CN1291233A - Human complement (3-degrading proteinase from i (streptococcus pheumoniae) - Google Patents
Human complement (3-degrading proteinase from i (streptococcus pheumoniae) Download PDFInfo
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- CN1291233A CN1291233A CN98809460A CN98809460A CN1291233A CN 1291233 A CN1291233 A CN 1291233A CN 98809460 A CN98809460 A CN 98809460A CN 98809460 A CN98809460 A CN 98809460A CN 1291233 A CN1291233 A CN 1291233A
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- protein
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- nucleic acid
- streptococcus pneumoniae
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Abstract
The present invention relates to the identification and use of a family of human complement C3-degrading proteinases expressed by S. pneumoniae. The proteinase has a molecular weight of about 15 kD to about 25 kD. A preferred proteinase of this invention includes the amino acid sequence of SEQ ID NO:2.
Description
Invention field
The present invention relates to streptococcus pneumoniae (Streptococcus pneumoniae) and be particularly related to a kind of evaluation of streptococcus pneumoniae protein, this protein can degradation of human complement protein C3.
Background of invention
The respiratory tract infection that streptococcus pneumoniae is caused causes pneumonia and 47,000 people's death of about 500,000 examples of estimation every year.To have high risk person be baby below two years old in streptococcal infection to bacterial pneumonia, individual and the elderly that function of immune system is clear.In these groups, streptococcus pneumoniae is the major cause of bacterial pneumonia and meningitis.And then streptococcus pneumoniae is the has age children's of institute an otic infections main cause.Children and old man invade breeding (colonization) on bacterium, carry out immunization after part or the systemic infection or with purified polysaccharide after, for the protection antibody of streptococcus pneumoniae capsular polysaccharide synthetic obstacle is arranged.Streptococcus pneumoniae is all the invasive bacterium and inhales the main cause of exhaling tract disease for the adult of HIV infection or children, and causes 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 capsular polysaccharide vaccine.As a result, there is four kinds of combinations (conjugate) vaccine to enter the stage of clinical trial at present.Combined vaccine comprises the streptococcus pneumoniae capsular polysaccharide that combines with protein carrier or adjuvant, and 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 for disadvantageous at a favourable vaccine in local region in another area.As for revising present available capsular polysaccharide vaccine or the development protein conjugate potential demand as the pod membrane vaccine that adapts to region serotype difference, then the money and the technical complicacy of stepping back made us in generation.Therefore, for the prevention of streptococcus pneumoniae infection and modulate for the extensive protectiveness Streptococcus pneumoniae vaccine, the immunity of looking for tool whole world conservative property in difference has the serotype of virulence produces the property surface, and to expose protein be most important.And then 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 capsular polysaccharide as single immunity.Existing report points out that being adhered to the relevant surface protein of airway epithelial cell with streptococcus pneumoniae comprises PsaA, PspC/CBPll2 and IgAl 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).But the antibody that resists these adhesion protein matter can suppress streptococcus pneumoniae and be adhered to airway epithelial cell, and therefore subtracts the intrusion breeding.Other is as lung lysin (pneumolysin), autolysin (autolysin), the tenuigenin streptococcus pneumoniae protein of nerve amines glycosides enzyme or sulphur glass Glycosylase is suggested as vaccine antigen, because antibody can potentiality stop these protein at streptococcus pneumoniae infection patient toxic action on one's body.Yet, be not the surface that is positioned at streptococcus pneumoniae on these protein typical case, but when cytolysis and when dead from the bacterium secretion or disengage (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, the antibody that resists these white matters neither can promote streptococcus pneumoniae death also can not prevent that initial or follow-up streptococcus pneumoniae from invading breeding.
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 the α of amido end spiral, then be the sequence of proline rich, and at the carboxylic end with 11 a series of choline binding tumor-necrosis factor glycoproteinss as end.Though be known with many information of its structurally associated, PspA is between different streptococcus pneumoniae serotype 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).Research has confirmed that PspA is at the immunity of animal generation property (people such as McDaniel, Microb.Pathogin.1994; 17:323-337).Produce property though PspA has immunity, its as the ability of vaccine antigen because of PspA be heterology, its with four structure groups exist with and do not give qualitative function and make 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-specific 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 instrumentality 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, the covalent attachment of C3b this moment (the opsonic activity fragment of C3) activates the identification of phagocytic cell 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.The method of right this control streptococcus pneumoniae infection is inefficiency completely.Excite the method for streptococcus pneumoniae opsonization can improve the lysis of this microorganism initiation.Method and treatment for the restriction streptococcus pneumoniae infection at present still has tight demand.
Summary of the invention
The present invention relates to identify and use the expressed a group human complement C 3 degradation property protein (proteolytic enzyme) of streptococcus pneumoniae.The preferable molecular weight with about 15kD to about 25kD of these protein, this molecular weight for example ties up on the 10%SDS polyacrylamide gel and measures.The present invention includes can separation from the different C3-degradation property bacterial strains of streptococcus pneumoniae numerous protein.
With regard on the one hand, the present invention relates to have the isolated protein of at least 80% sequence identity with SEQ ID NO:2.In preferable concrete example, this protein system separates from streptococcus pneumoniae, and perhaps this protein is a recombinant protein.Preferably, this isolated protein degradation of human complement protein C3.The preferable protein of the present invention is the isolated protein of aminoacid sequence with the SEQ of comprising ID NO:2, and more preferably this aminoacid sequence is SEQ ID NO:2.Term " separation " means by its physical environment at this paper and one of shifts out natural generation material or synthetic.Term " protein " comprises one or more functional unit in this article, and it comprises one or more peptide or polypeptide.
Also relevant peptide or the polypeptide that is separated from C3-degradation property proteolytic enzyme of the present invention of the present invention.Preferably, the invention provides the peptide or the polypeptide of at least 15 continuous amino acids, it is from having the isolated protein of 80% sequence identity with SEQ ID NO:2, and the peptide or the polypeptide of at least 15 continuous amino acids among the SEQ ID NO:2 more preferably are provided.In the present invention on the other hand, this peptide or polypeptide can degradation of human complement protein C3.
The preferable concrete example of the present invention comprise have among the SEQ ID NO:2 about the 1st to the isolated protein of about the 58th amino acid and tool SEQ ID NO:1 or its complementary strand the about the 1st to about the 174th Nucleotide.Preferably, this separating acid fragment comprises in SEQ ID NO:1 or its complementary strand the about the 150th to about the 174th Nucleotide.
On the other hand, the present invention relates to the isolated protein of degradation of human complement protein C3, the nucleic acid of this protein of wherein encoding can be hybridized under the hybridization part of highly strictness with SEQ ID NO:1 or its complementary strand.
The present invention also relates to immunity system irritating compositions (being preferably vaccine), it comprises the immunity system stimulatory peptides or the polypeptide of a significant quantity, this peptide or polypeptide have at least 15 continuous amino acids derived from a kind of protein, and this protein and SEQ ID NO:2 have at least 80% sequence identity and energy degradation of human complement protein C3.
Preferably, this protein system separates from streptococcus pneumoniae.In a concrete example, this immunity system irritating compositions or vaccine and then comprise at least a from other immunity system stimulatory peptides, polypeptide or protein that streptococcus pneumoniae separated.
The present invention so relate to a kind of can with SEQ ID NO:2 have at least 80% sequence identity and can degradation of human the antibody of protein bound (specificity combines on the typical case) of complement protein C3.In a concrete example, this antibody is monoclonal antibody, and in another concrete example, this antibody is polyclonal antibody.In another concrete example, this antibody is antibody fragment.This antibody or antibody fragment can derive from mouse, rat, goat, chicken, people, or rabbit.
In another concrete example, this antibody can be bonded at least one part of a protein, and the nucleic acid of this protein of wherein encoding can be hybridized under the hybridization part of highly strictness with SEQ ID NO:1 or its complementary strand.
The present invention also relate to can be with SEQ ID NO:1 or its complementary strand the separating acid fragment (polynucleotide) under the hybridization conditions of highly strictness, to hybridize.The hybridization conditions of the height strictness of indication comprises in this article, for example, with 6X SSC, 5X Denhardt, 0.5%SDS, and the salmon sperm dna of 100 mcg/ml fragmentations and sex change is hybridized a whole night at 65 ℃, and once at 2X SSC, under room temperature, cleaned about 10 minutes among the 0.1%SDS, then one inferior to about 15 minutes of 65 ℃ of cleanings, then have at least once at 0.2X SSC, under room temperature, cleaned among the 0.1%SDS 3-5 minute at least.In a concrete example, this nucleic acid fragment system separates from streptococcus pneumoniae, and in another concrete example, encode at least one part of a protein of this nucleic acid fragment.In a concrete example, this protein degradation human complement protein C 3.In another concrete example, this nucleic acid fragment encode a kind of peptide or polypeptide of non-degradable human complement C 3.
In another concrete example, this nucleic acid fragment ties up in the nucleic acid carrier, and this carrier is for can produce the expression vector of proteinic at least some.The cell that comprises this nucleic acid fragment is also planned in the present invention.In a concrete example, this cell is bacterium or eukaryotic cell.
The present invention and then relate to the separating acid fragment that comprises SEQ ID NO:1 nucleotide sequence or its complementary strand.The present invention and then relate to the RNA fragment of transcribing by the double-stranded DNA that comprises SEQ ID NO:1.
In the present invention on the other hand, the present invention relates to a kind of immunoreactive method Mammals (particularly human) streptococcus pneumoniae that creates antagonism, it comprises a composition is applied to the step of animal with this proteinic immune response that creates antagonism, said composition comprises one of treatment significant quantity proteinic at least one part, and the nucleic acid of this protein of wherein encoding can be hybridized under the hybridization conditions of highly strictness with SEQ ID NO:1 or its complementary strand.This immune response can be the B cell response, t cell responses, epithelial cell reaction or endotheliocyte reaction.In a preferable concrete example, said composition is a vaccine composition.Preferably this protein is at least 15 amino acid lengths, and also preferably said composition and then comprise at least a other immunity system stimulatory peptides, polypeptide or protein from streptococcus pneumoniae.In a concrete example, this protein comprises at least 15 amino acid among the SEQ ID NO:2.
The present invention so relate to from about 15kDa of streptococcus pneumoniae to about 25kDa can human complement C 3-degrading isolated protein, and relate to a kind of method that suppresses streptococcus pneumoniae-media C3 Degradation, it step that comprises is: the streptococcus pneumoniae bacterium is contacted an antibody, and this antibody can have the protein bound of at least 80% sequence identity with SEQ ID NO:2.
The method of repulsive interaction when the present invention also relates to a kind of inhibition C3-media inflammatory response and xenotransplantation is expressed the protein with SEQ ID NO:2 aminoacid sequence on the used animal organ surface when it is included in xenotransplantation.This method is particularly conducive at for example pig kidney and causes the expression of protein of the present invention, and suppresses the C3 media inflammation after the xenotransplantation by this.
The present invention also relates to an isolated nucleic acid molecule, and it comprises the zone of one at least 15 Nucleotide and can hybridize under the hybridization conditions of highly strictness with at least one zone in SEQID NO:1 or its complementary strand.In a concrete example, isolated nucleic acid molecule can be hybridized under the hybridization conditions of highly strictness with at least one zone in SEQ ID NO:1 or its complementary strand.Preferably, at least one zone is Nucleotide 1-174 or the 320-492 that comprises SEQ ID NO:1.
In another concrete example, the present invention also relates to an isolated nucleic acid molecule, and it comprises the zone of one at least 15 Nucleotide and can hybridize under the hybridization conditions of highly strictness with at least one zone in SEQ ID NO:4 or its complementary strand.In a concrete example, isolated nucleic acid molecule can be hybridized under the hybridization conditions of highly strictness with at least one zone in SEQ ID NO:4 or its complementary strand.Preferably, at least one zone is Nucleotide 507-681 or the 827-999 that comprises SEQ ID NO:4.
In another concrete example, nucleic acid molecule encoding one proteinic at least one part of at least one part among the SEQ ID NO:4.Preferably, this partly has the predicted amino acid sequence shown in SEQ ID NO:5, and has in SDS-PAGE for example measures the extremely molecular weight of about 85kDa of about 75kDa.
The present invention also relates to and has the NO:6 as SEQ ID, SEQ ID NO:7, the DNA isolation fragment or the primer of the nucleotide sequence of SEQ ID NO:8 and SEQ ID NO:9.
In the present invention on the other hand, the present invention relates to one immunity system-irritating compositions or vaccine, it comprises at least one part of one of treatment significant quantity protein, and the nucleic acid of this protein of wherein encoding can be hybridized under the hybridization part of highly strictness with SEQ ID NO:4 or its complementary strand.The present invention relates to a kind of immunoreactive method Mammals (particularly human) streptococcus pneumoniae that creates antagonism, it comprises a composition is applied to the step of animal with this proteinic immune response that creates antagonism, said composition comprises one of treatment significant quantity proteinic at least one part, and the nucleic acid of this protein of wherein encoding can be hybridized under the hybridization conditions of highly strictness with SEQ ID NO:4 or its complementary strand.
In another concrete example, the present invention relates to a vaccine or immunity system-irritating compositions, it comprises at least one part and the pharmaceutically acceptable carrier of one of treatment significant quantity protein, wherein this protein immunization or infection or the intrusion breeding of treatment Mammals to resist streptococcus pneumoniae effectively.This protein system derived from can with nucleotide sequence shown in the SEQ ID NO:4 or its complementary strand the nucleic acid molecule of hybridizing under the part of hybridizing in strictness highly.Preferably, this protein system is can effectively providing the deal of result of treatment to be supplied with for this mammalian subject (particularly human individual).
Brief description of drawingsfig
Fig. 1 provides the therefore nucleotide sequence of Translation Service's part (SEQ IDNO:1) of one of the present invention C3-degradation property protease-based.
Fig. 2 provides therefore aminoacid sequence (SEQ ID NO:2) of one of the present invention C3-degradation property protease-based.
Fig. 3 provides therefore aminoacid sequence of one of the present invention C3-degradation property protease-based, and one of itself and code book invention C3-degradation property protease-based is nucleotide sequence (SEQ ID NO:2-3) side by side therefore.
Fig. 4 provides prediction 79kDa the nucleotide sequence (SEQ ID NO:4) of aminoacid sequence.
Fig. 5 provides prediction 79kDa aminoacid sequence (SEQ ID NO:5).
Fig. 6 shows the sequence alignment of SEQ ID NO:1 and SEQ ID NO:4.
Fig. 7 shows the sequence alignment of corresponding amino acid/11 69-331 among SEQ ID NO:2 and the SEQ ID NO:5.
The detailed description of preferred embodiment
The present invention relates to identify and the isolated molecule amount is about 20kDa (± 5kDa) the human complement C 3 degradation property proteolytic enzyme of (on 10%SDS-PAGE glue) and the nucleic acid of coding C3 degradation property proteolytic enzyme.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).This type of degraded kenel that cutting does not take place is different in essence in the product of other microorganism, as elastoser of Pseudomonas aeruginosa (Pseudomonas aeruginosa) and the L-Cysteine HCL Anhydrous of entamoeba histolytica (Entamoeba histolytica).
Employed in this article term " degraded " means amino acid is removed from the protein molecule, produces peptide or polypeptide.As the observation on polyacrylamide gel, protein degradation C3 of the present invention and can not produce specific cutting fragment.The present invention's C3-degradation property proteolytic enzyme has the preferable of some degree for C3, and for example, can't degrade other protein of this C3-degradation property proteolytic enzyme is as albumin.
The C3-degradation property proteolytic enzyme of about 20kDa system identifies in the streptococcus pneumoniae gene pool of the property an inserted interruption and compares the clone with the C3 degrading activity that weakens with the wild-type streptococcus pneumoniae and separated.Embodiment 1 provides the exemplary test of assessment clone's C3-degrading activity.Evaluation has the clone that weakens the C3 degrading activity and selects the SmaI insert of a 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 therefore open reading frame of evaluation and C3-degrading activity dependency basis.The oligonucleotide (SEQ ID NO:10) that following and some PspA have a sequence identity be used to confirm in the distinctiveness hybridization the to encode gene of C3-degradation property proteolytic enzyme is completely different with the gene of coding PspA.
SEQ?ID?NO：10
GAAAACAATAATGTAGAAGACTACTTTAAAGAAGCTTTAGA
The complete open reading frame of 20kDa protein has 492 base pairs (SEQ ID NO:1), predicted molecular weight be about 20kDa (+/-protein of 163 amino acid (SEQ ID NO:2) 5kDa) or approximately arranged.The exemplary genes sequence of coding C3-degradation property protein is shown in Fig. 1 (SEQ ID NO:1), and the aminoacid sequence of this protein is shown in Fig. 2 (SEQ ID NO:2).Fig. 3 is combined into SEQ ID NO:3 with preferable gene order with corresponding preferable translated protein.
Use SEQ ID NO:2, determine that the aminoacid sequence of this protein is irrelevant with other protein in GenBank or Switzerland Prot database.The protein of prediction has the sequence signature of the proline rich (particularly between amino acid 80-108) of prokaryotic cell prokaryocyte membrane protein, and this advises that this protein system is expressed in cell surface.This aminoacid sequence there is no and shows tangible choline-associativity tumor-necrosis factor glycoproteins.To on SDS-PAGE glue, carry out electrophoresis from the streptococcus pneumoniae solute and the supernatant liquor of CP1200 culture with the C3 infiltration, both all identify about 20kDa (± 5kDa) band (band) confirm to have as SEQID NO:2 and predicts that big or small protein has C3-degrading activity (seeing embodiment 2) at supernatant liquor and solute.As described in embodiment 3, the gene of the C3-degradation property proteolytic enzyme 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 full-length gene of code book invention C3-degradation property proteolytic enzyme is embedded into an expression vector to express in intestinal bacteria (E.coli).As described in embodiment, regroup C3-degradation property proteolytic enzyme is separated.The general technology personage will understand that in this area, 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 well-known process different in this area to produce and separate the present invention's recombinant protein, and the general technology personage also can understand in this area, whether the particular expression system that the present invention's C3 degradation test will be measured except the expression system that is proposed in an embodiment has function, and needn't further test.In the basic technology document, can find many different molecules and immunological technique, as people such as Sambrook (Molecular Cloning-A LaboratoryManual, 1989 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and people (Antibodies such as Harlow; A Laboratory Manual.Cold Spring Harbor, NY; Cold Spring Harbor Laboratory Press, 1988).
The gene line of code book invention C3 degradation property protein uses to be identified from the obtained plasmid gene storehouse of the streptococcus pneumoniae genomic DNA fragment of CP1200 bacterial strain.Though known have many diverse ways can obtain the plasmid gene storehouse, with preferable strategy, constructing of plasmid gene storehouse is (to derive from D.A.Morrison from streptococcus pneumoniae CP1200 bacterial strain, 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 (O.5-4.0kb) through the Sau3A cutting, insert an imbedibility (shuttle) carrier pVA891 (erm that shuttles back and forth
r, cm
rHave colibacillary copy source) the BamHI position.Transformed into escherichia coli DH5 α MCR bacterial strain is removed in this gene pool utilization electric shock perforation.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 system uses by the property the inserted mutating technology of isomorphism type reorganization and finishes.
S. pneumoniae strains CP1200 cell can use the step transitions that moves pH with HCl in the CTM substratum be competent cell.To be competent at cell and store in-70 ℃ till needs use in a small amount of packing mode.
The present invention's isolated protein and human complement C 3 are cultivated 4 hours to detect the C3 Degradation in 37 ℃ in the presence of PBS.The control group sample that does not have separation streptococcus pneumoniae protein can be used as the control group of the comparative purpose of tool.
The present invention's protein on the 10%SDS-polyacrylamide gel, have the about 20kDa of obvious molecular weight (± 5kDa) and preferably have the about 15kDa of molecular weight to about 25kDa.One exemplary proteins sequence is shown in SEQ IDNO:2.The technology personage will understand as one of this area, have some variations to expect in aminoacid sequence, and these variations should not got rid of in the present invention's scope.For example, the conservative property sudden change is not got rid of in the scope of the invention, the variation that is less than about 80% consensus amino acid sequence (and preferably being less than about 90% consensus amino acid sequence) in the consensus amino acid sequence is not also got rid of in the scope of the invention, wherein this protein can degradation of human complement protein C3 and particularly this protein system separate or originally derive from a streptococcus pneumoniae bacterium.The fragment of this protein is also in the present invention's scope, particularly when these fragments energy degradation of human 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, L-LEU, L-iLeu, Xie Ansuan, proline(Pro), phenylalanine, and tryptophane.Polarity electric neutrality amino acid comprises glycine, Serine, Threonine, halfcystine, tyrosine, 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 molecular weight or iso-electric point that expection can impact polypropylene acid amides gel electrophoresis not be measured.Preferable especially conservative property metalepsy includes, but not limited to Lys is replaced into Arg (and vice versa) to keep positive electric charge; Glu is replaced into Asp (and vice versa) to keep negative electric charge; Ser is replaced into Thr to keep free state OH; And Gln is replaced into Asn to keep free state NH
2
The preferable protein of the present invention comprises the protein with aminoacid sequence SEQ ID NO:2.Other protein of planning in the present invention comprises the protein of degradable human complement protein C 3, and the nucleic acid of this protein of encoding can be hybridized under the hybridization conditions of highly strictness with SEQ ID NO:1, highly the hybridization conditions of strictness 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 a whole night at 65 ℃, and once at 2X SSC, under room temperature, cleaned about 10 minutes among the 0.1%SDS, then one inferior to about 15 minutes of 65 ℃ of cleanings, then have at least once at 0.2X SSC, under room temperature, cleaned among the 0.1%SDS 3-5 minute at least.On the typical case, SSC solution comprises sodium-chlor, and Trisodium Citrate and water are with the preparation stock solution.Also can use the peptide or the polypeptide of this protein.The present invention's preferable protein comprise among the SEQ ID NO:2 about the 1st to about the 50th amino acid.
Protein of the present invention can proteinic form be separated or be prepared.In other words, the nucleic acid of encode this protein or this protein some can be embedded into expression vector or embed in the karyomit(e) of cell, so that express this protein in this cell.This protein can be in bacterium or another cell (preferably eukaryotic cell, and more preferably zooblast) purifying.Perhaps, this protein can be expressed certainly in the cell of this protein and be separated, as the pneumonia streptococcus mycetocyte.Peptide or polypeptide also with among the present invention take into account.This peptide or polypeptide are preferably at least 15 amino acid whose length and preferable peptide or polypeptide and have at least 15 continuous amino acids from SEQ ID NO:2.
The nucleic acid of this 20kDa protein of encoding also is the present invention's some.SEQ ID NO:1 is the preferred nucleic acid fragment of coding C3-degradation property proteolytic enzyme.The general technology personage will understand in this area, and feature and character that some replacements can't make C3-degradation property proteolytic enzyme sequence change and cause this C3-degradation property proteolytic enzyme arrive the degree that changes in fact.For example, the present invention also plans with SEQ ID NO:1 and has at least 80% conforming nucleic acid.The method that determines a special nucleic acid sequence whether to fall within the scope of the invention should think deeply this special nucleic acid sequence whether encode C3-degradation property proteolytic enzyme and with SEQ ID NO:1 compare whether have at least 80% nucleic acid consistence.Other nucleotide sequence of coding C3 proteolytic enzyme comprises the nucleic acid of those codings C3 proteolytic enzyme, and wherein this C3 proteolytic enzyme and SEQ ID NO:2 compare and have identical sequence or at least 90% sequence identity, and comprise the degeneracy situation that relates to this nucleotide sequence.The degeneracy codon means different three word codons and can be used for specifying identical amino acid.For example, knowing following RNA codon (and therefore, relevant DNA codon wherein replaces U with T) in the art can intercourse and be used to encode each specific amino acids:
Phenylalanine (Phe or F) UUU, UUC, UUA or UUG
L-LEU (Leu or L) CUU, CUC, CUA or CUG
L-iLeu (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
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 revised and use the codon of a special cell classification institute preference.For example, for the use of the preference codons of intestinal bacteria, and the preference codon of animal (comprising the mankind) is known.These are changed in this area general skill personage and know, and therefore these gene orders are considered to the present invention's some.Other nucleotide sequence comprises from the length of the SEQ ID NO:1 nucleic acid fragment at least 15 (and being preferably 30) nucleic acid at least, or other length is the nucleic acid fragment of at least 15 (and being preferably at least 30) nucleic acid, wherein this fragment can be hybridized under the hybridization conditions of highly strictness with SEQ ID NO:1, highly the hybridization conditions of strictness 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 a whole night at 65 ℃, and once at 2X SSC, 0.1%SDS cleaned under room temperature about 10 minutes, then one inferior to about 15 minutes of 65 ℃ of cleanings, then, under room temperature, cleaned among the 0.1%SDS 3-5 minute at least at least once at 0.2X SSC.
The present invention's nucleic acid fragment codified SEQ ID NO:2 or SEQ ID NO:5 are all, can not contain SEQ ID NO:2 or SEQ ID NO:5 (the i.e. fragment that can't transcribe, the fragment or other fellow that comprise the generegulation zone) or codified SEQ ID NO:2 or SEQ ID NO:5 are partly, preferably comprise the continuity nucleic acid fragment of coding from least 9 amino acid of SEQ ID NO:2 or SEQ ID NO:5.Because the nucleic acid fragment of the some of planning coding C3 proteolytic enzyme in the present invention, with apprehensible be not to be all encode protein or the peptide or the polypeptide of tool C3-degrading activity of all nucleic acid fragments.And then the present invention's nucleic acid can suddenly change to remove the C3 degrading activity of (or otherwise not activating) this protein.Therefore, the present invention also plans and meets above-mentioned hybridization conditions and the fragment of tool C3-degrading activity.The method that makes nucleotide sequence sudden change or otherwise change nucleotide sequence describes in detail in this area, and produces the protein production that does not activate aspect property and the ferment characteristic in immunity and can test at therepic use.
The present invention's nucleic acid fragment can be merged in the nucleic acid carrier or stably incorporate in the host genome to produce recombinant protein, comprises regroup chimeric (chimeric) protein.In a concrete example, C3-degradation property protein is by the coded by said gene in carrier, and this carrier ties up in the cell.Preferably this cell is the prokaryotic cell, as bacterium.Gene and gene fragment can this gene whole or partly exist with the mode of another gene fusion, and the fused protein mode that this C3-degradation property protein can one or more protein exists, and this fused protein system expresses in single protein mode.That the present invention's many different nucleic acid carrier is known in the art and comprise many commercial available expression plasmids or virus vector.Using to general technology personage in this area of these carriers is known.The user of institute is some exemplary carriers in an embodiment, but should not be considered to the scope of the invention is limited to some extent.
The present invention also relates to can be in conjunction with (typical case on specificity combination) extremely from the antibody of the protein (preferably about 15kDa is the protein of about 25kDa extremely) of about 20kDa of streptococcus pneumoniae, and preferably wherein this protein can human complement C 3-degrading.Can be at the whole or a part of preparation polyclonal antibody of this protein.Similarly, can be at all or wherein a peptide or polypeptide (fragment) the preparation monoclonal antibody of the C3 degradation property protein of about 20kDa of the present invention.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 SpringHarbor Laboratory Press, 1988).In a preferable example, this antibody can be from human, rat, and mouse, goat, chicken, or rabbit derives.Protein bound antibody fragment and chimeric fragment also belong to be known and in the present invention's scope.
The present invention also relates to the use of immunostimulating composition.Term " immunostimulating " or " immunity system pungency " composition mean the present invention's protein, peptide or peptide composition, it activates at least a cell kenel in individual (as a Mammals) immunity system, preferably, this immunostimulating composition can provide a kind of immunization reaction or preventive effect in normal (not infecting) is individual, be a vaccine on its typical case.Yet any detectable immune response all helps individuality in treatment application or process.Preferably be activated cell in the immunity system and comprise and engulf sexual cell, as neutrophils or scavenger cell, T cell, B cell, epithelial cell and endotheliocyte.The peptide that comprises the present invention, the immunostimulating composition of polypeptide or protein can be used to produce antibody in animal, as rat, mouse, goat, chicken, the rabbit or the mankind, or be used to study the zootype of streptococcus pneumoniae infection.Preferable immunostimulating composition comprises at least a peptide or the polypeptide of immunostimulating deal (for example treating significant quantity), and it comprises at least 15 amino acid from this C3 degradation property proteolytic enzyme.
Term " vaccine " means the composition that is used for immunization.The process of immunization can comprise administration of protein, peptide, polypeptide; antigen; nucleotide sequence or complementarity (for example antisense) sequence, or antibody or its suspension are wherein used this molecule and can be produced active immne power and provide protection with antagonism streptococcus pneumoniae infection or intrusion breeding.On the typical case, these vaccines are made into the liquor or the suspension of injectable.Also can be made into dissolving before being adapted at injecting or be suspended in solid form in the liquid.This vaccine production thing is emulsification in addition optionally, or be wrapped in little fat body.
This immunostimulating composition (as a vaccine) can and then be included in other protein in pharmaceutically acceptable damping fluid or the supporting agent, these damping fluids or supporting agent such as PBS (phosphate buffered saline (PBS)), or other is considered to be fit to and be used safely in protein is imported the damping fluid of host with stimulating immune system in the art.This immunostimulating composition also can comprise other immunity system pungency protein, as adjuvant or from immunostimulating protein, peptide or the polypeptide of streptococcus pneumoniae or other biology.For example, the control streptococcus pneumoniae infection is best may be the allotment mixture (cocktail) of peptide or polypeptide.Preferably in a vaccine production thing, use one or more fragments of protein of the present invention to invade the result of causing a disease that breeding is brought with intrusion breeding or the streptococcus pneumoniae of antagonism or restriction streptococcus pneumoniae.
Use " treatment significant quantity " to mean the deal that effective generation one is intended to the result in this article.This deal is looked the health of individual immunity system or physics (being that antibody is synthetic) situation, the degree that is intended to protect, the formulation of preparation and other correlative factor and decide.Expected is that this deal will fall within a scope quite widely, and can determine via day to day tests.
Active immne pungency composition often and vehicle or mixing diluents, these vehicle or thinner be pharmaceutically acceptable supporting agent and with the active ingredient compatibility.Term " pharmaceutically acceptable supporting agent " mean with a composition in other composition compatible and do not endanger on recipient's the meaning and be " acceptable " supporting agent.Suitable vehicle is, for example, water, salt solution, dextrose, glycerine, ethanol or analogue, with and composition thereof.This immunostimulating composition (comprising vaccine) can comprise a small amount of complementary material according to hobby in addition, as moistening or emulsifying agent, and the pH buffer reagent, and/or can promote the adjuvant of this immunostimulating composition effect.
Effectively adjuvant or supporting agent include but not limited to aluminium hydroxide, N-ethanoyl-wall acyl group-L-different glutamine of amine acyl group-D-(thr-MDP) of reviving; N-ethanoyl-Xin-different glutamine (the CGP11637 of wall acyl group-L-propylamine acyl group-D-; be called as nor-UDP); the different paddy amine acyl group of N-acetyl basal wall acyl group-L-propylamine acyl group-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 walls framework (MPL+TDM+CWS).
The present invention also relates to the method that suppresses streptococcus pneumoniae-media C3 Degradation, comprise streptococcus pneumoniae is contacted with a protein (as an antibody), or another can with the protein that combines to the isolated protein of about 25kDa from about 15kDa of streptococcus pneumoniae.Can be an antibody or its fragment to the protein that the isolated protein of about 25kDa combines with about 15kDa, or this protein can be chimeric protein, this chimeric protein comprises the antibodies zone (as a mutability zone) from antibody, this antibody capable specificity identification from about 15kDa of streptococcus pneumoniae to about 25kDa and have the isolated protein of C3 degrading activity.
The present invention's separation streptococcus pneumoniae protein can be separated, and optionally give purifying, and can use this isolated protein or its immunity generation property fragment to produce immunological response, in one embodiment, it is included in the mankind or laboratory animal antibody response on one's body.This protein does not have the peptide or the polypeptide fragment of C3 degrading activity can be tested at its ability that limits the streptococcus pneumoniae infection influence.Similarly, the C3 degrading activity of this protein can be for example interrupted or not activate in the correction of the present invention's protein via sudden change.The antibody that can use the C3-degrading activity that can suppress protein of the present invention is as the strategy that prevents C3 Degradation and the removing of promotion streptococcus pneumoniae via conditioning property path.This isolated protein can make the antibody that is used for detecting the antagonism streptococcus pneumoniae in test, or the some of the vaccine of using as the streptococcus pneumoniae treatment, or as comprising a polyvalency or a plurality of protein, the vaccine of peptide or polypeptide.
Therefore, mean individual or round various streptococcus pneumoniae infections invasions at the term " processing " that this paper used for normal mammalian, be diagnosed as various streptococcus pneumoniae infections, or the mammalian subject of expressing 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 express and expresses the symptom of streptococcus pneumoniae infection or other relative disease very less or not.This type of processing can be followed the present invention's nucleic acid molecule (tool meaning or antisense person), protein, the using of peptide or polypeptide or antibody.
The protein that further is planned to the present invention can be expressed in the surface of vertebrate cells and be used to the C3 that degrades, for example, and when complement deposit (or activation) when becoming a difficult problem, for example in xenotransplantation or the brightic situation of complement-media.For example, complete streptococcus pneumoniae protein, recombinant protein, or above both arbitrary somes, can be merged in the xenotransplantation cell, and express to prevent complement deposit (and/or complement-media inflammatory response) or to make it reduce to minimum in the mode of surface protein or secretory protein.
Another special aspect of the present invention relates to use to express isolated protein and by it and the vaccine carrier of next peptide or polypeptide.In view of the above, so that on the one hand, the invention provides and a kind ofly cause immunoreactive method Mammals, it comprises that the vaccine carrier at least a or its mixture with expression isolated protein of the present invention and/or peptide or polypeptide provides to a Mammals.The present invention's protein and peptide or polypeptide can use vaccine carrier alive to be sent to Mammals, particularly use the recombinant bacteria of living, material that virus or other are lived, it comprises genetic material required when expressing this protein and/or peptide or polypeptide in external polypeptide mode.Particularly those bacteriums that in gi tract, breed oneself be 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 among the 75-88 (1992).
The present invention's extra concrete example relates at an individuality (as Mammals) and causes immunoreactive method; comprise with the isolated protein of the code book invention of a deal and/or from it and the dna molecular of next peptide or polypeptide (selectivity is united a kind of infection-assisting factor) is applied to this individuality; wherein this protein and/or peptide or polypeptide keep immunity generation property; and when this protein and/or peptide or polypeptide are incorporated an immunostimulating composition (for example vaccine) into and be applied to the mankind, 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.
The antisense sequences that further is planned to the gene of this 20kDa protein of coding can be used as the vaccine or the therapeutic treatment of anti-streptococcus pneumoniae infection.That antisense DNA is defined as is non--coding property sequence, its with SEQ ID NO:1 all or partly complementation (i.e. a complementary strand).For example, the antisense sequences of 5 '-ATGTCAAGC-3 ' is 3 '-TACAGTTCG-5 '.Antisense sequences or oligonucleotide are sent to animal can cause animal to produce antibody, or cause this sequence to incorporate into to cause in viable bacteria or other cell this 20kDa gene product all or the transcribing and/or translate and be suppressed of part.
The importing of anti sense nucleotide sequence can for example 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).On the typical case, have 8 or more the anti sense nucleotide sequence of polynucleotide can be bonded on bacterial nucleic acid or the bacterium message RNA.Comprise on the anti sense nucleotide sequence typical case at least about 15 Nucleotide (being preferably), provide required stability with hybridization product for bacterial nucleic acid or bacterium message RNA at least about 30 Nucleotide or polynucleotide more.The importing of sequence preferably suppresses transcribing or translating of at least one endogenous streptococcus pneumoniae nucleotide sequence.The method of loading antisense nucleic acid is in the art for what know, and for example the U.S. patent the 4th, 242, No. 046.
The present invention also provides the nucleic acid of the open reading frame (SEQ ID NO:4) with 2163 bases, and it comprises the nucleic acid of open reading frame (SEQ ID NO:1), and its coding has the protein of the about 20kDa of molecular weight (SEQ IDNO:2).At this 20kDa protein described herein and then be named as a C3-degradation property protein.Bigger open reading frame, 2163bp (SEQ ID NO:4) for example, the hypothetical protein of about 79kDa (the SEQ IDNO:5) that encode.
The reference of all references and deliver document and all incorporate this specification sheets in this article as the reference data.For haveing the knack of this operator, there are many different available substitute technologies and step can make the invention of implementer similarly according to this specification sheets successful implementation planning.Haveing the knack of this operator will understand that, though the present invention is in above being described according to specific embodiments and example, the present invention is not so limited certainly, and can carry out many other concrete examples, example, purposes is revised and at these concrete examples, situation beyond example and the purposes, and can not depart from the invention scope of the application's case.
Evaluation with imbedibility mutant strain of the C3-of weakening degrading activity
The imbedibility mutating strain series derives from Elaine doctor Tuomanen (Rockefeller Inst., New York, New York).Clone with insert tests with detection in test and weakens the C3-degrading activity.137 clones are via making these cells at room temperature go up growth a whole night and test in little dish of tiring in Todd Hewitt nutrient solution.These cells carry out 1: 10 dilution for the synthetic property substratum (seeing Sicard A.M., Genetics 50:31-44,1984) of streptococcus pneumoniae, and remaining cell is frozen in little dish of tiring.To add at the C3 of 63 nanograms (ng) in the substratum (comprising the 0.1%BSA of 1 mg/ml) of 100 microlitres or 83 nanograms at phosphate buffered saline (PBS) (PBS) about 200 microlitres in diluting cells.Cell was cultivated 4 hours in 37 ℃.To cultivate a whole night in the mixture adding ELISA dish of 100 microlitres and in 4 ℃.Should coil with cleaning buffer solution and clean three times, and the 0.05%Tween 20 in PBS was added in the hole, and before each the cleaning, carry out cultivating in 5 minutes.The 100 microlitre antibody of anti-C3 (to the human C3-IgG goat antibody of strain the West wasabi catalase-combination more than the tool specificity partly, ICN Cappel, Costa Mesa CA) carries out 1: 1200 dilution with the 3%BSA in PBS.This ELISA coils in 37 ℃ and cultivates about 30 minutes to 1 hour in the dark, and cleans with above-mentioned cleaning buffer solution.Should test and use 12 milligrams of OPD (in 30 milliliters of 0.1M sodium citrate buffer solutions) and 30% hydrogen peroxide of 12 microlitres to develop.Test-results is read to survey on the instrument in ELISA dish and is determined via the optical density (OD) reading in 490 micromillimeters.
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 partly sequence, and with the SmaI fragment of a 546bp with 32P carry out random primer demarcate (derive from the cover group of Stratagene, LaJolla, CA).From the SmaI fragment of the 546bp of SEQ ID NO:1 with on the Southern trace, hybridize through the cut substrate of EcoRI and Kpnl processing from many S. pneumoniae strains.This identical segments also is used for the Sau3A fragment gene storehouse that screening derives from the genomic dna of S. pneumoniae strains CP1200.
Identify the insert of 3.5kb from the CP1200 gene pool.This insert is carried out sequencing, and identify the open reading frame of one 492 base pairs, comprise terminator codon.These 163 amino acid of open reading frame coding and predicted molecular weight are about 18,500 dalton's protein.
Construct 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 insert that is amplified according to meet the coli expression carrier pQE30 that reads the frame mode and be connected to the His-mark (Qiagen, San Diego, CA).The plasmid that obtains is used for transformed into escherichia coli bacterial strain BL21, and (WI), it comprises lac inhibition (repressor) plasmid pREP4 (Qiagen) for Novagen, Madison.Culture of Escherichia coli is brought out expressing the protein of His-mark, and this protein carries out the tubing string purifying with Ni-NTA resin (Qiagen).The protein of this purifying is confirmed via SDS-PAGE glue.
The evaluation of the C3-degradation property proteolytic enzyme of 20kDa
In order to measure the C3-degradation capability of 20kDa protein, with the C3 of 0.5 mg/ml (according to people such as Tack, Meth.Enzymol.80:64-101,1984 are prepared) on the sodium laurylsulfonate that contains 15% acrylamide (SDS) glue (15%SDS-PAGE glue), 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 via 5 * 108 cells and 5%SDS at room temperature being cultivated 30 minutes and obtaining.Make apparatus 10 through solute, (Amicon, Beverly MA) concentrate 10 times 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 glue that comprises C3, and electrophoresis lies in 4 ℃ and carries out at 150V, till the dyestuff leading edge is run out of.Glue is cleaned (2 times, 10 minutes) with 50 milliliters of 2.5%Triton X-100 in water continuously, with the 2.5%Triton X-100 in 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, pH 7.4, pour in the dish ware that contains these glue, will coil that ware is added a cover and cultivate 1.5 hours and a whole night (about 16 hours) in 37 ℃.Glue with the blue dyeing of Kao Mashi (Coomassie) 10 minutes, is fully decoloured 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 protease activity in the streptococcus pneumoniae solute is observed after 1.5 hours in 37 ℃ of cultivations, yet the C3 protease activity is just observed after cultivating a whole night in the Pn supernatant liquor.Therefore, the C3 protease activity is obviously main relevant with cell.
Embodiment 3
Encode gene tool conservative property in some S. pneumoniae strains of this 20kD protein
From various S. pneumoniae strains (the 1st type, the 3rd type, L002 and L003 (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 protein of coding as probe.The separation chromosomal DNA cuts with EcoRI and separates via electrophoresis.DNA is transferred on the solid support, and hybridize with the SEQ ID NO:3 of end-demarcation, hybridization and cleaning condition are with 6X SSC, 5X Denhardt ' s, the salmon sperm dna of 0.5%SDS and 100 mcg/ml fragmentations and sex change is hybridized a whole night at 65 ℃, and once at 2X SSC, 0.1%SDS cleaned under room temperature about 10 minutes, then one inferior to about 15 minutes of 65 ℃ of cleanings, followed twice at 0.2X SSC, cleaned 3 minutes under room temperature among the 0.1%SDS.
The result points out that SEQ ID NO:3 is tried that at each hybridization is arranged in the dna sample equally, points out that this protein obviously has conservative property between each bacterial strain.In some bacterial strains, the DNA of C3-degradation property protein of coding 20kDa obviously is the some of a bigger 2163bp open reading frame, the hypothetical coding of this open reading frame 79kDa protein.
The Southern engram analysis of pneumococcal dna/5F1 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 agarose gel and via electrophoresis subsequently and resolve.Being included in sample in the glue is transferred to via capillary transfer subsequently and derives from Amersham (Upsafla is on Hybond-N+ film Sweden).The SmaI fragment that derives from one of 5F1 strain isolated 540bp use T7QuickPrime cover group (Pharmacia, Piscathaway, NJ) with
32P carries out random primer and demarcates, and uses NucTrap tubing string (Stragene, La Jolla, CA) purifying in non--Nucleotide of incorporating into, and hybridizing.
Hybridization conditions is with 6X SSC, 5X Denhardt, 0.5%SDS, reaching 100 mcg/ml fragmentations and denatured salmon sperm dna hybridizes a whole night at 65 ℃, and once at 2X SSC, 0.1%SDS cleaned under room temperature about 10 minutes, and then one inferior to about 15 minutes of 65 ℃ of cleanings, then, under room temperature, cleaned among the 0.1%SDS 3-5 minute at least at least once at 0.2XSSC.The stain film shows that this 20kDa gene line is present in the strain subject of all streptococcus pneumoniaes.
Make two dna primers and be used for the 20kDa gene order of amplification from SEQ ID NO:1 from streptococcus pneumoniae (serotype 3) genosome DNA.First primer is 5 '-primer (SEQ ID NO:6), and it is carried out from the ATG of this 20kDa gene initiator codon, wherein inserts a NcoI position, and after the ATG initiator codon tool Ala residue to keep correct reading frame.Second primer is 3 '-primer (SEQ ID NO:7), and its terminator codon from this 20kDa gene is carried out, and wherein inserts a BamHI position.
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 lie in Applied Biosystems 380A dna synthesizer, and (Foster City, CA) (Sterling, reagent VA) synthesizes available from Glen Research in middle use.Amplified reaction system uses Perkin Elmer Thermocycler (ABI) to carry out according to the indication of manufacturers.It is that the PCR choosing of tail end is grown carrier pCR2.1 (it is to derive from Invitrogen, and Carlsbad CA), and is used to transform the competent cell (Invitrogen) of OneShot Top10F ' that PCR product through identifying is connected to TA.Kantlex (Kanamycin) resistance transformant carries out restriction enzyme analysis via the plasmid DNA that alkaline dissolution method is made and is screened.Identifying an insert fragment that is about 500bp also cuts with restriction enzyme NcoI and BamHI subsequently.Will this 500bp fragment in low melting point agarose gel, separate, and be connected to subsequently and derive from Novagen (Madison, the NcoI-BamHI position of T7 activity expression vector pET28a WI).
This connection mixture is with after transform and to send into Top1OF ' cell (Invitrogen), and the Kanr transformant carries out restriction enzyme analysis via the plasmid DNA that alkaline dissolution method is made and screened.Regroup plasmid (pLP505) is with after transform and to send into BL21 (Novagen) cell and it is grown in the SOB substratum of the kantlex that adds 30 mcg/ml.Cell grows to 0.D.600 value 0.6, and (Boehringer Mannheirn, Indianapolis Indiana) bring out 2-4 hour with 0.4mMIPTG subsequently.Prepare full cytolysis thing and on 14%SDS-PAGE glue, carry out electrophoresis.With glue with the blue dyeing of Kao Mashi (Coomassie) and detect expression product.The blue painted glue of this Kao Mashi is presented at the band between 28kDa and the 18kDa molecular weight marker, and is about 20kDa after measured.
The dna sequence dna of insert uses ABI 370A dna sequencing instrument to obtain in this regroup pLP505 plasmid.(Oxford Molecular Group, Campbell CA) compares to use the MacVector DNA matrix dot of Pustell to paint feature the dna sequence dna of this dna sequence dna and SEQ ID NO:1.To derive from the pLP505 plasmid, the dna sequence dna of SEQ ID NO:1 and streptococcus pneumoniae (serotype 4) genome is compared, the open reading frame (ORF) of this 20kD protein of code displaying may for big ORF partly, promptly coding has the 2163bp (SEQ IDNO:4) of the protein of about 79kDa predicted molecular weight in the genome of serotype 4.The predicted amino acid 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 GenomicResearch 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 79kDa and carry out sequence relatively.Can observe among the SEQ ID NO:2 1-58 amino acid and 90-132 amino acid respectively with SEQ ID NO:5 in 170-227 amino acid and 258-300 amino acid whose sequence consistent basically.Comprising the protein of these special areas and peptide or polypeptide is the preferable concrete example of the present invention.
Based on the genomic dna that can get (serotype 4) sequence, design also uses ABI 380A dna synthesizer to synthesize (SEQ ID NO:8 and 9) at the primer of 2163bpORF both sides subsequently.SEQ ID NO:8 is a streptococcus pneumoniae 5 ＇-primer, and it has the NcoI position of insertion and adds the Glu residue to keep a correct open reading frame after the ATG initiator codon.SEQ ID NO:9 is a streptococcus pneumoniae 3 '-primer, and it has the HindIII position of insertion.
5’-AGA?GCT?CCT?CCC?ATG?GAA?GAT?CCG?AAT?TAT?CAG-3’；(SEQ?ID?NO：8)
5’-CCG?GGC?AAG?CTT?TTA?CTT?ACT?CTC?CT-3’；(SEQ?ID?NO：9)
One about 2100bpDNA fragment from 4 different streptococcus pneumoniae serotypes (serotype 3,5,6B and 7) amplification, obtains 4 fragments subsequently.4 fragments are connected to PCR choosing growing property carrier 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 370ADNA sequenator to obtain the clone's of serotype 7 dna sequence dna.This dna sequence dna is same as SEQ ID NO:4 basically and encodes a predicted amino acid sequence that is same as SEQ ID NO:5 basically.
Haveing the knack of this operator will understand that, though the present invention is in above being described according to specific embodiments and embodiment, the present invention is not so limited certainly, and can carry out many other concrete examples, example, purposes is revised and at these concrete examples, situation beyond example and the purposes, and can not depart from the application's case it
Invention scope.
Claims (61)
1. an isolated protein is characterized in that, the sequence identity of itself and SEQ ID NO:2 tool at least 80%, and it can degradation of human complement protein C3.
2. protein as claimed in claim 1 is characterized in that, it is to separate from streptococcus pneumoniae.
3. protein as claimed in claim 1 is characterized in that, it is a recombinant protein.
4. protein as claimed in claim 1 is characterized in that, its molecular weight is that about 15kDa is to about 25kDa.
5. isolated peptides or polypeptide is characterized in that it comprises from least 15 continuous amino acids in the protein as claimed in claim 1.
6. isolated peptides or polypeptide is characterized in that it comprises at least 15 continuous amino acids among the SEQ ID NO:2.
7. an isolated protein is characterized in that, it comprises SEQ ID NO:2.
8. protein as claimed in claim 7 is characterized in that, its molecular weight is that about 15kDa is to about 25kDa.
9. protein as claimed in claim 8 is characterized in that, it is to separate from streptococcus pneumoniae.
10. protein as claimed in claim 8 is characterized in that, its degradation of human complement protein C3.
11. protein as claimed in claim 7 is characterized in that, it is SEQ ID NO:2.
12. an isolated protein is characterized in that, it comprise among the SEQ ID NO:2 from about the 1st to about the 58th amino acid.
13. protein as claimed in claim 12 is characterized in that, itself so that comprise among the SEQ ID NO:2 from about the 90th to about the 132nd amino acid.
14. an isolated protein is characterized in that, it comprise among the SEQ ID NO:5 from about the 170th to about the 227th amino acid.
15. protein as claimed in claim 14 is characterized in that, itself so that comprise among the SEQ ID NO:5 from about the 258th to about the 300th amino acid.
16. the isolated protein of a degradation of human complement protein C3 is characterized in that, the nucleic acid of this protein of encoding can be hybridized under the height stringent hybridization condition with SEQ ID NO:1 or its complementary strand.
17. one kind from about 15kDa of streptococcus pneumoniae isolated protein to about 25kDa, it is characterized in that it can human complement C 3-degrading.
18. immunity system irritating compositions, it is characterized in that, it comprises the immunity system stimulatory peptides or the polypeptide of a significant quantity, this peptide or polypeptide have at least 15 continuous amino acids derived from a kind of protein, and this protein and SEQ ID NO:2 have at least 80% sequence identity and energy degradation of human complement protein C3.
19. immunity system irritating compositions as claimed in claim 18 is characterized in that, this protein system separates from streptococcus pneumoniae.
20. immunity system irritating compositions as claimed in claim 19 is characterized in that, itself so that comprise at least a other immunity system pungency protein, peptide or polypeptide from the streptococcus pneumoniae separation.
21. an immunity system irritating compositions is characterized in that, it comprises one of treatment significant quantity proteinic at least one part, and the nucleic acid of this protein of wherein encoding can be hybridized under the hybridization conditions of highly strictness with SEQ ID NO:1 or its complementary strand.
22. immunity system irritating compositions, it is characterized in that, it comprises at least one part and the pharmaceutically acceptable carrier of the protein as claimed in claim 17 of effective deal, wherein this protein immunity or infection or the intrusion breeding of treatment mammalian subject to resist streptococcus pneumoniae effectively.
23. immunity system irritating compositions as claimed in claim 22 is characterized in that, this protein system is can effectively providing the deal of result of treatment to be supplied with for this mammalian subject.
24. an antibody is characterized in that, it can have the protein bound of at least 80% sequence identity and energy degradation of human complement protein C3 with SEQ ID NO:2.
25. antibody as claimed in claim 24 is characterized in that, it is a monoclonal antibody.
26. antibody as claimed in claim 24 is characterized in that, it is to derive from mouse, rat, goat, chicken, the mankind, or rabbit.
27. one kind can with at least one part bonded antibody of a protein, it is characterized in that the nucleic acid of this protein of encoding can be hybridized with SEQ ID NO:1 or its complementary strand under the hybridization part of highly strictness.
28. a separating acid fragment is characterized in that, it can be hybridized under the hybridization part of highly strictness with SEQ ID NO:1 or its complementary strand.
29. nucleic acid fragment as claimed in claim 28 is characterized in that, it is to separate from streptococcus pneumoniae.
30. nucleic acid fragment as claimed in claim 28 is characterized in that, encode at least one part of a protein of this nucleic acid fragment.
31. nucleic acid fragment as claimed in claim 30 is characterized in that, this protein degradation human complement C 3.
32. nucleic acid fragment as claimed in claim 28 is characterized in that it is in nucleic acid carrier.
33. nucleic acid fragment as claimed in claim 32 is characterized in that, this carrier is the expression vector that can produce at least one part of a protein.
34. a cell is characterized in that it comprises nucleic acid as claimed in claim 28.
35. cell as claimed in claim 34 is characterized in that, this cell is bacterium or eukaryotic cell.
36. a separating acid fragment is characterized in that, it comprise in SEQ ID NO:1 or its complementary strand about the 1st to about the 174th Nucleotide.
37. separating acid fragment as claimed in claim 34 is characterized in that, itself so that comprise SEQ ID NO:1 or its complementary strand in about the 320th to about the 492nd Nucleotide.
38. a separating acid fragment is characterized in that it comprises the nucleotide sequence of SEQ ID NO:1 or its complementary strand.
39. a RNA fragment is characterized in that, it is transcribed by the double chain DNA sequence that comprises SEQ ID NO:1 or its complementary strand.
40. one kind in the create antagonism immunoreactive method of streptococcus pneumoniae of Mammals, it is characterized in that, it comprises a composition is applied to mammiferous step with this proteinic immune response that creates antagonism, said composition comprises one of treatment significant quantity proteinic at least one part, and the nucleic acid of this protein of wherein encoding can be hybridized under the hybridization conditions of highly strictness with SEQID NO:1 or its complementary strand.
41. method as claimed in claim 40 is characterized in that, this immune response can be the B cell response, t cell responses, epithelial cell reaction or endotheliocyte reaction.
42. method as claimed in claim 40 is characterized in that, at least one part of a protein is at least 15 amino acid lengths.
43. method as claimed in claim 40 is characterized in that, said composition and then comprise at least a other immunity system stimulatory peptides, polypeptide or protein from streptococcus pneumoniae.
44. method as claimed in claim 40 is characterized in that, at least one part of a protein comprises at least 15 amino acid among the SEQID NO:2.
45. a method that suppresses streptococcus pneumoniae-media C3 Degradation is characterized in that it step that comprises is: the streptococcus pneumoniae bacterium is contacted an antibody, and this antibody can have the protein bound of at least 80% sequence identity with SEQ ID NO:2.
46. the method for a repulsive interaction when suppressing C3-media inflammatory response and xenotransplantation is characterized in that, expresses the protein with SEQ ID NO:2 aminoacid sequence on the used animal organ surface when it is included in xenotransplantation.
47. an isolated nucleic acid molecule is characterized in that, it comprises the zone of one at least 15 Nucleotide and can hybridize under the hybridization conditions of highly strictness with at least one zone in SEQ ID NO:1 or its complementary strand.
48. an isolated nucleic acid molecule is characterized in that, it comprises the sequence that can hybridize with at least one zone in SEQ ID NO:1 or its complementary strand under the hybridization conditions of highly strictness, wherein should zone system be selected from Nucleotide 1-174 and 320-492.
49. an isolated nucleic acid molecule is characterized in that, it comprises the zone of one at least 15 Nucleotide and can hybridize under the hybridization conditions of highly strictness with at least one zone in SEQ ID NO:4 or its complementary strand.
50. an isolated nucleic acid molecule is characterized in that, it comprises the sequence that can hybridize with at least one zone in SEQ ID NO:4 or its complementary strand under the hybridization conditions of highly strictness, wherein should zone system be selected from Nucleotide 507-681 and 827-999.
51. nucleic acid molecule as claimed in claim 49 is characterized in that, its proteinic at least one part of encoding.
52. nucleic acid molecule as claimed in claim 51 is characterized in that, this protein has the predicted amino acid sequence shown in SEQ ID NO:5.
53. a separating acid fragment is characterized in that, it has to be selected from and comprises SEQ ID NO:6, SEQ IDNO:7, the nucleotide sequence of the group of SEQ ID NO:8 and SEQ ID NO:9.
54. an immunity system irritating compositions that comprises at least one part of one of treatment significant quantity protein, the nucleic acid of this protein of wherein encoding can be hybridized under the hybridization part of highly strictness with SEQ ID NO:4 or its complementary strand.
55. one kind in the create antagonism immunoreactive method of streptococcus pneumoniae of Mammals, it is characterized in that, it comprises a composition is applied to mammiferous step with this proteinic immune response that creates antagonism, said composition comprises one of treatment significant quantity proteinic at least one part, and the nucleic acid of this protein of wherein encoding can be hybridized under the hybridization conditions of highly strictness with SEQID NO:4 or its complementary strand.
56. immunity system irritating compositions, it is characterized in that, it comprises at least one part and the pharmaceutically acceptable carrier of a protein as claimed in claim 51 for the treatment of significant quantity, wherein this protein immunization or infection or the intrusion breeding of treatment Mammals to resist streptococcus pneumoniae effectively.
57. immunity system irritating compositions as claimed in claim 56 is characterized in that, this protein system is can effectively providing the deal of result of treatment to be supplied with for this mammalian subject.
58. immunity system irritating compositions as claimed in claim 56 is characterized in that, said composition is a vaccine.
59. a peptide species is characterized in that, its tool SEQ ID NO:5.
60. immunity system irritating compositions as claimed in claim 23 is characterized in that, the coded protein of this nucleotide sequence or its complementary strand can suppress transcribing or translating of at least one endogenous streptococcus pneumoniae nucleotide sequence.
61. immunity system irritating compositions as claimed in claim 56 is characterized in that, the coded protein of this nucleotide sequence or its complementary strand can suppress transcribing or translating of at least one endogenous streptococcus pneumoniae nucleotide sequence.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US5990797P | 1997-09-24 | 1997-09-24 | |
| US60/059,907 | 1997-09-24 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1291233A true CN1291233A (en) | 2001-04-11 |
Family
ID=22026061
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN98809460A Pending CN1291233A (en) | 1997-09-24 | 1998-09-24 | Human complement (3-degrading proteinase from i (streptococcus pheumoniae) |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP1017828A1 (en) |
| JP (1) | JP2001517449A (en) |
| KR (1) | KR20010024299A (en) |
| CN (1) | CN1291233A (en) |
| AU (1) | AU9510598A (en) |
| BR (1) | BR9812525A (en) |
| CA (1) | CA2305016A1 (en) |
| WO (1) | WO1999015675A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102731739A (en) * | 2012-07-05 | 2012-10-17 | 富阳经略化工技术有限公司 | Star polymer used as lubricating oil viscosity index improver, preparation method and application thereof |
Families Citing this family (26)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6245335B1 (en) | 1996-05-01 | 2001-06-12 | The Rockefeller University | Choline binding proteins for anti-pneumococcal vaccines |
| DE69737125T3 (en) | 1996-10-31 | 2015-02-26 | Human Genome Sciences, Inc. | Streptococcus pneumoniae antigens and vaccines |
| US6676943B1 (en) | 1997-04-24 | 2004-01-13 | Regents Of The University Of Minnesota | Human complement C3-degrading protein from Streptococcus pneumoniae |
| EP1790729A3 (en) * | 1998-07-27 | 2007-07-25 | Sanofi Pasteur Limited | Streptococcus pneumoniae proteins and nucleic acid molecules |
| US6936252B2 (en) | 1998-07-27 | 2005-08-30 | Microbial Technics Limited | Streptococcus pneumoniae proteins and nucleic acid molecules |
| EP1115875A1 (en) * | 1998-09-24 | 2001-07-18 | Regents Of The University Of Minnesota | Human complement c3-degrading polypeptide from streptococcus pneumoniae |
| ATE422899T1 (en) | 1998-12-21 | 2009-03-15 | Medimmune Inc | STREPTOCOCCUS PNEUMONIAE PROTEINS AND IMMUNOGENIC FRAGMENTS FOR VACCINES |
| AU2005209689B2 (en) * | 1998-12-23 | 2008-07-17 | Id Biomedical Corporation Of Quebec | Novel streptococcus antigens |
| TR200200633T2 (en) * | 1998-12-23 | 2002-06-21 | Shire Biochem Inc. | New streptococcus antigens |
| US7128918B1 (en) | 1998-12-23 | 2006-10-31 | Id Biomedical Corporation | Streptococcus antigens |
| EP1950302B1 (en) * | 1998-12-23 | 2012-12-05 | ID Biomedical Corporation of Quebec | Streptococcus antigens |
| CA2413450C (en) * | 2000-06-20 | 2014-02-18 | Shire Biochem Inc. | Streptococcus antigens |
| US7074415B2 (en) | 2000-06-20 | 2006-07-11 | Id Biomedical Corporation | Streptococcus antigens |
| GB0022742D0 (en) | 2000-09-15 | 2000-11-01 | Smithkline Beecham Biolog | Vaccine |
| EP1355918B9 (en) * | 2000-12-28 | 2012-01-25 | Wyeth LLC | Recombinant protective protein from $i(streptococcus pneumoniae) |
| US7262024B2 (en) | 2001-12-20 | 2007-08-28 | Id Biomedical Corporation | Streptococcus antigens |
| WO2007047995A2 (en) * | 2005-10-21 | 2007-04-26 | Catalyst Biosciences, Inc. | Modified proteases that inhibit complement activation |
| TWI457133B (en) | 2005-12-13 | 2014-10-21 | Glaxosmithkline Biolog Sa | Novel composition |
| EP3020411A1 (en) | 2005-12-22 | 2016-05-18 | GlaxoSmithKline Biologicals s.a. | Vaccine |
| GB0607088D0 (en) | 2006-04-07 | 2006-05-17 | Glaxosmithkline Biolog Sa | Vaccine |
| WO2009000825A2 (en) | 2007-06-26 | 2008-12-31 | Glaxosmithkline Biologicals S.A. | Vaccine comprising streptococcus pneumoniae capsular polysaccharide conjugates |
| EA201001479A1 (en) | 2008-04-16 | 2011-06-30 | Глаксосмитклайн Байолоджикалс С.А. | VACCINE |
| GB201003924D0 (en) | 2010-03-09 | 2010-04-21 | Glaxosmithkline Biolog Sa | Immunogenic composition |
| GB201003920D0 (en) | 2010-03-09 | 2010-04-21 | Glaxosmithkline Biolog Sa | Method of treatment |
| WO2012156391A1 (en) | 2011-05-17 | 2012-11-22 | Glaxosmithkline Biologicals S.A. | Vaccine against streptococcus pneumoniae |
| GB201518684D0 (en) | 2015-10-21 | 2015-12-02 | Glaxosmithkline Biolog Sa | Vaccine |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69737125T3 (en) * | 1996-10-31 | 2015-02-26 | Human Genome Sciences, Inc. | Streptococcus pneumoniae antigens and vaccines |
| CN1253589A (en) * | 1997-04-24 | 2000-05-17 | 美国明尼苏达州大学 | Human complement C3-degrading proteinase from streptococcus pneumoniae |
-
1998
- 1998-09-24 CN CN98809460A patent/CN1291233A/en active Pending
- 1998-09-24 WO PCT/US1998/020186 patent/WO1999015675A1/en not_active Application Discontinuation
- 1998-09-24 JP JP2000512965A patent/JP2001517449A/en active Pending
- 1998-09-24 AU AU95105/98A patent/AU9510598A/en not_active Abandoned
- 1998-09-24 KR KR1020007003198A patent/KR20010024299A/en not_active Application Discontinuation
- 1998-09-24 CA CA002305016A patent/CA2305016A1/en not_active Abandoned
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102731739A (en) * | 2012-07-05 | 2012-10-17 | 富阳经略化工技术有限公司 | Star polymer used as lubricating oil viscosity index improver, preparation method and application thereof |
| CN102731739B (en) * | 2012-07-05 | 2013-11-06 | 富阳经略化工技术有限公司 | Star polymer used as lubricating oil viscosity index improver, preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20010024299A (en) | 2001-03-26 |
| EP1017828A1 (en) | 2000-07-12 |
| WO1999015675A1 (en) | 1999-04-01 |
| BR9812525A (en) | 2000-07-25 |
| AU9510598A (en) | 1999-04-12 |
| CA2305016A1 (en) | 1999-04-01 |
| JP2001517449A (en) | 2001-10-09 |
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