CN106190943A - A kind of dual-gene disappearance Salmonella enteritidis, its construction method and the vaccine containing this dual-gene disappearance Salmonella enteritidis - Google Patents

A kind of dual-gene disappearance Salmonella enteritidis, its construction method and the vaccine containing this dual-gene disappearance Salmonella enteritidis Download PDF

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CN106190943A
CN106190943A CN201610626518.5A CN201610626518A CN106190943A CN 106190943 A CN106190943 A CN 106190943A CN 201610626518 A CN201610626518 A CN 201610626518A CN 106190943 A CN106190943 A CN 106190943A
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sop
rfa
salmonella
bacterium
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CN106190943B (en
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胡勇
林治华
舒茂
王远强
邱荣蓉
廖羚茜
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Chongqing University of Technology
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Abstract

The present invention discloses a kind of dual-gene disappearance Salmonella enteritidis, its construction method and the vaccine containing this dual-gene disappearance Salmonella enteritidis, and described dual-gene disappearance Salmonella enteritidis isrfaH gene andsopThe Salmonella enteritidis that 1 B gene is all knocked, described construction method uses the negative screening technique of methods of homologous recombination and sucrose sensitive gene in described Salmonella enteritidisrfaH gene andsop1 B gene knocks out, and the component of described vaccine includes the dual-gene disappearance Salmonella enteritidis using said method to prepare.The present invention dual-gene disappearance Salmonella enteritidis cellular immunization and mucosa-immune are strengthened, and counteracting toxic substances protected effect is good, and immune effect is excellent, and elimination effect is good in host, just can be gone by surely growing removing in vivo in 3 ~ 4 weeks, and security performance is excellent.

Description

A kind of dual-gene disappearance Salmonella enteritidis, its construction method and dual-gene containing this The vaccine of disappearance Salmonella enteritidis
Technical field
The invention belongs to gene Knockout field, be specifically related to a kind of dual-gene disappearance Salmonella enteritidis, its structure Method and the vaccine containing this dual-gene disappearance Salmonella enteritidis.
Background technology
Salmonella enteritidis is the important pathogen of a kind of Zoonosis, infect the poultry of Salmonella be Salmonella For common storage vault, the mankind are often as taking in contaminated eggs or the most well-done Fowl meat and infecting salmonella 's.In world wide, the alimentary toxicosis that the Fowl meat egg products polluted because eating Salmonella enteritidis cause, there are generation and number in each state Amount is continuously increased.Therefore, Salmonella enteritidis the most seriously hinders the development of aviculture, but also threatens human and livestock health, Become a major issue of medical science, veterinary and field of public health.At present, mainly use antibiotics pre- Prevent and treatment Salmonella, but being widely used along with antibiotic, result in the drug resistance of Salmonella and the continuous of Antibiotic Resistance Change and multidrug resistant phenomenon.
Vaccine is the another kind of effective measures of prevention enteritis Salmonella infection, and the vaccine nowadays used has inactivated vaccine and subtracts Virus live vaccine, inactivated vaccine need repeatedly inoculate through subcutaneous, in-convenience in use, and can not induce generation digestive tract localised protection power and Cellular immunization, thus strong immunity cannot be excited to resist the deficiencies such as the infection of wild pathogenic strain.And live vaccine because of Natural infection situation can be simulated, there is the feature of inducing cell, body fluid and mucosa-immune, the most in the world promotion live vaccine Controlling poultry SE to infect, the research and development of live vaccine have obtained the accreditation of World Health Organization (WHO), and complete as controlling salmonellosis A part for whole strategy.Live vaccine mainly by gene mutation or the attenuation mutant that knocks out structure, is widely used chicken at present Attenuated live vaccine, although have certain immune protection effectiveness, but in most experiments, be difficult to wild strain to produce stronger Determine grow depression effect, also can induce wild strain general propagate；Additionally, can not effectively induce, other nonhost is adapted to The cross protection of property serotype salmonella.Therefore, there is presently no a kind of very effective attenuated live vaccine to control poultry SE Infect.Along with people infects the continued popularity of Salmonella enteritidis, it is badly in need of developing significantly more efficient vaccine to control poultry SE antibacterial Propagate.
Summary of the invention
For deficiencies of the prior art, present invention solves the technical problem that and be: for enteritis in prior art Wild strain is difficult to produce by living salmonella vaccine stronger to be determined to grow depression effect, and easily the general of induction wild strain is propagated, And can not effectively induce the technical problem to other nonhost adaptability serotype salmonella cross protections, and provide one to exist In host, elimination effect is good, and the excellent dual-gene disappearance Salmonella enteritidis of immune effect, its construction method and containing this pair The vaccine of gene delection Salmonella enteritidis.
In order to solve above-mentioned technical problem, the present invention adopts the following technical scheme that a kind of dual-gene disappearance Salmonella Bacterium, the Salmonella enteritidis being all knocked for rfaH gene and sopB gene.
The construction method of a kind of dual-gene disappearance Salmonella enteritidis, uses the method for homologous recombination to described enteritis sramana RfaH gene and sopB gene in Salmonella knock out.Specifically include following steps:
1) according to the genome sequence of Salmonella enteritidis, the upstream of upstream homology arm sequence rU of rfaH gene is designed Primer rfaH-1F and downstream primer rfaH-1R, the forward primer rfaH-2F of downstream homology arm sequence rD of rfaH gene and under Trip primer rfaH-2R, the nucleotide sequence of described rfaH-1F as shown in SEQ ID NO.3, the nucleotides sequence of described rfaH-1R Row as shown in SEQ ID NO.4, the nucleotide sequence of described rfaH-2F as shown in SEQ ID NO.5, the core of described rfaH-2R Nucleotide sequence is as shown in SEQ ID NO.6；
2) use PCR amplification method, with the genomic DNA of Salmonella enteritidis as template, with rfaH-1F and rfaH-1R be Primer amplification obtains the upstream homology arm rU of rfaH, obtains the downstream homology of rfaH with rfaH-2F and rfaH-2R for primer amplification Arm rD；
3) with step 2) rU and rD that obtain of amplification is as template, rfaH-1F and rfaH-2R is primer, carries out overlapping PCR anti- Should, it is thus achieved that overlapping PCR primer rUD；
4) by step 3) overlapping PCR primer rUD that obtains is attached with pMD19-T carrier, will connect product and convert sense By state bacillus coli DH 5 alpha, use blue white screening and amicillin resistance screening to select positive colony, use the training of LB culture medium Support the positive colony picked out, extract plasmid, it is thus achieved that restructuring pMD-Δ rfaH plasmid；
5) with restricted enzyme Bgl II enzyme action suicide plasmid pYG4 respectively and restructuring pMD-Δ rfaH plasmid, it is recovered from The 1885bp fragment obtained after the 5797bp fragment obtained after killing plasmid pYG4 enzyme action and restructuring pMD-Δ rfaH plasmid enzyme restriction, will The 5797bp fragment and the 1885bp fragment that obtain connect overnight in metal bath；
6) by step 5) connect after product heat-shock transformed escherichia coli S17-l/ λ pir competent cell, to convert after Cell is cultivated, and cultivation bacterium solution is coated on kalamycin resistance LB flat board, picking list bacterium colony, extracts plasmid after increasing bacterium, Obtain restructuring suicide plasmid pYG4-Δ rfaH；
7) in the LB culture medium containing nalidixan, cultivate recipient bacterium Salmonella enteritidis, train at the LB containing kalamycin Support and base cultivated the donor bacterium escherichia coli S17-l/ λ pir containing restructuring suicide plasmid pYG4-Δ rfaH, to described recipient bacterium and Donor bacterium collects thalline resuspended respectively after cultivating, the donor bacterium after resuspended and recipient bacterium mix the LB training being incorporated in antibiotic-free Supporting and cultivate on base, bacterium solution cultivation obtained is coated on the dual anti-flat board containing kanamycin and nalidixan, screening positive clone Single bacterial strain of integrating, after list screening obtained integration bacterial strain is cultivated in the LB culture medium of antibiotic-free, screens with sucrose plate, Obtain occurring the Salmonella bacterial strain of dyeing In vivo recombination；With sequence rfaH-1 as shown in SEQ ID NO.7 and sequence such as RfaH-2 shown in SEQ ID NO.8 is primer, uses PCR method to enter the Salmonella bacterial strain that dyeing In vivo recombination occurs Row filter is identified, selects PCR and amplifies the bacterial strain of 631bp, obtains knocking out the Salmonella bacterial strain of rfaH gene；
8) according to the genome sequence of Salmonella enteritidis, the upstream of upstream homology arm sequence sU of sopB gene is designed Primer sopB-1F and downstream primer sopB-1R, the forward primer sopB-2F of downstream homology arm sequence sD of sopB gene and under Trip primer sopB-2R, the nucleotide sequence of described sopB-1F as shown in SEQ ID NO.9, the nucleotides sequence of described sopB-1R Row as shown in SEQ ID NO.10, the nucleotide sequence of described sopB-2F as shown in SEQ ID NO.11, described sopB-2R's Nucleotide sequence is as shown in SEQ ID NO.12；
9) use PCR amplification method, with the genomic DNA of Salmonella enteritidis as template, with sopB-1F and sopB-1R be Primer amplification obtains the upstream homology arm sU of sopB, obtains the downstream homology of sopB with sopB-2F and sopB-2R for primer amplification Arm sD；
10) with step 9) sU and sD that obtain of amplification is as template, it is anti-that sopB-1F and sopB-2R is that primer carries out overlapping PCR Should, it is thus achieved that overlapping PCR primer sUD；
11) by step 10) overlapping PCR primer sUD that obtains is attached with pMD19-T carrier, will connect product and convert Competence bacillus coli DH 5 alpha, uses blue white screening and amicillin resistance screening to select positive colony, uses LB culture medium Cultivate the positive colony picked out, extract plasmid, it is thus achieved that restructuring pMD-Δ sopB plasmid；
12) with restricted enzyme Bgl II enzyme action suicide plasmid pYG4 respectively and restructuring pMD-Δ sopB plasmid, reclaim The 1917bp fragment obtained after the 5797bp fragment obtained after suicide plasmid pYG4 enzyme action and restructuring pMD-Δ sopB plasmid enzyme restriction, 5797bp fragment and the 1917bp fragment of acquisition are connected overnight in metal bath；
13) by step 12) connect after product heat-shock transformed escherichia coli S17-l/ λ pir competent cell, to convert after Cell cultivate, cultivation bacterium solution is coated on kalamycin resistance LB flat board, picking list bacterium colony, increase and extract matter after bacterium Grain, it is thus achieved that restructuring suicide plasmid pYG4-Δ sopB；
14) in the LB culture medium containing nalidixan, recipient bacterium step 7 is cultivated) enteritis knocking out rfaH gene that obtains is husky Door Salmonella strain, cultivates the donor bacterium large intestine containing restructuring suicide plasmid pYG4-Δ sopB in the LB culture medium containing kalamycin Bacillus S17-l/ λ pir, collects thalline resuspended respectively after cultivating described recipient bacterium and donor bacterium, by the donor bacterium after resuspended Mixing with recipient bacterium and be incorporated in the LB culture medium of antibiotic-free cultivation, bacterium solution cultivation obtained is coated containing kanamycin and naphthalene On the dual anti-flat board of pyridine acid, bacterial strain integrated by screening positive clone list, and list screening obtained integrates bacterial strain in the LB of antibiotic-free After culture medium is cultivated, screen with sucrose plate, obtain occurring the Salmonella bacterial strain of dyeing In vivo recombination；With sequence such as SopB-1 shown in SEQ ID NO.13 and sequence sopB-2 as shown in SEQ ID NO.14 is primer, uses PCR method pair The Salmonella bacterial strain that dyeing In vivo recombination occurs carries out Screening and Identification, selects PCR and amplifies the bacterial strain of 379bp, is lacked Lose rfaH gene and the dual-gene disappearance Salmonella bacteria strain of sopB gene.
A kind of Salmonella bacteria vaccine, its component includes the dual-gene disappearance Salmonella using said method to prepare Bacterium.
Compared to existing technology, present invention have the advantage that
1, by research, the present invention finds that by the RfaH albumen of rfaH gene code be a kind of transcriptional antitermination albumen, and it can To assist RNA polymerase to cross ρ independent form tanscription termination minor structure during Bacterial transcript, it is achieved leading to of downstream gene Read, thus eliminate the polarity of long transcripton.In escherichia coli, rfaH albumen is a kind of virulence modulin, and it is many that it affects fat The synthesis of sugar (LPS) layer, hemolysin and pod membrane etc.；In Salmonella typhimurium, this albumen also can affect LPS core space and The expression of 0 antigen and the expression of other virulence genes, rfaH gene delection Salmonella typhimurium, invade epithelial cell and antigen The ability of presenting cell is the most all strengthened, and in resisting host cell, the resistance of the material such as antibacterial peptide weakens, thus Having a net increase of in host cell and grow quantity relatively wild strain and be substantially reduced, loss causes the ability of systemic disease, but can effectively protect The attack of street strain.Also there is rfaH gene in Salmonella enteritidis, knocks out Salmonella enteritidis rfaH gene and equally realize LPS and the attenuating effects of other Salmonella virulence gene, can excite effective immunoprotection to react simultaneously, is to build SE epidemic disease Seedling Candidate Strain preferably knock out target gene.Sop albumen is another virulence factor of Salmonella, has myo-inositol phosphates phosphoric acid Enzymatic activity, after transferring to host cell, energy inducing cell cytoskeleton rearrangement, symptom of diarrhea can be caused.SopB mutant decapacitation strengthens Outside humoral and cellular immune response, moreover it is possible to strengthen mucosal immunity, additionally reduce liquid secretion thus alleviating diarrhoea.The present invention invents People, on the basis of the studies above finds, uses suicide plasmid homologous recombination system, sensitive by homologous recombination and sucrose The reverse triage techniques of gene, first constructs Salmonella enteritidis-Δ rfaH gene-deleted strain that rfaH gene (1-489) lacks (D315), on this basis, utilize again same principle and technology, knock out another relevant to Salmonella enteritidis virulence Gene sopB (1-1686), it is thus achieved that the Salmonella enteritidis Salmonella Δ rfaH-dual-gene deletion mutation of Δ sopB of inheritance stability Strain (D316).
2, the present invention knocks out dual-gene Salmonella enteritidis cellular immunization and mucosa-immune is strengthened, and SE belongs to intestinal Pathogen, the humoral immunization of local to the elimination efficiency of the protection of intestinal and thalline higher than general immunity, the recovery of gastroenteritis with It is the most relevant that intestinal local produces sIgA, therefore can strengthen mucosal immunity and will assist in the removing of SE；SE is intracellular bacterium simultaneously, by force Cellular immunization to eliminate SE infect will play a significant role；Through verification experimental verification, the present invention is used to knock out dual-gene enteritis Salmonella counteracting toxic substances protected effect effect is obvious.
3, the present invention knocks out dual-gene Salmonella enteritidis and is improved the safety of host and environment, because gained is dashed forward Mutant resistance in host cell weakens, and causes time-to-live and shedding virus all to greatly reduce, in vivo in 3～4 weeks just Can be gone by surely growing removing, security performance is excellent.
4, the present invention knocks out dual-gene Salmonella enteritidis and protects the immunological cross of other serotypes and be improved, former Cause is shortening of core LPS (O-LPS) chain of this dual-gene gene-deleted strain, enhances the immunoreation to other surface proteins, and These albumen are guarded in different Salmonellas relatively, thus can improve Cross immunogenicity, improve other serum The preventive effect of type Salmonella.
5, the present invention knocks out the double sudden change of dual-gene Salmonella enteritidis and can reduce and return main risk, safer；And this Bright knock out dual-gene Salmonella enteritidis rfaH gene mutation after mutant strain can become half rough type, and wild strain is light Slip, provides convenient for the natural infection and vaccine immunity strain distinguishing SE.
Accompanying drawing explanation
Fig. 1 be rfaH upstream and downstream homology arm rU the pcr amplified fragment gel electrophoresis figure of rD.(1, DL-5000Marker DNA Marker(DL-5000)；2, ddH2O negative control；3, rU fragments；4, rD fragments)
Fig. 2 be rU rD overlap pcr amplified fragment rUD gel electrophoresis figure.(1, DL-5000Marker；2rU D fragment)
Fig. 3 is deletion mycopremna structure figure.
Fig. 4 is the PCR qualification figure of disappearance rfaH gene bacterial strain.(1, DL-5000Marker；2 deletion mycopremnas；3, parent bacterium Strain)
Fig. 5 be sopB upstream and downstream homology arm sU the pcr amplified fragment gel electrophoresis figure of sD.(1, DL-5000Marker DNA Marker(DL-5000)；2, sU fragments；3, sD sheets；4, ddH2O negative control section)
Fig. 6 be sU sD overlap pcr amplified fragment sUD gel electrophoresis figure.(1, DL-5000Marker；2sUD fragment)
Fig. 7 is disappearance rfaH gene and the PCR qualification figure of sopB gene bacterial strain.(1, DL-5000Marker；2 disappearance bacterium Strain；3, parent strain)
Detailed description of the invention
Below in conjunction with specific embodiment and Figure of description, the present invention is described in further detail.The implementation case with Implement under premised on the technology of the present invention, now provide detailed embodiment and concrete operating process to illustrate that the present invention has Creative, but protection scope of the present invention is not limited to below example.
The Salmonella enteritidis D1203 used in following embodiment is (by Chongqing University of Technology's pharmacy and Biological Engineering College Central laboratory separates, identifies and preserve)；Following step is to knock out rfaH gene and the concrete steps of sopB gene, described The nucleotide sequence of rfaH gene as shown in SEQ ID NO.1, the nucleotide sequence such as SEQ ID NO.2 institute of described sopB gene Show
The propagation of embodiment 1 Salmonella enteritidis and the extraction of genome
Salmonella enteritidis D1203 is inoculated in LB liquid medium, 37 DEG C of overnight incubation, takes 1.5mL and cultivate bacterium solution, put Being placed in Eppendorf centrifuge tube, 10000g is centrifuged 2min, abandons supernatant, adds the resuspended precipitation of 1.5mLPBS buffer, 10000g is centrifuged 2min, abandons supernatant, adds the 240 resuspended thalline of μ LTE, and add 15 μ L lysozyme, 0 DEG C of ice bath in precipitation 30min, adds mass concentration 10%SDS 15 μ L, adds E.C. 3.4.21.64,65 DEG C of water-bath 30min, add 5M after mix homogeneously NaCl solution 200 μ L, concussion, add 500 μ L phenol/chloroform/isoamyl alcohol (volume ratio 1:24:25) solution, mix homogeneously, 12000g Centrifugal 10min, takes supernatant in centrifuge tube, records its volume, add isopyknic freezing dehydrated alcohol, be positioned over-80 DEG C Refrigerator 10min, 12000g are centrifuged 10min, abandon supernatant, natural air drying, add 30 μ L ddH2O and dissolve, place-20 DEG C of refrigerators Preserve, for the template of PCR reaction.
The structure of embodiment 2rfaH gene knockout mutant strain
2.1 overlap PCR
According to the Salmonella enteritidis genome sequence delivered on GeneBank, design the upstream homology arm of rfaH gene The forward primer rfaH-1F and downstream primer rfaH-1R of sequence rU, the forward primer of downstream homology arm sequence rD of rfaH gene RfaH-2F and downstream rfaH-2R, the nucleotide sequence of described rfaH-1F as shown in SEQ ID NO.3, the core of described rfaH-1R Nucleotide sequence as shown in SEQ ID NO.4, the nucleotide sequence of described rfaH-2F as shown in SEQ ID NO.5, described rfaH- The nucleotide sequence of 2F is as shown in SEQ ID NO.6.With the Salmonella enteritidis D1203 genomic DNA of said extracted as template, PCR method amplification rfaH upstream and downstream homology arm sequence rU, rD.The 50 μ LPCR amplification system such as tables 1 of rU, rD:
Table 1PCR amplification system
PCR reaction condition: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 55 DEG C of annealing 40s, 72 DEG C extend 1min, and totally 25 Individual circulation；Last 72 DEG C extend 5min.
Upstream and downstream homology arm amplified production rU and rD obtained is expanded by using above-mentioned PCR reaction system and reaction condition Detect through 1.0% agarose gel, seen from result, obtain two bands (Fig. 1) being consistent with expection size (919bP and 933bp), Cutting two bands respectively, the operation instruction reclaiming test kit according to the gel of Omega company carries out reclaiming purification.
Due to rfaH-1R, rfaH-2F phase mutual partial complementarity sequence, mix rU and rD after purification, and with it as mould Plate, rfaH-1F/rfaH-2R is that primer carries out overlapping PCR.Overlapping PCR 50 μ LPCR amplification system is the same, PCR reaction condition: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 55 DEG C of annealing 45s, 72 DEG C extend 2min, totally 25 circulations；Last 72 DEG C of extensions 5min。
The structure of 2.2 intermediate transfer plasmid pMD-Δ rfaH
Overlapping PCR primer detects with 1.0% agarose gel, it is seen that about 2000bP band rUD (Fig. 2), cuts this band, The operation instruction reclaiming test kit according to the glue of Omega company carries out reclaiming purification, obtains overlapping PCR primer rUD.Mesh will be reclaimed Fragment rUD be connected with pMD19-T carrier.Linked system (10 μ L): 10 × ligase buffer 1 μ L, T4DNA ligase 1 μ L, PMD19-T carrier 1 μ L, rUD 5 μ L, sterilizing ddH2O 2 μ L, 16 DEG C of metal baths are overnight.
By connection product transformed competence colibacillus bacillus coli DH 5 alpha obtained above, select (based on blue white macula and ammonia benzyl penicillium sp Element resistance) positive colony, and in LB culture medium culturing positive colony, extract plasmid, carry out enzyme action, PCR qualification and sequence and survey Fixed.Correct recombiant plasmid named pMD-Δ rfaH will be measured.
Recombinant plasmid transformed bacillus coli DH 5 alpha specifically comprises the following steps that
Take bacillus coli DH 5 alpha competent cell, be placed in thawed on ice, draw restructuring and connect product 10 μ L in having melted In bacillus coli DH 5 alpha competent cell, ice bath 30min after mixing, 42 DEG C of heat shock 90s, ice bath 3min, add 1ml LB liquid Culture medium, 37 DEG C of shaking tables are cultivated 1h, 10000g and are centrifuged 2min, abandon supernatant, add the 100 μ resuspended thalline of L LB, by bacterium in precipitation Liquid coats 100 μ g/mL amicillin resistance LB flat boards, 37 DEG C of overnight incubation.
The structure of 2.3 restructuring suicide plasmid pYG4-Δ rfaH
Extract suicide plasmid pYG4 and recombiant plasmid pMD-Δ rfaH in a small amount, respectively with restricted enzyme Bgl II enzyme action, Enzyme action system (50 μ L): 10 × H Buffer buffer 5 μ L, plasmid (pMD-Δ rfaH or PYG4) 30 μ L, Bgl II 2 μ L, sterilizing ddH2O 13 μ L, 37 DEG C of water-bath enzyme action 2h.Digestion products becomes 1 fragment through 1% agarose gel electrophoresis, plasmid pYG4 enzyme action (5797bp), plasmid pMD-Δ rfaH enzyme action becomes 2 fragments (2707bp and 1885bp).DNA purification kit reclaims purification PYG4 fragment and the 1885bp fragment of pMD-Δ rfaH, above-mentioned two purified fragments connect overnight at 6 DEG C of metal baths.Metal bath connects System (10 μ l): 10 × ligase buffer 1 μ L, T4DNA ligase 1 μ L, pYG4 fragment 4 μ L, Δ rfaH fragment 3 μ L, sterilizing ddH2O 1μL。
Metal bath is connected product heat-shock transformed escherichia coli S17-l/ λ pir competent cell, is coated with kalamycin (100 μ g/ml) LB flat board, picking list bacterium colony, extracts plasmid after increasing bacterium, carries out enzyme action, PCR qualification and sequencing.To identify correct Restructuring suicide plasmid named pYG4-Δ rfaH.
Heat-shock transformed competent cell escherichia coli S17-l/ λ pir specifically comprises the following steps that
Take escherichia coli S17-l/ λ pir competent cell, be placed in thawed on ice, draw restructuring and connect product 20 μ L in In the escherichia coli S17-l/ λ pir competent cell melted, ice bath 30min after mixing, 42 DEG C of heat shock 90s, ice bath 3min, add Entering 1ml LB fluid medium, 37 DEG C of shaking tables are cultivated 1h, 10000g and are centrifuged 2min, abandon supernatant, add 100 μ L LB in precipitation Resuspended thalline, coats 100 μ g/mL kalamycin resistance LB flat boards, 37 DEG C of overnight incubation by bacterium solution.
2.4 engage transfer and the screening of the mono-gene-deleted strain of Salmonella enteritidis Δ rfaH
Structure route such as Fig. 3 of Δ rfaH gene-deleted strain, filters out gene mutation strain by two step method.Concrete steps: containing Have in the LB culture medium of how heavy stone used as an anchor acid (NaL, 40 μ g/ml) and cultivate recipient bacterium Salmonella enteritidis D1203 (NalR), containing OK a karaoke club The LB culture medium of mycin (Kan, 100 μ g/mL) is cultivated the escherichia coli S17-l/ λ pir containing pYG4-Δ rfaH suicide plasmid supply Body bacterium, collects thalline, the resuspended thalline of LB culture medium respectively after 37 DEG C of overnight incubation, respectively take 100 μ L bacterial suspension mixing, in nonreactive The LB plate overnight of raw element is cultivated, and collects thalline, after serial dilution, take appropriate bacterium solution coat containing kanamycin (Kan, 100 μ g/mL) and the dual anti-flat board of nalidixan (NaL, 40 μ g/ml) on, screening positive clone list integrate bacterial strain.
The bacterial strain of integrating obtained is placed in overnight incubation in the LB culture medium of antibiotic-free, in 5% sucrose plate after cultivation There is the bacterial strain of dyeing In vivo recombination in screening.Due to the Negative selection effect of sacB fragment, with sacB fragment integration strain without Method grows on the flat board containing 5% sucrose is carried out, and longer bacterium colony is all to there occurs dyeing In vivo recombination, thus removes The recombinant bacterial strain of carrier sequence (including sacB fragment).Restructuring has two kinds of situations, and wherein the probability of 50% produces and knocks out strain, The probability of 50% produces wild type strain.According to the Salmonella enteritidis genome sequence delivered on GeneBank, design one To primer rfaH-1/rfaH-2, rfaH-1 sequence as shown in SEQ ID NO.7, rfaH-2 sequence as shown in SEQ ID NO.8, Strain is knocked out by PCR mode Screening and Identification.PCR reaction system is the same, PCR reaction condition: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 55 DEG C of annealing 40s, 72 DEG C extend 1.5min, totally 25 circulations；Last 72 DEG C extend 5min.PCR reaction terminates, and 1.0% Agarose gel detects, and such as Fig. 4, knocks out strain and amplifies a band, be positioned at 631bp, and wild strain amplifies a band, position In about 1120bp.The pcr amplification product knocking out strain is delivered to Dalian Bao Shenggong biological engineering company limited and carries out sequencing. Sequencing result shows that successful knockout falls rfaH gene 489bp sequence, by named for this knock-out bacterial strain D315.
The structure of the double gene-deleted strain of embodiment 3 Salmonella enteritidis Δ rfaH Δ sopB
3.1 overlap PCR
According to the Salmonella enteritidis genome sequence delivered on GeneBank, design the upstream homology arm of sopB gene The forward primer sopB-1F and downstream primer sopB-1R of sequence sU, the forward primer of downstream homology arm sequence sD of sopB gene SopB-2F and downstream primer sopB-2R, the nucleotide sequence of described sopB-1F as shown in SEQ ID NO.9, described sopB-1R Nucleotide sequence as shown in SEQ ID NO.10, the nucleotide sequence of described sopB-2F is as shown in SEQ ID NO.11, described The nucleotide sequence of sopB-2R is as shown in SEQ ID NO.12.With Salmonella enteritidis D1203 genomic DNA as template, PCR side Method amplification sopB upstream and downstream homology arm sequence sU, sD.The 50 μ L PCR amplification system of sU, sD and PCR reaction condition are with rU and rD Amplification.
Upstream and downstream homology arm amplified production sU, sD of using said method amplification to obtain are examined through 1.0% agarose gel Surveying, obtain seen from result being consistent with expection size (920bP and 964bp) band (Fig. 5), cuts two bands respectively, according to Omega The gel of company reclaims the operation instruction of test kit to carry out reclaiming purification.
Due to sopB-1R, sopB-2F phase mutual partial complementarity sequence, mix sU and sD after purification, and with it as mould Plate, sopB-1F/sopB-2R is that primer carries out overlapping PCR.Overlapping PCR 50 μ L amplification system is the same.PCR reaction condition: 94 DEG C Denaturation 5min；94 DEG C of degeneration 30s, 55 DEG C of annealing 45s, 72 DEG C extend 2min, totally 25 circulations；Last 72 DEG C extend 5min, Obtain overlapping PCR product sUD.
The structure of 3.2 intermediate transfer plasmid pMD-Δ sopB
Overlapping PCR primer detects through 1.0% agarose gel electrophoresis, it is seen that obtain being consistent with expection size (1917bP) bar Band sUD (Fig. 6), cuts this band, and the operation instruction reclaiming test kit according to the glue of Omega company carries out reclaiming purification.To back Receive purpose fragment sUD to be connected with pMD19-T carrier, linked system (10 μ L): 10 × ligase buffer 1 μ L, T4DNA ligase 1 μ L, pMD19-T carrier 1 μ L, rUD 5 μ L, sterilizing ddH2O 2 μ L, 16 DEG C of metal baths are overnight.
Product transformed competence colibacillus bacillus coli DH 5 alpha will be connected, select (based on blue white macula and amicillin resistance) positive Clone, and in LB culture medium culturing positive colony, extract plasmid, carry out enzyme action, PCR qualification and sequencing.To measure correct Recombiant plasmid named pMD-Δ sopB.
The structure of 3.3 restructuring suicide plasmid pYG4-Δ sopB
Extract suicide plasmid pYG4 and recombiant plasmid pMD-Δ sopB in a small amount, respectively with restricted enzyme Bgl II enzyme action, Enzyme action system (50 μ L): 10 × H Buffer buffer 5 μ L, plasmid (pMD-Δ rfaH or PYG4) 30 μ L, Bgl 2 μ L, sterilizing ddH2O 13 μ L, 37 DEG C of water-bath enzyme action 2h.Digestion products becomes 1 fragment through 1% agarose gel electrophoresis, plasmid pYG4 enzyme action (5797bp), plasmid pMD-Δ rfaH enzyme action becomes 2 fragments (2707bp and 1917bp), and DNA purification kit reclaims purification Above-mentioned two purified fragments reclaimed are connected overnight by pYG4 fragment and the 1917bp fragment of pMD-Δ rfaH at 6 DEG C of metal baths.Even Junctor system (10ul): 10 × ligase buffer 1 μ L, T4DNA ligase 1 μ L, pYG4 fragment 4 μ L, Δ sopB fragment 3 μ L, goes out Bacterium ddH2O 1μL。
Metal bath is connected product heat-shock transformed escherichia coli S17-l/ λ pir competent cell, is coated with kalamycin (100 μ g/ml) LB flat board, picking list bacterium colony, extracts plasmid after increasing bacterium, carries out enzyme action, PCR qualification and sequencing.To identify correct Restructuring suicide plasmid named pYG4-Δ sopB.
3.4 screenings engaging transfer and the double gene-deleted strain of Salmonella enteritidis Δ rfaH Δ sopB
With the escherichia coli S17-l/ λ pir containing pYG4-Δ sopB suicide plasmid for donor bacterium, obtained in aforementioned 2.4 The bacterial strain D315 of disappearance rfaH gene is that recipient bacterium carries out engaging transfer.Donor bacterium and recipient bacterium are inoculated in containing OK a karaoke club mould respectively In the LB culture medium of element (Kan, 100 μ g/mL) and how heavy stone used as an anchor acid (NaL, 40 μ g/ml), collection thalline after 37 DEG C of overnight incubation, and use The resuspended thalline of LB culture medium, respectively takes 100 μ L bacterial suspension mixing, and the LB plate overnight in antibiotic-free is cultivated.Collect thalline After serial dilution, take that appropriate bacterium solution coats containing kanamycin (Kan, 100 μ g/mL) and nalidixan (NaL, 40 μ g/ml) is double On anti-flat board, bacterial strain integrated by screening positive clone list.The flat board that integration bacterial strain is placed in containing 5% sucrose is screened, with The integration strain of sacB fragment cannot grow on this flat board, and longer bacterium colony is all to there occurs dyeing In vivo recombination, thus Eliminate the recombinant bacterial strain of carrier sequence (including sacB fragment).
Restructuring has two kinds of situations, and wherein the probability of 50% produces and knocks out strain, and the probability of 50% produces wild type strain. According to the Salmonella enteritidis genome sequence delivered on GeneBank, design pair of primers sopB-1/sopB-2, sopB-1's Sequence is as shown in SEQ ID NO.13, and sopB-2 sequence, as shown in SEQ ID NO.14, is knocked out by PCR mode Screening and Identification Strain.PCR reaction system is the same, PCR reaction condition: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 55 DEG C of annealing 40s, 72 DEG C are prolonged Stretch 2.5min, totally 25 circulations；Last 72 DEG C extend 5min.PCR reaction terminates, 1.0% agarose gel detection, such as Fig. 7, strikes Except strain amplifies a band, being positioned at about 379bp, wild strain amplifies a band, is positioned at about 2065bp.Strain will be knocked out Pcr amplification product deliver to Dalian Bao Shenggong biological engineering company limited and carry out sequencing, sequencing result shows and successfully strikes Remove the 1686bp sequence of sopB gene, by the named D316 of this dual-gene knock-out bacterial strain.
Embodiment 4 antibacterial grows removing the most surely
Dual-gene knock out mutant strain D316 and wild strain inoculates LB fluid medium, 37 DEG C of shaken cultivation mistakes respectively At night, 2% inoculation fresh LB, bacterium solution, to OD600=0.7, is centrifuged 5min in 10000g, is resuspended in by 37 DEG C of shaken cultivation Sterilizing PBS, is adjusted to 10 by bacterial concentration8CFU/mL。
1 age in days Hai Lanbai chickling is randomly divided into 3 groups, often group 20.Wherein 1 group is administered orally 1 × 107CFU/0.1mL mutant bacteria Strain D316,1 group is administered orally 1 × 107CFU/0.1mL wild strain, 1 group is administered orally 0.1mLPBS as a control group.After immunity 1 In week, within 2 weeks, 3 weeks, 4 weeks, cut open at random and kill each immune group and each 5 of matched group chicken, aseptic take liver, spleen and caecum, weigh, nothing Bacterium PBS divides 2 parts after grinding, and is wherein coated with salmonella color culture medium flat board after 1 part of suitable dilution, counts after 37 DEG C of cultivations； After another part does Zengjing Granule, being coated with salmonella color culture medium flat board, PCR determines.
Antibacterial the most surely grows removing situation and is shown in Table 2.Postvaccinal 1st, 2,3,4 weeks, matched group chicken liver, spleen and blind Intestinal does not all detect Salmonella, and mutant bacteria is the most finally completely removed in liver, spleen and caecum, wild strain liver, Quantity in spleen and caecum is significantly higher than matched group, and during 4th week, host still can not be fully erased.
Bacterial strain distribution (mean ± SEM log10cfu/g) of table 2 each internal organs after inoculating D316
Embodiment 5 immune protective
40 female 1 Hai Lanbai chickling, are randomly divided into 2 groups, often group 20.Wherein 1 group in 1 age in days and 5 week old time-divisions It is not administered orally 1 × 107CFU/0.1mL mutant strain D316, another 1 group is administered orally 0.1mLPBS as right respectively when 1 age in days and 5 week old According to group.When 9 week old, two groups of chickens are all administered orally 1 × 109CFU/0.2mL wild strain carries out counteracting toxic substances.After counteracting toxic substances 1 week, 2 In week, cuing open and kill each immune group and each 10 of matched group chicken at random, aseptic take liver, spleen and caecum, weigh, aseptic PBS grinds Rear point 2 parts, wherein it is coated with salmonella color culture medium flat board after 1 part of suitable dilution, counts after 37 DEG C of cultivations；Another part is done and is increased After bacterium is cultivated, being coated with salmonella color culture medium flat board, PCR determines.
Protective rate and counteracting toxic substances bacterial strain are the most surely grown removing situation and are shown in Table 3.Postvaccinal 1st, 2 weeks, internal organs in immune group Official carries the chicken number of counteracting toxic substances bacterial strain and is below matched group, can reduce carrying disease germs of chicken Salmonella enteritidis after gene-deleted strain immunity is described Rate.Meanwhile, immune group is provided that also below matched group, the gene-deleted strain constructed by explanation at the content of molds of liver, spleen and caecum Good immune protective effect.
Bacterial strain distribution (mean ± SEM log10cfu/g) of table 3 counteracting toxic substances protective rate and each internal organs
Embodiment 6 Salmonella bacteria vaccine
According to above-described embodiment the result, the present invention knocks out dual-gene disappearance Salmonella enteritidis and has exempting from of excellence Epidemic disease protected effect, and can surely grow in vivo and dispose, can use as vaccine, further, the present embodiment provides a kind of intestinal Scorching Salmonella oral vaccine, its component includes that the present invention knocks out the dual-gene disappearance Salmonella of rfaH gene and sopB gene Bacterium, described dual-gene disappearance Salmonella concentration in described oral vaccine can be 1 × 107CFU/0.1mL。
Finally illustrating, above example is only in order to illustrate technical scheme and unrestricted, although with reference to relatively The present invention has been described in detail by good embodiment, it will be understood by those within the art that, can be to the skill of the present invention Art scheme is modified or equivalent, and without deviating from objective and the scope of technical solution of the present invention, it all should be contained at this In the middle of the right of invention.
<110>Chongqing University of Technology；
<120>one is dual-gene lacks Salmonella enteritidis, its construction method and contains this dual-gene disappearance Salmonella The vaccine of bacterium；
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 489
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of rfaH gene
<400> 1
atgcaatcct ggtatttact gtactgcaaa cgcgggcaac ttcagcgtgc tcaggaacac 60
ctcgaaagac aagcggtaag ttgcctgaca ccgatgatca ccctggaaaa aatggtacgc 120
ggaaaacgta cctccgtcag cgaaccgctc tttcctaatt atctgttcgt tgaatttgat 180
ccggaagtga tacataccac tacaatcaac gccacgcgcg gcgtcagcca ttttgtgcgc 240
tttggcgcgc atcctgcgat cgtgccttcc agcgttattc atcagctttc tatctacaag 300
cccgaaggcg ttgtcgatcc tgaaaccccc tatcccggcg atagcgtcat catcacggaa 360
ggcgcatttg aagggctgaa agcgattttt accgaaccgg atggcgaaac gcgttcgatg 420
ttactgctta atttactcaa taaagaagtg aagcagagcg taaaaaacac cggttttcgc 480
aagatttag 489
<210> 2
<211> 1686
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of sopB gene
<400> 2
atgcaaatac agagcttcta tcactcagct tcactaaaaa cccaggaggc ttttaaaagc 60
ctacaaaaaa ccttatacaa cggaatgcag attctctcag gccagggcaa agcgccggct 120
aaagcgcccg acgctcgccc ggaaattatt gtcctgcgag aacctggcgc gacatggggg 180
aattatctac agcatcagaa gacgtctaac cactcgctgc ataacctcta taacttacag 240
cgcgatcttc ttaccgtcgc ggcaaccgtt ctgggtaaac aagacccggt tctaacgtca 300
atggcaaacc aaatggagtt agccaaagtt aaagcggacc ggccagcaac aaaacaagaa 360
gaagccgcgg caaaagcatt gaagaaaaat cttatcgaac ttattgcagc acgcactcag 420
cagcaggatg gcttacctgc aaaagaagct catcgctttg cggcagtagc gtttagagat 480
gctcaggtca agcagcttaa taaccagccc tggcaaacca taaaaaatac actcacgcat 540
aacgggcatc actataccaa cacgcagctc cctgcagcag agatgaaaat cggcgcaaaa 600
gatatctttc ccagtgctta tgagggaaag ggcgtatgca gttgggatac caagaatatt 660
catcacgcca ataatttgtg gatgtccacg gtgagtgtgc atgaggacgg taaagataaa 720
acgctttttt gcgggatacg tcatggcgtg ctttccccct atcatgaaaa agatccgctt 780
ctgcgtcacg tcggcgctga aaacaaagcc aaagaagtat taactgcggc actttttagt 840
aaacctgagt tgcttaacaa agccttagcg ggcgaggcgg taagcctgaa actggtatcc 900
gtcgggttac tcaccgcgtc gaatattttc ggcaaagagg gaacgatggt cgaggaccaa 960
atgcgcgcat ggcaatcgtt gacccagccg ggaaaaatga ttcatttaaa aatccgcaat 1020
aaagatggcg atctacagac ggtaaaaata aaaccggacg tcgccgcatt taatgtgggt 1080
gttaatgagc tggcgctcaa gctcggcttt ggccttaagg catcggatag ctataatgcc 1140
gaggcgctac atcagttatt aggcaatgat ttacgccctg aagccagacc aggtggctgg 1200
gttggcgaat ggctggcgca atacccggat aattatgagg tcgtcaatac attagcgcgc 1260
cagattaagg atatatggaa aaataaccaa catcataaag atggcggcga accctataaa 1320
ctcgcacaac gccttgccat gttagcccat gaaattgacg cggtacccgc ctggaattgt 1380
aaaagcggca aagatcgtac aggaatgatg gattcagaaa tcaagcgaga gcacatttct 1440
ttacatcaga cccatatgtt aagtgcgcct ggtagtcttc cggatagcgg tggacagaaa 1500
attttccaaa aagtattact gaatagcggt aacctggaga ttcagaaaca aaatacgggc 1560
ggggcgggaa acaaagtaat gaaaaattta tcgccagagg tgctcaatct ttcctatcaa 1620
aaacgagttg gggatgaaaa tatttggcag tcagtaaaag gcatttcttc attaatcaca 1680
tcttga 1686
<210> 3
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<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of rfaH-1F
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gaagatctgc aaaggcatac tccgacagag ta 32
<210> 4
<211> 57
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of rfaH-1R
<400> 4
gttataaatt tggagtgtga aggttattgc gtgaatgact cttatccgct tgttcgg 57
<210> 5
<211> 59
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of rfaH-2F
<400> 5
cacgcaataa ccttcacact ccaaatttat aacctatcgt tcagaatacg acctcaaat 59
<210> 6
<211> 32
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of rfaH-2R
<400> 6
gaagatctga aattggcgtt ttctgctcac gc 32
<210> 7
<211> 21
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of rfaH-1
<400> 7
ttgcacagca ccggcatggc g 21
<210> 8
<211> 22
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of rfaH-2
<400> 8
aacgacgcgg gcttgagcta cg 22
<210> 9
<211> 32
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of sopB-1F
<400> 9
gaagatctgc agcagtataa gatggagcag ag 32
<210> 10
<211> 59
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of sopB-1R
<400> 10
gttataaatt tggagtgtga aggttattgc gtgagcgttt ttaatattcc tgaataggg 59
<210> 11
<211> 59
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of sopB-2F
<400> 11
cacgcaataa ccttcacact ccaaatttat aacgtcttga ggtaactata tggaaagtc 59
<210> 12
<211> 30
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of sopB-2R
<400> 12
gaagatctgt accggccgca tgcaaatatc 30
<210> 13
<211> 24
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of sopB-1
<400> 13
atgctgcaaa gtcaggatgt cgtc 24
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<211> 23
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of sopB-2
<400> 14
tgtaccgatc tcccccatga tcg 23

Claims (7)

1. a dual-gene disappearance Salmonella enteritidis, it is characterised in that forrfaH gene andsopThe intestinal that 1 B gene is all knocked Scorching Salmonella.
The most dual-gene disappearance Salmonella enteritidis, it is characterised in that describedrfaThe nucleotide of H gene Sequence is as shown in SEQ ID NO.1, describedsopThe nucleotide sequence of 1 B gene is as shown in SEQ ID NO.2.
3. the construction method of a dual-gene disappearance Salmonella enteritidis, it is characterised in that use methods of homologous recombination and sucrose The negative screening technique of sensitive gene is in described Salmonella enteritidisrfaH gene andsop1 B gene knocks out.
The most dual-gene disappearance Salmonella enteritidis construction method, it is characterised in that specifically include as Lower step:
1) according to the genome sequence of Salmonella enteritidis, designrfaThe forward primer of upstream homology arm sequence rU of H generfaH-1F and downstream primerrfaH-1R,rfaThe forward primer of downstream homology arm sequence rD of H generfaH-2F and downstream are drawn ThingrfaH-2R, describedrfaThe nucleotide sequence of H-1F is as shown in SEQ ID NO.3, describedrfaThe nucleotide sequence of H-1R is such as Shown in SEQ ID NO.4, describedrfaThe nucleotide sequence of H-2F is as shown in SEQ ID NO.5, describedrfaThe nucleotide of H-2R Sequence is as shown in SEQ ID NO.6；
2) PCR amplification method is used, with the genomic DNA of Salmonella enteritidis as template, withrfaH-1F andrfaH-1R is primer Amplification obtainsrfaThe upstream homology arm rU of H, withrfaH-2F andrfaH-2R is that primer amplification obtainsrfaThe downstream homology arm of H rD；
3) with step 2) rU and rD that obtain of amplification as template, rfaH-1F andrfaH-2R is primer, carries out overlapping PCR reaction, Obtain overlapping PCR primer rUD；
4) overlapping PCR primer rUD step 3) obtained is attached with pMD19-T carrier, will connect product transformed competence colibacillus Bacillus coli DH 5 alpha, uses blue white screening and amicillin resistance screening to select positive colony, uses LB culture medium culturing to choose The positive colony selected, extracts plasmid, it is thus achieved that restructuring pMD-△ rfaH plasmid；
5) with restricted enzyme Bgl II enzyme action suicide plasmid pYG4 respectively and restructuring pMD-△ rfaH plasmid, suicide matter is reclaimed The 1885bp fragment obtained after the 5797bp fragment obtained after grain pYG4 enzyme action and restructuring pMD-△ rfaH plasmid enzyme restriction, will obtain 5797bp fragment and 1885bp fragment connect overnight in metal bath；
6) by step 5) connect after product heat-shock transformed escherichia coli S17-l/ λ pir competent cell, to convert after thin Born of the same parents cultivate, and cultivation bacterium solution are coated on kalamycin resistance LB flat board, picking list bacterium colony, extract plasmid, obtain after increasing bacterium Must recombinate suicide plasmid pYG4-ΔrfaH；
7) in the LB culture medium containing nalidixan, recipient bacterium Salmonella enteritidis is cultivated, in the LB culture medium containing kalamycin Middle cultivation donor bacterium is containing restructuring suicide plasmid pYG4-ΔrfaThe escherichia coli S17-l/ λ pir of H, to described recipient bacterium and donor Bacterium collects thalline resuspended respectively after cultivating, the donor bacterium after resuspended and recipient bacterium mix the LB culture medium being incorporated in antibiotic-free Upper cultivation, bacterium solution cultivation obtained coats on the dual anti-flat board containing kanamycin and nalidixan, and screening positive clone list is whole Close bacterial strain, after list screening obtained integration bacterial strain is cultivated in the LB culture medium of antibiotic-free, screen with sucrose plate, obtain There is the Salmonella bacterial strain of dyeing In vivo recombination；With sequence as shown in SEQ ID NO.7rfaH-1 and sequence such as SEQ Shown in ID NO.8rfaH-2 is primer, uses PCR method to sieve the Salmonella bacterial strain that dyeing In vivo recombination occurs Choosing is identified, selects PCR and amplifies the bacterial strain of 631bp, is knocked outrfaThe Salmonella bacterial strain of H gene；
8) according to the genome sequence of Salmonella enteritidis, designsopThe forward primer of upstream homology arm sequence sU of 1 B genesopB-1F and downstream primersopB-1R,sopThe forward primer of downstream homology arm sequence sD of 1 B genesopB-2F and downstream are drawn ThingsopB-2R, describedsopThe nucleotide sequence of B-1F is as shown in SEQ ID NO.9, describedsopThe nucleotide sequence of B-1R is such as Shown in SEQ ID NO.10, describedsopThe nucleotide sequence of B-2F is as shown in SEQ ID NO.11, describedsopThe nucleoside of B-2R Acid sequence is as shown in SEQ ID NO.12；
9) PCR amplification method is used, with the genomic DNA of Salmonella enteritidis as template, withsopB-1F andsopB-1R is primer Amplification obtainssopThe upstream homology arm sU of B, withsopB-2F andsopB-2R is that primer amplification obtainssopThe downstream homology arm of B sD；
10) with step 9) sU and sD that obtain of amplification as template,sopB-1F andsopB-2R is that primer carries out overlapping PCR reaction, Obtain overlapping PCR primer sUD；
11) overlapping PCR primer sUD step 10) obtained is attached with pMD19-T carrier, will connect product and convert impression State bacillus coli DH 5 alpha, uses blue white screening and amicillin resistance screening to select positive colony, uses LB culture medium culturing The positive colony picked out, extracts plasmid, it is thus achieved that restructuring pMD-△sopB plasmid；
12) with restricted enzyme Bgl II enzyme action suicide plasmid pYG4 respectively and restructuring pMD-△sopB plasmid, reclaims and commits suiside The 5797bp fragment obtained after plasmid pYG4 enzyme action and restructuring pMD-△sopThe 1917bp fragment obtained after B plasmid enzyme restriction, will obtain The 5797bp fragment and the 1917bp fragment that obtain connect overnight in metal bath；
13) the product heat-shock transformed escherichia coli S17-l/ λ pir competent cell after step 12) being connected, after converting Cell is cultivated, and cultivation bacterium solution is coated on kalamycin resistance LB flat board, picking list bacterium colony, extracts plasmid after increasing bacterium, Obtain restructuring suicide plasmid pYG4-ΔsopB；
14) in the LB culture medium containing nalidixan, obtain knock out of recipient bacterium step 7) is cultivatedrfaThe Salmonella of H gene Bacterial strain, cultivates donor bacterium containing restructuring suicide plasmid pYG4-Δ in the LB culture medium containing kalamycinsopThe escherichia coli of B S17-l/ λ pir, collects thalline resuspended respectively after cultivating described recipient bacterium and donor bacterium, by the donor bacterium after resuspended be subject to Body bacterium mixes and is incorporated in the LB culture medium of antibiotic-free cultivation, and bacterium solution cultivation obtained is coated containing kanamycin and nalidixan Dual anti-flat board on, screening positive clone list integrate bacterial strain, the list that screening is obtained integrate bacterial strain in antibiotic-free LB cultivate After base is cultivated, screen with sucrose plate, obtain occurring the Salmonella bacterial strain of dyeing In vivo recombination；With sequence such as SEQ ID Shown in NO.13sopB-1 and sequence are as shown in SEQ ID NO.14sopB-2 is primer, uses PCR method to dyeing The Salmonella bacterial strain of In vivo recombination carries out Screening and Identification, selects PCR and amplifies the bacterial strain of 379bp, is lackedrfaH base Cause andsopThe dual-gene disappearance Salmonella bacteria strain of 1 B gene.
5. a Salmonella bacteria vaccine, it is characterised in that its component includes using claim 3 or 4 either method to prepare Dual-gene disappearance Salmonella enteritidis.
Salmonella bacteria vaccine the most according to claim 5, it is characterised in that described vaccine is oral vaccine.
Salmonella bacteria vaccine the most according to claim 5, it is characterised in that described dual-gene disappearance Salmonella enteritidis Concentration in described vaccine is 1 × 107 CFU/0.1mL。
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CN106754594A (en) * 2016-12-12 2017-05-31 扬州大学 A kind of Salmonella choleraesuls attenuated carrier bacterium and its construction method
CN109517776A (en) * 2018-11-16 2019-03-26 河北科技师范学院 A kind of Salmonella enteritidis icdA gene-deleted strain and its application
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