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

A strain of recombinant Escherichia coli and its application in the preparation of N-glycoprotein vaccine against O157:H7

technical field

The invention relates to a strain of recombinant Escherichia coli, its construction method and the application of the recombinant strain in the production of anti-Escherichia coli O157:H7N-glycoprotein vaccine. It belongs to the fields of biotechnology, genetic engineering and microbial fermentation.

Background technique

Enterohemorrhagic Escherichia coli O157:H7 often causes bloody diarrhea, anemia and renal failure in clinical infection, often endangering the lives of patients. Escherichia coli O157:H7 has been isolated in my country, indicating that there is also a potential risk of outbreaks of this disease in my country, and sufficient attention should be paid to it. my country has listed enterohemorrhagic Escherichia coli as one of the 12 pathogenic microorganisms that may have a significant impact on the health of the Chinese people in the 21st century, ranking second only to HIV. The use of antibiotic therapy can lead to the dissolution of the cell wall of E. coli O157:H7, which promotes the release of cytotoxins, thereby aggravating the course of the disease. The current clinical treatment for E. coli O157:H7 infection is mainly prevention and conservative therapy.

Glycoprotein vaccines are considered to be one of the most effective and safest anti-pathogenic vaccines and have broad application prospects. Glycoprotein vaccines, which elicit a T-cell-dependent immune response by linking bacterial polysaccharides such as O antigen or capsular polysaccharides to immunogenic carrier proteins, can be given to children against bacterial infections and can also be given to adults Humans provide a long-lasting immune response.

At present, the methods for synthesizing glycoprotein vaccines are mainly chemical methods. That is, the carrier protein is expressed and purified by genetic engineering technology, and then chemically coupled with the purified surface polysaccharide of pathogenic bacteria. However, due to the randomness of the chemical activation of sugar chains, the glycoproteins produced by cross-linking are highly heterogeneous; at the same time, this method also has problems such as complicated steps and difficult operation. Based on this, it is urgent to establish and develop new glycoprotein vaccine production methods that can greatly reduce production costs.

Contents of the invention

Aiming at the deficiencies of the existing methods, the problem to be solved by the present invention is to provide a recombinant Escherichia coli and its application in the preparation of an anti-O157:H7 N-glycoprotein vaccine.

The technical scheme of the present invention is based on the characteristics that the N-linked glycosylation pathway of Campylobacter jejuni is similar to the synthetic pathway of E. :3435170), and overexpress the malE gene (derived from NEB company's plasmid pMAL-p5X) mutant from Escherichia coli and the pglB gene (PglBNCBI-GeneID: 905417) derived from Campylobacter jejuni, in the improved TB medium Fermentative production of O157:H7-resistant N-glycoprotein.

The recombinant Escherichia coli described in the present invention is called recombinant Escherichia coli CBEB, and it is characterized in that: described recombinant Escherichia coli is made by following method: Construct the co-expression vector p-pglB-malE ^M that contains malE ^M and pglB gene, construct again The expression vector p-rfb containing the rfb gene cluster, and then the recombinant plasmid p-pglB-malE ^M and p-rfb constructed above were co-transformed into Escherichia coli CLM24 to obtain a recombinant that can simultaneously express the pglB, malE ^M gene and the rfb gene cluster Escherichia coli CBEB, whose genotype is W3110Δwaal/p-pglB-malE ^M +p-rfb;

Wherein, the malE ^M is derived from the plasmid pMAL-p5X, the pglB gene is derived from Campylobacter jejuni NCTC11168, and the vector co-expressing the malE ^M and pglB genes is pBAD24; the rfb gene cluster is derived from Escherichia coli O157:H7; expression The carrier of the rfb gene cluster is pYES1L;

The above-mentioned malE ^M is a mutant of the malE gene, which is obtained by inserting a nucleotide sequence such as a gene expressing a glycosylation sequence shown in SEQ ID No.1 at the 3' end of the malE gene;

The aforementioned Escherichia coli CLM24 is an Escherichia coli engineered bacterium constructed by knocking out the waal gene of the starting strain Escherichia coli W3110, its genotype is W3110Δwaal, and it is named Escherichia coli CLM24.

The construction method summary of above-mentioned recombinant escherichia coli CBEB:

1. Knockout of waal gene

The basic method is to knock out the gene waal (oligosaccharyltransferase) in Escherichia coli E.coliW3110 by the Red recombination system. The resulting Escherichia coli E. coli W3110Δwaal lacking the waal gene was named CLM24.

The above-mentioned Red recombination system expresses λ phage recombinase Gam, Bet and Exo through plasmid pSIM19, and amplifies the recombination fragment of kan (kanamycin resistance gene) with selection marker by designing primers with homology arms. Then, the recombinant fragments were transferred into E. coli expressing λ bacteriophage recombinases Gam, Bet and Exo by electroporation, and the recombinant fragments were recombined with the target genes on the genome under the action of the recombinases, so that the original genes Replacement, and the resistance gene is excised from the genome by expressing FLP endonuclease.

2. Construction of the expression vector of the rfb gene cluster

The basic method is to use Escherichia coli O157:H7 genome as a template to clone the rfb gene cluster and insert the obtained rfb gene cluster into the linear plasmid pYES1L to obtain the expression vector p-rfb of the rfb gene cluster.

3. Construction of the co-expression vector of malE ^M and pglB gene

The basic method is to use the Campylobacter jejuni genome as a template to clone the pglB gene, and insert the cloned pglB into the plasmid pBAD24 to obtain the expression vector p-pglB of pglB; use the plasmid pMAL-p5X as a template to clone the malE ^M gene, and insert The cloned malE ^M was inserted into the plasmid p-pglB, thereby obtaining the co-expression vector p-pglB-malE ^M of malE ^M and pglB.

4. Construction of N-glycoprotein fermentation recombinant strain

The basic method is to co-transform the constructed recombinant plasmids p-pglB and p-pglB-malE ^M into E. coli CLM24, so as to obtain the recombinant E. coli CBEB expressing rfb gene cluster and overexpressing malE ^M and pglB.

The application of the recombinant Escherichia coli described in the present invention in the preparation of N-glycoprotein vaccine against Escherichia coli O157:H7.

Further, the specific method for preparing the N-glycoprotein vaccine against Escherichia coli O157:H7 is:

1. Shake flask culture

Pick a single colony of the constructed recombinant strain CBEB into a 25mL Erlenmeyer flask filled with 3-5mL of modified TB medium, the final concentration of ampicillin is 100 μg/mL, the final concentration of spectinomycin is 50 μg/mL, at 37°C, 200-225r/min, cultivate for 12h.

The overnight cultured bacterial solution was inserted into a 100 mL Erlenmeyer flask equipped with 50 mL of improved TB medium according to the inoculum size of 0.5-3% (v/v), the final concentration of ampicillin was 100 μg/mL, and the final concentration of spectinomycin was 50μg/mL, 37°C, 200-225r/min.

When the bacterial liquid OD ₆₀₀ =0.4-0.6, according to the inoculum amount of 0.5-3% (v/v), transfer it into a 3L Erlenmeyer flask containing 1L of modified TB medium, and the final concentration of ampicillin is 100μg/mL, which is spectacular. The final concentration of mycin is 50 μg/mL, 37°C, 200-225r/min.

When the bacterial liquid OD ₆₀₀ =0.6-0.8, add L-arabinose with a final concentration of 0.2% (v/v), 16-37°C, 200-225r/min.

After the above-mentioned addition of L-arabinose, the fermentation temperature is preferably 28°C.

After 4-6 hours, add L-arabinose with a final concentration of 0.2% (v/v) once, at 16-37°C, 200-225r/min, and continue culturing for 14-16 hours.

It is preferable to add L-arabinose again at 6 hours above, the fermentation temperature is preferably 28° C., and the culture is continued for 14 hours.

2. N-glycoprotein purification

Centrifuge the bacterial solution fermented by the shake flask at 10,000-12,000r/min for 5-10min. The precipitate obtained by centrifugation was treated with 1 mg/ml lysozyme at 4°C for 1 h. Centrifuge at 10,000-12,000r/min for 5-10min, purify with an affinity nickel column, collect the eluate of N-glycoprotein, and desalt by ultrafiltration.

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

The recombinant Escherichia coli disclosed by the invention and its application in preparing an anti-O157:H7 N-glycoprotein vaccine have very important application value.

The preparation of anti-O157:H7N-glycoprotein vaccine by recombinant Escherichia coli provided by the present invention has simple production steps, short production cycle, high yield and low production cost, and can be applied to large-scale production of anti-O157:H7N-glycoprotein vaccine; At the same time, the anti-O157:H7N-glycoprotein vaccine produced by the method of the present invention has good immune effect, provides a new option for immunotherapy against bacterial infection, and suggests that it has good industrial development and application prospects.

Description of drawings

Figure 1. Schematic diagram of the principle of one-step fermentation production method of glycoprotein vaccine.

Figure 2. Schematic diagram of the principle steps of gene knockout and agarose gel electrophoresis detection of waal gene knockout.

Figure 3. Map of p-rfb expression vector.

Figure 4. Map of p-pglB-malE ^M expression vector.

Figure 5. SDS-PAGE analysis of MBP glycosylation. MBP is the protein expressed by malE ^M gene, and MBP-PS is glycosylated MBP.

Figure 6. Western blot analysis of MBP glycosylation. Lane 1 is MBP and lane 2 is MBP-PS.

Figure 7. MALDI-TOF analysis of MBP glycosylation.

Figure 8. ELISA detection of IgG antibody against O157 in mouse serum.

Figure 9. ELISA detection of mouse serum cytokines IL-2 and IL-4.

detailed description

General description: the enzymes involved in the following examples were all purchased from Thermo Company, the plasmid extraction kit and the DNA fragment recovery kit from agarose gel were purchased from Tiangen Company, and the operations were carried out in full accordance with the corresponding instructions. Gene sequencing in plasmid construction was completed by Huada Gene Company. The plasmid pYES1L was from Invitrogen; the plasmid pBAD24 and the starting bacteria Escherichia coli W3110 were from ATCC (American Type Culture Collection); DH5α competent cells were purchased from Quanshijin Biotechnology Co., Ltd. 4-6 weeks old Balb/C female mice were purchased from the New Drug Evaluation Center of Shandong University. Cytokine determination ELISA kit was purchased from Shenzhen Daco Co., Ltd. Other experimental methods and reagents in the examples are conventional methods and commercially available reagents in the art unless otherwise specified.

The LB medium is: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L.

The SOC medium is: peptone 2g/L, yeast powder 0.5g/L, NaCl 0.0585g/L, KCl 0.0186g/L, MgCl ₂ 0.203g/L, MgSO ₄ 0.246g/L, glucose 20mmol/L.

The improved TB medium is: peptone 10-20g/L, yeast powder 5-30g/L, glycerin 1-10ml/L, NaCl 5-10g/L.

Embodiment 1, knockout of Escherichia coli W3110waal gene

(1) Cloning of homologous fragments

The target gene was knocked out using the Red recombination system. Primers were designed according to the waal gene sequence published by Genbank:

pKD-waalF:

5'-TTGGAAAAGTTATCATCATTATAAAGGTAAAACATGCTAACATCCTTTAAACTTCATTCATTGAAACCTTACACTCTGGTGTAGGCTGGAGCTGCTTC-3'

pKD-waalR:

5'-GAGTTTTAACTCACTTCTTAAACTTGTTTATTCTTAATTAATTGTATTGTTACGATTATTAATGACGAGTAAGAGGACATGGGAATTAGCCATGGTCC-3'

The recombinant fragment with kanamycin resistance was amplified in vitro by PCR (polymerase chain reaction) with pKD4. The PCR reaction system is as follows: (primer concentration is 20 μmol/L)

Template DNA 1μl, add water to make up to 50μl.

PCR reaction conditions: pre-denaturation at 97°C for 10 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 90 s, extension at 72°C for 10 min after 30 cycles, and storage at 4°C. After digestion with DpnI endonuclease, the concentrated homologous recombination fragment was recovered and purified.

(2) Preparation of electroporation competent cells

(1) Pick Escherichia coli W3110 with the pSIM19 plasmid, transfer it to LB medium, add 100mg/L ampicillin at the same time, 30°C, 200r/min, culture OD ₆₀₀ to 0.4;

(II) 42°C water bath for 15 minutes, 200r/min;

(III) ice bath for 15min, centrifuge the thalline, then utilize 10% glycerol to wash 3 times;

(IV) Adding 10% glycerin, concentrating to 50 times, subpackaging competent state.

(3) Electrotransformation, screening of recombinants

(I) Aspirate 8 μg/ml homologous recombination fragments, add to 100 μl competent cells, mix well, transfer to a pre-cooled 2 mm electroporation cup, and gently shake to the bottom of the cup. Adjust the electroporator, 2.5Kv, electric shock;

(II) Add 900 μl of SOC medium, mix well and transfer to a sterile EP tube, culture at 37°C, 120r/min, for 1h;

(III) Coat the kanamycin-resistant plate and transfer recombinants for use

waaltestF:GGTATGTAGGGCTCCAAGAG

waaltestR:AATTTGGTCCCCGAATCATC

PCR detection was carried out to further confirm that the waal gene had been replaced by the kanamycin resistance gene by PCR product sequencing.

(IV) FLP site-specific recombination

Transform pCP20 into kanamycin-resistant clones, culture at 30°C for 8 hours, and then increase to 42°C overnight, heat-induced expression of FLP recombinase, and the plasmid is gradually lost. Use the inoculation loop to dip the bacterial solution and streak on the non-resistant medium, transfer the grown monoclonals to the non-resistant plate and the kanamycin-resistant plate at the same time, grow on the non-resistant plate and grow on the non-resistant plate. The kanamycin resistance gene that does not grow on the kanamycin resistance plate has been eliminated. The detection primers waaltestF and waaltestR were used for further identification.

(V) Obtain the Escherichia coli engineering bacterium constructed by knocking out the waal gene, its genotype is W3110Δwaal, and it is named Escherichia coli CLM24. The results are shown in Figure 2.

Example 2, Construction of rfb gene cluster (GI: 3435170) expression vector

Primers were designed according to the genome sequence of Escherichia coli O157:H7 published by NCBI:

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'

Using the O157:H7 genome as a template, using O157-1-F and O157-1-R, O157-2-F and O157-2-R, O157-3-F and O157-3-R as primers respectively, divided into three The rfb gene cluster was cloned by PCR. The PCR reaction system is as follows: (primer concentration is 20 μmol/L)

Template DNA 1μl, add water to make up to 50μl.

PCR reaction conditions: pre-denaturation at 97°C for 10 minutes, denaturation at 94°C for 30 seconds, annealing at 60°C (O157-1) or 54°C (O157-2) or 60°C (O157-3) for 30 seconds, extension at 72°C for 5 minutes, after 30 cycles Extend at 72°C for 10 minutes, and store at 4°C.

The cloned three PCR fragments were added to the ligation system. as follows:

After connecting to a water bath at 30°C for 30 minutes, add 35.5 μl DMSO and mix well, then heat shock at 42°C for 20 minutes. Centrifuge at 1,800r/min for 5min, resuspend the pellet with 1mL of 0.9% NaCl, and take 100μl to coat the CSM-Trp plate. After culturing at 30°C for 3 days, transformants were picked and plasmids were extracted for verification. Then further sequencing to verify the correctness of the rfb gene cluster. Thus the recombinant plasmid p-rfb was obtained.

Embodiment 3, the construction of recombinant plasmid p-pglB-malE ^M

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

Primers were designed according to the genome sequence of Campylobacter jejuni NCTC11168 published by NCBI:

pglB-F:

5'-CACGCCATGGTCTTGAAAAAAGAGTATTTAAAAAACCC-3'

pglB-R:

5'-ATTCCCGGGTCAATGATGATGATGATGATGAATTTTAAGTTTAAAAACTTTAG-3'

Using NCTC11168 genome as template, pglB gene was cloned by PCR. The PCR reaction system is as follows: (primer concentration is 20 μmol/L)

Template DNA 1μl, add water to make up to 50μl.

PCR reaction conditions: pre-denaturation at 97°C for 10min, denaturation at 94°C for 30s, annealing at 50°C for 30s, extension at 72°C for 2min, extension at 72°C for 10min after 30 cycles, and storage at 4°C.

The cloned pglB fragment was digested with endonucleases NcoI and SmaI respectively, and the plasmid vector pBAD24 was also digested with endonucleases NcoI and SmaI respectively. The digested pglB fragment and the pBAD24 plasmid vector were recovered using an agarose gel kit, and then ligated using T4 ligase.

The connection system is 10 μl:

pglB fragment: 6 μl;

pBAD24 vector: 2 μl;

10×Buffer: 1μl;

T4 ligase: 1 μl.

After connecting at 16°C for 12 hours, 10 μl of the connection solution was transformed into Escherichia coli DH5α competent cells. The transformation process is as follows: add 10 μl of the connection solution to 100 μl of DH5α competent cells, and mix well. Ice bath for 30min, heat shock at 42°C for 90s, ice bath for 2min, add 900μl LB medium, 37°C, 100r/min, incubate for 1h, coat ampicillin-resistant plates, culture for 16h, pick transformants, extract plasmids for verification . Then further sequencing to verify the correctness of the pglB gene. Thus the recombinant plasmid p-pglB was obtained.

(2) Construction of malE ^M (plasmid pMAL-p5X from NEB Company) and pglB gene expression vector

Primers were designed according to the sequence of plasmid pMAL-p5x published by NCBI:

malE ^M -F:

5'-TCCCCCGGGGGAAGGAGGCATAGATTATGAAAATAAAAAACAGGTGC-3'

malE ^M -R:

5'-GCGTCGACGTCTCAATGATGATGATGATGATGGGTCGCGTTCTGATCTCCTCCAGTGGCGTTCTGATCGCCGCCGGTCGCGTTCTGATCGCCGCCGGTCGCGTTCTGATCTTCCAGCTGCGCGTCTTTCAGGGC-3'

Wherein: the above-mentioned malE ^M -R contains a gene expressing a glycosylation sequence, and its nucleotide sequence is shown in SEQIDNo.1, specifically:

5'-CTGGAAGATCAGAACGCGACCGGCGGCGATCAGAACGCGACCGGCGGCGATCAGAACGCCACTGGAGGAGATCAGAACGCGACC-3'

Using the plasmid pMAL-p5x as a template, the malE ^M gene was cloned by PCR. The PCR reaction system is as follows: (primer concentration is 20 μmol/L)

Template DNA 1μl, add water to make up to 50μl.

PCR reaction conditions: pre-denaturation at 97°C for 10min, denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 90s, extension at 72°C for 10min after 30 cycles, and storage at 4°C.

The cloned pglB fragment was digested with endonucleases SmaI and SalI respectively, and the plasmid vector p-pglB was also digested with endonucleases SmaI and SalI at the same time. The digested pglB fragment and p-pglB plasmid vector were recovered using an agarose gel kit, and then ligated using T4 ligase.

The connection system is 10 μl:

pglB fragment: 6 μl;

pBAD24 vector: 2 μl;

10×Buffer: 1μl;

T4 ligase: 1 μl.

After connecting at 16°C for 12 hours, 10 μl of the connection solution was transformed into Escherichia coli DH5α competent cells. The transformation process is as follows: add 10 μl of the connection solution to 100 μl of DH5α competent cells, and mix well. Ice bath for 30min, heat shock at 42°C for 90s, ice bath for 2min, add 900μl LB medium, 37°C, 100r/min, incubate for 1h, coat ampicillin-resistant plates, culture for 16h, pick transformants, extract plasmids for verification . Further sequencing was then performed to verify the correctness of the malE ^M gene. Thus the recombinant plasmid p-pglB-malE ^M was obtained.

Embodiment 4, construction and fermentation of recombinant Escherichia coli strain and purification of N-glycoprotein

(1) Construction of recombinant Escherichia coli strain

CLM24 electroporation competent cells were prepared according to the method of Example 1 (2). Take 10 μl of the recombinant plasmid p-rfb, add it to 100 μl of competent cells, mix well, transfer to a pre-cooled 2mm electroporation cup, and gently shake it to the bottom of the cup. Adjust the electroporator, 2.5Kv, electric shock; then add 900 μl of SOC medium to the electroporation cup, mix well, transfer to a sterile EP tube, 37°C, 120r/min, and incubate for 1 hour; apply kanamycin-resistant After culturing at 37°C for 24 hours, the recombinants were collected for verification. Thus the recombinant strain CLM24/p-rfb was obtained.

Take 50ul prepared recombinant bacteria CLM24/p-rfb chemical (CaCl ₂ ) competent cells, add 5ul plasmid p-pglB-malE ^M , mix well, ice bath for 20-30 minutes, heat shock in 42°C water bath for 90 seconds, then ice Bath for 3 to 5 minutes; then add 900ulLB medium, resume cultivation at 37°C, 120r/min for 45min to 1h; centrifuge at 4,000r/min for 2min, pour off part of the supernatant, and spread the remaining bacteria on an ampicillin-resistant plate , After culturing at 37°C for 24 hours, the recombinants were taken for verification. Thus the recombinant strain CLM24/p-rfb+p-pglB-malE ^M was obtained.

(2) Fermentation of recombinant Escherichia coli

Pick a single colony of the constructed recombinant strain CBEB (CLM24/p-rfb+p-pglB-malE ^M ) into a 25mL Erlenmeyer flask filled with 5mL of modified TB medium, the final concentration of ampicillin is 100μg/mL, spectacular The final concentration of mycin was 50 μg/mL, cultured at 37°C, 200 r/min, for 12 hours.

Put the overnight cultured bacterial solution into a 100mL Erlenmeyer flask containing 50mL of modified TB medium according to the inoculation amount of 1% (v/v), the final concentration of ampicillin was 100 μg/mL, and the final concentration of spectinomycin was 50 μg/mL. mL, 37°C, 200-225r/min.

When the bacterial liquid OD600=0.6, according to the inoculum amount of 1% (v/v), insert it into a 3L Erlenmeyer flask equipped with 1L of improved TB medium, the final concentration of ampicillin is 100 μg/mL, and the final concentration of spectinomycin is 50μg/mL, 37°C, 200-225r/min.

When the bacterial solution OD ₆₀₀ =0.6, add L-arabinose with a final concentration of 0.2% (v/v), at 28°C, 200-225r/min.

After 6 hours, add L-arabinose with a final concentration of 0.2% (v/v) once, at 28°C, 200-225r/min, and continue to cultivate for 14 hours.

(3) Purification of N-glycoprotein

1L of fermentation broth was centrifuged at 10,000r/min for 10min, the bacteria were collected and extracted with 30mL of periplasmic space protein extract (20mM Tris-HCl, 20% (wt/vol) sucrose, 1mM EDTA, 1mg/ml lysozyme, pH7.5) 4 ℃ to treat the bacteria for 1h. Centrifuge at 12,000r/min for 30min, then collect the supernatant and add to 5mL Affinity which has been pre-equilibrated with binding buffer (10mM imidazole, 0.5MNaCl, 20mMNa ₂ HPO ₄ /NaH ₂ PO ₄ buffer, pH7.4) nickel column. After washing with 10 column volumes of binding buffer and eluting with elution buffer (250 mM imidazole, 0.5 M NaCl, 20 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer, pH 7.4), the elution containing N-glycoprotein was collected. dehydration.

Put the eluate containing the target protein into an ultrafiltration tube with a molecular weight cut-off of 10kDa, centrifuge at 5,000g at 4°C for 30min, discard the liquid in the lower tube, fill the tube with desalting buffer, and centrifuge at 5,000g at 4°C for 30min. Repeat 3 times until the target protein is concentrated to 2 mL and the imidazole is fully removed. The concentrated protein was frozen and stored in a -70°C freezer.

Embodiment 5, identification of N-glycoprotein

The purified N-glycoprotein was sampled for SDS-PAGE analysis. The molecular weight of MBP is about 46KDa, which is consistent with the theoretical molecular weight. N-glycosylated MBP forms a ladder-like band, and the molecular weight distribution of the main band is between 50KDa and 55KDa. Due to the limited sensitivity of SDS-PAGE, some glycoprotein bands were not displayed. The results are shown in Figure 5.

N-glycoprotein was identified by Western Blot, using anti-His, anti-MBP, anti-O157 antibodies as primary antibodies, and appropriate goat anti-mouse or goat anti-rabbit as secondary antibodies for detection and analysis. Due to the addition of O-antigens with different chain lengths, N-glycosylated MBP showed a ladder-like band with a larger molecular weight than non-glycosylated MBP, which proved that MBP was glycosylated. The results are shown in Figure 6.

MALDI-TOF was used to identify the molecular weight of N-glycosylated MBP and MBP. The molecular weight of MBP is 45823Da, and the main molecular weight distribution of MBP is around 48790Da. The results are shown in Figure 7.

Embodiment 6, N-glycoprotein immune effect

(1) Immunized mice

Take 6-week-old Balb/C female mice and divide them into three groups. N-glycosylated MBP (30 μg, 150 μl each, n=8) and MBP (30 μg, 150 μl each, n=8) were injected subcutaneously at multiple points on the back. 8) and PBS (150 μl each, n=5), immunized once every two weeks, and immunized three times in total. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for the second and third immunizations. Before immunization and one week after each immunization, 200ml of blood was collected from the saphenous vein of the mouse leg, and after standing at room temperature for 1 hour, it was centrifuged at 4,000r/min for 20 minutes, and the serum was taken out and stored at -20°C.

(2) Serum antibody titer detection

Escherichia coli O157:H7 lipopolysaccharide was coated on a 96-well plate, and the anti-titer of Escherichia coli O157:H7 lipopolysaccharide in mouse serum was detected by ELISA, and the results are shown in Figure 8.

(3) Serum cytokine detection

The levels of IL-2 and IL-4 in serum of mice after immunization were measured by cytokine ELISA kit (Shenzhen Daktronics). IL-2 level: MP group>M group>PBS group; IL-4 level: MP group<M group<PBS group. , indicating that MBP-PS can activate Th1 cells and inhibit the activity of Th2 cells. The results are shown in Figure 9.

According to the determination of the above N-glycoprotein immune effect, it can be concluded that the N-glycoprotein vaccine produced by the recombinant Escherichia coli of the present invention has obvious anti-Escherichia coli O157:H7 effect. This mouse model completely simulates the environment in the human body, so theoretically it will also have a good effect in the treatment of human tumors. It is predicted that the vaccine of the present invention can be used for human immunotherapy against Escherichia coli O157:H7.

The invention provides a new option for immunotherapy against bacterial infection and has good application prospects.

Claims (1)

1. a strain genotype is the preparation method of the recombinant escherichia coli of W3110Δwaal/p-pglB-malE ^M +p-rfb, it is characterized in that: construct the coexpression carrier p-pglB-malE ^M that contains malE ^M and pglB gene, then The expression vector p-rfb containing the rfb gene cluster was constructed, and the recombinant expression vector p-pglB-malE ^M and p-rfb constructed above were co-transformed into Escherichia coli CLM24 to obtain the pglB gene, malE ^M gene and rfb gene cluster simultaneously. Recombinant Escherichia coli W3110Δwaal/p-pglB-malE ^M +p-rfb; Wherein, the malE ^M is derived from the plasmid pMAL-p5X, the pglB gene is derived from Campylobacter jejuni NCTC11168, and the vector co-expressing the malE ^M and pglB genes is pBAD24; the rfb gene cluster is derived from Escherichia coli O157:H7, expressed The carrier of the rfb gene cluster is pYES1L; The above malE ^M is a mutant of the malE gene, which is obtained by inserting a nucleotide sequence such as a gene expressing a glycosylation sequence shown in SEQ ID No.1 at the 3' end of the malE gene, wherein the primer malE ^M used for amplification of malE ^M The nucleotide sequence of -F is shown in SEQIDNo.14, and the nucleotide sequence of primer ^malEM -R is shown in SEQIDNo.15; The above rfb gene cluster is based on the O157:H7 genome as a template, respectively O157-1-F and O157-1-R, O157-2-F and O157-2-R, O157-3-F and O157-3-R As a primer, the rfb gene cluster is cloned in three sections of PCR, and then connected to obtain; the nucleotide sequence of the primer O157-1-F is shown in SEQIDNo.6, and the nucleotide sequence of the primer O157-1-R is shown in SEQIDNo. Shown in .7, the nucleotide sequence of primer O157-2-F is shown in SEQIDNo.8, the nucleotide sequence of primer O157-2-R is shown in SEQIDNo.9, the nucleotide sequence of primer O157-3-F The acid sequence is shown in SEQIDNo.10, and the nucleotide sequence of primer O157-3-R is shown in SEQIDNo.11; The above-mentioned E. coli CLM24 is an engineering E. coli strain constructed by knocking out the waal gene of the starting strain E. coli W3110, and its genotype is W3110Δwaal.