombination method
Or the method for synthetic obtains.A kind of feasible method is to synthesize relevant sequence by the method for synthetic
Row, when especially fragment length is shorter.Generally, by first synthesizing multiple small fragment, it is attached the most again
The fragment that sequence is the longest can be obtained.Additionally, also can be by the coded sequence of heavy chain and expression label (such as 6His)
Merge, form fusion protein.
Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method.This leads to
It is often to be cloned into carrier, then proceeds to cell, then by conventional method from the host cell after propagation
The relevant sequence of isolated.Biomolecule (nucleic acid, albumen etc.) involved in the present invention includes the shape to separate
The biomolecule that formula exists.
At present, it is already possible to completely by chemosynthesis obtain code book invention albumen (or its fragment, or
Its derivant) DNA sequence.Then can this DNA sequence be introduced as known in the art various existing
In DNA molecular (or such as carrier) and cell.Additionally, sudden change is introduced egg of the present invention also by chemosynthesis
In Bai Xulie.
The invention still further relates to comprise above-mentioned suitable DNA sequence and suitable promoter or control the load of sequence
Body.These carriers may be used for converting suitable host cell, allows it to marking protein.
Host cell can be prokaryotic cell, such as bacterial cell；Or the eukaryotic cell such as low, such as yeast cells；
Or higher eucaryotic cells, such as mammalian cell.Representative example has: escherichia coli, streptomyces；
The bacterial cell of Salmonella typhimurium；Fungal cell's such as yeast；The insect cell of fruit bat S2 or Sf9；
CHO, COS7, the zooblast etc. of 293 cells.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.Work as place
When master is for prokaryote such as escherichia coli, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date,
Use CaCl2Method processes, and step used is generally well-known in the art.Another kind of method is to use MgCl2.As
Fruit needs, and converts and also can carry out by the method for electroporation.When host is eukaryote, can be selected for following DNA
Transfection method: calcium phosphate precipitation, conventional mechanical methods such as microinjection, electroporation, liposome is packed
Deng.
The transformant obtained can be cultivated by conventional method, expresses the polypeptide of the coded by said gene of the present invention.Root
According to host cell used, culture medium used in cultivation is selected from various conventional medium.Be suitable to host
Cultivate under conditions of cell growth.When after host cell growth to suitable cell density, with suitably
The promoter that method (such as temperature transition or chemical induction) induction selects, is further cultured for a period of time by cell.
Recombinant polypeptide in the above methods can be intracellular or express on cell membrane or be secreted into thin
Outside born of the same parents.If it is required, its physics, chemical being separated by various separation methods with other characteristic can be utilized
Albumen with purification of Recombinant.These methods are well-known to those skilled in the art.The example bag of these methods
Include but be not limited to: conventional renaturation processes, processes (salting-out method), centrifugal, infiltration with protein precipitant
Broken bacterium, super process, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography,
High performance liquid chroma-tography (HPLC) and other various liquid chromatography (LC) technology and the combination of these methods.
The antibody of the present invention can be used alone, it is possible to detectable (for diagnostic purpose), treatment
Agent, PK (protein kinase) modify part or the combination combination of any the above material or coupling.
Detectable for diagnostic purposes includes but not limited to: fluorescence or luminous marker, radioactivity
Label, MRI (nuclear magnetic resonance) or CT (CT technology) contrast agent,
Maybe can produce the enzyme that can detect product.
Present invention also offers a kind of compositions.In preference, described compositions is pharmaceutical composition,
It contains above-mentioned antibody or its active fragment or its fusion protein, and pharmaceutically acceptable carrier.Logical
Often, these materials can be formulated in nontoxic, in inert and pharmaceutically acceptable aqueous carrier medium,
Wherein pH ordinarily be about 5-8, and preferably pH is about 6-8, although pH value can be with the character being formulated material
And disease to be treated and be varied from.The pharmaceutical composition prepared can by conventional route carry out to
Medicine, including (but being not limited to): in tumor, intraperitoneal, intravenous or topical.
The pharmaceutical composition of the present invention can be directly used for combining GII.4 virus-like particle, thus can be used for preventing
It is to cause acute gastroenteritis with treatment norovirus (NoVs).Additionally, also can use other therapeutic agents simultaneously.
The pharmaceutical composition of the present invention contains safe and effective amount (such as 0.001-99wt%, preferably
0.01-90wt%, more preferably 0.1-80wt%) the above-mentioned monoclonal antibody (or its conjugate) of the present invention and
Pharmaceutically acceptable carrier or excipient.This kind of carrier includes (but being not limited to): saline, buffer,
Glucose, water, glycerol, ethanol, and combinations thereof.Pharmaceutical preparation should match with administering mode.The present invention
Pharmaceutical composition can be to be made into injection form, such as with normal saline or containing glucose and other adjuvant
Aqueous solution be prepared by conventional method.Pharmaceutical composition such as injection, solution is the most aseptically made
Make.The dosage of active component is therapeutically effective amount, such as about 1 microgram every day/kg body weight-about 5 milligram
/ kg body weight.Additionally, the polypeptide of the present invention also can be used together with other therapeutic agents.
When making pharmaceutical composition, it is that the immune conjugate of safe and effective amount is applied to mammal, wherein
This safe and effective amount typically at least about 10 micrograms/kg body weight, and in most of the cases it is no more than about 8
Mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-about 1 mg/kg body weight.
Certainly, concrete dosage is it is also contemplated that the factor such as route of administration, patient health situation, and these are all skilled practitioners
Within the scope of technical ability.
Hybridoma cell strain
Present invention also offers and can produce the hybridoma that the present invention is directed to anti-GII.4 viral monoclonal antibodies
Strain；Preferably, the hybridoma for anti-GII.4 viral monoclonal antibodies that the invention provides high-titer is thin
Born of the same parents' strain.
After obtaining the hybridoma of the anti-GII.4 viral monoclonal antibodies producing the present invention, art technology
Personnel can utilize this hybridoma cell strain to prepare antibody easily.Additionally, those skilled in the art also can be very
Know the structure (variable region of heavy chain of such as antibody and variable region of light chain) of the antibody of the present invention easily, then
The monoclonal antibody of the present invention can be prepared by recombination method.
The preparation of monoclonal antibody
The antibody of the present invention can be prepared by various technology known to a person skilled in the art.Example
As, antigen of the present invention, animal can be applied to induce the generation of monoclonal antibody.For monoclonal antibody,
Available hybridoma technology is prepared (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 available recombinant DNA method (U.S. Patent number 4,816,567) prepare.
Representational myeloma cell is effective integration, supports the steady of antibody by the antibody produced cell selected
Fixed high level produces and those myeloma cells sensitive to culture medium (HAT medium matrix), including bone
The myeloma cell strain of myeloma cells strain, such as muroid, including derived from MOPC-21 and MPC-11 mice
The myeloma cell strain of tumor (is purchased from Salk Institute Cell Distribution Center, sage
The sub-brother in ground, California, U.S.) and SP-2, NZ0 or X63-Ag8-653 cell (be purchased from
American Type Culture Collection, Luo Keweier, Maryland, the U.S.).Human myeloma
Human monoclonal antibodies [Kozbor, J. the most it has been described for mouse-human heteromyeloma's cell strain
Immunol., 133:3001 (1984)；Brodeur etc., the production technology of monoclonal antibody and application
(Monoclonal Antibodies Production Techniques and Applications), 51-63
Page (Marcel Dekker, Inc., New York, 1987)].
Growth of Hybridoma Cell is analyzed having required specific list with detection in culture medium therein
The generation of clonal antibody, e.g., by external binding analysis such as, Enzyme Linked Immunoadsorbent Assay (ELISA) or
Radioimmunoassay, RIA (RIA).The position of the cell expressing antibody can be detected with FACS.Then, can be by
Hybridoma clone forms sub-clone (subcloned) by limiting dilution procedures, and is grown by standard method
(Goding, monoclonal antibody (Monoclonal Antibodies): principle and put into practice (Principles and
Practice), Academic Press (1986) 59-103 page).Use to reach this purpose
The culture medium being suitable for includes, such as, DMEM or RPMI-1640 culture medium.Additionally, hybridoma can be
Grow as ascites tumor in animal body.
The monoclonal antibody secreted by sub-clone passes through conventional immune globulin from culture medium, ascites or serum
White purifying process is suitably separated, and these purifying process are such as, Protein A-agarose method (protein
A-Sepharose), hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatograph.
The invention provides a kind of monoclonal antibody for GII.4 virus.At one of the present invention preferably
In scheme, monoclonal antibody uses cultivation hybridoma method to prepare.Take the supernatant of Hybridoma Cell Culture
Liquid, slightly proposes IgG through saturated ammonium sulphate method, then by the antibody that slightly carries through affinity column (Protein
G-Sephrose) purification.
In one preferred scheme of the present invention, monoclonal antibody uses Balb/C mouse ascites to produce Dan Ke
Prepared by the method for grand antibody.In about hybridoma is inoculated into the mouse peritoneal of sensitization, about 10 days visible
Abdominal part substantially swells.Extraction ascites, after saturated ammonium sulphate method slightly carries, then by the antibody that slightly carries through parent
With chromatographic column (Protein G-Sephrose) purification.
The immunoglobulin (antibody) of labelling
In a preference of the present invention, described immunoglobulin is with detectable.More preferably,
Described label be selected from lower group: colloid gold label thing, horseradish peroxidase-labeled, colored labels or
Fluorescent marker.
Colloid gold label can use method known to those skilled in the art to carry out.At one of the present invention preferably
Scheme in, anti-GII.4 virus monoclonal antibody colloid gold label, obtain the Dan Ke of colloid gold label
Grand antibody.
The anti-GII.4 viral monoclonal antibodies of the present invention has good specificity, the highest titer.
Detection plate and material thereof
The detection plate of the present invention can use detection panel material commonly used in the art, uses conventional detection plate to prepare
Method is made.
The present invention detects the plate for detecting immunity of GII.4 virus, including test strip and the gripper shoe of support test strip,
As PVC polyester offset plate etc. can be used；Described test strip is by filtering sample paper, chromatographic material, nitrocellulose filter
Overlap composition successively with absorbent paper, overlapping part can use the method for routine, such as fixing connections such as adhesive tapes；
Wherein: the pre-coated colloid gold label of chromatographic material or the anti-GII.4 viral monoclonal antibodies or many of coloured label
Clonal antibody, preferably by the anti-GII.4 viral monoclonal antibodies of colloid gold label, nitrocellulose filter is inhaled
Attached detection line and nature controlling line；
In a preferred scheme: anti-GII.4 virus Dan Ke of pre-coated colloid gold label on chromatographic material
Grand antibody is that the anti-GII.4 viral monoclonal antibodies using concentration to be 0.5-1.5mg/ml colloid gold label is molten
Liquid carries out pre-coated, and package amount is 50 μ l/cm2；Preferably concentration is 0.5 or 1.5mg/ml,
50μl/cm2；
Detection method judges with result
Keep flat detection plate, sample is dropped on filter sample paper, sample about 120 μ l, observes chromatography in 3～5min
Result.Fringe position according to occurring carrys out judged result.
Negative: obvious colour band all occur in quality control region, detection zone, it is shown as negative；
Positive: only in quality control region, obvious colour band to occur, and at detection zone without colour band, it is shown as positive；
Invalid: without any colour band or in quality control region, quality control region, detection zone do not occur that colour band occurs at detection zone
Colour band, shows detection method mistake or detection plate is rotten or inefficacy, should again exchange detection plate detection for.
Method and sample
The present invention relates to for the method at the pattern detection norovirus with cell and/or histolysis.Should
Method step approximately as: obtain cell and/or tissue samples；Sample is dissolved in media as well；Detect
The level of GII.4 virus in the sample of described dissolving.The sample that the inventive method is used can be present in
Any sample including cell in cell-preservation liquid, as used in liquid basal cell detection method.
Test kit
Present invention also offers a kind of antibody (or its fragment) referred to containing the present invention or the detection plate of the present invention
Test kit, in a preference of the present invention, described test kit also include container, operation instructions,
Buffer agent etc..
The present invention is designed for detecting the detection kit of GII.4 virus levels further, and this test kit includes
Identify the antibody of anti-GII.4 virus, for dissolving the cracking medium of sample, the common reagent needed for detection and
Buffer, such as various buffer, detection labelling, detection substrate etc..This detection kit can be external examining
Disconnected device.
The present invention designs and develops further for examining the GII.4 virus infection correlation circumstance from solution sample
The test kit of disconnected assessment, this test kit can detect the GII.4 virus being present in sample solution, Qi Zhongbao
Storage sample cell-preservation liquid originally can be the cell-preservation liquid in such as liquid basal cell detection method.
The anti-GII.4 viral monoclonal antibodies of the present invention has the advantage such as high-affinity, high specific, can be wide
General apply preparing GII.4 virus detection field, as detectable or detection equipment preparation field etc.,
Have significantly than traditional detection method or detectable at aspects such as specificity, sensitivity and verification and measurement ratios
Advantage.
Main advantages of the present invention are:
(1) monoclonal antibody 8E1 of the present invention can specific identification norovirus sample granule；
(2) monoclonal antibody 8E1 of the present invention can specific combination norovirus GII.4, with promise as sick
Poison GI.1 no cross reaction, thus realize norovirus GI.1 and the qualification of norovirus GII.4.
(3) monoclonal antibody 8E1 of the present invention has powerful neutralization activity to norovirus.
Below in conjunction with specific embodiment, the further detailed old present invention.Should be understood that these embodiments are only used for
The bright present invention rather than restriction the scope of the present invention.The experiment side of unreceipted detailed conditions in the following example
Method, generally according to normal condition such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold
Spring Harbor Laboratory Press, 1989) condition described in, or built according to manufacturer
The condition of view.Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.Used by following example
Experiment material and reagent the most all can obtain from commercially available channel.
Material and method
1 antigen preparation and mouse immune
Utilize baculovirus-insect expression system, be prepared for virus by expressing norovirus GII.4 VP1
Sample granule[1].The virus-like particle (50ul volume) of 10ug is mixed with isopyknic aluminium adjuvant (500ug)
Close pneumoretroperitoneum immunity 6 weeks female Balb/c mices, 0 week, 2 weeks, each immunity in 4 weeks once.The 6th
Zhou Shi, takes mice serum detection to neutralize titre.When the 7th week, the mice neutralizing titre the highest passes through
Tail vein booster immunization 15ugGII.4 virus-like particle.After 3 days, take mouse spleen for preparing hybridoma
Cell.
The preparation of 2 hybridoma cell strains and screening
After mouse tail vein booster immunization 3 days, take Mouse spleen cells and pass through with myeloma cell SP2/0
PEG1500 merges, and prepares hybridoma.After 9 days, screened special by elisa
Property secretion for the antibody of GII.4 virus-like particle.In short, GII.4 virus-like particle is coated 96 orifice plates,
Every hole 100ng, 4 DEG C are coated overnight, close with the PBST containing 5% skim milk, and it is miscellaneous that every hole adds 50ul
Hand over tumor culture fluid to hatch 2 hours at 37 DEG C, then hatch 1 hour with the two of HRP labelling anti-(sigma),
Finally carry out chromogenic reaction, read the light absorption value of OD450.
3 ascites preparation and antibody purifications
Female Balb/c mouse peritoneal injection 500ul saxol, after two weeks, every mouse peritoneal note
Penetrate 300,000 hybridomies.After 7 days, ascites collected by No. 12 syringe needles, and 10,000rpm are centrifuged 10min,
Removing upper strata oils and fats and lower sediment, the ascites taking clarification carries out antibody purification.According to description, utilize
HiTrap HiTrapTM Protein G affinity column (GE health care) purification ascites obtains antibody.
4 enzyme-linked immunosorbent assays identify monoclonal antibody
Overnight it is coated 96 hole Elisa plates with every hole 100ngGI.1 or GII.4 virus-like particle 4 DEG C, identifies
The binding ability of monoclonal antibody.Elisa plate is closed after 1 hour at 37 DEG C through the PBST containing 5% skim milk,
By every hole 50ul, variable concentrations (5ug/ml, 2.5ug/ml, 1.25ug/ml and 0.625ug/ml) is added
Enter monoclonal antibody 37 DEG C to hatch 2 hours, then hatch with the anti-Mus two of HRP labelling is anti-, finally read extinction
Value OD450.
5 polyacrylamide gel electrophoresis and western blot analyze
After protein sample mixes with SDS-PAG sample-loading buffer, boil process 10min, through 12% polypropylene
Acrylamide gel separation protein sample.By coomassie brilliant blue staining display protein band or protein delivery is arrived
Western blot analysis is carried out on pvdf membrane.Monoclonal antibody is diluted to contain by ultimate density 1ug/ml
In the PBST of 1% skim milk.Mouse-anti GII.4 polyclonal antibody 1:1000 dilutes use, then uses HPR
The Mus two anti-(sigma) of labelling hatches, and finally carries out record with LAS-400 luminescence image analysis instrument.
6 rabbit anti-GI.1 virus-like particles or the preparation of GII.4 virus-like particle polyclonal antibody
GI.1 virus-like particle or GII.4 virus-like particle mix fully breast with Freund's complete adjuvant equal-volume
Changing, subcutaneous injection health rabbit 150ug/ is only.By the GI.1 virus-like particle of 150ug after 3 weeks and 6 weeks
Or GII.4 virus-like particle mixes with equivalent incomplete Freund's adjuvant adjuvant, fully emulsified after carry out reinforcement and exempt from
Epidemic disease.Within after last immunity 2 weeks, collecting serum, after subpackage ,-80 DEG C save backup.
7 sandwich Elisa detect GI.1 and GII.4 virus-like particle
Polyvalent antibody (preparation method is ibid) and the anti-GII.4 of rabbit with rabbit anti-GI.1 virus-like particle are sick respectively
Polyvalent antibody (preparation method is ibid) the 1:5000 dilution factor (50ul/ hole) 4 DEG C of poison sample granule is overnight coated
96 hole Elisa plates, Elisa plate, will after the PBST containing 5% skim milk closes 2 hours at 37 DEG C
Virus-like particle adds in Elisa plate, and 40ng/50ul/ begins in hole, 2 doubling dilution 12 concentration, and 37
DEG C hatch 2 hours, then 37 DEG C special for virus-like particle of monoclonal antibody 10ng/50ul/ hole is hatched 1 little
Time, then hatch with the Mus two of HPR labelling is anti-, finally read light absorption value OD450.
8 external replacements neutralize experiment
With porcine gastric mucin III (PGM) (Yuan Mu bio tech ltd, the Shanghai) (50ul/ of 10ug/ml
Hole) room temperature is coated 96 hole Elisa plates, and Elisa plate was closed at 4 DEG C through the PBST containing 5% skim milk
After night standby.Being begun by GII.4 virus-like particle specific monoclonal antibody 8ug/ml, 2 doubling dilutions, with body such as grade
The GII.4 virus-like particle incubated at room of long-pending 0.5ug/ml is added to be coated with 96 holes of PGM after 1 hour
On Elisa plate, incubated at room 1 hour, it is subsequently adding rabbit anti-GII.4 virus-like particle polyclonal antibody
(preparation method is ibid) 1:1000 diluent 37 DEG C is hatched 1 hour, then resists with the rabbit two of HPR labelling
(sigma) hatch, finally read light absorption value OD450.
The gene order amplification of 9 monoclonal antibodies and the structure of expression vector
First the cell of hybridoma cell strain Trizol reagent is extracted total serum IgE, then according to 5 ' RACE examinations
Agent box description amplifies heavy chain and light chain full-length gene.Utilize method that PCR expands at heavy chain and light chain
5 ' hold and 3 ' ends introducing HindIII and EcoRI restriction enzyme site respectively, and by amplification heavy chain out with light
The gene that chain is complete is cloned in pGEM-T (Promage) respectively, filters out positive colony order-checking, then will
Clone HindIII that sequence is correct and EcoRI double digestion, be purified into purpose sheet through agarose gel electrophoresis
Duan Hou, is connected with T4DNA ligase with the plasmid pcDNA3.1 (Promage) of same enzyme action, is built into eucaryon
Expression vector pcDNA3.1-(m8E1H) and pcDNA3.1-(m8E1L).
The recombinant expressed qualification of 10 monoclonal antibody genes
Method cotransfection pcDNA3.1-(m8E1H) and the pcDNA3.1-(m8E1L) that utilize liposome arrive
Chinese hamster ovary celI, collects culture supernatant after 72 hours and is analyzed, and uses ELISA to determine resisting in culture supernatant
The expression of body: with GI.1 virus-like particle wrapper sheet, close 1 hour in 37 DEG C with the PBST containing 5% milk,
Add different dilution culture supernatant to be measured 37 DEG C to hatch 2 hours, then with the anti-Mus IgG of HRP labelling
Two anti-hatch, and finally read light absorption value OD450.
Embodiment 1 secretes the screening of the hybridoma of GII.4 specific antibody
The spleen cell of immunity GII.4 virus-like particle mice is used for preparing hybridoma.Pass through Elisa
Experiment screening hybridoma supernatant, thus acquisition can be secreted and be had the hybridoma combining virus capable
Strain.Finally, the monoclonal antibody that five strains have excellent binding ability is screened out, and they can be sick in conjunction with GII.4
Poison sample granule.Hypotype identifies display, and G9, D11,7D8 and 8E1 belong to IgG1,2D8 and belong to IgG3.
Table 1. is secreted the hybridoma cell strain of monoclonal antibody and is identified
| 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 analyzing is 50ul hybridoma and cultivates cell.
* ,+: OD450nm ＞ 0.15；++: OD450nm ＞ 0.3；+++: OD450nm ＞ 0.5.
The specificity analyses of embodiment 2 anti-GII.4 monoclonal antibody
First pass through SDS-PAGE and identify purity and the integrity of the GII.4 monoclonal antibody of purification from ascites.Fig. 1
Show that heavy chain and the light chain of five kinds of monoclonal antibodies are respectively about 50KD and 25KD.Then, by Elisa method
Have detected the reactivity of monoclonal antibody and not synantigen, including GI.1 virus-like particle and GII.4 virus-like particle.
Fig. 2 shows that G9, D11,2D8 and 8E1 can not exist and GI.1 with specific recognition GII.4 virus-like particle
The cross reaction of virus-like particle, and some monoclonal antibody (e.g., 7D8), simultaneously with GI.1 virus-like particle and GII.4
Virus-like particle combines, it is impossible to specific recognition GII.4 virus.
Finally, being analyzed the combination situation of monoclonal antibody and GII.4 by Western blot, Fig. 3 shows, five kinds of antibody
In: D11, G9 and 8E1 are capable of identify that the GII.4 virus-like particle of natural structure, it is impossible to after enough identifying degeneration
GII.4 virus-like particle, prompting D11, G9 and 8E1 identify epi-position be probably comformational epitope.
Embodiment 3 sandwich Elisa based on monoclonal antibody special can detect GI.1 and GII.4 virus-like particle delicately
The monoclonal antibody minimum detectability degree to virus-like particle is measured (as OD450nm ＞ 0.15 by sandwich Elisa
Time, it is judged to the positive).Fig. 4 shows that G9, D11,7D8 and 8E1 monoclonal antibody all can special detect delicately
GII.4 virus-like particle, minimum detectability degree is respectively as follows: 0.3125ng, 0.3125ng, 0.3125ng, 0.625ng.
The potential neutralization activity of embodiment 4 monoclonal antibody
Tissue blood group antigen (HBGA) is present in the saccharide on mucosal tissue and erythrocyte, is that promise is as sick
Receptor needed for poison infection.The combination inhibition test of HBGA is widely used as antibody-mediated norovirus
Replacement neutralization test.Containing HBGA in porcine gastric mucin III (PGM), it is verified and may be used for
Substitute neutralization test[2].By substituting neutralization test respectively to five kinds of lists of G9, D11,2D8,7D8 and 8E1
Anti-potential neutralization activity detects.Fig. 5 shows, D11, G9 and 8E1 GII.4 is shown good in
And activity, they stop the EC50 that is combined with PGM of virus-like particles respectively: 0.0979ug/ml,
0.6948ug/ml and 0.04357ug/ml.The above results shows, 8E1 is in GII.4 and activity to be much better than other
Antibody strain, it is that the twice of D11 antibody strain is many that 8E1 monoclonal antibody neutralizes activity to GII.4, is G9 antibody strain
About 16 times.
The gene sequencing of embodiment 5 monoclonal antibody
The heavy chain of the monoclonal antibody of clone 8E1 out and sequence of light chain are following (wherein,Single underscorePart is letter
Number peptide sequence, italicized item is variable region sequences,For constant-region sequences):
8E1 monoclonal antibody heavy chain nucleotide sequence:
ATGGGATGGAGCTGGATCTTTCTCTTCCTTCTGTCAGGAACTGCAGG TGTCCACTCT (SEQ ID NO.:2)
8E1 monoclonal antibody heavy chain amino acid sequence:
MGWSWIFLFLLSGTAGVHS (SEQ ID NO.:3)
8E1 monoclonal antibody light chain nucleotide sequence:
ATGAGGGCTGCTGCACAGATTTTTGGCTTCTTGTTGCTCTTGTTTCCA GGTACCAGATGT (SEQ ID NO.:4)
8E1 monoclonal antibody light-chain amino acid sequence:
MRAAAQIFGFLLLLFPGTRC (SEQ ID NO.:5)
Analyze 8E1 monoclonal antibody variable region of heavy chain and light-chain variable sequence, 8E1 monoclonal antibody variable region of heavy chain ammonia further
Base acid following (underscore mark for heavy chain CDR region):
EIQLQQSGPELMKPGASVKISCKASGYSFTDYYMHWVKQSHGKSLEWIGYI DPFNGDTGYNQKFKVRATLTVDESSTTAYMHLSSLTSDDSAVYYCARDDWGSPFS YWGQGTLVTVSA(SEQ ID NO.:6)
Above-mentioned variable region of heavy chain belongs to IGHV1 subgroup.
8E1 monoclonal antibody chain variable region amino acid following (underscore mark for heavy chain CDR region):
DIQMTQSPSSLSASLGESVSLTCRASHDIGSNLDWLQQEPDGTIKRLIYAT STLDSGVPKRFSGSRSGSDYSLTISSLESEDFVEYYCLQYASSPRTFGGGTKLEI
K(SEQ ID NO.:7)
Above-mentioned variable region of light chain belongs to IGKV9 subgroup.
Each CDR region aminoacid sequence and nucleotide sequence are summarized in table 2.
Table 2
Recombinant expressed and the qualification of embodiment 6 monoclonal antibody gene
The most correct in order to verify the gene of cloned 8E1 monoclonal antibody, the coded sequence of heavy chain and light chain is inserted respectively
Enter in pcDNA3.1, construction of expression vector pcDNA3.1-(m8E1H) and pcDNA3.1-(m8E1L), so
Rear cotransfection Chinese hamster ovary celI, and by whether ELISA detection cell conditioned medium has specific binding GII.4 virus-like particle
Antibody exist.Fig. 6 shows, the cell conditioned medium expressing 8E1 monoclonal antibody sequence has a highest binding signal, and and supernatant
Extension rate be correlated with；And the supernatant not transfecting the compared with control cells of related plasmids is all not bound with letter in spite of dilution
Number.This result illustrates that the 8E1 monoclonal antibody of the present invention can successfully be expressed in host cell.
Discuss
Present invention obtains can monoclonal antibody 8E1 of specific bond GII.4 virion, its have powerful potential in
And activity, therapeutic monoclonal antibodies medicine can be used as after humanization or be used as detectable.By sandwich
ELISA detects, and monoclonal antibody 8E1 is 0.625ng to the minimum detectability degree of GII.4 virus-like particle, and this is by this
A little monoclonal antibodies are developed into norovirus detection kit and are provided favourable theoretical foundation.And obtained by the present invention
The EC50 of the interaction of GII.4 and PGM is by monoclonal antibody 8E1 for GII.4: 0.04357ug/ml, the most excellent
Monoclonal antibody in other.And, the epi-position of Western display 8E1 monoclonal antibody identification is comformational epitope, and this makes 8E1
The virion that space conformation is correct can only be identified, and depolymerization and degeneration virion can not be identified, it is possible to be used for
Reliably virion is quantitative.
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document by individually
It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, art technology
The present invention can be made various changes or modifications by personnel, and these equivalent form of values fall within the application appended claims equally
Limited range.
List of references
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. the variable region of heavy chain of an antibody, it is characterised in that described variable region of heavy chain includes following three
Individual 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, described variable region of heavy chain has the aminoacid sequence shown in SEQ ID NO:6.
2. the heavy chain of an antibody, it is characterised in that described heavy chain has as claimed in claim 1
Variable region of heavy chain and CH.
3. the variable region of light chain of an antibody, it is characterised in that described variable region of light chain has selected from lower group
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；
Preferably, described variable region of light chain has the aminoacid sequence shown in SEQ ID NO:7.
4. the light chain of an antibody, it is characterised in that described light chain has as claimed in claim 3
Variable region of light chain and constant region of light chain.
5. an antibody, it is characterised in that described antibody has:
(1) variable region of heavy chain as claimed in claim 1；And/or
(2) variable region of light chain as claimed in claim 3；
Or, described antibody has:
Heavy chain as claimed in claim 2；And/or as claimed in claim 4 light chain.
6. a recombiant protein, it is characterised in that described recombiant protein has:
(i) variable region of heavy chain as claimed in claim 1, heavy chain as claimed in claim 2, such as right
Require the variable region of light chain described in 3, light chain as claimed in claim 4 or as claimed in claim 5
Antibody；And
(ii) optional assistance is expressed and/or the sequence label of purification.
7. polynucleotide, it is characterised in that its coding is selected from the polypeptide of lower group:
(1) variable region of heavy chain as claimed in claim 1, heavy chain as claimed in claim 2, such as right
Require the variable region of light chain described in 3, light chain as claimed in claim 4 or as claimed in claim 5
Antibody；Or
(2) recombiant protein as claimed in claim 6.
8. a carrier, it is characterised in that it contains the polynucleotide described in the claims in the present invention 7.
9. a genetically engineered host cell, it is characterised in that it contains described in claim 8
Carrier or genome are integrated the polynucleotide having the right described in requirement 7.
10. a test kit, it is characterised in that described test kit includes:
Antibody described in claim 5.
11. 1 kinds of immune conjugates, it is characterised in that this immune conjugate contains:
Monoclonal antibody described in (a) claim 5 or the recombiant protein described in claim 6；With
B () is selected from the coupling moiety of lower group: detectable, medicine, toxin, cytokine, radioactive nucleus
Element or enzyme.
12. 1 kinds of pharmaceutical compositions, its spy is, described compositions comprise the antibody described in claim 5,
Immune conjugate described in recombiant protein described in claim 6 or claim 12；And
Pharmaceutically acceptable carrier.
The preparation method of 13. 1 kinds of recombinant polypeptides, it is characterised in that the method comprises:
A () under conditions suitable for the expression, cultivates the host cell described in claim 9；
B () isolates recombinant polypeptide from culture, described recombinant polypeptide is resisting described in claim 5
Recombiant protein described in body or claim 6.
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| CN108727488A (en) * | 2017-04-13 | 2018-11-02 | 中国科学院上海巴斯德研究所 | The preparation and application of anti-norovirus GII.17 monoclonal antibodies |
| CN109081868A (en) * | 2017-06-14 | 2018-12-25 | 中国科学院上海巴斯德研究所 | Target the monoclonal antibody and its application of zika virus envelope protein conserved epitope |
| CN109265542A (en) * | 2018-09-27 | 2019-01-25 | 国药中生生物技术研究院有限公司 | Specifically bind norovirus GII.4 genotype VP1 albumen and/or the antibody of VLP and its preparation method and application |
| CN109957014A (en) * | 2017-12-25 | 2019-07-02 | 中国科学院上海巴斯德研究所 | The preparation and application of anti-norovirus GII.3 source of mouse monoclonal antibody |
| CN110221080A (en) * | 2019-06-25 | 2019-09-10 | 上海交通大学 | A kind of source of people norovirus immune colloid gold reagent box and cell strain |
| CN112175912A (en) * | 2020-09-18 | 2021-01-05 | 广东省微生物研究所（广东省微生物分析检测中心） | Hybridoma cell strain 3G41D6, anti-GII.4 type norovirus P protein monoclonal antibody and application |
| WO2022143282A1 (en) * | 2020-12-30 | 2022-07-07 | 远大赛威信生命科学(南京)有限公司 | Norovirus virus-like particle, immune composition, or kit, and use thereof |
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| 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 |
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| 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 |
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| 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 |
| CN110221080A (en) * | 2019-06-25 | 2019-09-10 | 上海交通大学 | A kind of source of people norovirus immune colloid gold reagent box and cell strain |
| CN112175912A (en) * | 2020-09-18 | 2021-01-05 | 广东省微生物研究所（广东省微生物分析检测中心） | Hybridoma cell strain 3G41D6, anti-GII.4 type norovirus P protein monoclonal antibody and application |
| WO2022143282A1 (en) * | 2020-12-30 | 2022-07-07 | 远大赛威信生命科学(南京)有限公司 | Norovirus virus-like particle, immune composition, or kit, and use thereof |
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| CN106188281B (en) | 2019-10-08 |
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Address after: Building 2, No. 225, Chongqing South Road, Huangpu District, Shanghai, 200025 Patentee after: Shanghai Institute of Immunology and Infection, Chinese Academy of Sciences Address before: No. 411, Hefei Road, Huangpu District, Shanghai 200025 Patentee before: INSTITUT PASTEUR OF SHANGHAI, CHINESE ACADEMY OF SCIENCES |