CN106190943B - It is a kind of it is dual-gene missing Bacterium enteritidis, its construction method and contain this it is dual-gene missing Bacterium enteritidis vaccine - Google Patents
It is a kind of it is dual-gene missing Bacterium enteritidis, its construction method and contain this it is dual-gene missing Bacterium enteritidis vaccine Download PDFInfo
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Abstract
The present invention discloses the dual-gene missing Bacterium enteritidis of one kind, its construction method and the vaccine containing the dual-gene missing Bacterium enteritidis, the dual-gene missing Bacterium enteritidisrfaH gene andsopThe Bacterium enteritidis that 1 B gene is all knocked, the construction method is using the negative selection method of methods of homologous recombination and sucrose sensitive gene in the Bacterium enteritidisrfaH gene andsop1 B gene is knocked out, and the component of the vaccine includes using dual-gene missing Bacterium enteritidis made from the above method.The dual-gene missing Bacterium enteritidis cellular immunity of the present invention and mucosa-immune are enhanced, and attack that malicious protecting effect is good, and immune effect is excellent, and elimination effect is good in host, can be colonized removing in 3 ~ 4 weeks in vivo and gone, security performance is excellent.
Description
Technical field
The invention belongs to gene Knockout fields, and in particular to the dual-gene missing Bacterium enteritidis of one kind, its building
Method and contain this it is dual-gene missing Bacterium enteritidis vaccine.
Background technique
Bacterium enteritidis is a kind of important pathogen of Zoonosis, infect salmonella poultry be salmonella most
For common repository, the mankind are often as taking in contaminated eggs or not well-done poultry and infecting detection of Salmonella
's.In world wide, there are generation and number in food poisoning caused by the poultry egg products because eating Salmonella enteritidis pollution, each state
Amount is continuously increased.Therefore, Bacterium enteritidis not only seriously hinders the development of aviculture, but also threatens human and livestock health,
Have become a major issue of medicine, veterinary science and field of public health.Currently, mainly pre- using antibiotics
Anti- and treatment salmonella, but being widely used with antibiotic, result in salmonella drug resistance and Antibiotic Resistance it is continuous
Variation and multidrug resistant phenomenon.
Vaccine is another effective measures for preventing Bacterium enteritidis infection, and the vaccine nowadays used has inactivated vaccine and subtracts
Virus live vaccine, inactivated vaccine need subcutaneously repeatedly inoculation, it is inconvenient to use, and not can induce generate alimentary canal localised protection power and
Cellular immunity, thus the deficiencies of infection of the strong immunity to resist wild pathogenic strain can not be excited.And live vaccine because
Natural infection situation can be simulated, has the characteristics that inducing cell, body fluid and mucosa-immune, therefore advocates use live vaccine in the world
Poultry SE infection is controlled, the research and development of live vaccine have obtained the approval of the World Health Organization, and complete as control salmonellosis
A part of whole strategy.Live vaccine is mainly by gene mutation or to knock out the attenuation mutant constructed, is widely used at present in chicken
Attenuated live vaccine, although there is certain immune protection effectiveness, in most experiments, to wild strain be difficult generate it is stronger
Colonize depression effect, the whole body that can also induce wild strain spreads through sex intercourse；Other nonhosts are adapted in addition, can not effectively induce
The cross protection of property serotype detection of Salmonella.Therefore, there is presently no a kind of very effective attenuated live vaccines to control poultry SE
Infection.As people infects the continued popularity of Salmonella enteritidis, it is badly in need of developing significantly more efficient vaccine to control poultry SE bacterium
It propagates.
Summary of the invention
In view of the above shortcomings of the prior art, technical problem solved by the present invention is for enteritis in the prior art
Living salmonella vaccine to wild strain be difficult to generate it is stronger colonize depression effect, the whole body for being easy to induce wild strain spreads through sex intercourse,
And the technical issues of can not effectively inducing to other nonhost adaptability serotype detection of Salmonella cross protections, and one kind is provided and is existed
Elimination effect is good in host, and the excellent dual-gene missing Bacterium enteritidis of immune effect, its construction method and contains this pair
The vaccine of gene delection Bacterium enteritidis.
In order to solve the above-mentioned technical problem, the present invention adopts the following technical scheme: a kind of dual-gene missing Salmonella
Bacterium, the Bacterium enteritidis being all knocked for rfaH gene and sopB gene.
A kind of construction method of dual-gene missing Bacterium enteritidis, using the method for homologous recombination to the enteritis sramana
RfaH gene and sopB gene in Salmonella are knocked out.Specifically comprise the following steps:
1) according to the genome sequence of Bacterium enteritidis, the upstream of the upstream homology arm sequence rU of rfaH gene is designed
The upstream primer rfaH-2F of the downstream homology arm sequence rD of primer rfaH-1F and downstream primer rfaH-1R, rfaH gene is under
Primer rfaH-2R is swum, the nucleotide sequence of the rfaH-1F is as shown in SEQ ID NO.3, the nucleotides sequence of the rfaH-1R
Column are as shown in SEQ ID NO.4, and the nucleotide sequence of the rfaH-2F is as shown in SEQ ID NO.5, the core of the rfaH-2R
Nucleotide sequence is as shown in SEQ ID NO.6；
2) it is with rfaH-1F and rfaH-1R using the genomic DNA of Salmonella enteritidis as template using PCR amplification method
Primer amplification obtains the upstream homology arm rU of rfaH, and the downstream for obtaining rfaH using rfaH-2F and rfaH-2R as primer amplification is homologous
Arm rD；
3) for the rU and rD obtained using step 2) amplification as template, rfaH-1F and rfaH-2R are primer, and it is anti-to carry out overlapping PCR
It answers, obtains overlapping PCR product rUD；
4) the overlapping PCR product rUD and pMD19-T carrier for obtaining step 3) is attached, and connection product is converted and is felt
By state bacillus coli DH 5 alpha, positive colony is selected using blue and white screening and amicillin resistance screening, is trained using LB culture medium
The positive colony picked out is supported, plasmid is extracted, obtains recombination pMD- Δ rfaH plasmid；
5) digestion suicide plasmid pYG4 and recombination pMD- Δ rfaH plasmid are distinguished with restriction enzyme Bgl II, be recovered from
The 1885bp segment killing the 5797bp segment obtained after plasmid pYG4 digestion and obtaining after recombination pMD- Δ rfaH plasmid enzyme restriction, will
The 5797bp segment and 1885bp segment of acquisition connect overnight in metal bath；
6) by the heat-shock transformed Escherichia coli S17-l/ λ pir competent cell of product after step 5) connection, after conversion
Cell is cultivated, and culture bacterium solution is coated on kalamycin resistance LB plate, and picking single colonie extracts plasmid after increasing bacterium,
Obtain recombination suicide plasmid pYG4- Δ rfaH；
7) recipient bacterium Bacterium enteritidis is cultivated in the LB culture medium containing acidum nalidixicum, in the LB training containing kalamycin
The Escherichia coli S17-l/ λ pir for supporting culture donor bacterium suicide plasmid pYG4- Δ rfaH containing recombination in base, to the recipient bacterium and
Collect thallus respectively after the culture of donor bacterium and be resuspended, by after resuspension donor bacterium and the mixed LB for being incorporated in antibiotic-free of recipient bacterium train
It supports and is cultivated on base, the bacterium solution that culture obtains is coated on the dual anti-plate containing kanamycins and acidum nalidixicum, screening positive clone
Single integration bacterial strain is screened after cultivating single integration bacterial strain that screening obtains in the LB culture medium of antibiotic-free with sucrose plate,
Obtain occurring the Salmonella bacterial strain of dyeing In vivo recombination；Such as with sequence rfaH-1 as shown in SEQ ID NO.7 and sequence
RfaH-2 shown in SEQ ID NO.8 be primer, using PCR method to occur dyeing In vivo recombination Salmonella bacterial strain into
Row screening and identification selects the bacterial strain that PCR amplification goes out 631bp, obtains the Salmonella bacterial strain for knocking out rfaH gene；
8) according to the genome sequence of Bacterium enteritidis, the upstream of the upstream homology arm sequence sU of sopB gene is designed
The upstream primer sopB-2F of the downstream homology arm sequence sD of primer sopB-1F and downstream primer sopB-1R, sopB gene is under
Primer sopB-2R is swum, the nucleotide sequence of the sopB-1F is as shown in SEQ ID NO.9, the nucleotides sequence of the sopB-1R
Column as shown in SEQ ID NO.10, the nucleotide sequence of the sopB-2F as shown in SEQ ID NO.11, the sopB-2R's
Nucleotide sequence is as shown in SEQ ID NO.12；
9) it is with sopB-1F and sopB-1R using the genomic DNA of Salmonella enteritidis as template using PCR amplification method
Primer amplification obtains the upstream homology arm sU of sopB, and the downstream for obtaining sopB using sopB-2F and sopB-2R as primer amplification is homologous
Arm sD；
10) using step 9) the obtained sU of amplification and sD as template, it is anti-that sopB-1F and sopB-2R are that primer carries out overlapping PCR
It answers, obtains overlapping PCR product sUD；
11) the overlapping PCR product sUD and pMD19-T carrier for obtaining step 10) is attached, and connection product is converted
Competent E.coli DH5 α selects positive colony using blue and white screening and amicillin resistance screening, using LB culture medium
The positive colony picked out is cultivated, plasmid is extracted, obtains recombination pMD- Δ sopB plasmid；
12) digestion suicide plasmid pYG4 and recombination pMD- Δ sopB plasmid, recycling are distinguished with restriction enzyme Bgl II
The 1917bp segment obtained after the 5797bp segment and recombination pMD- Δ sopB plasmid enzyme restriction that are obtained after suicide plasmid pYG4 digestion,
The 5797bp segment of acquisition and 1917bp segment are connected overnight in metal bath；
13) by the heat-shock transformed Escherichia coli S17-l/ λ pir competent cell of product after step 12) connection, after conversion
Cell cultivated, will culture bacterium solution be coated on kalamycin resistance LB plate, picking single colonie, increase bacterium after extract matter
Grain obtains recombination suicide plasmid pYG4- Δ sopB；
14) enteritis that the knockout rfaH gene that recipient bacterium step 7) obtains is cultivated in the LB culture medium containing acidum nalidixicum is husky
Door Salmonella strain, cultivates the large intestine of donor bacterium suicide plasmid pYG4- Δ sopB containing recombination in the LB culture medium containing kalamycin
Bacillus S17-l/ λ pir, to collecting thallus respectively after the recipient bacterium and the culture of donor bacterium and being resuspended, by the donor bacterium after resuspension
It is cultivated with mixed be incorporated on the LB culture medium of antibiotic-free of recipient bacterium, the bacterium solution that culture obtains is coated on containing kanamycins and naphthalene
On the dual anti-plate of pyridine acid, screening positive clone list integration bacterial strain, single integration bacterial strain that screening is obtained is in the LB of antibiotic-free
It after being cultivated in culture medium, is screened with sucrose plate, obtains the Salmonella bacterial strain that dyeing In vivo recombination occurs；Such as with sequence
SopB-1 shown in SEQ ID NO.13 and the sequence sopB-2 as shown in SEQ ID NO.14 are primer, using PCR method pair
The Salmonella bacterial strain that dyeing In vivo recombination occurs carries out screening and identification, selects the bacterial strain that PCR amplification goes out 379bp, is lacked
Lose the dual-gene missing Salmonella bacteria strain of rfaH gene and sopB gene.
A kind of Salmonella bacteria vaccine, component include using dual-gene missing Salmonella made from the above method
Bacterium.
Compared with prior art, the present invention has the advantage that
1, the present invention is a kind of transcriptional antitermination albumen by studying the RfaH albumen that discovery is encoded by rfaH gene, it can
To assist RNA polymerase to cross ρ independent form tanscription termination minor structure during Bacterial transcript, the logical of downstream gene is realized
It reads, to eliminate the polarity of long transcripton.In Escherichia coli, rfaH albumen is a kind of virulence modulin, and it is more that it influences rouge
The synthesis of sugared (LPS) layer, hemolysin and pod membrane etc.；In salmonella typhimurium, this albumen can also influence 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 also all enhanced, and the resistance for resisting the substances such as antibacterial peptide in host cell weakens, thus
It is had a net increase of in host cell and grows quantity and substantially reduced compared with wild strain, loss causes the ability of systemic disease, but can effective protection
The attack of street strain.Also there is rfaH gene in Bacterium enteritidis, knocking out Bacterium enteritidis rfaH gene equally may be implemented
The attenuating effects of LPS and other salmonella virulence genes, while effective immunoprotection can be excited to react, it is building SE epidemic disease
The ideal knockout target gene of seedling Candidate Strain.Sop albumen is another virulence factor of salmonella, there is myo-inositol phosphates phosphoric acid
Enzymatic activity after being transferred to host cell, can induce cytoskeleton rearrangement, can cause symptom of diarrhea.SopB mutant strain, which removes, to be enhanced
Outside humoral and cellular immune response, moreover it is possible to enhance mucosal immunity, reduce liquid secretion additionally to alleviating diarrhoea.Present invention invention
People is sensitive by homologous recombination and sucrose using suicide plasmid homologous recombination system on the basis of the studies above is found
The reversed screening technique of gene constructs Bacterium enteritidis-Δ rfaH gene-deleted strain of rfaH gene (1-489) missing first
(D315), on this basis, but utilize same principle and technology, knocked out it is relevant to Bacterium enteritidis virulence another
Gene sopB (1-1686) obtains the dual-gene deletion mutation of Bacterium enteritidis Salmonella Δ rfaH- Δ sopB of inheritance stability
Strain (D316).
2, the present invention knocks out dual-gene Bacterium enteritidis cellular immunity and mucosa-immune and is enhanced, and SE belongs to enteron aisle
Pathogen, local humoral immunity are higher than general immunity to the elimination efficiency of the protection of enteron aisle and thallus, the recovery of gastroenteritis with
It is closely related that enteron aisle locally generates sIgA, therefore can enhance mucosal immunity and will be helpful to the removing of SE；SE is bacterium intracellular simultaneously, by force
Cellular immunity to eliminate SE infection will play a significant role；By verification experimental verification, dual-gene enteritis is knocked out using the present invention
It is obvious that salmonella attacks malicious protecting effect effect.
3, the present invention knocks out dual-gene Bacterium enteritidis and is improved to the safety of host and environment, because gained is prominent
Resistance of the mutant in host cell weakens, and time-to-live and shedding virus is caused all to greatly reduce, in vivo in 3~4 weeks just
Removing can be colonized to go, security performance is excellent.
4, the present invention knocks out dual-gene Bacterium enteritidis and is improved to the immunological cross protection of other serotypes, former
Cause is shortening for core LPS (O-LPS) chain of the dual-gene gene-deleted strain, enhances the immune response to other surfaces albumen, and
These albumen are relatively conservative in different salmonellas, thus can improve Cross immunogenicity, are improved to other serum
The preventive effect of type salmonella.
5, the double mutation of the dual-gene Bacterium enteritidis of present invention knockout, which can reduce, returns main risk, safer；And this hair
Mutant strain can become half rough type after the dual-gene Bacterium enteritidis rfaH gene mutation of bright knockout, and wild strain is light
Slip provides conveniently to distinguish natural infection and the vaccine immunity strain of SE.
Detailed description of the invention
Fig. 1 be rfaH upstream and downstream homology arm rU rD pcr amplified fragment gel electrophoresis figure.(1, DL-5000Marker
DNA Marker(DL-5000)；2, ddH2O negative control；3, rU segments；4, rD segments)
Fig. 2 be rU rD overlap pcr amplified fragment rUD gel electrophoresis figure.(1, DL-5000Marker；2rU D segment)
Fig. 3 is deletion mycopremna structure figures.
Fig. 4 is the PCR qualification figure for lacking rfaH gene bacterial strain.(1, DL-5000Marker；2 deletion mycopremnas；3, parent bacterium
Strain)
Fig. 5 be sopB upstream and downstream homology arm sU sD pcr amplified fragment gel electrophoresis figure.(1, DL-5000Marker
DNA Marker(DL-5000)；2, sU segments；3, sD pieces；4, ddH2O negative control section)
Fig. 6 be sU sD overlap pcr amplified fragment sUD gel electrophoresis figure.(1, DL-5000Marker；2sUD segment)
Fig. 7 is the PCR qualification figure for lacking rfaH gene and sopB gene bacterial strain.(1, DL-5000Marker；2 missing bacterium
Strain；3, parent strain)
Specific embodiment
Invention is further described in detail with Figure of description combined with specific embodiments below.The implementation case with
Implemented under premised on the technology of the present invention, provides detailed embodiment and specific operating process now to illustrate tool of the present invention
It is creative, but protection scope of the present invention embodiment not limited to the following.
Bacterium enteritidis D1203 is (by Chongqing University of Technology's pharmacy and Biological Engineering College used in following embodiments
Central laboratory's separation is identified and is saved)；Following step is to knock out the specific steps of rfaH gene and sopB gene, described
The nucleotide sequence of rfaH gene is as shown in SEQ ID NO.1, the nucleotide sequence of the sopB gene such as SEQ ID NO.2 institute
Show
The proliferation of 1 Salmonella enteritidis of embodiment and the extraction of genome
Salmonella enteritidis D1203 is inoculated in LB liquid medium, 37 DEG C of overnight incubations, takes 1.5mL to cultivate bacterium solution, put
It being placed in Eppendorf centrifuge tube, 10000g is centrifuged 2min, abandons supernatant, and 1.5mLPBS buffer is added, precipitating is resuspended,
10000g is centrifuged 2min, abandons supernatant, 240 μ LTE is added into precipitating, thallus is resuspended, and 15 μ L lysozymes, 0 DEG C of ice bath is added
15 μ L of mass concentration 10%SDS is added in 30min, and Proteinase K is added after mixing, and 5M is added in 65 DEG C of water-bath 30min
500 μ L phenol/chloroform/isoamyl alcohol (volume ratio 1:24:25) solution is added in 200 μ L of NaCl solution, concussion, is uniformly mixed, 12000g
It is centrifuged 10min, takes supernatant into centrifuge tube, records its volume, isometric freezing dehydrated alcohol is added, is placed in -80 DEG C
Refrigerator 10min, 12000g are centrifuged 10min, abandon supernatant, and natural air drying is added 30 μ L ddH2O dissolution, places -20 DEG C of refrigerators
It saves, the template for PCR reaction.
The building of embodiment 2rfaH gene knockout mutant strain
2.1 overlapping PCR
According to the Salmonella enteritidis genome sequence delivered on GeneBank, the upstream homology arm of rfaH gene is designed
The upstream primer of the downstream homology arm sequence rD of the upstream primer rfaH-1F and downstream primer rfaH-1R, rfaH gene of sequence rU
The nucleotide sequence of rfaH-2F and downstream rfaH-2R, the rfaH-1F are as shown in SEQ ID NO.3, the core of the rfaH-1R
Nucleotide sequence is as shown in SEQ ID NO.4, and the nucleotide sequence of the rfaH-2F is as shown in SEQ ID NO.5, the rfaH-
The nucleotide sequence of 2F is as shown in SEQ ID NO.6.Using the Salmonella enteritidis D1203 genomic DNA of said extracted as template,
PCR method expands the upstream and downstream rfaH 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 initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 40s, 72 DEG C of extension 1min, totally 25
A circulation；Last 72 DEG C of extensions 5min.
Upstream and downstream homology arm the amplified production rU and rD that will be expanded using above-mentioned PCR reaction system and reaction condition
It is detected through 1.0% Ago-Gel, as a result visible two bands (Fig. 1) for obtaining being consistent with expected size (919bP and 933bp),
Two bands are cut respectively, carry out recovery purifying according to the operation instruction of the gel reclaims kit of Omega company.
Due to rfaH-1R, rfaH-2F phase mutual partial complementarity sequence, rU and rD after purification is mixed, and using it as mould
Plate, rfaH-1F/rfaH-2R are that primer carries out overlapping PCR.Overlapping 50 μ LPCR amplification system of PCR is the same, PCR reaction condition:
94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C of extension 2min, totally 25 recycle；Last 72 DEG C of extensions
5min。
The building of 2.2 intermediate transfer plasmid pMD- Δ rfaH
Overlapping PCR product is detected with 1.0% Ago-Gel, it is seen that and about 2000bP band rUD (Fig. 2) cuts the band,
Recovery purifying is carried out according to the operation instruction of the plastic recovery kit of Omega company, obtains overlapping PCR product rUD.Mesh will be recycled
Segment rUD connect with pMD19-T carrier.Linked system (10 μ L): 10 × ligase buffer solution, 1 μ L, T4DNA ligase, 1 μ L,
15 μ L of μ L, rUD of pMD19-T carrier, sterilize ddH22 μ L of O, 16 DEG C of metal baths are stayed overnight.
Connection product transformed competence colibacillus bacillus coli DH 5 alpha obtained above is selected (based on blue hickie and ammonia benzyl mould
Plain resistance) positive colony, and in LB culture medium culture positive colony, plasmid is extracted, digestion, PCR identification and sequence is carried out and surveys
It is fixed.Correct recombinant plasmid will be measured and be named as pMD- Δ rfaH.
Specific step is as follows for recombinant plasmid transformed bacillus coli DH 5 alpha:
Bacillus coli DH 5 alpha competent cell is taken, is placed on ice to melt, draws recombination connection product 10 μ L in having melted
In bacillus coli DH 5 alpha competent cell, 1ml LB liquid is added in ice bath 30min after mixing, 42 DEG C of heat shock 90s, ice bath 3min
Culture medium, 37 DEG C of shaking table cultures 1h, 10000g are centrifuged 2min, abandon supernatant, 100 μ L LB are added into precipitating, thallus is resuspended, by bacterium
Liquid is coated on 100 μ g/mL amicillin resistance LB plates, 37 DEG C of overnight incubations.
The building of 2.3 recombination suicide plasmid pYG4- Δ rfaH
It is a small amount of to extract suicide plasmid pYG4 and recombinant plasmid pMD- Δ rfaH, II digestion of restriction enzyme Bgl is used respectively,
Digestion system (50 μ L): 10 × H Buffer buffer, 5 μ L, 30 II 2 μ L of μ L, Bgl of plasmid (pMD- Δ rfaH or PYG4), sterilizing
ddH2O 13 μ L, 37 DEG C of water-bath digestion 2h.Digestion products are through 1% agarose gel electrophoresis, and plasmid pYG4 digestion is at 1 segment
(5797bp), plasmid pMD- Δ rfaH digestion is at 2 segments (2707bp and 1885bp).DNA purification kit recovery purifying
The 1885bp segment of pYG4 segment and pMD- Δ rfaH, above-mentioned two purified fragments are stayed overnight in 6 DEG C of metal bath connections.Metal bath connection
System (10 μ l): 10 × ligase buffer solution, 1 μ L, T4DNA ligase, 1 μ L, pYG4 segment 4 μ L, 3 μ L of Δ rfaH segment, sterilizing
ddH2O 1μL。
By the heat-shock transformed Escherichia coli S17-l/ λ pir competent cell of metal bath connection product, it is coated with kalamycin (100
μ g/ml) LB plate, picking single colonie extracts plasmid after increasing bacterium, carries out digestion, PCR identification and sequencing.It will identify correct
Recombination suicide plasmid be named as pYG4- Δ rfaH.
Specific step is as follows by heat-shock transformed competent cell Escherichia coli S17-l/ λ pir:
Escherichia coli S17-l/ λ pir competent cell is taken, is placed on ice to melt, draws recombination 20 μ L of connection product in
In the Escherichia coli S17-l/ λ pir competent cell of thawing, ice bath 30min after mixing, 42 DEG C of heat shock 90s, ice bath 3min add
Enter 1ml LB liquid medium, 37 DEG C of shaking table cultures 1h, 10000g are centrifuged 2min, abandon supernatant, 100 μ L LB are added into precipitating
Thallus is resuspended, bacterium solution is coated on 100 μ g/mL kalamycin resistance LB plates, 37 DEG C of overnight incubations.
2.4 engagement transfers and the screening of the mono- gene-deleted strain of Salmonella enteritidis Δ rfaH
Building route such as Fig. 3 of Δ rfaH gene-deleted strain, filters out gene mutation strain by two step method.Specific steps: containing
Have and how to cultivate recipient bacterium Bacterium enteritidis D1203 (Nal in the LB culture medium of heavy stone used as an anchor sour (NaL, 40 μ g/ml)R), containing OK a karaoke club
The Escherichia coli S17-l/ λ pir that the rfaH of Δ containing pYG4- suicide plasmid is cultivated in the LB culture medium of mycin (Kan, 100 μ g/mL) is supplied
Body bacterium collects thallus after 37 DEG C of overnight incubations respectively, and thallus is resuspended in LB culture medium, respectively takes 100 μ L bacterial suspensions to mix, Yu Wukang
The LB plate overnight culture of raw element, collects thallus, after being serially diluted, take appropriate bacterium solution be coated on containing kanamycins (Kan, 100
μ g/mL) and the dual anti-plate of acidum nalidixicum (NaL, 40 μ g/ml) on, screening positive clone list integration bacterial strain.
The integration bacterial strain of acquisition is placed in overnight incubation in the LB culture medium of antibiotic-free, in 5% sucrose plate after culture
The bacterial strain of dyeing In vivo recombination occurs for screening.Since the Negative selection of sacB segment acts on, the integration strain nothing with sacB segment
Method carries out upper growth in the plate containing 5% sucrose, and longer bacterium colony is all that dyeing In vivo recombination has occurred, to remove
The recombinant bacterial strain of carrier sequence (including sacB segment).Recombination is there are two types of situation, wherein 50% a possibility that, generates and knocks out strain,
50% a possibility that, generates 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 initial denaturation 5min；94 DEG C of denaturation
30s, 55 DEG C of annealing 40s, 72 DEG C of extension 1.5min, totally 25 recycle；Last 72 DEG C of extensions 5min.PCR reaction terminates, and 1.0%
Ago-Gel detection, such as Fig. 4 knock out strain and amplify a band, be located at 631bp, wild strain amplifies a band, position
In 1120bp or so.The pcr amplification product for knocking out strain is sent to Dalian Bao Shenggong bioengineering Co., Ltd and carries out sequencing.
Sequencing result shows that successful knockout falls rfaH gene 489bp sequence, which is named as D315.
The building of the 3 bis- gene-deleted strains of Salmonella enteritidis Δ rfaH Δ sopB of embodiment
3.1 overlapping PCR
According to the Salmonella enteritidis genome sequence delivered on GeneBank, the upstream homology arm of sopB gene is designed
The upstream primer of the downstream homology arm sequence sD of the upstream primer sopB-1F and downstream primer sopB-1R, sopB gene of sequence sU
The nucleotide sequence of sopB-2F and downstream primer sopB-2R, the sopB-1F are as shown in SEQ ID NO.9, the sopB-1R
Nucleotide sequence as shown in SEQ ID NO.10, the nucleotide sequence of the sopB-2F is described as shown in SEQ ID NO.11
The nucleotide sequence of sopB-2R is as shown in SEQ ID NO.12.Using Salmonella enteritidis D1203 genomic DNA as template, the side PCR
Method expands the upstream and downstream sopB homology arm sequence sU, sD.The 50 μ L PCR amplification systems and PCR reaction condition of sU, sD are the same as rU and rD
Amplification.
Upstream and downstream homology arm amplified production sU, the sD expanded using the above method is examined through 1.0% Ago-Gel
It surveys, it is as a result visible to obtain and be expected size (920bP and 964bp) band that is consistent (Fig. 5), two bands are cut respectively, according to Omega
The operation instruction of the gel reclaims kit of company carries out recovery purifying.
Due to sopB-1R, sopB-2F phase mutual partial complementarity sequence, sU and sD after purification is mixed, and using it as mould
Plate, sopB-1F/sopB-2R are that primer carries out overlapping PCR.Overlapping 50 μ L amplification system of PCR is the same.PCR reaction condition: 94 DEG C
Initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C of extension 2min, totally 25 recycle；Last 72 DEG C of extensions 5min,
Obtain overlapping PCR reaction product sUD.
The building of 3.2 intermediate transfer plasmid pMD- Δ sopB
Overlapping PCR product is detected through 1.0% agarose gel electrophoresis, it is seen that obtains the item that is consistent with expected size (1917bP)
Band sUD (Fig. 6), cuts the band, carries out recovery purifying according to the operation instruction of the plastic recovery kit of Omega company.It will return
It receives target fragment sUD to connect with pMD19-T carrier, linked system (10 μ L): 10 × ligase buffer solution, 1 μ L, T4DNA ligase
1 μ L, pMD19-T carrier, 15 μ L of μ L, rUD, sterilize ddH22 μ L of O, 16 DEG C of metal baths are stayed overnight.
By connection product transformed competence colibacillus bacillus coli DH 5 alpha, select positive (based on blue hickie and amicillin resistance)
Clone, and in LB culture medium culture positive colony, plasmid is extracted, digestion, PCR identification and sequencing are carried out.It will measure correct
Recombinant plasmid be named as pMD- Δ sopB.
The building of 3.3 recombination suicide plasmid pYG4- Δ sopB
It is a small amount of to extract suicide plasmid pYG4 and recombinant plasmid pMD- Δ sopB, II digestion of restriction enzyme Bgl is used respectively,
Digestion system (50 μ L): 10 × H Buffer buffer, 5 μ L, 30 2 μ L of μ L, Bgl of plasmid (pMD- Δ rfaH or PYG4), sterilizing
ddH2O 13 μ L, 37 DEG C of water-bath digestion 2h.Digestion products are through 1% agarose gel electrophoresis, and plasmid pYG4 digestion is at 1 segment
(5797bp), plasmid pMD- Δ rfaH digestion is at 2 segments (2707bp and 1917bp), DNA purification kit recovery purifying
The 1917bp segment of pYG4 segment and pMD- Δ rfaH stays overnight above-mentioned two purified fragments of recycling in 6 DEG C of metal bath connections.Even
Junctor system (10ul): 10 × ligase buffer solution, 1 μ L, T4DNA ligase, 1 μ L, pYG4 segment 4 μ L, 3 μ L of Δ sopB segment go out
Bacterium ddH2O 1μL。
By the heat-shock transformed Escherichia coli S17-l/ λ pir competent cell of metal bath connection product, it is coated with kalamycin (100
μ g/ml) LB plate, picking single colonie extracts plasmid after increasing bacterium, carries out digestion, PCR identification and sequencing.It will identify correct
Recombination suicide plasmid be named as pYG4- Δ sopB.
3.4 engagement transfers and the screening of the bis- gene-deleted strains of Salmonella enteritidis Δ rfaH Δ sopB
Using the Escherichia coli S17-l/ λ pir of the sopB suicide plasmid of Δ containing pYG4- as donor bacterium, obtained in aforementioned 2.4
The bacterial strain D315 of missing rfaH gene is that recipient bacterium carries out engagement transfer.Donor bacterium and recipient bacterium are inoculated in mould containing OK a karaoke club respectively
Plain (Kan, 100 μ g/mL) and how in the LB culture medium of heavy stone used as an anchor acid (NaL, 40 μ g/ml), thallus is collected after 37 DEG C of overnight incubations, is used in combination
Thallus is resuspended in LB culture medium, respectively takes 100 μ L bacterial suspensions to mix, in the LB plate overnight culture of antibiotic-free.Collect thallus
After being serially diluted, appropriate bacterium solution is taken to be coated on pair containing kanamycins (Kan, 100 μ g/mL) and acidum nalidixicum (NaL, 40 μ g/ml)
On anti-plate, screening positive clone list integration bacterial strain.Integration bacterial strain is placed in the plate containing 5% sucrose to screen, is had
The integration strain of sacB segment can not be grown on the plate, and longer bacterium colony be all dyeing In vivo recombination has occurred, thus
Eliminate the recombinant bacterial strain of carrier sequence (including sacB segment).
Recombination is there are two types of situation, wherein 50% a possibility that generates and knocks out strain, 50% a possibility that generates wild type strain.
According to the Salmonella enteritidis genome sequence delivered on GeneBank, pair of primers sopB-1/sopB-2 is designed, sopB-1's
As shown in SEQ ID NO.13, sopB-2 sequence is knocked out as shown in SEQ ID NO.14 by PCR mode screening and identification sequence
Strain.PCR reaction system is the same, PCR reaction condition: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 40s, 72 DEG C are prolonged
2.5min is stretched, totally 25 circulations；Last 72 DEG C of extensions 5min.PCR reaction terminates, and 1.0% Ago-Gel detection, such as Fig. 7 strikes
Except strain amplifies a band, it is located at 379bp or so, wild strain amplifies a band, is located at 2065bp or so.Strain will be knocked out
Pcr amplification product send to Dalian Bao Shenggong bioengineering Co., Ltd and carry out sequencing, sequencing result shows and successfully strikes
The dual-gene knock-out bacterial strain is named as D316 by the 1686bp sequence for removing sopB gene.
4 bacterium of embodiment colonizes removing in vivo
Dual-gene knockout mutations bacterial strain D316 and wild strain are inoculated with LB liquid medium, 37 DEG C of shaken cultivation mistakes respectively
Bacterium solution is centrifuged 5min in 10000g, is resuspended in by night, 2% inoculation fresh LB, 37 DEG C of shaken cultivations to OD600=0.7
Sterilize PBS, and bacterial concentration is adjusted to 108CFU/mL。
1 age in days Hai Lanbai chick is randomly divided into 3 groups, every group 20.Wherein 1 group oral 1 × 107CFU/0.1mL mutant bacteria
Strain D316,1 group takes orally 1 × 107CFU/0.1mL wild strain, 1 group of oral 0.1mLPBS is as a control group.1 after immune
Week, 2 weeks, 3 weeks, 4 weeks are cutd open kill each immune group and control group chicken each 5 at random, sterile to take liver, spleen and caecum, weighing, nothing
Divide 2 parts after bacterium PBS grinding, wherein being coated with salmonella color culture medium plate after 1 part of appropriate dilution, is counted after 37 DEG C of cultures；
After another does Zengjing Granule, it is coated with salmonella color culture medium plate, PCR is determined.
Bacterium colonizes removing situation in vivo and is shown in Table 2.The the 1st, 2,3,4 week after inoculation, control group chicken liver, spleen and blind
Salmonella is not detected in intestines, and mutant bacteria is all finally completely removed in liver, spleen and caecum, wild strain liver,
Quantity in spleen and caecum is significantly higher than control group, and host still cannot be fully erased when 4th week.
Table 2 is inoculated with the bacterial strain distribution (mean ± SEM log10cfu/g) of each internal organs after D316
5 immune protective of embodiment
40 1 Hai Lanbai chick of female, are randomly divided into 2 groups, every group 20.Wherein 1 group in 1 age in days and 5 week old time-divisions
It Kou Fu 1 × 107CFU/0.1mL mutant strain D316, another 1 group takes orally 0.1mLPBS conduct pair in 1 age in days and 5 week old respectively
According to group.In 9 week old, two groups of chickens all oral 1 × 109CFU/0.2mL wild strain carries out attacking poison.1 week, 2 after attacking poison
It week cuts open kill each immune group and control group chicken each 10 at random, it is sterile to take liver, spleen and caecum, weighing, sterile PBS grinding
After divide 2 parts, wherein being coated with salmonella color culture medium plate after 1 part of appropriate dilution, counted after 37 DEG C of cultures；Another, which does, increases
After bacterium culture, it is coated with salmonella color culture medium plate, PCR is determined.
Protective rate and attack toxic bacterial strain colonize in vivo remove situation be shown in Table 3.The the 1st, 2 week after inoculation, internal organs in immune group
The chicken number that toxic bacterial strain is attacked in official's carrying is below control group, illustrates that carrying disease germs for chicken Salmonella enteritidis can be reduced after gene-deleted strain is immune
Rate.Meanwhile immune group liver, spleen and caecum content of molds also below control group, illustrate that constructed gene-deleted strain can provide
Good immune protective effect.
Table 3 attacks the bacterial strain distribution (mean ± SEM log10cfu/g) of malicious protective rate and each internal organs
6 Salmonella bacteria vaccine of embodiment
According to above-described embodiment verification result, the present invention, which knocks out dual-gene missing Bacterium enteritidis, has excellent exempt from
Epidemic disease protecting effect, and can colonize dispose in vivo, it can be used as vaccine use, further, the present embodiment provides a kind of intestines
Scorching salmonella oral vaccine, component include the dual-gene missing Salmonella that the present invention knocks out rfaH gene and sopB gene
Bacterium, concentration of the dual-gene missing salmonella in the oral vaccine can be 1 × 107CFU/0.1mL。
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with
Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the scope of the claims of invention.
<110>Chongqing University of Technology；
<120>a kind of dual-gene missing Bacterium enteritidis, its construction method and contain dual-gene missing enteritis sand
The vaccine of door Salmonella；
<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
<211> 32
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of rfaH-1F
<400> 3
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 (5)
1. a kind of Salmonella bacteria vaccine, which is characterized in that its component includes dual-gene missing Bacterium enteritidis；It is described double
Gene is rfaH gene and sopB gene, using the negative selection method of methods of homologous recombination and sucrose sensitive gene to the enteritis
RfaH gene and sopB gene in salmonella are knocked out.
2. Salmonella bacteria vaccine according to claim 1, which is characterized in that the nucleotide sequence of the rfaH gene is such as
Shown in SEQIDNO.1, the nucleotide sequence of the sopB gene is as shown in SEQIDNO.2.
3. Salmonella bacteria vaccine according to claim 1, which is characterized in that described to use methods of homologous recombination and sucrose
The negative selection method of sensitive gene specifically comprises the following steps:
1) according to the genome sequence of Bacterium enteritidis, the upstream primer of the upstream homology arm sequence rU of rfaH gene is designed
The upstream primer rfaH-2F of the downstream homology arm sequence rD of rfaH-1F and downstream primer rfaH-1R, rfaH gene and downstream are drawn
Object rfaH-2R, the nucleotide sequence of the rfaH-1F is as shown in SEQIDNO.3, and the nucleotide sequence of the rfaH-1R is such as
Shown in SEQIDNO.4, the nucleotide sequence of the rfaH-2F is as shown in SEQIDNO.5, the nucleotide sequence of the rfaH-2R
As shown in SEQIDNO.6；
2) PCR amplification method is used, using the genomic DNA of Salmonella enteritidis as template, using rfaH-1F and rfaH-1R as primer
Amplification obtains the upstream homology arm rU of rfaH, obtains the downstream homology arm of rfaH by primer amplification of rfaH-2F and rfaH-2R
rD；
3) for the rU and rD expanded using step 2 as template, rfaH-1F and rfaH-2R are primer, carry out overlapping PCR reaction,
Obtain overlapping PCR product rUD；
4) the overlapping PCR product rUD and pMD19-T carrier for obtaining step 3) is attached, by connection product transformed competence colibacillus
Bacillus coli DH 5 alpha is selected positive colony using blue and white screening and amicillin resistance screening, is chosen using LB culture medium culture
The positive colony selected extracts plasmid, obtains recombination pMD- △ rfaH plasmid；
5) digestion suicide plasmid pYG4 and recombination pMD- △ rfaH plasmid, recycling suicide matter are distinguished with restriction enzyme Bgl II
The 1885bp segment obtained after the 5797bp segment and recombination pMD- △ rfaH plasmid enzyme restriction that obtain after grain pYG4 digestion, will obtain
5797bp segment and 1885bp segment connected in metal bath overnight；
6) the heat-shock transformed Escherichia coli S17-l/ λ pir competent cell of product after connecting step 5), to the cell after conversion
It is cultivated, culture bacterium solution is coated on kalamycin resistance LB plate, picking single colonie extracts plasmid after increasing bacterium, obtains
Recombinate suicide plasmid pYG4- Δ rfaH；
7) recipient bacterium Bacterium enteritidis is cultivated in the LB culture medium containing acidum nalidixicum, in the LB culture medium containing kalamycin
The Escherichia coli S17-l/ λ pir of middle culture donor bacterium suicide plasmid pYG4- Δ rfaH containing recombination, to the recipient bacterium and donor
Collect thallus respectively after bacterium culture and be resuspended, by after resuspension donor bacterium and the mixed LB culture medium for being incorporated in antibiotic-free of recipient bacterium
The bacterium solution that culture obtains is coated on the dual anti-plate containing kanamycins and acidum nalidixicum by upper culture, and screening positive clone list is whole
Combined bacteria strain is screened with sucrose plate, is obtained after cultivating single integration bacterial strain that screening obtains in the LB culture medium of antibiotic-free
The Salmonella bacterial strain of dyeing In vivo recombination occurs；Such as with sequence rfaH-1 and sequence as shown in SEQIDNO.7
RfaH-2 shown in SEQIDNO.8 is primer, is carried out using PCR method to the Salmonella bacterial strain that dyeing In vivo recombination occurs
Screening and identification selects the bacterial strain that PCR amplification goes out 631bp, obtains the Salmonella bacterial strain for knocking out rfaH gene；
8) according to the genome sequence of Bacterium enteritidis, the upstream primer of the upstream homology arm sequence sU of sopB gene is designed
The upstream primer sopB-2F of the downstream homology arm sequence sD of sopB-1F and downstream primer sopB-1R, sopB gene and downstream are drawn
Object sopB-2R, the nucleotide sequence of the sopB-1F is as shown in SEQIDNO.9, and the nucleotide sequence of the sopB-1R is such as
Shown in SEQIDNO.10, the nucleotide sequence of the sopB-2F is as shown in SEQIDNO.11, the nucleotides sequence of the sopB-2R
Column are as shown in SEQIDNO.12；
9) PCR amplification method is used, using the genomic DNA of Salmonella enteritidis as template, using sopB-1F and sopB-1R as primer
Amplification obtains the upstream homology arm sU of sopB, obtains the downstream homology arm of sopB by primer amplification of sopB-2F and sopB-2R
sD；
10) for the sU and sD expanded using step 9) as template, sopB-1F and sopB-2R are that primer carries out overlapping PCR reaction,
Obtain overlapping PCR product sUD；
11) the overlapping PCR product sUD and pMD19-T carrier for obtaining step 10) is attached, and connection product is converted and is experienced
State bacillus coli DH 5 alpha selects positive colony using blue and white screening and amicillin resistance screening, using LB culture medium culture
The positive colony picked out extracts plasmid, obtains recombination pMD- △ sopB plasmid；
12) digestion suicide plasmid pYG4 and recombination pMD- △ sopB plasmid, recycling suicide matter are distinguished with restriction enzyme Bgl II
The 1917bp segment obtained after the 5797bp segment and recombination pMD- △ sopB plasmid enzyme restriction that obtain after grain pYG4 digestion, will obtain
5797bp segment and 1917bp segment connected in metal bath overnight；
13) the heat-shock transformed Escherichia coli S17-l/ λ pir competent cell of product after connecting step 12), to thin after conversion
Born of the same parents cultivate, and culture bacterium solution is coated on kalamycin resistance LB plate, and picking single colonie is extracted plasmid after increasing bacterium, obtained
Suicide plasmid pYG4- Δ sopB must be recombinated；
14) Salmonella for the knockout rfaH gene that recipient bacterium step 7) obtains is cultivated in the LB culture medium containing acidum nalidixicum
Bacterial strain cultivates the Escherichia coli of donor bacterium suicide plasmid pYG4- Δ sopB containing recombination in the LB culture medium containing kalamycin
S17-l/ λ pir, to collecting thallus respectively after the recipient bacterium and the culture of donor bacterium and be resuspended, by after resuspension donor bacterium and by
Mixed be incorporated on the LB culture medium of antibiotic-free of body bacterium is cultivated, and the bacterium solution that culture obtains is coated on containing kanamycins and acidum nalidixicum
Dual anti-plate on, screening positive clone list integration bacterial strain cultivates the obtained single integration bacterial strain of screening in the LB of antibiotic-free
It after being cultivated in base, is screened with sucrose plate, obtains the Salmonella bacterial strain that dyeing In vivo recombination occurs；Such as with sequence
The sopB-2 as shown in SEQIDNO.14 of sopB-1 and sequence shown in SEQIDNO.13 is primer, using PCR method to generation
The Salmonella bacterial strain for dyeing In vivo recombination carries out screening and identification, selects the bacterial strain that PCR amplification goes out 379bp, is lacked
The dual-gene missing Salmonella bacteria strain of rfaH gene and sopB gene.
4. Salmonella bacteria vaccine according to claim 1, which is characterized in that the vaccine is oral vaccine.
5. Salmonella bacteria vaccine according to claim 1, which is characterized in that the dual-gene missing Bacterium enteritidis
Concentration in the vaccine is 1 × 107CFU/0.1mL。
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| CN201610626518.5A CN106190943B (en) | 2016-08-02 | 2016-08-02 | It is a kind of it is dual-gene missing Bacterium enteritidis, its construction method and contain this it is dual-gene missing Bacterium enteritidis vaccine |
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| 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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| WO2012092226A1 (en) * | 2010-12-27 | 2012-07-05 | Biomune Company | Veterinary vaccine composition against infections caused by salmonella |
| CN103204936A (en) * | 2013-01-25 | 2013-07-17 | 天津商业大学 | Preparation method for polyclonal antibody to salmonella effect protein SopB |
| CN103436478A (en) * | 2013-09-06 | 2013-12-11 | 扬州大学 | Salmonella enteritidis double knockout attenuated mutant and preparation as well as application thereof |
| CN105483049A (en) * | 2015-12-31 | 2016-04-13 | 扬州大学 | Pullorum disease salmonella spiC-rfaH double-gene knockout attenuated strain and DIVA vaccine application thereof |
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| WO2012092226A1 (en) * | 2010-12-27 | 2012-07-05 | Biomune Company | Veterinary vaccine composition against infections caused by salmonella |
| CN103204936A (en) * | 2013-01-25 | 2013-07-17 | 天津商业大学 | Preparation method for polyclonal antibody to salmonella effect protein SopB |
| CN103436478A (en) * | 2013-09-06 | 2013-12-11 | 扬州大学 | Salmonella enteritidis double knockout attenuated mutant and preparation as well as application thereof |
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