CN1264425A - Syncytial respiratory virus epitopes and antibodies comprising them, useful in diagnosis and therapy - Google Patents

Syncytial respiratory virus epitopes and antibodies comprising them, useful in diagnosis and therapy Download PDF

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CN1264425A
CN1264425A CN98807304A CN98807304A CN1264425A CN 1264425 A CN1264425 A CN 1264425A CN 98807304 A CN98807304 A CN 98807304A CN 98807304 A CN98807304 A CN 98807304A CN 1264425 A CN1264425 A CN 1264425A
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seq
peptide
antibody
rsv
sequence
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T·N·恩谷元
U·泊尔
L·格斯特克
A·贝克
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Pierre Fabre Medicament SA
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Pierre Fabre Medicament SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Abstract

The invention concerns a polyclonal or monoclonal antibody directed against an epitope of the protein G of the syncytial respiratory virus corresponding to a sequence selected among one of the peptide sequences included respectively between the aminoacid residues 150-159, 176-189, 194-207 and 155-176 of the entire sequence of the protein G of the syncytial respiratory virus A or B, or of sequences having at least 98 % homology.

Description

Be used to diagnose and respiratory syncytial virus epi-position for the treatment of and the antibody that contains them
The present invention relates to respiratory syncytial virus, particularly identify new epi-position and the corresponding antibody that can be used for by treatment, prevention and the diagnostic field of this virus associated diseases.
Respiratory syncytial virus (RSV) is the pathogenic agent that is common among baby and the old man.Bronchiolitis often is a disease serious among the children, needs hospital care.Also not have now the method for the disease that prevention causes by RSV, the infection first time of RSV can not stop later infection.Antibiosis treatment (Ribayirine) and/or binding immunoassay treatment (human normal immunoglobulin) can not alleviate the state of an illness for the grave illness example.And this class treatment is very expensive always.The clinical trial of carrying out recently with the HNK20 monoclonal antibody (the F albumen of anti-RSV) of ORAVAX shows, compares children's the readily good therapeutic effect of rsv infection with placebo.Once attempted in order to the RSV vaccine of formalin-inactivated children to be inoculated in the sixties, the result is that the state of an illness increases the weight of but not protects the natural infection of lung antagonism RSV.Problem is relevant therewith, and present diagnostic reagent can not be identified the infection of RSV reliably, is like this at least in the adult.
RSV is attributed to Paramyxoviridae, and Pneumovirus contains non-sections negative polarity rna gene group, the 10 kinds of differential proteins of encoding.
Patent application WO 87/04185 proposes to use the structural protein of RSV in vaccine, as is called the envelope protein of albumen F (fusion rotein) or Protein G, the glycoprotein of 22Kd, the albumen of 9.5Kd or the main albumen (albumen N) of capsid.
Patent application WO 89/02935 has described the protection feature of the whole protein F of RSV, and it is modified or unmodified with monomeric form or desugar form.
A series of fragments of having cloned albumen F with study they in and characteristic.
The Protein G of WO 95/27787 proof RSV can be used for preparation and treats and/or prevents the product that infects due to the RSV (A or B hypotype).
The fragment that the present invention now finds to contain the RSV viral protein G of defined epitope has particularly advantageous character.Thereby can prepare the many new peptide fragment of rsv protein G, be used in particular for following application:
(i) constitute the effective vaccine that resists rsv infection by chemical process or the described peptide fragment by genetic engineering and carrier coupling or fusion, and regardless of the vaccine administration mode;
(ii) these same peptide fragment are used for being created in rsv infection host's prevention or effectively polyclonal antibody of treatment and monoclonal antibody;
(iii) these peptide fragment and monoclonal antibody are as the reactant in the diagnostic kit, and this test kit can be used for showing and confirming the host infection that RSV-A or RSV-B infect.
The polyclonal antibody or the monoclonal antibody that the purpose of this invention is to provide the epi-position of anti-rsv protein G, described epi-position be equivalent to be selected from RSV A or the B Protein G complete sequence peptide sequence between 150-159,176-189,194-207 and 155-176 amino-acid residue or have at least 80%, a kind of sequence of the sequence of preferred at least 98% homogeny.
The peptide that sequence contained between the 130-230 amino-acid residue of these antibody capable identification A hypotypes or B hypotype rsv protein G.
Corresponding to RSV A Protein G 130-230 amino acid whose should the zone hereinafter referred to as G2Na.G2Na produces in bacterium such as intestinal bacteria, thereby is nonglycosylated.It can provide the immunoprotection of anti-rsv infection; particularly when it and a kind of carrier coupling; carrier is as being OmpA (the albumen p40 of Gram-negative bacteria such as klebsiella; be described in WO 96/14415) or derived from streptococcic albumen (as with human serum albumin bonded albumen; hereinafter referred to as BB, be described in WO96/14416).
Prove unexpectedly, the cross protection of anti-A hypotype or B hypotype RSV is provided by a series of peptides of the present invention's preparation.
Especially; recombinant protein BBG2a1 provides the cross protection of RSV-A or RSV-B in BALB/c mouse; BB2a1 is a kind of derivative of BBG2Na, has wherein only modified 4 residue: residue A sn (aa191), Lys (aa192), Gly (195) and Thr (aa198) respectively by residue Ser, Asn, Lys and Pro displacement on the G2Na basis.In order to improve the identical purpose of cross protection, prepared two kinds of recruit: BBG2a2, wherein residue A sn (aa157), Asn (aa160), Asn (aa161) and Phe (aa163) are respectively by residue Lys, Lys, Asp and Tyr displacement; BBG2a3, it contains 8 modified amino acids of BBG2a1 and BBG2a2.
Has a kind of sequence that is selected from following sequence according to the particularly advantageous peptide of the present invention: the SEQ ID No.1 that in annex, provides, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ IDNo.14, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No.20, SEQ ID No.21 and/or SEQ ID No.22, this peptide can also contain at least one half Guang amino-acid residue at N-end or C-end in addition.
The reactivity of these peptides proves by mono-clonal or polyclone probe.Utilize this technology to disclose 4 zone: G5a (aa144-159), G11a (aa164-176), G4a (aa172-187) and the G9a (aa190-204) of G2Na.
Thereby the invention still further relates to the mono-clonal or the polyclonal antibody of the peptide of the anti-at least a sequence that contains SEQ ID No.1 to SEQ ID No.22.
Having the one or more unitary peptide corresponding to epi-position 150-159,176-189,194-207 and 155-176 in the rsv protein G sequence, will be very useful in different embodiments of the present invention.
Can be used as immunogenic agents with the present invention of carrier protein couplet.
Carrier proteins advantageously is selected from: fragment, the TT albumen (tetanus toxin) of the OmpA of gram negative bacterium and they, streptococcic human serum albumin is conjugated protein and its fragment, and the B subunit (CTB) of Toxins,exo-, cholera; Preferred vector albumen is the OmpA of Klebsiella bacterium.
According to an aspect of the present invention, peptide and carrier proteins are by a kind of albumen coupling that is connected; This connection albumen can be selected from the acceptor on albuminous acceptor of mammalian blood serum and mucomembranous cell surface especially.
Described coupling is covalent coupling preferably, and this can realize by chemical process or by the DNA recombinant technology.
On the other hand, the present invention relates to the encode nucleotide sequence of peptide as mentioned above or immunogenic agents.This can be by inserting in the proteic dna molecular of code carrier or the peptide of fusion coding claim 4 or 5 or the hybrid DNA molecule that its segmental DNA (merging with promotor) produces especially; It can also be a kind of RNA molecule.
Peptide of the present invention, antibody, immunogenic agents and nucleotide sequence useful as drug are especially for the composition that infects due to preparation treatment or prevention RSV A or the B hypotype.
For example, the monoclonal antibody that has prepared specific recognition G5a and G11a and G1 Δ Ca peptide.Passive transfer monoclonal antibody 5C2 (anti-G5a) and 18D1 in natural mouse (anti-G1 Δ Ca) can prevent the infection of RSV-A.On the other hand, same monoclonal antibody 5C2 can eliminate the chronic infection of RSV-A rapidly in immunodeficient mouse.
Therefore the present invention also relates to a kind of pharmaceutical composition, it is characterized in that it contains at least a as defined above mono-clonal or polyclonal antibody, peptide of the present invention or epi-position, immunogenic agents or nucleotide sequence, and pharmaceutically-acceptable excipients.
Monoclonal antibody is preferably humanized, and produces by recombination method.According to a further aspect in the invention, they obtain by the method for phage library.
According to one embodiment of the invention, peptide of the present invention, immunogenic agents, antibody and nucleotide sequence can enter in the composition of diagnostic kit.
As indicated above, immunogenic agents can be introduced nucleotide sequence of the present invention by the DNA recombinant technology and prepare in host cell.This nucleotide sequence can be a kind of fusion gene, introduces by the dna vector that derives from plasmid, phage, virus and/or clay.In a kind of embodiment of this preparation method, described fusion gene can be incorporated in the genome of host cell.
As is known to the person skilled in the art, described carrier can be a virus vector.
Host cell can be a protokaryon, is selected from especially: intestinal bacteria, genus bacillus, Bacterium lacticum, staphylococcus and suis.
But host cell can also be the cell or the insect cell of yeast, mammalian cell, plant origin.
According to an aspect of the inventive method, the fusion rotein of expression is: excretory, be in the endochylema or be exposed on the film of host cell.
The following example is used to illustrate the present invention.In these embodiments with reference to following accompanying drawing.
Fig. 1 and 2: the principle of clone gene G2a1, G2a2 and G2a3 in carrier.
Fig. 3: the 20%SDS-PAGE glue of A-Coomassie blue stain.M=molecular size standard, swimming lane 1 and 2 are the BBG2a1 albumen (theoretical molecular 38.7Kd) of affinity purification on HSA-Sepharose.
The B-BBG2a1 albumen immunoblotting of monoclonal antibody 18D1.
Fig. 4: the immunogenicity of A-peptide G5a and G9a.
B-peptide G5a and G9a render a service the protection of lung.
Fig. 5: the immunogenicity of A-peptide G7a and G8a.
B-peptide G7a and G8a render a service the protection of lung.
The protection of the immunogenicity of Fig. 6: BBG2a1 and RSV A (Fig. 6 A) and RSV B (Fig. 6 B) and antagonism RSVA (6C) and RSV B (6D) is renderd a service.
The result of treatment of Fig. 7: monoclonal antibody 18D1 and 5C2.
Fig. 8: the prevention of A-monoclonal antibody 18D1 is renderd a service.
The prevention of B-mono-clonal clone 5C2 is renderd a service.
Fig. 9: the evaluation of the G2Na B epi-position that exists in the BBG2Na immune serum.A: the overall demonstration; B: do not show the 155-176 district.
Figure 10: the identified region of determining monoclonal antibody 5B7 with Pepscan B method.
EXAMPLE Example 1: peptide synthetic
Peptide G5aCys and CysG5a synthetic example.Abbreviation: AA: amino acid Boc: tertbutyloxycarbonyl BHA: bromo hydrogenation succinimido acid (Bromo hydrosuccimidyl acid) CE: capillary electrophoresis ESMS: electrospray ionization mass spectrum FMOC: fluorenylmethyloxycarbonyl FZCE: free zone capillary electrophoresis HBTU:2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-urea hexafluoro
Phosphoric acid salt HMP: to hydroxy methyl phenyloxy methylated polystyrene MBHA: methyldiphenyl methylamine NMP:N-N-methyl-2-2-pyrrolidone N-Pmc:2; 2; 5; 7,8-pentamethyl-benzo dihydropyrane-6-alkylsulfonyl RP-HPLC: RPLC SPPS: solid-phase peptide is synthesized tBOC: tertbutyloxycarbonyl tBu: tertiary butyl TFA: trifluoroacetic acid Trt: trityl
Peptide G5a is a kind of 16 amino acid whose peptides, corresponding to a fragment (144-159) of RSV-A Protein G.Holding the N end to synthesize by the solid state chemistry synthesis method from C obtains.Peptide CysG5a and G5aCys increase a halfcystine corresponding to this peptide at N-or C-end respectively, are used for the single coupling with carrier proteins.The coupling direction that these two peptides can be used for studying peptide and carrier proteins may influence immunoreactive.
Utilize a kind of solid-phase peptide automatic DNA synthesizer DNA to hold N-end to synthesize these peptides (FMOC chemical method, scale are 0.1,0.25 or 1.0mmol) from C.Peptide GysG5a the synthetic proline(Pro) of preload from the HMP type resin begins to carry out, and can obtain the free acid functional group of C-end after cracking; Or, after cracking, can obtain the acid amides functional group that C-holds with Rink-acid amides MHBA type resin.Peptide G5aCys synthetic from these two kinds of resins on any halfcystine of preload begin.The reactive functional group of the used amino acid whose side chain suitable radical protection [Cys (Trt) of FMOC chemistry; Arg (Pmc); Asn (Trt); Gln (Trt); Lys (Soc); Ser (tBu); Thr (tBu)].Coupling circulation is carried out as follows: with first amino acid whose N-end amine function group deprotection, will treat that the amino acid whose acid function of second of link coupled group activates coupling then with HBTU/HMP with piperidines.When end of synthesis, peptide is taken off from resin, by with the reaction of water/TFA mixture with the side chain deprotection.With the ether precipitation of peptides that is chilled to-40 ℃ in advance, and centrifugal mixture.Precipitate three times with the ether wash-out, dry under nitrogen then.With the resuspended precipitation of the water that contains 0.1%TFA.Suspension is centrifugal again, will contain the supernatant liquor and the precipitate and separate of peptide, contain resin in the precipitation.Thick peptide is with preparation property reversed-phase HPLC purifying partly.The uniformity of purified peptide is by reversed-phase HPLC and capillary electrophoresis (FZCE) checking.By comparing the compatibility of ES-MS mass spectrum quality that records and the quality of calculating from the theoretical sequence of amino acid, confirmed theoretical construct.Peptide CysG5aNH 2Synthetic: as to be used for synthetic resin-peptide theoretical weight: the weight that records after the drying under the 593mg nitrogen: the quality of the thick peptide of cracking after 619mg resin-peptide cracking and thick peptide analysis: 200mg (crowd heavy 1/3) freeze-drying: 84mg (75% of peptide net weight
Be that yield is 97%) uniformity of thick peptide: 97% (RP-HPLC; UV210nm)
97% (FZCE/Microcoat TM) purifying: the quality of purified peptide: 50mg after the freeze-drying (75% of peptide net weight,
Be that yield is 58%) identify: the uniformity of purified peptide:＞99% (RP-HPLC; UV210nm)
＞99% (FZCE/Microcoat TMUV200nm) mass spectrum (ES-MS) calculated mass: 1951.29Da actual measurement quality: 1950.80Da ± 0.31.Embodiment 2: the coupling peptide G11 Δ Ca of peptide G5a, G7a, G8a, G9a, G11a and G11 Δ Ca and carrier proteins (P40, BB, TT, KLH) and albumen P40 link coupled example
Peptide G11 Δ Ca (SEQ ID No.14) is 13 amino acid whose peptides that are derived from rsv protein G, and it is corresponding to the sequence between this proteic 164-176.When synthetic, 173 Cys residue is replaced by the Ser residue, so that only keep a Cys residue of 176, avoids forming non-existent 1-2 disulphide bridges among the native protein G (1-4/2-3 pairing).
This peptide carries out single coupling (the Heterobifunctional reactant is with the thiol function group coupling of C-terminal position halfcystine) by glutaraldehyde coupling (all the difunctional reactant thing is rolled into a ball coupling with amine and thiol function) or by means of BHA.The dissolving of single coupling peptide G11 Δ Ca by BHA
2mg G11 Δ Ca is dissolved among 2ml pH7 0.1M phosphoric acid buffer+0.1%Zwittergent 3-14.By means of the single coupling of Heterobifunctional reactant (BHA) with albumen P40
The solution of 3mg BHA in 25 μ l DMF is joined in advance among the 2.5mg albumen P40 that pH7 0.1M phosphoric acid buffer+0.1%Zwittergent 3-14 was dialysed, and at room temperature stirred 1 hour.After desalination on the PD10 post; with pH7 0.1M phosphoric acid buffer+0.1%Zwittergent 3-14 wash-out; per 500 μ l, one pipe is collected, and the pipe that will contain the bromo acetylated protein merges (surveying OD under 280nm) and containing in the pipe of 0.95ml G11 Δ Ca solution.Reaction medium is saturated with nitrogen, stirred 2 hours in the dark under the room temperature.Then with this solution to pH7 0.1M phosphoric acid buffer+0.1%Zwittergent 3-14 dialysed overnight under+4 ℃ of stirrings, freezing preservation gained solution.By means of all couplings of difunctional reactant thing (glutaraldehyde) and albumen P40
10mg P40 is dialysed to pH7 0.1M phosphoric acid buffer+0.1Zwittergent 3-14.Regulate concentration to 2mg/ml with pH9 0.1M carbonate buffer solution+0.1%Zwittergent 3-14.Mouth is gone into 4% solution of 200mg SDS.
The glutaraldehyde of 55.5 μ l 2.5% is joined in the 2.5ml 1mg/ml solution (pH is between 9-10) of peptide G11 Δ Ca in pH9 0.1M carbonate buffer solution+0.11%Zwittergent 3-14.+ 4 ℃ of following stirring reaction media 24 hours rise to room temperature then.Add 25 μ l 1M Methionins and stop reaction.This solution was dialysed 24 hours to pH7 0.1M phosphate buffered saline buffer+0.1%Zwittergent 3-14 under+4 ℃ of stirrings.Collect dialyzate, remove SDS 6 times with 0.02M KCl solution precipitation.Contain P40-G11 Δ Ca glutaraldehyde conjugate in the final supernatant liquor, in-20 ℃ of freezing preservations down.The sterilization of conjugate
Conjugate is thawed, sterile filtration (0.22 μ m), portioning is preserved to avoid sedimentation problem down in+4 ℃.The Analysis and Identification of conjugate
Conjugate is identified by the following method: carry out protein determination with the Lowry method, with SDS-PAGE electrophoresis (Coomassie blue colour developing); Measure amino acid after the vapor phase acid hydrolysis, analyze with the PITC derivatize and through HPLC.
Conjugate Volume (ml) [albumen] mg/ml The Nb of immobilization G11 Δ Ca ??[G11ΔC] ????mg/ml
?rP40-G11ΔCa ????(BHA) ????2.7 ??0.74 ????2 ????0.05
RP40-G11 Δ Ca (glutaraldehyde) ????5.1 ??1.29 ????16 ????0.49
Embodiment 3: gene G2a1, the G2a2 clone in expression vector pvaBB308, fusion rotein BBG2a1 and the BBG2a2 generation in intestinal bacteria
The principle that gene G2a1 (SEQ ID No.15), G2a2 (SEQ ID No.16) and G2a3 (SEQ IDNo.17) clone in carrier is shown among Fig. 1 and 2.3.1 the structure of G2a1
Utilize plasmid pRIT28G2Na to make up the gene (SEQ ID No.15) of proteins encoded G2a1 by directed mutagenesis as parent material.For this reason, under following condition, RIT29/TH137 (PCR1) and RIT30/TH136 (PCR2) have been carried out two PCR reactions (polymerase chain reaction) with oligonucleotide:
PCR (94 ℃, 15 seconds
25 circulations (55 ℃, 30 seconds
(72 ℃, 30 seconds fixing on magnetic bead
To be fixed on the magnetic bead from reacting the fragment that obtains about 262bp and 208bp 1 and 2 respectively.25 μ l and streptavidin link coupled magnetic bead DYNAL  M-280 are used T.E. damping fluid (10mM Tris in advance; 1mM EDTA pH7.5) washes twice, is incubated 20 minutes at 37 ℃ with 90 μ l amplification reaction solutions 1 and 2 then.After fixing, magnetic bead and 50 μ l 0.5M NaOH at room temperature are incubated 10 minutes, fragment is by sex change.Collect 2 kinds of supernatant liquors,, be resuspended in 5 μ, 1 water with the dehydrated alcohol precipitation.Extension
This reaction is got each amplification reaction solution 10 μ l under the following conditions and is finished by PCR:
(95 ℃, and 5 circulations in 15 seconds (35 ℃, 30 seconds
(72 ℃, 30 seconds.
The fragment that produces is carried out pcr amplification with oligonucleotide RIT27 and RIT28:
(95 ℃, and 25 circulations in 15 seconds (55 ℃, 30 seconds
(72 ℃, 30 seconds.
The amplified fragments of 509 bp is digested with restriction enzyme Pst I and HindIII.With the fragment cloning of the 169bp that produces in carrier pRIT28 with same enzyme digestion.
Gained plasmid pRIT28G2a1 down presses the method order-checking that AppliedBiosystem (Perkin Elmer) describes with dyestuff deoxidation terminator chemistry.
The PstI/Hind III fragment (fragment in downstream, PstI site) of pRIT28G2a1 down is cloned in obtains plasmid pRIT28G2a1 among the carrier pRIT28G2Na.
Gene G2a1 is cloned on the restriction site EcoRI/HindIII of expression vector pvaBB308 then, generates carrier pyaBBG2a1.
The sequence of oligonucleotide is as follows:
RIT27：5′-GCTTCCGGCTCGTATGTTGTGTG-3′
RIT28：5′-AAAGGGGGATGTGCTGCAAGGCG-3′
TH136：5′-CCGAAGAAAAAACCGACGACCAAACCGACC-3′
TH137：5′-TTTTTTCTTCGGTTTGTTGCTCGGG-3′
The biotinylated RIT27 of RIT29:5 '.
The biotinylated RIT28 of RIT30:5 '.3.2.G2a2 structure (SEQ ID No.16)
Clone's principle schematic is seen the below of Fig. 2.
The oligonucleotide that uses in this makes up is to as follows: RIT29/TNG193 (PCR1), and TNG192/RIT30 (PCR2).The sequence of oligonucleotide is as follows: the structure (SEQ ID No.17) of TNG 192:5 '-CCGCCGAAAAAACCGAAAGACGAT-3 ' TNG 193:5 '-CGAAATGGTAATCGTCTTTCGG-3 ' 3.3 G2a3
Two fragments of single PstI site upstream and downstream are concentrated in the same carrier, produce pRIT28G2a3.
Three are inserted fragment G2a1, G2a2 and G2a3 and have been cloned in the different expression vectors in the intestinal bacteria, the carrier pvaBB308 among we embodiment particularly, and wherein BB is the gene of coding albumin acceptor.Fusion rotein BBG2a1, BBG2a2 that is produced and BBG2a3 can pass through HSA-Sepharose (human serum albumin) post affinity purification at an easy rate.3.4 the fermentation of fusion rotein BBG2a1 and BBG2a2 and purifying
In two erlenmeyer flasks, contain penbritin (100 μ g/ml, Sigma) and tsiklomitsin (8 μ g/ml, Sigma) 250ml TSB substratum (the tryptic soy nutrient solution, Difco) in, the intestinal bacteria RV308 that inoculation transforms with plasmid pvaBBG2a1 and pvaBBG2a2 respectively.T °=37 ℃ and stir under cultivated 16 hours.This nutrient solution of 200ml is seeded in (CHEMAP CF3000, ALFA LAVAL) in the fermentor tank that contains 2 liters of substratum.Contain (grams per liter) in the substratum: glycerine, 5; Ammonium sulfate, 2.6; Potassium primary phosphate, 3; Dipotassium hydrogen phosphate, 2; Trisodium Citrate, 0.5; Yeast extract, 1; Penbritin, 0.1; Tsiklomitsin, 0.008; VitB1,0.07; Sal epsom, 1 and 1ml/l trace element solution and 0.65ml/l vitamin solution.Controlled variable in the fermenting process is: pH, stirring, temperature, logical oxygen rate, associating feed (glycerine or glucose).PH transfers to 7.0, and temperature is fixed on 37 ℃.With glycerine (87%) the feed control growing of constant rate (12ml/h), keeping the tension signal of dissolved oxygen is 30%.When the turbidity of nutrient solution (580nm measures down) when reaching 80 (after cultivating about 24 hours), add indole acrylic acid (IAA) to final concentration 25mg/l induced protein and produce.Induced the back about 4 hours, centrifugal collecting cell.The yield of the biomass that obtains is about 200 grams (weight in wet base).The productive rate of BBG2a1 and BBG2a2 is about 4-6mg albumen/gram biomass.
The wet biomass of 30 grams is resuspended in 70mlTST solution (pH8.0 50mM Tris-HCl, 200mM NaCl, 0.05%Tween20 and 0.5mM EDTA).Ultrasonication cell (Vibracell72401, Sonics ﹠amp; Materials).Behind the eccentric cell lysate, filtration (1.2 μ M) supernatant liquor also dilutes in 500ml TST.The fusion rotein that obtains with solubilized form is by stating method (Stanl etc., immunological method magazine (J.Immunol.Methods), 1989; 124:43-52) go up purifying at affinity column HSA-Sepharose (human serum albumin).
After centrifugal, insoluble lysate is washed once (pH8.5 50mM Tris-HCl with damping fluid; 5mM MgCl 2).After the washing with resolution of precipitate in 30ml 7M Guanidinium hydrochloride, 25mM Tris-HCl (pH8.5), 10mM dithiothreitol (DTT) (DTT), then 37 ℃ the insulation 2 hours.Dissolved albumen is joined (25mM Tris-HCl (pH8.5) in the sex change damping fluid; 150mM NaCl and 0.05%Tween 20).Before adding the dissolved fusion rotein, the final concentration of regulating Guanidinium hydrochloride in the sex change damping fluid is 0.5M.Under the room temperature mixture is incubated 16 hours under the moderate agitation.After centrifugal, the purifying on the HSA-Sepharose post of the solvable fusion product in the supernatant liquor.The fusion rotein of purifying is used the analysis of SDS-PAGE (12%) glue on MINI PROTEAN II SYSTEM (BIORADS) instrument.With coomassie brilliant blue R250 albumen is developed the color.In addition, the immunoblotting that carries out with the RSV specific antibody is to the analysis revealed of recombinant protein, and these albumen are antigenic (seeing the SDS glue of BBG2a among Fig. 3 and the example of immunoblotting).Embodiment 4: render a service material and method with immunogenicity and the protection of P40 link coupled peptide G5a and G9a
Every group 7 organize mouse with 20 μ g P40-G9aCys, P40-CysG5a or P40-G5aCys intraperitoneal immunity 2 times.Control mice PBS immunity.Hydrogel (20%v/v) is as all immune adjuvants.Last immunity 2 weeks of back are got blood confirming the seroconversion to RSV-A from retro-orbital sinus, after one week the back with 10 5TCID 50(i.n.) attacks in the RSV-A nose, attacks and puts to death mouse in back 5 days.Take out lung and measure virus titer in the lung.Humoral immune reaction as a result
Described in Fig. 4 A, the immunogenicity of peptide G5a depends on the coupling direction with P40.During with the terminal coupling of the C-of peptide, in serum, induced weak anti--RSV-A antibody titers to moderate.And during with the terminal coupling of N-, G5a does not induce this antibody.With regard to inducing anti-RSV-A antibody, has weak immunogenicity when generally speaking, peptide G9a is with C-end and P40 coupling.In the P40-G9aCys mice immunized, 7 merely hit one has produced high titre serum antibody.The lung protection of peptide G5a and G9a is renderd a service
As shown in Fig. 4 B, directly related with anti-RSV-A detection of antibodies result, peptide G5a has induced protective immunological reaction when with the coupling of C-end, then can not during with the coupling of N-end.In fact, 7 with in P40-G5aCys (with the terminal coupling of the C-) mice immunized 6 protected, virus appears in lung, the last virus that occurs test detectability level one by one.And control group PBS immunity is arranged in lung and the same high virus titer of control mice with P40-CysG5a (with the terminal coupling of N-) mice immunized.These results confirm that the coupling direction of this peptide and carrier proteins is the key point that its protection is renderd a service.This lung of associated cue between this protection and anti-RSV-A antibody induction protect to small part by antibody-mediated.
Though P40-G9aCys has only weak immunogenicity in mouse with regard to inducing anti-RSV-A serum antibody, 7 lungs of merely hitting 5 mouse are subjected to resisting the protection that RSV-A attacks.In fact, virus appears in 7 mouse of merely hitting in lung, in addition in serum the antibody of anti-RSV-A not.Conclusion
Peptide G5a and G9a contain the protective epitope at the RSV-A pulmonary infection.It is crucial that the coupling direction of G5a and carrier proteins is renderd a service its protection.Embodiment 5: material and method are renderd a service in the immunogenicity of peptide G7a and G8a and protection
Every group of 3-4 only organize mouse with immune 2 times of 20 μ g G7a, G8a, BB-G7a or BB-G8a intraperitoneal.Control mice PBS immunity.Hydrogel (20%v/v) is as all immune adjuvants.Last immunity 2 weeks of back are got blood confirming the seroconversion to RSV-A from retro-orbital sinus, after one week the back with 10 5TCID 50(i.n.) attacks in the RSV-A nose, attacks and puts to death mouse in back 5 days.Take out lung and wash nasal meatus and measure virus titer in lung and the nasal meatus.Humoral immune reaction as a result
As shown in Fig. 5 A, whether two kinds of peptide G7a and G8a and BB coupling are immunogenic to RSV-A all.If consider the antibody titers of serum, non-link coupled peptide be it seems than the immunogenicity of coupling peptide more by force.The lung protection of peptide G7a and G8a is renderd a service
As shown in Fig. 5 B,, virus do not occur or the virus of test detectability level is only arranged with all protected attack that resists RSV-A of lung of all mouse of peptide G7a or G8a (with the BB coupling or not) immunity.Conclusion
Peptide G7a and G8a contain the protective epitope to lung.These peptides and BB coupling or not coupling are all effective.Embodiment 6: material and method are renderd a service in the immunogenicity of fusion rotein BBG 2a1 and protection
For definite BBG 2a1 renders a service immunogenicity and the protection of RSV-A and B,
Every group 3 the mouse of organizing is distinguished immunity 2 times and 3 times with 20 μ g albumen with the interval intraperitoneal in 2 weeks.Control mice PBS immunity.Hydrogel (20%v/v) is as all immune adjuvants.Last immunity 2 weeks of back are got blood confirming the seroconversion to RSV-A from retro-orbital sinus, after one week the back with 10 5TCID 50(i.n.) attacks in the RSV-A nose, attacks and puts to death mouse in back 5 days.Take out lung and measure virus titer in the lung.BBG2a1 is at the immunogenicity of RSV-A and B as a result
The result who is shown among Fig. 6 A and the B proves that BBG2a1 can induce the antibody of anti-RSV-A and B.According to expectation like that, the titre of anti-RSV-A antibody is more much higher than the titre of anti-RSV-B antibody.But the antibody titers of two strains of anti-RSV has all improved.BBG2a1 renders a service at the protection of RSV-A and RSV-B
As shown in Fig. 6 C and D, induced the immunne response that to protect lung antagonism RSV-A attack and antagonism RSV-B to attack with the BBG2a1 immune mouse.The mouse of attacking with RSV-A is protected, and makes the virus (1/3 mouse) that virus (2/3 mouse) do not occur or the detectability level is only arranged in lung.The mouse of attacking with RSV-B is protected, and makes virus (1/3 mouse) not occur in lung, or has only the virus (1/3 mouse) of detectability level or the virus (1/3 mouse) that is slightly larger than this detectability is arranged.Conclusion
Fusion rotein BBG2a1 is a high immunogenicity to RSV-A with to RSV-B.BBG2a1 can induce replying of protection lung antagonism two hypotypes attacks of RSV.Obtaining of embodiment 7:18D1,5C2 and 5B7 antibody
The peptide of immunization is: (i) with KLH (keyhole limpet hemocyanin) link coupled G1 Δ Ca, and (ii) G2 Δ Ca and (iii) BBG2aNa.
The 0th day with 50 μ g antigen intraperitoneal immune mouses among the CFA (complete Freund's adjuvant), the 14th day with each antigen intraperitoneal immunity of 10 μ g among the IFA (incomplete Freund's adjuvant), do not have the immunity of adjuvant intravenously at the 38th day with 10 each peptide of μ g then.Took out spleen at the 42nd day, merge with 1/1 ratio with the SP2-0 medullary cell.Keep anti-each antigenic positive hybridoma.These hybridomas are injected to mouse to obtain ascites, and the different antibodies of gained is with different peptide screenings, with the specificity of definite gained antibody then.Discern RSV-A specifically with screened monoclonal antibody 18D1, the 5C2 that comes out of its anti-peptide G1 Δ Ca, G5a specificity.Obtaining also from BBG2Na, the monoclonal antibody 5B7 of identification polypeptide G11a can discern RSV-A.Embodiment 8: monoclonal antibody 18D1 and 5C2 are to chronically infected result of treatment material of RSV-A in the SCID mouse and method
With 10 5TCID 50RSV-A (50 μ l) nose in (i.n.) attack C.B-17 scid/scid mouse.After 26 days, every group of 7 mouse, (i.n.) is 10 by way of accepting anti-RSV-A ELISA titre in the nose 450 μ l 18D1 antibody or 5C2 Antibody Preparation liquid.It is 10 that control mice is accepted anti-BBELISA titre 4Anti-BB serum.Put to death animal after 5 days, and the lung that takes out them is used for virus and measures.The result
Result shown in Fig. 7 proves that monoclonal antibody 18D1 and 5C2 can eliminate the RSV-A chronic infection of SCID mouse, and is the character of killing virus.When putting to death animal, do not detect any viral vestige.The result who obtains in the mouse lung of handling with 5C2 is corresponding to the average detected limit of this experiment, and this detectability is higher owing to lack sample, does not exist but be not equivalent to virus.Conclusion:
Anti-clonal antibody 5C2 and 18D1 can be used for the treatment in RSV-A pulmonary infection field.Embodiment 9: the preventive effect material perception method that monoclonal antibody 18D1 infects mouse RSV-A
One group of seronegative natural BALB/c mouse of RSV-A is accepted anti-RSV-A ELISA titre and is transferred to 10 5The peritoneal injection of 18D1 Antibody Preparation liquid 200 μ l.The control mice intraperitoneal is accepted anti-P40 ELISA titre and is transferred to 10 4The parallel injection of the anti-P40 serum of 200 μ l (uncorrelated serum).Next day, (i.n.) is with containing 10 in all mouse noses 5TCID 50The 50 μ l viral suspensions of RSV-A infect.Put to death animal after 5 days, be used for the detection of lung virus.The result
Shown in Fig. 8 A, the mouse lung protection that antibody 18D1 can induce antagonism RSV-A to attack after intraperitoneal shifts.In injection 10 5Behind the 18D1 300 μ l of titre, all mouse have all obtained protection.7 degree of protections of merely hitting 3 are not occur virus in the lung.Show the trace virus (3/7) that only reaches the experiment detectability in remaining mouse, or a little more than this detectability (1/7).The titre of control mice is at every gram lung log 103.70-4.45 between.Conclusion:
Antibody 18D1 can prevent the pulmonary infection of RSV-A to BALB/c mouse, has proved its strong prevention effectiveness.Embodiment 10: preventive effect material and method that monoclonal antibody 5C2 infects mouse RSV-A
(i.n.) shifts the anti-RSV-A ELISA titre of acceptance and transfers to 10 in the natural mouse nose of RSV-A negative serum 45C2 prepare liquid 50 μ l.The anti-BB ELISA of the parallel acceptance of control mice titre transfers to 10 4Anti-BB serum.
Next day, all mouse are with containing 10 5TCID 50(i.n.) infects in the viral suspension 50 μ l noses of RSV-A.Put to death animal after 5 days and be used for lung virus mensuration.The result
As shown in Fig. 8 B, institute useful 10 4The lung of the mouse that the 5C2 of titre handles is all protected.7 merely hit has only viral vestige.The virus titer of control mice is at every gram lung log 103.70 to 4.70.Conclusion
5C2 antibody can prevent the RSV-A pulmonary infection of BALB/c mouse, has proved its strong prevention effectiveness.Embodiment 11:Pepscan: cover the aa130-230 sequence of G2Na and the multiple synthetic example of 94 octapeptides overlapping each other with an amino acid
Press Geysen etc., institute of NAS newspaper (Proc.Natl.Acad.Sci), 1984,81, the MULTIPIN that 3998-4002 describes TMTechnology is prepared " PEPSCAN " plate (peptides of 2 control peptide+94 covering RSV-A Protein G 130-230 sequences) of parallel synthetic 96 octapeptides.
These peptides are synthetic on the terminal solid support of complementation 96 " pin " (8 * 12) of ELISA titer plate, will directly carry out the screening of mono-clonal or polyclone (serum) antibody in this plate.The station-keeping system in pin and hole
Use following numbering system: A1 (1) position=A plate, 1 row (totally 12 row), 1 row (the H1 position among the ELISA) A2 (1) position=A plate, 2 row, 1 row (the H2 position among the ELISA) A1 (1): control peptide #1:PLAQGGGG A2 (1): control peptide #2:PLAQGGGG A3 (1): octapeptide #1:TVKTKNTT A4 (1): octapeptide #2:VKTKNTTT or the like A12 (8): octapeptide #94:KEVPTTKP.Synthetic
Should be synthetic corresponding to a plurality of circulations of deprotection, washing and link coupled, up to obtaining the purpose peptide sequence.When end of synthesis, with peptide N-acetylize, again with the side chain deprotection.
Be used on pin synthetic amino acid with group Fmoc (9-fluorenylmethyloxycarbonyl) and the protection of following side chain protected group: tertbutyl ether (tBu) is used for Serine, Threonine and tyrosine; tertiary butyl ester (OtBu) is used for aspartic acid and L-glutamic acid; tertbutyloxycarbonyl (Boc) is used for Methionin; Histidine and tryptophane, 2,2; 5; 7,8-pentamethyl-benzo dihydropyrane-6-alkylsulfonyl (Pmc) is used for arginine, and trityl (Trt) is used for halfcystine, l-asparagine and glutamine.The activation of acid function group is carried out with the DIC (DIC) and the I-hydroxybenzotriazole (HOBt) that are dissolved among the DMF.Fmoc deprotection and washing: under stirring at room., needle plate be immersed in the bath that contains 200ml 20% piperidines DMF solution (40/160ml) reach 20 minutes.From bath, take out these pins then.These pins are patted on paper handkerchief remove unnecessary solvent.In 200ml DMF, under stirring at room, wash pin.These pins are patted on paper handkerchief, and immerse fully methyl alcohol molten in 2 minutes, do not have to stir.Then these pins are immersed in (3 baths, each 2 minutes, 200ml/ bathed) in the 200ml methyl alcohol.With dry 30 minutes of plate.The amino acid whose coupling of Fmoc-: Fmoc-amino acid will be through an activation step before can coupling.The time length of coupling step is 2 hours for concentration, 1.5 equivalent HOBt and the 1.2 equivalent DIC of 100mM.There is software can calculate the amount of the required reactant of coupling step.In by the tactic test tube of amino acid individual character code letter, weigh.Fill test tube on a piece of paper towel outside balance, the indicated weight on obtaining of weighing then near pan paper.This weight is not compared with theoretical value should lack 0.2mg, also should not have more 0.9mg.Coupling step: pin is put into each hole lightly.Close box carefully, (amino acid concentration is 100mM) is placed in the ventilating kitchen in 2 hours coupling process.Can carry out 3 such couplings in 2 hours every day.The processing of pin heap after the coupling: pin is taken out from the plate that contains coupling solution, stir and use 200ml methanol wash 5 minutes down.Pin is piled up the paper handkerchief arsis packed airing 2 minutes.Pin is placed 200mlDMF, and under agitation washed 5 minutes, and then carry out ensuing deprotection circulation.Routine is washed plate, carries out the step of cracking Fmoc group as mentioned above after last coupling.The acetylize of terminal amino group: syringe needle is incubated in the plate hole that contains the following reaction mixture of 150 μ l: DMF/ diacetyl oxide/triethylamine 50/5/1 (v/v/v).These pins are enclosed in the box, and room temperature was assigned 90 minutes.These pins washed 15 minutes in 200ml methyl alcohol then, dry 154 minutes then.The deprotection of side chain:, remove the blocking group of side chain with pin soaking at room temperature 2.5 hours in the mixture of 200ml TFA/ phenylmethylether/dithioglycol 190/5/5ml.After this deprotection steps, pin is taken out from acid solution.Box with washed with methanol once charges into methyl alcohol then, and pin soaked 10 minutes more therein.Then pin is patted on paper handkerchief, in 200ml methanol/acetate (100/100/1ml) mixture, soaked 1 hour then, on paper handkerchief, pat again.These pins are placed in the moisture eliminator vacuum to spend the night.Embodiment 12:ELISA
The titer plate that will have a pin in saturated damping fluid (PBS, Tween 0.1%, gelatin 1%) 37 ℃ saturated 1 hour, washing is 10 minutes in PBS, stir down in 4 ℃ with prediluted serum incubated overnight to be analyzed.Wash plate 4 times with PBS then, each 10 minutes, at room temperature with second antibody (1/5000 the dilution proportion) insulation of peroxidase labelling 1 hour.After PBS washing 4 times, add substrate TMB.Add the sulfuric acid termination reaction.
The data that produce with the anti-BBG2Na mouse of Pepscan B methods analyst serum are summarised among Fig. 9.
Fig. 9 A and 9B show the reactivity of the anti-BBG2Na mice serum in 4 zones of anti-G2Na molecule (amino acid that the residue representative of black matrix plays an important role) in Ac/Ag identification :-the first district is positioned between the 150-159 residue, and its sequence is QRQNKPPNKP.This zone is contained among the peptide G5a (144-159), and corresponding to the reaction active region of monoclonal antibody 5C2;-the second district is positioned between the 176-189 residue, and its sequence is CSNNPTC WAICKPI.This is a zone that is positioned at peptide G1 Δ Ca (174-187) position, and corresponding to the reactive behavior of monoclonal anti 18D1 and 5D3;-Di 3 districts are positioned between the 194-207 residue, and its sequence is PGKKTTTKPTKKPT.This sequence is corresponding to the reactive behavior of peptide G9 (194-204) level, and this reactive behavior is confirmed in the monoclonal antibody that produces anti-BBG2Na; The wide region from 155 residues to 176 residues is crossed in-Di 4 districts, it seems that it is various reactive total result.Cover one (G11a) of the high hydrophobic region of G2Na molecule identified region corresponding to monoclonal antibody 5B7 (with obtaining after the candidate vaccine BBG2Na immunity BALB/c mouse) in these reactive behavioies, its Pepscan B is shown among following Figure 10.
This monoclonal antibody recognition sequence FEVFNFVP (165-172).
The summation of above-mentioned four reactive behavioies is confirmed by the mensuration of using the different ELISA antagonism BBG2Na serum that each reactive behavior is carried out in addition.Table I shows that anti-BBG2Na serum represented " anti--G4a, G5aCys, G9aCys and G11a " activity well.Table I: with the log of anti-BBG2Na serum ref.BE-02 10Anti-G4a, G5aCys, G9aCys and the G11 Δ Ca titre of expression
Titre (log 10) BE-02
?G2Na ?KLH-G4a ?KLH-G9aCys ?KLH-G5aCys ?P40-G11ΔCa ?5.9 ?4.7 ?5.0 ?3.8 ?3.5
Conclusion:
With studies show that of Pepscan technical antagonism BBG2Na serum, produced the antibody that resists 4 epi-positions with the BBG2Na immune mouse, these 4 epi-positions lay respectively at 150-159,176-189,194-207 and 155-176 district.Thereby utilize this technology to confirm the importance of 164-176 district (G11 Δ Ca).
In addition, these results are with in full accord about the ELISA data of anti-BBG2Na serum and peptide G4a, G5aCys, G9aCys and G11 Δ Ca reactive behavior.
The signal G1 Δ Ca sequence type of sequence table SEQ ID No.1: amino acid and nucleotide sequence length: 14 amino acid, 42 nucleotide chain numbers: strand configuration: linear molecule type: peptide
Signal G1 ' the Δ Ca sequence type of 174 176 182 186 187N-Ser Ile Cys Ser Asn Asn Pro Tnr Cys Trp Ala Ile Ser Lys-C5 '-AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC AGC AAA-3 ' SEQ ID No.2: length amino acid sequence: 14 amino acid chain numbers: strand configuration: linear molecule type: the signal G1 Δ Cb sequence type of peptide 174 176 182 186 187N-Ser Ile Asp Ser Asn Asn Pro Thr Orn Trp Ala Ile Ser Lys-CSEQ ID No.3: amino acid and nucleotide sequence length: 14 amino acid, 42 nucleotide chain numbers: strand configuration: linear molecule type: peptide
Signal G1 ' the Δ Cb sequence type of 174 176 182 186 187N-Ser Ile Cys Gly Asn Asn Gln Ieu Cys Lys Ser Ile Ser Lys-C5 '-AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC AGC AAA-3 ' SEQ ID No.4: length amino acid sequence: 14 amino acid chain numbers: strand configuration: linear molecule type: the signal G5a sequence type of peptide 174 176 182 186 187 N-Ser Ile Asp Gly Asn Asn Gln Leu Orn Lys Ser Ile Ser Lys-CSEQ ID No.5: amino acid and nucleotide sequence length: 16 amino acid, 48 nucleotide chain numbers: strand configuration: linear molecule type: peptide
The signal GSb sequence type of 144 159 N-Ser Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro Pro Asn Lys Pro-C 5 '-AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG-5 ＇ SEQ ID No.6: amino acid and nucleotide sequence length: 16 amino acid, 48 nucleotide chain numbers: strand configuration: linear molecule type: peptide
The signal G7a sequence type of 144 159 N-Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro Pro Lys Lys Pro-C 5 '-AAC AAA CCG AGC ACC AAA AGC CGT AGC AAA AAC CCG CCG AAA AAA CCG-3 ' SEQ ID No.7: amino acid and nucleotide sequence length: 33 amino acid, 99 nucleotide chain numbers: strand configuration: linear molecule type: protein
The signal G7b sequence type of 158 173N-Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile5 '-AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC GTG CCG TGC AGC ATC176 182 186 190Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro-CTGC AGC AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA CGT ATC CCG-3 ' SEQ ID No.8: amino acid and nucleotide sequence length: 33 amino acid, 99 nucleotide chain numbers: strand configuration: linear molecule type: protein
158?????????????????????????????????????????????????????????173?????????176?N??-Lys?Pro?Lys?Asp?Asp?Tyr?His?Phe?Glu?Val?Phe?Asn?Phe?Val?Pro?Cys?Ser?Ile?Cys?Gly?5′-AAA?CCG?AAA?GAT?GAT?TAC?CAC?TTC?GAA?GTG?TTC?AAC?TTC?GTG?CCC?TGC?AGC?ATC?TGC?GGC
The signal G8a sequence type of 182 186 190 Asn Asn Gln Leu Cys Lys Ser Ile Cys lys Thr Ile Pro-C AAC AAC CAG CTG TGC AAA AGC ATC TGC AAA ACC ATC CCG-3 ' SEQ ID No.9: amino acid and nucleotide sequence length: 43 amino acid, 129 nucleotide chain numbers: strand configuration: linear molecule type: protein
158?????????????????????????????????????????????????????????173?????????176?N??-Lys?Pro?Asn?Asn?Asp?Phe?His?Phe?Glu?Val?Phe?Asn?Phe?Val?Pro?Cys?Ser?Ile?Cys?Ser?5′-AAA?CCG?AAC?AAC?GAT?TTC?CAT?TTC?GAA?GTG?TTC?AAC?TTC?GTG?CCG?TGC?AGC?ATC?TGC?AGC
182?????????????186??????????????????????????????????????????197?Asn?Asn?Pro?Thr?Cys?Trp?Ala?Ile?Cys?Lys?Arg?Ile?Pro?Asn?Lys?Lys?Pro?Gly?Lys?Lys?Thr?AAC?AAC?CCG?ACC?TGC?TGG?GCG?ATC?TGC?AAA?CGT?ATC?CCG?AAC?AAA?AAA?CCG?GGC?AAA?AAA?ACC
The signal G8b sequence type of 200 Thr Thr-C ACG ACC-3 ' SEQ ID No.10: amino acid and nucleotide sequence length: 43 amino acid, 129 nucleotide chain numbers: strand configuration: linear molecule type: protein
158?????????????????????????????????????????????????????????173?????????176N??-Lys?Pro?Lys?Asp?Asp?Tyr?His?Phe?Glu?Val?Phe?Asn?phe?Val?Pro?Cys?Ser?Ile?Cys?Gly5′-AAA?CCG?AAA?GAT?GAT?TAC?CAC?TTC?GAA?GTG?TTC?AAC?TTC?GTG?CCC?TGC?AGC?ATC?TGC?GGC178?????????????182?????????????186?????????????????????????????????????????????198Asn?Asn?Gln?Leu?Cys?Lys?Ser?Ile?Cys?Lys?Thr?Ile?Pro?Ser?Asn?Lys?Pro?Lys?Lys?Lys?ProAAC?AAC?CAG?CTG?TGC?AAA?AGC?ATC?TGC?AAA?ACC?ATC?CCG?AGC?AAC?AAA?CCG?AAA?AAG?AAA?CCG
The signal G9a sequence type of 200Thr Ile-CACC ATC-3 ' SEQ ID No.11: amino acid and nucleotide sequence length: 15 amino acid, 45 nucleotide chain numbers: strand configuration: linear molecule type: protein
The signal G9b sequence type of 190 204N-Pro Asn Lys Lys Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys-C5 '-CCG AAC AAA AAA CCG GGC AAA AAA ACC ACG ACC AAA CCG ACC AAA-3 ' SEQ ID No.12: amino acid and nucleotide sequence length: 15 amino acid, 45 nucleotide chain numbers: strand configuration: linear molecule type: protein
The signal G11a sequence type of 190 204N-Pro Ser Asn Lys Pro Lys Lys Lys Pro Thr Ile Lys Pro Thr Asn-C5 ＇-CCG AGC AAC AAA CCG AAA AAG AAA CCG ACC ATC AAA CCG ACC AAC-3 ' SEQ ID No.13: amino acid and nucleotide sequence length: 13 amino acid, 39 nucleotide chain numbers: strand configuration: linear molecule type: protein
The signal G11 Δ Ca sequence type of 164 176N-His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys-C5 '-CAT TTC GAA GTG TTC AAC TTC GTG CCG TGC AGC ATC TGC-3 ' SEQ ID No.14: amino acid and nucleotide sequence length: 13 amino acid, 39 nucleotide chain numbers: strand configuration: linear molecule type: protein
The signal G2a1 sequence type of 164 176N-His Phe Glu Val Phe Asn Phe Val Pro Ser Ser Ile Cys-C5 '-CAT TTC GAA GTG TTC AAC TTC GTG CCG AGC AGC ATC TGC-3 ' SEQ ID No.15: amino acid and nucleotide sequence length: 101 amino acid, 303 nucleotide chain numbers: strand configuration: linear molecule type: protein
130N-Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys5′-ACC GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG CCG AGC AAA CCG ACC ACC AAA150Gln Arg Gln Asn Lys Pro Pro Asn Lys Pro Asn Asn Asp Phe His Phe Glu Val Phe Asn PheCAG CGT CAT AAC AAA CCG CCG AAC AAA CCG AAC AAC GAT TTC CAT TTC GAA GTG TTC AAC TTC171 173 176 182 186 191Val Pro Cys Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro SerGTG CCG TGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA CGT ATC CCG AGC192 195 198Asn Lys Pro Lys Lys Lys Pro Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys Thr Thr LysAAC AAA CCG AAG AAA AAA CCG ACG ACC AAA CCG ACC AAA AAA CCG ACC TTC AAA ACC ACC AAA213 230Lys Asp His Lys Pro Gln Thr Thr Lys Pro Lys Glu Val Pro Thr Thr Lys Pro-CAAA GAT CAT AAA CCG CAG ACC ACC AAA CCG AAA GAA GTG CCG ACC ACC AAA CCG-3′SEQ ID No.16 G2a2：：101,303：：：
130N-Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys5′-ACC GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG CCG AGC AAA CGG ACC ACC AAA150 157 160 161 163Gln Arg Gln Asn Lys Pro Pro Lys Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn PheCAG CGT CAG AAC AAA CCG CCG AAA AAA CCG AAA GAC GAT TAC CAT TTC GAA GTG TTC AAC TTC171 173 176 182 186Val Pro Cys Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro AsnGTG CCG TGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG CCG ATC TGC AAA CGT ATC CCG AAC192Lys Lys Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys Thr Thr LysAAA AAA CCG GGC AAA AAA ACC ACG ACC AAA CCG ACC AAA AAA CCG ACC TTC AAA ACC ACC AAA213 230Lys Asp His Lys Pro Gln Thr Thr Lys Pro Lys Glu Val Pro Thr Thr Lys Pro-CAAA GAT CAT AAA CCG CAG ACC ACC AAA CCG AAA GAA GTG CCG ACC ACC AAA CCG-3′SEQ ID No.17 G2a3：：101,303：：：
130N-Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gln Thr Gln Pro Ser Lys Pro Thr Thr Lys5′-ACC GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG CCG AGC AAA CCG ACC ACC AAA150 157 160 161 163Gln Arg Gln Asn Lys Pro Pro Lys Lys Pro Lys Asp Asp Tyr His Phe Glu Val Phe Asn PheCAG CGT CAG AAC AAA CCG CCG AAA AAA CCG AAA GAC GAT TAC CAT TTC GAA GTG TTC AAC TTC171 173 176 182 186 191Val Pro Cys Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro SerGTG CCG TGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA CGT ATC CCG AGC192 195 198Asn Lys Pro Lys Lys Lys Pro Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys Thr Thr LysAAC AAA CCG AAG AAA AAA CCG ACG ACC AAA CCG ACC AAA AAA CCG ACC TTC AAA ACC ACC AAA 213 230Lys Asp His Lys Pro Gln Thr Thr Lys Pro Lys Glu Val Pro Thr Thr Lys Pro-CAAA GAT CAT AAA CCG CAG ACC ACC AAA CCG AAA GAA GTG CCG ACC ACC AAA CCG-3′SEQ ID No.18 G9v：：15,45：：：
The signal G5v sequence type of 190 204N-Thr Glu Arg Ala Pro Ser Arg Ala Pro Thr Ile Thr Leu Lys Lys-C5 '-ACG GAA AGA GCA CCA AGC AGA GCA CCA ACA ATC ACC CTC AAA AAG-3 ' SEQ ID No.19: amino acid and nucleotide sequence length: 16 amino acid, 48 nucleotide chain numbers: strand configuration: linear molecule type: peptide
The signal G4A sequence type of 144 159N-Arg Lys Pro Pro Ile Asn Pro Ser Gly Ser Ile Pro Pro Glu Asn His-C5 '-AGA AAA CCA CCA ATT AAT CCA TCA GGA AGC ATC CCA CCA GAA AAC CAT-3 ' SEQ ID No.20: amino acid and nucleotide sequence length: 17 amino acid, 42 nucleotide chain numbers: strand configuration: linear molecule type: peptide
The signal G4B sequence type of 171 173 176 182 186 187 N-Val Pro Cys Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys-C5 '-GTG CCG TGC AGC ATC TGC AGC AAC AAC CCG ACC TGC TGG GCG ATC TGC AAA-3 ' SEQ ID No.21: amino acid and nucleotide sequence length: 17 amino acid, 42 nucleotide chain numbers: strand configuration: linear molecule type: peptide
The signal G4V sequence type of 171 173 176 182 186 187N-Val Pro Cys Ser Ile Cys Gly Asn Asn Gln Leu Cys Lys Ser Ile Cys Lys-C5 '-GTG CCC TGC AGC ATC TGC GGC AAC AAC CAG CTG TGC AAA AGC ATC TGC AAA-3 ' SEQ ID No.22: amino acid and nucleotide sequence length: 17 amino acid, 51 nucleotide chain numbers: strand configuration: linear molecule type: peptide
171?????173?????????176?????????????????????182?????????????186?187N??-Val?Pro?Cys?Ser?Thr?Cys?Glu?Gly?Asn?Leu?Ala?Cys?Leu?Ser?Leu?Cys?His-C5′-GTT?CCC?TGC?AGT?ACA?TGT?GAA?GGT?AAT?CTT?GCA?TGC?TTA?TCA?CTC?TGC?CAT-3

Claims (33)

1. the polyclone of the epi-position of anti-rsv protein G or monoclonal antibody, described epi-position is corresponding to being selected from RSV A or the B Protein G complete sequence peptide sequence between 150-159,176-189,194-207 and 155-176 amino-acid residue or having a kind of sequence of the sequence of at least 80% homogeny.
2. according to the antibody of claim 1, it is characterized in that it is the antibody of the contained peptide of sequence between anti-RSV A hypotype or B subtype protein G 130-230 amino-acid residue.
3. according to the antibody of claim 1 or 2, it is characterized in that it is anti-antibody with peptide of one of following at least sequence: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ IDNo.13, SEQ ID No.14, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No.20, SEQ ID No.21 and/or SEQ ID No.22.
4. can produce the peptide of the antibody of one of claim 1-3, it is characterized in that it has at least a sequence that is selected from following sequence: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ IDNo.13, SEQ ID No.14, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No.20, SEQ ID No.21 and/or SEQ ID No.22.
5. according to the peptide of claim 4, it is characterized in that it contains at least one cysteine residues at N-end or C-terminal position in addition.
6. immunogenic agents is characterized in that it contains and at least one claim 4 of carrier protein couplet or 5 peptide.
7. according to the immunogenic agents of claim 6, it is characterized in that carrier proteins is selected from: the OmpA of gram negative bacterium and its fragment, TT albumen, streptococcic human serum albumin is conjugated protein and its fragment and choleratoxin B subunit (CTB).
8. according to the immunogenic agents of claim 6 or 7, it is characterized in that carrier proteins is the OmpA of Klebsiella bacterium.
9. according to the immunogenic agents of one of claim 6～8, it is characterized in that peptide and carrier proteins are by being connected albumen coupling.
10. according to the immunogenic agents of claim 9, it is characterized in that connecting albumen is selected from: mammalian blood serum albumin acceptor and the acceptor that is present in the mucomembranous cell surface.
11., it is characterized in that described coupling is a covalent coupling according to the immunogenic agents of one of claim 6-10.
12. the nucleotide sequence of the immunogenic agents of one of coding claim 6-11.
13., it is characterized in that it is a kind of coding claim 4 that insertion or fusion and promotor merge in the proteic dna molecular of code carrier or 5 the hybrid DNA molecule that peptide or its segmental DNA produced according to the nucleotide sequence of claim 12.
14., it is characterized in that it is a kind of RNA molecule according to the nucleotide sequence of claim 12.
15. nucleotide sequence as one of the immunogenic agents of one of the peptide of the antibody of one of claim 1-3 of medicine, claim 4 or 5, claim 6-11 or claim 12-14.
16. a pharmaceutical composition is characterized in that it contains the nucleotide sequence of one of the immunogenic agents of one of the peptide of the antibody of one of claim 1-3, claim 4 or 5, claim 6-11 or claim 12-14, and pharmaceutically-acceptable excipients.
17. the nucleotides sequence of one of the immunogenic agents of one of the peptide of the antibody of one of at least a claim 1-3, claim 4 or 5, claim 6-11 or claim 12-14 is listed in that preparation is used for the treatment of or the composition that prevents to infect due to RSV hypotype A or the B in purposes.
18. a diagnostic kit is characterized in that it comprises the nucleotide sequence of one of the immunogenic agents of one of the peptide of the antibody of one of claim 1-3, claim 4 or 5, claim 6-11 or claim 12-14.
19. the method for the immunogenic agents of one of preparation claim 6-11 is characterized in that the coupling between peptide and carrier proteins realizes through chemical process.
20. the method for the immunogenic agents of one of preparation claim 6-11 is characterized in that the coupling between peptide and carrier proteins realizes through the DNA recombinant technology.
21., it is characterized in that it comprises the step of introducing the nucleotide sequence of one of claim 12-14 in host cell according to the preparation method of claim 19.
22., it is characterized in that nucleotide sequence is the fusion gene of introducing by means of the dna vector that is derived from plasmid, phage, virus and/or clay according to the preparation method of claim 21.
23., it is characterized in that fusion gene is incorporated in the host cell gene group according to the method for claim 21 or 22.
24., it is characterized in that host cell is a protokaryon according to the method for one of claim 21-23.
25., it is characterized in that host cell is selected from: intestinal bacteria, genus bacillus, Bacterium lacticum, staphylococcus and suis according to the method for claim 24.
26., it is characterized in that host cell is a primary yeast according to the method for one of claim 21-23.
27., it is characterized in that host cell is a kind of mammalian cell according to the method for one of claim 21-23.
28., it is characterized in that host cell is a kind of cell of plant origin according to the method for one of claim 21-23.
29., it is characterized in that host cell is a kind of insect cell according to the method for one of claim 21-23.
30., it is characterized in that using virus vector according to the method for one of claim 21-23.
31. according to the method for one of claim 20-27, it is characterized in that fusion molecule is expressed, grappling and being exposed on the host cell membrane.
32., it is characterized in that monoclonal antibody is humanized, and produce through recombination method according to the composition of claim 16.
33., it is characterized in that monoclonal antibody obtains through the phage library method according to the composition of claim 16.
CN98807304A 1997-07-17 1998-07-17 Syncytial respiratory virus epitopes and antibodies comprising them, useful in diagnosis and therapy Pending CN1264425A (en)

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