CN103740632B - One strain recombination bacillus coli and the application in the N-glucoprotein vaccine of the anti-O157:H7 of preparation thereof - Google Patents

Abstract

The invention discloses a strain recombination bacillus coli, called after recombination bacillus coli CBEB, is the recombinant plasmid p-pglB-malE that will build ^mobtain with in p-rfb cotransformation intestinal bacteria CLM24, its genotype is W3110 Δ waal/p-pglB-malE ^m+ p-rfb.Utilize recombination bacillus coli fermentation of the present invention for anti-O157:H7N-glucoprotein vaccine, production stage is simple, and with short production cycle, output is high, and production cost is low, can be applied to the scale operation of anti-O157:H7N-glucoprotein vaccine; And the anti-O157:H7N-glucoprotein vaccine that the inventive method is produced has good immune effect, the immunotherapy for bacterial-infection resisting provides new selection, and prompting has good commercial exploitation and application prospect.

Description

One strain recombination bacillus coli and the application in the N-glucoprotein vaccine of the anti-O157:H7 of preparation thereof

Technical field

The present invention relates to the application in production Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7N-glucoprotein vaccine of a strain recombination bacillus coli and construction process and this recombinant bacterial strain.Belong to biotechnology, genetically engineered and field of microbial fermentation.

Background technology

Enterorrhagia Bacillus coil 0157: H7 usually causes blood sample diarrhoea, anaemia and renal failure in clinical infection, usually jeopardizes patient vitals.China isolates Escherichia coli O157, shows that also there is the potentially danger of this sick eruption and prevalence in China, should cause enough attention.Enterohemorrhagic Escherichia coli has been classified as 21 century and to one of compatriots' hygiene and health 12 kinds of pathogenic micro-organisms having great effect, may be only second to hiv virus (HIV) by China, arrangement second.Escherichia coli O 157 can be caused: the cell wall lysis of H7, promotes cytotoxic release, thus increase the weight of the course of disease owing to adopting antibiotic therapy.At present in clinical for Escherichia coli O 157: the treatment that H7 infects is based on prevention and conservative treatment.

Glucoprotein vaccine is considered to one of the most effective and safest anti-microbial pathogen vaccine, has broad prospect of application.Glucoprotein vaccine is by having on immunogenic carrier proteins by bacterial polysaccharides as O antigen or capsular polysaccharide are connected to, this glycoprotein can cause the immunne response depending on T cell, can give children to resist bacteriological infection and also can provide the long-time immunne response continued to grownup.

At present, the method mainly chemical method of glucoprotein vaccine is synthesized.Namely by genetic engineering technique expression and purification carrier proteins, then carry out chemistry with the pathogenic bacteria surface polysaccharide of purifying and be coupled.But due to the chemically sensitized randomness of sugar chain, the crosslinked glycoprotein produced has height unhomogeneity; Meanwhile, this method also also exists the problems such as step is numerous and diverse, operational difficulty.Based on this, suddenly wait to set up and develop the new glucoprotein vaccine production method that significantly can reduce production cost.

Summary of the invention

For existing methodical deficiency, the problem to be solved in the present invention is to provide a strain recombination bacillus coli and the application in the N-glucoprotein vaccine of the anti-O157:H7 of preparation thereof.

Technical scheme of the present invention connects glycosylation pathway differ with the similar feature of e. coli lipopolysaccharide route of synthesis based on campylobacter jejuni N-, employing derives from the rfb gene cluster (O157:H7rfb gene cluster: GI:3435170) in intestinal bacteria at expression in escherichia coli, and overexpression derives from malE gene (the plasmid pMAL-p5X of the source NEB company) mutant in intestinal bacteria and derives from the pglB gene (PglBNCBI-GeneID:905417) of campylobacter jejuni, the N-glycoprotein of the anti-O157:H7 of fermentative production in improvement TB substratum.

Recombination bacillus coli called after recombination bacillus coli CBEB of the present invention, is characterized in that: described recombination bacillus coli obtains by the following method: build containing malE ^mwith the co-expression carrier p-pglB-malE of pglB gene ^m, then build the expression vector p-rfb containing rfb gene cluster, then by the recombinant plasmid p-pglB-malE of above-mentioned structure ^mwith in p-rfb cotransformation intestinal bacteria CLM24, obtain to express pglB, malE simultaneously ^mthe recombination bacillus coli CBEB of gene and rfb gene cluster, its genotype is W3110 Δ waal/p-pglB-malE ^m+ p-rfb;

Wherein, described malE ^mderive from plasmid pMAL-p5X, described pglB gene source in campylobacter jejuni NCTC11168, coexpression malE ^mbe pBAD24 with the carrier of pglB gene; Described rfb gene cluster derives from Escherichia coli O 157: H7; The carrier of expressing rfb gene cluster is pYES1L;

Above-mentioned malE ^mfor malE gene mutation body, it is the gene gained by inserting the expression glycosylation sequences of one section of nucleotide sequence as shown in SEQIDNo.1 at 3 ' end of malE gene;

Above-mentioned intestinal bacteria CLM24 is waal gene by knocking out starting strain intestinal bacteria W3110 and the colibacillus engineering built, and its genotype is W3110 Δ waal, called after intestinal bacteria CLM24.

The construction process general introduction of above-mentioned recombination bacillus coli CBEB:

Knocking out of 1.waal gene

Basic skills in E. coli W3110, knocks out gene waal(oligosaccharyl transferase by Red recombination system).The E. coli W3110 Δ waal called after CLM24 of gained disappearance waal gene.

Above-mentioned Red recombination system expresses lambda particles phage recombinase Gam, Bet and Exo by plasmid pSIM19, by design with the primer amplification of homology arm with the kan(kalamycin resistance gene of selection markers) recombinant fragment.Then shocked by electricity by electroporation apparatus, recombinant fragment is proceeded in the intestinal bacteria of expressing lambda particles phage recombinase Gam, Bet and Exo, recombinant fragment is recombinated with the goal gene on genome under the effect of recombinase, thus original gene substitution is got off, and resistant gene is by expressing FLP restriction endonuclease, cut away from genome.

The structure of the expression vector of 2.rfb gene cluster

Basic skills is with Escherichia coli O 157: H7 genome is template, and clone rfb gene cluster, is inserted into obtained rfb gene cluster in linear plasmid pYES1L, thus obtains the expression vector p-rfb of rfb gene cluster.

3.malE ^mwith the structure of the co-expression carrier of pglB gene

Basic skills is with campylobacter jejuni gene group for template, clone's pglB gene, by clone the pglB obtained and inserts in plasmid pBAD24, thus the expression vector p-pglB of acquisition pglB; With plasmid pMAL-p5X for template, clone malE ^mgene, by clone obtain malE ^minsert in plasmid p-pglB, thus obtain malE ^mwith the co-expression carrier p-pglB-malE of pglB ^m.

The structure of 4.N-glycoprotein fermentation recombinant bacterial strain

Basic skills is by constructed recombinant plasmid p-pglB and p-pglB-malE ^min cotransformation intestinal bacteria CLM24, thus obtain expression rfb gene cluster, overexpression malE ^mwith the recombination bacillus coli CBEB of pglB.

The application of recombination bacillus coli of the present invention in the N-glucoprotein vaccine of preparation Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7.

Further, the N-glucoprotein vaccine method of concrete preparation Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 is:

1. shake-flask culture

The mono-bacterium colony of recombinant bacterial strain CBEB constructed by picking to be equipped with 3-5mL improvement TB substratum 25mL triangular flask in, penbritin final concentration is 100 μ g/mL, and spectinomycin final concentration is 50 μ g/mL, 37 DEG C, 200-225r/min, cultivate 12h.

The bacterium liquid of incubated overnight is equipped with 50mL according to the inoculum size access of 0.5-3% (v/v) to be improved in the triangular flask of the 100mL of TB substratum, penbritin final concentration is 100 μ g/mL, spectinomycin final concentration is 50 μ g/mL, 37 DEG C, 200-225r/min.

Treat bacterium liquid OD ₆₀₀during=0.4-0.6, according to the inoculum size access of 0.5-3% (v/v), 1L is housed and improves in the triangular flask of the 3L of TB substratum, penbritin final concentration is 100 μ g/mL, and spectinomycin final concentration is 50 μ g/mL, 37 DEG C, 200-225r/min.

Treat bacterium liquid OD ₆₀₀during=0.6-0.8, add the L-arabinose that final concentration is 0.2% (v/v), 16-37 DEG C, 200-225r/min.

Above-mentioned add L-arabinose after, leavening temperature preferably 28 DEG C.

After 4-6h, add the L-arabinose that final concentration is 0.2% (v/v), 16-37 DEG C, 200-225r/min, continue to cultivate 14-16 hour.

Above-mentioned preferred 6h adds L-arabinose again, leavening temperature preferably 28 DEG C, continues to cultivate 14h.

2.N-glycoprotein purification

By the centrifugal 5-10min of bacterium liquid 10,000-12,000r/min of shake flask fermentation.Centrifugal gained precipitation 1mg/ml N,O-Diacetylmuramidase 4 DEG C process 1h.The centrifugal 5-10min of 10,000-12,000r/min, uses affine ni-sepharose purification, collects the elutriant of N-glycoprotein, ultrafiltration desalination.

Wherein, in shake flask fermentation, the output of N-glycoprotein is 3mg/L.

Recombination bacillus coli disclosed by the invention and the application in the N-glucoprotein vaccine of the anti-O157:H7 of preparation thereof have very important using value.

Utilize recombination bacillus coli fermentation provided by the invention for anti-O157:H7N-glucoprotein vaccine, production stage is simple, and with short production cycle, output is high, and production cost is low, can be applied to the scale operation of anti-O157:H7N-glucoprotein vaccine; Meanwhile, the anti-O157:H7N-glucoprotein vaccine that the inventive method is produced has good immune effect, and the immunotherapy for bacterial-infection resisting provides new selection, and prompting has good commercial exploitation and application prospect.

Accompanying drawing explanation

Fig. 1. glucoprotein vaccine one-step fermentation production method principle schematic.

Fig. 2. gene knockout principle steps schematic diagram and waal gene knockout agarose gel electrophoresis detect.

Fig. 3 .p-rfb expression vector collection of illustrative plates.

Fig. 4 .p-pglB-malE ^mexpression vector collection of illustrative plates.

Fig. 5 .SDS-PAGE analyzes MBP glycosylation.MBP is malE ^mthe albumen of genetic expression, MBP-PS is glycosylated MBP.

Fig. 6 .Westernblot analyzes MBP glycosylation.Swimming lane 1 is MBP, and swimming lane 2 is MBP-PS.

Fig. 7 .MALDI-TOF analyzes MBP glycosylation.

Fig. 8. the IgG antibody ELISA of the anti-O157 of mice serum detects.

Fig. 9. the ELISA of mice serum cytokine IL-2, IL-4 detects.

Embodiment

General explanation: following enzyme involved by embodiment is all purchased from Thermo company, and plasmid extraction kit and sepharose reclaim DNA fragmentation test kit purchased from Tian Gen company, and operation is carried out according to respective description book completely.In plasmid construction, gene sequencing is completed by Hua Da genome company.Plasmid pYES1L derives from invitrogen company; Plasmid pBAD24 and the bacterium intestinal bacteria W3110 that sets out derives from ATCC (American Type Culture preservation center); DH5 α competent cell is purchased from Quan Shijin Bioisystech Co., Ltd.4-6 Balb/C female mice in age in week, is purchased from Shandong University's new drug evaluation center.Cytokine assay ELISA kit reaches purchased from Shenzhen and can be company limited.Other experimental techniques in embodiment and reagent if no special instructions, are this area ordinary method and commercial reagent.

Described LB substratum is: peptone 10g/L, yeast powder 5g/L, NaCl10g/L.

Described SOC substratum is: peptone 2g/L, yeast powder 0.5g/L, NaCl0.0585g/L, KCl0.0186g/L, MgCl ₂0.203g/L, MgSO ₄0.246g/L, glucose 20mmol/L.

Described improvement TB substratum is: peptone 10-20g/L, yeast powder 5-30g/L, glycerine 1-10ml/L, NaCl5-10g/L.

Knocking out of embodiment 1, intestinal bacteria W3110waal gene

(1) clone of homologous fragment

Red recombination system is utilized to knock out goal gene.Waal gene order design primer according to Genbank announces:

pKD-waalF：

5’-TTGGAAAAGTTATCATCATTATAAAGGTAAAACATGCTAACATCCTTTAAACTTCATTCATTGAAACCTTACACTCTGGTGTAGGCTGGAGCTGCTTC-3’

pKD-waalR：

5’-GAGTTTTAACTCACTTCTTAAACTTGTTTATTCTTAATTAATTGTATTGTTACGATTATTAATGACGAGTAAGAGGACATGGGAATTAGCCATGGTCC-3’

With pKD4 by PCR(polymerase chain reaction) amplification in vitro obtains recombinant fragment with kalamycin resistance.PCR reaction system is as follows: (primer concentration is 20 μm of ol/L)

Template DNA 1 μ l, adds water and mends to 50 μ l.

PCR reaction conditions: 97 DEG C of denaturation 10min, 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 90s, 30 rear 72 DEG C of extension 10min of circulation, 4 DEG C of preservations.After DpnI endonuclease digestion, reclaim purified concentration homologous recombination fragment.

(2) preparation of Electroporation-competent cells

(I) picking is with the intestinal bacteria W3110 of pSIM19 plasmid, proceeds in LB substratum, adds 100mg/L penbritin simultaneously, 30 DEG C, 200r/min, cultivates OD ₆₀₀to 0.4;

(II) 42 DEG C of water-bath 15min, 200r/min;

(III) ice bath 15min, centrifugal thalline, then utilizes the glycerine of 10% to wash 3 times;

(IV) add the glycerine of 10%, be concentrated into 50 times, packing competence.

(3) electricity transforms, screening recon

(I) draw the homologous recombination fragment of 8 μ g/ml, add in the competent cell of 100 μ l, be transferred to after mixing in the 2mm electricity revolving cup of precooling, gently get rid of to cup.Regulate electroporation apparatus, 2.5Kv, electric shock;

(II) add the SOC substratum of 900 μ l, be transferred in aseptic EP pipe after mixing, 37 DEG C, 120r/min, cultivate 1h;

(III) be coated with kalamycin resistance dull and stereotyped, transfer recon and utilize

waaltestF:GGTATGTAGGGCTCCAAGAG

waaltestR:AATTTGGTCCCCGAATCATC

Carry out PCR detection, confirm that waal gene is replaced by kalamycin resistance gene further by PCR primer order-checking.

(IV) FLP Site-specific recombinase

PCP20 is proceeded to kalamycin resistance clone, cultivates 8h for 30 DEG C, after be increased to 42 DEG C and spend the night, thermal induction FLP recombinase is expressed, and plasmid is also lost gradually.Utilize transfering loop to pick bacterium liquid to rule on non-resistant substratum, the mono-clonal grown is proceeded on non-resistant flat board and kalamycin resistance flat board simultaneously and cultivate, kalamycin resistance gene being eliminated of not growing on kalamycin resistance flat board at non-resistant grow on plates.Detection primer waaltestF and waaltestR is utilized to identify further.

(V) obtain and knock out waal gene and the colibacillus engineering that builds, its genotype is W3110 Δ waal, called after intestinal bacteria CLM24.The results are shown in Figure 2.

The structure of embodiment 2, rfb gene cluster (GI:3435170) expression vector

Escherichia coli O 157 according to NCBI announces: H7 genome sequence design primer:

O157-1-F:

5’-GTACCCGGGGATCCTCTAGAGTCGACGCATAAATTTTAATGCTTATCAAAACTATTAGC-3’

O157-1-R:

5’-CATGGATGTCCGTATAAATGGACACACATAATAGCTTTAGTTTTATTAGTGA-3’

O157-2-F:

5’-TCACTAATAAAACTAAAGCTATTATGTGTGTCCATTTATACGGACATCCATG-3’

O157-2-R:

5’-TATGCGCGACAATTATAATGCACATCGTC-3’

O157-3-F:

5’-TGACGATGTGCATTATAATTGTCGCGCATATTTTAACAATAAAACAAATGATGC-3’

O157-3-R:

5’-GAATTTCTTCTCTCATCCGCCAAAACAGAAGCTTTGTTTACTCCTGTCAGGGGTTACC-3’

With O157:H7 genome for template, respectively with O157-1-F and O157-1-R, O157-2-F and O157-2-R, O157-3-F and O157-3-R for primer, point three sections of PCR clone rfb gene cluster.PCR reaction system is as follows: (primer concentration is 20 μm of ol/L)

Template DNA 1 μ l, adds water and mends to 50 μ l.

PCR reaction conditions: 97 DEG C of denaturation 10min, 94 DEG C of sex change 30s, 60 DEG C (O157-1) or 54 DEG C (O157-2) or 60 DEG C (O157-3) anneal 30s, and 72 DEG C extend 5min, and latter 72 DEG C of 30 circulations extend 10min, 4 DEG C of preservations.

Three PCR fragment of clone are joined linked system.As follows:

After 30 DEG C of water-baths connect 30min, add 35.5 μ lDMSO and mix, then 42 DEG C of thermal shock 20min.The centrifugal 5min of 1,800r/min, by the resuspended precipitation of 1mL0.9%NaCl, gets 100 μ l and is coated with CSM-Trp flat board.Cultivate picking transformant after 3 days for 30 DEG C, extract plasmid checking.Then further sequence verification rfb gene cluster is correct.Thus obtained recombinant plasmid p-rfb.

Embodiment 3, recombinant plasmid p-pglB-malE ^mstructure

(1) structure of pglB gene (PglBNCBI-GeneID:905417) expression vector

Campylobacter jejuni NCTC11168 genome sequence design primer according to NCBI announces:

pglB-F:

5’-CACGCCATGGTCTTGAAAAAAGAGTATTTAAAAAACCC-3’

pglB-R:

5’-ATTCCCGGGTCAATGATGATGATGATGATGAATTTTAAGTTTAAAAACTTTAG-3’

With NCTC11168 genome for template, PCR clones pglB gene.PCR reaction system is as follows: (primer concentration is 20 μm of ol/L)

Template DNA 1 μ l, adds water and mends to 50 μ l.

PCR reaction conditions: 97 DEG C of denaturation 10min, 94 DEG C of sex change 30s, 50 DEG C of annealing 30s, 72 DEG C extend 2min, 30 rear 72 DEG C of extension 10min of circulation, 4 DEG C of preservations.

The pglB fragment of clone is utilized endonuclease NcoI and SmaI digestion process respectively, plasmid vector pBAD24 is also utilized endonuclease NcoI and SmaI digestion process respectively simultaneously.Utilize sepharose test kit to reclaim the pglB fragment of digestion process and pBAD24 plasmid vector, then utilize T4 ligase enzyme to connect.

Linked system is 10 μ l:

PglB fragment: 6 μ l;

PBAD24 carrier: 2 μ l;

10×Buffer：1μl；

T4 ligase enzyme: 1 μ l.

After 16 DEG C of connection 12h, by the connecting fluid transformation of E. coli DH5 α competent cell of 10 μ l.Conversion process is: the connecting fluid of 10 μ l added in the DH5 α competent cell of 100 μ l, mixing.Ice bath 30min, 42 DEG C of thermal shock 90s, ice bath 2min, add the LB substratum of 900 μ l, 37 DEG C, 100r/min, hatching 1h, and coating amicillin resistance is dull and stereotyped, cultivates 16h, picking transformant, extracts plasmid checking.Then further sequence verification pglB gene is correct.Thus obtained recombinant plasmid p-pglB.

(2) malE ^mthe structure of (the plasmid pMAL-p5X of source NEB company) and pglB expression vector

Plasmid pMAL-p5x primers according to NCBI announces:

malE ^M-F:

5’-TCCCCCGGGGGAAGGAGGCATAGATTATGAAAATAAAAACAGGTGC-3’

malE ^M-R:

5’-GCGTCGACGTCTCAATGATGATGATGATGATGGGTCGCGTTCTGATCTCCTCCAGTGGCGTTCTGATCGCCGCCGGTCGCGTTCTGATCGCCGCCGGTCGCGTTCTGATCTTCCAGCTGCGCGTCTTTCAGGGC-3’

Wherein: above-mentioned malE ^mcontaining expressing the gene of glycosylation sequences in-R, its nucleotide sequence as shown in SEQIDNo.1, specifically:

5’-CTGGAAGATCAGAACGCGACCGGCGGCGATCAGAACGCGACCGGCGGCGATCAGAACGCCACTGGAGGAGATCAGAACGCGACC-3’

With plasmid pMAL-p5x for template, PCR clones malE ^mgene.PCR reaction system is as follows: (primer concentration is 20 μm of ol/L)

Template DNA 1 μ l, adds water and mends to 50 μ l.

PCR reaction conditions: 97 DEG C of denaturation 10min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 90s, 30 rear 72 DEG C of extension 10min of circulation, 4 DEG C of preservations.

The pglB fragment of clone is utilized endonuclease SmaI and SalI digestion process respectively, plasmid vector p-pglB is also utilized endonuclease SmaI and SalI digestion process respectively simultaneously.Utilize sepharose test kit to reclaim the pglB fragment of digestion process and p-pglB plasmid vector, then utilize T4 ligase enzyme to connect.

Linked system is 10 μ l:

PglB fragment: 6 μ l;

PBAD24 carrier: 2 μ l;

10×Buffer：1μl；

T4 ligase enzyme: 1 μ l.

After 16 DEG C of connection 12h, by the connecting fluid transformation of E. coli DH5 α competent cell of 10 μ l.Conversion process is: the connecting fluid of 10 μ l added in the DH5 α competent cell of 100 μ l, mixing.Ice bath 30min, 42 DEG C of thermal shock 90s, ice bath 2min, add the LB substratum of 900 μ l, 37 DEG C, 100r/min, hatching 1h, and coating amicillin resistance is dull and stereotyped, cultivates 16h, picking transformant, extracts plasmid checking.Then further sequence verification malE ^mgene correct.Thus obtained recombinant plasmid p-pglB-malE ^m.

The purifying of the structure of embodiment 4, recombinant escherichia coli strain and fermentation and N-glycoprotein

(1) structure of recombinant escherichia coli strain

With reference to embodiment 1(2) method prepares CLM24 electricity and turns competent cell.Draw the recombinant plasmid p-rfb of 10 μ l, add in the competent cell of 100 μ l, be transferred to after mixing in the 2mm electricity revolving cup of precooling, gently get rid of to cup.Regulate electroporation apparatus, 2.5Kv, electric shock; In electric revolving cup, add the SOC substratum of 900 μ l subsequently, mixing, is transferred in aseptic EP pipe, 37 DEG C, 120r/min, cultivates 1h; Coating kalamycin resistance is dull and stereotyped, transfers recon checking after 37 DEG C of cultivation 24h.Thus obtain recombinant bacterium CLM24/p-rfb.

Get recombinant bacterium CLM24/p-rfb chemistry (CaCl prepared by 50ul ₂) add 5ul plasmid p-pglB-malE in competent cell ^m, mixing ice bath after 20 ~ 30 minutes, 42 DEG C of water-bath heat shocks ice bath 3 ~ 5 minutes after 90 seconds; Then 900ulLB substratum is added, 37 DEG C, 120r/min renewal cultivation 45min ~ 1h; The centrifugal 2min of 4,000r/min, pours out part supernatant, by dull and stereotyped for remaining thalline coating amicillin resistance, transfers recon checking after 37 DEG C of cultivation 24h.Thus obtain recombinant bacterium CLM24/p-rfb+p-pglB-malE ^m.

(2) fermentation of recombination bacillus coli

Recombinant bacterial strain CBEB(CLM24/p-rfb+p-pglB-malE constructed by picking ^m) single bacterium colony to be equipped with 5mL improvement TB substratum 25mL triangular flask in, penbritin final concentration is 100 μ g/mL, and spectinomycin final concentration is 50 μ g/mL, 37 DEG C, 200r/min, cultivate 12h.

The bacterium liquid of incubated overnight is equipped with 50mL according to the inoculum size access of 1% (v/v) improve in the triangular flask of the 100mL of TB substratum, penbritin final concentration is 100 μ g/mL, and spectinomycin final concentration is 50 μ g/mL, 37 DEG C, 200-225r/min.

When bacterium liquid OD600=0.6, according to the inoculum size access of 1% (v/v), 1L is housed and improves in the triangular flask of the 3L of TB substratum, penbritin final concentration is 100 μ g/mL, and spectinomycin final concentration is 50 μ g/mL, 37 DEG C, 200-225r/min.

Treat bacterium liquid OD ₆₀₀when=0.6, add the L-arabinose that final concentration is 0.2% (v/v), 28 DEG C, 200-225r/min.

After 6h, add the L-arabinose that final concentration is 0.2% (v/v), 28 DEG C, 200-225r/min, continue to cultivate 14h.

(3) purifying of N-glycoprotein

The centrifugal 10min of 1L fermentation liquor 10,000r/min, collects thalline and processes thalline 1h with the periplasmic space protein extract (20mMTris-HCl, 20% (wt/vol) sucrose, 1mMEDTA, 1mg/ml N,O-Diacetylmuramidase, pH7.5) 4 DEG C of 30mL.The centrifugal 30min of 12,000r/min, then collects supernatant and joins and use binding buffer liquid (10mM imidazoles, 0.5MNaCl, 20mMNa ₂hPO ₄/ NaH ₂pO ₄damping fluid, pH7.4) in the affine nickel post of 5mL crossed of pre-equilibration.Binding buffer liquid through 10 column volumes washs, with elution buffer (250mM imidazoles, 0.5MNaCl, 20mMNa ₂hPO ₄/ NaH ₂pO ₄damping fluid, pH7.4) wash-out, collect the elutriant containing N-glycoprotein.

It is 10kDa super filter tube that elutriant containing target protein is loaded molecular weight cut-off, and 4 DEG C of 5,000g centrifugal 30min, discards lower pipe liquid, fill it up with desalination damping fluid in sleeve pipe, 4 DEG C of 5,000g centrifugal 30min.Repeat 3 times, be concentrated into 2mL to target protein, and imidazoles is fully removed.Albumen after concentrated is frozen in-70 DEG C of refrigerators.

The qualification of embodiment 5, N-glycoprotein

SDS-PAGE analysis is carried out in the N-glycoprotein sampling of purifying.MBP molecular weight, at about 46KDa, conforms to theoretical molecular.The glycosylated MBP of N-forms scalariform band, and main band molecular weight distribution is between 50KDa and 55KDa.Because SDS-PAGE sensitivity is limited, the band of some glycoprotein does not show.The results are shown in Figure 5.

Utilize WesternBlot to identify N-glycoprotein, adopt anti-His, anti-MBP, anti-O157 antibody as primary antibodie respectively, select suitable sheep anti mouse or goat-anti rabbit to resist as two, carry out detection and analyze.The glycosylated MBP of N-due to the addition of chain length not wait O-antigen and demonstrate the large scalariform band of the nonglycosylated MBP of molecular weight ratio, proves MBP generation glycosylation.The results are shown in Figure 6.

MALDI-TOF is utilized to carry out the qualification of molecular weight to glycosylated MBP and MBP of N-.MBP molecular weight is that 45823Da, MBP host molecule amount is distributed near 48790Da.The results are shown in Figure 7.

Embodiment 6, N-glycoprotein immune effect

(1) immune mouse

Get the female mouse of Balb/C in 6 week age, be divided into three groups, be respectively dorsal sc multi-point injection N-glycosylation MBP(every only 30 μ g, 150 μ l, n=8), MBP(every only 30 μ g, 150 μ l, n=8) and the every only 150 μ l of PBS(, n=5), every immunity in two weeks once, immunity three times are total to.First immunisation uses complete Freund's adjuvant, and second and third immunity is cannotd be used up full freund's adjuvant.Before immunity and after each immunity one week, get blood 200ml from mouse leg saphena, after room temperature leaves standstill 1h, the centrifugal 20min of 4,000r/min, taking-up serum, packing was stored in-20 DEG C.

(2) serum antibody titer detects

Escherichia coli O 157: H7 lipopolysaccharides bag is by 96 orifice plates, and ELISA detects Escherichia coli O 157 in mice serum: the anti-titre of H7 lipopolysaccharides, the results are shown in Figure 8.

(3) serum cytokines detects

IL-2 and the IL-4 level in immunized mice serum is measured by cytokine ELISA kit (Shenzhen reaches section).IL-2 level: MP group > M group > PBS group; IL-4 level: MP group < M group < PBS group., show that MBP-PS can activating Th 1 cell, suppress Th2 cytoactive.The results are shown in Figure 9.

Measure can reach a conclusion according to above N-glycoprotein immune effect, the N-glucoprotein vaccine that recombination bacillus coli of the present invention is produced has the effect of obvious Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7.This mouse model simulates the environment in human body completely, therefore also has good effect for human tumor treatment in theory.Indicate that vaccine of the present invention can be used for the mankind and prevents Escherichia coli O 157: the immunotherapy of H7.

The present invention is that the immunotherapy of bacterial-infection resisting provides new selection, has a good application prospect.

Claims (1)

1. a pnca gene type is W3110 Δ waal/p-pglB-malE ^mthe preparation method of the recombination bacillus coli of+p-rfb, is characterized in that: build containing malE ^mwith the co-expression carrier p-pglB-malE of pglB gene ^m, then build the expression vector p-rfb containing rfb gene cluster, by the recombinant expression vector p-pglB-malE of above-mentioned structure ^mwith p-rfb cotransformation intestinal bacteria CLM24, obtain expressing pglB gene, malE simultaneously ^mthe recombination bacillus coli W3110 Δ waal/p-pglB-malE of gene and rfb gene cluster ^m+ p-rfb;

Wherein, described malE ^mderive from plasmid pMAL-p5X, described pglB gene source in campylobacter jejuni NCTC11168, coexpression malE ^mbe pBAD24 with the carrier of pglB gene; Described rfb gene cluster derives from Escherichia coli O 157: H7, and the carrier of expressing rfb gene cluster is pYES1L;

Above-mentioned malE ^mfor malE gene mutation body, be the gene gained by inserting the expression glycosylation sequences of one section of nucleotide sequence as shown in SEQIDNo.1 at 3 ' end of malE gene, wherein malE ^mthe primer malE that amplification adopts ^mthe nucleotide sequence of-F as shown in SEQIDNo.14, primer malE ^mthe nucleotide sequence of-R is as shown in SEQIDNo.15;

Above-mentioned rfb gene cluster is with O157:H7 genome for template, respectively with O157-1-F and O157-1-R, O157-2-F and O157-2-R, O157-3-F and O157-3-R for primer, point three sections of PCR clone rfb gene cluster, then connect acquisition; The nucleotide sequence of described primer O157-1-F is as shown in SEQIDNo.6, the nucleotide sequence of primer O157-1-R is as shown in SEQIDNo.7, the nucleotide sequence of primer O157-2-F is as shown in SEQIDNo.8, the nucleotide sequence of primer O157-2-R is as shown in SEQIDNo.9, the nucleotide sequence of primer O157-3-F is as shown in SEQIDNo.10, and the nucleotide sequence of primer O157-3-R is as shown in SEQIDNo.11;

Above-mentioned intestinal bacteria CLM24 is waal gene by knocking out starting strain intestinal bacteria W3110 and the colibacillus engineering built, and its genotype is W3110 Δ waal.