CN101124241A - Lawsonia intracellularis 26 kd subunit vaccine - Google Patents
Lawsonia intracellularis 26 kd subunit vaccine Download PDFInfo
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- CN101124241A CN101124241A CNA2004800367439A CN200480036743A CN101124241A CN 101124241 A CN101124241 A CN 101124241A CN A2004800367439 A CNA2004800367439 A CN A2004800367439A CN 200480036743 A CN200480036743 A CN 200480036743A CN 101124241 A CN101124241 A CN 101124241A
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- nucleic acid
- albumen
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Abstract
The present invention relates i.a. to nucleic acids encoding novel Lawsonia intracellularis proteins. It furthermore relates to DNA fragments, recombinant DNA molecules and live recombinant carriers comprising these sequences. Also it relates to host cells comprising such nucleic acids, DNA fragments, recombinant DNA molecules and live recombinant carriers. Moreover, the invention relates to proteins encoded by these nucleotide sequences and to their use for the manufacturing of vaccines. The invention also relates to vaccines for combating Lawsonia intracellularis infections and methods for the preparation thereof. Finally the invention relates to diagnostic tests for the detection of Lawsonia intracellularis antigens and of antibodies against Lawsonia intracellularis.
Description
The present invention's proteic nucleic acid of new lawsonia intracellularis (Lawsoniaintracellularis) that is specifically related to encode, comprise these sequences dna fragmentation, recombinant DNA molecules and the recombinant vectors of living, comprise these nucleic acid, dna fragmentation, recombinant DNA molecules and the host cell of the recombinant vectors of living, the vaccine and its preparation method that infect by labor Sonia bacterium in purposes in vaccine production of these nucleotide sequence coded albumen and its, the anti-cell and the diagnostic check method that is used for the detection of antibodies of labor Sonia bacterium in lawsonia intracellularis antigen and the anti-cell.
Pig hyperplasia enteropathy (PPE or PE) has become the major disease of modern pig industry in the world wide.Described sickness influence 15% to 50% the drove of growing and in the problem drove of having determined influence up to 30% animal individual.Nowadays on the extra feed and the cost of facility time of every affected pig, year financial loss estimates to have reached the 5-10 dollar.PPE be have many different clinical symptom (animal of dead, pale (pale) and anaemia, water sample, black or scarlet diarrhoea ight soil, dispirited, lose the appetite and be slow in action, the FCR of growthing lag and increase) one group of chronic and acute disease.Yet, the feature of two unanimities of existence.First feature (only observed pathology change when ptomatopsia) is thickening of small intestine and mucous membrane of colon.The secondth, little crooked bacterium (small-curved bacteria) in the kytoplasm appears in the intestinal epithelial cells (enterocytes) of infected intestines.These bacteriums have been confirmed as the pathogenic factor (etiological agent) of PPE now and have been called as lawsonia intracellularis.
For many years, found that lawsonia intracellularis infects the animal of numerous species, comprised other big animals as monkey, rabbit, ferret (ferrets), hamster, fox, horse and difference such as ostrich and the emoe.Lawsonia intracellularis is a bacterium gram-negative, the band flagellum, and it is bred in Eukaryotic intestinal epithelial cells and the cultivation of acellular system also was not described.For existence in cell and breeding, lawsonia intracellularis must penetrate just at splitted (dividing) pit cell.Bacterium combines with cytolemma and enters intestinal epithelial cells apace by entering vacuole.This vacuole promptly breaks (in 3 hours) then, and bacterium just grows in tenuigenin and breeding freely vigorously.Bacterium causes the infected cells can not be ripe, carries out mitotic division constantly and form the mechanism of undergrown pit cell still unclear.
At present labor Sonia bacterium infects, the understanding of this treatment of diseases and control is hindered because lawsonia intracellularis can not cultivate in the acellular substratum always in the pair cell.Although reported labor Sonia bacterium in the co-cultured cell successful in the rat intestine epithelial cell, this does not cause the developing vaccines through deactivation of labor Sonia bacterium in the anti-cell yet, although there is the demand to these vaccines significantly.
The purpose of this invention is to provide the vaccine that labor Sonia bacterium infects in the anti-cell.
Be surprisingly found out that now lawsonia intracellularis produces the new albumen that can induce the protective immunity of labor Sonia bacterium in the anti-cell.
Described new albumen is called 26kD albumen.
This new proteic aminoacid sequence is shown in sequence flag symbol SEQ ID NO:2.This proteic gene of encoding is checked order, and its nucleotide sequence is shown in sequence flag symbol SEQ ID NO:1.This gene is also referred to as " gene 5608 " in an embodiment.
Well-known in this area, many different nucleotide sequence codifieds are with a kind of albumen.Wave phenomenon on the second and the particularly the 3rd bit base of so-called each triplet at coded amino acid of this phenomenon.This phenomenon can cause the heterology of two nucleotide sequences about 30%, the identical albumen but it still can be encoded.Therefore, two nucleotide sequences with about 70% sequence homology still codified with a kind of albumen.
Therefore, embodiment relates to the part of this proteic immunogenic fragments of coding of the nucleic acid of labor Sonia mycoprotein in the Codocyte and this nucleic acid, and wherein these nucleic acid or its sequence of partial sums nucleic acid that is shown in SEQ ID NO:1 has at least 90% homology level.
Preferably, the encode part of proteic nucleic acid of this lawsonia intracellularis or described nucleic acid and nucleic acid with sequence shown in the SEQ ID NO:1 has at least 92%, preferably 94%, more preferably 95% and even 96% homology more preferably.More preferably 98% or even 100% homology level.
Available computers program " BLAST 2 SEQUENCES ", by select can
Www.ncbi. Nlm.nih.gov/blast/b12seq/b12.htmlOn the sub-routine " BLASTN " that finds determine the level of nucleotide homology.
The bibliography of this program is Tatiana A.Tatusova, Thomas L.MaddenFEMS Microbiol.Letters174:247-250 (1999).The parameter of using is a default parameter: coupling reward is divided (Reward for match) :+1, mispairing point penalty :-2, open the room: 5, extend the room: 2, Gap x_dropoff:50.
Determine that whether certain nucleic acid be that the other method of nucleic acid of the present invention relates to such problem, promptly this specific nucleic acid whether under stringent condition really with the nucleic acid hybridization with the nucleotide sequence shown in the SEQ ID NO:1.
If nucleic acid under stringent condition with the nucleotide sequence hybridization shown in the SEQ ID NO:1, it is considered to nucleic acid of the present invention so.
Formula definition stringent condition according to Meinkoth and Wah1 (1984.Hybridization of nucleic acidsimmobilized on solid supports.Anal.Biochem.138:267-284.)
T
m-1 ℃/1% mispairing in=[(%GC)-0.61,81.5 ℃+16.6 (log M)+0.41 (% methane amide)-500/L]
In formula, M is the volumetric molar concentration of univalent cation; %GC is the per-cent of guanine and cytidylic acid(CMP) among the DNA; L is the hybridization length on base pair.
Stringent condition is such condition, and promptly under this condition, if nucleic acid or its fragment have 10% mispairing at the most with the nucleic acid with sequence shown in the SEQ ID NO:1, it still can be hybridized.
Because the invention discloses the new proteic nucleic acid of lawsonia intracellularis of coding, therefore for the first time might obtain these albumen now with enough amounts.This can realize by the gene that for example uses expression system to express encoding said proteins.
Therefore, in a more preferred embodiment, the present invention relates to comprise the dna fragmentation of nucleic acid of the present invention.Such dna fragmentation can be the plasmid of for example having cloned nucleic acid of the present invention.As described below, these dna fragmentations for example can be in order to increase the amount of DNA as primer.
The prerequisite of express nucleic acid is the appropriate promotor that connects described nucleic acid effectively, and described like this nucleic acid is just under the control of promotor.The selection of promotor extends to and can instruct any eucaryon, protokaryon or the viral promotors of genetic transcription apparent to those skilled in the art in cell (as the host cell of protein expression).
Therefore, the more preferred form of this embodiment relates to recombinant DNA molecules, and described recombinant DNA molecules comprises dna fragmentation of the present invention or the nucleic acid under the promotor control that places effective connection.This can by the Protocols in Molecular Biology of standard for example (Sambrook, J. and Russell, D.W., Molecular cloning:a laboratory manual, method 2001.ISBN0-87969-577-3) realizes.Effectively the promotor that connects is to control the promotor of the transcribed nucleic acid of its connection.
Such promotor can be labor Sonia Bordetella (Lawsonia) promotor for example participate in the encoding promotor of expression in vivo of the proteic gene of 26kD, if this promotor is effective at the cell that is used for expressing.It also can be an allogeneic promoter.When host cell was bacterium, spendable useful expression control sequenc comprised Trp promotor and operon (Goeddel waits the people, Nucl.Acids Res., 8,4057,1980); Lac promotor and operon (Chang waits the people, Nature, 275,615,1978); Outer membrane protein promotor (Nakamura, K. and Inouge, M., EMBO J., 1,771-775,1982); Phage promotor and operon (Remaut, people such as E., Nuc l.Acids Res., 11,4677-4688,1983); αDian Fenmei (subtilis (B.subtilis)) promotor and operon, the sequence that the terminator sequence compatible with the host cell of selecting and other enhancings and control are expressed.
When host cell was yeast, useful expression control sequenc comprised, for example α-mating factor (mating factor).For insect cell, can use polyhedrin and the p10 promotor (Smith, people such as G.E., Mol.Cell.Biol.3,2156-65,1983) of baculovirus.When host cell was the host cell in Mammals source, the useful expression control sequenc of illustrative comprised SV-40 promotor (Berman, people such as P.W., Science, 222,524-527,1983) or metallothionein promoter (Brinster, R.L., Nature, 296,39-42,1982) or heat-inducible promoter (people such as Voellmy, Proc.Natl.Acad.Sci.USA, 82,4949-53,1985).
Bacterium, yeast, fungi, insect and mammalian cell expression system are the very frequent systems that uses.These systems are that know and normally obtainable in this area, for example from Invitrogen (www.invitrogen.com), Novagen (www.merckbiosciences.de) or Clontech Laboratories, Inc.4030 Fabian Way, Palo Alto, California 94303-4607, USA is commercially available.Except these expression systems, be very attractive expression system based on parasitic expression system.These system descriptions are 2714074 french patent application and US NTIS publication number US08/043109 (Hoffman, S. and Rogers, W.:Public.1993 December 1) in for example publication number.
Other more preferred form of this embodiment of the present invention relate to live-weight group carrier (LRC), and this recombinant vectors comprises coding 26kD albumen of the present invention or the nucleic acid of its immunogenic fragments, dna fragmentation of the present invention or recombinant DNA molecules of the present invention.These carriers are for example bacterium and virus.These LRC are such microorganism or virus, have promptly cloned extra genetic information therein, are the nucleic acid of coding 26kD albumen of the present invention or its immunogenic fragments in this case.To produce such immunne response with these LRC infected animals, promptly this immunne response not only resists the immunogen of described carrier but also the immunogen part of the albumen (for example 26kD albumen) that anti-genetic code additionally is cloned into LRC.
As the example of LRC, can use salmonella (Salmonella) the strain system of attractive attenuation known in the art.
Vermeulen, A.N. (Int.Journ.Parasitol.28:1121-1130 (1998)) have specifically described the recombinant vectors parasite that lives.LCR also can be used as the means that nucleic acid transported into target cell.The recombinant vectors virus of living is also referred to as vector virus.Usually be vaccinia virus (people such as Panicali-as the virus of carrier; Proc.Natl.Acad.Sci.USA, 79:4927 (1982), simplexvirus (E.P.A.0473210A2) and retrovirus (Valerio, people such as D.; In Baum, S.J., Dicke, K.A., Lotzova, E. and Pluznik, D.H. (writing), Experimental Haematology today-1988.Springer Verlag, New York:pp.92-99 (1989)).
Can use the genome of homologous recombination technique is selected the recombinant nucleic acid importing in the body well known in the art bacterium, parasite or virus, described bacterium, parasite or virus can induce the nucleic acid of insertion of the present invention to express in host animal.
Other forms of last this embodiment of the present invention relate to such host cell, the coding proteic nucleic acid of the present invention under promptly its promotor that is included in effective connection is controlled, the recombinant DNA molecules that comprises the dna fragmentation of such nucleic acid or comprise such nucleic acid.This form also relates to the host cell that contains recombinant vectors alive, and described carrier contains coding 26kD albumen of the present invention or its segmental nucleic acid molecule.
Host cell can be to contain based on plasmid such as pBR322 or bacterial expression vector such as the pGEX of bacterium or contain the cell cell of intestinal bacteria, subtilis (Bacillus subtilis) and lactobacillus genus (Lactobacillus) kind for example of the bacterial origin of phage.Host cell also can be the cell in eucaryon source, for example contains the yeast cell of yeast idiosyncratic carrier molecule, or the higher eucaryotic cells that contains carrier or recombinant baculovirus insect cell (people such as Luckow for example; Bio-technology 6:47-55 (1988)) and for example contain vegetable cell (Barton, people such as K.A. based on Ti-plasmids carrier or plant viral vector; Cell32:1033 (1983)), the mammalian cell such as Hela cell, Chinese hamster cell (CHO) or the Crandell Feline nephrocyte that also contain suitable carriers or recombinant virus.
Another embodiment of the invention relates to new albumen of the present invention and its immunogenic fragments.
Define the notion of immunogenic fragments below.
A kind of form of this embodiment is specifically related to have lawsonia intracellularis albumen and the described proteic immunogenic fragments that has the aminoacid sequence of at least 90% homology with the aminoacid sequence shown in the SEQ ID NO:2.
In a preferred form, described embodiment relates to such lawsonia intracellularis albumen and described proteic immunogenic fragments, and the aminoacid sequence shown in promptly described albumen and the SEQ ID NO:2 has at least 92%, preferably 94%, more preferably 96% sequence homology.Even more preferably have 98% or even 100% homology level.
Available computers program " BLAST 2 SEQUENCES ", by the sub-routine of selecting to find on www.ncbi.nlm.nih.gov/blast/b12seq/b12.html.: " BLASTP " determines the level of albumen homology.
The bibliography of this program is Tatiana A.Tatusova, Thomas L.MaddenFEMS Microbiol.Letters 174:247-250 (1999).The matrix that uses: " blosum62 ".The parameter of using is a default parameter: open room: 11, extend the room: 1, Gap x_dropoff:50.
It being understood that for the specific protein that comprises herein, can have natural variation between the independent lawsonia intracellularis strain system.These variations can show by amino acid whose disappearance, replacement, insertion, inversion or interpolation in the described sequence of amino acid difference XOR on total sequence.For example, people such as Neurath has described the amino acid that does not change biology and immunologic competence basically and has replaced in " The Proteins " Academic Press New York (1979).Among other things, amino-acid substitution between the cognate amino acid or in evolution the frequent displacement that takes place particularly Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (referring to Dayhof, M.D., Atlas of protein sequence and structure, Nat.Biomed.Res.Found., Washington D.C., 1978, the 5th volume, suppl.3).Other amino acid are replaced and are comprised Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Thr/Phe, Ala/Pro, Lys/Arg, Leu/Ile, Leu/Val and Ala/Glu.Based on this information, Lipman and Pearson have developed and have been used for fast and the amino acid method of the functional similarity between (Science, 227,1435-1441,1985) and the definite homologous protein relatively delicately.These amino acid of exemplary of the present invention replace and have disappearance and/or the variation of inserting within the scope of the present invention, as long as the albumen of gained keeps its immunoreactivity.This has explained lawsonia intracellularis albumen of the present invention, when separating the open-air strain isolated (field isolate) from difference, can have about 90% homology level, but representative has the reason of the same protein of identical immunological characteristic.These variations on the aminoacid sequence of specific protein of the present invention are considered to " not influencing immunogenicity substantially ", and promptly described variation still can provide the albumen of the immunne response of the clinical manifestation that can induce interior microbial infection of labor Sonia of anti-cell or anti-at least described infection.
Yet, when albumen is used for for example inoculating purpose or is used to produce antibody, needn't use complete albumen.Also may use this proteic fragment (so-called immunogenic fragments), described fragment can be induced anti-this proteic immunne response equally or is coupled to the carrier KLH for example that can induce anti-this proteic immunne response." immunogenic fragments " is understood to be in the fragment of the full-length proteins of the ability that still keeps its induce immune response among the host, and promptly described fragment comprises B or t cell epitope.Now, can utilize the various technology dna fragmentation of identification code antigen fragment (determinant) easily.By people such as Geysen (patent application WO 84/03564, patent application WO 86/06487, United States Patent (USP) 4,833,092, Proc.Natl Acad.Sci.81:3998-4002 (1984), the method (so-called PEPSCAN method) that J.Imm.Meth.102,259-274 (1987) describe be carry out easily, the fast and good method that is used to detect important area on epi-position, the proteic immunology of foundation.Described method is used widely and knows equally for a person skilled in the art.(experimental) method of being somebody's turn to do is particularly suitable for the detection of B cell epitope.In addition, the sequence of any proteic gene of given coding, computerized algorithm can be appointed as former epi-position important on the immunology with specific protein fragments on basis consistent with present known anti-epi-position on its sequence and/or the structure.Determining based on according to the wetting ability standard of Hopp and Woods (Proc.Nat1.Acad.Sci.78:38248-3828 (1981)) with according to Chou and Pasman (Advancesin Enzymology 47:45-148 (1987) and United States Patent (USP) 4 of these zones, the combination of secondary structure situation 554,101).Equally can be by computer according to the sequence prediction t cell epitope by means of the amphipathic standard (Science235,1059-1062 (1987) and U.S. Patent application NTIS US 07/005,885) of Berzofsky.Can: Shan Lu is about the epitope of people such as general principle: Tibtech9:238-242 (1991), Good about malaria; The summary of Science235:1059-1062 (1987), Lu; Vaccine 10:3-7 (1992), Berzowsky are about the HIV-epi-position; Find the general introduction of simplifying among the The FASEB Journal 5:2412-2418 (1991).
Therefore, a kind of form of other embodiments of the present invention relates to can protect the vaccine that labor Sonia bacterium infects in the pig anti-cell, and this vaccine comprises aforesaid albumen of the present invention or its immunogenic fragments and medicine acceptable carrier.
Other embodiments of the present invention relate to the albumen of the present invention that is used for vaccine.
Other embodiments relate to the purposes on the albumen of the present invention vaccine that labor Sonia bacterium infects in producing anti-cell.
A kind of method for preparing vaccine of the present invention is to be purified into albumen of the present invention or its immunogenic fragments by the biological chemistry method of purification from bacterium (mucous membrane that obtains from the intestines wall that infects is scraped and got acquisition thing (scrapings)).Yet this is the method for preparing vaccine very consuming time.
Therefore in vaccine, use the expression of gene product of coding albumen of the present invention or its immunogenic fragments will make things convenient for manyly.The invention provides the nucleic acid of the proteic gene of coding 26kD.
According to the present invention,, as described below by albumen of the present invention or its immunogenic fragments are mixed these vaccines that can easily prepare based on these expression of gene products with drug acceptable carrier.
Perhaps, vaccine of the present invention can comprise the recombinant vectors of living as described above, and described carrier can be expressed albumen of the present invention or its immunogenic fragments.These vaccines, for example based on infect enteric epithelium or for example the salmonella carrier of respiratory epithelium or the vaccine of virus vector have the favourable aspect that is better than subunit vaccine, it is the natural mode of the infection of labor Sonia bacterium in the analog cell better.In addition, its self-reproduction is favourable, because immunity only needs a spot of recombinant vectors.
Above-mentioned vaccine all helps initiatively inoculation, that is, host's immunity system causes by albumen of the present invention or its immunogenic fragments, produces anti-these proteic antibody.
In addition, these antibody can for example produce maybe and can obtain from antibody generation sexual cell system as described below in the rabbit.These antibody can be used to host animal then.This inoculation method (passive inoculation) is the inoculation that immunne response infected when animal and that not free permission is natural is selected when being triggered.It also is the preferable methods of the animal of immunoprophylaxis function impaired (immune-compromised).The antibody of labor Sonia bacterium can be in these cases directly in conjunction with bacterium in the anti-cell of using.This has its favourable aspect that has reduced or stoped the growth of lawsonia intracellularis immediately.
Therefore, a kind of other form of this embodiment of the present invention relates to the vaccine of the antibody that comprises anti-26kD lawsonia intracellularis of the present invention.
Vaccine also can be based on aforesaid host cell, and it comprises albumen of the present invention or its immunogenic fragments.
Effectively vaccination ways is that direct DNA with the coding related antigen inoculates in addition.Dna direct inoculation with proteins encoded is succeedd on many different albumen.(, being summarized among the The Immunologist 2:20-26 (1993)) as people such as for example Donnelly.The inoculation of the pig that this inoculation method antagonism lawsonia intracellularis infects is very attractive.
Therefore, other forms of this embodiment of the present invention relate to the vaccine of the nucleic acid that comprises coding albumen of the present invention or its immunogenic fragments and relate to the vaccine that comprises the dna fragmentation that contains these nucleic acid.
Other forms of this embodiment relate to the vaccine that comprises recombinant DNA molecules of the present invention.
For example use needleless injector (needle-less injector) can easily use dna vaccination by intradermal administration.This application process is delivered to dna direct in the cell of the animal that is inoculated.DNA amount in the microgram scope between the 1 and 100 μ g provides extraordinary result.
In other embodiments, vaccine of the present invention has comprised one or more antigen or these antigenic genetic information of encoding that derive from other pig pathogenic organisms and virus extraly.
These organisms and virus preferably are selected from pseudorabies virus (Pseudorabiesvirus), swine influenza virus (Porcine influenza virus), pig parvoviral (Porcine parvo virus), Transmissible gastroenteritis virus (Transmissiblegastro-erteritis virus), rotavirus (Rotavirus), intestinal bacteria, erysipelothrix rhusiopathiae (Erysipelothrix rhusiopathiae), bordetella bronchiseptica (Bordetella bronchiseptica), Salmonella choleraesuls (Salmonellacholerasuis), haemophilus parasuis (Haemophilus parasuis), multocida (Pasteurella multocida), swine streptococcus (Streptococcus suis), mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae), swine dysentery spirochete (Brachyspira hyodysenteriae) and actinobacillus pleuropneumoniae (Actinoba cillus pleuropneumoniae).
All vaccines of the present invention comprise the medicine acceptable carrier.The medicine acceptable carrier can be for example sterilized water or sterile physiological salts solution.More the carrier of complex form can be a buffer reagent for example.
The method that is used to prepare vaccine comprises mixes albumen of the present invention or its immunogenic fragments with the medicine acceptable carrier.
Vaccine of the present invention preferably also can comprise adjuvant.Adjuvant comprises the material that strengthens host's immunne response in non-specific mode usually.Many different adjuvants are known in this area.The example of adjuvant be Fu Shi fully and Freund, vitamin-E, nonionic block polymer, Muramyl dipeptide, QuillA
(R), mineral oil Bayol for example
(R)Or Markol
(R), vegetables oil and Carbopol
(R)(homopolymer), or Diluvac
(R)Forte.Vaccine also can comprise so-called " carrier ".Carrier is that polypeptide adheres to but the non-compound that is total to the terrace in conjunction with it.Often the carrier compound that uses is for example aluminium hydroxide, aluminum phosphate or aluminum oxide, silicon-dioxide, kaolin and bentonite.The special shape of such carrier is so-called ISCOM (EP 109.942, EP180.564, EP242.380), is embedded in the carrier to antigen part in described form.In addition, vaccine can comprise one or more suitable surface active cpd or emulsifying agents, for example Span or Tween.Usually, vaccine mixes the polypeptide that for example is easy to degrade with protection and avoids degraded with stablizer, thereby prolongs the shelf-lives (shelf-life) of vaccine, or improves lyophilize efficient.Useful stablizer is SPGA (people such as Bovarnik particularly; J.Bacteriology 59:509 (1950)), carbohydrate Sorbitol Powder, N.F,USP MANNITOL, trehalose, starch, sucrose, dextran or glucose, albumen albumin or casein or its degraded product and buffer reagent alkali metal phosphate for example for example for example.
In addition, vaccine can be suspended in the physiology acceptable diluent.Much less, the method for other adjuvantizations, interpolation carrier compound or thinner, emulsification or stabilization polypeptide also within the scope of the invention.
Amount in very suitably can 1 to 100 microgram scope is used vaccine of the present invention, although can use the dosage of less amount in principle.The dosage that surpasses 100 micrograms, although most suitable in immunity, less attractive for business reason.
Vaccine based on the recombinant vectors (for example LRC virus and above-mentioned bacterium) of the attenuation of living can be used by much lower dosage, because himself can be bred in course of infection.Therefore, for bacterium and the most suitable amount of virus respectively 10
3To 10
9Between the CFU/PFU scope.
Can use many methods of application.Dosage forms for oral administration is very attractive method of application, is gastral infection because infect.In the preferred dosage forms for oral administration mode of known in the art and frequent use is that vaccine is incapsulated, and described capsule only disintegrates behind its environment by the highly acidic of stomach.In addition, also can be with vaccine and compound known in the art pH value with interim increase stomach.The intramuscular administration that general is for example used by vaccine also is suitable.If use this approach, known in the art to be used for the standard method that general uses be suitable.
From the protectiveness aspect of anti-disease, diagnosing lawsonia intracellularis is very important fast and correctly.Therefore another object of the present invention provides and is suitable for the diagnostic tool that lawsonia intracellularis infects detection.
The diagnostic check method that is used for detecting serum lawsonia intracellularis antibody can be for example simple standard sandwich ELISA method of inspection, in described method with 26kD albumen of the present invention or its antigenicity fragment bag by the hole wall of elisa plate.The method that is used to detect these antibody is by for example using from mammiferous serum incubation 26kD albumen to be measured or its antigenicity fragment, then with for example carrying out through the antibody incubation of the mammiferous antibody of decorrelation of mark.Color reaction can show the existence of the antibody of labor Sonia bacterium in the anti-cell or not exist then.Another example of diagnostic check system is for example to comprise 26kD albumen of the present invention or the segmental Western trace of its antigenicity and mammiferous serum to be measured incubation together, then carries out engram analysis.
Therefore, another embodiment of the present invention relates to the diagnostic check method that is used for the detection of antibodies of labor Sonia bacterium in the anti-cell.These methods of inspection comprise albumen of the present invention or its fragment.
Thereby the diagnostic check method that is suitable for the lawsonia intracellularis INFECTION IN DETECTION based on the detection of the proteic antigenicity material of the antigenic specific 26kD of lawsonia intracellularis also can be the ELISA method of inspection of standard.In an embodiment of such method of inspection, with the hole wall of the proteic antibody sandwich elisa plate of anti-26kD.After with the detected materials incubation, Xiang Kongzhong adds the antibody of labor Sonia bacterium in the anti-cell of mark.Color reaction shows the existence from the antigenicity material of lawsonia intracellularis then.
Therefore, another embodiment of the present invention relates to the diagnostic check method of the detection of the antigenicity material that is used for lawsonia intracellularis.These methods of inspection comprise anti-albumen of the present invention or its segmental antibody.
Can use polypeptide of the present invention or its immunogenic fragments of the expression that as above characterizes to produce antibody, described antibody can be polyclonal, monospecific or monoclonal (or derivatives thereof).If polyclonal antibody is wanted, the technology that is used to produce and process polyclonal serum is (Mayer and Walter (writing) the ImmunochemicalMethodsin Cell and Molecular Biology for example that knows in this area, Academic Press, London, 1987).
Can be by prepare the monoclonal antibody of anti-polypeptide of the present invention (or its variant or fragment) of the present invention with the immune inbreeding mouse of technology also known in the art (Kohler and Milstein, Nature, 256,495-497,1975).
The method that is used for scale operation antibody of the present invention also is known in this area.These methods depend on the clone of coding proteic genetic information of the present invention (its fragment) the filobactivirus that is used for phage display.In " Antibody Engineering Page " under special " the filamentous phagedisplay " on http://aximtl.imt.uni-marburg.de/～rek/aepphage.html. and Cortese, R. wait the people, (1994) in Trends Biotechn.12:262-267., Clackson, T.﹠amp; Wells, J.A. (1994) in Trends Biotechn.12:173-183, Marks, J.D. wait the people, (1992) in J.Biol.Chem.267:16007-16010, Winter, G. wait the people, (1994) in Annu.Rev.Immunol.12:433-455 and Little, people such as M. have described these technology in the summary paper of (1994) Biotechn.Adv.12:539-555.Use phage selection to express the camel expression library of camel (camelid) heavy chain antibody subsequently.(Muyldermans, S. and Lauwereys, M., Journ.Molec.Recogn.12:131-140 (1999) and Ghahroudi, people such as M.A., FEBS Letters 414:512-526 (1997)).The cell in the library of the reproducible antibody of wanting from expression is used it for the extensive expression of antibody then.
Embodiment
Embodiment 1:
The hang oneself separation of lawsonia intracellularis of the pig ileum that infects
From the pig that suffers from PE death, collect through histopathology and the definite ileum of the antiacid Buddhist nun's staining of withering (acid-fast Zieh1-Neelsen staining), and store down at-80 ℃ through the lawsonia intracellularis infection.After thawing, scrape from the mucous membrane that picks up from intestines wall and to get the thing labor Sonia bacterium in the isolated cell through infecting.Described as people such as Lawson (Vet.Microbiol.10:303-323 (1985)), in general pressure-even pulp crusher, in PBS, ileum scraped and get thing and carry out homogenate repeatedly to discharge intracellular bacterium.Low-speed centrifugal is removed the supernatant liquor that obtains behind the cell debris to be filtered by 5.0,3.0,1.2 and 0.8 μ m strainer (Millipore).With filtrate under 8000g centrifugal 30 minutes, produce the little precipitation of lawsonia intracellularis then.Use the Percoll gradient centrifugation to be further purified these bacteriums.Detect identity people such as (, J.Clin.Microbiol.31:2611-2615 (1993)) Jones of purified bacterium by PCR, the purity (＞95%) by phase microscope assessment bacterium is to show existing of any contaminative bacterium or ramenta intestinonun simultaneously.
Bacterial strain system and plasmid
Labor Sonia mycetocyte in the isolated cell from ileum material as mentioned above through infecting.(USA) buying the e. coli strains that contains carrier pLysSrare and plasmid pET22b is BL21star (DE3) for Madison, Wisconsin from Novagen.E. coli strains is that TOP10F ' is available from Invitrogen (Groningen, the Netherlands).The original seed (containing 30% glycerine) of all bacterial strain systems is stored down at-70 ℃.Method according to standard prepares LuriaBertani liquid nutrient medium (LB) and LB flat board.
DNA separates
In order to obtain highly purified lawsonia intracellularis chromosomal DNA, use Biorad chromosomal DNA separating kit (Biorad, Veenendaal, the Netherlands) from bacterial cell, to prepare DNA.Use Qiagen product isolated plasmid dna.
Pcr amplification
Use contains 52U/ml Expand High Fidelity Enzyme Mix, has 2.5mM MgCl
2, (Expand HF damping fluid USA), the primer of 20 picomole and 15ng carry out pcr amplification as the PCR mixture of the chromosomal DNA of the lawsonia intracellularis of template to 16mM dNTPs for Promega, Wisconsin.Application (being bacterium colony PCR) for standard, the PCR mixture contains 20U/ml Supertaq and contains 8mM dNTPs (Promega, Wisconsin, (the HT Biotechnology Ltd of Supertaq damping fluid USA), Cambridge, UK), the primer and the 15ng template of 10 picomole.
Connect and conversion
Under 16 ℃, (Gibco BRLLife Technologies Inc. USA) connects and spends the night to connect in the buffering ligase enzyme with 1 unit at 1x.By the ligation thing conversion large intestine sense bacterium competence cell of heat shock method with 1 μ l.The method of use standard prepares competent BL21star (DE3) competent escherichia coli cell and TOP10F ' competent escherichia coli cell.
The 10xHIS Expression of Fusion Protein
Determine the dna sequence dna of expression vector, then expression vector is transformed into the BL21star (DE3) that contains pLysSrar.The strain of gained tied up under 37 ℃, 200rpm in 5ml contain overnight incubation among the LB of 100 μ g/ml penbritins.Contain among the LB of 100 μ g/ml penbritins overnight culture by dilution in 1: 100 at 50ml.This culture is cultivated until OD under identical condition
600Reach 0.5.Cultivated again 3 hours with IPTG inducing culture thing to final concentration and the continuation of 1mM.Taking out 100 μ l samples analyzes.The e. coli strains that cultivation contains pLysSrare is BL21star (DE3) and induces under identical condition, obtains sample as negative control.By SDS PAGE electrophoretic analysis sample.
Poly-propionic acid amide gel electrophoresis and western trace
(Invitrogen, Bis-Tris gel www.invitrogen.com) carries out SDS-PAGE from the NuPAGE electrophoresis system to use 4-12%.Use partial desiccation trace method to carry out the Western trace.Use the anti-Lawsonia polyclonal serum of chicken or use porcine blood serum that the Western trace is developed the color, the anti-Lawsonia polyclonal serum of described chicken is that (water: the intact cell preparation oil=45: 55) produces, and described porcine blood serum infects the animal of (post-mortem) damage typically after death available from attack and produce clinical symptom and lawsonia intracellularis with the lawsonia intracellularis per os of purifying at emulsion.Under 4 ℃, used isopyknic rough cell extract preadsorption serum from the BL21star that contains carrier pLysSrare (DE3) 4 hours.
The result
Lawsonia intracellularis gene 5608 is based on the clone in the statement carrier of T7
Use primer 2 179 (CATGCCATGGATTTGATGGAACAGGATTAAAG) and 2180 (CCGCTCGAGCCATAACCCCTTTTCGATAC) amplification gene 5608.In this process, 5 ' NcoI and 3 ' XhoI site are introduced in the PCR product.The PCR product that uses Restriction Enzyme NcoI and XhoI digestion to be obtained.PCR product with digestion is connected to the pET22b that has cut with two identical Restriction Enzymes subsequently.To connect mixture transformed into escherichia coli TOP10F and be incubated overnight at 37 ℃.Use colony PCR to detect the transformant of supposition with regard to correct plasmid.Detect the plasmid inset of colony PCR positive transformant by nucleotide sequence analysis.Selection contains based on one among the clone of the sequence of clone's strategy expection clones and is referred to as pET5608.
Lawsonia intracellularis gene 5608 passes through the expression of T7 promotor in intestinal bacteria
Plasmid pET5608 is transformed into BL21Star (DE3) pLysSrare.Detect the recombinant protein product of the bacterial strain of gained as mentioned above.By sample and the control sample (Figure 1A) of SDS-PAGE gel electrophoresis analysis through the inductive culture.With compare without inductive sample (Figure 1A, swimming lane 2), in the sample of after 3 hours induce, gathering (Figure 1A, swimming lane 3), observe the clear protein band of about 26kDa.Also use pig to pass through the identical sample of Western engram analysis with chicken serum.The serum (Fig. 1 C, swimming lane 3) of serum (Figure 1B, swimming lane 3) of the pig that the lawsonia intracellularis cell per os that using controls oneself uses purifying is attacked and the interior labor Sonia bacterium of chicken anti-cell is observed the reaction with albumen 5608.
Conclusion: the 26kD vaccine component can successfully carry out great expression and the pig anti-cell attacked by per os really in labor Sonia bacterium serum and the chicken anti-cell labor Sonia bacterium blood discern significantly.
Description of drawings
Fig. 1. by the analysis of the overexpression of labor Sonia bacterium gene 5608 in e. coli bl21 STAR/pLysSRARE in the Western trace pair cell of SDS-PAGE (A) and use polyclone porcine blood serum (B) and polyclone chicken serum (C).Swimming lane 1, molecular weight marker; Swimming lane 2, pET5608 T=0; Swimming lane 3, pET5608 T=3.Arrow is represented the position of expression product.
Sequence table
<110>AKZO?Nobel?N.V.
＜120〉Lawsonia intracellularis 26 kd subunit vaccine
<130>2003.023
<160>2
<170>PatentIn?vetsion?3.2
<210>1
<211>856
<212>DNA
＜213〉lawsonia intracellularis (Lawsonia intracellularis)
<220>
<221>CDS
<222>(80)..(823)
<400>1
atggctataa?gcgattgaat?aacagaaaat?aacacctatg?cctgaaattt?tcgacgcgtc 60
gaaattttta?gaggaaacc?atg?aaa?aaa?cta?ctc?ctt?ttg?tta?tct?att?ctg 112
Met?Lys?Lys?Leu?Leu?Leu?Leu?Leu?Ser?Ile?Leu
1 5 10
ttt?cta?acc?cca?agt?att?acc?ttg?gcg?gaa?ggt?aat?act?ttc?aat?gat 160
Phe?Leu?Thr?Pro?Ser?Ile?Thr?Leu?Ala?Glu?Gly?Asn?Thr?Phe?Asn?Asp
15 20 25
agt?ttc?aac?aag?gct?aag?cgc?ata?ctg?caa?gat?gag?gtg?tat?tac?gac 208
Ser?Phe?Asn?Lys?Ala?Lys?Arg?Ile?Leu?Gln?Asp?Glu?Val?Tyr?Tyr?Asp
30 35 40
cac?caa?gtt?aca?cta?tac?tgc?gga?tat?gaa?tat?gat?gac?caa?aaa?agg 256
His?Gln?Val?Thr?Leu?Tyr?Cys?Gly?Tyr?Glu?Tyr?Asp?Asp?Gln?Lys?Arg
45 50 55
ata?tgt?ctc?cct?gat?gga?ttt?ata?gca?gag?aaa?cat?caa?aaa?aga?tca 304
Ile?Cys?Leu?Pro?Asp?Gly?Phe?Ile?Ala?Glu?Lys?His?Gln?Lys?Arg?Ser
60 65 70 75
tat?aaa?att?gag?tgg?gaa?cat?agt?gtg?cct?gct?gag?aat?ttt?ggc?aga 352
Tyr?Lys?Ile?Glu?Trp?Glu?His?Ser?Val?Pro?Ala?Glu?Asn?Phe?Gly?Arg
80 85 90
gct?ttt?act?gaa?tgg?cgc?gaa?ggt?cat?cct?ctt?tgt?gta?gat?aat?aaa 400
Ala?Phe?Thr?Glu?Trp?Arg?Glu?Gly?His?Pro?Leu?Cys?Val?Asp?Asn?Lys
95 100 105
ggt?aaa?agt?ttc?aaa?gga?cga?aaa?tgt?gca?gaa?aaa?gta?aat?aaa?aca 448
Gly?Lys?Ser?Phe?Lys?Gly?Arg?Lys?Cys?Ala?Glu?Lys?Val?Asn?Lys?Thr
110 115 120
tat?aga?tat?atg?cag?tct?gat?atg?tac?aat?ttg?ttt?cca?gca?gtc?ggg 496
Tyr?Arg?Tyr?Met?Gln?Ser?Asp?Met?Tyr?Asn?Leu?Phe?Pro?Ala?Val?Gly
125 130 135
tct?gtc?aat?gct?gcg?aga?agc?aat?aag?caa?tac?tca?gag?tta?ctt?gga 544
Ser?Val?Asn?Ala?Ala?Arg?Ser?Asn?Lys?Gln?Tyr?Ser?Glu?Leu?Leu?Gly
140 145 150 155
gtt?caa?tct?gct?ttt?gga?acg?tgt?gag?gca?aaa?ata?gat?ggg?aat?aga 592
Val?Gln?Ser?Ala?Phe?Gly?Thr?Cys?Glu?Ala?Lys?Ile?Asp?Gly?Asn?Arg
160 165 170
ttc?gaa?cca?ccg?gat?aga?gct?aaa?ggt?caa?gta?gcc?cgt?gct?gct?ctt 640
Phe?Glu?Pro?Pro?Asp?Arg?Ala?Lys?Gly?Gln?Val?Ala?Arg?Ala?Ala?Leu
175 180 185
tat?atg?gat?aaa?gag?tac?aag?gaa?tac?aat?cta?agt?cgt?cag?caa?aga 688
Tyr?Met?Asp?Lys?Glu?Tyr?Lys?Glu?Tyr?Asn?Leu?Ser?Arg?Gln?Gln?Arg
190 195 200
aga?ctt?ttt?gag?gct?tgg?agt?aat?atg?tat?cca?gtc?gat?gaa?tgg?gag 736
Arg?Leu?Phe?Glu?Ala?Trp?Ser?Asn?Met?Tyr?Pro?Val?Asp?Glu?Trp?Glu
205 210 215
tgt?aca?cga?gcc?aaa?cga?atc?gaa?tct?ata?cag?gga?aat?gaa?aat?att 784
Cys?Thr?Arg?Ala?Lys?Arg?Ile?Glu?Ser?Ile?Gln?Gly?Asn?Glu?Asn?Ile
220 225 230 235
ttt?gta?aaa?aat?atg?tgt?atc?gaa?aag?ggg?tta?tgg?taa?acaaacgagg 833
Phe?Val?Lys?Asn?Met?Cys?Ile?Glu?Lys?Gly?Leu?Trp
240 245
acaatataaa?tactacctaa?gta 856
<210>2
<211>247
<212>PRT
＜213〉lawsonia intracellularis
<400>2
Met?Lys?Lys?Leu?Leu?Leu?Leu?Leu?Ser?Ile?Leu?Phe?Leu?Thr?Pro?Ser
1 5 10 15
Ile?Thr?Leu?Ala?Glu?Gly?Asn?Thr?Phe?Asn?Asp?Ser?Phe?Asn?Lys?Ala
20 25 30
Lys?Arg?Ile?Leu?Gln?Asp?Glu?Val?Tyr?Tyr?Asp?His?Gln?Val?Thr?Leu
35 40 45
Tyr?Cys?Gly?Tyr?Glu?Tyr?Asp?Asp?Gln?Lys?Arg?Ile?Cys?Leu?Pro?Asp
50 55 60
Gly?Phe?Ile?Ala?Glu?Lys?His?Gln?Lys?Arg?Ser?Tyr?Lys?Ile?Glu?Trp
65 70 75 80
Glu?His?Ser?Val?Pro?Ala?Glu?Asn?Phe?Gly?Arg?Ala?Phe?Thr?Glu?Trp
85 90 95
Arg?Glu?Gly?His?Pro?Leu?Cys?Val?Asp?Asn?Lys?Gly?Lys?Ser?Phe?Lys
100 105 110
Gly?Arg?Lys?Cys?Ala?Glu?Lys?Val?Asn?Lys?Thr?Tyr?Arg?Tyr?Met?Gln
115 120 125
Ser?Asp?Met?Tyr?Asn?Leu?Phe?Pro?Ala?Val?Gly?Ser?Val?Asn?Ala?Ala
130 135 140
Arg?Ser?Asn?Lys?Gln?Tyr?Ser?Glu?Leu?Leu?Gly?Val?Gln?Ser?Ala?Phe
145 150 155 160
Gly?Thr?Cys?Glu?Ala?Lys?Ile?Asp?Gly?Asn?Arg?Phe?Glu?Pro?Pro?Asp
165 170 175
Arg?Ala?Lys?Gly?Gln?Val?Ala?Arg?Ala?Ala?Leu?Tyr?Met?Asp?Lys?Glu
180 185 190
Tyr?Lys?Glu?Tyr?Asn?Leu?Ser?Arg?Gln?Gln?Arg?Arg?Leu?Phe?Glu?Ala
195 200 205
Trp?Ser?Asn?Met?Tyr?Pro?Val?Asp?Glu?Trp?Glu?Cys?Thr?Arg?Ala?Lys
210 215 220
Arg?Ile?Glu?Ser?Ile?Gln?Gly?Asn?Glu?Asn?Ile?Phe?Val?Lys?Asn?Met
225 230 235 240
Cys?Ile?Glu?Lys?Gly?Leu?Trp
245
Claims (16)
1.) the part of the described nucleic acid of the immunogenic fragments of coding 26kD lawsonia intracellularis (Lawsonia intracellularis) proteic nucleic acid or encoding said proteins, described nucleic acid or its described part have at least 90%, preferably 92%, more preferably 94% even more preferably 96% homology with the nucleic acid with the sequence that is shown in SEQ ID NO:1.
2.) contain the dna fragmentation of the nucleic acid of claim 1.
3.) contain the recombinant DNA molecules of the dna fragmentation of the nucleic acid of claim 1 or claim 2, described recombinant DNA molecules is under the control of the promotor that effectively connects.
4.) the recombinant vectors of Huoing, it comprises the nucleic acid of claim 1, the dna fragmentation of claim 2 or the recombinant DNA molecules of claim 3.
5.) host cell, it contains the recombinant vectors of the work of the recombinant DNA molecules of dna fragmentation, claim 3 of nucleic acid, the claim 2 of claim 1 or claim 4.
6.) the lawsonia intracellularis albumen of 26kD or described proteic immunogenic fragments, described albumen comprise the aminoacid sequence that has at least 90%, preferably 92%, more preferably 94% even more preferably 96% homology with the aminoacid sequence shown in the SEQ ID NO:2.
7.) be used for the lawsonia intracellularis albumen of the claim 6 of vaccine.
8.) the purposes in the lawsonia intracellularis albumen of claim 6 vaccine that labor Sonia bacterium infects in the preparation anti-cell.
9.) the vaccine that labor Sonia bacterium infects in the anti-cell is characterized in that the host cell that it comprises the recombinant vectors of the work of the recombinant DNA molecules of the dna fragmentation of the nucleic acid of claim 1, claim 2, claim 3, claim 4, claim 5 or the albumen and the medicine acceptable carrier of claim 6.
10.) the vaccine of claim 9 is characterized in that it contains adjuvant.
11.) claim 9 or 10 vaccine, it is characterized in that it contains the antigen of other pathogenic virus that derives from pig or microorganism or the described antigenic genetic information of encoding.
12.) vaccine of claim 11, it is characterized in that pathogenic virus of described pig or microorganism are selected from pseudorabies virus (Pseudorabies virus), swine influenza virus (Porcineinfluenza virus), pig parvoviral (Porcine parvo virus), Transmissible gastroenteritis virus (Transmissible gastro-enteritis virus), rotavirus (Rotavirus), intestinal bacteria (Escherichia coli), erysipelothrix rhusiopathiae (Erysipelothrix rhusiopathiae), bordetella bronchiseptica (Bordetellabronchiseptica), Salmonella choleraesuls (Salmonella cholerasuis), haemophilus parasuis (Haemophilus parasuis), multocida (Pasteurellamultocida), swine streptococcus (Streptococcus suis), mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae), swine dysentery spirochete (Brachyspirahyodysenteriae) and actinobacillus pleuropneumoniae (Actinobacilluspleuropneumoniae).
13.) labor Sonia bacterium infects in the anti-cell vaccine, it is characterized in that it contains the proteic antibody of anti-claim 6.
14.) method of the vaccine of preparation claim 9-13, described method comprises mixes the proteic antibody of the albumen of the host cell of the recombinant vectors of the work of the recombinant DNA molecules of the dna fragmentation of the nucleic acid of claim 1, claim 2, claim 3, claim 4, claim 5, claim 6 or anti-claim 6 and medicine acceptable carrier.
15.) be used for the diagnostic check method of the detection of antibodies of labor Sonia bacterium in the anti-cell, it is characterized in that described method of inspection comprises albumen or its fragment of definition in the claim 6.
16.) be used for the diagnostic check method of detection of the antigenicity material of lawsonia intracellularis, it is characterized in that described method of inspection comprises albumen or its segmental antibody of definition in the anti-claim 6.
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| EP03104603 | 2003-12-09 | ||
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| CNA2004800367439A Pending CN101124241A (en) | 2003-12-09 | 2004-12-08 | Lawsonia intracellularis 26 kd subunit vaccine |
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| EP (1) | EP1694698A1 (en) |
| JP (1) | JP2007537715A (en) |
| KR (1) | KR20060112674A (en) |
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| AU (1) | AU2004297018A1 (en) |
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| CN110029079A (en) * | 2019-03-27 | 2019-07-19 | 南京农业大学 | The recombined bacillus subtilis in expression secretion porcine pseudorabies toxalbumin dominant antigen area and application |
| CN113994006A (en) * | 2019-02-28 | 2022-01-28 | 萨斯喀彻温大学 | Lawsonia intracellularis compositions and methods of using the same |
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| CN103157100B (en) * | 2011-12-08 | 2014-09-24 | 普莱柯生物工程股份有限公司 | hemophilus parasuis disease, swine streptococcosis bivalent inactivated vaccine and preparation method thereof |
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| US5885823A (en) * | 1995-06-05 | 1999-03-23 | Nobl Laboratories, Inc. | Lawsonia intracellularis cultivation, anti-Lawsonia intracellularis vaccines and diagnostic agents |
| CA2372102A1 (en) * | 1999-05-13 | 2000-11-23 | Australian Pork Limited | Lawsonia derived gene and related omph polypeptides, peptides and proteins and their uses |
| EP1094070A3 (en) * | 1999-10-22 | 2002-01-09 | Pfizer Products Inc. | Lawsonia intracellularis proteins, and related methods and materials |
| CA2501238A1 (en) * | 2002-10-04 | 2004-04-22 | Regents Of The University Of Minnesota | Nucleic acid and polypeptide sequences from lawsonia intracellularis and methods of using |
-
2004
- 2004-12-08 KR KR1020067013035A patent/KR20060112674A/en not_active Application Discontinuation
- 2004-12-08 WO PCT/EP2004/053342 patent/WO2005056586A1/en active Application Filing
- 2004-12-08 AU AU2004297018A patent/AU2004297018A1/en not_active Abandoned
- 2004-12-08 CA CA002548750A patent/CA2548750A1/en not_active Abandoned
- 2004-12-08 CN CNA2004800367439A patent/CN101124241A/en active Pending
- 2004-12-08 JP JP2006543544A patent/JP2007537715A/en not_active Withdrawn
- 2004-12-08 EP EP04820075A patent/EP1694698A1/en not_active Withdrawn
- 2004-12-08 BR BRPI0417440-2A patent/BRPI0417440A/en not_active IP Right Cessation
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113994006A (en) * | 2019-02-28 | 2022-01-28 | 萨斯喀彻温大学 | Lawsonia intracellularis compositions and methods of using the same |
| CN110029079A (en) * | 2019-03-27 | 2019-07-19 | 南京农业大学 | The recombined bacillus subtilis in expression secretion porcine pseudorabies toxalbumin dominant antigen area and application |
| CN110029079B (en) * | 2019-03-27 | 2022-10-11 | 南京农业大学 | Recombinant bacillus subtilis for expressing and secreting porcine pseudorabies virus protein dominant antigen region and application |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2004297018A1 (en) | 2005-06-23 |
| WO2005056586A1 (en) | 2005-06-23 |
| JP2007537715A (en) | 2007-12-27 |
| BRPI0417440A (en) | 2007-03-06 |
| EP1694698A1 (en) | 2006-08-30 |
| CA2548750A1 (en) | 2005-06-23 |
| KR20060112674A (en) | 2006-11-01 |
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