CN101182516B - Bacterium surface displaying novel system, method and applications - Google Patents
Bacterium surface displaying novel system, method and applications Download PDFInfo
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Abstract
The invention provides a novel bacterial surface display system, which comprises the gene sequence of coding target protein and the nucleic acid sequence of Vibrio anguillarum which is responsible for the transmembrane location and transportation. The gene sequence of coding target protein is at the C end of the nucleic acid sequence of Vibrio anguillarum; the proteins which are coded by the nucleic acid sequence of Vibrio anguillarum comprise the signal peptide of outer membrane lipoprotein of Vibrio anguillarum and the transmembrane anchoring sequence of outer membrane protein of Vibrio anguillarum; the transmembrane anchoring sequence connects the signal peptide with the target protein; the outer membrane lipoprotein of Vibrio anguillarum is Wza protein; the outer membrane proteins of Vibrio anguillarum are Omporf1protein, OmpU protein or Omp26Laprotein. The bacterial surface display system of the invention can successfully display the target protein on the surface of bacteria cell, which can be used in areas such as the development of vaccines, the preparation of antibody, the screening of peptide library, the development of environmental biological adsorbent and whole cell biocatalysis. The invention is especially suitable for the construction of safe and effective polyvalent vector vaccines of Vibrio anguillarum on the basis of the attenuated strain of Vibrio anguillarum.
Description
Technical field
The present invention relates to gene engineering technology field, more specifically, relate to the bacterium surface displaying technical field, be meant a kind of bacterium surface displaying novel system, method and application especially.
Background technology
The cell surface localized molecules is at the occurring in nature ubiquity, and its surface alignment process is regulated and control by different cell surface proteins, and has formed a lot of biological phenomenas, such as intercellular identification, and signal transduction, surface adsorption is grown surely, immune response or the like.The researchist utilizes these surface proteins as polypeptide and albumen and its fusion of anchoring element with external source, thereby its surface that is shown to bacterium or virus is reached certain research and application purpose.With the foreign protein localization and expression behind bacterium surface, can directly carry out the subsequent experimental operation with recombinant microorganism, the product of having removed target protein from extracts, sequence of operations such as purifying, if what show is antigen protein, because antigen-exposed is at cell surface, so more help immune identification, and the factors such as lipopolysaccharides on the adventitia can enhancing immunity be reacted, also may be and (the Lee J.S. that plays a role as the cofactor of target protein that is anchored on cell surface, Shin K.S., PanJ.G., and Kim C.J.2000.Surface-displayed viral antigens on Salmonella carrier vaccine.Nat.Biotechnol.18:645-648).First piece about utilizing genetic method to be fused on the carrier proteins allogenic polypeptide or albumen, thereby successfully the report that they are showed is respectively by (Freudl R. such as Freudl in 1986, MacIntyre S., Degen M., Henning U., 1986.Cell surface exposure of the outer membraneprotein OmpA of Escherichia coli K-12.J.Mol.Biol.188,491-494) and (Charbit A. such as Charbit, Boulain J.C., Ryter A., Hofnung be the topology of abacterial membrane protein by genetic insertion of a foreign epitope M.1986.Probing; J.5, expression at the cellsurface.EMBO 3029-3037) finishes.Afterwards, many different bacterium surface exhibiting systems are studied and develop, and make that the surface display technology is developed rapidly.
In all surface display systems that are developed and study, a most widely used system is the Lpp-OmpA system, it is that Georgiou and his co-worker made up in 1992, what they utilized the success of Lpp-OmpA fusant is shown to β-Nei Xiananmei intestinal bacteria surface (Francisco J.A., Earhart C.F., Georgiou is and anchoring of beta-lactamase to the external surface of Escherichia coli.Proc Natl Acad Sci USA 89 (7) G.1992.Transport: 2713-7), this system is made up of two portions: 1) nine amino acid of E.coli major outer membrane lipoprotein Lpp signal peptide and N end; 2) the 46-159 amino acids of E.coli outer membrane protein OmpA (comprise and stride film district B3-B7), the foreign protein C-terminal of system therewith merge.Afterwards, the investigator has utilized this system demonstration many foreign proteins make this system in biocatalysis, Antibody Preparation, and immobilized cell, all there is application aspects such as biosensor preparation.Because having avoided sandwich assay to merge foreign protein, this system carries out the many restriction of surface display to foreign protein, make with E.coli the size of the foreign protein that to be the host show with this system reach 70kDa (Wan H.M., Chang B.Y., Lin S.C.2002.Anchorage of cyclodextringlucanotransferase on the outer membrane of Escherichia coli.Biotechnol Bioeng79 (4): 457-64).Utilizing this system to carry out surface display also has many good qualities, such as, the Lpp-OmpA system has secreting signal peptide and unique membrane structure of striding efficiently, and suitable target protein position of fusion is provided, and its firm anchoring structure can be positioned at cell surface with foreign protein accurately.
Vibrio anguillarum is a kind of important fish bacterial cause of disease, can cause the epidemic disease of sea water fish and fresh-water fishes in worldwide, and fish farming is caused very big loss.(preserving number is CCTCC NO:M204066 to the attenuated eel vibrio strain MVAV6203 that makes up by the DNA recombinant technology on Vibrio anguillarum virulent strain MVM425 basis; preservation day is on September 15th, 2004; specifically see also Chinese patent ZL200410089496.0; the applying date is on December 14th, 2004); this attenuated strain virulence attenuation of 10000 times; nontoxic to target and non-target animal safety, and vibriosis had higher immune protective efficiency, be the outstanding attenuated live vaccine candidate strain of a strain.
Therefore, the inventor attempts making up and developing bacterium surface exhibiting system efficiently, and the protective antigen factor that is intended to derive from other pathogenic micro-organisms is showed in this attenuated eel vibrio strain surface, makes up the multiple-effect valency carrier bacterin of Vibrio anguillarum safely and efficiently.
Summary of the invention
Main purpose of the present invention is exactly the problems and shortcomings at above existence, a kind of bacterium surface displaying novel system, method and application are provided, this bacterium surface displaying novel system can successfully be showed target protein on the bacterial cell surface, can be used for the living vaccine exploitation, Antibody Preparation, the polypeptide libraries screening, exploitation of environmental organism sorbent material and whole-cell biological catalysis aspect are used in particular for making up Vibrio anguillarum multiple-effect valency carrier bacterin safely and efficiently on the basis of attenuated eel vibrio strain.
In a first aspect of the present invention, a kind of bacterium surface displaying novel system is provided, the gene order that comprises the target protein of encoding, be characterized in, also comprise the Vibrio anguillarum nucleotide sequence of being responsible for striding film location and transhipment, described gene order is positioned at the C end of described Vibrio anguillarum nucleotide sequence, what the nucleotide sequence coded albumen of described Vibrio anguillarum comprised the signal peptide of Vibrio anguillarum outer membrane lipoprotein and Vibrio anguillarum outer membrane protein strides the film anchor series, and the described film anchor series of striding connects described signal peptide and described target protein.
Described Vibrio anguillarum outer membrane lipoprotein is a Wza albumen, and described Vibrio anguillarum outer membrane protein is an Omporf1 albumen, OmpU albumen or Omp26La albumen.
Described Vibrio anguillarum nucleotides sequence is classified the nucleotide fragments of the proteic 80-158 amino acids of He Gansuanpianduan coding Omp26La of the proteic 180-238 amino acids of He Gansuanpianduan coding OmpU of preceding 11 amino acid whose nucleotide sequences of the coding proteic signal peptide of described Wza and N end and the proteic 6-50 amino acids of the described Omporf1 of coding as.
Described target protein is the EseB antigen protein of blunt tarda.
In a second aspect of the present invention, a kind of method of utilizing above-mentioned bacterium surface displaying novel system at the cell surface display target protein of bacterium is provided, be characterized in, may further comprise the steps:
A. described bacterium surface displaying novel system is built on the expression vector, obtains recombinant expression vector;
B. described recombinant expression vector is imported in the host bacterium and express, obtain fusion rotein, described fusion rotein comprises described signal peptide, described film anchor series and the described target protein of striding.
Described Vibrio anguillarum outer membrane lipoprotein is a Wza albumen, and described Vibrio anguillarum outer membrane protein is an Omporf1 albumen, OmpU albumen or Omp26La albumen.
Described Vibrio anguillarum nucleotides sequence is classified the nucleotide fragments of the proteic 80-158 amino acids of He Gansuanpianduan coding Omp26La of the proteic 180-238 amino acids of He Gansuanpianduan coding OmpU of preceding 11 amino acid whose nucleotide sequences of the coding proteic signal peptide of described Wza and N end and the proteic 6-50 amino acids of the described Omporf1 of coding as, and described host bacterium is intestinal bacteria or Vibrio anguillarum.
Described expression vector is pUC18, and described host bacterium is intestinal bacteria Top10 or attenuated eel vibrio strain MVAV6203, and described target protein is the EseB antigen protein of blunt tarda.
In a third aspect of the present invention, provide above-mentioned bacterium surface displaying novel system to develop, Antibody Preparation, polypeptide libraries screening, the application of exploitation of environmental organism sorbent material and whole-cell biological catalysis aspect at living vaccine.
Described living vaccine is a Vibrio anguillarum multiple-effect valency carrier bacterin.
Beneficial effect of the present invention is as follows:
1) the invention belongs to surface display system based on Vibrio anguillarum self element, adopt Vibrio anguillarum outer membrane lipoprotein Wza albumen and Vibrio anguillarum outer membrane protein Omporf1, the fusant of OmpU or Omp26La is a carrier, utilize the N-end signal peptide of Wza, Omporf1, OmpU or Omp26La stride the film anchor series, successfully the EseB antigen protein of blunt tarda is shown to the cell surface of attenuated eel vibrio strain MVAV6203, Vibrio anguillarum multiple-effect valency carrier bacterin lays a solid foundation in order further to develop safely and efficiently.
2) utilize bacterium surface displaying novel system of the present invention; N-end signal peptide by Wza; Omporf1; OmpU or Omp26La stride the film anchor series; heterologous protein (antigen protein that particularly has immune protective efficiency) successfully can be shown to cell surface; to strengthen and the proteic biological function of optimization aim; increase the biological function of used host strain; the present invention is in Antibody Preparation; the polypeptide libraries screening, other various fields such as the exploitation of environmental organism sorbent material and whole-cell biological catalysis aspect have the potential using value.
Description of drawings
Fig. 1 is a plasmid pUC18 of the present invention
ΔThe carrier collection of illustrative plates of E is that the 4th bit base in the EcoR I site in the pUC18 carrier multiple clone site method by the nonsense point mutation is become C from T, thereby makes the product that EcoR I site is removed.
Fig. 2 is the carrier collection of illustrative plates of plasmid porf1G of the present invention and pUG, be with the fragment behind omporf1 or ompU gene fragment and the goal gene gfp gene fragment overlap select for use Kpn I-BamH I restriction enzyme site respectively with plasmid pUC18 shown in Figure 1
ΔE connects the product that obtains, wherein the site of having introduced Nsi I and EcoR I in the middle of omporf1 or ompU gene fragment and gfp gene fragment.
Fig. 3 is the carrier collection of illustrative plates of plasmid pWorf1G of the present invention and pWUG, is to select for use Sac I-Kpn I restriction enzyme site to be connected the product that obtains with plasmid porf1G or pUG shown in Figure 2 respectively the wza gene fragment.
Fig. 4 is the carrier collection of illustrative plates of plasmid pW26La of the present invention, is to select the fragment behind wza gene fragment and the omp26La gene fragment overlap for use Sac I-BamH I restriction enzyme site and plasmid pUC18 shown in Figure 1
ΔE connects the product that obtains, and has wherein introduced Nsi I in omp26La gene fragment downstream primer, the site of EcoR I and BamH I.
Fig. 5 is plasmid pWorf1B of the present invention, pWUB and pW26LaB, be with target gene eseB fragment select for use EcoR I-BamH I restriction enzyme site respectively with plasmid pWorf1G shown in Figure 3, pWUG or plasmid pW26La shown in Figure 4 connect the product that obtains.
Fig. 6 is the structure schema of recombinant plasmid pWorf1G of the present invention and pWUG.
Fig. 7 is the structure schema of recombinant plasmid pW26LaB of the present invention.
Fig. 8 a contains the reorganization bacterium Top10/pWorf1G of plasmid pWorf1G and pWUG and ELISA that Top10/pWUG is shown to cell surface checking result respectively.OM: outer membrane protein component; CP: intracellular protein component; E/Worf1G: bacterial strain Top10/pWorf1G; E/WUG: bacterial strain Top10/pWUG; Expression strain Top10/pG in the E/G:GFP born of the same parents.
Fig. 8 b contains the reorganization bacterium Top10/pWorf1G of plasmid pWorf1G and pWUG and Western-blot that Top10/pWUG is shown to cell surface checking result respectively.OM: outer membrane protein component; CP: intracellular protein component; E/Worf1G: bacterial strain Top10/pWorf1G; E/WUG: bacterial strain Top10/pWUG; Expression strain Top10/pG in the E/G:GFP born of the same parents.
Fig. 9 a contains reorganization bacterium Top10/pWorf1B, the Top10/pWUB of plasmid pWorf1B, pWUB and pW26LaB and ELISA that Top10/pW26LaB is shown to cell surface checking result respectively.OM: outer membrane protein component; CP: intracellular protein component; E/Worf1B: bacterial strain Top10/pWorf1B; E/WUB: bacterial strain Top10/pWUB; E/W26LaB: bacterial strain Top10/pW26LaB; Expression strain Top10/pB in the E/B:EseB born of the same parents.
Fig. 9 b contains reorganization bacterium Top10/pWorf1B, the Top10/pWUB of plasmid pWorf1B, pWUB and pW26LaB and Western-blot that Top10/pW26LaB is shown to cell surface checking result respectively.OM: outer membrane protein component; CP: intracellular protein component; E/Worf1B: bacterial strain Top10/pWorf1B; E/WUB: bacterial strain Top10/pWUB; E/W26LaB: bacterial strain Top10/pW26LaB; Expression strain Top10/pB in the E/B:EseB born of the same parents.
Figure 10 a is the reorganization bacterium AV/pWorf1B that contains plasmid pWorf1B, pWUB and pW26LaB respectively, and AV/pWUB or AV/pW26LaB are shown to the ELISA checking result of cell surface.OM: outer membrane protein component; CP: intracellular protein component; A/Worf1B: strains A V/pWorf1B; A/WUB: strains A V/pWUB; A/W26LaB: strains A V/pW26LaB; Expression strain AV/pB in the A/B:EseB born of the same parents.
Figure 10 b is the reorganization bacterium AV/pWorf1B that contains plasmid pWorf1B, pWUB and pW26LaB respectively, and AV/pWUB or AV/pW26LaB are shown to the Western-blot checking result of cell surface.OM: outer membrane protein component; CP: intracellular protein component; A/Worf1B: strains A V/pWorf1B; A/WUB: strains A V/pWUB; A/W26LaB: strains A V/pW26LaB; Expression strain AV/pB in the A/B:EseB born of the same parents.
Embodiment
The present invention at first makes up bacterium surface displaying novel system: at first obtain required Vibrio anguillarum nucleotide sequence and target protein gene order, connect successively then, obtain fusion sequence.
Described target protein gene order is meant the desired proteic gene order at cell surface display of coding.
The above-mentioned Vibrio anguillarum nucleotide sequence and the acquisition of target protein gene order can be adopted pcr amplification, directly synthetic or other suitable methods obtain, and can adopt enzyme to cut connection, over-lap pcr amplification or other suitable methods above-mentioned sequence is connected the acquisition fusion rotein.
Vibrio anguillarum outer membrane lipoprotein Wza albumen of the present invention, Vibrio anguillarum outer membrane protein Omporf1, OmpU or Omp26La derive from Vibrio anguillarum Vibrio anguillarum MVM425, and this bacterial strain has following feature:
Amphimicrobian, arc shape is extremely given birth to single flagellum, Gram-negative, 25～28 ℃ of optimum growth temperatures, 0%NaCl and be higher than 6～7%NaCl and do not grow, catalase and oxidase positive can be utilized Citrate trianion, lysine decarboxylase and ornithine decarboxylase feminine gender, glucose is aerogenesis not, the sucrose fermentation and acid can produce amylase, lipase and casease, urease negative, the penicillin resistance positive, O1 serotype.
The used proteic molecular weight of Wza of the present invention is 42.8kDa, preceding 11 amino acid of its signal peptide and N end are shown in SEQ ID NO:1, the proteic molecular weight of used Omporf1 is 7.4kDa, its 6-50 amino acids is shown in SEQ IDNO:2, the proteic molecular weight of OmpU is 35.4kDa, its 180-238 amino acids is shown in SEQ ID NO:3, and the proteic molecular weight of Omp26La is 28.2kDa, and its 80-158 amino acids is shown in SEQ ID NO:4.
Utilize above-mentioned bacterium surface displaying novel system when the cell surface display target protein of host bacterium, implementation can be placed on the carrier, such as plasmid pUC18; Used host bacterium can be intestinal bacteria Escherichiacoli. (E.coli), Vibrio anguillarum or other host bacterium, such as intestinal bacteria Top10 or attenuated eel vibrio strain MVAV6203.
A series of surface displaying novel systems that the present invention utilizes Vibrio anguillarum outer membrane lipoprotein and Vibrio anguillarum outer membrane protein to make up, target protein successfully can be shown to bacterium surface, can strengthen and the proteic biological function of optimization aim, or increase the biological function of used host strain.The present invention has widened the range of application of attenuated eel vibrio strain, provides technical support for making up attenuated live vaccine, has also expanded the development research of bacterium surface system, for its widespread use in various fields lays the foundation.
Content for a better understanding of the present invention, with Vibrio anguillarum outer membrane lipoprotein Wza and Vibrio anguillarum outer membrane protein Omporf1, OmpU or Omp26La are example, are described further below.
The structure of embodiment 1 bacterium surface displaying novel system and the detection of target protein surface display
1. the structure of bacterium surface displaying type system
1.1 choose each gene fragment that will make up display systems and clone by PCR;
1.2 reclaim each gene fragment:
Reclaim the target gene fragment from the object that will study, adopt the glue recovery test kit of TIANGEN company to reclaim.After agarose gel electrophoresis finished, the purpose band was downcut in EB dyeing rapidly on long-wave ultra violet lamp, the Eppendorf that packs into pipe also takes by weighing weight, adds the sol solutions PN of 3 times of volumes, and 50 ℃ of water-baths were placed 10 minutes, constantly leniently spin upside down centrifuge tube therebetween, fully dissolve to guarantee blob of viscose; The solution that obtains is added in the adsorption column centrifugal (12, the centrifugal 30sec of 000rpm), outwell the waste liquid in the collection tube, adsorption column is reentered in the collection tube; In adsorption column, add 700 μ l rinsing liquid PW, 12, the centrifugal 30sec of 000rpm outwells waste liquid, uses 500 μ l rinsing liquid PW12 again, and the centrifugal 30sec of 000rpm outwells waste liquid.Centrifugal adsorption column is put back in the collection tube, 12, the centrifugal 2min of 000rpm goes out rinsing liquid as far as possible, again adsorption column is placed room temperature or 50 ℃ of incubator numbers minute, after drying up hill and dale, put it to one neatly in the centrifuge tube, be no less than the elution buffer EB buffer of 30 μ l to the unsettled dropping in adsorption film mid-way, room temperature is placed 2min, 12, the centrifugal 1min of 000rpm collects and contains the segmental dna solution of purpose, and electrophoresis detection reclaims DNA concentration.
1.3 the 4th bit base in the EcoR I site in the pUC18 carrier multiple clone site method by the nonsense point mutation is become C from T.PUC18 fragment in the amplification AflIII-Sac I site, and in the downstream primer of pcr amplification, the 4th bit base of EcoR I site is become C from T, again this fragment is passed through AflIII-Sac I double digestion, pUC18 is also cut by these two enzymes, reclaim big fragment of purifying and the purpose fragment that contains AflIII-Sac I site, plasmid after purpose fragment and the cutting is connected for 16 ℃ with the T4 dna ligase and spends the night CaCl
2Conversion method transforms Top10, obtains plasmid vector pUC18
ΔE.
1.4 with omporf1 or ompU gene and gfp gene while forward and pUC18
ΔThe E plasmid connects.Ompof1 is connected together with the method for gfp gene by overlap PCR with gfp gene or ompU, again this junction fragment is cut by Kpn I and BamH I enzyme, pUC18
ΔE also cuts by these two enzymes, reclaim the big fragment of purifying and contain the purpose fragment in Kpn I-BamH I site, with the purpose fragment with cut after plasmid be connected for 16 ℃ with the T4 dna ligase and spend the night CaCl
2Conversion method transforms Top10, obtain plasmid porf1G or pUG, plasmid porf1G and pUG are used Sac I-Kpn I double digestion respectively, with wza also after these two enzymes are cut, reclaim the purpose fragment that purifying obtains containing Sac I-Kpn I site, plasmid after purpose fragment and the cutting is connected for 16 ℃ with the T4 dna ligase and spends the night CaCl
2Conversion method transforms Top10, obtains intestinal bacteria reorganization bacterium.
1.5 with wza gene and omp26La gene while forward and pUC18
ΔThe E plasmid connects.Wza is connected together with the method for omp26La gene by overlap PCR, again this junction fragment is cut by Sac I and BamH I enzyme, pUC18
ΔE also cuts by these two enzymes, reclaim the big fragment of purifying and contain the purpose fragment in Sac I-BamH I site, with the purpose fragment with cut after plasmid be connected for 16 ℃ with the T4 dna ligase and spend the night CaCl
2Conversion method transforms Top10, obtains plasmid pW26La, plasmid pW26La is cut with EcoR I and BamH I enzyme, with also with these two enzymes cut and reclaim behind the purifying target gene fragment with cut after plasmid be connected for 16 ℃ with the T4 dna ligase and spend the night CaCl
2Conversion method transforms Top10, obtains intestinal bacteria reorganization bacterium.
1.6 the structure of Vibrio anguillarum reorganization bacterium
Inoculation Vibrio anguillarum MVAV6203 incubated overnight in the high salt LB of 5ml nutrient solution, again by 1: the 100 high salt LB of switching 100ml nutrient solution, in 30 ℃ with the 200rpm shaking culture to OD
600Value be 0.6 o'clock, centrifugal collection thalline is placed thalline for some time in ice bath, makes cell as far as possible near 0 ℃, and with the sucrose damping fluid of 272mM washing thalline three times, uses an amount of sucrose damping fluid (1ml) suspension thalline again, making its bacterial concentration is 1 * 10
9/ ml obtains electric transformed competence colibacillus cell.Get 100 μ l competence bacteria suspensions and transform in the cup (Bio-Rad company product), add certain density donor plasmid DNA10 μ l, on ice bath, place 10min behind the mixing, use Genepulser in the electricity of a 0.2cm
TMType electric impulser (Bio-Rad company product) carries out electricimpulse.Electric pulse parameter is chosen to be: pulse field intensity V=2kV/cm, the burst length is 3ms.After electricimpulse finishes, add 800 μ l LB nutrient solutions in electric revolving cup, and change in the centrifuge tube of 1.5ml after 30 ℃ of 200rpm recover to cultivate 3h, coat LB agar resistant panel, place 30 ℃ of cultivations.The ammonia benzyl is 200 μ g/ml.
The detection of 2 target protein surface displays
2.1 the above-mentioned Vibrio anguillarum reorganization bacterium that obtains is carried out the extracting of outer membrane protein component
Tropina is prepared by following method: the intestinal bacteria Top10 inoculation that will contain plasmid is at the LB less salt liquid nutrient medium that contains 100 μ g/ml penbritins, 37 ℃ of overnight incubation of 200rpm, as the one-level kind, be inoculated in the one-level kind in the 250ml triangular flask of LB less salt (Amp) substratum that contains the 50ml working concentration by 1: 100 (v/v) in second day, as the secondary kind, 37 ℃ of 200rpm shaking tables are cultured to OD
600=0.6, add inductor IPTG to final concentration 0.5mmol/l, in 37 ℃ of abduction delivering 12h, the results thalline.The Vibrio anguillarum MVAV6203 inoculation that contains plasmid is being contained the high salt liquid of the LB substratum of 200 μ g/ml penbritins, 30 ℃ of overnight incubation of 200rpm, as the one-level kind, be inoculated in the one-level kind in the 250ml triangular flask of the high salt of the LB that contains the 50ml working concentration (Amp) substratum by 1: 100 (v/v) in second day, as the secondary kind, 30 ℃ of 200rpm shaking tables are cultivated 24h, the results thalline.The centrifugal 2min of thalline 10000g to results, after the PBS washing three times, be resuspended in 1.5ml Tris-HCl-NaCl (50mM, pH8.0, containing 0.3%NaCl) in the damping fluid, carrying out ultrasonic bacteria breaking 5min in ice bath is broken fully to thalline with this resuspended liquid, with the cellular lysate liquid that obtains at 4 ℃ of centrifugal 5min of 10000g to remove uncracked cell and cell debris.For obtaining whole membranins, (Germany), the supernatant that then obtains is soluble intracellular protein composition, is precipitated as whole membranins for Sigma, Osterode at 4 ℃ of 20000g ultracentrifugation 1h with supernatant liquor.In order to obtain outer film component, precipitation is resuspended in 0.4ml HEPES (10mM, pH7.4) in the damping fluid, HEPES damping fluid (the 10mM that contains 2%SLS again with 0.4ml, pH7.4) mixing is with the dissolving inner membrance, this mixed solution at room temperature placed behind the 30min again at 4 ℃ of 20000g ultracentrifugation 1h, thereby obtained the outer membrane protein particle, be inner membrane protein in the supernatant, the extraction step that repeats once outer film component again will obtain purer outer membrane protein.
2.2 ELISA detects adventitia, intracellular protein component
The intracellular protein component all is diluted to identical OD (OD earlier with the outer membrane protein component
280=1.0).In each elisa plate hole, add 50 each component solution of μ l then, 4 ℃ of bags are spent the night, next day, discard solution in the hole, wash 3 times with the PBST damping fluid, every hole adds 200 μ l and contains the PBST of 3%BSA in 37 ℃ of sealing 1h, after discard solution in the hole, wash 3 times with the PBST damping fluid, adding the rabbit that is diluted to suitable multiple resists, hatch 1.5h for 37 ℃, wash 3 later cell antibody mixtures with the PBST damping fluid and hatch 1h with horseradish catalase bonded goat-anti rabbit two anti-(the Jackson company products) 37 ℃ of 1: 5000 (v/v) dilution again, PBST washes after 3 times, and every hole adds 37 ℃ of effect 10-30min of the solvable type tmb substrate colour developing of 100 μ l single components liquid (day root company product), and every hole adds 100 μ l 1MH again
2SO
4Termination reaction, the light absorption value in every hole detects down with the wavelength of microplate reader (Bio-Red company product) in 450am.Wherein to express the negative contrast of this proteic bacterium in the born of the same parents.
2.3 the preparation of SDS-PAGE gel
Get 30% gel mother liquor (29.2% acrylamide, 0.8% methylene diacrylamide) 2ml, 1.5M Tris-HCl, pH8.8,1.3ml, 10%SDS 50 μ l, ultrapure water 1.6ml, 10% ammonium persulphate, 50 μ l, TEMED 2 μ l, inject vertical slab electrophoresis glue device behind the mixing, seal up one deck water, in the separation gel that is 12% under the room temperature about polymerization 1h.The incline water on upper strata is got 30% gel mother liquor, 330 μ l, 1M Tris-HCl, pH6.8,250 μ l, 10%SDS20 μ l, ultrapure water 1.4ml, 10% ammonium persulphate, 20 μ l, TEMED 2 μ l inject the separation gel upper strata behind the mixing, insert the electrophoresis pecten, polymerization 1h under room temperature.
2.4 Western-blot detects each protein ingredient
Identical OD (OD will be diluted to
280=1.0) get equal-volume and sample-loading buffer (10%SDS with the outer membrane protein component in the born of the same parents, 10%2-mercaptan, pH6.8 0.3M Tris-HCl, 0.05% bromophenol indigo plant, 50% glycerine) mixing, in 12% SDS-PAGE glue, carry out electrophoresis, to excise the separation gel that concentrates glue afterwards and change damping fluid (25mmol/l Tris-HCl with the electricity of electroporation (Bio-Red company product) under 90V voltage, the 192mmol/l glycine, 20% methyl alcohol) electrotransfer 3h is to pvdf membrane in, the film that electricity is taken a turn for the better takes out, immunodetection is carried out in sealing after the 1h in 37 ℃ of PBS-T-BSA solution (the PBS damping fluid that contains 0.05% soil temperature and 1%BSA): pvdf membrane is resisted with the rabbit that is diluted to suitable multiple at 37 ℃ hatch 1.5h, use the horseradish catalase bonded goat-anti rabbit two anti-marks of 1: 5000 (v/v) dilution then, hatch 1h for 37 ℃, after washing with PBS-T solution, add 100 μ l single component sedimentation type tmb substrates colour developing liquid, 37 ℃ of effect 15min develop the color, and use 1M H again
2SO
4Termination reaction, observations.Wherein to express the negative contrast of this proteic bacterium in the born of the same parents.
The structure of the bacterium surface displaying novel system of embodiment 2 green fluorescent proteins, displaying and detection
According to target protein and other required nucleotide sequence design primer of required displaying, and in primer two ends introducing proper restriction site.With the plasmid pUC18 is template, primer pUCF and pUCR amplification obtain one section sequence on the pCU18 plasmid, and in downstream primer, the 4th bit base in EcoR I site is suddenlyd change, utilize the AflII and the Sac I site at two ends it to be linked in the pCU18 fragment of cutting through same enzyme the pCU18 in the EcoR I site that obtained suddenling change
ΔE (Fig. 1); With Vibrio anguillarum MVM425 genome is template, utilize primer omporf1F and omporf1R amplification to obtain the proteic one section encoding sequence of required Omporf1, utilize primer ompUF and ompUR amplification to obtain the proteic one section encoding sequence of needed OmpU, with the mTn5gusA-pgfp12 plasmid is template, gfpF and gfpR are that primer amplification obtains the gfp sequence, method by overlap PCR is fused to ompof1 fragment and gfp fragment or ompU fragment and gfp fragment together again, and Kpn I and BamH I site are used to merge fragments with these two and link pCU18 respectively
ΔIn the corresponding site of E, obtain recombinant plasmid porf1G and pUG (Fig. 2); With Vibrio anguillarum MVM425 genome is template, utilize primer wzaF and wzaR amplification to obtain the proteic one section encoding sequence of required Wza, utilize Sac I and Kpn I site, it is linked on plasmid porf1G or the pUG, obtain recombinant expression plasmid pWorf1G and pWUG (Fig. 3).PWorf1G and pWUG are changed among the Top10 respectively.By to Top10/pWorf1G and the Top10/pWUG bacterium extracting outer membrane protein component expressed and be respectively ELISA (Fig. 8 a) and Western-blot (Fig. 8 b) verify and know that GFP albumen successfully is shown on the adventitia.Proved that Wza-Omporf1 of system and Wza-OmpU can be shown to the external source intracellular protein on the adventitia in escherichia coli host, in Antibody Preparation, the polypeptide libraries screening, there is the potential using value in fields such as the development of environmental organism sorbent material and the catalytic development of whole-cell biological.
Design synthetic primer:
pUCF：5’-CCACATGTTCTTTCCTGCGT-3’
pUCR：5’-TCCGAGCTCGAGTTCGTAATCATGGTC-3’
Reaction conditions is: 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 55s, totally 30 circulations, 72 ℃ of 7min.
wzaF：5’-GGTGAGCTCAATGTCAAAAAAGTAT-3’
wzaR：5’-CGGGGTACCTGCGGCATTTTTTTCGCCTGTA-3’
Reaction conditions is: 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 20s, totally 30 circulations, 72 ℃ of 7min.
omporf1F：5’-CAGCGGGGTACCCCGATCGCACTATTAGCATCTT-3’
omporf1R：5’-AGCCATGAATTCCGGATGCATTGCCGTTACTGTACCTACT-3’
Reaction conditions is: 94 ℃ of 30s, 51 ℃ of 30s, 72 ℃ of 20s, totally 30 circulations, 72 ℃ of 7min.
ompUF：5’-CAGCGGGGTACCCCGAATGCAGATGGCTACTCT-3’
ompUR：5’-AGCCATGAATTCCGGATGCATTTGACCATCAACAAAAGTACC-3’
Reaction conditions is: 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 20s, totally 30 circulations, 72 ℃ of 7min.
gfpF：5’-CCGGAATTCATGGCTAGCAAAGGAG-3’
gfpR：5’-CGCGGATCCTTATTTGTACAGTTCATCCATGCCATG-3’
Reaction conditions is: 94 ℃ of 30s, 51 ℃ of 30s, 72 ℃ of 55s, totally 30 circulations, 72 ℃ of 7min.
The structure of the bacterium surface displaying novel system of embodiment 3 EseB antigen proteins, displaying and detection
Blunt tarda be a kind of can the septicemic cemia pathogenic bacteria of causing bleeding property in freshwater fish and marine fishes, it has an infection host scope very widely in water surrounding, and for many animals even people certain infectivity is arranged all.EseB albumen comes from pathogenic blunt tarda, be the membrane element albumen in the bacterium III type excretory system, have and report that the proteic disappearance of EseB can make the toxicity of blunt tarda reduce, prove pathogenic relevant (the Srinivasa Rao P.S. of itself and bacterium, Yamada Y., Tan Y.P., Leung K.Y.2004.Use ofproteomics to identify novel virulence determinants that are required for Edwardsiellatarda pathogenesis.Mol Microbiol 53 (2), 573-586).
With Vibrio anguillarum MVM425 genome is template, utilize primer omp26LaF and omp26LaR amplification to obtain the proteic one section encoding sequence of needed Omp26La, utilize the proteic one section encoding sequence of the required Wza that obtains previously, method by overlap PCR is fused to wza fragment and omp26La fragment together again, and Sac I and BamH I site are used to that this is merged fragment and link pCU18
ΔIn the corresponding site of E, obtain recombinant plasmid pW26La (Fig. 4); With the clone plasmid pPRoD of blunt tarda EseB protein coding gene eseB being arranged is template, the gene of pcr amplification eseB, acquisition meets the specific amplification products of expection size (600bp), reclaim this product, utilize the EcoR I and the BamH I site at its two ends directly to be cloned into the pWorf1G that also cuts respectively through same enzyme, on pWUG or the pW26La plasmid, obtain containing fusion gene wza/omporf1/eseB, the recombinant plasmid pWorf1B of wza/ompU/eseB or wza/omp26La/eseB, pWUB or pW26LaB (as shown in Figure 5).With pWorf1B, pWUB or pW26LaB change over to respectively among Top10 and the Vibrio anguillarum MVAV6203.By Top10/pWorf1B to expressing, Top10/pWUB, Top10/pW26LaB and AV/pWorf1B, AV/pWUB, AV/pW26LaB bacterium extracting outer membrane protein component and be respectively ELISA (Fig. 9 a and Figure 10 a) and Western-blot (Fig. 9 b and Figure 10 b) checking know that EseB albumen is regardless of all successfully being shown on the adventitia in intestinal bacteria Top10 host or among the Vibrio anguillarum MVAV6203 host, proved that the Wza-Omp26La system can be shown to foreign protein on the adventitia in intestinal bacteria and Vibrio anguillarum host, proved that simultaneously Wza-Omporf1 and Wza-OmpU system can also be shown to the external source intracellular protein on the adventitia in the Vibrio anguillarum host.This research has EseB the antigen protein of protection as a potential; it is merged mutually with the Wza-Omporf1/OmpU/Omp26La display systems; and be showed in the cell surface of attenuated eel vibrio strain, be expected to develop and develop simultaneously at Vibrio anguillarum and blunt tarda infect multiple-effect valency carrier living vaccine.
Design synthetic primer:
omp26LaF：5’-GCCGCAGGTACCCCGTTCTTTGGTAACACA-3’
omp26LaR：5’-CGGGATCCAGCCATGAATTCCGGATGCATGACATGGAAGTAGTTAACG-3’
Reaction conditions is: 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 25s, totally 30 circulations, 72 ℃ of 7min.
eseBF：5’-CCGGAATTCATGACTGTCAATAC-3’
eseRR：5’-CGGGATCCTTAGCGGATATTCTGGG-3’
Reaction conditions is: 94 ℃ of 30s, 57 ℃ of 30s, 72 ℃ of 45s, totally 30 circulations, 72 ℃ of 7min.
So, the fusant that utilizes Wza-Omporf1, Wza-OmpU or Wza-Omp26La is as carrier, can be with the target protein localization and expression in bacterium surface, both can strengthen and the proteic biological function of optimization aim, also can increase the biological function of used host strain, in living vaccine development, Antibody Preparation, the polypeptide libraries screening, various fields such as the development of environmental organism sorbent material and the catalytic development of whole-cell biological all have the potential using value.
In sum, bacterium surface displaying novel system of the present invention can successfully be showed target protein on the bacterial cell surface, can be used for the living vaccine exploitation, Antibody Preparation, the polypeptide libraries screening, exploitation of environmental organism sorbent material and whole-cell biological catalysis aspect are used in particular for making up Vibrio anguillarum multiple-effect valency carrier bacterin safely and efficiently on the basis of attenuated eel vibrio strain.
Need to prove, all quote in this application as a reference, just quoted as a reference separately as each piece document at all documents that the present invention mentions.Should understand in addition, above-described is specific embodiments of the invention and the know-why used, after having read above-mentioned teachings of the present invention, those skilled in the art can make various changes or modifications and not deviate from spirit of the present invention and scope the present invention, and these equivalent form of values fall within the scope of the invention equally.
Sequence table
＜110〉East China University of Science
＜120〉bacterium surface displaying novel system, method and application
<160>4
<210>1
<211>44
<212>PRT
＜213〉Vibrio anguillarum (Vibrio anguillarum)
<220>
<221>SIGNAL
<222>(1)..(44)
＜223〉proteic signal peptide of outer membrane lipoprotein Wza and N hold preceding 11 aminoacid sequences
<400>1
Met?Thr?Met?Ile?Thr?Asn?Ser?Ser?Ser?Met?Ser?Lys?Lys?Tyr?Leu?Pro
1 5 10 15
Leu?Ile?Ile?Ala?Ser?Val?Val?Leu?Thr?Gly?Cys?Thr?Ile?Pro?Gly?Ser
20 25 30
His?Leu?Pro?Thr?Gly?Glu?Lys?Asn?Ala?Ala?Gly?Thr
35 40
<210>2
<211>45
<212>PRT
＜213〉Vibrio anguillarum (Vibrio anguillarum)
<220>
<221>TRANSMEM
<222>(1)..(45)
＜223〉the proteic 6-50 amino acids of outer membrane protein Omporf1 sequence
<400>2
Ile?Ala?Leu?Leu?Ala?Ser?Phe?Ala?Phe?Gly?Gly?Val?Ala?Met?Ala?Ala
1 5 10 15
Val?Glu?Glu?Thr?Thr?Thr?Ala?Ser?Thr?Thr?Gly?Gly?Ala?Ala?Gly?Gly
20 25 30
Thr?Ala?Ala?Thr?Thr?Ala?Ala?Val?Gly?Thr?Val?Thr?Ala
35 40 45
<210>3
<211>59
<212>PRT
＜213〉Vibrio anguillarum (Vibrio anguillarum)
<220>
<221>TRANSMEM
<222>(1)..(59)
＜223〉the proteic 180-238 amino acids of outer membrane protein OmpU sequence
<400>3
Asn?Ala?Asp?Gly?Tyr?Ser?Leu?Ser?Ala?Ile?Tyr?Ala?Ile?Gly?Asp?Thr
1 5 10 15
Gly?Val?Lys?Leu?Gly?Ala?Gly?Tyr?Ala?Asp?Gln?Asp?Thr?Ala?Ala?Asn
20 25 30
Ala?Ser?Ser?Asp?Gln?Tyr?Met?Leu?Ala?Ala?Ser?Tyr?Ala?Ile?Ser?Asp
35 40 45
Phe?Tyr?Phe?Ala?Gly?Thr?Phe?Val?Asp?Gly?Gln
50 55
<210>4
<211>79
<212>PRT
＜213〉Vibrio anguillarum (Vibrio anguillarum)
<220>
<221>TRANSMEM
<222>(1)..(79)
＜223〉the proteic 80-158 amino acids of outer membrane protein Omp26La sequence
<400>4
Phe?Phe?Gly?Asn?Thr?Gly?Asp?Val?Val?Asn?Leu?Gly?Thr?Tyr?Leu?Thr
1 5 10 15
Gly?Ser?Gly?Val?Thr?Tyr?Asp?Gln?Asp?Ser?Ala?Asn?Ser?Val?Lys?Gly
20 25 30
Met?Asp?Lys?Arg?Lys?Ala?Thr?Ile?Asp?Leu?Gly?Leu?Asn?Ala?Asp?Ile
35 40 45
Ala?Leu?Gly?Asp?Gly?Thr?Val?Ser?Thr?Tyr?Phe?Gln?His?Asp?Ile?Leu
50 55 60
Asn?Glu?Asn?Lys?Gly?Tyr?Lys?Thr?Gly?Val?Asn?Tyr?Phe?His?Val
65 70 75
Claims (8)
1. bacterium surface exhibiting system, the gene order and the host bacterium that is used at the described target protein of cell surface display that comprise the target protein of encoding, it is characterized in that, described bacterium surface exhibiting system also comprises the Vibrio anguillarum nucleotide sequence of being responsible for striding film location and transhipment, the gene order of described coding target protein is positioned at the C end of described Vibrio anguillarum nucleotide sequence, what the nucleotide sequence coded albumen of described Vibrio anguillarum comprised the signal peptide of Vibrio anguillarum outer membrane lipoprotein and Vibrio anguillarum outer membrane protein strides the film anchor series, the described film anchor series of striding connects described signal peptide and described target protein, described Vibrio anguillarum outer membrane lipoprotein is a Wza albumen, described Vibrio anguillarum outer membrane protein is an Omporf1 albumen, OmpU albumen or Omp26La albumen, described Vibrio anguillarum nucleotides sequence is classified nucleotide fragments or the nucleotide fragments of the proteic 180-238 amino acids of coding OmpU shown in SEQ ID NO:3 or the nucleotide fragments of the proteic 80-158 amino acids of coding Omp26La shown in SEQ ID NO:4 of the proteic 6-50 amino acids of Omporf1 as described in preceding 11 amino acid whose nucleotide sequences of the proteic signal peptide of Wza and N end as described in the coding shown in SEQ ID NO:1 and the coding shown in SEQ ID NO:2 as, and described host bacterium is intestinal bacteria or Vibrio anguillarum.
2. bacterium surface exhibiting system as claimed in claim 1 is characterized in that, described host bacterium is intestinal bacteria Top10 or attenuated eel vibrio strain MVAV6203.
3. bacterium surface exhibiting system as claimed in claim 1 is characterized in that, described target protein is the EseB antigen protein of blunt tarda.
4. method of utilizing the described bacterium surface exhibiting system of claim 1 at the cell surface display target protein of bacterium.
5. method as claimed in claim 4 is characterized in that, described host bacterium is intestinal bacteria Top10 or attenuated eel vibrio strain MVAV6203.
6. method as claimed in claim 4 is characterized in that, described host bacterium is intestinal bacteria Top10 or attenuated eel vibrio strain MVAV6203, and described target protein is the EseB antigen protein of blunt tarda.
7. bacterium surface exhibiting system as claimed in claim 1 is developed at living vaccine, Antibody Preparation, polypeptide libraries screening, the application of exploitation of environmental organism sorbent material and whole-cell biological catalysis aspect.
8. application as claimed in claim 7 is characterized in that, described living vaccine is a Vibrio anguillarum multiple-effect valency carrier bacterin.
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| CN1623601A (en) * | 2003-12-03 | 2005-06-08 | 华东理工大学 | Attenuated live vaccine of vibrion from eel |
| CN1637135A (en) * | 2004-12-14 | 2005-07-13 | 华东理工大学 | Attenuated eel vibrio strain and its application |
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| CN1646020A (en) * | 2002-01-31 | 2005-07-27 | 圣必健公司 | Methods and composition for delivering nucleic acids and/or proteins to the respiratory system |
| CN1623601A (en) * | 2003-12-03 | 2005-06-08 | 华东理工大学 | Attenuated live vaccine of vibrion from eel |
| CN1637135A (en) * | 2004-12-14 | 2005-07-13 | 华东理工大学 | Attenuated eel vibrio strain and its application |
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| .细菌表面展示技术及其在环境重金属污染修复中的意义.生态与农村环境学报22 4.2006,22(4),74-79. |
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