CN102676421B - Bordetella bronchiseptica gene deletion strain, vaccine prepared from Bordetella bronchiseptica gene deletion strain and application - Google Patents
Bordetella bronchiseptica gene deletion strain, vaccine prepared from Bordetella bronchiseptica gene deletion strain and application Download PDFInfo
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Abstract
The invention relates to a Bordetella bronchiseptica gene deletion strain, vaccine prepared from the Bordetella bronchiseptica gene deletion strain and application of the strain. The aroA gene deletion strain, namely aroA-LY1 (Bordetella bronchiseptica), is preserved in the China Center for Type Culture Collection (CCTCC) on 23 December 2011, and the preservation number is CCTCCNO: M 2011479. The Bordetella bronchiseptica gene deletion strain disclosed by the invention has excellent immune protection efficacy and weak toxicity without any adverse side effect on immunized pigs and is safer compared with the traditional vaccine. Therefore, the vaccine which is prepared from the gene deletion strain built by a parent strain has wide market application prospect. In addition, the Bordetella bronchiseptica gene deletion strain disclosed by the invention retains the immune protective efficacy aiming at Bordetella bronchiseptica infection, and has clear genetic background and stable characteristics, so that the Bordetella bronchiseptica gene deletion strain has better biosecurity. The Bordetella bronchiseptica gene deletion strain disclosed by the invention does not contain a resistance tag and totally accords with the biological security requirement of vaccine.
Description
Technical field
The present invention relates to a kind ofly not containing the bordetella bronchiseptica gene deleted bacterial strain of resistance marker, the application by vaccine and this vaccine of its structure, belongs to animal bacteria gene engineering technology field.
Background technology
Segmental bronchus sepsis bordetella bacilli (Bordetella bronchiseptica, Bb) be a kind of gram-negative aerobic dialister bacterium, Whooping cough bordetella bacilli (B.pertussis with obligate infection people, Bp), parapertussis bordetella bacilli (B.parapertussis, Bpp) belongs to Podbielniak Pseudomonas.Bb can extensively infect multiple Mammals, and the infection of pig, rabbit, dog is especially general, shows as the acute or chronic inflammation of respiratory tract, is referred to as Podbielniak bacterium sick.In swinery, segmental bronchus sepsis bordetella bacilli can cause pig generation pneumonia and atrophic rhinitis (atrophic rhinitis, AR), be also the important virulence factor of PRDC (porcine respiratory disease complex) (porcine respiratory disease complex, PRDC).The pig Podbielniak bacterium disease that the atrophic rhinitis of take is representative has now spread all over pig industry developed country, world swinery approximately has 25-50% infected, cause serious financial loss to world's pig industry, be one of important respiratory infectious disease of harm intensive culture efficiency (Chen Puyan. animal doctor's loimology. the 5th edition. Beijing: Chinese agriculture press, 2010.p.260-264.).The more important thing is, the infection in advance of segmental bronchus sepsis bordetella bacilli is easy to cause other multiple cause of diseases, as the secondary infection of pasteurella multocida, suis, haemophilus parasuis, porcine reproductive and respiratory syndrome virus, swine influenza virus etc., thereby improve sickness rate and the severity of swinery respiratory tract disease, cause heavy economic losses (Soensen V, Jorsal SE, Mousing J.Diseases of the respiratory system.In:Straw BE, Zimmerman JJ, d ' Allaire S, Taylor DJ.Diseases of Swine (9
thedition), Blackwell Publishing, IA, USA, 2006, p.161-194.).Between 2003 to 2008, Zhao Zhanqin etc. are separated to 652 strain Bb from there are 3506 parts of lungs pathological material of diseases of respiratory symptom pig all parts of the country, separation rate is up to 18.6%, between each province, there is no notable difference, show Bb China pig farm (Zhao Z that is widely current and causes a disease, Wang C, Xue Y, et al.The occurrence of Bordetella bronchiseptica in pigs with clinical respiratory disease.Vet J.2011,188 (3): 337-340.).
Segmental bronchus sepsis bordetella bacilli is a kind of ancient microorganism, it extensively infects multiple Mammals, jointly evolved 1 years with host, formed unique infection and immunologic mechanism (Parkhill J, Sebaihia M, Preston A, et al.2003.Comparative analysis of the genome sequences of Bordetellapertussis, Bordetella parapertussis and Bordetella bronchiseptica.Nat Genet, 35 (1): 32-40.).According to different living environments (in host or external, animal or human etc.), Bb has the complicated regulation mechanism of different levels to adapt with it, causes the variation of the even whole bacterium phase of various surface antigens, virulence factor (I phase～II phase～III phase).Have benefited from a pattern bacterium as people's Whooping cough bordetella bacilli, and the carrying out of genome sequencing and annotation work, at present distincter to the research of segmental bronchus sepsis bordetella bacilli virulence factor.Have now found that with the pathogenic relevant virulence factor of segmental bronchus sepsis bordetella bacilli and mainly comprise that lipopolysaccharides (Lipopolysaccharide, LPS), adhesin, extracellular toxin, iron utilize associated protein, III type excretory system, urine enzyme, proteolytic enzyme etc.Wherein, first three class virulence factor is all also its important protective antigens.Foreign scholar uses study in vitro, as cloning screens, regulatory screenses etc. identify multiple adhesin and the extracellular toxin of segmental bronchus sepsis bordetella bacilli, comprise filamentous hemagglutinin (filamentous hemagglutinin, FHA), PRN (pertactin, PRN), pili (fimbriae, FIM), tracheae colonizing factor (tracheal colonization factor, TCF), dermatonecrotoxin (dermonecrotic toxin, DNT), tracheal cell toxin (tracheal cytotocin, TC), adenylate cyclase toxin (adenylate cyclase toxin, CyaA), bordetella bacilli colonization factor A (Bordetella colonization factorA, BcfA) etc.Meanwhile, the method such as the vivoexpression by gene, deletion mutantion, has carried out preliminary explaination to the relation between the coded product of these genes and immune response.The virulence factor of segmental bronchus sepsis bordetella bacilli regulates and expresses by BvgA/S double factor regulation system.The activation of Bvg can significantly change expression level (the Cotter PA of segmental bronchus sepsis bordetella bacilli antigen gene, Jones AM.2003.Phosphorelay control of virulence gene expression in Bordetella.Trends Microbiol, 11 (8): 367-373.).
Because the harm understanding to segmental bronchus sepsis bordetella bacilli is not enough, cause people far to lag behind other diseases to the control of pig Podbielniak bacterium disease.At present, (the I phase bacterium culture of segmental bronchus sepsis bordetella bacilli is prepared through deactivation in world wide, to be widely used the conventional deactivation vaccine of segmental bronchus sepsis bordetella bacilli, part is segmental bronchus sepsis bordetella bacilli and pasteurella multocida connection seedling), but clinical investigation result for many years shows, although such vaccine can induce piglet to produce higher somatic antibody level, but protection effect is generally poor, the generation of pig Podbielniak bacterium disease is very general (United States Department of Agriculture (USDA) .Part II:reference of swine health and health management in the United States still, 2000.CO:USDA:APHIS:VS, CEAH, National Animal Health Monitoring System, Fort Collins, #N355.0202, 2002.p.29-32.).Afterwards, various countries scholar once attempted using the natural less-virulent strain of segmental bronchus sepsis bordetella bacilli and chemical process to cause weak bacterial strain as attenuated vaccine bacterial strain, but its genetic background is unclear, and existed virulence to return strong risk, was not used widely.Along with molecular biological development, the new generation vaccines such as gene-deleted vaccine become the important directions of its research.At present, the target gene that the strain of structure mutant bacteria lacks mainly contains two classes, and a class is the relevant virulence gene of disappearance, as toxin and adhesin; Another kind of is " looking after the house " gene that lacks bacterial metabolism approach or conform in regulating.Because segmental bronchus sepsis bordetella bacilli is containing a plurality of virulence factors, between different strains, there is obvious virulence and pathogenic difference, and also not clear and definite which or which kind of virulence associated gene plays conclusive effect in pathogenic and Immune interrelation function at present, therefore, select the some or several target genes of disappearance, have certain blindness.And pathways metabolism or environment " looking after the house " gene in regulating is that each bacterium contains, by molecular biological method, lacks this genoid, and then a little less than reaching street strain being caused, there is better universality.Aromatic series (aro) compound biosynthetic pathway is the total pathways metabolism of Gram-negative bacteria and positive bacteria.AroA genes encoding 5-enol pyruvylshikimate-3-phosphate synthase, lacking this gene can affect the synthetic of die aromatischen Aminosaeuren, benzaminic acid and other aromatics, makes the bacterium can only limited breeding in host, and then makes its virulence attenuation of; Meanwhile, such gene-deleted strain tool also has other advantages, as various immunogenic genes that can effective expression segmental bronchus sepsis bordetella bacilli; Within several weeks of host's Colonization inside plants, both can the powerful immunne response of excitating organism, can by host, be removed in time again, there is better security and immune efficacy; By nasal membrane immunization route, even if containing a small amount of microbiotic, animal body is also difficult to affect vaccine strains at immunne response (the Spreng S of local multiplication and induction, Dietrich G, Weidinger is design of Salmonella-based vaccination strategies.Methods G.2006.Rational, and 38 (5): 133-143.).It is research object that the segmental bronchus sepsis bordetella bacilli virulent strain LY1 that has an independent intellectual property right is take in the present invention; the homologous recombination technique mediating by suicide plasmid has built and has not contained the segmental bronchus sepsis bordetella bacilli attenuated vaccine bacterial strain of resistant maker gene and other foreign genes; this vaccine strains has been lost pathogenic to host pig, but the inoculation protection completely that piglet infects for segmental bronchus sepsis bordetella bacilli can be provided.
Summary of the invention
One object of the present invention is to provide a kind of immunogenicity better and the stronger bordetella bronchiseptica gene deleted bacterial strain of security.
Further, second object of the present invention has been to provide a kind of bordetella bronchiseptica gene deleted vaccine that utilizes bordetella bronchiseptica gene deleted bacterial strain to prepare.
Further, the 3rd object of the present invention is to provide the application of a kind of bordetella bronchiseptica gene deleted bacterial strain in preparing bordetella bronchiseptica gene deleted vaccine.
To achieve these goals, the technical solution used in the present invention is: a kind of segmental bronchus sepsis bordetella bacilli (Classification And Nomenclature: Bordetella bronchiseptica) aroA genetically deficient bacterial strain aroA that does not contain resistance marker
-lY1 (is called for short aroA
-lY1, lower same), on December 23rd, 2011, be deposited in Chinese Typical Representative culture collection center (CCTCC), deposit number: CCTCC NO:M 2011479.
The described segmental bronchus sepsis bordetella bacilli that does not contain resistance marker has lacked the Nutrition and Metabolism gene aroA of segmental bronchus sepsis bordetella bacilli LY1 strain, and after disappearance, segmental bronchus sepsis bordetella bacilli virulence significantly weakens, but still has fine immunogenicity.
Of the present invention not containing the bordetella bronchiseptica gene deleted bacterial strain aroA of resistance marker
-lY1, its engineering strain is derived from segmental bronchus sepsis bordetella bacilli LY1 strain (being called for short LY1, lower same).Wherein, LY1 is the separated wild-type segmental bronchus sepsis bordetella bacilli virulent strain from pig, on December 23rd, 2011, is deposited in Chinese Typical Representative culture collection center (CCTCC), and deposit number is CCTCC NO:M 2011478.
Described bordetella bronchiseptica gene deleted bacterial strain aroA
-lY1 has lacked the DNA fragmentation of its Nutrition and Metabolism gene aroA 1315bp, lack this gene and can affect the synthetic of die aromatischen Aminosaeuren, benzaminic acid and other aromatics, make the bacterium can only limited breeding in host, growth fraction is slower, and then make its virulence attenuation of, and virulence reduces greatly, thereby there is very high security.Through animal experiment, confirm, this genetically deficient bacterial strain still has good immune protective efficiency.
Basic construction method of the present invention is: utilize genetic engineering technique to lack the Nutrition and Metabolism gene aroA of segmental bronchus sepsis bordetella bacilli wild-type virulent strain LY1, thereby be built into new less-virulent strain aroA
-lY1.The genetically deficient bacterial strain aroA preparing by a large amount of biological experiment digital proof the present invention
-lY1 can be used for the attenuated live vaccines that preparation is infected for B.bronchisepticai.
Bordetella bronchiseptica gene deleted bacterial strain of the present invention has good immune protective, and virulence a little less than, to immune swine without any adverse side effect, with tradition such vaccine compare safer.Therefore, with the genetically deficient bacterial strain that this parent strain builds, make vaccine, there is wide market application foreground.In addition, the bordetella bronchiseptica gene deleted bacterial strain of the present invention has retained the immune protection effectiveness infecting for segmental bronchus sepsis bordetella bacilli, and genetic background is clear, and proterties is stable, thereby has better biological safety.Bordetella bronchiseptica gene deleted bacterial strain of the present invention, containing resistance marker, does not meet the requirement of vaccine biological safety completely.
Accompanying drawing explanation
Fig. 1 is the physical map of the transferring plasmid pREaroA12 for preparing of the present invention;
Fig. 2 is the schema that the transferring plasmid pREaroA12 for preparing of the present invention builds;
Fig. 3 is homologous recombination principle schematic used in the present invention;
Fig. 4 does not contain the segmental bronchus sepsis bordetella bacilli aroA genetically deficient bacterial strain aroA of resistance marker in the present invention
-the PCR screening of LY1.In figure, M is DNAmarker (DL 2,000); 1 is H
2o blank; 2 and 3 is parent strain LY1 contrast (3127bp); 4 aroA for screening
-lY1 strain (1813bp);
Fig. 5 be in the present invention not containing the segmental bronchus sepsis bordetella bacilli strain aroA of resistance marker
-the colonial morphology that LY1 grows on the LB solid medium that contains 10% calf serum; In figure, A and B are respectively parent strain LY1 (bacterium colony is larger, about 1.5mm left and right) and deletion mycopremna aroA
-the colonial morphology that LY1 (bacterium colony is less, about 0.7mm left and right) grows after 36h on the LB solid medium that contains 10% calf serum;
Fig. 6 is the segmental bronchus sepsis bordetella bacilli strain aroA in the present invention
-the growth characteristics test-results of LY1;
Fig. 7 is a kind of segmental bronchus sepsis bordetella bacilli deletion mycopremna aroA in the present invention
-the genetic stability experimental result of LY1.
Note: be that primer ar1/ar4 is to deletion mycopremna aroA
-the PCR of LY1 genetic stability identifies figure, M:DNAmarker in figure (DL 2,000); 1-6 be 1,10,20,30,40 and 50 generation deletion mycopremna template amplification; 7 is H
2o blank.
Embodiment
The Construction and identification of embodiment 1 segmental bronchus sepsis bordetella bacilli LY1aroA genetically deficient bacterial strain
1, design of primers (for gene clone and Molecular Detection)
AroA gene order (GenBank No:AF315119) design 2 couples of primers (ar1/ar2 and ar3/ar4 with reference to the segmental bronchus sepsis bordetella bacilli strain RB50 having reported, in Table 1), from segmental bronchus sepsis bordetella bacilli virulent strain LY1, (LY1 is the separated wild-type segmental bronchus sepsis bordetella bacilli virulent strain from pig, on December 23rd, 2011, be deposited in Chinese Typical Representative culture collection center (CCTCC), deposit number: CCTCC NO:M2011478.) aroA gene upstream and downstream fragment aroA1 (upper arm) and aroA2 (underarm) increase respectively in genome, amplified fragments size is respectively 927bp and 887bp, Kpn I and BamH I restriction enzyme site are introduced respectively in upper arm two ends, and BamH I and Sac I restriction enzyme site are introduced respectively in underarm two ends.Primer is synthetic by Shanghai Sangon company.
Table 1:PCR primer
2, the clone of segmental bronchus sepsis bordetella bacilli LY1 aroA gene upstream and downstream gene fragment
The segmental bronchus sepsis bordetella bacilli LY1 of freeze-drying is rule on the TSA solid that contains 10% calf serum (40g, distilled water is settled to 1000mL, 121 ℃ of high pressure 15min) substratum, cultivate 36h for 37 ℃.Picking list colony inoculation is in the TSB liquid nutrient medium that contains 10% calf serum (40g, distilled water is settled to 1000mL, 121 ℃ of high pressure 15min), and 37 ℃, 200r/min jolting are cultivated 16h.By bacterial genomes, extracting test kit (purchased from Beijing TIANGEN company) specification sheets extraction genome is pcr template.
AroA1 and aroA2 amplified reaction all carry out in the system of 25 μ L, and reaction system (PCR related reagent is all purchased from Shanghai Sangon company) is as follows: template DNA 1 μ L, 10 * PCR damping fluid, 2.5 μ L, 25mmol/L MgCl
22 μ L, each 1 μ L of 10 μ mol/L upstream and downstream primers, 2mmol/L dNTPs 1 μ L, 2U/ μ L Taqase 0.5 μ L, ddH
2o 16 μ L.
AroA1 and aroA2 amplification condition are: after 95 ℃ of sex change 5min, enter circulation, loop parameter is 94 ℃ of 1min, 58 ℃ of 1min, 72 ℃ of 1min.After 30 circulations, 72 ℃ are extended 10min.The PCR product of amplification is through 0.8% agarose gel electrophoresis analysis, 2 clip size that increases with expect sizableness, be respectively 927bp and 887bp.The goal gene obtaining is cloned into pMD18-T carrier (purchased from Dalian TaKaRa company), serves the mensuration that extra large Sangon company carries out gene order.
3, the structure of transferring plasmid pREaroA12
With Kpn I and BamH I double digestion aroA1 and pBluescriptSK (+) carrier (purchased from U.S. Stratagene company), after recovery purifying, use T
4dNAligase (purchased from Shanghai Sangon company) connects, and 16 ℃ of water-bath 12h transform DH5
αcompetence bacterium (purchased from Dalian TaKaRa company), on solid LB flat board, cultivate 12h for 37 ℃, choose bacterium to liquid LB substratum, 37 ℃, 200r/min jolting cultivation 12h, used plasmid extraction kit (purchased from Beijing TIANGEN company) thereby preparing in a small amount plasmid obtains plasmid pSKaroA1.Use again BamH I and Sac I double digestion aroA2 and plasmid pSKaroA1.After recovery, with T4DNAligase, connect, transform DH5 α competence bacterium, cultivate 12h for 37 ℃, choose bacterium to liquid LB substratum, 37 ℃, 200r/min jolting cultivation 12h, obtain plasmid pSKaroA12 thereby prepare in a small amount plasmid.With Kpn I and Sac I double digestion transferring plasmid pSKaroA12 and suicide plasmid pRE112, (by Dr.Roy Curtiss professor III of Washington, DC university, be so kind as to give; Miller VL, Mekalanos JJ 1988.A novel suicide vector and its use in construction on insertion mutations:osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR.J.Bacteriol, 170:2575-2583), reclaim " aroA1+aroA2 " fragment and plasmid pRE112, use T
4dNA ligase connects, 16 ℃ of water-bath 12h, electricity conversion (Huang Peitang etc. translate. Pehanorm Brooker J, Russell DW work. molecular cloning experiment guide (third edition). Beijing: Science Press, 2002) intestinal bacteria χ 7213 (is so kind as to give by Dr.Roy Curtiss professor III of Washington, DC university; Edwards RA, Keller LH, Schifferli DM.1998.Improved allelic exchange vectors and their use to analyze 987Pfimbria gene expression.Gene, 207:149-157) build recombinant bacterial strain χ 7213 (pREaroA12), cultivate 12h for 37 ℃ and choose bacterium to liquid LB substratum, 37 ℃, 200r/min jolting cultivation 12h, obtain pREaroA12 transferring plasmid thereby prepare in a small amount plasmid, and its physical map is shown in Fig. 1.Enzyme is cut qualification result and is confirmed that the transferring plasmid pREaroA12 building is correct." aroA1+aroA2 " DNA fragmentation in pREaroA12 contains the aroA gene fragment that has lacked 1315bp.Transferring plasmid pREaroA12 builds flow process as shown in Figure 2.
4, segmental bronchus sepsis bordetella bacilli LY1aroA genetically deficient bacterial strain aroA
-the Construction and identification of LY1
Homologous recombination principle used in the present invention as shown in Figure 3.Take χ 7213 (pREaroA12) as donor bacterium, and LY1 is that recipient bacterium carries out conjugal transfer.Donor bacterium and recipient bacterium be overnight incubation in the TSB substratum that contains 10% calf serum respectively, sterile phosphate damping fluid (PBS, NaCl 8.0g, KCl 0.2g, Na
2hPO
4.12H
2o 2.9g, KH
2pO
40.2g, distilled water adds to 1000mL, through 121 ℃ of autoclaving 30min) wash twice, adjust bacteria concentration to OD
600be 0.8, respectively get 100 μ L bacteria suspensions and mix.Aseptic nitrocellulose is affixed on to solid containing on the TSA flat board of 10% calf serum, mixed bacterium drop, on filter membrane, is cultivated to 12h for 37 ℃, do donor and acceptor contrast simultaneously.Wash bacterium liquid on lower filter membrane, aseptic PBS washes twice, coating is containing paraxin (Cm, final concentration 30 μ g/mL) TSA dull and stereotyped (being added with 10% calf serum), cultivate 24h for 37 ℃, Cm resistance bacterium colony is transferred containing the TSA dull and stereotyped (being added with 10% calf serum) of Cm and 5% sucrose simultaneously, screening Cm resistance (Cm
r) zygote, extract genome, with primer upper arm upstream primer and underarm downstream primer ar1/ar4 (in Table 1) amplification, to identify, wild-type amplification goes out the band of 3127bp, and absence type amplifies the band (as Fig. 5) of 1813bp.Positive zygote is at non-resistant, cultivate 24h containing in the TSB liquid nutrient medium of 10% calf serum, continuous 10 times of dilutions, coating is containing the solid TSA dull and stereotyped (being added with 10% calf serum) of 5% sucrose, and picking list bacterium colony is replicated in the solid plate containing Cm and 5% sucrose, the responsive (Cm of screening Cm
s) bacterium colony.Extract genome, again with primer ar1/ar4 amplification, identify, wild-type amplification goes out the band of 3127bp, and absence type amplifies the band (seeing Fig. 4) of 1813bp.The fragment that absence type amplification is obtained is carried out sequencing (Sangon company completes by Shanghai), and sequencing result shows, constructed LY1aroA genetically deficient bacterial strain aroA
-lY1 is correct, has lacked the full gene fragment of aroA of 310bp.
The bordetella bronchiseptica gene deleted bacterial strain aroA of embodiment 2
-the growth characteristics analysis of LY1
Genetically deficient bacterial strain aroA
-lY1 is 37 ℃ of cultivation 36h on the TSA flat board that contains 10% calf serum, and its diameter of bacterium colony is about 0.7mm, compared with parent strain LY1 (1.5mm), significantly reduces (seeing Fig. 5).Parent strain LY1 and genetically deficient bacterial strain aroA
-lY1 is from 10
6cFU/mL starts to cultivate, every 1h sampling, and carry out live bacterial count.Result shows, genetically deficient bacterial strain aroA
-the LY1 speed of growth is significantly slower than parent strain LY1 (seeing Fig. 6), its average length of generation (Mean generation time) is 38.7min, compared with parent strain (26.1min), extend 12.6min, illustrate that the genetically deficient bacterial strain LY1 speed of growth is significantly slower than parent strain LY1.
Genetically deficient bacterial strain aroA prepared by the present invention
-lY1 is streak culture on the TSA flat board that contains 10% calf serum, picking list bacterium colony is on the TSA that contains 10% calf serum, 37 ℃ of incubators are cultivated 24h, then on cultured flat board again picking list bacterium colony to the TSA flat board that contains 10% calf serum, carry out continuously 50 switchings, observe bacterium colony size.Result shows genetically deficient bacterial strain aroA
-the colony diameter of LY1 is still about 0.7mm, compared with parent strain LY1 (being about 1.5mm), significantly reduces, and still meets aroA
-lY1 genetically deficient strain growth speed is partially slow growth characteristics obviously.Use primer ar1/ar4 (in Table 1) to carry out PCR evaluation, genetically deficient bacterial strain aroA simultaneously
-aroA missing gene heredity situation in LY1 is shown in Fig. 7.Fig. 7 shows, deletion mycopremna 1,10,20,30,40 and 50 generation template the equal band that can amplify absence type 1763bp, show genetically deficient bacterial strain aroA prepared by the present invention
-lY1 can genetic stability gene deletion type fragment.
By the bordetella bronchiseptica gene deleted bacterial strain aroA obtaining
-lY1 is cultivating containing on the TSA solid medium of 10% calf serum, and picking list bacterium colony is cultivated in the TSB liquid nutrient medium that contains 10% calf serum, until viable bacteria concentration reaches 5.0 * 10
9cFU/mL.In bacterium liquid: gelatin protective material (volume: be volume) that the ratio of 7: 1 adds gelatin protective material (this gelatin protective material compound method is: in every 100mL deionized water with sucrose 40g; gelatin 8g; after fully melting; put at 121 ℃ and save backup after sterilizing 30min); in sterilizing freeze-drying bottle, press the packing of 2.0mL/ bottle; put freeze-drying in-50 ℃ of freeze driers; freeze-drying 36h rear pressing cover; with 10% aluminium glue physiology salt, dissolve and carry out live bacterial count (CFU); and determine there is no living contaminants; put-20 ℃ and save backup, as the vaccine strains of development gene-deleted vaccine.
1, genetically deficient bacterial strain aroA of the present invention
-the toxicity test of LY1
For measuring the genetically deficient bacterial strain aroA building
-the virulence of LY1 to BALB/c mouse, is equally divided into 8 groups by 32 BALB/c mouse.1-4 group mouse carries out intraperitoneal inoculation genetically deficient bacterial strain aroA
-lY1, every of every group injects respectively 4.2 * 10
8, 4.2 * 10
7, 4.2 * 10
6with 4.2 * 10
5cFU; 5-8 group is carried out intraperitoneal inoculation parent strain LY1, and every of every group injects respectively 5.6 * 10
7, 5.6 * 10
6, 5.6 * 10
5with 5.6 * 10
4cFU.Record death condition and according to Reed and Muench method (Reed L J, Muench is simple method of estimating fifty percent endpoints.Am J Hyg H.1938.A, 27:493-497) calculate mouse mld (50%lethal dose, LD
50).Evaluate genetically deficient bacterial strain aroA
-lY1 compares virulence attenuation of degree with parent strain LY1, the results are shown in Table 2.Parent strain and the deletion mycopremna LD to mouse
50be respectively 5.6 * 10
6cFU and 4.2 * 10
7cFU.AroA is described
-the virulence of LY1 has reduced by 7.5 times compared with parent strain LY1.This shows genetically deficient bacterial strain aroA of the present invention
-lY1 compares its virulence with parent strain LY1 and obviously reduces.
Table 2 gene-deleted vaccine bacterial strain of the present invention and the comparison test of parent strain virulence
2, genetically deficient bacterial strain aroA of the present invention
-the safety evaluation of LY1 to pig
10 of the weanling pigs of selection 22-25 age in days, segmental bronchus sepsis bordetella bacilli feminine gender, aroA prepared by the present invention
-lY1 gene-deleted vaccine (contains 4.0 * 10 by every pig 2mL
9cFU viable count) dosage is inoculated piglet by ear vein, after inoculation there is heating, the fervescence reaction of a slight property crossed in 12-48h piglet, after 48h, all recover normal, the spiritual appetite of piglet of injecting during this period gene-deleted vaccine prepared by the present invention is normal, have no abnormal changes, inoculate latter 14 days and segmental bronchus sepsis bordetella bacilli antibody all can be detected.Genetically deficient bacterial strain aroA prepared by the present invention
-lY1 (contains 4.0 * 10 by every pig 2mL
9cFU viable count) dosage is inoculated 10 of pregnant sows by ear vein, and the spiritual appetite of all pregnant sows is normal, has no abnormal changes, and inoculates latter 14 days and segmental bronchus sepsis bordetella bacilli antibody all can be detected; With nonvaccinated pregnant sow comparison, nest litter size is substantially suitable, does not all occur stillborn foetus, mummy tire etc.The genetically deficient bacterial strain aroA of these two test proved invention preparations
-lY1 is all safe to weanling pig and pregnant sow.
1, the immune programme for children of mouse
By 30 of age in 5-6 week, the negative BALB/c mouse of segmental bronchus sepsis bordetella bacilli, be equally divided into 2 groups, be respectively aroA prepared by the present invention
-lY1 gene-deleted vaccine group and PBS blank group.Immunization route is that intranasal inoculation 0.1mL is (containing 1.0 * 10
8cFU viable bacteria amount) bacterium liquid and PBS, 1 (intranasal inoculation method reference: Zhao Z of booster immunization after 14 days, Xue Y, Wu B, et al.2008.Subcutaneous vaccination with attenuated Salmonella enterica serovar Choleraesuis C500expressing recombinant filamentous hemagglutinin and pertactin antigens protects mice against fatal infections with both S.enterica serovar Choleraesuis and Bordetella bronchiseptica.Infect Immun, 76 (5): 2157-2163.).Respectively at first 0 day of immunity, head, exempt to detect for latter 14 days and 28 days segmental bronchus sepsis bordetella bacilli serum antibody (standard ELISA method, reference: Zhao Zhanqin. the recombinant salmonella recombinant vaccine research [A] of segmental bronchus sepsis bordetella bacilli. Hua Zhong Agriculture University Library .2008,97-107.).
2, immune mouse humoral immunization antibody horizontal detects
Blood sampling before mouse immune, second and third blood sampling carried out respectively for 14,28 days after head exempts from, and chooses 5 through tail vein negative pressure hemostix for every group, and separation of serum is got equivalent and is mixed the anti-segmental bronchus sepsis of rear detection bordetella bacilli average serum antibody horizontal.The results are shown in Table 3.As can be seen from Table 3, head exempts from rear the 14th day aroA of the present invention
-the segmental bronchus sepsis bordetella bacilli antibody of LY1 gene-deleted vaccine group mouse is 1: 32, two exempt from latter 14 days (being that head exempts from latter 28 days), and the segmental bronchus sepsis bordetella bacilli antibody titer of bordetella bronchiseptica gene deleted vaccine group mouse of the present invention rises at 1: 256.And the PBS synchronously carrying out contrasts all negative (< 1: 10).Above-mentioned test-results shows aroA prepared by the present invention
-after LY1 gene-deleted vaccine immune mouse, can induce body to produce the immune response of specific anti-segmental bronchus sepsis bordetella bacilli.
AroA prepared by table 3 the present invention
-lY1 gene-deleted vaccine immune serum antibody test (ELISA method)
3, aroA
-the protectiveness test of LY1 gene-deleted vaccine immunity BALB/c mouse
After mouse immune 30 days, by above-mentioned aroA
-lY1 gene-deleted vaccine group and PBS blank group mouse BALB/c mouse are respectively got 10, all use 5 * LD
50(in Table 2, contain 2.8 * 10
7cFU) segmental bronchus sepsis bordetella bacilli virulent strain LY1 attacks poison to mouse peritoneal, observes 30 days.PBS control group mice attack poison after 8h start to present spirit depressed, do not eat, flock together; Within the 3rd day, start to occur death, peak mortality appears at the 5th day, all dead by the 10th day.AroA prepared by the present invention
-there is not obvious disease symptom after attacking poison in LY1 gene-deleted vaccine group, does not have death condition to occur.AroA prepared by this explanation the present invention
-lY1 gene-deleted vaccine immune mouse can be resisted 5 * LD
50the attack of segmental bronchus sepsis bordetella bacilli intensity strain LY1.After attacking poison, protection ratio situation is shown in Table 4.
AroA prepared by table 4 the present invention
-lY1 gene-deleted vaccine is attacked the protectiveness evaluation of BALB/c small white mouse to segmental bronchus sepsis bordetella bacilli LY1 virulent strain
AroA prepared by embodiment 6 the present invention
-the immune efficacy of LY1 gene-deleted vaccine in piglet body detects
1, the immune programme for children of pig:
12 of the piglets of selection 7-8 age in days, segmental bronchus sepsis bordetella bacilli feminine gender, test divides 3 groups, and the 1st group is aroA of the present invention
-lY1 gene-deleted vaccine group, numbering 1-4, the 2nd group be segmental bronchus sepsis bordetella bacilli inactivated vaccine (preparation method's reference: Zhao Zhanqin. the recombinant salmonella recombinant vaccine research [A] of segmental bronchus sepsis bordetella bacilli. the .2008 of Library of Hua Zhong Agriculture University, 97-107) group, numbering 5-8.The 3rd group is PBS control group, numbering 9-12.By collunarium, inoculate aroA of the present invention for the 1st group
-lY1 genetically deficient attenuated vaccine 2mL (viable bacteria content 3.0 * 10
9cFU), by musculi colli, inject 2mL inactivated vaccine (thalline content 3.0 * 10 for the 2nd group
9cFU), every pig of the 3rd group of PBS control group is injected the aseptic PBS of 2mL by musculi colli.All piglets are exempted from rear 21d in head and take same method to carry out two to exempt from.Within 20 days and 30 days after head exempts from, respectively take a blood sample once, by ELISA method, detect segmental bronchus sepsis bordetella bacilli serum antibody (standard ELISA method respectively, method is with embodiment 5, with reference to Zhao Z, Xue Y, Wu B, et al.2008.Subcutaneous vaccination with attenuated Salmonella enterica serovar CholeraesuisC500expressing recombinant filamentous hemagglutinin and pertactin antigens protects mice against fatal infections with both S.enterica serovar Choleraesuis and Bordetella bronchiseptica.Infect Immun, 76 (5): 2157-2163.).Test grouping, immunization route and immunizing dose details are in Table 5.
2, immune piglet ELISA antibody horizontal detects
In immunity, every group of piglet pig carried out to precaval vein blood sampling in latter 20 days and 30 days respectively, separation of serum, gets equivalent and mixes rear employing indirect ELISA method detection segmental bronchus sepsis bordetella bacilli serum average antibody level, the results are shown in Table 5.Latter 20 days aroA of the present invention of immunity
-the average antibody of LY1 gene-deleted vaccine group and inactivated vaccine group is tired and is respectively 1: 40 and 1: 80, latter 30 days of immunity, aroA of the present invention
-lY1 gene-deleted vaccine group average antibody is tired and is risen at 1: 160 and 1: 640.And the PBS synchronously carrying out contrasts all negative (< 1: 10).The above results shows aroA prepared by the present invention
-after LY1 gene-deleted vaccine immunity piglet, can induce body to produce the specific humoral immunoresponse(HI) of specific anti-segmental bronchus sepsis bordetella bacilli.
AroA prepared by table 5 the present invention
-lY1 gene-deleted vaccine immunity piglet Serum Antibody Detection
3, the aroA that prepared by the present invention
-the protectiveness test of LY1 gene-deleted vaccine immunity piglet:
Head exempts from rear 31d, by aroA
-lY1 gene-deleted vaccine group, inactivated vaccine group, all piglets of PBS control group carry out the respiratory tract of pig source segmental bronchus sepsis bordetella bacilli virulent strain and attack.Every piglet is injected 2mL by pars cervicalis tracheae and will contain 5.2 * 10
10the injection of solution of CFU segmental bronchus sepsis bordetella bacilli virulent strain LY1 viable bacteria, in respiratory tract, allows it absorb completely.Attack the rear Continuous Observation 30d of poison.PBS control group piglet all shows fervescence (table 6) after attacking poison, and spirit is depressed, fluffy and disorderly by hair, cough, and expiratory dyspnea, nasal cavity has mucus secretion in various degree.There is congestion in part pig, breathing is the devil, and is sitting position of dog gesture, and cry is sharp-pointed, appetite stimulator or absolutely useless.Continuous high temperature always in after attacking poison 5 days of PBS control group piglet, then just slowly declines and occurs the phenomena of mortality, and to attacking 22d after poison, in blank group, 4 piglets have 3 death, and its mortality ratio is 75%.A pig of survival shows as retarded growth.Dead pig is dissected and finds that tracheae has a large amount of mucus, and hemorrhage, necrosis or atrophy in various degree occurs lungs.AroA
-only there is temporary fervescence in 4 piglets of LY1 gene-deleted vaccine group, the time length, in 2-3d, then recovers by normal (table 6) gradually after attacking poison.Except a pig occurs that, slight respiratory symptom, obvious disease symptom does not appear in other pig, and occur death, protection ratio is 100%.After observing 30d, dissect all 4 piglets, find that obvious pathology (table 6) does not appear in its respiratory tract and lungs.What is interesting is, although inactivated vaccine group compares aroA
-lY1 gene-deleted vaccine group has produced higher antibody horizontal, but its protection effect is but inferior to gene-deleted vaccine immune group.4 immune piglets have all shown certain respiratory symptom, and have a death, and pneumonia and trachitis pathology (table 6) to a certain degree all appears in 4 piglets.
The different immune group piglets of table 6 are attacked Symptoms and the death condition after poison
Note:
[1] fervescence: "-" surpasses 40.0 ℃ in 1d for body temperature, and "+" be at 2-3d, and " ++ " be at 3-4d, and " +++ " is over 4d;
[2] mental status: "-" is normal, and "+", for slightly poor, " ++ " is poor, and " +++ " is extreme difference;
[3] appetite situation: "-" is normal for searching for food, and "+" is that food consumption is less, and " ++ " is that food consumption is few;
[4] expiratory dyspnea (after attacking malicious 48h): "-", for normal, "+" is slight, and " ++ ", for more serious, " +++ " is for extremely serious;
[5] have loose bowels: "-", for not occurring the symptom of having loose bowels, "+" is for occurring;
[6] lesion degree of pneumonia: "-" is normal, "+", for there being slight pneumonia, " ++ " is serious, there is necrotic area on lungs surface, but area is less than 10%, and " +++ " is for extremely serious; Necrotic area, lungs surface area surpasses 10%;
[7] lesion degree of trachitis: "-" is normal, "+", for there being slight pneumonia, " ++ " is moderate, " +++ " is serious;
[8] death condition: "-" is survival, and "+" is dead;
The test-results of the embodiment of the present invention shows, tracheae sepsis bordetella bacilli gene-deleted vaccine (aroA of the present invention
-lY1) after immune piglet, can produce high-titer for the specific antibody of tracheae sepsis bordetella bacilli by excitating organism, very low to the toxicity of mouse and piglet, security is good.In immune protective efficiency test, immune swine can be resisted the attack of lethal dose tracheae sepsis bordetella bacilli virulent strain.In addition, tracheae sepsis bordetella bacilli gene-deleted vaccine (aroA of the present invention
-lY1) genetic background is clear, and not containing resistance marker, proterties is stable, meets the biological safety requirement of live vaccine completely.
Sequence table
<110> University Of Science and Technology Of He'nan
<120> bordetella bronchiseptica gene deleted bacterial strain and vaccine prepared therefrom and application
<170>patentin version 3.3
<210>1
<212>DNA
<213> synthetic
<223> upstream primer
<400>1
tccggcgctg ccatattgtt tttctgcagg tatgagcgcg aacaattcgg caaacgggca 60
ggggcccctg gtccccgtgc tggctgtggc cggcgtaggc ttgatcggtg gctcgttcgc 120
cgccgcgttg cgccacgccg ggcaggtcgg caccattctc ggcgtgggcc gcaacccggc 180
ctcgctggcg cgtgcgcgcg agctgggcct gatcgacgag gccgtctcgc ccgaggaagc 240
ggcggcgcgc gcagacctgg tgctgctgtc caccccggtg ggcgggctgg gcgcgatgct 300
ggcgcgcatg cgcgaccatc tgcggccggg ctgcctgctg accgatgccg gcagcaccaa 360
atcgcaggtc gtcatggcgg cgcgccaggc gctgggcgag caggtttcct gtttcgtgcc 420
ggggcatccg atcgccggag gcgagcgcac cgggcccgag gcggccgacg cggggctgta 480
cgtacggcgt gcggtagtgc ttacgcccct gccggagaac gcggccgcat cggtggcgcg 540
cgtgcgcgcc tgctggcacg catgcggggc gcacgtggtc gagatggacg acgtggcgca 600
cgaccggctg ctggcttcgg tcagccacat gccgcacttc ctggccgccg tctacatggc 660
ccaggtggcc ggctccgatg acgcccaggc gcgcatggat ctggccggta gcggttttcg 720
ggatttcacg cgcatcgcgg ccggttcgcc ggaaatgtgg cgcgatatct ttttgtccaa 780
ccaggccgcc atgcagtccg agctggcggc cctgcgacgg gtgctcgacg aggccgagca 840
ggcattgggc gccggcgacg gcgcgggcct gcaggccttg ctggagcgcg cggcgcacgc 900
gcggcgcaat tggcgcaagg attccaa 927
<210>2
<212>DNA
<213> synthetic
The aroA gene order of disappearance part in <223>LY1
<400>2
aatgagcgga ttggcatatc tcgacctgcc cgcggcgcgc ctggcgcgcg gcgaggtggc 60
cctgccgggt tccaagagca tctccaacag ggtattgctg ctggccgcgc tggccgaagg 120
cagcaccgaa atcacgggcc tgctcgattc cgatgacacc cgcgtcatgc tggccgcgtt 180
gcgccagctg ggcgtatcgg tgggcgaggt ggccgatggc cgcgtgacca tcgaaggcgt 240
ggcgcgcttt ccgaccgaac aggccgagct gttcctaggc aacgccggca ccgcgttccg 300
gccgctgacc gcggcgctgg cgttgatggg cggcgattac cgcctgtccg gcgtgccgcg 360
catgcacgag cggcccatcg gcgacctggt cgacgccttg cgccagttcg gcgccgggat 420
cgaatatctg gggcaggcgg ggtatccgcc gctgcgcatc ggcggcggca gcattcgcgt 480
cgacgggccg gtgcgggtgg agggctcggt gtccagccag ttcctgaccg ccttgctgat 540
ggccgccccc gtgctggctc ggcgcagcgg ccaggacatc accatcgagg tggtgggcga 600
gctgatttcc aaaccctata tcgagatcac gctcaatctg atggcgcgtt ttggcgtgtc 660
ggtgcggcgc gacggctggc gcgccttcac gatcgcgcgc gatgcggcct accgcggccc 720
gggccgcatg gcgatcgagg gcgatgcctc gacggcgtcg tacttcctgg ccctgggcgc 780
catcggcggc gggccggtgc gcgtcaccgg cgtgggcgag gacagcatcc agggcgacgt 840
ggcgttcgcc gcgacgctgg cggcgatggg cgccgacgtg cgctatggcc cgggctggat 900
cgagacgcgc ggcgtgcggg tggccgaggg cggacgcctg aaggcgttcg acgctgactt 960
caacctgatt cccgacgccg ccatgacggc cgcgacgctg gcgctgtacg ccgacggccc 1020
atgccgcctg cgcaacatcg gcagctggcg cgtcaaggag accgaccgca tccacgccat 1080
gcacaccgag ctggagaagc tgggggcggg cgtgcaaagc ggggcggact ggctggaggt 1140
ggcgccgccg gcgcccggcg gctggcgcga cgcccacatc ggcacctggg acgaccaccg 1200
catggccatg tgcttctcgc tggccgcgtt cggtccggct gcggtgcgca tcctggatcc 1260
gggttgcgtc agcaagactt tccccgatta tttcgacgtg tacgcgggcc tgctg 1315
<210>3
<212>DNA
<213> synthetic
<223> downstream primer
<400>3
ggccgcgcgg gactgaacgg cgcctgcccc atggctattt ccgcatccgc cggcgccgcg 60
ccggtcatca ccatcgacgg ccccacggct tccggcaagg gcaccatcgc gcaccgggtg 120
gccaagcagc tgggctggga cgtgctcgac agcggcgccc tgtaccggct gaccgccctg 180
gccgcgttgc ggcgcggcct gccggccacc gacgaaccgg cggtggccgc cgtggcccag 240
gcgctggacg tacggttcga cgggccgcat gtctacctgg aagggcggga tgccgggcac 300
gaaatccgcc aggaagaggt cggaaactac gcttcgcgca tcgccgccta cccgggcgtt 360
cgtcaggcct tgctggagcg ccagcgggct ttccggcagc cgccgggcct ggtcgccgac 420
ggccgcgaca tggggacggt cgtgtttcct gatgcgtccc tgaaaatatt tctggtggcc 480
gatgtcgagg cgcgcgcgca aagacgctgt aagcagttga tcgaaaaggg aatttctgct 540
aatctagatg acctgctacg cgatatgcgt gaacgcgatg cgcgcgacac gcagcgtgcg 600
gtggcaccgc ttgccccggc tgccgatgcg catgtgctgg attcttccgg cctgaccatc 660
gaacagaccg tacaggccgt gctggatttc tggcgcgcct agccacggcg cgtgttgttt 720
gggcagtacc tcagtacctg tttgaggccc tgacggccaa tctccagccc ccacgggaca 780
ccccgcaagg gccggcgaag ccggcaggtt ttaccactcc actgctgcgg gcaatccgtt 840
gcggtgtgtt ttgaacggcc aatcccggcc aaatggattc caaccta 887
Claims (3)
- One kind not containing the segmental bronchus sepsis bordetella bacilli of resistance marker ( bordetella bronchiseptica) genetically deficient bacterial strain aroA-LY1, it is characterized in that: described bacterial strain is deposited in Chinese Typical Representative culture collection center C CTCC on December 23rd, 2011, and deposit number is CCTCC NO:M2011479.
- One kind as claimed in claim 1 not containing the segmental bronchus sepsis bordetella bacilli of resistance marker ( bordetella bronchiseptica) genetically deficient bacterial strain aroA-LY1 is in the application of preparing aspect bordetella bronchiseptica gene deleted vaccine.
- One kind by claimed in claim 1 not containing the segmental bronchus sepsis bordetella bacilli of resistance marker ( bordetella bronchiseptica) bordetella bronchiseptica gene deleted vaccine prepared of genetically deficient bacterial strain aroA-LY1.
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| CN106620685B (en) * | 2016-12-30 | 2020-07-03 | 青岛易邦生物工程有限公司 | Rabbit bordetella bronchiseptica subunit vaccine |
| BR112021013241A2 (en) * | 2019-01-04 | 2021-09-14 | Boehringer Ingelheim Animal Health USA Inc. | ATTENUATED STRAINS OF BORDETELLA BRONCHISEPTICA, ORAL VACCINES CONTAINING THE ATTENUATED STRAINS AND METHODS OF PREPARATION AND USE THEREOF |
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