CN101328211B - Candidates outer membrane proteins of two leptospira vaccine - Google Patents
Candidates outer membrane proteins of two leptospira vaccine Download PDFInfo
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- CN101328211B CN101328211B CN200710042222XA CN200710042222A CN101328211B CN 101328211 B CN101328211 B CN 101328211B CN 200710042222X A CN200710042222X A CN 200710042222XA CN 200710042222 A CN200710042222 A CN 200710042222A CN 101328211 B CN101328211 B CN 101328211B
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Abstract
The invention provides two leptospira outer membrane proteins which can be taken as candidate vaccines, polynucleotide for coding the outer membrane proteins and a method for producing the outer membrane proteins through the recombination technology. The outer membrane proteins are useful immunogens for preparing vaccines. The invention also discloses an application of the proteins and a coded sequence for the proteins in preparing the vaccines.
Description
Technical field
The invention belongs to biotechnology and medical field, be specifically related to can be used as two Leptospira outer membrane proteins of candidate vaccine.
Background technology
Leptospiral is a kind of elongate curved shape, active unicellular prokaryotic micro-organisms of motion in the shape of a spiral, belongs to spirochaetale (Spirochaetals) Leptospiraceae (Leptospiraceae) leptospira.Pathogenic leptospiral hook end spiral sick (Leptospirosis) is the legal Category B notifiable disease of China, also is the threat human health of the extensive distribution in the whole world and the common Amphixenosis that livestock industry is produced.There have been 77 countries and regions reports that this sick existence is arranged, the tens thousand of examples of annual morbidity.The leptospirosis main clinical manifestation is high heat, headache, shiver with cold, weak, lymphadenectasis and tangible myalgia.Weight person can concurrent pulmonary apoplexy, jaundice, meningoencephalitis and renal failure etc.China is one of hotspot of leptospirosis in the world.
Vaccine is the best method of preventing and treating leptospirosis safely and effectively.The research of leptospira vaccine starts from Japanese Ido in 1916 etc. and manufactures experimently leptospiral whole cell vaccine first, has successively experienced concentrated vaccine and leptospiral adventitia vaccine period of traditional whole cell vaccine, improvement.No matter be the adventitia vaccine of thalline vaccine or purifying, the common shortcoming is that immunizing power is for a long time strong inadequately, needs the annual immunization of carrying out in the epidemic-stricken area.Simultaneously used vaccine all has certain spinoff, and can not cause the cross immunity protection to the leptospiral of different serotypes, these drawbacks limit the application of leptospira vaccine.
Pathogenic leptospiral immunogenic protein especially surface exposes protein, because most possibly contact with host's immunity system and produce antibody, might be effective vaccine candidate molecule.Therefore identify those conservative protein matter that surface exposes in pathogenic leptospiral, thereby the leptospiral that can produce different serotypes produces the cross immunity protection, becomes the emphasis of leptospiral research.Since nineteen ninety generation, there have been many leptospiral surface proteins to be identified out, wherein some protein shows in animal model and can cause protective immunological reaction.The leptospiral vaccine candidate antigen that has identified up to now mainly contains OmpL1, LipL41, LipL32, LipL21, LigA and LigB etc., though some results are arranged, does not solve this difficult problem so far as yet fully.
Summary of the invention
The purpose of this invention is to provide two leptospiral outer membrane proteins that can be used as candidate vaccine and fragment thereof, analogue, verivate.
Another object of the present invention provides these proteic polynucleotide of coding.
Another object of the present invention provides the purposes of these proteic methods of production and albumen and encoding sequence.
In first aspect of the present invention, the leptospiral outer membrane protein of separating is provided, like polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative of SEQIDNO:1,3 aminoacid sequence.Preferably do not contain signal peptide sequence.
In second aspect of the present invention, the polynucleotide of the coding aforementioned polypeptides of separating are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group:
(a) polynucleotide of the above-mentioned Leptospira outer membrane protein of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of SEQ ID NO:1,3 aminoacid sequences.More preferably, the sequence of these polynucleotide is be selected from down group a kind of:
(a) have SEQ ID NO:2,4 sequence.
In the third aspect of the invention, the carrier that contains above-mentioned polynucleotide is provided, and by this carrier transformed host cells or by the direct transformed host cells of above-mentioned polynucleotide.
In fourth aspect of the present invention, the method for preparing the Leptospira outer membrane protein polypeptide is provided, this method comprises:
(a) under the condition that is fit to the expression Leptospira outer membrane protein, cultivate above-mentioned by transformed host cells;
(b) from culture, isolate the Leptospira outer membrane protein polypeptide.
Aspect the of the present invention the 5th, provide and above-mentioned leptospiral ospa polypeptide specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided.
Aspect the of the present invention the 6th; The method that whether has outer membrane protein in the test sample is provided; It comprises: with the specific anti precursor reactant of sample and outer membrane protein, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample outer membrane protein.
Aspect the of the present invention the 7th, the purposes of polypeptide of the present invention and encoding sequence is provided.For example the encoding sequence of Leptospira outer membrane protein of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.
In eight aspect of the present invention, a kind of vaccine composition is provided, it contains leptospiral ospa polypeptide of the present invention or its fragment and the pharmaceutically acceptable carrier of safe and effective amount.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Description of drawings
Figure 1A and Figure 1B are respectively the agarose gel electrophoresis analytical results figure of two gene PCR amplified productions of LA0957LA1495 of the present invention;
Fig. 2 A and Fig. 2 B are respectively that the enzyme of two gene PQE31 of LA0957LA1495 of the present invention recombinant expression plasmid is cut qualification result figure;
Fig. 3 A and Fig. 3 B are respectively SDS-PAGE figure (the 1:uninduced recombinant protein as a result of two recombinant proteins of LA0957LA1495 of the present invention; 2:induced recombinant protein; 3:supernatant of recombinant protein; 4:precipitation of recombinantprotein; 5:purified protein; M:protein marker);
Fig. 4 A and Fig. 4 B are respectively the figure as a result that FACS of the present invention detects LA0957 and LA1495;
Fig. 5 A and Fig. 5 B are respectively the figure as a result that Western blot detects vaccine candidate antigen conservative property in 15 serogroups representative strains.
Embodiment
The inventor is through extensive and deep research, relies the strain separation and purification and identified one type of new outer membrane protein from leptospiral, and this outer membrane protein can be used as design and the preparation that antigen is used for vaccine.Accomplished the present invention on this basis.
In the present invention, term " outer membrane protein ", " ospa polypeptide " or " Leptospira outer membrane protein " interchangeable use, all refer to have the Leptospira outer membrane protein aminoacid sequence albumen or the polypeptide of (SEQ ID NO:1,3).They comprise the Leptospira outer membrane protein that contains or do not contain initial methionine, and the outer membrane protein that contains or do not contain signal peptide.
" isolating " as used herein is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification like polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
" isolating outer membrane protein or polypeptide " as used herein is meant that ospa polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying outer membrane protein of standard.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell), to produce.The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, but preferably nonglycosylated.
The present invention also comprises Leptospira outer membrane protein, proteic fragment, verivate and analogue.As used herein, term " fragment ", " verivate " are meant with " analogue " and keep identical biological function of natural Leptospira outer membrane protein of the present invention or active polypeptide basically.Polypeptide fragment of the present invention, verivate or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue); And so substituted amino-acid residue can be also can not encoded by genetic code; Or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical; Or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period; Polyoxyethylene glycol for example) merges formed polypeptide; Or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (like leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, verivate and analogue belong to the known scope of those skilled in the art.
In the present invention, term " leptospiral ospa polypeptide " refers to have the active or immunogenic SEQ ID NO:1 of Leptospira outer membrane protein, 3 polypeptide of sequence.This term also comprises having and the variant form Leptospira outer membrane protein identical function, above-mentioned sequence.These variant forms comprise (but being not limited to): several (are generally 1-50; Preferably 1-30; More preferably 1-20; 1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several (being generally in 10, more preferably is in 5) amino acid at C-terminal and/or N-terminal.This term also comprises the active fragments and the reactive derivative of Leptospira outer membrane protein.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of Leptospira outer membrane protein DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-leptospiral ospa polypeptide to obtain.The present invention also provides other polypeptide, as comprises leptospiral ospa polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of leptospiral ospa polypeptide.Usually; This fragment have leptospiral ospa polypeptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids; More preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.These fragments also can be used as immunogen to induce to leptospiral immune response.
Invention also provides the analogue of Leptospira outer membrane protein or polypeptide.The difference of these analogues and natural leptospiral ospa polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain through various technology, as through radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(like D-amino acid), and has non-natural analogue that exist or synthetic amino acid (like β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to above-mentioned representational polypeptide of giving an example.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, in the synthetic and processing of polypeptide or further, carries out glycosylation modified and polypeptide that produce in the procedure of processing like those.Modified forms also comprises have the phosphorylated amino acid residue sequence of (like Tyrosine O-phosphate, Serine O-phosphate, phosphothreonine).Thereby also comprise and modified the polypeptide that has improved its anti-proteolyze performance or optimized solubility property.
In the present invention; " Leptospira outer membrane protein conservative property variation polypeptide " refers to compare with SEQ ID NO:1,3 aminoacid sequence; There are 10 at the most; Preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.
The polynucleotide of encoding mature polypeptide comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the verivate of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it possibly be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.Giving invention is particularly related under stringent condition and the interfertile polynucleotide of polynucleotide according to the invention.In the present invention, " stringent condition " is meant: (1) is than hybridization under LIS and the comparatively high temps and wash-out, like 0.2 * SSC; 0.1%SDS; 60 ℃: or (2) hybridization the time is added with denaturing agent, like 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll; 42 ℃ etc.: or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with sophisticated Leptospira outer membrane protein.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (like PCR) of nucleic acid to confirm and/or to separate the polynucleotide of coding outer membrane protein.
Leptospiral adventitia Nucleotide full length sequence of the present invention or its fragment can use the method for pcr amplification method, recombination method or synthetic to obtain usually.For the pcr amplification method; Can be disclosed according to the present invention about nucleotide sequence; Especially open reading frame sequence designs primer, and with commercially available DNA library or by made each the DNA library of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually need carries out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can come to obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell again over to, from the host cell after the propagation, separates obtaining relevant sequence then through ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, through first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) through chemosynthesis.Can this dna sequence dna be introduced in various existing dna moleculars as known in the art (or like carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention through chemosynthesis.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or outer membrane protein encoding sequence, and produce the method for polypeptide according to the invention through recombinant technology.
Through the recombinant DNA technology of routine, polymerized nucleoside acid sequence of the present invention capable of using can be used to express or produce the ospa polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding leptospiral ospa polypeptide of the present invention, or with recombinant expression vector conversion that contains these polynucleotide or transduction proper host cell;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, the sweet acid sequence of leptospiral adventitia multinuclear can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell is viral, mammalian cell is viral like adenovirus, retrovirus or other carriers.The carrier that is suitable in the present invention includes but not limited to: the expression vector based on T7 of in bacterium, expressing; The pMSXND expression vector of in mammalian cell, expressing and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can in host, duplicate and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains leptospiral adventitia DNA sequences encoding and suitable transcribing/the translate expression vector of wave.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition; Expression vector preferably comprises one or more selected markers; To be provided for selecting the phenotypic character of transformed host cells; Cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness like eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, like bacterial cell; Or eukaryotic cell such as low, like yeast cell or higher eucaryotic cells, like mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurtum; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO etc.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (like temperature transition or chemically induced), cell is cultivated for some time again.
The extracellular can expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating through various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, ultraly handle, ultra centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) is technological with other various LCs and the combination of these methods.
The Leptospira outer membrane protein or the polypeptide of reorganization are of use in many ways, comprising (but being not limited to): be used to prepare vaccine as antigen.
On the other hand, the present invention also comprises leptospiral adventitia DNA or the polypeptide of its segment encoding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into leptospiral ospa gene product or fragment.Preferably, refer to that those can combine with leptospiral ospa gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.The present invention also comprise those can with modify or without the leptospiral ospa gene product bonded antibody of modified forms.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, like Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody: or chimeric antibody.
Antibody of the present invention can prepare through the known various technology of those skilled in that art.For example, the leptospiral ospa gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing Leptospira outer membrane protein or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.
Vaccine composition
Vaccine of the present invention can be preventative (being preventing infection) or curative (promptly after infection, treating disease).
These vaccines comprise immunity antigen or immunogen, immunogenic polypeptide, albumen or protein fragments or nucleic acid (like Yeast Nucleic Acid or thymus nucleic acid); Usually with " pharmaceutically acceptable carrier " combination, these carriers comprise itself does not induce any carrier of generation to the individual deleterious antibody of accepting said composition.Suitable carriers normally big, metabolism macromole slowly, like the virion of protein, polysaccharide, POLYACTIC ACID, Sodium bromoacetate homopolymer, SRU, aminoacid polymers, amino acid copolymer, lipid agglutinator (like oil droplet or liposome) and non-activity.
These carriers are well known to those of ordinary skill in the art.In addition, these carriers can play immunostimulant (adjuvant) effect.In addition, antigen or immunogen can and bacterial toxoid (like the toxoid of pathogenic agent such as diphtheria, tetanus, cholera, helicobacter pylori) coupling.
The preferable adjuvant of enhancing composition effect is including, but not limited to (1) aluminium salt (alum), like white lake, phosphagel phosphaljel, Tai-Ace S 150 etc.; (2) the oil-in-water emulsion prescription (is with or without other specific immunostimulant; Like muramylpeptides (seeing below) or bacteria cell wall composition), for example, (a) MF59 its contain 5% shark alkene, 0.5% tween 80 and the 0.5%Span85 (MTP-PE (seeing below) that randomly contains different amounts; Though do not need); (process submicron particles with micro-fluidisation device like 110Y type trace fluidisation device (Microfluidics, Newton, MA)); (b) SAF; It contains 10% shark alkene, 0.4% tween 80,5% Pluronic (pluronic) block polymer L121 and thr-MDP (seeing below); Trace stream changes into industry micron order emulsion or eddy oscillating produces the bigger emulsion of particle diameter; (c) Ribi adjuvant system (RAS) (RibiImmunochem, Hamilton, MT); One or more bacterial cell wall fractions that it contains 2% shark alkene, 0.2% tween 80 and takes from monophosphoryl lipid A (MPL), two mycolic acid marine alga sugar esters (TDM) and cell wall skeleton (CWS), preferably MPL+CWS (Detox); (3) saponin adjuvant, for example can adopt stimulon (cambridge Bioscience, worcester is MA) or from the particle of its generation, like ISCOM (immunostimulating mixture); (4) Freund Freund's complete adjuvant (CFA) and Freund Freund (IFA); (5) cytokine; (M-CFS), tumour necrosis factor (TNF) etc.: (6) bacterium ADP-ribosylation toxin is (like Toxins,exo-, cholera CT like interleukin (like IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 etc.), Interferon, rabbit (like IFN-), M-CSF; Toxins, pertussis PT or intestinal bacteria heat-labile toxin LT) the detoxification varient; Especially LT-K63, LT-R72, CT-S109, PT-K9/G129; Referring to for example WO93/13302 and WO92/19265; And other material of enhancing composition effect is come in (7) as immunostimulant.
As stated; Muramylpeptides is including, but not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-go muramyl-L-alanyl-D-isoglutamine (nor-MDP), the different glutaminase acyl group of N-acetyl muramyl-L-alanyl-D--L-L-Ala-2-(1 '-2 '-two palmityls-sn-glycerine-3-hydroxyl phosphinylidyne oxygen)-ethamine (MTP-PE) etc.
Comprise the vaccine composition (as comprising antigen, pharmaceutically acceptable carrier and adjuvant) of immunogenic composition, contain thinner usually, like water, salt solution, glycerine, ethanol etc.In addition, complementary material can be present in this type vector like wetting agent or emulsifying agent, pH buffer substance etc.In addition, the vaccine composition that comprises immunogenic composition can contain antigen, polypeptide, albumen, protein fragments or nucleic acid in pharmaceutically acceptable carrier.
More specifically, comprise the vaccine of immunogenic composition, comprise the immunogenic polypeptide of immunology significant quantity, and above-mentioned other required component." immunology significant quantity " refers to that giving individual amount with a single agent or a continuous agent part is effective to treatment or prevention.This consumption according to the individual healthy state of treat and physiological situation, institute treat the ability of classification (like non-human primates etc.), the individual immunity system synthetic antibody of individuality, required degree of protection, vaccine preparation, treat the doctor assessment of medical conditions, the correlative factor that reaches other decided.Estimate that this consumption will can confirm through normal experiment in the scope of relative broad.
Usually, can vaccine composition or immunogenic composition be processed the injectable agent, for example liquor or suspension; Also can be made into the solid form that before injection, is fit to allocate into solution or suspension, liquid excipient.But also emulsification or be encapsulated in the liposome of said preparation strengthens adjuvant effect under above-mentioned pharmaceutically acceptable carrier.
Ordinary method is to give immunogenic composition from parenteral (subcutaneous or intramuscular) approach through injection.Other prescription that is fit to other administering mode comprises oral prepns.Therapeutic dose can be single agent scheme or multi-agent scheme.Vaccine can combine other immunomodulator to give together.
A kind of replacement scheme as with protein being the vaccine on basis is to adopt the dna vaccination inoculation.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook at " molecular cloning: laboratory manual " (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.
One, the genome method based on information biology and gene chip screens the leptospira vaccine candidate gene
1, bioinformatic analysis prediction leptospiral coded surface exposes proteic gene
2, the comparative genomics screening by hybridization goes out gene conservative in ten popular serogroups leptospiral representative strains of Chinese popular
3, cance high-expression gene in the transcription group Analysis and Identification leptospiral genome
Through above-mentioned 3 methods, Screening and Identification goes out 226 vaccine candidate genes from the bad pnca gene group of leptospiral at last.The protein of these 226 vaccine candidate genetic expressions is still not surperficial to expose albumen, and is conservative in the representative strains of the most of serogroups of Chinese popular, and has higher transcriptional level.(this part content has been published in BMC genomics2006 the 7th volume) two, two new vaccine candidate gene 1a0957 of leptospiral, the clonal expression of 1a1495 and Screening and Identification
Table one
Two new vaccine candidate gene 1a0957, the clonal expression of 1a1495
Through these two genes of conventional molecular biology method clonal expression, expression vector is pQE31, and expressing bacterium is E.coli M15.The recombinant plasmid transformed that order-checking is successful, adds in the LB liquid nutrient medium behind the two anti-screening positive clones of Amp and Kan to competence M15, and 37 ℃ of 250rpm amplifications are spent the night.Inoculum size by 1:100 goes to the LB nutrient solution, 37 ℃ be cultured to OD and be about 0.6～0.8 after, adopt the IPTG inducible protein to express.Different protein induced temperature and IPTG concentration have nothing in common with each other.Sonicated is the E.coli M15 cell of expressing protein, difference collecting precipitation and supernatant, and 10%～15%SDS-PAGE and Western blot identify.Great expression recombinant protein then, and recombinant protein carried out purifying, detect protein concentration with BCA (bicinchoninic acid) method.1a0957; The agarose gel electrophoresis analysis of two gene PCR amplified productions of 1a1495 such as Figure 1A and Figure 1B; The enzyme of PQE31 recombinant expression plasmid is cut qualification result such as Fig. 2 A and Fig. 2 B, the SDS-PAGE result of two recombinant proteins of LA0957LA1495 such as Fig. 3 A and Fig. 3 B.
The immunogenicity of recombinant protein detects
The polyclonal antibody that at first prepares above-mentioned 2 recombinant proteins with BALB/c mouse, detecting serum titer is 1: 32000, shows good immunogenicity.Western-blot detects the immunoreactivity of recombinant protein, shows that recombinant protein can react with corresponding polyvalent antibody, explains that these two vaccine candidate antigens have good immunoreactivity
Western blot detects vaccine candidate antigen and relies the antibody in the strain rabbit anteserum leptospiral
Method is that 2 μ g recombinant proteins are changeed film behind SDS-PAGE, and (1% skim-milk-0.1%Tween20-PBS, pH7.4) sealing is 2 hours, PBS-T liquid flushing 3 times, each 10 minutes with confining liquid.Anti-as one with the dilution of rabbit antileptospira serum, extension rate is 1:50,1:100, and 1:200 and 1:400 reacted 3 hours, and PBS-T liquid is given a baby a bath on the third day after its birth inferior, each 10 minutes.Two anti-are Goat-anti-Rabbit IgG HRP (1:2000), reacts after 1 hour, and PBS-T gives a baby a bath on the third day after its birth inferior.The chemical luminous substrate colour developing.
Detected result shows that the antibody of LA0957 and LA1495 is present in the full bacterium rabbit anti-serum of leptospiral.These 2 leptospira vaccine candidate molecules have to express in infecting leptospiral animal body also can stimulate body to produce corresponding antibodies.
FACS (flow cytometer) detects LA0957 and LA1495 is that the leptospiral surface exposes protein
Method: it is 2 * 10 that leptospiral relies strain to be cultured to concentration
8/ ml, PBS damping fluid wash 2 times and adjust concentration to 3 * 10
7/ ml.Get BALB/c mouse serum or PBS immunity BALB/c mouse gained antiserum(antisera) (final concentration is 1:50) that 300 μ l leptospiral cell suspensions add recombinant protein; Hatched 30 minutes for 37 ℃; Centrifugal 10 minutes of 10000r/min; The PBS damping fluid is washed 2 times, adds 6 μ l FITC anti-mouse IgG again
2a/2b, 37 ℃ of lucifuges were hatched 30 minutes, PBS washing 2 times, and PBS is resuspended.FACS detects fluorescent value, statistics software SPSS10.0 analytical results.Negative control is normal BALB/c mouse serum, and positive control is the BALB/c mouse antiserum(antisera) of recombinant protein LipL32 and LipL41.Each sample duplicate detection 4～5 times.
The result sees Fig. 4 A and Fig. 4 B, and wherein grey peak figure is normal mouse serum and leptospiral cell response, to get rid of mice serum and leptospiral non-specific binding.White peak figure is the association reaction of specificity outer membrane protein antiserum(antisera) or PBS immunity BALB/c mouse antiserum(antisera) and leptospiral cell.The two otherness shows the combination degree of specificity leptospiral protein and leptospiral cell.SPSS10.0 software carries out the nonparametric statistics of two independent samples and adds up (Kolmorov-Smirnov test, K-Stest), whether the MV of analyzing vaccine candidate antigen immune mouse antiserum(antisera) and normal mouse serum fluorescent signal has statistical significance.The result shows: the K-S of the known albumen LipL32 that is exposed to adventitia and LipL41 and vaccine candidate antigen LA0957 and LA1495 check P 0.05, statistical significance is arranged.Explain that vaccine candidate antigen LA0957 and LA1495 are that leptospiral surface exposes protein.
Western Blot detects the conservative property of vaccine candidate antigen in different strains
The leptospiral thalline is handled 15 serogroups representative strains of China popular leptospiral with 28 ℃ of shaking culture of EMJH substratum; Cultivated after 3 days 10000rpm centrifugal 10 minutes; Weighing weight in wet base behind the unnecessary PBS is removed in PBS washing 2 times, adds aseptic double-distilled water according to 100mg/ml; Fully mixing is subsequent use.
Western Blot changes film after detecting 12%SDS-PAGE, and (1% skim-milk-0.1%Tween20-PBS, pH7.4) sealing is 2 hours, PBS-T liquid flushing 3 times, each 10 minutes with confining liquid.BALB/c mouse serum 1:1000 dilution with recombinant protein is anti-as one, reacts 3 hours, and PBS-T liquid is given a baby a bath on the third day after its birth inferior, each 10 minutes.Two anti-are Goat-anti-Mouse IgG HRP (1:2000), reacts 1 hour, and PBS-T gives a baby a bath on the third day after its birth inferior.The chemical luminous substrate colour developing.
Result such as Fig. 5 A and Fig. 5 B all have expression with LA1495 at vaccine candidate antigen LA0957 in the different serogroups representative strains of 15 strains, be the vaccine candidate antigen of guarding.
The present invention uses the theory of reverse vaccinology, through bioinformatics method, and has integrated the result of comparative genomics hybridization and transcription group, filters out 226 leptospira vaccine candidate genes.
Through LA0957 and these 2 vaccine candidate genes of LA1495 further being screened and identifying; Show that they not only have better immunogenicity; In the bad strain rabbit anteserum of leptospiral, there is antibody; FACS detects and confirms that the surface that is the leptospiral cell exposes protein, and in the representative strains of 15 serogroupss, has strong conservative property, is two new leptospira vaccine candidate genes.
Sequence table
< 110>Medical College, Shanghai Communication Univ.
< 120>outer membrane protein of two vaccine candidates of leptospiral
<130>
<160>4
<170>PatentIn?version3.3
<210>1
<211>557
<212>PRT
< 213>Leptospira interrogans (leptospiral)
<400>1
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<211>1674
<212>DNA
< 213>Leptospira interrogans (leptospiral)
<400>2
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<212>PRT
< 213>Leptospira interrogans (leptospiral)
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<211>996
<212>DNA
< 213>Leptospira interrogans (leptospiral)
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Claims (2)
1. ability and Leptospira outer membrane protein specificity bonded antibody is characterized in that the aminoacid sequence of said Leptospira outer membrane protein is shown in SEQ ID No:3.
2. a vaccine composition is characterized in that, it contains the Leptospira outer membrane protein polypeptide and the pharmaceutically acceptable carrier of safe and effective amount, and the aminoacid sequence of said Leptospira outer membrane protein is shown in SEQ IDNo:3.
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| WO2003072698A2 (en) * | 2002-02-28 | 2003-09-04 | Fundação de Amparo a Pesquisa Do Estado de São Paulo | Surface proteins of leptospira |
| US6852322B2 (en) * | 2002-02-28 | 2005-02-08 | Fundacao De Amparo A Pesquisa Do Estado De Sao Paulo | Surface proteins of Leptospira |
Non-Patent Citations (3)
| Title |
|---|
| Hong-Liang Yang ET AL.In silico and microarray-based genomic approaches to identifying potential vaccine candidates against Leptospira interrogans.《BMC Genomics》.2006,第7卷(第293期),第1-12页. * |
| Ren S.-X.ET AL.Q8F617-1.《UniProt》.2003, * |
| Ren S.-X.ET AL.Q8F7I8-1.《UniProt》.2003, * |
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