CN106801029A - One plant of recombination bacillus coli and its application in the saccharide vaccines for preparing anti-Shigella - Google Patents
One plant of recombination bacillus coli and its application in the saccharide vaccines for preparing anti-Shigella Download PDFInfo
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Abstract
The invention discloses a kind of recombination bacillus coli of the lipopolysaccharides synthase gene cluster that can express the type pathogen of shigella dysenteriae 1, the bacterium contains the rfbR (rhamnosyltransferase) in the type pathogen rfb gene clusters of shigella dysenteriae 1, rfbQ (rhamnosyltransferase) and orf9 (galactosyltransferase), its genotype is W3110 Δ wbbl/p rfp+p O Ag.The invention also discloses application of the bacterium in the glucoprotein vaccine for preparing the type pathogen of anti-shigella dysenteriae 1.This recombination bacillus coli can produce the lipopolysaccharides structure of the type pathogen of shigella dysenteriae 1, with the bacterium as platform, can the further anti-type pathogen of Shigella 1 of cost-efficiently production glucoprotein vaccine, with good commercial development and application prospect.
Description
Technical field
Dysentery will he can be expressed the present invention relates to one plant of recombination bacillus coli and construction method and application, more particularly to one kind
The recombination bacillus coli of the lipopolysaccharides synthase gene cluster of the type pathogen of Salmonella 1 and its preparing the type of anti-shigella dysenteriae 1 disease
Application in the glucoprotein vaccine of opportunistic pathogen.Belong to biotechnology, genetic engineering and field of microbial fermentation.
Background technology
Bacillary dysentery is the enteric infectious disease caused by Shigella (shigella dysenteriae), with serious colon inflammation as special
Levy, while with whole body toxemia synptom, severe patient can trigger infectious shock and (or) toxic encephalopathy.The whole world is big every year
1.65 hundred million bacillary dysentery cases are there are about, causes dead more than 1,100,000 people, and 70% or so breaking-out Symptoms are being less than
With the children of 5 years old.Bacillary dysentery is the common disease of China, frequently-occurring disease, and the incidence of disease is located in legal first, Category B notifiable disease
4th, at least there is different scales prevalence in 10 provinces, area, enough attention should be caused.
Different with biochemical reaction by antigenic structure, Shigella is divided into 4 serotypes, the wherein type of shigella dysenteriae 1
Pathogen (Shigella dysenteriae serotype 1, S.dysenteriae 1) is popular serious bacillary dysentery
The topmost pathogen of disease outburst, can cause serious complication (such as hemorrhagic colitis, septicemia, haemolytic uraemic
Syndrome and purpura) and high mortality.The vaccine effective to the type pathogen of shigella dysenteriae 1 is that World Health Organization's suggestion is excellent
One of vaccine for first developing.
The lipopolysaccharides of S.dysenteriae 1 (Lipopolysaccharide, LPS) is that a class can cause host immune anti-
The surface antigen answered, can be used to prepare the vaccine of bacteria resistance dysentery.The biosynthesis gene position of S.dysenteriae 1LPS
In two gene locis of onrelevant, a gene rfp site is present on the multicopy plasmid of 9kb, another gene position
Point is present in the rfb gene clusters on chromosome.Research imports 8 genes and rfp genes of whole rfb gene clusters nontoxic jointly
Mouse enteron aisle detection of Salmonella enteron aisle subspecies antityphoid sera type bacterial strain (the Salmonella enterica subspecies of property
Enterica serotype Typhi, S.Typhi) in, the LPS of typical S.dysenteriae 1 can be produced, and it is right
Host has certain protection to be immunized.Have the disadvantage that gene cluster is excessive and be not easy conversion and Late reformation.
Through retrieval, the route of synthesis of Wzy is depended on, S.dysenteriae 1rfbR will be contained
(rhamnosyltransferase), rfbQ (rhamnosyltransferase), orf9 (galactosyltransferase)
Portion gene cluster and rfp (galactosyltransferase) channel genes E.coli K-12W3110 of gene, it is right to realize
The document or patent that S.dysenteriae 1LPS carry out heterogenous expression yet there are no report.
The content of the invention
For the deficiency of existing method, the problem to be solved in the present invention is to provide one plant of recombination bacillus coli, and it can be with table
It is that platform can prepare anti-dysentery will with it up to the exocellular polysaccharide of shigella dysenteriae 1 type (S.dysenteriae 1) pathogen
The glucoprotein vaccine of the type pathogen of Hayes bacterium 1.
The technical scheme is that the lipopolysaccharides (LPS) based on the type pathogen of shigella dysenteriae 1 and Escherichia coli
The characteristics of structure is similar with route of synthesis, the type pathogenic bacteria gene of shigella dysenteriae 1 is derived from using in expression in escherichia coli
RfbR (rhamnosyltransferase) in rfb gene clusters in group, rfbQ (rhamnosyltransferase) and
Three genes of orf9 (galactosyltransferase), from the multicopy of the 9kb of the type pathogen of shigella dysenteriae 1
Rfp (galactosyltransferase) gene on free plasmid, the nucleotides oligosaccharides by the use of Escherichia coli is obtained as donor
The lipopolysaccharides structure of the type pathogen of shigella dysenteriae 1 is obtained, is the sugared epidemic disease for further producing the type pathogen of anti-shigella dysenteriae 1
Seedling provides platform.
Recombination bacillus coli of the present invention is a kind of lipopolysaccharides synthesis that can express the type pathogen of shigella dysenteriae 1
The recombination bacillus coli of enzyme gene cluster, it is characterised in that：The recombination bacillus coli contains the type pathogen of shigella dysenteriae 1
RfbR (rhamnosyltransferase) in rfb gene clusters, rfbQ (rhamnosyltransferase) and orf9
(galactosyltransferase), its genotype is W3110 Δs wbbl/p-rfp+p-O-Ag.
The restructuring large intestine bar of the lipopolysaccharides synthase gene cluster that can express the type pathogen of shigella dysenteriae 1 of the present invention
The construction method of bacterium, step is：
Build containing the rfbR (rhamnosyltransferase) in the type pathogen rfb gene clusters of shigella dysenteriae 1,
The expression vector p-O- of three genes of rfbQ (rhamnosyltransferase) and orf9 (galactosyltransferase)
Ag, then the expression vector p-rfp of rfp genes is built, it is then that recombinant plasmid p-O-Ag and the p-rfp cotransformation of the structure is big
Enterobacteria K12W3110, that is, the restructuring of the lipopolysaccharides synthase gene cluster for obtaining that the type pathogen of shigella dysenteriae 1 can be expressed is big
Enterobacteria；
Wherein, it is described to include rfbR (rhamnosyltransferase), rfbQ (rhamnosyltransferase)
Rfb gene clusters O-Ag with three genes of orf9 (galactosyltransferase) derives from the type cause of disease of shigella dysenteriae 1
The genome of bacterium, the carrier of its expression rfb gene cluster is pACT3；The rfp genes are present in the type cause of disease of shigella dysenteriae 1
9Kb free plasmids in bacterium, the carrier of its expression rfp gene is pET15b；The e. coli k-12 W3110, due to insertion
So as to interrupt the function of WbbL, its genotype is W3110 Δs wbbl for the insertion of sub- IS5.
Specifically, the restructuring large intestine of the above-mentioned lipopolysaccharides synthase gene cluster that can express the type pathogen of shigella dysenteriae 1
The construction method of bacillus is：
The structure of 1.O-Ag gene cluster expression vectors
Basic skills is, with the type pathogenic bacteria gene group of shigella dysenteriae 1 as template, to clone O-Ag gene clusters, will be obtained
O-Ag portion gene clusters be inserted into linearisation pACT3 plasmids in, so as to obtain the expression vector p-O-Ag of O-Ag gene clusters.
The structure of 2.rfp expression vectors
Basic skills is, with the type pathogenic bacteria gene group of Shigella 1 as template, to clone rfp genes, will clone what is obtained
In rfp insertion plasmids pET15b, so as to obtain the expression vector p-rfp of rfp.
3. the structure of recombinant escherichia coli strain
Basic skills be by constructed recombinant plasmid p-O-Ag and p-rfp cotransformation e. coli k-12 W3110 so that
Obtain expression O-Ag gene clusters, the recombination bacillus coli of overexpression rfp.
The restructuring large intestine of the lipopolysaccharides synthase gene cluster that can express the type pathogen of shigella dysenteriae 1 of the present invention
Application of the bacillus in the glucoprotein vaccine for preparing the type pathogen of anti-shigella dysenteriae 1.
The E.coli expression platforms of the pathogen LPS of structure, can be used for producing saccharide vaccines or more effective and safety sugar
Protein vaccine.The glucoprotein vaccine of bioanalysis synthesis is in E.coli expression platforms that bacterial polysaccharides such as lipopolysaccharides or pod membrane is more
Sugar is connected on the carrier protein with immunogenicity, and the immune response of T cell is depended on by triggering so as to provide for a long time
Lasting immune response.Glucoprotein vaccine is considered as one of most effective and safest anti-microbial pathogen vaccine, with wide
Application prospect.
Recombination bacillus coli disclosed by the invention is based on E.coli K-12W3110 and S.dysenteriae 1LPS
Synthesis belongs to the route of synthesis for depending on Wzy, therefore the present invention will contain S.dysenteriae 1rfbR
(rhamnosyltransferase), rfbQ (rhamnosyltransferase), orf9 (galactosyltransferase)
Portion gene cluster and rfp (galactosyltransferase) the channel genes E.coli K-12W3110 of gene are successfully right
S.dysenteriae 1LPS have carried out heterogenous expression.This method is also applied for the LPS of heterologous other pathogens of synthesis, has
Very important application value.
Brief description of the drawings
The schematic diagram of Fig. 1 .S.dysenteriae 1LPS structure of modification.
Fig. 2 .p-O-Ag expression vector collection of illustrative plates.
Fig. 3 .p-rfp expression vector collection of illustrative plates.
Fig. 4 silver stainings and Western Bloting analyze the lipopolysaccharides of recombination bacillus coli.
Wherein：M：LMWP standard
1：S.dysenteriae 1LPS
2：E. coli k12 W3110LPS
3：Recombination bacillus coli (W3110 Δ wbbl/p-rfp+p-O-Ag) LPS.
The O- antigens composition of Fig. 5 ion chromatography recombination bacillus colis.
Wherein：1：Rha；2：Gal；3：Glc；4：GlcNAc.
Specific embodiment
General explanation：Restriction enzyme, archaeal dna polymerase, nucleic acid molecular weight standard 1kb involved by following examples
Marker, Protein Marker (12-120kDa) are purchased from Thermo companies；T4ligase is purchased from Takara companies；Plasmid is carried
Take kit and Ago-Gel reclaims DNA fragmentation kit purchased from Omega companies, operation is entered fully according to corresponding instructions
OK.Gene sequencing is completed by Huada gene company in plasmid construction.Plasmid pACT3 and pET15b derive from Novagen companies；
The bacterium e. coli k-12 W3110 that sets out derives from Invitrogen companies；Top10 competent cells have purchased from ancient cooking vessel state biotechnology
Limit company；LPS (Lipopolysaccharide) Extraction kits are purchased from iNtRON BIOTECHNOLOGY companies；Dysentery
Disease Shigella O- diagnostic antigens serum is purchased from Tianjin Biochip Technology Co., Ltd；HRP mark goat anti-rabbit iggs are purchased from
KPL companies；The type pathogen (S.dysenteriae 1) of shigella dysenteriae 1 is purchased from Shandong Center for Disease Control ＆ Prevention.
CaCl2Purchased from Sigma companies, other reagents and consumptive material are purchased from domestic each Reagent Company.Other experimental techniques in embodiment and
Reagent unless otherwise specified, is this area conventional method and commercial reagent.
The LB culture mediums are：Peptone 10g/L, dusty yeast 5g/L, NaCl 10g/L.
The SOC culture mediums are：Peptone 2g/L, dusty yeast 0.5g/L, NaCl 0.0585g/L, KCl 0.0186g/L,
MgCl20.203g/L, MgSO40.246g/L, glucose 20mmol/L.
The structure of embodiment 1, O-Ag gene cluster expression vectors
According to the type pathogenic bacteria gene group primers of shigella dysenteriae 1 that NCBI is announced：
S.d-F-SmalI：5’-TCCCCCGGGATGAATAAATATTGTATCTTAGTA-3’
S.d-R-XbaI：5’-GCTCTAGATCACATTAATGCTACCAAAAAGAGT-3’
With the type pathogenic bacteria gene group of shigella dysenteriae 1 as template, PCR clones part O-Ag gene clusters, the gene cluster
Contain tri- genes of rfbR, rfbQ and orf9.PCR reaction systems are as follows：(primer concentration is 20 μm of ol/L)
PCR reaction conditions：95 DEG C of predegenerations 3min, 95 DEG C of denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C extend 2.5min, 30
72 DEG C of extension 10min, 16 DEG C of preservations after individual circulation.
The O-Ag gene cluster fragments of clone are utilized respectively endonuclease SmalI and XbaI digestion process, while by matter
Grain carrier pACT3 is also utilized respectively endonuclease SmalI and XbaI digestion process.By the O-Ag fragments of digestion process and
PACT3 plasmid vectors are reclaimed using Ago-Gel kit, then using the connection of T4 ligases.
Linked system is 10 μ l：
O-Ag fragments：6μl；
PACT3 carriers：2μl；
10×Buffer：1μl；
T4 ligases：1μl.
After 16 DEG C of connection 12h, the connection liquid of 10 μ l is converted into Escherichia coli Top10 competent cells.Conversion process is：Will
The connection liquid of 10 μ l is added in the Top10 competent cells of 100 μ l, is mixed.Ice bath 30min, 42 DEG C of thermal shock 90s, ice bath 2min,
The SOC culture mediums of 900 μ l are added, 37 DEG C, 100r/min hatches 1h, is coated with chlorampenicol resistant flat board, cultivate 16h, picking conversion
Son, extracts plasmid checking.Then further sequence verification O-Ag gene clusters is correct, so as to obtain recombinant plasmid p-O-Ag.See
Fig. 2.
The structure of embodiment 2, rfp expression vectors
According to the type pathogenic bacteria gene group primers of shigella dysenteriae 1 that NCBI is announced：
15b-F-NdeI：5’-GGAATTCCATATGATGAAGATCTCAATAATAGGGAA-3’
15b-R-BamHI：5’-CGGGATCCTTAATCAGGAATCCCTAGTA-3’
With the type pathogenic bacteria gene group of shigella dysenteriae 1 as template, PCR clones rfp genes.PCR reaction systems are as follows：
(primer concentration is 20 μm of ol/L)
PCR reaction conditions：95 DEG C of predegenerations 3min, 95 DEG C of denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C extend 2.5min, 30
72 DEG C of extension 10min, 16 DEG C of preservations after individual circulation.
The rfp genetic fragments of clone are utilized respectively endonuclease NdeI and BamHI digestion process, while plasmid is carried
Body pET15b is also utilized respectively endonuclease NdeI and BamHI digestion process.By the rfp fragments and pET15b matter of digestion process
Grain carrier is reclaimed using Ago-Gel kit, then using the connection of T4 ligases.
Linked system is 10 μ l：
Rfp genetic fragments：6μl；
PET15b carriers：2μl；
10×Buffer：1μl；
T4 ligases：1μl.
After 16 DEG C of connection 12h, the connection liquid of 10 μ l is converted into Escherichia coli Top10 competent cells.Conversion process is：Will
The connection liquid of 10 μ l is added in the Top10 competent cells of 100 μ l, is mixed.Ice bath 30min, 42 DEG C of thermal shock 90s, ice bath 2min,
The SOC culture mediums of 900 μ l are added, 37 DEG C, 100r/min hatches 1h, is coated with amicillin resistance flat board, cultivates 16h, picking
Transformant, extracts plasmid checking.Then further sequence verification rfb genes is correct, so as to obtain recombinant plasmid p-rfp.
See Fig. 3.
The structure of embodiment 3, recombinant escherichia coli strain
(1) preparation of competence E.coli K-12W3110
(I) thalline E.coli K-12W3110 37 DEG C of 200r/min incubated overnights in 5mL test tubes；
(II) above-mentioned bacterium solution is inoculated in by 50mL LB culture mediums with 1% inoculum concentration, 37 DEG C, 200r/min cultivates 2-3h,
To OD600=0.3-0.4, (0.36 is optimal, general no more than 0.4)；
(III) in bacterium solution being transferred into 50mL centrifuge tubes (centrifuge tube aseptic and 4 DEG C to the cold), ice bath 20min allows cell to stop
Only grow, 4 DEG C of 4000r/min are centrifuged 10min；
(IV) precipitate with the CaCl of appropriate precooling2Resuspended, 4 DEG C of 4000r/min are centrifuged 5-10min, abandon supernatant；
(V) precipitation is again with the CaCl of appropriate precooling2Resuspended, ice bath 1h, 4 DEG C of 4000r/min centrifugation 10min abandon supernatant；
(VI) with the CaCl of 2mL precoolings2It is resuspended, dispense competence.
(2) structure of recombinant escherichia coli strain
Respectively by above-mentioned constructed plasmid p-O-Ag and p-rfp cotransformation e. coli k-12 W3110 competent cells,
Screened using ampicillin (final concentration of 100 μ g/mL) and chloramphenicol (final concentration of 50 μ g/mL) and obtain recombinant bacterial strain K-
12W3110/p-O-Ag+p-rfp。
Embodiment 4, the extraction of recombination bacillus coli lipopolysaccharides (LPS) and analysis
(1) fermentation of recombination bacillus coli
Recombinant bacterial strain (K-12W3110/p-O-Ag+p-rfp) single bacterium constructed by picking drops down onto the LB culture mediums equipped with 5mL
25mL test tube in, the final concentration of 100 μ g/mL of ampicillin, chloramphenicol concentration is 50 μ g/mL, 37 DEG C, 200r/min,
Culture 12h.
The bacterium solution of incubated overnight is accessed three of the 100mL equipped with 50mL LB culture mediums according to the inoculum concentration of 1% (v/v)
In the bottle of angle, the final concentration of 100 μ g/mL of ampicillin, chloramphenicol concentration is 50 μ g/mL, 37 DEG C, 200r/min.
When bacterium solution OD600=0.6, final concentration of 0.2mM IPTG, 16 DEG C, 200r/min culture 20h induction tables are added
Reach.
E. coli k12 W3110 starting strains are induced as negative control simultaneously；Pathogen S.dysenteriae 1 exists
37 DEG C, 200r/min incubated overnights are as positive control.
(2) extraction of lipopolysaccharides (LPS)
The bacterium solution normal temperature 13000r/min of incubated overnight is centrifuged 30s, all supernatants are discarded, LPS extracts reagents are used
Box extracts the LPS of recombination bacillus coli.
(I) thalline adds the Lysis Buffer of 1mL and is fully vortexed until cell mass disappears；
(II) 200 μ L chloroforms are added, be fully vortexed 10-20s, in incubation at room temperature 5min；
(III) 4 DEG C of 13,000r/min centrifugation 10min, the supernatant for shifting 400 μ L is managed to new EP, is careful not to be drawn onto
Precipitation；
(IV) 800 μ L Purification Buffer are added, is fully mixed, -20 DEG C of incubation 10min；
(V) 4 DEG C of 13,000r/min centrifugation 15min, abandon supernatant；
(VI) LPS sediments are washed with 1mL 70% (w/v) ethanol, 4 DEG C of 13,000r/min are centrifuged 3min, abandon supernatant, room
Temperature is dried；
(VII) add 50 μ L 10mM Tris-HCl buffer solutions (pH 8.0) to dissolve LPS in LPS sediments, boil
2min；
(VIII) 2.5 μ L Proteinase K Solutions (30mg/mL), 50 DEG C for the treatment of 30min are added.
(3) analysis of lipopolysaccharides (LPS)
The lipopolysaccharides sampling of extraction purification carries out SDS-PAGE analyses, is reflected using silver staining and Western Blotting
It is fixed.Western Blotting detections are using shigella dysenteriae O diagnostic antigens serum as primary antibody, the goat-anti rabbit of HRP marks
IgG is tested and analyzed as secondary antibody.Lipopolysaccharides because due to the addition of chain length not wait O antigens and show scalariform band,
The LPS that only S.dysenteriae 1 and recombination bacillus coli are extracted can be with specific by shigella dysenteriae O diagnostic antigens
Serum is recognized.Negative control E.coli K-12W3110 have interrupted the function of WbbL due to the insertion of intron IS5, form
O16 deficiencies.Such result is exactly the end only one of which GlcNAc residues of E.coli K-12W3110LPS, can not be by
Shigella dysenteriae O diagnostic antigen serum is recognized.Result is shown in Fig. 4.
Embodiment 5, the extraction of recombination bacillus coli O- antigens (O-PS) and analysis
(1) extraction of recombination bacillus coli O- antigens (O-PS)
(I) 20g cell precipitations (5g/L) extract 15min with (w/v) phenol of 200mL 50% at 65 DEG C；
(II) 10,000g centrifugations 30min at 4 DEG C；
(III) the supernatant aqueous solution is collected, is dialysed with tri-distilled water and is removed phenol, then dialyzate is freezed；
(IV) freeze-dried powder is dissolved in 20mL 0.02M sodium acetates (pH7.0), respectively with DNase, RNase and Proteinase K
2h is processed at 37 DEG C；
(V) removal precipitation (10,000g centrifugations 30min at 4 DEG C), solution uses 110,000g, 4 DEG C of ultrahigh speeds from 12h；
(VI) centrifugation gained jelly tri-distilled water dissolves, and freezes and prepares LPS；
(VII) purifying LPS 1% acetic acid, 100 DEG C for the treatment of 1.5h；
(VIII) 12,000g centrifugations 30min removal precipitations；
(IX) purifying supernatant with Bio-Gel P-2column can prepare O-PS.
(2) the monosaccharide component analysis of recombination bacillus coli O- antigens (O-PS)
The O-PS of extraction 110 DEG C for the treatment of 6h of 3M TFA, are then determined the monosaccharide component of O-PS using chromatography of ions
Property detection.
Detection method：The mixture of standard items, sample, sample and standard items is eluted under identical condition, according to reservation
Whether contain this mark product in the change judgement sample of time, peak value and peak area.
Detector：Dionex ED-50, USA；Splitter：Dionex Carbo PacTM PA1BioLCTM(2X250mm)；
Guard column：Carbo PacTM PA-100G(2X50mm)。
Mobile phase：10mM NaOH, 200mM NaAc；Flow velocity：0.3mL/min.Result is shown in Fig. 5.
According to above recombination bacillus coli extract LPS and O-PS measure it could be assumed that, restructuring of the present invention
Escherichia coli can produce the lipopolysaccharides structure of the type pathogen of shigella dysenteriae 1, can further produce anti-as platform
The glucoprotein vaccine of the type pathogen of Shigella 1.
Claims (3)
1. a kind of recombination bacillus coli of the lipopolysaccharides synthase gene cluster that can express the type pathogen of shigella dysenteriae 1, it is special
Levy and be：The recombination bacillus coli contains the rfbR in the type pathogen rfb gene clusters of shigella dysenteriae 1
(rhamnosyltransferase), rfbQ (rhamnosyltransferase) and orf9
(galactosyltransferase), its genotype is W3110 Δs wbbl/p-rfp+p-O-Ag.
2. the restructuring large intestine of the lipopolysaccharides synthase gene cluster of the type pathogen of shigella dysenteriae 1 can be expressed described in claim 1
The construction method of bacillus, step is：
Build containing the rfbR (rhamnosyltransferase), rfbQ in the type pathogen rfb gene clusters of shigella dysenteriae 1
(rhamnosyltransferase) and three genes of orf9 (galactosyltransferase) expression vector p-O-Ag,
The expression vector p-rfp of rfp genes is built again, then by recombinant plasmid p-O-Ag and p-rfp the cotransformation large intestine of the structure
Bacillus K12W3110, that is, the restructuring large intestine of the lipopolysaccharides synthase gene cluster for obtaining that the type pathogen of shigella dysenteriae 1 can be expressed
Bacillus；
Wherein, it is described to include rfbR (rhamnosyltransferase), rfbQ (rhamnosyltransferase) and
The rfb gene clusters O-Ag of three genes of orf9 (galactosyltransferase) derives from the type pathogen of shigella dysenteriae 1
Genome, its expression rfb gene clusters carrier be pACT3；The rfp genes are present in the type pathogen of shigella dysenteriae 1
In 9Kb free plasmids, its expression rfp genes carrier be pET15b；The e. coli k-12 W3110, due to intron
So as to interrupt the function of WbbL, its genotype is W3110 Δs wbbl for the insertion of IS5.
3. the restructuring large intestine of the lipopolysaccharides synthase gene cluster of the type pathogen of shigella dysenteriae 1 can be expressed described in claim 1
Application of the bacillus in the glucoprotein vaccine for preparing the type pathogen of anti-shigella dysenteriae 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710046262.5A CN106801029B (en) | 2017-01-22 | 2017-01-22 | Recombinant escherichia coli and application thereof in preparation of sugar vaccine for resisting shigella |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710046262.5A CN106801029B (en) | 2017-01-22 | 2017-01-22 | Recombinant escherichia coli and application thereof in preparation of sugar vaccine for resisting shigella |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106801029A true CN106801029A (en) | 2017-06-06 |
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| CN109486844A (en) * | 2018-10-12 | 2019-03-19 | 中南民族大学 | A kind of specific marker method of enterotoxigenic escherichia coli |
| CN114636781A (en) * | 2020-12-15 | 2022-06-17 | 北京泰德制药股份有限公司 | Method for evaluating sialylation level of glycoprotein oligosaccharide |
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| CN109486844A (en) * | 2018-10-12 | 2019-03-19 | 中南民族大学 | A kind of specific marker method of enterotoxigenic escherichia coli |
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| CN114636781B (en) * | 2020-12-15 | 2023-07-04 | 北京泰德制药股份有限公司 | Method for evaluating sialylation level of glycoprotein oligosaccharide |
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