CN101298473B

CN101298473B - Conservative neisserial antigens

Info

Publication number: CN101298473B
Application number: CN200810092797.7A
Authority: CN
Inventors: R·拉普奥利
Original assignee: Novartis Vaccines and Diagnostics SRL
Current assignee: GSK Vaccines SRL
Priority date: 1999-04-30
Filing date: 2000-04-28
Publication date: 2015-08-12
Anticipated expiration: 2020-04-28
Also published as: GB9910168D0; CN101298473A

Abstract

The invention provides conservative neisserial antigens.In order to ensure identity and reactivity between maximum bacterial strain, can utilize in different Neisseria species, protein domain conservative between serotype and bacterial strain.The invention provides the protein containing amino acid sequence segment, these sections, at most of neisserial, are especially total in Neisseria meningitidis and neisseria gonorrhoeae.

Description

Conservative neisserial antigens

Patent application of the present invention is international application no is PCT/IB 00/00642, international filing date is on April 28th, 2000, the application number entering National Phase in China is 00809624.4, the divisional application of the application for a patent for invention that name is called " conservative neisserial antigens ".

The content of all documents quoted herein is all included in herein as a reference.

Technical field

The present invention relates to the conservative antigen of neisseria (Neisseria) bacterium.

Background technology

Neisseria meningitidis (Neisseria meningitidis) be immotile, have pathogenic gram-negative diplococcus to people.

According to the capsular polysaccharide of this bacterium, identify the serotype of 12 kinds of Neisseria meningitidis.Modal pathogenic agent in A Xing Shiya the Sahara-African Territories prevailing disease.Type B and C type serotype bacterium are the reasons causing most case in the U.S. and most of developed country.W135 and Y type serotype bacterium is the reason of all the other cases causing the U.S. and developed country.

The tetravalence polysaccharide vaccine that the meningococcus vaccine of current use is made up of serotypes A, C, Y and W135.But this method can not be used for Type B meningococcus, because menB capsular polysaccharide is the polymkeric substance of N-acetylneuraminic acid of α (2-8)-be connected, it is also present in mammalian tissues.A kind of menB vaccine approach adopts the mixture of outer membrane protein (OMP).In order to overcome antigenic variation, construct containing the polyvalent vaccine (such as, Poolman JT (1992) " development of meningococcus vaccine " Infect.Agents Dis.4:13-28) up to 9 kinds of different porins.Other albumen for outer membrane vaccines is opa and opc albumen, but these methods all can not overcome antigenic variation (as Ala ' Aldeen and Borriello (1996) " meningococcus TBP1 and 2 all exposes, and produces the bactericidal antibody response that can kill and wound homology and heterologous strain " Vaccine 14 (1): 49-53).

A large amount of neisserial proteins and nucleotide sequence are disclosed in WO99/24578, WO99/36544, WO99/57280 and WO00/22430.The content of these 4 applications is incorporated herein by reference.The Comprehensive sequence data of bacterial strain MC58 is disclosed in Science (2000) 287:1809-1815 of the people such as Tettelin, and the content of the document is also incorporated herein by reference.

Summary of the invention

In order to ensure the maximum identity between bacterial strain and reactivity, the protein domain that can be used in different Neisseria species, guard between serotype and bacterial strain.Therefore, the invention provides protein, these protein contain amino acid sequence segment total in most of neisserial (especially Neisseria meningitidis and neisseria gonorrhoeae).

The invention provides the protein containing Neisserial protein fragment, wherein said fragment comprises n continuous print conserved amino acid, and condition is that the present invention does not comprise total length Neisserial protein within the scope of it.According to concrete protein, n is 7 or higher (such as, 8,10,12,14,16,18,20 or higher).Described fragment should comprise an antigen or the immunogenic region of Neisserial protein.

" conservative " amino acid is the amino acid existed in the specific Neisserial protein of at least x% neisserial.The value of x can be 50% or higher, such as 66%, 75%, 80%, 90%, 95% or even 100% (namely this amino acid is present in the protein involved of all neisseriales).

In order to determine whether an amino acid is " guarding " in specific Neisserial protein, need this amino acid compared in the associated proteins sequence of multiple different neisserial (" with reference to group ").Neisseria species (preferred Neisseria meningitidis and neisseria gonorrhoeae) different in a large number can be comprised with reference to group, or can single species be comprised.Different in a large number serotype (A, B, C of such as Neisseria meningitidis of a certain specific species can be comprised with reference to group., W135, X, Y, Z and 29E serotype), or comprise single serotype.With reference to group also can comprise a certain particular serotype different in a large number bacterial strain (NG6/88, the BZ198 of such as Type B Neisseria meningitidis, NG3/88,297-0, BZ147, BZ169,528, BZ133, NGE31, NGH38, NGH15, BZ232, BZ83 and 44/76 strain).A kind of preferred reference group is made up of 5 kinds of modal N. meningitidis strains and/or 5 kinds of modal neisseria gonorrhoeae bacterial strains.

Should contain the k bacterial strain of the k difference branch from suitable phylogenetic tree with reference to group, such as those disclosed in the following documents: people (1992) the Epidemiol Infect 109:227-239 such as (a) Ni; People (1992) nucleic acids research (Nucl.Acids Res.) 20:46757 such as (b) Wolff; (c) Bygraves & Maiden (1992) hereditary JOURNAL OF MICROBIOLOGY (J.Gen.Microbiol.); The people such as (d) Caugant, (1987) Bacteriology (J.Bacteriol.) 69:2781-2792.Another kind of operational phylogenetic tree is as shown in the application Fig. 8, and another kind as shown in figure 9b.

Should be understood that a certain specific species, serotype or bacterial strain, when only having its coded protein and there is relevant amino acid in this protein, be just included in reference in group.Such as, in the amino acid example in following ORF40, neisseria gonorrhoeae should not be comprised with reference to group, because these species are not containing ORF40.

Therefore, for the protein all existed in Neisseria meningitidis and neisseria gonorrhoeae, preferred reference group comprises:

■ Neisseria meningitidis A, Z2491 bacterial strain

■ Neisseria meningitidis B, NG6/88 bacterial strain

■ Neisseria meningitidis W, A22 bacterial strain

■ neisseria gonorrhoeae, Ng F62 bacterial strain

These bacterial strains have description in the following documents: people (1996) molecular microbiologies (Mol.Microbiol.) 19 (4:841-856) such as (a) Seiler A.; The people such as (b) Maiden, institute of (1998) American Academy of Sciences report (Proc.Natl.Acad.Sci.USA) 95:3140-3145; People (1992) molecular microbiology (Mol.Microbiol.) 6:1271-1279 such as (c) Virji; The people such as (d) Dempsey, (1991) Bacteriology (J.Bacteriol.) 173:5476-5486.

But for the protein existed only in Neisseria meningitidis, preferred reference group comprises:

■ Neisseria meningitidis A, Z2491 bacterial strain

■ Neisseria meningitidis B, NG6/88 bacterial strain

■ Neisseria meningitidis W, A22 bacterial strain

The aminoacid sequence of different neisserial can compare easily with computer.This is usually directed to such as, with algorithm, CLUSTAL [Thompson etal (1994) nucleic acids research (Nucl.Acids Res.) 22:4673-4680; TrendsBiochem Sci (1998) 23:403-405] or PILEUP algorithm [integral part of GCG Wisconsin software package, preferably 9.0 editions], multiple sequence is compared.

Conserved amino acid is clearly in Multiple Sequence Alignment, and at relevant amino acid position place, the sequence that great majority are compared can contain specific amino acid.Can also use program, such as BOXSHADE [can obtain online from such as NIH], PRETTYBOX [GCG Wisconsin, version 10] or JALVIEW [can obtain online from EB], conserved amino acid can more clearly with the naked eye be found out.

The fragment of described protein preferably containing one of protein disclosed in WO99/24578, WO99/36544, WO99/57280 and WO00/22430 or disclosed in people Science (2000) 287:1809-1815 such as Tettelin the fragment of one of 2158ORF.More specifically, the fragment of one or more albumen in its ORF4, ORF40 preferably containing open (see embodiment herein) herein, ORF46, protein 225, protein 235, protein 519, protein 726, protein 919 and protein 953.Usually, albumen of the present invention is not containing disclosed protein sequence clear and definite in people's documents such as WO99/24578, WO99/36544, WO99/57280, WO00/22430 or Tettelin.

Present invention also offers the protein containing one of sequence shown in drawings attached.

The certain available various method of albumen of the present invention (such as recombinant expressed, natural expression, purifying, chemosynthesis etc. from cell cultures) makes various forms (such as natural, fusion rotein etc.).They should make substantially pure form (being namely substantially free of the albumen of other neisserial or host cell).

On the other hand, the invention provides can in conjunction with the antibody of these albumen.They may be polyclonal or monoclonal, and available any suitable method obtains.

On the other hand, the invention provides the nucleic acid of code book invention albumen.Should also be understood that nucleic acid provided by the invention comprises and the sequence of these complementary (such as antisense or probe object).

In addition, the invention provides the nucleic acid with the nucleic acid hybridization of Neisseria meningitidis disclosed in embodiment, preferably under " High stringency " condition (as 65 DEG C, at 0.1 × SSC, 0.5%SDS solution).

Certainly, nucleic acid of the present invention can obtained by many modes (such as chemosynthesis, from genome or cDNA library or obtain from organism itself), and can adopt various forms (such as strand, double-strand, carrier, probe etc.).

In addition, term " nucleic acid " comprises DNA and RNA, and their analogue, as containing those analogues modifying skeleton, also comprises peptide nucleic acid(PNA) (PNA) etc.

On the other hand, the invention provides the carrier (as expression vector) containing nucleotide sequence of the present invention and the host cell with these vector.

On the other hand, the invention provides the composition comprising albumen of the present invention, antibody and/nucleic acid.Such as, these compositions are suitable as vaccine, or as diagnostic reagent, or as immunogenic composition.

Present invention also offers nucleic acid of the present invention, albumen or antibody as medicament (such as vaccine) or as diagnostic reagent.Present invention also offers nucleic acid of the present invention, albumen or antibody and producing the application in following material: (i) is used for the treatment of or prevents the medicament of Neisserial infections affect; (ii) for detecting neisserial or detecting the diagnostic reagent whether anti-neisserial antibody exist; And/or (iii) is for generation of the reagent of the antibody of anti-Neisserial.This purposes should be applicable to Neisserial all species.

When Neisserial protein contains the amino acid guarded more than q%, present invention provides the purposes of Neisserial protein (or its fragment) as the albumen of non-strain specificity, this albumen is at multiple species, show cross reactivity between serotype and bacterial strain.Q value can be 50%60%, 75%, 80%, 90%, 95% or even 100%.

Present invention also offers a kind of method for the treatment of patient, the method comprises the nucleic acid of the present invention, albumen and/or the antibody that give bacterium.

On the other hand, the invention provides various method.

Provide a kind of method of producing albumen of the present invention, the method comprising the steps of: under the condition that inducible protein is expressed, cultivate host cell of the present invention.

Provide a kind of method of producing albumen of the present invention or nucleic acid, the method comprising the steps of: part or all of chemically synthetic protein or nucleic acid.

Provide a kind of method detecting polynucleotide of the present invention, the method comprises the following steps: that (a) makes nucleic acid probe of the present invention contact with biological sample under hybridization conditions, forms duplex; (b) described duplex is detected.

Provide a kind of detection method of protein of the present invention, the method comprises the following steps: that (a) makes antibody of the present invention contact with biological sample under the condition of applicable formation Antibody-antigen complex; (b) described mixture is detected.

Accompanying drawing explanation

Fig. 1-7 show provided by BOXSHADE (1) ORF40, (2) ORF4, (3) 225, (4) 235, (5) 287, (6) 519, the sequence alignment of (7) 919.Conserved amino acid has black background.

Fig. 8 shows phylogenetic tree.

Fig. 9 A show in Neisseria meningitidis ORF4, ORF40,225,235,287,519, the variability of the aminoacid sequence of 919.These sequences are used to the phylogenetic tree shown in design of graphics 9B.

Figure 10-19 show provided by BOXSHADE (10) ORF4, (11) ORF40, (12) ORF46, (13) 225, (14) 235, (15) 287, (16) 519, (17) 726, (18) 919, the sequence alignment of (19) 953.

Figure 20 show ORF4,225,235, the western blot result of 519 and 919.

Embodiment

Following is a list and can be used for implementing the standard technique of the present invention's (being such as in immunization or the sequence disclosed in diagnostic purpose use) and the general introduction of program.This general introduction does not limit the present invention, but which gives available and nonessential example on the contrary.

Summary

Unless otherwise described, enforcement of the present invention will adopt molecular biology, microbiology, recombinant DNA and immunologic routine techniques, and these are all known to those skilled in the art.These technology have complete description in the following documents: such as, Sambrook " Molecular Cloning: A Laboratory guide " the 2nd edition (1989); " DNA clone " I and II rolls up (D.N.Glover compiles 1985); " oligonucleotide synthesis " (M.J.Gait compiles, 1984); " nucleic acid hybridization " (B.D.Hames and S.J.Higgins compiles .1984); " transcribe and translate " (B.D.Hames and S.J.Higgins compiles, 1984); " animal cell culture " (R.I.Freshney compiles, 1986); " immobilized cell and enzyme " (IRL press, 1986); B.Perbal, " molecular cloning practical guide " (1984); " Enzymology method " series of books (Academic Press, Inc.), especially 154 and 155 volumes; " gene transfer vector of mammalian cell " (J.H.Miller and M.P.Calos compiles, 1987, Cold SpringHarbor Laboratory); Mayer and Walker compiles (1987), " immuno-chemical method of Cytobiology and molecular biology " (Academic Press, London); Scopes, (1987) " protein purification: principle and put into practice " the 2nd edition (Springer-Verlag, N.Y.), and " experiment immunization learns to do volume " I-IV rolls up (D.C.Weir and C.C.Blackwell compiles 1986).

Have employed Nucleotide and amino acid whose standardized abbreviations in this manual.

The all publications quoted herein, patent and patent application are all included in herein incorporated by reference.Particularly, the content of international patent application WO99/24578, WO99/36544, WO99/57280 and WO00/22430 is merged in herein.

Definition

When at least 85% of X+Y weight total in composition is X, then the composition containing X is claimed " to there is no Y ".Preferably, X account for X+Y gross weight in composition at least about 90%, better for 95% or even 99% (weight).

Term " comprise " refer to " containing " and " by ... form ", the composition such as " comprising " X can be made up of X completely, or can contain the material outside X, such as X+Y.

Term " allos " refers to find two kinds of biological components of being not together at occurring in nature.This component can be that host cell, gene or control region are as promotor.Although heterologous component finds to be not together at occurring in nature, they can work together, such as, when being connected with this genetic manipulation with a kind of promotor of certain gene allos.Another example is Neisserial sequence and mouse host cell allos.Another example is two epi-positions from identical or different protein, and the spread pattern that they do not have with nature is assembled on single protein.

A kind of polynucleotide sequence that " replication orgin " is startup and regulates polynucleotide (such as expression vector) to copy.Replication orgin can be used as the autonomous unit that intracellular polynucleotide copies, and can copy under the control of himself.It is required that replication orgin is that carrier copies in particular host cell.Had a certain replication orgin, expression vector just can in cell appropriate proteins existence under the copying of high copy number.The example of replication orgin is effective autonomously replicating sequence in yeast; And in COS-7 cell effective viral T-antigen.

" mutant " sequence definition is different from natural or disclosed sequence but has DNA, RNA or the aminoacid sequence of sequence thereto.According to concrete sequence, sequence thereto degree between natural or disclosed sequence and mutant sequence should be greater than 50% (such as 60%, 70%, 80%, 90%, 95%, 99% or higher, wherein sequence thereto calculates with Smith-Waterman algorithm as mentioned above).As described herein, the nucleic acid molecule of nucleotide sequence provided herein or " allele variant " in region are nucleic acid molecule on locus substantially the same in the genome of another or second strain isolated or region, due to such as sudden change or the natural variation caused of recombinating, they have similar but not identical nucleotide sequence.The albumen that coding region allelic variant is encoded usually has the activity with protein similar coded by its icp gene.Allele variant also can comprise the change in gene 5 ' or 3 ' non-translational region, such as, change (such as seeing United States Patent (USP) 5,753,235) in regulable control district.

Expression system

Neisserial nucleotide sequences can be expressed in various different expression system; Those systems such as used together with yeast with mammalian cell, baculovirus, plant, bacterium.

I. mammlian system

Mammalian expression system is as known in the art.Mammalian promoter can start in conjunction with mammalian RNA polymerase any DNA sequence dna that downstream (3 ') encoding sequence (as structure gene) is transcribed into mRNA.Promotor has a transcription initiation region, and it is close to 5 ' end of encoding sequence usually, and also have a TATA box, it is usually located at base pair (bp) place, 25-30, transcription initiation site upstream.Think that TATA box guide RNA polymerase II synthesizes at correct site starting rna.Mammalian promoter is also containing a upstream promoter element, and it is usually located in TATA box upstream 100 to 200bp.This upstream promoter element determines the speed of transcripting starting, and can work in one of both direction [people (1989) " expression of clone gene in mammalian cell " " molecular cloning: laboratory manual " such as Sambrook, the 2nd edition].

Mammalian viral genes normally high expression level, has a wide host range; Therefore, the sequence of encoding mammalian viral gene provides useful especially promoter sequence.Example comprises SV40 early promoter, MMTV LTR promotor, adenovirus major late promoter (Ad MLP) and herpes simplex virus promoters.In addition, useful promoter sequence is also provided from non-viral gene (as mouse metallothionein gene) derivative sequence.Expression can be composing type or modulated (induction type), depends on that can promotor induce with glucocorticoid in hormone response sexual cell.

Strengthen the existence of element (enhanser), combine above-mentioned promoter element and usually can improve expression level.Enhanser is so a kind of control DNA sequence dna, and when it is connected with homology or allogeneic promoter, synthesize when normal RNA initiation site starts, it can stimulate transcribes raising 1000 times.When enhanser is positioned at upstream or the downstream of transcription initiation site, be in normal or reverses direction, or during more than distance promotor 1000 Nucleotide distance, it all has activity [people (1987) the Science 236:1237 such as Maniatis; The people such as Alberts (1989) " Cell. Mol ", the 2nd edition].It may be useful especially for deriving from virus the enhancer element obtained, because they have wider host range usually.Example comprises SV40 early gene enhanser [people (1985) EMBO such as Dijkema J.4:761] and the enhancers/promoters [people (1982b) Proc.Natl.Acad.Sci.79:6777 such as Gorman] derived from the sarcoma viral long terminal repeat of Rous (LTR) and the enhancers/promoters [people (1985) the Cell 41:521 such as Boshart] from human cytomegalic inclusion disease virus.In addition, some enhansers are only adjustable and have activity [Sassone-Corsi and Borelli (1986) Trends Genet.2:215 under the existence of inductor (such as hormone or metal ion); The people such as Maniatis (1987) Science 236:1237].

DNA molecular can in mammalian cell intracellular expression.Promoter sequence directly can be connected with DNA molecular, and in this case, the N of recombinant protein holds first amino acid to be methionine(Met) all the time, and it is encoded by ATG initiator codon.If needed, hold by cutting this N with cyanogen bromide extracorporeal culture from albumen.

In addition, foreign protein also can be secreted in growth medium from cell, and method produces chimeric DNA molecular, and the fusion rotein of this DNA molecule encode comprises a leader fragment, and this fragment provides the secretion of foreign protein in mammalian cell.Preferably, can have between leader fragment and foreign gene can in vivo or the Processing position of external fracture.Leader fragment is encoded a kind of signal peptide usually, and the hydrophobic amino acid that this signal peptide secretes cell by pilot protein forms.Adenovirus tripartite leader sequence is the example of the leader sequence secreting foreign protein in mammalian cell.

Usually, the Transcription Termination of mammalian cell identification and polyadenylation se-quence are the regulation and control regions being positioned at translation stop codon 3 ', and therefore it is connected to the side of encoding sequence together with promoter element.3 ' of ripe mRNA holds and forms [people (1985) the Cell 41:349 such as Birnstiel by the posttranscriptional cleavage of fixing a point and polyadenylation; Proudfoot and Whitelaw (1988) " terminal of eucaryotic RNA and 3 ' end processing " " transcribing and montage " (B.D.Hames and D.M.Glover volume); Proudfoot (1989) Trends Biochem.Sci.14:105].These sequence-directed mRNA transcribe, and mRNA can be translated into the polypeptide of this DNA encoding.The example of transcription terminator/polyadenylation signal comprises from derivative those of SV40 people (1989) " expression of clone gene the mammalian cell cultivated " " Molecular Cloning: A Laboratory guide " such as [] Sambrook.

Usually, said modules, comprises promotor, polyadenylation signal and transcription termination sequence and is placed in expression constructs together.If needed, in expression constructs, also comprise enhanser, the intron with functional splice donor and acceptor site and leader sequence.Expression constructs maintains with replicon form usually, such as, be can at the extra-chromosomal element (as plasmid) of stable maintenance in host (as mammalian cell or bacterium).Mammals dubbing system comprises from those derivative systems of animal virus, and it needs trans-acting factor to copy.Such as, the plasmid containing papovavirus dubbing system, as SV40 [Gluzman (1981) Cell 23:175] or polyomavirus, copies high copy number under suitable viral T antigen exists.Other example of Mammals replicon comprises the replicon derived from bovine papilloma virus and Epstein-Barr virus.In addition, replicon can have two dubbing systems, thus can maintain in such as mammalian cell and carry out expression can clone in prokaryotic hosts and increase.The example of these mammalian-bacteria shuttle carriers comprises pMT2 [people (1989) Mol.Cell.Biol.9:946 such as Kaufman] and pHEBO [people (1986) Mol.Cell.Biol.6:1074 such as Shimizu].

Transformation Program used depends on host to be transformed.Method heterologous polynucleotide imported in mammalian cell is known in the art, it comprise dextran mediation transfection, calcium phosphate precipitation, Polybrene (1,5-dimethyl-1,5-phenodiazine 11 methylene radical gathers Methobromide) transfection, protoplast fusion, the electroporation that mediate, polynucleotide to be wrapped in liposome and by direct for DNA microinjection in karyon.

It is as known in the art for can be used as the mammal cell line that host carries out expressing, it comprises many immortal cell lines obtained from American type culture collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cell, HeLa cell, newborn hamster kidney (BHK) cell, monkey-kidney cells (COS), human liver cell cancer cells (as HepG2) and other much clone.

Ii. rhabdovirus system

Also the polynucleotide of coded protein can be inserted in suitable insect expression vector, and be connected with the controlling elements operability in this carrier.Vector construction adopts technology known in the art.Generally speaking, the component of expression system comprises a kind of transfer vector, normally bacterial plasmid, and it contains Baculovirus Gene pack section and is convenient to insert the restriction site of heterologous gene to be expressed; Wild-type baculovirus, the baculovirus specific fragment homology (this makes heterologous gene energy homologous recombination in Baculovirus Gene group) in its sequence and transfer vector; And suitable insect host cell and growth medium.

The DNA sequence dna of coded protein is inserted after in transfer vector, carrier and wild-type virus genome are transfected in insect host cell, make carrier and viral genome restructuring.Express the recombinant virus of packaging, qualification is purification of Recombinant plaque also.Baculovirus/insect cell expression system material and method thereof, among other, can in a kit form purchased from Invitrogen, San Diego CA (" MaxBac " test kit).These technology are normally known to those skilled in the art, in Texas Agricultural Experiment Station Bulletin No.1555 (1987) (rear claim " article of Summer and Smith ") of Summers and Smith, have abundant description.

Before the DNA sequence dna of coded protein is inserted shaft-like viral genome, usually by said modules, comprise promotor, leader sequence (if needs), interested encoding sequence and transcription termination sequence and be assemblied in Intermediate transplacement construction (transfer vector).The controlling element that this construction can be connected containing individual gene and operability; Multiple gene, each gene has its oneself a set of operability linked regulatory elements; Or the multiple genes to be regulated and controled by same group of controlling element.Intermediate transplacement construction remains in a replicon usually, such as, can stablize the extra-chromosomal element (as plasmid) kept in host (as bacterium).Replicon will have a dubbing system, thus can remain in suitable host and carry out cloning and increasing.

At present, the most frequently used transfer vector being used for foreign gene to import AcNPV is pAc373.Also can design other many carrier well known by persons skilled in the art.These carriers such as comprise, and (initiator codon of polyhedrin is become ATT from ATG to pVL985 by it, and in ATT downstream, 32 base pair places introduce a BamHI cloning site; See Luckow and Summers, Virology (1989) 17:31).

Plasmid is also selected in intestinal bacteria containing polyhedrin polyadenylation signal people (1988) Ann.Rev.Microbiol., 42:177 such as () Miller and being used for and protokaryon amicillin resistance (amp) gene of breeding and replication orgin usually.

Baculovirus transfer vector is usually containing bacilliform virus promoter.Bacilliform virus promoter can start in conjunction with baculovirus RNA polymerase the DNA sequence dna that downstream (5 ' to 3 ') encoding sequence (as structure gene) is transcribed into mRNA.Promotor has a transcription initiation region, and this district is close to 5 ' end of encoding sequence usually.This transcription initiation region generally includes a RNA polymerase binding site and a transcription initiation site.Baculovirus transfer vector also may have the secondth district being called enhanser, if this region exists, it is usually away from structure gene.Expression can be regulation type or composing type.

The structure gene of to transcribe in a large number late period at viral infection cycle provides useful especially promoter sequence.Example comprises and derives from the gene of encode viral polyhedrin the sequence obtained, the people such as Friesen (1986) " regulation and control that Baculovirus Gene is expressed " " baculovirus molecular biology " (Walter Doerfler edits); EPO publication number 127 839 and 155476; And the gene of coding p10 albumen, the people such as Vlak (1988), J.Gen.Virol.69:765.

Encoding the DNA of suitable signal sequence can derived from gene people such as (, (1988) Gene, 73:409) Carbonell of the insect of secretion or baculovirus protein (as baculovirus polyhedrin gene).In addition, because the signal of mammalian cell posttranslational modification is (as signal peptide fracture, proteolytic cleavage and phosphorylation) it seems can by insect cell identification, and secretion and the signal needed for karyon accumulation it seems between spinal zooblast and vertebrate cells it is conservative, therefore secretion in insect also can be provided with the leader sequence in non-insect source, these leader sequences are such as from the encoding human alpha-interferon (people (1985) such as Maeda, Nature 315:592), peptide (the people (1988) such as Lebacq-Verheyden of people's gastrin release, Molec.Cell.Biol.8:3129), human IL-2 (people (1985) PNAS such as Smith, 82:8404), mouse IL-3 (people (1987) the Gene 58:273 such as Miyajima) and hGCD (people (1988) DNA such as Martin, gene 7:99) derives acquisition.

Recombinant polypeptide or polyprotein can at intracellular expressions, if or it express with suitable regulating and controlling sequence, it can be secreted.The heterologous gene with short leader sequence that usually needs that the good intracellular expression of nonfused foreign protein is desirable has suitable translation initiation signal before ATG start signal.If needed, hold methionine(Met) by cutting N with cyanogen bromide extracorporeal culture from maturation protein.

In addition, by producing restructuring polyprotein that non-natural secretes by chimeric DNA molecular or albumen is secreted from insect cell, this chimeric fusion rotein coded by DNA molecular comprises a leader fragment, and this fragment provides in insect the effect of secreting foreign protein.This leader fragment is encoded a kind of signal peptide usually, and the hydrophobic amino acid pilot protein matter that this signal peptide comprises is transferred in endoplasmic reticulum.

After the DNA sequence dna inserting this protein expressioning product precursor of coding and/or gene, with the allogeneic dna sequence DNA of transfer vector and genomic dna cotransformation (normally cotransfection) the insect cell host of wild-type baculovirus.The promotor of this construction and transcription termination sequence comprise the Baculovirus Gene pack section of 2-5kb usually.The method introduced by allogeneic dna sequence DNA in baculovirus in required site is known in the art.(see the article of Summers and Smith, the same; The people such as Ju (1987); The people such as Smith, Mol.Cell.Biol. (1983) 3:2156; With Luckow and Summers (1989)).Such as, insertion can be recombinated by homologous double-crossover to insert a gene as in polyhedron gene; Insertion can also be insert engineered enter in restriction enzyme site in required Baculovirus Gene.The people such as Miller (1989), Bioessays 4:91.When DNA sequence dna is cloned in expression vector polyhedron gene position, its 5 ' and 3 ' equal side joint polyhedrin specific sequence, and be positioned at the downstream of polyhedrin promoter.

Subsequently the rhabdovirus expression vector newly formed is packaged in infectious recombinant baculovirus.There is the frequency very low (about between 1% and 5%) of homologous recombination; Therefore, the most of viruses produced after cotransfection are still wild-type virus.Therefore, need to differentiate recombinant virus by a kind of method.An advantage of this expression system is that naked eyes screening can distinguish recombinant virus.In the virus infection later stage, the level that the polyhedrin that natural viral produces is being subject to produce in the karyon of its cells infected is very high.The inclusion body that the polyhedrin of accumulation is formed also contains embedded particles.The size of these inclusion bodys is 15 microns, and they have the refractivity of height, thus make them present bright shiny appearance, is easy under an optical microscope observe.The cell having infected recombinant virus lacks inclusion body.In order to distinguish recombinant virus and wild-type virus, by technology known in the art transfection supernatant being inoculated on individual layer insect cell and forming plaque.That is, screen existence (being shown to be wild-type virus) under an optical microscope or there is not the plaque of (being shown to be recombinant virus) inclusion body." Current Protocols in Microbiology " the 2nd volume people such as (edit) Ausubel, 16.8 (supplements 10,1990); Summers and Smith, the same; The people such as Miller (1989).

Develop the recombination rhabdovirus expression vector infecting and enter several insect cell.Such as, the recombinant baculovirus for infecting following insect cell has been developed: Aedes aegypti, Autographa californica multicapsid nucleopolyhedrosisvirus, silkworm, black-tailed fruit flies, fall army worm and cabbage looper (WO 89/046699; The people such as Carbonell (1985) J.Virol.56:153; Wright (1986) Nature 321:718; The people such as Smith (1983) Mol.Cell.Biol.3:2156; Summary is shown in the people such as Fraser (1989) InVitro Cell.Dev.Biol.25:225).

Cell and cell culture medium can be buied for directly expressing and amalgamation and expression heterologous polypeptide in baculovirus/expression system; Cell culture technology is that those skilled in the art are usually known.Such as see Summers and Smith, the same.

Then, modified insect cell can grow in suitable nutritional medium, and this substratum can stably keep this plasmid in the insect host modified.When the gene of expression product is under derivable control, host growth can be made to high-density, and abduction delivering.In addition, when expression is constitutive expression, product will by continuous expression in substratum, and the required constantly circulation of nutritive medium, takes out interested product and supplement the nutrition consumed simultaneously.Product can carry out purifying by these technology following: such as chromatography, as HPLC, affinity chromatography, ion exchange chromatography etc.; Electrophoresis; Density gradient centrifugation; Solvent extraction etc.Product can be further purified as required, substantially to remove all insect proteins being also secreted in substratum or being produced by insect cell cracking, is at least substantially free of the product of host debris as protein, lipid and polysaccharide to provide a kind of.

In order to carry out protein expression, cultivate deriving the recombinant host cell obtained from transformant under the condition allowing the encoding sequence of recombinant protein to express.These conditions become with selected host cell.But those skilled in the art easily determine this condition according to knowledge known in the art.

Iii. Plant cell expression systems

Known in the art have many plant cell culture systems and whole plant genetic expression system.Typical plant cell gene expression system is included in those that describe in following patent, such as: US 5,693,506; US 5,659,122; And US5,608,143.Other example of genetic expression in plant cell cultures is described in Zenk, Phytochemistry 30:3861-3863 (1991).Except above-mentioned reference, the description about plant protein signal peptides also can be found in the following documents: the people such as Vaulcombe, Mol.Gen.Genet.209:33-40 (1987); The people such as Chandler, Plant MolecularBiology 3:407-418 (1984); Rogers, J.Biol.Chem.260:3731-3738 (1985); The people such as Rothstein, Gene 55:353-356 (1987); The people such as Whittier, Nucleic Acids Research 15:2515-2535 (1987); The people such as Wirsel, Molecular Microbiology 3:3-14 (1989); The people such as Yu, Gene 122:247-253 (1992).About can at R.L.Jones and J.MacMillin with the description of enzyme regulating plant genetic expression of plant hormone, Plant hormones regulators,gibberellins acid and the sour secretion inducing of Plant hormones regulators,gibberellins, Gibberellins, " plant physiology progress ", Malcolm B.Wilkins edits, 1984Pitman Publishing Limited, find in London, 21-52 page.Describing other regulates the reference of the gene of metabolism see Sheen, Plant Cell, 2:1027-1038 (1990); The people such as Maas, European Molecular Biology association magazine (EMBO J.) 9:3447-3452 (1990); Benkel and Hickey, institute of American Academy of Sciences report (Proc.Natl.Acad.Sci.) .84:1337-1339 (1987).

Usually, utilize technology known in the art, inserted by required polynucleotide sequence in an expression cassette, this expression cassette is the gene regulatory elements of design containing promising operation in plant.In expression vector needed for being inserted by this expression cassette, the upstream and downstream of expression cassette has the companion sequence being adapted at expressing in plant host.These companion sequences can from plasmid or virus, and provide required performance for carrier, is moved in required plant host by DNA to allow carrier from cloning host (as bacterium) originally.Bacterium/the plant vector construct on basis preferably can provide wide host range prokaryotic origin of replication; Protokaryon selectable markers; And, for Agrobacterium-mediated Transformation, T DNA sequence dna should be provided to be transferred to plant chromosome for agriculture bacillus mediated.When heterologous gene not easily detects, this construction preferably also has a selectable markers gene being applicable to determine whether vegetable cell has transformed.Summary about appropriate flags (such as grass family member) can at Wilmink and Dons, and 1993, Plant Mol.Biol.Reptr, finds in 11 (2): 165-185.

Also suggestion adopts the sequence being applicable to being incorporated into by heterologous sequence in Plant Genome.These sequences may comprise for homologous recombination transposon sequence and allow by the Ti sequence in heterogenous expression box radom insertion Plant Genome.Suitable protokaryon selectable markers comprises microbiotic (as penbritin or tsiklomitsin) resistance marker.Other DNA sequence dna of other function of encoding also can be present in carrier, and this is known in the art.

Nucleic acid molecule of the present invention can be included in an expression cassette and express interested protein.As long as an expression cassette usually, but two or more expression cassette is also feasible.Except the sequence of encoding heterologous albumen, recombinant expression cassettes is also containing following elements: promoter region, plant 5 ' non-translated sequence, initiator codon (originally whether have according to structure gene and determine) and transcribe and translation termination sequence.The unique restriction enzyme sites that expression cassette 5 ' and 3 ' is held can make expression cassette insert easily in the carrier be pre-existing in.

Allogeneic coding sequence may be used for any albumen relevant with the present invention.The sequence of the albumen of interest encodes is by a signal peptide of encoding out, and this signal Toplink is suitably processed and translocator matter, and usually lacks and may cause desirable proteins of the present invention and membrane-bound sequence.Due to for major part, the gene that transcription initiation region will express for duration of germination and transport, adopts the signal peptide providing transhipment, also can provide transhipment interested protein.In this way, interested albumen transports by from the transit cell of expressing this albumen, and can effectively be gathered in the crops.Usually, the secretion in seed is the endosperm being entered seed by aleuron or pelta body epithelial lining.Although do not need to make albumen be secreted from the cell producing this albumen, this secretion is conducive to the abstraction and purification of recombinant protein.

Because the final expression of required gene product will be carried out in eukaryotic cell, therefore need the Gene Partial of determining to clone whether containing the sequence of being processed by host's spliceosome mechanism as intron.If like this, need to carry out site-directed mutagenesis to " intron " district, lose, Reed and Maniatis, Cell 41:95-105,1985 as the intron password of mistake to prevent a part of genetic information.

Available trace transfer pipet mechanically shifts recombinant DNA, by direct for carrier microinjection in vegetable cell.Crossway，Mol.Gen.Genet，202：179-185。Also available polyoxyethylene glycol by transfer of genetic material in vegetable cell, the people such as Krens, Nature, 296,72-74,1982.The another kind of method importing nucleic acid fragment carries out high velocity ballistic with small-particle to run through, in the matrix of these globules or particle or on the surface with nucleic acid, the people such as Klein, Nature, 327,70-73,1987, Knudsen and Muller, 1991, Planta, 185:330-336 proposition by partickle bombardment barley endosperm to produce transgene barley.A kind of introduction method is also had to be that protoplastis and other entity (minicell (minicell), cell, lysosome or other lipid sur body that can merge) are merged, the people such as Fraley, institute of American Academy of Sciences report (Proc.Natl.Acad.Sci.USA), 79,1859-1863,1982.

Carrier also imports in vegetable cell by electroporation.(people such as Fromm, PNAS 82:5824,1958).In the art, Electroporated plant protoplastis under the plasmid containing gene constructs exists.The electricimpulse of high strength of electric field makes microbial film reversibly penetrating, thus allows to import plasmid.The plant protoplast of electroporation forms cell walls again, divides and forms plant callus.

Can isolate protoplastis and all plants of helping aftergrowth can be cultivated, can transform with the present invention, thus recovery obtains containing genetically modified whole plant.Known and in fact can regenerate all plants from the cell or tissue cultivated, it is including, but not limited to all main species of, sugarcane, beet, cotton, fruit and other tree, leguminous plants and vegetables.Some suitable plants comprise, such as, Fragaria, lotus flower belongs to, Medicago, donkey food Macroptilium, Trifolium, Trigonella, Vigna, Citrus, linum, Geranium, cassava (Manihot), Daucus (Daucus), mouse ear mustard belongs to, Btassica, Rhaphanus, sinapsis alba belongs to, Atropa, Capsicum, Datura, poison tobacco, tomato belongs to, Nicotiana, Solanum, green winter Solanum, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Brome, Asparagus, antirrhinum, keel angle belongs to, Nemesia belongs to (Nemesia), Pelargonium, millet belongs to, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, lolium, Zea, wheat, Sorghum and Datura various types of.

Regeneration is different with each kind of plant, but normally first provides the protoplast suspension of the conversion containing heterologous gene copy.Form callus, from callus, induce out branch, follow by root.In addition, formation embryo can be induced from protoplast suspension.These embryos resemble to germinate natural embryo and form plant.Substratum contains each seed amino acid and hormone usually, as plant hormone and phytokinin.Especially for corn and Medicago, L-glutamic acid is added in the medium and proline(Pro) is also very favourable.Branch and root are grown usually simultaneously.Effective regeneration is depended on substratum, genotype and is cultivated history.If control this three variablees, so Regenerated energy reproduces completely and repeats.

In some plant cell culture systems, albumen required for the present invention may be excreted, or albumen can extract from whole plant.When albumen required for the present invention is secreted into after in substratum, just can collect.Or, can mechanically broken embryo and without embryo-half seed or other plant tissue, to discharge the albumen be secreted between biological cells and tissues.This mixture is suspended from damping fluid, to regain soluble proteins.Then conventional albumen sepn and purification process purification of recombinant proteins is used.By parameters such as ordinary method regulating time, temperature, pH, oxygen and volumes, to optimize expression and the recovery of heterologous protein.

Iv. bacterial system

Bacterial expression techniques is known in the art.Promoters can start in conjunction with bacterial RNA polymerase the DNA sequence dna that downstream (3 ') encoding sequence (as structure gene) is transcribed into mRNA.Promotor has a transcription initiation region, and it is usually located near 5 ' end of encoding sequence.This transcription initiation region generally includes RNA polymerase binding site and a transcription initiation site.Promoters may also have second functional area to be called operon, and it may to synthesize the RNA polymerase binding site started overlapping with the RNA adjoined.This operon allows (can induce) to the negative regulator of transcribing, because thus gene repressor protein also may suppress transcribing of specific gene in conjunction with operon.When negative regulation element (as operon) does not exist, constitutive expression may be there is.In addition, just regulating and realizing by gene activation albumen binding sequence, if any, this binding sequence is close to (5 ') of RNA polymerase binding sequence usually.The example of gene activation albumen is catabolite activator albumen (CAP), and its helps to start the transcribing of the lac operon in intestinal bacteria (E.coli) people (1984) Annu.Rev.Genet.18:173 such as [] Raibaud.Therefore, the regulation and control of expression may be positive interaction or negative interaction, thus strengthen or weaken and transcribe.

The sequence of the enzyme in encoding metabolic pathway provides useful especially promoter sequence.Example comprises the promoter sequence derived from sugar (as semi-lactosi, lactose (lac) people (1977) Nature 198:1056 such as [] Chang and maltose) metabolic enzyme.Other example comprises derived from biosynthetic enzyme (as tryptophane (trp)) [people (1980) the Nuc.Acids Res.8:4057 such as Goeddel; The people such as Yelverton (1981) Nucl.Acids Res.9:731; United States Patent (USP) 4,738,921; EP-A-0036776 and EP-A-0121775] promoter sequence.β-lactamase (bla) promoter systems [Weissmann (1981) " clone of Interferon, rabbit and other mistake " " Interferon, rabbit 3 " (I.Gresser edits)], λ is addicted to thalline PL [people (1981) the Nature 292:128 such as Shimatake] and T5 [United States Patent (USP) 4,689,406] promoter systems also provides useful promoter sequence.

In addition, the promotor of the synthesis of non-natural existence also can bacteria-like promotor equally work.Such as, a kind of bacterium or the transcriptional activating sequence addicted to bacteriophage promoter can link together with another kind of bacterium or addicted to the operon sequence of bacteriophage promoter, form the hybrid promoters [United States Patent (USP) 4,551,433] of synthesis.Such as, tac promotor is the trp-lac promotor of heterozygosis, and it forms [people (1983) the Gene 25:167 such as Amann by trp promotor and by the lac operon sequence that lac aporepressor regulates; The people such as de Boer, (1983) Proc.Natl.Acad.Sci.80:21].In addition, promoters can comprise non-bacterial source but can also start the naturally occurring promotor of transcribing in conjunction with bacterial RNA polymerase.Together with the promotor in naturally occurring non-bacterial source can also be coupled at compatible RNA polymerase, thus in prokaryotic cell prokaryocyte, express some gene [people (1986) J.Mol.Biol.189:113 such as Studier at a high level; The people such as Tabor (1985) Proc.Natl.Acad.Sci.82:1074].In addition, the promotor of heterozygosis also can form (EP-A-0267851) by addicted to bacteriophage promoter and E. coli operator region.

Except the promoter sequence having function, effective ribosome bind site is also useful for the expression of alien gene in prokaryotic cell prokaryocyte.In intestinal bacteria, ribosome bind site is called Shine-Dalgarno (SD) sequence, and it comprises initiator codon (ATG) and is the sequence [people (1975) the Nature 254:34 such as Shine] of 3-9 Nucleotide in the length at upstream from start codon 3-11 Nucleotide place.Think that SD sequence is [people (1979) " Genetic signals in messenger RNA(mRNA) and the nucleotide sequence " biology such as Steitz regulates and grows: genetic expression " (editor R.F.Goldberger)] that between 3 ' end by SD sequence and intestinal bacteria 16SrRNA, base pairing promotes mRNA to be combined with rrna.In order to express the prokaryotic gene and eukaryotic gene [people (1989) " expression of clone gene in intestinal bacteria " " Molecular Cloning: A Laboratory handbook " such as Sambrook] with weak ribosome bind site.

DNA molecular can at intracellular expression.Promoter sequence can directly be connected with DNA molecular, and in this case, first amino acid of N end is methionine(Met) all the time, and it is encoded by ATG initiator codon.If need, by with cyanogen bromide extracorporeal culture or by and bacterial methionine N-peptide ending enzyme body in or extracorporeal culture, by N hold methionine(Met) cut (EP-A-0 219 237) from protein.

Fusion rotein provides another kind of method for directly expressing.Usually, the DNA sequence dna of N end portion of encoding endogenous bacterioprotein or other stable albumen and 5 ' of allogeneic coding sequence are held merge.When expressing, this construction will provide the fusions of these two aminoacid sequences.Such as, lambda particles phage cytogene can be connected with 5 ' end of foreign gene and express in bacterium.Gained fusion rotein should retain enzyme (factor Xa) Processing position, phage protein and foreign gene to be cut [people (1984) the Nature 309:810 such as Nagai].Fusion rotein also can use lacZ [people (1987) the Gene 60:197 such as Jia], trpE [people (1987) J.Biotechnol.5:93 such as Allen; The people such as Makoff (1989), J.Gen.Microbiol.135:11] and the sequence composition of Chey [EP-A-0324 647] gene.The DNA sequence dna of two aminoacid sequence junctions can be encoded or not encode a site that can cut.Another example is ubiquitin fusion rotein.This fusion rotein is made up of ubiquitin region, and this region should retain an enzyme (such as ubiquitin specific processing protease) Processing position, foreign protein and ubiquitin to be cut.By this method, can be separated and obtain natural foreign protein people (1989) Bio/Technology 7:698 such as [] Miller.

In addition, also by producing chimeric DNA molecular, heterologous protein secretion is gone out cell, the fusion rotein of this chimeric DNA molecule encode contains a signal peptide sequence fragment, and this sequence fragment can make the heterologous protein secretion out [United States Patent (USP) 4 in bacterium, 336,336].Signal sequence fragment is encoded a signal peptide usually, and this signal peptide contains hydrophobic amino acid, and protein excretion can be guided to go out cell.Protein is secreted in the periplasmic space in growth medium (gram positive organism) or between intercellular membrane and adventitia (gram-negative bacteria).Should have between the signal peptide fragment and foreign gene of coding can in vivo or the Processing position of In vitro digestion.

The DNA of encoding suitable signal sequence can obtain from the gene of secretion property bacterioprotein is derivative, and these genes are such as escherichia coli outer membrane protein gene (ompA) [people (1983) such as Masui, " experimental implementation of genetic expression "; The people such as Ghrayeb (1984) EMBO is J.3:2437] and escherichia coli alkaline phosphatase signal sequence (phoA) [people (1985) Proc.Natl.Acad.Sci.82:7212 such as Oka].Another example is, the signal sequence of the α-amylase gene of various Bacillus strain can be adopted secrete heterologous proteins to be gone out subtilis [people (1982) the Proc.Natl.Acad.Sci.USA 79:5582 such as Palva; EP-A-0244 042].

Usually, the transcription termination sequence that bacterium identifies is the control region being positioned at translation stop codon 3 ', it together with promotor side joint in the both sides of encoding sequence.These sequence-directed mRNA transcribe, and mRNA can be translated into the polypeptide coded by this DNA.Transcription termination sequence generally includes the DNA sequence dna of about 50 Nucleotide, and this sequence can form the loop-stem structure helping to stop transcribing.Example comprises the transcription termination sequence derived from the gene with strong promoter (as the trp gene in intestinal bacteria and other biosynthetic gene).

Said modules, comprises promotor, signal sequence (if needs), interested encoding sequence and transcription termination sequence and to be usually placed in together in expression constructs.Expression constructs maintains with the form of replicon usually, such as can at the extra-chromosomal element (as plasmid) of stable maintenance in host (as bacterium).Replicon has a dubbing system, thus allows it to maintain in prokaryotic hosts or carry out expressing or carry out to clone and increase.In addition, replicon can be the plasmid of high copy number or low copy number.The copy number of high copy number plasmid roughly between about 5 to 200, usually between about 10 to 150.Host containing high copy number plasmid should be contained at least about 10 plasmids, and better contains at least about 20 plasmids.According to carrier and foreign protein on the impact of host, the carrier of high copy number or low copy number can be selected.

In addition, expression constructs can be integrated in bacterial genomes together with an integrative vector.Integrative vector usually containing at least one sequence and bacterial chromosome homology, thus allows this vector integration.Integrate and it seems it is the result of recombinating between homologous dna in carrier and bacterial chromosome.Such as, (EP-A-0127 328) in Bacillus chromosome is incorporated into the integrative vector that the DNA of different Bacillus strain builds.Integrative vector also can comprise phage or transposon sequence

Usually, the expression constructs of the outer construction of karyomit(e) and integration can contain selectable mark, to select the bacterial strain transformed.Selectable markers can be expressed in host bacterium, and it comprises gives bacterium to the gene of medicine (as penbritin, paraxin, erythromycin, kantlex (Liu Suanyan NEOMYCIN SULPHATE) and tsiklomitsin) resistance people (1978) Annu.Rev.Microbiol.32:469 such as [] Davies.Selectable markers also can comprise biosynthesizing gene, as those genes in Histidine, tryptophane and leucine biosynthetic pathway.

In addition, some assembly above-mentioned can be placed in conversion carrier together.Conversion carrier comprises a selectable markers usually, and as mentioned above, this carrier maintains with replicon form or develops into an integrative vector.

Have developed for being transformed into expression in many bacteriums and conversion carrier (no matter being extrachromosomal replication or integrative vector).Such as, have developed the expression vector for following bacterium: subtilis [people such as Palva, (1982) Proc.Natl.Acad.Sci.USA 79:5582; EP-A-0036 259 and 063 953; PCT publication WO 84/04541], intestinal bacteria [people such as Shimatake, (1981) Nature 292:128; The people such as Amann, (1985) Gene 40:183; The people such as Studier, (1986) J.Mol.Biol.189:113; EP-A-0036 776,136 829 and 136907], junket suis [people such as Powell, (1988) Appl.Environ.Microbiol.54:655]; Shallow livid purple suis [people such as Powell, (1988) Appl.Environ.Microbiol.54:655], shallow Streptomyces glaucoviolaceus [US patent4,745,056].

Be well known in the art by the method for Exogenous DNA transfered host bacterium, generally include and with calcium chloride or other reagent (as divalent cation and DMSO) process, bacterium is transformed.DNA also imports bacterial cell by electroporation method.Transformation Program is usually different because of bacterial species to be transformed.[see, such as use bacillus: the people such as Masson, (1989) FEMS Microbiol.Lett.60:273; The people such as Palva, (1982) Proc.Natl.Acad.Sci.USA79:5582; EP-A-0036 259 and 063 953; WO 84/04541; Use Campylobacter: the people such as Miller, (1988) Proc.Natl.Acad.Sci.85:856; With people such as Wang, (1990) J.Bacteriol.172:949; Use dust Xi Shi intestinal bacteria: the people such as Cohen, (1973) Proc.Natl.Acad.Sci.69:2110; The people such as Dower, (1988) NucleicAcids Res.16:6127; Kushner (1978) " with improving one's methods of ColE1-plasmid transformation of E. coli " Genetic Engineering:Proceedings of the International Symposium on Genetic Engineering (H.W.Boyer and S.Nicosia edits); The people such as Mandel, (1970) J.Mol.Biol.53:159; Taketo (1988) Biochim.Biophys.Acta 949:318; Use lactobacillus: the people such as Chassy, (1987) FEMS Microbiol.Lett.44:173; Use pseudomonas: the people such as Fiedler, (1988) Anal.Biochem 170:38; Use staphylococcus: the people such as Augustin, (1990) FEMS Microbiol.Lett.66:203; Usage chain coccus: the people such as Barany, (1980) J.Bacteriol.144:698; Harlander (1987) " with Electroporation Transformation suis lactics " Streptococcal Genetics (J.Ferretti and R.Curtiss III edits); The people such as Perry, (1981) Infect.Immun.32:1295; The people such as Powell, (1988) Appl.Environ.Microbiol.54:655; The people such as Somkuti, (1987) Proc.4th Evr.Cong.Biotechnology 1:412].

V. yeast expression

Yeast expression system is also known to those skilled in the art.Yeast promoter is energy combining yeast RNA polymerase and starts the DNA sequence dna that downstream (3 ') encoding sequence (as structure gene) is transcribed into mRNA.Promotor has a transcription initiation region, it be usually located at encoding sequence 5 ' end near.This transcription initiation region generally includes RNA polymerase binding site (" TATA " box) and a transcription initiation site.Yeast promoter may also have second functional area to be called upstream activating sequence (UAS), and if present, it is usually away from structure gene.UAS can regulate expression (can induce).When UAS does not exist, there is constitutive expression.The regulation and control expressed may be positive interaction or negative interaction, thus strengthen or weaken and transcribe.

Yeast is a kind of fermenting organisms, has active pathways metabolism, and the sequence of the enzyme therefore in encoding metabolic pathway provides useful especially promoter sequence.Example comprises alcoholdehydrogenase (ADH) (EP-A-0284 044), enolase, glucokinase, GPI, GAPDH (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phoshoglyceric acid mutase and pyruvate kinase (PyK) (EP-A-0329 203).The yeast PHO5 gene of encoding acid phosphatase also provides useful promoter sequence [people (1983) the Proc.Natl.Acad.Sci.USA 80:1 such as Myanohara].

In addition, the promotor of the synthesis of non-natural existence also can work as Yeast promoter.Such as, a kind of UAS sequence of Yeast promoter can link together with the transcriptional activation domain of another kind of Yeast promoter, forms the hybrid promoter of synthesis.The example of this hybrid promoter comprises the ADH regulating and controlling sequence (U.S. Patent No. 4,876,197 and 4,880,734) be connected with GAP transcriptional activation domain.Other example of hybrid promoter comprises the promotor and glycolytic enzyme gene that are made up of the regulating and controlling sequence of ADH2, GAL4, GAL10 or PHO5 gene as the combination (EP-A-0164 556) of the transcriptional activation domain of GAP or PyK.In addition, Yeast promoter can comprise non-yeast sources but combining yeast RNA polymerase can also start the naturally occurring promotor of transcribing.The example of these promotors comprises, especially, and [people such as Cohen, (1980) Proc.Natl.Acad.Sci.USA 77:1078; The people such as Henikoff, (1981) Nature 283:835; The people such as Hollenberg, (1981) Curr.Topics Microbiol.Immunol.96:119; The people such as Hollenberg, (1979) " expression of bacterial antibiotic resistance gene in yeast saccharomyces cerevisiae " Plasmids of Medical, Environmental andCommercial Importance (K.N.Timmis and A.Puhler edits); The people such as Mercerau-Puigalon, (1980) Gene 11:163; The people such as Panthier, (1980) Curr.Genet.2:109].

DNA molecular can at yeast intracellular expression.Promoter sequence can directly be connected with DNA molecular, and in this case, first amino acid of recombinant protein N end is methionine(Met) all the time, and it is encoded by ATG initiator codon.If needed, by the methionine(Met) that N holds being cut from protein with cyanogen bromide extracorporeal culture.

As in Mammals, plant, baculovirus and bacterial expression system, fusion rotein is that yeast expression system provides another kind of method.Usually, the DNA sequence dna of N end portion of encoding endogenous Yeast protein or other stable albumen and 5 ' of allogeneic coding sequence are held merge.When expressing, this construction will provide the fusions of these two aminoacid sequences.Such as, yeast or human superoxide dismutase (SOD) gene can be connected with foreign gene 5 ' end and express in yeast.The DNA sequence dna of two aminoacid sequence junctions can be encoded or not encode the site that can cut.For example, see EP-A-0196 056.Another example is ubiquitin fusion rotein.This fusion rotein is made up of ubiquitin region, and this region should retain an enzyme (such as ubiquitin specific processing protease) Processing position, foreign protein and ubiquitin to be cut.Therefore, by this method, the natural foreign protein (such as WO88/024066) of acquisition can be separated.

In addition, also by producing chimeric DNA molecular by during foreign protein is from emiocytosis to growth medium, the fusion rotein of this chimeric DNA molecule encode contains a leader fragment, and this leader fragment can make heterologous protein secretion in yeast out.Preferably, should have between the leader fragment and alien gene of coding can in vivo or the Processing position of In vitro digestion.This leader fragment encodes the signal peptide containing hydrophobic amino acid usually, and its pilot protein from emiocytosis out.

The DNA of encoding suitable signal sequence can obtain from the gene of secretion property Yeast protein is derivative, and these genes such as have yeast invertase gene (EP-A-0012 873; JPO 62:096,086) and A-factor gene (United States Patent (USP) 4,588,684).In addition, the existence of the leader sequence (as Interferon, rabbit leader sequence) of non-yeast sources also can provide the effect of secreting yeast (EP-A-0060 057).

Preferably a class secretion leader sequence have employed the fragment of yeast alpha-factor gene, and it contains " pre " signal sequence and " pro " district.The type of adoptable alpha factor fragment comprises α-factor leaders (usually about 25 to 50 amino-acid residues) (United States Patent (USP) 4 of total length pre-pro alpha factor leader sequence (about 83 amino-acid residues) and brachymemma, 546,083 and 4,870,008; EP-A-0324 274).Adopt α-factor leader fragment to provide other leader sequence secretory to comprise the α-factor leaders of heterozygosis, it is made up of (such as seeing WO 89/02463) the pre sequence of first yeast and the pro region of second yeast alpha factor.

Usually, the transcription termination sequence of yeast identification is the control region being positioned at translation stop codon 3 ', its together with promotor side joint in the both sides of encoding sequence.These sequence-directed mRNA transcribe, and mRNA can be translated into the polypeptide coded by this DNA.The example of the terminator sequence of transcription termination sequence and other yeast identification is those transcription termination sequences of encoding glycolytic enzymes.

Said modules, comprises promotor, leader sequence (if needs), interested encoding sequence and transcription termination sequence, is usually placed in expression constructs together.Expression constructs keeps with the form of replicon usually, such as, can stablize the extra-chromosomal element (as plasmid) kept in host (as yeast or bacterium).Replicon may have two dubbing systems, thus allows it can maintain in such as yeast to express, and can maintain prokaryotic hosts and carry out cloning and increasing.The example of these yeast-bacteria shuttle carriers comprises YEp24 [people (1979) the Gene 8:17-24 such as Botstein], pCL/1 [people such as Brake, (1984) Proc.Natl.Acad.Sci.USA 81:4642-4646] and YRp17 [people (1982) J.Mol.Biol.158:157 such as Stinchcomb].In addition, replicon can be the plasmid of high copy number or low copy number.The copy number of high copy number plasmid roughly between about 5 to 200, usually between about 10 to 150.Host containing high copy number plasmid should be contained at least about 10 plasmids, and better contains at least about 20 plasmids.According to carrier and foreign protein on the impact of host, the carrier of high copy number or low copy number can be selected.For example, see people such as Brake, the same.

In addition, expression constructs can be integrated in Yeast genome together with an integrative vector.Integrative vector usually containing at least one sequence and yeast chromosomal homology, thus allows this vector integration, preferably containing two these expression constructs of homologous sequence side joint.Integrate and it seems it is result people (1983) Methods in Enzymol.101:228-245 such as [] Orr-Weaver of recombinating between homologous dna in carrier and yeast chromosomal.By selecting in suitable homologous sequence insertion vector, integrative vector can be imported a certain specific locus in yeast.See the people such as Orr-Weaver, the same.Can be integrated into one or more expression constructs, this may affect level people (1983) Proc.Natl.Acad.Sci.USA 80:6750 such as [] Rine that recombinant protein produces.Individual chip form in carrier can there is (thus causing the integration of whole carrier) in the chromosome sequence in carrier, or with two fragments of the adjacent segment homology in karyomit(e), these two fragments in the carrier side joint, in expression constructs both sides, thus can cause the stability of only expression constructs to be integrated.

Usually, the expression of the outer construction of karyomit(e) and integration can build thing all containing selectable mark, to select the yeast strain transformed.Selectable markers can comprise the biosynthesis gene (as ADE2, HIS4, LEU2, TRP1 and ALG7 and G418 resistant gene) can expressed in yeast host, and these genes give the resistance of yeast cell to tunicamycin and G418 respectively.In addition, suitable selectable markers also may provide energy for growth for yeast under toxic chemical (as metal) exists.Such as, the existence of CUP1 makes yeast can growth under cupric ion exists people such as [, (1987) Microbiol, Rev.51:351] Butt.

Have developed for being transformed into expression in many yeast and conversion carrier (no matter being extrachromosomal replication or integrative vector).Such as, develop for following saccharomycetic expression vector with by the method for Exogenous DNA transfered yeast host: Candida albicans [Kurtz, Deng people, (1986) Mol.Cell.Biol.6:142], [Kunze, waits people to maltose candidiasis, (1985) J.Basic Microbiol.25:141], [Gleeson, waits people to multiple-shaped nuohan inferior yeast, (1986) J.Gen.Microbiol.132:3459; The people such as Roggenkamp, (1986) Mol.Gen.Genet.202:302], Kluyveromyces fragilis [Das, Deng people, (1984) J.Bacteriol.158:1165], Kluyveromyces lactis [people such as DeLouvencourt, (1983) J.Bacteriol.154:737; The people such as Van den Berg, (1990) Bio/Technology 8:135], Pichia guilliermondii [the people such as Kunze, (1985) J.Basic Microbiol.25:141], pichia pastoris phaff [Cregg, Deng people, (1985) Mol.Cell.Biol.5:3376; U.S. Patent No. 4,837,148 and 4,929,555], yeast saccharomyces cerevisiae [people such as Hinnen, (1978) Proc.Natl.Acad.Sci.USA 75:1929; The people such as Ito, (1983) J.Bacteriol.153:163], chestnut wine splits plants yeast [Beach and Nurse (1981) Nature300:706], and Yarrowia lipolytica [Davidow, Deng people, (1985) Curr.Genet.10:380471Gaillardin, waits people, (1985) Curr.Genet.10:49].

Be well known in the art by the method for Exogenous DNA transfered yeast host, generally include and transform spheroplast or Whole yeast cells with base cations process.Transformation Program is usually different because of yeast specie to be transformed.For example, see, [people such as Kurtz, (1986) Mol.Cell.Biol.6:142; The people such as Kunze, (1985) J.Basic Microbiol.25:141; Candidiasis]; [people such as Gleeson, (1986) J.Gen.Microbiol.132:3459; The people such as Roggenkamp, (1986) Mol.Gen.Genet.202:302; Debaryomyces hansenii]; [people such as Das, (1984) J.Bacteriol.158:1165; The people such as DeLouvencourt, (1983) J.Bacteriol.154:1165; The people such as Van den Berg, (1990) Bio/Technology 8:135; Kluyveromyces]; [people such as Cregg, (1985) Mol.Cell.Biol.5:3376; The people such as Kunze, (1985) J.Basic Microbiol.25:141; U.S. Patent No. 4,837,148 and 4,929,555; Pichia spp]; [people such as Hinnen, (1978) Proc.Natl.Acad.Sci.USA 75; 1929; The people such as Ito, (1983) J.Bacteriol.153:163 yeast saccharomyces cerevisiae]; [Beach and Nurse (1981) Nature 300:706; Fission yeast]; [people such as Davidow, (1985) Curr.Genet.10:39; The people such as Gaillardin, (1985) Curr.Genet.10:49; Yarrowia].

Antibody

Term used herein " antibody " refers to one or one group of polypeptide being made up of at least one antibody combining site." antibody combining site " is a three-dimensional binding space, the feature complementary of its inner surface configuration and charge distribution and epitope, thus antibody is combined with antigen." antibody " such as comprises, vertebrate antibodies, hybrid antibody, chimeric antibody, humanized antibody, modified antibody, univalent antibody, Fab albumen and single domain antibody.

Antibody for albumen of the present invention can be used for affinity chromatography, immunity test and difference/qualification Neisserial protein.

Obtain for the polyclone of albumen of the present invention and monoclonal antibody using conventional procedures.Usually, first immune suitable animal, preferably mouse, rat, rabbit or goat is come with albumen.Because obtainable serum volume is many, can obtain the anti-rabbit of mark and anti-goat antibody, therefore for preparing polyclonal antiserum, rabbit and goat are preferably.Immunity is carried out usually like this: mixed or emulsification with salt solution (preferably with adjuvant as FreundShi Freund's complete adjuvant) by albumen, then parenteral (normally subcutaneous or intramuscular) injects this mixture or emulsion.The dosage of per injection 50-200 microgram is just enough.Use protein injection one or many that salt solution (preferably using FreundShi Freund's incomplete adjuvant) is joined with reinforced immunological in 2-6 week afterwards.Ion vitro immunization can be carried out by methods known in the art in addition and produce antibody, from object of the present invention, think immunity equivalence in itself and body.Animal blood after immunity is drawn in glass or plastic containers, cultivates this blood 1 hour for 25 DEG C, then cultivate 2-18 hour for 4 DEG C, obtain polyclonal antiserum.Centrifugal (such as 1000g 10 minutes) reclaims serum.Rabbit gets blood at every turn can obtain about 20-50 milliliter.

By standard method [Nature (1975) 256:495-96] or its obtained monoclonal antibody of improving one's methods of Kohler and Milstein.Usually, as mentioned above to mouse or rat immunity.But, be not that blood then extracting serum is got to animal, but take out spleen (and optionally taking out several large lymphoglandula), be isolated into unicellular.If needed, cell suspension (after the cell of removing non-specific adhesion) can be added bag by the plate of proteantigen or hole, splenocyte is screened.The B cell of the specific membrane bound immunoglobulin of antigen expressed is attached on plate, does not resemble other material of suspension and is washed away.Then make gained B cell or all splenocytes of dissociating and myeloma cell fusion form hybridoma, cultivate in selective medium (as xanthoglobulin, aminopterin, thymidine medium, " HAT ").By limiting dilution inoculation gained hybridoma, and measure the generation of the antibody of specific binding immunizing antigen (and not in conjunction with irrelevant antigen).Then, in external (such as in tissue culture flasks or hollow fiber reactor) or body (as in mouse ascites) cultivate selected by the hybridoma of secrete monoclonal antibody.

If needed, antibody (no matter being polyclone or monoclonal antibody) can mark with routine techniques.Suitable mark comprises fluorophore, chromophoric group, radioactive atoms (specifically ³²p and ¹²⁵i), close electrophile, enzyme and there is the part of Specific binding partner.Enzyme detects by its activity usually.Such as, horseradish peroxidase normally detects it by 3,3 ', and 5,5 '-tetramethyl benzidine (TMB) is transformed into blue ability, available spectrophotometer quantitative assay." Specific binding partner " refers to can with the protein of high specific binding partner molecule, such as antigen and have specific monoclonal antibody to it.Other Specific binding partner comprises vitamin H and avidin or streptavidin, IgG and albumin A, and many receptor-ligands pair known in the art.Same mark should be understood that foregoing does not really want various mark to be divided into different classes, because can work in several different model.Such as, ¹²⁵i can be used as radioactivity mark, or as close electrophile.HRP can be used as the antigen of enzyme or monoclonal antibody.In addition, a kind of material can with various marker combination to obtain required effect.Such as, in the embodiment of this invention, monoclonal antibody and avidin also need mark, therefore, can use biotin labeling monoclonal antibody, and with marked ¹²⁵the avidin of I detects it to be existed, or detects its existence with the antibiotin monoclonal antibody of mark HRP.Other is replaced and possibility is apparent to those skilled in the art, so should take that Equivalent belongs to scope of the present invention as.

Pharmaceutical composition

Pharmaceutical composition can comprise polypeptide of the present invention, antibody or nucleic acid.This pharmaceutical composition will comprise the treatment polypeptide of the present invention of significant quantity, antibody or polynucleotide.

Term used herein " treatment significant quantity " refers to therapeutic agent treats, alleviation or prevents the amount of target disease or situation, or shows the amount of detectable treatment or preventive effect.This effect such as detects by chemical labeling or antigen levels.Result for the treatment of also comprises the minimizing of physical symptoms, such as, cause body temperature to reduce.Accurate significant quantity for a certain object depend on the build of this object and healthy state, illness nature and extent and select the combination of therapeutical agent and/or the therapeutical agent given.Therefore, significant quantity is accurately specified to be useless in advance.But for the situation that certain is given, can determine this significant quantity with normal experiment, clinicist can judge.

For the purposes of the present invention, effective dosage is the DNA construction giving individual about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to 10 mg/kg.

Pharmaceutical composition also can contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to the carrier being used for the treatment of agent (such as antibody, polypeptide, gene or other therapeutical agent) administration.This term refers to some medicament carriers like this: they itself are not induced and produce the antibody harmful to the individuality accepting said composition, and do not have undue toxicity after administration.Suitable carrier may be large, metabolism macromole slowly, as the virion of protein, polysaccharide, poly(lactic acid) (polylactic acid), polyglycolic acid, aminoacid polymers, amino acid copolymer and non-activity.These carriers are well known to those of ordinary skill in the art.

Available pharmacy acceptable salt such as has herein: inorganic acid salt, example hydrochloric acid salt, hydrobromate, phosphoric acid salt, vitriol etc.; And organic acid salt, as acetate, propionic salt, malonate, benzoate etc.Discussing fully about pharmaceutically acceptable vehicle can be found in Remington ' sPharmaceutical Sciences (Mack Pub.Co., N.J.1991).

In therapeutic composition Chinese materia medica, acceptable carrier can contain liquid, as water, salt solution, glycerine and ethanol.In addition, in these carriers, also may there is complementary material, as wetting agent or emulsifying agent, pH buffer substance etc.Usually, therapeutic composition can be made injectable agent, such as liquor or suspension; Also can be made into be applicable to allocating in solution or suspension before the injection, the solid form of liquid vehicle.Liposome is also included within the definition of pharmaceutically acceptable carrier.

Delivery methods

Once be made into composition of the present invention, directly object can be given by it.Object to be treated can be animal; Especially people's object can be treated.

Direct conveying said composition is usually by subcutaneous, intraperitoneal, intravenously or intramuscularly or be delivered to tissue space to realize.Composition also can be delivered to focal zone.Other administering mode comprises oral with lung administration, suppository and transdermal or through dermal administration (see such as WO98/20734), use pin, particle gun or manual sprayer (hypospray).Therapeutic dose scheme can be single dose regimen or multiple doses.

Vaccine

Vaccine of the present invention can be preventative (i.e. preventing infection) or curative (namely after infection disease therapy).

These vaccines comprise immunising antigen or immunogen, immunogenic polypeptide, albumen or protein fragments or nucleic acid, usually combine with " pharmaceutically acceptable carrier ", these carriers comprise any carrier of itself not inducing and producing the antibody that the individuality accepting said composition is harmful to.Suitable carrier normally large, metabolism macromole slowly, as the virion of protein, polysaccharide, poly(lactic acid), polyglycolic acid, aminoacid polymers, amino acid copolymer, lipid aggregates (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 with bacterial toxoid (toxoid as pathogenic agent such as diphtheria, tetanus, cholera, helicobacter pyloris) coupling.

The preferably adjuvant of enhancing composition effect including, but not limited to (1) aluminium salt (alum), as aluminium hydroxide, aluminum phosphate, Tai-Ace S 150 etc., (2) oil-in-water emulsion formula (being with or without other specific immunostimulant, as muramylpeptides (seeing below) or bacterial cell wall component), such as, (a) MF59 (WO 90/14837, 10th chapter vaccine design: subunit and adjuvant approach, Powell & Newman compiles, Plenum Press, 1995), it contains 5% MF59,0.5% tween 80 and 0.5%Span 85 (MTP-PE (seeing below) optionally containing different amount, although do not need), with micro-Fluidizer (as 110Y type trace Fluidizer (Microfluidics, Newton, MA)) make submicron particles, (b) SAF, it contains 10% MF59, 0.4% tween 80, 5% Pluronic (pluronic) block polymer L121 and thr-MDP (seeing below), trace stream changes into submicron order emulsion or eddy oscillating produces the larger emulsion of particle diameter, (c) RibiTM adjuvant system (RAS) (Ribi Immunochem, Hamilton, MT), it contains 2% MF59, 0.2% tween 80 and take from Monophosphoryl lipid A (MPL), two mycolic acid marine alga sugar esters (TDM), with one or more bacterial cell wall components of cell wall skeleton (CWS), preferably MPL+CWS (Detox ^tM), (3) saponin adjuvant, such as, can adopt Stimulon ^tM(Cambridge Bioscience, Worcester, MA) or the particle produced from it, as ISCOM (immunostimulating mixture), (4) Freund Freund's complete adjuvant (CFA) and Freund Freund's incomplete adjuvant (IFA), (5) cytokine, as interleukin (as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 etc.), Interferon, rabbit (as IFN-γ), macrophage colony stimulating factor (M-CFS), tumour necrosis factor (TNF) etc., and (6) carry out other material of enhancing composition effect as immunostimulant.Alum and MF59 is preferably.

As mentioned above; muramylpeptides including, but not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-remove the different glutaminase acyl group-ALANINE-2-of muramyl-L-alanyl-D-isogluatme (nor-MDP), N-acetylmuramyl-L alanyl-D-(1 '-2 '-two palmityl-sn-glycerine-3-hydroxyl phosphinylidyne oxygen)-ethamine (MTP-PE) etc.

Immunogenic composition (as the antigen/immunogen/polypeptide/albumen/nucleic acid for immunity, pharmaceutically acceptable carrier and adjuvant), usually containing thinner, as water, salt solution, glycerine, ethanol etc.In addition, auxiliary substances, as wetting agent or emulsifying agent, pH buffer substance etc. can be present in this kind of vehicle.

Usually, immunogenic composition can be made injectable agent, such as liquor or suspension; Also can be made into the solid form being applicable to allocating into solution or suspension, liquid excipient before the injection.Said preparation also can emulsification or be encapsulated in liposome, under above-mentioned pharmaceutically acceptable carrier, strengthen adjuvant effect.

As the immunogenic composition of vaccine, comprise the antigenicity of immunological effective amount or immunogenic polypeptide and above-mentioned component needed for other." immunological effective amount " refers to that it is effective for giving individual amount to treatment or prevention with single dose or a continuous agent part.This consumption according to treat that the classification (as non-human primates etc.) of individuality is treated by individual healthy state and physiological situation, institute, the ability of individual immunity system synthesis antibody, required degree of protection, the preparation of vaccine, treating physician determine the assessment of medical conditions and other correlative factor.Estimate that this consumption is by relatively wide scope, determines by normal experiment.

Ordinary method gives immunogenic composition from parenteral route by injection, such as subcutaneous, intramuscular or transdermal administration (such as WO98/20734).Other formula being applicable to other administering mode comprises oral and lung preparation, suppository and transdermal application.Therapeutic dose can be single dose regimen or multiple doses.Vaccine can give together in conjunction with other immunomodulator.

A kind of replacement scheme as the vaccine based on protein is, DNA vaccination can be adopted to inoculate [such as, Robinson and Torres (1997) Seminars in Immunology 9:271-283; The people such as Donnelly (1997) AnnuRev.Immunol 15:617-648; See below].

Gene delivery carrier

For carrying the gene therapy vector of construction oral or general to give, wherein said construction comprises the encoding sequence of therapeutical agent of the present invention, is delivered to Mammals so that at Mammals expression in vivo.These constructions can to utilize in body or virus in vitro in mode or non-virus carrier method.The available endogenous mammalian promoter of expression of these encoding sequences or allogeneic promoter induction.The expression in vivo of encoding sequence can be composing type or modulated.

The present invention includes the gene delivery vehicles of the nucleotide sequence involved by expressing.Gene delivery vehicles is preferably virus vector, more preferably retrovirus, adenovirus, adeno associated virus (AAV), simplexvirus or alphavirus vectors.Virus vector can also be the virus vector of Astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus or togavirus.Usually see Jolly (1994) Cancer GeneTherapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) HumanGene Therapy 6:185-193; And Kaplitt (1994) Nature Genetics 6:148-153.

Retroviral vector is well known in the art and we think that any retroviral gene therapy carrier all can be used for the present invention, and they comprise B, C and D type retrovirus, (the wide preferendum retrovirus of such as NZB-X1, NZB-X2 and NZB9-1 (see O ' Neill (1985) J.Virol.53:160) is as MCF and MCF-MLV (see Kelly (1983) J.Virol 45:291), foamy virus and slow virus for xenotropic retrovirus.See " RNA tumour virus " the 2nd edition, Cold Spring Harbor Laboratory, 1985.

All parts of retroviral gene therapy carrier can obtain from different retrovirus is derivative.Such as, Retroviral Vector LTR can obtain from mouse sarcoma virus is derivative, and tRNA binding site can obtain from Rous sarcoma virus is derivative, and packaging signal obtains from mouse leukaemia virus, and the synthesis starting point of the second chain obtains from avian leukosis viruses.

These recombinant retroviral vectors can be imported suitable package cell line, be used for producing transduction competent state retroviral vector particle (see United States Patent (USP) 5,591,624).By chimeric integrase enzyme is mixed retroviral particle, build retroviral vector, so that by its site-directed integration to (see WO96/37626) in host cell DNA.Preferably recombinant viral vector is replication defect type recombinant virus.

The package cell line being applicable to using together with above-mentioned retroviral vector is well known in the art, be easy to obtain (see WO95/30763 and WO92/05266), and can be used for producing can the type of production clone (also referred to as carrier cell system or " VCL ") of Restruction carrier granule.Package cell line should be produced, to eliminate the deactivation of human serum from people's parental cell (as HT1080 cell) or ermine parental cell system.

Be used for building the preferably retrovirus of retroviral gene therapy carrier, comprise avian leukosis viruses, bovine leukemia virus, murine leukemia virus, Mink-Cell stove elicit virus, murine sarcoma virus, reticuloendotheliosis virus and Rous sarcoma virus.Murine leukemia virus good especially comprises 4070A and 1504A (Hartley and Rowe (1976) J Virol 19:19-25), Abelson (ATCC No.VR-999), Friend (ATCC No.VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey sarcoma virus and Rauscher (ATCC No.VR-998) and Moloney murine leukemia virus (ATCC No.VR-190).These retrovirus can obtain from preservation mechanism or preservation center such as the American type culture collection (ATCC) of Rockville, Maryland, or obtain from the separation of known source by conventional technology.

Typical known retroviral gene therapy carrier used in the present invention is included in those carriers described in following patent application: GB2200651, EP0415731, EP0345242, EP0334301, WO89/02468; WO89/05349, WO89/09271, WO90/02806, WO90/07936, WO94/03622, WO93/25698, WO93/25234, WO93/11230, WO93/10218, WO91/02805, WO91/02825, WO95/07994, US 5,219,740, US 4,405,712, US 4,861,719, US 4,980,289, US 4,777,127, US 5,591,624. is separately shown in Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Ram (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Natl Acad Sci81:6349; And Miller (1990) Human Gene Therapy 1.

Human adenoviral gene therapy's carrier is also as known in the art, and can be used for the present invention.For example, see Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and WO93/07283, WO93/06223 and WO93/07282.Those examples described in above-mentioned document and following patent are included in: WO94/12649 for typically known adenoviral gene therapy carrier of the present invention, WO93/03769, WO93/19191, WO94/28938, WO95/11984, WO95/00655, WO95/27071, WO95/29993, WO95/34671, WO96/05320, WO94/08026, WO94/11506, WO93/06223, WO94/24299, WO95/14102, WO95/24297, WO95/02697, WO94/28152, WO94/24299, WO95/09241, WO95/25807, WO95/05835, WO94/18922 and WO95/09654.In addition, giving and the method for killing the DNA that adenovirus is connected of describing in Curiel (1992) Hum.Gene Ther.3:147-154 can be adopted.Gene delivery vectors of the present invention also comprises adeno-associated virus (AAV) carrier.Mainly and preferably example for this carrier of the present invention is the carrier based on AAV-2 disclosed in Srivastava, WO93/09239.Best AAV carrier comprises two AAV inverted terminal repeats, wherein modify by replacing nucleotide pair native D-sequence, make at least 5-18 natural Nucleotide (preferably at least 10-18 natural nucleotide, 10 best natural nucleotides) be retained, and remaining nucleotide deletion of D-sequence or replace with non-natural nucleotide.The native D-sequence of AAV inverted terminal repeat is the sequence (namely there is a sequence every one end) not participating in 20 tandem nucleotides that HP is formed in each AAV inverted terminal repeat.Non-natural replacement Nucleotide can be any Nucleotide beyond Nucleotide seen in this position of native D-sequence.Other adoptable exemplary AAV vectors is pWP-19, pWN-1, and both are all disclosed in Nahreini (1993) Gene 124:257-262.Another example of such AAV carrier is psub201 (see Samulski (1987) J.Virol.61:3096).Another typical AAV carrier is Double-D ITR carrier.The constructing plan of Double-D ITR carrier is disclosed in United States Patent (USP) 5,478, in 745.The carrier also having other is disclosed in the carrier in the United States Patent (USP) 4,797,368 of Carter and the WO94/288157 of the United States Patent (USP) 5,139,941 of Muzyczka, the United States Patent (USP) 5,474,935 of Chartejee and Kotin.Another AAV Examples of carriers used in the present invention is SSV9AFABTKneo, and it contains AFP enhanser and albumin promoter, and mainly instructs Liver hydatid.Its structure and constructing plan are disclosed in Su (1996) Human Gene Therapy 7:463-470.Other AAV gene therapy vector at United States Patent (USP) 5,354,678,5,173,414,5,139,941,5,252, described by having in 479.

Gene therapy vector of the present invention also comprises herpes vectors.Mainly and preferably example is the herpes simplex virus vector of the sequence containing encoding thymidine kinase polypeptide, as being disclosed in US5,288,641 and EP0176170 (Roizman) in those.Other typical herpes simplex virus vector comprises HFEM/ICP6-LacZ disclosed in WO95/04139 (WistarInstitute), pHSVlac disclosed in Geller (1988) Science 241:1667-1669 and WO90/09441 and WO92/07945, the HSV Us3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19, the HSV 7134 described in EP0453242 (Breakefield), 2RH 105 and GAL4 and be preserved in ATCC, preserving number is those viruses of ATCC VR-977 and ATCC VR-260.

Also consider that alpha virus gene therapy carrier also can be used for the present invention.Preferably alphavirus vectors is Sindbis viral vectors.Togavirus, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532) and at United States Patent (USP) 5,091,309,5,217,879 and WO92/10578 in describe those.More particularly, the U. S. application 08/405,627 that can adopt submit to March 15 nineteen ninety-five, WO94/21792, WO92/10578, WO95/07994, US 5,091,309 and US 5,217, those alphavirus vectors described in 879.These Alphaviruses can obtain from preservation mechanism or preservation center such as the American type culture collection (ATCC) of Rockville, Maryland, or obtain from the separation of known source by conventional technology.It is preferred that adopt the alphavirus vectors (see USSN08/679640) that cytotoxicity reduces.

DNA vector system, (layered) expression system as eucaryon layering also can be used for expressing nucleic acid of the present invention.Expression system about eucaryon layering refers to WO95/07994.Preferably, eucaryon layered expression systems of the present invention should obtain from alphavirus vectors is derivative, and better obtains from Sindbis viral vectors is derivative.

Be applicable to other virus vector of the present invention comprise: from the carrier that poliovirus is derivative, such as ATCCVR-58 and describe in Evans, Nature 339 (1989) 385 and Sabin (1973) J.Biol.Standardization 1:115 those; Rhinovirus, such as ATCC VR-1110 and describe in Arnold (1990) J Cell Biochem L401 those; Poxvirus, as golden yellow poxvirus or vaccinia virus, such as ATCC VR-111 and ATCCVR-2010, and at Fisher-Hoch (1989) Proc Natl Acad Sci 86:317; Flexner (1989) Ann NYAcad Sci 569:86, Flexner (1990) Vaccine 8:17; Describe in US 4,603,112, US 4,769,330 and WO89/01973 those; SV40 virus, such as ATCC VR-305 and describe in Mulligan (1979) Nature 277:108 and Madzak (1992) J Gen Virol 73:1533 those; Influenza virus, such as ATCC VR-797 and with such as US 5,166,057 and Enami (1990) Proc Natl Acad Sci87:3802-3805; Enami and Palese (1991) J Virol 65:2711-2713; The recombinant influenza (separately seeing McMichael (1983) NEJMed 309:13, Yap (1978) Nature 273:238 and Nature (1979) 277:108) that antigene technology described in Luytjes (1989) Cell 59:110 is obtained; The human immunodeficiency virus described in EP-0386882 and Buchschacher (1992) J.Virol.66:2731; Measles virus, such as ATCC VR-67 and VR-1247, and those description in EP-0440219; Aura virus, such as ATCC VR-368; Bebaru virus, such as ATCC VR-600 and ATCC VR-1240; Cabassou virus, such as ATCC VR-922; Chikungunya virus, such as ATCC VR-64 and ATCC VR-1241; Fort Morgan virus, such as ATCC VR-924; Getah virus, such as ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, such as ATCC VR-927; Mayaro virus, such as ATCC VR-66; Mucambo virus, such as ATCC VR-580 and ATCC VR-1244; Ndumu virus, such as ATCC VR-371; Pixuna virus, such as ATCC VR-372 and ATCC VR-1245; Tonate virus, such as ATCC VR-925; Triniti virus, such as ATCC VR-469; Una virus, such as ATCC VR-374; Whataroa virus, such as ATCC VR-926; Y-62-33 virus, such as ATCC VR-375; O ' Nyong virus, east encephalitis, such as ATCC VR-65 and ATCC VR-1242; WE virus, such as ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; And coronavirus, such as ATCC VR-740 and describe in Hamre (1966) Proc Soc Exp Biol Med 121:190 those.

Above-mentioned virus vector is not limited to by delivery of composition of the present invention to cell.Also can adopt other carrying method and medium, such as nucleic acid expression vector, with the DNA that killed adenovirus is connected or disjunct independent polycation is condensing (for example, see on December 30th, 1994 U. S. application No.08/366, 787 and Curiel (1992) HumGene Ther 3:147-154), the DNA (for example, see Wu (1989) J.Biol.Chem.264:16985-16987) that part connects, eukaryotic cell delivery vehicles cell is (for example, see the U. S. application No.08/240 that on May 9th, 1994 submits to, 030 and U. S. application No.08/404, 796), the precipitation of photopolymerized hydrogel material, hand-held gene transfer particle rifle is (as United States Patent (USP) 5, 149, described in 655), ionizing rays is (as US5, 206, 152 and WO92/11033 described in), nuclear charge neutralization or and cell membrane fusion.Described by other method has in Philip (1994) Mol Cell Biol 14:2411-2418 and Woffendin (1994) Proc.Natl.Acad.Sci.91:1581-1585.

The transgenosis that particle mediates can be adopted, for example, see U. S. application No.60/023,867.In brief, can sequence be inserted in the conventional carrier containing the routine sequence controlling high level expression, then cultivate together with synthetic gene transfer molecules, these gene transfer molecules be such as polymerizability DNA-in conjunction with positively charged ion (as polylysine, protamine and albumin), itself and cell targeting ligands are (as asialoorosomucoid (as described in Wu and Wu (1987) J.Biol.Chem.262:4429-4432), Regular Insulin (as described in Hucked (1990) Biochem Pharmacol 40:253-263), semi-lactosi (as described in Plank (1992) Bioconjugate Chem 3:533-539), lactose or transferrin) be connected.

Naked DNA also can be used for transformed host cell.Typical naked DNA introduction method at WO 90/11902 and US5,580, described by having in 859.Picked-up effect can be improved by biodegradable latex bead.After starting the endocytosis of bead, the latex bead of DNA bag quilt is transported in cell.The method can be improved further by process bead to improve its hydrophobicity, thus promote the destruction of endosome and DNA is discharged in tenuigenin.

Can be used as the liposome of gene delivery vehicles at US 5,422,120, WO95/13796, WO94/23697, WO91/14445 and EP-524, described by having in 968.As USSN 60/023, described in 867, when non-viral conveying, can the nucleotide sequence of coded polypeptide be inserted in the conventional carrier containing the routine sequence controlling high level expression, then cultivate together with synthetic gene transfer molecules, these gene transfer molecules be such as polymerizability DNA-in conjunction with positively charged ion (as polylysine, protamine and albumin), it is connected with cell targeting ligands (as asialoorosomucoid, Regular Insulin, semi-lactosi, lactose or transferrin).Other delivery system comprises employing liposome to wrap up DNA, and gene contained by this DNA is under various tissue specificity or active ubiquitous promotor control.Other Non-viral delivery systems be suitable for comprises mechanical conveying system, as the people such as Woffendin (1994) Proc.Natl.Acad.Sci.USA91 (24): the method described in 11581-11585.In addition, this kind of encoding sequence and expression product can be carried by the precipitation of photopolymerisable hydrogel material.Can be used to carry other gene of encoding sequence conveying ordinary method such as to comprise, with hand-held gene transfer particle rifle (as United States Patent (USP) 5,149, described in 655); With ionizing rays activate transfer gene (as US 5,206,152 and WO92/11033 described in).

Described by typical liposome and polycationic gene delivery vehicles have in the following documents: US 5,422,120 and 4,762,915; WO 95/13796; WO94/23697; WO91/14445; EP-0524968; Stryer, Biochemistry, 236-240 page (1975) W.H.Freeman, San Francisco; Szoka (1980) Biochem Biopys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem 176:420.

Polynucleotide compositions can comprise the gene therapy vehicle for the treatment of significant quantity, and its definition as mentioned above.For purposes of the present invention, effective dosage is the DNA construction giving individual about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to 10 mg/kg.

Carrying method

Once after preparation, polynucleotide compositions of the present invention can three kinds of modes give below: (1) directly gives object; (2) cell that object is derivative is in vitro flowed to; Or (3) vivoexpression recombinant protein.Pending object can be Mammals or birds.In addition, also can treat people.

Direct conveying said composition is usually by subcutaneous, intraperitoneal, intravenously or intramuscularly or be delivered to tissue space to realize.Said composition also can be delivered to lesion.Other administering mode comprises oral with lung administration, suppository and transdermal or through dermal application (such as seeing WO98/20734), use pin, particle gun or manual sprayer (hypospray).Therapeutic dose scheme can be single dose regimen or multiple doses.

In vitro conveying and be known in the art by the method that the cell of conversion is implanted in subject again, described by having in such as WO93/14778.Cell example in vitro applying comprises such as stem cell, especially hematopoietic cell, lymphocyte, scavenger cell, dendritic cell or tumour cell.

Usually, for in vitro with external application, the conveying of nucleic acid realizes by following steps, such as dextran mediation transfection, calcium phosphate precipitation, Polybrene mediation transfection, protoplast fusion, electroporation, polynucleotide are wrapped in in karyon in liposome and by direct for DNA microinjection, all these are all known in the art

Polynucleotide and polypeptide drug composition

Except above-mentioned pharmaceutically acceptable carrier and salt, in polynucleotide and peptide composition, also can adopt following additive reagent.

A. polypeptide

An example is polypeptide, and it is including, but not limited to asialoorosomucoid (ASOR); Transferrin; Asialoglycoprotein; Antibody; Antibody fragment; Ferritin; Interleukin; Interferon, rabbit; Granulocyte-macrophage colony stimutaing factor (GM-CSF); Granulocyte colony-stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), STEM CELL FACTOR and erythropoietin.Also virus antigen can be used, as envelope protein.In addition, can be used to the albumen from other aggressive biology, 17 amino acid whose peptides (being called RII) of the circumsporozoite protein of such as plasmodium pernicious malaria

B. hormone, VITAMIN etc.

Other kind that can comprise is such as: hormone, steroid, male sex hormone, oestrogenic hormon, Triiodothyronine or VITAMIN, folic acid.

C. polyalkylene, polysaccharide etc.

In addition, polyalkylene glycol can together with required polynucleotide or polypeptides in combination.In one preferably embodiment, polyalkylene glycol is polyoxyethylene glycol.In addition, monose, disaccharides or polysaccharide can be added.In a better embodiment in this regard, polysaccharide is dextran or DEAE-dextran.There are chitosan and polylactide-polyglycolide lactone copolymers in addition.

D. lipid and liposome

Required polynucleotide or polypeptide also can be wrapped in lipid or be wrapped in liposome before flowing to object or the derivative cell of object.

Lipid encapsulation is usually with can stable bond or to catch and the liposome retaining nucleic acid realizes.Concentrated polynucleotide or polypeptide can be different from the ratio of lipid formulations, but usually between about 1: 1 (milligram DNA: micromoles lipid), or lipid is more.About liposome as the summary of carrier of conveying nucleic acid see Hug and Sleight (1991) Biochim.Biophys.Acta.1097:1-17; Straubinger (1983) Meth.Enzymol.101:512-527.

Positively charged ion (positively charged), negatively charged ion (electronegative) and neutral preparations is comprised for Liposomal formulation of the present invention.Cationic-liposome has demonstrated can carry (Felgner (1987) Proc.Natl.Acad.Sci.USA 84:7413-7416) to have in the born of the same parents of the form mediation plasmid DNA of function; MRNA (Malone (1989) Proc.Natl.Acad.Sci.USA 86:6077-6081); (Debs (1990) J.Biol.Chem.265:10189-10192) is carried with in the born of the same parents of the transcription factor of purifying.

Cationic-liposome is easy to buy.Such as, N [1-2,3-bis-oleyl oxygen) propyl group]-N, N, N-tri-second ammonium (DOTMA) liposome can with the trade mark of Lipofectin from GIBCO BRL, Grand Island, NY buys.(separately seeing Felgner, the same).Other commodity liposome comprises transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger).Other cationic-liposome can obtain from the material easily buied by method well known in the art.For example, see, Szoka (1978) PNAS 75:4194-4198; The description that WO90/11092 synthesizes about DOTAP (1,2-bis-(oleoyl oxygen)-3-(trimethyl ammonium is molten) propane) liposome.

Equally, negatively charged ion and neutral liposome are also easily obtain, and such as, purchased from Avanti PolarLipids (birmingham, AL), or easily obtain with the material easily buied.This material comprises phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleyl phosphatidyl choline (DOPC), DOPG (DOPG), DOPE (DOPE).These materials can also mix with DOTMA and DOTAP raw material with proper ratio.The method preparing liposome with these materials is well known in the art.

Liposome can comprise multilamellar liposome (MLV), little unilamellar liposome (SUV) or large unilamellar liposome (LUV).Various liposome-nucleic acid mixture can obtain by methods known in the art.For example, see Straubinger (1983) Meth.Immunol.101:512-527; Szoka (1978) Proc.Natl.Acad.Sci.USA 75:4194-4198; Papahadjopoulos (1975) Biochim.Biophys.Acta 394:483; Wilson (1979) Cell 17:77); Deamer and Bangham (1976) Biochim.Biophys.Acta 443:629; Ostro (1977) Biochem.Biophys.Res.Commun.76:836; Fraley (1979) Proc.Natl.Acad.Sci.USA 76:3348); Enoch and Strittmatter (1979) Proc.Natl.Acad.Sci.USA 76:145; Fraley (1980) J.Biol.Chem. (1980) 255:10431; Szoka and Papahadjopoulos (1978) Proc.Natl.Acad.Sci.USA 75:145; And Schaefer-Ridder (1982) Science 215:166.

E. lipoprotein

In addition, lipoprotein also can add in polynucleotide to be conveyed or polypeptide.The example of the lipoprotein adopted comprises: chylomicron, HDL, IDL, LDL and VLDL.Also can adopt the mutant of these albumen, fragment or fusion rotein.In addition, the modifier of naturally occurring lipoprotein can be adopted, such as acetylizad LDL.These lipoprotein can make the conveying of polynucleotide point to the cell of expressing lipoprotein receptor.Preferably, if add lipoprotein in polynucleotide to be conveyed, then the part of other target-seeking is not added in composition.

Naturally occurring lipoprotein comprises lipid and protein part.Protein part is called apoprotein.At present, be separated and identified apo A, B, C, D and E.Wherein have two at least containing several albumen, with Roman number AI, AII, AIV; CI, CII, CIII name.

Lipoprotein can comprise multiple apoprotein.Such as, naturally occurring chylomicron comprises A, B, C and E, and As time goes on, these lipoprotein lose A, obtain C and E apoprotein.VLDL comprises A, B, C and E apoprotein, and LDL comprises apoB; HDL comprises apo A, C and E.

The amino acid of these apoproteins is known, and described by having in the following documents: Breslow (1985) Annu Rev.Biochem 54:699; Law (1986) Adv.Exp Med.Biol.151:162; Chen (1986) J BiolChem 261:12918; Kane (1980) Proc Natl Acad Sci USA 77:2465; And Utermann (1984) Hum Genet 65:232.

Lipoprotein contains various lipid, comprises triglyceride level, cholesterol (free and ester type) and phosphatide.In naturally occurring lipoprotein, the composition of lipid is different.Such as, chylomicron is mainly containing triglyceride level.Lipid content more detailed description about naturally occurring lipoprotein can find in such as Meth.Enzymol.128 (1986).Select the composition of lipid, be conducive to receptor-binding activity to make the configuration of apoprotein.Also can select the composition of lipid, to promote and the hydrophobic interaction in polynucleotide binding molecule and combination.

Naturally occurring lipoprotein can be separated from serum with such as ultracentrifugation.These methods are at Meth.Enzymol. (the same); Described by having in Pitas (1980) J.BioChem.255:5454-5460 and Mahey (1979) J Clin.Invest64:743-750.Lipoprotein also can produce in vitro, or is produced by the recombination method of expressing apoprotein genes in required host cell.For example, see Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acta 30:443.Lipoprotein also can available from commercial supplier, as BiomedicalTechniologies, Inc., Stoughton, Massachusetts, USA.Can find in WO98/06437 about further describing of lipoprotein.

F. polycationic agents

Polycationic agents can with or be not included in containing required polynucleotide to be conveyed or polypeptide composition together with lipoprotein in.

Clean positive charge is shown under the pH that polycationic agents is correlated with in physiological usually, and in energy and the electric charge of nucleic acid, and contribute to being delivered to desired location.These reagent have external, in vitro with the purposes in body.Polycationic agents can be used to nucleic acid to be delivered to object alive by intramuscular or subcutaneous etc.

Here is the polypeptide example being used as polycationic agents: polylysine, poly arginine, poly ornithine and protamine.Other example comprises the coat protein of histone, protamine, human serum albumin, DBP, nonhistone chromosomal protein, DNA virus, as (X174, transcription factor also containing the structural domain in conjunction with DNA, therefore can be used as nucleic acid condensing agent.In brief, transcription factor such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP and TFIID contains basic structure territory in conjunction with DNA sequence dna.

Polycation organic reagent comprises: spermine, spermidine and putrescine.

Size and the physiological property of polycationic agents can be released, to build other polypeptide polycationic agents or to produce the polycationic agents synthesized from inventory above.

Useful synthesis polycationic agents such as comprises, DEAE-dextran, Polybrene.Lipofectin ^tMand lipofectAMINE ^tMbe and the monomer forming polycation complexes during polynucleotide/polypeptides in combination.

Immunodiagnostic Assays

Neisserial antigens of the present invention can be used for immunity test to detect antibody horizontal (or on the contrary, can carry out detectable antigens level with anti-neisserial antibody).According to clear and definite immunity test, recombinant antigen can be developed, to replace invasive diagnostic method.The antibody of the anti-Neisserial protein in biological sample (such as comprising blood or serum sample) can be detected.The design of immunity test can make wide variation, and its various scheme is all as known in the art.The scheme of immunity test can take such as competitiveness or direct reaction or sandwich type assays.Scheme such as also can adopt solid support, maybe can adopt immuno-precipitation.Great majority test relates to the markd antibody of employing or polypeptide; This mark can be such as fluorescent mark, chemiluminescent labeling, radioactivity mark or dye molecule.The test of amplification probe signal is also known; Its example is the test adopting vitamin H and avidin, enzyme labelling with the immunity test of mediation, as ELISA test.

Suitable material (comprising composition of the present invention) and other reagent needed for testing and material (such as suitable damping fluid, salts solution etc.) and suitable test specification sheets are packaged in suitable container, form and be applicable to immunodiagnosis and test kit containing the suitable reagent of mark.

Nucleic acid hybridization

" hybridization " refers to that two nucleotide sequences are combined by hydrogen bond each other.Usually, a sequence is fixed on solid carrier, and another will be free in solution.Then, be conducive under the condition forming hydrogen bond, two sequences being contacted with each other.The factor affecting this combination comprises: the type of solvent and volume; Temperature of reaction; Hybridization time; Stir; The reagent (Denhardt ' s reagent or bovine lacto transfer technique optimizer) of confining liquid facies-suite and solid carrier non-specific binding; The concentration of each sequence; Whether use the speed (T 500 or polyoxyethylene glycol) that compound combines to increase sequence; And the Stringency of post-hybridization wash conditions.See the people such as Sambrook [the same] the 2nd volume, the 9th chapter, 9.47 to 9.57 pages.

" preciseness " refers to that being conducive to closely similar sequence combines and be unfavorable for the hybridization condition that different sequence combines.Such as, answer the combination of selective temperature and salt concn, make temperature lower than the Tm calculated value of the hybridization studied about 120 to 200 DEG C.Temperature and salt concn, often by determining by rule of thumb in preliminary experiment, in preliminary experiment, being fixed on the genome DNA sample on filter membrane and interested sequence hybridization, then washing under different stringency conditions.See the people such as Sambrook the 9.50th page.

When carrying out such as Southern trace, the parameter that consider is that (1) treats the complicacy of the DNA of trace and the homology between (2) probe and examined sequence.For the single copy gene in the eukaryotic gene group of high complexity, the total amount of fragment to be studied can change in a number order magnitude range of 10, and plasmid is 0.1 to 1 microgram, or by phage digestion to 10-9 to 10-8 gram.For the polynucleotide that complicacy is lower, can in fact shorter trace, hybridization and duration of contact be adopted, the starting polynucleotide of less amount, and the probe that specific activity is lower.Such as, from 1 microgram cerevisiae dna, use the duration of contact of only 1 hour, trace 2 hours, then with 10 ⁸the probe hybridization 4-8 hour of cpm/ μ g, just can detect and singly copy yeast genes.For single copy mammalian genes, a kind of conservative method is from 10 micrograms of DNA, blot overnight, with 10 under 10% T 500 exists ⁸the probe hybridization of cpm/ more than μ g spends the night, and causes being about 24 hours duration of contact.

There is several factor may affect the melting temperature(Tm) (Tm) of the DNA-DNA hybrid between probe and fragment interested, the conditions suitable of thus impact hybridization and washing.In many cases, probe is not and fragment 100% homology to be measured.Other variable usually run into comprises length and the G+C total content of hybridization sequences, and the ionic strength of hybridization buffer and formamide content.The effect of all of these factors taken together can be approximated by an equation:

Tm=81+16.6 (log ₁₀ci)+0.4 [% (G+C)]-0.6 (% methane amide)-600/n-1.5 (% mispairing)

Wherein Ci is salt concn (univalent ion), and n is the length (to having done slightly amendment in Meinkoth and Wahl (1984) Anal.Biochem.138:267-284) of hybrid base pair.

When designing hybrid experiment, some factors affecting nucleic acid hybridization can be changed easily.The adjustment of salt concn when temperature when hybridization and washing and washing is the simplest.Along with the rising of hybridization temperature (i.e. rigor), the possibility occurring between the chain of not homology to hybridize becomes less, and result background value reduces.If radiolabeled probe is not and the fixing complete homology of fragment (this is common in gene family and species hybridization are tested), then hybridization temperature must be reduced, and background value will increase.Wash temperature affects the intensity of hybrid belt and the degree of background value in a similar fashion.The preciseness of washing also raises with the reduction of salt concn.

Usually, the hybridization temperature easily under 50% methane amide exists is: reaching for the probe of 95% to 100% for target fragment homology, is 42 DEG C; Being the probe of 90% to 95% for homology, is 37 DEG C; For the probe that homology is 85% and 90%, it is 32 DEG C.For lower homology, application aforesaid equation correspondingly should reduce formamide content and regulate temperature.If the homology between probe and target fragment is unknown, then the simplest method is from non-rigorous hybridization and wash conditions.If found nonspecific band or high background value after radioautograph, then can wash filter membrane under high preciseness, and again expose.If exposure required time makes the method unrealistic, then answer several hybridization of parallel testing and/or washing preciseness.

Embodiment

Embodiment 1

The embodiment 1 of WO99/36544 discloses the cloning and expressing of the Neisserial protein being called " ORF40 ".Disclose the protein from A and B serotype Neisseria meningitidis and DNA sequence dna, complete protein sequence has 83.7% identical in 601 amino acid whose overlapping districts.

For the reference group be made up of 21 bacterial strains of Neisseria meningitidis, ORF40 is checked order.

The comparison of these 21 sequences is shown in Fig. 1.The section of conserved amino acid is obvious.Such as front 17 amino acid are conservative (MNKIYRIIWNSALNAWV), although the Serine at 11 residue places is not present in 100% neisserial.Being the amino acid do not guarded after this, is again 16 conserved amino acids (BSELTRNHTKRASATV) subsequently.The C end of this protein is made up of 116 conserved amino acids.

The conservative region identified in the present embodiment confirms, the fragment of total length ORF40 albumen is suitable as vaccine or the diagnostic reagent of polyspecific.

Again the ORF40 of 31 bacterial strains is altogether checked order, and sequence is compared.Result is shown in Figure 11.

Interested especially conservative region is:

-MNKIYRIIWNSALNAWV

-VSELTRNHTKRASATV

-TAVLATLL

-TLKAGDNLKIKQ

-FTYSLKKDLTDLTSV

-TEKLSFGANG

-KVNITSDTKGLNFAKETAGTNGD

-TVHLNGIGSTLTDTL

-RAAS(V/I)KDVLNAGWNIKGVK

-NVDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGK

-KGENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGT

-GTTATVSKDDQGNITV

-YDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETVNINAGNNIEITRNGKNIDIATSM

-PQFSSVSLGAGADAPTLSVD

-NKPVRITNVApGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKSMMAI

GGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW.

Embodiment 2

The embodiment 26 of W099/24578 discloses the cloning and expressing of the Neisserial protein being called " ORF4 ".Disclose the protein from A and B serotype Neisseria meningitidis and DNA sequence dna, and the sequence of Neisseria meningitidis.Homogeny on amino acid levels between sequence is as follows:

For the reference group be made up of 32 bacterial strains of Neisseria meningitidis, ORF4 is checked order.

Fig. 2 is shown in the sequence alignment that PILEUP produces.The section of conserved amino acid is obvious.Such as front 34 amino acid are conservative, although the Serine at 26 residue places is not present in 100% neisserial.The C end of this protein is made up of 228 conserved amino acids.

The conservative region identified in the present embodiment confirms, the fragment of total length ORF4 albumen is suitable as vaccine or the diagnostic reagent of polyspecific.

Again the ORF4 of 35 bacterial strains is altogether checked order, and sequence is compared.Result is shown in Figure 10.

Interested especially conservative region is:

-MKTFFKTLSAAALALILAACGGQKDSAPAASASAAADNGA

-KKEIVFGTTVGDFGDMVKE

-ELEKKGYTVKLVEFTDYVRPNLALAEGELDINVFQHKPYLDDFKKEHNLDITEVFQVPTAPLGLYPGK

LKSLEEVKDGSTVSAPNDPSNFARVLVMLDELGWIKLKDGINPLTASKADIAENLKNIKIVELEAAQL

PRSRADVDFAVVNGNYAISSGMKLTEALFQEPSFAYVNWSAVKTADKDSQWLKDVTEAYNSDAFKAYA

HKRFEGYKSPAAWNEGAAK

Embodiment 3

The embodiment 16 of WO99/57280 discloses the cloning and expressing of the Neisserial protein being called " 225 ".Disclose the protein from A and B serotype Neisseria meningitidis and DNA sequence dna, and from the sequence of neisseria gonorrhoeae.

For the reference group be made up of 34 bacterial strains of Neisseria meningitidis, check order to 225.

Fig. 3 is shown in the sequence alignment that PILEUP produces.The section of conserved amino acid is obvious.Such as front 74 amino acid are conservative, although the Isoleucine at 51 residue places is not present in 100% neisserial.The C end of this protein is made up of 148 conserved amino acids.Similar comparison is shown in Figure 13.

The conservative region identified in the present embodiment confirms, the fragment of total length 225 albumen is suitable as vaccine or the diagnostic reagent of polyspecific.

Embodiment 4

The embodiment 16 of WO99/57280 discloses the cloning and expressing of the Neisserial protein being called " 235 ".Disclose the protein from A and B serotype Neisseria meningitidis and DNA sequence dna, and from the sequence of neisseria gonorrhoeae.

For the reference group be made up of 31 bacterial strains of Neisseria meningitidis, check order to 235.

Fig. 4 is shown in the sequence alignment that PILEUP produces.The section of conserved amino acid is obvious.Protein is conservative on the whole, although show some change at the Serine at 168 residue places.

The conservative region identified in the present embodiment confirms, the fragment of total length 235 albumen is suitable as vaccine or the diagnostic reagent of polyspecific.

Again 235 of 35 bacterial strains are altogether checked order, and sequence is compared.Result is shown in Figure 14.

Embodiment 5

The embodiment 16 of WO99/57280 discloses the cloning and expressing of the Neisserial protein being called " 287 ".Disclose the protein from A and B serotype Neisseria meningitidis and DNA sequence dna, and from the sequence of neisseria gonorrhoeae.

For the reference group be made up of 6 bacterial strains of Neisseria meningitidis, check order to 287.

Fig. 5 is shown in the sequence alignment that PILEUP produces.The section of conserved amino acid is obvious.Such as front 42 amino acid are conservative, and can from seeing a long conservative region at the C end of protein.

The conservative region identified in the present embodiment confirms, the fragment of total length 287 albumen is suitable as vaccine or the diagnostic reagent of polyspecific.

Again 287 of 35 bacterial strains (comprising C11, a kind of C serotype N. meningitidis strains) are altogether checked order, and sequence is compared.Result is shown in Figure 15.

Interested especially conservative region is:

-MFKRSVIAMACI

-ALSACGGGGGGSPDVKSADT

-SKPAAPVV

-QDMAAVS

-ENTGNGGAATTD

-QNDMPQ

-DGPSQNITLTHCK

-KSEFE

-RRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKL

-GGSYAL

-VQGEPAKGEMLAGTAVYNGEVLHFH

-GRFAAKVDFGSKSVDGIIDSGDDLHMG

-QKFKAAIDGNGFKGTWTENGGGDVSG(R/K)FYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKDRD

Embodiment 6

The embodiment 16 of WO99/57280 discloses the cloning and expressing of the Neisserial protein being called " 519 ".Disclose the protein from A and B serotype Neisseria meningitidis and DNA sequence dna, and from the sequence of neisseria gonorrhoeae.

For the reference group be made up of 22 bacterial strains of Neisseria meningitidis, check order to 519.

Fig. 6 is shown in the sequence alignment that PILEUP produces.The section of conserved amino acid is obvious, and the display of this protein is conservative over the entire length thereof.

The conservative region identified in the present embodiment confirms, the fragment of total length 519 albumen is suitable as vaccine or the diagnostic reagent of polyspecific.

Again 519 of 33 bacterial strains are altogether checked order, and sequence is compared.Result is shown in Figure 16.

Interested especially conservative region is:

-MEFFIILL

-AVAVFGFKSFVVIPQQEVHVVERLGRFHRALTAGLNILIPFIDRVAYRHSLKEIPLDVPSQVCITRDN

TQLTVDGIIYFQVTDPKLASYGSSNYIMAITQLAQTTLRSVIGRMELDKTFEERDEINSTVV

-ALDEAAGAWGVKVLRYEIKDLVPPQEILRSMQAQITAEREKRARIAESEGRKIEQINLASGQREAEIQ

QSEGEAQAAVNASNAEKIARINRAKGEAESLRLVAEANAEANRQIAAALQTQSGADAVNLKIAGQYVT

AFKNLAKEDNTRIKPAKVAEIGNPNFRRHEKFSPEAKTAK

Embodiment 7

The embodiment 16 of WO99/57280 discloses the cloning and expressing of the Neisserial protein being called " 919 ".Disclose the protein from A and B serotype Neisseria meningitidis and DNA sequence dna, and from the sequence of neisseria gonorrhoeae.

For the reference group be made up of 35 bacterial strains of Neisseria meningitidis, check order to 919.

Fig. 7 is shown in the sequence alignment that PILEUP produces.Another comparison is shown in Figure 18.The section of conserved amino acid is obvious.The display of this protein is almost all guarded.

The conservative region identified in the present embodiment confirms, the fragment of total length 919 albumen is suitable as vaccine or the diagnostic reagent of polyspecific.

Interested especially conservative region is:

-MKKYLFRAAL

-GIAAAILAACQSKSIQTFPQPDTSVINGPDRPVGIPDPAGTTV(G/A)GGGAVYTVVPHLSLPHWAAQ

DFAKSLQSFRLGCANLKNRQGWQDVCAQAFQTPVHSFQAKQFFERYFTPWQVAGNGSLAGTVTGYYEP

VLKGDDRRTAQARFPIYGIPDDFISVPLPAGLRSGKALVRIRQTGKNSGTIDN

-GGTHTADLS

-FPITARTTAIKGRFEGSRFLPYHTRNQINGGALDGKAPILGYAEDPVELFFMHIQGSGRLKTPSGKYI

RIGYADKNEHPYVSIG(R/K)YMADKGYLKLGQTSMQGIK

-YMRQNPQRLAEVLGQNPSYIFFREL

-NDGPVGALGTPLMGEYAGAVDRHYITLGAPLFVATAHPVTRKALNRLIMAQDTGSAIKGAVRVDYFWG

YGDEAGELAGKQKTTGYVWQLLPNGMKPEYRP

Embodiment 8

The embodiment 55 of WO99/23578 discloses the cloning and expressing of the Neisserial protein being called " ORF46 ".Disclose the protein from A and B serotype Neisseria meningitidis and DNA sequence dna, and from the sequence of neisseria gonorrhoeae.

For the reference group be made up of 6 bacterial strains of B serotype Neisseria meningitidis, total length ORF46 is checked order.The comparison of these sequences is shown in Figure 12, therefrom obviously can find out the section of conserved amino acid.

Interested especially conservative region is:

-RKISLILSILAVCLPMHAHASDLANDSFIRQVLDRQHFEPDGKYHLFGSRGELAERSGHIGLG

-IQSHQLGNLMIQQAAIKGNIGYIVRFSDHGHEVHSPFDNHASHSDSDEAGSPVDGFSLYRIHWDGYEH

HPADGYDGPQGGGYPAPKGARDIYSYDIKGVAQNIRLNLTDNRSTGQRLADRFHNAG

-MLTQGVGDGFKRATRYSPELDRSGNAAEAFNGTADIVKNIIGAAGEIVGAGDAVQGISEGSNIAVMHG

LGLLSTENKMARINDLADMAQLKDYAAAAIRDWAVQNPNAAQGIEAVSNIF

-IPIKGIGAVRGKYGLGGITAHP(V/I)KRSQMGEIALPKGKSAVS

-NFADAAYAKYPSPYHSRNIRSNLEQRYGKENITSSTVPPSNGKNVKLANKRHPKTKVPFDGKGFPNFE

KDVKY

The conservative region of ORF46 confirms, the fragment of this albumen is suitable as vaccine or the diagnostic reagent of polyspecific.

Embodiment 9

WO99/57280 discloses the cloning and expressing of the Neisserial protein being called " 726 ".Disclose the protein from A and B serotype Neisseria meningitidis and DNA sequence dna.

For the reference group be made up of 7 bacterial strains of A, B and C serotype Neisseria meningitidis, check order to 726.The comparison of these sequences is shown in Figure 17, therefrom obviously can find out the section of conserved amino acid.

Interested especially conservative region is:

-IYFKNGFYDDTLG

-IPEGAVAVRAEEYAALLAGQAQGGQIAADSDGRPVLTPPRPS(D/E)YHEWDGKKW

-AAAAARFAEQKTATAFRLA

-KADELKNSLLAGYPQVEIDSFYRQEKEALARQADNNAPTPMLAQIAAARGVELDVLIEKV(I/V)EKS

ARLAVAAGAIIGKRQQLEDKLN

-IETAPGLDALEKEIEEWT

The conservative region of 726 confirms, the fragment of this albumen is suitable as vaccine or the diagnostic reagent of polyspecific.

Embodiment 10

WO99/57280 discloses the cloning and expressing of the Neisserial protein being called " 953 ".Disclose the protein from A and B serotype Neisseria meningitidis and DNA sequence dna, and from the sequence of neisseria gonorrhoeae.

For the reference group be made up of 8 bacterial strains of A, B and C serotype Neisseria meningitidis, check order to 953.The comparison of these sequences is shown in Figure 19, therefrom obviously can find out the section of conserved amino acid.This protein is high conservative.

Interested especially conservative region is:

-MKKIIFAALAAAAVGTASAATYKVDEYHANARFAIDMFNTSTNVGGFYGLTGSVEFDQAKRDGKIDIT

IP(I/V)ANLQSGSQHFTDHLKSADIFDAAQYPDIRFVSTKFNFNGKKLVSVDGNLTMHGKTAPVKLK

AEKFNCYQSPM

-ATYKVDEYHANARFAIDHFNTSTNVGGFYGLTGSVEFDQAKRDGKIDITIP(I/V)ANLQSGSQHFTD

HLKSADIFDAAQYPDIRFVSTKFNFNGKKLVSVDGNLTMHGKTAPVKLKAEKFNCYQSPM

-KTEVCGGDFSTTIDRTKWG(M/V)DYLVNVGMTKSVRIDIQIEAAKQ

The conservative region of 953 confirms, the fragment of this albumen is suitable as vaccine or the diagnostic reagent of polyspecific.

Phylogenetic tree

Fig. 8 is the arborization figure of the genetic affinity of display 107 kinds of N. meningitidis strains, and it is based on the MLST analytical results [adapting from the people such as Maiden (1998) PNAS USA 95:3140] to 6 gene fragments.This arborization figure can be used for selecting representational B meningococcus serogroup bacterial strain (arrow).5 kinds of extra bacterial strains are (for these 5 kinds of bacterial strains, the people [Mol.Microbiol. (1992) 6:1271] such as people [Mol.Microbiol. (1996) 19:841] and Virji such as the people such as Wang [J.Infect.Dis (1993) 167:1320], Seiler determine them independently in heredity, are classified as super malicious pedigree), to be superimposed on arborization figure and to mark with an asterisk.Except 22 kinds of MenB bacterial strains, also use 2 kinds of MenA bacterial strains, 2 kinds of MenC bacterial strains, each a kind of bacterial strain in MenY, X, Z and W135.The bold-type letter of these bacterial strains before title represents.When not having system generation data, bacterial strain is just listed in outside phylogenetic tree.Super strain ET-5, ET-37 and IV-1 are marked.

Sequence variability

Fig. 9 a be protein 225 in Neisseria meningitidis, 235,287,519,919, the schematic diagram of the amino acid sequence variability of ORF4 and ORF40.Transverse axis represents the sequence of MC58.Amino acid difference in MenB bacterial strain represents with the vertical line above transverse axis.The line of difference among A, C, Y, X, Z and W135 serotype below axle represents.The height of vertical line represents the number of strains with amino acid difference.Like this, peak represents Variable Area.Strigula below 225 and 287 represents the sequence section lacked in some bacterial strain.

Fig. 9 b is the arborization figure of the N. meningitidis strains with identical 7 kinds of albumen acquisitions.Arborization figure that conform to Fig. 8, that include super strain is provided according to the Phylogenetic analysis of these genes.The bacterial strain that lines mark includes analyzes bacterial strain that conform to, that obtain 100% guiding support (bootstrap support) with MLST.Number below each node guides numerical value (only reporting the numerical value being greater than 80%).The PILEUP program comparison of gene order in GCG software package of different strains.With adjacent algorithm [Saitou & Nei (1987) Mol.Biol.Evol.4:406], use the NEIGHBOR program in PHYLIP software package, carry out Phylogenetic analysis.With Kimura-two-parameter [Kimura (1980), J.Mol.Evol.16:111], calculate the distance between two of 31 kinds of N. meningitidis strains.The tandem repeat region of the N end regions of ORF40, total length 27 and 225 has been discharged in analysis.1000 guiding are altogether allowed to copy (replicate) to assess support level.The gathering bunchy of super strain obtains the confirmation of maximum Analysis on Minimal.

Western blot

By western blot analyze various different strains ORF4,225,235,519 and 919 antigens.Result is shown in Figure 20.For 225, trace display different strains has the fragment of different size, and wherein arrow indicates the band of correct size.The repetition Insert Fragment that 225 albumen contain absent region and define, the clip size on trace conforms to gene variability data.

Bacterial strain for Figure 20 is as follows:

B serotype Neisseria meningitidis:

1＝NG6/88 2＝BZ198 3＝NG3/88 4＝297-0 5＝1000

6＝BZ147 7＝BZ169 8＝528 9＝NGP165 10＝BZ133

11＝NGE31 12＝NGF26 13＝NGE28 14＝NGH38 15＝SWZ107

16＝NGH15 17＝NGH36 18＝BZ232 19＝BZ83 20＝44/76

21＝MC58 96＝2996

A serotype Neisseria meningitidis:

22＝205900 23＝F6124

C serotype Neisseria meningitidis:

24＝90/18311 25＝93/4286

Other Neisseria meningitidis

26=A22 (W serotype)

27=E26 (X serotype)

28=860800 (Y serotype)

29=E32 (Z serotype)

Other neisseriales

30=neisseria cinerea

L17=neisseria lactamica

L19=neisseria lactamica

31=neisseria gonorrhoeae F62

32=neisseria gonorrhoeae SN4

Should be understood that the present invention is described by means of only way of example, and can change within the spirit and scope of the present invention.

Claims

1. a protein, is characterized in that, it is the fragment of protein 287, and wherein, described protein 287 is made up of the aminoacid sequence of Neisseria meningitidis B serogroups MC58 strain shown below:

MFKRSVIAMA CIFALSACGG GGGGSPDVKS ADTLSKPAAP VVSEKETEAK

EDAPQAGSQG QGAPSAQGSQ DMAAVSEENT GNGGAVTADN PKNEDEVAQN

DMPQNAAGTD SSTPNHTPDP NMLAGNMENQ ATDAGESSQP ANQPDMANAA

DGMQGDDPSA GGQNAGNTAA QGANQAGNNQ AAGSSDPIPA SNPAPANGGS

NFGRVDLANG VLIDGPSQNI TLTHCKGDSC SGNNFLDEEV QLKSEFEKLS

DADKISNYKK DGKNDKFVGL VADSVQMKGI NQYIIFYKPK PTSFARFRRS

ARSRRSLPAE MPLIPVNQAD TLIVDGEAVS LTGHSGNIFA PEGNYRYLTY

GAEKLPGGSY ALRVQGEPAK GEMLAGAAVY NGEVLHFHTE NGRPYPTRGR

FAAKVDFGSK SVDGI IDSGD DLHMGTQKFK AAIDGNGFKG TWTENGSGDV

SGKFYGPAGE EVAGKYSYRP TDAEKGGFGV FAGKKEQD，

Wherein, described fragment is made up of 7 or more continuous print conserved amino acids, described conserved amino acid is present in the neisserial of at least 50% with reference in the protein 287 of group, and described reference group is made up of following bacterial strain: BZ198, NGP165, NGH38, MC58, Z2491, FA1090.

2. a protein, is characterized in that, it is 7 or more continuous print conserved amino acid fragments, and wherein, described fragment appears in the aminoacid sequence of at least two shown in Fig. 5 and Figure 15.

3. protein as claimed in claim 2, it is characterized in that, described fragment appears in the aminoacid sequence of at least 3 shown in Fig. 5.

4. protein as claimed in claim 3, it is characterized in that, described fragment appears in all aminoacid sequences shown in Fig. 5.

5. protein as claimed in claim 2, it is characterized in that, described fragment appears in the aminoacid sequence of at least 10 shown in Figure 15.

6. protein as stated in claim 5, it is characterized in that, described fragment appears in all aminoacid sequences shown in Figure 15.

7. the protein described in aforementioned any one claim, is characterized in that, described fragment is made up of 20 or more continuous print conserved amino acids.

8. a protein, is characterized in that, it is made up of less than one or more aminoacid sequence:

MFKRSVIAMACI

ALSACGGGGGGSPDVKSADT

SKPAAPVV

QDMAAVS

ENTGNGGAATTD

QNDMPQ

DGPSQNITLTHCK

KSEFE

RRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKL

GGSYAL

VQGEPAKGEMLAGTAVYNGEVLHFH

GRFAAKVDFGSKSVDGIIDSGDDLHMG

QKFKAAIDGNGFKGTWTENGGGDVSG(R/K)FYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKDRD，

Condition is, described protein is not containing following aminoacid sequence:

MFKRSVIAMA CIFALSACGG GGGGSPDVKS ADTLSKPAAP VVSEKETEAK

EDAPQAGSQG QGAPSAQGSQ DMAAVSEENT GNGGAVTADN PKNEDEVAQN

DMPQNAAGTD SSTPNHTPDP NMLAGNMENQ ATDAGESSQP ANQPDMANAA

DGMQGDDPSA GGQNAGNTAA QGANQAGNNQ AAGSSDPIPA SNPAPANGGS

NFGRVDLANG VLIDGPSQNI TLTHCKGDSC SGNNFLDEEV QLKSEFEKLS

DADKISNYKK DGKNDKFVGL VADSVQMKGI NQYIIFYKPK PTSFARFRRS

ARSRRSLPAE MPLIPVNQAD TLIVDGEAVS LTGHSGNIFA PEGNYRYLTY

GAEKLPGGSY ALRVQGEPAK GEMLAGAAVY NGEVLHFHTE NGRPYPTRGR

FAAKVDFGSK SVDGIIDSGD DLHMGTQKFK AAIDGNGFKG TWTENGSGDV

SGKFYGPAGE EVAGKYSYRP TDAEKGGFGV FAGKKEQD。

9. a nucleic acid, is characterized in that, the protein of its coding according to any one of claim 1-8.

10. the purposes of the protein according to any one of claim 1-8, is characterized in that, for the preparation of the medicine of the infection that treatment or prevention neisserial cause.

The purposes of 11. nucleic acid according to claim 9, is characterized in that, for the preparation of the medicine of the infection that treatment or prevention neisserial cause.

The purposes of the protein according to any one of 12. claim 1-8, is characterized in that, for the preparation of multi-specific diagnostic reagent.

The purposes of 13. nucleic acid according to claim 9, is characterized in that, for the preparation of multi-specific diagnostic reagent.