CN109402036A - The application of ox source Salmonella enteritidis trxB gene delection - Google Patents
The application of ox source Salmonella enteritidis trxB gene delection Download PDFInfo
- Publication number
- CN109402036A CN109402036A CN201811321305.7A CN201811321305A CN109402036A CN 109402036 A CN109402036 A CN 109402036A CN 201811321305 A CN201811321305 A CN 201811321305A CN 109402036 A CN109402036 A CN 109402036A
- Authority
- CN
- China
- Prior art keywords
- salmonella enteritidis
- trxb
- secl
- source
- trxb gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- 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
- C07K14/255—Salmonella (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/025—Enterobacteriales, e.g. Enterobacter
- A61K39/0275—Salmonella
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses the construction methods of ox source Salmonella enteritidis trxB gene-deleted strain, and ox source Salmonella enteritidis trxB gene delection can reduce the virulence of ox source Salmonella enteritidis, and ox source Salmonella enteritidis trxB gene delection can be applied to Salmonella enteritidis vaccine.The present invention constructs ox source Salmonella enteritidis trxB gene-deleted strain using Red methods of homologous recombination, and its biological characteristics and virulence are studied, specify that trxB gene delection causes ox source Salmonella enteritidis virulence to decline, reference is provided for design ox source Salmonella enteritidis vaccine target spot, and then Salmonella enteritidis infection is prevented and treated, while can further explore the physiologic function of Salmonella enteritidis TrxB albumen.
Description
Technical field
The present invention relates to the applications of gene delection, specifically, being related to a kind of ox source Salmonella enteritidis trxB gene delection
Application and trxB gene-deleted strain construction method, belong to field of biotechnology.
Background technique
Ox salmonellosis is also known as the paratyphoid of ox, be meat, milk cow (especially calf) cultivation in common disease, the disease lead
Will by Salmonella in Salmonella typhimurtum (S.typhi murium), Salmonella dublin (S.dublin) and enteritis
Caused by detection of Salmonella (S.enteritidis) infection, mainly using gastroenteritis, septicemia, toxaemia as main feature, ox can be seriously caused
It is dead.Clinically all ages and classes cattle infected detection of Salmonella symptom differs greatly, and young ox disease incidence is high and in popularity, except fever, intestines
It is scorching outer, it is mishandling to develop as septicemia, it causes death；Adult Bovine morbidity is anxious, characterized by chronic enteritis and diarrhea, pregnant domestic animal
It can miscarry.The disease is generally existing in ox cultivation at present, causes extensive damage to clinic production.More importantly suffering from
Infected cattle can carry disease germs for a long time, and the meat and dairy produce of production can be infected by food source approach to people, jeopardize human health.At present
Detection of Salmonella is that the whole world and the most important food source pathogenic microorganism in China, annual Salmonella infection case occupy in bacillary food
The first place of poison, can not be ignored.
Salmonella infection is difficult to prevention and control and is that it has strong tolerance for external environment.Ox salmonellosis mainly leads to
The infection of alimentary canal approach is crossed, cause of disease needs to be resistant to the extreme acid-base environment of alimentary canal, and detection of Salmonella is needed resistance to after infecting foundation
Necessary nutriment is synthesized by under conditions of extremely barren in vivo, while also to resist killing for various unfavorable factors in host
Wound.Detection of Salmonella is typical bacterial parasite intracellular, causes to infect the acidic environment that must be resistant to host's application, antibacterial to host
Matter, nutrition is barren and the killing of immunocyte.At present in the research of detection of Salmonella, many virulence have been had found in succession and have been answered
Swash GAP-associated protein GAP.Early-stage study detection of Salmonella intracellular infection discovery, albumen TrxB high expression during detection of Salmonella intracellular infection, takes off
Show itself and the close phase of bacterium intracellular infection.TrxB is the thioredoxin oxidoreducing enzyme of bacterial identification, is catalyzed NADP+It generates
NADPH plays key effect in bacterial respiratory metabolism, but the research about the albumen and bacterium infection yet there are no report at present
Road, in view of it in detection of Salmonella unconventionality expression intracellular, thus it is speculated that it participates in Salmonella infection pathogenic course, may be with Pathogenicity of Bacteria phase
It closes.
Based on this, the present invention constructs ox source Salmonella enteritidis trxB gene-deleted strain using Red methods of homologous recombination, and right
Its biological characteristics and virulence are studied, and the relationship between trxB gene delection and ox source Salmonella enteritidis virulence is specified, and are visited
Study carefully the influence that TrxB albumen causes a disease to detection of Salmonella, ox source Salmonella enteritidis vaccine development provides tool and basis is ability to explore
The problem of field technique personnel's urgent need to resolve.
Summary of the invention
It is an object of the invention to the relationships between clear trxB gene delection and ox source Salmonella enteritidis virulence, and visit
Study carefully the influence that TrxB albumen causes a disease to detection of Salmonella.
To achieve the above object, the present invention adopts the following technical scheme:
Ox source Salmonella enteritidis trxB gene delection for reducing ox source Salmonella enteritidis virulence.
Further, application of the ox source Salmonella enteritidis trxB gene delection in the Salmonella enteritidis vaccine of ox source.
Further, the construction method of ox source Salmonella enteritidis trxB gene-deleted strain, the specific steps are as follows:
(1) primer P1 and P2 are utilized, through PCR amplification, constructs trxB gene knockout target practice segment；The primer sequence are as follows:
P1:5 '-CTCATGGGCACGACCAAACACAGTAAGCTGCTTATTCTGGGCTCAGGACCGTGTAGGCTGGA
GCTGCTTCG-3 ', SEQ ID NO.1；
P2:5 '-AAAGTCCGGCGATGTCCAGCGTCTCGATATTATCGCTCTGCTGCGTGTCGATGGGAATTAGC
CATGGTCC-3 ', SEQ ID NO.2；
(2) plasmid pKD46 is transferred to SECL-5 competent cell, obtains SECL-5/pKD46 positive strain, and will be described
Positive strain SECL-5/pKD46 is prepared into competent cell；
(3) trxB gene knockout target practice segment electrotransformation is entered into SECL-5/pKD46 competent cell, obtains positive weight
The sub- SECL-5trxB::cat of group；
(4) competent cell is prepared using positive SECL-5trxB::cat bacterial strain, electrotransformation imports pCP20 plasmid, eliminates
Chloramphenicol resistance marker obtains purpose bacterial strain SECL-5 Δ trxB；
(5) PCR verifying is carried out to purpose bacterial strain SECL-5 Δ trxB.
Further, the reaction system of step (1) described PCR amplification each 2.5 μ l of 1 μ l, dNTPs of totally 50 μ l: primer P1, P2,
15 μ l of μ l, 10 × buffer of rTaq enzyme, 100 times of diluted 1 μ l of pKD3 plasmid, remaining is supplied with water；
PCR response procedures are as follows: 94 DEG C of initial denaturation 5min, 25 circulations (94 DEG C of 45s, 54 DEG C of 30s, 72 DEG C of 60s), 72 DEG C of ends
Extend 10min.
Further, the condition of step (3) described electrotransformation are as follows: voltage 1.8KV, pulse 25 μ F, 200 Ω of resistance.
The beneficial effects of the present invention are: the present invention constructs ox source Salmonella enteritidis trxB base using Red methods of homologous recombination
It is studied because of gene-deleted strain, and to its biological characteristics and virulence, specifies that trxB gene delection causes ox source Salmonella enteritidis
Virulence decline provides reference for design ox source Salmonella enteritidis vaccine target spot, and then prevents and treats the infection of ox source Salmonella enteritidis, simultaneously
The physiologic function of ox source Salmonella enteritidis TrxB albumen can further be explored.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 is ox source Salmonella enteritidis trxB gene-deleted strain PCR of the present invention verifying；
M:DL5000Marker；Swimming lane 1:SECL-5 wild-type strain；Swimming lane 2:SECL-5trxB::cat；Swimming lane 3:
SECL-5ΔtrxB；
Fig. 2 is growth curve measurement of the ox source Salmonella enteritidis trxB gene-deleted strain of the present invention on LB culture medium；
Fig. 3 is that growth curve of the ox source Salmonella enteritidis trxB gene-deleted strain of the present invention on Minimal culture medium is surveyed
It is fixed；
Fig. 4 be ox source Salmonella enteritidis trxB gene-deleted strain of the present invention for acidity stress resistance measurement；
Fig. 5 is measurement of the ox source Salmonella enteritidis trxB gene-deleted strain of the present invention for oxidative stress resistance；
Fig. 6 is for ox source Salmonella enteritidis trxB gene delection of the present invention to SECL-5 bacterial strain in macrophage Raw264.7 born of the same parents
The influence of interior survival；
In Fig. 2-Fig. 6, WT: wild-type strain SECL-5；KO:trxB gene-deleted strain SECL-5 Δ trxB；RS: covering bacterium
Strain SECL-5 Δ trxB/pBR322-trxB.
Specific embodiment
Combined with specific embodiments below and attached drawing further describes the present invention, and the advantages and features of the present invention will be with
Description and it is apparent.It should be understood that described, examples are merely exemplary, does not constitute any restrictions to the scope of the present invention.
It will be understood by those skilled in the art that without departing from the spirit and scope of the invention can be to technical solution of the present invention
Details and form are modified or are replaced, but these modifications or substitutions each fall within protection scope of the present invention.
Embodiment 1 constructs trxB gene knockout target practice segment
According to Salmonella enteritidis genome sequence (CP023436.1) design primer issued on NCBI.
Using pKD3 plasmid as template, PCR amplification is carried out with primer P1 and P2, obtains trxB gene knockout target practice segment, PCR
Reaction system is 1 μ l, 10 × buffer 5 μ l, 100 times of diluted pKD3 of each 2.5 μ l, rTaq enzyme of 1 μ l, dNTPs of primer P1, P2
1 μ l of plasmid complements to 50 μ l with water；
PCR response procedures are as follows: 94 DEG C of initial denaturation 5min, 25 circulations (94 DEG C of 45s, 54 DEG C of 30s, 72 DEG C of 60s), 72 DEG C of ends
Extend 10min；Agarose gel electrophoresis is carried out after the reaction was completed, is recycled PCR product with gel reclaims kit, is obtained 1133bp
The band of size；Wherein, primer P1 and P2 are as follows:
P1:5 '-CTCATGGGCACGACCAAACACAGTAAGCTGCTTATTCTGGGCTCAGGACCGTGTAGGCTGGA
GCTGCTTCG-3 ', SEQ ID NO.1；
P2:5 '-AAAGTCCGGCGATGTCCAGCGTCTCGATATTATCGCTCTGCTGCGTGTCGATGGGAATTAGC
CATGGTCC-3 ', SEQ ID NO.2；
Primer P1, P2 are made of front and back two parts respectively, the part and gene to be knocked out trxB sequence that 5 ' ends underline
Homologous, it is complementary with plasmid pKD3 chloramphenicol resistance gene cat two sides sequence that 3 ' ends do not underline part.
The building of 2 Ns of source Salmonella enteritidis trxB gene-deleted strains of embodiment
TrxB gene is knocked out using λ-Red methods of homologous recombination, the specific steps are as follows: be transferred to plasmid pKD46
Salmonella enteritidis SECL-5 competent cell obtains SECL-5/pKD46 positive strain；By positive strain SECL-5/pKD46 system
For at competent cell；The trxB gene knockout target practice segment electrotransformation that the amplification of embodiment 1 obtains is entered into SECL-5/pKD46
Competent cell replaces trxB gene using chloromycetin gene cat；Wherein electrotransformation step are as follows: by beating for P1, P2 amplification purification
1 μ l of target segment and 50 μ l Electroporation-competent cells mix, and mixture is transferred in the electric revolving cup of pre-cooling by ice bath 5-10min,
Outside droplet is wiped, is put into electroporation groove, adjusting electroporation parameter is 1.8KV, and 25 μ F of pulse, shocks by electricity by 200 ohm of resistance
After converted product is moved into immediately in the LB culture mediums of 37 DEG C of preheatings, 1h are cultivated in 37 DEG C of shakings, and it is mould that product coating contains chlorine
The nutrient agar plate of element.PCR identification, the reaction of PCR amplification are carried out through resistance screening, and using specificity identification primer P3, P4
System is 25 μ l, comprising: 0.5 2.5 μ l of μ l, 10 × buffer of each 2.5 μ l, rTaq enzyme of 1 μ l, dNTPs of primer P3, P4, bacterium solution
1 μ l of culture is template, remaining is supplied with water；Response procedures are as follows: 94 DEG C of initial denaturation 5min, 25 circulations (94 DEG C of 45s, 54 DEG C
30s, 72 DEG C of 90s), 72 DEG C extend 10min eventually；The band (as shown in Figure 1) of 2089bp size is obtained, screening obtains positive restructuring
Sub- SECL-5trxB::cat；Competent cell is prepared using positive SECL-5trxB::cat bacterial strain, electrotransformation imports pCP20 matter
Grain eliminates chloramphenicol resistance marker, and obtaining secondary recombinant bacterial strain is trxB gene-deleted strain SECL-5 Δ trxB.Utilize primer
P3, P4 carry out PCR identification to it, obtain the band (as shown in Figure 1) of 1159bp size, are further confirmed by sequencing and are lacked
Lose bacterial strain；The result shows that trxB gene deletion strains SECL-5 Δ trxB (KO) is constructed successfully.
Wherein, primer P3 and P4 are as follows:
P3:5 '-TATGTCCGCAGTTACGTTGGC-3 '；SEQ ID NO.3；
P4:5 '-CAGGATACCTCTTACACCCTCACC-3 '；SEQ ID NO.4.
The building of 3 Ns of source Salmonella enteritidis trxB gene covering bacterial strains of embodiment
Using ox source Salmonella enteritidis trxB gene as template, P5, P6 primer amplification trxB genetic fragment, PCR amplification are utilized
Reaction system 15 μ l, SECL-5 bacterium of μ l, 10 × buffer of each 2.5 μ l, rTaq enzyme of 1 μ l, dNTPs of totally 50 μ l: primer P5, P6
1 μ l of pnca gene group template, remaining is supplied with water；Response procedures are as follows: 94 DEG C of initial denaturation 5min, 30 circulations (94 DEG C of 45s, 52 DEG C
30s, 72 DEG C of 90s), 72 DEG C extend 10min eventually；The band for obtaining 1347bp size, is cloned on pBR322 plasmid, obtains
Cover pBR322 plasmid-trxB.Covering pBR322 plasmid-trxB is converted into SECL-5 Δ trxB mutant strain, is constructed back
It mends bacterial strain SECL-5 Δ trxB/pBR322-trxB (RS).
Wherein, primer P5 and P6 are as follows:
P5:5 '-CGGGATCCATGGGCACGACCAAACACAGTAAGC-3'；BamHI；SEQ ID NO.5；
P6:5 '-GCGTCGACCTATTTGCTCGCGTCGGCCAGA-3'；Sal I；SEQ ID NO.6.
4 growth rate measurment of embodiment
Wild-type strain SECL-5 (WT), trxB gene-deleted strain (KO) and covering bacterial strain (RS) are inoculated with liquid LB respectively
In, 37 DEG C of overnight incubations, transferring by 1:50 next day, (1L culture medium needs MgSO to fresh LB or Minimal culture medium42mmol/L、
CaCl20.1mmol/L、Na2PO4·7H2O 12.8g、KH2PO43.0g、NaCl 0.5g、NH4Cl 1.0g, glucose 4g), 37
DEG C synchronous shaking culture；Bacteria liquid sample is drawn every 2h, measures its absorbance OD600, draw growth curve.
The results show that under 37 DEG C of condition of culture, 3 plants of bacterium are raw in LB culture medium (Fig. 2) or Minimal culture medium (Fig. 3)
Long speed no significant difference.The above results show that the missing of trxB gene does not influence Salmonella enteritidis growth rate, not shadow
Ring the basic metabolism of Salmonella enteritidis.
The measurement of 5 biochemical characteristic of embodiment
Using Mei Liai (ATB) automatic biochemical identification systems to wild-type strain, trxB gene-deleted strain and covering bacterial strain
It carries out Analysis of Biochemical Characteristics: strain to be tested being cultivated in crossing on nutrient agar, scraping bacterium colony is resuspended in sterile saline, adjusts
Whole cell concentration to Maxwell turbidity is 0.5, by bacterium solution drop in ID32E biochemical identification carton detection hole, the every hole 40L/.It is placed in wet box
In 37 DEG C of culture 16h；Machine in test card is identified.
The results are shown in Table 1, and three's biochemical reaction spectrum is consistent, and showing trxB gene delection not influences Salmonella enteritidis biochemistry
Characteristic.
1. biochemical identification of table
Embodiment 6trxB gene delection influences ox source Salmonella enteritidis environment resistant stress ability
The single bacterium colony for picking them separately wild-type strain, trxB gene-deleted strain and covering bacterial strain is inoculated in liquid LB culture
Base, thalline were collected by centrifugation after 37 DEG C of overnight incubations, and thallus, adjustment bacterial concentration to 10 is resuspended with sterilizing PBS7cfu/mL.It is acid
Under stressed condition (pH 3.5), 37 DEG C of shaken cultivation 1h；Oxidative stress condition (10mmoL/L H2O2) under, 37 DEG C of shaken cultivations
30min.To stress under the conditions of bacterial cultures count.Test is repeated 3 times, and experimental data statistics uses prism 5.0
Software analysis.
As a result, it has been found that acidic environment (pH3.5, Fig. 4) does not significantly affect the growth of trxB gene-deleted strain, and lack
Losing strain, treated, and tolerance is remarkably decreased with 10mmol/L hydrogen peroxide (Fig. 5), shows that TrxB albumen participates in regulation enteritis
Detection of Salmonella is to anti-oxidation stress.
Influence of the embodiment 7trxB gene delection to survival ability in Salmonella enteritidis macrophage
Recovery source of mouse macrophage Raw264.7, it is good to be passaged to cultivation conditions, and Raw264.7 macrophage is inoculated with 12
Porocyte culture plates digest macrophage when cell density reaches 60%-70% from culture dish, and sterilizing PBS is washed
Wash primary, progress cell count.By SECL-5 wild-type strain, trxB gene-deleted strain and covering strain culturing to logarithmic growth
Phase carries out count of bacteria after sufficiently being washed three times with sterilizing PBS, is 10:1 dosage infection cell according to MOI, bacterium and thin
Born of the same parents mix well, 37 DEG C of incubation 45min.4 removing free bacterias of cell are washed with sterilizing PBS afterwards, replacement is celebrated containing 50g/mL
The culture medium of big mycin.Different time points (3h and 23h) after infection is thin with the sterilizing water-splitting containing 1%TritonX-100
Born of the same parents, cell cracking drop are counted on LB agar, calculate the endogenous multiplication rate (23h bacterium number/3h bacterium number) of Salmonella enteritidis.
As a result as shown in fig. 6, trxB gene-deleted strain Raw264.7 survive into the cell and proliferative capacity far below wild-type strain and
Bacterial strain is covered, shows that TrxB albumen participates in survival and proliferation of the regulation Salmonella enteritidis in macrophage.
8 Pathogenic Tests of embodiment
The Kunming mouse of 65 half male and half females is grouped at random, every group 5, totally 13 groups, wherein 6 groups are SECL-5 infection experiment
Group, another 6 groups are SECL-5 Δ trxB gene-deleted strain infection experiment group, and remaining 1 group is control group.Collect logarithmic growth phase Bacteria Culture
Object preparation attacks poison and uses bacterium.
When challenge test, every group of mouse presses 0.5mL/ respectively, and only (bacterial concentration is 1.0 × 104cfu/mL、1.0×
105cfu/mL、1.0×106cfu/mL、1.0×107cfu/mL、1.0×108cfu/mL、1.0×109Cfu/mL dosage) into
Row intraperitoneal injection, control group inject equivalent sterile saline.It is normally fed after attacking poison, it is primary every 6h observation after injection, even
Continuous observation 13d records each group death toll in detail and calculates LD50Value.
The results are shown in Table 2, the LD of SECL-5 and deletion mycopremna50Respectively 1.99 × 105With 7.94 × 106Cfu is increased
About 35 times, show that trxB gene-deleted strain virulence is remarkably decreased, it is in close relations between trxB gene and Salmonella enteritidis virulence.
The LD of table 2 Salmonella enteritidis SECL-5 wild-type strain and Δ trxB gene-deleted strain50Measurement result
Each embodiment in this specification is described in a progressive manner, the highlights of each of the examples are with other
The difference of embodiment, the same or similar parts in each embodiment may refer to each other.To the upper of the disclosed embodiments
It states bright, enables those skilled in the art to implement or use the present invention.Various modifications to these embodiments are to ability
Will be apparent for the professional technician in domain, the general principles defined herein can not depart from it is of the invention
In the case where spirit or scope, realize in other embodiments.Therefore, the present invention be not intended to be limited to it is shown in this article these
Embodiment, and it is to fit to the widest scope consistent with the principles and novel features disclosed herein.
Sequence table
<110>Hebei Science & Technology Normal College
<120>application of ox source Salmonella enteritidis trxB gene delection
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 71
<212> DNA
<213> Artificial Sequence
<400> 1
ctcatgggca cgaccaaaca cagtaagctg cttattctgg gctcaggacc gtgtaggctg 60
gagctgcttc g 71
<210> 2
<211> 70
<212> DNA
<213> Artificial Sequence
<400> 2
aaagtccggc gatgtccagc gtctcgatat tatcgctctg ctgcgtgtcg atgggaatta 60
gccatggtcc 70
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 3
tatgtccgca gttacgttgg c 21
<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 4
caggatacct cttacaccct cacc 24
<210> 5
<211> 33
<212> DNA
<213> Artificial Sequence
<400> 5
cgggatccat gggcacgacc aaacacagta agc 33
<210> 6
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 6
gcgtcgacct atttgctcgc gtcggccaga 30
Claims (5)
1. N source Salmonella enteritidis trxB gene delection is for reducing the virulence of ox source Salmonella enteritidis.
2. N application of the source Salmonella enteritidis trxB gene delection in the Salmonella enteritidis vaccine of ox source.
3. the construction method of N source Salmonella enteritidis trxB gene-deleted strain, which is characterized in that specific step is as follows:
(1) primer P1 and P2 are utilized, through PCR amplification, constructs trxB gene knockout target practice segment；The primer sequence are as follows:
P1:5 '-CTCATGGGCACGACCAAACACAGTAAGCTGCTTATTCTGGGCTCAGGACCGTGTAGGCTGGAGCTG
CTTCG-3 ', SEQ ID NO.1；
P2:5 '-AAAGTCCGGCGATGTCCAGCGTCTCGATATTATCGCTCTGCTGCGTGTCGATGGGAATTAGCCATG
GTCC-3 ', SEQ ID NO.2；
(2) plasmid pKD46 is transferred to SECL-5 competent cell, obtains SECL-5/pKD46 positive strain, and by the positive
Bacterial strain SECL-5/pKD46 is prepared into competent cell；
(3) trxB gene knockout target practice segment electrotransformation is entered into SECL-5/pKD46 competent cell, obtains positive recombinant
SECL-5trxB::cat；
(4) competent cell is prepared using positive SECL-5trxB::cat bacterial strain, electrotransformation imports pCP20 plasmid, and it is mould to eliminate chlorine
Plain resistance marker obtains purpose bacterial strain SECL-5 Δ trxB；
(5) PCR verifying is carried out to purpose bacterial strain SECL-5 Δ trxB.
4. the construction method of ox source Salmonella enteritidis trxB gene-deleted strain according to claim 3, which is characterized in that step
Suddenly each 2.5 μ l, rTaq enzyme of 1 μ l, dNTPs of the reaction system of (1) described PCR amplification totally 50 μ l, primer P1, P2 1 μ l, 10 ×
5 μ l of buffer, 100 times of diluted 1 μ l of pKD3 plasmid, remaining is supplied with water；
PCR response procedures are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of 45s, 54 DEG C of 30s, 72 DEG C of 60s, totally 25 recycle；72 DEG C of ends prolong
Stretch 10min.
5. the construction method of ox source Salmonella enteritidis trxB gene-deleted strain according to claim 3, which is characterized in that step
Suddenly the condition of (3) described electrotransformation are as follows: voltage 1.8KV, pulse 25 μ F, 200 Ω of resistance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811321305.7A CN109402036A (en) | 2018-11-07 | 2018-11-07 | The application of ox source Salmonella enteritidis trxB gene delection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811321305.7A CN109402036A (en) | 2018-11-07 | 2018-11-07 | The application of ox source Salmonella enteritidis trxB gene delection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109402036A true CN109402036A (en) | 2019-03-01 |
Family
ID=65472353
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811321305.7A Pending CN109402036A (en) | 2018-11-07 | 2018-11-07 | The application of ox source Salmonella enteritidis trxB gene delection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109402036A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1326466A (en) * | 1998-09-14 | 2001-12-12 | 拉斯·奥斯特加德·佩德森 | Method of producing functional immunoglubulin superfaminly protein |
| CN1966526A (en) * | 2006-11-23 | 2007-05-23 | 中国人民解放军第二军医大学 | Evolved immunoglobulin binding molecule, and its preparation method and uses |
| WO2008076806A9 (en) * | 2006-12-15 | 2008-08-07 | Univ Boston | Compositions and methods to potentiate colistin activity |
| CN103396485A (en) * | 2005-12-15 | 2013-11-20 | 健泰科生物技术公司 | Method and composition for targeting polyubiquitin |
| CN108486029A (en) * | 2018-04-24 | 2018-09-04 | 河北科技师范学院 | The application of Salmonella enteritidis sufB gene delections |
-
2018
- 2018-11-07 CN CN201811321305.7A patent/CN109402036A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1326466A (en) * | 1998-09-14 | 2001-12-12 | 拉斯·奥斯特加德·佩德森 | Method of producing functional immunoglubulin superfaminly protein |
| CN103396485A (en) * | 2005-12-15 | 2013-11-20 | 健泰科生物技术公司 | Method and composition for targeting polyubiquitin |
| CN1966526A (en) * | 2006-11-23 | 2007-05-23 | 中国人民解放军第二军医大学 | Evolved immunoglobulin binding molecule, and its preparation method and uses |
| WO2008076806A9 (en) * | 2006-12-15 | 2008-08-07 | Univ Boston | Compositions and methods to potentiate colistin activity |
| CN108486029A (en) * | 2018-04-24 | 2018-09-04 | 河北科技师范学院 | The application of Salmonella enteritidis sufB gene delections |
Non-Patent Citations (3)
| Title |
|---|
| EVA BJUR等: "Thioredoxin 1 Promotes Intracellular Replication and Virulence of Salmonella enterica Serovar Typhimurium", 《INFECTION AND IMMUNITY》 * |
| PRASANTA KUMAR K. MISHRA等: "Cloning and Sequencing of Thioredoxin Reductase (trxB) Gene of Salmonella enterica serovar Typhimurium Isolated from Poultry", 《JOURNAL OF ANIMAL RESEARCH》 * |
| 黄静玮等: "沙门氏菌分子生物学研究进展", 《中国***共患病学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108486029B (en) | Application of salmonella enteritidis sufB gene deletion | |
| Jung et al. | Comparison of pathogenic and non-pathogenic Enterococcus cecorum strains from different animal species | |
| CN109439634B (en) | Pseudorabies virus gene engineering attenuated vaccine strain and application thereof | |
| CN103981139B (en) | One strain brucella low virulent strain and vaccine thereof | |
| CN106479924A (en) | The preparation of a kind of Clostridium butyricum and clostridium butyricum active bacteria preparation and application | |
| CN110527669B (en) | Porcine pseudorabies virus gene deletion strain and construction method and application thereof | |
| CN113862230A (en) | Porcine pseudorabies virus gene deletion strain, vaccine composition, preparation method and application thereof | |
| CN104845916A (en) | Rough type Brucella melitensis attenuated strain and vaccine thereof | |
| CN102344892B (en) | Chinese isolate of Leachiii mycoplasma, isolation culture medium and purpose thereof | |
| CN111304119B (en) | Feeding bacillus subtilis for degrading fumonisins and application thereof | |
| KR101873682B1 (en) | Novel Lactobacillus acidophilus KNU-02 having immune enhancing activity and use thereof | |
| CN105132310B (en) | One plant of cold water fish probiotics Bacillus strain and application thereof | |
| CN108865931A (en) | One bacillus and its application | |
| CN107828741A (en) | The dual-gene missing low virulent strain of pseudorabies virus and its application | |
| CN109402036A (en) | The application of ox source Salmonella enteritidis trxB gene delection | |
| Zhao et al. | Postharvest decay of the cultivated mushroom Pleurotus eryngii caused by Lactococcus lactis subsp. lactis | |
| CN104498417A (en) | Streptococcus suis chorismate-synthase gene deletion strain, and construction method and application thereof | |
| CN109517773A (en) | The application of ox source Salmonella enteritidis nmpC gene delection | |
| CN113512559B (en) | Mycoplasma bovis Mbov _0701 mutant gene and mutant strain and application thereof | |
| CN106916768A (en) | A kind of entomopathogenic nematode symbiotic bacteria and application with inhibition of potato tar spot | |
| CN105039221A (en) | Cold water fish probiotics arthrobacter strain and application thereof | |
| CN105483049A (en) | Pullorum disease salmonella spiC-rfaH double-gene knockout attenuated strain and DIVA vaccine application thereof | |
| CN105524862A (en) | Salmonella pullorum spiC-rfaJ double-gene-knockout attenuated strain and DIVA vaccine application | |
| CN108004181B (en) | Bacillus methylotrophicus and culture and application thereof | |
| CN112481188A (en) | Preparation method of mixed bacterium agent for efficiently producing beta-glucuronidase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190301 |
|
| RJ01 | Rejection of invention patent application after publication |