CN106191091A - Structure can express method and the gained mutant thereof of the negative bacterium of positive bacteria polysaccharide - Google Patents
Structure can express method and the gained mutant thereof of the negative bacterium of positive bacteria polysaccharide Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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- C12N15/09—Recombinant DNA-technology
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- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
Abstract
A kind of method that the invention provides gram negative bacteria building and expressing Gram-positive granulose, it includes step: low for Gram-positive granulose copy expression plasmid is transformed into disappearanceasdAndrfbPIn the gram negative bacteria of gene, screened by balanced lethal system, obtain producing the gram negative bacteria of Gram-positive granulose.Present invention also offers the gram negative bacteria that can produce Gram-positive granulose prepared by said method.Present invention also offers a strain Salmonella typhimuriumSalmonella entericaSerovar Typhimurium P0005 mutant, Classification And Nomenclature isSalmonella enterica subsp.enterica Serovar Typhimurium P0005, preserving number is CCTCC NO:M 2016344.The present invention enables to gram negative bacteria and expresses the polysaccharide of gram positive bacteria, and the inventive method does not contains any resistance marker simultaneously, meets follow up vaccine and the bio-safety demand of other application.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of leather building and expressing Gram-positive granulose
The method of Lan Shi negative bacterium and a strain can produce the gram negative bacteria mutant of Gram-positive granulose.
Background technology
Streptococcus pneumoniae is the main leather orchid causing infant and old people to suffer from the diseases such as pneumonia, meningitis, otitis media
One of family name's positive pathogen, and its capsular polysaccharide is owing to can induce body to produce specific antibody, this antibody can be to pneumonia chain
Coccus infects provides serotype specificity protection, so being largely used to the research and development of its vaccine.And simple polysaccharide vaccine is deposited
In purification difficult and be difficult to provide enough immune protection effectiveness, so the method having researcher to utilize albumen to be fitted together to polysaccharide is come
Research polysaccharide vaccine, but effect is the most notable.Present stage, numerous studies report is had to utilize gram negative bacteria to express polysaccharide
Antigen achieves good effect, but utilizes Gram-negative bacteria conventional engineering bacteria such as such as escherichia coli or Salmonella etc. all
It is the polysaccharide expressing gram negative pathogenic bacteria, if Gram-positive granulose can be expressed in gram negative bacteria, is then
Initiative research.
At present, the report utilizing gram negative bacteria Salmonella to express synthesis Gram-positive granulose is not yet found
Road.
Therefore, this area is needed badly and is sought a kind of method that gram negative bacteria can be made to express Gram-positive granulose,
Need method and the mutant of foundation the method gained that Salmonella can be made to express streptococcus pneumoniae capsular polysaccharide especially badly.
Summary of the invention
For the shortcoming of prior art, syllabus of the present invention one of be to provide a kind of structure can express gram positive bacteria
The method of the gram negative bacteria of polysaccharide, the method comprises the steps:
The low copy expression plasmid of synthesis Gram-positive granulose is transformed into the leather having lacked asd and rfbP gene
In Lan Shi negative bacterium, screened by balanced lethal system, obtain producing the gram negative bacteria of Gram-positive granulose；
Described gram-positive bacterium includes one or more in streptococcus pneumoniae, A type streptococcus, Type B streptococcus；
Described gram negative bacteria includes one or more in Salmonella typhimurium, escherichia coli, shigella.
It was found by the inventors of the present invention that by the O o antigen polysaccharide o of itself in above-mentioned gram negative bacteria is knocked out, then
The disappearance carrying out rfbP gene processes, and can block the synthesis of O o antigen polysaccharide o, and remain WaaL polysaccharide synthesis ligase.Logical
Cross the interconnection function of this enzyme, the capsular polysaccharide of gram positive bacteria can be connected on core oligosaccharide, thus utilize gene work
The method of journey enables gram negative bacteria to produce Gram-positive granulose.Meanwhile, the present invention has knocked out gram negative bacteria
Asd gene, this gene code aspartate-semialdehyde dehydrogenase (DAP), with in carrier DAP express formed balanced lethal system mutual
Mend, so that the method for this generation Gram-positive granulose is without any resistance marker.
Described gram negative bacteria is Salmonella typhimurium；Described Gram-positive granulose low copy expression plasmid is
Streptococcus pneumoniae capsular polysaccharide low copy expression plasmid.Described streptococcus pneumoniae capsular polysaccharide low copy expression plasmid contains SEQ
Nucleotide sequence shown in ID No.1 or there is the sequence iden of at least 80% with nucleotide sequence shown in SEQ ID No.1
The low copy expression plasmid of nucleotide sequence, the nucleotides sequence shown in described SEQ ID No.1 is classified as gram positive bacteria pneumonia
Streptococcus 6A serotype (Streptococcus pneumoniae 6A) capsular polysaccharide gene bunch.
As shown in the embodiment of the present invention, the method utilizing the present invention, Salmonella typhimurium successful expression pneumonia chain
Coccus capsular polysaccharide.
By comparing discovery, in gram negative bacteria and Gram-positive granulose route of synthesis, there is similar synthesis
Approach, is required for being connected on glycosyl seat by polysaccharide by polysaccharide synthase.This is for express and to produce in gram negative bacteria
Gram-positive granulose provides theoretical basis.The pod membrane of streptococcus pneumoniae 6A serotype is expressed by low copy expression plasmid
Polysaccharide full genome bunch, then by the WaaL ligase of Salmonella typhimurium self, the polysaccharide of expression is connected to Salmonella
On core oligosaccharide, by such genetic engineering modified capsular polysaccharide that Salmonella can be made to produce streptococcus pneumoniae, for rear
The gram positive bacteria polysaccharide vaccine of continuous research novelty provides theoretical basis, also provides a kind of efficient epidemic disease for polysaccharide vaccine
Seedling carrier platform.
Gram negative bacteria Salmonella is utilized to have following to produce gram positive bacteria streptococcus pneumoniae capsular polysaccharide
Feature: 1, owing to Salmonella genetic background is clear and genetic manipulation is easy, can carry out subsequent adaptation on this basis, from
And build more precisely efficient polysaccharide submission vaccine；2, due to Salmonella, there is intracellular parasitic character, be a kind of maturation
Antigen presentation platform, it is possible to stimulate body to cause strong mucosa and humoral immune reaction, efficient capsular polysaccharide can be become
Submission vaccine；3, the capsular polysaccharide vaccine that this kind is produced by Salmonella is prone to cultivate, and can be substantially reduced capsular polysaccharide vaccine
Production cost；4, this kind produces the construction strategy of Gram-positive granulose in gram negative bacteria and can be used for other lungs
In the structure of scorching streptococcus serum type capsular polysaccharide, there is application prospect widely.
It is worthy of note, those skilled in the art easily know, and the inventive method is not limited to Salmonella typhimurium.This
Escherichia coli and shigella that inventive method is mentioned are also common gram negative pathogenic bacteria, and it is many to have similar fat
Sugar structure, is formed by lactone A, core oligosaccharide and O antigen superposition, carries out synthesis by similar synzyme and assembles, so this
Inventive method goes for escherichia coli and shigella, or even other multiple gram negative bacteria, especially has
Negative bacteria medicinal, that prepared by vaccine or other valuable material produces.
Gram-positive bacterium of the present invention can be that extracellular polysaccharide is virulence factor or has medical value or food
The gram-positive bacterium that product are worth, but it is not limited to this, as long as the positive bacteria that its polysaccharide has practical use is all applicable to this
Invention.
Preferably, electricity conversion it is converted into described in.
Preferably, described low copy expression plasmid builds with pYA3337 for plasmid vector.
Preferably, the construction method of described streptococcus pneumoniae capsular polysaccharide low copy expression plasmid is:
1) design of primers
Vector-F:5 '-AAGAAAATATTTGAATAACGCTCATGAGACAATAACCCT-3 '
Vector-R:5 '-ACTATTTTTCCATTCATGGTCTGTTTCCTGTGTGAAATTG-3 '
6A-1F:5 '-TCACACAGGAAACAGACCATGAATGGAAAAATAGTAAAG-3 '
6A-1R:5 '-TGTTTGAGACCTAATACTATC-3 '
6A-2F:5 '-TATAATGGTGAGCGATATTTG-3 '
6A-2R:5 '-GTTATTGTCTCATGAGCGTTATTCAAATATTTTCTTTCT-3 '
2) extract and be in the pneumococcal dna of exponential phase as template, be utilized respectively 6A-1F, 6A-1R and 6A-
The total length gene cluster of its capsular polysaccharide is expanded by 2F, 6A-2R, obtains its amplified production, utilizes primer Vector-F simultaneously
Expand with template plasmid pYA3337 with Vector-R, obtain its carrier segments；
3) utilize Gibson to assemble test kit purpose fragment and carrier segments are assembled, and transgene engineering bacteria
In, it is thus achieved that the low copy expression plasmid of streptococcus pneumoniae capsular polysaccharide.
Preferably, described genetic engineering bacterium is TOP10 escherichia coli.
Further object is that provide by said method prepare can to produce gram positive bacteria many
The gram negative bacteria mutant of sugar.
Further object is that offer one strain can produce gram positive bacteria streptococcus pneumoniae 6A serotype
Capsular polysaccharide Salmonella typhimurium Salmonella enterica serovar Typhimurium P0005 sudden change
Body, has lacked asd and rfbP gene, has existed containing containing SEQ simultaneously in this mutant in described mutant chromosomal DNA
Nucleotide sequence shown in ID No.1 or there is the sequence iden of at least 80% with nucleotide sequence shown in SEQ ID No.1
The low copy expression plasmid of nucleotide sequence, the nucleotides sequence shown in described SEQ ID No.1 is classified as gram positive bacteria pneumonia
Streptococcus 6A serotype (Streptococcus pneumoniae 6A) capsular polysaccharide gene bunch；The classification life of described mutant
Entitled Salmonella enterica subsp.enterica serovar Typhimurium P0005, is preserved in China's allusion quotation
Type culture collection center (Luo Jia Shan, wuchang, wuhan, Wuhan University, postcode: 430072), preserving number is CCTCC NO:M
2016344, the preservation time is on June 23rd, 2016.
What this mutant was initiative produces Gram-positive granulose by gram negative bacteria, can be efficient as one
Gram positive bacteria polysaccharide vaccine carrier and novel polysaccharide vaccine provide precondition.
The invention has the beneficial effects as follows:
1, Gram-positive granulose is produced during the present invention can make gram negative bacteria Salmonella typhimurium；
2, it has been employed successfully in vaccine carrier due to Salmonella typhimurium, and has been prone to genetic engineering modified, the present invention
The research and development of the gram positive bacteria polysaccharide vaccine of novelty and Gram-positive based on gram negative bacteria submission can be promoted
Granulose vaccine applied research；
3, the method for the generation Gram-positive granulose that the present invention provides, does not contains any resistance marker, meets follow-up
The bio-safety requirement of vaccine research.
Accompanying drawing explanation
Fig. 1: the inventive method schematic diagram；
Fig. 2: express aspartate-semialdehyde dehydrogenase (DAP) and the low copy of streptococcus pneumoniae 6A serotype capsular polysaccharide
Plasmid pYA337-6A collection of illustrative plates；
The qualification result of the low copy expression plasmid that Fig. 3: the present invention builds, M:DNAmarker III 1 in figure: pneumonia streptococcus
Bacterium 6A serotype capsular polysaccharide total length gene cluster amplification qualification result；2: streptococcus pneumoniae 6A serotype capsular polysaccharide gene bunch half
Long amplification.
Fig. 4: the Salmonella typhimurium silver staining producing streptococcus pneumoniae 6A serotype capsular polysaccharide is identified, in figure 1: contain
The Salmonella typhimurium mutant of streptococcus pneumoniae 6A serotype capsular polysaccharide expression plasmid；2: containing the mouse typhus of empty plasmid
Mutant salmonella body；
Fig. 5: produce the Immunized With Salmonella. Typhimurium trace checking of streptococcus pneumoniae 6A serotype capsular polysaccharide, in figure 1:
Salmonella typhimurium mutant containing streptococcus pneumoniae 6A serotype capsular polysaccharide expression plasmid；2: containing the Mus of empty plasmid
Salmonella typhi mutant；
Detailed description of the invention
Below by embodiment, the present invention is specifically described, it is necessary to it is pointed out here that be that following example are simply used
In the present invention is further detailed, it is impossible to be interpreted as limiting the scope of the invention, being skilled in technique of this field
Some nonessential improvement and adjustment that personnel are made according to foregoing invention content, still fall within protection scope of the present invention.
Embodiment 1
According to the streptococcus pneumoniae 6A serotype capsular polysaccharide full genome bunch strain sequence reported for work (with reference to GenBank sequence
Number for GCA_001088685.1) design two to there is the PCR primer of overlapping fragments respectively by total length that total length is 7267bp fragment
Gene cluster divides two sections to expand, and amplified fragments size is respectively 3420bp and 3847bp, and above-mentioned primer is by Beijing Hua Da base
Because company synthesizes, primer sequence is as follows:
6A-1F:5 '-TCACACAGGAAACAGACCATGAATGGAAAAATAGTAAAG-3 '
6A-1R:5 '-TGTTTGAGACCTAATACTATC-3 '
6A-2F:5 '-TATAATGGTGAGCGATATTTG-3 '
6A-2R:5 '-GTTATTGTCTCATGAGCGTTATTCAAATATTTTCTTTCT-3 '
Simultaneously need to carrier pYA3337 as template, design the PCR primer amplification vector sheet a pair with overlapping fragments
Section, primer is synthesized by Beijing Hua Da genome company, and primer sequence is as follows:
Vector-F:5 '-AAGAAAATATTTGAATAACGCTCATGAGACAATAACCCT-3 '
Vector-R:5 '-ACTATTTTTCCATTCATGGTCTGTTTCCTGTGTGAAATTG-3 '
Plasmid construction needs to use Gibson and assembles test kit (Gibson Assembly Master Mix, NEB), passes through
In fragment, add overlap fragment when designing primer, thus fragment can pass through overlapping fragments, and assembled by gibson
Enzyme connects into a complete plasmid.Specifically comprise the following steps that and fragment is controlled cumulative volume at 10 microlitres according to identical mole ratio
Having added reaction system, system is as follows:
Acting on 1 hour at 50 degrees Celsius after having added system, junctional complex is transformed into by the method taking 2 microlitre electricity consumptions conversions
In TOP10 competent cell, coating on Kan flat board, placement is chosen bacterium full genome bunch qualification after bacterium colony grows with 37 DEG C and is drawn
Thing carries out PCR qualification, and PCR is accredited as after positive bacterium colony extraction plasmid proceeds to Salmonella typhimurium and again carries out expressing it
The follow-up qualification of streptococcus pneumoniae capsular polysaccharide.Qualification result confirms that the expression plasmid built is correct, such as Figure of description 3
Shown in, the plasmid map of polysaccharide expression plasmid is as shown in Figure of description Fig. 2.
Boiling lysis prepares the genomic DNA template of streptococcus pneumoniae, 37 DEG C of incubated overnight of picking list bacterium colony.Take
0.5ml bacterium solution 12000r/min is centrifuged 3min, abandons supernatant, collects thalline with ultrapure washing once, and resuspended.It is put in boiling water and boils
10min, cooled rear 12000r/min is centrifuged 3min, standby using supernatant as template.With its genome as template, use LAtaq
Polymerase expands,
The amplified reaction of PCR is carried out in the system of 50 μ L, and reaction system is as follows: template DNA 1 μ L, 2 × high-fidelity DNA
Enzyme premixed liquid, purchased from precious biological (Dalian) company limited 25 μ L, forward primer 1 μ L, downstream primer 1 μ L, ddH2O 22μL。
Amplification condition is: entering circulation after 94 DEG C of degeneration 5min, loop parameter is 94 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C
10sec.After 35 circulations, 72 DEG C extend 10min.The PCR primer of amplification, through the agarose gel electrophoresis analysis of 1%, expands two
Individual clip size is respectively 3420bp and 3847bp, with expection sizableness.
TOP10 Electroporation-competent cells Making programme is as follows:
Picking list bacterium colony escherichia coli TOP10 is inoculated in LB fluid medium, and 37 DEG C of shaken cultivation are overnight.Take 400 μ L
Mother solution joins in the 100ml triangular flask equipped with LB culture medium, 37 DEG C, 180r/min shaken cultivation to OD600=0.8-1, ice
Bath 30min, 4 DEG C, 4500r/min is centrifuged 10min and collects thalline, the ultrapure washing of precipitation ice bath pre-cooling twice, final step
Wash one time with 10% glycerol of pre-cooling, resuspended the most standby with 10% glycerol of 60 μ L pre-coolings.
Produce the structure of the Salmonella typhimurium mutant of streptococcus pneumoniae capsular polysaccharide
By in the expression plasmid built conversion to Salmonella typhimurium mutants which had, (this mutant has lacked asd
Gene, needs DAP to grow, and complementary can express DAP in expression plasmid, thus the screening of beneficially mutant), utilize electricity
Expression plasmid is transformed in mutant by the method converted, and grow on common LB flat board is positive bacterium colony, and picking list
Bacterium colony carries out PCR qualification.The present embodiment mutant is identified and is successfully constructed, as shown in Figure of description Fig. 3.
Embodiment 2
LPS map identification: for the mutant of embodiment 1, incubated overnight 5ml PCR be accredited as the positive include expression
The salmonella typhimurium strain of plasmid, centrifugal collection thalline, (component is: 0.5M Tris-Cl pH to take 200 μ l buffer A
6.8,10%glycerol, 10%SDS and 5%Beta-mercaptoethanol) resuspended thalline, sample fully mix after
Boiling water boils 10 minutes, after sample cools down centrifugal 15 minutes to remove undissolved impurity, after being centrifuged, by supernatant according to
The ratio of 1:10 (10 μ l add to 90 μ l) add buffer B (component is: 0.5M Tris-Cl pH 6.8,10%glycerol,
0.05%Bromophenol blue) in, and to add 1 μ l concentration be the E.C. 3.4.21.64 of 20mg/ml.Fully after mixing, sample is put
It is placed in 37 DEG C.After one hour, sample is taken 15 μ l at the SDS-PAGE glue of 15% concentration and carry out electrophoresis, after glue is run through, will
Glue carries out silver ammonia dyeing, if expressing streptococcus pneumoniae capsular polysaccharide, then it can be seen that there is band, as shown in Figure of description Fig. 4.
If not expressing, owing to Salmonella typhimurium itself has lacked O antigen, then show as rough type.
Staining procedure is as follows:
1) fixing: glue to be put in fixative and fix overnight, rinse three times with ultra-pure water afterwards, each 10min；
2) sensitization: adding sensitizing solution in dyeing dish, jog 10min, afterwards with ultrapure washing three times, each 10min；
3) upper silver: adding silver staining liquid in dyeing dish, jog 10min, afterwards with ultrapure washing three times, each 10min；
4) colour developing: add nitrite ion in dyeing dish, vibrate gently, after showing band, change ultra-pure water immediately, glue is floated
Till being washed till invariant color, carry out photograph and preserve analysis.
Embodiment 3
Immunoblotting (Western blot) is identified: is separated by SDS-PAGE glue by the lipopolysaccharide sample handled well, treats
After electrophoresis completes, LPS band is transferred on nitrocellulose filter (nitrocellulose membrane) by transfer instrument.
Treat that transferring film completes, with the Tris buffer blind containing 5% skim milk 2 hours, add specificity immediately for pneumonia streptococcus
The rabbit anti-serum (1:100 dilution) of bacterium 6A serotype capsular polysaccharide is positioned over incubated at room 2 hours.After having hatched, by alkalescence
The anti-dilution with 1:10000 times of goat anti-rabbit igg two of phosphatase enzyme mark is hatched, and washes three times with TBS buffer after two hours.Exempt from
Epidemic disease marking band develops the color by adding BCIP nitrite ion.Reaction terminating then by putting into cleaning in ultra-pure water, then eventually by film
Only reaction.The Salmonella typhimurium wherein not proceeding to plasmid is negative control.If there being band, then illustrate that the polysaccharide produced is then
Specificity is for the polysaccharide of streptococcus pneumoniae capsular polysaccharide, as shown in Figure of description Fig. 5.
Claims (10)
1. the method building the gram negative bacteria that can express Gram-positive granulose, it is characterised in that: described method
Comprise the following steps:
The low copy expression plasmid of synthesis Gram-positive granulose is transformed into the gram having lacked asd and rfbP gene
In negative bacterium, screened by balanced lethal system, obtain producing the gram negative bacteria of Gram-positive granulose；
One or more included in streptococcus pneumoniae, A type streptococcus, Type B streptococcus described；
Described gram negative bacteria includes one or more in Salmonella typhimurium, escherichia coli, shigella.
Method the most according to claim 1, it is characterised in that described gram negative bacteria is Salmonella typhimurium.
Method the most according to claim 1, it is characterised in that described in be converted into electricity conversion.
Method the most according to claim 1 and 2, it is characterised in that matter expressed by the low copy of described Gram-positive granulose
Grain is streptococcus pneumoniae capsular polysaccharide low copy expression plasmid.
Method the most according to claim 4, it is characterised in that described streptococcus pneumoniae capsular polysaccharide low copy expression plasmid
Containing nucleotide sequence shown in SEQ ID No.1 or with nucleotide sequence shown in SEQ ID No.1, there is the sequence of at least 80%
The low copy expression plasmid of the nucleotide sequence of homogeneity, the nucleotides sequence shown in described SEQ ID No.1 is classified as gram sun
Property bacterium streptococcus pneumoniae 6A serotype (Streptococcus pneumoniae 6A) capsular polysaccharide gene bunch.
6. according to the method described in any one of claim 1-5, it is characterised in that described low copy expression plasmid is with pYA3337
Build for plasmid vector.
Method the most according to claim 5, it is characterised in that described streptococcus pneumoniae capsular polysaccharide low copy expression plasmid
Construction method be:
1) design of primers
Vector-F:5 '-AAGAAAATATTTGAATAACGCTCATGAGACAATAACCCT-3 '
Vector-R:5 '-ACTATTTTTCCATTCATGGTCTGTTTCCTGTGTGAAATTG-3 '
6A-1F:5 '-TCACACAGGAAACAGACCATGAATGGAAAAATAGTAAAG-3 '
6A-1R:5 '-TGTTTGAGACCTAATACTATC-3 '
6A-2F:5 '-TATAATGGTGAGCGATATTTG-3 '
6A-2R:5 '-GTTATTGTCTCATGAGCGTTATTCAAATATTTTCTTTCT-3 '
2) extract and be in the pneumococcal dna of exponential phase as template, be utilized respectively 6A-1F, 6A-1R and 6A-2F,
The total length gene cluster of its capsular polysaccharide is expanded by 6A-2R, obtains its amplified production, utilize simultaneously primer Vector-F and
Vector-R expands with template plasmid pYA3337, obtains its carrier segments；
3) utilize Gibson to assemble test kit purpose fragment and carrier segments are assembled, and in transgene engineering bacteria,
Obtain the low copy expression plasmid of streptococcus pneumoniae capsular polysaccharide.
Method the most according to claim 7, it is characterised in that described genetic engineering bacterium is TOP10 escherichia coli.
9. blue by the leather that can produce Gram-positive granulose prepared according to method described in any one of claim 1-8
Family name's negative bacterium mutant.
10. a strain can produce the Salmonella typhimurium of capsular polysaccharide of gram positive bacteria streptococcus pneumoniae 6A serotype
Salmonella enterica serovar Typhimurium P0005 mutant, it is characterised in that described mutant dyes
Body DNA lacks asd and rfbP gene, this mutant has existed containing containing nucleotide shown in SEQ ID No.1 simultaneously
Sequence or have with nucleotide sequence shown in SEQ ID No.1 at least 80% the low copy of nucleotide sequence of sequence iden
Expression plasmid, the nucleotides sequence shown in described SEQ ID No.1 is classified as gram positive bacteria streptococcus pneumoniae 6A serotype
(Streptococcus pneumoniae 6A) capsular polysaccharide gene bunch；The Classification And Nomenclature of described mutant is Salmonella
Enterica subsp.enterica serovar Typhimurium P0005, is preserved in China typical culture collection
The heart, preserving number is CCTCC NO:M 2016344, and the preservation time is on June 23rd, 2016.
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| CN111793591A (en) * | 2020-06-22 | 2020-10-20 | 南昌大学 | Salmonella mutant strain capable of efficiently stimulating immune response and construction method and application thereof |
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Non-Patent Citations (4)
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| QINGKE KONG ET AL.: "Effect of Deletion of Genes Involved in Lipopolysaccharide Core and O-Antigen Synthesis on Virulence and Immunogenicity of Salmonella enterica Serovar Typhimurium.", 《INFECTION AND IMMUNITY》 * |
| QIONG LIU ET AL.: "Immunogenicity and Cross-Protective Efficacy Induced by Outer Membrane Proteins from Salmonella Typhimurium Mutants with Truncated LPS in Mice.", 《INT. J. MOL. SCI.》 * |
| XUEYAN LIU ET AL.: "Attenuated Salmonella Typhimurium delivery of a novel DNA vaccine induces immune responses and provides protection against duck enteritis virus.", 《VETERINARY MICROBIOLOGY》 * |
| YUE HAN ET AL.: "The waaL gene is involved in lipopolysaccharide synthesis and plays a role on the bacterial pathogenesis of avian pathogenic Escherichia coli.", 《VETERINARY MICROBIOLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111793591A (en) * | 2020-06-22 | 2020-10-20 | 南昌大学 | Salmonella mutant strain capable of efficiently stimulating immune response and construction method and application thereof |
| CN111793591B (en) * | 2020-06-22 | 2022-11-25 | 南昌大学 | Salmonella mutant strain capable of efficiently stimulating immune response and construction method and application thereof |
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