CN101830985B - Recombination protein of enterohemorrhagic Escherichia coli O157:H7Tir and Tccp - Google Patents
Recombination protein of enterohemorrhagic Escherichia coli O157:H7Tir and Tccp Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology and relates to a preparation method and application of a recombination protein of enterohemorrhagic Escherichia coli O157:H7Tir and Tir couple cytoskeleton protein (Tccp). The preparation method comprises the steps of: connecting sequences at the C ends of the genes of tir and tccp for cloning to obtain a recombination plasmid of pGEM-T-tir-tccp; constructing a recombination plasmid of pGEX-4T-1-tir-tccp, and converting into a competent cell BL21 (DE3) to obtain a recombination strain; and introducing by IPTG to achieve high-efficiency expression. The recombination protein Tir-Tccp has good immunogenicity, and can induce an organism to generate a high-titre protective antibody. Tir-Tccp can be used as a candidate multivalent fusion protein for developing subunit vaccines and provides a technology platform and a prevention and control means for preventing and treating the infection of enterohemorrhagic Escherichia coli O157:H7.
Description
One, technical field
The present invention relates to EHEC (Enterohaemorrhagic Escherichia coli; EHEC) the plain acceptor of O157:H7 transposition tight adhesion (Tir) and interior membranogen acceptor coupling cytoskeletal protein (TccP) recombinant protein preparation method and application belong to biological technical field.
Two, background technology
EHEC (EHEC) O157:H7 is familiar with the history that year surplus in the of 20 is arranged as a kind of food-borne pathogens; It is the The main pathogenic fungi that causes people's hemorrhagic colitis (HC); Also can in 5%～10% case, cause hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura severe complications such as (TTP), severe patient can cause death and die.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.The infection of this bacterium has at present become a global public health problem, receives extensive concern.But it is infected still not to have effectively prevent and treat method, research proof microbiotic can impel the O157 bacterium to discharge lethality shiga toxin (Stx), thereby makes the danger increase of the concurrent HUS of patient.Also do not have at present the ideal vaccine to can be used for the control that EHEC infects, for this reason, develop as early as possible and effective can cut off contagium becomes current this field with the effective vaccine of thorough removing pathogenic bacteria research emphasis.
(Translocation intimin receptor Tir) strides the film adaptin for the transposition of O157:H7 to the plain acceptor of transposition tight adhesion, is closely plain principal recipient, by the tir genes encoding on the EHEC O157:H7 pathogenicity island LEE island.(type III Secretion System TTSS) secretes and is indexed in the host cell this albumen, and tight adhesion, the actin polymerization pedestal of participating in bacterium form the A/E damage through EHEC III type excretory system.The full gene of Tir is 1677bp, and coding contains 559 amino-acid residues, and molecular weight is the protein of 72kDa.
Interior membranogen acceptor coupling cytoskeletal protein (Tir couple cytoskeleton protein; TccP) be a kind of virulence factor that newly discovered is also confirmed in EHEC O157:H7 in recent years; By the open reading frame sequence coding that is numbered Z3072 in the O157:H7 genome; Intramolecularly contains the repeated fragment of several proline rich, and this repeated fragment is made up of 47 amino-acid residues, the repeated fragment that 15 amino acid of C-terminal also are brachymemmas.The repeated fragment number of the TccP in different strains source is different, does not wait by 2-8.This albumen gets into host cell through the transduction of III type excretory system, in EHEC O157:H7 field planting course of infection, plays an important role.International reference strain EDL933EHEC TccP full length gene 1155bp, coding contains 384 amino-acid residues, and molecular weight is the protein of 42.5kDa.
Three, find content
Technical problem the object of the invention is to express plain acceptor of EHEC O157:H7 transposition tight adhesion (Tir) and interior membranogen acceptor coupling cytoskeletal protein (TccP) recombinant protein; Study its immunogenicity; And then, technique means is provided for more safe and effective prevention and control O157:H7 infects for the genetic engineering subunit vaccine of development EHECO157:H7 lays the foundation.
Technical scheme specific embodiments of the present invention is following:
EHEC (Enterohaemorrhagic Escherichia coli; EHEC) the tight plain acceptor of O157:H7 transposition (Tir) and interior membranogen acceptor coupling cytoskeletal protein (TccP) recombinant protein preparation method and application; It is characterized in that said recombinant protein is expressed for the reorganization bacterium BL21 (pGEX-4T-1-tir-tccp) that makes up.The preparation method of recombinant protein comprises:
(1) obtain tir and tccp gene C terminal sequence, parallel-series is cloned into the pGEM-T carrier
EHEC O157:H7 EDL933 bacterial strain Tir gene order (AF125993) and the TccP sequence (NP_288437) announced according to GenBank design and synthesize two pairs of Auele Specific Primers respectively; The DNA that boils preparation with O157:H7 EDL933 bacterial strain is a template; Increase respectively tir and tccp gene C end 1243bp and 825bp sequence, primer sequence is following:
Tir-P1：5’-tac
ggatccactcttaacaggcagattgg-3’
Tir-P2：5’-cga
aagcttcgttatcagtagtatccc-3’
TccP-P?1：5’-acc
aagcttcccattcggcatcatttc-3’
TccP-P2：5’-cgg
ctcgaggcttagatgtattaatgcc-3
Tir-P1 and TccP-P2 primer two ends add restriction enzyme site BamH I and XhoI and protectiveness base respectively, and Tir-P2 adds identical restriction enzyme site Hind III and protectiveness base with TccP-P1 primer two ends, and underscore partly is restriction enzyme site.Tir/tccp gene PCR reaction conditions is: 94 ℃ of preparatory sex change 5min, and 94 ℃ of 1min, 60 ℃/58 ℃ 1min, 72 ℃ of 1.5min, totally 30 circulations, last 72 ℃ are extended 10min, with the negative contrast of distilled water;
The purpose band that Tir-P1 and Tir-P2, TccP-P1 and two pairs of primer amplifications of TccP-P2 obtain corresponding size is 1243bp and 825bp, and uses the agarose electrophoresis of mass ratio 1% respectively, cuts glue and weighs, 56 ℃ of effect 10min behind the Extraction buffer of 3 times of volumes of adding; The liquid that shifts thawing is in miniature pillar (2mL), and the centrifugal 1min of 6000g, abandons liquid, adds the Extraction buffer of 500ul; The centrifugal 30s of 12000g abandons liquid again, adds wash buffer 750ul; The centrifugal 30s of 12000g abandons liquid, the centrifugal 30s of blank pipe 12000g; Shift pillar in the eppendorf of clean 1.5ml pipe, and add 30ul Elution buffer, the centrifugal 30s of 12000g; Collect centrifugate, be purified 1243bp and 825bp fragment, called after tir414 and tccp275 fragment;
Hind III enzyme is cut tir414 and each 1.6 μ L of tccp275 fragment, and T4DNA connects buffer and each 1.0 μ L of T4DNA ligase enzyme, and distilled water 4.8 μ L add an eppendorf pipe simultaneously, and behind the mixing, 4 ℃ of connections are spent the night; Get spending the night of tir414-tccp275 and connect product 3.0 μ L, together add an eppendorf pipe with 2 * Rapid Ligation Buffer5.0 μ L, T4DNALigase 1.0 μ L and pGEM-T carrier 1.0 μ L, behind the mixing; 4 ℃ connect 18h-24h, transform the Top10 competent cell, and the single bacterium colony of picking carries out incubated overnight; Extract plasmid, with BamHI and XhoI double digestion, 37 ℃ of water-baths 3 hours; Agarose gel electrophoresis is identified; The band that can see a 2068bp occurs, and double digestion is identified the order-checking of male bacterium, obtains recombinant plasmid pGEMT-tir414-tccp275;
(2) pGEX-4T-1-tir-tccp construction of recombinant plasmid
PGEMT-4T-1-tir414-tccp275 recombinant plasmid and expression vector pGEX-4T-1 plasmid are used BamH I, Xho I double digestion respectively, reclaim tir-tccp recombination and pGEX-4T-1 endonuclease bamhi, and the T4DNA enzyme connects; Construction recombination plasmid pGEX-4T-1-tir-tccp; Ligation system: 10 * T4 DNA Ligasebuffer1.0 μ L, T4 DNALigase1.0 μ L, pGEX-4T-1 endonuclease bamhi 3.0 μ L; Purpose fragment 1.2 μ L, sterilization ddH
2O 3.8 μ L.In 4 ℃ of connection 18h～24h, will connect product and transform in competence BL21 (DE3) behind the mentioned reagent mixing, structure contains recombinant bacterial strain BL21 (pGEX4T-1-tir-tccp); The single bacterium colony of picking carries out incubated overnight, extracts plasmid, with BamH I and Xho I double digestion; 37 ℃ of water-baths 2 hours; Agarose gel electrophoresis is identified, the 2068bp fragment can occur, promptly obtains positive recombinant plasmid pGEX-4T-1-tir-tccp;
(3) expression of Tir-TccP recombination fusion protein
Picking is cut through enzyme and is accredited as male list bacterium colony, and 37 ℃ of shaking culture are spent the night, and transform the pGEX-4T-1 empty plasmid simultaneously and do feminine gender.Culture bacteria liquid is inoculated in LB (the Amp working concentration the is 100 μ g/mL) liquid nutrient medium by 2% volume ratio, and 37 ℃ of concussions are cultured to OD
600≈ 0.4～0.6, add IPTG to final concentration be 1mM, continue to cultivate 7h at 30 ℃ afterwards and carry out abduction delivering, collect bacterium liquid and carry out SDS-PAGE.Induce the centrifugal collection thalline of bacterium liquid to be resuspended in the PBS damping fluid, behind the ultrasonic degradation, the centrifugal 20min of 10000g; Cleer and peaceful precipitation is not carried out SDS-PAGE in the collection; To confirm the expressing target protein existence form, can clearly see that in supernatant the molecular weight size for the 102kD target protein, shows that expressing protein mainly is present in the tropina with soluble form; Account for 25% of bacterial protein, be the Tir-TccP recombinant protein.
Beneficial effect characteristics 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 tir and two gene fragments of tccp, and, finally be cloned among the expression plasmid pGEX-4T-1, successfully make up pGEX-4T-1-tir-tccp, be transformed into and obtain reorganization bacterium, i.e. BL21 (pGEX-4T-1-tir-tccp) among the BL21 through series connection.
2, the present invention successfully obtains BL21 (pGEX-4T-1-tir-tccp); Cultivate through 37 ℃ of high density fermentations, inducing of 30 ℃ of IPTG obtains to efficiently express, and expression amount accounts for 25% of whole bacterial protein; Through obtaining reorganization pattern of fusion albumen, i.e. Tir-TccP recombinant protein behind the purifying.
3, the Tir-TccP recombinant protein of the present invention after to purifying prepared recombinant vaccine; The immunity BALB/c mouse; 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: tir and tccp gene PCR amplified fragments.
M:markerDL5000; The 1:tir gene; The 2:tccp gene
Fig. 2: pGEX-4T-1-tir-tccp recombinant plasmid enzyme is cut the evaluation collection of illustrative plates.
1:marker DL15000; 2:marker DL2000; 3:pGEX-4T-1-tir-tccp recombinant plasmid BamH I/Xho I
Fig. 3: pGEX-4T-1-tir-tccp reorganization bacterium SDS-PAGE.
M: lower molecular weight standard protein; After the 1:pGEX-4T-1 empty carrier is induced; 2:pGEX-4T-1-tir-tccp induces preceding whole bacterial protein; 3-6: be respectively pGEX-4T-1-tir-tccp and induce whole bacterial protein
Fig. 4: Tir-TccP fusion protein immunization mice serum IgG antibody growth and decline law curve.
Fig. 5: stool in mice discharge of bacteria quantitative change trend map.
Five, embodiment
1, obtain tir and tccp gene, parallel-series is cloned into the pGEM-T carrier
EHEC O157:H7 EDL933 bacterial strain (bio tech ltd is stepped in Shanghai three) Tir gene order (AF125993) and the TccP sequence (NP_288437) announced according to GenBank design and synthesize two pairs of Auele Specific Primers respectively; DNA with the preparation of O157:H7 EDL933 bacterial strain is a template; Increase respectively tir and tccp gene C end 1243bp and 825bp sequence, primer sequence is following:
Tir-P1：5’-tac
ggatccactcttaacaggcagattgg-3’
Tir-P2：5’-cga
aagcttcgttatcagtagtatccc-3’
TccP-P?1：5’-acc
aagcttcccattcggcatcatttc-3’
TccP-P2：5’-cgg
ctcgaggcttagatgtattaatgcc-3
Tir-P1 and TccP-P2 primer two ends add restriction enzyme site BamH I and XhoI and protectiveness base respectively, and Tir-P2 and TccP-P1 primer two ends add same restriction enzyme site Hind III and protectiveness base, and underscore partly is restriction enzyme site.Tir/tccp gene PCR reaction conditions is: 94 ℃ of preparatory sex change 5min, and 94 ℃ of 1min, 60 ℃/58 ℃ 1min, 72 ℃ of 1.5min, totally 30 circulations, last 72 ℃ are extended 10min, with the negative contrast of distilled water;
The purpose band that Tir-P1 and Tir-P2, TccP-P1 and two pairs of primer amplifications of TccP-P2 obtain corresponding size is 1243bp and 825bp, and uses quality respectively: the agarose electrophoresis of volume (W/V) ratio 1%, cut glue to weigh 56 ℃ of effect 10min behind the Extraction buffer of 3 times of volumes of adding; The liquid that shifts thawing is in miniature pillar (2mL), and the centrifugal 1min of 6000g, abandons liquid, adds the Extraction buffer of 500ul; The centrifugal 30s of 12000g abandons liquid again, adds wash buffer 750ul; The centrifugal 30s of 12000g abandons liquid, the centrifugal 30s of blank pipe 12000g; Shift pillar in the eppendorf of clean 1.5ml pipe, and add 30ul Elution buffer, the centrifugal 30s of 12000g; Collect centrifugate, be purified 1243bp and 825bp fragment, called after tir414 and tccp275 fragment;
Hind III enzyme is cut tir414 and each 1.6 μ L of tccp275 fragment, and T4 DNA connects buffer and each 1.0 μ L of T4 dna ligase, and distilled water 4.8 μ L add an eppendorf pipe simultaneously, and behind the mixing, 4 ℃ of connections are spent the night; Get spending the night of tir414-tccp275 and connect product 3.0 μ L, together add an eppendorf pipe with 2 * Rapid Ligation Buffer5.0 μ L, T4 DNALigase 1.0 μ L and pGEM-T carrier 1.0 μ L, behind the mixing; 4 ℃ connect 18h-24h, transform the Top10 competent cell, and the single bacterium colony of picking carries out incubated overnight; Extract plasmid, with BamHI and XhoI double digestion, 37 ℃ of water-baths 3 hours; Agarose gel electrophoresis is identified; The band that can see a 2068bp occurs, and double digestion is identified the order-checking of male bacterium, obtains recombinant plasmid pGEM-T-tir414-tccp275;
2, pGEX-4T-1-tir-tccp construction of recombinant plasmid
PGEMT-4T-1-tir414-tccp275 recombinant plasmid and expression vector pGEX-4T-1 plasmid (Novagen company) are used BamH I, Xho I double digestion respectively, reclaim tir-tccp recombination and pGEX-4T-1 endonuclease bamhi, and the T4DNA enzyme connects; Construction recombination plasmid pGEX-4T-1-tir-tccp; Ligation system: 10 * T4 DNALigase buffer1.0 μ L, T4 DNA Ligase1.0 μ L, pGEX-4T-1 endonuclease bamhi 3.0 μ L; Purpose fragment 1.2 μ L, sterilization ddH
2O 3.8 μ L.In 4 ℃ of connection 18h～24h, will connect product and transform in competence BL21 (DE3) behind the mentioned reagent mixing, structure contains recombinant bacterial strain BL21 (pGEX4T-1-tir-tccp); The single bacterium colony of picking carries out incubated overnight, extracts plasmid, with BamH I and Xho I double digestion; 37 ℃ of water-baths 2 hours; Agarose gel electrophoresis is identified, the 2068bp fragment can occur, promptly obtains positive recombinant plasmid pGEX-4T-1-tir-tccp;
3, the structure of reorganization bacterium, the expression and the purifying of recombination fusion protein
Picking is cut through enzyme and is accredited as male list bacterium colony, and 37 ℃ of shaking culture are spent the night, and transform the pGEX-4T-1 empty plasmid simultaneously and do feminine gender.Culture bacteria liquid is inoculated in LB (the Amp working concentration the is 100 μ g/mL) liquid nutrient medium by 2% volume ratio, and 37 ℃ of concussions are cultured to OD
600≈ 0.4～0.6, add IPTG to final concentration be 1mM, continue to cultivate 7h at 30 ℃ afterwards and carry out abduction delivering, collect bacterium liquid and carry out SDS-PAGE.Induce the centrifugal collection thalline of bacterium liquid to be resuspended in the PBS damping fluid, behind the ultrasonic degradation, the centrifugal 20min of 10000g; Cleer and peaceful precipitation is not carried out SDS-PAGE in the collection; To confirm the expressing target protein existence form, can clearly see that in supernatant the molecular weight size for the 102kD target protein, shows that expressing protein mainly is present in the tropina with soluble form; Account for 25% of bacterial protein, be the Tir-TccP recombinant protein.
4, the preparation of EHEC O157:H7 recombinant protein Tir-TccP recombinant protein recombinant vaccine
Above-mentioned purified recombinant albumen and adjuvant (ISA 50v, French Seppic company produces) are carried out mixing and emulsifying according to 4.6: 5.4 ratios process oil-emulsion, wherein the final concentration of recombinant protein is 150 μ g/mL.
5, immunity test
(1) laboratory animal is chosen 20 of male BALB/c mouses (Yangzhou University), and 18g-22g/ only.
(2) pre-treatment of laboratory animal all groups before attacking poison all give 5g/L Streptomycin sulphate solution and drink 3d, 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 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.
(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 gene engineering vaccine of preparation, and control group is alternative with the aseptic PBS of equivalent.Adopt back and subcutaneous abdomen multi-point injection mode to carry out immunity, immunity is three times altogether, and the immunity time was 0,2,4 weeks, and immunizing dose is 30ug/.Three exempt to irritate stomach after 7 days attacks 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 Tir-TccP specific IgG antibodies in the serum of immunity back
After little mouse's head is exempted from, week about 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.
It is following that ELISA detects concrete steps: the preparation of (1) ELISA antigen coated microplate: antigen is diluted to 2.5ug/mL with coating buffer, and 100 μ l/ holes encapsulate elisa plate, and 4 ℃ are spent the night.PBST washes plate 3-4 time.Every hole adds 5% skim-milk of 200 μ l, and after 37 ℃ of incubators sealing 2h, PBST washed plate 3-4 time ,-20 ℃ of preservations were subsequent use.(2) detection of serological specificity IgG antibody: with antibody diluent two doubling dilutions serum to be checked and negative serum, from 1: 50 to 1: 102400, add the elisa plate that encapsulates by (1) respectively, 100 μ l/ holes place 37 ℃ to hatch 1.5h simultaneously.PBST washes plate 3-4 time, adds the sheep anti-mouse igg (1: 20000) of HRP mark, and 100 μ l/ holes place 37 ℃ to hatch 45-60min, and PBST washes plate 3-4 time.Add tmb substrate colour developing liquid, 100 μ l/ holes, room temperature lucifuge reaction 5-10min.2molL
-1Sulfuric acid stop buffer 50 μ L termination reactions are made blank with the PBS hole, on enzyme mark determinator, measure each hole OD450 value.Be judged as the positive with OD mensuration/OD feminine gender＞2.1.
(5) immune effect is confirmed foundation
1. the antibody variation utilizes ELISA to detect twice immunity back serum antibody variation, and with the recombinant antigen coated elisa plate, the result shows; Head exempts from can detect the Tir-TccP specific antibody behind the 14d of back, and rises gradually, exempts from back 14d detection antibody two and reaches high value; Compare with control group, P/N＞2.1, it is positive that immune group serum becomes; Explain that this recombinant protein can bring out mouse through this kind immunization ways and produce Tir-TccP specific IgG antibodies, Fig. 4.
2. attack malicious protection situation three and exempt to attack in back 7 days poison, the result shows that immune group mouse survival rate is 10/10, and the control group survival rate is 5/10.
3. 24h began to gather ight soil after the discharge of bacteria quantitative changeization was attacked poison, gathered once every three days afterwards, and is dull and stereotyped through coating screening property Mai Kangkai after suitably handling; Cultivate 18～24h 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 8d detected less than O157:H7 after immune group was attacked poison, and control group still can detect discharge of bacteria in 14d, Fig. 5.
Sequence table
< 110>Jiangsu Province Agriculture Science Institute
< 120>enterorrhagia Bacillus coil 0157: the recombinant protein of H7 Tir and Tccp
< 130>specification sheets
<160>4
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<210>1
<211>29
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< 213>manual work
<220>
<221>Tir-P1
<222>(1)..(29)
<223>
<400>1
tacggatcca?ctcttaacag?gcagattgg 29
<210>2
<211>27
<212>DNA
< 213>manual work
<220>
<221>Tir-P2
<222>(1)..(27)
<223>
<400>2
cgaaagcttc?gttatcagta?gtatccc 27
<210>3
<211>27
<212>DNA
< 213>manual work
<220>
<221>TccP-P1
<222>(1)..(27)
<223>
<400>3
accaagcttc?ccattcggca?tcatttc 27
<210>4
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< 213>manual work
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<222>(1)..(28)
<223>
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cggctcgagg?cttagatgta?ttaatgcc 28
Claims (3)
1. tight plain acceptor Tir of EHEC (Escherichia coli) O157:H7 transposition and interior membranogen acceptor coupling cytoskeletal protein TccP recombinant protein is characterized in that, are obtained by following method:
(1) obtain tir and tccp gene C terminal sequence, parallel-series is cloned into the pGEM-T carrier
Design and synthesize two pairs of Auele Specific Primers, the DNA for preparing with O157:H7EDL933 is a template, increase respectively tir and tccp gene C end 1243bp and 825bp sequence, and primer sequence is following:
Tir-P1：5’-tac
ggatccactcttaacaggcagattgg-3’
Tir-P2：5’-cga
aagcttcgttatcagtagtatccc-3’
TccP-P1：5’-acc
aagcttcccattcggcatcatttc-3’
TccP-P2：5’-cgg
ctcgaggcttagatgtattaatgcc-3
Tir-P1 and TccP-P2 primer two ends add restriction enzyme site BamH I and XhoI and protectiveness base respectively, and Tir-P2 and TccP-P1 primer two ends add same restriction enzyme site HindIII and protectiveness base, and underscore partly is restriction enzyme site; Tir/tccp gene PCR reaction conditions is: 94 ℃ of preparatory sex change 5min, 94 ℃ of 1min, 60 ℃/58 ℃ 1min; 72 ℃ of 1.5min; Totally 30 circulations, last 72 ℃ are extended 10min, with the negative contrast of distilled water;
The purpose band that Tir-P1 and Tir-P2, TccP-P1 and two pairs of primer amplifications of TccP-P2 obtain corresponding size is 1243bp and 825bp, and uses quality respectively: the agarose electrophoresis of volume ratio 1%, and cut glue and weigh, 56 ℃ of effect 10min behind the Extraction buffer of 3 times of volumes of adding; The liquid that shifts thawing is in the miniature pillar of 2mL, and the centrifugal 1min of 6000g, abandons liquid, adds the Extraction buffer of 500ul; The centrifugal 30s of 12000g abandons liquid again, adds wash buffer 750ul; The centrifugal 30s of 12000g abandons liquid, the centrifugal 30s of blank pipe 12000g; Shift pillar in the eppendorf of clean 1.5ml pipe, and add 30ul Elution buffer, the centrifugal 30s of 12000g; Collect centrifugate, be purified 1243bp and 825bp fragment, called after tir414 and tccp275 fragment;
Hind III enzyme is cut tir414 and each 1.6 μ L of tccp275 fragment, and T4DNA connects buffer and each 1.0 μ L of T4DNA ligase enzyme, and distilled water 4.8 μ L add an eppendorf pipe simultaneously, and behind the mixing, 4 ℃ of connections are spent the night; Get spending the night of tir414-tccp275 and connect product 3.0 μ L, together add an eppendorf pipe with 2 * Rapid Ligation Buffer5.0 μ L, T4DNALigase 1.0 μ L and pGEM-T carrier 1.0 μ L, behind the mixing; 4 ℃ connect 18h-24h, transform the Top10 competent cell, and the single bacterium colony of picking carries out incubated overnight; Extract plasmid, with BamHI and XhoI double digestion, 37 ℃ of water-baths 3 hours; Agarose gel electrophoresis is identified; The band that can see a 2068bp occurs, and double digestion is identified the order-checking of male bacterium, obtains recombinant plasmid pGEMT-tir414-tccp275;
(2) pGEX-4T-1-tir-tccp construction of recombinant plasmid
PGEMT-4T-1-tir414-tccp275 recombinant plasmid and expression vector pGEX-4T-1 plasmid are used BamH I, Xho I double digestion respectively, reclaim tir-tccp recombination and pGEX-4T-1 endonuclease bamhi, and the T4DNA enzyme connects; Construction recombination plasmid pGEX-4T-1-tir-tccp; Ligation system: 10 * T4DNA Ligase buffer1.0 μ L, T4DNA Ligase1.0 μ L, pGEX-4T-1 endonuclease bamhi 3.0 μ L; Purpose fragment 1.2 μ L, sterilization ddH
2O 3.8 μ L in 4 ℃ of connection 18h～24h, will connect product and transform in competence BL21 (DE3) behind the mentioned reagent mixing; The single bacterium colony of picking carries out incubated overnight, extracts plasmid, with BamH I and Xho I double digestion; 37 ℃ of water-baths 2 hours; Agarose gel electrophoresis is identified, the 2068bp fragment can occur, promptly obtains the positive reorganization bacterium BL21 that contains the pGEX4T-1-tir-tccp plasmid;
(3) expression of Tir-TccP recombination fusion protein
Picking is cut through enzyme and is accredited as male list bacterium colony; 37 ℃ of shaking culture are spent the night; Transform the pGEX-4T-1 empty plasmid simultaneously and do feminine gender, culture bacteria liquid 2% is inoculated in the LB liquid nutrient medium that the Amp working concentration is 100 μ g/mL by volume, 37 ℃ of concussions are cultured to OD
600=0.4～0.6, add IPTG to final concentration be 1mM, continue to cultivate 7h at 30 ℃ afterwards and carry out abduction delivering; Collect bacterium liquid and carry out SDS-PAGE, induce the centrifugal collection thalline of bacterium liquid to be resuspended in the PBS damping fluid, behind the ultrasonic degradation; The centrifugal 20min of 10000g; Cleer and peaceful precipitation is not carried out SDS-PAGE in the collection, to confirm the expressing target protein existence form, can clearly see that in supernatant the molecular weight size is the 102kD target protein; Show that expressing protein mainly is present in the tropina with soluble form, is the Tir-TccP recombinant protein.
2. the vaccine that is used for prevention and control enterorrhagia Bacillus coil 0157: H7 infection for preparing with the said recombinant protein of claim 1.
3. with the detection enterorrhagia Bacillus coil 0157 of the said recombinant protein of claim 1: the test kit of H7 as the detection antigen prepd.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1631438A (en) * | 2004-03-27 | 2005-06-29 | 中国人民解放军第三军医大学 | 0157 bacterium gene engineering multivalence subunit vaccine of human and sensitive animals and its preparing method |
| CN1748791A (en) * | 2005-08-08 | 2006-03-22 | 中国人民解放军第三军医大学 | Haemorrhagic E, coli 0157:H7 vaccine for human and livestock prevention and preparing method |
| CN101062410A (en) * | 2007-02-05 | 2007-10-31 | 中国人民解放军第三军医大学 | Genetic engineering vaccine of enterohemorrhagic escherichia coli 0157:H7 and the preparing method thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1631438A (en) * | 2004-03-27 | 2005-06-29 | 中国人民解放军第三军医大学 | 0157 bacterium gene engineering multivalence subunit vaccine of human and sensitive animals and its preparing method |
| CN1748791A (en) * | 2005-08-08 | 2006-03-22 | 中国人民解放军第三军医大学 | Haemorrhagic E, coli 0157:H7 vaccine for human and livestock prevention and preparing method |
| CN101062410A (en) * | 2007-02-05 | 2007-10-31 | 中国人民解放军第三军医大学 | Genetic engineering vaccine of enterohemorrhagic escherichia coli 0157:H7 and the preparing method thereof |
Non-Patent Citations (3)
| Title |
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| 张雪寒等.出血性大肠杆菌O157:H7的Tir与stx1/2B融合基因的构建和表达.《内蒙古农业科技》.2009,(第03期), * |
| 杨琴等.肠出血性大肠杆菌O157∶H7 TccP基因克隆、表达及其抗原性鉴定.《中国人兽共患病学报》.2007,(第04期), * |
| 王玲等.大肠埃希菌O157:H7 Tir基因的生物信息学分析.《热带医学杂志》.2009,(第05期), * |
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