CN1325731A - Living toxic-reduced bacteria as vaccine - Google Patents

Abstract

The present invention relates to the living attenuating bacterium used for medicine and the vaccine based on said attenuating bacterium for preventing microbetic diseases. The living attenuating recombinant bacterium, and the vaccine based on said bacterium; and their preparing process to said vaccines and bacteriums are disclosed.

Description

The living toxic-reduced bacteria that is used for vaccine

The present invention relates to be used for a kind of living toxic-reduced bacteria of medicament, based on to the useful antibacterial of prophylaxis of microbial mechanism of causing a disease and the preparation method of the vaccine that produces, the living toxic-reduced bacteria that carries heterologous gene and these vaccines and antibacterial.

It is the process of a complexity that homoiothermic animal is defeated the morbific mode of microorganism.The immunity of the disease that microorganism is caused is that homoiothermic animal is avoided morbidity or suffered strong morbific a kind of mode.When the population exposed pathogen, the incomplete immunity of giving pathogen is caused morbidity and causes death.It is generally acknowledged the immunne response of inducing a kind of heavy duty detergent based on the vaccine (attenuated vaccine alive) of the attenuated microorganisms of living.This vaccine has individual advantage, in case the animal reservoir is by vaccination, the microorganism cause of disease bacterium that enters the host is induced the memory of an early stage acceleration cell-mediated or humoral immunization, its can be before infection presents clinical remarkable ratio the further growth of controlling microbial.Vaccine (killed vaccine) based on dead pathogen in general can not obtain such immunne response.Yet, contain the vaccines of pathogenic bacterium alive, depend on the level of attenuation, such danger can appear, that is and, the inoculation host may suffer from the disease that people attempt to resist after prophylactic immunization.Therefore, people wish to obtain having the immune attribute of viable microbial but the vaccine that can not cause undesirable side effect after prophylactic immunization.

The basic skills of antibacterial attenuation is to remove one or more virulence factors.Yet under most situation, virulence factor also plays important effect aspect induction of immunity.Under those situations, the disappearance of virulence factor has weakened the immunogenicity ability of antibacterial inevitably.This yes a kind of undesirable situation.Live vaccine preferably should keep the antigen composition of wild-type strain.

In addition, live vaccine should have sufficient a-virulence to avoid unacceptable pathology effect, and but then, it must cause the immunity of the enough levels of host.At last, the attenuated live vaccine bacterial strain preferably should not have answer to be the probability of virulence wild-type strain basically.

Be surprisingly found out that now a known proteinic gene that works of coding can be lacked in the metabolism of the main carbohydrate that many antibacterials belong to, cause attenuation behavior in vivo and do not weaken these antibacterials viability in vivo.The antibacterial that this gene is lacked unexpectedly demonstrates a kind of attenuation feature really.In addition, because what effect is encoded protein matter do not play in the inducing of immunity, lacked the identical of the antigen load of antibacterial of this gene and wild type.Therefore, this antibacterial can be unexpectedly by the field of priority application in the medicament preparation.It more specifically is the preparation that is used for attenuated vaccine alive.

Indication of the present invention can be lacked and be caused the gene of attenuation behavior in the body of deletion mutant to be called as the fruR gene in the past, yet is called the cra gene now.The known mutant that lacks this gene can be in growth in vitro, but only when growing when the active and grown culture medium of defective that cause of cra is compensated by lacking.This means the cra deletion mutant can rely in the growth nutritional labeling must be present in the growth medium.

In general, pathogenic bacterium can the oneself be supported to be meant that its metabolism can adapt to available nutrient.(as follows) cra gene plays a kind of like this adaptability effect in many main metabolic pathway.Yet the mutant that the cra gene has been lacked can rely on glucose and a lot of other sugar as carbon source to grow well.In host animal, such sugar is utilizable, so people can not expect under the cra gene condition in vivo function is arranged.Therefore, people can not expect that the mutant of cra disappearance demonstrates the attenuation characteristic in the host.Though this why just explained these mutants be well known to a person skilled in the art never the people disclose its potentiality as the attenuated live vaccine candidate.

One embodiment of the invention relate to the living toxic-reduced bacteria that is used for vaccine, and it is because the sudden change of Cra gene causes no longer expressing a kind of functional Cra albumen.

Gene outcome (was called FruR in the past; The fructose repressor protein), being also referred to as Cra (catabolite repression/activator protein) now, is the adjusting albumen in many carbohydrate metabolism main paties.

Cra gene outcome Cra is regulating main carbon metabolism.More specifically be, Cra is by (for example: the promoter upstream of the gene key enzyme in TCA circulation, glyoxylate bypass, gluconeogenesis approach and the electron transfer) is just being regulated these gene transcription, negatively (for example: the gene transcription key enzyme in EM approach and the ED approach) regulates the coding glycolytic ferment in conjunction with encoding human synzyme and oxidase.

Because its key position in carbohydrate metabolism, cra gene and gene outcome Cra thereof are present in the antibacterial widely.Cra albumen is a kind of albumen of high conservative.For example, it may reside in escherichia coli, the intestinal Salmonella is mouse typhus for example, enteritis and Dublin serotype, Actinobacillus is the lobar pneumonia Actinobacillus for example, and haemophilus is haemophilus paragallinarum for example, aeromonas salmonicida genus, pasteurella be bar fish pasteurella for example, and multocida, Streptococcus is streptococcus equi, Streptococcus suis for example, and Yersinia is Yersinia pestis for example.

Jahreis, people such as K have set forth Salmonella and Escherichia in 1991 gene itself and complete nucleotide sequence (Mol.Gen.Genet.226:332-336 (1991)) .Jahreis thereof illustrate the intestinal Salmonella, mouse typhus serotype is only different four positions with colibacillary Cra albumen, and wherein two only is the conservative exchange.These meet the desired albumen of playing a role for a kind of certainly in like this numerous general approach of antibacterial carbohydrate metabolism, especially under Salmonella and the divergent during evolution situation so not far away of escherichia coli.Ramseier, people such as T.M. have set forth the protein bound mechanism of Cra (molecular biology magazine 234:28-44 (1993)) to small part.Proteic effect of Cra and function formally are documented on the document, for example: on the little summary that Saier, M.H. and Ramseier, T.M. deliver recently (bacteriology's magazine 178:3411-3417 (1996)).

This sudden change can be insertion, disappearance, replacement or several combinations, and condition is the failure that sudden change has caused functional Cra protein expression.Functional Cra albumen is considered to a kind of albumen with control characteristic of wild-type protein.Therefore, the Cra albumen of its a kind of functional defect is considered to non-functional Cra albumen at least.

The living toxic-reduced bacteria that the present invention uses can obtain by number of ways.A kind of possible approach that obtains this antibacterial be by traditional method for example with mutagenic agent for example base analogue handle the wild-type strain have the cra gene, or with UV treatment or Temperature Treatment.

Not producing the proteic bacterial strain of functional Cra is easy to be chosen.They only contain glucose and other sugar as the minimal medium of carbon source in growth (this makes they and cya make a distinction with the crp mutant) but they can not utilize with gluconeogenesis substrate grow on as the culture medium of sole carbon source people such as (, bacteriology's magazine 169:897-899 (1987)) Chin.Therefore they be easy to external selected.

The person's character of the sudden change that the tradition mutating technology causes is unknown.This may be a point mutation, though can not certain take place, may finally reply by point mutation and is wild type.For fear of this little danger, transposon mutagenesis will be a good selection.The sudden change that transposon mutagenesis causes also is a known mutating technology of those of ordinary skill in the art.This is a sudden change of finishing in chromosomal localized site.The insertion of transposon is not at a special genes.Because the cra-mutant is can not be in growth in vitro to lacking that the Cra activity carries out under the condition of nutritional compensation, so be easy to screened come out.Therefore they are easy to screened come out the antibacterial of transposon mutant at random from a group.

Recombinant DNA technology provides wittingly rather than has imported at predetermined site randomly the probability of a sudden change.This sudden change can be the insertion, disappearance of nucleotide or nucleotide is replaced by another nucleotide or their combination, and unique condition is the gene encoding function Cra no longer of sudden change.This sudden change can for example be the disappearance of some nucleic acid.Even the disappearance of for example few one section sequence to 10 nucleic acid can cause Cra not have function.Even for example the disappearance of a mononucleotide can cause Cra not have function, because this results of mutation, other nucleotide is no longer in correct reading frame.The disappearance that a large amount of nucleic acid of being made up of three indivisible nucleic acid inserts causes this frameing shift.More preferably segment length's sequence for example 100 nucleic acid lacked, further preferably whole cra gene is lacked.Be very easy to find, in open reading frame, imported the specific sudden change of a terminator, or cause that in open reading frame the sudden change of frameing shift is very suitable for obtaining the no longer bacterial strain of encoding function Cra.

The technology of all structure Cra-deletion mutants is known standard technique.It comprises the clone of cra gene, and gene order that direct mutagenesis causes is modified, and the degraded of Restriction Enzyme is to connect or PCR-method and replace the cra gene (equipotential exchange or equipotential replace) of wild type with mutant gene then again.The recombinant DNA technology of standard is as clone cra gene in plasmid, use the Restriction Enzyme degrading genes, handle with endonuclease then, connect again and in host strain, carry out homologous recombination, be technology well known in the art and be described in Maniatis/Sambrook and go up (Sambrook, J. wait the people, molecular cloning: laboratory manual.ISBN?0-87969-309-6)。Direct mutagenesis can be by for example using the Transformer available from Clontech.PCR-techniques ^Test kit is at external direct mutagenesis, and it is described in (Dieffenbach ﹠amp; Dreksler; The PCR primer, laboratory manual.ISBN 0-87969-447-3 and ISBN 0-87969-447-5) on.

The cra gene not only contains the proteic coded sequence of coding Cra, and contains the regulating and controlling sequence just like promoter, and this gene also contains the necessary site of correct translation of Cra mRNA, for example ribosome binding site.

Therefore, not only the sudden change of coding region but also the correct sudden change of transcribing and translating necessary sequence all are considered to belong within the scope of the present invention.

In a preferred embodiment, the present invention relates to be used for the Escherichia of vaccine, Salmonella, Actinobacillus, haemophilus, Aeromonas, pasteurella, the living toxic-reduced bacteria of Streptococcus and Yersinia.

In a more preferred form of the present invention, living toxic-reduced bacteria of the present invention is selected from intestinal Salmonella mouse typhus, enteritis, hog cholera, Dublin, typhoid fever, chicken, sheep miscarriage, equine abortion, Pullorum Disease serotype, escherichia coli or Yersinia pestis.These antibacterials contain in a large number to human and the morbific kind of different animals kind.Living toxic-reduced bacteria of the present invention is preferably the intestinal Salmonella, escherichia coli or Yersinia pestis.

In its more preferred form, living toxic-reduced bacteria of the present invention is intestinal Salmonella, escherichia coli or Yersinia pestis.

In a more preferred form, this embodiment relates to living toxic-reduced bacteria of the present invention, has wherein utilized recombinant DNA technology to cause the sudden change of cra gene.

Better limit and carefully carry out comprise the cra genetic fragment or even the disappearance of whole gene or sudden change or the two simultaneous sudden change of the segmental insertion of allogeneic dna sequence DNA following advantage is arranged: compare with traditional induced mutation, it can not be returned to wild-type status.

Therefore, in a more preferred form, this embodiment of the present invention is meant living toxic-reduced bacteria, and wherein the cra gene contains insertion and/or disappearance.

Nowadays in a single day a large amount of vaccines are used for house pet and farm-animals, and iff in order to reduce the cost of vaccine, the co-administered of several vaccines will be desirable obviously.Therefore using living toxic-reduced bacteria is very attractive as the recombinant vector of heterologous gene, and the antigen of heterologous gene coding wherein is selected from other pathogenic microorganism or virus.The administration of this recombinant vector has an advantage promptly to induce the immunity of resisting two or more diseases simultaneously.Because the proteic gene of coding Cra can be used as heterologous gene and inserts the site, the living toxic-reduced bacteria that is used for vaccine of the present invention just provides the carrier that is fit to that is used for this heterologous gene.The cra gene as the heterologous gene of the application of the inserting the site same stylish importing that an advantage is arranged is the cra gene inactivation can be expressed (with homologous bacterial gene together).The Protocols in Molecular Biology that the structure of this recombinant vector can use standard for example equipotential exchange carries out routinely.Therefore, another embodiment of the present invention's attenuation recombinant bacteria alive is preferably Escherichia, Salmonella, Actinobacillus, haemophilus, Aeromonas, pasteurella, Streptococcus and Yersinia have wherein inserted heterologous gene but can not produce functional Cra albumen.So heterologous gene can for example be the antigenic gene that coding is selected from other pathogenic microorganism or virus as mentioned above.This gene can derive from the herpesvirus (genes of the structural protein of the herpesvirus of for example encoding) that for example causes a disease, retrovirus (for example gp160 envelope protein), adenovirus etc.Heterologous gene also can derive from malignant bacteria.As an example, the coding bacteriotoxin is lobar pneumonia Actinobacillus toxin for example, and clostruidium toxin, the gene of outer membrane protein etc. are very suitable antibacterial heterologous genes.Another kind of probability is to insert coding and trigger the immune system proteins associated, the gene of interleukin or interferon for example, or another relates to the gene of immunoregulation.

It is useful inserting heterologous gene in the cra gene, because it does not need to seek the insertion site of heterologous gene, and the cra gene is knocked out simultaneously.

Thereby in a preferred embodiment, heterologous gene is inserted into the cra gene.Heterologous gene can be inserted in arbitrary position of cra gene or be inserted into the cra Gene Partial or the site when all lacking.

Because they are attenuation unexpectedly except causing immunological characteristic in vivo, the antibacterial that the present invention is used for vaccine is suitable as the basis of attenuated vaccine alive very much.Thereby another embodiment of the invention relates to and is used to watch for animals and human attenuated vaccine alive of resisting the wild-type bacterium infection that contains the cra gene.

These vaccines contain living toxic-reduced bacteria or the described live-weight group carrier antibacterial of the present invention and the pharmaceutically acceptable carrier that is used for vaccine of the present invention of immune effective dose.

More preferably, the vaccine that contains living toxic-reduced bacteria according to the present invention is selected from Escherichia, Salmonella, Actinobacillus, haemophilus, Aeromonas, pasteurella, Streptococcus and Yersinia.

The immunity effectiveness is meant that the amount of the living toxic-reduced bacteria that is used for immune administration is enough to induce the effective immunne response that causes the antibacterial toxic forms the host.

Except the living toxic-reduced bacteria of immune effective dose as mentioned above, vaccine of the present invention also contains pharmaceutically acceptable carrier.This carrier can be simply as water, and still, it also can contain for example culture fluid of culture of bacteria.Another kind of suitable carriers for example is the solution of physiological salt concentration.

The useful dosage of administration depends on the animal of age, body weight, inoculation, the difference of the type of the pathogen of the mode of administration and vaccine effect and changing.

Vaccine can contain the antibacterial that is enough to fully evoke immunne response of any dosage.Dosage range is 10 ³With 10 ¹⁰It between the antibacterial unusual proper dosage.

Selectively, one or more have the chemical compound of adjuvanticity to be added in the vaccine.Adjuvant is immune nonspecific stimulation thing.It strengthens the immunne response of host to vaccine.The example that well known to a person skilled in the art adjuvant is the complete and Freund of Fu Shi, vitamin E, nonionic block polymer, muramyldipeptide, ISCOMs (the immunostimulating complex is for example referring to EP 109942), saponin, mineral oil, vegetable oil and Carbopol.

Be specially adapted to the adjuvant that mucosa uses and be for example escherichia coli heat-labile toxin (LT) or cholera toxin (CT).

Other suitable adjuvant is for example aluminium hydroxide, aluminum phosphate or aluminium oxide, oil emulsion (for example, Bayol F (R) or Marcol 52 (R)) saponin or vitamin E soluble-salt.

Therefore, vaccine of the present invention preferably contains adjuvant.

Other the example that is used for pharmaceutically acceptable carrier of the present invention or diluent comprises for example SPGA, carbohydrate (sorbitol for example, mannitol, starch, sucrose, glucose, glucosan), as albumin or caseic protein, (for example: protein phosphate buffer) contain Ox blood serum or defatted milk and buffer agent.

Especially when this stabilizing agent added vaccine, vaccine was very suitable for lyophilization.Therefore, in a kind of preferred form, vaccine is a kind of freeze dried form.

In order to give the administration of animal or human's class, but vaccine intranasal administration of the present invention, the intradermal administration, subcutaneous administration, oral, suck or intramuscular administration.During the poultry administration, wing administration and eye drip administration are very suitable.

Another embodiment relates to application or of the present invention recombinant bacteria of antibacterial in vaccine and is used for preparing the application that watches for animals with the human pathogenic sex vaccine of resisting the wild-type bacterium infection or infecting.

Another embodiment of the invention relates to the preparation method of vaccine of the present invention.The method comprises mixes living toxic-reduced bacteria of the present invention or recombinant vector antibacterial of the present invention with pharmaceutically acceptable carrier.

Embodiment

The evaluation of embodiment 1. Salmonella typhimurium SR-11 Fad-mutant genes, clone and order-checking

The gene of the transposon mutant of Salmonella typhimurium SR-11 Fad-mutant is identified, clone and order-checking.

Provide the nucleotide sequence of the mutant gene of SR-11 Fad-a-virulence to be disclosed among the SEQ ID NO:1.

SEQ ID NO:2 discloses the aminoacid sequence of the nucleotide sequence coded protein molecular of SEQ ID NO:1.

Having identified Salmonella typhimurium SR-11 Fad-now is cra gene mutation strain.The point that transposon inserts has been found about 45 the base pair places that are positioned at apart from 3 ｀ ends of cra translation stop codon.

One contains the Pst1 fragment that 1.5kb Tn 10d Cam inserts the 4.5kb of son (distolateral connect one section 1.9kb Salmonella typhimurium SR-11 dna fragmentation and one section 1.1kb Salmonella typhimurium of other end side joint SR-11 dna fragmentation) and is inserted in the Pst1 site of pBluescript II SK (+).The plasmid pJHA 7 that the result obtains enters into E.Coli HB101 by electroporation.Insert sub zone by the two deoxidation thermal cycling method order-checking of Sanger side joint Tn 10d Cam.The nucleotide sequence that direct side joint Tn 10d Cam inserts sub every side is found to be had 100% homology with Salmonella typhimurium cra (fruR) gene and finds that the insertion point is apart from terminal 45 nucleotide of 3 ｀ of cra translation stop codon.These have shown that Salmonella typhimurium SR-11 Fad-is a cra mutant.

With SR-11 contrast, SR-11 Fad ^-Can not contain citrate, oleate, pyruvate, acetate is grown on the basic agar culture medium flat board of the M9 of succinate and fumarate.

By pcr amplification Salmonella typhimurium SR-11 wild type cra (fruR) and be inserted into the pst1 site of the ampicillin resistance gene of pBR322.Gained pJHA8 plasmid has recovered the ability of Salmonella typhimurium SR-11 Fad and also can utilize each above-mentioned chemical compound as carbon source as wild-type strain.These experiments determine that Salmonella typhimurium SR-11-is cra (fruR) mutant.

The structure of SR-11 Fad is that chlorampenicol resistant by little transposon mutant strain that will come from LT-2d is by phage P22HT105int ^-Transduction is gone into and to be realized among the SR-11.Although may not, SR-Fad ^-Avirulent probability depends on some SR-11 DNA the losing of when transduction, for example, and the losing of pathogenic island, rather than depend on defective cra gene.Therefore, as described below, a bacterial strain that is equal to SR-11, SR-11 Cra hereinafter ^ModAX-2, except containing the identical cra gene mutation that is present among the SRFad-, it exchanges by equipotential and makes up.

Contain SR-11 Fad ^-The Pst1 SR-11 Fad of the 4.3kb of sudden change cra gene (the cra gene contains chloramphenicol resistance gene) ^-Dna fragmentation is inserted into a Pst1 site of containing the suicide vector pLD55 of ampicillin resistance gene and tetracycline resistance gene.It is called as pMJN10.PMJN10 enters among the E.coli S17-1 λ pir by electroporation.E.coli S17-1 λ pir (pMJN10) has detected some ampicillin with after SR-11 engages, and tetracycline and chloromycetin SR-11 transconjugant utilize oleate, citrate, acetate, pyruvate, succinate and fumarate are as the ability of sole carbon source.If pMJN10 has changed by the single cross of homologous sequence and has been incorporated in the chromosome, then all transconjugants can both as desiredly utilize above-mentioned salt, promptly seem to suddenly change and the cra allele of wild type all is present in the chromosome.Like this five " integrons " are used to detect the pMJN10 that wherein whether exists as free plasmid, and the result does not exist, thereby determine that further this plasmid has been inserted in the SR-11 chromosome.In five integrons each is being contained on the Luria agar plate of chloromycetin by streak culture.In this case, as long as just to stay cra allele in the chromosome be that the cell that the secondary exchange then takes place for the mutation allele that contains chloramphenicol resistance gene can be survived.Streak culture conjugon sample is streak culture then on sensitive tetracycline screening agar (TSS agar).TSS agar contains Fumaric acid and sensitive tetracycline sexual cell, and the cell of promptly having lost suicide plasmid grows up to very big colony with respect to the tetracyclin resistance cell that still contains plasmid in chromosome.34 big colonies are used to detect chlorampenicol resistant altogether, the sensitivity of ampicillin and tetracycline and utilize oleate, and acetate, pyruvate, citrate, succinate and fumarate are as the ability of sole carbon source.In 34 separators, 6 have chlorampenicol resistant, but to ampicillin and sensitive tetracycline, can not utilize above-claimed cpd as sole carbon source.One of them separator is called as SR-11Cra ^ModDAX-2 (AX is meant the allele exchange), transformed with pBR322 or pJHA8 (pJHA8 contains wild type cra gene) and two bacterial strains all detected use oleate, acetate, pyruvate, citrate, succinate and fumarate are as the ability of sole carbon source.Contrast SR-11Cra ^ModAX-2 (pBR322), SR-11Cra ^ModAX-2 (pJHA8) can utilize above-claimed cpd as sole carbon source, and this shows SR-11Cra ^ModAX-2 is the cra mutant.These two bacterial strains, positive according to expectation, can utilize glucose and glycerol as sole carbon source.

In order to determine whether functional cra (fruR) gene makes Salmonella typhimurium have the SR-11 virulence, carried out following experiment.

Four BALB/c mouse SR-11 (2.1 * 10 ⁸The cfu/ mice) peroral infection and five mices are used SR-11Cra ^ModAX-2 (2.8 * 10 ⁸The cfu/ mice) infects.To infecting the back the 8th day, all 4 mices that infect SR-11 are all dead, yet 5 are infected SR-11Cra ^ModThe mice of AX-2 still healthy and active (table 1).Because except being present in SR-11Cra ^ModIn the identical sudden change of cra outside, SR-11Cra ^ModAX-2 is the same with SR-11, is making up SR-11Cra by transduceing from the LT-2 bacterial strain ^ModThe irrelevant probability of some anomalous events that take place in the process and cra is excluded, and this may be the reason of virulence forfeiture.

Also might the insertion of chlorampenicol resistant element in the cra gene cause polarity effect to downstream gene, so SR-11 Fad ^-Attenuation do not depend on defective cra gene.Therefore, SR-11Cra ^ModCompensate with the pJHA8 that only contains wild type cra gene, whether to detect SR-11Cra ^Mod(pJHAB) recover virulence.As a contrast, SR-11Cra ^ModCompensate with pBR332, this carrier is used for making up pJHA8.Four BALB/c mouse are by with 3.1 * 10 ⁸The SR-11Cra of cfu/ mice ^Mod(pBR322) peroral infection and other four BALB/c mouse are by with 4.3 * 10 ⁸The SR-11 Fad of cfu/ mice ^-(pJHA8) infect.To infecting the back the 9th day, use SR-11Cra for four ^Mod(pJHA8) three death in the mice infected and use SR-11Cra ^Mod(pBR322) 4 mices of Gan Raning still healthy and active (table 1).Liver and the spleen of all dead mices all contain greater than 10 ⁸The SR-11 Fad of cfu/ organ ^-(pJHA8).This result has got rid of the inactivation of the cra gene that has the chlorampenicol resistant element causes that thereby downstream effect causes avirulent probability and proved that functional cra gene is that the SR-11 virulence is necessary.Salmonella typhimurium strain infects number ^aSurvival number SR-11 4 0SR-11Cra ^ModAX-2 5 5SR-11Cra ^Mod(pBR322) 4 4SR-11Cra ^Mod(pJHA8) 41

^aAccording to the difference of bacterial strain, with 2.0 * 10 ⁸Cfu/ mice～5.0 * 10 ⁸The oral dose infecting mouse of cfu/ mice.All dead Mus all are to infecting back death in the 9th day.All survival pindones recover entirely.Table 1 detects the cra gene in the different bacterium genus

Salmonella typhimurium four strains, the intestinal Salmonella, Salmonella gallinarum, each strain of salmonella dublin and Salmonella choleraesuls is hybridized by Southern and is detected the cra gene.In all cases, the cra gene is found the Pst1 dna fragmentation of the same 4.3kb of having size with SR-11.Six different pathogenic E.Coli bacterial strains are also detected all the cra gene, though the cra gene in the six strain bacterium is present in the Pst1 fragments of three kinds of different sizes.In addition, a strain aeromonas salmonicida and an Actinobacillus, haemophilus, pasteurella, some bacterial strains of Streptococcus and Yersinia all demonstrate the existence of cra gene after testing.

The proof that the cra gene is present in above-mentioned antibacterial is as follows:

The genomic DNA of these bacterial strains is in 37 ℃ of Pst1 with 20 units (Promega) degraded of spending the night.(0.7% agarose, 1 * TAE) is used for separating the Pst1DNA fragment of different sizes to gel electrophoresis.Separated DNA S﹠amp; S Turboblotter system (Schleicher and Schuell) transfers to last 3 hour of nylon membrane of positively charged under the condition of alkalescence.Film cures under 90 ℃ and DNA was connected on the film in 30 minutes.Then, 700 base pair fragments of Salmonella typhimurium cra gene are by the DIG labelling and be used for detection membrane.Film is containing 5 * SSC under 62 ℃, 0.1% N-Hamposyl L, and 0.02%SDS, prehybridization (rolling in bottle hybrid heater at) is 2～4 hours in 1.5% closed reagent (DIG of band CSPD detects bottle opener medicine box II, Boehringer Mannheim).The probe of labelling is by degeneration and add in the fresh hybridization buffer, and trace was cultivated 16-20 hour down at 62 ℃.Trace 0.1%SDS, 2 * SSC washed twice at 62～65 ℃ in following 5 minutes.Use 0.1%SDS then, 0.1 * SSC washed twice at 60 ℃ in following 15 minutes.Launch trace by following modification: 2% closed reagent is used to lock solution (using 1% usually), and the low concentration (using 70% concentration usually) of trace is closed one hour (usually sealing time be 30 minutes) and antibody is used to the detection of DIG-label probe.These change manufactured merchant and are proposed to be used in lower background signal.

The salmonella typhimurium strain SR11Cra of embodiment 2 usefulness Cra-feminine genders ^ModThe inoculation chicken

Inoculation is renderd a service.The growth conditions of Salmonella bacterial strain is similar with description in embodiment 2.In an experiment, two groups of 20 chickens (three day age) are with being dissolved in 6 * 10 among the PBS ⁷CFU Salmonella typhimurium SR11Cra ^ModThe oral administration inoculation.One group after 11 days with 8.3 * 10 ⁷The same bacterial strain booster immunization inoculation of CFU.After 18 days, two groups respectively by subcutaneous, intramuscular, oral 1.9 * 10 ⁹The virulence wild-type strain attack.Table 2 has provided the result.

SR11Cra ^ModSR11Cra ^ModDosage 6.0 * 10 when contrasting inoculation in the 1st day ⁷6.0 * 10 ⁷Dosage-8.3 * 10 during-Di inoculation in 11 days ⁷Dosage 1.9 * 10 when-Di attacked in 18 days ⁹1.9 * 10 ⁹1.9 * 10 ⁹The safety of mortality rate (%) 15 10 100 table 2 simultaneous inoculations and effectiveness experiment.In second experiment, detect the effectiveness of vaccine and the safety of vaccine.The safety of vaccine records on the basis of retarded growth.One group of 15 chicken is dissolved in 2.7 * 10 in the culture medium by oral vaccination ⁸CFU Salmonella typhimurium SR11Cra ^Mod15 chicken oral vaccinations of another group are dissolved in 1.3 * 10 among the PBS ⁸The same bacterial strain of CFU.After 18 days, all use 6.5 * 10 for two groups ⁸The virulence wild-type strain is attacked.Table 3 has shown the result.

SR11Cra ^Mod(ⅰ) SR11Cra ^ModDosage 2.7 * 10 when (ⅱ) contrast was inoculated on the 1st day ⁸1.3 * 10 ⁸--the dosage 6.5 * 10 when the 7th day the 18th day weight of weight 178 ND 185 733 722 749 the 18th days is attacked ⁸6.5 * 10 ⁸6.5 * 10 ⁸Mortality rate (%) 0 13 100 tables 3: ⅰ=culture medium; ⅱ=PBS result: two experiments show; although used high challenge dose, the salmonella typhimurium strain of Cra-feminine gender has obtained the protection of high level.In addition, find that vaccinated result does not have significant retarded growth.Therefore can conclude Cra ^ModNegative salmonella typhimurium strain is very suitable for being used to protect poultry to resist the attenuated vaccine alive of the infection of wild-type bacterium.

Embodiment 3: preface

A pig Attack Research is finished to detect negative gene knockout (KO) bacterial strain 34682 of Salmonella choleraesuls Cra-and 35276 safeties with respect to cra male 34682 and 35276 parent strains.These knockout mutant strains be by use plasmid and with above-mentioned Cra ^ModThe identical method of the structure of AX-2 obtains.1. pig safety testing A. animal and feeding manner

There is not the farm of Salmonella history to buy 20 (20) 5-6 pig that is not inoculated Salmonella in all ages from one.Pig is divided into four groups, five every group.Whole experimental session, pig are enclosed at 4 independently in the isolation room.B. attack

5 pigs of each group are attacked during age in week at 5-6.Pig is attacked and oral attack (1.0ml culture+4.0ml antibacterial diluent) by intranasal (0.5ml/ nose).Attack culture and be approximately 9.0 * 10 ⁸CFU/ml.Then, pig is monitored every day, and the typical clinical symptoms of Salmonella infection comprises body weight loss, the temperature of the rectum of diarrhoea and rising.C. euthanasia

Attacked the back the 7th day, pig is implemented euthanasia.Pig is by necropsy and lung, liver, and spleen, mesentery lymph node (MLN) and ileum are used for the growth of Salmonella choleraesuls by cultivation.D. weight increase

Increasing (ADG) in average day is by deducting originally body weight again divided by obtaining from the natural law of attacking to euthanasia from final body weight.Group ADG is the average of individual pig ADG.2. mice safety testing

A mice is attacked the LD that experiment is finished to detect different Salmonella choleraesuls bacterial strains ₅₀, i.e. 34682 and 35276 of the positive parent 34682 of Cra and 35276 bacterial strains and cra feminine gender LD that knock out (KO) bacterial strain ₅₀A. animal

The CF-1Sasco mice of 200 (200) 16-20 grams is divided into the security test that 20 groups every group 10 mices are used for mice.Be placed in the same room whole experimental session mice, but in different buckets.B. attack

Each bacterial strain is for the subgroup of trying to contain 10 mices in 5.These subgroups 5 kinds of different diluents (10 of 0.25ml bacterial strain ^-3-10 ^-7) intraperitoneal (lP) attack.

The freezing seed of each of four kinds of bacterial strains is diluted as 10 ^-3-10 ^-7Each of these diluents by peritoneal injection (0.25ml) in 10 mices.I as a result. pig safety testing A. weight increase

The weight increase of pig is shown among Fig. 1.These data clearly illustrate the different of the pig attacking and attack with parent strain with the KO bacterial strain.Data also reflect the difference of the total health situation of animal.Two groups of pigs of attacking with parent strain have all alleviated body weight to euthanasia when attacking; Yet the pig of attacking with the KO bacterial strain has increased about 0.75 kilogram every day.II. mice safety experiment A. death

The results are shown in the following table 4 of mouse experiment.Right hurdle has shown the half lethal dose of different strains CFU.Knock-out bacterial strain clearly shows high-caliber attenuation.Bacterial strain LD ₅₀Cfu35276 parent＜7.7cfu35276 knocks out 1.5E+04cfu34682 parent 12.5cfu34682 and knocks out＞9.0E+04cfu table 4 conclusion

Shown that from the pig safety experiment knocking out (KO) bacterial strain through attack Cra has produced obvious higher weight increase with respect to parent strain.It has proved the attenuation characteristic of CraKO mutant.Except the pig safety testing, the mice safety testing shows that also Cra KO bacterial strain is attenuated: the LD of KO mutant ₅₀The LD that is higher than parent strain significantly ₅₀

Embodiment 4: preface

The purpose of this embodiment is to estimate the safety of KO-34682 Cra mutant with respect to its parent strain, and detects the effectiveness that Salmonella bacterial strain KO-34682 opposing allos toxicity Salmonella bacterial strain 35276 is attacked.In addition, KO-34682 cra mutant is with respect to the not immune effectiveness that contrasts.Animal and feeding manner

There are not the farm purchase ages in 20 3 weeks of Salmonella history and the pig that is not inoculated from one.Pig is divided into four groups, and circle is at 4 independently in the isolation room.Vaccine and inoculation

Pig is in 3 weeks during ages oral about 1 * 10 ⁹The Salmonella choleraesuls bacterial strain KO-34682 of CFU/ml, parent's 34682 inoculations, or do not inoculate.Attack

Inoculate after 21 days, pig attacks with toxicity Salmonella choleraesuls bacterial strain 35276 intranasal (0.5ml/ nose) and oral (1.0ml culture+4.0ml dilution of bacteria) attacked.Attack bacterial strain 35276 produces nalidixic acid (Nal) and can be used to distinguish the attack bacterial strain so resistance contains the plate of Nal from vaccine strains before attack.After the attack, pig is monitored that the typical clinical symptoms of Salmonella infection comprises every day and is lost weight, the rising of diarrhoea and rectal temperature.The persistent period that Salmonella discharges is estimated by the feces of cultivating every day.

Attack after 9 days, pig is implemented euthanasia, and cultivates lung by necropsy and containing on Hektoen intestinal (HE) agar plate of 80 μ g/ml nalidixic acids, liver, and spleen, mesentery lymph node (MLN) and ileum are bred Salmonella choleraesuls.The diarrhoea scoring

Diarrhoea scoring is by calculating for point of diarrhoea of attacking every the pig in back every day.When off-test, always the counting divided by from attacking the natural law of euthanasia of experimental session.This number multiply by 100 percents that just obtain viewed diarrhoea natural law.Salmonella discharges

The Excreta of getting pig every day detects after immunity and the time that Salmonella choleraesuls discharge after attacking.Behind two days negative findings, stop to get Excreta.Excreta is titrated separates on the HE plate.After carrying out different dilutions, sample finds the point relevant with growth.Count following the distribution:

10 ^-11 point

10 ^-22 points

10 ^-33 points

10 ^-44 points

10 ^-55 weight increase

Increasing (ADG) in average day is by deducting originally body weight again divided by obtaining from the natural law of attacking to euthanasia from final body weight.Group ADG is the average of individual pig ADG.The result: the immunity back is dead

There is two dead after inoculation with the pig of Salmonella choleraesuls wild-type parent bacterial strain 34682 inoculations.Other three pigs in this group are all living from start to finish that experiment stops.The scoring of Salmonella separation/necropsy

Fig. 2 has shown the isolating result of each organ Salmonella choleraesuls.Contrast parent and non-inoculation group all are not separated to Salmonella choleraesuls with any organ in the group of the pig of KO vaccine immunity.All be separated to Salmonella choleraesuls among ileum of 5 pigs of all of non-inoculation group and the MLN and be not separated in those organs of parent group.Be labeled as a point by the organ that each is separated to the cholera Salmonella, every component is calculated from scoring.The result has proved with the pig of KO vaccination has lower scoring with respect to the pig of parent strain inoculation, and is starkly lower than the scoring of non-inoculation group.The diarrhoea scoring

Per day diarrhoea scoring is shown in Fig. 3.This figure demonstrates KO scoring has tangible difference with respect to other three groups.The KO group has clearly illustrated minimum scoring.Salmonella discharges

After vaccination and attacking, the release of the Salmonella of pig is shown in the Figure 4 and 5.After the vaccination, figure demonstrates the release that the parent has top level.Among the figure after attack, the Salmonella choleraesuls that non-vaccinated animal discharges continue the longest time, therefore obtain the highest scoring.The increase of body weight

The increase of pig body weight is shown among Fig. 6.Difference between these data have clearly illustrated not on the same group.They also reflect health level whole between the different animals.0.15 kilogram of non-vaccination group average loss of weight every day, and KO organizes and increases weight average every day 0.6 kilogram.Discuss

Salmonella choleraesuls knock-out bacterial strain 34682 is proved to be and is effective and safe vaccine strains.The separation of Salmonella when necropsy is negative fully for knock-out bacterial strain.Non-vaccinated scoring is the highest in secondary separation.In view of the per day weightening finish after attacking, the animal of gene knock-out bacterial strain inoculation has the weightening finish of the highest weightening finish and non-inoculation animal obviously lower.Conclusion

The KO-34682 Salmonella choleraesuls vaccine strain of living is safe and efficient.

The description of the drawings: Fig. 1: attack the poundage that every pig average weight every day in back increases.This weight multiply by 0.4536 and has just obtained the kilogram number.Fig. 2: the percent (MLN=mesentery lymph node) that can from different tissues, isolate the pig of Salmonella choleraesuls again.Fig. 3: the average percent of attacking back diarrhoea scoring.Fig. 4: immunity back Salmonella discharges the daily average of scoring.Fig. 5: attack the daily average that the back antibacterial discharges scoring.Fig. 6: the per day poundage of attacking every the pig weightening finish in back.

( 2 ) SEQ ID NO:1： ( ⅰ ) ： ( A ) ：1200 ( B ) ： ( C ) ： ( D ) ： ( ⅱ ) ：DNA ( ⅸ ) ： ( A ) /：CDS ( B ) ：118..1119 ( ⅸ ) ： ( A ) /：misc- ( B ) ：118..120 ( C ) ：/=“” ( ⅹ ) ：SEQ ID NO:1GTGGTAACCC GGAATAAAAG CGGTTGCCGG AGTGATCAAA CTGCGCTTAG ATGTTAACGA 60TTTTAACCCA TGCCGACATA AAGGTTATGG TTTGTACAAT TTACACAAGG GGCAATT 117GTG AAA CTG GAT GAA ATC GCT CGG CTG GCC GGT GTC TCG CGC ACA ACT 165Val Lys Leu Asp Glu Ile Ala Arg Leu Ala Gly Val Ser Arg Thr Thr 1 5 10 15GCA AGC TAC GTT ATA AAC GGT AAA GCA AAG CAA TAC CGC GTG AGC GAC 213Ala Ser Tyr Val Ile Asn Gly Lys Ala Lys Gln Tyr Arg Val Ser Asp

20 25 30AAA?ACC?GTA?GAA?AAA?GTC?ATG?GCG?GTA?GTG?CGT?GAG?CAC?AAT?TAC?CAT 261Lys?Thr?Val?Glu?Lys?Val?Met?Ala?Val?Val?Arg?Glu?His?Asn?Tyr?His

35 40 45CCT?AAC?GCT?GTG?GCT?GCC?GGG?CTG?CGT?GCT?GGA?CGC?ACA?CGT?TCC?ATT 309Pro?Asn?Ala?Val?Ala?Ala?Gly?Leu?Arg?Ala?Gly?Arg?Thr?Arg?Ser?Ile

50 55 60GGT?CTG?GTG?ATC?CCG?GAC?CTT?GAA?AAC?ACG?AGC?TAC?ACC?CGT?ATC?GCA 357Gly?Leu?Val?Ile?Pro?Asp?Leu?Glu?Asn?Thr?Ser?Tyr?Thr?Arg?Ile?Ala?65 70 75 80AAC?TAT?CTT?GAG?CGC?CAG?GCA?CGC?CAG?CGT?GGC?TAC?CAA?CTG?CTG?ATC 405Asn?Tyr?Leu?Glu?Arg?Gln?Ala?Arg?Gln?Arg?Gly?Tyr?Gln?Leu?Leu?Ile

85 90 95?GCC?TGT?TCT?GAA?GAT?CAG?CCG?GAT?AAC?GAA?ATG?CGC?TGC?ATT?GAG?CAC 453Ala?Cys?Ser?Glu?Asp?Gln?Pro?Asp?Asn?Glu?Met?Arg?Cys?Ile?Glu?His

100 105 110CTT?TTG?CAA?CGC?CAG?GTG?GAT?GCA?ATC?ATT?GTT?TCA?ACT?TCG?TTA?CCG 501Leu?Leu?Gln?Arg?Gln?Val?Asp?Ala?Ile?Ile?Val?Ser?Thr?Ser?Leu?Pro

115 120 125CCG?GAG?CAT?CCC?TTC?TAT?CAG?CGC?TGG?GCC?AAC?GAT?CCG?TTC?CCC?ATC 549Pro?Glu?His?Pro?Phe?Tyr?Gln?Arg?Trp?Ala?Asn?Asp?Pro?Phe?Pro?Ile

130 135 140GTC?GCG?CTC?GAC?CGC?GCG?CTG?GAT?CGC?CAA?CAT?TTC?ACC?AGC?GTG?GTC 597Val?Ala?Leu?Asp?Arg?Ala?Leu?Asp?Arg?Glu?His?Phe?Thr?Ser?Val?Val145 150 155 160GGC?GCC?GAT?CAG?GAT?GAT?GCC?GAG?ATG?TTG?GCG?GAA?GAG?CTG?CGT?AAA 645Gly?Ala?Asp?Gln?Asp?Asp?Ala?Glu?Met?Leu?Ala?Glu?Glu?Leu?Arg?Lys

165 170 175TTC?CCG?GCG?GAA?ACG?GTG?CTT?TAT?TTG?GGC?GCG?CTG?CCG?GAG?TTG?TCC 693Phe?Pro?Ala?Glu?Thr?Val?Leu?Tyr?Leu?Gly?Ala?Leu?Pro?Glu?Leu?Ser

180 185 190GTC?AGT?TTC?CTG?CGC?GAG?CAG?GGG?TTC?CGC?ACC?GCA?TGG?AAA?GAC?GAT 741Val?Ser?Phe?Leu?Arg?Glu?Gln?Gly?Phe?Arg?Thr?Ala?Trp?Lys?Asp?Asp

195 200 205CCG?CGG?GAG?GTG?AAT?TTC?TTA?TAT?GCC?AAC?AGC?TAT?GAG?CGC?GAA?GCC 789Pro?Arg?Glu?Val?Asn?Phe?Leu?Tyr?Ala?Asn?Ser?Tyr?Glu?Arg?Glu?Ala

210 215 220GCC?GCG?CAG?TTG?TTT?GAG?AAA?TGG?CTG?GAA?ACG?CAT?CCT?ATG?CCG?CAG 837Ala?Ala?Gln?Leu?Phe?Glu?Lys?Trp?Leu?Glu?Thr?His?Pro?Met?Pro?Gln225 230 235 240GCG?CTC?TTT?ACG?ACA?TCG?TTC?GCG?CTA?TTA?CAG?GGC?GTG?ATG?GAC?GTA 885Ala?Leu?Phe?Thr?Tnr?Ser?Phe?Ala?Leu?Leu?Gln?Gly?Val?Met?Asp?Val

245 250 255ACG?CTG?CGG?CGC?GAT?GGA?AAA?CTG?CCT?TCG?GAT?TTA?GCG?ATT?GCG?ACC 933Thr?Leu?Arg?Arg?Asp?Gly?Lys?Leu?Pro?Ser?Asp?Leu?Ala?Ile?Ala?Thr

260 265 270TTC?GGC?GAT?CAT?GAG?CTG?CTG?GAT?TTT?CTG?CAA?TGC?CCG?GTA?CTG?GCG 981Phe?Gly?Asp?His?Glu?Leu?Leu?Asp?Phe?Leu?Gln?Cys?Pro?Val?Leu?Ala

275 280 285GTG?GCG?CAG?CGT?CAT?CGT?GAT?GTC?GCG?GAA?CGC?GTG?CTG?GAG?ATT?GTG 1029Val?Ala?Gln?Arg?His?Arg?Asp?Val?Ala?Glu?Arg?Val?Leu?Glu?Ile?Val

290 295 300CTG?GCA?AGT?CTT?GAT?GAA?CCG?CGT?AAA?CCG?AAA?CCC?GGC?TTA?ACG?CGT 1077Leu?Ala?Ser?Leu?Asp?Glu?Pro?Arg?Lys?Pro?Lys?Pro?Gly?Leu?Thr?Arg305 310 315 320ATT?CGG?CGA?AAC?CTT?TAT?CGT?CGC?GGC?ATT?CTG?AGC?CGT?AGC 1119Ile?Aro?Arg?Asn?Leu?Tyr?Arg?Arg?Gly?Ile?Leu?Ser?Arg?Ser

325 330TAAAGGACCG?GCGGTAAAAG?ACTCTCTCTT?CTGCCGCCGT?CAAACAAATG?CGTATCAGTA 1179AAAATATCCC?TTAAATAATT?A 1200

(2) data of SEQ ID NO:2:

(ⅰ) sequence signature

(A) length: 334 aminoacid

(B) type: aminoacid

(C) topological structure: linearity

(ⅱ) molecule type: protein

(ⅹ ⅰ) sequence description: SEQ ID NO:2Val Lys Leu Asp Glu Ile Ala Arg Leu Ala Gly Val Ser Arg Thr Thr 15 10 15Ala Ser Tyr Val Ile Asn Gly Lys Ala Lys Gln Tyr Arg Val Ser Asp

20 25 30Lys?Thr?Val?Glu?Lys?Val?Met?Ala?Val?Val?Arg?Glu?His?Asn?Tyr?His

35 40 45Pro?Asn?Ala?Val?Ala?Ala?Gly?Leu?Arg?Ala?Gly?Arg?Thr?Arg?Ser?Ile

50 55 60Gly?Leu?Val?Ile?Pro?Asp?Leu?Glu?Asn?Thr?Ser?Tyr?Thr?Arg?Ile?Ala?65 70 75 80Asn?Tyr?Leu?Glu?Arg?Gln?Ala?Arg?Gln?Arg?Gly?Tyr?Gln?Leu?Leu?Ile

85 90 95Ala?Cys?Ser?Glu?Asp?Gln?Pro?Asp?Asn?Glu?Met?Arg?Cys?Ile?Glu?His

100 105 110Leu?Leu?Gln?Arg?Gln?Val?Asp?Ala?Ile?Ile?Val?Ser?Thr?Ser?Leu?Pro

115 120 125Pro?Glu?His?Pro?Phe?Tyr?Gln?Arg?Trp?Ala?Asn?Asp?Pro?Phe?Pro?Ile

130 135 140Val?Ala?Leu?Asp?Arg?Ala?Leu?Asp?Arg?Glu?His?Phe?Thr?Ser?Val?Val145 150 155 160Gly?Ala?Asp?Gln?Asp?Asp?Ala?Glu?Met?Leu?Ala?Glu?Glu?Leu?Arg?Lys

165 170 175Phe?Pro?Ala?Glu?Thr?Val?Leu?Tyr?Leu?Gly?Ala?Leu?Pro?Glu?Leu?Ser

180 185 190Val?Ser?Phe?Leu?Arg?Glu?Gln?Gly?Phe?Arg?Thr?Ala?Trp?Lys?Asp?Asp

195 200 205Pro?Arg?Glu?Val?Asn?Phe?Leu?Tyr?Ala?Asn?Ser?Tyr?Glu?Arg?Glu?Ala

210 215 220Ala?Ala?Gln?Leu?Phe?Glu?Lys?Trp?Leu?Glu?Thr?His?Pro?Met?Pro?Gln225 230 235 240Ala?Leu?Phe?Thr?Thr?Ser?Phe?Ala?Leu?Leu?Gln?Gly?Val?Met?Asp?Val

245 250 255Thr?Leu?Arg?Arg?Asp?Gly?Lys?Leu?Pro?Ser?Asp?Leu?Ala?Ile?Ala?Thr

260 265 270Phe?Gly?Asp?His?Glu?Leu?Leu?Asp?Phe?Leu?Gln?Cys?Pro?Val?Leu?Ala

275 280 285Val?Ala?Gln?Arg?His?Arg?Asp?Val?Ala?Glu?Arg?Val?Leu?Glu?Ile?Val

290 295 300Leu?Ala?Ser?Leu?Asp?Glu?Pro?Arg?Lys?Pro?Lys?Pro?Gly?Leu?Thr?Arg305 310 315 320Ile?Arg?Arg?Asn?Leu?Tyr?Arg?Arg?Gly?Ile?Leu?Ser?Arg?Ser

325 330

Claims (10)

1 is used to watch for animals avoids that malignant bacteria infects or the attenuated vaccine alives of its pathogenic effects, its contain owing to the cra gene mutation causes it can not proteic living toxic-reduced bacteria of expressive function Cra and pharmaceutically acceptable carrier.

2. attenuated vaccine alive as claimed in claim 1 is characterized in that malignant bacteria wherein belongs to Escherichia, Salmonella, Actinobacillus, haemophilus, Aeromonas, pasteurella, any of Streptococcus and Yersinia.

3. attenuated vaccine alive as claimed in claim 1 is characterized in that living toxic-reduced bacteria carries heterologous gene.

4. attenuated vaccine alive as claimed in claim 3 is characterized in that malignant bacteria wherein belongs to Escherichia, Salmonella, Actinobacillus, haemophilus, Aeromonas, pasteurella, any in Streptococcus and the Yersinia.

5. as the described attenuated vaccine alive of claim 1-4, it is characterized in that containing adjuvant.

6. as the described attenuated vaccine alive of claim 1-5, it is characterized in that being freeze dried form.

7. because the cra gene mutation causes it can not the proteic living toxic-reduced bacteria of expressive function Cra to be used to prepare watch for animals and to avoid that malignant bacteria infects or the purposes of the vaccine of its pathogenic effects.

8. purposes as claimed in claim 7 is characterized in that living toxic-reduced bacteria carries heterologous gene.

9. the preparation method of the described vaccine of claim 1 comprises living toxic-reduced bacteria is mixed with pharmaceutically acceptable carrier.

10. use the malignant bacteria immune animal to support method of combating infection, comprise to animal and use the described vaccine of claim 1.

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