CN1352691A - Human complement C3-degrading polypeptide from i(streptococcus pneumoniae) - Google Patents
Human complement C3-degrading polypeptide from i(streptococcus pneumoniae) Download PDFInfo
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- CN1352691A CN1352691A CN99813016A CN99813016A CN1352691A CN 1352691 A CN1352691 A CN 1352691A CN 99813016 A CN99813016 A CN 99813016A CN 99813016 A CN99813016 A CN 99813016A CN 1352691 A CN1352691 A CN 1352691A
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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
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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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
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Abstract
The present invention relates to the identification and use of a family of human complement C3-degrading polypeptides expressed by i(S.pneumoniae). The polypeptides have molecular weights of about 15 kD to about 25 kD or about 75 kDa to about 95 kDa, for example. Preferred polypeptides of this invention include the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:5.
Description
Abstract of invention
This relates to evaluation and uses the human complement c 3 degradation property peptide family that streptococcus pneumoniae showed.This polypeptide has for example about 15kDa to about 25kDa or the about 75kDa molecular weight of about 95kDa extremely.The preferred polypeptide of the present invention comprises the aminoacid sequence of SEQ ID NO:2 and SEQ ID NO:5.
Invention field
The present invention relates to streptococcus pneumoniae (Streptococcus pneumoniae) and be particularly related to a kind of evaluation of streptococcus pneumoniae polypeptides, this polypeptide people's complement proteins C3 that can degrade.
Background of invention
The respiratory tract infection that streptococcus pneumoniae is caused causes every year estimates about 500,000 routine pneumonia and 47,000 people's death.To have high risk person be baby below two years old in streptococcal infection to bacterial pneumonia, individual that function of immune system is impaired and the elderly.In these groups, streptococcus pneumoniae is bacterial pneumonia and meningitic major cause.And then streptococcus pneumoniae is the has age children's of institute an otic infections main cause.Children and old man be at bacterial colonization (colonization), after part or the systemic infection or with after the purifying polysaccharide immunization, for the protection antibody of streptococcus pneumoniae pod membrane polysaccharide synthetic obstacle arranged.Be all the invasive bacterium for adult that streptococcus pneumoniae infects for HIV or the children and inhale the main cause of exhaling tract disease, and cause courageous and upright infection the (people such as Connor, Current Topics in AIDS1987 on one's body these patients; People such as 1:185-209 and Janoff, Ann.Intern.Med.1992:117 (4): 314-324).
The individuality that shows the greatest danger for severe infections can't produce antibody to existing pod membrane polysaccharide vaccine.As a result, there is four kinds of combinations (conjugate) vaccine to enter clinical experimental stage at present.Combined vaccine comprises and protein carrier or adjuvant bonded streptococcus pneumoniae pod membrane polysaccharide that hope can be strengthened antibody response.Yet the combined vaccine in clinical trial has other potential difficult problem in addition.For example, U.S.'s streptococcus pneumoniae serotype very in vogue with in for example Israel, West Europe, the modal serotype difference in ground such as South Africa or Scandinavia.Therefore, may be disadvantageous at a favourable vaccine in local region in another area.As for modifying present available pod membrane polysaccharide vaccine or development protein conjugate potential demand as the pod membrane vaccine that adapts to region serotype difference, the generation money and the technical complicacy of making us halting then.Therefore, for the prevention of streptococcus pneumoniae infection and prepare for the extensive protectiveness Streptococcus pneumoniae vaccine, looking for the immunogenicity with whole world conservative property in the different serotype that virulence is arranged, to expose protein be most important.And the plain resistance streptococcus pneumoniae of penicillin and head-germ spore appears in ecumenicity ground, makes for the demand of effective vaccine most urgent (people such as Baquero, J.Antimicrob.Chemother.1991; 28S; 31-8).
There are several streptococcus pneumoniae protein to be suggested the immune response that combines or excited originally the antagonism streptococcus pneumoniae with streptococcus pneumoniae pod membrane polysaccharide as single immunity.Existing report is pointed out, adheres to the relevant surface protein of airway epithelial cell with streptococcus pneumoniae and comprises PsaA, PspC/CBP112 and IgA1 proteolytic enzyme (people such as Sampson, Infect.Immun.1994; 62:319-324, people such as Sheffield, Microb.Pathogen.1992; People such as 13:261-9 and Wani Infect.Immun.1996; 64:3967-3974).The antibody that resists these attachment proteinses can suppress streptococcus pneumoniae and adhere to airway epithelial cell, moves life thereby reduce.Other tenuigenin streptococcus pneumoniae protein as pneumolysin, autolysin, neuraminidase or Unidasa is suggested as vaccine antigen, because antibody can potentiality stop these protein at streptococcus pneumoniae infection patient toxic action on one's body.Yet these protein are not the surface that is positioned at streptococcus pneumoniae usually, but when cytolysis and when dead from bacterium secrete or discharge (people such as Lee, Vaccine 1994; People such as 12:875-8 and Berry, Infect.Immun.1994; 62:1101-1108).Though use these cytoplasmic proteins can relax the Late Effect of streptococcus pneumoniae infection as immunogen, resist these proteinic antibody and neither can promote streptococcus pneumoniae death, can not prevent that initial or follow-up streptococcus pneumoniae from moving life yet.
The prototype surface protein that is carrying out the Streptococcus pneumoniae vaccine test is pneumococcal surface protein matter A (PspA).PspA is the heterologous protein of about 70-140kDa.The structure of PspA comprises: at N-terminal α spiral, then be the sequence of proline rich, and at the carboxylic end with a series of 11 choline binding tumor-necrosis factor glycoproteinss as end.Though be known with many information of its structurally associated, PspA is between different streptococcus pneumoniae serotypes and the non-structure conservative property, and its function unknown fully (people such as Yother, J.Bacteriol.1992; 174:601-9 and Yother, J.Bacteriol.1994; 176:2976-2985).Studied immunogenicity (people such as McDaniel, the Microb.Pathogin.1994 of PspA verified animal; 17:323-337).Though PspA has immunogenicity, its as the ability of vaccine antigen because of the heterology of PspA, its with four structure groups exist with and do not give qualitatively that function makes situation become complicated.
Can't produce on one's body the patient of protection antibody the third composition C3 of complement and substitute the relevant protein in complement path and constitute the first line of defence that the host resists streptococcus pneumoniae infection to type-specificity polysaccharide pod membrane.Because complement protein can not penetrate the firm cell walls of streptococcus pneumoniae, the deposition of conditioning property C3b on the streptococcus pneumoniae surface becomes the main vehicle of removing streptococcus pneumoniae.Interaction in streptococcus pneumoniae and the blood plasma between the C3 has been notified when streptococcus pneumoniae property microbemia and has been taken place, and the covalent attachment of C3b this moment (the opsonic activity fragment of C3) activates cytophagous identification and engulfs (people such as Johnston, J.Exp.Med.1969; 129:1275-1290, Hasin HE, J.Immunol.1972; People such as 109:26-31 and Hostetter, J.Infect.Dis.1984; 150:653-61).C3b is deposited on streptococcus pneumoniae pod membrane and the cell walls.Yet the effect of the method for this control streptococcus pneumoniae infection is on duty mutually.Excite the method for streptococcus pneumoniae opsonization can improve the lysis that this microorganism causes.Method and treatment for the restriction streptococcus pneumoniae infection at present still has tight demand.
Summary of the invention
The present invention relates to human complement c 3 degradation property polypeptide (protein) family that identifies and use that streptococcus pneumoniae shows.These polypeptide should have about 15kDa to about 25kDa or the about 75kDa molecular weight of about 95kDa extremely, and this molecular weight for example records on the 10%SDS polyacrylamide gel electrophoresis.The present invention includes the isolating numerous protein of different C3-degradation property bacterial strain institute's energy from streptococcus pneumoniae.
On the one hand, the present invention relates to have the isolated polypeptide of at least 80% sequence homogeny with SEQ ID NO:2 or SEQ ID NO:5.In preferable embodiment, this polypeptid system separates from streptococcus pneumoniae, and perhaps this polypeptide is a recombinant polypeptide.Preferably, this isolated polypeptide degraded people complement proteins C3.The preferred polypeptide of the present invention is the isolated polypeptide with the aminoacid sequence that comprises SEQ ID NO:2 or SEQ ID NO:5, more preferably this aminoacid sequence, i.e. SEQ ID NO:2 or SEQ ID NO:5.Term " isolating " means naturally occurring material or the synthetic material that has been shifted out by its physical environment at this paper.Term " polypeptide " comprises peptide, polypeptide and protein in this article, no matter its length how.Preferably, polypeptide of the present invention comprises one or more functional element, and these unit comprise the proteinic polypeptide of human complement C 3-degrading.
Therefore, the invention still further relates to the polypeptide fragment that goes out from C3-degradation property albumen sepn of the present invention.This type of fragment is included in the term as used in this specification " polypeptide ".Preferably, the invention provides the polypeptide of at least 15 continuous amino acids, it is derived from having the protein of 80% sequence homogeny with SEQ ID NO:2 or SEQ ID NO:5, and the polypeptide of at least 15 continuous amino acids among SEQ ID NO:2 or the SEQ ID NO:5 more preferably is provided.In the present invention on the other hand, this preferable polypeptide people's complement proteins C3 that can degrade.
On the other hand, the isolated polypeptide of people's complement proteins C3 that the present invention relates to preferably to degrade, the nucleic acid of this isolated polypeptide of wherein encoding can be hybridized under highly rigorous hybridization conditions with the part of SEQ ID NO:1 or SEQ ID NO:4 or its complementary strand.Preferably, this polypeptide comprises at least 15 continuous amino acids, more preferably at least 15 continuous amino acids among SEQ ID NO:2 or the SEQ ID NO:5.
On the other hand, the present invention relates to have the polypeptide of minimizing of people C3 degrading activity or non-degradable human C3; Yet, the nucleic acid of this group polypeptide of encoding each self-contained can with the coding human C3 degradation property polypeptide nucleic acid or can with the nucleotide sequence of indivedual nucleic acid hybridizations.That back one polypeptide group has a reduction or do not have human C3 degrading activity, be called as " nondegradation " polypeptide at this specification sheets.The difference of nondegradation polypeptide and C3 degradation property polypeptide can be one or more amino acid.Amino acid whose change can be displacement, changes or lacks one or more amino acid.Dissimilar amino acid changes will be discussed in this manual.The nucleic acid of coding nondegradation polypeptide is the other preferable embodiment of the present invention.
The invention still further relates to immunity system irritating compositions (preferably vaccine), it comprises the immunity system pungency polypeptide of the present invention of significant quantity and treats and go up acceptable carrier that this polypeptide should separate from streptococcus pneumoniae.In one embodiment, this immunity system irritating compositions or vaccine also comprise at least a separation other immunity system pungency polypeptide from streptococcus pneumoniae.
The invention still further relates to the antibody of a kind of can the combination (being generally the specificity combination) with polypeptide of the present invention (at least a portion).In one embodiment, this antibody is monoclonal antibody, and in another embodiment, this antibody is polyclonal antibody.In another embodiment, this antibody is antibody fragment.This antibody or antibody fragment can derive from mouse, rat, goat, chicken, people, or rabbit.
The invention still further relates to the isolated nucleic acid molecule that can hybridize under highly rigorous hybridization conditions with SEQ ID NO:1 or SEQ ID NO:4 or its complementary strand (is polynucleotide, it can be strand or two strands, and it can be the part or the fragment of macromole (as carrier)).The rigorous hybridization conditions of the height of indication comprises in this article, for example, with 6X SSC, 5X Denhardt, 0.5%SDS, reaching 100 mcg/ml fragmentations and sex change salmon sperm DNA spends the night 65 ℃ of hybridization, and at 2X SSC, cleaned once about 10 minutes under the room temperature among the 0.1%SDS, then in once about 15 minutes of 65 ℃ of cleanings, then, cleaned at least once under the room temperature 3-5 minute at least among the 0.1%SDS at 0.2X SSC.In one embodiment, this nucleic acid fragment system separates from streptococcus pneumoniae, and in another embodiment, polypeptide of this nucleic acid molecule encoding.In one embodiment, this polypeptide degraded people complement proteins C3.In another embodiment, the polypeptide of a kind of non-degradable human complement C 3 of this nucleic acid molecule encoding.
In another embodiment, this nucleic acid molecule is in carrier (that is, it is the fragment of nucleic acid carrier), and this carrier is can production polypeptide expression carrier.The cell that comprises this nucleic acid molecule also comprises in the present invention.In one embodiment, this cell is bacterium or eukaryotic cell.
The invention still further relates to the isolated nucleic acid molecule that comprises SEQ ID NO:1 or SEQ ID NO:4 nucleotide sequence or its complementary strand.The invention still further relates to the RNA molecule of transcribing by the double-stranded DNA that comprises SEQ ID NO:1 or SEQ ID NO:4.
The present invention relates to the immunoreactive method of a kind of streptococcus pneumoniae that creates antagonism on the other hand in Mammals (particularly human).This method comprises and gives Mammals with a composition that with the immune response of this polypeptide that creates antagonism, said composition comprises the polypeptide of the present invention and the pharmaceutically acceptable carrier for the treatment of significant quantity.This immune response can be B cell response, t cell responses, epithelial cell reaction or endotheliocyte reaction.In a preferable embodiment, said composition is a vaccine composition.Preferably, this protein is at least 15 amino acid longs, and also preferably said composition also comprises at least a other immunity system pungency polypeptide from streptococcus pneumoniae.In one embodiment, this polypeptide comprises at least 15 amino acid among SEQ ID NO:2 or the SEQ ID NO:5.
The invention still further relates to from about 15kDa of streptococcus pneumoniae to about 25kDa or the about 75kDa isolated polypeptide of the energy human complement C 3-degrading of about 95kDa extremely, and relate to a kind of method that suppresses the C3 Degradation of streptococcus pneumoniae-mediation.This method comprises makes the streptococcus pneumoniae bacterium contact an antibody, and this antibody capable combines with polypeptide of the present invention (its at least one part).
The method of repulsive interaction when the invention still further relates to the inflammatory response of a kind of C3-of inhibition mediation and xenotransplantation.When being included in xenotransplantation, this method expresses polypeptide of the present invention on the used animal organ surface.This method is particularly conducive at for example pig kidney and expresses polypeptide of the present invention, thereby suppresses the inflammation of the C3 mediation after the xenotransplantation.
The invention still further relates to a kind of isolated nucleic acid molecule, it comprises the zone of one at least 15 Nucleotide, and this zone can be hybridized under highly rigorous hybridization conditions with at least a portion in SEQ ID NO:1 or SEQ ID NO:4 or its complementary strand.
The invention still further relates to and have NO:6, SEQ ID NO:7, the isolated DNA molecule or the primer of nucleotide sequence shown in SEQ ID NO:8 and the SEQ ID NO:9 as SEQ ID.
The accompanying drawing summary
Fig. 1 provides the translation nucleotide sequence (SEQ IDNO:1) partly of C3-degradation property polypeptide of the present invention (about 20kDa) gene.
Fig. 2 provides the aminoacid sequence (SEQ ID NO:2) of C3-degradation property polypeptide of the present invention (about 20kDa) gene.
Fig. 3 provides the aminoacid sequence of C3-degradation property polypeptide of the present invention (about 20kDa) gene, itself and code book are invented the nucleotide sequence (two strands) (SEQ ID NOS:1-3, wherein SEQ ID NOS:3 is the complementary sequence of SEQ IDNOS:1) arranged side by side of C3-degradation property polypeptide gene.
Fig. 4 provides the nucleotide sequence (SEQ ID NO:4) of the 92kDa aminoacid sequence of prediction.
Fig. 5 provides the 92kDa aminoacid sequence (SEQ ID NO:5) of prediction.
Fig. 6 shows the sequence alignment of SEQ ID NO:1 and part SEQ ID NO:4.
Fig. 7 shows the sequence alignment of SEQ ID NO:2 and part SEQ ID NO:5.
Fig. 8: the full cytolysis thing of several streptococcus pneumoniaes with polyclone anti--～Western engram analysis that r20kDa serum carries out.Molecular weight marker is at swimming lane 1; From the regroup 20kDa polypeptide of pDF122 at swimming lane 2; From the regroup 92kDa polypeptide of the 7th type at swimming lane 3; Swimming lane 4 is blank; The full cytolysis thing of CP1200 is at swimming lane 5; The full cytolysis thing of the 3rd type is at swimming lane 6; The full cytolysis thing of the 7th type is at swimming lane 7.But the regroup polypeptide of the about 20kDa of this antiserum(antisera) identification and about 92kDa, but in full cytolysis thing, can only discern bigger polypeptide.
Fig. 9: show autoradiogram(ARGM) via the biotinylation C3 of 20kDa of the present invention and the degraded of 92kDa polypeptide.
Figure 10: the 7.5%SDS-PAGE via the Coomassie blue stain of 20kDa of the present invention (swimming lane A8) and 92kDa (swimming lane C8) polypeptide degraded analyzes.
Figure 11 is for showing SC (subcutaneous) inoculation r79kDa protein (adjuvant is MPL) and carrying out the figure that IN (in the nose) attacks back CBA/CAHN xid/J mouse survival rate with streptococcus pneumoniae the 3rd type.
Figure 12 is embodiment 8 described SDS-PAGE gels.
Preferable embodiment describes in detail
The present invention relates to identify and separate the greatly human complement c 3 degradation property polypeptide fragment of polypeptide of about 75kDa to about 95kDa.This fragment has molecular weight and is about 20kDa (± 5kDa) (on 10% SDS-PAGE).The invention still further relates to the nucleic acid of coding human complement C 3 degradation property polypeptide.
Observed from the streptococcus pneumoniae of several serotypes at the culture of logarithmic phase growth phase α-chain of C3 beta chain of degrading subsequently of at first degrading, can not produce the C3 cutting fragment (people such as Angel of existing definition, J.Infect.Dis.170:600-608,1994).The degraded form that this type of does not cut is different in essence in the product of other microorganism, as elastoser of Pseudomonas aeruginosa (Pseudomonas aeruginosa) and the L-Cysteine HCL Anhydrous of the Nei Ami of histolytica (Entamoebahistolytica).
Employed in this article term " degraded " means amino acid is removed from the protein molecule, produces peptide or polypeptide.Polypeptide of the present invention is degraded C3 and can not produced known C3b, iC3b or C3d cutting fragment.C3-degradation property polypeptide of the present invention has the preference of some degree for C3, and for example, can't degrade other protein of this C3-degradation property polypeptide is as albumin.
The C3-degradation property polypeptid system of about 20kDa identifies in the streptococcus pneumoniae gene pool of the property an inserted interruption, compares with the wild-type streptococcus pneumoniae, has the clone of the C3 degrading activity that weakens and is separated.Embodiment 1 provides the exemplary test of assessment clone's C3-degrading activity.Identify clone that the C3 degrading activity weakens and select the SmaI insert of 546bp based on the clone that this demonstration weakens the C3 degrading activity.This SmaI fragment is as the probe of the streptococcus pneumoniae gene pool that is made by the CP1200 bacterial strain.From the streptococcus pneumoniae gene pool and can be separated with the positive colony of this SmaI fragment hybridization, and the open reading frame of evaluation and C3-degrading activity genes involved.The oligonucleotide (SEQ ID NO:10) that following and some PspA have a sequence homogeny be used to confirm in the differential hybridization reaction the to encode gene of C3-degradation property polypeptide is completely different with the gene of coding PspA.
SEQ?ID?NO：10
GAAAACAATAATGTAGAAGACTACTTTAAAGAAGCTTTAGA
The complete open reading frame of 20kDa polypeptide has 500 base pairs (SEQ ID NO:1), predicted molecular weight be about 20kDa (+/-polypeptide of 168 amino acid (SEQ ID NO:2) 5kDa) or approximately arranged.The exemplary genes sequence of coding C3-degradation property polypeptide is shown in Fig. 1 (SEQ ID NO:1), and this amino acid sequence of polypeptide is shown in Fig. 2 (SEQ IDNO:2).Fig. 3 is combined into SEQ ID NOS:1-3 with preferable gene order with corresponding preferable translation polypeptide.
Use SEQ ID NO:2, determine that this about 20kDa amino acid sequence of polypeptide is irrelevant with other polypeptide in GenBank or Switzerland Prot database.The polypeptide of prediction has the sequence signature of the proline rich (particularly between amino acid 80-108) of prokaryotic cell prokaryocyte membrane protein, and this hints that this expression of polypeptides is on cell surface.This aminoacid sequence does not show tangible choline-associativity tumor-necrosis factor glycoproteins.To on the SDS-PAGE gel that soaks into C3, carry out electrophoresis from the streptococcus pneumoniae solute and the supernatant liquor of CP1200 culture, both all identify about 20kDa (± 5kDa) band confirm to have as SEQ ID NO:2 and predicts that big or small polypeptide has C3-degrading activity (seeing embodiment 2) at supernatant liquor and solute.As described in embodiment 3, the gene of the C3-degradation property polypeptide of this 20kDa that encodes all has conservative property in 24 streptococcus pneumoniae strain isolateds representing 5 serotypes ( serotype 1,3,4,14, and 19F) at least.
The nucleotide sequence of code book invention C3-degradation property polypeptide is inserted into expression vector and is used for expressing intestinal bacteria (E.coli).As described in embodiment, regroup C3-degradation property polypeptide is separated.One skilled in the art will understand that, with regard to a specific gene sequence (as the SEQ ID NO:1 supplier of institute), can use different expression vectors to express this gene.And then, can use currently known methodss different in this area to produce and separate recombinant polypeptide of the present invention, and one skilled in the art also can understand, C3 degradation test of the present invention will determine whether the particular expression system except the expression system that is proposed in an embodiment has function, and need not further test.In the basic technology document, can find many different molecules and immunological technique, as people such as Sambrook (Molecular Cloning-A Laboratory Manual, 1989 Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY) and people (Antibodies such as Harlow; A Laboratory Manual.Cold Spring Harbor, NY; Cold Spring Harbor Laboratory Press, 1988).
The polynucleotide of code book invention C3 degradation property polypeptide are to identify with the prepared plasmid gene of the streptococcus pneumoniae genomic DNA fragment storehouse of CP1200 bacterial strain.Though known have many diverse ways can obtain the plasmid gene storehouse, with preferable strategy, the structure in plasmid gene storehouse is to use from streptococcus pneumoniae CP1200 bacterial strain (to derive from D.A.Morrison, University of Illinois, Champagne-Urbana, Illinois, and as people Proc.Natl.Acad.Sci. (USA) 1995 such as Havarstein LF; 92:11140-11144 describes) the streptococcus pneumoniae genomic DNA fragment (0.5-4.0kb) through Sau3A cutting, and insert imbedibility shuttle vectors pVA891 (erm
r, cm
rHave colibacillary replication orgin) the BamHI position.Utilize electroporation to remove transformed into escherichia coli DH5 α MCR bacterial strain this gene pool.In the intestinal bacteria transformant that some are selected at random, extract plasmid, show that all transformants all contain the regroup plasmid.
Plasmid gene storehouse DNA can extract in the intestinal bacteria transformant, and is used to transform CP1200 parental generation streptococcus pneumoniae strain, and this conversion realizes with the sudden change of the homologous recombination property inserted.
The step transitions that S. pneumoniae strains CP1200 cell can be in the CTM substratum moves pH in order to HCl is a competent cell.Competent cell is frozen in-70 ℃ till needs use in a small amount of packing mode.
Isolated polypeptide of the present invention and human complement C 3 were cultivated 4 hours or more of a specified duration to detect the C3 Degradation in 37 ℃ in the presence of PBS.The control sample that does not have the separation streptococcus pneumoniae polypeptides can be used as the control group of comparison purpose.
Polypeptide of the present invention has the about 15kDa of apparent molecular weight to about 25kDa, preferable about 20kDa on 10% SDS-polyacrylamide gel electrophoresis; Perhaps has the about 75kDa of apparent molecular weight to about 95kDa, preferable about 92kDa.Typical peptide sequence is shown in SEQ ID NO:2 and SEQ ID NO:5.One of ordinary skill in the art will be understood, and have some variations to expect in aminoacid sequence, and these variations should not got rid of outside scope of the present invention.For example, the conservative property sudden change also is not precluded within outside the scope of the invention, the variation thing that is less than about 80% aminoacid sequence homogeny (and preferably being less than about 90% aminoacid sequence homogeny) in the aminoacid sequence homogeny also is contained in the scope of the invention, wherein can degrade people's complement proteins C3 and particularly separate or originally derive from a streptococcus pneumoniae bacterium when this polypeptide of this polypeptide.Protein and fragment thereof (all being called polypeptide) are also in scope of the present invention, particularly when these polypeptide can be degraded people's complement proteins C3.
Can expect in S. pneumoniae strains and serotype has some variant nucleic acid sequences, as expection has some amino acid variations.It is as known in the art that conservative amino acid replaces, and comprises, for example, uses with this amino acid to belong to of a sort other member's aminoacid replacement.For example, nonpolar amino acid comprises L-Ala, leucine, Isoleucine, Xie Ansuan, proline(Pro), phenylalanine, and tryptophane.Polar neutral amino acid comprises glycine, Serine, Threonine, halfcystine, tyrosine, l-asparagine and glutamine.The amino acid of positive electric charge (alkalescence) comprises arginine, Methionin and Histidine.The amino acid of negative electric charge (acidity) comprises aspartic acid and L-glutamic acid.This type of changes expection can impact polypropylene acrylamide gel electrophoresis or measured molecular weight or iso-electric point.Good especially conservative property metalepsy includes, but not limited to Lys is replaced into Arg (vice versa) to keep positive electricity nuclear; Glu is replaced into Asp (vice versa) to keep negative electricity nuclear; Ser is replaced into Thr to keep free OH; And Gln is replaced into Asn to keep free NH
2
The preferable polypeptide of the present invention comprises the polypeptide with aminoacid sequence SEQ ID NO:2 and SEQ ID NO:5.Other polypeptide comprises the polypeptide of degradable human complement peptide C 3, and the nucleic acid of this polypeptide of encoding can be hybridized under highly rigorous hybridization conditions with SEQ IDNO:1 and SEQ ID NO:4, highly rigorous hybridization conditions is for for example with 6X SSC, 5X Denhardt, 0.5%SDS (sodium laurylsulfonate), and the salmon sperm DNA of 100 mcg/ml fragmentations and sex change is hybridized at 65 ℃ and is spent the night, and at 2X SSC, under room temperature, cleaned once about 10 minutes among 0.1% SDS, then in once about 15 minutes of 65 ℃ of cleanings, then, under room temperature, cleaned at least once 3-5 minute at least among the 0.1%SDS at 0.2 * SSC.Usually, SSC solution comprises sodium-chlor, and Trisodium Citrate and water are with the preparation stock solution.
Polypeptide of the present invention can recombinant polypeptide form separated or prepared.In other words, the nucleic acid of encode this protein or this protein some can be inserted into expression vector or embed in the karyomit(e) of cell, so that express this polypeptide in this cell.This polypeptide can be in bacterium or another cell (preferably eukaryotic cell and more preferably zooblast) purifying.Perhaps, this polypeptide can be expressed middle separation of cell (as the pneumonia streptococcus mycetocyte) of this polypeptide certainly.Therefore, when using term " polypeptide ", protein, peptide or polypeptide also are considered to be within the purview.This peptide or polypeptide are preferably at least 15 amino acid whose length, and preferable peptide or polypeptide have at least 15 continuous amino acids from SEQ ID NO:2 and SEQ ID NO:5.
The nucleic acid of this about 15kDa to about 20 polypeptide and about 75kDa to about 95kDa polypeptide of encoding also is some of the present invention.SEQ ID NOS:1 and 4 preferable nucleic acid molecule for coding C3-degradation property polypeptide.One skilled in the art will understand, and feature and character that some replacements can't make C3-degradation property peptide sequence change and cause this C3-degradation property polypeptide arrive the degree that changes in fact.For example, the present invention also comprises the nucleic acid that has at least 80% homogeny with SEQ ID NO:1.Determine that method that whether a concrete nucleotide sequence falls within the scope of the invention should see whether whether encode C3-degradation property polypeptide and compare with SEQ ID NO:1 and SEQ ID NO:4 of this concrete nucleotide sequence has at least 80% nucleic acid homogeny.Other nucleotide sequence of coding C3 polypeptide comprises the nucleic acid of those codings C3 polypeptide, and wherein this C3 polypeptide is compared the sequence homogeny with identical sequence or at least 90% with SEQ ID NO:2 or SEQ ID NO:5, and comprises the degeneracy of this nucleotide sequence.The codon of degeneracy means different three-letter code and is used for determining identical amino acid.For example, being known in the art following RNA codon (and therefore, relevant DNA codon wherein replaces U with T) can intercourse and be used to encode each specific amino acids:
Phenylalanine (Phe or F) UUU, UUC, UUA or UUG
Leucine (Leu or L) CUU, CUC, CUA or CUG
Isoleucine (Ile or I) AUU, AUC or AUA
Methionine(Met) (Met or M) AUG
Xie Ansuan (Val or V) GUU, GUC, GUA, GUG
Serine (Ser or S) AGU or AGC
Proline(Pro) (Pro or P) CCU, CCC, CCA, CCG
Threonine (Thr or T) ACU, ACC, ACA, ACG
L-Ala (Ala or A) GCU, GCG, GCA, GCC
Tryptophane (Trp) UGG
Tyrosine (Tyr or Y) UAU or UAC
Histidine (His or H) CAU or CAC
Glutamine (GIn or Q) CAA or CAG
L-asparagine (Asn or N) AAU or AAC
Methionin (Lys or K) AAA or AAG
Aspartic acid (Asp or D) GAU or GAC
L-glutamic acid (Glu or E) GAA or GAG
Halfcystine (Cys or Q) UGU or UGC
Arginine (Arg or R) AGA or AGG
Glycine (Gly or G) GGU or GGC or GGA or GGG
Terminator codon UAA, UAG or UGA
And then, a special dna sequence dna can be modified, to use the preferable codon of a special cell classification.For example, the use of colibacillary preferable codon, and the preferable codon of animal (comprising the mankind) is known.It is known that these change into one skilled in the art, so these gene orders are considered to some of the present invention.Other nucleotide sequence comprises from the length of SEQ ID NO:1 or the SEQ ID NO:4 nucleic acid fragment at least 15 (and preferable 30) nucleic acid at least, or other length is the nucleic acid fragment of at least 15 (and preferable at least 30) nucleic acid, wherein this fragment can be hybridized under highly rigorous hybridization conditions with SEQ ID NO:1 or SEQ ID NO:4, highly rigorous hybridization conditions is for example with 6X SSC, 5X Denhardt, 0.5% SDS (sodium laurylsulfonate), and the salmon sperm DNA of 100 mcg/ml fragmentations and sex change is hybridized at 65 ℃ and is spent the night, and at 2X SSC, 0.1%SDS cleaned once under room temperature about 10 minutes, then in once about 15 minutes of 65 ℃ of cleanings, then, under room temperature, cleaned at least once 3-5 minute at least among 0.1% SDS at 0.2X SSC.
Nucleic acid molecule codified SEQ ID NO:2 of the present invention or SEQ ID NO:5's is whole, can not contain SEQ IDNO:2 or SEQ ID NO:5 (the i.e. fragment that can't transcribe, comprise the fragment in generegulation zone etc.) or the part of codified SEQ ID NO:2 or SEQ ID NO:5, at least 9 the amino acid whose continuity nucleic acid fragments of coding preferably comprised from SEQ ID NO:2 or SEQ IDNO:5.Because comprised the nucleic acid molecule of the some of coding C3 degradation property polypeptide in the present invention, be not to be all encode protein or the peptide or the polypeptide of tool C3-degrading activity of all nucleic acid fragments with apprehensible.In addition, nucleic acid of the present invention can suddenly change with the C3 degrading activity (or deactivation otherwise) that removes this polypeptide.Therefore, the present invention has also considered to meet above-mentioned hybridization conditions and has encoded and do not had a nucleic acid molecule of C3-degrading activity polypeptide.The method that makes nucleotide sequence sudden change or otherwise change nucleotide sequence describes in detail in this area to some extent, and in the tool immunogenicity but the generation of not having a polypeptide of enzymatic activity can test at therepic use.
Nucleic acid molecule of the present invention can be inserted in the nucleic acid carrier or stably insert in the host genome to produce the regroup polypeptide, comprises the regroup chimeric polyeptides.In one embodiment, C3-degradation property protein is by the coded by said gene in the carrier, and this carrier is in cell.Preferably, this cell is a prokaryotic cell prokaryocyte, as bacterium.Gene and gene fragment can this gene whole or partly exist with the mode of another gene fusion, and this C3-degradation property polypeptide can one or more proteinic fused protein modes exist, and this fused protein is expressed in single proteinic mode.Various nucleic acid carrier of the present invention is known in the art, comprises many commercially available expression plasmids or virus vector.The use of these carriers is that persons skilled in the art are known.Employed in an embodiment is some typical carriers, but should not be considered to the scope of the invention is limited to some extent.
The invention still further relates to can be in conjunction with about 15kDa of (be generally specificity in conjunction with) streptococcus pneumoniae polypeptide of about 20kDa (preferable about 20kDa) and about 75kDa antibody of the polypeptide of about 95kDa (preferable about 92kDa) extremely extremely, and preferably wherein this polypeptide can human complement C 3-degrading.Can prepare polyclonal antibody or monoclonal antibody at the whole of polypeptide of the present invention or part.The method for preparing antibody at protein see for details in, for example, people such as Harlow, (Antibodies; A Laboratory Manual.Cold Spring Harbor, NY; Cold Spring HarborLaboratory Press, 1988).In a preferred embodiment, this antibody can be derived from the mankind, rat, mouse, goat, chicken, or rabbit.Polypeptide binding antibodies fragment and chimeric fragment also are known and in scope of the present invention.
The invention still further relates to the use of immunostimulating composition.Term " immunostimulating " or " immunity system pungency " composition mean protein of the present invention, peptide or peptide composition, at least a cell type in individual (as the Mammals) immunity system of their activation.Preferably, this immunostimulating composition can provide a kind of immunization reaction or preventive effect in normal (not infecting) is individual, it typically is a vaccine.Yet any detectable immune response all helps individuality in treatment application or process.Active cells preferable in the immunity system comprises phagocytic cell, as neutrophil or scavenger cell, and T cell, B cell, epithelial cell and endotheliocyte.Comprise peptide of the present invention, polypeptide or proteinic immunostimulating composition are used in and produce antibody in the animal (as rat, mouse, goat, chicken, the rabbit or the mankind), or are used to study the animal model of streptococcus pneumoniae infection.Preferable immunostimulating composition comprises at least a peptide or the polypeptide of immunostimulating (for example treating significant quantity), and it comprises at least 15 amino acid from this C3 degradation property polypeptide.
Term " vaccine " means the composition that is used for immunization.The process of immunization can comprise and gives protein, peptide, polypeptide; antigen, nucleotide sequence or complementation (for example antisense) sequence, or antibody or its suspension; after wherein giving this molecule, this molecule will produce active immne power and provide protection to resist streptococcus pneumoniae infection or to move life.Usually, these vaccines are made into injectable liquor or suspension.Also can be made into dissolving before being adapted at injecting or be suspended in solid pattern in the liquid.This vaccine production thing is emulsification in addition optionally, or be wrapped in little fat body.
This immunostimulating composition (as vaccine) also can be included in other polypeptide in acceptable damping fluid in the pharmacy or the carrier, these damping fluids or carrier for example are PBS (phosphate buffered saline (PBS)s), or other is considered to be fit to be used safely in polypeptide is imported the damping fluid of host with stimulating immune system in the art.This immunostimulating composition also can comprise other immunity system pungency polypeptide, as adjuvant or from streptococcus pneumoniae or other biological immunostimulating protein, peptide or polypeptide.For example, the most suitable mixture of control streptococcus pneumoniae infection with peptide or polypeptide.Preferably, use one or more polypeptide of the present invention or its fragment in the vaccine production thing, moving of streptococcus pneumoniae given birth to or streptococcus pneumoniae moves the living pathogenic result who is brought to resist or to limit.
Employed in this article term " treatment significant quantity " means the required result's of effective generation deal.This deal is looked the health of individual immunity system or physics (being that antibody is synthetic) situation, the degree of required protection, the formulation of preparation and other correlative factor and decide.Can estimate that this deal will fall within a scope quite widely, and can decide via routine test.
Active immne pungency composition frequent and vehicle or mixing diluents, these vehicle or thinner are acceptable carrier and compatible with active ingredient in the pharmacy.Term " acceptable carrier in the pharmacy " mean with a composition in other composition compatible and do not endanger and be " acceptable " carrier on recipient's the meaning.Suitable vehicle is, for example, and water, salt solution, dextrose, glycerine, ethanol etc., and composition thereof.In addition, if desired, this immunostimulating composition (comprising vaccine) can comprise a spot of complementary material, as wetting agent or emulsifying agent, and the pH buffer reagent, and/or can strengthen the adjuvant of this immunostimulating composition effect.
Effectively adjuvant or carrier include but not limited to aluminium hydroxide, N-ethanoyl-muramyl-L-Threonyl-D-isoglutamine (thr-MDP); N-ethanoyl-Xin-muramyl-L-alanyl-D-isoglutamine (CGP11637; be called as nor-UDP); the different glutamyl of N-ethanoyl muramyl-L-alanyl-D--L-L-Ala-2-(1 '; 2 '-two palmitoyl-sn-glycerine-3-hydroxyl phosphoryl oxygen)-ethylamine (CGP 19835A; be called as MTP-PE); and RIBI; it comprises three kinds of compositions that extract from bacterium in squalene/Tween 80 emulsions of 2%; single phosphoryl fat A, two mycolic acid trehaloses and cell wall skeleton (MPL+TDM+CWS).
The invention still further relates to the method for the C3 Degradation that suppresses streptococcus pneumoniae-mediation, comprise streptococcus pneumoniae is contacted with polypeptide, if can with from about 15kDa of streptococcus pneumoniae to about 25kDa or about 75kDa extremely isolated polypeptide (typical at least one part) bonded antibody or another polypeptide of about 95kDa.Can with about 15kDa to about 25kDa or about 75kDa extremely the isolated polypeptide bonded protein of about 95kDa can be an antibody or its fragment, or this protein can be chimeric protein, this chimeric protein comprises the antibodies district (as the variable region) from antibody, this antibody capable specific recognition from about 15kDa of streptococcus pneumoniae to about 25kDa or about 75kDa about 95kDa and have the isolated polypeptide of C3 degrading activity extremely.
Isolating streptococcus pneumoniae polypeptides of the present invention can be separated, and optionally give purifying, and can use this isolated polypeptide or its immunogenic fragments to produce immunological response, in one embodiment, it is included in the mankind or laboratory animal antibody response on one's body.The polypeptide that does not have the C3 degrading activity can be tested at its ability that limits the streptococcus pneumoniae infection influence.Similarly, the modification of polypeptide of the present invention can be for example be interrupted or the C3 degrading activity of this polypeptide of deactivation via sudden change.The antibody that can use the C3-degrading activity that can suppress polypeptide of the present invention is as preventing the C3 Degradation via the conditioning approach and promoting the strategy that streptococcus pneumoniae is removed.This isolated polypeptide can be used in test, detecting the antibody of antagonism streptococcus pneumoniae, or as the some of the vaccine of streptococcus pneumoniae treatment usefulness, or as comprising multivalence or a plurality of protein, the vaccine of peptide or polypeptide.
Therefore, mean normal mammalian subject or suffer various streptococcus pneumoniae infections invasions, be various streptococcus pneumoniae infections after diagnosing in term as used herein " processing ", or the mammalian subject that shows the feature of various streptococcus pneumoniae infections or symptom prevents or treats.Term " treatment " means for mammalian subject therapeutic effect is provided, and makes this individuality show less or does not show the symptom of streptococcus pneumoniae infection or other relative disease.This type of processing can be followed nucleic acid molecule of the present invention (tool meaning or antisense person), protein, the giving of peptide or polypeptide or antibody.
Further planning is, polypeptide of the present invention can be expressed in the vertebrate cells surface and be used to the C3 that degrades, for example, and when complement deposit (or activation) when becoming a difficult problem, for example under the brightic situation of xenotransplantation or complement-mediation.For example, complete streptococcus pneumoniae polypeptides, regroup polypeptide, or above both arbitrary somes, can be merged in the xenotransplantation cell, and express, to prevent complement deposit (and/or inflammatory response of complement-mediation) or to make it reduce to minimum in the mode of surperficial polypeptide or secrete polypeptide.
Another concrete aspect of the present invention relates to use to express isolating protein and by it and the peptide that comes or the vaccine carrier of polypeptide.In view of the above, the present invention provides a kind of immunoreactive method that causes on the other hand in Mammals, and it comprises that the vaccine carrier of will express at least a or its mixture of isolating protein of the present invention and/or peptide or polypeptide provides to Mammals.Protein of the present invention and peptide or polypeptide can use vaccine carrier alive to flow to Mammals, the material of the recombinant bacteria of particularly use living, virus or other work, it comprises genetic material required when expressing this protein and/or peptide or polypeptide in external polypeptide mode.Particularly those bacteriums that breed in gi tract have been developed into vaccine carrier, as Salmonella (Salmonella), shiga bacillus (Shigella), Yale Xin Shi bacillus (Yersinia), vibrios (Vibrio), Escherichia (Escherichia) and BCG more than reach other example people such as J.Holmgren, Immunobiol.184, people such as 157-179 (1992) and J.McGhee, Vaccine, 10, discuss to some extent among the 75-88 (1992).
Extra embodiment of the present invention relates to the immunoreactive method of initiation in individual (as Mammals); comprise and give this individuality a certain amount of code book invention isolated protein and/or the peptide that comes from it or the dna molecular (randomly co-infection-assisting factor) of polypeptide; wherein this protein and/or peptide or polypeptide keep immunogenicity; and when this protein and/or peptide or polypeptide mix immunostimulating composition (for example vaccine) and administration of human time-like, protection can be provided and can when the mankind cause follow-up infection by the streptococcus pneumoniae cause of disease, not increase the weight of the state of an illness.Infection-assisting the factor is as known in the art.
Further planning is, this about 15kDa that encodes can be used as the vaccine or the therapeutic treatment of anti-streptococcus pneumoniae infection to the antisense sequences of the gene of about 95kDa polypeptide to about 25kDa and about 75kDa.That antisense DNA is defined as is non--coding property sequence, and itself and the whole of SEQ ID NO:1 or SEQ ID NO:4 or part complementation (being complementary strand).For example, 5 '-antisense sequences of ATGTCAAGC-3 ' is 3 '-TACAGTTCG-5 '.Antisense sequences or oligonucleotide are flowed to animal can make animal produce antibody, or this sequence is inserted in viable bacteria or other cell, cause transcribing and/or translate and being suppressed of the whole of this 92kDa gene product or part.
The importing of anti sense nucleotide sequence for example can be gone up and import in streptococcus pneumoniae or the infected cell via antisense nucleic acid being loaded into suitable carriers (as little fat body).Usually, have 8 or more the anti sense nucleotide sequence of polynucleotide can be incorporated on bacterial nucleic acid or the bacterium messenger RNA(mRNA).Anti sense nucleotide sequence comprises usually at least about 15 Nucleotide (preferable be at least about 30 Nucleotide or more), thinks that the hybridization product of bacterial nucleic acid or bacterium messenger RNA(mRNA) provides required stability.The importing of sequence should suppress transcribing or translating of at least one endogenous streptococcus pneumoniae nucleotide sequence.The method of loading antisense nucleic acid is well known in the art, and for example the U.S. patent the 4th, 242, No. 046.
The present invention also provides the have 2478 base open reading frame nucleic acid of (SEQ ID NO:4), and it comprises the open reading frame of nucleotide sequence (SEQ ID NO:1), the polypeptide of the about 20kDa of its coding molecule amount (SEQ ID NO:2).20kDa polypeptide as herein described and then be named as C3-degradation property protein.Bigger open reading frame, 2163bp (SEQ ID NO:4) for example, the polypeptide of inferring of about 92kDa that encodes (SEQ ID NO:5).
The reference of all references and deliver document and all incorporate this specification sheets in this article as the reference data.For the people who knows this technology, there are many different substitute technologies and step can make the implementer come the invention of successful implementation planning according to this specification sheets similarly.Haveing the knack of this operator will understand that, though the present invention is in above being described according to particular and embodiment, the present invention is not so limited certainly, and can carry out many other embodiments, embodiment, purposes, improvement and the situation beyond these embodiments, embodiment and purposes, and can not depart from the invention scope of the application's case.
The evaluation of the insertion mutant strain that the C3-degrading activity weakens
The property inserted mutating strain series derives from Elaine doctor Tuomanen (Rockefeller Inst., New York, New York).Clone with inset tests in test, the C3-degrading activity that weakens with detection.137 clones via make these cells in Todd Hewitt nutrient solution under the room temperature on microtiter plate grow overnight and testing.These cells carry out dilution in 1: 10 for the synthetic property substratum (seeing Sicard A.M., Genetics50:31-44,1984) of streptococcus pneumoniae, and remaining cell is frozen in the microtiter plate.Will be at the C3 of 63 nanogram(ng)s in the 100 microlitre substratum (comprising the 0.1%BSA of 1 mg/ml) or 83 nanogram(ng)s (according to people such as Tack at phosphate buffered saline (PBS) (PBS), Meth.Enzymol.80:64-101,1984 method purifying in blood plasma) add in the diluted cell of about 200 microlitres.Cell was cultivated 4 hours for 37 ℃.The mixture of 100 microlitres is added in the elisa plate, and spend the night in 4 ℃ of cultivations.This plate is cleaned three times with cleaning buffer solution, and the 0.05%Tween 20 in PBS is added in the hole, before each the cleaning, carry out cultivating in 5 minutes.The 100 microlitre antibody of anti-C3 (to the human C3-IgG narrow spectrum polyclone horseradish peroxidase of tool-link coupled goat antibody partly, ICN Cappel, Costa Mesa CA) carries out 1: 1200 dilution with the 3%BSA in PBS.This elisa plate is cultivated about 30 minutes to 1 hour in the dark in 37 ℃, and cleans with above-mentioned cleaning buffer solution.This test is developed with 12 milligrams of OPD (in 30 milliliters of 0.1M sodium citrate buffer solutions) and 30% hydrogen peroxide of 12 microlitres.Test-results is read to survey on the instrument at elisa plate and is measured via the optical density (OD) reading under 490 nanometers.
Each clone tests four times.Compare with not mutated control group and to have 19 clones that are less than 40% C3 Degradation and be selected.These 19 clones screen six times with above-mentioned test, and are selected to compare with not mutated control group by the result and have 6 clones that are less than 40% C3 Degradation.Clone each for these 6 and screen 11 times, and select 2 clones and do further research with minimum C3-degrading activity.
Obtain one of them clone's part sequence, and with the SmaI fragment of 546bp with
32P carry out random primer labelling (derive from the test kit of Stratagene, LaJolla, CA).From the SmaI fragment of the 546bp of SEQ ID NO:1 with hybridize on the Southern trace from the EcoRI and the Kpnl digestion product of many S. pneumoniae strains.This identical segments also is used to screen the Sau3A fragment gene storehouse of the genomic dna that derives from S. pneumoniae strains CP1200.
Identify the inset of 3.5kb from the CP1200 gene pool.This inset is checked order, and identify the open reading frame of one 492 base pairs, comprise terminator codon.These 168 amino acid of open reading frame coding and estimated molecular weight are about 18,500 daltonian polypeptide.
Make up the PCR primer with the amplification open reading frame; This 5 ' PCR primer comprises a BamHI position; This 3 ' primer comprises a PstI position.The inset that is amplified according to meet the coli expression carrier pQE30 that the mode of translating the frame frame is connected to the His-tailing (Qiagen, San Diego, CA).The plasmid that obtains is used for transformed into escherichia coli bacterial strain BL21, and (WI), it comprises lac inhibition plasmid pREP4 (Qiagen) for Novagen, Madison.Induce culture of Escherichia coli to express the polypeptide of His-tailing, this polypeptide carries out column purification with Ni-NTA resin (Qiagen).The protein of this purifying is confirmed with the SDS-PAGE gel.
The evaluation of the C3-degradation property polypeptide of 20kDa
In order to determine the proteinic C3-degradation capability of 20kDa, the C3 that makes 0.5 mg/ml is (according to people such as Tack, Meth.Enzymol.80:64-101,1984 are prepared) on the sodium laurylsulfonate that contains 15% acrylamide (SDS) gel (15%SDS-PAGE gel), carry out copolymerization.Streptococcus pneumoniae supernatant liquor system is obtained from the S. pneumoniae strains CP1200 culture that grows to logarithmic phase in the Todd Hewitt nutrient solution, and the streptococcus pneumoniae solute is by with 5 * 10
8Individual cell and 5%SDS at room temperature cultivate and obtained in 30 minutes.Apparatus 10, (Amicon, Beverly MA) concentrate 10 times with solute to the Centricon filtration unit of 000MW separation value.Sample does not heat before electrophoresis.The sample of supernatant liquor and solute is added the 15% SDS-PAGE gel that contains C3, and electrophoresis carries out at 150V in 4 ℃, till the dyestuff leading edge is run out of.Gel cleans (2 times, 10 minutes) with 50 milliliters of 2.5%TritonX-100 in water continuously, is used in the 2.5%Triton X-100 among the 50Mm Tris-HCl, pH7.4 (2 times, 10 minutes) cleans, and with 50mm Tris-HCl, pH7.4 cleans (2 times, 10 minutes), to remove SDS.After the cleaning, with 50 milliliters 50mM Tris-HCl, pH7.4 pours in the dish ware that contains these gels, will coil that ware is added a cover and cultivates 1.5 hours and spend the night (about 16 hours) in 37 ℃.With glue with Coomassie blue stain 10 minutes, decolouring fully subsequently.
To impinging upon the faint blue background in solute and the supernatant liquor, can observe two solvability bands, one of them is about the 20kDa size.C3 degrading activity in the streptococcus pneumoniae solute is observed after 1.5 hours in 37 ℃ of cultivations, yet the C3 degrading activity is just observed after cultivating through spending the night in the Pn supernatant liquor.Therefore, the C3 degrading activity is obviously main relevant with cell.
Encode gene tool conservative property in some S. pneumoniae strains of this 20kD polypeptide
From various S. pneumoniae strains (the 1st type, the 3rd type, LOO2 and LOO3 (the 3rd type), the 4th type, the clinical separation strain of the 14th type and CP1200, WU2, R6X, 6303,109,110, JY1119, JY182, and the laboratory strain isolated of JY53) obtain DNA, and whether SEQ ID NO:3 is present among the DNA from these bacterial strains with the nucleic acid that detects this 20kD polypeptide of coding as probe.Separate that chromosomal DNA cuts with EcoRI and via electrophoretic separation.DNA is transferred on the solid support, and with the SEQ ID NO:3 of end-mark hybridization, hybridization and cleaning condition are for using 6X SSC, 5X Denhardt ' s, the salmon sperm DNA of 0.5%SDS and 100 mcg/ml fragmentations and sex change spends the night 65 ℃ of hybridization, and uses 2X SSC, 0.1%SDS cleaned once under room temperature about 10 minutes, then,, under room temperature, clean 3 minutes twice among the 0.1%SDS then at 0.2X SSC in once about 15 minutes of 65 ℃ of cleanings.
The result shows that SEQ ID NO:3 is tried that at each same hybridization is arranged in the DNA sample, and this shows that this polypeptide obviously has conservative property between each bacterial strain.In some bacterial strains, the DNA of C3-degradation property polypeptide of coding 20kDa obviously is the some of a bigger 2478bp open reading frame, the hypothetical coding of this open reading frame 92kDa polypeptide.
The Southern trace of pneumococcal dna probe
Obtain 5 microgram samples of genomic dna from 11 S. pneumoniae strains.Each sample cuts with restriction enzyme KpnI.These samples are loaded on the sepharose and via electrophoresis subsequently and split.Be included in sample in the gel glue and be passed to via capillary subsequently that (Upsafla is on Hybond-N+ film Sweden) available from Amersham.The SmaI fragment that derives from the 540bp of open reading frame is used
T7The QuickPrime test kit (Pharmacia, Piscathaway, NJ) with
32P carries out random primer labelling, and with NucTrap post (Stragene, La Jolla, CA) purifying from the non-Nucleotide that mixes, and hybridizing.
Hybridization conditions is with 6X SSC, 5X Denhardt, 0.5%SDS, and 100 mcg/ml fragmentations and sex change salmon sperm DNA are hybridized at 65 ℃ and are spent the night, and at 2X SSC, 0.1% SDS cleaned once under room temperature about 10 minutes, then in once about 15 minutes of 65 ℃ of cleanings, then, under room temperature, cleaned at least once 3-5 minute at least among 0.1% SDS at 0.2X SSC.Trace shows that this 20kDa gene line is present in the strain subject of all streptococcus pneumoniaes.
Make two dna primers from SEQ ID NO:1, and be used for the nucleotide sequence of amplification coding 20kDa polypeptide from streptococcus pneumoniae (serotype 3) genosome DNA.First primer is 5 '-primer (SEQ ID NO:6), it comprises the ATG initiator codon of this nucleotide sequence 5 ' end, has wherein inserted a NcoI site, and has the Ala residue to read frame to keep correct translating after the ATG initiator codon.Second primer be 3 '-primer (SEQ ID NO:7), it comprises the terminator codon of 3 of this nucleotide sequence ' end, has wherein inserted a BamHI site.
5′-GGGGG?CCA?TGG?CC?TCA?AGC?CTT?TTA?CGT?GAA?TTG-3′；(SEQ?ID?NO：6)
5′-GGGGG?GGA?TCC?CTA?GCT?ATA?TGA?GAT?AAA?CTT?TCC?TGC?T-3′；(SEQ?ID?NO：7)
These two primers are that (Foster City uses that (Sterling, reagent VA) is synthetic to be obtained available from Glen Research in CA) at Applied Biosystems 380A dna synthesizer.Amplification is to carry out according to manufacturer specification with Perkin ElmerThermocycler (ABI).It is that (available from Invitrogen, Carlsbad CA), and is used to transform OneShot Top10F ' competent cell (Invitrogen) for the PCR cloning vector PCR2.1 of tail end that PCR product through identifying is connected to TA.The Kanr transformant carries out restriction enzyme analysis via the plasmid DNA that alkaline dissolution method is made and is screened.Identifying one is about the insertion fragment of 500bp and uses restriction enzyme NcoI and BamHI cutting subsequently.This 500bp fragment is isolated from low melting point agarose gel, and be connected to (Madison, the NcoI-BamHI position of T7 activity expression vector pET28a WI) subsequently available from Novagen.
This connection mixture subsequent transformation is gone into Top10F ' cell (Invitrogen), and the Kanr transformant is to carry out restriction enzyme analysis by the plasmid DNA that alkaline dissolution method is made to filter out.Regroup plasmid (pLP505) is with after transform and to send into BL21 (Novagen) cell and it is grown in the SOB substratum that adds 30 mcg/ml kantlex.Cell grows to O.D.
600Value 0.6, and (Boehringer Mannheim, Indianapolis Indiana) induce 2-4 hour to use 0.4mM IPTG subsequently.Prepare full cytolysis thing and go up electrophoresis in 14%SDS-PAGE.To gel-colored, and detect expression product with Coomassie blue.The gel of this Coomassie blue stain demonstrates the band between 28kDa and 18kDa molecular weight marker, and is about 20kDa after measured.
The dna sequence dna of inset is measured with ABI 370A dna sequencing instrument in this regroup pLP505 plasmid.The dna sequence dna of this dna sequence dna and SEQ ID NO:1 is painted feature with the MacVector DNA matrix dot of Pustell, and (Oxford Molecular Group, Campbell CA) compares.To derive from the pLP505 plasmid, the genomic dna sequence dna of SEQ ID NO:1 and streptococcus pneumoniae (serotype 4) is compared, the open reading frame (ORF) of this 20kDa polypeptide of code displaying may be the part of big ORF, and promptly coding has the 2478bp (SEQ ID NO:4) of the polypeptide of about 92kDa estimated molecular weight in the genome of serotype 4.The expectation aminoacid sequence of DNA SEQ ID NO:4 coding shown in SEQ ID NO:5.
Streptococcus pneumoniae (serotype 4) genome sequence system uses ClustalW feature (the OxfordMolecular Group of MacVector, Campbell CA) is the Institute for Genomic Research of www.tigr.org and/or is that the NCBI of www.ncbi.nlm.nih.gov obtains via network address from network address.At the aminoacid sequence (SEQ ID NO:2) of this 20kDa and be predicted as between the aminoacid sequence (SEQ ID NO:5) of 92kDa and carry out sequence relatively.
According to the genomic dna that can get (serotype 4) sequence, design also uses ABI 380A dna synthesizer to synthesize (SEQ ID NOS:8 and 9) at the primer of the ORF both sides of 2478bp subsequently.SEQ ID NO:8 is streptococcus pneumoniae 5 '-primer, and it has the NcoI site of insertion and adds the Glu residue to keep a correct open reading frame after the ATG initiator codon.SEQ ID NO:9 is streptococcus pneumoniae 3 '-primer, and it has the HindIII site of insertion.
5′-CCC?GGG?CCA?TGG?CTA?AAA?TTA?ATA?AAA?AAT?ATC?TAG-3’；(SEQ?ID?NO：8)
5′-CCG?GGC?AAG?CTT?TTA?CTT?ACT?CTC?CT-3′；(SEQ?ID?NO：9)
About 2400bp dna fragmentation from 4 different streptococcus pneumoniae serotypes ( serotype 3,5,6B and 7) amplification, obtains 4 fragments subsequently.4 fragments are connected to PCR cloning vector PCR2.1 (Invitrogen) subsequently separately, and are used to transform OneShot Top10F ' cell (Invitrogen).The Kanr transformant carries out restriction enzyme analysis via the plasmid DNA that alkaline dissolution method is made and is screened.The regroup plasmid that contains the PCR product of serotype 7 is identified out, for example is pLP512.Use ABI model 370A DNA sequencing instrument to obtain the clone's of serotype 7 dna sequence dna.This dna sequence dna is same as SEQ ID NO:4 and identical with the SEQ IDNO:5 basically predicted amino acid sequence of coding basically.
The Western trace of 92kDa polypeptide detects in the full pneumonia streptococcus mycetocyte
Be purified into the about 20kDa C3 of regroup degradation property polypeptide at the large intestine bacterial strain BLR that contains plasmid pDF122.Plasmid pDF122 contains the polynucleotide that are shown in SEQ ID NO:1, and its expression is subjected to the T7 phage to activate the control of son.Bacterial cell grows to mid-log phase in Hy-Soy/yeast Extract substratum, this substratum contains penbritin in order to select this plasmid.This regroup polypeptide expression is by adding IPTG to 1mM concentration and continuing to cultivate and induced in extra 3 hours.Bacterial cell via centrifugal collect and resuspending cushion salt solution in Tris, among the pH7.2.With cell mechanically dissolving in French Pressure container, and the insoluble substance that contains inclusion body precipitates in centrifugation step.Throw out is dissolved in the NaPO with 100mM
4, the pH8.0 damping fluid also contains in the 3M urea of 0.1%Triton X-100.Through precipitation and discard behind the insoluble substance (～100,000xg is centrifugal), this solubility r20kDa polypeptide is replaced to 0.1% pair of property sanitising agent (Zwittergent) 3-12 (Calbiochem-Behrng) with substituted ureas, and is replaced to 100mM NaPO subsequently
4, pH8.0 is to replace sanitising agent.The SDS-PAGE analysis confirmation should～the 20kDa material still is soluble.The recombinant polypeptide of this His-tailing is at 50mM NaPO
4Damping fluid is dialysed among the pH8.0, and is adsorbed in on the same damping fluid equilibrated Ni post.With this post in regular turn with 50mM NaPO
4At pH7.0,6.6 and 5.5 are cleaned, and all reach baseline absorbance (OD up to each damping fluid
280) till.Combined～r20kDa polypeptide is with 50mM NaPO
4, the pH4.5 elution.Analyze the polypeptide that is gone out by elution as can be known by SDS-PAGE and be about 90% uniformity.
This polypeptide is used to inoculate the Swiss-Webster mouse.The polypeptide of five micrograms dose mixes with single phosphoryl fat A (Ribi Immunochemicals) of 50 micrograms, and intramuscularly is to mouse.Animal was inoculating and is taking a blood sample in the 8th week in the 0th, 4 and 6 weeks.Serum merged and find that it contains this immunogenic height of antagonism antiserum(antisera) of tiring.
Streptococcus pneumoniae strain CP1200, T3 and T7 system grows in the Todd-Hewitt nutrient solution that contains the yeast extract, and cell is through centrifugal and precipitate.The pneumonia streptococcus mycetocyte is dissolved through boiling 5 minutes under reductive condition in the SDS-PAGE lysis buffer.Solute added carry out electrophoresis in the 10%SDS-PAGE gel.Isolated polypeptide through electroblotting on nitrocotton, and with this filter membrane with at the phosphate buffered salt solution, the 5%BLOTTO among the pH7.4 blocks.With polyclonal antiserum in bovine lacto transfer technique optimizer with dilution in 1: 2000 and be used to detect isolated polypeptide.Bonded antibody is to detect with alkaline phosphatase link coupled mountain sheep anti-mouse igg.The Western trace the results are shown in Fig. 8 and and then is described in this figure summary.
The evidence of 20kDa and 92kDa polypeptide degraded C3
The gel that is shown in Fig. 9 is Western trace result.This data system is via 20kDa polypeptide and the 92kDa polypeptide C3 with the biotinylation shown in the following table and methylamine-processing is cultivated and gets.These expression of polypeptides are from the T7 carrier.
Methylamine-processing C3: the people C3 of 500 microlitre purifying of concentration 4.46 mg/ml (according to people such as Tack, the method for Meth.Enzymol.80:64 101,1984 is prepared) and 55 microlitres are at 0.1M TRIS/0.01M EDTA, the 1M methylamine (CH among the pH8.0
3NH
2) 37 ℃ of common cultivations 80 minutes, at 0.1M TRIS/0.01M EDTA, carry out dialysed overnight at 4 ℃ among the pH8.0 subsequently.Next day, the 1M methylamine of C3 and 60 microlitres in 0.1 TRIS/0.01M EDTA was cultivated 80 minutes at 37 ℃, subsequently at 50mM NaHCO
3In 0 ℃ of dialysed overnight.
The biotinylation of the C3 of methylamine-processing: (it is dissolved in 1 milliliter of H with 1 milligram of NSC vitamin H with the C3 of 2 milligrams of as above prepared methylamine-processing and the NSC-vitamin H (Pierce) of 75 microlitres
2The O mode prepares) cultivated 30 minutes in room temperature.Biotinylated C3 is at 50mM NaHCO
3, carry out dialysed overnight in 4 ℃ among the pH8.0.
| Reagent | Test tube A-20kDa | Test tube C-92kDa |
| Polypeptide | 34 microlitre 20kDa polypeptide 5.6 * 10
-9 | 100 microlitre 92kDa polypeptide 5.6 * 10
-9 |
| Biotinylated C3 | ||
| 3 microlitre C3 71 * 10
-12 | 3 microlitre C3 71 * 10 -12Mole | |
| Damping fluid | 63 microlitres, 0.01% BSA/PBS | 63 microlitre 0.01%BSA/PBS |
Cultivate from the sample of test tube A and C and to shift out after 66 hours, in the reductibility damping fluid, carried out reduction reaction in 2 minutes via boiling with beta-mercaptoethanol, electrophoresis on 7.5%SDS-PAGE, and be transferred to and carry out the Western trace on the nitrocellulose filter.The Western blotting was carried out 1 hour according to standard program (people PNAS USA76:4350 4354,1979 such as Tobin).With filter paper and common cultivation of horseradish-peroxidase-avidin (1: 10,000 dilution), and develop to come the C3 fragment of detection of biological elementization with Supersignal CL-HRP Substrate System (Amersham).
In Fig. 9, the 1st swimming lane only has C3 (control group), and the 2nd and 3 swimming lanes are 20 and the 92kDa polypeptide, and the 4th and 5 swimming lanes only have C3 (control group).Right margin is presented at C3 α chain, C3 β chain and the segmental position of C3 in the control sample.To confirm in a C3 fragment of 20kDa cutting and in several segmental positions of 92kDa cutting.
Second first experiment reagent (through polypeptide expressed, biotinylated C3, and damping fluid) that experiment is used as above to show described same ratio.The C3 control sample 8 hours, shifted out when reaching 24 hours at 0,2 hour.Yet these samples do not carry out the Western trace, but at 7.5%SDS-PAGE reduction reaction take place, and illustrate that some extra degradation fragments may not be biotinylated, and therefore may be shown on the SDS-PAGE gel of Coomassie blue stain.In fact, real viewed situation that Here it is.Only there are 20 (A8 swimming lanes) and 92 (C8 swimming lane) kDa polypeptide on this gel, to analyze.The gel upper edge right margin of Figure 10 shows the position of C3 α chain and C3 β chain, and shows the position of 92kDa polypeptide at the C8 swimming lane.Have two fragments at least at the C8 swimming lane, its size is from about 90kDa to 75kDa.Below the C3 of 75kDa β chain, several fragments are also arranged.It in the A8 swimming lane the main degradation fragment that just in time is lower than C3 β chain.So, at the 20kDa polypeptide of the 92kDa of C8 swimming lane polypeptide and the A8 swimming lane C3 that all degrades.
The preparation of 92kDa recombinant protein and the generation of polyclonal antiserum
Inset in plasmid pLP512 (seeing embodiment 5) is sheared with NcoI and HindIII.As expect fragment purifying in low melting point agarose of size, and (Novagen, Madison is in NcoI-HindIII position W1) to be connected to the expression vector pET28a that T7 drives subsequently.
To connect mixture subsequently and be transformed into Top 10F ' cell (Invitrogen), and as described in previous embodiment 5, filter out kantlex (kanamycin) resistance transformant.Subsequently recombinant plasmid (pLP515) is transformed into BL21 cell (Novagen), and in the SOB substratum that adds 30 mcg/ml kantlex, cultivates.Make cell grow to O.D.
600Value reaches 0.6, and (Boehringer Marinheim, Indianapolis IN) induce 2-4 hour with 0.4mM IPTG subsequently.Prepare full cytolysis thing and on the 10%SDS-PAGE gel, carry out electrophoresis.Gel is after Coomassie blue stain, with the new colour band that relatively demonstrates about 80kDa without the sample that brings out.
The recombinant protein that about 92kDa ORF is coded separates in the e. coli strains BL21 (Novagen) that contains plasmid pLPS 15.Make bacterial cell in the SOB substratum that contains 30 mcg/ml kantlex, grow to mid-log phase, to select this plasmid.The expression of recombinant polypeptide is through adding IPTG to 0.4mM and continuing to cultivate and induced in 3 hours.Bacterial cell is cushioned salt solution via centrifugal collection and resuspending in Tris, among the pH7.2.Cell is carried out mechanical type dissolving in French Pressure Cell, the insoluble substance that contains inclusion body 7700xg in 4 ℃ centrifugal 10 minutes and precipitate.The throw out that will contain inclusion body dissolves again, and this soluble protein via ion exchange chromatography purifying.Recombinant protein is used to produce polyclonal antibody in mouse.In brief, 5 micrograms of protein are adjuvant subcutaneous injection 6-8 Swiss Webster mouse neck in age in week with 20 microgram QS21 for every dose.When the 0th week,, carry out preventive vaccination, take a blood sample and bloodletting in the 6th week subsequently in the 4th week with mouse blood sampling and preventive vaccination.10 mouse carry out preventive vaccination with the regroup 92kDa protein of helping with QS21.The serum that merges is through dilution in 1: 1000, is used for the Western blotting to check full cytolysis thing and from the concentrated culture supernatant of several serotype streptococcus pneumoniaes.These serum are with full cytolysis thing and concentrate protein generation specificity reaction in the culture supernatant, and this protein molecular weight is about 90kDa.
Embodiment 9
Chart among Figure 11 has been summed up from the CBA/CAHNxid/J mouse with r92kDa protein protective inoculation is carried out the result that (IN) attacks in the nose, and r92kDa protein is helped with single phosphoryl fat A (MPL) as making as described in the embodiment 8 (SEQ ID NO:5).
10 mouse become one group in the 0th, 3 and 5 weeks at neck area with the 92kDa of per 5 micrograms assistant with 50 microgram MPL, per 0.1 microgram assistant is with the 3rd type pod membrane (it is connected with protein carrier CRM197) of 100 microgram aluminum phosphates, or phosphate buffered salt solution (PBS) only arranged, carry out subcutaneous preventive vaccination with the sample volume of 10 microlitres.CRM 197 is a genetically engineered toxicide diphtheria toxin.Every mouse the 7th week to include 1 * 10
6The 10 microlitre samples of cfu the 3rd type streptococcus pneumoniae are attacked.Attack and isolated nose inner tissue in back three days, and via planting on selective medium and determine the 3rd type streptococcus pneumoniae colony forming single-digit order in every gram nose tissue.The 3rd type pod membrane system separates from streptococcus pneumoniae the 3rd serotype, and (preparation about the 3rd type pod membrane control group can be referring to U.S. Patent No. 5 to be connected to protein carrier CRM 197 via reductibility activation (reductive animation) subsequently, 360,897; Can be about genetically engineered toxicide diphtheria toxin referring to U.S. Patent No. 5,614,382; About using the CRM 197 can be referring to United States Patent (USP) 4,902,506) as carrier.
Pictorialization among Figure 11 goes out; in this pattern; negative control group (mouse is injected with MPL) and be untreated mouse when 6 weeks are big; when attacking in the nose, all have about 30% survival rate, but positive controls (the 3rd type pod membrane connector help with aluminum phosphate) so far study termination (about 14 days) 100% protection that avoids death then is provided.When with the premunitive mouse of r92kDa under fire the time, research so far stops 100% survival.This result shows that r92kDa protein can provide protection to attacking the death that causes in the 3rd type streptococcus pneumoniae nose really.
Method
92kDa (r92kDa) polypeptide (SEQ ID NO:5) of reorganization was cultivated 2 hours at 37 ℃ with purified human C3, and 6 hours, and 26 hours, ratio is as follows:
Test tube A (control group): C3-3 microlitre [7.2 * 10
-12Mole] PBS/0.01% bovine serum albumin (BSA)-100 microlitre; Test tube B:r92kDa-50 microlitre [5.6 * 10
-10Mole] C3-3 microlitre [7.2 * 10
-12Mole] the PBS/0.01%BSA-50 microlitre.
In each time point (2,6, reach 26 hours), 20 microlitre samples are shifted out from test tube A and B, and add the reductibility damping fluid, and this sample was boiled 2 minutes in 100 ℃.After boiling sample is upward carried out electrophoresis in 7.5%SDS-PAGE under reductive condition.With this SDS-PAGE gel with Coomassie blue stain.The gel of gained is shown in Figure 12.
The 1st swimming lane-molecular weight standard (as follows by gel top reading: 200kDa, 116kDa, 97kDa, 66kDa, 45kDa); Hour cultivation of the 2nd and 4 swimming lanes-2; The 2nd swimming lane-C3 control group (test tube A); The 4th swimming lane-r92kDa sample (test tube 13); Hour cultivation of the 5th and 7 swimming lanes-6; The 5th swimming lane-C3 control group (test tube A); The 7th swimming lane-r92kDa sample (test tube 13); Hour cultivation of the 8th and 10 swimming lanes-26; The 8th swimming lane-C3 control group (test tube A); The 10th swimming lane-r92kDa sample (test tube B).
The deciphering of gel
The α chain electrophoresis showed of C3 is about 115kDa.The β chain electrophoresis showed of C3 is about 75kDa.These form mark on gel.Protein band at about 66kDa is an albumin.The 2nd and 4 swimming lanes are cultivation in 2 hours.Compare the 4th swimming lane demonstration C3 α chain generation cutting phenomenon-new protein band occurs at about 97kDa with the 2nd swimming lane (C3 control group).The 5th and 7 swimming lanes are cultivation in 6 hours.New 97kDa cutting fragment also appears at the 7th swimming lane.The 8th and 10 swimming lanes are cultivations in 26 hours.Compare with the 8th swimming lane (C3 control group), the 10th swimming lane shows that C3 α chain continues cutting.
Those skilled in the art will understand that, though the present invention is in above being described according to particular and embodiment, the present invention is not so limited certainly, and can carry out many other embodiments, embodiment, purposes is improved and in these embodiments, situation beyond embodiment and the purposes, and can not depart from the invention scope of the application's case.
Sequence table＜110〉HOSTETTER, Margaret K.
FINKEL，David?J.
CHENG，Qi
MASI，Amy?W.
REGENTS?OF?THE?UNIVERSITY?OF?MINNESOTA
AMERICAN CYNAMID COMPANY＜120〉from the human complement c, 3 degradability polypeptide of streptococcus pneumonia＜130〉11000570230＜140〉transfer the possession of＜141〉1999-09-24＜150〉60/101; 736＜151〉1998-09-24＜150〉09/283,094＜151〉1999-03-31＜160〉10＜170〉PatentIn Ver.2.0＜210〉1＜211〉504＜212〉DNA＜213〉＜400〉1atgtcaagcc ttttacgtga attgtatgct aaacccttat cagaacgcca tgtagaatct 60gatggcctta ttttcgaccc agcgcaaatc acaagtcgaa ccgccaatgg tgttgctgta 120ccgcacggag accattatca ctttattcct tattcacaac tgtcaccttt ggaagaaaaa 180ttggctcgta ttattcccct tcgttatcgt tcaaaccatt gggtaccaga ttcaagacca 240gaacaaccaa gtccacaatc gactccggaa cctagtccaa gtccgcaacc tgcaccaaat 300cctcaaccag ctccaagcaa tccaattgat gagaaattgg tcaaagaagc tgttcgaaaa 360gtaggcgatg gttatgtctt tgaggagaat ggagttcctc gttatatccc agccaaggat 420ctttcagcag aaacagcagc aggcattgat agcaaactgg ccaagcagga aagtttatct 480cataagctgc agttagatcc atta 504＜210〉2＜211〉168＜212〉＜213〉＜400〉2Met Ser Ser Leu Leu Arg Glu Leu Tyr Ala Lys Pro Leu Ser Glu Arg 1 5 10 15His Val Glu Ser Asp Gly Leu Ile Phe Asp Pro Ala Gln Ile Thr Ser
20??????????????????25??????????????????30Arg?Thr?Ala?Asn?Gly?Val?Ala?Val?Pro?His?Gly?Asp?His?Tyr?His?Phe
35??????????????????40??????????????????45Ile?Pro?Tyr?Ser?Gln?Leu?Ser?Pro?Leu?Glu?Glu?Lys?Leu?Ala?Arg?Ile
50??????????????????55??????????????????60Ile?Pro?Leu?Arg?Tyr?Arg?Ser?Asn?His?Trp?Val?Pro?Asp?Ser?Arg?Pro?65??????????????????70??????????????????75??????????????????80Glu?Gln?Pro?Ser?Pro?Gln?Ser?Thr?Pro?Glu?Pro?Ser?Pro?Ser?Pro?Gln
85??????????????????90??????????????????95Pro?Ala?Pro?Asn?Pro?Gln?Pro?Ala?Pro?Ser?Asn?Pro?Ile?Asp?Glu?Lys
100?????????????????105?????????????????110Leu?Val?Lys?Glu?Ala?Val?Arg?Lys?Val?Gly?Asp?Gly?Tyr?Val?Phe?Glu
115?????????????????120?????????????????125Glu?Asn?Gly?Val?Pro?Arg?Tyr?Ile?Pro?Ala?Lys?Asp?Leu?Ser?Ala?Glu
130?????????????????135?????????????????140Thr?Ala?Ala?Gly?Ile?Asp?Ser?Lys?Leu?Ala?Lys?Gln?Glu?Ser?Leu?Ser145?????????????????150?????????????????155?????????????????160His?Lys?Leu?Gln?Leu?Asp?Pro?Leu
165＜210〉3＜211〉504＜212〉DNA＜213〉＜400〉3taatggatct aactgcagct tatgagataa actttcctgc ttggccagtt tgctatcaat 60gcctgctgct gtttctgctg aaagatcctt ggctgggata taacgaggaa ctccattctc 120ctcaaagaca taaccatcgc ctacttttcg aacagcttct ttgaccaatt tctcatcaat 180tggattgctt ggagctggtt gaggatttgg tgcaggttgc ggacttggac taggttccgg 240agtcgattgt ggacttggtt gttctggtct tgaatctggt acccaatggt ttgaacgata 300acgaagggga ataatacgag ccaatttttc ttccaaaggt gacagttgtg aataaggaat 360aaagtgataa tggtctccgt gcggtacagc aacaccattg gcggttcgac ttgtgatttg 420cgctgggtcg aaaataaggc catcagattc tacatggcgt tctgataagg gtttagcata 480caattcacgt aaaaggcttg acat 504＜210〉4＜211〉2478＜212〉DNA＜213〉＜400〉4atgaaaatta ataaaaaata tctagcaggt tcagtggcag tccttgccct aagtgtttgt 60tcctatgaac ttggtcgtca ccaagctggt caggttaaga aagagtctaa tcgagtttct 120tatatagatg gtgatcaggc tggtcaaaag gcagaaaact tgacaccaga tgaagtcagt 180aagagggagg ggatcaacgc cgaacaaatc gtcatcaaga ttacggatca aggttatgtg 240acctctcatg gagaccatta tcattactat aatggcaagg tcccttatga tgccatcatc 300agtgaagagc tcctcatgaa agatccgaat tatcagttga aggattcaga cattgtcaat 360gaaatcaagg gtggttatgt tatcaaggta gatggaaaat actatgttta ccttaaggat 420gcagctcatg cggataatat tcggacaaaa gaagagatta aacgtcagaa gcaggaacac 480agtcataatc acgggggtgg ttctaacgat caagcagtag ttgcagccag agcccaagga 540cgctatacaa cggatgatgg ttatatcttc aatgcatctg atatcattga ggacacgggt 600gatgcttata tcgttcctca cggcgaccat taccattaca ttcctaagaa tgagttatca 660gctagcgagt tagctgctgc agaagcctat tggaatggga agcagggatc tcgtccttct 720tcaagttcta gttataatgc aaatccagct caaccaagat tgtcagagaa ccacaatctg 780actgtcactc caacttatca tcaaaatcaa ggggaaaaca tttcaagcct tttacgtgaa 840ttgtatgcta aacccttatc agaacgccat gtggaatctg atggccttat tttcgaccca 900gcgcaaatca caagtcgaac cgccagaggt gtagctgtcc ctcatggtaa ccattaccac 960tttatccctt atgaacaaat gtctgaattg gaaaaacgaa ttgctcgtat tattcccctt 1020cgttatcgtt caaaccattg ggtaccagat tcaagaccag aacaaccaag tccacaatcg 1080actccggaac ctagtccaag tccgcaacct gcaccaaatc ctcaaccagc tccaagcaat 1140ccaattgatg agaaattggt caaagaagct gttcgaaaag taggcgatgg ttatgtcttt 1200gaggagaatg gagtttctcg ttatatccca gccaaggatc tttcagcaga aacagcagca 1260ggcattgata gcaaactggc caagcaggaa agtttatctc ataagctagg agctaagaaa 1320actgacctcc catctagtga tcgagaattt tacaataagg cttatgactt actagcaaga 1380attcaccaag atttacttga taataaaggt cgacaagttg attttgaggc tttggataac 1440ctgttggaac gactcaagga tgtcccaagt gataaagtca agttagtgga tgatattctt 1500gccttcttag ctccgattcg tcatccagaa cgtttaggaa aaccaaatgc gcaaattacc 1560tacactgatg atgagattca agtagccaag ttggcaggca agtacacaac agaagacggt 1620tatatctttg atcctcgtga tataaccagt gatgaggggg atgcctatgt aactccacat 1680atgacccata gccactggat taaaaaagat agtttgtctg aagctgagag agcggcagcc 1740caggcttatg ctaaagagaa aggtttgacc cctccttcga cagaccatca ggattcagga 1800aatactgagg caaaaggagc agaagctatc tacaaccgcg tgaaagcagc taagaaggtg 1860ccacttgatc gtatgcctta caatcttcaa tatactgtag aagtcaaaaa cggtagttta 1920atcatacctc attatgacca ttaccataac atcaaatttg agtggtttga cgaaggcctt 1980tatgaggcac ctaaggggta tactcttgag gatcttttgg cgactgtcaa gtactatgtc 2040gaacatccaa acgaacgtcc gcattcagat aatggttttg gtaacgctag cgaccatgtt 2100caaagaaaca aaaatggtca agctgatacc aatcaaacgg aaaaaccaag cgaggagaaa 2160cctcagacag aaaaacctga ggaagaaacc cctcgagaag agaaaccgca aagcgagaaa 2220ccagagtctc caaaaccaac agaggaacca gaagaatcac cagaggaatc agaagaacct 2280caggtcgaga ctgaaaaggt tgaagaaaaa ctgagagagg ctgaagattt acttggaaaa 2340atccaggatc caattatcaa gtccaatgcc aaagagactc tcacaggatt aaaaaataat 2400ttactatttg gcacccagga caacaatact attatggcag aagctgaaaa actattggct 2460ttattaaagg agagtaag 2478＜210〉5＜211〉826＜212〉＜213〉＜400〉5Met Lys Ile Asn Lys Lys Tyr Leu Ala Gly Ser Val Ala Val Leu Ala 1 5 10 15Leu Ser Val Cys Ser Tyr Glu Leu Gly Arg His Gln Ala Gly Gln Val
20??????????????????25??????????????????30Lys?Lys?Glu?Ser?Asn?Arg?Val?Ser?Tyr?Ile?Asp?Gly?Asp?Gln?Ala?Gly
35??????????????????40??????????????????45Gln?Lys?Ala?Glu?Asn?Leu?Thr?Pro?Asp?Glu?Val?Ser?Lys?Arg?Glu?Gly
50??????????????????55??????????????????60Ile?Asn?Ala?Glu?Gln?Ile?Val?Ile?Lys?Ile?Thr?Asp?Gln?Gly?Tyr?Val?65??????????????????70??????????????????75??????????????????80Thr?Ser?His?Gly?Asp?His?Tyr?His?Tyr?Tyr?Asn?Gly?Lys?Val?Pro?Tyr
85??????????????????90??????????????????95Asp?Ala?Ile?Ile?Ser?Glu?Glu?Leu?Leu?Met?Lys?Asp?Pro?Asn?Tyr?Gln
100?????????????????105?????????????????110Leu?Lys?Asp?Ser?Asp?Ile?Val?Asn?Glu?Ile?Lys?Gly?Gly?Tyr?Val?Ile
115?????????????????120?????????????????125Lys?Val?Asp?Gly?Lys?Tyr?Tyr?Val?Tyr?Leu?Lys?Asp?Ala?Ala?His?Ala
130?????????????????135?????????????????140Asp?Asn?Ile?Arg?Thr?Lys?Glu?Glu?Ile?Lys?Arg?Gln?Lys?Gln?Glu?His145?????????????????150?????????????????155?????????????????160Ser?His?Asn?His?Gly?Gly?Gly?Ser?Asn?Asp?Gln?Ala?Val?Val?Ala?Ala
165?????????????????170?????????????????175Arg?Ala?Gln?Gly?Arg?Tyr?Thr?Thr?Asp?Asp?Gly?Tyr?Ile?Phe?Asn?Ala
180?????????????????185?????????????????190Ser?Asp?Ile?Ile?Glu?Asp?Thr?Gly?Asp?Ala?Tyr?Ile?Val?Pro?His?Gly
195?????????????????200?????????????????205Asp?His?Tyr?His?Tyr?Ile?Pro?Lys?Asn?Glu?Leu?Ser?Ala?Ser?Glu?Leu
210?????????????????215?????????????????220Ala?Ala?Ala?Glu?Ala?Tyr?Trp?Asn?Gly?Lys?Gln?Gly?Ser?Arg?Pro?Ser225?????????????????230?????????????????235?????????????????240Ser?Ser?Ser?Ser?Tyr?Asn?Ala?Asn?Pro?Ala?Gln?Pro?Arg?Leu?Ser?Glu
245?????????????????250?????????????????255Asn?His?Asn?Leu?Thr?Val?Thr?Pro?Thr?Tyr?His?Gln?Asn?Gln?Gly?Glu
260?????????????????265?????????????????270Asn?Ile?Ser?Ser?Leu?Leu?Arg?Glu?Leu?Tyr?Ala?Lys?Pro?Leu?Ser?Glu
275?????????????????280?????????????????285Arg?His?Val?Glu?Ser?Asp?Gly?Leu?Ile?Phe?Asp?Pro?Ala?Gln?Ile?Thr
290?????????????????295?????????????????300Ser?Arg?Thr?Ala?Arg?Gly?Val?Ala?Val?Pro?His?Gly?Asn?His?Tyr?His305?????????????????310?????????????????315?????????????????320Phe?Ile?Pro?Tyr?Glu?Gln?Met?Ser?Glu?Leu?Glu?Lys?Arg?Ile?Ala?Arg
325?????????????????330?????????????????335Ile?Ile?Pro?Leu?Arg?Tyr?Arg?Ser?Asn?His?Trp?Val?Pro?Asp?Ser?Arg
340?????????????????345?????????????????350Pro?Glu?Gln?Pro?Ser?Pro?Gln?Ser?Thr?Pro?Glu?Pro?Ser?Pro?Ser?Pro
355?????????????????360?????????????????365Gln?Pro?Ala?Pro?Asn?Pro?Gln?Pro?Ala?Pro?Ser?Asn?Pro?Ile?Asp?Glu
370?????????????????375?????????????????380Lys?Leu?Val?Lys?Glu?Ala?Val?Arg?Lys?Val?Gly?Asp?Gly?Tyr?Val?Phe385?????????????????390?????????????????395?????????????????400Glu?Glu?Asn?Gly?Val?Ser?Arg?Tyr?Ile?Pro?Ala?Lys?Asp?Leu?Ser?Ala
405?????????????????410?????????????????415Glu?Thr?Ala?Ala?Gly?Ile?Asp?Ser?Lys?Leu?Ala?Lys?Gln?Glu?Ser?Leu
420?????????????????425?????????????????430Ser?His?Lys?Leu?Gly?Ala?Lys?Lys?Thr?Asp?Leu?Pro?Ser?Ser?Asp?Arg
435?????????????????440?????????????????445Glu?Phe?Tyr?Asn?Lys?Ala?Tyr?Asp?Leu?Leu?Ala?Arg?Ile?His?Gln?Asp
450?????????????????455?????????????????460Leu?Leu?Asp?Asn?Lys?Gly?Arg?Gln?Val?Asp?Phe?Glu?Ala?Leu?Asp?Asn465?????????????????470?????????????????475?????????????????480Leu?Leu?Glu?Arg?Leu?Lys?Asp?Val?Pro?Ser?Asp?Lys?Val?Lys?Leu?Val
485?????????????????490?????????????????495Asp?Asp?Ile?Leu?Ala?Phe?Leu?Ala?Pro?Ile?Arg?His?Pro?Glu?Arg?Leu
500?????????????????505?????????????????510Gly?Lys?Pro?Asn?Ala?Gln?Ile?Thr?Tyr?Thr?Asp?Asp?Glu?Ile?Gln?Val
515?????????????????520?????????????????525Ala?Lys?Leu?Ala?Gly?Lys?Tyr?Thr?Thr?Glu?Asp?Gly?Tyr?Ile?Phe?Asp
530?????????????????535?????????????????540Pro?Arg?Asp?Ile?Thr?Ser?Asp?Glu?Gly?Asp?Ala?Tyr?Val?Thr?Pro?His545?????????????????550?????????????????555?????????????????560Met?Thr?His?Ser?His?Trp?Ile?Lys?Lys?Asp?Ser?Leu?Ser?Glu?Ala?Glu
565?????????????????570?????????????????575Arg?Ala?Ala?Ala?Gln?Ala?Tyr?Ala?Lys?Glu?Lys?Gly?Leu?Thr?Pro?Pro
580?????????????????585?????????????????590Ser?Thr?Asp?His?Gln?Asp?Ser?Gly?Asn?Thr?Glu?Ala?Lys?Gly?Ala?Glu
595?????????????????600?????????????????605Ala?Ile?Tyr?Asn?Arg?Val?Lys?Ala?Ala?Lys?Lys?Val?Pro?Leu?Asp?Arg
610?????????????????615?????????????????620Met?Pro?Tyr?Asn?Leu?Gln?Tyr?Thr?Val?Glu?Val?Lys?Asn?Gly?Ser?Leu625?????????????????630?????????????????635?????????????????640Ile?Ile?Pro?His?Tyr?Asp?His?Tyr?His?Asn?Ile?Lys?Phe?Glu?Trp?Phe
645?????????????????650?????????????????655Asp?Glu?Gly?Leu?Tyr?Glu?Ala?Pro?Lys?Gly?Tyr?Thr?Leu?Glu?Asp?Leu
660?????????????????665?????????????????670Leu?Ala?Thr?Val?Lys?Tyr?Tyr?Val?Glu?His?Pro?Asn?Glu?Arg?Pro?His
675?????????????????680?????????????????685Ser?Asp?Asn?Gly?Phe?Gly?Asn?Ala?Ser?Asp?His?Val?Gln?Arg?Asn?Lys
690?????????????????695?????????????????700Asn?Gly?Gln?Ala?Asp?Thr?Asn?Gln?Thr?Glu?Lys?Pro?Ser?Glu?Glu?Lys705?????????????????710?????????????????715?????????????????720Pro?Gln?Thr?Glu?Lys?Pro?Glu?Glu?Glu?Thr?Pro?Arg?Glu?Glu?Lys?Pro
725?????????????????730?????????????????735Gln?Ser?Glu?Lys?Pro?Glu?Ser?Pro?Lys?Pro?Thr?Glu?Glu?Pro?Glu?Glu
740?????????????????745?????????????????750Ser?Pro?Glu?Glu?Ser?Glu?Glu?Pro?Gln?Val?Glu?Thr?Glu?Lys?Val?Glu
755?????????????????760?????????????????765Glu?Lys?Leu?Arg?Glu?Ala?Glu?Asp?Leu?Leu?Gly?Lys?Ile?Gln?Asp?Pro
770?????????????????775?????????????????780Ile?Ile?Lys?Ser?Asn?Ala?Lys?Glu?Thr?Leu?Thr?Gly?Leu?Lys?Asn?Asn785?????????????????790?????????????????795?????????????????800Leu?Leu?Phe?Gly?Thr?Gln?Asp?Asn?Asn?Thr?Ile?Met?Ala?Glu?Ala?Glu
805?????????????????810?????????????????815Lys?Leu?Leu?Ala?Leu?Leu?Lys?Glu?Ser?Lys
820 825＜210〉6＜211〉34＜223〉oligonucleotide＜400〉10.gaaaacaata atgtagaaga ctactttaaa gaaggttaga 40
Claims (35)
1. isolated polypeptide, it has the monoamino-acid sequence, wherein encode the nucleotide sequence of this isolated polypeptide aminoacid sequence under highly rigorous hybridization conditions with at least one part hybridization of following sequence: (a) Nucleotide among the SEQ ID NO:1 or its complementary strand, or (b) Nucleotide or its complementary strand among the SEQ ID NO:4.
2. polypeptide according to claim 1, it separates from streptococcus pneumoniae.
3. polypeptide according to claim 1, it is a recombinant polypeptide.
4. polypeptide according to claim 1, its molecular weight are 15kDa to 25kDa.
5. polypeptide according to claim 1, its molecular weight are 75kDa to 95kDa.
6. polypeptide according to claim 1, its degradable people complement proteins C3.
7. polypeptide according to claim 1, it comprises at least 15 successive amino acid.
8. isolated polypeptide, it comprises at least 15 successive amino acid among SEQ ID NO:2 or the SEQ ID NO:5.
9. isolated polypeptide, it is SEQ ID NO:2 or SEQ ID NO:5.
10. immunity system irritating compositions, it comprise significant quantity the described polypeptide of claim 1 and and treatment go up acceptable carrier.
11. immunity system irritating compositions according to claim 10, wherein this polypeptide separates from streptococcus pneumoniae.
12. immunity system irritating compositions according to claim 11, it also comprises at least a separation other immunity system pungency polypeptide from streptococcus pneumoniae.
13. immunity system irritating compositions according to claim 10, wherein this polypeptide effectively immunization or the treatment mammalian subject with the antagonism streptococcus pneumoniae infection or move life.
14. immunity system irritating compositions according to claim 13, wherein this polypeptide is being that mammalian subject effectively provides the amount of result of treatment to be supplied with.
15. an antibody, it can combine with the described polypeptide of claim 1.
16. antibody according to claim 15, it is a monoclonal antibody.
17. antibody according to claim 16, it obtains from mouse, rat, goat, chicken, people or rabbit.
18. an isolated nucleic acid molecule, it under highly rigorous hybridization conditions can with at least one part hybridization of following sequence: (a) Nucleotide among the SEQ ID NO:1 or its complementary strand, or (b) Nucleotide or its complementary strand among the SEQ ID NO:4.
19. nucleic acid molecule according to claim 18, it separates from streptococcus pneumoniae.
20. nucleic acid molecule according to claim 19, the polypeptide of its coding degraded human complement c 3.
21. a carrier, it comprises the described nucleic acid molecule of claim 18.
22. a cell, it comprises the described nucleic acid of claim 18.
23. cell according to claim 22, wherein this cell is bacterium or eukaryotic cell.
24. an isolated nucleic acid molecule, it comprises the nucleotide sequence of SEQ ID NO:1 or its complementary strand or SEQ ID NO:4 or its complementary strand.
25. a RNA molecule, it is to be transcribed by the double chain DNA sequence that comprises SEQ ID NO:1 or SEQ ID NO:4 to obtain.
Give Mammals a kind of composition 26. the immunoreactive method of the streptococcus pneumoniae that creates antagonism in Mammals, this method comprise, said composition comprises:
(a) the described polypeptide of the claim 1 of significant quantity in the treatment; And
(b) pharmaceutically acceptable carrier.
27. method according to claim 26, wherein immune response is B cell response, t cell responses, epithelial cell reaction or endotheliocyte reaction.
28. method according to claim 26, wherein the length of this polypeptide is at least 15 amino acid.
29. method according to claim 26, wherein said composition also comprises at least a other immunity system pungency polypeptide from streptococcus pneumoniae.
Make the streptococcus pneumoniae bacterium and can contact 30. a method that suppresses the C3 Degradation of streptococcus pneumoniae-mediation, this method comprise in conjunction with the antibody of the described polypeptide of claim 1.
31. one kind is suppressed the inflammatory response of the mediation of C3-in the xenotransplantation and the method for repulsive interaction, expresses the described polypeptide of claim 1 on the used animal organ surface when this method is included in xenotransplantation.
32. an isolating nucleic acid fragment, it has the SEQ of being selected from ID NO:6, SEQ ID NO:7, the nucleotide sequence of SEQ IDNO:8 and SEQ ID NO:9.
33. an isolated polypeptide, it and SEQ IDNO:2 or SEQ IDNO:5 have at least 80% sequence homogeny.
34. the isolated polypeptide from the 15kDa to 25kDa of streptococcus pneumoniae, its people's complement proteins C3 that can degrade.
35. the isolated polypeptide from the 75kDa to 95kDa of streptococcus pneumoniae, its people's complement proteins C3 that can degrade.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US10173698P | 1998-09-24 | 1998-09-24 | |
| US28309499A | 1999-03-31 | 1999-03-31 | |
| PCT/US1999/022362 WO2000017370A1 (en) | 1998-09-24 | 1999-09-24 | HUMAN COMPLEMENT C3-DEGRADING POLYPEPTIDE FROM $i(STREPTOCOCCUS PNEUMONIAE) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1352691A true CN1352691A (en) | 2002-06-05 |
Family
ID=26798575
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN99813016A Pending CN1352691A (en) | 1998-09-24 | 1999-09-24 | Human complement C3-degrading polypeptide from i(streptococcus pneumoniae) |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP1115875A1 (en) |
| JP (1) | JP2002526082A (en) |
| KR (1) | KR20010089280A (en) |
| CN (1) | CN1352691A (en) |
| AU (1) | AU6060899A (en) |
| BR (1) | BR9914066A (en) |
| CA (1) | CA2343931A1 (en) |
| IL (1) | IL142017A0 (en) |
| MX (1) | MXPA01003073A (en) |
| WO (1) | WO2000017370A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102258784A (en) * | 2003-05-15 | 2011-11-30 | 唐纳士公司 | Methods and compositions for prevention and treatment of sepsis |
Families Citing this family (25)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69739981D1 (en) * | 1996-10-31 | 2010-10-14 | Human Genome Sciences Inc | Streptococcus pneumoniae antigens and vaccines |
| US6800744B1 (en) | 1997-07-02 | 2004-10-05 | Genome Therapeutics Corporation | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| US6582706B1 (en) | 1998-12-21 | 2003-06-24 | Medimmune, Inc. | Vaccine compositions comprising Streptococcus pneumoniae polypeptides having selected structural MOTIFS |
| CA2356836C (en) * | 1998-12-23 | 2011-09-13 | Shire Biochem Inc. | Novel streptococcus antigens |
| EP1950302B1 (en) | 1998-12-23 | 2012-12-05 | ID Biomedical Corporation of Quebec | Streptococcus antigens |
| US7128918B1 (en) | 1998-12-23 | 2006-10-31 | Id Biomedical Corporation | Streptococcus antigens |
| US7074415B2 (en) | 2000-06-20 | 2006-07-11 | Id Biomedical Corporation | Streptococcus antigens |
| TR200704553T2 (en) * | 2000-06-20 | 2007-11-21 | Id Biomedical Corporation | Streptococcal antigens. |
| GB0022742D0 (en) | 2000-09-15 | 2000-11-01 | Smithkline Beecham Biolog | Vaccine |
| WO2003054007A2 (en) | 2001-12-20 | 2003-07-03 | Shire Biochem Inc. | Streptococcus antigens |
| US7955605B2 (en) | 2005-04-08 | 2011-06-07 | Wyeth Llc | Multivalent pneumococcal polysaccharide-protein conjugate composition |
| US7709001B2 (en) | 2005-04-08 | 2010-05-04 | Wyeth Llc | Multivalent pneumococcal polysaccharide-protein conjugate composition |
| EP2425854A1 (en) | 2005-04-08 | 2012-03-07 | Wyeth LLC | Multivalent pneumococcal polysaccharide-protein conjugate composition |
| US20070184072A1 (en) | 2005-04-08 | 2007-08-09 | Wyeth | Multivalent pneumococcal polysaccharide-protein conjugate composition |
| US8008453B2 (en) | 2005-08-12 | 2011-08-30 | Amgen Inc. | Modified Fc molecules |
| US20070093443A1 (en) * | 2005-10-21 | 2007-04-26 | Madison Edwin L | Modified proteases that inhibit complement activation |
| TWI457133B (en) | 2005-12-13 | 2014-10-21 | Glaxosmithkline Biolog Sa | Novel composition |
| EP1973564B1 (en) | 2005-12-22 | 2016-11-09 | GlaxoSmithKline Biologicals S.A. | Vaccine comprising streptococcus pneumoniae capsular polysaccharide conjugates |
| GB0607088D0 (en) | 2006-04-07 | 2006-05-17 | Glaxosmithkline Biolog Sa | Vaccine |
| US20100183662A1 (en) | 2007-06-26 | 2010-07-22 | Ralph Leon Biemans | Vaccine comprising streptococcus pneumoniae capsular polysaccharide conjugates |
| PT2271360E (en) | 2008-04-16 | 2015-12-07 | Glaxosmithkline Biolog Sa | Vaccine |
| GB201003920D0 (en) | 2010-03-09 | 2010-04-21 | Glaxosmithkline Biolog Sa | Method of treatment |
| GB201003924D0 (en) | 2010-03-09 | 2010-04-21 | Glaxosmithkline Biolog Sa | Immunogenic composition |
| AU2012257771C1 (en) | 2011-05-17 | 2015-12-03 | Glaxosmithkline Biologicals S.A. | Vaccine against Streptococcus pneumoniae |
| GB201518684D0 (en) | 2015-10-21 | 2015-12-02 | Glaxosmithkline Biolog Sa | Vaccine |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5846547A (en) * | 1996-01-22 | 1998-12-08 | Regents Of The University Of Minnesota | Streptococcal C5a peptidase vaccine |
| DE69739981D1 (en) * | 1996-10-31 | 2010-10-14 | Human Genome Sciences Inc | Streptococcus pneumoniae antigens and vaccines |
| EP0977872A1 (en) * | 1997-04-24 | 2000-02-09 | Regents Of The University Of Minnesota | HUMAN COMPLEMENT C3-DEGRADING PROTEINASE FROM $i(STREPTOCOCCUS PNEUMONIAE) |
| CA2305016A1 (en) * | 1997-09-24 | 1999-04-01 | Regents Of The University Of Minnesota | Human complement c3-degrading proteinase from streptococcus pneumoniae |
-
1999
- 1999-09-24 KR KR1020017003811A patent/KR20010089280A/en not_active Application Discontinuation
- 1999-09-24 BR BR9914066-7A patent/BR9914066A/en not_active Application Discontinuation
- 1999-09-24 CA CA002343931A patent/CA2343931A1/en not_active Abandoned
- 1999-09-24 MX MXPA01003073A patent/MXPA01003073A/en unknown
- 1999-09-24 CN CN99813016A patent/CN1352691A/en active Pending
- 1999-09-24 IL IL14201799A patent/IL142017A0/en unknown
- 1999-09-24 WO PCT/US1999/022362 patent/WO2000017370A1/en not_active Application Discontinuation
- 1999-09-24 EP EP99969446A patent/EP1115875A1/en not_active Withdrawn
- 1999-09-24 AU AU60608/99A patent/AU6060899A/en not_active Abandoned
- 1999-09-24 JP JP2000574269A patent/JP2002526082A/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102258784A (en) * | 2003-05-15 | 2011-11-30 | 唐纳士公司 | Methods and compositions for prevention and treatment of sepsis |
Also Published As
| Publication number | Publication date |
|---|---|
| IL142017A0 (en) | 2002-03-10 |
| JP2002526082A (en) | 2002-08-20 |
| AU6060899A (en) | 2000-04-10 |
| KR20010089280A (en) | 2001-09-29 |
| WO2000017370A1 (en) | 2000-03-30 |
| MXPA01003073A (en) | 2002-04-24 |
| EP1115875A1 (en) | 2001-07-18 |
| BR9914066A (en) | 2002-04-23 |
| CA2343931A1 (en) | 2000-03-30 |
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