CN1179181A - GnRH-leukotoxin chimeras - Google Patents

GnRH-leukotoxin chimeras Download PDF

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CN1179181A
CN1179181A CN96192680A CN96192680A CN1179181A CN 1179181 A CN1179181 A CN 1179181A CN 96192680 A CN96192680 A CN 96192680A CN 96192680 A CN96192680 A CN 96192680A CN 1179181 A CN1179181 A CN 1179181A
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gnrh
sequence
leukotoxin
chimeric protein
polypeptide
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安德鲁·A·波特
约翰·G·曼斯
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University of Saskatchewan
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University of Saskatchewan
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Abstract

The present invention discloses new immunological carrier systems, DNA encoding the same, and the use of these systems. The carrier systems include chimeric proteins which include a leukotoxin polypeptide fused to one or more selected GnRH multimers which comprise at least one repeating GnRH decapeptide sequence, or at least one repeating unit of a sequence corresponding to at least one epitope of a selected GnRH molecule. Under the invention, the selected GnRH sequences may all be the same, or may correspond to different derivatives, analogues, variants or epitopes of GnRH so long as the GnRH sequences are capable of eliciting an immune response. The leukotoxin functions to increase the immunogenicity of the GnRH multimers fused thereto.

Description

GnRH-leukotoxin mosaic
Generally speaking the present invention relates to the immunology carrier system.Specifically, the present invention relates to leukotoxin-GnRH mosaic, it comprises the GnRH polypeptide of copy more than.Compare it demonstrates and has stronger immunogenicity with single GnRH polypeptide.
In vertebrates, two kinds of gonad-stimulating hormone, the synthetic and release of lutropin (LH) and follitropin (FSH) is by the polypeptides for modulating that is called gonadotropin releasing hormone (GnRH) (past called after LHRH).Thereby an approach of control animal population breeding is for example to reduce the GnRH level by anti-GnRH immunization, causes the attenuating of LH and FSH level and is accompanied by the oestrus cycle and the destruction of spermatogenesis.Referring to for example., Adams et al., J.Anim.Sci. (1990) 68:2793-2802.
Specifically, the early stage research to the GnRH molecule has shown that duplicate injection synthetic GnRH peptide may increase antiserum(antisera) (Arimura et al., incretology (1973) 93 (5): 1092-1103).In addition, in numerous species,, produced GnRH antibody (Carelli et al., Proc.Natl.Acad.Sci. (1982) 79:5392-5395) by with the Chemical bond of GnRH and suitable carrier and will be present in this binding substances administration in the proper adjuvant.The recombinant fusion protein that will comprise GnRH or GnRH analogue is used for peptide vaccine with to various domesticate animals and farm-animals immunity castrating or suppress the existing report of method (Meloen et al., the vaccine (1994) 12 (8): 741-746 of its reproductive function; Hoskinson et al., Aust.J.Biotechnol. (1 990) 4:166-170; With disclosed international publication number WO92/19746 on November 12nd, 1992; On March 7th, 1991 disclosed WO 91/02799; October 4 nineteen ninety disclosed WO 90/11298; On December 18th, 1986 disclosed WO 86/07383).
Yet, because the extremely low immunogenicity of GnRH peptide and, provide the effort of suitable immune sterilization product to fail, and obtained inhomogenous basically very little deterministic GnRH binding substances because the Chemical bond process is restive.In addition, it is also very low to obtain the success ratio of homogeneous effect based on the peptide vaccine of GnRH even after to the animal individual repeated inoculation.In this, because GnRH is the molecule of little " self ", usually can not be by the immune system recognition of broken examination animal, just the less and immunne response that can not induce significantly anti-endogenous GnRH of its immunogenicity, existing GnRH construct can't become the immune sterilization vaccine product of success.
It has been generally acknowledged that virus antigen, the immunogenicity of small protein matter or endogenous substance can strengthen by the immunogen form of these molecules that preparation comprises the selected epitope of multiple copy.In this, 2 or 4 repetition (Ploeget al. of the peptide 9-21 of I herpes simplex virus type glycoprotein D have been proved, J. immunization method magazine (1989) 124:211-217), 2-6 repetition (the Lowell et al. of the antigen ring sporozoite tetrapeptide NPNA of Plasmodium falciparum, science (1988) 240:800-802), 2 or 4 copy (Broekhuijsen etal. in the main immunogens site of the VP1 of mouth tellurium eqpidemic disease poison, J.gen.Virol. (1987) 68:3137-3143) and the series connection of class GnRH polypeptide repeat construct (Meloen et al., vaccine (1994) 12 (8): 741-746) can improve the immunogenicity of these molecules effectively on the basis.
And, in order to be excited to cause tangible immunne response in the animal, small protein or endogenous substance also can with the appropriate carriers combination.Appropriate carriers generally comprises proteinic antigenic region or the carrier peptides polypeptide of sequence that originates from as the infected material of virus surface proteins.The effect right and wrong of these carriers stimulate t helper cell active and combine with the molecule of main histocompatibility complex (MHC) to guide antigen presents sexual cell at antigen cell surface to the processing of this peptide with present specifically.
For this purpose has been developed several carrier systems.For example, little peptide antigen usually with as key hole chirp serum protein (Bittle et al., nature (1982) 298:30-33), Toxoid,tetanus (Mulleret al., Proc.Natl.Acad.Sci.USA (1982) 79:569-573), the protein carrier coupling of ovalbumin and Physter macrocephalus myohaemoglobin produces immunne response.These linked reactions cause mixing several moles of peptide antigens usually in every mole of carrier proteins.Though the general enhancing immunity originality of the antigenic appearance of the peptide of multiple copy, but carrier can cause and the irrelevant strong immunity of peptide antigen, when this may suppress second immunisation to the immunne response (Schutze et al, Journal of Immunology (1985) 135:2319-2322) of peptide vaccine.
The antigen release system also is based on special carrier.For example, preformed particle is as antigen coupling and the platform that mixes.Developed based on proteoplast (Lowell et al., science (1988) 240:800-802), immunostimulating complex (Morein et al., nature (1984) 308:457-460) with as virion (the Neurath et al. of HBsAg, (1989) 26:53-62) and the system of rotavirus inner shell albumen (Redmond et al., Mol.Immunol. (1991) 28:269-278) Mol.Immunol..
Also utilize the oneself of recombinant production to be assembled into the particulate chimeric protein and designed carrier system.For example, the yeast retrotransponsons, Ty, a series of protein (Ty-VLPs that are assembled into viruslike particle encode; Kingsman, S.M. and A.J.Kingsman Vacc. (1988) 6:304-306).Foreign gene has been inserted the TyA gene and in yeast, expressed as fusion rotein.Fusion rotein keeps the particulate ability that the oneself is assembled into the homogeneous size.
After testing other chimeric protein particle such as HBsAg (Valenzuela et al., Bio/Technol. (1985) 3:323-326; U.S. Patent number .4,722,840; Delpeyroux et al., science (1986) 233:472-475), hepatitis B core antigen (Clarke et al., vaccine 88 (Ed.H.Ginsberg, et al., 1988) pp.127-131), poliovirus (Burke et al., nature (1988) 332:81-82), and tobacco mosaic virus (TMV) (Haynes et al., Bio/Technol. (1986) 4:637-641).But the purposes of these carriers is subjected to can insert structure albumen and do not interfere the restriction of limited size of the active agent of particle assembling.
At last, utilize haemolysis bus moral Salmonella leukotoxin (LKT) polypeptide that merges with selected antigen to design chimeric system.Referring to, for example, on April 29th, 1993 disclosed international publication number WO 93/08290 and on March 5th, 1992 disclosed WO 92/03558, and U.S. Patent number 5,238,823 and 5,273,889.Have the reactive T-cell epitope of wide range kind by providing, thereby cause a T-cell dependent immune response in immune main body, the peptide antigen mosaic that comprises the LKT carrier part provides this mosaic enhanced immunogenicity.In this, inducing suitable T-cell help is crucial to the immunne response that produces this mosaic peptide antigen part, and particularly working as this antigen is the endogenous minute period of the day from 11 p.m. to 1 a.m.But, till now, also do not have the description of the multiple epi-position bonded purposes of leucocytotoxicity peptide carrier and GnRH peptide.
The present invention is based on haemolysis bus moral Salmonella leukotoxin gene, and the structure of the new fusion gene between the nucleotide sequence of varient and the multiple GnRH polypeptide of coding.When comparing with the immunne response of the individually dosed initiation of GnRH, these constructs have produced and have shown the shockingly chimeric protein of enhancing immunity originality.
So in one embodiment, the present invention relates to a chimeric protein that comprises the leukotoxin polypeptide that merges with many bodies, these many bodies are made up of GnRH polypeptide selected more than basically, and the effect of mosaic leukotoxin part is the immunogenicity that strengthens the GnRH polypeptide.Specifically, the many bodies of GnRH can be corresponding to selected the GnRH polypeptide or the epi-position of copy more than, or the series multiple of selected GnRH polypeptide or epi-position repeats.In addition, the many bodies of GnRH can be positioned at carboxyl or the N-terminal or the inner site of leukotoxin polypeptide.The many bodies of GnRH also can be corresponding to the molecule of general formula GnRH-X-GnRH, and wherein X is selected from by peptide bond, amino acid spacer groups and (GnRH) nThe group of forming, wherein n is more than or equal to 1, and in addition wherein " GnRH " can comprise any GnRH polypeptide.
Simultaneously disclosed is the method that comprises the vaccine composition of this chimeric protein and pharmaceutical acceptable carrier and selected many bodies of GnRH are provided in pin main body, comprises this vaccine composition of using significant quantity.
In another embodiment, the present invention relates to the to encode DNA construct of this chimeric protein.This DNA construct comprises first Nucleotide of the coding leukotoxin polypeptide that is operably connected with second nucleotide sequence of coding GnRH epi-position of copy more than.
In another embodiment, the present invention relates to comprise the expression cassette of the DNA construct of above-mentioned (a) and the control sequence that (b) instructs this construct to transcribe, thereby this construct can be transcribed and translate in host cell.
In another embodiment, the present invention relates to the host cell that transforms with these expression cassettes.
Another embodiment of the invention provides the method for producing recombinant polypeptide.This method comprises that (a) provides above-described host cell group and (b) cultivate this cell mass under the condition that the expression cassette encoded polypeptides is expressed.
According to content disclosed herein, those of ordinary skills are easy to expect these and other embodiment of the present invention.Brief description of drawings
Figure 1A and 1B have shown the nucleotide sequence and the aminoacid sequence of the GnRH construct that is used for chimeric leukotoxin-GnRH polypeptide fusion gene.Figure 1A has described the GnRH-1 of the GnRH decapeptide that comprises single copy; Figure 1B has described the GnRH decapeptide that comprises four copies when n=1 and the GnRH-2 of the GnRH of 8 copies when n=2.
Fig. 2 has described the structure of plasmid pAA352, and wherein tac is from colibacillary heterozygosis trp ∷ lac promotor; Bla represents β-Nei Xiananmei gene (amicillin resistance); Ori is based on the plasmid replication initial point of ColE1; LktA is a haemolysis bus moral Salmonella leukotoxin structure gene; With lac1 be E.coli lac operon repressor.Arrow is represented leukotoxin gene transcription/translation direction.The size of each composition is not drawn in proportion.
Fig. 3-1 has represented the nucleotide sequence of leukotoxin 352 (LKT352) and the aminoacid sequence of supposition to 3-9.The structure gene of LKT352 and the sequence in side joint carrier district have been shown.
Fig. 4 has represented to carry the structure of the plasmid pCB113 of leukotoxin-GnRH (LKT-GnRH) fusion gene.
Fig. 5-1 has represented from the nucleotide sequence of the LKT-GnRH chimeric protein of pCB113 and the aminoacid sequence of supposition to 5-8.Aminoacid sequence from the nucleotide sequence of the LKT-GnRH chimeric protein of pCB112 and supposition is identical with the sequence of the chimeric protein that originates from pCB113, except the sequence of the multiple copy of twice insertion GnRH, as described in top Fig. 4.
Fig. 6 has represented to carry the structure of the plasmid pCB111 of leukotoxin-GnRH (LKT-GnRH) fusion gene.
Fig. 7-1 has represented from the nucleotide sequence of the LKT-GnRH chimeric protein of pCB111 and the aminoacid sequence of supposition to 7-5.The identical sequence except the multiple copy of twice insertion GnRH of sequence from the described chimeric protein that originates from pCB111 of the aminoacid sequence of the nucleotide sequence of the LKT-GnRH chimeric protein of pCB114 and supposition and top Fig. 6 is different.
Fig. 8 has represented the nucleotide sequence of merging point of concordant end of leukotoxin gene of brachymemma of plasmid pCB111 and the aminoacid sequence (Fig. 8-2) of supposition, removes an inner dna fragmentation (length is about 1300bp) (Fig. 8-1) by the digestion of restriction enzyme BstB1 and Nael from LKT352.
Except as otherwise noted, method of the present invention will be used molecular biology, microbiology, and virusology, recombinant DNA technology and immunologic routine techniques, they are within the known scope of those skilled in the art.The explanation fully of these technology is arranged in the literature.Referring to for example, Sambrook, Fritsch and Maniatis, Molecular cloning: laboratory manual; Dna clone, Vol.I and II (D.N.Glover ed.); Oligonucleotide is synthetic(M.J.Gait ed.); Nucleic acid hybridization(B.D.Hames and S.J.Higgins eds.); Animal cell culture(R.K.Freshney ed.); The fixed cell And enzyme(IRL publication); B.Perbal, The molecular cloning experiment instructionBook series, Enzymology method(S.Colowick and N.Kaplan eds., Academic press, Inc.) and Experiment immunization learns to do volume, Vols.I-IV (D.M.Weir and C.C.Blackwell eds., Blackwell scientific publication).A. Definition
When description is of the present invention, will use following term, and as described below the definition.
Term " gonadotropin releasing hormone " or " GnRH " are meant hypothalamus excretory decapeptide, and it controls the release (Fink, G., Britain medical science circular (1979) 35:155-1 60) of lutropin (LH) and follitropin (FSH) in vertebrates.The aminoacid sequence of GnRH is a high conservative between vertebrates, particularly in Mammals.In this, originate from most of Mammalss and comprise the people, ox, the GnRH of pig and sheep GnRH (past called after LHRH) has aminoacid sequence pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2(Murad et al., hormone and hormone antagonist, Therapeutic pharmacology principle, sixth version (1980) and Seeburg et al., nature (1984) 311:666-668).
As used herein, " GnRH polypeptide " comprises the molecule that originates from natural GnRH sequence, and as described belowly has basically with natural GnRH homology and keep GnRH polypeptide recombinant production or chemosynthesis of immunogenic aminoacid sequence.So this term is included in peptide inside or amino or C-terminal single or multiple amino acid stacks, derivative and the analogue of the GnRH that substitutes and/or lack takes place.Therefore, in the present invention, " GnRH polypeptide " comprises the molecule with native sequences, molecule (having terminal Gin residue of a N-rather than burnt Glu (pyro Glu) residue) as Figure 1A description, with to have other amino acid additional, the molecule that substitutes and/or lack, it has kept the ability of initiation with the formation of the antibody of naturally occurring GnRH cross reaction.This paper is particularly related to is the tumor-necrosis factor glycoproteins (wherein each selected GnRH polypeptide comprises the terminal Gln of a N-and substitutes, and wherein each other GnRH polypeptide 2 comprises an Asp residue and substitutes in the position in addition) of the oligomeric GnRH polypeptide described as Figure 1B.The epi-position of GnRH is also in the scope of this definition.
Term " epi-position " refers to the site on specific antibody molecule bonded antigen or the haptens.Because GnRH is very little molecule, utilizes technology known in the art to be easy to finish to wherein causing the evaluation of the epi-position of antibody response.Referring to for example, Geysen et al.Proc.Natl.Acad.Sci.USA (1984) 81:3998-4002 (synthetic fast peptide is determined the general method of the position of immunogen epi-position in given antigen); U.S. Patent number 4,708,871 (methods of evaluation and the antigenic epi-position of chemosynthesis); With Geysen et al., molecular immunology (1986) 23:709-715 (identify given antibody is had the technology of the peptide of high-affinity).
As used herein, term " T-cell epitope " is meant the feature structure that can induce a kind of peptide of peptidic constructs or relevant haptenic T-cellular immunity.In this, those of ordinary skill has been accepted the T-cell epitope and has been included in the peptide of MHC molecule in conjunction with the linear peptides determinant that has expanded configuration in the crack (Unanue et al., science (1987) 236:551-557).Polypeptide is converted into mhc class ii dependent linearity peptide determinant (length is generally between 5-14 amino acid) is defined as " antigen processing ", this presents cell (APC) by antigen and finishes.Specifically, the T-cell epitope is by the local feature of small peptide structure such as one-level aminoacid sequence characteristic, and the secondary structure that comprises electric charge and hydrophobicity and do not rely on some folding type of whole polypeptide determines as spirality.In addition, it is generally acknowledged that the small peptide that can be discerned by helper T-cell generally is amphipathic structure, comprise a hydrophobic side chain (with the MHC molecular reaction) and a hydrophilic side chain (with the T-cell receptors reaction), (Margalit etal., computer is inferred the T-cell epitope, the new vaccine Marcel-Dekker that produces, Inc, ed.G.C.Woodrow et al., (1990) pp.109-116), amphipathic in addition structure have an alpha-helical conformation (referring to for example, Spouge et al., J.Immunol. (1987) 138:204-212; Berkower et al., J.Immunol. (1986) 136:2498-2503).
So, utilize many computer programs can easily infer the protein fragments that comprises the T-cell epitope.(referring to for example, Margalit et al., the computer of T-cell epitope is inferred, is newly produced vaccine Marcel-Dekker, Inc, ed.G.C.Woodrow et al., (1990) pp.109-116).Usually these programs are compared the aminoacid sequence of peptide with the known array of inducing the T-cell response, seek and be sure of to be the required amino acid spectral pattern of T-cell epitope.
" immunogenic protein " or " immunogenicity aminoacid sequence " is respectively protein or the amino acid that causes immunne response in the main body that it is applied.In the present invention, " GnRH immunogen " be meant, when entering the host, and the GnRH molecule that immune stimulatory is replied.In this, the GnRH immunogen comprises the many bodies corresponding to GnRH peptide sequence selected more than; Specifically, have many bodies of multiple or the GnRH peptide sequence that the series connection multiple is selected, many bodies of the multiple or GnRH epi-position that the series connection multiple is selected, or its their many bodies of combination that can be contemplated to.
" immunne response " to antigen or vaccine is to produce the cell of compositions for use or vaccine and/or antibody-mediated immunne response in the host.Usually, a kind of like this result who replys below one or more; The anti-specifically generation that is included in antigenic antibody, B cell, helper cell, repressor T cell and/or cytotoxic T cell and/or gamma delta T cells in composition therefor or the vaccine.Can utilize any one detection immunne response of a plurality of immunoassay known in the art.
Term " leukotoxin polypeptide " or " LKT polypeptide " mean and comprise at least one T-cell epitope and originate from that to belong to C-terminal same feeling aminoacid sequence Gly-Gly-X-Gly-X-Asp be proteinic polypeptide (the Highlander et al. of family of the molecule of feature, DNA (1989) 8:15-28), wherein X is Lys, Asp, Val or Asn.These protein comprise the leukotoxin that originates from haemolysis bus moral Salmonella and Actinobacillus pleuropneumoniae, and intestinal bacteria AH (Strathdee et al., Infect.Immun. (1987) 55:3233-3236; Lo, Can.J.Vet.Res. (1990) 54:S33-S35; Welch, Mol.Microbiol. (1991) 5:521-528) and other.Known this toxin family is " RTX " family (Lo, Can.J.Vet.Res. (1990) 54:S33-S35) of toxin.In addition, term " leukotoxin polypeptide " is meant chemosynthesis, separates from the organism of expressing it or the leukotoxin polypeptide of recombinant production.And, this term mean have basically with specific natural leukotoxin molecule in the immunogen protein of the contiguous sequence homologous aminoacid sequence that exists.So this term comprises total length and partial sequence and analogue thereof.Though natural total length leukotoxin has the leukotoxin activity, term " leukotoxin " also means and keeps the molecule that immunogenicity lacks the cytotoxicity characteristic of natural leukotoxin.The nucleotide sequence of several leukotoxins and corresponding amino acid sequence are known.Referring to for example, U.S. Patent number 4,957,739 and 5,055,400; Lo et al., Infect.Immun. (1985) 50:667-67; Lo et al., Infect.Immun. (1987) 55:1987-1996; Strathdee et al., Infect.Immun. (1987) 55:3233-3236; Highlander et al., DNA (1989) 8:15-28; Welch, Mol.Microbiol. (1991) 5:521-528.In mosaic produced according to the invention, selected leukotoxin peptide sequence has improved the immunogenicity that merges the many bodies of GnRH, this is to contain T-cell epitope and other thing that length is 5-14 amino acid whose little peptide fragment and realize by providing, described little peptide fragment can be compound with MHC II quasi-molecule, to be presented in and to activate the T-helper.As following further discussion, these T-cell epitopes are present in the whole leukotoxin molecule and think to concentrate on the N-terminal portions of leukotoxin, promptly between amino-acid residue 1-199.
As used herein, the leukotoxin polypeptide that " lacks the leukotoxin activity " is meant that aforesaid and natural total length leukotoxin is (as U.S. Patent number 5,055,400 and 4,957, the total length haemolysis bus moral Salmonella leukotoxin of describing in 739) compares the shortage significant cytotoxicity, but still keep the leukotoxin polypeptide of immunogenicity and at least one T-cell epitope.Can utilize known detection method such as Korzeniewski et al., the serum lactic dehydrogenase that immunological method magazine 64:313-320 describes discharges the white corpuscle cytotoxic activity that detects the leukotoxin polypeptide, wherein by measuring cytotoxicity from the release of ox neutrophil serum lactic dehydrogenase.If compare the release that causes the serum lactic dehydrogenase with statistical significance with the non-leucocytotoxicity molecule of contrast, then this molecule is accredited as and has leucocytotoxicity.
In the present invention, comprise the chimeric construct of LKT-GnRH that lacks the active leukotoxin polypeptide of leukotoxin several important benefits are provided.At first, cause local cells death (PMN and scavenger cell) and cause serious inflammatory reaction and abscess in injection site successively, thereby need to lack the active leukotoxin polypeptide of leukotoxin because the injection active toxin enters a physical efficiency.In this, the leukotoxin activity that causes killing scavenger cell can cause reducing antigen and present, thereby causes suboptimal immunne response.As finding at the non-leucocytotoxicity LKT polypeptide that is used for production fusion rotein of the present invention, the LKT gene of removing the brachymemma that the cytotoxicity part produced can be higher than the horizontal expression of total length LKT far away.In addition, use non-leucocytotoxicity LKT polypeptide to reduce the antigenic whole amount of leucocytotoxicity-GnRH in the syzygy that the present invention who keeps enough T-cell antigen makes up, this antigen amount is that to make the host produce enough B-cell responses to selected GnRH polypeptide necessary.The specific example that lacks the active immunogenicity leukotoxin of leukotoxin polypeptide, the LKT352 and the LKT111 that describe in detail very much below comprising.
" LKT352 " means the protein (Fig. 2, ATCC registration number 68283) of the lktA gene that originates among the plasmid pAA352.The nucleotide sequence of this gene and corresponding amino acid sequence have been described as shown in Figure 3 in international publication number WO91/15237.931 amino acid of this genes encoding, molecular weight is about the leukotoxin of the brachymemma of 99kDa, and it lacks the cytotoxicity part of molecule.Purifying is expressed and be easier to the gene of the brachymemma of the Chan Shenging level (surpass 40% total cell protein, and the total length form being 1% total cell protein) that is higher than full-length molecule far away like this.In the present invention, derivative LKT352 not necessarily physically originates from the sequence among the plasmid pAA352.On the contrary, it may produce by any way, for example comprises chemosynthesis or recombinant production.In addition, this proteinic aminoacid sequence only need with described sequence homology basically.So as long as the LKT polypeptide can strengthen its institute's bonded immunogenicity of antigens and also lack the leukotoxin activity, sequence variations can exist.
" LKT111 " means the leucocytotoxicity polypeptide (Fig. 6, ATCC registration number 69748) of the gene that originates among the plasmid pCB111.The nucleotide sequence and the corresponding amino acid sequence of this gene are shown in Fig. 7.A clipped form of this genes encoding leukotoxin, it be by remove inside dna fragmentation that a length is about 1300bp from be present in plasmid pAA352 recombination leukocyte toxin gene (Fig. 2, ATCC registration number 68283) development and come.The molecular weight of LKT111 polypeptide is about 52kDa (comparing with 99kDa LKT 352 polypeptide), but kept from containing of LKT352 enough necessary T-cell epitopes of T-cell immunogenicity the N-terminal portions and be useful on the C-terminal portions of the suitable restriction site of production fusion rotein of the present invention from containing of LKT352.In the present invention, LKT111 leucocytotoxicity peptide is not to originate from the sequence that is present among the plasmid pCB111.On the contrary, it can produce by any way, for example comprises chemosynthesis or recombinant production.In addition, this proteinic aminoacid sequence only need with described sequence homology basically.So as long as this protein can strengthen its institute's bonded immunogenicity of antigens and also lack the leukotoxin activity, sequence variations can exist.
When leukotoxin-GnRH polypeptide mosaic manys body when having the ability of higher initiation immunne response than corresponding independent GnRH, it demonstrates " enhanced immunogenicity ".By using the contrast of the many bodies of specific leukotoxin-GnRH polypeptide and GnRH to animal and utilizing as known in the art radioimmunoassay and relatively more anti-both antibody titer of the standard test of ELISA can determine these enhanced immunogenicities.
" reorganization " albumen or polypeptide be meant by recombinant DNA technology, i.e. the polypeptide of producing from the foreign DNA construct cell transformed of the required polypeptide of encoding." synthetic " albumen or polypeptide are those chemosynthesis preparations.
" nucleotide coding sequence " of DNA " encoding sequence " or specific protein is in the control lower body of suitably regulating sequence or in-vitro transcription and the dna sequence dna of translating into polypeptide.Determine the boundary of encoding sequence by terminal translation stop codon of the terminal initiator codon and 3 ' (carboxyl) of 5 ' (amino).Encoding sequence can include, but are not limited to, the prokaryotic organism sequence, and from the cDNA of eukaryote mRNA, from the genomic dna sequence of eukaryote (as, Mammals) DNA, even the synthetic dna sequence dna.Transcription termination sequence is usually located at 3 ' end of encoding sequence.
DNA " control sequence " is a promoter sequence, ribosome bind site, and polyadenylation signal, transcription termination sequence, the upstream regulation district, enhanser, the general name that waits, they regulate transcribing and translating of encoding sequence jointly in host cell.
An encoding sequence " is operably connected " with another encoding sequence and is meant that working as RNA polymerase is transcribed into mRNA with two encoding sequences, when translating into the chimeric polyeptides of these two encoding sequences codings subsequently.Encoding sequence does not need adjacency one by one, as long as finally processed the required chimeric protein of generation by transcription sequence.When control sequence was controlled transcribing of encoding sequence, it " was operably connected " with encoding sequence.
Control sequence " instructs transcribing of encoding sequence " and is meant that working as RNA polymerase is transcribed into mRNA in conjunction with promoter sequence and encoding sequence, translates into this encoding sequence encoded polypeptides subsequently in cell.
" host cell " is or can be by the exogenous DNA array cell transformed.
When foreign DNA was in the transfered cell film, cell was by foreign DNA " conversion ".Foreign DNA can or can unconformability (covalently bound) be formed the genome of cell in the chromosomal DNA.In prokaryotic organism and yeast, for example, foreign DNA can exist by episome, as plasmid.For eukaryotic cells, stable transformant is that wherein foreign DNA has been integrated in the karyomit(e) so that can have entailed daughter cell by chromosome duplication.This stability by eukaryotic cells can form the clone that contains this foreign DNA daughter cell group or clone confirm.
When in determining the molecule of length during at least about the Nucleotide of 80% (preferably at least about 90%, most preferably at least about 95%) or amino acid coupling, two DNA or peptide sequence " basic homology ".In the Southern hybrid experiment, for example, can identify basic homologous dna sequence dna under the definite tight Jin condition of particular system.Determine that suitable hybridization conditions is well known by persons skilled in the art.Referring to for example, Sambrook et a., as mentioned above; Dna clone, Vol I and II, as mentioned above; Nuclear Acid hybridization, as mentioned above.
" allos " of DNA construct district is attached to or is present in discernible DNA section among another dna molecular, and this another molecule does not combine existence with other molecule in native state.So, when allos district coding bacterial gene, this gene usually with the DNA side joint of the bacterial gene of side joint not in the derived bacterium genome.Another example of allogeneic coding sequence is itself construct at the non-existent encoding sequence of occurring in nature (composition sequence that for example, has the codon that is different from natural gene).As used herein, allelotrope variation or abiogenous catastrophic event do not produce the allos district of DNA.
" vertebrates main body " is meant any member of chordate subphylum, include, but are not limited to, and as rodent, ox, pig, sheep, goat, horse and people's Mammals; As dog, the performing animal of cat; Comprise as comprising chicken the raising and train of cock of turkey and hen and other chicken class bird, wild and can hunt the bird of bird.The special age do not represented in this term.So adult and new born animal is all included.B. group method
Center of the present invention is to have found that the leukotoxin polypeptide when with the coupling of selected GnRH polypeptide iteron (or many bodies), can give the bonded GnRH component higher immunogenicity.In this, the effect of leukotoxin polypeptide is the carrier proteins of selected many bodies of GnRH to be provided for the immunity system of main body with high immunogen form.So the chimeric protein that the present invention makes up can be mixed with vaccine composition, it provides the enhanced immunogenicity for the GnRH polypeptide that exists.The leukotoxin gene also helps from this chimeric protein of cell purification of expressing it with the fusion of the GnRH polypeptide of selecting.
Therefore, be the leukotoxin mosaic that comprises the leukotoxin that merges with an above GnRH peptide sequence as the example of this paper.The specially designed embodiment of the present invention comprises the mosaic that comprises the leukotoxin polypeptide that merges with the many bodies of GnRH, wherein said many bodies repeat GnRH decapeptide sequence by at least one substantially to be formed, or is made up of at least one repeating unit corresponding to the sequence of the epi-position of at least one selected GnRH molecule.In addition, selected GnRH peptide sequence can be all identical, or can be corresponding to the epi-position of the different derivative of GnRH, analogue, mutant or GnRH, as long as they keep causing the ability of immunne response.Figure 1A has described the nucleotide sequence of a representative GnRH decapeptide.GnRH sequence of the present invention is to modify by the Pyrrolidonecarboxylic acid that exists in the alternative native sequences of the glutamine of N-end.This is special alternatively to make molecule keep natural L-glutamic acid structure but has also kept the no charge structure of Pyrrolidonecarboxylic acid.Therefore, the peptide that obtains does not need the cyclisation of glutaminic acid residue and can take place to produce under the necessary condition of cyclisation in shortage.
Because the GnRH sequence is quite short, can easily produce with the synthetic technology of describing in detail below.In the present invention, in order in vertebrates, to help to cause suitable immunne response, give bonded GnRH polypeptide (as carrier proteins) immunogenicity with the leukotoxin peptide sequence to endogenous GnRH.By this way, can in by the inoculation animal, regulate reproductivity with the immunization of GnRH by destroying oestrus cycle or spermatogenesis.At U.S. Patent number 4,975,420 can find going through of relevant GnRH.
In addition, this paper is noticeable especially provided at present raise and train with farm livestock industry in the invasive methods of sterilisation such as the surgery that use castrate surgery hystero-oophorectomy and suchlike effective alternative method reliably.Leukotoxin-GnRH the mosaic that utilizes in vertebrates according to the present invention provides an effective alternative method to the immunosuppression regeneration activity, and wherein this construct is implemented in the homogeneous inactivation that produces regeneration activity in the immune animal.In this, for the means that successfully substitute surgical method are provided, sterilization vaccine product suitable when individual animals is replied a small amount of vaccination must make the regenerative power inactivation equably.This characteristic is to the immune sterilization particularly important of compoundanimal, and particularly, it is willing to the male piggy of ground immunity castrating as the people, with " not castrating pig is infected with " of the synthetic generation that prevents male steroid in the normal function testis of male piggy.Referring to for example Meloen et al., vaccine (1994) 12 (8): 741-746.Because the inadequate immunogenicity of GnRH peptide and/or relevant carrier system, can not induce enough immunne responses to endogenous GnRH with consequent various existing vaccines based on GnRH, the effort of this product of exploitation in past does not also produce the constant result.
Therefore, in order to obtain the more GnRH peptide antigen of high immunogenicity, the leukotoxin-GnRH polypeptide mosaic of the present invention's design contains the GnRH part corresponding to GnRH peptide sequence selected more than.This characteristic is based on such understanding, intrinsic protein generally can by they epi-position diversity and give himself antigenicity effectively, as mentioned above.Specifically, the new chimeric GnRH part of the present invention's design can comprise multiple or the selected GnRH sequence of series connection multiple, the multiple or selected GnRH epi-position of series connection multiple, or its any suitable composition.In this, the technology that utilization is described in detail above can be identified the GnRH epi-position, perhaps can test the proteic segmental immunogenicity of GnRH and can substitute whole polypeptide be used for composition active fragments.Figure 1B has described the specific GnRH sequence partly of the present invention, wherein four GnRH sequences, respectively by (1), (2), (3) and (4) expression, separated by ternary amino acid intervening sequence, this intervening sequence contains various Serines and glycine residue combination.In this oligopolymer, contain a non-conserved amino acid every one GnRH sequence (use respectively (2) and (4) expression) in the second position of GnRH decapeptide and substitute, substitute the His residue that exists in the natural GnRH sequence by the Asp residue.Alternate many bodies of GnRH sequence of Chan Shenging provides a high immunogenicity GnRH antigen peptide that can be used for fusion rotein of the present invention like this.The inventor has also imagined other additional corresponding to any single or multiple amino acid, as to substitute and/or the lack GnRH analogue that can be used for repetition or alternate many bodies sequence especially.
In addition, the specific GnRH part of Figure 1B description contains intervening sequence between the GnRH part.In order to give this construct enhanced immunogenicity, the inventor has imagined the usage policy of the various intervening sequences between the selected GnRH polypeptide especially.Therefore, in the present invention, selected intervening sequence can code length be all kinds of one or more amino acid whose parts.Selected spacer groups can preferably provide the enzyme cleavage site so that can process the mosaic of this expression by proteolytic ferment (APC ' s or like that) in vivo, produces multiple peptide---respectively containing the T-cell epitope that at least one originates from carrier part (leukotoxin part)---and preferably merge with complete basically GnRH peptide sequence.In addition, can make up spacer groups so that it is tangible external sequence that the connection district between selected GnRH component is contained concerning the animal of immunization, thereby give the GnRH peptide enhanced immunogenicity of associating.In addition,, can make up intervening sequence for T-is provided cell antigen, as the sequence amphipathic and/or the alpha-helix peptide sequence of encoding, this area it is generally acknowledged that this sequence provides immunogenic T-cell helper epitope.In this, the selection of the specific T-cell epitope that is provided by such intervening sequence can depend on the particular ridge Vertebrate kind of being inoculated and change.Though, for example understanding the specific GnRH part that contains intervening sequence, the inventor has also imagined the many bodies of GnRH of the GnRH sequence (not getting involved intervening sequence) that comprises direct adjacency.
The leukotoxin of recombinant production chimeric protein form-GnRH polypeptide complex easily.Chimeric GnRH part can be blended in 5 ' or 3 ' end of the leukotoxin part of this molecule, and perhaps the GnRH part can be positioned the site, inside of leukotoxin molecule.The nucleotide sequence of coding total length haemolysis bus moral Salmonella A1 leukotoxin is determined, referring to for example, and Lo, infection immunity (1987) 55:1987-1996; U.S. Patent number 5,055,400.In addition, herein disclosed is several anomaly leukotoxin gene orders.
Equally, pig, ox, the encoding sequence of sheep GnRH is determined, (Murad et al., hormone and hormone antagonist, Therapeutic pharmacology principle, sixth version (1980)), and the cDNA of people GnRH cloned, so set up its sequence (Seeburg et al., nature (1984) 311:666-668) well.Other GnRH polypeptide of known array is open, as is present in the GnRH molecule (the open No.WO86/07383 of disclosed international patent application on December 18th, 1986) in salmon and the chicken.In this, it should be noted that vertebrates, particularly GnRH is a high conservative in the Mammals, in addition, pig, ox, sheep and people GnRH sequence are identical.Utilize that the known recombinant technology of persons skilled in the art can gene clone separates and connects together with GnRH with needed leukotoxin.Referring to for example, Sambrook et al., as mentioned above.
Another kind is method optionally, can synthesize the dna sequence dna of preparation rather than clones coding chimeric protein.Suitable codon with coding specific amino acids sequence can design this dna sequence dna.Generally speaking, will be used for expressing as infructescence, people will be the selected preferred codon of predetermined host.From assembling complete sequence and be assembled into complete encoding sequence by the overlapping oligonucleotide of standard method preparation.Referring to for example, Edge, nature (1981) 292:756; Nambair et al. science (1984) 223:1299; Jay et al.J.Biol.Chem. (1984) 259:6311.
In case prepare or separated the encoding sequence of chimeric protein, can clone into any suitable carriers or replicon to them.The known numerous cloning vectors of those skilled in the art, using which kind of carrier is the problem of a selection.The example of the host cell that the recombinant DNA carrier that is used to clone and they transform comprises lambda phage (intestinal bacteria), pBR322 (intestinal bacteria), pACYC177 (intestinal bacteria), pKT230 (Gram-negative bacteria), pGV1106 (Gram-negative bacteria), pKAFR1 (Gram-negative bacteria), pME290 (non-intestinal bacteria Gram-negative bacteria), pHV14 (intestinal bacteria and subtilis), pBD9 (genus bacillus), pIJ61 (streptomycete), pUC6 (streptomycete), YIp5 (yeast), YCp19 (yeast) and bovine papilloma virus (mammalian cell).Generally can referring to Dna clone: Vols.I and II, as mentioned above; T.Maniatis et al., as mentioned above; B.Perbal, as mentioned above.
Fusion gene is placed promotor, under the control of ribosome bind site (being used for bacterial expression) and optional operator gene (this paper is generically and collectively referred to as " control " element), so that the dna sequence dna of coding chimeric protein is transcribed into RNA in the host cell that is transformed by the carrier that contains this expression construct.This encoding sequence can or can not contain signal peptide or homing sequence.For example utilize, natural haemolysis bus moral Salmonella promotor, intestinal bacteria tac promotor or a-protein gene (spa) promotor and signal sequence can be expressed chimeric protein of the present invention.Homing sequence can be removed by the bacterium host in the translation post-treatment.Referring to for example, U.S. Patent number 4,431,739; 4,425,437; 4,338,397.
Except control sequence, also may need add to regulate sequence, regulate with growth the relative host of expression of this protein sequence.It is well known by persons skilled in the art regulating sequence, example comprises replys chemistry or physical stimulation, comprises whether regulate compound exists, and those sequences that expression of gene begun or close, the regulatory element of other type also may reside in the carrier, for example enhancer sequence.
Having made up expression vector makes specific fusion encoding sequence be positioned to have in the carrier of suitable adjusting sequence, encoding sequence should make this encoding sequence transcribe (that is, on control sequence with this encoding sequence of dna molecular bonded rna polymerase transcribe) under " control " of control sequence with respect to the position of control sequence and direction.For reaching this purpose, may need to modify coding the sequence of interested specific chimeric protein.For example, in some cases, may need to modify this sequence and make it be connected in control sequence, promptly keep single open reading frame with suitable direction.Control sequence is regulated sequence with other and can be connected with encoding sequence before at insertion carrier (aforesaid cloning vector).Another kind of optionally method, encoding sequence can directly be cloned the expression vector that has into contained control sequence and suitable restriction site.
In some cases, need adding can cause that polypeptide is from the secretion of host organism, the sequence of cutting secretion signal subsequently.Also may need to prepare the mutant or the analogue of required chimeric protein.By the insertion of the disappearance of the sequence of a part of coded protein, sequence and/or in sequence, substitute one or more Nucleotide and can prepare mutant or analogue.The technology of modified nucleotide sequence as site-specific mutagenesis, is well known by persons skilled in the art.Referring to for example, T.Maniatis et al., as mentioned above; Dna clone, Vols.I and II, as mentioned above; Nucleic acid hybridization, as mentioned above.
Many prokaryotic expression carriers are known in the art.Referring to for example, U.S. Patent number 4,440,859; 4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246; 4,342,832; Simultaneously enclose patent application GB2,121,054 referring to English; GB2,008,123; GB2,007,675; With european patent application 103,395.Yeast expression carrier also is known in the art.Referring to for example, U.S. Patent number 4,446,235; 4,443,539; 4,430,428; Simultaneously referring to european patent application 103,409; 100,561; 96,491.
According to selected expression system and host, produce protein of the present invention by the host cell that above-described expression vector transforms by growth under the condition of expressing desired protein.Separate this chimeric protein and purifying from host cell then.If the expression system secretory protein enters growth medium, can be from substratum direct purification protein.If protein is not secreted, separate by cell lysate.The suitable growth conditions and the selection of recovery method are the known technologies of this area.
According to the aminoacid sequence of determining, by also producing chimeric protein of the present invention as solid-phase peptide synthetic chemical synthesis process.These methods are well known by persons skilled in the art.Referring to for example, J.M.Stewart and J.D.Young, solid-phase peptide is synthetic, second edition, Pierce Chemical Co., Rockford, IL (1984) and G.Barany and R.B.Merrifield, peptide: analysis, synthetic, biology, editor E.Gross and J.Meienhofer, Vol.2, the academic press, New York (1980), pp.3-254 is used for the solid-phase peptide synthetic technology; And M.Bodansky, the peptide composition principle, Springer-Verlag, Berlin (1984) and E.Gross and J.Meienhofer, Eds., peptide: analysis, synthetic, biology, above-mentioned document, Vol.1, it is synthetic to be used for typical solutions.
Can comprise said proteinic vaccine composition and carry out immunization by using with the chimeric protein that makes up according to the present invention animal individual.Before immunization, may need further to strengthen the immunogenicity of specific chimeric protein.This can be with any the finishing in the several method well known by persons skilled in the art.For example, leukotoxin-GnRH polypeptide amalgamation protein can combine administration with one two grade carrier.For example, a fragment can combine with macromolecular carrier.The macromole that the metabolism that appropriate carriers is big typically is slow, as: protein; Polysaccharide, as agarose, agar, Mierocrystalline cellulose, Mierocrystalline cellulose globule and like that; Polymeric amino acid, as polyglutamic acid, polylysine and like that; Amino acid copolymer; Virion with inactivation.Useful especially protein substrate is a serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin and other protein well known to those skilled in the art.
These protein substrates can use with their natural form or can react the functional group of modifying them by the succinylation of for example lysine residue or with the Cys-thiolactone.Also can mix carrier (or selected GnRH polypeptide) to sulfydryl by for example function amino with the reaction of the N-hydroxy-succinamide ester of 2-imino-thiolane or 3-(4-two thiopyridines bases) propionic salt.Suitable carriers also can be modified so that mix spacerarm (as hexamethylenediamine or other onesize bifunctional molecule) is used for binding peptide.
Other suitable carrier that is used for chimeric protein of the present invention comprises as U.S. Patent number 5,071, the VP6 polypeptide of 651 disclosed rotaviruss, or its function fragment.While is virus protein and the immunogenic fusion product of leukotoxin-GnRH usefully, and wherein this fusion product is by U.S. Patent number 4,722, and 840 disclosed methods are made.Also have other suitable carriers to comprise cell such as lymphocyte, because the presenting to have imitated and produce the natural presentation mode that exists in the individuality of immunization state of this form.Another kind of optionally method, fusion rotein of the present invention can with red corpuscle, preferably the red corpuscle coupling of intrasubject.Peptide and protein or cell link coupled method are well known by persons skilled in the art.
New chimeric protein of the present invention also can be by expressing their vector virus administration.Can be used for vector virus of the present invention and include, but are not limited to, bovine vaccine and other poxvirus, adenovirus, simplexvirus.For example, vaccine virus recombinant chou that can following this new chimeric protein of construction expression.At first the DNA of coding particular leukocyte GnRH chimeric protein is inserted appropriate carriers and make it and bovine vaccine promotor adjacency, and and as the dna sequence dna side joint of the thymidine kinase of encoding.The cell that infects with bovine vaccine simultaneously with this carrier transfection then.By homologous recombination the bovine vaccine promotor is added that the gene of code book invention chimeric protein inserts viral genome.By when 5-bromouracil deoxyribose exists, cultivating this cell and selecting viral plaque and can select the TK-recombinant chou that obtains with resistance.
Separately or with pharmaceutical acceptable carrier or mixed with excipients administration, also be possible with chimeric protein immunization individuality produced according to the invention.Typically, vaccine is prepared to injectable liquor or suspension; Also can be prepared into and before injection, be suitable for the solid form that is dissolved in liquid vehicle or is suspended in liquid vehicle.Also can be wrapped in the liposome vectors with preparation emulsification or with active ingredient.The former sexual element of the active immne often carrier of the vehicle acceptable and compatible with active ingredient with containing pharmacy mixes.Suitable carriers for example is, water, salt, glucose, glycerine, ethanol, or like that and their associating.In addition, if desired, carrier can contain auxiliary substance such as the moistening agent or the emulsifying agent of minimum, pH buffer reagent, or the adjuvant of enhancing vaccine effect.Adjuvant for example can comprise, Muramyl dipeptide, avridine, aluminium hydroxide, oil, Saponin/TSM and other material known in the art.The concrete grammar for preparing these medicine types is known, or those skilled in the art are conspicuous.Referring to for example, the pharmaceutical science of Remington, Mack publishing company, Easton, Pennsylvania, the 18th edition, 1990.Under any circumstance, will contain a certain amount of protein that is suitable in the individuality of treatment, obtaining desirable immunization state in the composition of administration or the prescription.
Other the vaccine preparation that is suitable for other administering mode comprise suppository and, under some situation, in the smoke substance, nose, oral prescription and lasting release formulation.For suppository, carrier form will comprise conventional junction compound and carrier as, poly-alkaline ethylene glycol, or triglyceride.Can form these suppositorys in the mixture of the active ingredient of about 2% scope of preferably about 1%-from containing about 10% (w/w) of the 0.5%-that has an appointment.Oral carrier comprises those normally used vehicle, for example, and the N.F,USP MANNITOL of pharmaceutical grade, lactose, starch, magnesium, stearate, asccharin sodium cellulosate, magnesiumcarbonate and like that.These oral vaccine compositions can be with solution, suspension, and tablet, pill, capsule is kept release formulation, or form of powder takes, and contains the active ingredient of the 1%-that has an appointment about 30%, preferably about 2%-about 20%.
The prescription of intranasal administration generally includes and causes that neither nasal mucosa stimulates the also not obvious carrier of upsetting fibre function.Thinner such as water, salt solution or other known substance can be used for the present invention.The nose prescription also can contain sanitas as, but be not limited to trichloro-butyl alcohol, benzalkonium chloride.Tensio-active agent may show and strengthen nasal mucosa to the proteic absorption of main body.
By chimeric protein being mixed carrier such as liposome, the polymer of non-absorbent sealing such as ethylene-vinyl acetate multipolymer and Hytrel  multipolymer, but the polymkeric substance of imbibition such as hydrogel, or absorbable polymer such as collagen and some polyprotonic acid or as those be used to make absorbable suture the polyester production control or sustained release formulation.Chimeric protein also can provide with the little pump of implantation known in the art.
In addition, chimeric protein (or its mixture) can be mixed with vaccine composition with neutrality or salt form.Pharmaceutically acceptable salt comprises acid salt (free amine group of usefulness active polypeptide forms) and those usefulness mineral acid example hydrochloric acid or phosphoric acid, or organic acid such as acetate, oxalic acid, tartrate, phenylglycollic acid and formation like that.The salt that forms from free carboxy also can originate from mineral alkali as, sodium hydroxide, potassium, ammonium, calcium, or iron and organic bases such as Isopropylamine, Trimethylamine 99,2-ethylamino-ethanol, Histidine, PROCAINE HCL, PHARMA GRADE and like that.
For the immunization individuality, with selected GnRH-leukotoxin mosaic with parenteral in the suitable carriers, intramuscular injection administration usually.But release also is acceptable in other administering mode such as subcutaneous, intravenous injection and nose.The injectable vaccine preparation will contain the significant quantity active ingredient that is present in the carrier, and accurately amount is determined by those skilled in the art easily.The scope of typical active ingredient is for the composition of about 1%-about 95% (w/w), if or suitable even higher or lower.The amount of administration is according to the animal of being treated, and the ability of the immuning system synthesising antibody of animal and the degree of protection that needs are determined.
For this vaccine preparation, for producing immunne response, every ml injection solution contains about 1 μ g-1mg when administration, and more generally 5 μ g-200 μ g GnRH polypeptide are suitable.In this, the cytotoxic ratio of GnRH dialogue will change according to particular leukocyte toxin and the GnRH polypeptide fraction that being used to of selecting makes up these molecules in the white corpuscle of this vaccine preparation-GnRH antigen.Specifically, at the leukotoxin that is used for producing vaccine preparation according to the present invention-GnRH polypeptide, the nearly 1-25%GnRH of each fusion molecule, preferably about 3-20% and most preferably about 7-1 7%GnRH polypeptide.The increase that is present in the percentage ratio of the GnRH in the LKT-GnRH antigen has reduced the total antigen amount that must use to individuality the B-cell response of GnRH in order to cause effectively.By setting up the routine test of dose response curve, those of ordinary skills can easily establish effective dose.By using at least one dosage and preferably the white corpuscle of two dosage-GnRH polypeptide can the immunization individuality.In addition, can carry out multiple dosing to animal, to keep immunological status.
Be the example of realizing particular embodiment of the present invention below.These examples do not plan to limit the scope of the invention by any way only for illustration purpose.
C. Experiment Material and method
Enzyme is bought from commercial channels and is used according to the explanation of manufacturers.Buy simultaneously radioactive nuleus thuja acid and nitrocellulose filter paper from commercial channels.
In the clone of dna fragmentation, except that explanation was arranged, all DNA operations were all carried out according to standard method.Referring to Sambrook et al., as mentioned above.Use restriction enzyme, T from the commercial supplier purchase and according to the explanation of manufacturers 4Dna ligase, e. coli dna polymerase I Klenow fragment and other biological reagent.On sepharose, separate double chain DNA fragment.
In pUC13 and λ gt11 phage, prepare cDNA and genomic library respectively with standard technique.Referring to dna clone: Vol I and lI, as mentioned above.
Separate obtaining haemolysis bus moral Salmonella biotype A from the lung of the ox of the bus moral bacterium disease that dies of pneumonia, serotype 1 (" A1 ") bacterial strain B122 also is stored in the defibrinated blood at-70 ℃.At blood agar dull and stereotyped or with 5% horse serum (Canada) the additional brain heart leaches meat soup (Difco laboratory, Detroit carries out routine in MI) and breed for Gibco Canada Ltd., Burlington.All cultivations are at 37 ℃ of incubations.
Embodiment 1
Separate haemolysis bus moral Salmonella leukotoxin gene
In order to separate the leukotoxin gene, utilize standard technique to make up the gene library of haemolysis bus moral Salmonella A1 (bacterial strain B122).Referring to, Lo et al., Infect.Immun., as mentioned above; DNA Clone: Vol.I and II, as mentioned above; With Sambrook et al., as mentioned above.In plasmid vector pUC13, make up genomic library and in phage gt11, made up the DNA library.The clone who obtains is used for transformed into escherichia coli and collection and screened and from surviving that haemolysis bus moral Salmonella infects and strengthening so that increase the single clone of sero-reaction of the ox of antileukocidin antibody horizontal with the concentrated culture supernatant of haemolysis bus moral Salmonella.By cell lysate and ox neutrophilic leukocyte are cultivated the ability that the release of also measuring latter's serum lactic dehydrogenase subsequently detects positive colony generation leukotoxin.
Identified several positive colonies and make restriction endonuclease map and analyze these recombinant chous.As if the leukotoxin gene with the past clone is identical for a clone.Referring to, Lo etal., Infect.Immun., as mentioned above.In order to prove, littler fragment and restriction figure have relatively been cloned again.The DNA that has confirmed about 4kb is cloned.For the total length recombinant chou of the about 8kb of separation length, separated bigger clone gradually by carrying out chromosome walking (5 '-3 ' direction).The construct called after pAA114 that this is last.This construct contains whole leukotoxin gene order.
Klenow fragment with dna polymerase i adds nucleoside triphosphate acid treatment lktA, a MaeI restriction endonuclease fragment from whole leukotoxin gene of containing of pAA114, and be connected to the SmaI site of cloning vector pUC13.With this plasmid called after pAA179.Thus, with SmaI digestion based on the carrier pGH432:lacI of ptac in two expression construct of preparation.One is pAA342, contain 5 '-AhaIII fragment of lktA gene, and another is pAA345, contains above-described whole M aeI fragment.Clone pAA342 is with the leukotoxin peptide of high level expression brachymemma, and pAA345 is with low expression level total length leukotoxin.So 3 ' terminal pAA342 with StyI BamHI digestion of lktA gene (from the StyI BamHI fragment of pAA345) is connected, and produces plasmid pAA352.Fig. 2 has represented the structure of pAA352, and Fig. 3 has represented to originate from the nucleotide sequence of haemolysis bus moral Salmonella leukotoxin of pAA352 construct (hereinafter being called LKT 352) and the aminoacid sequence of supposition.
Embodiment 2
Make up the LKT-GnRH syzygy
Representational LKT-GnRH syzygy makes up as follows.Phosphoramidate (phosphoramidite) chemical process with standard has made up the oligonucleotide that contains corresponding to the sequence of single copy GnRH and four multiple multiple GnRH on Pharmacia gene assembler.Figure 1A and 1B have shown the sequence of these oligonucleotide.The annealing of this oligonucleotide and be connected to carrier pAA352 (ATCC numbers 68283, as mentioned above) with restriction endonuclease BamH1 digestion.This carrier contains haemolysis bus moral Salmonella leukotoxin gene.Utilize the DNA transformed into escherichia coli bacterial strain MH3000 that connects.Make restriction endonuclease map and identify the transformant that contains this oligonucleotide insertion body.
By being connected to the GnRH tandem repetitive sequence for preparing eight copies on the carrier that digests with restriction endonuclease BamH1 with the annealing of the GnRH oligonucleotide of four copies and them.Oligopolymer is designed to make BamH1 site, upstream lose function when inserting and guarantee that the insertion of the oligopolymer of other copy will be oriented to correct single open reading frame.Figure 1B has shown the sequence of this oligonucleotide.Separate from the plasmid DNA of intestinal bacteria MH3000 bacterial strain then and be used to transform bacterial strain JM105.With this recombinant plasmid called after pCB113 (GnRH of LKT352:4 copy, ATCC registration number 69749) and pCB112 (GnRH of LKT352:8 copy).Fig. 4 has shown recombinant plasmid pCB113, and plasmid pCB112 is identical with pCB113, and different is that a plurality of copy GnRH sequences (corresponding to the oligopolymer of Figure 1B) are inserted twice as mentioned above.Fig. 5 has shown the nucleotide sequence of the reorganization LKT-GnRH syzygy of pCB113.The nucleotide sequence of the reorganization LKT-GnRH syzygy of pCB112 is identical, and the GnRH sequence of different is a plurality of copies is inserted twice.
Embodiment 3
Make up the LKT carrier peptides of brachymemma
Make up a clipped form of recombination leukocyte toxin peptide from the recombination that is present in plasmid pAA352 (as mentioned above).Produce the LKT gene of brachymemma by the inside dna fragmentation that resection length is about 1300bp from reorganization LKT gene as described below.
With restriction enzyme BstBl (New England Biolabs) digested plasmid pCB113 (ATCC registration number 69749), it contains the LKT352 polypeptide that merges with the GnRH polypeptide of four copies.The linearization plasmid of using mung bean (mungbean) nuclease (Pharmacia) digestion to obtain then is to remove the strand protruding terminus that BstB1 digestion produces.Use restriction enzyme Nae1 (New England Biolabs) to digest this concordant DNA then.And the DNA of this digestion is added on 1% the sepharose electrophoretic separation dna fragmentation.With the big dna fragmentation of gene cleaning agents box (Bio 101), and use phage T from sepharose separation and the about 6190bp of purifying 4Dna ligase (Pharmacia) connects purifying fragment oneself.With the connection mixture transformed competence colibacillus E.coli JM105 cell that obtains, the proteic ability of gathering that has the about 57KDa of molecular weight according to generation is identified positive colony.The recombinant plasmid called after pCB111 (ATCC registration number 69748) of Xing Chenging like this, its produces the leucocytotoxicity polypeptide (LKT111 hereinafter referred to as) of the brachymemma that the GnRH polypeptide with four copies merges.Fig. 6 has shown the structure of pCB111.Plasmid pCB114 is identical with pCB111, and different is that multiple copy GnRH sequence (corresponding to the oligopolymer of Figure 1B) is inserted twice.Fig. 7 has shown the nucleotide sequence of the reorganization LKT-GnRH syzygy of pCB111, and the nucleotide sequence of the reorganization LKT-GnRH syzygy of pCB114 is identical, and different is that multiple copy GnRH sequence is inserted twice.
With phage t7 polysaccharase sequencing kit (Pharmacia) order-checking, the nucleotide sequence of verified connection merging point that should the clone.Fig. 8 has shown the nucleotide sequence of these merging points.
Embodiment 4
The purifying of LKT-antigen syzygy
Purifying embodiment 2 and 3 reorganization LKT-GnRH syzygy in the following method.To each syzygy, the E.coli bacterial strain bacterium colony of 5-10 conversion of inoculation in the TB meat soup that 10ml replenishes with the penbritin of 100mg/ml, and at 37C at the G10 shaking table, the last incubation of 220rpm 6 hours.This culture of 4ml is diluted in (batfled) Fernbach flask of two stopping offs that contain 400ml TB meat soup+penbritin, and be incubated overnight as mentioned above.With Sorvall GS3 whizzer in the polypropylene vial of 500ml volume, with 4000rpm, centrifugal 10 minutes collecting cells.With pellet resuspended in isopyknic be preheating to 37 ℃ the TB meat soup that contains penbritin (that is, and 2 * 400ml), the incubation cell is 2 hours as mentioned above.
In order to induce the synthetic of recombination fusion protein, in each culture, add 3.2ml sec.-propyl-B soluble in water, D-sulfydryl galactopyranoside (IPTG, Gibco/BRL), 500mM (final concn=4mM).Incubation culture 2 hours.Centrifugal collecting cell is resuspended to 30ml 50mMTris-HCl as mentioned above, 25% (w/v) sucrose, and pH8.0 ,-70C is freezing.After 60 minutes, melt freezing cell in room temperature-70 ℃ of placements, (Sigma, 20mg/ml is in 250mM Tris-HCl, pH8.0) to add the 5ml N,O-Diacetylmuramidase.This mixture of high speed vortex 10 seconds was put in 15 minutes on ice then.Then cell is added in the 500ml dissolving damping fluid in the 1000ml beaker and mix with the transfer pipet of 2ml.The beaker that contains the dissolved cell suspending liquid is positioned on ice, and uses the Braun ultrasonic sound appratus, big probe is set in 100 watts, sonication totally 2.5 minutes (handle 5-30 intermittently 1 minute second).Be placed on isopyknic solution in the Teflon SS34 centrifuge tube and in Sorvall SS34 rotary head centrifugal 20 minutes at 10000rpm.Precipitating the repeated centrifugation step by high speed vortex resuspension in the aseptic double distilled water of 100ml altogether.Abandoning supernatant, precipitation is integrated with 20ml 10mMTris-HCl, 150mM NaCl, pH8.0 (Tris buffering salt), and spend the night at-20 ℃ of freezing these suspension.
At room temperature melt this reorganization suspension and join 100ml and be dissolved in 8M Guanidinium hydrochloride (Sigma) and violent mixing the in the Tris-buffering salt.In bottle, put a magnetic stirring bar, at room temperature with dissolved sample mix 30 minutes.Solution is transferred in the 2000ml Erlenmyer flask, added 1200ml Tris-damping fluid fast.At room temperature the restir mixture is 2 hours.The 500ml aliquots containig is put into dialysis tubing (action spectrum (Spectrum), 63.7mm diameter, 6000-8000MW cutoff (cutoff), #132670, from the Fisher science), these are put into the 4000ml beaker that contains 3500ml Tris-buffer saline+0.SM Guanidinium hydrochloride.Beaker is put in 4 ℃ indoorly on magnetic stirrer, spends the night, after this, replace dialysis buffer liquid and continue dialysis 12 hours with Tris-buffer saline+0.1M Guanidinium hydrochloride.Then damping fluid is replaced with Tris-damping fluid+0.05M Guanidinium hydrochloride, and continued dialyzed overnight.Damping fluid is replaced with Tris-damping fluid (not containing guanidine), and continued dialysis 12 hours.Repeat this way more than 3 times.Last solution is poured in the plastic drum bottle (Coming) of 2000ml and added 13ml 100mM PMSF (in ethanol) arrestin enzymic activity.Solution is stored in-20 ℃ with the 100ml aliquots containig.
In order to prove that fusion rotein separates, the aliquots containig of each preparation is diluted 20 times in double distilled water, mix with isopyknic SDS-PAGE sample buffer, be positioned over boiling water bath 5 minutes and electrophoresis on 12% polyacrylamide gel.Simultaneously also to reorganization leukotoxin contrast carrying out electrophoresis.
All fusion roteins are with inclusion body form high level expression.The molecular weight of inferring according to the fusion rotein dna sequence dna is 104869 (GnRH of 4 copies of LKT 352 ∷ is from pCB113); 110392 (GnRH of 8 copies of LKT352 ∷ is from pCB112); 57542 (GnRH of 4 copies of LKT111 ∷ is from pCB111); 63241 (GnRH of 8 copies of LKT111 ∷ is from pCB114).The supposition molecular weight of recombinant chou LKT352 molecule is 99338, and the supposition molecular weight of recombinant chou LKT111 molecule is 51843.
Embodiment 5
Immunocompetence in the body of LKT-GnRH syzygy
In order to induce the ability of anti-GnRH immunne response in the LKT-GnRH syzygy body in the test body, and for it is compared replying of other GnRH carrier conjugates, the immunization trial below having carried out.Utilized the male hog in three groups 8 about 8 ages in week (35-50kg), their SFFs.In the facility of easy infection disease least, feed these animals and inoculate with following prescription the 0th and 21 day in test:
Group 1---by the placebo of forming with the salt of Emulsigen Plus adjuvant preparation (2ml), this adjuvant contains 15mgDAA;
Group 2---with the LKT352-GnRH (250 μ g LKT prepare as previously described in the embodiment) of same adjuvant preparation (2ml);
Group 3---VP6-GnRH, 0.5 μ g VP6 and 5 μ g GnRH are with same adjuvant preparation (2ml).With U.S. Patent number 5,071,651 binding peptides of describing prepare the VP6 preparation.
Taked blood sample at the 0th, 21 and 35 day, aggegation, centrifugal at 1500g, remove serum deprivation.With Silversides et al., the RIA method of J.Reprod.Immunol. (1985) 7:171-184 is measured the serum antibody titer of anti-GnRH.
The result of this test shows tire (the tiring＞1: 70) of having only those to produce significant anti-GnRH with the animal of LKT 352-GnRH prescription immunization.Placebo and VP6-GnRH group do not produce anti-GnRH and tire.Past needs to use the GnRH dosage that surpasses 100 μ g in order to induce hormone antagonist to tire, and combines repeated inoculation with other carrier proteins.These results show that the LKT-GnRH carrier system provides the immunogen of the advanced greatly carrier system in the past.
Embodiment 6
Immune effect in the series multiple GnRH multiple body that is connected with LKT
Contain the ability of inducing anti-GnRH immunne response in the multiple GnRH polypeptide multiple reorganization LKT-GnRH fusion rotein body, the inoculation test below having carried out in order to test.Prepared as mentioned above and contained plasmid pCB113 and pCB175 (GnRH that has 4 and 8 copies that are connected with LKT352 respectively), with the culture of the E.coli of the plasmid of the GnRH with 1 copy that is connected with LKT352.The vaccine of each culture makes it to contain 5 μ g GnRH equivalents above the preparation in 0.2ml Emulsigen Plus.Be separated by for twice for 3 groups of 10 female mouse and carried out subcutaneous injection and the 23rd, 35 and 44 day collection blood sample behind initial injection in 23 days.With the serum antibody titer of standard radioimmunoassay method at final extent of dilution 1: 100 and 1: 1000 anti-GnRH of mensuration.Be less than 5% iodate GnRH if combine, think that antibody is immesurable.The antibody titer that obtains like this is summarized in table 1.
The result of this research shows, the antibody that the isodose GnRH that repeats to exist with series multiple (GnRH of 4 or 8 copies) is produced significantly improves (as be bonded to the natural GnRH of iodate mensuration) than the GnRH of single copy.Further, top result shows, contains 4 copy GnRH series connection multiple fusion roteins that are connected with LKT352 and has represented best immunogen GnRH antigen form, though immunogenicity may be subjected to dosage or be tried individual kind to influence.
Sample time Group 1 Group 2 Group 3
LKT 352 ∷ 1 copy GnRH LKT 352 ∷ 4 copy GnRH LKT 352 ∷ 8 copy GnRH
The number of individuals of replying On average reply (%) * The number of individuals of replying On average reply (%) * The number of individuals of replying On average reply (%) *
??1∶100 ??1∶1000 ??1∶100 ??1∶1000 ??1∶100 ??1∶1000 ??1∶100 ??1∶1000 ??1∶100 ??1∶1000 ??1∶100 ??1∶1000
??23 ????0 ????0 ????- ????- ????3 ????1 ????16 ????9 ????2 ????0 ????33 ????-
??35 ????2 ????2 ????45 ????20 ????9 ????9 ????75 ????30 ????7 ????5 ????48 ????41
??44 ????2 ????2 ????60 ????39 ????10 ????10 ????55 ????43 ????8 ????7 ????57 ????46
*On average reply is that those are to surpass 5% in conjunction with I 125The average combination of the animal of-GnRH.
Table 1
Embodiment 7
Immunocompetence and biological effect in the body of LKT352 ∷ GnRH and LKT111 ∷ GnRH syzygy
In order to test ability and the intravital biological effect of proof that the fusion rotein (being connected with LKT352 or LKT111) that contains series multiple multiple GnRH causes anti-GnRH immunne response in vivo, the inoculation test below having carried out.Prepared the culture that contains plasmid pCB113 and the E.coli of pCB111 (GnRH of 4 copies that are connected with LKT352 or LKT111 respectively) as mentioned above.The vaccine of each culture above the preparation; Make and contain 5 μ g GnRH etc. in the 0.2ml VSA-3 adjuvant (the Emulsigen Plus adjuvant of improvement) and work as thing, also prepared the contrast immunity seedling that contains the 0.2ml adjuvant.Give 3 groups 5 male Swiss mouse with subcutaneous injection twice at interval in 21 days, give to inject first (0 day) during age in week at 5-6.Killed these animal subjects at 49 days.
Use the standard radioimmunoassay method, measure the immunocompetence of this GnRH-LKT syzygy by measuring anti-GnRH antibody titer with 1: 1000 serum dilution.Susceptibility with 25pg/ml is tested the biological effect of this GnRH-LKT syzygy of detection by quantitative by the radioimmunity of serum testosterone level, and weighs and the histology testis tissue.The result of this test is summarized in table 2.
In test, the animal individual of useful GnRH:LKT antigen injection have the antibody horizontal that can detect easily; Yet, as reply and the homogeneity of tiring shows that LKT111 ∷ GnRH syzygy (from plasmid pCB111) shows outstanding immunogenicity.Serum testosterone (being produced by testis Leydig cell) is secreted in the mode of fluctuation, therefore, can expect that in the intact animal individuality serum level value is low and variable especially.In test, control group (accept 0.2ml Adjuvanted vaccines injection) has normal serum testosterone level, and two handle group of individuals have detect basically less than the serum testosterone.
In addition, in test, the histology of testis tissue appraisal result shows that the Leydig cell presents decline in various degree, and spermatogenic tubule diameter reduces the interruption with the spermatogenesis of processed individuality; But, though the testicular weight of processed animal still near normal-even when having high anti-GnRH antibody titer-accepting in 5 individualities of 4 copies of LKT111 ∷ GnRH syzygy 2 tangible testis degeneration sign is arranged.
Therefore, the GnRH of the multiple copy that is connected with LKT352 or LKT111 of these demonstrations has as a result formed strong immunogen; But shown inoculation energy of initiation with the fusion rotein of the present invention production of antibodies of endogenous GnRH that neutralizes in vivo in addition, and the animal individual of accepting such inoculation can be attended by the generation of biological effect in the discernible body.
Animal Group 1 Group 2 Group 3
Contrast 5 μ g LKT, 352 ∷, 4 copy GnRH 5 μ g LKT, 111 ∷, 4 copy GnRH
Antibody titer * The weight of testis (mg) Testosterone in the serum Antibody titer * The weight of testis (mg) Testosterone in the serum Antibody titer * The weight of testis (mg) Testosterone in the serum
??1 ????7?0 ????252 ????.04 ????73.0 ????282 ????.13 ????75.0 ????163 ????.00
??2 ????4.0 ????327 ????.18 ????14.0 ????334 ????.10 ????59.0 ????296 ????.07
??3 ????0.0 ????276 ????2.73 ????18.0 ????254 ????.03 ????54.0 ????260 ????.24
??4 ????0.0 ????220 ????.36 ????55.0 ????222 ????.05 ????66.0 ????265 ????.03
??5 ????1.0 ????232 ????1.44 ????61.0 ????226 ????.19 ????64.0 ????50 ????.00
On average ????2.4 ????261 ????.95 ????44 ????263 ????.10 ????64 ????206 ????.07
Standard error ????1.4 ????19 ????.51 ????12 ????21 ????.03 ????4 ????45 ????.04
*I when 1: 1000 serum dilution 125The % combination of-GnRH
Table 2
Embodiment 8
Immunocompetence in the body of LKT ∷ GnRH syzygy in the pig individuality
In order to test the ability of anti-GnRH immunne response in fusion rotein (being connected) primosome that contains series multiple multiple GnRH in the pig individuality with LKT352 or LKT111, the inoculation test below having carried out.Prepared as mentioned above and contained plasmid pCB113, pCB111, pCB175 and pCB114 (are respectively 4 copies of LKT352 ∷ GnRH, the GnRH of 4 copies of LKT111 ∷, the GnRH of the GnRH of 8 copies of LKT352 ∷ and 8 copies of LKT111 ∷) culture of Escherichia coli.The vaccine of each culture above the preparation makes it to contain 50 μ g GnRH equivalents, and with the form administration in the VSA-3 adjuvant that is dissolved in of 2.0ml volume.Injection in the 0th day of test and 4 groups 5 of injections in 21 days the weanling piggy in male and 5 35 day female ages (at the 0th day time).Collecting blood sample at 0,21 and 35 day, is 1: 1000 anti-GnRH antibody titer with the final extent of dilution of standard radioimmunoassay.Measurement result is summarised in table 3.
In test, before immunization, any individuality all can not record anti-GnRH antibody, and most of individualities all easily measure (irrelevant infection is died from and handled to the body one by one of treatment group 4) in the time of the 35th day.This test-results shows, contains the multiple GnRH multiple fusion rotein that is connected with LKT352 or LKT111 carrier polypeptide is formed with usefulness in the pig individuality immunogen.According to decapeptide GnRH (1,200), the supposition molecular weight of LKT111 polypeptide (52,000) and LKT352 polypeptide (100,000), the percentage ratio of GnRH in LKT-GnRH antigen syzygy is as follows: 4.9% (GnRH of 4 copies of LKT352 ∷); 8.5% (GnRH of 4 copies of LKT111 ∷); 9.3% (GnRH of 8 copies of LKT352 ∷); With 15.7% (GnRH of 8 copies of LKT111 ∷).Therefore, the actual result that obtains like this shows with containing the LKT-GnRH syzygy of LKT111 peptide carrier, give the whole amount of individual administration of antigens (LKT-GnRH) can reduce by half (with comparing) with the vaccine composition of LKT352 carrier polypeptide system can obtain identical resisting-GnRH and reply.
The animal individual number Group 1 Group 2 Group 3 Group 4
LKT 352 ∷ 4 copy GnRH 50 μ g LKT 111 ∷ 4 copy GnRH 50 μ g LKT 352 ∷ 8 copy GnRH 50 μ g LKT 111 ∷ 8 copy GnRH 50 μ g
The 35th day 1: 1000 extent of dilution The 35th day 1: 1000 extent of dilution The 35th day 1: 1000 extent of dilution The 35th day 1: 1000 extent of dilution
??1 ????♂47.7 ????♀46.0 ????♂68.3 ????♂51.0
??2 ????♀50.3 ????♂71.6 ????♂65.1 ????♂31.7
??3 ????♀66.0 ????♀21.4 ????♀50.7 ????♀35.7
??4 ????♀70.2 ????♂46.2 ????♂4.7 ????♀65.9
??5 ????♂17.3 ????♀48.9 ????♀38.3 ????♀
??6 ????♂18.3 ????♂69.4 ????♀17.4 ????♂11.3
??7 ????♀14.7 ????♂47.9 ????♀51.4 ????♀28.3
??8 ????♂37.0 ????♀44.4 ????♂18.0 ????♂43.0
??9 ????♂26.0 ????♂70.8 ????♂83.5 ????♀78.7
??10 ????♀2.7 ????♀37.8 ????♀24.2 ????♂55.9
On average ?????35.0 ?????50.4 ?????42.2 ?????44.6
Standard deviation ?????7.3 ?????5.1 ?????8.1 ?????6.9
Respondent's ratio ?????9/10 ?????10/10 ?????9/10 ?????9/9
Table 3
Embodiment 9
The prediction of the T-cell epitope in reorganization LKT352 and LKT111 molecule
In order to predict the potential T-cell epitope that is used for the chimeric leukotoxin peptide sequence of LKT-GnRH of the present invention, describing as table 4 will be corresponding to the aminoacid sequence enforcement Margalit of LKT molecule 1-199 and the method (Margalit et al., J.Immunol (1987) 138:2213) of co-worker's suggestion.In the method, the sequence of the known T-of the inducing cell response of the aminoacid sequence of leukotoxin peptide sequence and other be sure of that the needed amino acid spectral pattern of T-cell epitope compares.Table 4 has been described comparative result.
Predict the outcome just as can be seen, identified that according to the principle of Margalit et al (as mentioned above) suggestion the several short sequence of leucocytotoxicity polypeptide is a potential T-cell epitope.Specifically, identify 9 sequences have (charged/Gly-is hydrophobic-sequence (as spectral pattern " 1 " indication of table 4) of hydrophobic-polarity/Gly), and identified 3 sequences have (charged/Gly-is hydrophobic-sequence (as spectral pattern " 2 " indication of table 4) of hydrophobic-hydrophobic/Pro-polarity/Gly).Anti-GnRH is active in the body that the LKT352 of these data and top embodiment 7 and 8 and LKT111 carrier system are produced combines, can find out, crucial T-cell epitope is retained in the LKT111 molecule of brachymemma, and those epi-positions may be included in the N-terminal portions of LKT352 and LKT111 molecule.
Table 4
The LKT aminoacid sequence that shows spectral pattern " 1 " corresponding to the LKT sequence map of potential T-cell epitope
GTID????????????(aa′s?27-30)
GITG????????????(aa′s?66-69)
GVIS????????????(aa′s?69-72)
HVAN????????????(aa′s?85-88)
KIVE????????????(aa′s?93-96)
DLAG????????????(aa′s?152-155)
KVLS????????????(aa′s?162-165)
DAFE????????????(aa′s?171-174)
KLVQ????????????(aa′s?183-186)
GIID (aa ' s 192-195) shows the LKT aminoacid sequence of spectral pattern " 2 "
RYLAN???????????(aa′s?114-118)
KFLLN???????????(aa′s?124-128)
KAYVD (aa ' s 167-171) D. industrial application
When giving vertebrates host administration, leukotoxin of the present invention-GnRH mosaic can be used as immunogen and carries out immunity at endogenous GnRH, thereby suppresses host's reproductive function or reproductive performance.
Although the illustrational special purposes of this specification sheets, new chimeric molecule disclosed herein has been provided by the mode that contains the fusion rotein of the multiple or series connection GnRH peptide sequence that repeats to exist more than that provides, wherein the immunogen epi-position that provides of the leukotoxin polypeptide portion of this GnRH peptide sequence and molecule (in some situation, being the spacer peptide sequence that is present between selected GnRH sequence) merges.This chimeric protein that the present invention makes up offers and merges GnRH peptide sequence enhanced immunogenicity, makes the vertebrates host who is inoculated strengthen effective immunne response to endogenous GnRH; Make two kinds of gonad-stimulating hormone, the synthetic and release of lutropin (LH) and follitropin (FSH) stops, thereby makes the host temporarily sterile.By this way, new leukotoxin-GnRH construct can be used for immune sterilization vaccine, is used for substituting the invasive methods of sterilisation that is used at present to raise and train with farm-animals livestock industry.
The purposes of other of fusion molecule of the present invention comprises colony's control, for example destroys the regenerative power of wild rodent colony.In this, the LKT-GnRH fusion molecule can be used for substituting colony's measure of control of using at present as poisoning etc.Fusion product of the present invention also can have the slow and fast construct form administration that discharges composition.By this way, can avoid the needs repeatedly inoculated.In addition, because the aminoacid sequence of GnRH is a high conservative, can produce a kind of leukotoxin-GnRH fusion bacterin product of kind intersection effect widely that has between kind.
So, herein disclosed is the various chimeric proteins that contain the leukotoxin that merges with selected GnRH polypeptide.Though described the preferred embodiments of the invention in detail, but still can make some significantly change and do not break away from accompanying Claim institute restricted portion.Be used to implement the preservation of bacterial strain of the present invention
At American type culture collection (ATCC), 12301 Parklawn Drive, Rockville, Maeyland has carried out preservation to the biology pure growth of following bacterial strain.The registration number of being indicated is in the test of the viability of success, paid to register after the filing fee.This preservation is to carry out under the regulation of internationally recognized microbial preservation budapest treaty that is used for patented procedure and detailed rules for the implementation thereof.This guaranteed from the culture of preservation day survival kept 30 years and require depositary institution that the preservation sample is provided after kept at least 5 years.Can obtain these microorganisms from ATCC under the clause of budapest treaty, it is permanent and can obtain these cultures without restriction that this treaty guarantees to give the people of qualification by United States Patent (USP) and the trade mark council according to rule (the comprising 37 CFR § 1.12) decision of the 35 USC § 122 and the council.
These preservations only are those skilled in the art for convenience, are not to admit the preservation carried out for the requirement of satisfying 35 USC § 112.During being incorporated herein, the nucleotide sequence of these plasmids and their encoded polypeptides aminoacid sequences as a reference, and be contrast when inconsistent with existing of the description of this paper.Make, use or sell preserved material all to obtain permission, but such permission can not authorized.
Bacterial strain preservation date ATCC No. haemolysis bus moral Salmonella serotype 1B122 pAA352 nineteen ninety March 30 day 68283 pCB113 nineteen ninety-five February 1 day 69749 the pCB111 nineteen ninety-five February 1 day 69748 among Escherichia coli JM105s among Escherichia coli JM105s of 1 day 53863 February in 1989 in Escherichia coli W1485

Claims (14)

1. chimeric protein that contains the leukotoxin polypeptide that merges with many bodies, these many bodies are made up of more than one selected GnRH polypeptide basically, and wherein the effect of the leukotoxin of said chimeric protein part is the immunogenicity that strengthens the many bodies of said GnRH.
2. according to the chimeric protein of claim 1, wherein said leukotoxin polypeptide has lacked the leukotoxin activity.
3. according to the chimeric protein of claim 2, wherein said leukotoxin is LKT352.
4. according to the chimeric protein of claim 2, wherein said leukotoxin is LKT111.
5. according to the chimeric protein of claim 1, the many bodies of wherein said GnRH contain the molecule that general formula is GnRH-X-GnRH, and wherein X is selected from by peptide bond, amino acid spacer groups, leukotoxin polypeptide and (GnRH) nThe group of forming, wherein n is more than or equal to 1, and wherein GnRH comprises any GnRH polypeptide in addition.
6. according to the chimeric protein of claim 5, wherein X comprises the amino acid spacer groups that contains at least one T-cell helper epitope.
7. according to the chimeric protein of claim 1, wherein said chimeric protein comprises the aminoacid sequence that Fig. 5 describes, or homology basic with it aminoacid sequence suitable with function.
8. according to the chimeric protein of claim 1, wherein said chimeric protein comprises the aminoacid sequence aminoacid sequence suitable with function with homology basic with it that Fig. 7 describes.
9. contain the chimeric protein of any one and the vaccine composition of pharmaceutical acceptable carrier among the with good grounds claim 1-8.
10. one kind provides the method for selected many bodies of GnRH to animal subject, comprises the vaccine composition according to claim 9 of using significant quantity to said animal.
11. the DNA construct of any one chimeric protein among the coding claim 1-8.
12. an expression cassette comprises: (a) according to the DNA construct of claim 11; (b) control sequence of transcribing of the said construct of guidance, wherein said construct can transcribe in host cell and translate.
13. the host cell that transforms with the expression cassette of claim 12.
14. produce the method for recombinant polypeptide, comprising: the host cell group that claim 13 (a) is provided; (b) under the condition that said expression cassette encoded polypeptides is expressed, cultivate said cell mass.
CN96192680A 1995-02-10 1996-01-24 GnRH-leukotoxin chimeras Pending CN1179181A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101553500B (en) * 2006-06-16 2014-01-01 医疗研究局 GnRH peptide derivative (gonadotropin-releasing hormone)
CN107073086A (en) * 2014-07-25 2017-08-18 美国联合生物医学公司 Immunity LHRH compositions and its application in pig
CN112190705A (en) * 2020-09-25 2021-01-08 广州源博医药科技有限公司 RSV-SH subunit vaccine and preparation method and application thereof
CN115960184A (en) * 2022-09-28 2023-04-14 华中农业大学 Mannheimia haemolytica A6 serotype leukotoxin antigen protein, antibody and application thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101553500B (en) * 2006-06-16 2014-01-01 医疗研究局 GnRH peptide derivative (gonadotropin-releasing hormone)
CN107073086A (en) * 2014-07-25 2017-08-18 美国联合生物医学公司 Immunity LHRH compositions and its application in pig
CN112190705A (en) * 2020-09-25 2021-01-08 广州源博医药科技有限公司 RSV-SH subunit vaccine and preparation method and application thereof
CN115960184A (en) * 2022-09-28 2023-04-14 华中农业大学 Mannheimia haemolytica A6 serotype leukotoxin antigen protein, antibody and application thereof
CN115960184B (en) * 2022-09-28 2023-12-12 华中农业大学 Leucobacillus mannii A6 serotype leukotoxin antigen protein, antibody and application thereof

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