CN106801029B - Recombinant escherichia coli and application thereof in preparation of sugar vaccine for resisting shigella - Google Patents
Recombinant escherichia coli and application thereof in preparation of sugar vaccine for resisting shigella Download PDFInfo
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Abstract
The invention discloses a recombinant escherichia coli capable of expressing a lipopolysaccharide synthase gene cluster of Shigella dysenteriae type 1 pathogenic bacteria, which contains rfbR (rhamnosylransferase), rfbQ (rhamnosylransferase) and orf9 (galactosylransferase) in an rfb gene cluster of Shigella dysenteriae type 1 pathogenic bacteria, and the genotype of the recombinant escherichia coli is W3110 delta wbbl/p-rfp + p-O-Ag. The invention also discloses application of the strain in preparation of glycoprotein vaccines for resisting Shigella dysenteriae type 1 pathogenic bacteria. The recombinant escherichia coli can produce lipopolysaccharide structures of Shigella dysenteriae type 1 pathogenic bacteria, and by taking the bacterium as a platform, glycoprotein vaccines for resisting Shigella dysenteriae type 1 pathogenic bacteria can be further produced with low cost and high efficiency, so that the recombinant escherichia coli has good industrial development and application prospects.
Description
Technical Field
The invention relates to a recombinant escherichia coli, a construction method and application thereof, in particular to a recombinant escherichia coli capable of expressing a lipopolysaccharide synthase gene cluster of Shigella dysenteriae type 1 pathogenic bacteria and application thereof in preparation of glycoprotein vaccines for resisting Shigella dysenteriae type 1 pathogenic bacteria. Belongs to the fields of biotechnology, genetic engineering and microbial fermentation.
Background
Bacillary dysentery is an intestinal infectious disease caused by Shigella (Shigella dysenteriae), characterized by severe colonic inflammation accompanied by systemic toxemia, which, in severe cases, can cause septic shock and/or toxic encephalopathy. There are approximately 1.65 billion cases of bacillary dysentery worldwide each year, causing over 110 million deaths, and about 70% of the onset symptoms are manifested in children under the age of 5. Bacillary dysentery is a common disease and frequently encountered disease in China, the morbidity rate is in the fourth place of the legal A and B infectious diseases, and the bacillary dysentery is popular in different scales in at least 10 provinces and regions and needs to be paid sufficient attention.
Shigella is classified into 4 serotypes according to antigenic structure and biochemical reactions, with Shigella dysenteriae type 1 pathogen (Shigella dysenteriae serotype 1, s.dysenteriae 1) being the leading causative agent of severe epidemics of bacillary dysentery, leading to serious complications (e.g., hemorrhagic colitis, sepsis, hemolytic uremic syndrome, and purpura) and high mortality. A vaccine effective against Shigella dysenteriae type 1 pathogenic bacteria is one of the vaccines proposed by the world health organization to be developed first.
By searching, depending on the synthesis pathway of Wzy, a partial gene cluster containing the s.dysenteriae 1rfbr (rhamsytransferase), rfbq (rhamsytransferase), orf9 (galactosylransferase) gene and the rfp (galactosylransferase) gene were introduced into e.coli K-12W3110, and no document or patent was reported for achieving heterologous expression of s.dysenteriae 1 LPS.
Disclosure of Invention
Aiming at the defects of the existing method, the invention aims to provide a recombinant escherichia coli which can express exopolysaccharide of pathogenic bacteria of Shigella dysenteriae type 1 (S.dysenteriae type 1) and can be used as a platform for preparing glycoprotein vaccines for resisting the pathogenic bacteria of Shigella dysenteriae type 1.
The technical scheme of the invention is based on the characteristic that the structure and the synthesis route of Shigella dysenteriae type 1 pathogenic bacteria and Lipopolysaccharide (LPS) of Escherichia coli are similar, three genes of rfbR (rhamsstransferase), rfbQ (rhamsstransferase) and orf9 (galactosylstransferase) in rfb gene clusters on the genome of the Shigella dysenteriae type 1 pathogenic bacteria are expressed in the Escherichia coli, the rfp (galactosylstransferase) gene on 9kb multicopy free plasmid of the Shigella dysenteriae type 1 pathogenic bacteria is obtained, nucleotide oligosaccharide of the Escherichia coli is used as a donor to obtain the lipopolysaccharide structure of the Shigella dysenteriae type 1 pathogenic bacteria, and a platform is provided for further producing the sugar vaccine for resisting the Shigella dysenteriae type 1 pathogenic bacteria.
The recombinant escherichia coli of the invention is a recombinant escherichia coli capable of expressing lipopolysaccharide synthetase gene clusters of shigella dysenteriae type 1 pathogenic bacteria, and is characterized in that: the recombinant Escherichia coli contains rfbR (rhamsylyltransferase), rfbQ (rhamsylyltransferase) and orf9 (galactosylyltransferase) in the rfb gene cluster of pathogenic bacteria of Shigella dysenteriae type 1, and the genotype of the recombinant Escherichia coli is W3110 delta wbbl/p-rfp + p-O-Ag.
The invention relates to a construction method of recombinant escherichia coli capable of expressing lipopolysaccharide synthetase gene clusters of shigella dysenteriae type 1 pathogenic bacteria, which comprises the following steps:
constructing an expression vector p-O-Ag containing three genes of rfbR (rhamsylyltransferase), rfbQ (rhamsylyltransferase) and orf9 (galactosylyltransferase) in the rfb gene cluster of the Shigella dysenteriae 1 pathogenic bacteria, then constructing an expression vector p-rfp of the rfp gene, and then co-transforming the constructed recombinant plasmid p-O-Ag and the p-rfp into escherichia coli K12W3110 to obtain the recombinant escherichia coli capable of expressing the lipopolysaccharide synthase gene cluster of the Shigella dysenteriae 1 pathogenic bacteria;
wherein the rfb gene cluster O-Ag containing three genes of rfbR (rhamsylyltransferase), rfbQ (rhamsylyltransferase) and orf9 (galactosylyltransferase) is derived from the genome of Shigella dysenteriae type 1 pathogenic bacteria, and the vector for expressing the rfb gene cluster is pACT 3; the rfp gene exists in 9Kb free plasmid in Shigella dysenteriae type 1 pathogenic bacteria, and a vector for expressing the rfp gene is pET15 b; the E.coli K-12W3110, whose genotype was W3110. delta. WbbL, disrupted in WbbL function by insertion of the insert IS 5.
Specifically, the construction method of the recombinant escherichia coli capable of expressing the lipopolysaccharide synthase gene cluster of the shigella dysenteriae type 1 pathogenic bacteria comprises the following steps:
construction of O-Ag Gene Cluster expression vector
The basic method is to clone an O-Ag gene cluster by taking Shigella dysenteriae type 1 pathogenic bacterium genome as a template, and insert the obtained O-Ag partial gene cluster into a linearized pACT3 plasmid so as to obtain an expression vector p-O-Ag of the O-Ag gene cluster.
Construction of rfp Gene expression vector
The basic method is to clone rfp gene by taking Shigella 1 pathogenic bacterium genome as a template, and insert the cloned rfp into a plasmid pET15b, thereby obtaining an expression vector p-rfp of the rfp.
3. Construction of recombinant E.coli strains
The basic method is to co-transform the constructed recombinant plasmid p-O-Ag and p-rfp into Escherichia coli K-12W3110, thereby obtaining recombinant Escherichia coli expressing O-Ag gene cluster and over-expressing rfp.
The recombinant escherichia coli capable of expressing the lipopolysaccharide synthase gene cluster of the Shigella dysenteriae type 1 pathogenic bacteria is applied to preparation of glycoprotein vaccines for resisting the Shigella dysenteriae type 1 pathogenic bacteria.
Coli expression platform of pathogenic bacteria LPS can be used for producing sugar vaccine or more effective and safe glycoprotein vaccine. Coli expression platform bacterial polysaccharides such as lipopolysaccharides or capsular polysaccharides are linked to immunogenic carrier proteins to provide a long lasting immune response by eliciting a T cell dependent immune response. The glycoprotein vaccine is considered to be one of the most effective and most safe anti-pathogenic bacteria vaccines, and has wide application prospect.
The recombinant Escherichia coli disclosed by the invention is based on that the synthesis of E.coli K-12W3110 and S.dysenteriae 1LPS belongs to a synthesis path depending on Wzy, so that the recombinant Escherichia coli successfully carries out heterologous expression on the S.dysenteriae 1LPS by introducing a partial gene cluster containing S.dysenteriae 1rfBR (rhamsystem transferase), rfbQ (rhamsystem transferase), orf9 (galactosysytransferase) genes and rfp (galactosyltransferase) genes into the E.coli K-12W 3110. The method is also suitable for the heterologous synthesis of LPS of other pathogenic bacteria, and has very important application value.
Drawings
Figure 1 is a schematic representation of the structural engineering of s.dysenteriae 1 LPS.
FIG. 2 is a map of p-O-Ag expression vector.
FIG. 3. map of p-rfp expression vector.
FIG. 4 analysis of lipopolysaccharide of recombinant E.coli by silver staining and Western blotting.
Wherein: m: low molecular weight protein standards
1：S.dysenteriae 1LPS
2: escherichia coli K12W3110LPS
3: recombinant Escherichia coli (W3110. delta. wbbl/p-rfp + p-O-Ag) LPS.
FIG. 5 ion chromatography analysis of recombinant E.coli O-antigen composition.
Wherein: 1: rha; 2: gal; 3: glc; 4: GlcNAc.
Detailed Description
General description: restriction enzymes, DNA polymerase, nucleic acid molecular weight standard 1kbMarker, protein molecular weight standard (12-120kDa) as referred to in the following examples were purchased from Thermo corporation; t4ligase was purchased from Takara; the plasmid extraction kit and the agarose gel DNA fragment recovery kit were purchased from Omega, and the procedures were performed completely according to the corresponding instructions. Gene sequencing in plasmid construction was accomplished by Huada Gene. Plasmids pACT3 and pET15b were both from Novagen; the Escherichia coli K-12W3110 is derived fromInvitrogen corporation; top10 competent cells were purchased from Dingguo Biotechnology Ltd; LPS (Lipopolysaccharide) Extraction kit was purchased from iNtRON BIOTECHNOLOGY; shigella dysenteriae O-antigen diagnostic serum was purchased from Tianjin biochip technology, Inc.; HRP-labeled goat anti-rabbit IgG was purchased from KPL corporation; shigella dysenteriae type 1 pathogenic bacteria (s.dysenteriae 1) were purchased from the centers for disease prevention and control in shandong province. CaCl2Purchased from Sigma, and other reagents and consumables from various domestic reagent companies. Other experimental methods and reagents in the examples are conventional in the art and commercially available, unless otherwise specified.
The LB culture medium is: 10g/L of peptone, 5g/L of yeast powder and 10g/L of NaCl.
The SOC culture medium is as follows: peptone 2g/L, yeast powder 0.5g/L, NaCl 0.0585g/L, KCl 0.0186g/L, MgCl20.203g/L，MgSO40.246g/L and glucose 20 mmol/L.
Example 1 construction of O-Ag Gene Cluster expression vector
Designing a primer according to a genome sequence of Shigella dysenteriae type 1 pathogenic bacteria published by NCBI:
S.d-F-SmalI：5’-TCCCCCGGGATGAATAAATATTGTATCTTAGTA-3’
S.d-R-XbaI：5’-GCTCTAGATCACATTAATGCTACCAAAAAGAGT-3’
a part of O-Ag gene cluster is cloned by PCR by taking Shigella dysenteriae type 1 pathogenic bacterium genome as a template, and the gene cluster contains three genes of rfbR, rfbQ and orf 9. The PCR reaction system is as follows: (primer concentration 20. mu. mol/L)
And (3) PCR reaction conditions: pre-denaturation at 95 deg.C for 3min, denaturation at 95 deg.C for 30s, annealing at 50 deg.C for 30s, extension at 72 deg.C for 2.5min, extension at 72 deg.C for 10min after 30 cycles, and storage at 16 deg.C.
The cloned O-Ag gene cluster fragment was digested with restriction endonucleases SmaI and XbaI, respectively, and the plasmid vector pACT3 was also digested with restriction endonucleases SmaI and XbaI, respectively. The digested O-Ag fragment and pACT3 plasmid vector were recovered using an agarose gel kit and then ligated using T4 ligase.
The linker was 10 μ l:
O-Ag fragment: 6 mu l of the solution;
pACT3 vector: 2 mu l of the solution;
10×Buffer：1μl；
t4 ligase: 1 μ l.
After 12h of ligation at 16 ℃ 10. mu.l of the ligation solution were transformed into competent cells of E.coli Top 10. The transformation process is as follows: add 10. mu.l of ligation solution to 100. mu.l of Top10 competent cells and mix well. Ice-bath for 30min, heat shock for 90s at 42 ℃, ice-bath for 2min, adding 900 mul SOC culture medium, incubating for 1h at 37 ℃ and 100r/min, coating a chloramphenicol resistant plate, culturing for 16h, selecting a transformant, and extracting a plasmid for verification. Then further sequencing verifies the correctness of the O-Ag gene cluster, thereby obtaining the recombinant plasmid p-O-Ag. See fig. 2.
Example 2 construction of rfp Gene expression vector
Designing a primer according to a genome sequence of Shigella dysenteriae type 1 pathogenic bacteria published by NCBI:
15b-F-NdeI：5’-GGAATTCCATATGATGAAGATCTCAATAATAGGGAA-3’
15b-R-BamHI：5’-CGGGATCCTTAATCAGGAATCCCTAGTA-3’
the rfp gene is cloned by PCR by taking Shigella dysenteriae type 1 pathogenic bacterium genome as a template. The PCR reaction system is as follows: (primer concentration 20. mu. mol/L)
And (3) PCR reaction conditions: pre-denaturation at 95 deg.C for 3min, denaturation at 95 deg.C for 30s, annealing at 50 deg.C for 30s, extension at 72 deg.C for 2.5min, extension at 72 deg.C for 10min after 30 cycles, and storage at 16 deg.C.
The cloned rfp gene fragment was digested with endonuclease NdeI and BamHI, respectively, and plasmid vector pET15b was also digested with endonuclease NdeI and BamHI, respectively. The digested rfp fragment and the pET15b plasmid vector were recovered using an agarose gel kit and then ligated using T4 ligase.
The linker was 10 μ l:
rfp gene fragment: 6 mu l of the solution;
pET15b vector: 2 mu l of the solution;
10×Buffer：1μl；
t4 ligase: 1 μ l.
After 12h of ligation at 16 ℃ 10. mu.l of the ligation solution were transformed into competent cells of E.coli Top 10. The transformation process is as follows: add 10. mu.l of ligation solution to 100. mu.l of Top10 competent cells and mix well. Ice-bath for 30min, heat shock for 90s at 42 ℃, ice-bath for 2min, adding 900 mul SOC culture medium, incubating for 1h at 37 ℃ and 100r/min, coating ampicillin resistant plates, culturing for 16h, selecting transformants, and extracting plasmids for verification. Then, the rfb gene is further sequenced to verify the correctness, so that the over-recombinant plasmid p-rfp is obtained. See fig. 3.
Example 3 construction of recombinant E.coli strains
(1) Preparation of competence E.coli K-12W3110
(I) Coli K-12W3110 was cultured overnight at 37 ℃ at 200r/min in a 5mL test tube;
(II) inoculating the strain into 50mL LB medium at 1%, culturing at 37 deg.C and 200r/min for 2-3h until OD600 is 0.3-0.4(0.36 is the best, generally not more than 0.4);
(III) transferring the bacterial liquid into a 50mL centrifuge tube (the centrifuge tube is sterile and is cooled at 4 ℃), carrying out ice bath for 20min to stop the growth of cells, and centrifuging for 10min at 4000r/min at 4 ℃;
(IV) precipitation with a suitable amount of precooled CaCl2Resuspending, centrifuging at 4000r/min at 4 deg.C for 5-10min, and discarding the supernatant;
(V) precipitating with a suitable amount of precooled CaCl2Resuspending, carrying out ice bath for 1h, centrifuging at 4 ℃ at 4000r/min for 10min, and discarding the supernatant;
(VI) use of 2mL of precooled CaCl2Resuspending and packaging to be competent.
(2) Construction of recombinant E.coli strains
The plasmids p-O-Ag and p-rfp constructed above were co-transformed into E.coli K-12W3110 competent cells, respectively, and screened using ampicillin (final concentration of 100. mu.g/mL) and chloramphenicol (final concentration of 50. mu.g/mL) to obtain a recombinant strain K-12W3110/p-O-Ag + p-rfp.
Example 4 extraction and analysis of recombinant Escherichia coli Lipopolysaccharide (LPS)
(1) Fermentation of recombinant E.coli
The constructed recombinant strain (K-12W3110/p-O-Ag + p-rfp) was singly cloned into a 25mL tube containing 5mL of LB medium, ampicillin was 100. mu.g/mL at a final concentration, chloramphenicol was 50. mu.g/mL at 37 ℃ at 200r/min, and the tube was cultured for 12 hours.
The overnight-cultured bacterial suspension was inoculated into a 100mL Erlenmeyer flask containing 50mL of LB medium at an inoculum size of 1% (v/v) to give a final concentration of 100. mu.g/mL ampicillin and 50. mu.g/mL chloramphenicol at 37 ℃ at 200 r/min.
When the OD600 of the bacterial liquid is equal to 0.6, 0.2mM IPTG is added, and the bacterial liquid is cultured at 16 ℃ and 200r/min for 20h for induction expression.
Simultaneously inducing starting strains of Escherichia coli K12W3110 as negative controls; pathogen S.dysenteriae 1 was cultured overnight at 37 ℃ at 200r/min as a positive control.
(2) Extraction of Lipopolysaccharide (LPS)
Centrifuging overnight-cultured bacteria liquid at normal temperature of 13000r/min for 30s, discarding all supernatant, and extracting recombinant Escherichia coli LPS by using LPS extraction kit.
(I) Adding 1mL Lysis Buffer into the thallus and fully whirling until cell mass disappears;
(II) adding 200. mu.L chloroform, vortexing thoroughly for 10-20s, and incubating at room temperature for 5 min;
(III) centrifugation at 13,000r/min at 4 ℃ for 10min, transferring 400. mu.L of the supernatant to a new EP tube, taking care not to suck the pellet;
(IV) adding 800. mu.L of Purification Buffer, mixing well, incubating at-20 ℃ for 10 min;
(V) centrifuging at 4 ℃ for 15min at 13,000r/min, and discarding the supernatant;
(VI) washing the LPS precipitate with 1mL of 70% (w/v) ethanol, centrifuging at 4 ℃ for 3min at 13,000r/min, discarding the supernatant, and air-drying at room temperature;
(VII) adding 50. mu.L of 10mM Tris-HCl buffer (pH 8.0) to the LPS precipitate to dissolve LPS, and boiling for 2 min;
(VIII) 2.5. mu.L of proteinase K solution (30mg/mL) was added and the mixture was treated at 50 ℃ for 30 min.
(3) Analysis of Lipopolysaccharide (LPS)
The purified lipopolysaccharide samples were extracted and analyzed by SDS-PAGE and identified by silver staining and Western Blotting. Western Blotting detection adopts Shigella dysenteriae O antigen diagnostic serum as a primary antibody, and HRP-labeled goat anti-rabbit IgG as a secondary antibody for detection and analysis. Lipopolysaccharide shows ladder-shaped strips due to the addition of O antigen with different chain lengths, and only LPS extracted from S.dysenteriae 1 and recombinant Escherichia coli can be specifically recognized by Shigella dysenteriae O antigen diagnostic serum. Coli K-12W3110 was deficient in O16 due to insertion of the insert IS5 that disrupted WbbL function. This resulted in the presence of only a single GlcNAc residue at the end of the LPS of E.coli K-12W3110 which was not recognized by Shigella dysenteriae O antigen diagnostic sera. The results are shown in FIG. 4.
Example 5 extraction and analysis of recombinant E.coli O-antigen (O-PS)
(1) Extraction of recombinant Escherichia coli O-antigen (O-PS)
(I)20g of the cell pellet (5g/L) was extracted with 200mL of 50% (w/v) phenol at 65 ℃ for 15 min;
(II) centrifugation at 10,000g for 30min at 4 ℃;
(III) collecting the supernatant, dialyzing with triple distilled water to remove phenol, and freeze-drying the dialysate;
(IV) dissolving the lyophilized powder in 20mL of 0.02M sodium acetate (pH7.0), and treating with DNase, RNase and proteinase K at 37 deg.C for 2 h;
(V) removing the precipitate (centrifugation at 10,000g for 30min at 4 ℃), and ultracentrifugation of the solution at 110,000g at 4 ℃ for 12 h;
(VI) dissolving the jelly obtained by centrifugation with triple distilled water, and freeze-drying to prepare LPS;
(VII) purifying LPS and treating with 1% acetic acid at 100 deg.C for 1.5 h;
(VIII) centrifugation at 12,000g for 30min to remove the precipitate;
(IX) O-PS was prepared by purifying the supernatant with Bio-Gel P-2 column.
(2) Monosaccharide composition analysis of recombinant Escherichia coli O-antigen (O-PS)
The extracted O-PS was treated with 3M TFA at 110 ℃ for 6h, and then the monosaccharide component of O-PS was qualitatively detected using ion chromatography.
The detection method comprises the following steps: and (3) eluting the standard substance, the sample, the mixture of the sample and the standard substance under the same condition, and judging whether the sample contains the standard substance according to the changes of retention time, peak value and peak area.
A detector: dionex ED-50, USA; separating the column: dionex carbon PacTMPA1BioLCTM(2X250 mm); protection of the column: carbo PacTMPA-100G(2X50mm)。
Mobile phase: 10mM NaOH, 200mM NaAc; flow rate: 0.3 mL/min. The results are shown in FIG. 5.
According to the determination of the LPS and O-PS extracted from the recombinant escherichia coli, the recombinant escherichia coli can produce the lipopolysaccharide structure of Shigella dysenteriae type 1 pathogenic bacteria, and can further produce glycoprotein vaccines for resisting Shigella dysenteriae type 1 pathogenic bacteria by taking the lipopolysaccharide structure as a platform.
Claims (3)
1. A recombinant Escherichia coli capable of expressing lipopolysaccharide synthase gene cluster of Shigella dysenteriae type 1 pathogenic bacteria, characterized in that: the recombinant Escherichia coli contains rfbR (rhamsylyltransferase), rfbQ (rhamsylyltransferase) and orf9 (galactosylyltransferase) in an rfb gene cluster of pathogenic bacteria of Shigella dysenteriae type 1, and the genotype of the recombinant Escherichia coli is W3110 delta wbbl/p-rfp + p-rfbR-rfbQ-orf 9.
2. The method for constructing recombinant Escherichia coli capable of expressing lipopolysaccharide synthase gene cluster of Shigella dysenteriae type 1 pathogenic bacteria according to claim 1, comprising the steps of:
constructing an expression vector p-O-Ag containing three genes of rfbR (rhamsylyltransferase), rfbQ (rhamsylyltransferase) and orf9 (galactosylyltransferase) in the rfb gene cluster of the Shigella dysenteriae 1 pathogenic bacteria, then constructing an expression vector p-rfp of the rfp gene, and then co-transforming the constructed recombinant plasmid p-O-Ag and the p-rfp into escherichia coli K12W3110 to obtain the recombinant escherichia coli capable of expressing the lipopolysaccharide synthase gene cluster of the Shigella dysenteriae 1 pathogenic bacteria;
wherein the rfb gene cluster O-Ag containing three genes of rfbR (rhamsylyltransferase), rfbQ (rhamsylyltransferase) and orf9 (galactosylyltransferase) is derived from the genome of Shigella dysenteriae type 1 pathogenic bacteria, and the vector for expressing the rfb gene cluster is pACT 3; the rfp gene exists in 9Kb free plasmid in Shigella dysenteriae type 1 pathogenic bacteria, and a vector for expressing the rfp gene is pET15 b; the E.coli K-12W3110, whose genotype was W3110. delta. WbbL, disrupted in WbbL function by insertion of the insert IS 5.
3. Use of the recombinant escherichia coli expressing the lipopolysaccharide synthase gene cluster of shigella dysenteriae type 1 pathogenic bacteria of claim 1 for preparing a glycoprotein vaccine against shigella dysenteriae type 1 pathogenic bacteria.
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