CN101544689A - Leptospira vaccine candidate outer membrane protein - Google Patents
Leptospira vaccine candidate outer membrane protein Download PDFInfo
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- CN101544689A CN101544689A CN200910050588A CN200910050588A CN101544689A CN 101544689 A CN101544689 A CN 101544689A CN 200910050588 A CN200910050588 A CN 200910050588A CN 200910050588 A CN200910050588 A CN 200910050588A CN 101544689 A CN101544689 A CN 101544689A
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Abstract
The invention provides a leptospira outer membrane protein capable of serving as a candidate vaccine, a polynucleotide for coding the outer membrane protein and a method for generating the outer membrane protein through a recombinant technique. The outer membrane protein is a useful immunogen for preparing the vaccine. The invention also discloses application of the protein and a coded sequence thereof for preparing the vaccine.
Description
Technical field
The invention belongs to biotechnology and medical field, be specifically related to can be used as the Leptospira outer membrane protein of candidate vaccine.
Background technology
Leptospira be a kind of elongated, bending shape, motion are active in the shape of a spiral, belong to the prokaryotic microorganism of spirochaetale (Spirocheatals) Spirochaetaceae (Spirocaetaleae) leptospira (leptospira).In the past, Leptospira was divided into 2 kinds: pathogenic leptospira interrogans (L.interrogans broad sense) and saprophytic form leptospira biflexa (L.biflexa broad sense).Leptospira interrogans can be divided into different serotypes again according to the cross agglutination experiment, can be divided into 25 serogroupss at least, a serotype surplus in the of 200.There are 18 groups 75 types in China, is the country that finds that in the world serotype is maximum.According to the result of DNA-DNA hybridization Leptospira has been carried out classification according to the gene kind in recent years.Being divided at present is 17 gene kinds, and wherein pathogenic Leptospira mainly contains L.interrogans (narrow sense), L.kirschneri, L.noguchii, L.borgpetersenii, gene kinds such as L.santarosai and L.weilii.The hook end spiral disease (Leptospirosis) that pathogenic Leptospira can cause is called for short the coupler body disease, is the legal Category B notifiable disease of China.This disease distribution on global, be the common Amphixenosis who threatens human health and livestock industry to produce, had 77 countries and regions reports that the existence of this disease is arranged, China is critically ill by coupler body to do harm to one of the most serious country, case reaches more than 250 ten thousand since nineteen fifty-five, dead people more than 20,000.This disease mainly concentrates on the Yangtze valley and other most of area, south in China, based on rice field type and flood type.What are closely related for sickness rate and flood, rainfall amount.To send out the patient numerous whenever summer and autumn, and patient's condition is rapid.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.In recent years, occurring in abroad of this disease risen to some extent, even comprises some areas of some developed countries.At present, owing to lack effective molecular biology research means, mechanism pathogenic to it and immunity is understood also more superficial.
Vaccine is the best method of preventing and treating leptospirosis safely and effectively.Leptospira vaccine has successively experienced concentrated vaccine and adventitia vaccine period of traditional whole cell vaccine, improvement, has made certain contribution to preventing and treating leptospirosis.But it is lasting inadequately that their common shortcomings are immunizing power, and it is low to tire, and sphere of action is narrow; need the annual immunization of carrying out in the epidemic-stricken area; simultaneously all have certain side effect, and can not cause the cross immunity protection, these drawbacks limit the application of leptospira vaccine.One of emphasis of Leptospira research field is the development of new generation vaccine now, and its core is to seek suitable vaccine candidate gene.In recent years; some vaccine candidate molecules such as Hap-1 (LipL32), LipL41, OmpL1, Lig, FlaB, Hsp58, SphH etc. are studied in succession; though in some experimentation on animalies, have immune protective effect preferably; but there do not have suitable Leptospira subunit vaccine to enter so far to be clinical, therefore needs new research ideas and methods to carry out leptospira vaccine research.
Leptospira vaccine has successively experienced concentrated vaccine and adventitia vaccine period of traditional whole cell vaccine, improvement, has made certain contribution to preventing and treating leptospirosis.But it is lasting inadequately that their common shortcomings are immunizing power, and it is low to tire, and sphere of action is narrow; need the annual immunization of carrying out in the epidemic-stricken area; simultaneously all have certain side effect, and can not cause the cross immunity protection, these drawbacks limit the application of leptospira vaccine.
Summary of the invention
The purpose of this invention is to provide the leptospiral outer membrane protein that can be used as candidate vaccine and fragment thereof, analogue, derivative.
Another object of the present invention provides these proteic polynucleotide of coding.
Another object of the present invention provides the purposes of this proteic method of production and albumen and encoding sequence.
In a first aspect of the present invention, provide the leptospiral outer membrane protein of separating, as polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative of the aminoacid sequence of SEQ IDNO:1.Preferably do not contain signal peptide sequence.
In a 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 aminoacid sequence.More preferably, the sequence of these polynucleotide is be selected from down group a kind of:
(a) has the sequence of SEQ ID NO:2.
In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been stopped host cell that transforms or transduce or the host cell that is directly transformed or transduce by above-mentioned polynucleotide this year.
In a 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 the above-mentioned host cell that is transformed or transduce;
(b) from culture, isolate the Leptospira outer membrane protein polypeptide.
In a fifth aspect of the present invention, provide and above-mentioned Leptospira ospa polypeptide specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided.
In a sixth aspect of the present invention, the method that whether has outer membrane protein in the test sample is provided, it comprises: sample is contacted with the specific antibody of outer membrane protein, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample outer membrane protein.
In a seventh aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.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 a eighth aspect of the present invention, a kind of vaccine composition is provided, it contains Leptospira 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
Fig. 1 is LA3744 gene cloning and expression of the present invention figure as a result;
Fig. 2 is the agarose gel electrophoresis analytical results figure of LA3744 gene PCR amplified production of the present invention;
Fig. 3 is the SDS-PAGE analytical results figure of LA3744 purification of recombinant proteins of the present invention;
Fig. 4 is that LA3744 recombinant protein of the present invention carries out mass spectroscopy qualification result figure;
Fig. 5 is the conservative property qualification result figure of LA3744 of the present invention in 15 serogroups representative strains;
Fig. 6 is the conservative property qualification result figure of PL40 albumen of the present invention in 15 serogroups representative strains;
Fig. 7 is whether PL40 albumen of the present invention is that the Leptospira surface exposes albumen figure as a result with the FACS detection;
Fig. 8 is the distribution plan of PL40 albumen of the present invention in the different membrane tissue compositions of Triton X-114 purification coupler body;
Fig. 9 is the proteic expression of results figure of PL40 when coupler body infects Mammals among the present invention;
Figure 10 infects coupler body mouse, cavy or Golden Hamster anti-PL40 antibody titers figure as a result in the serum after 0,7,14 and 28 days among the present invention.
Embodiment
The inventor is through extensive and deep research, relies the strain separation and purification and identified the new outer membrane protein of a class from Leptospira, and this outer membrane protein can be used as design and the preparation that antigen is used for vaccine.Finished the present invention on this basis.
In the present invention, term " outer membrane protein ", " ospa polypeptide " or " Leptospira outer membrane protein " are used interchangeably, all refer to have the Leptospira outer membrane protein aminoacid sequence albumen or the polypeptide of (SEQ ID NO:1).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 as 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 to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).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, derivative and analogue.As used herein, term " fragment ", " derivative " 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, derivative 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 the amino-acid residue of such replacement 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 (as 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, derivative and analogue belong to the known scope of those skilled in the art.
In the present invention, term " Leptospira ospa polypeptide " refers to have the active or immunogenic SEQ ID NO:1 polypeptide of sequence of Leptospira outer membrane protein.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 at C-terminal and/or N-terminal interpolation one or several (being generally in 10, more preferably is in 5) amino acid.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-Leptospira ospa polypeptide to obtain.The present invention also provides other polypeptide, as comprises Leptospira ospa polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of Leptospira ospa polypeptide.Usually, this fragment have Leptospira 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 at leptospiral immune response.
Invention also provides the analogue of Leptospira outer membrane protein or polypeptide.The difference of these analogues and natural Leptospira 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 by various technology, as by 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-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(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, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, " Leptospira outer membrane protein conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:1, 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 following table and produce.
| 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 derivative 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 may 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 of the present invention.In the present invention, " stringent condition " is meant: (1) is than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, 0.1%SDS, 60 ℃: or (2) hybridization the time is added with denaturing agent, as 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 (as PCR) of nucleic acid to determine and/or to separate the polynucleotide of coding outer membrane protein.
Leptospira adventitia Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic 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 needs to carry 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 obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by 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) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by 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 the method that produces polypeptide of the present invention through recombinant technology.
By the recombinant DNA technology of routine, can utilize polymerized nucleoside acid sequence of the present invention to 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 of the present invention Leptospira ospa polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(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 Leptospira adventitia multinuclear can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: the expression vector based on T7 of expressing in bacterium; 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 duplicate in host 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 Leptospira adventitia DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.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 as 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, as bacterial cell; Or eukaryotic cell such as low, as yeast cell or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CH0 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 optional white various conventional substratum of 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 (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be 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 by 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, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and 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 Leptospira adventitia DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into Leptospira ospa gene product or fragment.Preferably, refer to that those can combine with Leptospira 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 Leptospira 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, as Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody: or chimeric antibody.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the Leptospira 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 treating disease after infection).
These vaccines comprise immunity antigen or immunogen, immunogenic polypeptide, albumen or protein fragments or nucleic acid (as 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, as the virion of protein, polysaccharide, poly(lactic acid), polyglycolic acid, aminoacid polymers, amino acid copolymer, lipid agglutinator (as 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 (as 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), as aluminium hydroxide, aluminum phosphate, Tai-Ace S 150 etc.; (2) the oil-in-water emulsion prescription (is with or without other specific immunostimulant, as muramylpeptides (seeing below) or bacteria cell wall composition), for example, (a) MF59 its contain 5% shark alkene, tween 80 O.5% and the 0.5%Span85 (MTP-PE (seeing below) that randomly contains different amounts, though do not need), (make submicron particles with micro-fluidisation device as 110Y type trace fluidisation device (Microfluidics, Newton, MA)); (b) SAF, it contains 1O% 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) (Ribi Immunochem, Hamilton, MT), it contains 2% shark alkene, 0.2% tween 80 and take from monophosphoryl lipid A (MPL), two mycolic acid marine alga sugar esters (TDM), and one or more bacterial cell wall fractions of 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, as 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 (as Toxins,exo-, cholera CT as interleukin (as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 etc.), Interferon, rabbit (as IFN-), macrophage colony stimulating factor, 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 W093/13302 and W092/19265; And (7) come other material of enhancing composition effect as immunostimulant.
As mentioned above; 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, as water, salt solution, glycerine, ethanol etc.In addition, complementary material can be present in this class vehicle as 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 the 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 individual classification (as non-human primates etc.), individual immunity system synthetic antibody, 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 determine by normal experiment in the scope of relative broad.
Usually, vaccine composition or immunogenic composition can be made 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 by injection.Other prescription that is fit to other administering mode comprises oral preparations.Therapeutic dose can be single agent scheme or multi-agent scheme.Vaccine can give together in conjunction with other immunomodulator.
Be to adopt the dna vaccination inoculation as a kind of replacement scheme based on the vaccine of protein.
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 and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, 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 Leptospira coded surface exposes proteic gene
2, the comparative genomics screening by hybridization goes out gene conservative in ten popular serogroups Leptospira representative strains of Chinese popular
3, cance high-expression gene in the transcription group Analysis and Identification Leptospira genome
Through above-mentioned 3 methods, Screening and Identification goes out 226 vaccine candidate gene dosages from the bad pnca gene group of Leptospira 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.
Two, clonal expression and the Screening and Identification of two new vaccine candidate gene LA3744 of Leptospira
The used primer of table 1 amplification corresponding gene
Embodiment 1Clonal expression and the evaluation of new vaccine candidate gene LA3744
By conventional molecular biology method clonal expression LA3744 gene, expression vector is pET28b+, and expression strain is E.coli BL21-3E, behind the Kan resistance screening positive colony, adds in the LB liquid nutrient medium, and 37 ℃ of 250rpm amplifications are spent the night.Inoculum size by 1:100 goes to the LB nutrient solution, cultivates 2.5hr to OD for 37 ℃ and is about about 0.6, adds IPTG to final concentration 0.5mM, and 30 ℃ of 250rpm 8hr inducible proteins are expressed.Ultrasonication is the E.coli BL21-3E cell of expressing protein, collecting precipitation.The method that provides according to Qiagen company is by the Ni-NTA affinity chromatography, collects to contain the component of recombinant protein, and the renaturation of dialysing.The result that the enzyme of pcr amplification product, recombinant plasmid is cut evaluation and purifying protein sees Fig. 1, Fig. 2 and shown in Figure 3 respectively.
Recombinant protein is carried out SDS-PAGE electrophoresis gained band, after decolouring fully with 1% acetate,, use 8 μ g/ml tryptic digestions 8 hours then with the ammonium bicarbonate soln flushing.Lyophilization recovering peptide section is resuspended in 50% acetonitrile (CAN)/0.1% trifluoroacetic acid (TFA) solution that 2ml contains 10mg/mL α cyano group-4-hydroxycinnamic acid (CHCA).Get 0.7ml point target and carry out the evaluation of MALDI-TOF/TOF biological mass spectrometry, use peptide quality fingerprinting spectrum (the Pep tide mass fingerp rinting of peptide mixt, PMF) analytical results is used Mascot (http://www.matrixscience.com) search engine and is retrieved in the NCBInr database.With the LA3744 gene coded protein very high matching degree (Fig. 4) is arranged.Purified albumen target protein just is described.
Embodiment 2The immunogenicity of recombinant protein detects
400 μ g recombinant proteins add the immune 3kg new zealand rabbit of equal-volume complete Freund's adjuvant (Sigma company), the negative control group of PBS+ adjuvant, every interval 15 days adds the equal-volume incomplete Freund's adjuvant with 200 μ g recombinant proteins and strengthens once behind the initial immunity, strengthen the blood sampling in the 10th day of twice back, ELISA surveys it and tires.Recombinant protein PL40 produces antibody titer greater than 1:16000., show good immunogenicity.Western-blot detects the immunoreactivity of recombinant protein, shows that the polyclonal antibody of preparation can effective and corresponding recombinant protein combination.
Embodiment 3LA3744 and proteins encoded thereof conservative property in Chinese coupler body epidemic strain detects
Conservative property in nucleic acid level detects: with 15 strain Chinese epidemic strains and the total DNA of spirochaeta biflexa is template, conservative region design primer carries out pcr amplification, all have special amplified band to produce therein in 14 strains, and no specific band produce (Fig. 5) in L 105 strains of Wei Shi Leptospira clear water type and the non-virulent leptospira biflexa.
Conservative property at protein level detects: carry out Westernblot with the PL40 antiserum(antisera) and detect its expression at 15 serogroups representative strains of Chinese popular.Have 14 strains can both detect corresponding hybridization band, the molecular weight size conforms to theoretical value, and showing all has this protein expression, and does not detect respective strap (Fig. 6) in type L105 strain of Wei Shi Leptospira clear water and the non-virulent leptospira biflexa.PCR detected result and western detected result are on all four.
15 serogroups representative strains of used Chinese popular among the present invention
*Nat'l Pharmaceutical ﹠ Biological Products Control Institute bacterium number
1, whether FACS detection vaccine candidate antigen is that the Leptospira surface exposes protein
With the normal rabbit serum is blank, LipL31 immunize rabbit gained antiserum(antisera) is as negative control, LipL32 and LipL41 immunize rabbit gained antiserum(antisera) are as positive control, and whether FACS detects vaccine candidate antigen is that the Leptospira surface exposes the protein result as shown in Figure 7.Grey peak figure is the detected result of anti-rabbit LipL31 serum and Leptospira cell among the figure, and white peak figure is the detected result of specificity outer membrane protein antiserum(antisera) and Leptospira cell.The fluorescence mean value (mean) of Jian Ceing sees Table 2 each time.It is carried out the student-t check, is significant difference with P＜0.05 all, and whether the mean value of analyzing vaccine candidate antigen immunize rabbit antiserum(antisera) and negative control fluorescent signal has statistical significance.The result shows: known albumen LipL32 and the average of LipL41 and PL40 and t check P＜0.05 of normal rabbit serum that is exposed to adventitia, statistical significance is arranged, and the check P of inner membrane protein LipL31 0.05.Illustrating that PL40 is similar with LipL41 to LipL32, can combine with antibodies specific, is that the surface exposes albumen.
Whether table 2.FACS detects vaccine candidate antigen is that the Leptospira surface exposes protein
2, Triton X-114 extraction separation Subcellular Localization:
The Leptospira cell dissolves with 1% Triton X-114, centrifugal removal insolubles, and soluble part is further divided into organic phase and water.Full cell, precipitation, organic phase are separated in SDS-PAGE with water four parts, and carry out immunoblotting with anti-PL40, anti-LipL32, anti-LipL31 antiserum(antisera) and detect.LipL32 is known outer membrane protein, and LipL31 is known inner membrane protein, is used as contrast.As shown in Figure 8, LipL32, major part is present in the organic phase, and minority is present in the precipitation, and LipL31 exists only in the precipitation.PL40 is the same with LipL32 all to be that major part is present in the organic phase, illustrates that this albumen also may be the albumen that exists in the adventitia.
Embodiment 5Infect recombinant protein antibody generation in coupler body cavy, mouse and the Golden Hamster
1, Westernblot detected result
To infect coupler body 14 days cavy, mouse and Golden Hamster serum is one anti-, and Goat-anti-mouse IgGHRP, goat anti-guinea pig IgG HRP or goat anti-hamster IgG HRP are two anti-, have all detected special luminous band.As shown in Figure 9, illustrate at coupler body after infecting mouse, cavy, Golden Hamster, all have anti-PL40 to produce.
2, ELISA detected result
In the present invention, rely type to rely strain intraperitoneal injection of mice, cavy or Golden Hamster with the pass at the low Leptospira in generation of sublethal dose respectively, heart extracting blood is gathered 0,7,14 and 28 day blood.With 0.3ug/ml reorganization PL40 albumen coated elisa plate, carry out ELISA and detect.As shown in figure 10, the result shows that antibody titers is than normal serum height in the mouse that infects after 7 days and the suslik serum, and antibody titers significantly increases after 28 days and infect.This presentation of results PL40 albumen is the main immunogenic albumen in the leptospiral infection process.Therefore, PL40 albumen not only can be used as leptospiral vaccine candidate gene, and can play a role in the detection of coupler body disease.
According to the full genome annotation of Leptospira, the long 1074bp of LA3744, proteins encoded contain 357 amino acid, predict that its molecular weight is 39.0KD, and in conjunction with the recombinant protein electrophoresis result, we are with its proteins encoded called after PL40.TMHMM carries out secondary structure prediction and shows that this albumen is membrane spaning domain at the C-end, and rest part is mainly the film foreign lands, and BLASTN and BLASTP software analysis are found, except Leptospira, do not found the LA3744 similar sequence in other biology.In finishing the pathogenic Leptospira of 3 strains of order-checking, the sequence of LA3744 is very conservative, is respectively 99% with Copenhagen type Fiocruz L1-130 strain similarity, with the spirochetal similarity of other two strain Bo Shi also up to 90%.At the saprophytic form leptospira biflexa homologous sequence of LA3744 is arranged also, but similarity is very low, only is 37%.Carry out PCR with the total length primer of LA3744 and detected the conservative property of PL40 15 serogroups representative strains of Chinese popular from gene level.The result shows except that Wei Shi spirochete clear water type and non-virulent leptospira biflexa, has all detected specific band in other 14 strain bacterium.The result shows that PL40 may not have homogenic existence in this two strains bacterium.This result also obtains confirming that only this two strains bacterium does not have PL40 at protein level by Western blot method.
Immunofluorescence technique, Triton X-114 extraction process and FACS method confirm that all PL40 albumen is that Leptospira relies type to rely the surface of strain to expose protein, and be similar to LipL32, is present in the adventitia.
We find that PL40 albumen is an albumen newfound, that conservative surface exposes in the Leptospira of causing a disease by the experiment of front, but its whether when the leptospiral infection human body, express be decision its can be as valuable diagnostic antigen and the proteic key of vaccine candidate.After pathogenic coupler body infects mammalian hosts, have only the bacterial antigens of expression, just can become the target site of host immune system identification.Because rodent is main contagium of pathogenic Leptospira and reservoir host, research leptospiral infection Mammals, we have selected for use mouse, Golden Hamster and cavy as the situation of leptospiral infection host research PL40 albumen in Mammals expression in vivo and antibody generation.Mouse behind the coupler body infecting mouse, does not generally have tangible disadvantageous effect as the animal model of persistence symptomless infection, does not also cause acute onset, but coupler body settles down in mouse kidney for a long time, and constantly breeding, excretes with urine.Golden Hamster and cavy then are the susceptible hosts of pathogenic coupler body, and children cavy in age and Golden Hamster symptoms such as typical jaundice, pulmonary apoplexy, liver injury of spleen can occur after infecting coupler body, and mortality ratio is high.In the research of experiment of a large amount of immunoprotection and mechanism of causing a disease all they animal models as acute infection.In this test, find in the research, in infecting leptospiral mouse, cavy and Golden Hamster serum, can both detect PL40 antibody, beginning in the 7th day just can obviously detect PL40 antibody and produce, along with time lengthening, its antibody titers increases gradually in the serum, and the titre of antibody was apparently higher than the 7th day in the time of the 28th day.The result shows when Mammals is infected in pathogenic coupler body 56601 strains the PL40 protein expression is arranged and have antibody to produce.And infer that this albumen may play a role in coupler body causes a disease.Because it has the conservative property of height in pathogenic strain, it is the peculiar albumen in the Leptospira simultaneously, is a very valuable diagnostic antigen and vaccine candidate albumen therefore.
SEQUENCE?LISTING
＜110〉Medical College, Shanghai Communication Univ.
＜120〉leptospira vaccine candidate's outer membrane protein
<130>/
<160>2
<170>PatentIn?version?3.3
<210>1
<211>357
<212>PRT
＜213〉Leptospira interrogans (Leptospira)
<400>1
<210>2
<211>1074
<212>DNA
＜213〉Leptospira interrogans (Leptospira)
<400>2
Claims (10)
1. an isolating Leptospira outer membrane protein polypeptide is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with the aminoacid sequence that is selected from SEQ ID No:1.
2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with aminoacid sequence of SEQ ID No:1.
3. isolating polynucleotide is characterized in that, it comprises 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) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3 is characterized in that this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ IDNo:1.
5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down group:
(a) has sequence among the SEQID No:2.
6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.
8. the preparation method of a Leptospira outer membrane protein polypeptide is characterized in that, this method comprises:
(a) be fit to express under the condition of Leptospira outer membrane protein, cultivating the described host cell of claim 7:
(b) from culture, isolate the Leptospira outer membrane protein polypeptide.
9. energy and the described Leptospira outer membrane protein specificity of claim 1 bonded antibody.
10. a vaccine composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.
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