CN102206607A - Three genes deleted mutant of enterohemorrhagic escherichia coli O157:H7 - Google Patents
Three genes deleted mutant of enterohemorrhagic escherichia coli O157:H7 Download PDFInfo
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Abstract
The invention relates to a three genes deleted mutant of enterohemorrhagic escherichia coli (EHEC) O157:H7, and belongs to the biotechnology field. A preparation method for the three genes deleted mutant of EHEC O157:H7 comprises: 1), inducing resistance to obtain an EHEC O157:H7 strain with streptomycin resistance; 2) sequential constructing deleted mutants of EHEC O157:H7 (delta hly), EHEC O157:H7 (delta hly delta stx) and EHEC O157:H7 (delta hly delta stx delta toxB). Compared to a parent strain, the EHEC O157:H7 (delta hly delta stx delta toxB) has lower pathogenicity, lower colonization capability in the mice and better immunogenicity. According to the present invention, the EHEC O157:H7 (delta hly delta stx delta toxB) is constructed, and the pathogenicity and the colonization capability of the EHEC O157:H7 (delta hly delta stx delta toxB) are identified. The three genes deleted mutant of the EHEC O157:H7 (delta hly delta stx delta toxB) provided by the present invention can be applicable for developing an EHEC gene-deleted vaccine so as to vaccinate animals to reduce the colonization of the EHEC O157:H7, such that a possibility of EHEC O157:H7 contracting of people is reduced so as to guarantee physical and mental health of the people.
Description
One, technical field
The present invention relates to enterorrhagia Bacillus coil 0157: H7 three genetically deficient bacterial strains are specifically related to enterohemorrhagic Escherichia coli (enterohaemorrhagic
Escherichia coli, EHEC) structure and the characteristic research of O157:H7 hemolysin (Hly), shiga toxin (Stx) and ToxB deletion mycopremna belong to biological technical field.
Two, background technology
Enterohemorrhagic Escherichia coli (enterohaemorrhagic
Escherichia coli, EHEC) O157:H7 is that important food source property Amphixenosis is former.Over year, the food poisoning that EHEC O157:H7 causes comprises that all over the world all there is the outbreak of epidemic of different scales in China surplus in the of nearly 20.EHEC O157:H7 infected patient and asymptomatic carrier can be used as contagium.Very extensive in animal host's scope, wherein the ruminating animal bacterial bearing rate is higher.Comparatively speaking, animal is more even more important than the mankind as contagium, and it is the root of animal-derived food pollution often.The infection of EHEC O157:H7 because of have outbreak of epidemic trend, intensive is pathogenic and characteristics such as lethality and antibiotic therapy can aggravate one's illness, and has become global public health problem.At present it is infected shortage effectively preventing method, its pathogenesis is still needed and will be furtherd investigate.
Studies show that, EHEC O157:H7 adheres to and the correlation factor of causing a disease has (Locus of enterocyte effacement, LEE) the III type excretory system albumen of pathogenicity island coding, the shiga toxin I and the II (Stx I and II) of prophage coding, big plasmid pO157 coding such as some virulence factors such as hemolysin (Hly) and ToxB.In bacterial body, the Stx II is a secretion type expression, and the Stx I is to express in the born of the same parents.Two kinds of toxin are formed by 1 A subunit and 5 B subunits, and A subunit has toxicity in the cell, can cause protein synthesis to stop with 28S rRNA effect, is the pathologic basis that EHEC O157:H7 causes clinical manifestation; B subunit has the cell binding characteristic, can combine with the cell with special receptor (Gb3), thereby guiding A subunit plays a role.Stx can cause infected people's diarrhoea, and hemorrhagic colitis (Hc) or hemolytic urinary tract syndromes general complication such as (Hus) may occur.Nearly all O157 bacterial strain EHEC hemolysin albumen of all encoding, and EHEC O157:H7 can be a raw material with oxyphorase and protoheme, growth and breeding produces more toxin and causes pathology fast.Hly produces the product that needs 4 kinds of continuous gene HlyC, A, B, D, and wherein HlyA is a structure gene.Hly and intestinal bacteria alpha hemolysin (α-Hly) belong to together RTX(Repeat toxin) toxin family, base sequence has 60% similarity.By the big plasmid pO157 coding of EHEC O157:H7, because of with clostridium difficile (Clostridium difficile) toxin B homology being arranged, called after ToxB, whole reading frame 9501bp.ToxB is by the indirect synthetic and secretion that influences LEE pathogenicity island proteins encoded of some mechanism, thus the adhesion field planting effect of reduction EHEC O157:H7.
With EHEC O157:H7(
Stx1
- Stx2+
Hly+
ToxB+) be parent plant, at
Stx2A,
HlyWith
ToxBIt is right that three full gene upstream and downstream sequences design Auele Specific Primer respectively, and amplification upstream and downstream homology arm is chosen spectinomycin simultaneously, gentamicin and kanamycin gene expression cassette are as selection markers, successively make up three gene-deleted strains, finally obtain three genetically deficient bacterial strains, i.e. O157:H7(△
Hly△
Stx△
ToxB) gene-deleted strain.The strain of two aspect evaluations disappearances is to adhering to and the influence of field planting in external and the body respectively to choose Balb/c mouse that HEp-2 cell and Streptomycin sulphate handle, and the gene-deleted vaccine that is used for prevention and control EHEC O157:H7 for development lays the foundation and technology platform is provided.
Three, find content
Technical problem the object of the invention is to make up enterohemorrhagic Escherichia coli (enterohaemorrhagic
Escherichia coli, EHEC) O157:H7
Hly, stxWith
ToxBThree gene-deleted strains are studied the ability of its field planting in enteron aisle and pathogenic, and then for development EHEC O157:H7 gene-deleted vaccine lays the foundation, reduce the animal bacterial bearing rate, and then reduce the chance of human infection EHEC, ensure the human physical and mental health.
Technical scheme specific embodiments of the present invention is as follows:
Enterorrhagia Bacillus coil 0157: the H7 deletion mycopremna is characterized in that said bacterium disappearance
HlyWhole A gene,
Stx2 whole A gene, and
ToxBWhole gene.
Make up the method for above-mentioned deletion mycopremna, comprising:
(1) makes up EHEC O157:H7 hlyA gene-deleted strain, i.e. EHEC O157:H7(△
Hly) structure
1. obtain
HlyHN and HC and
SpcGene, parallel-series are cloned into the pGEM-T carrier
Land according to GenBank
HlySequence, sequence number are NC_007414, design two pairs of Auele Specific Primers, and the DNA for preparing with EHEC O157:H7 bacterial strain is a template, respectively amplification
HlyGene 5 ' end upstream and 3 ' end downstream 655bp and 834bp sequence, wherein
Hly-NR comprises
HlyInitial 3 bases of gene 5 ' end,
Hly-CF comprises
Hly3 bases in 3 ' end end:
hly-NF：5-tcg
ggatcctctaatgcttttgacgtc-3
hly-NR：5-gcc
gaattcgtcatattagttttctttc-3
hly-CF：5-?gct
gaattcgagactaaaagaggaggt-3
hly-CR：5-aat
gcatgctagcacccagttcgacgt-3
Hly-NF and
Hly-CR primer two ends add restriction enzyme site respectively
BamHI and
SphI and protectiveness base,
Hly-NR and
Hly-CF primer two ends add same restriction enzyme site
EcoRI and protectiveness base, underscore partly is restriction enzyme site, and the PCR reaction conditions is: 95 ℃ of pre-sex change 5min, 94 ℃ of 1min, 55 ℃ of 30s, 72 ℃ of 1min, totally 30 circulations, last 72 ℃ are extended 5min, with the negative contrast of distilled water;
According to the pSET4s sequence that GenBank lands, sequence number is AB055650, designs a pair of Auele Specific Primer S
Pc-F and S
Pc-R is that template amplification is whole with the pSET4s plasmid
SpcExpression cassette:
Spc-F：5-cgt
gaattccgttcgtgaatacatgttat-3
Spc-R：5-?atg
gaattcgcgcttaccaattagaatga-3
Spc-F and
Spc-R primer two ends add same restriction enzyme site
EcoRI and protectiveness base, underscore partly is restriction enzyme site.The PCR reaction conditions is: 95 ℃ of pre-sex change 5min, and 94 ℃ of 1min, 54 ℃ of 30s, 72 ℃ of 1.2min, totally 30 circulations, last 72 ℃ are extended 7min, with distilled water and the negative contrast of EHEC O157:H7 DNA;
Hly-NF and
Hly-NR,
Hly-CF and
Hly-CR,
Spc-F and
SpcThree pairs of primer amplifications of-R obtain the purpose band 655bp of corresponding size, 834bp and 1137bp, and use 1% agarose electrophoresis respectively, cutting glue weighs, 75 ℃ of effect 10min after the DE-A damping fluid of 3 times of volumes of adding, the DE-B damping fluid that then adds 0.5 times of volume DE-A damping fluid, put upside down mixing, transfer liquid is in miniature pillar, and the centrifugal 1min of 12000g, abandons liquid, add lavation buffer solution I 500ul, and the centrifugal 1min of 12000g, abandon liquid, add lavation buffer solution II 750 ul, the centrifugal 1min of 12000g, abandon liquid, repeated washing damping fluid II is washed once, then the centrifugal 1min of blank pipe 12000g, shift pillar in the eppendorf of clean 1.5ml pipe, and adding the 60ul elution buffer, the centrifugal 1min of 12000g collects centrifugate, be purified 655bp, 834bp and 1137bp fragment, called after HN, HC and
SpcFragment;
Will
EcoRThe I enzyme is cut HN and each 3ul(6ng/ul of HC fragment), T4 DNA connects damping fluid and each 1.0ul of T4 dna ligase (350U/ul), and distilled water 2.0ul adds an eppendorf pipe simultaneously, and behind the mixing, 4 ℃ of connections are spent the night; Get spending the night of HN-HC and connect product 3.5ul(4ng/ul) and pGEM-T 1.0ul(50ng/ul), T4 DNA 2 * connection damping fluid 5.0 ul and T4 dna ligase 0.5ul(350U/ul) together add eppendorf pipe, behind the mixing, 4 ℃ of connections are spent the night, be used to transform the Top10 competent cell, the single bacterium colony of picking carries out incubated overnight, extracts plasmid, uses
BamHI and
SphI double digestion, 37 ℃ of water-bath 3h, agarose gel electrophoresis are identified, can see that the band of a 1489bp occurs, and double digestion is identified the order-checking of male bacterium, obtain the pGEM-T-HN-HC recombinant plasmid, and glue reclaims
SpcFragment 4.5ul(3.6ng/ul), together add an eppendorf pipe with pMD18-T 0.5ul and solution I 4.5ul, behind the mixing, 16 ℃ connect 3h, are used to transform the Top10 competent cell, and the single bacterium colony of picking carries out incubated overnight, extracts plasmid, uses
EcoRI single endonuclease digestion, 37 ℃ of water-bath 2h, agarose gel electrophoresis are identified, see that the band of a 1137bp occurs, and double digestion is identified the order-checking of male bacterium, obtain the pMD18-T-spc recombinant plasmid;
2. the pMEG-375-HSH reorganization knocks out the structure of plasmid
PGEM-T-HN-HC recombinant plasmid and pMD18-T-
SpcRecombinant plasmid is used respectively
ECo
RThe I single endonuclease digestion, the alkaline phosphatase dephosphorylation is handled, and 4 ℃ of connections transform the Top10 competent cell, process
ECo
RThe I enzyme is cut and is obtained positive recombinant plasmid pGEM-T-HN-
Spc-HC, pGEM-T-HN-
Spc-the HC and the quick plasmid pMEG375 warm in nature that commits suiside use
BamHI and
SphThe I double digestion, respectively get 2ul(7.1ng/ul), be connected damping fluid and each 1.0ul of T4 dna ligase (350U/ul) with T4 DNA, distilled water 4.0ul adds an eppendorf pipe simultaneously, and behind the mixing, 4 ℃ of connections are spent the night, transform the sm10 competent cell, with the screening of paraxin plate, obtain positive recombinant plasmid, called after pMEG375-HN-
Spc-HC, the positive gene that is acquisition knocks out plasmid pHSH;
3. bacterium solid phase mating
Adopt solid phase filter hybridization method, inoculate recipient bacterium EHEC O157:H7 respectively and contain the donor bacterium sm10 of pHSH in 5mL LB liquid nutrient medium, spend the night in 37 ℃ of shaking culture, get the overnight culture of sm10 and EHEC O157:H7 then respectively in the 4:1 ratio, mixing, filter with filter, bacterium is collected on the filter membrane, filter membrane is positioned on the nonresistant LB agar plate, the germy one side of filter membrane upwards in 37 ℃ of overnight incubation, washes bacterium on the filter membrane with physiological saline then, after 1000 times of dilutions, separate application is in having Streptomycin sulphate and chlorampenicol resistant Strep
RCm
ROn the LB agar plate, 37 ℃ of overnight incubation, the single colony inoculation of picking is in Strep
RCm
RIncrease bacterium among the liquid LB, then bacterium liquid is carried out O157 serotype encoding gene
RfbEWith
SpcCan encode simultaneously the two bacterium colony of gene amplification, acquisition, promptly
RfbE+
Spc+;
4. paraxin concentration method
The chemical sproof hybrid strain of paraxin of inoculating above-mentioned screening is in the LB liquid nutrient medium, 37 ℃ of overnight incubation, transfer in the fresh LB liquid nutrient medium with volume ratio 2% inoculum size, cultivate 1-2h for 37 ℃, add paraxin and continue to cultivate 1-2h, press 1mg/mL and add the D-seromycin, put 37 ℃ more rapidly and continue to cultivate 1-2h, the centrifugal collection thalline of 8000r/min, wash once with physiological saline, coat on the 10% sucrose flat board that contains 100ug/ml Spc after 100 times of dilutions, cultivate 16-20h for 30 ℃, the bacterium colony that grows is inoculated the dull and stereotyped and common LB flat board of the LB that contains paraxin respectively, screening paraxin sensitive strain;
5. EHEC O157:H7(△
Hly) screening and acquisition
The centrifugal collection thalline of the responsive single bacterium colony culture of paraxin, resuspended, boil 7min, 4 ℃ of centrifugal collection supernatants are template to be detected, prepare the template supernatant of EHEC O157:H7 simultaneously, use
Spc-F and
Spc-R, design
Hly-F and
HlyThe a pair of primer of-R:
Hly-F:5-cacacggagcttataatattctgtca-3
Hly-R:5-aatgttatcccattgacatcatttgact-3
Carry out pcr amplification respectively, filtering out reorganization has
SpcThe EHEC O157:H7 of gene, i.e. EHEC O157:H7(△
Hly); With full bacterium polyclonal antiserum respectively with EHEC O157:H7 and EHEC O157:H7(△
Hly) carry out the protein immunoblotting detection, through the evaluation of gene level and protein level, successfully obtain the EHEC O157:H7 bacterial strain of Hly disappearance, i.e. EHEC O157:H7(△
Hly) bacterial strain;
6. the full bacterium polyclonal antiserum preparation of EHEC O157:H7
EHEC O157:H7 inoculation LB liquid nutrient medium 3mL cultivates 8h for 37 ℃, gets the 1mL culture, and centrifugal collection thalline with physiological saline washing 2 times, after 10 times of dilutions, carries out emulsification with equal-volume Fu Shi Freund's complete adjuvant, subcutaneous multi-point injection rabbit; After 3 weeks, after the emulsification of equal-volume freund 's incomplete adjuvant, subcutaneous multi-point injection rabbit; After 2 weeks, serum is collected in the heart blood sampling, is the full bacterium polyclonal antiserum of EHEC O157:H7;
(2) EHEC O157:H7(△
Hly△
Stx) structure
1. at first obtain
Stx2The SN of A and SC reach
GmGene, parallel-series are cloned into the pGEM-T carrier
Land according to GenBank
Stx2A sequence, sequence number are NC_007414, design two pairs of Auele Specific Primers, and the DNA for preparing with EHEC O157:H7 bacterial strain is a template, respectively amplification
Stx2A gene 5 ' end upstream and 3 ' end downstream 397bp and 229bp sequence, wherein
Stx2A-NR comprises
Stx2 Initial 3 bases of A gene 5 ' end,
Stx2A-CF comprises
Stx23 bases in A3 ' end end:
stx2A-NF：5-aat
ggatcctacaaatccagcaagggcc-3
stx2A-NR：5-gcc
gaattccgaaaagtctatcgtaaac-3
stx2A-CF：5-tta
gaattcgcggcggattgtgctaaag-3
stx2A-CR：5-ccc
gcatgccattattaaactgcacttca-3
Stx2A-NF and
Stx2A-CR primer two ends add restriction enzyme site respectively
BamHI and
SphI and protectiveness base,
Stx2A-NR and
Stx2A-CF primer two ends add same restriction enzyme site
EcoRI and protectiveness base, underscore partly is restriction enzyme site, and the PCR reaction conditions is: 95 ℃ of pre-sex change 5min, 94 ℃ of 1min, 56 ℃ of 30s, 72 ℃ of 30s, totally 30 circulations, last 72 ℃ are extended 5min, with the negative contrast of distilled water;
The sequence number that lands according to GeneBank is the Fastbac of AY598466.1
TM1Sequence designs a pair of Auele Specific Primer
Gm-F and
Gm-R, it is whole to increase
GmExpression cassette, 811bp;
Gm-F：5-
gaattcaccgtggaaacgcatgaag-3
Gm-R：5
-gaattcacggcttgaacgaattgtt-3
Gm-F and
Gm-R primer two ends add restriction enzyme site
EcoRI, underscore partly are restriction enzyme site, and the PCR reaction conditions is: 95 ℃ of pre-sex change 5 min, and 94 ℃ of 1 min, 56 ℃ of 30s, 72 ℃ of 50s, totally 30 circulations, last 72 ℃ are extended 5 min, with the negative contrast of distilled water;
Gm-F and
Gm-R primer two ends add same restriction enzyme site
EcoRI and protectiveness base, underscore partly is restriction enzyme site, and the PCR reaction conditions is: 95 ℃ of pre-sex change 5min, 94 ℃ of 1min, 56 ℃ of 30s, 72 ℃ of 1.0min, totally 30 circulations, last 72 ℃ are extended 7min, with distilled water and the negative contrast of EHEC O157:H7 DNA;
Stx2A-NF and
Stx2A-NR,
Stx2A-CF and
Stx2A-CR,
Gm-F and
GmThree pairs of primer amplifications of-R obtain the purpose band 397bp of corresponding size, 229bp and 811bp, and use 1% agarose electrophoresis respectively, cutting glue weighs, 75 ℃ of effect 10min after the DE-A damping fluid of 3 times of volumes of adding, the DE-B damping fluid that then adds 0.5 times of volume DE-A damping fluid, put upside down mixing, transfer liquid is in miniature pillar, and the centrifugal 1min of 12000g, abandons liquid, add lavation buffer solution I 500ul, and the centrifugal 1min of 12000g, abandon liquid, add lavation buffer solution II 750 ul, the centrifugal 1min of 12000g, abandon liquid, repeated washing damping fluid II is washed once, then the centrifugal 1min of blank pipe 12000g, shift pillar in the eppendorf of clean 1.5ml pipe, and adding the 60ul elution buffer, the centrifugal 1min of 12000g collects centrifugate, be purified 397bp, 229bp and 811bp fragment, called after SN, SC and
GmFragment;
Will
EcoRThe I enzyme is cut SN and each 3ul of SC fragment (4ng/ul), and T4 DNA connects damping fluid and each 1.0 ul of T4 dna ligase (350U/ul), and distilled water 2.0ul adds an eppendorf pipe simultaneously, and behind the mixing, 4 ℃ of connections are spent the night; Get spending the night of SN-SC and connect product 3.5ul (2.7ng/ul) and pGEM-T 1.0ul (50ng/ul), T4 DNA 2 * connection damping fluid 5.0 ul and T4 dna ligase 0.5ul(350U/ul) together add eppendorf pipe, behind the mixing, 4 ℃ of connections are spent the night, be used to transform the Top10 competent cell, the single bacterium colony of picking carries out incubated overnight, extracts plasmid, uses
BamHI and
SphI double digestion, 37 ℃ of water-bath 3h, agarose gel electrophoresis are identified, can see that the band of a 626bp occurs, and double digestion is identified the order-checking of male bacterium, obtain the pGEM-T-SN-SC recombinant plasmid, and glue reclaims
GmFragment 4.5ul(5ng/ul), together add an eppendorf pipe with pMD18-T0.5ul (50ng/ul) and solution I 4.5ul, behind the mixing, 16 ℃ connect 3h, are used to transform the Top10 competent cell, and the single bacterium colony of picking carries out incubated overnight, extract plasmid, use
EcoRI single endonuclease digestion, 37 ℃ of water-bath 2h, agarose gel electrophoresis are identified, can see that the band of a 811bp occurs, and double digestion is identified the order-checking of male bacterium, obtain pMD18-T-
GmRecombinant plasmid;
2. the pMEG-375-SGS reorganization knocks out the structure of plasmid
PGEM-T-SN-SC recombinant plasmid and pMD18-T-
GmRecombinant plasmid is used respectively
ECo
RThe I single endonuclease digestion, the alkaline phosphatase dephosphorylation, 4 ℃ of connections transform the Top10 competent cell, process
ECo
RThe I enzyme is cut and is obtained positive recombinant plasmid pGEM-T-SN-
Gm-SC, pGEM-T-SN-
Gm-the SC and the quick plasmid pMEG375 warm in nature that commits suiside use
BamHI and
SphEach 2ul(11ng/ul of I double digestion), be connected damping fluid and each 1.0ul of T4 dna ligase (350U/ul) with T4 DNA, distilled water 4.0ul adds an eppendorf pipe simultaneously, behind the mixing, 4 ℃ of connections are spent the night, and transform the sm10 competent cell, with the screening of paraxin plate, obtain positive recombinant plasmid, called after pMEG375-SN-
Gm-SC, the positive gene that is acquisition knocks out plasmid pSGS;
3. bacterium solid phase mating
Adopt solid phase filter hybridization method, inoculate recipient bacterium EHEC O157:H7(△ respectively
Hly) and the donor bacterium sm10 that contains pSGS in 5mL LB liquid nutrient medium, spend the night in 37 ℃ of shaking culture, the 4:1 ratio is got sm10 and the EHEC O157:H7(△ that contains pSGS respectively by volume then
Hly) overnight culture, mixing, filter with filter, bacterium is collected on the filter membrane, filter membrane is positioned on the nonresistant LB agar plate, and the germy one side of filter membrane makes progress, in 37 ℃ of overnight incubation, wash after bacterium on the filter membrane carries out 1000 times of dilutions with physiological saline then, separate application is in having Streptomycin sulphate and chlorampenicol resistant Strep
RCm
ROn the LB agar plate, 37 ℃ of overnight incubation, the single colony inoculation of picking is in Strep
RCm
RIncrease bacterium among the liquid LB, then bacterium liquid is carried out O157 serotype encoding gene
RfbEWith
GmCan encode simultaneously the two bacterium colony of gene amplification, acquisition, promptly
RfbE+
Gm+;
4. paraxin concentration method
The chemical sproof hybrid strain of paraxin of inoculating above-mentioned screening is in the LB liquid nutrient medium, 37 ℃ of overnight incubation, transfer in the fresh LB liquid nutrient medium with 2% inoculum size, cultivate 1-2h for 37 ℃, add paraxin and continue to cultivate 1-2h, press 1mg/mL and add the D-seromycin, put 37 ℃ more rapidly and continue to cultivate 1-2h, the centrifugal collection thalline of 8000r/min is washed once with physiological saline, after 100 times of dilutions, coat on the 10% sucrose flat board that contains 50ug/ml Gm, cultivate 16-20h for 30 ℃, the bacterium colony that grows is inoculated the dull and stereotyped and common LB flat board of the LB that contains paraxin respectively, screening paraxin sensitive strain;
5. EHEC O157:H7(△
Hly△
Stx2A) screening and acquisition
The centrifugal collection thalline of the responsive single bacterium colony culture of paraxin, resuspended, boil 7min, 4 ℃ of centrifugal collection supernatants are template to be detected, prepare the template supernatant of EHEC O157:H7 simultaneously, use
Gm-F and
Gm-R, design
Stx2A-F and
Stx2The a pair of primer of A-R:
Stx2A-F:5-gcgttaatggagttcagtggt-3
Stx2A-R:5-cttaactcctttatttacccgttgt-3
Carry out pcr amplification respectively, filtering out reorganization has
GmThe EHEC O157:H7 of gene, i.e. EHEC O157:H7(△
Hly△
Stx2A); With full bacterium polyclonal antiserum respectively with EHEC O157:H7(△
Hly) and EHEC O157:H7(△
Hly△
Stx2A) carry out protein immunoblotting and detect,, successfully obtain the EHEC O157:H7 bacterial strain of Hly and Stx2A disappearance, i.e. EHEC O157:H7(△ through the evaluation of gene level and protein level
Hly△
Stx2A) bacterial strain;
(3) EHEC O157:H7(△
Hly△
Stx△
ToxB) structure
1. at first obtain
ToxBBN and BC and
KanGene, parallel-series are cloned into the pET32 carrier
Land according to GenBank
ToxBSequence, sequence number are NC_007414, design two pairs of Auele Specific Primers, and the DNA for preparing with EHEC O157:H7 bacterial strain is a template, respectively amplification
ToxBGene 5 ' end upstream and 3 ' end downstream 831bp and 863bp sequence, wherein
ToxB-NR is
ToxBGene a 5 ' end upstream 36-54 base,
ToxB-CF is
ToxBGene a 3 ' end downstream 32-50 base;
toxB-NF：5-tta
ggatccatccatcaggcgttgtgc-3
toxB-NR：5-ggg
gaattctttgattagcgacaacct-3
toxB-CF：5-gcc
gtcgactaatcttacccagcatca-3
toxB-CR：5-att
gcggccgcgagttatcagcccgtcat-3
ToxB-NF,
ToxB-NR,
ToxB-CF and
ToxB-CR primer two ends add restriction enzyme site respectively
BamThe H I,
EcoRI,
SalI and
NotI and and the protectiveness base, underscore partly is restriction enzyme site.The PCR reaction conditions is: 95 ℃ of pre-sex change 5 min, and 94 ℃ of 1 min, 58 ℃ of 30 s, 72 ℃ of 1 min, totally 30 circulations, last 72 ℃ are extended 5 min, with the negative contrast of distilled water;
The sequence number that lands according to GeneBank is the pET28 sequence of EF442785.1, designs a pair of Auele Specific Primer
Kan-F and
Kan-R, it is whole to increase
KanExpression cassette, 1192bp;
Kan-F：5-ccg
gaattcacggctacactagaaggac-3
Kan-R：5-?aga
gtcgacaaatgtgcgcggaaccccta-3
Kan-F and
Kan-R primer two ends add restriction enzyme site
EcoRI and
SalI and protectiveness base, underscore partly is restriction enzyme site, and the PCR reaction conditions is: 95 ℃ of pre-sex change 5 min, 94 ℃ of 1 min, 56 ℃ of 30s, 72 ℃ of 1.2 min, totally 30 circulations, last 72 ℃ are extended 7 min, with the negative contrast of distilled water;
ToxB-NF and
ToxB-NR,
ToxB-CF and
ToxB-CR,
Kan-F and
KanThree pairs of primer amplifications of-R obtain the purpose band 831bp of corresponding size, 863bp and 1192bp, and use 1% agarose electrophoresis respectively, cutting glue weighs, 75 ℃ of effect 10min after the DE-A damping fluid of 3 times of volumes of adding, the DE-B damping fluid that then adds 0.5 times of volume DE-A damping fluid, put upside down mixing, transfer liquid is in miniature pillar, and the centrifugal 1min of 12000g, abandons liquid, add lavation buffer solution I 500ul, and the centrifugal 1min of 12000g, abandon liquid, add lavation buffer solution II 750 ul, the centrifugal 1min of 12000g, abandon liquid, repeated washing damping fluid II is washed once, then the centrifugal 1min of blank pipe 12000g, shift pillar in the eppendorf of clean 1.5ml pipe, and adding the 60ul elution buffer, the centrifugal 1min of 12000g collects centrifugate, be purified 831bp, 863bp and 1192bp fragment, called after BN, BC and
KanFragment;
Will
BamThe H I and
EcoRThe I enzyme is cut BN fragment 3ul (9.6ng/ul) and pET32a carrier 4ul (10ng/ul), T4 DNA connects damping fluid and each 1.0ul of T4 dna ligase (350U/ul), distilled water 1.0ul adds an eppendorf pipe simultaneously, behind the mixing, 4 ℃ of connections are spent the night, and transform the Top10 competent cell, and the single bacterium colony of picking carries out incubated overnight, extract plasmid, use
BamThe H I and
EcoRI double digestion, 37 ℃ of water-bath 3h, agarose gel electrophoresis are identified, can see that the band of a 831bp occurs, and obtain the pET32a-BN recombinant plasmid, then will
EcoRI and
SalThe Kan that the I enzyme is cut is connected with the pET32a-BN recombinant plasmid, obtains pET32a-BN-Kan, at last will
SalI and
NotThe BC that the I enzyme is cut is connected with the pET32a-BN-Kan recombinant plasmid, obtains pET32a-BN-Kan-BC, transforms the Top10 competent cell, cultivates 16h for 37 ℃, and the dull and stereotyped single bacterium colony of picking is identified.To identify that male recombinant plasmid and pMEG375 carrier use respectively
BamHI and
NotAfter the I enzyme was cut, 4 ℃ of connections were spent the night, and transformed competence colibacillus cell sm10 with the screening of paraxin plate, obtains positive recombinant plasmid, called after pMEG375-BN-
Kan-BC, the positive gene that is acquisition knocks out plasmid pBKB;
2. bacterium solid phase mating
Adopt solid phase filter hybridization method, inoculate recipient bacterium EHEC O157:H7(△ respectively
Hly△
Stx) and the donor bacterium sm10 that contains pBKB in 5mL LB liquid nutrient medium, spend the night in 37 ℃ of shaking culture, get sm10 and the EHEC O157:H7(△ that contains pBKB respectively in the 4:1 ratio then
Hly△
Stx) overnight culture, mixing, filter with filter, bacterium is collected on the filter membrane, and filter membrane is positioned on the nonresistant LB agar plate, and the germy one side of filter membrane upwards, in 37 ℃ of overnight incubation, wash bacterium on the filter membrane with physiological saline then, after 1000 times of dilutions, separate application is in having Streptomycin sulphate and chlorampenicol resistant Strep
RCm
ROn the LB agar plate, 37 ℃ of overnight incubation, the single colony inoculation of picking is in Strep
RCm
RIncrease bacterium among the liquid LB, then bacterium liquid is carried out O157 serotype encoding gene
RfbEWith
KanCan encode simultaneously the two bacterium colony of gene amplification, acquisition, promptly
RfbE+
Kan+;
3. paraxin concentration method
The chemical sproof hybrid strain of paraxin of inoculating above-mentioned screening is in the LB liquid nutrient medium, 37 ℃ of overnight incubation, transfer in the fresh LB liquid nutrient medium with 2% inoculum size, cultivate 1-2h for 37 ℃, add paraxin and continue to cultivate 1-2h, press 1mg/mL and add the D-seromycin, put 37 ℃ more rapidly and continue to cultivate 1-2h, the centrifugal collection thalline of 8000r/min is washed once with physiological saline, after 100 times of dilutions, coat and contain on the 100ug/ml kantlex 10% sucrose flat board, cultivate 16-20h for 30 ℃, the bacterium colony that grows is inoculated the dull and stereotyped and common LB flat board of the LB that contains paraxin respectively, screening paraxin sensitive strain;
4. EHEC O157:H7(△
Hly△
Stx△
ToxB) screening and acquisition
The centrifugal collection thalline of the responsive single bacterium colony culture of paraxin, resuspended, boil 7min, 4 ℃ of centrifugal collection supernatants are template to be detected, prepare the template supernatant of EHEC O157:H7 simultaneously, use
Kan-F and
Kan-R, design
ToxB-F and
ToxBThe a pair of primer of-R:
ToxB-F:5-aattcaatggaattatcagaat-3
ToxB-R:5-taaacgattttctacctctgg-3
Carry out pcr amplification respectively, filtering out reorganization has
KanThe EHEC O157:H7 of gene, i.e. EHEC O157:H7(△
Hly△
Stx△
ToxB); With full bacterium polyclonal antiserum respectively with EHEC O157:H7(△
Hly△
Stx2A) and EHEC O157:H7(△
Hly△
Stx2A △
ToxB) carry out the protein immunoblotting detection, through the evaluation of gene level and protein level, successfully obtain the EHEC O157:H7 bacterial strain of Hly, Stx and ToxB disappearance, i.e. EHEC O157:H7(△
Hly△
Stx△
ToxB) bacterial strain.
Beneficial effect characteristics of the present invention and advantage are as follows:
1, the present invention is directed to EHEC O157:H7 main toxin gene (
StxWith
Hly) and have ability of regulation and control
ToxBGene has made up three genetically deficient bacterial strains.Utilize round pcr, obtain respectively
Hly,
StxWith
ToxBThe upstream and downstream homology arm of three genes, and contact successively, finally be cloned into suicide plasmid pMEG375, successfully made up three gene knockout plasmids, be respectively pMEG375-HN-Spc-HC, pMEG375-SN-Gm-SC and pMEG375-BN-Kan-BC, i.e. called after pHSH, pSGS and pBKB.
2, the present invention successfully obtains EHEC O157:H7(△
Hly△
Stx2A △
ToxB) bacterial strain.PHSH, pSGS and pBKB gene knockout plasmid and corresponding parent plant hybridization obtain the genetically deficient bacterial strain successively by homologous recombination, the screening of sucrose plate, through gene level and protein level screening and evaluation, successfully obtain disappearance
Hly,
StxWith
ToxBThe EHEC O157:H7 bacterial strain of three genes, i.e. EHEC O157:H7(△
Hly△
Stx2A △
ToxB) bacterial strain.
3, the present invention is to EHEC O157:H7(△
Hly△
Stx2A △
ToxB) disappearance strain cultural characters, cell adhesion ability and mouse is pathogenic and the field planting ability is analyzed, and its immune immunogenicity has been done preliminary research.This deletion mycopremna to the adhesive capacity of HEp-2 cell and in the mouse body field planting ability obviously reduce, can be used as one of candidate strain of development EHEC O157:H7 gene-deleted vaccine.
Four, description of drawings
Fig. 1: EHEC O157:H7(△
Hly△
Stx2A △
ToxB) the structure collection of illustrative plates
Gene knockout plasmid pBKB(figure top) pass through homologous recombination with EHEC O157:H7 karyomit(e) (in the middle of the figure), obtain disappearance
Hly,
Stx2A and
ToxBEHEC O157:H7(△
Hly△
Stx2A △
ToxB) karyomit(e) (figure bottom).Among the figure black rectangle icon represent kantlex (
KanGene), arrow blank parts and blank arrow representative
ToxBGene.The alternate arrow in point-like arrow and black and white side represent respectively pMEG375 replication origin and paraxin (
CatGene) representative.Black adds the thick lines representative
ToxBGene both sides sequence.
Fig. 2: pMEG375-HN-
Spc-HC's
BamHI/
SphThe I double digestion is identified figure
Fig. 3: pMEG375-SN-
Gm-SC's
BamHI/
SphThe I double digestion is identified figure
Fig. 4: pMEG375-BN-
Kan-BC's
BamHI/
NotThe I double digestion is identified figure
Fig. 5: EHEC O157:H7 and disappearance strain EHEC O157:H7(△
Hly△
Stx△
ToxB) growth characteristics relatively
Fig. 6: EHEC O157:H7 and EHEC O157:H7(△
Hly△
StxΔ
ToxB) adhesive capacity relatively
Fig. 7: EHEC O157:H7 and EHEC O157:H7(△
Hly△
Stx△
ToxB) the discharge of bacteria detection
Five, embodiment
Enterorrhagia Bacillus coil 0157: the structure of H7 deletion mycopremna comprises
HlyWhole A gene,
Stx2 whole A gene, and
ToxBTherefore knocking out of whole gene, made up EHEC O157:H7(△ successively
Hly), EHEC O157:H7(△
Hly△
Stx) and EHEC O157:H7(△
Hly△
Stx△
ToxB), be divided into three steps and finish.
, EHEC O157:H7(△
Hly△
Stx△
ToxB) structure of deletion mycopremna is as follows:
(1) makes up EHEC O157:H7 hlyA gene-deleted strain, i.e. EHEC O157:H7(△
Hly) structure
1. obtain
HlyHN and HC and
SpcGene, parallel-series are cloned into the pGEM-T carrier
Land according to GenBank
HlySequence, sequence number are NC_007414, design two pairs of Auele Specific Primers, and the DNA for preparing with EHEC O157:H7 bacterial strain is a template, respectively amplification
HlyGene 5 ' end upstream and 3 ' end downstream 655bp and 834bp sequence, wherein
Hly-NR comprises
HlyInitial 3 bases of gene 5 ' end,
Hly-CF comprises
Hly3 bases in 3 ' end end:
hly-NF：5-tcg
ggatcctctaatgcttttgacgtc-3
hly-NR：5-gcc
gaattcgtcatattagttttctttc-3
hly-CF：5-?gct
gaattcgagactaaaagaggaggt-3
hly-CR：5-aat
gcatgctagcacccagttcgacgt-3
Hly-NF and
Hly-CR primer two ends add restriction enzyme site respectively
BamHI and
SphI and protectiveness base,
Hly-NR and
Hly-CF primer two ends add same restriction enzyme site
EcoRI and protectiveness base, underscore partly is restriction enzyme site, and the PCR reaction conditions is: 95 ℃ of pre-sex change 5min, 94 ℃ of 1min, 55 ℃ of 30s, 72 ℃ of 1min, totally 30 circulations, last 72 ℃ are extended 5min, with the negative contrast of distilled water;
According to the pSET4s sequence that GenBank lands, sequence number is AB055650, designs a pair of Auele Specific Primer S
Pc-F and S
Pc-R is that template amplification is whole with the pSET4s plasmid
SpcExpression cassette:
Spc-F：5-cgt
gaattccgttcgtgaatacatgttat-3
Spc-R：5-?atg
gaattcgcgcttaccaattagaatga-3
Spc-F and
Spc-R primer two ends add same restriction enzyme site
EcoRI and protectiveness base, underscore partly is restriction enzyme site.The PCR reaction conditions is: 95 ℃ of pre-sex change 5min, and 94 ℃ of 1min, 54 ℃ of 30s, 72 ℃ of 1.2min, totally 30 circulations, last 72 ℃ are extended 7min, with distilled water and the negative contrast of EHEC O157:H7 DNA;
Hly-NF and
Hly-NR,
Hly-CF and
Hly-CR,
Spc-F and
SpcThree pairs of primer amplifications of-R obtain the purpose band 655bp of corresponding size, 834bp and 1137bp, and use 1% agarose electrophoresis respectively, cutting glue weighs, 75 ℃ of effect 10min after the DE-A damping fluid of 3 times of volumes of adding, the DE-B damping fluid that then adds 0.5 times of volume DE-A damping fluid, put upside down mixing, transfer liquid is in miniature pillar, and the centrifugal 1min of 12000g, abandons liquid, add lavation buffer solution I 500ul, and the centrifugal 1min of 12000g, abandon liquid, add lavation buffer solution II 750 ul, the centrifugal 1min of 12000g, abandon liquid, repeated washing damping fluid II is washed once, then the centrifugal 1min of blank pipe 12000g, shift pillar in the eppendorf of clean 1.5ml pipe, and adding the 60ul elution buffer, the centrifugal 1min of 12000g collects centrifugate, be purified 655bp, 834bp and 1137bp fragment, called after HN, HC and
SpcFragment;
Will
EcoRThe I enzyme is cut HN and each 3ul(6ng/ul of HC fragment), T4 DNA connects damping fluid and each 1.0ul of T4 dna ligase (350U/ul), and distilled water 2.0ul adds an eppendorf pipe simultaneously, and behind the mixing, 4 ℃ of connections are spent the night; Get spending the night of HN-HC and connect product 3.5ul(4ng/ul) and pGEM-T 1.0ul(50ng/ul), T4 DNA 2 * connection damping fluid 5.0 ul and T4 dna ligase 0.5ul(350U/ul) together add eppendorf pipe, behind the mixing, 4 ℃ of connections are spent the night, be used to transform the Top10 competent cell, the single bacterium colony of picking carries out incubated overnight, extracts plasmid, uses
BamHI and
SphI double digestion, 37 ℃ of water-bath 3h, agarose gel electrophoresis are identified, can see that the band of a 1489bp occurs, and double digestion is identified the order-checking of male bacterium, obtain the pGEM-T-HN-HC recombinant plasmid, and glue reclaims
SpcFragment 4.5ul(3.6ng/ul), together add an eppendorf pipe with pMD18-T 0.5ul and solution I 4.5ul, behind the mixing, 16 ℃ connect 3h, are used to transform the Top10 competent cell, and the single bacterium colony of picking carries out incubated overnight, extracts plasmid, uses
EcoRI single endonuclease digestion, 37 ℃ of water-bath 2h, agarose gel electrophoresis are identified, see that the band of a 1137bp occurs, and double digestion is identified the order-checking of male bacterium, obtain the pMD18-T-spc recombinant plasmid;
2. the pMEG-375-HSH reorganization knocks out the structure of plasmid
PGEM-T-HN-HC recombinant plasmid and pMD18-T-
SpcRecombinant plasmid is used respectively
ECo
RThe I single endonuclease digestion, the alkaline phosphatase dephosphorylation is handled, and 4 ℃ of connections transform the Top10 competent cell, process
ECo
RThe I enzyme is cut and is obtained positive recombinant plasmid pGEM-T-HN-
Spc-HC, pGEM-T-HN-
Spc-the HC and the quick plasmid pMEG375 warm in nature that commits suiside use
BamHI and
SphThe I double digestion, respectively get 2ul(7.1ng/ul), be connected damping fluid and each 1.0ul of T4 dna ligase (350U/ul) with T4 DNA, distilled water 4.0ul adds an eppendorf pipe simultaneously, and behind the mixing, 4 ℃ of connections are spent the night, transform the sm10 competent cell, with the screening of paraxin plate, obtain positive recombinant plasmid, called after pMEG375-HN-
Spc-HC, the positive gene that is acquisition knocks out plasmid pHSH;
3. bacterium solid phase mating
Adopt solid phase filter hybridization method, inoculate recipient bacterium EHEC O157:H7 respectively and contain the donor bacterium sm10 of pHSH in 5mL LB liquid nutrient medium, spend the night in 37 ℃ of shaking culture, get the overnight culture of sm10 and EHEC O157:H7 then respectively in the 4:1 ratio, mixing, filter with filter, bacterium is collected on the filter membrane, filter membrane is positioned on the nonresistant LB agar plate, the germy one side of filter membrane upwards in 37 ℃ of overnight incubation, washes bacterium on the filter membrane with physiological saline then, after 1000 times of dilutions, separate application is in having Streptomycin sulphate and chlorampenicol resistant Strep
RCm
ROn the LB agar plate, 37 ℃ of overnight incubation, the single colony inoculation of picking is in Strep
RCm
RIncrease bacterium among the liquid LB, then bacterium liquid is carried out O157 serotype encoding gene
RfbEWith
SpcCan encode simultaneously the two bacterium colony of gene amplification, acquisition, promptly
RfbE+
Spc+;
4. paraxin concentration method
The chemical sproof hybrid strain of paraxin of inoculating above-mentioned screening is in the LB liquid nutrient medium, 37 ℃ of overnight incubation, transfer in the fresh LB liquid nutrient medium with volume ratio 2% inoculum size, cultivate 1-2h for 37 ℃, add paraxin and continue to cultivate 1-2h, press 1mg/mL and add the D-seromycin, put 37 ℃ more rapidly and continue to cultivate 1-2h, the centrifugal collection thalline of 8000r/min, wash once with physiological saline, coat on the 10% sucrose flat board that contains 100ug/ml Spc after 100 times of dilutions, cultivate 16-20h for 30 ℃, the bacterium colony that grows is inoculated the dull and stereotyped and common LB flat board of the LB that contains paraxin respectively, screening paraxin sensitive strain;
5. EHEC O157:H7(△
Hly) screening and acquisition
The centrifugal collection thalline of the responsive single bacterium colony culture of paraxin, resuspended, boil 7min, 4 ℃ of centrifugal collection supernatants are template to be detected, prepare the template supernatant of EHEC O157:H7 simultaneously, use
Spc-F and
Spc-R, design
Hly-F and
HlyThe a pair of primer of-R:
Hly-F:5-cacacggagcttataatattctgtca-3
Hly-R:5-aatgttatcccattgacatcatttgact-3
Carry out pcr amplification respectively, filtering out reorganization has
SpcThe EHEC O157:H7 of gene, i.e. EHEC O157:H7(△
Hly); With full bacterium polyclonal antiserum respectively with EHEC O157:H7 and EHEC O157:H7(△
Hly) carry out the protein immunoblotting detection, through the evaluation of gene level and protein level, successfully obtain the EHEC O157:H7 bacterial strain of Hly disappearance, i.e. EHEC O157:H7(△
Hly) bacterial strain;
6. the full bacterium polyclonal antiserum preparation of EHEC O157:H7
EHEC O157:H7 inoculation LB liquid nutrient medium 3mL cultivates 8h for 37 ℃, gets the 1mL culture, and centrifugal collection thalline with physiological saline washing 2 times, after 10 times of dilutions, carries out emulsification with equal-volume Fu Shi Freund's complete adjuvant, subcutaneous multi-point injection rabbit; After 3 weeks, after the emulsification of equal-volume freund 's incomplete adjuvant, subcutaneous multi-point injection rabbit; After 2 weeks, serum is collected in the heart blood sampling, is the full bacterium polyclonal antiserum of EHEC O157:H7;
(2) EHEC O157:H7(△
Hly△
Stx) structure
1. at first obtain
Stx2The SN of A and SC reach
GmGene, parallel-series are cloned into the pGEM-T carrier
Land according to GenBank
Stx2A sequence, sequence number are NC_007414, design two pairs of Auele Specific Primers, and the DNA for preparing with EHEC O157:H7 bacterial strain is a template, respectively amplification
Stx2A gene 5 ' end upstream and 3 ' end downstream 397bp and 229bp sequence, wherein
Stx2A-NR comprises
Stx2Initial 3 bases of A gene 5 ' end,
Stx2A-CF comprises
Stx23 bases in A3 ' end end:
stx2A-NF：5-aat
ggatcctacaaatccagcaagggcc-3
stx2A-NR：5-gcc
gaattccgaaaagtctatcgtaaac-3
stx2A-CF：5-tta
gaattcgcggcggattgtgctaaag-3
stx2A-CR：5-ccc
gcatgccattattaaactgcacttca-3
Stx2A-NF and
Stx2A-CR primer two ends add restriction enzyme site respectively
BamHI and
SphI and protectiveness base,
Stx2A-NR and
Stx2A-CF primer two ends add same restriction enzyme site
EcoRI and protectiveness base, underscore partly is restriction enzyme site, and the PCR reaction conditions is: 95 ℃ of pre-sex change 5min, 94 ℃ of 1min, 56 ℃ of 30s, 72 ℃ of 30s, totally 30 circulations, last 72 ℃ are extended 5min, with the negative contrast of distilled water;
The sequence number that lands according to GeneBank is the Fastbac of AY598466.1
TM1Sequence designs a pair of Auele Specific Primer
Gm-F and
Gm-R, it is whole to increase
GmExpression cassette, 811bp;
Gm-F：5-
gaattcaccgtggaaacgcatgaag-3
Gm-R：5
-gaattcacggcttgaacgaattgtt-3
Gm-F and
Gm-R primer two ends add restriction enzyme site
EcoRI, underscore partly are restriction enzyme site, and the PCR reaction conditions is: 95 ℃ of pre-sex change 5 min, and 94 ℃ of 1 min, 56 ℃ of 30s, 72 ℃ of 50s, totally 30 circulations, last 72 ℃ are extended 5 min, with the negative contrast of distilled water;
Gm-F and
Gm-R primer two ends add same restriction enzyme site
EcoRI and protectiveness base, underscore partly is restriction enzyme site, and the PCR reaction conditions is: 95 ℃ of pre-sex change 5min, 94 ℃ of 1min, 56 ℃ of 30s, 72 ℃ of 1.0min, totally 30 circulations, last 72 ℃ are extended 7min, with distilled water and the negative contrast of EHEC O157:H7 DNA;
Stx2A-NF and
Stx2A-NR,
Stx2A-CF and
Stx2A-CR,
Gm-F and
GmThree pairs of primer amplifications of-R obtain the purpose band 397bp of corresponding size, 229bp and 811bp, and use 1% agarose electrophoresis respectively, cutting glue weighs, 75 ℃ of effect 10min after the DE-A damping fluid of 3 times of volumes of adding, the DE-B damping fluid that then adds 0.5 times of volume DE-A damping fluid, put upside down mixing, transfer liquid is in miniature pillar, and the centrifugal 1min of 12000g, abandons liquid, add lavation buffer solution I 500ul, and the centrifugal 1min of 12000g, abandon liquid, add lavation buffer solution II 750 ul, the centrifugal 1min of 12000g, abandon liquid, repeated washing damping fluid II is washed once, then the centrifugal 1min of blank pipe 12000g, shift pillar in the eppendorf of clean 1.5ml pipe, and adding the 60ul elution buffer, the centrifugal 1min of 12000g collects centrifugate, be purified 397bp, 229bp and 811bp fragment, called after SN, SC and
GmFragment;
Will
EcoRThe I enzyme is cut SN and each 3ul of SC fragment (4ng/ul), and T4 DNA connects damping fluid and each 1.0 ul of T4 dna ligase (350U/ul), and distilled water 2.0ul adds an eppendorf pipe simultaneously, and behind the mixing, 4 ℃ of connections are spent the night; Get spending the night of SN-SC and connect product 3.5ul (2.7ng/ul) and pGEM-T 1.0ul (50ng/ul), T4 DNA 2 * connection damping fluid 5.0 ul and T4 dna ligase 0.5ul(350U/ul) together add eppendorf pipe, behind the mixing, 4 ℃ of connections are spent the night, be used to transform the Top10 competent cell, the single bacterium colony of picking carries out incubated overnight, extracts plasmid, uses
BamHI and
SphI double digestion, 37 ℃ of water-bath 3h, agarose gel electrophoresis are identified, can see that the band of a 626bp occurs, and double digestion is identified the order-checking of male bacterium, obtain the pGEM-T-SN-SC recombinant plasmid, and glue reclaims
GmFragment 4.5ul(5ng/ul), together add an eppendorf pipe with pMD18-T0.5ul (50ng/ul) and solution I 4.5ul, behind the mixing, 16 ℃ connect 3h, are used to transform the Top10 competent cell, and the single bacterium colony of picking carries out incubated overnight, extract plasmid, use
EcoRI single endonuclease digestion, 37 ℃ of water-bath 2h, agarose gel electrophoresis are identified, can see that the band of a 811bp occurs, and double digestion is identified the order-checking of male bacterium, obtain pMD18-T-
GmRecombinant plasmid;
2. the pMEG-375-SGS reorganization knocks out the structure of plasmid
PGEM-T-SN-SC recombinant plasmid and pMD18-T-
GmRecombinant plasmid is used respectively
ECo
RThe I single endonuclease digestion, the alkaline phosphatase dephosphorylation, 4 ℃ of connections transform the Top10 competent cell, process
ECo
RThe I enzyme is cut and is obtained positive recombinant plasmid pGEM-T-SN-
Gm-SC, pGEM-T-SN-
Gm-the SC and the quick plasmid pMEG375 warm in nature that commits suiside use
BamHI and
SphEach 2ul(11ng/ul of I double digestion), be connected damping fluid and each 1.0ul of T4 dna ligase (350U/ul) with T4 DNA, distilled water 4.0ul adds an eppendorf pipe simultaneously, behind the mixing, 4 ℃ of connections are spent the night, and transform the sm10 competent cell, with the screening of paraxin plate, obtain positive recombinant plasmid, called after pMEG375-SN-
Gm-SC, the positive gene that is acquisition knocks out plasmid pSGS;
3. bacterium solid phase mating
Adopt solid phase filter hybridization method, inoculate recipient bacterium EHEC O157:H7(△ respectively
Hly) and the donor bacterium sm10 that contains pSGS in 5mL LB liquid nutrient medium, spend the night in 37 ℃ of shaking culture, the 4:1 ratio is got sm10 and the EHEC O157:H7(△ that contains pSGS respectively by volume then
Hly) overnight culture, mixing, filter with filter, bacterium is collected on the filter membrane, filter membrane is positioned on the nonresistant LB agar plate, and the germy one side of filter membrane makes progress, in 37 ℃ of overnight incubation, wash after bacterium on the filter membrane carries out 1000 times of dilutions with physiological saline then, separate application is in having Streptomycin sulphate and chlorampenicol resistant Strep
RCm
ROn the LB agar plate, 37 ℃ of overnight incubation, the single colony inoculation of picking is in Strep
RCm
RIncrease bacterium among the liquid LB, then bacterium liquid is carried out O157 serotype encoding gene
RfbEWith
GmCan encode simultaneously the two bacterium colony of gene amplification, acquisition, promptly
RfbE+
Gm+;
4. paraxin concentration method
The chemical sproof hybrid strain of paraxin of inoculating above-mentioned screening is in the LB liquid nutrient medium, 37 ℃ of overnight incubation, transfer in the fresh LB liquid nutrient medium with 2% inoculum size, cultivate 1-2h for 37 ℃, add paraxin and continue to cultivate 1-2h, press 1mg/mL and add the D-seromycin, put 37 ℃ more rapidly and continue to cultivate 1-2h, the centrifugal collection thalline of 8000r/min is washed once with physiological saline, after 100 times of dilutions, coat on the 10% sucrose flat board that contains 50ug/ml Gm, cultivate 16-20h for 30 ℃, the bacterium colony that grows is inoculated the dull and stereotyped and common LB flat board of the LB that contains paraxin respectively, screening paraxin sensitive strain;
5. EHEC O157:H7(△
Hly△
Stx2A) screening and acquisition
The centrifugal collection thalline of the responsive single bacterium colony culture of paraxin, resuspended, boil 7min, 4 ℃ of centrifugal collection supernatants are template to be detected, prepare the template supernatant of EHEC O157:H7 simultaneously, use
Gm-F and
Gm-R, design
Stx2A-F and
Stx2The a pair of primer of A-R:
Stx2A-F:5-gcgttaatggagttcagtggt-3
Stx2A-R:5-cttaactcctttatttacccgttgt-3
Carry out pcr amplification respectively, filtering out reorganization has
GmThe EHEC O157:H7 of gene, i.e. EHEC O157:H7(△
Hly△
Stx2A); With full bacterium polyclonal antiserum respectively with EHEC O157:H7(△
Hly) and EHEC O157:H7(△
Hly△
Stx2A) carry out protein immunoblotting and detect,, successfully obtain the EHEC O157:H7 bacterial strain of Hly and Stx2A disappearance, i.e. EHEC O157:H7(△ through the evaluation of gene level and protein level
Hly△
Stx2A) bacterial strain;
(3) EHEC O157:H7(△
Hly△
Stx△
ToxB) structure
1. at first obtain
ToxBBN and BC and
KanGene, parallel-series are cloned into the pET32 carrier
Land according to GenBank
ToxBSequence, sequence number are NC_007414, design two pairs of Auele Specific Primers, and the DNA for preparing with EHEC O157:H7 bacterial strain is a template, respectively amplification
ToxBGene 5 ' end upstream and 3 ' end downstream 831bp and 863bp sequence, wherein
ToxB-NR is
ToxBGene a 5 ' end upstream 36-54 base,
ToxB-CF is
ToxBGene a 3 ' end downstream 32-50 base;
toxB-NF：5-tta
ggatccatccatcaggcgttgtgc-3
toxB-NR：5-ggg
gaattctttgattagcgacaacct-3
toxB-CF：5-gcc
gtcgactaatcttacccagcatca-3
toxB-CR：5-att
gcggccgcgagttatcagcccgtcat-3
ToxB-NF,
ToxB-NR,
ToxB-CF and
ToxB-CR primer two ends add restriction enzyme site respectively
BamThe H I,
EcoRI,
SalI and
NotI and and the protectiveness base, underscore partly is restriction enzyme site.The PCR reaction conditions is: 95 ℃ of pre-sex change 5 min, and 94 ℃ of 1 min, 58 ℃ of 30 s, 72 ℃ of 1 min, totally 30 circulations, last 72 ℃ are extended 5 min, with the negative contrast of distilled water;
The sequence number that lands according to GeneBank is the pET28 sequence of EF442785.1, designs a pair of Auele Specific Primer
Kan-F and
Kan-R, it is whole to increase
KanExpression cassette, 1192bp;
Kan-F：5-ccg
gaattcacggctacactagaaggac-3
Kan-R：5-?aga
gtcgacaaatgtgcgcggaaccccta-3
Kan-F and
Kan-R primer two ends add restriction enzyme site
EcoRI and
SalI and protectiveness base, underscore partly is restriction enzyme site, and the PCR reaction conditions is: 95 ℃ of pre-sex change 5 min, 94 ℃ of 1 min, 56 ℃ of 30s, 72 ℃ of 1.2 min, totally 30 circulations, last 72 ℃ are extended 7 min, with the negative contrast of distilled water;
ToxB-NF and
ToxB-NR,
ToxB-CF and
ToxB-CR,
Kan-F and
KanThree pairs of primer amplifications of-R obtain the purpose band 831bp of corresponding size, 863bp and 1192bp, and use 1% agarose electrophoresis respectively, cutting glue weighs, 75 ℃ of effect 10min after the DE-A damping fluid of 3 times of volumes of adding, the DE-B damping fluid that then adds 0.5 times of volume DE-A damping fluid, put upside down mixing, transfer liquid is in miniature pillar, and the centrifugal 1min of 12000g, abandons liquid, add lavation buffer solution I 500ul, and the centrifugal 1min of 12000g, abandon liquid, add lavation buffer solution II 750 ul, the centrifugal 1min of 12000g, abandon liquid, repeated washing damping fluid II is washed once, then the centrifugal 1min of blank pipe 12000g, shift pillar in the eppendorf of clean 1.5ml pipe, and adding the 60ul elution buffer, the centrifugal 1min of 12000g collects centrifugate, be purified 831bp, 863bp and 1192bp fragment, called after BN, BC and
KanFragment;
Will
BamThe H I and
EcoRThe I enzyme is cut BN fragment 3ul (9.6ng/ul) and pET32a carrier 4ul (10ng/ul), T4 DNA connects damping fluid and each 1.0ul of T4 dna ligase (350U/ul), distilled water 1.0ul adds an eppendorf pipe simultaneously, behind the mixing, 4 ℃ of connections are spent the night, and transform the Top10 competent cell, and the single bacterium colony of picking carries out incubated overnight, extract plasmid, use
BamThe H I and
EcoRI double digestion, 37 ℃ of water-bath 3h, agarose gel electrophoresis are identified, can see that the band of a 831bp occurs, and obtain the pET32a-BN recombinant plasmid, then will
EcoRI and
SalThe Kan that the I enzyme is cut is connected with the pET32a-BN recombinant plasmid, obtains pET32a-BN-Kan, at last will
SalI and
NotThe BC that the I enzyme is cut is connected with the pET32a-BN-Kan recombinant plasmid, obtains pET32a-BN-Kan-BC, transforms the Top10 competent cell, cultivates 16h for 37 ℃, and the dull and stereotyped single bacterium colony of picking is identified.To identify that male recombinant plasmid and pMEG375 carrier use respectively
BamHI and
NotAfter the I enzyme was cut, 4 ℃ of connections were spent the night, and transformed competence colibacillus cell sm10 with the screening of paraxin plate, obtains positive recombinant plasmid, called after pMEG375-BN-
Kan-BC, the positive gene that is acquisition knocks out plasmid pBKB;
2. bacterium solid phase mating
Adopt solid phase filter hybridization method, inoculate recipient bacterium EHEC O157:H7(△ respectively
Hly△
Stx) and the donor bacterium sm10 that contains pBKB in 5mL LB liquid nutrient medium, spend the night in 37 ℃ of shaking culture, get sm10 and the EHEC O157:H7(△ that contains pBKB respectively in the 4:1 ratio then
Hly△
Stx) overnight culture, mixing, filter with filter, bacterium is collected on the filter membrane, and filter membrane is positioned on the nonresistant LB agar plate, and the germy one side of filter membrane upwards, in 37 ℃ of overnight incubation, wash bacterium on the filter membrane with physiological saline then, after 1000 times of dilutions, separate application is in having Streptomycin sulphate and chlorampenicol resistant Strep
RCm
ROn the LB agar plate, 37 ℃ of overnight incubation, the single colony inoculation of picking is in Strep
RCm
RIncrease bacterium among the liquid LB, then bacterium liquid is carried out O157 serotype encoding gene
RfbEWith
KanCan encode simultaneously the two bacterium colony of gene amplification, acquisition, promptly
RfbE+
Kan+;
3. paraxin concentration method
The chemical sproof hybrid strain of paraxin of inoculating above-mentioned screening is in the LB liquid nutrient medium, 37 ℃ of overnight incubation, transfer in the fresh LB liquid nutrient medium with 2% inoculum size, cultivate 1-2h for 37 ℃, add paraxin and continue to cultivate 1-2h, press 1mg/mL and add the D-seromycin, put 37 ℃ more rapidly and continue to cultivate 1-2h, the centrifugal collection thalline of 8000r/min is washed once with physiological saline, after 100 times of dilutions, coat and contain on the 100ug/ml kantlex 10% sucrose flat board, cultivate 16-20h for 30 ℃, the bacterium colony that grows is inoculated the dull and stereotyped and common LB flat board of the LB that contains paraxin respectively, screening paraxin sensitive strain;
4. EHEC O157:H7(△
Hly△
Stx△
ToxB) screening and acquisition
The centrifugal collection thalline of the responsive single bacterium colony culture of paraxin, resuspended, boil 7min, 4 ℃ of centrifugal collection supernatants are template to be detected, prepare the template supernatant of EHEC O157:H7 simultaneously, use
Kan-F and
Kan-R, design
ToxB-F and
ToxBThe a pair of primer of-R:
ToxB-F:5-aattcaatggaattatcagaat-3
ToxB-R:5-taaacgattttctacctctgg-3
Carry out pcr amplification respectively, filtering out reorganization has
KanThe EHEC O157:H7 of gene, i.e. EHEC O157:H7(△
Hly△
Stx△
ToxB); With full bacterium polyclonal antiserum respectively with EHEC O157:H7(△
Hly△
Stx2A) and EHEC O157:H7(△
Hly△
Stx2A △
ToxB) carry out the protein immunoblotting detection, through the evaluation of gene level and protein level, successfully obtain the EHEC O157:H7 bacterial strain of Hly, Stx and ToxB disappearance, i.e. EHEC O157:H7(△
Hly△
Stx△
ToxB) bacterial strain.
, EHEC O157:H7(△
Hly△
Stx△
ToxB) cultural characters research, as described below:
EHEC O157:H7(△
Hly△
Stx△
ToxB) single colony inoculation in 5ml LB nutrient solution, incubated overnight.Then get the 16h culture with 1% ratio (about 1.5 * 10
9CFU) be inoculated in once more among the fresh LB of 100ml, cultivate 30h, respectively at 2h, 4h, 6h, 8h, 10h, 12h, 16h, 20h, 24h and 30h get 1ml bacterium liquid and measure OD600, with the growth curve (Fig. 5) of monitoring bacterium.EHEC O157:H7(△
Hly△
Stx△
ToxB) there is not notable difference with the parent plant EHEC O157:H7 speed of growth and bacterial concentration.
, EHEC O157:H7(△
Hly△
Stx△
ToxB) the cell adhesion capability study, as described below:
EHEC O157:H7 is inoculated in the 3mL LB substratum (containing NaI 150 mg/L), in 37 ℃ of shaking culture 16-20h in advance.Get this an amount of culture and be added in the cell adhesion nutrient solution (1%D-seminose, do not contain antibiotic DMEM cell culture fluid), regulating bacterial count is 2 * 10
7CFUmL
-1, 37 ℃ of static cultivation 30min.The EHEC O157:H7 inoculum 1ml of above-mentioned processing is joined in 24 orifice plates that contain the HEp-2 cell, after hatching 2h under 37 ℃, 5%CO2 condition, the sucking-off nutrient solution, discard,, add new aseptic DMEM nutrient solution with Hank ' s liquid washing 1 time, after hatching 2h once more, abandon liquid, with Hank ' s liquid washing 4 times, each washing will be moved gently.Use trypsin digestion cell 15min, coat after suitably diluting on the Mai Kangkai culture plate, calculate adherent bacterium number, ask three multiple mean values and standard deviation.EHEC O157:H7(△
Hly△
StxΔ
ToxB) compare parent plant EHEC O157:H7 the adhesive capacity of HEp-2 cell is had tangible reduction (P=0.0002) (Fig. 6).
, EHEC O157:H7(△
Hly△
Stx△
ToxB) to the Study on Pathogenicity of mouse, as described below:
40 Balb/C mouse are divided into 2 big groups at random.I winding kind EHEC O157:H7 parent plant; II winding kind EHEC O157:H7 disappearance strain (△
Hly△
Stx△
ToxB), two groups are provided with 10 respectively
10CFU/, 10
9CFU/, 10
8CFU/ is only with 10
7CFU/ is four gradients only.Choosing and attacking the toxic agent amount is 10
9CFU/ mouse only detects from ight soil and carries disease germs and discharge of bacteria.All mouse all give 5g/L Streptomycin sulphate solution and drink 3d before attacking poison, and excrement inspection enteron aisle discharge of bacteria is less than 10 behind the 3d
3CFU/g irritates stomach and attacks the Streptomycin sulphate solution that all gives 0.5g/L behind the bacterium and drink.To get rid of normal intestinal flora, for EHEC O157:H7 tames an environment of settling down and growing in the mouse body, in enteron aisle, become dominant microflora, increase the susceptibility of mouse.And the jejunitas 12h that before attacking poison, cuts off the water supply, attack poison back 6h and recover drinking water diet, prevent to attack poison back bacterium and discharge animal intestinal fast, prolong action time in its body.
EHEC O157:H7(△
Hly△
StxΔ
ToxB) the disappearance strain has the mouse ability that causes death and necessarily weaken (table 1), the ability of field planting has comparatively significantly and reduces in the mouse body, and the discharge of bacteria amount is few, and the discharge of bacteria time shortens (Fig. 7).
SEQUENCE?LISTING
＜110〉Jiangsu Province Agriculture Science Institute
＜120〉enterorrhagia Bacillus coil 0157: H7 three genetically deficient bacterial strains
＜130〉specification sheets
<160> 24
<170> PatentIn?version?3.1
<210> 1
<211> 27
<212> DNA
＜213〉artificial
<220>
<221> hly-NF
<222> (1)..(27)
<223>
<400> 1
tcgggatcct?ctaatgcttt?tgacgtc 27
<210> 2
<211> 28
<212> DNA
＜213〉artificial
<220>
<221> hly-NR
<222> (1)..(28)
<223>
<400> 2
gccgaattcg?tcatattagt?tttctttc 28
<210> 3
<211> 27
<212> DNA
＜213〉artificial
<220>
<221> hly-CF
<222> (1)..(27)
<223>
<400> 3
gctgaattcg?agactaaaag?aggaggt 27
<210> 4
<211> 27
<212> DNA
＜213〉artificial
<220>
<221> hly-CR
<222> (1)..(27)
<223>
<400> 4
aatgcatgct?agcacccagt?tcgacgt 27
<210> 5
<211> 29
<212> DNA
＜213〉artificial
<220>
<221> Spc-F
<222> (1)..(29)
<223>
<400> 5
cgtgaattcc?gttcgtgaat?acatgttat 29
<210> 6
<211> 29
<212> DNA
＜213〉artificial
<220>
<221> Spc-R
<222> (1)..(29)
<223>
<400> 6
atggaattcg?cgcttaccaa?ttagaatga 29
<210> 7
<211> 26
<212> DNA
＜213〉artificial
<220>
<221> hly-F
<222> (1)..(26)
<223>
<400> 7
cacacggagc?ttataatatt?ctgtca 26
<210> 8
<211> 28
<212> DNA
＜213〉artificial
<220>
<221> hly-R
<222> (1)..(28)
<223>
<400> 8
aatgttatcc?cattgacatc?atttgact 28
<210> 9
<211> 28
<212> DNA
＜213〉artificial
<220>
<221> stx2A-NF
<222> (1)..(28)
<223>
<400> 9
aatggatcct?acaaatccag?caagggcc 28
<210> 10
<211> 28
<212> DNA
＜213〉artificial
<220>
<221> stx2A-NR
<222> (1)..(28)
<223>
<400> 10
gccgaattcc?gaaaagtcta?tcgtaaac 28
<210> 11
<211> 28
<212> DNA
＜213〉artificial
<220>
<221> stx2A-CF
<222> (1)..(28)
<223>
<400> 11
ttagaattcg?cggcggattg?tgctaaag 28
<210> 12
<211> 29
<212> DNA
＜213〉artificial
<220>
<221> stx2A-CR
<222> (1)..(29)
<223>
<400> 12
cccgcatgcc?attattaaac?tgcacttca 29
<210> 13
<211> 25
<212> DNA
＜213〉artificial
<220>
<221> Gm-F
<222> (1)..(25)
<223>
<400> 13
gaattcaccg?tggaaacgca?tgaag 25
<210> 14
<211> 25
<212> DNA
＜213〉artificial
<220>
<221> Gm-R
<222> (1)..(25)
<223>
<400> 14
gaattcacgg?cttgaacgaa?ttgtt 25
<210> 15
<211> 21
<212> DNA
＜213〉artificial
<220>
<221> stx2A-F
<222> (1)..(21)
<223>
<400> 15
gcgttaatgg?agttcagtgg?t 21
<210> 16
<211> 25
<212> DNA
＜213〉artificial
<220>
<221> stx2A-R
<222> (1)..(25)
<223>
<400> 16
cttaactcct?ttatttaccc?gttgt 25
<210> 17
<211> 27
<212> DNA
＜213〉artificial
<220>
<221> toxB-NF
<222> (1)..(27)
<223>
<400> 17
ttaggatcca?tccatcaggc?gttgtgc 27
<210> 18
<211> 27
<212> DNA
＜213〉artificial
<220>
<221> toxB-NR
<222> (1)..(27)
<223>
<400> 18
ggggaattct?ttgattagcg?acaacct 27
<210> 19
<211> 27
<212> DNA
＜213〉artificial
<220>
<221> toxB-NR
<222> (1)..(27)
<223>
<400> 19
gccgtcgact?aatcttaccc?agcatca 27
<210> 20
<211> 29
<212> DNA
＜213〉artificial
<220>
<221> toxB-CR
<222> (1)..(29)
<223>
<400> 20
attgcggccg?cgagttatca?gcccgtcat 29
<210> 21
<211> 28
<212> DNA
＜213〉artificial
<220>
<221> Kan-F
<222> (1)..(28)
<223>
<400> 21
ccggaattca?cggctacact?agaaggac 28
<210> 22
<211> 29
<212> DNA
＜213〉artificial
<220>
<221> Kan-R
<222> (1)..(29)
<223>
<400> 22
agagtcgaca?aatgtgcgcg?gaaccccta 29
<210> 23
<211> 22
<212> DNA
＜213〉artificial
<220>
<221> toxB-F
<222> (1)..(22)
<223>
<400> 23
aattcaatgg?aattatcaga?at 22
<210> 24
<211> 21
<212> DNA
＜213〉artificial
<220>
<221> toxB-R
<222> (1)..(21)
<223>
<400> 24
taaacgattt?tctacctctg?g 21
Claims (3)
1. enterorrhagia Bacillus coil 0157: H7 three genetically deficient bacterial strains is characterized in that, said enterorrhagia Bacillus coil 0157: H7 three genetically deficient bacterial strains are enterorrhagia Bacillus coil 0157: H7 bacterium disappearance
HlyWhole A gene,
Stx2 whole A gene, and
ToxBWhole gene.
2. deletion mycopremna as claimed in claim 1, its construction process comprises:
(1)Make up EHEC O157:H7 hlyA gene-deleted strain, i.e. EHEC O157:H7(△
Hly) structure
1. obtain
HlyHN and HC and
SpcGene, parallel-series are cloned into the pGEM-T carrier
Land according to GenBank
HlySequence, sequence number are NC_007414, design two pairs of Auele Specific Primers, and the DNA for preparing with EHEC O157:H7 bacterial strain is a template, respectively amplification
HlyGene 5 ' end upstream and 3 ' end downstream 655bp and 834bp sequence, wherein
Hly-NR comprises
HlyInitial 3 bases of gene 5 ' end,
Hly-CF comprises
Hly3 bases in 3 ' end end:
hly-NF：5-tcg
ggatcctctaatgcttttgacgtc-3
hly-NR：5-gcc
gaattcgtcatattagttttctttc-3
hly-CF：5-?gct
gaattcgagactaaaagaggaggt-3
hly-CR：5-aat
gcatgctagcacccagttcgacgt-3
Hly-NF and
Hly-CR primer two ends add restriction enzyme site respectively
BamHI and
SphI and protectiveness base,
Hly-NR and
Hly-CF primer two ends add same restriction enzyme site
EcoRI and protectiveness base, underscore partly is restriction enzyme site, and the PCR reaction conditions is: 95 ℃ of pre-sex change 5min, 94 ℃ of 1min, 55 ℃ of 30s, 72 ℃ of 1min, totally 30 circulations, last 72 ℃ are extended 5min, with the negative contrast of distilled water;
According to the pSET4s sequence that GenBank lands, sequence number is AB055650, designs a pair of Auele Specific Primer S
Pc-F and S
Pc-R is that template amplification is whole with the pSET4s plasmid
SpcExpression cassette:
Spc-F：5-cgt
gaattccgttcgtgaatacatgttat-3
Spc-R：5-?atg
gaattcgcgcttaccaattagaatga-3
Spc-F and
Spc-R primer two ends add same restriction enzyme site
EcoRI and protectiveness base, underscore partly is restriction enzyme site; The PCR reaction conditions is: 95 ℃ of pre-sex change 5min, and 94 ℃ of 1min, 54 ℃ of 30s, 72 ℃ of 1.2min, totally 30 circulations, last 72 ℃ are extended 7min, with distilled water and the negative contrast of EHEC O157:H7 DNA;
Hly-NF and
Hly-NR,
Hly-CF and
Hly-CR,
Spc-F and
SpcThree pairs of primer amplifications of-R obtain the purpose band 655bp of corresponding size, 834bp and 1137bp, and use 1% agarose electrophoresis respectively, cutting glue weighs, 75 ℃ of effect 10min after the DE-A damping fluid of 3 times of volumes of adding, the DE-B damping fluid that then adds 0.5 times of volume DE-A damping fluid, put upside down mixing, transfer liquid is in miniature pillar, and the centrifugal 1min of 12000g, abandons liquid, add lavation buffer solution I 500ul, and the centrifugal 1min of 12000g, abandon liquid, add lavation buffer solution II 750 ul, the centrifugal 1min of 12000g, abandon liquid, repeated washing damping fluid II is washed once, then the centrifugal 1min of blank pipe 12000g, shift pillar in the eppendorf of clean 1.5ml pipe, and adding the 60ul elution buffer, the centrifugal 1min of 12000g collects centrifugate, be purified 655bp, 834bp and 1137bp fragment, called after HN, HC and
SpcFragment;
Will
EcoRThe I enzyme is cut HN and each 3ul(6ng/ul of HC fragment), T4 DNA connects damping fluid and each 1.0ul of T4 dna ligase (350U/ul), and distilled water 2.0ul adds an eppendorf pipe simultaneously, and behind the mixing, 4 ℃ of connections are spent the night; Get spending the night of HN-HC and connect product 3.5ul(4ng/ul) and pGEM-T 1.0ul(50ng/ul), T4 DNA 2 * connection damping fluid 5.0 ul and T4 dna ligase 0.5ul(350U/ul) together add eppendorf pipe, behind the mixing, 4 ℃ of connections are spent the night, be used to transform the Top10 competent cell, the single bacterium colony of picking carries out incubated overnight, extracts plasmid, uses
BamHI and
SphI double digestion, 37 ℃ of water-bath 3h, agarose gel electrophoresis are identified, can see that the band of a 1489bp occurs, and double digestion is identified the order-checking of male bacterium, obtain the pGEM-T-HN-HC recombinant plasmid, and glue reclaims
SpcFragment 4.5ul(3.6ng/ul), together add an eppendorf pipe with pMD18-T 0.5ul and solution I 4.5ul, behind the mixing, 16 ℃ connect 3h, are used to transform the Top10 competent cell, and the single bacterium colony of picking carries out incubated overnight, extracts plasmid, uses
EcoRI single endonuclease digestion, 37 ℃ of water-bath 2h, agarose gel electrophoresis are identified, see that the band of a 1137bp occurs, and double digestion is identified the order-checking of male bacterium, obtain the pMD18-T-spc recombinant plasmid;
2. the pMEG-375-HSH reorganization knocks out the structure of plasmid
PGEM-T-HN-HC recombinant plasmid and pMD18-T-
SpcRecombinant plasmid is used respectively
ECo
RThe I single endonuclease digestion, the alkaline phosphatase dephosphorylation is handled, and 4 ℃ of connections transform the Top10 competent cell, process
ECo
RThe I enzyme is cut and is obtained positive recombinant plasmid pGEM-T-HN-
Spc-HC, pGEM-T-HN-
Spc-the HC and the quick plasmid pMEG375 warm in nature that commits suiside use
BamHI and
SphThe I double digestion, respectively get 2ul(7.1ng/ul), be connected damping fluid and each 1.0ul of T4 dna ligase (350U/ul) with T4 DNA, distilled water 4.0ul adds an eppendorf pipe simultaneously, and behind the mixing, 4 ℃ of connections are spent the night, transform the sm10 competent cell, with the screening of paraxin plate, obtain positive recombinant plasmid, called after pMEG375-HN-
Spc-HC, the positive gene that is acquisition knocks out plasmid pHSH;
3. bacterium solid phase mating
Adopt solid phase filter hybridization method, inoculate recipient bacterium EHEC O157:H7 respectively and contain the donor bacterium sm10 of pHSH in 5mL LB liquid nutrient medium, spend the night in 37 ℃ of shaking culture, get the overnight culture of sm10 and EHEC O157:H7 then respectively in the 4:1 ratio, mixing, filter with filter, bacterium is collected on the filter membrane, filter membrane is positioned on the nonresistant LB agar plate, the germy one side of filter membrane upwards in 37 ℃ of overnight incubation, washes bacterium on the filter membrane with physiological saline then, after 1000 times of dilutions, separate application is in having Streptomycin sulphate and chlorampenicol resistant Strep
RCm
ROn the LB agar plate, 37 ℃ of overnight incubation, the single colony inoculation of picking is in Strep
RCm
RIncrease bacterium among the liquid LB, then bacterium liquid is carried out O157 serotype encoding gene
RfbEWith
SpcCan encode simultaneously the two bacterium colony of gene amplification, acquisition, promptly
RfbE+
Spc+;
4. paraxin concentration method
The chemical sproof hybrid strain of paraxin of inoculating above-mentioned screening is in the LB liquid nutrient medium, 37 ℃ of overnight incubation, transfer in the fresh LB liquid nutrient medium with volume ratio 2% inoculum size, cultivate 1-2h for 37 ℃, add paraxin and continue to cultivate 1-2h, press 1mg/mL and add the D-seromycin, put 37 ℃ more rapidly and continue to cultivate 1-2h, the centrifugal collection thalline of 8000r/min, wash once with physiological saline, coat on the 10% sucrose flat board that contains 100ug/ml Spc after 100 times of dilutions, cultivate 16-20h for 30 ℃, the bacterium colony that grows is inoculated the dull and stereotyped and common LB flat board of the LB that contains paraxin respectively, screening paraxin sensitive strain;
5. EHEC O157:H7(△
Hly) screening and acquisition
The centrifugal collection thalline of the responsive single bacterium colony culture of paraxin, resuspended, boil 7min, 4 ℃ of centrifugal collection supernatants are template to be detected, prepare the template supernatant of EHEC O157:H7 simultaneously, use
Spc-F and
Spc-R, design
Hly-F and
HlyThe a pair of primer of-R:
Hly-F:5-cacacggagcttataatattctgtca-3
Hly-R:5-aatgttatcccattgacatcatttgact-3
Carry out pcr amplification respectively, filtering out reorganization has
SpcThe EHEC O157:H7 of gene, i.e. EHEC O157:H7(△
Hly); With full bacterium polyclonal antiserum respectively with EHEC O157:H7 and EHEC O157:H7(△
Hly) carry out the protein immunoblotting detection, through the evaluation of gene level and protein level, successfully obtain the EHEC O157:H7 bacterial strain of Hly disappearance, i.e. EHEC O157:H7(△
Hly) bacterial strain;
6. the full bacterium polyclonal antiserum preparation of EHEC O157:H7
EHEC O157:H7 inoculation LB liquid nutrient medium 3mL cultivates 8h for 37 ℃, gets the 1mL culture, and centrifugal collection thalline with physiological saline washing 2 times, after 10 times of dilutions, carries out emulsification with equal-volume Fu Shi Freund's complete adjuvant, subcutaneous multi-point injection rabbit; After 3 weeks, after the emulsification of equal-volume freund 's incomplete adjuvant, subcutaneous multi-point injection rabbit; After 2 weeks, serum is collected in the heart blood sampling, is the full bacterium polyclonal antiserum of EHEC O157:H7;
(2) EHEC O157:H7(△
Hly△
Stx) structure
1. at first obtain
Stx2The SN of A and SC reach
GmGene, parallel-series are cloned into the pGEM-T carrier
Land according to GenBank
Stx2A sequence, sequence number are NC_007414, design two pairs of Auele Specific Primers, and the DNA for preparing with EHEC O157:H7 bacterial strain is a template, respectively amplification
Stx2A gene 5 ' end upstream and 3 ' end downstream 397bp and 229bp sequence, wherein
Stx2A-NR comprises
Stx2Initial 3 bases of A gene 5 ' end,
Stx2A-CF comprises
Stx23 bases in A3 ' end end:
stx2A-NF：5-aat
ggatcctacaaatccagcaagggcc-3
stx2A-NR：5-gcc
gaattccgaaaagtctatcgtaaac-3
stx2A-CF：5-tta
gaattcgcggcggattgtgctaaag-3
stx2A-CR：5-ccc
gcatgccattattaaactgcacttca-3
Stx2A-NF and
Stx2A-CR primer two ends add restriction enzyme site respectively
BamHI and
SphI and protectiveness base,
Stx2A-NR and
Stx2A-CF primer two ends add same restriction enzyme site
EcoRI and protectiveness base, underscore partly is restriction enzyme site, and the PCR reaction conditions is: 95 ℃ of pre-sex change 5min, 94 ℃ of 1min, 56 ℃ of 30s, 72 ℃ of 30s, totally 30 circulations, last 72 ℃ are extended 5min, with the negative contrast of distilled water;
TM1 GmGmGm811bp；
Gm-F：5-
gaattcaccgtggaaacgcatgaag-3?
Gm-R：5
-gaattcacggcttgaacgaattgtt-3
Gm-F and
Gm-R primer two ends add restriction enzyme site
EcoRI, underscore partly are restriction enzyme site, and the PCR reaction conditions is: 95 ℃ of pre-sex change 5 min, and 94 ℃ of 1 min, 56 ℃ of 30s, 72 ℃ of 50s, totally 30 circulations, last 72 ℃ are extended 5 min, with the negative contrast of distilled water;
Gm-F and
Gm-R primer two ends add same restriction enzyme site
EcoRI and protectiveness base, underscore partly is restriction enzyme site, and the PCR reaction conditions is: 95 ℃ of pre-sex change 5min, 94 ℃ of 1min, 56 ℃ of 30s, 72 ℃ of 1.0min, totally 30 circulations, last 72 ℃ are extended 7min, with distilled water and the negative contrast of EHEC O157:H7 DNA;
Stx2A-NF and
Stx2A-NR,
Stx2A-CF and
Stx2A-CR,
Gm-F and
GmThree pairs of primer amplifications of-R obtain the purpose band 397bp of corresponding size, 229bp and 811bp, and use 1% agarose electrophoresis respectively, cutting glue weighs, 75 ℃ of effect 10min after the DE-A damping fluid of 3 times of volumes of adding, the DE-B damping fluid that then adds 0.5 times of volume DE-A damping fluid, put upside down mixing, transfer liquid is in miniature pillar, and the centrifugal 1min of 12000g, abandons liquid, add lavation buffer solution I 500ul, and the centrifugal 1min of 12000g, abandon liquid, add lavation buffer solution II 750 ul, the centrifugal 1min of 12000g, abandon liquid, repeated washing damping fluid II is washed once, then the centrifugal 1min of blank pipe 12000g, shift pillar in the eppendorf of clean 1.5ml pipe, and adding the 60ul elution buffer, the centrifugal 1min of 12000g collects centrifugate, be purified 397bp, 229bp and 811bp fragment, called after SN, SC and
GmFragment;
Will
EcoRThe I enzyme is cut SN and each 3ul of SC fragment (4ng/ul), and T4 DNA connects damping fluid and each 1.0 ul of T4 dna ligase (350U/ul), and distilled water 2.0ul adds an eppendorf pipe simultaneously, and behind the mixing, 4 ℃ of connections are spent the night; Get spending the night of SN-SC and connect product 3.5ul (2.7ng/ul) and pGEM-T 1.0ul (50ng/ul), T4 DNA 2 * connection damping fluid 5.0 ul and T4 dna ligase 0.5ul(350U/ul) together add eppendorf pipe, behind the mixing, 4 ℃ of connections are spent the night, be used to transform the Top10 competent cell, the single bacterium colony of picking carries out incubated overnight, extracts plasmid, uses
BamHI and
SphI double digestion, 37 ℃ of water-bath 3h, agarose gel electrophoresis are identified, can see that the band of a 626bp occurs, and double digestion is identified the order-checking of male bacterium, obtain the pGEM-T-SN-SC recombinant plasmid, and glue reclaims
GmFragment 4.5ul(5ng/ul), together add an eppendorf pipe with pMD18-T0.5ul (50ng/ul) and solution I 4.5ul, behind the mixing, 16 ℃ connect 3h, are used to transform the Top10 competent cell, and the single bacterium colony of picking carries out incubated overnight, extract plasmid, use
EcoRI single endonuclease digestion, 37 ℃ of water-bath 2h, agarose gel electrophoresis are identified, can see that the band of a 811bp occurs, and double digestion is identified the order-checking of male bacterium, obtain pMD18-T-
GmRecombinant plasmid;
2. the pMEG-375-SGS reorganization knocks out the structure of plasmid
PGEM-T-SN-SC recombinant plasmid and pMD18-T-
GmRecombinant plasmid is used respectively
ECo
RThe I single endonuclease digestion, the alkaline phosphatase dephosphorylation, 4 ℃ of connections transform the Top10 competent cell, process
ECo
RThe I enzyme is cut and is obtained positive recombinant plasmid pGEM-T-SN-
Gm-SC, pGEM-T-SN-
Gm-the SC and the quick plasmid pMEG375 warm in nature that commits suiside use
BamHI and
SphEach 2ul(11ng/ul of I double digestion), be connected damping fluid and each 1.0ul of T4 dna ligase (350U/ul) with T4 DNA, distilled water 4.0ul adds an eppendorf pipe simultaneously, behind the mixing, 4 ℃ of connections are spent the night, and transform the sm10 competent cell, with the screening of paraxin plate, obtain positive recombinant plasmid, called after pMEG375-SN-
Gm-SC, the positive gene that is acquisition knocks out plasmid pSGS;
3. bacterium solid phase mating
Adopt solid phase filter hybridization method, inoculate recipient bacterium EHEC O157:H7(△ respectively
Hly) and the donor bacterium sm10 that contains pSGS in 5mL LB liquid nutrient medium, spend the night in 37 ℃ of shaking culture, the 4:1 ratio is got sm10 and the EHEC O157:H7(△ that contains pSGS respectively by volume then
Hly) overnight culture, mixing, filter with filter, bacterium is collected on the filter membrane, filter membrane is positioned on the nonresistant LB agar plate, and the germy one side of filter membrane makes progress, in 37 ℃ of overnight incubation, wash after bacterium on the filter membrane carries out 1000 times of dilutions with physiological saline then, separate application is in having Streptomycin sulphate and chlorampenicol resistant Strep
RCm
ROn the LB agar plate, 37 ℃ of overnight incubation, the single colony inoculation of picking is in Strep
RCm
RIncrease bacterium among the liquid LB, then bacterium liquid is carried out O157 serotype encoding gene
RfbEWith
GmCan encode simultaneously the two bacterium colony of gene amplification, acquisition, promptly
RfbE+
Gm+;
4. paraxin concentration method
The chemical sproof hybrid strain of paraxin of inoculating above-mentioned screening is in the LB liquid nutrient medium, 37 ℃ of overnight incubation, transfer in the fresh LB liquid nutrient medium with 2% inoculum size, cultivate 1-2h for 37 ℃, add paraxin and continue to cultivate 1-2h, press 1mg/mL and add the D-seromycin, put 37 ℃ more rapidly and continue to cultivate 1-2h, the centrifugal collection thalline of 8000r/min is washed once with physiological saline, after 100 times of dilutions, coat on the 10% sucrose flat board that contains 50ug/ml Gm, cultivate 16-20h for 30 ℃, the bacterium colony that grows is inoculated the dull and stereotyped and common LB flat board of the LB that contains paraxin respectively, screening paraxin sensitive strain;
5. EHEC O157:H7(△
Hly△
Stx2A) screening and acquisition
The centrifugal collection thalline of the responsive single bacterium colony culture of paraxin, resuspended, boil 7min, 4 ℃ of centrifugal collection supernatants are template to be detected, prepare the template supernatant of EHEC O157:H7 simultaneously, use
Gm-F and
Gm-R, design
Stx2A-F and
Stx2The a pair of primer of A-R:
Stx2A-F:5-gcgttaatggagttcagtggt-3
Stx2A-R:5-cttaactcctttatttacccgttgt-3
Carry out pcr amplification respectively, filtering out reorganization has
GmThe EHEC O157:H7 of gene, i.e. EHEC O157:H7(△
Hly△
Stx2A); With full bacterium polyclonal antiserum respectively with EHEC O157:H7(△
Hly) and EHEC O157:H7(△
Hly△
Stx2A) carry out protein immunoblotting and detect,, successfully obtain the EHEC O157:H7 bacterial strain of Hly and Stx2A disappearance, i.e. EHEC O157:H7(△ through the evaluation of gene level and protein level
Hly△
Stx2A) bacterial strain;
(3) EHEC O157:H7(△
Hly△
Stx△
ToxB) structure
1. at first obtain
ToxBBN and BC and
KanGene, parallel-series are cloned into the pET32 carrier
The toxB sequence of landing according to GenBank, sequence number is NC_007414, design two pairs of Auele Specific Primers, DNA with the preparation of EHECO157:H7 bacterial strain is a template, toxB gene 5 ' end upstream and 3 ' end downstream 831bp and 863bp sequence increase respectively, wherein toxB-NR is toxB gene 5 ' end upstream 36-54 base, and toxB-CF is toxB gene 3 ' end downstream 32-50 base;
toxB-NF：5-tta
ggatccatccatcaggcgttgtgc-3
toxB-NR：5-ggg
gaattctttgattagcgacaacct-3
toxB-CF：5-gcc
gtcgactaatcttacccagcatca-3
toxB-CR：5-att
gcggccgcgagttatcagcccgtcat-3
ToxB-NF,
ToxB-NR,
ToxB-CF and
ToxB-CR primer two ends add restriction enzyme site respectively
BamThe H I,
EcoRI,
SalI and
NotI and and the protectiveness base, underscore partly is restriction enzyme site; The PCR reaction conditions is: 95 ℃ of pre-sex change 5 min, and 94 ℃ of 1 min, 58 ℃ of 30 s, 72 ℃ of 1 min, totally 30 circulations, last 72 ℃ are extended 5 min, with the negative contrast of distilled water;
KanKanKan
Kan-F：5-ccg
gaattcacggctacactagaaggac-3
Kan-R：5-?aga
gtcgacaaatgtgcgcggaaccccta-3
KanKanEcoRSal?
ToxB-NF and
ToxB-NR,
ToxB-CF and
ToxB-CR,
Kan-F and
KanThree pairs of primer amplifications of-R obtain the purpose band 831bp of corresponding size, 863bp and 1192bp, and use 1% agarose electrophoresis respectively, cutting glue weighs, 75 ℃ of effect 10min after the DE-A damping fluid of 3 times of volumes of adding, the DE-B damping fluid that then adds 0.5 times of volume DE-A damping fluid, put upside down mixing, transfer liquid is in miniature pillar, and the centrifugal 1min of 12000g, abandons liquid, add lavation buffer solution I 500ul, and the centrifugal 1min of 12000g, abandon liquid, add lavation buffer solution II 750 ul, the centrifugal 1min of 12000g, abandon liquid, repeated washing damping fluid II is washed once, then the centrifugal 1min of blank pipe 12000g, shift pillar in the eppendorf of clean 1.5ml pipe, and adding the 60ul elution buffer, the centrifugal 1min of 12000g collects centrifugate, be purified 831bp, 863bp and 1192bp fragment, called after BN, BC and
KanFragment;
Will
BamThe H I and
EcoRThe I enzyme is cut BN fragment 3ul (9.6ng/ul) and pET32a carrier 4ul (10ng/ul), T4 DNA connects damping fluid and each 1.0ul of T4 dna ligase (350U/ul), distilled water 1.0ul adds an eppendorf pipe simultaneously, behind the mixing, 4 ℃ of connections are spent the night, and transform the Top10 competent cell, and the single bacterium colony of picking carries out incubated overnight, extract plasmid, use
BamThe H I and
EcoRI double digestion, 37 ℃ of water-bath 3h, agarose gel electrophoresis are identified, can see that the band of a 831bp occurs, and obtain the pET32a-BN recombinant plasmid, then will
EcoRI and
SalThe Kan that the I enzyme is cut is connected with the pET32a-BN recombinant plasmid, obtains pET32a-BN-Kan, at last will
SalI and
NotThe BC that the I enzyme is cut is connected with the pET32a-BN-Kan recombinant plasmid, obtains pET32a-BN-Kan-BC, transforms the Top10 competent cell, cultivates 16h for 37 ℃, and the dull and stereotyped single bacterium colony of picking is identified; To identify that male recombinant plasmid and pMEG375 carrier use respectively
BamHI and
NotAfter the I enzyme was cut, 4 ℃ of connections were spent the night, and transformed competence colibacillus cell sm10 with the screening of paraxin plate, obtains positive recombinant plasmid, called after pMEG375-BN-
Kan-BC, the positive gene that is acquisition knocks out plasmid pBKB;
2. bacterium solid phase mating
Adopt solid phase filter hybridization method, inoculate recipient bacterium EHEC O157:H7(△ respectively
Hly△
Stx) and the donor bacterium sm10 that contains pBKB in 5mL LB liquid nutrient medium, spend the night in 37 ℃ of shaking culture, get sm10 and the EHEC O157:H7(△ that contains pBKB respectively in the 4:1 ratio then
Hly△
Stx) overnight culture, mixing, filter with filter, bacterium is collected on the filter membrane, and filter membrane is positioned on the nonresistant LB agar plate, and the germy one side of filter membrane upwards, in 37 ℃ of overnight incubation, wash bacterium on the filter membrane with physiological saline then, after 1000 times of dilutions, separate application is in having Streptomycin sulphate and chlorampenicol resistant Strep
RCm
ROn the LB agar plate, 37 ℃ of overnight incubation, the single colony inoculation of picking is in Strep
RCm
RIncrease bacterium among the liquid LB, then bacterium liquid is carried out O157 serotype encoding gene
RfbEWith
KanCan encode simultaneously the two bacterium colony of gene amplification, acquisition, promptly
RfbE+
Kan+;
3. paraxin concentration method
The chemical sproof hybrid strain of paraxin of inoculating above-mentioned screening is in the LB liquid nutrient medium, 37 ℃ of overnight incubation, transfer in the fresh LB liquid nutrient medium with 2% inoculum size, cultivate 1-2h for 37 ℃, add paraxin and continue to cultivate 1-2h, press 1mg/mL and add the D-seromycin, put 37 ℃ more rapidly and continue to cultivate 1-2h, the centrifugal collection thalline of 8000r/min is washed once with physiological saline, after 100 times of dilutions, coat and contain on the 100ug/ml kantlex 10% sucrose flat board, cultivate 16-20h for 30 ℃, the bacterium colony that grows is inoculated the dull and stereotyped and common LB flat board of the LB that contains paraxin respectively, screening paraxin sensitive strain;
4. EHEC O157:H7(△
Hly△
Stx△
ToxB) screening and acquisition
The centrifugal collection thalline of the responsive single bacterium colony culture of paraxin, resuspended, boil 7min, 4 ℃ of centrifugal collection supernatants are template to be detected, prepare the template supernatant of EHEC O157:H7 simultaneously, use
Kan-F and
Kan-R, design
ToxB-F and
ToxBThe a pair of primer of-R:
ToxB-F:5-aattcaatggaattatcagaat-3
ToxB-R:5-taaacgattttctacctctgg-3
Carry out pcr amplification respectively, filtering out reorganization has
KanThe EHEC O157:H7 of gene, i.e. EHEC O157:H7(△
Hly△
Stx△
ToxB); With full bacterium polyclonal antiserum respectively with EHEC O157:H7(△
Hly△
Stx2A) and EHEC O157:H7(△
Hly△
Stx2A △
ToxB) carry out the protein immunoblotting detection, through the evaluation of gene level and protein level, successfully obtain the EHEC O157:H7 bacterial strain of Hly, Stx and ToxB disappearance, i.e. EHEC O157:H7(△
Hly△
Stx△
ToxB) bacterial strain.
3. the application of claim 1 or 2 described enterorrhagia Bacillus coil 0157: H7, three genetically deficient bacterial strains.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108018228A (en) * | 2017-09-28 | 2018-05-11 | 江苏省农业科学院 | One plant of Escherichia coli O 157:H7 low virulent strains and its application |
| CN108220213A (en) * | 2017-03-13 | 2018-06-29 | 安徽安龙基因医学检验所有限公司 | A kind of construction method for lacking ompA gene riemerella anatipestifer CH3 attenuated strains |
| CN108362629A (en) * | 2018-02-09 | 2018-08-03 | 中国计量科学研究院 | Escherichia coli O 157:The rapid detection method and kit of the single viable bacterias of H7 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1600849A (en) * | 2003-09-24 | 2005-03-30 | 冯书章 | Bacilluscoli 0157 gene deficiency bacterin of intestinal hemorrhage |
| CN100478028C (en) * | 2007-02-05 | 2009-04-15 | 中国人民解放军第三军医大学 | Genetic engineering vaccine of enterohemorrhagic escherichia coli O157:H7 and the preparing method thereof |
| WO2009088403A2 (en) * | 2007-10-08 | 2009-07-16 | Rutgers, The State University | Nontoxic shiga-like toxin mutant compositions and methods |
-
2011
- 2011-04-08 CN CN 201110087699 patent/CN102206607B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1600849A (en) * | 2003-09-24 | 2005-03-30 | 冯书章 | Bacilluscoli 0157 gene deficiency bacterin of intestinal hemorrhage |
| CN100478028C (en) * | 2007-02-05 | 2009-04-15 | 中国人民解放军第三军医大学 | Genetic engineering vaccine of enterohemorrhagic escherichia coli O157:H7 and the preparing method thereof |
| WO2009088403A2 (en) * | 2007-10-08 | 2009-07-16 | Rutgers, The State University | Nontoxic shiga-like toxin mutant compositions and methods |
Non-Patent Citations (5)
| Title |
|---|
| HAIQING SHENG ET AL.: "Role of Escherichia coli O157:H7 Virulence Factors in Colonization at the Bovine Terminal Rectal Mucosa", 《AMERICAN SOCIETY FOR MICROBIOLOGY》 * |
| MMAITE MUNIESA ET AL.: "Occurrence of Escherichia coli O157:H7 and Other Enterohemorrhagic Escherichia coli in the Environment", 《ENVIRON. SCI. TECHNOL》 * |
| VALERIE BURLAND ET AL.: "The complete DNA sequence and analysis of the large virulence plasmid of Escherichia coli O157:H7", 《NUCLEIC ACIDS RESEARCH》 * |
| 张雪寒 等: "出血性大肠杆菌O157∶ H7 Stx2B-Tir-Stx1B多价融合蛋白表达及免疫原性研究", 《华北农学报》 * |
| 潘劲草 等: "肠出血性大肠埃希菌O157毒力因子研究进展", 《预防医学情报杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108220213A (en) * | 2017-03-13 | 2018-06-29 | 安徽安龙基因医学检验所有限公司 | A kind of construction method for lacking ompA gene riemerella anatipestifer CH3 attenuated strains |
| CN108018228A (en) * | 2017-09-28 | 2018-05-11 | 江苏省农业科学院 | One plant of Escherichia coli O 157:H7 low virulent strains and its application |
| CN108362629A (en) * | 2018-02-09 | 2018-08-03 | 中国计量科学研究院 | Escherichia coli O 157:The rapid detection method and kit of the single viable bacterias of H7 |
| CN108362629B (en) * | 2018-02-09 | 2021-02-05 | 中国计量科学研究院 | Rapid detection method and kit for single viable bacteria of Escherichia coli O157H 7 |
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