CN1531653A - Method for identification of proteins from intracellular bacteria - Google Patents

Method for identification of proteins from intracellular bacteria Download PDF

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CN1531653A
CN1531653A CNA028091140A CN02809114A CN1531653A CN 1531653 A CN1531653 A CN 1531653A CN A028091140 A CNA028091140 A CN A028091140A CN 02809114 A CN02809114 A CN 02809114A CN 1531653 A CN1531653 A CN 1531653A
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阿兰·克里斯蒂安·肖
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布赖恩·伯格·旺达赫尔
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Abstract

The present invention relates to a novel combination of methods that enables identification of proteins secreted from intracellular bacteria regardless of the secretion pathway. The invention further provides proteins that are identified by these methods. Secreted proteins are known to be suitable candidates for inclusion in immunogenic compositions and/or diagnostic purposes. The invention also provides peptide epitopes (T-cell epitopes) from the identified secreted proteins, as well as nucleic acid compounds that encode the proteins. The invention further comprises various applications of the proteins or fragments thereof, such as pharmaceutical and diagnostic applications.

Description

Be used to identify the method for proteins from intracellular bacteria
Technical field
The present invention relates to a kind of new combined method, this method can be identified with the protein of any secretory pathway by the intracellular bacteria secretion.The present invention further provides the protein that identifies by these methods.Known secretory protein is to be suitable for being contained in the immunogenic composition and/or the suitable candidate of diagnostic purpose.The present invention also provides the peptide epitopes (T-cell epitope) that is derived from certified secretory protein and the nucleic acid substances of code for said proteins.The present invention further comprises the multiple application of described protein or its fragment, such as medicine or diagnostic application.
Technical background
Chlamydia (chlamydia) is the obligate intracellular bacteria, and it is bred in eukaryotic host cell and is important human pathogen.Chlamydiales comprises a section of containing a genus (chlamydiaceae), and this genus is divided into four kinds: chlamydia trachomatis (C.trachomatis), Chlamydia pneumoniae (C.pneumoniae), chlamydia psittaci (C.psittaci) and cat heart Chlamydia (C.pecorum).
The serotype of the human cause of disease of chlamydia trachomatis is divided into: the A-C that causes eye illness; With trafficability characteristic D to K that propagate and that cause urethritis or complication such as salpingitis, epididymitis and ectopic pregnancy; And the L1 to L3 that causes whole body severe infections, lymphogranuloma venereum (LGV).The human pathogen Chlamydia pneumoniae causes causing the respiratory tract infection of bronchitis and pneumonia, and thinks recently and cause atherosclerotic relevant (Saikku etc., 1988[1.], Shor etc., 1992[2.]).
If choamydiae infection may develop into chronic disease without treatment and with severe complication such as formation infertile, blind and that may cause thrombus.
Reply because the choamydiae infection that the growth circulation continues in the born of the same parents may cause abnormal immune, it can not remove biosome.Many immunogenicity I (chlamydia) protein matter have been considered as vaccine candidate object, and especially protein such as the major outer membrane protein (MOMP) [7.] that immunodominant surface exposes in chlamydia trachomatis also has stress protein matter such as Hsp60[8.].Yet, do not have in these material standed fors a kind ofly in vaccine test, to be proved to be effective.
Explain it is character in the born of the same parents of biosome for a kind of possibility of the faint immunity of limited humoral response and protectiveness.Therefore a kind of alternative method be seek can by cell-mediated immune system recognition, be proved the protein that in the process of main effect elimination choamydiae infection by CTL (CTL), plays a crucial role (Iguitseme etc., 1994[9.]).
Since these secretory proteins can be at host cell proteins matter enzyme body (proteasome) thus in process and offered cell surface and have the effect of tangible vaccine target as MHC-I type antigen, so these secretory proteins have caused people's very big concern (Hess and Kaufmann, 1993[45]).A cell that example is infected by Yersinia ruckeri of relevant this respect, this cell are offered the epi-position of YopH effector molecules to MHC restrictive cell poison T lymphocyte (CTL) (Starnbach ﹠amp; Bevan, 1995[14.]).Interaction between Chlamydia and the host cell is survived in born of the same parents and bred for bacterium is essential.
(the Stephens etc. at chlamydia trachomatis serotype D have been arranged, 1998 [4.]) the searchable chlamydia trachomatis gene group of complete sum of (comprise 894 prediction open read frames (ORF)) and Chlamydia pneumoniae VR1310 (comprising 1073 ORF) (Kalman etc., 1999[5.]).In addition, the complete genome group of chlamydia trachomatis MoPn (Read etc., 2000[12.]), Chlamydia pneumoniae AR39 (Read etc. [12.] and Chlamydia pneumoniae J138 (Shirai etc., 2000[13.]) is that the public can access.Can learn that from genome sequence Chlamydia has the gene relevant with mechanism of secretion, comprise several and the III type secretor that is derived from other biosome have the gene of homology (Stephens etc., 1998[4.] and Kalman etc., 1999[5.]).
The effect protein matter of candidate's secretion may be present in the III type secretion subgroup (subclusters) (Subtil etc., 2000) [10.]).This viewpoint is illustrated (Fields ﹠amp by the III type secretion characteristic of finding CopN recently; Hackstadt, 2000) [11.].Yet III type secretory protein lacks discernible signal peptidase cleavage site, but also does not have the consensus sequence of fixed protein by the secretion of this system in the Chlamydia, and this may be confined to specific biosome to be measured.And secretory protein may be to be positioned at the protein group that the function on the unpredictable site is different in the genome (Subtil, 2000) [10.].
Be confined to be present in member (Rockey etc., 1995) [1 5.] [16.], CopN (the Fields ﹠amp that comprises Inc family with the existing knowledge of the Chlamydia effect protein qualitative correlation of being secreted; Hackstadt .2000[11.]) and CT529 (Fling etc., 2001) [37.] at the interior protein that includes in the film.
Verifiedly be specific to chlamydial CD8+T-cell and in course of infection, occur, mean that the kytoplasm that I (chlamydia) protein matter is exposed to host cell is the condition precedent of offering MHC I type antigen.From genomic library, identify CT529 (Probst) [41] by in eukaryotic, expressing and discerning by the Chlamydia specificity T cell line/lineage.Confirmed that CT529 contains epi-position, this epi-position can provide anti-infectious protective effect in the experiment of mouse vaccine.
Put down in writing by utilizing viral vectors to carry out transfection in eukaryotic the genomic library of expressing chlamydia trachomatis serotype L2 in the International Patent Application WO 00/34483 and utilize the Chlamydia specific T-cells to screen subsequently and measure the protein (Probst) [41] that contains I type MHC restricted epitope.Identify five positive colonies by this method, one of them clone contains CT529, and another clone contains three open read frames and its excess-three clone is not also further described.The shortcoming of this screening technique is that the eukaryotic expression of bacterioprotein is different from bacterial expression, the mode of this eukaryotic expression has changed the processing of protein enzyme body and as the probability of MHC angtigen presentation, and the T cell clone keep and stimulation is different from situation in the body, and unapproachable clone's identification of protein may produce false positive in conventional course of infection.Other method in the above-mentioned patented claim relates to the evaluation at the candidate vaccine of humoral immunity defence.
When seeking the protein of being secreted by intracellular bacteria, simple method is to isolate kytoplasm from infected host cell, seeks bacterioprotein then.Yet, because the fragility of Chlamydia reticulate body, and make this scheme can not be used for Chlamydia.Another kind method is to identify virulence factor by the transposons analysis, and this virulence factor is secretory protein normally.Yet the transfection Chlamydia is impossible.Also do not have the method for can predict what protein being secreted, and the gene of the coding effect protein matter that is suitable for developing vaccine may be positioned on the unknown site in the genome.
Therefore, need a kind of reliable system, this system can limit the number of candidate vaccine and comprise minimum experimental procedure in the cost-effective mode of cost.
In [18], by [35S]-methionine/halfcystine mark chlamydia trachomatis protein and after the 2D gel electrophoresis, carry out radioautograph and study the effect of IFN-γ to chlamydia trachomatis A and L2 protein expression.The IFN-γ that adds in the process with chlamydia trachomatis A infection HeLa cell culture produces tangible downward modulation effect to several chlamydia trachomatis A protein, and this effect is not obvious for chlamydia trachomatis L2.In chlamydia trachomatis A and L2, all observed～30 and～the IFN-γ dependence of 40kDa protein induces phenomenon.The L-tryptophane by add excusing from death reason (super-fysiological) amount to growth medium of inducing of these protein is inhibited.This shows relevant to the tremble rise of amine 2,3 dioxygenases of the inducing action of these chlamydia trachomatis protein and tryptophane degraded host cell enzymes Yin by IFN-γ mediation by IFN-γ mediation.L2 compares with chlamydia trachomatis, and the chlamydia trachomatis protein that a kind of IFN among the chlamydia trachomatis A induces is with lower molecular weight migration.
In [19], chlamydia trachomatis A that above-mentioned IFN-γ induces and L2 protein (Shaw etc., 1999) are by further qualitative.Utilize the MALDI-TOF mass spectroscopy and the database retrieval that carries out subsequently is defined as chlamydia trachomatis tryptophan synthetase α (TrpA) and β (TrpB) subunit with protein by preparation type 2D gel.Described protein can also be induced by IFN-γ in chlamydia trachomatis D, and described inducing by the L-tryptophane that adds super physiological amount is inhibited in three kinds of serotypes.Compare with L2 with chlamydia trachomatis D, the TrpA among the chlamydia trachomatis A is with lower molecular weight migration.By the trpA gene that derives from these chlamydia trachomatis serotypes is analyzed, show with chlamydia trachomatis D and compare that chlamydia trachomatis A and C TrpA are by brachymemma～7.7kDa with L2TrpA.May reduce the synthesis capability of tryptophane and make these serotypes be more vulnerable to the influence of disappearance of the tryptophane of IFN-γ mediation in the brachymemma that causes tryptophan synthetase in the serotype of trachoma (chlamydia trachomatis A, B and C) or disappearance.This point can be used for explaining the difference of pathogenesis between the human chlamydia trachomatis serotype.
In [43], studied the degradation of host protein enzyme body to the secretory protein (p60) of aforementioned intracellular bacteria monocyte Listeria monocytogenes (Listeria monocytogenes).Normally used method is to carry out on the basis of the pulse-chase method of utilizing [35S]-methionine/halfcystine mark under the situation that has or do not have two kinds of peptide aldehyde (peptidealdehude) to exist, described two kinds of peptide aldehyde are N-acetyl-Leu-Leu-nor-leucine (LLnL) and (phenyloxycarbonyl)-Leu-Leu-phenylalanine (Z-LLF), and it suppresses the proteolysis activity of eukaryotic protein enzyme body.The polyclonal antibody that produces at p60 be used to from handle by protein enzyme body inhibitor with untreated through the lysate of the J774 cell that monocyte Listeria monocytogenes infects immunoprecipitation p60.The radioautographic analysis that the immunoprecipitation mark p60 that separates by one-dimensional (one-dimensional) SDS PAGE is carried out shows that protein enzyme body inhibitor can suppress the protein enzyme body degradation of p60.By utilizing LLnL and Z-LLF to handle, the p60-CTL epi-position decreased number on each infection cell.Relevant between the generation of this protein enzyme body degradation that shows p60 and p60-CTL epi-position.
In [44], put down in writing defensive immunity mechanism and at the general characteristic of cells of microorganisms immune response in the born of the same parents, purpose is to develop the effective recombinant vaccine at microorganism in the born of the same parents.Attach most importance to Mycobacterium bovis (Mycobacterium bovis) BCG and salmonella typhi (Salmonellatyphi) aroA-the method for exploitation antigen transmission system has been discussed.Can carry out genetic modification so that transmit antigen to these avirulence intracellular bacterias, thereby can be used as the target thing of vaccine immunity identification.The literature author has pointed out that having advantage with secretory protein as the target thing that develops vaccine is because these protein can be processed and be offered to by cell-mediated immune system, and bacterium still can be duplicated in host cell.
The invention summary
The present invention includes by new combined method and identify secretory protein.The protein group that deducts proteins from intracellular bacteria in the gross protein group (proteome) of described combined method by the bacterioprotein from be present in infection cell constitutes secretory protein group (secretome) (aggregate of secretory protein).
Also by bidimensional electrophoresis and radioautograph bacterioprotein being carried out selectivity subsequently by pulse labeling under the situation that has the eukaryotic protein synthetic inhibitor to exist shows.The figure (profile) of the bacterioprotein of the purifying protein figure (profile) with total lysate of infected cell is compared, measuring method of mass spectrum by the advanced person identifies the protein spot that is present in the differential image, the secretory protein group from the gel of total lysate of having loaded infection cell.
Thereby provide to estimate it is the peptide sequence of effective t cell epitope by the secretory protein that the further Analysis and Identification of advanced person's artificial neural network goes out.
And, select its renewal (turnover) to be subjected to the protein that host cell proteins matter enzyme body inhibitor postpones, because these protein degraded might take place in host cell proteins matter enzyme body very much and offered host cell surface as MHC I type antigen.
Compare with other method of identifying vaccine candidate protein and epi-position, the present invention has realized that it only can reach by new combined method to the restriction of candidate albumen prime number purpose.
The present invention is based on following observations:
● be secreted in the bacterium that protein the host cell will not be present in purifying from intracellular bacteria and but be present in the complete lysate of infected cell.
● the 2D protein figure of purification of bacterial and the complete lysate of infected cell can utilize bacterioprotein special burst mark to carry out the bidimensional electrophoresis and obtain showing.
● the 2D protein figure that deducts purification of bacterial in the 2D protein figure of the bacterioprotein from be present in host cell can identify secretory protein by measuring method of mass spectrum.
● by the processing by host cell proteins matter enzyme body of the protein of intracellular bacteria secretion may produce can activated T cell MHC I type antigen.
● owing to the secretory protein that the adding of the inhibitor of eukaryotic protein enzyme body postpones to upgrade may be offered host cell surface.The evaluation of this protein realizes by the graphical analysis of 2D protein.
● the T-cell epitope can be predicted by artificial neural network, can be transformed into and can discern the epi-position that I type MHC compound is had the height affinity.
The present invention has used following definition:
Definition
Secretory protein group (Secretome)
Probably be the protein of secreting out from intracellular bacteria.
(III type) secretion subgroup
One group of chlamydia trachomatis gene comprises the gene that has very big homology with the III type secretor that derives from other biosome.Secretion group's definition is meant the aggregate of the ORF (open read frame) with inhibition or unknown function, it is positioned at the upstream of four genes, any gene that described four genes have known homology away from the relevant gene of III type secretory protein with other bacterium (for example salmonella, Shigella, Yersinia ruckeri).
Protein enzyme body inhibitor
Chemosynthesis or the naturally occurring compound of any 26S eukaryotic protein enzyme body protein hydrolysing activity that can reversible or irreversible inhibition be activated.Those skilled in the art can learn that the proteolysis activity of protein enzyme body comprises several different activities (chymotrypsin protein matter enzyme-sample activity of for example cutting after big hydrophobic residue, the tryptose matter enzyme-sample activity of behind alkaline residue, cutting, back-glutamy the hydrolytic enzyme activities that after acidic residues, cuts, the preferential BrAAP that behind branched-chain amino acid, cuts, the SNAAP that behind the little neutral amino acid of subunit, cuts).Several compounds of known Profilin matter enzyme body can be obtained by commercial sources, and mostly contain the permeable inhibitor based on peptide of cell (for example peptide aldehyde, peptide polyvinyl alcohol (PVA) sulfone).Inhibitor based on peptide can form a kind of adduct with protein enzyme body avtive spot as transition state analog, and is inhibited to protein enzyme body yet naturally occurring clasto-Lactacystin-β-lactone carries out irreversible modification by the avtive spot to protein enzyme body subunit.These compounds and composition thereof may be used to the function of Profilin matter enzyme body effectively and the processing of I type MHC-antigen (for example MG115, MG132, MG262, PSI, clasto-Lactacystin-β-lactone, epoxy rhzomorph (Epoxymycin)).Protein enzyme body inhibitor can be before carrying out pulse labeling or following the trail of, in the process or after any time point use in chlamydial whole growth cycle.
Host cell
Host cell is any eukaryotic, and this eukaryotic can be infected by intracellular bacteria.Those skilled in the art can know and comprise epithelial cell line (for example HeLa, Hep-2, bhk cell) or infinite multiplication monocytic series that for example U-937 is the suitable host of choamydiae infection at interior many immortal cell lines.The infinite multiplication cell can by abiogenous cancer obtain or by the virus that the carcinogenicity gene is carried in utilization transform primary cell, the result causes the unlimited differentiation of cell and growth (for example SV40) to obtain.The definition of host cell also comprises elementary epithelium or endothelium mammal cell line, and these clones can be obtained or be obtained by postmortem by the mammal that lives, at external breeding certain hour, and organ cell's culture.
The genetic modification host cell
Those skilled in the art should know proper host cell and also comprise the genetically modified host cell that can carry out overexpression or suppressor, the development of described gene and chlamydia vaccine is relevant, and for example the gene of coded protein enzyme body subunit or other are coded in the gene of the protein that I type MHC offers to play an important role in the process.
Protein enzyme body
Protein enzyme body is the important enzyme compound of non-lysosomal protein degraded, is an essential component in the ATP-dependence protein matter enzyme hydrolysis approach, the quick degraded of the multiple rate-limiting enzyme of catalysis, transcriptional regulatory agent and important adjusting protein.In most eukaryotes, be necessary to remove fast improper protein, aggegation, non-folding or normal host-derived cell protein and come from protein in the intracellular bacteria body that is positioned in the host cell.Protein enzyme body in the higher eucaryote body is delivered to peptide by protein degradation is become peptide the process that T-cell epitope that host cell surface is used as being offered participates in MHC I type antigen.
Pulse labeling
Labelled protein is meant that in a period of time (for example 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours) will contain radioactive isotope (L-[for example 35S]-methionine, the L-[methyl- 3H]-methionine, the L[methyl- 14C]-methionine, [ 35S]-halfcystine, [ 3H]-tryptophane or its composition) amino acid be incorporated in the bacterioprotein, in this process, adopt the eukaryotic protein synthetic inhibitor of enough high concentrations to suppress the synthetic of host cell proteins matter.Mark can carry out in the cycle in chlamydial whole growth.Thereby the mark nutrient culture media must have enough nutrition makes host cell and pathogen grow at infected cell growing period.In labeling process, can realize the inhibition synthetic, thereby make radioactivity amino acid only be incorporated in the intracellular bacteria of synthetic protein by adding synthetic other inhibitor (for example ipecine) of Cyclohexamide or host cell proteins matter to host cell proteins matter.Can prolong the degraded of protein by the cell-perviousness inhibitor that in nutrient culture media, adds protein degradation between mark phase.It should be noted that the present invention is not limited to radiolabeled use.
Pulse-chase
The protein that is labeled by tracking behind the protein synthesis can be assessed update time, for example in albumen quality synthetic between mark phase (for example 80%, 90%, 95%, 99%, 100%) time range of being degraded preferably more than 75%.The optical density of the given protein spot in the gel before or after during described assessment is followed the trail of by mensuration is carried out, and has shown the time length that protein exists in infected cell.By not replacing mark nutrient culture media and the different time points behind mark and reclaim infected cell and carry out described tracking with not containing the amino acid whose nutrient culture media of radioactivity.Can follow the trail of so that determine the time length that protein exists in infected cell in the different time points behind different times behind the mark (for example 0.5 hour, 1 hour, 1.5 hours, 2 hours, 6 hours, 12 hours, 24 hours, 72 hours) and the mark.The protein of some maturity state can be processed by propetide, thereby generating capacity accumulates and do not reduce during following the trail of.In this case, accumulate before observing the mature protein degraded in can be during following the trail of.Protein generation degradation time scope can be extended by add cell permeability protein degradation inhibitor in nutrient culture media in tracing process.
Lysis buffer
The lysis buffer that the present invention uses is the damping fluid that is used for the infected cell of cracking and makes the protein solubilising before carrying out the bidimensional gel electrophoresis.
Described lysis buffer contains the urea of 9M, the 3-[(3-courage amido propyl of 4%w/v) dimethylammonium]-1-propane sulfonic acid salt (CHAPS; Roche, Germany), the DTE of Tris Base, the 65mM of 40mM and the Pharmalyte 3-10 (Amersham Pharmacia Biotech) of 2%v/v.For the hydrophobic protein of enrichment high molecular, lysis buffer contains the urea of 7M, the sulphur urine of 2M, the 3-[(3-courage amido propyl of 4%w/v alternatively) dimethylammonium]-1-propane sulfonic acid salt (CHAPS; Boehringer Mannheim, Germany), the Pharmalyte 3-10 (AmershamPharmacia Biotech) of the dithioerythritol (DTE) of Tris Base, the 65mM of 40mM and 2%v/v.
The known solubleness (for example sulphur urine can increase hydrophobic and solubleness high molecular weight protein) that can increase some protein by the change lysis buffer.
The effect protein matter of secretion
The effect protein matter of term secretion is meant and anyly is secreted into protein in host cell kytoplasm or any born of the same parents' inner cell organ by bacterium.The effect protein matter of secretion may produce very big influence to the relation between host/pathogen, and because the existence in the host cell kytoplasm may and be offered the surface of host cell by targeting proteins matter enzyme body as MHC I type antigen.The effect protein matter of secretion can be secreted by some kinds of Sec-dependences putting down in writing in the document or dependent/non-dependent system (for example I type, II type, III type, IV type).
Intracellular bacteria
Any bacterium that in eukaryotic host cell (for example Chlamydia, salmonella, Shigella, Listera, Legionella, Yersinia ruckeri), has infection and fertility.This definition comprises intracellular bacteria, and this intracellular bacteria is an obligate in the born of the same parents, and its implication is that they only can utilize eukaryotic host cell survival or breeding, or facultative in the born of the same parents, and its implication is that it both can also can be survived by environment in born of the same parents in the outer survival of born of the same parents.
Substance (EB)
That obtain by the ultracentrifugation purifying and qualitative for the about 300nm of diameter and have the set of the Chlamydia bacterium of condensed nucleus (condensed nucleus) by electron microscopy.
Reticulate body (RB)
That obtain by the ultracentrifugation purifying and qualitative for the about 1000nm of diameter and have the set of the Chlamydia bacterium of standard bacterium nuclear by electron microscopy.
Analyze gel
Any 2D-PAGE gel that shows the protein example of protein requirement that loaded.The amount that in the embodiment that this paper put down in writing, is used for analysis purpose normally 200.000 to 300.000 reading/minute (cpm) or＞100ug be used to dye the protein (for example silver dyes, coomassie dyeing) of gel.
Significantly reduced intensity/content
Be preferably greater than 10% (for example 20%, 35%, 50%, 65%, 80%, 90%, 100%) but can repeat photometry density reduction, described optical density is to being positioned the integrated value of the given spot total area on the 2D-PAGE gel.
Remarkable intensity/the content that increases
Be preferably greater than 10% (for example 20%, 30%, 45%, 60%, 75%, 90%, 100%, 150%, 200%, 300% or higher) but can repeat photometry density recruitment, described optical density is to being positioned the integrated value of the given spot total area on the 2D-PAGE gel.
Preparation type gel
A kind of 2D-PAGE gel has loaded on it and can pass through the protein example that one of authentication method described herein (for example MALDI-MS, ESI-Q-TOF, Edman edman degradation Edman) is identified specified protein spot aequum.According to the Stationary pH gradient that is adopted, usually be used to prepare the amount that loads on the gel of purpose greater than 500 μ g.The definition of the preparation type gel that uses among the present invention comprise contain loose, utilize colouring method (for example silver dyes, coomassie dyeing) or electricity to be imprinted onto on the pvdf membrane and the gel of fixing protein.By with the labelled protein background applications to preparation type gel, might observe the protein on the preparation type gel, it is what to separate with non-marked protein.Thereby this gel can also be compared with the analysis gel and accurately cut destination protein matter.
Candidate vaccine
Based on the protein of the possibility of result that obtains by the inventive method by the intracellular bacteria secretion.Secretory protein is subjected to host cell proteins matter enzyme body easily and is degraded by the effect of the peptide of these protein derived, therefore can be used as the surface that MHC I type antigen is offered infected cell, thereby is discerned by the T-cell.This protein thus can become the development anti-intracellular bacteria vaccine clear and definite target material.The protein that is described to candidate vaccine can also be as the useful component of diagnostic test.
Vaccine
In the present invention, the term vaccine should be understood that a kind of immunogenic composition that can excite acquired immunity to reply (body fluid or cell) with implication the most widely.The candidate vaccine that can excite acquired immunity to reply can adopt injectable solution, suspension or emulsion form to be applied to the animal or human recipient.Candidate vaccine as the immunogenic composition active component can mix with pharmaceutical excipient such as water, salt solution, glycerine and ethanol before the recipient is given in injection.Can inject (for example hypodermic injection or intramuscular injection) in different ways.Candidate vaccine can i) with the form of total length, perhaps ii) so that immunogenic fragments to be provided, for example the source formation of T-cell epitope is as vaccine.Known can with specific protein or peptide separately or with other protein or peptide combined administration in the animal or human recipient and as vaccine.
And, can with coding candidate vaccine protein DNA fragment cloning in carrier, be introduced into the animal or human recipient by injection then.Described dna fragmentation is absorbed by for example muscle cell and can be in most eukaryotes expressing under the control of promoters active.In this so-called dna vaccination, the dna fragmentation of expression can stimulating immune system.
MHC I type antigen
Main histocompatbility I type antigen, contain the peptide different with the protein that is exposed to the host cell kytoplasm, the MHC I type molecule of the different dimerization in this peptide and the endoplasmic reticulum is puted together and is offered cell surface as the T-cell epitope, and described cell is connected in the groove (grove) of HLA compound (in the human body).Most MHC-I type is offered the peptide protein processed with passing through to activate 26S-protein enzyme body in the host cell kytoplasm different.
HLA
Human leucocyte antigen (HLA), the title of the main histocompatibility complex of people.
The T-cell epitope
The small peptide that combines with the MHC I type molecule of cell surface dimeric forms can for example be made up of 8-10 amino acid usually by the identification of the acceptor of specific cytotoxic T cell.
Full cell lysate
Directly in lysis buffer, reclaim, the infected host cell of purifying or separation in advance not.The lysate of known full cell can obtain at any time in Chlamydia growth cycle point, the lysate of described full cell contains the potpourri that is present in all protein in the infected cell, comprises that those derive from bacterium.
The bacterium of purifying
The bacterium of purifying from infected cell.With the Chlamydia is example, according to the different time points that reclaims in the chlamydial growth cycle, and can purifying RB and the Chlamydia of EB and intermediate forms by the density gradient ultracentrifugal method.Purity can be measured by the electron microscopic microscopy.In the present invention by a large amount of host cell proteins matter (for example actin matter, 'beta '-tubulin matter, alpha-tubulin matter, calprotectin matter) of dying on the 2D gel assessment hyperinfection at silver account for all proteins on the gel total optical density ratio and promptly detect purity.
The protein of identifying
The title of the protein that utilization is identified based on the authentication method of mass spectrum or Edman edman degradation Edman be according to be used for the chlamydia trachomatis gene batch total and draw Http:// socrates.berkeley.edu:Corresponding with the suitable mutually nomenclature of the nomenclature of the gene that obtains in 4231/ indicates, and described gene is documented in the following document:
i)Stephens，R.S.，Kalman，S.，Lammel，C.，Fan，J.，Marathe，R.，Aravind，L.，Mitchell，W.，Olinger，L.，Tatusov，R.L.，Zhao，Q.，Koonin，E.V.，Davis，R.W.(1998)Genome?sequence?of?an?obligateintracellular?pathogen?of?humans：Chlamydia?trachomatis.Science282：754-759
ii)Kalman，S.，Mitchell，W.，Marathe，R.，Lammel，C.，Fan，J.，Hyman，R.W.，Olinger，L.，Grimwood，J.，Davis，R.W.，Stephens，R.S(1999).Comparative?genomes?of?Chlamydia?pneumoniae?and?C.trachomatis.Nat?Gent.21：385-389。
EIISPOT
In the ELISPOT method, bag in advance by antibacterial agent (IFN-g for example, IL-6, TNF-a)-cultivate external through the T-of antigenic stimulus cell in the droplet hole (microtitter wells) of antibody.After cultivating a period of time, the T-cell peripheral part that the second antibody of having puted together enzyme such as horseradish peroxidase alkaline peroxide enzyme by adding may be displayed on activation has produced cell factor.Can be that the substrate of coloured product shows that thus the cell that produces cell factor estimates the output of cell factor by enzymatic conversion by adding at last.
Adjuvant
Adjuvant is meant a kind of specific immunogenic, emulsion (for example Freund) that can cause immune response in the mammal recipient's body that contains.Have realized that the adjuvant with immunogene can add such as compositions such as drying bacteria or bacterial products so that the immune response of enhancing in the mammalian body of quilt immunity.Perhaps, can also with immunomodifier such as lymphokine (IFNg for example, IL12) or polyinosinic acid (poly I:C) use with immunogene and adjuvant.
Seroconversion
Respond antigen and form antibody dissimilar or hypotype.
Microimmunofluorescence (MIF or little-IF)
A kind of detection of antibodies method of utilizing the immunofluorescence microscopy surveyingpin to immobilization bacterium or protein.
Immunogenicity
If protein or peptide are injected into and can cause the so described protein of adaptive immune response in human body or the animal body or peptide is exactly immunogenic.
Accompanying drawing is described
Figure 1AExpression comes from the gel figure example of chlamydia trachomatis D protein of the lysate of full cell, and described chlamydia trachomatis D protein is to carry out [35S]-mark in 22-24 hour and reclaim rapidly behind mark and separate by 2D-PAGE at (h.p.i.) after the infection.Black arrow refers to the significantly reduced protein of the intensity of protein on gel of the EB (substance) that comes from purifying, and is that the EB of described purifying is that (h.p.i.) carried out mark in 22-24 hour after infection and infect and carried out purifying in back 72 hours.The PI and the Mw characteristic of indication spot (DT1-77) are listed in the middle of the Table I.
Figure 1B-ERepresenting that certified candidate vaccine and the different time after synthetic thereof appear at the example in the chlamydia oculogenitale body, is to show after amplifying by the area that will select from Figure 1A.By following the trail of the update time of evaluating protein matter at whole growth cycle different time behind mark to the process of purifying EB.Above scale represent time point behind the zero point mark; Following scale representative was from 24 hours metainfective hourages.Figure 1B: DT1 and DT2 (CT668).Fig. 1 C:DT8.Fig. 1 D:DT7 (top arrow) and DT11 (following arrow) (both are confirmed as CT610).Fig. 1 E:DT3 (following arrow, CT783), DT4 (top arrow, CT858).
Fig. 2 AExpression comes from the gel figure example of Chlamydia pneumoniae VR1310 protein of the lysate of full cell, described Chlamydia pneumoniae VR1310 protein be (h.p.i.) after the infection 55-57 hour by [35S]-mark and also behind mark, reclaim rapidly and separate by 2D-PAGE.Black arrow refers to the significantly reduced protein of intensity on the gel of the protein that comes from EB, and is that this protein is that 6,12,24,36,42,48,54,60 hours whole growth is carried out mark with two hours cycle in the cycle after infection and infect and carried out purifying in back 72 hours.Arrow refers to the significantly reduced protein of intensity on the gel of the protein that comes from pure EB.The PI and the Mw characteristic of indication spot (CP1-91) are listed in the middle of the Table II.
Fig. 2 BThe part of presentation graphs 2A is amplified.
Fig. 2 C represents the corresponding enlarged image of the EB protein that 2D-PAGE separates, and wherein EB protein has carried out mark according to the method described above.The amplifier section of Fig. 2 B and Fig. 2 C has provided the example of two kinds of protein C P63 (being confirmed as CPN1016) and CP65, and described two kinds of protein all are present in the pyrolysis product of full cell and are not present among the EB.The circled spot that comes both had been present in the lysate that also is present in full cell in the middle of the EB and had been accredited as the polypeptide deformylase.
Fig. 3 AExpression is used for the DT1 spot is accredited as the peptide matter finger print (peptide mass fingerprint) of hypothetical protein matter CT668.Dotted arrow is represented the peptide that the autodigestion by tryptose matter enzyme produces.These peaks are used to make mark in the spectrum.Black arrow represents to be equivalent to the peptide material of CT668 sequence.Double-head arrow represents to derive from the peptide of people's infection protein matter.
Fig. 3 BThe result that the peptide material that expression utilizes evaluation software MS-Fit to be obtained by Fig. 3 A by analysis obtains shows that the highest (higest-ranking) chlamydia trachomatis protein is CT668.
Fig. 4 AThe peptide mass spectrum that expression is produced by the ESI-Q-TOF MS of spot DT1, wherein said spot DT1 contain is with (R) KIVDWVSSGEEILNR (A) (black arrow) of doubly charged 1744.9Da parent ion (parention), is equivalent to the amino acid sequence of CT668.Dotted line is meant the Y-peptide ion ion that is produced by the parent ion cracking.The amino acid sequence of inferring is represented with the runic one-letter code.
Fig. 4 BExpression is by the peptide mass spectrum of the PSD MALDI MS generation of spot CP63.1919.8Da parent ion (K) ELLFGWDLSQQTQQAR (L) is cleaved and produce the peptide of several displaying sequences.Wherein two kinds of peptides are illustrated as the 243.35Da b that quality is different from leucine quality (113Da) 2-ion (EL) and 356.34Da b 3-ion (ELL).
Fig. 5Represent the nucleotide sequence of novel chlamydia trachomatis specific candidate vaccine 3-protein d T8 and the corresponding amino acid sequence of representing by one-letter code.The runic amino acid sequence has the sequence mark thing that is obtained by ESI-Q-TOF MS.
Fig. 6The pulse-chase research that expression utilizes a kind of protein enzyme body inhibitor MG-132 to carry out simultaneously. Fig. 6 .1: loaded the complete gel figure of the 2D-gel of chlamydia trachomatis D protein, described chlamydia trachomatis D protein has carried out mark at metainfective 22-24 hour and has had under the 20 μ M MG132 existence conditions following the trail of 4 hours in addition. Fig. 6 A, 6B and 6C: contain the partial enlarged drawing of chlamydia trachomatis D protein, this figure show when with adopt (tracking+when the follow that MG-132) or not adopts (tracking) MG-132 to carry out compares, make prolongation update time owing to adopt MG-132 to handle.The protein figure (profiles) of the first displacement table infection cell that reclaim rapidly the back between two hours mark phase.Show and the gel phase ratio that has loaded the full cell lysate that reclaims rapidly behind the mark under the condition that does not have the MG132 existence, DT9 on the gel that has loaded the full cell lysate that carries out mark and tracking under the condition that has MG132 to exist, DT10 and DT11 intensity have had significant raising.
Fig. 7 AExpression utilizes the complete gel figure of IMB of the full cell lysate of PAb 245. A1: IMB represents the reaction with DT4 spot and DT48 spot, and DT4 spot and DT48 spot are respectively C-end and the N-end fragments of CT858. A2: the corresponding radioactive label background of IMB.
Fig. 7 B: anti-YscN ( B1) PAb241 and the IMB (spot DT1 and DT2) of the PAb238 of anti-CT668 ( B4).The corresponding radioautograph of IMB, the expression with B2: YscN and B5: CT668 is total to-location.YscN ( B3) and CT668 ( B6) in the location of analyzing on the 2D-gel.
Fig. 7 CThe IMB that represents the PAb255 of anti-CT610. C1: the enlarged drawing of 2D gel trace shows that PAb255 dyes to the spot of arranging, last displacement table DT7 and following displacement table DT9,10,11 and 12. C2: enlarged drawing show infection carried out in back 30 hours cell reclaim before with MG132 with 6 hours effect of infected cell processing.Notice that the MG132 processing makes the amount of DT9, DT10 and DT11 with respect to the amount of representing DT7 one row tangible increase arranged. C3: be labeled the analysis gel accordingly. C4: the SDS-PAGE of the IMB of PAb255: the complete infected product of cell lysis that the infection of swimming lane a and c:20 μ g and 10 μ g (difference) was reclaimed in back 30 hours.The complete infected product of cell lysis of swimming lane b and d:10 μ g and 5 μ g (difference), this complete infected product of cell lysis was handled 6 hours with 50 μ MMG132 before infection was reclaimed in back 30 hours.
Fig. 8Represent the testing result of indirect immunofluorescence microscopy, this result has shown the Subcellular Localization of candidate vaccine.A and B: infected by chlamydia trachomatis D and at back 24 hours HeLa 229 cells of infection with formalin fixed. C: by infection involving chlamydia pneumoniae and at back 72 hours HEp-2 cells of infection with formalin fixed.The 1st row's expression Normasky image.The reaction of the 2nd row expression and the MAb 32.3 of desertification chlamydia oculogenitale MOMP, this reaction are to show by the rhodamine that GAM IgG antibody has been puted together in employing.The 3rd row expression and the reaction that is specific to the rabbit polyclonal antibody for the treatment of candidate vaccine, this reaction are to show by the FITCH that GAR IgG antibody has been puted together in employing.The candidate vaccine that is studied is A: DT8, B: CT858, C: CPN1016 (the CT858 homolog (homologue) in the Chlamydia pneumoniae).The edge of the Chlamydia inclusion body in the infected cell of white single head arrow points.Empty arrow refers to non-infected cells.Notice that CPN1016 represents Subcellular Localization identical with CT858 and secretion characteristic.
Fig. 9The genome location of the candidate vaccine example that expression is identified from chlamydia trachomatis D, described vaccine comprises certified protein, this protein positioning is in inferior bunch 1,2 and 3 of III type secretion.
Figure 10The position of expression chlamydia trachomatis D candidate's secretion in the 2D gel images, described candidate's secretion derives from the lysate of complete infected cell, this infected cell carried out mark in 22-24 hour after infection, carried out RB mark and purifying and carried out the EB purifying in back 72 hours in infection at identical time point.All gels are to adopt non-linear pH3-10Immobiline Drystrips preparation.22-24hpi: the enlarged image of the chlamydia trachomatis D protein gel figure of the lysate that derives from complete infected cell shown in Figure 1A, described infected cell is to carry out [35S]-mark in 22-24 hour after infection, is reclaimed rapidly behind the mark and separates by 2D-PAGE.
The RB of purifying: derive from the respective regions on the gel images of chlamydia trachomatis D protein of bacterium, described chlamydia trachomatis D protein carried out mark in 22-24 hour and promptly has been engraved on later on to infect and was purified as RB in back 24 hours after infection.
The EB of purifying: the respective regions on the gel images of bacterioprotein, this protein carried out mark and were purified as EB in back 72 hours in infection after infection in 22-24 hour.
In row A-G, secretion material standed for DT4, DT48, DT23, DT76, DT77, DT47 and DT75 are circled next.
Figure 11Shown the position of Chlamydia pneumoniae VR1310 candidate secretion on the 2D-gel images, described candidate's secretion derives from the lysate of complete infected cell, described infected cell advanced mark in 55-57 hour after infection, the EB of purifying, the lysate of complete infected cell carried out mark and carried out RB mark and purifying at identical time point after infection in 34-36 hour:
55-57hpi: the enlarged image of the gel figure of the Chlamydia pneumoniae VR1310 protein that derives from full cell lysate shown in Fig. 2 A, described cell carried out [35S]-mark in 55-57 hour after infection, reclaimed rapidly behind the mark and utilize non-linear pH3-10ImmobilineDrystrip to separate by 2D-PAGE.
The EB of purifying: the respective regions on the bacterioprotein gel images (non-linear pH3-10Immobiline Drystrip), described bacterium promptly infected in back 6,12,24,36,42,48,54,60 hours to carry out mark two hours period and to be purified as EB in back 72 hours in infection in the whole growth cycle.
34-36hpi: derive from the respective regions (linear pH4-7Immobiline Drystrip) on the gel images of Chlamydia pneumoniae VR1310 protein of lysate of complete infected cell, described infected cell has carried out mark at 34-36 after the infection hour and has been reclaimed rapidly behind mark.
RB 36hpi: derive from the respective regions (linear pH4-7Immobiline Drystrip) on the gel images of Chlamydia pneumoniae VR1310 protein of bacterium, described protein promptly is engraved in to infect after carrying out mark in 34-36 after the infection hour and was purified as RB in back 36 hours.
In A-F, secretion material standed for CP34, CP37, CP46, CP47, CP52, CP63 and CP75 are circled next.
G is illustrated in the zone on the original image that does not contain the zone of secreting material standed among the A-F.
Detailed Description Of The Invention
Derive from the 2D-PAGE protein figure (profile) of the bacterium of full cell lysate and purifying Comparison
Be present in the complete infected cell and the protein that is not present in the purification of bacterial may be by bacterium, for example Chlamydia is secreted. Therefore, initial methods of the present invention be with the different time points of the comfortable growth cycle protein figure of 2D-PAGE protein figure and the 2D-PAGE of the purification of bacterial that carries out [35S]-mark at corresponding time point of I (chlamydia) protein matter of [35S]-mark of full cell lysate that carried out the infected cell of mark compares. The method provides the detection method of several protein, and described protein clearly appears on the protein figure of full cell lysate, and can only faintly detect or can't detect on the protein figure of purification of bacterial.
Studies show that of carrying out of about 600 protein spots that showed in full cell lysate at 22-24 after the infection hour by high-resolution 2D-PAGE (IPG) altogether wherein existed 77 kinds of chlamydia trachomatis D protein, and its intensity in substance (EB) obviously reduces. Similarly, when the full cell lysate of 55-57 hour-symbols and purifying EB compared after will infecting, the intensity of 91 kinds of protein in CPN VR1310 significantly reduced. Protein detected and note has Mw and the pI that describes such as the CP1-CP91 protein of the CPN listed in the Mw of the DT1-DT77 protein of the chlamydia trachomatis D that lists in the Table I and pI and the Table II.
The method has been summarized the necessary possible secretory protein of further research. Embodiment compares the EB of full cell lysate and purifying, the EB of described full cell lysate and purifying or at i) carry out the corresponding time point (chlamydia trachomatis of mark with full cell lysate, Fig. 1) or ii) (CPN Fig. 2) is labeled for each time point in whole growth cycle.
The purifying of RB can be differentiated secretory protein and RB-specific protein. Can with the protein figure of the complete lysate of infection cell with compare in order to identify secretory protein at the RB of same time point purifying protein figure. In the method, do not detect false-positive RB specific protein. The protein that detection is synthesized and secreted at the time point of studying. Protein also may be in At All Other Times synthetic and secretion.
Described method comprises that also the protein of described secretion may just be synthesized out in the early stage of growth cycle to the at once detection of the protein of secretion after infecting. These protein are by adopting the EB be labeled in the early stage of growth cycle to infect, then whole product of cell lysis being carried out 2D-PAGE and observe. Growth cycle before EB is divided into RB is early stage, obtains host cell kytoplasm [30,31] by the saponin permeates cell membranes. Owing to can not come the pollution of the I (chlamydia) protein matter of self destruction RB this moment, this only is a kind of possibility.
The evaluation of candidate vaccine
Identify by advanced mass spectrometry method from the candidate vaccine protein that preparation type 2D-gel cuts out.
Cut spot digests with enzyme such as tryptose matter enzyme, thereby produces the zymolytic peptide of many tryptose matter. By MALDI-MS or other method, the quality of these peptides can be higher than 100/1000000ths accuracy (ppm) and be determined. Utilize MS-Fit or Peptidesearch software that the theoretic tryptose matter enzymatic lysis product of all protein in the quality that obtains and the database is compared, thereby on statistical basis, analyzed protein is identified.
When protein spot is cut out from the gel that has loaded full cell lysate, polluting host cell proteins matter may be positioned on the identical position with bacterioprotein, and as complicated situation more, a spot can contain more than one bacterioprotein. In order to clearly identify, if necessary, for example can adopt ESI-Q-TOF or opisthogenesis decline (post source decay) (PSD) to obtain the sequence information of bacterioprotein for fear of the interference of pollutant.
Described method comprises the protein by Mass Spectrometric Identification, as in the Table III by (being respectively A and B) as illustrated in chlamydia trachomatis D or the CPN VR1310.
Therefore, first aspect present invention relates to the method for protein of identifying the intracellular bacteria secretion, comprises the following steps:
1) by the intracellular bacterial infections host cell,
2) mark is present in infected intracellular intracellular bacteria,
3) prepare a) the full cell lysate of infected cell
B) bacterium of purifying and cracking from infected cell,
4) to i) by step 3a) the full cell lysate and the ii that obtain) by step 3b) purifying that obtains and the 2D-gel electrophoresis protein figure of cracking bacterium compare,
5) detect by step 4) protein spot that obtains, described protein spot be present in the complete cell lysate and be not present in the bacterium of purifying or the content in the bacterium of purifying very low,
6) identify in step 5) in protein in the spot of selection.
Pulse/the follow-up analysis of candidate vaccine
The purpose of the method is to detect the secretory protein that is degraded in host cell. For the time that the candidate vaccine of assessing evaluation exists, carried out a series of pulse/follow in host cell. Monitor at 2D-gel by the follow-up analysis that behind mark protein is carried out different time the update time of [35S]-labelled protein. Be the degradation rate of definite protein this update time, and determine that thus the time that described protein exists in infected cell provides valuable information.
The method provides cited estimation update time that can secernent chlamydia trachomatis protein in the Table I.
Therefore, another aspect of the present invention relates to for the identification of the method for protein by the intracellular bacteria secretion, comprises the following steps:
1) use the intracellular bacterial infections host cell,
2) pulse labeling is present in infected intracellular intracellular bacteria,
3) follow step 2), behind the follow-up analysis that has carried out different time, prepare the full cell lysate of infected cell,
4) comparison step 3) the 2D-gel electrophoresis protein figure of full cell lysate behind the follow-up analysis of different time of preparation,
5) detect by step 4) protein spot that obtains, this protein spot is along with step 3) in its content of prolongation of tracking time also reducing,
6) identify in step 5) in protein in the spot of selection.
The pulse-chase analysis of conjugated protein enzyme body inhibitor
In order to limit the quantity of the material standed for that is suitable for use as vaccine, the present invention includes the proteasome inhibitor method in conjunction with pulse-chase research. These experiments provide favourable instrument for monitoring host cell proteins matter enzyme body to the impact of the update time of the I (chlamydia) protein matter of secretion.
Being offered the eukaryotic surface must be by at many-catalytic proteins complex as the immunogenic protein of MHC-I type antigen, i.e. proteolysis and by ubiquitin materialization (abiquitiny-lated) and cracking in the protease body. Described protease body is cracked into immunogenic protein the peptide of 8-10 amino acid typical length. These peptides are combined with different dimerization MHC I type molecule after being transported to ER (endoplasmic reticulum), and then the MHC-antigenic compound is transported to cell surface. At cell surface, the MHC-antigenic compound is identified (having carried out commenting [6.] in Rock and Goldberg) by the specific receptor on the cytotoxic T-lymphocytes.
By in cell culture, adding cell-penetrating proteasome inhibitor such as peptide aldehyde, might suppress the active of eukaryotic protein enzyme body and suppress the offering of MHC I type (Rock etc., 1994) [36.]. Being extended the I (chlamydia) protein matter of update time by the adding proteasome inhibitor can secrete from bacterium, is then processed by the protease body. In addition, this part content of the present invention can detect I (chlamydia) protein matter, because these I (chlamydia) protein matter are degraded rapidly by protease after being secreted in the host cell, so only under the condition that has proteasome inhibitor to exist, just can be detected.
The present invention includes chlamydia trachomatis D and CPN VR1310 candidate vaccine, it is subjected to the impact of proteasome inhibitor.
3-protein d T1, DT2, DT3, DT5, DT9, DT10, DT11, DT13, DT14, DT36, DT47, DT59, DT60, DT61, DT62 (listing in the following Table IV) are the examples of chlamydia trachomatis D candidate vaccine, prolong the update time of these protein during follow-up analysis by adding proteasome inhibitor.
The present invention also provides the method for purifying RB, and this RB processes through the protease body between mark phase before results. Purifying RB with the proteasome inhibitor processing compares to the full cell lysate processing through the protease body and carried out simultaneously mark, further specifies protein and is secreted in the host cell kytoplasm and by the protease body and degrades. In addition, the host cell system in these experiments can be become can carry out expression to gene by genetic modification, and this is in the process of the restricted T-cell epitope of MHC I type extremely important [38,39,40] (Sijts, 2000, Van Hall, 2000, Shockett, 1995). By utilize this clone clearer and more definite the effect of proteasome inhibitor. Therefore the present invention also comprises the purposes of the host cell system that is commercially available, and these host cells systems have carried out the gene genetic transformation, with MHC I type antigen offer relevant.
Therefore, the present invention relates on the other hand for the identification of the method for protein by the intracellular bacteria secretion, comprises the following steps:
1) use the intracellular bacterial infections host cell,
2) under the condition that has and do not have proteasome inhibitor to exist, cultivate host cell respectively,
3) intracellular bacteria in the infected cell of mark, this infected cell are cultivated under the condition that has and do not have proteasome inhibitor to exist respectively,
4) the full cell lysate of the infected cell of preparation,
The 2D-gel electrophoresis protein figure of the full cell lysate of the infected cell of 5) under the condition that has and do not have proteasome inhibitor to exist, cultivating more respectively,
6) detect by step 5) protein spot that obtains, this protein spot is present in the full cell lysate of cultivating in the presence of proteasome inhibitor is arranged, and the amount that does not exist or exist in the full cell lysate of cultivating in the presence of without proteasome inhibitor obviously reduces
7) identify in step 6) in protein in the spot of selection.
The generation of polyclonal antibody
The invention provides the polyclonal antibody that is specific to candidate vaccine. Utilize the gene that for example connects dependent/non-dependent clone (LIC)-system clones coding candidate vaccine protein. Thereby the fused protein that contains the candidate vaccine sequence that is expressed is used to the serum that immune rabbit obtains to contain the polyclonal antibody (PAb) that is specific to candidate vaccine. The present invention utilizes Pab with the correct specificity that confirms described antibody by common location or the isotype candidate vaccine of identifying the unknown in the 2D-PAGE Western blotting. The invention provides by Western blotting and be total to the location and verify/identify candidate vaccine, listed such as Table III. The present invention utilizes PAb to measure the Subcellular Localization of candidate vaccine by for example indirect immunofluorescence microscopy.
Therefore the present invention also provides a kind of alternative of said method, and the method also comprises the following steps:
1) obtain the anti-of the anti-described proteins from intracellular bacteria of identifying according to above-mentioned any method,
2) utilizing step 1) antibody that obtains carries out the 2D-PAGE Western blotting to the full cell lysate of the cell that infected by bacterium,
3) be determined at step 2) in the protein spot that reacts,
4) identify in step 3) in protein in the spot selected.
The combination of described four kinds of systems of selection also belongs to a part of the present invention.
In the optimal technical scheme of the inventive method, the mark of intracellular bacteria is to be undertaken by the method for radiation, such as [35S] cysteine, and [35S] methionine, the amino acid of [14C] mark or its composition.
Can be based on Edman edman degradation Edman or any measuring method of mass spectrum for the identification of the method for protein in the protein spot of selecting, such as MALDI TOF MS (Matrix-Assisted Laser Desorption/lonisation Time-Of-Flight Mass Spectrometry), the combination of ESI Q-TOF MS (Electrospray lonisation QuadrupIe Time-Of-Flight Mass Spectrometry), PSD-MALDI MS (Post Source Decay MALDI Mass Spectrometry) or these methods. And, described protein can carry out the chemical method cracking before evaluation, process or the azanol processing such as cyanogen bromide, perhaps utilize any suitable enzyme such as tryptose matter enzyme, slymotrypsin, chymotrypsin protein matter enzyme or stomach cardia matter enzyme or its composition to carry out enzymatic lysis.
Described intracellular bacteria can be facultative intracellular bacteria or obligate intracellular bacteria, and be derived from chlamydiaceae such as CPN, chlamydia trachomatis, chlamydia psittaci or the chlamydial bacterium of the cat heart, comprise that any specific serum type or the bacterial strain of these bacteriums especially receives publicity. Yet other intracellular bacteria such as salmonella, Shigella, Yersinia ruckeri or Listeria also receive concern of the present invention.
The host cell that the present invention uses can be common host cell known in the art, such as immortalized cell be, HeLa for example, Hep2, McCoy or U937, by mammal donor or the primary cell that obtains by becoming celestial system, the clone of genetic modification, or organ cell's culture or even the cell of other bacterium that growing. Described host cell can be before by intracellular bacterial infections or during utilize IFN-γ to process and/or become expressing gene by genetic modification or suppressed this gene expression, described gene is relevant with the development of chlamydia vaccine.
When method of the present invention had been used one or more proteasome inhibitors, any known inhibitor such as MG132, MG262, MG115, epoxy mycin, PSI and clasto-Lactacystin-β-lactone all were suitable for using.
Method of the present invention is particularly suited for identification of protein, the total length form of this protein or its immunogenic fragments form all are suitable for being included in the immunogenic composition and/or are used for diagnostic purpose, especially such protein, it contains the T-cell epitope, as MHC-I type or II type, preferred-material standed for that the restricted antigen of I type is offered, be suitable for being included in the immunogenic composition.
Therefore, another importance of the present invention relates to protein or its immunogenic fragments that can be identified by any desired method, and preferred this protein or its fragment are to be suitable for being included in immunogenic composition and/or to be suitable for diagnostic purpose.
Protein of the present invention can be the protein by chlamydia trachomatis and CPN secretion. This protein be for example those be accredited as respectively as shown in Table I DT1-77 and protein or its immunogenic fragments of CP1-CP91 as shown in Table II, have respectively the pI shown in Table I and the Table II and Mw value, the mean error of the pI that measures and Mw value is+/-10 %.
Table I
Protein spot      Pi      Mw
  DT1     4.45     23.5
  DT2     4.55     23.5
  DT3     4.55     34.5
  DT4     4.75     36.1
  DT5     4.83     11.4
Protein spot      Pi      Mw
  DT6     9.3     9.27
  DT7     4.85-4.9     24.8
  DT8     5.1     7.8
  DT9     4.73     23.7
  DT10     4.8     23.7
  DT11     4.85     23.7
  DT12     4.93     23.7
  DT13     6.05     24.3
  DT14     6.2     27.5
  DT15     6.1     32.4
  DT16     5.98     39
  DT17     6.28     55.2
  DT18     6.1     41.1
  DT19     6.1     47.9
  DT20     7.4     37.6
  DT21     7.7     34.7
  DT22     8.2     22.4
  DT23     4.83     30.4
  DT24     5     29.5
  DT25     5     12.6
  DT26     4.7     10.9
  DT27     5.15     13.5
  DT28     5.7     31.9
  DT29     4.97     54.8
  DT30     5.86     36
  DT31     5.78     36.2
  DT32     6.4     10.4
  DT33     6.3     13.3
  DT34     9.5     32.4
Protein spot      Pi      Mw
  DT35     8     46.5
  DT36     7.49     40.6
  DT37     7.15     37.6
  DT38     7.24     34.5
  DT39     7.44     46.5
  DT40     6.4     67.2
  DT41     5.04     56.4
  DT42     8.5     32.9
  DT43     8.5     30.5
  DT44     8.66     42.6
  DT45     8.85     43.1
  DT46     4.4     87.6
  DT47     5.4     41
  DT48     7.36     24.2
  DT49     9.25     47.4
  DT50     5.0     94
  DT51     5.35     100.5
  DT52     5.41     59.7
  DT53     5.97     23
  DT54     6.12     25.5
  DT55     5.34     36.4
  DT56     4.88     10.5
  DT57     4.87     18.5
  DT58     6.14     97
  DT59     4.5     19.7
  DT60     5.5     40.9
  DT61     5.5     39.9
  DT62     5.98     41.1
  DT63     6.9     46.8
Protein spot      Pi      Mw
  DT64     5.5     34.5
  DT65     4.5     68.2
  DT66     4.35     57.6
  DT67     6.13     66.5
  DT68     6     62.9
  DT69     5.85     65.6
  DT70     5.72     70.4
  DT71     5.5     44
  DT72     5.85     10.2
  DT73     4.45     30.5
  DT74     5.02     48.2
  DT75     4.37     21.9
  DT76     5.14     23.3
  DT77     5.64     23.0
Listed after infecting 24 hours the protein of the chlamydia trachomatis D secretion that contains in may be by full cell lysate, but in EB, significantly reduced, and the pi/Mw that assesses, the mean error of+/-10 %.
Table II
Title      Pi      Mw
  CP01     5.5     100.4
  CP02     6.7     91.0
  CP03     5.3     75.2
  CP04     5.4     68.7
  CP05     5.4     72.8
  CP06     5.5     68.6
  CP07     5.6     80.5
  CP08     5.7     74.1
  CP09     6.0     77.8
  CP10     6.0     71.2
  CP11     6.1     82.6
  CP12     6.2     68.4
  CP13     6.2     72.0
  CP14     5.4     64.8
  CP15     5.5     63.7
  CP16     5.8     61.1
  CP17     5.8     102.4
  CP18     6.1     63.6
  CP19     6.1     61.0
  CP20     6.5     64.0
  CP21     6.6     63.7
  CP22     5.0     60.9
  CP23     5.0     60.5
  CP24     5.6     60.4
  CP25     5.7     50.2
  CP26     6.3     57.0
  CP27     6.3     52.8
Title      Pi      Mw
    CP28     6.4     48.5
    CP29     4.9     50.6
    CP30     5.0     48.6
    CP31     4.9     473
    CP32     4.7     42.1
    CP33     4.9     439
    CP34     5.0     39.3
    CP35     5.1     42.9
    CP36     5.2     42.1
    CP37     5.3     40.7
    CP38     5.4     40.7
    CP39     5.6     40.3
    CP40     5.6     41.6
    CP41     5.9     41.8
    CP42     6.4     38.4
    CP43     6.2     44.3
    CP44     6.5     45.3
    CP45     6.8     45.3
    CP46     4.6     38.6
    CP47     4.6     37.8
    CP48     5.1     35.8
    CP49     5.3     38.9
    CP50     5.5     38.9
    CP51     5.6     33.6
    CP52     5.7     33.7
    CP53     5.9     34.9
    CP54     6.2     34.8
    CP55     6.2     34.7
    CP56     6.3     34.8
Title      Pi      Mw
    CP57     8.3     36.0
    CP58     8.7     36.1
    CP59     4.5     29.7
    CP60     4.8     26.0
    CP61     5.2     27.6
    CP62     5.4     30.6
    CP63     6.2     25.2
    CP64     6.6     26.3
    CP65     5.9     22.8
    CP66     4.7     24.2
    CP67     4.8     22.4
    CP68     5.1     24.1
    CP69     5.2     24.3
    CP70     5.3     22.3
    CP71     5.6     21.4
    CP72     6.9     17.8
    CP73     4.8     12.0
    CP74     5.0     8.9
    CP75     5.1     11.9
    CP76     6.5     9.3
    CP77     7.0     10.5
    CP78     7.2     10.4
    CP79     8.7     13.0
    CP80     5.7     93.3
    CP81     6.4     37.2
    CP82     6.9     45.0
    CP83     7.0     41.6
    CP84     7.1     38.6
    CP85     6.3     32.2
Title      Pi      Mw
    CP86     6.4     32.0
    CP87     8.8     31.3
    CP88     5.0     23.8
    CP89     4.7     73.0
    CP90     7.4     40.1
    CP91     7.8     37.7
Listed after infecting 55 hours the protein of the CPN secretion that contains in may be by full cell lysate, but in EB, significantly reduced, and the pI/Mw that assesses, the mean error of+/-10 %.
The preferred protein of the present invention is chlamydia trachomatis protein and CPN protein and immunogenic fragments thereof, described chlamydia trachomatis protein number is defined as CT017 (gene title CT017) such as what provide among those Table III A by the calculated corresponding gene of chlamydia trachomatis gene group, CT044 (gene title ssp), CT243 (gene title lpxD), CT263 (gene title CT263), CT265 (gene title accA), CT286 (gene title clpC), CT292 (gene title dut), CT407 (gene title dksA), CT446 (gene title euo), CT460 (gene title SWIB), CT541 (gene title mip), CT610 (gene title CT610), CT650 (gene title recA), CT655 (gene title kdsA), CT668 (gene title CT668), CT691 (gene title CT691), CT734 (gene title CT734), CT783 (gene title CT783), CT858 (gene title CT858), the protein of CT875 (gene title CT875) or ORF5 (gene title ORF5), or protein name is called the protein of DT8, described CPN be such as those as provide among the Table III B by corresponding gene called after CPN0152 (gene title CPN0152), CPN0702, CPN0705 (gene title CPN0705), CPN0711 (gene title CPN0711), CPN0998 (gene title ftsH), CPN0104 (gene title CPN0104), CPN0495 (gene title aspC), CPN0684 (gene title parB), CPN0796 (gene title CPN0796), CPN0414 (gene title accA), CPN1016 (gene title CPN1016), CPN1040 (gene title CPN1040), CPN0079 (gene title R110), CPN0534 (gene title dksA), CPN0619 (gene title ndk), CPN0711 (gene title CPN0711), CPN0628 (gene title rs13), CPN0926 (gene title CPN0926), the protein of CPN1063 (gene title tpiS) or CPN0302 (gene title IpxD).
Table III A
Protein spot Gene number* The gene title Authentication method      pl      Mw
    DT1     CT668     CT668     M，Q，I     4.45     23.5
    DT2     CT668     CT668     M，Q，I     4.55     23.5
    DT3     CT783     CT783     M，Q，I     4.55     34.5
    DT4     CT858     CT858     M，Q，I     4.75     36.1
    DT48     CT858     CT858     M.I     7.36     24.2
    DT7     CT610     CT610     M，E，I     4.85-4.9     24.8
    DT9     CT610     CT610     I     4.73     23.7
    DT10     CT610     CT610     I     4.8     23.7
    DT11     CT610     CT610     I     4.85     23.7
    DT12     CT610     CT610     I     4.93     23.7
    DT8     None     DT8 *     Q，I     5.1     7.8
    DT6     CT460     SWIB     M     9.3     9.27
    DT14     ORF5     ORF5     M     6.2     27.5
    DT22     CT446     euo     Q     8.2     22.4
    DT23     CT541     mip     M     4.83     30.4
    DT24     CT541     mip     M     5     29.5
    DT25     CT407     dksA     M，Q     5     12.6
    DT26     CT734     CT734     Q，I     4.7     10.9
    DT27     CT292     dut     M，Q     5.15     13.5
    DT28     CT655     kdsA     M，E     5.7     31.9
    DT30     CT265     accA     M     5.86     36
    DT35     CT017     CT017     M     8     46.5
    DT39     CT017     CT017     M     7.44     46.5
    DT36     CT243     lpxD     M     7.49     40.6
    DT37     CT650     recA     M，E     7.15     37.6
    DT57     CT044     ssp     M     4.87     18.5
    DT58     CT286     clpC     M     6.14     97
    DT69     CT875     CT875     M     5.85     65.6
    DT76     CT691     CT691     Q     5.14     23.3
    DT77     CT263     CT263     Q     5.64     23.0
Table III B
Protein spot Gene number The gene title Authentication method     pl      Mw
    CP34   CPN1016   CPN1016     I     5.0     39.3
    CP37   CPN0998   ftsH     M     5.3     40.7
    CP42   CPN0104   CPN0104     M     6.4     38.4
    CP46   CPN0796   CPN0796     Q     4.6     38.6
    CP47   CPN0705   CPN0705     M     4.6     37.8
    CP50   CPN0495   aspC     M     5.5     38.9
    CP52   CPN0152   CPN0152     M     5.7     33.7
    CP55   CPN0684   parB     M     6.2     34.7
    CP56   CPN0414   accA     M     6.3     34.8
    CP63   CPN1016   CPN1016     M     6.2     25.2
    CP71   CPN1040   CPN1040     M     5.6     21.4
    CP72   CPN0079   rl10     M     4.8     12.0
    CP73   CPN0534   dksA     M     5.0     8.9
    CP75   CPN0619   ndk     M     5.1     11.9
    CP76   CPN0711   CPN0711     M     6.5     9.3
    CP78   CPN0628   rs13     M     7.2     10.4
    CP79   CPN0926   CPN0926     M     8.7     13.0
    CP88   CPN1063   tpiS     M     5.0     23.8
    CP91   CPN0302   lpxD     M     7.8     37.7
Listed the A that identifies: chlamydia trachomatis D and B: the example of CPN candidate vaccine. M:MALDI-MS, Q:ESI-Q-TOFMS, P:PSD-MALDI MS,
I: Western blotting (Western blotting).
*: the DT8 representative is by the marking protein of novel open read frame coding, and it is not found in chlamydia trachomatis D genome.
CP34, CP37, CP46, CP47, CP52, CP63 and CP75 shown in 3-protein d T4, DT23 shown in Figure 10, DT47, DT48, DT75, DT76 and DT 77 and Figure 11 is relevant especially.
Other preferred protein of the present invention is chlamydia trachomatis protein, this protein is the protein that has the update time of prolongation under the condition that proteasome inhibitor exists, it is characterized in that having DT1, the DT2, DT3, DT5, DT9, DT10, DT11, DT13, DT14, DT17, DT47, DT59, the DT60 that provide in the Table IV, the pI of a kind of protein among DT61 or the DT62 and Mw, mean error is+/-10%. The CPN protein of being regulated with the same manner by proteasome inhibitor also is the preferred technical solution of the present invention.
Table IV
Spot nr. The gene title    pl    Mw The impact of MG115 The impact of MG132 The impact of PSI The impact of epoxy mycin The impact of B-clasto lactacy-stin
  DT1  CT668   4.45   23.5     +     +
  DT2  CT668   4.55   23.5     +     +
  DT3  CT783   4.55   34.5     +     +
  DT5   4.83   11.4     +     +
  DT9  CT610   4.73   23.7     +     +
  DT10  CT610   4.8   23.7     +     +
  DT11  CT610   4.85   23.7     +     +
  DT13   6.05   24.3     +     +     +
  DT14  ORF5   6.2   27.5     +     +
  DT17   6.28   55.2     +     +
  DT47   5.4   41     +     +     +     +
  DT59   4.5   19.7     +     +
  DT60   5.5   40.9     +     +     +     +
  DT61   5.5   39.9     +     +     +     +
  DT62   5.98   41.1     +     +     +     +
Listed the example of the chlamydia trachomatis D protein of identifying, in mark and follow-up analysis process, this protein is by adopting different proteasome inhibitors to process so that be extended update time.
Another preferred protein of the present invention is chlamydia trachomatis polypeptide DT8, and this polypeptide contains in the claims defined sequence SEQ ID NO:1 and immunogenic fragments thereof.
In other optimal technical scheme of this respect of the present invention, described protein and protein of the present invention or its fragment have at least 40% sequence homogeny, at least 60%, more preferably at least 70% even more preferably at least 80%, still more preferably 90%, most preferably at least 95% sequence homogeny preferably, and perhaps described protein contains at least 7 continuous amino acids of protein of the present invention.
Another aspect of the present invention relates to nucleic acid compound, and this compound contains the sequence of coding protein of the present invention or its immunogenic fragments.
Preferred nucleic acid compound is a kind of nucleic acid compound that contains the sequence (SEQ ID NO:2) of coded polypeptide TD8, and described peptide T D8 contains sequence SEQ ID NO:1.
Another aspect of the present invention relates to the carrier that contains nucleic acid compound of the present invention and transforms or the host cell of transfection with this carrier.
The present invention also provides protein of the present invention or its immunogenic fragments to be used to produce the purposes of the antibody that resists described protein, prepare the method for antibody of anti-intracellular bacteria and the antibody that can obtain by this method, protein wherein of the present invention or its immunogenic fragments are applied to be produced in the animal body, is purified into described antibody then in this animal body.
And, the invention provides the medicine or the diagnosis composition that contain protein of the present invention or its fragment, antibody of the present invention or nucleic acid compound on the other hand, and described protein or its fragment, described antibody or nucleic acid compound are used to prepare the purposes of diagnostic reagent.
The evaluation of the T-cell epitope on the candidate vaccine
The invention provides the T-cell epitope, this epi-position can be MHC I type antigen by surface expression and have the effect that stimulates the T-cell, predict as the protein of identifying by the present invention by computer-based method, or by further by the analytical test checking of embodiment record.
Therefore, to relate to be MHC I type antigen and peptide epitopes that have the effect of T-cytositimulation by surface expression to another importance of the present invention.According to the present invention, a kind of method that is used to identify the T-cell epitope on the intracellular bacteria secretory protein is provided, this method comprises such step, such as the protein identified through method of the present invention or its immunogenic fragments and peptide epitopes are carried out computer forecast, MHC type molecule in conjunction with test and/or ELISPOT test.Also provide nucleic acid compound as a part of the present invention, this compound contains the sequence of described peptide epitopes of encoding, and contains the carrier of described nucleic acid compound and by described carrier transformed host cells.
Preferred peptide epi-position of the present invention contains 4 to 25 continuous amino acids, preferably 6 to 15 even the amino acid of 7 to 10 protein of the present invention more preferably.
In a preferred technical scheme of the present invention, described epi-position contains 7 to 10 continuous amino acids of chlamydia trachomatis or Chlamydia pneumoniae protein.
Another preferred peptide epi-position of the present invention is a kind of 4 to 25 continuous amino acids of the polypeptide that comprises sequence SEQ ID NO:1, more preferably 6 to 15 and 7 to 10 amino acid whose epi-positions most preferably of containing.
The chlamydia trachomatis peptide epitopes that contains the amino acid sequence that is selected from sequence SEQ ID NO.3-SEQ ID NO.45, the Chlamydia pneumoniae peptide epitopes that contains the amino acid sequence that is selected from sequence SEQ ID NO.46-SEQ ID NO.121, the chlamydia trachomatis peptide epitopes that contains the Chlamydia pneumoniae peptide epitopes of the amino acid sequence that is selected from sequence SEQ ID NO.122-SEQ IDNO.148 and contain the amino acid sequence that is selected from sequence SEQ IDNO.149-SEQ ID NO.194 are relevant especially.The epi-position that the present invention identifies is further property description in Table V-VIII.
Table V
Protein ID The position Peptide sequence The A2 combination
CT263 ??181 ????KLAEAIFPI ????8
CT263 ??170 ????FLKNNKVKL ????123
CT263 ??56 ????ALSPPPSGY ????210
CT263 ??141 ????FIAKQASLV ????210
CT263 ??17 ????TLSLFPFSL ????286
CT263 ??147 ????SLVACPCSM ????332
CT263 ??6 ????LIFADPAEA ????386
CT263 ??4 ????LLLIFADPA ????438
CT541 ??4 ????ILSWMLMFA ????38
CT541 ??94 ????KQMAEVQKA ????89
CT541 ??9 ????LMFAVALPI ????122
CT541 ??135 ????KLQYRVVKE ????221
CT541 ??118 ????FLKENKEKA ????222
CT541 ??46 ????KLSRTFGHL ????239
CT541 ??223 ????SLLIFEVKL ????265
CT541 ??148 ????VLSGKPTAL ????352
CT541 ??204 ????VLYIHPDLA ????398
CT541 ??54 ????LLSRQLSRT ????472
CT691 ??172 ????LLQRELMKV ????9
CT691 ??25 ????STINVLFPL ????66
CT691 ??15 ????PLQAHLELV ????114
CT691 ??6 ????SLFGQSPFA ????194
CT691 ??212 ????KLAYRVSMT ????251
CT691 ??194 ????VLWMQIIKG ????284
CT691 ??29 ????VLFPLFSAL ????298
CT691 ??122 ????FLQTVQSF ????468
CT691 ????8 ????FGQSPFAPL ????480
CT858 ????85 ????VLADFIGGL ????33
CT858 ????177 ????RMASLGHKV ????52
CT858 ????92 ????GLNDFHAGV ????90
CT858 ????490 ????FSCADFFPV ????90
CT858 ????379 ????MLTDRPLEL ????101
CT858 ????408 ????LLENVDTNV ????121
CT858 ????391 ????RMILTQDEV ????132
CT858 ????491 ????SCADFFPVV ????132
CT858 ????519 ????FVFNVQFPN ????132
CT858 ????372 ????YLYALLSML ????247
CT858 ????539 ????SLAVREHGA ????288
CT858 ????109 ????YLPYTVQKS ????350
CT858 ????219 ????ATIAPSIRA ????358
CT858 ????140 ????LLEVDGAPV ????375
CT858 ????512 ????RTAGAGGFV ????384
CT858 ????250 ????SLFYSPMVP ????431
The epi-position of the chlamydia trachomatis protein of the evaluation of prediction
Table VI
Protein ID The position Peptide sequence The A2 combination
CPN0152 ????6 ????FLVSCLFSV ????18
CPN0152 ????135 ????YLRDAQTIL ????28
CPN0152 ????237 ????LLIRIQDHV ????48
CPN0152 ????100 ????KLGRKFAAV ????51
CPN0152 ????266 ????LVSRTQQTL ????164
CPN0152 ????10 ????CLFSVAIGA ????190
CPN0152 ????222 ????GFGPPPIIV ????354
CPN0152 ????249 ????SLPTKPYIL ????387
CPN0152 ????240 ????RIQDHVTAN ????408
CPN0152 ????15 ????AIGASAAPV ????410
CPN0152 ????156 ????RLGISGFSL ????444
CPN0619 ????64 ????FMVSGPVVV ????31
CPN0619 ????73 ????LVLEGANAV ????398
CPN0705 ????164 ????FVGANLTLV ????24
CPN0705 ????89 ????CLAENAFAG ????114
CPN0705 ????233 ????KIEEVQTPL ????116
CPN0705 ????211 ????ALKGHQLTL ????178
CPN0705 ????190 ????QMAEAADLV ????358
CPN0796 ????583 ????FMGAHVFAS ????15
CPN0796 ????419 ????LLIQHSAKV ????31
CPN0796 ????372 ????FLCPFQAPS ????39
CPN0796 ????376 ????FQAPSPAPV ????50
CPN0796 ????211 ????AMNACVNGI ????86
CPN0796 ????548 ????FMGIQVLHL ????112
CPN0796 ????74 ????RHAAQATGV ????134
CPN0796 ????328 ????FQYADGQMV ????148
CPN0796 ????618 ????SVSAMGNFV ????212
CPN0796 ????460 ????FLSYRSQVH ????214
CPN0796 ????53 ????FLLTAIPGS ????218
CPN0796 ????38 ????VLTPWIYRK ????219
CPN0796 ????656 ????SVVMNQQPL ????221
CPN0796 ????408 ????SLKNSQQQL ????279
CPN0796 ????162 ????MLPDTLDSV ????284
CPN0796 ????511 ????ALPYTEQGL ????295
CPN0796 ????523 ????VLSGFGGQV ????399
CPN0998 ????22 ????LLFGVVFGV ????8
CPN0998 ????174 ????SLQERYPTL ????29
CPN0998 ????416 ????MLLKGQNKV ????33
CPN0998 ????379 ????FTFLPIILV ????53
CPN0998 ????754 ????FLGDISSGA ????56
CPN0998 ????36 ????FLAGKKARV ????66
CPN0998 ????824 ????LLDAAYQRA ????66
CPN0998 ????374 ????YLGYLFTFL ????78
CPN0998 ????377 ????YLFTFLPII ????109
CPN0998 ????717 ????SLGATHFLP ????124
CPN0998 ????96 ????ELIDQGHRL ????134
CPN0998 ????381 ????FLPIILVLL ????197
CPN0998 ????386 ????LVLLFVYLV ????219
CPN0998 ????161 ????VTGPATPQL ????223
CPN0998 ????319 ????SLEKQDPEV ????224
CPN0998 ????567 ????ILMAATNRP ????236
CPN0998 ????230 ????LTQETDTEA ????237
CPN0998 ????823 ????MLLDAAYCF ????238
CPN0998 ????639 ????LLNEAALLA ????254
CPN0998 ????736 ????ELYDQLAVL ????256
CPN0998 ????199 ????LIGKYLSPV ????294
CPN0998 ????454 ????SLGGRIPKG ????303
CPN0998 ????781 ????GMSPQLGNV ????306
CPN0998 ????645 ????LLAARKDRT ????315
CPN0998 ????424 ????VTFADVAGI ????427
CPN0998 ????154 ????VLTEPLVVT ????439
CPN0998 ????66 ????KIALNDNLV ????470
CPN1016 ????5 ????KLGAIVFGL ????7
CPN1016 ????135 ????YLGDEILEV ????34
CPN1016 ????284 ????FLPTFGPIL ????99
CPN1016 ????439 ????SLQNFSQSV ????108
CPN1016 ????414 ????FTDEQAVAV ????145
CPN1016 ????92 ????SLNDYHAGI ????164
CPN1016 ????392 ????RMIFTQDEV ????175
CPN1016 ????64 ????TQQARLQLV ????294
CPN1016 ????217 ????SLVAPLIPE ????312
CPN1016 ????255 ????YMVPYFWEE ????358
CPN1016 ????576 ????YVEAVKTIV ????389
CPN1016 ????395 ????FTQDEVSSA ????444
CPN1016 ????516 ????GAGGFVFQV ????491
CPN1016 ????464 ????LLGFAQVRP ????498
The epi-position of the Chlamydia pneumoniae protein of the evaluation of prediction
Table VII
Protein ID The position Peptide sequence The A2 combination
CPN0412(CT263) ????186 ????RLEEVSQKL ????80
CPN0412(CT263) ????103 ????LTTDTPPVL ????103
CPN0412(CT263) ????147 ????KLLDMEGYA ????167
CPN0412(CT263) ????110 ????VLSEDPPYI ????183
CPN0412(CT263) ????62 ????ALQSYCQAY ????215
CPN0412(CT263) ????193 ????KLTQTLVEL ????248
CPN0412(CT263) ????81 ????FVGACSPEI ????267
CPN0412(CT263) ????102 ????NLTTDTPPV ????286
CPN0412(CT263) ????205 ????LMERAIPPK ????410
CPN0661(CT541) ????103 ????KMAEVQKLV ????46
CPN0661(CT541) ????199 ????ALGMQGMKE ????221
CPN0661(CT541) ????54 ????KLSRTFGHL ????239
CPN0661(CT541) ????232 ????LLIFEINLI ????334
CPN0661(CT541) ????8 ????VLATVALAL ????391
CPN0661(CT541) ????187 ????ILLPLGQTI ????396
CPN0661(CT541) ????212 ????VLYIHPDLA ????398
CPN0661(CT541) ????7 ????LVLATVALA ????413
CPN0681(CT691) ????29 ????YMLPIFTAL ????40
CPN0681(CT691) ????136 ????LLHEFNQLL ????66
CPN0681(CT691) ????172 ????VLQRELMQI ????91
CPN0681(CT691) ????15 ????PLQAHLEMV ????169
CPN0681(CT691) ????6 ????RLFGQSPFA ????197
CPN0681(CT691) ????73 ????GLFMPISRA ????223
CPN0681(CT691) ????212 ????KLAHRINMT ????229
CPN0681(CT691) ????194 ????YLWLQVIRR ????322
CPN0681(CT691) ????135 ????TLLHEFNQL ????474
CPN0681(CT691) ????8 ????FGQSPFAPL ????480
The epi-position of the Chlamydia pneumoniae homolog of the chlamydia trachomatis protein of the evaluation of prediction
Table VIII
Protein ID The position Peptide sequence The A2 combination
CT149(CPN0152) ????274 ????FLGAAPAQM ????17
CT149(CPN0152) ????237 ????FLGIQDHIL ????29
CT149(CPN0152) ????101 ????LLTANGIAV ????31
CT149(CPN0152) ????248 ????SLPRRIPVL ????86
CT149(CPN0152) ????42 ????GLQEHCRGV ????107
CT149(CPN0152) ????160 ????SLGCHTTIH ????170
CT149(CPN0152) ????307 ????ILTHFQSNL ????181
CT149(CPN0152) ????52 ????VLSCGYNLV ????202
CT149(CPN0152) ????195 ????LLKEICATI ????248
CT149(CPN0152) ????272 ????RLFLGAAPA ????318
CT149(CPN0152) ????141 ????ATVAKYPEV ????338
CT149(CPN0152) ????11 ????LLSGSGFAA ????343
CT149(CPN0152) ????102 ????LTANGIAVA ????373
CT149(CPN0152) ????15 ????SGFAAPVEV ????397
CT500(CPN0619) ????64 ????FMISGPVVV ????20
CT500(CPN0619) ????103 ????ALFGESIGV ????121
CT500(CPN0619) ????119 ????SLENAAIEV ????212
CT500(CPN0619) ????87 ????LMGATNPKE ????313
CT500(CPN0619) ????31 ????RIAAMKMVH ????385
CT671(CPN0705) ????102 ????ALVETPMAV ????13
CT671(CPN0705) ????167 ????FCGANLTLV ????49
CT671(CPN0705) ????214 ????SLKARQLNL ????151
CT671(CPN0705) ????193 ????QLTEATQLV ????239
CT671(CPN0705) ????127 ????DLQWVEQLV ????403
CT671(CPN0705) ????155 ????IVLDNSNTV ????423
CT841(CPN0998) ????22 ????LLFGVIFGV ????9
CT841(CPN0998) ????415 ????LLAKGQNKV ????14
CT841(CPN0998) ????378 ????FTFMPIILV ????29
CT841(CPN0998) ????753 ????FLGDVSSGA ????43
CT841(CPN0998) ????824 ????LLDAAYQRA ????66
CT841(CPN0998) ????780 ????GMSDHLGTV ????110
CT841(CPN0998) ????716 ????SLGATHFLP ????124
CT841(CPN0998) ????170 ????NLAALENRV ????153
CT841(CPN0998) ????376 ????YLFTFMPII ????160
CT841(CPN0998) ????15 ????FPTAFFFLL ????167
CT841(CPN0998) ????566 ????ILMAATNRP ????236
CT841(CPN0998) ????66 ????KTALNDNLV ????244
CT841(CPN0998) ????638 ????LLNEAALLA ????254
CT841(CPN0998) ????735 ????ELYDQLAVL ????256
CT841(CPN0998) ????318 ????ALEKQDPEV ????264
CT841(CPN0998) ????453 ????SLGGRIPKG ????303
CT841(CPN0998) ????380 ????FMPIILVLL ????314
CT841(CPN0998) ????644 ????LLAARKDRT ????315
CT841(CPN0998) ????423 ????VTFADVAGI ????427
CT841(CPN0998) ????142 ????YTISPRTDV ????467
CT841(CPN0998) ????464 ????LIGAPGTGK ????495
The epi-position of the chlamydia trachomatis homolog of the Chlamydia pneumoniae protein of the evaluation of prediction
Peptide epitopes can be used as the part of fused protein or carries out coupling with carrier part.
A kind of method that is commonly used to predict the epi-position that combines with MHC comprises motif retrieval (motifsearch).The most complicated accurate motif retrieval has utilized all matrix (entive matrices) of representing MHC to prolong motif.Although sequence-independent manner combination specificity may be correct as average Consideration, obviously is incorrect for individual peptides.And crystal structure has shown that an interaction on the sublocus can influence the interaction on other sublocus.
Artificial neural networks (ANN) is particularly suitable for handling and discerning any non-linear sequence information.Information can be carried out processing and handle then to be assigned to and be furnished with input layer, hide in the middle of the computer system of layer and output layer, and described each layer concerns by weighting and is connected in the ad hoc structure.This ANN can be processed into and can discern and the relevant input signal (peptide) of specific output signal (being the MHC combination).In case after handling, this network should be discerned the complicated model peptide that is suitable for combination.Utilize the ANN method, the size for the treatment of apparatus has become a key factor with quality.This is even more important for HLA because only have an appointment 1% one overlap at random that peptide could combine with any given HLA.
Therefore, if peptide is at random screened, fewly will need synthetic and test about 10000 peptides to 100 binding peptides in order to produce.Even for the suitable bond of number in the treating apparatus, this also is a method that expends very big financial resources and labour, and this method need be carried out repetition to each HLA to be measured.
Therefore, the matrix method of forecasting relevant with the present invention be used to scan SWISS-PROT (http://www.expasy.ch/sprot/) thus database finds potential high affinity in conjunction with epi-position.Synthetic and tested a large amount of this epi-positions in conjunction with test in biological chemistry.Just as expected, obtained the high affinity bond (binder) (about 80%) of higher proportion.Next these data are used to handle ANN.
For four kinds in four kinds of detected MHC I type molecules, ANN shows better than matrix-driving predicted method.This predicted method forms a kind of pattern, can predict that actual value rather than the arbitrary classification in conjunction with IC50 becomes " bond " and " non-binding thing ".In fact, thus might one on a large scale in the prediction bond identify bond and the low-affinity and the non-binding thing of high affinity.
The present invention comprises that further peptide epitopes of the present invention is used to prepare the purposes of vaccine, and the vaccine that contains peptide epitopes of the present invention, and this vaccine optionally contains acceptable excipient.
Another aspect of the present invention relate to protein of the present invention, antibody of the present invention, nucleic acid compound of the present invention or peptide epitopes of the present invention preparation be used for the treatment of or the pharmaceutical composition of the infection that prevents to cause such as choamydiae infection by intracellular bacteria in purposes, perhaps be used for detecting the purposes of the antibody that whether has intracellular bacteria such as chlamydial diagnostic reagent or produce at intracellular bacteria in preparation.
The present invention further provides the method for induce immune response in human body, comprise protein of the present invention, antibody, nucleic acid compound or the peptide epitopes of described human body being used the immunology effective dose, provide a kind of so especially and be used for the treatment of or prevent intracellular bacteria such as Chlamydia pneumoniae or chlamydia trachomatis to cause the method for human infection.
At last, the invention provides the method for producing protein of the present invention or its fragment or peptide epitopes of the present invention respectively, described method comprises that the carrier with the nucleic acid compound that contains code for said proteins or peptide epitopes transforms, the transfection host cells infected, can express at described host cell under the condition of described protein or its fragment and cultivate host cell.
The present invention is further specified by following non-limiting examples.
Embodiment
Embodiment 1:
The infection of mammalian cell cultures
According to the method for having put down in writing in [19.] and [17.], form HeLa, HEp-2 or the McCoy (ATCC that unit (IFU) infects partly-converges with Chlamydia pneumoniae VR1310, chlamydia trachomatis serotype A (HAR-13), D (UW-3/Cx) or L2. (434/Bu) inclusion body (ATCC), Rockville, MD, USA) cell monolayer.The infection nutrient culture media that is used for chlamydia trachomatis A and D is made up of RPMI 1640,25mM HEPES, 10%FCS, 1%w/v glutamine, 10mg/ml gentamicin, and the infection nutrient culture media that is used for chlamydia trachomatis L2 is made up of RPMI 1640,25mM HEPES, 5%FCS, 1%w/v glutamine, 10mg/ml gentamicin.
Embodiment 2:
Pulse labeling/follow-up analysis
For with two hours time of I (chlamydia) protein matter mark, (Shaw etc. according to the method described above, 1999,2000) [18.] [19.], containing RPMI 1640,10mg/ml gentamicin, 40 μ g/ml cycloheximides, 100 μ Ci/ml[35S]-methionine/halfcystine (Promix, Amersham Pharmacia Biotech, Uppsala cultivates infected cell in nutrient culture media Sweden).Behind the mark, after washing twice, change the mark nutrient culture media into ordinary culture medium, and the different time points behind mark is collected infected cell with ordinary culture medium.Similarly, by the Chlamydia mark being cultivated the metainfective EB protein that obtained mark in 72 hours after two hours.Collect the EB of mark then, and utilize the density gradient ultracentrifugation of two consecutive steps that described EB is carried out purifying, described density gradient ultracentrifugation is carried out according to the density gradient ultracentrifugation method that is used for chlamydia trachomatis (Schacter and Wyrick, 1994) [22.] and Chlamydia pneumoniae (Knudsen et al.1999[17.]) basically.Protein in EB preparation and the pulse-chase preparation be derived from complete lysate preparation in identical time interval of protein carry out mark, thereby help correctly carrying out the comparison of 2D-PAGE protein figure.
Embodiment 3
The preparation of sample
Behind [35S]-mark, cell is dissolved in the standard lysis buffer after washing twice with PBS, and this damping fluid contains 9M urea, 4%w/v 3-[(3-courage acylamino-propyl group) Dimethyl Ammonium]-1-propane sulfonic acid salt (CHAPS; Roche, Germany), 40mM Tris Base, 65mM DTE and Pharmalyte 3-10 (Amersham Pharmacia Biotech).For the hydrophobic protein of enrichment high molecular, adopt 7M urea, 2M thiocarbamide, 4%w/v 3-[(3-courage acylamino-propyl group) Dimethyl Ammonium]-1-propane sulfonic acid salt (CHAPS; Boehringer Mannheim, Germany), 40mM Tris Base, 65mM dithioerythritol (DTE) and 2%v/vPharmalyte 3-10 (Amersham Pharmacia Biotech), basically according to described carrying out such as (Harder .1999[23.]).Sonicated contains the sample of complete thin lysate or the EB of purifying, and with 10000 * g centrifugal 10 minutes.Sample is stored under-70 ℃ until use.
Embodiment 4:
The separation of I (chlamydia) protein matter
By basically as the two dimensional gel electrophore-sis separation source described in (Shaw et al., 1999,2000) [18.] [19.] from the I (chlamydia) protein matter of the EB of full cell lysate and purifying.
In order to carry out the isoelectric focusing on first direction, pass through IPGphor TmThe fixing dried band of pH-gradient (Amersham Pharmacia Biotech) of pH3-10NL (non-linear), 4-7L (linearity) that 18cm is long or 6-11 (linearity) is with swelling (reswelled) 12 hours repeatedly under 20 ℃ in the protein (in 350 μ l lysis buffers) of sample size (cpm) mark of 200.00 readings of per minute.Other band that the present invention uses comprises super narrow ipg strip band.This band is as described in the Table IX.If necessary, this makes us focus on the specific pH interval of containing destination protein matter.The voltage program of carrying out isoelectric focusing when utilizing 3-10NL, 4-7L and the dried band of 6-11L under 20 ℃ is as follows: following 1 hour of 300V, following 2 hours of 300-500V (linear increasing), following 1 hour of 1000V, following 1 hour of 2000V, following 24 hours of following 3 hours of 3500V and 5000V.
Table I X:
Ipg strip band name Linear (Liniarity) The pH spacing that covers Band length
??Immobiline?Drystrip?pH?3-10 Non--linear (non-liniar) ????3-10 ????18cm
??Immobiline?Drystrip?pH?3-10 Linear (liniar) ????3-10 ????18cm
??Immobiline?Drystrip?pH?4-7 Linear ????4-7 ????18cm
??Immobiline?Drystrip?pH?6-11 Linear ????6-11 ????18cm
??Immobiline?Drystrip?pH?6-9 Linear ????6-9 ????18cm
??Immobiline?Drystrip?pH?3.5-4.5 Linear ????3.5-4.5 ????18cm
??Immobiline?Drystrip?pH?4-5 Linear ????4-5 ????18cm
??Immobiline?Drystrip?pH?4.5-5.5 Linear ????4.5-5.5 ????18cm
??Immobiline?Drystrip?pH?5-6 Linear ????5-6 ????18cm
??Immobiline?Drystrip?pH?5.5-6.7 Linear ????5.5-6.7 ????18cm
Listed the example that is purchased the dried band of IPG that the present invention uses
After having carried out the electrophoresis of first direction, with dried band balance 15 minutes in the damping fluid that contains 6M urea, 30%v/v glycerine, 2%w/v DTE, 2%w/v SDS, 0.05M Tris-Hci pH6.8.Balance 15 minutes again in the damping fluid that described then band DTE has therein been replaced by the 2.5%w/v iodoacetamide.In order to carry out the electrophoresis of second direction, (CA USA) comes that (18cm * 20cm * 1mm) goes up isolated protein at 9-16% linear gradient SDS-PAGE gel for Bio-Rad, Richmond with Protean II xi Multicell system.Gel is fixed 30 minutes in the solution that contains 10% acetic acid and 25%2-propyl alcohol and use AmpIlfy (Amersham Pharmacia Biotech) to handle 30 minutes with analyzing.After 8-10 days, show the protein that is labeled through Kodak Biomax-MR film (Amersham Pharmacia Biotech) exposure by autoradiography in-70 times ℃.
Figure 1A is the autoradiographic example of the Analytical high resolution 3-10 IPG 2D-gel (having used the lysis buffer of standard) of the chlamydia trachomatis D protein of 22-24 hour-symbols after infecting.Fig. 2 A is the autoradiographic example of the 3-10IPG2D-gel (having used the lysis buffer that contains thiocarbamide) of the Chlamydia pneumoniae protein of 55-57 hour-symbols after infecting.As estimated, on every clotting glue, can show about 600 protein spots altogether by Melanie II software.
In order to prepare the sample that is used for mass spectrophotometry, carry out the 2D-gel electrophoresis with the full cell lysate of 500-1000 μ g.In order to show the protein for preparing on the gel, 2 * 10 on the x-ray film 6The protein of [35S]-protein labeling of cpm runs glue on same gel, the protein source of described [35S]-protein labeling in the parallel cultured cells of unmarked sample.Use ddH 2O was with preparation type gel flushing 10 minutes and be dried to not fixed form.Mark on the gel side with the radioactivity China ink, so that after exposure, can compare accurately to desiccant gel and corresponding x-ray film.Downcut the destination protein matter and combine from least three blocks of identical gels.Identify a central problem that exists if the host cell pollutant is a mass spectrum, use the gel of narrow or super narrow dried band (Table I X) to increase separating distance.
Embodiment 5:
Utilize MAIDI MS, ESI-Q-TOF MS, PSD MAIDI MS and Edman Edman degradation Edman is identified candidate vaccine
Carry out digesting in the gel with the protein spot of tryptose matter enzyme to the representative candidate vaccine on the preparation type gel of full product of cell lysis.Reversed-phase column that the peptide utilization that obtains is made up of Poros R2 material (Gobom etc., 1999[20.]) or pearl (Gevaert etc., 1997[21.]) carry out purifying.Utilize subsequently with Bruker REFLEX MALDI flight time (timeof flight) mass spectrum (Bruker-Daltonik, GmbH, Bremen, Germany) of electronic pattern operation sample is analyzed.Peptide mapping method by having put down in writing (Schevchenko 1996[24.]) compares gained material and the peptide material that produces by the protein in the theoretical tryptose matter enzymatic lysis database.
Utilize example that MALDI-MS is accredited as CT668 with DT1 as shown in Figure 3.Fig. 3 A represents the peptide matter finger print by the acquisition of MALDI mass spectrum.Utilize Prospector software MS-Fit that the material that obtained and the theoretical tryptose matter enzymatic lysis product of all proteins in the database are compared.If with retrieval be limited in gel on the protein found near the pI/Mw scope, the highest (higest-ranking) protein is CT668 (Fig. 3 B).Yet the protein of host cell is present in the spot on the gel of full cell lysate sometimes, and evaluation may be correct.Therefore, if necessary, adopt tandem mass spectrum and opisthogenesis decline (post sourcedecay) (PSD) analytic approach verify described result (commentary of Mann and Wilm, 1995[25.], Gevaert, 1997[21.]).
(Micromass, Manchester UK) carry out the tandem mass spectrum analysis to the peptide that is produced by digestion in the gel to utilize Electrospray Ionization Quadrupie Time-Of-Flight (ESI-QTOF) mass spectrum.Utilize this method, can from sample, isolate single Ionized peptide.Produced several new ions by colliding this parent ion of cracking, and these several new ions are recorded on the finger print of new peptide material with air.These new ions only differ from an amino acid in size, and therefore the detailed amino acid sequence (Mann and Wilm, 1995) [25.] of original peptide is provided.
Fig. 4 A represents the example by the peptide parent ion of DT1 cracking, utilizes BLAST or MS Tag searching database to find that the sequence that obtains is corresponding with the fragment of CT668.From the CT668 sample, also identify the sequence mark that produces by people's PBP matter.
If retrieval is limited to human protein, also can in MALDI MS peptide material finger print (double-head arrow, Fig. 3 A), detect the peptide that obtains by this protein.This shows that the spot undetermined that contains more than one protein can be identified accurately by ESI-Q-TOF, thereby has confirmed to exist the MALDI MS of query to identify.
The another kind that the present invention uses confirms that the method for MALDI is PSD.PSD has utilized the peptide that carries out metastable decay after the ionization, means that promptly the fragments of peptides with phase same rate has different quality, thereby has different kinetic energy.The difference of kinetic energy aspect can decide by reflection fragment in magnetic field.It is darker thereby delayed time that the high energy fragment penetrates magnetic field than low energy fragment.As the cracking that mainly occurs on the peptide bond, the collection of illustrative plates that is obtained by the separation of single peptide can be used to infer the amino acid sequence (Mann etc., 1993) [28.] of peptide sequence label (PST).PST can compare with Protein Data Bank, thereby can identify the protein (Wilkins etc., 1996 [27.]) that therefrom produces.
By PSD MALDI MS the example that the CP63 spot is accredited as CPN1016 is shown in Fig. 4 B.In the PSD collection of illustrative plates in observed altogether 36 kinds of materials, wherein 16 kinds of materials that produce with the peptide that is had 1919.80Da parent ion sequence (R) ELLFGWDLSQQTQQAR (L) by theoretical cracking in a mass unit scope are suitable, promptly are equivalent to the CPN1016 protein of Chlamydia pneumoniae.
Utilize mass spectroscopy, the evaluation example of chlamydia trachomatis D (Table III A) and Chlamydia pneumoniae (Table III b) candidate vaccine is provided.Mw and Pi value by electrophoretic determination have+/-10% average error.
CT858 has the theoretical Mw of 67kDa, therefore shows that certified protein is the processing fragment of macro-molecular protein.In fact, analyze the C-end parts that the whole three kinds of peptides that obtain are positioned at described protein by DT4 being carried out ESI-Q-TOF.
Another spot DT48 that is in the gel alkalescence zone also contains CT858.All peptides that DT48 is accredited as CT858 are equivalent to the N-end parts of described protein, show that DT48 represents the N-end fragment of CT858.
In chlamydia trachomatis D and L2, identify CT610 by MALDI MS and Edman edman degradation Edman by EB.Yet, to compare with full cell lysate, this protein obviously reduces in EB, thereby still is considered as candidate vaccine.From chlamydia trachomatis L2 CT610, identify CT610 by the Edman edman degradation Edman.Described N-end is measured as MNFLDQ, and it is different from MMEVFMNFLDQ sequence (Stephens etc., 1998b) [35.] that doped by chlamydia trachomatis gene group engineering.
On the basis of four kinds of sequence mark things that produce by ESI TOF MS, identify DT8.These sequences not with chlamydia trachomatis D genome in any prediction open read frame corresponding [35.].Yet, retrieving chlamydia trachomatis gene group in all 6 open read frames by utilizing BLAST, can produce and the suitable counter pair of coupling of four kinds of sequence marks.To the dna sequence dna of the described peptide of encoding and on every side the analysis result of sequence show that the novel open read frame that comprises ribosome bind site contains a kind of 7.2kDa protein.The dna sequence dna of the DT8 that is translated as shown in Figure 5.How the mass spectrometry method that this discovery explanation the present invention adopts discerns the important ORF of potential coding candidate vaccine, and this ORF may be left in the basket in big genome project.
It is the N-end fragment of CPN1016 that the spot CP63 that derives from Chlamydia pneumoniae is identified, and the Chlamydia pneumoniae homolog of CT858 (Fig. 2 B and C, Table III B) shows that these protein process in chlamydial two kinds.
The following example has shown being carried out more detailed research by the characteristic of identification of protein example.
Embodiment 6
The comparison of the whole lysates of infection cell and the RB of purifying
According to embodiment 2 described methods, under the condition that has cycloheximide to exist, with [35S] methionine/halfcystine with the cell marking of chlamydia trachomatis or infection involving chlamydia pneumoniae two hours.After mark was intact, infection cell (as described in example 3 above) was directly collected in lysis buffer or is used for direct purification Chlamydia RB.Basically according to Schachter and Wyrick, 1994[22.] the super centrifugal method of described density gradient carries out the purifying of RB.
In Figure 10, respective regions on the gel picture of example region on the gel picture of chlamydia trachomatis D protein and RB and EB is compared, wherein chlamydia trachomatis D protein source carried out whole lysates of the infected HeLa cell of mark in 22-24 after infection hour, and described RB and EB are purifying from HeLa cell chlamydia trachomatis infection and carried out mark in 22-24 hour after infection.The DT4 listed (the C-end fragment of CT858), DT48 (the N-end fragment of CT858), DT23 (Mip), DT76 (hypothetical protein matter CT691) and DT77 (hypothetical protein matter CT263) have been identified by mass spectrum as Table III.
In Figure 11, to derive from the gel picture of HEp-2 cell infection involving chlamydia pneumoniae and carried out mark in 55-57 hour after infection respectively and compare at the example region of the gel picture of the EB of the purifying of each time point mark in the whole growth cycle respective regions with the gel picture of the cell culture that derives from infection involving chlamydia pneumoniae, the cell culture of wherein said infection involving chlamydia pneumoniae carries out mark in 34-36 hour and has been collected or was purified as RB in back 36 hours in infection in back 36 hours whole lysate forms with infected cell of infection after infection.
The protein spot that Figure 11 centre circle gets up is identified in such a way.Figure 11 A and E represent CP34 and CP63, and it is accredited as is two fragments of CPN1016.Figure 11 B represents CP37, and it is accredited as CPN0998.Figure 11 C represents CP46 and CP47, and it is accredited as CPN0796 and CPN0705 respectively.Figure 11 D represents CP52, and it is accredited as CPN0152.Figure 11 F represents CP75, and it is accredited as CPN0619.
Embodiment 7:
Be positioned at the detection and the evaluation of the protein in inferior bunch of the III type secretor
Because the III type secretor of most of intracellular bacterias is positioned in the gene cluster as pathogenicity island (island), so Chlamydia III type secretor is determined in the genome that is present in chlamydia trachomatis and Chlamydia pneumoniae (Stephens etc. among different inferior bunch of three on the different loci, 1998[4.] and Kalman etc., 1999[5.]).As the part of the whole protein analysis of chlamydia trachomatis A, D and L2 and Chlamydia pneumoniae VR1310, be present in protein in the III type secretion bunch from the EB gel of purifying and obtain identifying.
The III type secretory protein of certified chlamydia trachomatis comprises Yop secretion ATP enzyme (yscN), Yop transport protein matter L (YscL) and secretes chaperone protein (SycE), and these protein are that protein is transported to excretory system from the bacterium kytoplasm is necessary.The Unknown Function of being identified in addition but the chlamydia trachomatis D protein that is positioned in inferior bunch of the III type secretion comprises CT560 and a large amount of CT577 and CT579.CT668 obviously is present in the full cell lysate, but is not present among the EB of purifying, and because its position neighbour YscN, so this protein may be secreted.The position of described protein in genome as shown in Figure 9.
Chlamydia pneumoniae III type secernent protein LcrE (CPN0324), YscC (CPN0702), YscN (CPN0707) and YscL (CPN0826) have been identified.YscC (CP89 schemes II, Table II) is not present among the EB of purifying, and this may be because YscC is positioned in the inclusion body film, from then on is exposed to the kytoplasm of host cell.Be present in the III type bunch, be positioned at YscC (CPN0702) and YscN (CPN0707) on every side the content of the content of two kinds of protein in full cell lysate in the EB that is being purified.These two kinds of protein are CPN0705 (CP90, figure II, Table II) and CPN0711 (CP76, figure II, table 11).These protein also may be positioned in the inclusion body film or be present in the kytoplasm of host cell, and in both cases, these two kinds of protein may be vulnerable to the effect of protein enzyme body.
Embodiment 8:
The pulse-chase research of candidate's secretory protein
In order to assess the life period of candidate albumen matter in infected cell of evaluation, the invention provides the series of pulses follow.In the following example, infected cell culture quilt [35S] mark 22-24 hour.Behind the mark, nutrient culture media changed into the common RPMI nutrient culture media that do not contain [35S]-methionine and the different time collecting cell behind mark.Showing by several independently results of research may be because the nuance of the effectiveness aspect of host cell density and/or infection have produced less variation.
Figure 1B, C, D and E provide the example of pulse/chase experiment.CT668 and DT8 intensity (are respectively Fig. 1: A, B) obviously reduced at synthetic back 1.5 hours and disappeared and until the EB stage from gel fully in 4.5 hours.(Fig. 1: C D) obviously reduces, but still can be detected in 4.5 hours and until the EB stage for CT610 and CT783.The C-end fragment of CT858 (D) (with the N-end fragment of CT858) progressively increased at the tracking initial stage, but did not exist among the EB, showed that pyrolysis product accumulates in the Chlamydia growth course.There is not (Fig. 2) among the EB in the N-end fragment CPN1016 of the Chlamydia pneumoniae homolog of CT858 yet.
Embodiment 9:
Be used in combination the pulse-chase research that protein enzyme body inhibitor carries out
This embodiment shows how to determine which candidate vaccine processes in protein enzyme body.At mark and track phase cell-penetrating protein enzyme body inhibitor is added in the infected cells culture, and carries out mark/viewed protein of tracking to the update time of protein with when not adding protein enzyme body inhibitor and compare update time.The important example of this method as shown in Figure 6.In metainfective 22-24 hour, under the condition that the protein enzyme body inhibitor MG-132 that 10-100 μ M is arranged exists or under the condition that does not have protein enzyme body inhibitor to exist described protein is being carried out mark.Metainfective 24-28 hour, contain or do not contain the growth medium of MG132 and follow the trail of with after twice of the common growth medium flushing mark nutrient culture media being changed into.Cell lysate carries out 2D-PAGE (IPG) to be analyzed, compare with the contrast that does not contain MG-132, and with mark after the gel of the protein that reclaims at once compare (Fig. 6 A, B and C).The invention provides the example of 15 kinds of chlamydia trachomatis D protein, these protein obtain prolonging owing to the processing through protein enzyme body inhibitor makes update time.
Content height in the control sample that DT9, DT10 and the DT11 content in tracked+MG132 gel reclaims after in fact than mark at once.This shows that DT9, DT10 and DT11 have been degraded soon by protein enzyme body.On the contrary, little by adding protein enzyme body inhibitor to the content influence of DT7.
The present invention includes other some protein enzyme body inhibitor (MG115 for example, MG262, PSI and lactocystein) purposes, the different catalytically active part of this some protein enzyme body inhibitor Profilin matter enzyme body, it can be illustrated by other protein (Table IV) of protein enzyme body degraded.
Embodiment 10:
Utilizing genetically-altered cells is to come analysing protein enzyme body inhibitor chlamydia protein The influence of update time
The present invention also provides and has been purchased the purposes that mouse embryo cell is MEC-PA28 clone PW8875.MEC-PA28 is by the IFN-γ induction type PA28 α of protein enzyme body and the transfection of institute of β subunit, and MEC217 clone is by IFN-γ induction type LMP2, the LMP7 of protein enzyme body and the transfection of MECL institute.MEC-PA28 and MEC217 cultivate partly degree of converging and with chlamydia trachomatis or infection involving chlamydia pneumoniae.Do not infected simultaneously by the control mice embryo cell line of protein enzyme body subunit transfection.
Because the rotaring redyeing gene that Codocyte is the mistake of MEC-PA28 and MEC217 expresses subunit is the processing of MHC I type antigen and offers necessaryly, as previously mentioned these host cells utilized the experiment of aleuroplast inhibitor in conjunction with pulse labeling/tracking.
Embodiment 11:
The clone and the expression of coding candidate vaccine open read frame (ORF)
PET-30LIC support agent box (Novagen, Madison, the USA) operation instructions that provide according to the manufacturer clone and the expression of candidate vaccine open read frame (ORF) of encoding are provided.
The primer that is used for gene PCR have following 5 ' end and 3 '-the LIC jag held:
Forward primer: 5 ' GACGACGACAAGATX-gene specific sequence 3 '
Reverse primer: 5 ' GAGGAGAAGCCCGGT-gene specific sequence 3 '
(X: first nucleotide that inserts distinguished sequence)
Full-length gene or the gene that does not contain homing sequence increase by the PCR that utilizes amplification high-fidelity PCR (Expand High Fidelity PCR) (Roche, Germany).Utilize DNA thermal cycler (GeneAmp PCR system 9600, Perkin Elmer) to carry out 35 PCR-circulations: 92 ℃ 15 seconds (sex change), 55 ℃ 15 seconds (primer annealing) and 68 ℃ 4 minutes (extension) according to follow procedure.The PCR product that obtains comprises the LIC-jag contiguous with the gene specific sequence.The PCR product carries out Wizard (Promega, Madison, USA) purifying and being connected in the pET-30 carrier.Described pET-30 carrier contains the gene of the kalamycin resistance of encoding and the upstream group propylhomoserin label of LIC cloning site.
The pET-30 carrier that contains candidate gene is transformed in the competent E.coli NovaBlue bacterial strain.On the kanamycins agar plate, select the clone, and utilize carrier special primer and insertion fragment special primer that selecteed clone is contrasted PCR.Plasmid DNA purification from the positive colony that contains the special insertion fragment of candidate gene.Plasmid DNA is transformed among the E.coli (BL21) then.On that agar plate of card, select to insert positive colony.(Apollo Scientific GB) induces the expression of the fused protein that contains the specific insetion sequence of gene (comprising the histidine mark thing that is positioned at the N-end) to IPTG by adding 1mM in the LB of 500ml nutrient culture media.
Produced the E.coli (BL21) of the recombinant fusion protein of the CPN1016 of CT668, the CT858, CT783, CT610, YscN and the DT8 that express chlamydia trachomatis and Chlamydia pneumoniae VR1310 and YscC.
Utilize foregoing nickel resin column (High Trap Sepharose, AmershamPharmacia Biotech) described recombinant fusion protein of purifying from the bacterium of cracking.Protein purification is carried out the SDS-PAGE of coomassie dyeing.Thereby the coomassie dyeing electrophoresis band of representing fused protein has carried out clear and definite evaluation proof by MALDI MS and has produced correct fused protein.Described in Knudsen etc. [17], be dissolved in the 50 μ g fused proteins that 50 μ g fused proteins in the Freund and twice intravenous injection be dissolved among the PBS by three intramuscular injections and come New Zealand white rabbit is carried out immunity, thereby obtain to contain the serum of polyclonal antibody.
Embodiment 12:
The Western blotting that utilizes the PAb of anti-candidate vaccine to carry out
In order to determine that PAb discerns correct candidate vaccine and in order to show contingent posttranslational modification or processing, carried out the 2D-PAGE Western blotting.By 2D-PAGE separate that 500 μ g from full cell lysate are not labeled with 2 * 10 6The chlamydia trachomatis D of cpm mark or Chlamydia pneumoniae protein, and with the protein electroblotting to pvdf membrane.Be used in and contain 150mM NaCl (or high salt concentration, 400mM), (Dako, the PAb of 1/500-1/1000 dilution carries out Western blotting in damping fluid GlostrupDenmark) for 20mM Tris, 0.2%w/v gelatin, 0.05%v/v Tween 20 (Bio-Rad) and 2%v/v standard lowlenthal serum.
Employed second antibody be puted together the goat of alkaline phosphatase anti--IgG (Bio-Rad) of rabbit, this IgG in the antibody damping fluid with 1/2000 dilution.Trace (Bio-Rad) dyes up to detecting tangible reaction with 5-bromo-4-chloro-3-Yin tricresyl phosphate ammonium (the BCIP)/nitroblue tetrazolium (NBT) of trembling.Western blotting carries out behind the computer scanning x-ray film being exposed about 8 days.Come being compared with Melanie II software by the homologue of the protein of immunostaining and its [35S]-mark.
In Fig. 7 A1 and 7A2, shown example with the complete 2D-PAGEIMB image of the PAb245 of anti-CT858.Be labeled shown in the background (Fig 7A2) as corresponding, the repeatability reaction takes place in PAb245 and DT4 (CT858C-end fragment) and DT48 (CT858N-end fragment) (Fig. 7 A1).
Fig. 7 B1 and 7B4 represent the IMB of CT668 and YscN.Immune-reactive protein matter spot on the gel and corresponding with the protein position on analyzing gel (Fig. 7 B3 and B6) in the location that is labeled on the background (Fig. 7 B2 and B5) has confirmed that thus reaction has taken place for PAb and correct protein.
The invention provides an IMB example, it shows with untreated control compares, and the content of some the isotype CT610 in the infection cell that MG132 handles obviously increases.DT7 is accredited as CT610 and just in time is positioned at the top (Fig. 6 A) of DT9, DT10, DT11 and DT12.Significantly reaction, wherein a displacement table DT7 and another displacement table DT9, DT10, DT11 and DT12 take place in the IMB of the PAb255 of the anti-CT610 on the 2D-gel trace of full cell lysate and two row's spots.The chances are owing to modification and processing after the different translations, and CT610 exists with different isotypes.If cell is handled with protein enzyme body inhibitor MG132, the amount of DT9, DT10 and DT11 then obviously increases (Fig. 7 C2).
Carry out the IMB of PAb255 on the SDS-PAGE of routine PVDF trace, described trace contains the protein that derives from the full cell lysate of handling respectively under the condition that has or do not have MG132 to exist.Fig. 7 C4 clearly illustrates that and compares (swimming lane a and c) with untreated contrast, in the swimming lane (swimming lane b and d) that contains the protein that comes from the infection cell that MG132 handles, represent DT7 arrange spot electrophoresis band below have more a band.Represent the level of DT7 row's top band obvious variation not to take place through the processing of protein enzyme body inhibitor.
Embodiment 13:
The expression of material standed in different chlamydia trachomatis serotypes and different host cells
The present invention has considered the specificity difference that serotype/host cell exists aspect may the secretory protein expression, this be because the interaction between host cell/Chlamydia for other serotype or may be different by in other host cell, cultivate.This is that the selection of candidate vaccine of conventional chlamydia trachomatis vaccine is relevant with most probable.
CT668, CT858, CT783 and CT610 have all been detected on the same position in chlamydia trachomatis A, D and L2.This is consistent with the high conservative that the gene of these protein of coding has between chlamydia trachomatis D and L2.In addition, when cultivate chlamydia trachomatis A in McCoy or Hep-2 cell rather than HeLa cell, when D and L2, express all proteins, this shows that these protein expressions do not rely on employed cell type.Yet, based on containing at metainfective 22-24 hour or carried out the gel of the I (chlamydia) protein matter of mark in 34-36 hour, in serotype A and D the pI of detected DT8 be 5.1 and Mw be 7.5.Yet, in serotype L2, the PI of detected DT8 be 6.4 and Mw be 7.5, the chances are for this because less aminoacid replacement has changed the static charge and the isoelectric point of protein.
Embodiment 14:
The indirect immunofluorescence microscopy of candidate vaccine
The cell (wells) that has cover glass contains the partly individual layer HeLa cell of degree of converging, and infects described individual layer HeLa cell with Chlamydia pneumoniae or chlamydia trachomatis D.So owing to used only have an appointment 50% cell of the Chlamydia of low titre infected, therefore can differentiate infected cells and infected cells not significantly.Fixedly go forward side by side with twice of cell flushing and with formaldehyde or methyl alcohol with PBS at metainfective different time and to connect immunofluorescence microscopy in the ranks.If necessary, with the HeLa protein pre-absorbing PAb of acetone precipitation so as to make and human body protein between the cross reaction minimum.
In example shown in Figure 8, adopted dilutability be 1/200 be 1/25 mouse monoclonal antibody by the rabbit PAb of pre-absorbing and dilutability, the Mab 18.3 of this monoclonal antibody desertification chlamydia oculogenitale MOMP (MAb 32.3) or anti-Chlamydia pneumoniae wherein.Puted together goat antirabbit (GAR) the IgG antibody of fluorescein isothiocynate (FITC) and puted together the goat of rhodamine anti--mouse (GAM) IgG antibody (Jackson, Trichem, Denmark) is used as second antibody.Carry out two immunostainings for the Subcellular Localization of measuring the candidate vaccine relevant with the Chlamydia inclusion body.
Faint reaction (Fig. 8 A3) takes place at the PAb 249 of the DT8 with short update time and the RB in the Chlamydia inclusion body.Although outside inclusion body, MIN cross reaction has taken place, between discovery and the host cell structure tangible reaction is not arranged with the HeLa cell protein.
On the contrary, CT858 to the kytoplasm in infected cell dyeing obviously and to the dyeing of non-infected cells not obvious (Fig. 8 B3).In a word, stronger in the dyeing of the edge of inclusion body.The dyeing of host cell kytoplasm can manifest at metainfective 12 to 72 hours, and this is consistent with the long update time of being predicted by pulse-chase analysis.When the Hep-2 cell of the PAb of the anti-Chlamydia pneumoniae CPN1016 that is produced and infection involving chlamydia pneumoniae reacts, observe reaction, wherein said Chlamydia pneumoniae CPN1016 and CT858 homology with same characteristic features.
To by the description of identification of protein example
Embodiment 15:
Spot period CT668 (DT1 and DT2)
CT668 is placed the tight upstream of the YscN ATP enzyme of an Asia bunch, and wherein said Asia bunch is contained with III type secretor and to be had the gene of homology and not contain the discernible restriction enzyme site of the signal peptidase of being predicted.Only there be about 4-6 hour in CT668 in chlamydia trachomatis D, its content is gradually reducing during this.The PAb of anti-CT668 as if just with inclusion body in RB faint reaction takes place in IMF, but lack the update time of considering this protein, this can be interpreted as having taken place quick degraded in host cell.Detected CT668 at metainfective 12-40 hour, this shows all produced this protein in the most of the time of growing in born of the same parents.Therefore may constantly CT668 be flowed to host cell by chlamydia trachomatis, here it has been brought into play effect and has been degraded fast.
Identified the variant of two kinds of CT668.Basic variations content on the gel of chlamydia trachomatis protein was the highest, and wherein said protein has carried out mark and has been recovered subsequently at metainfective 22-24 hour.What is interesting is that in all researchs of carrying out, the intensity of basic variations reduces along with the intensity increase of acid variant of time.When following the trail of CT668, in the time of 1 hour, just detected the increase (Figure 1B) of acid variant with 30 minutes the time interval.
This discovery shows described change and the conditional independence of running glue, runs the adhesive tape part and can only produce such as carbamylation or the such variation of amidation.On the contrary, described change seemingly produces by a kind of unknown enzyme, and this unknown enzyme is modified described protein.
Described the change of secretory protein in ornithosis virus, wherein IncA is after inserting on the inclusion body film, by the Ser/Thr kinases institute phosphorylated (Rockey etc., 1997) [29.] of host cell.By adopting protein enzyme body inhibitor to handle, be extended the update time of CT668, shows that limited amount at least CT668 can obtain processing in protein enzyme body.
Embodiment 16:
Spot period DT8 (DT8)
DT8 is a kind of novel 7.2kDa protein, by the homology retrieval, proves that it is a kind of chlamydia trachomatis specific protein.The psort analysis result shows the homing sequence discerned that does not have this protein.It is quite consistent that theoretic equal pI5.21/7.2kDa and experiment are measured.The numerous characteristics DT8 of known CT668 also has.Find that update time is very short, it is less than 6 hours, and IMF only shows and with RB faint reaction takes place.
After the terminator codon in DT8, can predict a possible stem-ring zone, this zone is a Rho-dependent/non-dependent tanscription termination of protein.
Embodiment 17:
Spot period DT7, DT9, DT10, DT11, DT12 (CT610)
CT610 is not positioned near the gene of the relevant dna homolog of secretion any and in other biosome.From full cell lysate and EB, detected described protein, after still assessing, found that the protein content in the full cell lysate has more 30 times at least by Melanie II software.Detect several isotypes of described protein, wherein these isotypes show as the polymorphism of two kinds of molecular weight, and this polymorphism is determined by the Pab255 of anti-CT610.These a few row's spots are represented DT7 (row of going up) and DT9,10,11,12 (row down).In pulse-chase research, these rows have short update time.What is interesting is that according to pulse-chase/MG132 research, the content of DT9, DT10 and DT11 significantly improves by the processing of protein enzyme body inhibitor.Verified the result of study of pulse-chase by the SDS PAGE IMB of Pab255.
Therefore the invention provides the evidence of in protein enzyme body, secreting and processing about some isotype of CT610.The Different Results of CT610 isotype represents to utilize the Pab of anti-candidate vaccine to detect the relation of these isotypes.
Embodiment 18:
Spot period DT3 (CT783)
CT783 has been proved to be a kind of chlamydia trachomatis protein disulfide isomerase.CT783 and thioredoxin matter disulfide isomerase (CT780) have homology, and also have homology with the protein disulfide-isomerase that derives from hot autotrophic methane bacteria.Can predict 33 amino acid whose homing sequences by PSORT and SignalP, and the theoretical PI of crack protein matter conforms to the result that molecular weight and experiment record.In addition, the polyclonal antibody that anti-CT783 produces in utilizing the 2D-PAGE IMB of 4-7L IPG dyes to correct spot.
Because N-end homing sequence generation cracking, this protein may be secreted by I or III type system, is to secrete by III type system more possibly.Although most PDI is positioned in the pericentral siphon usually, weak PSORT prediction shows the Subcellular Localization on the bacterium inner membrance.CT783 has short update time and by about 4-6 hour tracking after synthetic, finds not contain CT783 substantially in the Chlamydia.
Be extended by protein enzyme body inhibitor the update time of CT783, shows in protein enzyme body and may process.
Embodiment 19:
Spot period DT4 and DT48 (CT858) and CP63 (CPN1016)
CT858 have a N-end homing sequence that can cut and predicted be the periplasm protein matter of a kind of 67kDa, but on two diverse locations on the gel, detected CT858.The C-end that DT4 is accredited as the sequence mark thing of CT858 and CT858 is coupling fully.Among the IMB, the reaction of tangible repeatability has taken place in PAb245 and this C-short-movie section on containing the 2D-PAGE pvdf membrane of full cell lysate.In addition, Pab245 and protein spot DT48 reaction, it also has the update time of the length the same with DT4 in the pulse-chase research process.
Also DT48 is defined as the N-end fragment of CT858 by MALDI MS.The molecule homologue (coordinate) of DT48 is pI7.3/Mw25.8.The N-end parts of CT858 produces a kind of peptide that contains homologue, and it conforms to the homologue that utilizes the pI/Mw instrument to N-end (not containing signal peptide) measuring of different length.This analysis showed that at K 233S 234M 235Near exist cleavage site.
Hold and the C-end fragment until the EB phase (metainfective 72 hours) can both detect the N-of CT858 on the 2D-gel, and do not detect in the EB of purifying, this fact shows in the kytoplasm of host cell to have very long update time.This is consistent with the result that can both most clearly detect CT858 in IMF analyzes up to 72 hours in the host cell kytoplasm.CT858 and colibacillary afterbody-specific protein enzyme (tsp) has very little homology, wherein said enzyme is relevant with the processing of penicillin-binding protein and comprise the IRBP domain that derives from light receptor retinoid-like-conjugated protein (interphotoreceptor retionoid-bindingproteins) in the middle of the people, wherein said conjugated protein is in conjunction with hydrophobicity aglucon (Silber etc., 1992) [32.].The output relevant (referring to the commentary of Hobbs and Mattick, 1993) [33.] of the degradability protein in II type secretion and several Gram-negative bacterias that comprise P.aeroginosa and Aeromonas hydrophila.In Aerosiginosa hydrophila, a kind of elastin laminin matter enzyme (ahpB) of secretion is proved to be recently for the toxicity of microbial body extremely important (Cascon etc., 2000) [34.].
These protein have the result similar to CT858.But they mostly are synthesized to containing the propetide form of a cleavable signal peptide, are processed to mature protein then.The chlamydia trachomatis protein that another kind of and tail specific protein enzyme also has homology is false albuminoid CT441.Still need to determine whether this protein has the secretion characteristic identical with CT858.
Utilize the Pab253 of anti-CPN1016 to observe the identical Subcellular Localization of CT858 of Chlamydia pneumoniae homolog CPN1016, thereby show that this gene is that function is conservative between two Chlamydia kinds.
Embodiment 20:
Chlamydia trachomatis D and Chlamydia pneumoniae protein by the definite secretion of its gene number are analyzed by ANN, and described ANN can discern the peptide that has affinity with people HLA-A2.In Table V-VIII, listed the peptide that the expectation selected by ANN combines with HLA-A2 and provided the affinity of representing with nM (Kd).Value is lower better.The peptide that Kd is lower than 50nM mostly is immunogenic.The peptide that Kd is lower than 500nM (but being higher than 50nM) has potential immunogenicity.Can improve by the appropriate amino acid in Asia on the anchor station that replaces P2 or P9 with the adhesion of given peptide-a kind of common reservation is at the specific scheme of native peptides.
Embodiment 21:
Candidate vaccine produces specific cytotoxic CD8+T cell in the experimental animal model body Ability is measured
In one approach, use the described candidate vaccine immunization experiment animal (mouse or cavy) of total length recombinant protein, wherein said animal used as test comprises the transgenosis A2 mouse of expressing human HLA I type molecule.
In another approach, by the T-cell epitope of computerized algorithm screening candidate vaccine, synthesize the peptide that contains these epi-positions then, and be used for immunity according to the immunization method that is used for the total length candidate vaccine.
In the third method, with the peptide of synthetic 8-10 the amino acid long of a kind of overlapping method, so these peptides have covered the full length sequence of candidate vaccine, and the MHC I type that is used for competing with radio-labeled middle junction zoarium combines test.To be used for immunity for the peptide of good combination thing according to the immunization method that is used for the total length candidate vaccine.
Candidate vaccine both can be used also and can use with the single peptide/protein form in adjuvant with the form of peptide/protein combination.Candidate vaccine can also be used or use by the virus of expressing candidate vaccine as the DNA-vaccine.
By purify in immune animal body peripheral blood lymphocytes (PBMC) and come purifying CD8+ cell with the sub antibody that combines of magnetic bead or by other method of density gradient centrifugation by utilizing.The CD8+ cytoactive is by such as ELISPOT with mix the such proliferation test of tritiate thymidine and measure by specificity breaking test (chromium release).
The PBMC or the CD8+ cell of purifying placed on the droplet plate by tiling with the limiting dilution degree in the immunized animal body, together with antigen presenting cell, growth factor and specificity or the nonspecific stimulation thing of irradiation.In order to carry out differential stimulus, adopted that to be predicted to be good T-cell epitope or single candidate vaccine protein or the peptide that is found to be MHC I type molecule good combination thing.In order to carry out nonspecific stimulation, used by the cell of choamydiae infection.With cellular incubation 9-14 days, antigen-specific sexual cell bred during this.The generation of specific cytotoxic CD8+T-cell is by utilizing the ELISPOT test determination to be determined by the cytokine secretion of irritation cell.The propagation of T-cell is by mixing the tritiate thymidine, carrying out scintillation counting then and measure.The cytotoxicity of proliferative cell utilizes cytotoxicity test to measure, and this test is as with the cell of choamydiae infection or express the chromium-release test [42] of the recombinant cell of candidate vaccine proteins/peptides as target cell.
Embodiment 21:
Candidate vaccine is protected the detection of the ability of mouse and cavy opposing choamydiae infection
Adopt the candidate vaccine of simple protein/peptide form or adopt the candidate vaccine combination that animal used as test is carried out immunity (as mentioned above).After the immunity, the described animal sexuality that experimentizes is dyed (intranasal infection Chlamydia pneumoniae and genital infection chlamydia trachomatis) with Chlamydia.By chlamydia cultivate, immunohistochemistry, quantitative PCR and the metainfective seroconversion of research measure anti-infectious protective capability.
Embodiment 23:
In human body, produce the choamydiae infection of candidate vaccine-specific cytotoxic CD8+T cell The mensuration of ability
Adopt ELISA (Medac) method to detect among the human serum sample whether have chlamydial antibody.Select the serum-positive individuals that contains candidate vaccine-specific cytotoxic CD8+T-cell.
Utilize density from detected be purifying peripheral blood lymphocytes (PBMC) and utilize antibody and magnetic bead or other method purifying CD8+ cell the anti-chlamydial antibody sample human body.The activity of the anti-candidate vaccine proteins/peptides of CD8+T-cell-specific is to measure by the method described in the embodiment 19.
Embodiment 24
The ELISA that utilizes candidate vaccine to be used for diagnostic purpose detects
Because secretory protein of the present invention does not exist or content is very low, thereby can not detect antibody at this protein based on the immunity test of purifying substance in the microbial body that is purified.Therefore secretory protein can be represented unrecognized major antigen, and this antigen is also relevant with humoral immunoresponse(HI).In addition and since secretory protein only in the born of the same parents of Chlamydia growth the stage express, so based on being detected chlamydial persistent infection by the ELISA of secretion I (chlamydia) protein matter.
1) secretory protein produces with the recombinant protein form of purifying.Perhaps also produce the over lapping synt hetic peptides of representing secretory protein.
2) the dull and stereotyped Bao Beiliao of ELISA represent the recombinant protein synthetic peptide of secretory protein generation (or by) of the purifying of secretory protein.The dull and stereotyped hyclone sealing with 15% of ELISA to avoid non-specific binding.
3) utilize little-IF or ELISA (Medac) screening among the patients serum the desertification chlamydia oculogenitale or the antibody of Chlamydia pneumoniae.Represent at Bao Beiliao on the ELISA flat board of recombinant antigen of secretory protein and detect positive serum.
4) use anti-human IgG, IgA or IgM to detect antibodies.With the serum of infected mouse with comparing.
5) will by little-IF or ELISA (Medac) to the result with compare based on the ELISA of the recombinant protein of representing secretory protein.
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Sequence table
<110>??Vandahl，Brian?Berg
＜120〉be used to identify the method for proteins from intracellular bacteria
<130>??P200100157WO
<150>??DK?PA?200100581
<151>??2001-04-09
<160>??194
<170>??PatentIn?version?3.1
<210>??1
<211>??67
<212>??PRT
＜213〉chlamydia trachomatis (Chlamydia trachomatis)
<400>??1
Met?Gln?His?Thr?Ile?Met?Leu?Ser?Leu?Glu?Asn?Asp?Asn?Asp?Lys?Leu
1???????????????5???????????????????10??????????????????15
Ala?Ser?Met?Met?Asp?Arg?Val?Val?Ala?Ala?Ser?Ser?Ser?Ile?Leu?Ser
20??????????????????25??????????????????30
Ala?Ser?Lys?Asp?Ser?Glu?Ser?Asn?Arg?Gln?Phe?Thr?Ile?Ser?Lys?Ala
35??????????????????40??????????????????45
Pro?Asp?Lys?Glu?Ala?Pro?Cys?Arg?Val?Ser?Tyr?Val?Ala?Ala?Ser?Ala
50??????????????????55??????????????????60
Leu?Ser?Glu
65
<210>2
<211>204
<212>DNA
＜213〉chlamydia trachomatis
<400>2
atgcaacaea?caattatgct?gtctttagag?aacgataatg?ataagcttgc?ttctatgatg
60
gatcgagttg?ttgctgcgtc?atcaagcatt?ctttctgctt?ccaaagattc?tgagtccaat
120
agacagttta?ctatttctaa?agctccggat?aaagaagctc?cttgcagagt?atcttatgta
180
gctgcaagtg?cactttcaga?atag
204
<210>3
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT263 immunogenic peptide
<400>3
Lys?Leu?Ala?Glu?Ala?Ile?Phe?Pro?Ile
1???????????????5
<210>4
<211>9
<212>PRT
＜213〉chlamydia trachomatis
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<222>(1)..(9)
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Phe?Leu?Lys?Asn?Asn?Lys?Val?Lys?Leu
1???????????????5
<210>5
<211>9
<212>PRT
＜213〉chlamydia trachomatis
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＜221〉combination
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<400>5
Ala?Leu?Ser?Pro?Pro?Pro?Ser?Gly?Tyr
1???????????????5
<210>6
<211>9
<212>PRT
＜213〉chlamydia trachomatis
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＜221〉combination
<222>(1)..(9)
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Phe?Ile?Ala?Lys?Gln?Ala?Ser?Leu?Val
1???????????????5
<210>7
<211>9
<212>PRT
＜213〉chlamydia trachomatis
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<400>7
Thr?Leu?Ser?Leu?Phe?Pro?Phe?Ser?Leu
1???????????????5
<210>8
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT263 immunogenic peptide
<400>8
Ser?Leu?Val?Ala?Cys?Pro?Cys?Ser?Met
1???????????????5
<210>9
<211>9
<212>PRT
＜213〉chlamydia trachomatis
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＜221〉combination
<222>(1)..(9)
＜223〉CT263 immunogenic peptide
<400>9
Leu?Ile?Phe?Ala?Asp?Pro?Ala?Glu?Ala
1???????????????5
<210>10
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT263 immunogenic peptide
<400>10
Leu?Leu?Leu?Ile?Phe?Ala?Asp?Pro?Ala
1???????????????5
<210>11
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT263 immunogenic peptide
<400>11
Ile?Leu?Ser?Trp?Met?Leu?Met?Phe?Ala
1???????????????5
<210>12
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT541 immunogenic peptide
<400>12
Lys?Gln?Met?Ala?Glu?Val?Gln?Lys?Ala
1???????????????5
<210>13
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT541 immunogenic peptide
<400>13
Leu?Met?Phe?Ala?Val?Ala?Leu?Pro?Ile
1???????????????5
<210>14
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT541 immunogenic peptide
<400>14
Lys?Leu?Gln?Tyr?Arg?Val?Val?Lys?Glu
1???????????????5
<210>15
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT541 immunogenic peptide
<400>15
Phe?Leu?Lys?Glu?Asn?Lys?Glu?Lys?Ala
1???????????????5
<210>16
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT541 immunogenic peptide
<400>16
Lys?Leu?Ser?Arg?Thr?Phe?Gly?His?Leu
1???????????????5
<210>17
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT541 immunogenic peptide
<400>17
Ser?Leu?Leu?Ile?Phe?Glu?Val?Lys?Leu
1???????????????5
<210>18
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT541 immunogenic peptide
<400>18
Val?Leu?Ser?Gly?Lys?Pro?Thr?Ala?Leu
1???????????????5
<210>19
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT541 immunogenic peptide
<400>19
Val?Leu?Tyr?Ile?His?Pro?Asp?Leu?Ala
1???????????????5
<210>20
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT541 immunogenic peptide
<400>20
Leu?Leu?Ser?Arg?Gln?Leu?Ser?Arg?Thr
1???????????????5
<210>21
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT691 immunogenic peptide
<400>21
Leu?Leu?Gln?Arg?Glu?Leu?Met?Lys?Val
1???????????????5
<210>22
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT691 immunogenic peptide
<400>22
Ser?Thr?Ile?Asn?Val?Leu?Phe?Pro?Leu
1???????????????5
<210>23
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT691 immunogenic peptide
<400>23
Pro?Leu?Gln?Ala?His?Leu?Glu?Leu?Val
1???????????????5
<210>24
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT691 immunogenic peptide
<400>24
Ser?Leu?Phe?Gly?Gln?Ser?Pro?Phe?Ala
1???????????????5
<210>25
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT691 immunogenic peptide
<400>25
Lys?Leu?Ala?Tyr?Arg?Val?Ser?Met?Thr
1???????????????5
<210>26
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT691 immunogenic peptide
<400>26
Val?Leu?Trp?Met?Gln?Ile?Ile?Lys?Gly
1???????????????5
<210>27
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT691 immunogenic peptide
<400>27
Val?Leu?Phe?Pro?Leu?Phe?Ser?Ala?Leu
1???????????????5
<210>28
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT691 immunogenic peptide
<400>28
Phe?Leu?Gln?Lys?Thr?Val?Gln?Ser?Phe
1???????????????5
<210>29
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT691 immunogenic peptide
<400>29
Phe?Gly?Gln?Ser?Pro?Phe?Ala?Pro?Leu
1???????????????5
<210>30
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<400>30
Val?Leu?Ala?Asp?Phe?Ile?Gly?Gly?Leu
1???????????????5
<210>31
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<400>31
Arg?Met?Ala?Ser?Leu?Gly?His?Lys?Val
1???????????????5
<210>32
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<400>32
Gly?Leu?Asn?Asp?Phe?His?Ala?Gly?Val
1???????????????5
<210>33
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<400>33
Phe?Ser?Cys?Ala?Asp?Phe?Phe?Pro?Val
1???????????????5
<210>34
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<400>34
Met?Leu?Thr?Asp?Arg?Pro?Leu?Glu?Leu
1???????????????5
<210>35
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<400>35
Leu?Leu?Glu?Asn?Val?Asp?Thr?Asn?Val
1???????????????5
<210>36
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<400>36
Arg?Met?Ile?Leu?Thr?Gln?Asp?Glu?Val
1???????????????5
<210>37
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<400>37
Ser?Cys?Ala?Asp?Phe?Phe?Pro?Val?Val
1???????????????5
<210>38
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<400>38
Phe?Val?Phe?Asn?Val?Gln?Phe?Pro?Asn
1???????????????5
<210>39
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<400>39
Tyr?Leu?Tyr?Ala?Leu?Leu?Ser?Met?Leu
1???????????????5
<210>40
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<400>40
Ser?Leu?Ala?Val?Arg?Glu?His?Gly?Ala
1???????????????5
<210>41
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<400>41
Tyr?Leu?Pro?Tyr?Thr?Val?Gln?Lys?Ser
1???????????????5
<210>42
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<400>42
Ala?Thr?Ile?Ala?Pro?Ser?Ile?Arg?Ala
1???????????????5
<210>43
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<220>
<221>BINDING
<222>(1)..(9)
<223>
<400>43
Leu?Leu?Glu?Val?Asp?Gly?Ala?Pro?Val
1???????????????5
<210>44
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<400>44
Arg?Thr?Ala?Gly?Ala?Gly?Gly?Phe?Val
1???????????????5
<210>45
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT858 immunogenic peptide
<400>45
Ser?Leu?Phe?Tyr?Ser?Pro?Met?Val?Pro
1???????????????5
<210>46
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae (Chlamydia pneumoniae)
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0152 immunogenic peptide
<400>46
Phe?Leu?Val?Ser?Cys?Leu?Phe?Ser?Val
1???????????????5
<210>47
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0152 immunogenic peptide
<400>47
Tyr?Leu?Arg?Asp?Ala?Gln?Thr?Ile?Leu
1???????????????5
<210>48
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0152 immunogenic peptide
<400>48
Leu?Leu?Ile?Arg?Ile?Gln?Asp?His?Val
1???????????????5
<210>49
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0152 immunogenic peptide
<400>49
Lys?Leu?Gly?Arg?Lys?Phe?Ala?Ala?Val
1???????????????5
<210>50
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0152 immunogenic peptide
<400>50
Leu?Val?Ser?Arg?Thr?Gln?Gln?Thr?Leu
1???????????????5
<210>51
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0152 immunogenic peptide
<400>51
Cys?Leu?Phe?Ser?Val?Ala?Ile?Gly?Ala
1???????????????5
<210>52
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0152 immunogenic peptide
<400>52
Gly?Phe?Gly?Pro?Pro?Pro?Ile?Ile?Val
1???????????????5
<210>53
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0152 immunogenic peptide
<400>53
Ser?Leu?Pro?Thr?Lys?Pro?Tyr?Ile?Leu
1???????????????5
<210>54
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0152 immunogenic peptide
<400>54
Arg?Ile?Gln?Asp?His?Val?Thr?Ala?Asn
1???????????????5
<210>55
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0152 immunogenic peptide
<400>55
Ala?Ile?Gly?Ala?Ser?Ala?Ala?Pro?Val
1???????????????5
<210>56
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0152 immunogenic peptide
<400>56
Arg?Leu?Gly?Ile?Ser?Gly?Phe?Ser?Leu
1???????????????5
<210>57
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0619 immunogenic peptide
<400>57
Phe?Met?Val?Ser?Gly?Pro?Val?Val?Val
1???????????????5
<210>58
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0619 immunogenic peptide
<400>58
Leu?Val?Leu?Glu?Gly?Ala?Asn?Ala?Val
1???????????????5
<210>59
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0705 immunogenic peptide
<400>59
Phe?Val?Gly?Ala?Asn?Leu?Thr?Leu?Val
1???????????????5
<210>60
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0619 immunogenic peptide
<400>60
Cys?Leu?Ala?Glu?Asn?Ala?Phe?Ala?Gly
1???????????????5
<210>61
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0705 immunogenic peptide
<400>61
Lys?Ile?Glu?Glu?Val?Gln?Thr?Pro?Leu
1???????????????5
<210>62
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0705 immunogenic peptide
<400>62
Ala?Leu?Lys?Gly?His?Gln?Leu?Thr?Leu
1???????????????5
<210>63
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0705 immunogenic peptide
<400>63
Gln?Met?Ala?Glu?Ala?Ala?Asp?Leu?Val
1???????????????5
<210>64
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>64
Phe?Met?Gly?Ala?His?Val?Phe?Ala?Ser
1???????????????5
<210>65
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>65
Leu?Leu?Ile?Gln?His?Ser?Ala?Lys?Val
1???????????????5
<210>66
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>66
Phe?Leu?Cys?Pro?Phe?Gln?Ala?Pro?Ser
1???????????????5
<210>67
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>67
Phe?Gln?Ala?Pro?Ser?Pro?Ala?Pro?Val
1???????????????5
<210>68
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>68
Ala?Met?Asn?Ala?Cys?Val?Asn?Gly?Ile
1???????????????5
<210>69
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<220>
＜221〉combination
<222>(1)..(9)
<223>
<400>69
Phe?Met?Gly?Ile?Gln?Val?Leu?His?Leu
1???????????????5
<210>70
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>70
Arg?His?Ala?Ala?Gln?Ala?Thr?Gly?Val
1???????????????5
<210>71
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>71
Phe?Gln?Tyr?Ala?Asp?Gly?Gln?Met?Val
1???????????????5
<210>72
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>72
Ser?Val?Ser?Ala?Met?Gly?Asn?Phe?Val
1???????????????5
<210>73
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>73
Phe?Leu?Ser?Tyr?Arg?Ser?Gln?Val?His
1???????????????5
<210>74
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>74
Phe?Leu?Leu?Thr?Ala?Ile?Pro?Gly?Ser
1???????????????5
<210>75
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>75
Val?Leu?Thr?Pro?Trp?Ile?Tyr?Arg?Lys
1???????????????5
<210>76
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>76
Ser?Val?Val?Met?Asn?Gln?Gln?Pro?Leu
1???????????????5
<210>77
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>77
Ser?Leu?Lys?Asn?Ser?Gln?Gln?Gln?Leu
1???????????????5
<210>78
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>78
Met?Leu?Pro?Asp?Thr?Leu?Asp?Ser?Val
1???????????????5
<210>79
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>79
Ala?Leu?Pro?Tyr?Thr?Glu?Gln?Gly?Leu
1???????????????5
<210>80
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0796 immunogenic peptide
<400>80
Val?Leu?Ser?Gly?Phe?Gly?Gly?Gln?Val
1???????????????5
<210>81
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>81
Leu?Leu?Phe?Gly?Val?Val?Phe?Gly?Val
1???????????????5
<210>82
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>82
Ser?Leu?Gln?Glu?Arg?Tyr?Pro?Thr?Leu
1???????????????5
<210>83
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>83
Met?Leu?Leu?Lys?Gly?Gln?Asn?Lys?Val
1???????????????5
<210>84
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>84
Phe?Thr?Phe?Leu?Pro?Ile?Ile?Leu?Val
1???????????????5
<210>85
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>85
Phe?Leu?Gly?Asp?Ile?Ser?Ser?Gly?Ala
1???????????????5
<210>86
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>86
Phe?Leu?Ala?Gly?Lys?Lys?Ala?Arg?Val
1???????????????5
<210>87
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>87
Leu?Leu?Asp?Ala?Ala?Tyr?Gln?Arg?Ala
1???????????????5
<210>88
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>88
Tyr?Leu?Gly?Tyr?Leu?Phe?Thr?Phe?Leu
1???????????????5
<210>89
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>89
Tyr?Leu?Phe?Thr?Phe?Leu?Pro?Ile?Ile
1???????????????5
<210>90
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>90
Ser?Leu?Gly?Ala?Thr?His?Phe?Leu?Pro
1???????????????5
<210>91
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>91
Glu?Leu?Ile?Asp?Gln?Gly?His?Arg?Leu
1???????????????5
<210>92
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>92
Phe?Leu?Pro?Ile?Ile?Leu?Val?Leu?Leu
1???????????????5
<210>93
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>93
Leu?Val?Leu?Leu?Phe?Val?Tyr?Leu?Val
1???????????????5
<210>94
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>94
Val?Thr?Gly?Pro?Ala?Thr?Pro?Gln?Leu
1???????????????5
<210>95
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>95
Ser?Leu?Glu?Lys?Gln?Asp?Pro?Glu?Val
1???????????????5
<210>96
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>96
Ile?Leu?Met?Ala?Ala?Thr?Asn?Arg?Pro
1???????????????5
<210>97
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>97
Leu?Thr?Gln?Glu?Thr?Asp?Thr?Glu?Ala
1???????????????5
<210>98
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>98
Met?Leu?Leu?Asp?Ala?Ala?Tyr?Gln?Arg
1???????????????5
<210>99
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>99
Leu?Leu?Asn?Glu?Ala?Ala?Leu?Leu?Ala
1???????????????5
<210>100
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>100
Glu?Leu?Tyr?Asp?Gln?Leu?Ala?Val?Leu
1???????????????5
<210>101
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>101
Leu?Ile?Gly?Lys?Tyr?Leu?Ser?Pro?Val
1???????????????5
<210>102
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>102
Ser?Leu?Gly?Gly?Arg?Ile?Pro?Lys?Gly
1???????????????5
<210>103
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>103
Gly?Met?Ser?Pro?Gln?Leu?Gly?Asn?Val
1???????????????5
<210>104
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>104
Leu?Leu?Ala?Ala?Arg?Lys?Asp?Arg?Thr
1???????????????5
<210>105
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>105
Val?Thr?Phe?Ala?Asp?Val?Ala?Gly?Ile
1???????????????5
<210>106
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>106
Val?Leu?Thr?Glu?Pro?Leu?Val?Val?Thr
1???????????????5
<210>107
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0998 immunogenic peptide
<400>107
Lys?Ile?Ala?Leu?Asn?Asp?Asn?Leu?Val
1???????????????5
<210>108
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN1016 immunogenic peptide
<400>108
Lys?Leu?Gly?Ala?Ile?Val?Phe?Gly?Leu
1???????????????5
<210>109
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN1016 immunogenic peptide
<400>109
Tyr?Leu?Gly?Asp?Glu?Ile?Leu?Glu?Val
1???????????????5
<210>110
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN1016 immunogenic peptide
<400>110
Phe?Leu?Pro?Thr?Phe?Gly?Pro?Ile?Leu
1???????????????5
<210>111
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN1016 immunogenic peptide
<400>111
Ser?Leu?Gln?Asn?Phe?Ser?Gln?Ser?Val
1???????????????5
<210>112
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN1016 immunogenic peptide
<400>112
Phe?Thr?Asp?Glu?Gln?Ala?Val?Ala?Val
1???????????????5
<210>113
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN1016 immunogenic peptide
<400>113
Ser?Leu?Asn?Asp?Tyr?His?Ala?Gly?Ile
1???????????????5
<210>114
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN1016 immunogenic peptide
<400>114
Arg?Met?Ile?Phe?Thr?Gln?Asp?Glu?Val
1???????????????5
<210>115
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN1016 immunogenic peptide
<400>115
Thr?Gln?Gln?Ala?Arg?Leu?Gln?Leu?Val
1???????????????5
<210>116
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN1016 immunogenic peptide
<400>116
Ser?Leu?Val?Ala?Pro?Leu?Ile?Pro?Glu
1???????????????5
<210>117
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN1016 immunogenic peptide
<400>117
Tyr?Met?Val?Pro?Tyr?Phe?Trp?Glu?Glu
1???????????????5
<210>118
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN1016 immunogenic peptide
<400>118
Tyr?Val?Glu?Ala?Val?Lys?Thr?Ile?Val
1???????????????5
<210>119
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN1016 immunogenic peptide
<400>119
Phe?Thr?Gln?Asp?Glu?Val?Ser?Ser?Ala
1???????????????5
<210>120
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN1016 immunogenic peptide
<400>120
Gly?Ala?Gly?Gly?Phe?Val?Phe?Gln?Val
1???????????????5
<210>121
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN1016 immunogenic peptide
<400>121
Leu?Leu?Gly?Phe?Ala?Gln?Val?Arg?Pro
1???????????????5
<210>122
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0412 immunogenic peptide
<400>122
Arg?Leu?Glu?Glu?Val?Ser?Gln?Lys?Leu
1???????????????5
<210>123
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0412 immunogenic peptide
<400>123
Leu?Thr?Thr?Asp?Thr?Pro?Pro?Val?Leu
1???????????????5
<210>124
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0412 immunogenic peptide
<400>124
Lys?Leu?Leu?Asp?Met?Glu?Gly?Tyr?Ala
1???????????????5
<210>125
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0412 immunogenic peptide
<400>125
Val?Leu?Ser?Glu?Asp?Pro?Pro?Tyr?Ile
1???????????????5
<210>126
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0412 immunogenic peptide
<400>126
Ala?Leu?Gln?Ser?Tyr?Cys?Gln?Ala?Tyr
1???????????????5
<210>127
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0412 immunogenic peptide
<400>127
Lys?Leu?Thr?Gln?Thr?Leu?Val?Glu?Leu
1???????????????5
<210>128
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0412 immunogenic peptide
<400>128
Phe?Val?Gly?Ala?Cys?Ser?Pro?Glu?Ile
1???????????????5
<210>129
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0412 immunogenic peptide
<400>129
Asn?Leu?Thr?Thr?Asp?Thr?Pro?Pro?Val
1???????????????5
<210>130
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0412 immunogenic peptide
<400>130
Leu?Met?Glu?Arg?Ala?Ile?Pro?Pro?Lys
1???????????????5
<210>131
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0661 immunogenic peptide
<400>131
Lys?Met?Ala?Glu?Val?Gln?Lys?Leu?Val
1???????????????5
<210>132
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0661 immunogenic peptide
<400>132
Ala?Leu?Gly?Met?Gln?Gly?Met?Lys?Glu
1???????????????5
<210>133
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0661 immunogenic peptide
<400>133
Lys?Leu?Ser?Arg?Thr?Phe?Gly?His?Leu
1???????????????5
<210>134
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0661 immunogenic peptide
<400>134
Leu?Leu?Ile?Phe?Glu?Ile?Asn?Leu?Ile
1???????????????5
<210>135
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0661 immunogenic peptide
<400>135
Val?Leu?Ala?Thr?Val?Ala?Leu?Ala?Leu
1???????????????5
<210>136
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0661 immunogenic peptide
<400>136
Ile?Leu?Leu?Pro?Leu?Gly?Gln?Thr?Ile
1???????????????5
<210>137
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0661 immunogenic peptide
<400>137
Val?Leu?Tyr?Ile?His?Pro?Asp?Leu?Ala
1???????????????5
<210>138
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
<221>HOMOLOGY
<222>(1)..(9)
＜223〉Homolog to CT541 immunogenic peptide
<400>138
Leu?Val?Leu?Ala?Thr?Val?Ala?Leu?Ala
1???????????????5
<210>139
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0681 immunogenic peptide
<400>139
Tyr?Met?Leu?Pro?Ile?Phe?Thr?Ala?Leu
1???????????????5
<210>140
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0681 immunogenic peptide
<400>140
Leu?Leu?His?Glu?Phe?Asn?Gln?Leu?Leu
1???????????????5
<210>141
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0681 immunogenic peptide
<400>141
Val?Leu?Gln?Arg?Glu?Leu?Met?Gln?Ile
1???????????????5
<210>142
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0681 immunogenic peptide
<400>142
Pro?Leu?Gln?Ala?His?Leu?Glu?Met?Val
1???????????????5
<210>143
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0681 immunogenic peptide
<400>143
Arg?Leu?Phe?Gly?Gln?Ser?Pro?Phe?Ala
1???????????????5
<210>144
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0681 immunogenic peptide
<400>144
Gly?Leu?Phe?Met?Pro?Ile?Ser?Arg?Ala
1???????????????5
<210>145
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0681 immunogenic peptide
<400>145
Lys?Leu?Ala?His?Arg?Ile?Asn?Met?Thr
1???????????????5
<210>146
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0681 immunogenic peptide
<400>146
Tyr?Leu?Trp?Leu?Gln?Val?Ile?Arg?Arg
1???????????????5
<210>147
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0681 immunogenic peptide
<400>147
Thr?Leu?Leu?His?Glu?Phe?Asn?Gln?Leu
1???????????????5
<210>148
<211>9
<212>PRT
＜213〉Chlamydia pneumoniae
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0681 immunogenic peptide
<400>148
Phe?Gly?Gln?Ser?Pro?Phe?Ala?Pro?Leu
1???????????????5
<210>149
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT149 immunogenic peptide
<400>149
Phe?Leu?Gly?Ala?Ala?Pro?Ala?Gln?Met
1???????????????5
<210>150
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT149 immunogenic peptide
<400>150
Phe?Leu?Gly?Ile?Gln?Asp?His?Ile?Leu
1???????????????5
<210>151
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CPN0681 immunogenic peptide
<400>151
Leu?Leu?Thr?Ala?Asn?Gly?Ile?Ala?Val
1???????????????5
<210>152
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT149 immunogenic peptide
<400>152
Ser?Leu?Pro?Arg?Arg?Ile?Pro?Val?Leu
1???????????????5
<210>153
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT149 immunogenic peptide
<400>153
Gly?Leu?Gln?Glu?His?Cys?Arg?Gly?Val
1???????????????5
<210>154
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT149 immunogenic peptide
<400>154
Ser?Leu?Gly?Cys?His?Thr?Thr?Ile?His
1???????????????5
<210>155
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT149 immunogenic peptide
<400>155
Ile?Leu?Thr?His?Phe?Gln?Ser?Asn?Leu
1???????????????5
<210>156
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT149 immunogenic peptide
<400>156
Val?Leu?Ser?Cys?Gly?Tyr?Asn?Leu?Val
1???????????????5
<210>157
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT149 immunogenic peptide
<400>157
Leu?Leu?Lys?Glu?Ile?Cys?Ala?Thr?Ile
1???????????????5
<210>158
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT149 immunogenic peptide
<400>158
Arg?Leu?Phe?Leu?Gly?Ala?Ala?Pro?Ala
1???????????????5
<210>159
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT149 immunogenic peptide
<400>159
Ala?Thr?Val?Ala?Lys?Tyr?Pro?Glu?Val
1???????????????5
<210>160
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT149 immunogenic peptide
<400>160
Leu?Leu?Ser?Gly?Ser?Gly?Phe?Ala?Ala
1???????????????5
<210>161
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT149 immunogenic peptide
<400>161
Leu?Thr?Ala?Asn?Gly?Ile?Ala?Val?Ala
1???????????????5
<210>162
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT149 immunogenic peptide
<400>162
Ser?Gly?Phe?Ala?Ala?Pro?Val?Glu?Val
1???????????????5
<210>163
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT500 immunogenic peptide
<400>163
Phe?Met?Ile?Ser?Gly?Pro?Val?Val?Val
1???????????????5
<210>164
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT500 immunogenic peptide
<400>164
Ala?Leu?Phe?Gly?Glu?Ser?Ile?Gly?Val
1???????????????5
<210>165
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT500 immunogenic peptide
<400>165
Ser?Leu?Glu?Asn?Ala?Ala?Ile?Glu?Val
1???????????????5
<210>166
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT500 immunogenic peptide
<400>166
Leu?Met?Gly?Ala?Thr?Asn?Pro?Lys?Glu
1???????????????5
<210>167
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT500 immunogenic peptide
<400>167
Arg?Ile?Ala?Ala?Met?Lys?Met?Val?His
1???????????????5
<210>168
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT671 immunogenic peptide
<400>168
Ala?Leu?Val?Glu?Thr?Pro?Met?Ala?Val
1???????????????5
<210>169
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT671 immunogenic peptide
<400>169
Phe?Cys?Gly?Ala?Asn?Leu?Thr?Leu?Val
1???????????????5
<210>170
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT671 immunogenic peptide
<400>170
Ser?Leu?Lys?Ala?Arg?Gln?Leu?Asn?Leu
1???????????????5
<210>171
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT671 immunogenic peptide
<400>171
Gln?Leu?Thr?Glu?Ala?Thr?Gln?Leu?Val
1???????????????5
<210>172
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT671 immunogenic peptide
<400>172
Asp?Leu?Gln?Trp?Val?Glu?Gln?Leu?Val
1???????????????5
<210>173
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT671 immunogenic peptide
<400>173
Ile?Val?Leu?Asp?Asn?Ser?Asn?Thr?Val
1???????????????5
<210>174
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉in conjunction with G
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>174
Leu?Leu?Phe?Gly?Val?Ile?Phe?Gly?Val
1???????????????5
<210>175
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>175
Leu?Leu?Ala?Lys?Gly?Gln?Asn?Lys?Val
1???????????????5
<210>176
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>176
Phe?Thr?Phe?Met?Pro?Ile?Ile?Leu?Val
1???????????????5
<210>177
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉in conjunction with G
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>177
Phe?Leu?Gly?Asp?Val?Ser?Ser?Gly?Ala
1???????????????5
<210>178
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>178
Leu?Leu?Asp?Ala?Ala?Tyr?Gln?Arg?Ala
1???????????????5
<210>179
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>179
Gly?Met?Ser?Asp?His?Leu?Gly?Thr?Val
1???????????????5
<210>180
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉in conjunction with G
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>180
Ser?Leu?Gly?Ala?Thr?His?Phe?Leu?Pro
1???????????????5
<210>181
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>181
Asn?Leu?Ala?Ala?Leu?Glu?Asn?Arg?Val
1???????????????5
<210>182
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>182
Tyr?Leu?Phe?Thr?Phe?Met?Pro?Ile?Ile
1???????????????5
<210>183
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉in conjunction with G
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>183
Phe?Pro?Thr?Ala?Phe?Phe?Phe?Leu?Leu
1???????????????5
<210>184
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>184
Ile?Leu?Met?Ala?Ala?Thr?Asn?Arg?Pro
1???????????????5
<210>185
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>185
Lys?Thr?Ala?Leu?Asn?Asp?Asn?Leu?Val
1???????????????5
<210>186
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉in conjunction with G
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>186
Leu?Leu?Asn?Glu?Ala?Ala?Leu?Leu?Ala
1???????????????5
<210>187
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>187
Glu?Leu?Tyr?Asp?Gln?Leu?Ala?Val?Leu
1???????????????5
<210>188
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>188
Ala?Leu?Glu?Lys?Gln?Asp?Pro?Glu?Val
1???????????????5
<210>189
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉in conjunction with G
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>189
Ser?Leu?Gly?Gly?Arg?Ile?Pro?Lys?Gly
1???????????????5
<210>190
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>190
Phe?Met?Pro?Ile?Ile?Leu?Val?Leu?Leu
1???????????????5
<210>191
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>191
Leu?Leu?Ala?Ala?Arg?Lys?Asp?Arg?Thr
1???????????????5
<210>192
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉in conjunction with G
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>192
Val?Thr?Phe?Ala?Asp?Val?Ala?Gly?Ile
1???????????????5
<210>193
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>193
Tyr?Thr?Ile?Ser?Pro?Arg?Thr?Asp?Val
1???????????????5
<210>194
<211>9
<212>PRT
＜213〉chlamydia trachomatis
<220>
＜221〉combination
<222>(1)..(9)
＜223〉CT841 immunogenic peptide
<400>194
Leu?Ile?Gly?Ala?Pro?Gly?Thr?Gly?Lys
1???????????????5

Claims (72)

1. one kind is used to identify the method for protein by the intracellular bacteria secretion, and this method comprises the following steps:
1) use the intracellular bacteria host cells infected,
2) mark is present in the intracellular bacteria in the infected cell,
3) preparation
A) the full cell lysate of infected cell
B) bacterium that derives from infected cell of purifying and cracking,
4) to i) derive from step 3a) full cell lysate and ii) derive from step 3b) the 2D-gel electrophoresis protein figure that is purified with the bacterium of cracking compare,
5) detect the protein spot derive from step 4), it is present in the full cell lysate, and is not present in the bacterium that is purified or the content in the bacterium that is purified obviously reduces,
6) authentication step 5) protein in the spot selected.
2. one kind is used to identify the method for protein by the intracellular bacteria secretion, and this method comprises the following steps:
1) use the intracellular bacteria host cells infected,
2) pulse labeling is present in the intracellular bacteria in the infected cell,
3) preparation is in step 2) after the difference tracking time after the full cell lysate of infected cell,
4) the 2D-gel electrophoresis protein figure of the later full cell lysate for preparing of tracking of different time has been carried out in comparison in step 3),
5) detect the protein spot that derives from step 4), its content reduces along with the increase of the tracking time in the step 3),
6) authentication step 5) protein in the spot selected.
3. one kind is used to identify the method for protein by the intracellular bacteria secretion, and this method comprises the following steps:
1) use the intracellular bacteria host cells infected,
2) under the condition that has and do not have protein enzyme body inhibitor to exist, cultivate described host cell respectively,
3) the infected intracellular intracellular bacteria of cultivating is respectively carried out mark under the condition that has and do not have protein enzyme body inhibitor to exist,
4) the full cell lysate of the infected cell of preparation,
5) the 2D-gel electrophoresis protein figure to the full cell lysate of the infected cell cultivated under the condition that has and do not have protein enzyme body inhibitor to exist respectively compares,
6) detect the protein spot that derives from step 5), it is present in the full cell lysate of cultivating in the presence of protein enzyme body inhibitor is arranged, but be not present in the full cell lysate of in the presence of not having protein enzyme body inhibitor, cultivating, perhaps the content in the full cell lysate of cultivating in the presence of not having protein enzyme body inhibitor obviously reduces
7) authentication step 6) protein in the spot selected.
4. each the described method among the claim 1-3 further comprises the following steps:
1) obtain the antibody from the protein of described intracellular bacteria of any evaluation among the anti-claim 1-3,
2) utilize the antibody that obtains in the step 1) to being carried out the 2D-PAGE Western blotting by the full cell lysate of the cell of described bacterial infection,
3) detect step 2) the middle protein spot that reacts,
4) authentication step 3) protein in the spot selected.
5. one kind is used to identify the method for protein by the intracellular bacteria secretion, comprises the combination of the described method of claim 1 to 4.
6. each described method among the claim 1-5, wherein said mark has used amino acid or its combination of radioactivity means as [35S] halfcystine, [35S] methionine, [14C] mark.
7. the described method that is used for identification of protein of each among the claim 1-6, wherein whole length protein or its immunogenic fragments are suitable for being included in the immunogenic composition and/or are applicable to diagnostic purpose.
8. each described method among the claim 1-7, wherein authentication method is based on Edman edman degradation Edman or any mass spectrometry method, such as the combination of MALDI TOF MS (Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight MassSpectrometry), ESI Q-TOF MS (Electrospray Ionisation QuadrupoleTime-Of-Flight Mass Spectrometry), PSD-MALDI MS (Post SourceDecay MALDI Mass Spectrometry) or these methods.
9. each described method among the claim 1-8, wherein protein is handled by chemical method such as cyanogen bromide before identifying or cracking is carried out in the azanol processing, perhaps by utilizing any suitable enzyme such as the enzymatic method of tryptose matter enzyme, slymotrypsin, chymotrypsin protein matter enzyme or stomach cardia matter enzyme or its composition to carry out cracking.
10. each described method among the claim 1-9, wherein said intracellular bacteria is facultative intracellular bacteria or obligate intracellular bacteria.
11. the described method of claim 10, wherein said bacterial origin comprise these chlamydial any special serotype or strains in chlamydiaceae such as Chlamydia pneumoniae, chlamydia trachomatis, ornithosis virus or cat heart Chlamydia.
12. the described method of claim 11, wherein said intracellular bacteria is a chlamydia trachomatis.
13. the described method of claim 11, wherein said intracellular bacteria is a Chlamydia pneumoniae.
14. the described method of each among the claim 1-13, wherein said host cell is an immortalized cell system, such as HeLa, Hep2, McCoy or U937, primary cell that obtain from the mammal donor or that obtain by becoming celestial is the clone of genetic modification or organ cell's culture.
15. the described method of claim 14, wherein host cell was become expression or inhibition and the relevant gene of chlamydia vaccine development by genetic modification, encoded such as the gene of coded protein enzyme body subunit or other and offered the gene of the relevant protein with critical function with MHC I type.
16. each described method among the claim 1-15, wherein said host cell is before infecting with intracellular bacteria or handle through IFN-γ in the course of infection.
17. each described method among claim 2 or the 4-16 has wherein been used protein enzyme body inhibitor such as MG132, MG262, MG115, epoxy mycin, PSI and clasto-Lactacystin-β-lactone or its composition.
18. each described method is identified among the claim 1-17 protein or its immunogenic fragments.
19. the described protein of claim 18 or its immunogenic fragments, it is suitable for being included in the immunogenic composition and/or is applicable to diagnostic purpose.
20. the described protein of claim 19, it contains the T-cell epitope, and this epi-position is the material standed for of offering with MHC-I type or the restricted antigen form of II type, is suitable for being included in the immunogenic composition.
21. the described protein of claim 20, it contains the T-cell epitope, and this epi-position is the material standed for of offering with the restricted antigen form of MHC-I type, is suitable for being included in the immunogenic composition.
22. the described chlamydia trachomatis protein of each among the claim 18-21 or its immunogenic fragments have pI and the Mw feature of one of listed 3-protein d T1-DT77 of table 1, measure average error to be+/-10%.
23. the described chlamydia trachomatis protein of each among the claim 18-21 or its immunogenic fragments, it is by number be confirmed as CT017 (gene title CT017) as the corresponding gene that provides among the Table III A, CT044 (gene title ssp), CT243 (gene title lpxD), CT263 (gene title CT263), CT265 (gene title accA), CT286 (gene title clpC), CT292 (gene title dut), CT407 (gene title dksA), CT446 (gene title euo), CT460 (gene title SWIB), CT541 (gene title mip), CT610 (gene title CT610), CT650 (gene title recA), CT655 (gene title kdsA), CT668 (gene title CT668), CT691 (gene title CT691), CT734 (gene title CT734), CT783 (gene title CT783), CT858 (gene title CT858), CT875 (gene title CT875) or ORF5 (gene title ORF5), or determined by gene title DT8.
24. the described chlamydia trachomatis protein of each among the claim 18-21 or its immunogenic fragments, pI and Mw feature with one of the listed 3-protein d T1 of Table IV, DT2, DT3, DT5, DT9, DT10, DT11, DT13, DT14, DT17, DT47, DT59, DT60, DT61 or DT62, measure average error and be+/-10%.
25. the described chlamydia trachomatis protein of claim 22 or its immunogenic fragments are selected from 3-protein d T4 (gene title CT858), DT23 (gene title mip), DT47, DT48 (gene title CT858), DT75, DT76 (gene title CT691) and DT77 (gene title CT263).
26. the described Chlamydia pneumoniae protein of each among the claim 18-21 or its immunogenic fragments have pI and the Mw feature of one of listed protein C P1-CP91 of Table II, measure average error to be+/-10%.
27. the described Chlamydia pneumoniae protein of each among the claim 18-21 or its immunogenic fragments, it is by number be confirmed as CPN0152 (gene title CPN0152) as the listed corresponding gene of Table III B, CPN0702, CPN0705 (gene title CPN0705), CPN0711 (gene title CPN0711), CPN0796 (gene title CPN0796), CPN0998 (gene title ftsH), CPN0104 (gene title CPN0104), CPN0495 (gene title aspC), CPN0684 (gene title parB), CPN0414 (gene title accA), CPN1016 (gene title CPN1016), CPN1040 (gene title CPN1040), CPN0079 (gene title rl10), CPN0534 (gene title dksA), CPN0619 (gene title ndk), CPN0711 (gene title CPN0711), CPN0628 (gene title rs13), CPN0926 (gene title CPN0926), CPN1016 (gene title CPN1016) CPN1063 (gene title tpiS) or CPN0302 (gene title lpxD).
28. the described Chlamydia pneumoniae protein of claim 26 or its immunogenic fragments are selected from protein C P34 (gene title CPN1016), CP37 (gene title CPN0998), CP46 (gene title CPN0796), CP47 (gene title CPN0705), CP52 (gene title CPN0152), CP63 (gene title CPN1016) and CP75 (gene title ndk).
29. chlamydia trachomatis polypeptide or its immunogenic fragments is characterized in that it is DT8, and contain following sequence (SEQ ID NO:1): MQHTIMLSLENDNDKLASMMDRVVAASSSILSASKDSESNRQFTISKAPDKEAPCR VSYVAASALSE
30. each the described protein in the protein, itself and claim 18-29 or its immunogenic fragments have at least 40% sequence homogeny, preferably at least 60%, more preferably at least 70% even more preferably at least 80%, further 90% and most preferably at least 95% sequence homogeny more preferably.
31. a protein or its immunogenic fragments, it contains at least 7 continuous amino acids of each the described protein among the claim 18-30.
32. the described chlamydia trachomatis protein of claim 31 or its immunogenic fragments, it contains the amino acid sequence that is selected from SEQ ID NO.3-SEQ ID NO.45 sequence.
33. the Chlamydia pneumoniae homolog of the described chlamydia trachomatis protein of claim 32 or its immunogenic fragments, it contains the amino acid sequence that is selected from SEQ ID NO.122-SEQ ID NO.148 sequence.
34. the described Chlamydia pneumoniae protein of claim 31 or its immunogenic fragments, it contains the amino acid sequence that is selected from SEQ ID NO.46-SEQ ID NO.121 sequence.
35. the chlamydia trachomatis homolog of the described Chlamydia pneumoniae protein of claim 34 or its immunogenic fragments, it contains the amino acid sequence that is selected from SEQ ID NO.149-SEQ ID NO.194 sequence.
36. a nucleic acid compound, it contains each the described protein among the coding claim 18-35 or the sequence of its immunogenic fragments.
37. a nucleic acid compound, it contains the sequence of the described polypeptide of coding claim 29.
38. the described nucleic acid compound of claim 37, it contains following sequence (SEQ IDNO:2):
ATGCAACACACAATTATGCTGTCTTTAGAGAACGATAATGATAAGCTTGCTTCTATGATG
GATCGAGTTGTTGCTGCGTCATCAAGCATTCTTTCTGCTTCCAAAGATTCTGAGTCCAAT
AGACAGTTTACTATTTCTAAAGCTCCGGATAAAGAAGCTCCTTGCAGAGTATCTTATGTA
GCTGCAAGTGCACTTTCAGAATAG
Or its fragment or its degenerate sequence.
39. contain the carrier of each the described nucleic acid compound among the claim 36-38.
40. with the carrier conversion of claim 39 or the host cell of transfection.
41. the described protein of each among the claim 18-35 or its immunogenic fragments are used to produce the purposes of the antibody that resists described protein or fragment.
42. a method that is used to produce the antibody of anti-intracellular bacteria, wherein each the described protein among the claim 18-35 or its immunogenic fragments are applied to and produce animal, therefrom antibody purification.
43. antibody by the described method acquisition of claim 42.
44. medicine or diagnosis composition, it contains each described protein among the claim 18-35 or each the described nucleic acid compound among its fragment, the described antibody of claim 43 or the claim 36-38.
45. the purposes of each the described nucleic acid compound among the described protein of each among the claim 18-35 or its fragment, the described antibody of claim 43 or the claim 36-38 in the preparation diagnostic reagent.
46. a method that is used to identify the T-cell epitope on the intracellular bacteria secretory protein comprises that step is such as protein or its immunogenic fragments of identifying by each the described method among the claim 1-17 carried out computer forecast, MHC type molecule in conjunction with test and/or ELISPOT test.
47. by the peptide epitopes that the described method of claim 46 obtains, this peptide epitopes can be that offer on the surface.
48. a peptide epitopes, it contains 4 to 25 continuous amino acids of each the described protein among the claim 18-31, preferably 6 to 15 amino acid and 7 to 10 amino acid most preferably.
49. a peptide epitopes, it contains 7 to 10 continuous amino acids of chlamydia trachomatis or Chlamydia p.
50. a peptide epitopes, it contains 4 to 25 continuous amino acids of the polypeptide with sequence SEQ ID NO:1, preferably 6 to 15 amino acid and 7 to 10 amino acid most preferably.
51. the described chlamydia trachomatis peptide epitopes of claim 47, it contains the amino acid sequence that is selected from SEQ IDNO.3-SEQ ID NO.45 sequence.
52. the Chlamydia pneumoniae peptide epitopes of the chlamydia trachomatis peptide epitopes of claim 51, it contains the amino acid sequence that is selected from SEQ ID NO.122-SEQ ID NO.148 sequence.
53. the described Chlamydia pneumoniae peptide epitopes of claim 47, it contains the amino acid sequence that is selected from SEQ IDNO.46-SEQ ID NO.121 sequence.
54. the chlamydia trachomatis peptide epitopes of the described Chlamydia pneumoniae peptide epitopes of claim 53, it contains the amino acid sequence that is selected from SEQ ID NO.149-SEQ ID NO.194 sequence.
55. the described peptide epitopes of each among the claim 47-54 is characterized in that it is a part of melting a protein.
56. the described peptide epitopes of each among the claim 47-54 is characterized in that it puts together carrier part.
57. a nucleic acid compound is characterized in that it contains the sequence of each the described peptide epitopes among the coding claim 47-56.
58. contain the carrier of the described nucleic acid compound of claim 57.
59. host cell with described carrier conversion of claim 58 or transfection.
60. the described peptide epitopes of each among the claim 47-56 is used to prepare the purposes of immunogenic composition.
61. immunogenic composition, it contains the described peptide epitopes of each claim among the claim 47-56, and this immunogenic composition randomly contains pharmaceutical excipient.
62. each the described nucleic acid compound among the described protein of each among the claim 18-35, the described antibody of claim 43, the claim 36-38 or 57 or each the described peptide epitopes among the claim 47-56 preparation be used for the treatment of or the pharmaceutical composition of the infection that prevents to cause by intracellular bacteria in purposes.
63. each the described nucleic acid compound among the described protein of each among the claim 22-35, the described antibody of claim 43, the claim 36-38 or 57 or each the described peptide epitopes among the claim 47-56 preparation be used for the treatment of or the pharmaceutical composition of the infection that prevents to cause by Chlamydia in purposes.
64. each described nucleic acid compound among the described protein of each among the claim 18-35, the described antibody of claim 43, the claim 36-38 or 57 or the described peptide epitopes of each claim among the claim 47-56 are used for detecting the purposes of diagnostic reagent of the antibody of born of the same parents' inner cell bacterium or anti-intracellular bacteria in preparation.
65. the purposes that each the described nucleic acid compound among the described protein of each among the claim 22-35, the described antibody of claim 43, the claim 36-38 or 57 or each the described peptide epitopes among the claim 47-56 are used for detecting Chlamydia or resist the diagnostic reagent of chlamydial antibody in preparation.
66. the method for induce immune response in human body comprises described human body is used each described nucleic acid compound among each described protein among a kind of claim 18-35 of immune effective dose, the described antibody of claim 43, the claim 36-38 or 57 or each the described peptide epitopes among the claim 47-56.
67. the method that claim 66 is described, the human body or the zoogenetic infection that are used for the treatment of or prevent to cause by intracellular bacteria.
68. each described method among the claim 66-67, wherein intracellular bacteria is derived from chlamydiaceae.
69. the described method of claim 68, wherein intracellular bacteria is a chlamydia trachomatis.
70. the described method of claim 68, wherein intracellular bacteria is a Chlamydia pneumoniae.
71. each the described protein among the production claim 18-35 or the method for its fragment, it comprises with the described carrier conversion of claim 39, transfection or host cells infected and making host cell to express cultivates described host cell under the condition of described protein or its fragment.
72. a method of producing each the described peptide epitopes among the claim 47-54, it comprises with the described carrier conversion of claim 58, transfection or host cells infected and making host cell to express cultivates described host cell under the condition of described peptide epitopes.
CNA028091140A 2001-04-09 2002-04-09 Method for identification of proteins from intracellular bacteria Pending CN1531653A (en)

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CN110402392A (en) * 2017-03-02 2019-11-01 拜斯帕尔有限公司 Diagnostic method and diagnostic system
CN112175955A (en) * 2020-10-29 2021-01-05 西南大学 Strong promoter CP09 specifically expressed in plant pollen and application thereof

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GB0409748D0 (en) * 2004-04-30 2004-06-09 Chiron Srl Lactoferrin cleavage
US7468383B2 (en) 2005-02-11 2008-12-23 Cephalon, Inc. Proteasome inhibitors and methods of using the same
JP2010526070A (en) 2007-05-01 2010-07-29 ザ ボード オブ リージェンツ オブ ザ ユニバーシティ オブ テキサス システム Chlamydia antigens as agents for diagnosis and treatment of Chlamydia infections and diseases
US7892567B2 (en) 2007-10-01 2011-02-22 Board Of Regents, The University Of Texas System Methods and compositions for immunization against chlamydial infection and disease
CA2739111A1 (en) 2008-10-09 2010-04-15 Board Of Regents, University Of Texas System Methods and compositions for chlamydial antigens for diagnosis and treatment of chlamydial infection and disease
CN102725300B (en) 2009-12-22 2015-03-11 赛福伦公司 Proteasome inhibitors and processes for their preparation, purification and use

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CA2307846A1 (en) * 1997-11-21 1999-06-03 Genset S.A. Chlamydia pneumoniae genomic sequence and polypeptides, fragments thereof and uses thereof, in particular for the diagnosis, prevention and treatment of infection
EP2218731A1 (en) * 1997-11-28 2010-08-18 Merck Serono Biodevelopment Chlamydia trachomatis genomic sequence and polypeptides, fragments thereof and uses thereof, in particular for the diagnosis, prevention and treatment of infection

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Publication number Priority date Publication date Assignee Title
CN102749233A (en) * 2012-06-04 2012-10-24 王新华 Pretreatment method for directly detecting infection urine pathogen by MALDI-TOF MS
CN110402392A (en) * 2017-03-02 2019-11-01 拜斯帕尔有限公司 Diagnostic method and diagnostic system
CN112175955A (en) * 2020-10-29 2021-01-05 西南大学 Strong promoter CP09 specifically expressed in plant pollen and application thereof

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EP1412757A2 (en) 2004-04-28
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