CN105483051A - Pullorum disease salmonella spiC-rfaL double-gene knockout attenuated strain and DIVA vaccine application thereof - Google Patents
Pullorum disease salmonella spiC-rfaL double-gene knockout attenuated strain and DIVA vaccine application thereof Download PDFInfo
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Abstract
The invention belongs to the field of fowl salmonellosis prevention and control and particularly relates to a pullorum disease salmonella spiC-rfaL double-gene knockout attenuated strain and preparation and application thereof. The attenuated strain is pullorum disease salmonella in which a spiC gene or an rfaL gene is not expressed. It is found through animal experiments that virulence of the mutant strain is remarkably reduced, and colonization time in a host body is shortened obviously. The attenuated strain is adopted for preparing pullorum disease salmonella live vaccine, after chickens are inoculated with the vaccine, no adverse reaction is generated, and strong virulence strain infection can be effectively eliminated. The host does not generate an anti-body aiming at O-antigen, by means of pullorum disease fowl typhoid multi-valence detection antigen, the attenuated stain can be effectively applied to glass plate agglutination experiments to distinguish immunized chickens and naturally infected chickens, and the characteristic of the DIVA vaccine is achieved.
Description
Technical field
The invention belongs to the prevention and control field of fowl salmonellosis, be specifically related to that a kind of Salmonella Pullorm is dual-gene knocks out attenuated strain and preparation and application thereof.
Background technology
White dysentery is the multiple transmissible disease of one caused by Salmonella Pullorm (SalmonellaPullorum), very harmful to the poultry husbandry of China.This pathogenic bacteria, except can horizontal transmission, also can cause the reduction of hatching rate and chickling-surviving rate through egg vertical transmission, chick within many infringements 20 ages in days, with white diarrhea for feature, and performance acute sepsis process, sickness rate and case fatality rate are quite high; Adult Chicken mostly is chronic or inapparent infection, generally without obvious clinical symptom.The traditional method of preventing and treating this disease uses antibiotics, and life-time service easily causes many impacts such as drug residue and bacterial drug resistance enhancing, therefore, is quite paid attention to the research of vaccine prevention white dysentery.
According to for a long time to the exploration and practice of Salmonella Pullorm prevention and control measure, the control of Salmonella Pullorm relies on a regional gradient of infection.Infection level is low or can the area of quarantine elimination system Serologic detection and the way of slaughtering can be taked to control white dysentery, and infection level is high or cannot carry out eliminating the area of slaughtering and consider to use vaccine.Therefore, desirable Salmonella Pullorm attenuated live vaccine is except should possessing security and validity, preferably also should possess DIVA (DifferentiationofInfectedandVaccinatedAnimals, DIVA) feature, namely by simple and effective Serology test, just can distinguish vaccine immunity animal and natural infected animal, use with the vaccine at Instructing manufacture scene.O-antigen is the chief component of Grain-negative mycetocyte outermost layer structured lipid polysaccharide (Lipopolysaccharide, LPS), is not only the virulence factor of pathogenic bacteria, and the number difference of its specific polysaccharide repeating unit is also the foundation of O-serotype.Based on the agglutination test of LPS specific glass plate with its simple operating features, be widely used in the bacteriology of white dysentery at present at home and abroad.In sum, O-antigen can be used as operation target spot and the selection marker of novel Salmonella Pullorm attenuated live vaccine, in order to build DIVA vaccine.
DIVA living vaccine has been successfully applied to and has prevented virus infection in livestock and poultry.
Restructuring suicide plasmid technology and Red homologous recombination technique have been widely used in the gene knockout of bacterium as transgenation method and gene is replaced.Salmonella spiC and pathogenic closely related, rfaL encode O-antigen ligase, participate in the biosynthesizing of bacterium LPS.Dual-gene knocking out significantly reduces the possibility that reverse mutation occurs attenuated live vaccine in actual application, not affecting under immunogenic prerequisite, farthest evading the risk of virulence reversion, ensure that the security of vaccine.
Summary of the invention
In view of the situation of above prior art, the object of the present invention is to provide that a kind of Salmonella Pullorm is dual-gene knocks out attenuated strain and preparation and application thereof.
To achieve these goals and other relevant objects, the present invention adopts following technical scheme:
A first aspect of the present invention, provides that a kind of Salmonella Pullorm is dual-gene knocks out attenuated strain, is the Salmonella Pullorm that spiC gene and rfaL gene are not expressed.
Preferably, described Salmonella Pullorm is dual-gene knocks out attenuated strain, is the Salmonella Pullorm that spiC gene and rfaL gene are all knocked.
Salmonella Pullorm is prior art, and specifying information is: Accession:NC_021984.1; GI:529218781.SpiC gene is prior art, and specifying information is: Accession:JN673268.1; GI:374304599.RfaL gene is prior art, and specifying information is: ACCESSION:CP006575; REGION:3935096..3936310.
As long as the spiC gene in Salmonella Pullorm and rfaL gene are all knocked out, spiC gene and rfaL gene are not expressed, can obtain that Salmonella Pullorm is dual-gene knocks out attenuated strain.
Preferably, in one embodiment of the invention, the sequence of the spiC gene be knocked, as shown in SEQIDNO.1, is specially:
TCTGAGGAGGGATTCATGCTGGCAGTTTTAAAAGGCATTCCATTAATTCAGGATATCAAGGCCGAAGGTAATAGCCGATCCTGGATAATGACTATTGATGGGCATCCTGCCAGAGGAGAAATTTTCTCAGAAGCATTTTCTATTTCTTTGTTCTTAAATGACCTGGAAAGCTTACCTAAGCCTTGTCTTGCCTATGTGACACTACTGCTTGCAGCACACCCGGACGTACATGATTATGCTATACAGCTCACAGCGGATGGGGGATGGTTAAACGGTTATTATACCACAAGTAGTAGCTCTGAGCTTATTGCTATTGAGATAGAAAAACACCTGGCTTTAACTTGCATTTTAAAAAATGTAATACGCAATCACCATAAACTTTATTCGGGTGGGGTATAA。
Preferably, in one embodiment of the invention, the sequence of the rfaL gene be knocked, as shown in SEQIDNO.2, is specially:
ATGCTAACCACATCATTAACGTTAAATAAAGAGAAATGGAAACCGATCTGGAATAAAGCGCTGGTTTTTCTTTTTGTTGCCACGTATTTTCTGGATGGTATTACGCGTTATAAACATTTGATAATCATACTTATGGTTATCACCGCGATTTATCAGGTCTCACGCTCACCGAAAAGTTTCCCCCCTCTTTTCAAAAATAGCGTATTTTATAGCGTAGCAGTATTATCATTAATCCTTGTTTATTCCATACTCATATCGCCAGATATGAAAGAAAGCTTCAAGGAATTTGAAAATACGGTACTGGAGGGTTTCTTATTATATACTTTATTAATTCCCGTACTATTAAAAGATGAAACAAAAGAAACGGTTGCGAAAATAGTACTTTTCTCCTTTTTAACAAGTTTAGGACTTCGCTGCCTTGCAGAGAGTATTCTGTATATCGAGGACTATAATAAAGGGATTATGCCATTCATAAGCTATGCGCATCGACATATGTCCGATTCCATGGTTTTCTTATTTCCAGCATTATTGAATATTTGGCTGTTTAGAAAAAATGCAATTAAGTTGGTTTTTTTGGTGCTTAGCGCCATCTACCTTTTCTTTATCCTGGGAACCCTATCGCGAGGGGCATGGTTGGCGGTGCTTATAGTAGGTGTTCTGTGGGCAATACTGAACCGCCAATGGAAGTTAATAGGAGTTGGTGCCATTTTATTAGCCATTATCGGCGCTTTGGTTATCACTCAACATAATAACAAACCAGACCCAGAACATTTACTGTATAAATTACAGCAGACAGATAGCTCATATCGTTATACTAACGGAACCCAGGGCACCGCGTGGATACTGATTCAGGAAAACCCGATCAAGGGCTACGGCTATGGTAATGATATGTATGATGGTGTTTATAATAAACGCGTTGTCGATTATCCAACGTGGACCTTTAAAGAATCTATCGGTCCGCATAATACCATTCTGTACATCTGGTTTAGTGCAGGCATATTGGGTCTGGCGAGCCTGGCCTATTTATATGGCGCTATCATCAGGGAAACAGCCAGCTCTACCTTCAGGAAAGTAGAGATAAGCCCCTACAATGCTCATCTCTTGCTATTTTTATCTTTCGTCGGTTTTTATATCGTTCGTGGCAATTTTGAACAGGTCGATATTGCTCAAATTGGTATCATTACTGGTTTTCTGTTGGCGCTAAGAAATAGATAA。
Further, described attenuated strain also can be passed through other genetic modifications.
Other described genetic modifications refer to the genetic modification except spiC gene and rfaL Gene Double knock out.Other genetic modifications described can be the transformation of single target gene or the transformation for multiple interesting target gene.Interesting target gene does not refer in particular to, and can need set and transform according to research.Such as, can be the transformation of one, two, more than three target gene.Constantly can carry out genetic modification to it in theory, the transformation quantity for target gene can operate as required, is not particularly limited.
Genetic modification structurally changes before specifically referring to and making gene compare transformation by the means of biological or chemical or physics.This change mainly refers to the change that base pair forms, and includes but not limited to the replacement of one or more base pair, increases, lacks the change caused.Described genetic modification includes but not limited to the knocking in of target gene, the knocking out of target gene.The knocking in of target gene, knocking out of target gene can adopt the technology such as gene targeting, homologous recombination.
Preferably, in another embodiment of the present invention, the rfaL gene as shown in SEQIDNO.2 is knocked and replaces with FRT site sequence.Described FRT site sequence, as shown in SEQIDNO.3, is specially:
Tgggaattagccatggtccatatgaatatcctccttagttcctattccgaagttcctattctctagaagtataggaacttcgaagctgctccagcctaca。
The introducing of above-mentioned FRT site sequence, does not affect the dual-gene performance knocking out attenuated strain of Salmonella Pullorm.
A second aspect of the present invention, provides the dual-gene construction process knocking out attenuated strain of aforementioned Salmonella Pullorm, comprises step: the spiC gene in Salmonella Pullorm and rfaL gene are all knocked out.
Existing gene editing method can be adopted to knock out described spiC gene and rfaL gene.Preferably, the method for homologous recombination can be adopted to knock out spiC gene and rfaL gene.In a preferred embodiment, the method for Red homologous recombination is adopted to knock out spiC gene and rfaL gene.Additive method also can be adopted to realize gene knockout, be not limited to the mode cited by embodiment.
Based on knocking out of spiC gene, the complete sequence of spiC gene is removed from chromosomal DNA; Based on knocking out of rfaL gene, the complete sequence of rfaL gene is removed from chromosomal DNA.
In one embodiment, first build the Salmonella Pullorm mutant strain of spiC gene knockout, and knock out rfaL gene on this basis.
A third aspect of the present invention, provides that aforementioned Salmonella Pullorm is dual-gene to be knocked out attenuated strain and preparing the purposes in Salmonella Pullorm living vaccine.
A fourth aspect of the present invention, provides a kind of Salmonella Pullorm living vaccine, knocks out attenuated strain containing aforementioned Salmonella Pullorm is dual-gene.
Further, also adjuvant is contained in described Salmonella Pullorm living vaccine.
A fifth aspect of the present invention, provides spiC gene and rfaL gene is screening the purposes in Salmonella Pullorm treatment of infection medicine as target gene jointly.
Salmonella Pullorm treatment of infection medicine of the present invention be with spiC gene and rfaL gene for therapeutic target gene, make the medicine that spiC gene and rfaL gene knockout, the medicine of not expressing or the albumen with spiC gene and rfaL genetic expression are antagonism object; Or using spiC gene, rfaL gene and other genes jointly as the medicine that target gene is in addition reticent.With spiC gene and rfaL gene for target spot, screening the medicine that this two genes are not expressed, for making Salmonella Pullorm perform toxic attenuation, infecting to treat Salmonella Pullorm.Such as, this Salmonella Pullorm treatment of infection medicine can be disturbed by RNA or the method for gene recombination makes spiC gene and rfaL gene knockout or do not express; Or by preparation spiC albumen and rfaL protein antagonist, the albumen of spiC gene and rfaL genetic expression is not played a role.
Compared with prior art, the present invention has following beneficial effect:
Purport of the present invention have developed the dual-gene Salmonella Pullorm attenuated strain knocked out of a kind of spiC-rfaL; the present invention is found by animal experiment; described mutant strain virulence significantly reduces; obviously shorten in host's Colonization inside plants time; after inoculation, chicken has no adverse reaction; there is provided good provide protection to strong virus force Salmonella Pullorm, effectively can remove virulent isolates and infect.
Further, the Salmonella Pullorm living vaccine adopting described attenuated strain to do to prepare, its virulence significantly reduces relative to wild-type Salmonella Pullorm, shows that it significantly improves the lethal dose of 1 age in days Hai Lanbai chick.After inoculating Salmonella Pullorm attenuated live vaccine of the present invention, host does not produce the antibody for O-antigen, by white dysentery Salmonella gallinarum multivalence detectable antigens, effectively can apply the agglutination test of glass plate and be distinguished immunization chicken and natural infection chicken.Described vaccine has good immunogenicity, can provide solid immune protective effect to chick, and can distinguish vaccine inoculation chicken and natural infection chicken rapidly by the agglutination test of glass plate.That is described vaccine has the function distinguishing immunization chicken and natural infection chicken, can be distinguished by the agglutination test of glass plate, vaccine application does not affect the enforcement of white dysentery positive chicken quarantine elimination system.
Accompanying drawing explanation
The pcr amplification of Fig. 1: Salmonella Pullorm rfaL gene targeting fragment.M:DL2000DNAMarker; 1:P1/P2 amplified production.
The PCR qualification of Fig. 2: attenuated strain S06004 Δ spiC Δ rfaL.M：λ-EcoT14DNAMarker；1：S06004；2：S06004ΔspiCΔrfaL::Cm。
Fig. 3: the acridine orange agglutination test knocking out strain.1：S06004；2：S06004ΔspiCΔrfaL。
Fig. 4: the SDS-PAGE silver dye knocking out strain LPS structure is analyzed.1：S06004；2：S06004ΔspiCΔrfaL。
Fig. 5: Salmonella Pullorm attenuated strain determining in chick liver, spleen is grown.A: liver; B: spleen." Δ " represents attenuated strain.
Fig. 6: chick body weight change after immunity." Δ " represents attenuated strain.
Fig. 7: immunized chicks removes the efficiency of strong virus force Salmonella infection." Δ " represents attenuated strain.
Fig. 8: knock out strain and there is the DIVA performance distinguishing vaccinated animals and natural infected animal.1：S06004；2：S06004ΔspiCΔrfaL。" d.p.i " represents post-immunized day.
Embodiment
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, instead of in order to limit the scope of the invention.The test method of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that each manufacturers advises.
When embodiment provides numerical range, should be understood that except non-invention is otherwise noted, between two end points of each numerical range and two end points, any one numerical value all can be selected.Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology and those skilled in the art of the present technique understand usually.Except the concrete grammar used in embodiment, equipment, material, according to those skilled in the art to the grasp of prior art and record of the present invention, any method of prior art that is similar with the method described in the embodiment of the present invention, equipment, material or that be equal to, equipment and material can also be used to realize the present invention.
Unless otherwise indicated, disclosed in the present invention experimental technique, detection method, preparation method all adopt the routine techniques of the molecular biology of the art routine, biological chemistry, chromatin Structure and analysis, analytical chemistry, cell cultures, recombinant DNA technology and association area.These technology existing improving in existing document illustrates, specifically can see Sambrook etc.
MOLECULARCLONING:ALABORATORYMANUAL, Secondedition, ColdSpringHarborLaboratoryPress, 1989andThirdedition, 2001; Ausubel etc., CURRENTPROTOCOLSINMOLECULARBIOLOGY, JohnWiley & Sons, NewYork, 1987andperiodicupdates; TheseriesMETHODSINENZYMOLOGY, AcademicPress, SanDiego; Wolffe, CHROMATINSTRUCTUREANDFUNCTION, Thirdedition, AcademicPress, SanDiego, 1998; METHODSINENZYMOLOGY, Vol.304, Chromatin (P.M.WassarmanandA.P.Wolffe, eds.), AcademicPress, SanDiego, 1999; And METHODSINMOLECULARBIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) HumanaPress, Totowa, 1999 etc.
The dual-gene structure knocking out attenuated strain S06004 Δ spiC Δ rfaL of embodiment 1 Salmonella Pullorm
1. materials and methods
1.1 bacterial strains and plasmid
Salmonella Pullorm S06004 Δ spiC mutant strain utilizes the restructuring gene constructed acquisition of the seamless spiC of knocking out of suicide plasmid method by the present inventor laboratory and preserves that (described Salmonella Pullorm S06004 Δ spiC mutant strain is prior art, its concrete construction process refers to " Construction and identification of Salmonella Pullorm S06004 Δ spiC mutant strain; Chinese veterinary science, 04 phase in 2014 ").
Salmonella Pullorm S06004 (Nal
r), E.coliDH5 α, E.coliSpy372 (λ pir) and plasmid pKD46 (Amp
r), pKD3 (Cm
r), pCP20 (Amp
r, Cm
r) preserve by the present inventor laboratory, these bacterial strains and plasmid are prior art, specifically see structure and the biological characteristics thereof of Salmonella Pullorm S06004 strain pathogenicity island-2 gene-deleted strain, and microorganism journal, 2015,55 (5).
PMD20-T cloning vector is purchased from precious biotechnology (Dalian) company limited.
1.2 main agents
GenomicDNAExtractionKit, dNTP, general T aq enzyme, DNAmarker are purchased from precious biotechnology (Dalian) company limited, DNAFragmentPurificationKit is purchased from Tiangen company, bacterium micro biochemical fermentation tube is purchased from sky, Hangzhou and biotech firm, L-arabinose available from Sigma, LPSExtractionKit is purchased from iNtRON company, PierceSilverStrainKit is purchased from Thermo company, and all the other conventional reagent are domestic or Import Analysis straight product.Primer synthesis is completed by Nanjing Jin Sirui biotech firm.
The 1.3 dual-gene construction processs knocking out attenuated strain
1.3.1 primer
According to the Salmonella Pullorm genome sequence that GenBank has announced, be designed for the primer of homologous recombination with primer-design software Primer5.0.
Table 1 is for the primer sequence of gene knockout
Primer P1/P2 is used for homologous recombination, and this primer is made up of two portions respectively, 5 ' terminal sequence and rfaL gene both wings sequence homology, and 3 ' holds the chloramphenicol resistance gene both sides complementary on the sequence of italic and template plasmid pKD3.YZ1/YZ2 is a pair checking primer of rfaL homologous recombination areas outside, and the sequence between this primer is be about 1150bp after 1360bp, rfaL gene is replaced by chloramphenicol resistance gene in parent, and chloramphenicol resistance gene is knocked rear about 250bp.
1.3.2 the amplification of target practice fragment and purifying
With pKD3 plasmid for template, carry out pcr amplification, amplification condition with primer P1/P2 to rfaL homologous recombination fragment: 94 DEG C of sex change 5min, 94 DEG C of 45s, 58 DEG C of 45s, 72 DEG C of 60s, carry out 35 circulations, last 72 DEG C extend 10min, 4 DEG C of preservations.Adopt 1% agarose gel electrophoresis qualification PCR primer, use glue to reclaim kits, reclaim fragment as rfaL gene targeting fragment.
1.3.3 electroporated target practice fragment is to competent cell
The pKD46 plasmid electricity of coding Red recombination system is transformed into Salmonella Pullorm S06004 Δ spiC to be knocked out in strain, obtains S06004 Δ spiC (pKD46).Picking list colony inoculation 30 DEG C of shaking culture in the LB substratum containing penbritin are spent the night, contain in the LB substratum of penbritin in 1:100 ratio switching culture to 10mL, add the L-arabinose of final concentration 30mmol/L, 30 DEG C of shaking culture are to OD simultaneously
600be 0.5 ~ 0.6, ice bath 10min, 4 DEG C, 4000rpm centrifugal 10min collection bacterium, wash 1 time with the aseptic deionized water of ice bath, then wash 2 times with 10% glycerine of precooling, finally resuspended with 10% glycerine of the precooling of 1:200 volume, namely prepares competent cell.
Joining diameter after target practice fragment concentration being not less than 150ng/ μ L mixes with 50 μ L competent cells is in the electric shock cup of 1mm, carries out electroporated after ice bath 2min.Shock parameters: resistance 200 Ω, electric capacity 25 μ F, voltage 1800V.After electric shock terminates, add 1mLSOC substratum immediately, 180rpm cultivates 1.5 ~ 2h, subsequently the LB substratum of coating containing paraxin, 37 DEG C of cultivations.Picking mono-clonal carries out PCR qualification, correct i.e. called after S06004 Δ spiC Δ rfaL::Cm.
1.3.4 the elimination of resistant gene
The pCP20 plasmid carrying FLP site-specific recombinase is imported in S06004 Δ spiC Δ rfaL::Cm mutant strain, by the Double LB substratum 30 DEG C cultivation containing penbritin and paraxin, screening obtains positive colony S06004 Δ spiC Δ rfaL::Cm (pCP20), picking list bacterium colony cultivates about 12h in 42 DEG C of LB liquid nutrient mediums, can obtain and knock out strain, called after S06004 Δ spiC Δ rfaL not containing pCP20 plasmid and chloramphenicol resistance gene.
1.3.5 the biological characteristics of strain is knocked out
By biochemical identification pipes such as inoculation glucose, sucrose, maltose, seminose, rhamnosyl, wood sugar, galactitol, sorbyl alcohol, lactose, lysine decarboxylase, ethanamide, hydrogen sulfide, peptone water, side golden flower alcohol, biochemical characteristic mensuration is carried out to Salmonella Pullorm S06004 Δ spiC Δ rfaL gene-deleted strain.
1.3.6 the phenotypic evaluation of strain is knocked out
The agglutination test of employing acridine orange, bacterium colony violet staining carry out the qualification of S-R type to knocking out strain.Extract test kit and silver-colored transfection reagent box specification sheets according to LPS, mutant strain LPS structure is analyzed.
2 experimental results
The pcr amplification of 2.1 target practice fragments
With pKD3 plasmid for template, use P1/P2 primer amplification to go out two ends and rfaL gene upstream and downstream sequence homology, centre is the DNA fragmentation of chloramphenicol resistance gene Cm, identifies through 1% agarose gel electrophoresis, and expects that 1100bp's is in the same size, as shown in Figure 1.
2.2 PCR knocking out strain identify
The mutant strain that checking primer pair resistant gene in use table 1 eliminates front and back carries out PCR qualification, as shown in Figure 2.The fragment amplified to be cloned on pMD20-T carrier by Outside primer and to send order-checking, sequence alignment proves that knocking out strain successfully constructs.The FLP recombinase that plasmid pCP20 expresses can identify the FRT site of resistant gene both sides, is removed by the resistant gene of centre by Site-specific recombinase, only leaves the FRT site of single copy at action site.
That is, as above method is adopted in the present embodiment, by in Salmonella Pullorm as shown in SEQIDNO.1 spiC gene knockout, and the rfaL gene as shown in SEQIDNO.2 replaces with FRT site sequence (achieving knocking out of rfaL gene) as shown in SEQIDNO.3, spiC gene and rfaL gene are not expressed, have successfully been obtained that Salmonella Pullorm is dual-gene knocks out attenuated strain.
2.3S06004 the biochemical characteristic qualification of Δ spiC Δ rfaL
The biochemical identification pipes such as bacterium switching glucose, rhamnosyl, wood sugar, maltose, N.F,USP MANNITOL, galactitol, sorbyl alcohol, lactose, sucrose, lysine dehydrogenase, potassium cyanide, urea, ethanamide, Vitamin C2, hydrogen sulfide, peptone water, side golden flower alcohol, 37 DEG C of incubated overnight, biochemical identification result (table 2) shows that mutant strain is consistent with wild strain bacteria bio characteristic.
The biochemical characteristic of table 2 Salmonella Pullorm S06004 Δ spiC Δ rfaL mutant strain and wild strain S06004 compares
Note: "+" represents the positive, "-" represents negative, and " WT " refers to Salmonella Pullorm S06004, and " Δ spiC Δ rfaL " refers to that Salmonella Pullorm S06004 Δ spiC Δ rfaL knocks out strain.
2.4 knock out the phenotype of strain
Knock out strain fully to mix with 0.1% acridine orange solution, can be observed somatic agglutination particle (Fig. 3), the institute LPS that carries is through SDS-PAGE gel silver dye, and result shows O-antigenic structure disappearance (Fig. 4) of LPS, meets the feature of rough type Salmonella Pullorm.
The dual-gene DIVA vaccine application knocking out attenuated strain S06004 Δ spiC Δ rfaL of embodiment 2 Salmonella Pullorm
The Salmonella Pullorm of test materials constructed by embodiment 1 is dual-gene knocks out S06004 Δ spiC Δ rfaL attenuated strain.
The single bacterium colony of picking bacterium is full plate line on LB culture plate, and 37 DEG C of quiescent culture 20h, use aseptic PBS carefully to be scraped by the bacterium on LB flat board, wash 3 times with aseptic PBS, and adjustment bacterial concentration is 10
9cFU/mL, namely makes attenuation Salmonella Pullorm vaccine.
The dual-gene safety testing knocking out strain of test example 1 Salmonella Pullorm
1. test method
110 1 age in days Hai Lanbai chick are divided into 11 groups at random, often organize 10, respectively with 1 × 10
10, 1 × 10
9, 1 × 10
8, 1 × 10
7, 1 × 10
6the S06004 Δ spiC Δ rfaL and 1 × 10 of CFU
9, 1 × 10
8, 1 × 10
7, 1 × 10
6, 1 × 10
5the S06004 intramuscular injection 0.1mL bacterial suspension of CFU, one group is blank group.Continuous Observation two weeks record death condition, calculate the LD of bacterium
50, evaluate attenuated strain to the security of chick.
2. test-results
Calculate according to Karber-Behrens method, S06004 Δ spiC Δ rfaL is to the LD of 1 Japanese instar chickling
50be 3.98 × 10
9cFU, S06004 are to the LD of chick
50be 1.0 × 10
7cFU (table 3).S06004 Δ spiC Δ rfaL knocks out strain compared to wild strain LD
50raise 398 times, its virulence significantly declines.
Table 3 Salmonella Pullorm S06004 Δ spiC Δ rfaL knocks out the LD of strain
50measure
Test example 2 attenuation Salmonella Pullorm determining in host grows test
1. test method
Salmonella Pullorm S06004 Δ spiC Δ rfaL mutant strain is with 1 × 10
7cFU/0.1mL/ dosage intramuscular injection immunity 1 age in days Hai Lanbai chick only, sets up S06004 wild strain positive controls simultaneously.7d, 14d, 21d, 28d cut open and kill experimental chicken after infection respectively, often organize 3, asepticly get its spleen, liver, weigh, the PBS adding 1mL aseptic fully grinds, and gets the continuous 10 times of dilutions of 100 μ L lapping liquids, choose 3 suitable extent of dilution, each extent of dilution is got 100 μ L and is coated with LB flat board (Nal30 μ g/mL), respectively does 3 repetitions, bacterial count.Calculate the dynamic rule of bacterium in internal organs (spleen, liver).
2. test-results
Salmonella Pullorm attenuated strain determines the situation of growing as shown in Figure 5 in chick liver, spleen.The each time point detected, the bacterium amount of carrying of wild strain infected group is all apparently higher than attenuated strain group, and attenuated strain is finally completely removed in chick internal organ.
Body weight change after test example 3 attenuation Salmonella Pullorm immunized chicks
1. test method
Salmonella Pullorm S06004 Δ spiC Δ rfaL mutant strain is with 1 × 10
7cFU/0.1mL/ dosage intramuscular injection immunity 1 age in days Hai Lanbai chick only, sets up blank group simultaneously.7d, 14d, 21d, 28d after immunity, weigh, record body weight change, the impact on chick weight gains after the immunity of evaluation mutant strain.
2. test-results
By comparing the impact on chick weight gains after the immunity of attenuation Salmonella Pullorm, immunization group body weight compared with blank group, without significant difference (Fig. 6), shows that vaccine inoculation does not affect chick growth.
Pathologic examination after test example 4 immunized chicks
1. test method
Salmonella Pullorm S06004 Δ spiC Δ rfaL mutant strain is with 1 × 10
7cFU/0.1mL/ dosage intramuscular injection immunity 1 age in days Hai Lanbai chick only, sets up blank group simultaneously.Observe chick state, and respectively after immunity 7d, 14d, 21d, 28d cut open and kill each group of 3 chick, pathologic examination is carried out to liver, spleen, caecum.
2. test-results
Salmonella Pullorm S06004 Δ spiC Δ rfaL mutant strain does not cause any clinical symptom of chick, to examine internal organs identical with control group, all without pathology.Test-results shows that this attenuated strain has good security to chick further.
Test example 5 Immunoprotection test
1. test method
With the mode of intramuscular injection immunity 1 age in days Hai Lanbai chick, intramuscular injection dosage 10
7cFU.After immunity the 14th day, adopt intramuscular injection mode to attack poison to immune chicken respectively with Salmonella Pullorm wild strain S06004, Salmonella gallinarum Sg9, Salmonella enteritidis C50041, attacking toxic agent amount was 10
9cFU.Attack the rear Continuous Observation record chick death condition of poison, to evaluate the immanoprotection action of Attenuated Salmonella.Set up the strong poison of not immune chicken to attack poison group and normal healthy controls group when attacking poison, often organize 10.
2. test-results
Attenuated strain S06004 Δ spiC Δ rfaL shows the desirable immune protection effectiveness resisting Salmonella Pullorm infection, and virulent strain attacks the rear chick survival rate 100% of poison.There is good cross-protection ability simultaneously, the infection of Salmonella gallinarum (survival rate is 100%) can be protected completely, Salmonella enteritidis is also had to the Vaccine effectiveness (table 4) of 70%.
The immune protection effectiveness of table 4 attenuated strain
Test example 6 immunized chicks removes the efficiency of strong virus force Salmonella infection
1. test method
With the mode of intramuscular injection immunity 1 age in days Hai Lanbai chick, intramuscular injection dosage 10
7cFU.After immunity the 14th day, adopt intramuscular injection mode to attack poison to immune chicken respectively with Salmonella Pullorm wild strain S06004, Salmonella gallinarum Sg9, Salmonella enteritidis C50041, attacking toxic agent amount was 10
9cFU.Attack poison latter 14th day, cut open and kill all survival live chickens, with the malicious bacterium of attacking in caecum, counting is separated to liver, spleen.
2. test-results
After attacking poison, the amount of bacteria in immunized chicks internal organs is obviously less than nonimmune group (Fig. 7), shows that attenuation Salmonella Pullorm of the present invention can induce body to remove the infection of virulent strain effectively.
Test example 7 mutant strain DIVA function
1. test method
Salmonella Pullorm S06004 Δ spiC Δ rfaL mutant strain is with 1 × 10
7cFU/0.1mL/ dosage intramuscular injection immunity 1 age in days Hai Lanbai chick only, sets up wild-type to infect control group (intramuscular injection 10 simultaneously
6cFU Salmonella Pullorm S06004).The aseptic Culling heart blood of 7d, 14d, 21d, 28d after immunity respectively, separation of serum.Use white dysentery Salmonella gallinarum multivalence detectable antigens to carry out the agglutination test of glass plate, observe aggegation situation, judged result.
2. test-results
After Salmonella Pullorm attenuated strain S06004 Δ spiC Δ rfaL immunity, chick serum all the time not with detectable antigens generation aggegation, and positive group can produce aggegation (Fig. 8) at 21d, very easily distinguish vaccine immunity group and wild-type infected group, there is obvious DIVA feature.
The present inventor also adopts the methods of homologous recombination of restructuring suicide plasmid mediation, seamless knocking out is carried out to rfaL gene, that is, in Salmonella Pullorm, as shown in SEQIDNO.1, spiC gene and the rfaL gene as shown in SEQIDNO.2 are all knocked, do not introduce other any gene, have successfully been obtained that Salmonella Pullorm is dual-gene knocks out attenuated strain, the attenuated strain performance obtained is consistent with the attenuated strain performance in embodiment 1.
The above; be only preferred embodiment of the present invention; not to any formal and substantial restriction of the present invention; should be understood that; for those skilled in the art; under the prerequisite not departing from the inventive method, also can make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.All those skilled in the art, without departing from the spirit and scope of the present invention, a little change made when utilizing disclosed above technology contents, the equivalent variations of modifying and developing, be Equivalent embodiments of the present invention; Meanwhile, all according to substantial technological of the present invention to the change of any equivalent variations that above-described embodiment is done, modify and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (10)
1. Salmonella Pullorm is dual-gene knocks out an attenuated strain, is the Salmonella Pullorm that spiC gene and rfaL gene are not expressed.
2. Salmonella Pullorm according to claim 1 is dual-gene knocks out attenuated strain, is the Salmonella Pullorm that spiC gene and rfaL gene are all knocked.
3. Salmonella Pullorm according to claim 2 is dual-gene knocks out attenuated strain, it is characterized in that, the sequence of the spiC gene be knocked is as shown in SEQIDNO.1.
4. Salmonella Pullorm according to claim 2 is dual-gene knocks out attenuated strain, it is characterized in that, the sequence of the rfaL gene be knocked is as shown in SEQIDNO.2.
5. the dual-gene construction process knocking out attenuated strain of Salmonella Pullorm as described in claim as arbitrary in Claims 1 to 4, comprises step: the spiC gene in Salmonella Pullorm and rfaL gene are all knocked out.
6. method according to claim 5, is characterized in that, adopts the method for homologous recombination to knock out spiC gene and rfaL gene.
7. Salmonella Pullorm as described in as described in claim as arbitrary in Claims 1 to 4 is dual-gene to be knocked out attenuated strain and is preparing the purposes in Salmonella Pullorm living vaccine.
8. a Salmonella Pullorm living vaccine, knocks out attenuated strain containing, for example the Salmonella Pullorm described in the arbitrary claim of Claims 1 to 4 is dual-gene.
9. Salmonella Pullorm living vaccine according to claim 8, is characterized in that, also containing adjuvant.
10.spiC gene and rfaL gene are jointly as the purposes of target gene in screening Salmonella Pullorm treatment of infection medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112546210A (en) * | 2020-12-15 | 2021-03-26 | 南京农业大学 | Preparation method and application of salmonella inactivated vaccine |
| CN113265358A (en) * | 2021-05-25 | 2021-08-17 | 山东省滨州畜牧兽医研究院 | Salmonella pullorum and mycoplasma synoviae double plate agglutination antigen and preparation method and application thereof |
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| CN1315871A (en) * | 1998-07-24 | 2001-10-03 | 梅根保健股份有限公司 | Live attenuated i(salmonella) vaccines to control avian pathogens |
| WO2011092185A1 (en) * | 2010-01-28 | 2011-08-04 | Universiteit Gent | Salmonella vaccine |
| WO2015179270A2 (en) * | 2014-05-19 | 2015-11-26 | Board Of Regents, The University Of Texas System | Combinatorial platform for the display of surface adjuvants and antigens |
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| CN1315871A (en) * | 1998-07-24 | 2001-10-03 | 梅根保健股份有限公司 | Live attenuated i(salmonella) vaccines to control avian pathogens |
| WO2011092185A1 (en) * | 2010-01-28 | 2011-08-04 | Universiteit Gent | Salmonella vaccine |
| WO2015179270A2 (en) * | 2014-05-19 | 2015-11-26 | Board Of Regents, The University Of Texas System | Combinatorial platform for the display of surface adjuvants and antigens |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112546210A (en) * | 2020-12-15 | 2021-03-26 | 南京农业大学 | Preparation method and application of salmonella inactivated vaccine |
| CN113265358A (en) * | 2021-05-25 | 2021-08-17 | 山东省滨州畜牧兽医研究院 | Salmonella pullorum and mycoplasma synoviae double plate agglutination antigen and preparation method and application thereof |
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