CN102586311A - Enterohemorrhage Escherichia coli (EHEC) O157:H7 multivalence fliC-hcpA-tir-eae recombinant strain and vaccine - Google Patents
Enterohemorrhage Escherichia coli (EHEC) O157:H7 multivalence fliC-hcpA-tir-eae recombinant strain and vaccine Download PDFInfo
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Abstract
The invention relates to an Enterohemorrhage Escherichia coli (EHEC) O157:H7 multivalence fliC-hcpA-tir-eae recombinant strain and a vaccine, which belong to the field of biopharmacy. H7 fliC gene of EHECO157:H7, hcpA genes, tir genes and eae genes are respectively amplified, sequentially connected in series, cloned into a expression vector pCold to obtain recombinant plasmid pCold-fliC-hapA-tir-eae, and efficiently expressed in BL21(DE3). Highly-purified fusion protein molecue vaccines are obtained through high-density fermentation and a serial of purification programs. The preparation process of the vaccine is fast, convenient, easy to amplify and good in repeatability and has an immunoprophylaxis protection and treatment function. The obtained target protein is high in purity, and according to animal experiments, high-efficiency immune response in terms of humoral immunity, cellular immunity and mucosal immunity of organisms can be simulated.
Description
One, technical field
The present invention relates to enterorrhagia Bacillus coil 0157: the reorganization of H7 multivalence pattern of fusion bacterium, recombinant protein preparation method and subunit vaccine belong to field of biological pharmacy.
Two, background technology
EHEC (enterohaemorrhagic
Escherichia coli, be that important food source property Amphixenosis is former EHEC), O157:H7 is its important serotype.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 human infection can cause serious gi tract and blood circulation diseases; Like hemorrhagic colitis (hemorrhagic Colitis; HC), (Haemolytic uraemic syndrome, HUS) etc., individual patient can be because of acute dead with chronic renal failure for hemolytic uremic syndrome.Zoogenetic infection does not generally cause tangible clinical symptom, but can carry for a long time even throughout one's life.EHEC animal host scope is very extensive, and wherein the ruminating animal bacterial bearing rate is higher.Therefore, the most important thing of prevention and control EHEC O157:H7 is that minimizing and control animal carry disease germs and discharge of bacteria.
At present, the infection shortage effectively preventing method to O157:H7 can only give symptomatic treatment and suitable antibacterial therapy.Because the outbreak of epidemic characteristics of O157:H7 and the difficulty of treating, it is very urgent that vaccine research just seems.Effectively vaccine development is that prevention and treatment O157:H7 infect the simplest, economical and means efficiently.
Choose and adhere to EHEC O157:H7 that to distinguish design specific primers right with the closely-related flagellum of field planting (H7), pili (HCP) and tight four genes of element (Intimin) and the tight plain acceptor of transposition (Tir); The amplification corresponding gene fragment; And series connection successively, make up the reorganization bacterium, obtain recombinant protein; The preparation subunit vaccine is used to prevent EHEC O157:H7 field planting in animal body.
Three, find content
Technical problemThe object of the invention is to make up EHEC O157:H7 multivalence pattern of fusion reorganization bacterium, express recombinant protein, and then development subunit vaccine.The present invention directly is directed against the important adhesion factor of EHEC O157:H7 on antigen selection; The combined utilization of stressing a plurality of adhesion factors is to produce maximum immanoprotection action; Reduce the animal bacterial bearing rate, and then reduce the chance of human infection EHEC, ensure the human physical and mental health.
Technical schemeSpecific embodiments of the present invention is following:
The reorganization of enterorrhagia Bacillus coil 0157: H7 (EHEC O157:H7) multivalence pattern of fusion bacterium, recombinant protein preparation method and subunit vaccine; It is characterized in that said polyvalent recombinant bacterium and recombinant protein are BL21 (pCold I-fliC-hcpA-tir-eae).The preparation method of polyvalent recombinant bacterium and recombinant protein comprises:
(1) enterorrhagia Bacillus coil 0157: H7 immunity multivalence pattern of fusion reorganization bacterium, its construction process is:
According to O157:H7 EDL933's among the GenBank
FliC,
HcpA,
TirWith
EaeGene order designs primer respectively, and adds restriction enzyme site (underscore part) at upstream and downstream primer two ends respectively,
HcpABehind the gene with
EaeAdding length before the gene is the junction fragment (dash area) of 24 Nucleotide, and primer sequence is following:
fliC-P1：5-ggg
gagctcactattaccaacaaa-3
Sac?Ⅰ
fliC-P2：5-tta
ctcgagggtcgttgcagaacc-3
Xhol?Ⅰ
hcpA-P1：5-ggg
ctcgagtttacacttatcgaactgat-3
Xhol?Ⅰ
hcpA-P2：5-ttt
gaattctttagcagcagctttagcagcagcgttggcgtcatc-3
EcoRⅠ
tir-P1：?5-gat
gaattcgagccggatagccca-3
EcoRⅠ
tir-P2：?5-tta
gtcgacagcccccgatgaaac-3
Sal?Ⅰ
eae-P1：5-tta
gtcgacgctgctgctaaagctgctgctaaatttgatcaaacc-3
Sal?Ⅰ
eae-P2：5-?ggc
tctagattattctacacaaaccgc-3
Xba?Ⅰ
The O157:H7 overnight culture, 1mL/ pipe, centrifugal 2 minutes of 12000r abandons supernatant, collects thalline and is resuspended in 1/5 volume distilled water, boils 10min, and centrifugal 10 minutes of 4 ℃ of 12000r draw frozen-20 ℃ of supernatant, are used for pcr amplification and use;
Amplification
FliCSegmental upstream and downstream primer is added with respectively
SacI with
XholThe I restriction enzyme site, amplification
EaeSegmental upstream and downstream primer is added with respectively
SalI with
XbaThe I restriction enzyme site, the PCR reaction conditions is: 95 ℃ of preparatory sex change 5 minutes, 94 ℃ 1 minute, 55 ℃ 30 seconds, 72 ℃ 1 minute, 30 circulations, 72 ℃ were extended 10 minutes again;
Amplification
HcpASegmental upstream and downstream primer respectively is added with
XholI with
EcoRThe I restriction enzyme site, amplification
TirSegmental upstream and downstream primer is added with respectively
EcoRI with
SalThe I restriction enzyme site, the PCR reaction conditions is: 95 ℃ of preparatory sex change 5 minutes, 94 ℃ 1 minute, 56 ℃ 30 seconds, 72 ℃ 30 seconds, 30 circulations, 72 ℃ were extended 6 minutes again.
FliC-P1 with
FliC-P2,
HcpA-P1 with
HcpA-P2,
Tir-P1 with
Tir-P2,
Eae-P1 with
Eae-P2 expanding fragment length is respectively 945bp, 438bp, 327bp and 891bp.The fliC fragment of amplification is being read between the frame 538-1483 Nucleotide; The hcpA fragment of amplification is being read between the frame 19-441 Nucleotide; The tir fragment of amplification is being read between the frame 775-1083 Nucleotide; The eae fragment of amplification is being read between the frame 1995-2805 Nucleotide.
With the agarose electrophoresis of amplification PCR products with mass ratio 1.2%, cut glue and weigh, add after 3 times of volume DE-A damping fluids 65 ℃ of effects 10 minutes, treat that glue all melts after; The DE-B damping fluid that adds 0.5 volume of DE-A damping fluid again, put upside down mixing after, the liquid that melts is shifted in miniature pillar, and centrifugal 1 minute of 12000g; Abandon liquid, add lavation buffer solution I 500uL, centrifugal 30 seconds of 12000g abandons liquid again; Add lavation buffer solution II 750uL, centrifugal 1 minute of 12000g abandons liquid, centrifugal 1 minute of blank pipe 12000g; Shift pillar in clean 1.5mL centrifuge tube, and add 60uL elution buffer, centrifugal 1 minute of 12000g; Collect centrifugate, be purified 945bp, 438bp, 327bp and 891bp fragment, called after fliC, hcpA, tir and eae fragment.
Will
SacI with
XholThe carrier pCold I fragment 4uL that fliC fragment 4uL that the I enzyme is cut and same enzyme are cut, T4 DNA connects damping fluid and each 1.0uL of T4 dna ligase, adds a centrifuge tube simultaneously; Behind the mixing; 4 ℃ of connections are spent the night, and are used to transform the Top10 competent cell, and the single bacterium colony of picking carries out incubated overnight; Extract plasmid, use
SacI with
XholThe I double digestion, 37 ℃ of water-baths 2 hours, agarose gel electrophoresis can see that the band of a 945bp occurs, obtain the pCold I-
FliCRecombinant plasmid; Use
XhoI with
EcoRThe I enzyme is cut hcpA fragment and pCold I-fliC recombinant plasmid, after glue reclaims, and the pCold I-
FliCGet 4.0uL, the hcpA fragment is got 2 uL, and T4 DNA connects damping fluid and each 1.0ul of T4 dna ligase, moisturizing 2 uL; Together add the eppendorf pipe, behind the mixing, 4 ℃ of connections are spent the night, and transform the Top10 competent cell; The single bacterium colony of picking carries out incubated overnight, extracts plasmid, and enzyme is cut, 37 ℃ of water-baths 2 hours; Agarose gel electrophoresis can see that the band of 438bp occurs, and obtains pCold I-fliC-hcpA recombinant plasmid; Use
EcoRI with
SalThe I enzyme is cut tir fragment and pCold I-fliC-hcpA recombinant plasmid, and after glue reclaimed, pCold I-fliC-hcpA got 2.0uL, and the tir fragment is got 3.0 uL; T4 DNA connects damping fluid and each 1.0ul of T4 dna ligase, and moisturizing 3 uL together add in the centrifuge tube; 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, enzyme is cut, 37 ℃ of water-baths 2 hours; Agarose gel electrophoresis can see that the band of 327bp occurs, and obtains pCold I-fliC-hcpA-tir recombinant plasmid; Use
SalI with
XbaThe I enzyme is cut eae fragment and pCold I-fliC-hcpA-tir recombinant plasmid, and after glue reclaimed, pCold I-fliC-hcpA-tir and eae respectively got 4.0uL; T4 DNA connects damping fluid and each 1.0ul of T4 dna ligase, together adds in the pipe, 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, enzyme is cut, 37 ℃ of water-baths 2 hours; Agarose gel electrophoresis can see that the band of 891bp occurs, and obtains pCold I-fliC-hcpA-tir-eae recombinant plasmid; Correct positive plasmid pCold I-fliC-hcpA-tir-eae is transformed in BL21 (DE3) competent cell with order-checking, and acquisition polyvalent recombinant bacterium BL21 (the pCold I-fliC-hcpA-tir-eae).
(2) structure of polyvalent recombinant bacterium, polyvalent recombinant Expression of Fusion Protein and purifying
(culture of pCold I-fliC-hcpA-tir-eae) 2% is inoculated in and contains 100 μ g/mL and have among the antibiotic LB of ammonia benzyl by volume with BL21; 37 ℃ of shaking culture to A=0.6 ~ 0.8 o'clock add inductor IPTG to working concentration be 0.5mM, continues 15 ℃ of concussion cultivations 24 hours, induce the centrifugal collection thalline of bacterium liquid to be resuspended in the TE damping fluid; Behind the ultrasonic degradation; Centrifugal 25 minutes of 12000g, cleer and peaceful deposition is carried out SDS-PAGE in the collection, can clearly see that the molecular weight size is the 88kD target protein; Mainly be present in the thalline supernatant, obtain the fliC-hcpA-tir-eae polyvalent recombinant protein.
Induce reorganization bacterium BL21 (pCold I-fliC-hcpA-tir-eae) was through centrifugal 10 minutes of 12000 g above-mentioned; Collect thalline, resuspended with the chelation buffer of 1/10 volume, add the N,O-Diacetylmuramidase of 1mg/mL again; 30 ℃ act on 2 hours behind the mixing; UW carries out ultrasonication, and 4 ℃ of 12000 g collected supernatant in centrifugal 30 minutes, and 0.45 μ M filter filters the back and goes up the Ni+ affinity column through pre-treatment (chelation buffer of 4 times of column volumes is washed pillar); Add elution buffer and collect albumen, be purified fliC-hcpA-tir-eae recombinant protein.
(3) preparation of EHEC O157:H7 multivalence fusion rotein subunit vaccine
The multivalence fusion rotein of above-mentioned purifying is measured protein concentration, be controlled at 0.2 μ g/ μ L.Antigen and adjuvant (ISA50V, French Seppic company produces) carry out dispersion treatment according to 1:1 (V/V) and process oil-emulsion.
Beneficial effectCharacteristics of the present invention and advantage are following:
1, the present invention is directed to the important adhesion factor series connection of EHEC O157:H7 and made up pattern of fusion reorganization bacterium.Utilize PCR, obtain fliC, hcpA, tir and four gene fragments of eae, and through series connection; Finally be cloned among the expression plasmid pCold; Success makes up pCold-fliC-hcpA-tir-eae, is transformed into and obtains reorganization bacterium, i.e. BL21 (pCold-fliC-hcpA-tir-eae) among the BL21.
2, the present invention successfully obtains BL21 (pCold-fliC-hcpA-tir-eae); Cultivate through 37 ℃ of high density fermentations; Inducing of 15 ℃ of IPTG obtains to efficiently express; Expression amount accounts for 30% of whole bacterial protein, through obtaining reorganization pattern of fusion albumen, i.e. fliC-hcpA-tir-eae recombinant protein behind the purifying.
3, the fliC-hcpA-tir-eae recombinant protein of the present invention after with purifying prepared subunit vaccine; The immunity Balb/c mouse and goat; The recombinant protein of clear and definite above-mentioned expression has good antigenicity, is the infection of more safe and effective prevention and control EHEC and causes a disease effective technical is provided.
Four, description of drawings
Fig. 1: pCold-fliC-hcpA-tir-eae recombinant plasmid enzyme is cut evaluation.
M, DL-2000; 1-3, pCold-fliC-hcpA-tir-eae recombinant plasmid plasmid
SacI/
XbaI
The electrophoretic analysis of Fig. 2: BL21 (pCold-fliC-hcpA-tir-eae) reorganization bacterium.
M, middle molecular weight protein matter Marker; 1, BL21 (pCold-fliC-hcpA-tir-eae) induces the back whole bacterial protein; 2, BL21 (pCold-fliC-hcpA-tir-eae) induces preceding whole bacterial protein; 3, BL21 (pCold-fliC-hcpA-tir-eae) induces back whole bacterial protein supernatant
Fig. 3: IgG titer determination in the mice serum of immunity back
Fig. 4: immune group and control group mice are attacked discharge of bacteria and are changed
Fig. 5: IgG titer determination in the goat ight soil of immunity back
Fig. 6: immune group and control group goat are attacked discharge of bacteria and change
Five, embodiment
1, obtain fliC, hcpA, tir and eae gene, parallel-series is cloned into the pCold carrier
According to O157:H7 EDL933's among the GenBank (bio tech ltd is stepped in Shanghai three)
FliC(sequence number: L07388),
HcpA(sequence number: NC_013008),
Tir(sequence number: AF125993) with
Eae(sequence number: Z11541) gene order designs primer respectively, and adds restriction enzyme site (underscore part) at upstream and downstream primer two ends respectively,
HcpABehind the gene with
EaeAdding length before the gene is the junction fragment (dash area) of 24 Nucleotide, and primer sequence is following:
fliC-P1：5-ggg
gagctcactattaccaacaaa-3
Sac?Ⅰ
fliC-P2：5-tta
ctcgagggtcgttgcagaacc-3
Xhol?Ⅰ
hcpA-P1：5-ggg
ctcgagtttacacttatcgaactgat-3
Xhol?Ⅰ
hcpA-P2：5-ttt
gaattctttagcagcagctttagcagcagcgttggcgtcatc-3
EcoRⅠ
tir-P1：?5-gat
gaattcgagccggatagccca-3
EcoRⅠ
tir-P2：?5-tta
gtcgacagcccccgatgaaac-3
Sal?Ⅰ
eae-P1：5-tta
gtcgacgctgctgctaaagctgctgctaaatttgatcaaacc-3
Sal?Ⅰ
eae-P2：5-?ggc
tctagattattctacacaaaccgc-3
Xba?Ⅰ
The O157:H7 overnight culture, 1mL/ pipe, centrifugal 2 minutes of 12000r abandons supernatant, collects thalline and is resuspended in 1/5 volume distilled water, boils 10min, and centrifugal 10 minutes of 4 ℃ of 12000r draw frozen-20 ℃ of supernatant, are used for pcr amplification and use;
Amplification
FliCSegmental upstream and downstream primer is added with respectively
SacI with
XholThe I restriction enzyme site, amplification
EaeSegmental upstream and downstream primer is added with respectively
SalI with
XbaThe I restriction enzyme site, the PCR reaction conditions is: 95 ℃ of preparatory sex change 5 minutes, 94 ℃ 1 minute, 55 ℃ 30 seconds, 72 ℃ 1 minute, 30 circulations, 72 ℃ were extended 10 minutes again; Amplification
HcpASegmental upstream and downstream primer respectively is added with
XholI with
EcoRThe I restriction enzyme site, amplification
TirSegmental upstream and downstream primer is added with respectively
EcoRI with
SalThe I restriction enzyme site, the PCR reaction conditions is: 95 ℃ of preparatory sex change 5 minutes, 94 ℃ 1 minute, 56 ℃ 30 seconds, 72 ℃ 30 seconds, 30 circulations, 72 ℃ were extended 6 minutes again.
FliC-P1 with
FliC-P2,
HcpA-P1 with
HcpA-P2,
Tir-P1 with
Tir-P2,
Eae-P1 with
Eae-P2 expanding fragment length is respectively 945bp, 438bp, 327bp and 891bp.The fliC fragment of amplification is being read between the frame 538-1483 Nucleotide; The hcpA fragment of amplification is being read between the frame 19-441 Nucleotide; The tir fragment of amplification is being read between the frame 775-1083 Nucleotide; The eae fragment of amplification is being read between the frame 1995-2805 Nucleotide.
With the agarose electrophoresis of amplification PCR products with mass ratio 1.2%, cut glue and weigh, add the back 65 ℃ of effects of 3 times of volume DE-A damping fluids (day root company) 10 minutes, treat that glue all melts after; The DE-B damping fluid that adds 0.5 volume of DE-A damping fluid again, put upside down mixing after, the liquid that melts is shifted in miniature pillar (day root company), and centrifugal 1 minute of 12000g; Abandon liquid, add lavation buffer solution I 500uL, centrifugal 30 seconds of 12000g abandons liquid again; Add lavation buffer solution II 750uL, centrifugal 1 minute of 12000g abandons liquid, centrifugal 1 minute of blank pipe 12000g; Shift pillar in clean 1.5mL centrifuge tube, and add 60uL elution buffer, centrifugal 1 minute of 12000g; Collect centrifugate, be purified 945bp, 438bp, 327bp and 891bp fragment, called after fliC, hcpA, tir and eae fragment.
Will
SacI with
XholThe carrier pCold I that fliC fragment 4uL that the I enzyme is cut and same enzyme are cut (the precious biotech firm in Dalian) fragment 4uL, T4 DNA connects damping fluid and each 1.0uL of T4 dna ligase, adds a centrifuge tube simultaneously; Behind the mixing; 4 ℃ of connections are spent the night, and are used to transform the Top10 competent cell, and the single bacterium colony of picking carries out incubated overnight; Extract plasmid, use
SacI with
XholThe I double digestion, 37 ℃ of water-baths 2 hours, agarose gel electrophoresis can see that the band of a 945bp occurs, obtain the pCold I-
FliCRecombinant plasmid; Use
XhoI with
EcoRThe I enzyme is cut hcpA fragment and pCold I-fliC recombinant plasmid, after glue reclaims, and the pCold I-
FliCGet 4.0uL, the hcpA fragment is got 2 uL, and T4 DNA connects damping fluid and each 1.0ul of T4 dna ligase, moisturizing 2 uL; Together add the eppendorf pipe, behind the mixing, 4 ℃ of connections are spent the night, and transform the Top10 competent cell; The single bacterium colony of picking carries out incubated overnight, extracts plasmid, and enzyme is cut, 37 ℃ of water-baths 2 hours; Agarose gel electrophoresis can see that the band of 438bp occurs, and obtains pCold I-fliC-hcpA recombinant plasmid; Use
EcoRI with
SalThe I enzyme is cut tir fragment and pCold I-fliC-hcpA recombinant plasmid, and after glue reclaimed, pCold I-fliC-hcpA got 2.0uL, and the tir fragment is got 3.0 uL; T4 DNA connects damping fluid and each 1.0ul of T4 dna ligase, and moisturizing 3 uL together add in the centrifuge tube; 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, enzyme is cut, 37 ℃ of water-baths 2 hours; Agarose gel electrophoresis can see that the band of 327bp occurs, and obtains pCold I-fliC-hcpA-tir recombinant plasmid; Use
SalI with
XbaThe I enzyme is cut eae fragment and pCold I-fliC-hcpA-tir recombinant plasmid, and after glue reclaimed, pCold I-fliC-hcpA-tir and eae respectively got 4.0uL; T4 DNA connects damping fluid and each 1.0ul of T4 dna ligase, together adds in the pipe, behind the mixing; 4 ℃ of connections are spent the night, and transform Top10 (Nanjing hundred is thought triumphant) competent cell, and the single bacterium colony of picking carries out incubated overnight; Extract plasmid, enzyme is cut, 37 ℃ of water-baths 2 hours; Agarose gel electrophoresis can see that the band of 891bp occurs, and obtains pCold I-fliC-hcpA-tir-eae recombinant plasmid; The correct positive plasmid pCold I-fliC-hcpA-tir-eae of order-checking is transformed in BL21 (DE3) (Nanjing hundred the is thought triumphant) competent cell, and acquisition polyvalent recombinant bacterium BL21 (the pCold I-fliC-hcpA-tir-eae).
2, the structure of polyvalent recombinant bacterium, polyvalent recombinant Expression of Fusion Protein and purifying
(culture of pCold I-fliC-hcpA-tir-eae) 2% is inoculated in and contains 100 μ g/mL antibiotic LB substratum (the peptone 10g/L of ammonia benzyl is arranged by volume with BL21; Yeast extract 5g/L; Sodium-chlor 5g/L) in, 37 ℃ of shaking culture to A=0.6 ~ 0.8 o'clock add inductor IPTG to working concentration be 0.6mM, continues 15 ℃ of concussion cultivations 24 hours; Induce the centrifugal collection thalline of bacterium liquid to be resuspended in the TE damping fluid, behind the ultrasonic degradation, centrifugal 25 minutes of 12000g; Cleer and peaceful deposition is carried out SDS-PAGE in the collection; Can clearly see that the molecular weight size for the 88kD target protein, mainly is present in the thalline supernatant, obtain the fliC-hcpA-tir-eae polyvalent recombinant protein.
Induce reorganization bacterium BL21 (pCold I-fliC-hcpA-tir-eae) is collected thalline through centrifugal 10 minutes of 12000 g, and is resuspended with the chelation buffer of 1/10 volume above-mentioned; The N,O-Diacetylmuramidase that adds 1mg/mL again; 30 ℃ act on 2 hours behind the mixing, and UW carries out ultrasonication, and 4 ℃ of 12000 g collected supernatant in centrifugal 30 minutes; After 0.45 μ M filter filters; Join Ni+ affinity column (the handsome company in Shanghai), add elution buffer and collect albumen, be purified fliC-hcpA-tir-eae recombinant protein.
3, the preparation of EHEC O157:H7 multivalence fusion rotein subunit vaccine
The multivalence fusion rotein of above-mentioned purifying is measured protein concentration, be controlled at 0.2 μ g/ μ L.Antigen and adjuvant ISA50V (French Seppic company produce) carry out dispersion treatment according to 1:1 (V/V) and process oil-emulsion.
4, immunity test
(1) mouse test
(1) laboratory animal is chosen 20 of male BALB/c mouses (Yangzhou University's purchase), and 18g-22g/ only.
(2) pre-treatment of laboratory animal all groups before attacking poison all give 5g/L Streptomycin sulphate solution and drank 3 days, and excrement is examined the enteron aisle discharge of bacteria and is less than 10 after 3 days
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 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 before attacking poison, cut off the water supply jejunitas 12 hours, attack poison and recovered drinking water diet in back 6 hours, prevent to attack poison back bacterium and discharge animal intestinal fast, prolong action time in its body.
(3) immunity is attacked poison and with the grouping situation 20 mouse is divided into 2 groups at random, and 10 every group, promptly experimental group is used the subunit vaccine of preparation, and control group is alternative with the sterile phosphate damping fluid of equivalent.Adopt back and subcutaneous abdomen multi-point injection mode to carry out immunity, immunity 1 time, immunizing dose is 25ug/.Immunity is irritated stomach after back 35 days and is attacked poison, and dosage is about 1 * 10
10CFU/ only.Attack close observation behind the bacterium and respectively organize the variation of the behavior of mouse, the situation of ingesting and the mental status, detail statistics is respectively organized the dead mouse situation, and collects the mouse fresh excreta every three days, measures the changing conditions of discharge of bacteria time and discharge of bacteria amount in the ight soil.
(4) detection of fliC-hcpA-tir-eae specific IgG antibodies in the serum of immunity back
Before and after the mouse immune, mouse is taked the docking mode preparation serum of taking a blood sample, serum IgG antibody is tired after using indirect ELISA method and detecting mouse immune.
(5) immune effect is confirmed foundation
1. the antibody variation utilizes ELISA to detect the serum antibody variation of immunity back, and with the recombinant antigen coated elisa plate, the result shows: can induce the IgG of high titre, reach 3.89 * 10
5More than, and consistence is good.Fig. 3.
2. attack malicious protection situation and attacked poison in 35 days, the result shows: immune group mouse survival rate is 10/10, and the control group survival rate is 6/10.
3. the discharge of bacteria quantitative changeization is attacked and begun to gather ight soil in malicious back 24 hours, gathers once every three days afterwards, and is dull and stereotyped through coating screening property Mai Kangkai after suitably handling; Cultivated 18～24 hours for 37 ℃; Take out plate count, and bacterium colony is identified with double PCR, be O157:H7.The immune group discharge of bacteria time shortens, and immune group is attacked poison and detected less than O157:H7 in the 4th day afterwards, and control group still can detect up to 10 in 14d
4The discharge of bacteria that CFU is above, Fig. 4.
(2) goat test
(1) laboratory animal is chosen 10 of male goats (Jiangsu Province Agriculture Science Institute Yang Chang purchase), 3 monthly ages.
(2) all goats are jejunitas cut off the water supply 12 hours before attacking poison in the pre-treatment of laboratory animal, attack poison and recover drinking water diet in back 6 hours, prevent to attack the back bacterium and discharge animal intestinal fast, prolongation action time in its body.
(3) immunity is attacked poison and with the grouping situation 10 goats is divided into 2 groups at random, 5/organize.The immunity of neck multi-point injection, immunizing dose are 200ug/.After 21 days, carry out 2 immunity at interval, irritate stomach after 14 days behind the second immunisation and attack poison, dosage is about 1 * 10
10CFU/ only.Close observation was respectively organized the behavior of sheep after attacking bacterium, and whenever collected the fresh excreta of goat, the changing conditions of discharge of bacteria time and discharge of bacteria amount in the mensuration ight soil at a distance from 2 days.
(4) detection of fliC-hcpA-tir-eae specific IgG antibodies in the serum of immunity back
After exempting from two before the goat immunity, ear vein blood sampling preparation serum is used indirect ELISA method and is detected immunity back serum IgG antibody and tire.
(5) immune effect is confirmed foundation
1. the antibody variation utilizes ELISA to detect the serum antibody variation of immunity back, and with the recombinant antigen coated elisa plate, the result shows: can induce the IgG of high titre, reach 2.48 * 10
4More than.Fig. 5.
2. attack malicious protection situation contrast goat and immune goat and have no untoward reaction, do not have dead.
3. the discharge of bacteria quantitative changeization is attacked and begun to gather ight soil in malicious back 24 hours, gathers once in per afterwards 2 days, and is dull and stereotyped through coating screening property Mai Kangkai after suitably handling; Cultivated 18～24 hours for 37 ℃; Take out plate count, and bacterium colony is identified with double PCR, be O157:H7.Immune goat is discharge of bacteria not, and contrast goat the 12nd day is still at discharge of bacteria.Fig. 6.
SEQUENCE?LISTING
< 110>Jiangsu Province Agriculture Science Institute
< 120>enterorrhagia Bacillus coil 0157: H7 multivalence fliC-hcpA-tir-eae reorganization bacterium and vaccine
<130> 0
<160> 8
<170> PatentIn?version?3.1
<210> 1
<211> 24
<212> DNA
< 213>manual work
<220>
<221> fliC-P1
<222> (1)..(24)
<223>
<400> 1
ggggagctca?ctattaccaa?caaa 24
<210> 2
<211> 24
<212> DNA
< 213>manual work
<220>
<221> fliC-P2
<222> (1)..(24)
<223>
<400> 2
ttactcgagg?gtcgttgcag?aacc 24
<210> 3
<211> 29
<212> DNA
< 213>manual work
<220>
<221> hcpA-P1
<222> (1)..(29)
<223>
<400> 3
gggctcgagt?ttacacttat?cgaactgat 29
<210> 4
<211> 45
<212> DNA
< 213>manual work
<220>
<221> hcpA-P2
<222> (1)..(45)
<223>
<400> 4
tttgaattct?ttagcagcag?ctttagcagc?agcgttggcg?tcatc 45
<210> 5
<211> 24
<212> DNA
< 213>manual work
<220>
<221> tir-P1
<222> (1)..(24)
<223>
<400> 5
gatgaattcg?agccggatag?ccca 24
<210> 6
<211> 24
<212> DNA
< 213>manual work
<220>
<221> tir-P2
<222> (1)..(24)
<223>
<400> 6
ttagtcgaca?gcccccgatg?aaac 24
<210> 7
<211> 45
<212> DNA
< 213>manual work
<220>
<221> eae-P1
<222> (1)..(45)
<223>
<400> 7
ttagtcgacg?ctgctgctaa?agctgctgct?aaatttgatc?aaacc 45
<210> 8
<211> 27
<212> DNA
< 213>manual work
<220>
<221> eae-P2
<222> (1)..(27)
<223>
<400> 8
ggctctagat?tattctacac?aaaccgc 27
Claims (7)
1. enterorrhagia Bacillus coil 0157: H7 multivalence pattern of fusion reorganization bacterium, its construction process is:
According to O157:H7 EDL933's among the GenBank
FliC,
HcpA,
TirWith
EaeGene order designs primer respectively, and adds restriction enzyme site at upstream and downstream primer two ends respectively,
HcpABehind the gene with
EaeAdding length before the gene is the junction fragment of 24 Nucleotide, and primer sequence is following:
fliC-P1：5-ggg
gagctcactattaccaacaaa-3
Sac?Ⅰ
fliC-P2：5-tta
ctcgagggtcgttgcagaacc-3
Xhol?Ⅰ
hcpA-P1：5-ggg
ctcgagtttacacttatcgaactgat-3
Xhol?Ⅰ
hcpA-P2：5-ttt
gaattctttagcagcagctttagcagcagcgttggcgtcatc-3
EcoRⅠ
tir-P1：?5-gat
gaattcgagccggatagccca-3
EcoRⅠ
tir-P2：?5-tta
gtcgacagcccccgatgaaac-3
Sal?Ⅰ
eae-P1：5-tta
gtcgacgctgctgctaaagctgctgctaaatttgatcaaacc-3
Sal?Ⅰ
eae-P2：5-?ggc
tctagattattctacacaaaccgc-3
Xba?Ⅰ
The O157:H7 overnight culture, 1mL/ pipe, centrifugal 2 minutes of 12000r abandons supernatant, collects thalline and is resuspended in 1/5 volume distilled water, boils 10min, and centrifugal 10 minutes of 4 ℃ of 12000r draw frozen-20 ℃ of supernatant, are used for pcr amplification and use;
Amplification
FliCSegmental upstream and downstream primer is added with respectively
SacI with
XholThe I restriction enzyme site, amplification
EaeSegmental upstream and downstream primer is added with respectively
SalI with
XbaThe I restriction enzyme site, the PCR reaction conditions is: 95 ℃ of preparatory sex change 5 minutes, 94 ℃ 1 minute, 55 ℃ 30 seconds, 72 ℃ 1 minute, 30 circulations, 72 ℃ were extended 10 minutes again; Amplification
HcpASegmental upstream and downstream primer respectively is added with
XholI with
EcoRThe I restriction enzyme site, amplification
TirSegmental upstream and downstream primer is added with respectively
EcoRI with
SalThe I restriction enzyme site, the PCR reaction conditions is: 95 ℃ of preparatory sex change 5 minutes, 94 ℃ 1 minute, 56 ℃ 30 seconds, 72 ℃ 30 seconds, 30 circulations, 72 ℃ were extended 6 minutes again;
FliC-P1 with
FliC-P2,
HcpA-P1 with
HcpA-P2,
Tir-P1 with
Tir-P2,
Eae-P1 with
Eae-P2 expanding fragment length is respectively 945bp, 438bp, 327bp and 891bp; The fliC fragment of amplification is being read between the frame 538-1483 Nucleotide; The hcpA fragment of amplification is being read between the frame 19-441 Nucleotide; The tir fragment of amplification is being read between the frame 775-1083 Nucleotide; The eae fragment of amplification is being read between the frame 1995-2805 Nucleotide;
With the agarose electrophoresis of amplification PCR products with mass ratio 1.2%, cut glue and weigh, add after 3 times of volume DE-A damping fluids 65 ℃ of effects 10 minutes, treat that glue all melts after; The DE-B damping fluid that adds 0.5 volume of DE-A damping fluid again, put upside down mixing after, the liquid that melts is shifted in miniature pillar, and centrifugal 1 minute of 12000g; Abandon liquid, add lavation buffer solution I 500uL, centrifugal 30 seconds of 12000g abandons liquid again; Add lavation buffer solution II 750uL, centrifugal 1 minute of 12000g abandons liquid, centrifugal 1 minute of blank pipe 12000g; Shift pillar in clean 1.5mL centrifuge tube, and add 60uL elution buffer, centrifugal 1 minute of 12000g; Collect centrifugate, be purified 945bp, 438bp, 327bp and 891bp fragment, called after fliC, hcpA, tir and eae fragment;
Will
SacI with
XholThe carrier pCold I fragment 4uL that fliC fragment 4uL that the I enzyme is cut and same enzyme are cut, T4 DNA connects damping fluid and each 1.0uL of T4 dna ligase, adds a centrifuge tube simultaneously; Behind the mixing; 4 ℃ of connections are spent the night, and are used to transform the Top10 competent cell, and the single bacterium colony of picking carries out incubated overnight; Extract plasmid, use
SacI with
XholThe I double digestion, 37 ℃ of water-baths 2 hours, agarose gel electrophoresis can see that the band of a 945bp occurs, obtain the pCold I-
FliCRecombinant plasmid; Use
XhoI with
EcoRThe I enzyme is cut hcpA fragment and pCold I-fliC recombinant plasmid, after glue reclaims, and the pCold I-
FliCGet 4.0uL, the hcpA fragment is got 2 uL, and T4 DNA connects damping fluid and each 1.0ul of T4 dna ligase, moisturizing 2 uL; Together add the eppendorf pipe, behind the mixing, 4 ℃ of connections are spent the night, and transform the Top10 competent cell; The single bacterium colony of picking carries out incubated overnight, extracts plasmid, and enzyme is cut, 37 ℃ of water-baths 2 hours; Agarose gel electrophoresis can see that the band of 438bp occurs, and obtains pCold I-fliC-hcpA recombinant plasmid; Use
EcoRI with
SalThe I enzyme is cut tir fragment and pCold I-fliC-hcpA recombinant plasmid, and after glue reclaimed, pCold I-fliC-hcpA got 2.0uL, and the tir fragment is got 3.0 uL; T4 DNA connects damping fluid and each 1.0ul of T4 dna ligase, and moisturizing 3 uL together add in the centrifuge tube; 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, enzyme is cut, 37 ℃ of water-baths 2 hours; Agarose gel electrophoresis can see that the band of 327bp occurs, and obtains pCold I-fliC-hcpA-tir recombinant plasmid; Use
SalI with
XbaThe I enzyme is cut eae fragment and pCold I-fliC-hcpA-tir recombinant plasmid, and after glue reclaimed, pCold I-fliC-hcpA-tir and eae respectively got 4.0uL; T4 DNA connects damping fluid and each 1.0ul of T4 dna ligase, together adds in the pipe, 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, enzyme is cut, 37 ℃ of water-baths 2 hours; Agarose gel electrophoresis can see that the band of 891bp occurs, and obtains pCold I-fliC-hcpA-tir-eae recombinant plasmid; Correct positive plasmid pCold I-fliC-hcpA-tir-eae is transformed in BL21 (DE3) competent cell with order-checking, and acquisition polyvalent recombinant bacterium BL21 (the pCold I-fliC-hcpA-tir-eae).
2. the said enterorrhagia Bacillus coil 0157 of claim 1: the H7 multivalence pattern of fusion reorganization bacterium BL21 (recombinant protein of pCold I-fliC-hcpA-tir-eae) express.
3. recombinant protein according to claim 2 is characterized in that,
(culture of pCold I-fliC-hcpA-tir-eae) 2% is inoculated in and contains 100 μ g/mL and have among the antibiotic LB of ammonia benzyl by volume with the said BL21 of claim 1; 37 ℃ of shaking culture to A=0.6 ~ 0.8 o'clock add inductor IPTG to working concentration be 0.5mM, continues 15 ℃ of concussion cultivations 24 hours, induce the centrifugal collection thalline of bacterium liquid to be resuspended in the TE damping fluid; Behind the ultrasonic degradation; The centrifugal 25min of 12000g, cleer and peaceful deposition is carried out SDS-PAGE in the collection, can clearly see that the molecular weight size is the 88kD target protein; Mainly be present in the thalline supernatant, obtain the fliC-hcpA-tir-eae polyvalent recombinant protein.
4. recombinant protein according to claim 3 is characterized in that, said (the bacterium liquid of pCold I-fliC-hcpA-tir-eae) was through centrifugal 10 minutes of 12000 g through 0.6mM IPTG inductive reorganization bacterium BL21 with claim 3; Collect thalline, resuspended with the chelation buffer of 1/10 volume, add the N,O-Diacetylmuramidase of 1mg/mL again; 30 ℃ act on 2 hours behind the mixing; Ultrasonication, 4 ℃ of 12000 g collected supernatant in centrifugal 30 minutes, and 0.45 μ M filter filters the back and goes up affinity column; Add elution buffer and collect albumen, be purified fliC-hcpA-tir-eae recombinant protein.
5. with the said enterorrhagia Bacillus coil 0157 of claim 1: the vaccine of H7 multivalence pattern of fusion reorganization bacterium preparation.
6. the vaccine for preparing with the said recombinant protein of one of claim 2-4.
7. vaccine according to claim 6 is characterized in that, the said recombinant protein of one of claim 2-4 as antigen, is carried out dispersion treatment with adjuvant ISA50V according to volume ratio 1:1 and processes oil-emulsion.
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| CN201310099542.4A CN103275912B (en) | 2012-03-15 | 2013-03-10 | Enterorrhagia Bacillus coil 0157: H7 multivalence fliC-hcpA-tir-eae recombinant bacterium and vaccine |
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| CN201310099542.4A Active CN103275912B (en) | 2012-03-15 | 2013-03-10 | Enterorrhagia Bacillus coil 0157: H7 multivalence fliC-hcpA-tir-eae recombinant bacterium and vaccine |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102747024A (en) * | 2012-06-19 | 2012-10-24 | 江苏省农业科学院 | EHEC 0157:H7 multivalent fliC-hcpA-tir-eae recombinant strain and vaccine |
| CN103804474A (en) * | 2013-12-31 | 2014-05-21 | 李越希 | Expression, purification and application of E.coli O157:H7 flagellin H7 antigen segment |
| CN109055415A (en) * | 2018-08-16 | 2018-12-21 | 湖北大学 | A kind of new expression vector of effective expression tryptophan synthetase and its application |
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| CN103540606B (en) * | 2013-11-06 | 2015-09-23 | 湖南大学 | Express the colibacillus engineering of lignin peroxidase, preparation method and application thereof |
| CN110156881A (en) * | 2019-05-28 | 2019-08-23 | 余文庚 | A kind of improved enterohemorrhagic escherichia coli vaccine composition |
| CN112481236B (en) * | 2020-11-25 | 2023-06-06 | 武汉理工大学 | Recombinant protein INP-AidH and preparation method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102747024A (en) * | 2012-06-19 | 2012-10-24 | 江苏省农业科学院 | EHEC 0157:H7 multivalent fliC-hcpA-tir-eae recombinant strain and vaccine |
| CN103804474A (en) * | 2013-12-31 | 2014-05-21 | 李越希 | Expression, purification and application of E.coli O157:H7 flagellin H7 antigen segment |
| CN103804474B (en) * | 2013-12-31 | 2016-01-27 | 李越希 | E. the expression of coli O157:H7 flagellin H7 antigen fragment, purifying and application |
| CN109055415A (en) * | 2018-08-16 | 2018-12-21 | 湖北大学 | A kind of new expression vector of effective expression tryptophan synthetase and its application |
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| CN103275912B (en) | 2016-03-02 |
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