CN101195823A

CN101195823A - Novel bacterium surface exhibiting system, method and application of the same

Info

Publication number: CN101195823A
Application number: CNA2007101706337A
Authority: CN
Inventors: 刘琴; 张元兴; 杨朝; 王启要
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2007-11-20
Filing date: 2007-11-20
Publication date: 2008-06-11
Anticipated expiration: 2027-11-20
Also published as: CN101195823B

Abstract

The invention provides a novel showing system of the bacterial surface, which comprises a positioning transporting sequence of bacteria transmembrane and a gene sequence of encoding target albumen. The positioning transporting sequence of the bacteria transmembrane comprises a nucleotide sequence of the out membrane lipoprotein signal peptide of escherichia coil, and also comprises a nucleotide sequence of vibrio anguillarum, and the nucleotide sequence of the out membrane lipoprotein signal peptide of the escherichia coil and the gene sequence of encoding target protein are connected with the nucleotide sequence of the vibrio anguillarum, encoding protein comprises a transmembrane grappling sequence of the vibrio anguillarum outer-membrane protein, the out membrane lipoprotein of the escherichia coil out membrane lipoprotein is Lpp protein, and the out membrane protein of the vibrio anguillarum is Omporf1 protein, OmpU protein, or Omp26La protein, the invention of the novel showing system of the bacterial surface which can successfully shows target protein on the bacterial surface, and the invention can be used in factors of live vaccine exploitation, antibody preparation, polypeptide library sieving, environment biological absorbent exploitation, and full cell biological catalysis, in particular to multifactor value carrier vaccine of the vibrio anguillarum with secure and highly effective construction based on the vibrio anguillarum poison gene stock.

Description

Novel bacterium surface exhibiting system, method and application

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 novel bacterium surface exhibiting 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 novel bacterium surface exhibiting system, method and application are provided, this novel bacterium surface exhibiting 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 novel bacterium surface exhibiting system is provided, comprise that bacterium strides the gene order of film location transit sequence and coding target protein, described bacterium strides the nucleotide sequence that film location transit sequence comprises intestinal bacteria outer membrane lipoprotein signal peptide, be characterized in, described bacterium strides film location transit sequence and also comprises the Vibrio anguillarum nucleotide sequence, described Vibrio anguillarum nucleotide sequence connects the gene order of the nucleotide sequence and the described coding target protein of described intestinal bacteria outer membrane lipoprotein signal peptide, and what the nucleotide sequence coded albumen of described Vibrio anguillarum comprised the Vibrio anguillarum outer membrane protein strides the film anchor series.

Described intestinal bacteria outer membrane lipoprotein is a Lpp albumen, and described Vibrio anguillarum outer membrane protein is an Omporf1 albumen, OmpU albumen or Omp26La albumen.

Described bacterium stride preceding 9 amino acid whose nucleotide sequences that film location transit sequence is coding proteic signal peptide of described Lpp and N end and the proteic 6-50 amino acids of the described Omporf1 of coding nucleotide fragments the proteic 180-238 amino acids of coding OmpU nucleotide fragments the nucleotide fragments of the proteic 80-158 amino acids of coding Omp26La.

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 novel bacterium surface exhibiting system at the cell surface display target protein of bacterium is provided, be characterized in, may further comprise the steps:

A. described novel bacterium surface exhibiting 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 bacterium stride preceding 9 amino acid whose nucleotide sequences that film location transit sequence is coding proteic signal peptide of described Lpp and N end and the proteic 6-50 amino acids of the described Omporf1 of coding nucleotide fragments the proteic 180-238 amino acids of coding OmpU nucleotide fragments the nucleotide fragments of the proteic 80-158 amino acids of coding Omp26La, 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 novel bacterium surface exhibiting 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 intestinal bacteria outer membrane lipoprotein Lpp albumen and Vibrio anguillarum outer membrane protein Omporf1, the fusant of OmpU or Omp26La is a carrier, utilize the N-end signal peptide of Lpp, 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 novel bacterium surface exhibiting system of the present invention; N-end signal peptide by Lpp; 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 pLorf1 of the present invention, pLU and pL26La, is with lpp gene fragment and omporf1, and the fragment behind ompU or the omp26La gene fragment overlap is selected Sac I-BamH I restriction enzyme site and plasmid pUC18 shown in Figure 1 for use _ΔE connects the product that obtains, and wherein at omporf1, has introduced Nsi I in ompU and the omp26La gene fragment downstream primer, the site of EcoR I and BamH I.

Fig. 3 is the carrier collection of illustrative plates of plasmid pLUG of the present invention and pL26LaG, is to select for use EcoR I-Ba mH I restriction enzyme site to be connected the product that obtains with plasmid pLU or pL26La shown in Figure 2 respectively goal gene gfp fragment.

Fig. 4 is the carrier collection of illustrative plates of plasmid pLorf1B of the present invention, pLUB and pL26LaB, be with target gene eseB fragment select for use EcoR I-BamH I restriction enzyme site respectively with plasmid pLorf1 shown in Figure 2, pLU or pL26La connect the product that obtains.

Fig. 5 is recombinant plasmid pLUG shown in Figure 3 of the present invention and the structure schema of pL26LaG.

Fig. 6 is the structure schema of recombinant plasmid pLorf1B shown in Figure 4 of the present invention.

Fig. 7 a contains the reorganization bacterium Top10/pLUG of plasmid pLUG and pL26LaG and ELISA that Top10/pL26LaG is shown to cell surface checking result respectively.OM: outer membrane protein component; CP: intracellular protein component; E/LUG: bacterial strain Top10/pLUG; E/L26LaG: bacterial strain Top10/pL26LaG; Expression strain Top10/pG in the E/G:GFP born of the same parents.

Fig. 7 b contains the reorganization bacterium Top10/pLUG of plasmid pLUG and pL26LaG and Western-blot that Top10/pL26LaG is shown to cell surface checking result respectively.OM: outer membrane protein component; CP: intracellular protein component; E/LUG: bacterial strain Top10/pLUG; E/L26LaG: bacterial strain Top10/pL26LaG; Expression strain Top10/pG in the E/G:GFP born of the same parents.

Fig. 8 a contains reorganization bacterium Top10/pLorf1B, the Top10/pLUB of plasmid pLorf1B, pLUB and pL26LaB and ELISA that Top10/pL26LaB is shown to cell surface checking result respectively.OM: outer membrane protein component; CP: intracellular protein component; E/Lorf1B: bacterial strain Top10/pLorf1B; E/LUB: bacterial strain Top10/pLUB; E/L26LaB: bacterial strain Top10/pL26LaB; Expression strain Top10/pB in the E/B:EseB born of the same parents.

Fig. 8 b contains reorganization bacterium Top10/pLorf1B, the Top10/pLUB of plasmid pLorf1B, pLUB and pL26LaB and Western-blot that Top10/pL26LaB is shown to cell surface checking result respectively.OM: outer membrane protein component; CP: intracellular protein component; E/Lorf1B: bacterial strain Top10/pLorf1B; E/LUB: bacterial strain Top10/pLUB; E/L26LaB: bacterial strain Top10/pL26LaB; Expression strain Top10/pB in the E/B:EseB born of the same parents.

Fig. 9 a contains plasmid pLorf1B respectively, the ELISA checking result that reorganization bacterium AV/pLorf1B, the AV/pLUB of pLUB and pL26LaB and AV/pL26LaB are shown to cell surface.OM: outer membrane protein component; CP: intracellular protein component, A/Lorf1B: strains A V/pLorf1B; A/LUB: strains A V/pLUB; A/L26LaB: strains A V/pL26LaB; Expression strain AV/pB in the A/B:EseB born of the same parents.

Fig. 9 b contains plasmid pLorf1B respectively, the Western-blot checking result that reorganization bacterium AV/pLorf1B, the AV/pLUB of pLUB and pL26LaB and AV/pL26LaB are shown to cell surface.OM: outer membrane protein component; CP: intracellular protein component, A/Lorf1B: strains A V/pLorf1B; A/LUB: strains A V/pLUB; A/L26LaB: strains A V/pL26LaB; Expression strain AV/pB in the A/B:EseB born of the same parents.

Embodiment

The present invention at first makes up novel bacterium surface exhibiting system, at first obtains required bacterium and strides film location transit sequence and target protein gene order, connects successively then, obtains fusion sequence.

Described target protein gene order is meant the desired proteic gene order at cell surface display of coding.

Above-mentioned bacterium strides the nucleotide sequence and the Vibrio anguillarum nucleotide sequence of the intestinal bacteria outer membrane lipoprotein signal peptide that film location transit sequence comprises, 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.

Lpp dietary protein origin of the present invention is in intestinal bacteria Escherichia coli. (E.coli), and Vibrio anguillarum outer membrane protein Omporf1, OmpU or Omp26La derive from Vibrio anguillarum Vibrio anguilarum 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.

Preceding 9 amino acid of proteic signal peptide of Lpp that the present invention is used 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 ID NO:2, the proteic molecular weight of OmpU is 35.4kDa, its 180-238 amino acids is shown in SEQ ID NO:3, 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 novel bacterium surface exhibiting 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, Vibrio anguillarum or other host bacterium, such as intestinal bacteria Top10 or attenuated eel vibrio strain MVAV6203.

A series of novel bacterium surface exhibiting systems that the present invention utilizes intestinal bacteria 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 intestinal bacteria outer membrane lipoprotein Lpp and Vibrio anguillarum outer membrane protein Omporf1, OmpU or Omp26La are example, are described further below.

The structure of embodiment 1 novel bacterium surface exhibiting system and the detection of target protein surface display

1. the structure of novel bacterium surface exhibiting 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 lpp gene and omporf1, ompU or omp26La gene be forward and pUC18 simultaneously _ΔThe E plasmid connects.With lpp and omporf1, ompU or omp26La gene connect together by the method for overlap PCR, these junction fragments are cut by Sac I and BamH I enzyme respectively, with pUC18 again _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 pLorf1, pLU and pL26La, with plasmid pLorf1, pLU and pL26La cut with EcoR I and BamH I enzyme respectively, are connected for 16 ℃ with the T4 dna ligase and spend the night CaCl also cutting and reclaim plasmid after target gene fragment and the cutting behind the purifying with these two enzymes ₂Conversion method transforms Top10, obtains intestinal bacteria reorganization bacterium.

1.5 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, pH 7.4) mixing is with the dissolving inner membrance, and this mixed solution is 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 l1MH again ₂SO ₄Termination reaction, the light absorption value in every hole detects down with the wavelength of microplate reader (Bio-Red company product) in 450nm.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, pH 8.8,1.3ml, 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 novel bacterium surface exhibiting 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 AflIII 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 ompUF and ompUR amplification to obtain the proteic one section encoding sequence of required OmpU, utilize primer omp26LaF and omp26LaR amplification to obtain the proteic one section encoding sequence of needed Omp26La, with pTX101 plasmid (Francisco J.A., Earhart C.F., Georgiou is and anchoring of beta-lactamase to the external surface ofEscherichia coli.Proc Natl Acad Sci USA 89 (7) G.1992.Transport: 2713-7) be template, lppF and lppR are that primer amplification obtains needed one section lpp sequence, method by overlap PCR is fused to lpp fragment and ompU fragment or omp26La fragment together again, and Sac 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 pLU and pL26La (Fig. 2); With the mTn5gusA-pgfp12 plasmid is template, and gfpF and gfpR are that primer amplification obtains the gfp sequence, utilizes EcoR I and BamH I site, and it is linked on plasmid pLU or the pL26La, obtains recombinant expression plasmid pLUG and pL26LaG (Fig. 3).PLUG and pL26LaG are changed among the Top10 respectively.By to Top10/pLUG and the Top10/pL26LaG bacterium extracting outer membrane protein component expressed and be respectively ELISA (Fig. 7 a) and Western-blot (Fig. 7 b) verify and know that GFP albumen successfully is shown on the adventitia.Proved that Lpp-OmpU of system and Lpp-Omp26La 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.

lppF：5’-GGTGAGCTCAATGAAAGCTACTAAACTGGTACTGG-3’；

lppR：5’-CGGGGTACCCCGCTGATCAATTTTAGCGTTG-3’；

Reaction conditions is: 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 20s, totally 30 circulations, 72 ℃ of 7min.

ompUF：5’-CAGCGGGGTACCCCGAATGCAGATGGCTACTCT-3’；

ompUR：5’-CGGGATCCAGCCATGAATTCCGGATGCATTTGACCATCAACAAAAGTACC-3’；

Reaction conditions is: 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 20s, totally 30 circulations, 72 ℃ of 7min.

omp26LaF：5’-CAGCGGGGTACCCCGTTCTTTGGTAACACAGG-3’；

omp26LaR：5’-CGGGATCCAGCCATGAATTCCGGATGCATGACATGGAAGTAGTTAACG-3’；

Reaction conditions is: 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 25s, 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 novel bacterium surface exhibiting 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).

According to target protein and other required nucleotide sequence design primer of required displaying, and in primer two ends introducing proper restriction site.With Vibrio anguillarum MVM425 genome is template, utilize primer omporf1F and omporf1R amplification to obtain the proteic one section encoding sequence of needed Omporf1, utilize embodiment 2 methods to obtain the proteic one section encoding sequence of required Lpp, method by overlap PCR is fused to lpp fragment and omporf1 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 pLorf1 (Fig. 2); Method according to embodiment 1 obtains recombinant plasmid pLU and pL26La; With the clone plasmid pPRoD of blunt tarda EseB protein coding gene eseB being arranged is template, utilize primer eseBF and eseBR to carry out 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 pLorf1 that also cuts respectively through same enzyme, on pLU or the pL26La plasmid, obtain containing fusion gene lpp/omporf1/eseB, the recombinant plasmid pLorf1B of lpp/ompU/eseB or lpp/omp26La/eseB, pLUB or pL26LaB (as shown in Figure 4).With pLorf1B, pLUB or pL26LaB change over to respectively among Top10 and the Vibrio anguillarum MVAV6203.By Top10/pLorf1B to expressing; Top10/pLUB; Top10/pL26LaB and AV/pLorf1B; AV/pLUB; AV/pL26LaB bacterium extracting outer membrane protein component also is ELISA (Fig. 8 a respectively; Fig. 9 a) and Western-blot (Fig. 8 b; Fig. 9 b) checking is known EseB albumen no matter all successfully be shown on the adventitia in intestinal bacteria Top10 host or among the Vibrio anguillarum MVAV6203 host; proved that the Lpp-Omporf1 system can be shown to foreign protein on the adventitia in intestinal bacteria or Vibrio anguillarum host; proved that simultaneously Lpp-OmpU and Lpp-Omp26La 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 Lpp-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, lay the foundation for making up Vibrio anguillarum multiple-effect valency attenuated live vaccine.

Design synthetic primer:

omporf1F：5’-CAGCGGGGTACCCCGATCGCACTATTAGCATCTT-3’；

omporf1R：5’-CGGGATCCAGCCATGAATTCCGGATGCATTGCCGTTACTGTACCTACT-3’；

Reaction conditions is: 94 ℃ of 30s, 51 ℃ of 30s, 72 ℃ of 20s, totally 30 circulations, 72 ℃ of 7min.

eseBF：5’-CCGGAATTCATGACTGTCAATAC-3’；

eseBR：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 Lpp-Omporf1, Lpp-OmpU or Lpp-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, novel bacterium surface exhibiting 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〉novel bacterium surface exhibiting system, method and application

<160>4

<210>1

<211>42

<212>PRT

＜213〉intestinal bacteria (Escherichia coli)

<220>

<221>SIGNAL

<222>(1)..(33)

＜223〉proteic signal peptide of outer membrane lipoprotein Lpp and N hold preceding 9 aminoacid sequences

<400>1

Met?Thr?Met?Ile?Thr?Asn?Ser?Ser?Ser?Met?Lys?Ala?Thr?Lys?Leu?Val

1 5 10 15

Leu?Gly?Ala?Val?Ile?Leu?Gly?Ser?Thr?Leu?Leu?Ala?Gly?Cys?Ser?Ser

20 25 30

Asn?Ala?Lys?Ile?Asp?Gln?Arg?Gly?Thr?Pro

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

1. novel bacterium surface exhibiting system, comprise that bacterium strides the gene order of film location transit sequence and coding target protein, described bacterium strides the nucleotide sequence that film location transit sequence comprises intestinal bacteria outer membrane lipoprotein signal peptide, it is characterized in that, described bacterium strides film location transit sequence and also comprises the Vibrio anguillarum nucleotide sequence, described Vibrio anguillarum nucleotide sequence connects the gene order of the nucleotide sequence and the described coding target protein of described intestinal bacteria outer membrane lipoprotein signal peptide, and what the nucleotide sequence coded albumen of described Vibrio anguillarum comprised the Vibrio anguillarum outer membrane protein strides the film anchor series.

2. novel bacterium surface exhibiting system as claimed in claim 1 is characterized in that, described intestinal bacteria outer membrane lipoprotein is a Lpp albumen, and described Vibrio anguillarum outer membrane protein is an Omporf1 albumen, OmpU albumen or Omp26La albumen.

3. novel bacterium surface exhibiting system as claimed in claim 2, it is characterized in that, described bacterium stride preceding 9 amino acid whose nucleotide sequences that film location transit sequence is coding proteic signal peptide of described Lpp and N end and the proteic 6-50 amino acids of the described Omporf1 of coding nucleotide fragments the proteic 180-238 amino acids of coding OmpU nucleotide fragments the nucleotide fragments of the proteic 80-158 amino acids of coding Omp26La.

4. novel bacterium surface exhibiting system as claimed in claim 3 is characterized in that, described target protein is the EseB antigen protein of blunt tarda.

5. a method of utilizing novel bacterium surface exhibiting system as claimed in claim 1 at the cell surface display target protein of bacterium is characterized in that, may further comprise the steps:

6. method as claimed in claim 5 is characterized in that, described intestinal bacteria outer membrane lipoprotein is a Lpp albumen, and described Vibrio anguillarum outer membrane protein is an Omporf1 albumen, OmpU albumen or Omp26La albumen.

7. method as claimed in claim 6, it is characterized in that, described bacterium stride preceding 9 amino acid whose nucleotide sequences that film location transit sequence is coding proteic signal peptide of described Lpp and N end and the proteic 6-50 amino acids of the described Omporf1 of coding nucleotide fragments the proteic 180-238 amino acids of coding OmpU nucleotide fragments the nucleotide fragments of the proteic 80-158 amino acids of coding Omp26La, described host bacterium is intestinal bacteria or Vibrio anguillarum.

8. method as claimed in claim 7 is characterized in that 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.

9. novel 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.

10. application as claimed in claim 9 is characterized in that, described living vaccine is a Vibrio anguillarum multiple-effect valency carrier bacterin.