CN104962541A - Purifying process for oral recombinant helicobacter pylori vaccine - Google Patents
Purifying process for oral recombinant helicobacter pylori vaccine Download PDFInfo
- Publication number
- CN104962541A CN104962541A CN201510144744.5A CN201510144744A CN104962541A CN 104962541 A CN104962541 A CN 104962541A CN 201510144744 A CN201510144744 A CN 201510144744A CN 104962541 A CN104962541 A CN 104962541A
- Authority
- CN
- China
- Prior art keywords
- chromatography
- vaccine
- protein
- purifying process
- ureb
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a purifying process for an oral recombinant helicobacter pylori vaccine. The effective component of the vaccine is fusion protein of Escherichia coli heat labile enterotoxin B subunit (LTB) and urase subunit B (UreB). LTB-UreB vaccine protein with high purity is obtained by subjecting a collected fermented genetically-engineered bacterium expressing the LTB-UreB fusion protein to high pressure bacterium breaking, washing, cleavage, anion-exchange column chromatography, ultrafiltration, gel filtration chromatography, etc. The purifying process has the advantages of simplicity, high efficiency, good repeatability, high yield and easy realization of industrial production; the obtained target protein has a purity of more than 98% according to detection results of high performance liquid chromatography; and animal test proves that the oral recombinant helicobacter pylori vaccine protein prepared by using the purifying process has good immunocompetence and immunoprotectivity.
Description
Technical field
The invention belongs to field of biological pharmacy, particularly relate to a kind of purifying process of oral administration recombinant helicobacterpylori vaccine.
Technical background
Helicobacter pylori (
helicobacter pylori,
h.pylori) be a kind of Gram-negative, microaerophilic bacterium, closely related with chronic gastritis, peptide ulceration and adenocarcinoma of stomach.
h.pyloriinfection is one of modal chronic infection disease, has caused the population in the whole world more than 50% to infect, in China,
h.pylorithe infected is more than 600,000,000.
h.pyloritoxin and toxic substance can not only destroy stomach mucous membrane, body can also be made to produce inflammation and immune response, affect the secretion of hydrochloric acid in gastric juice, finally cause the formation of a series of disease.
Within 1994, the World Health Organization will
h.pyloribe classified as I class carcinogen (grade carinogen).Present stage mainly adopts acid inhibitor and two kinds or three kinds of antibiotic therapies clinically
h.pyloriinfect, but exist obviously not enough, comprise curative effect instability, easily recurrence, easily produce resistance, toxic side effects is large and medical expense is high, maximum defect to eradicate
h.pylori.If a kind of existing prophylactic function medicative vaccine again successfully can be developed, will become and prevent
h.pylorithe high-performance bio preparation infected, and there is very great social effect and economic benefit.
Due to
h.pylorihuge Social benefit and economic benefit contained by vaccine, just has the eighties as far back as twentieth century
h.pylorithe research of vaccine.Existing vaccine is primarily of following five kinds of forms: (1) full bacterium living vaccine, be comparatively traditional vaccine, its antigenic component is complicated, contains
h.pyloriall antigenic components, immunne response efficiency is high, but its specificity not high, easily there is cross-immune reaction, there is carcinogenic Potential feasibility, therefore, numerous investigator not encourage growth
h.pyloriwhole-bacterial-vaccine; (2) subunit vaccine, is
h.pylorithe emphasis of vaccine research, its antigenic component is clear, security good, high specificity, and the antigen of common subunit vaccine mainly comprises urease (UreA, UreB), vacuolating cytotoxin (VacA), Cytotoxin associated antigen A (CagA), adhesin (BabA, HpaA) etc.According to statistics, urease is selected to account for as the research report of vaccine antigen
h.pylorimore than 70% of vaccine research document, therefore urease becomes
h.pylorithe first-selected subunit antigen of vaccine; (3) combined vaccine, has two or more antigenic components.There is the shortcomings such as immunogenicity is weak, immune effect is poor in univalent vaccine, and combined vaccine can obtain immune protective effect more good than univalent vaccine, can significantly improve the immune protective rate of animal; (4) nucleic acid vaccine, belongs to third generation of vaccine.Have compared with conventional vaccine that manufacture is easy, efficient, cheap, no pathogenicity, can the advantage such as activated cell and humoral immunoresponse(HI); (5) epiposition vaccine is the new direction of Vaccine Development.Epiposition vaccine is the vaccine adopting epitope to be prepared from.Epiposition vaccine has the features such as specificity is high, immunogenicity is strong, is
h.pylorithe new approaches of vaccine design, have good application prospect (Yang Wuchen, Guo Hong. the Advances on Vaccine of helicobacter Pylori urease B subunit. Chinese Amphixenosis's journal [J], 2011,27(12): 1139-1142).
Existing
h.pylorivaccine immunity prevention or result for the treatment of all undesirable, its possible reason is: the selection of (1) vaccine antigen is unreasonable with composition; (2) immunolgical adjuvant activity is not enough, there is potential safety hazard; (3) immune delivery approach and mode uncertain, make immune response strength more weak (Wu Chao, Li Haibo etc. the polyepitope vaccines of a kind of helicobacter pylori Multi-Epitope Fusion Protein and preparation thereof. China, 201210330695.0,212.12.26).Due to
h.pylorivaccine Project difficulty is comparatively large, and the overwhelming majority also concentrates on mouse and primate is tested with it, and up to now, Jin Youjijia research unit has carried out I or the II clinical trial phase of vaccine respectively.The restructuring of being researched and developed by Orovax company for 1999
h.pyloriurease therapeutic vaccine enters II clinical trial phase, although have good conservative property antigen and tolerance, needs adjuvant, and needs multi-agent to take; The supervision salmonella of the expression urease of Oravax company research enters I clinical trial phase, and it is taken simply, does not produce clear and definite mucosal immunoreaction; The full bacterium antigen vaccine of deactivation of Antex Biologics company development enters II clinical trial phase, there is more full immunity, but preparation difficulty, security need be investigated further (Zou Quanming. the progress of helicobacter pylori vaccine. gastroenterology [J], 2007,12(9): 567-570).Therefore, develop a kind of for
h.pylorithe medicine of vaccine immunity prevention just seems particularly important.
Summary of the invention
The object of this invention is to provide a kind of purification process of oral administration recombinant helicobacterpylori vaccine.The inclusion body protein of cracking is carried out the gradient elution of NaCl concentration by the method in anion-exchange chromatography post.Advantage of the present invention is: the method is simple and direct, save time, purity is high, be easy to suitability for industrialized production, be applicable to the preparation of oral administration recombinant helicobacterpylori vaccine, the target protein purity that obtains detects through high performance liquid chromatography (HPLC), purity can reach more than 98%, and application will have good economic benefit in the industrial production.
In order to achieve the above object, the invention provides a kind of purifying process of oral administration recombinant helicobacterpylori vaccine, after fermentation mode High Cell Density And High Expression LTB-UreB, adopt and highly crush bacterium, washing, cracking, anion exchange chromatography, ultrafiltration, gel filtration chromatography, obtain highly purified LTB-UreB vaccine protein.
The concrete operation step of further improvement is as follows:
(1) height crushes bacterium: suspended by coli somatic Tutofusin tris (Tris-HCl) damping fluid of high expression LTB-UreB fusion rotein, after fully mixing, high pressure homogenizer is adopted to break bacterium, collecting precipitation after high speed centrifugation, i.e. inclusion body;
(2) wash: step (1) gained inclusion body employing washings I (10mmol/L Tris-HCl, 1% Tween 80, pH 6.0 ~ 9.0), washings II (10mmol/L Tris-HCl, 2 ~ 3mol/L Urea, pH 6.0 ~ 9.0) are washed, high speed centrifugation, collecting precipitation;
(3) cracking: the precipitation that step (2) obtains utilized lysate (50mmol/L Tris-HCl, 6 ~ 8mol/L Urea, pH 6.0 ~ 9.0) denatured lysis to spend the night;
(4) Q FF chromatography purification: select Q FF chromatography to carry out preliminary purification, balance liquid (50mmol/L Tris-HCl, 6 ~ 8mol/L Urea, pH 6.0 ~ 9.0) carry out Q-FF chromatography column balance, utilize the balance liquid containing different concns NaCl to carry out gradient elution;
(5) ultrafiltration: protein sample step (4) obtained uses ultra-filtration membrane bag to concentrate;
(6) G-50 chromatography purification: adopt G-50 chromatography to carry out purifying the protein sample of above-mentioned gained, utilize NaHCO
3damping fluid carries out balancing and wash-out.
Further improvement is: described step (1) uses that 600 ~ 800bar is high crushes bacterium technology, and broken bacterium rate >=99%, high speed centrifugation obtains inclusion body.
Further improvement is: the described employing washings I of described step (2), washings II wash inclusion body secondary.
Further improvement is: described step (3) adopts the lysate of pH 6.0 ~ 9.0 to carry out denatured lysis, spends the night in 10 ~ 18 DEG C of stirrings.
Further improvement is: the filler that the described anion-exchange chromatography of step (4) uses is Q Sepharose Fast Flow, adopts Q-FF chromatography to carry out the purifying of target protein LTB-UreB.
Further improvement is: target protein ultrafiltration and concentration to the protein concentration of step (5) purifying is 3 ~ 4 mg/mL.
Further improvement is: step (6) adopts G-50 chromatography to carry out desalination and except urea, makes protein renaturation.
The target protein prepared by above-mentioned art breading carries out SDS-PAGE detection, and result shows its purity >=98%, and molecular weight is about 75kD ± 10%; HPLC detects purity >=98%, and target protein collection of illustrative plates repeatability is good; Isoelectric focusing electrophoresis shows, and the iso-electric point of target protein is between 5.0 ~ 7.0.Described fusion rotein being made oral restructuring helicobacter pylorus vaccine by freeze-dry process, oral immunity BALB/c mouse, evaluating by attacking the effect of malicious mode to vaccine prevention helicobacter pylori infection after first immunity.Result shows, and mouse can be stimulated to produce specificity sIgA secretion, and effectively can reduce the field planting of helicobacter pylori in Mouse Stomach, have significant protected effect, can prevent helicobacter pylori infection by the target protein of above-mentioned technique purifying.
In sum, high by the LTB-UreB fusion rotein purity of above-mentioned technique purifying, be easy to suitability for industrialized production, and effectively can prevent helicobacter pylori infection.
Accompanying drawing explanation
Fig. 1 is that target protein LTB-UreB washs and lysate sample SDS-PAGE detection figure, wherein, and swimming lane 1,2: inclusion body secondary washing supernatant liquor result; Swimming lane 3,4: inclusion body lysate (before purifying sample); Swimming lane 5: protein molecular weight standard (Marker).
Fig. 2 is the Q-FF chromatography SDS-PAGE detection figure of target protein LTB-UreB, wherein, and swimming lane 1: protein molecular weight standard (Marker); Swimming lane 2,3,4: adopt the target protein that the balance liquid wash-out of 42mmol/L NaCl obtains; Swimming lane 5,6: adopt the target protein that the balance liquid wash-out of 55mmol/L NaCl obtains.
Fig. 3 is the G-50 chromatography SDS-PAGE detection figure of target protein LTB-UreB.Wherein, swimming lane 1: protein molecular weight standard (Marker); Swimming lane 2: be target protein after purifying.
Fig. 4 is that the HPLC of target protein LTB-UreB detects collection of illustrative plates.
Embodiment
In order to deepen the understanding of the present invention, below in conjunction with embodiment, the invention will be further described, and this embodiment only for explaining the present invention, does not form limiting the scope of the present invention.
Embodiment one
Bacterial cell disruption
Take engineering bacteria 2000g, add 10mmol/L pH 8.0 Tris-HCl damping fluid in the ratio of 1:10, with high-pressure homogeneous crusher machine thalline after fully mixing, bacterium is broken 3 times in 700bar, adopt tubular-bowl centrifuge 9000rpm centrifugal, flow velocity 1L/min, precipitation is thick inclusion body.Microscopy bacterium breakdown ratio >=99%.
The washing of inclusion body and cracking
(1) take 300g inclusion body, add washings I in the ratio of 1:10, after high speed disperser disperses completely, the centrifugal 40min of mix and blend 20min, 5000rpm, collecting precipitation.
(2) in precipitation, washings II is added, after high speed disperser disperses completely, mixing and stirring, the centrifugal 40min of 5000rpm, collecting precipitation.
(3) in precipitation, add inclusion body lysate in the ratio of 1:10, be placed in 15 DEG C of stirrings and spend the night, the centrifugal 50min of 7000rpm, collect supernatant, for Q-FF chromatography.
After secondary washing, the foreign protein in inclusion body significantly reduces, cracking spend the night after inclusion body exist with the form of soluble proteins, through SDS-PAGE testing goal purity of protein > 40%.
Anion exchange chromatography (Q-FF chromatography)
(1) adopt 1 times of column volume 1 mol/L NaCl, 1mol/L NaOH solution process Q-FF chromatography column, and connect Ultraviolet Detector and portable record instrument.Utilize the balance liquid balance Q-FF chromatography column of 3 times of column volumes.
(2), after loading, the balance liquid containing 30mmol/L, 42mmol/L, 55mmol/L NaCl is adopted to carry out gradient elution, under ultraviolet detection condition, Fractional Collections protein sample.The balance liquid Main Function of 30mmol/L NaCl is removal of impurities albumen, after target protein LTB-UreB mainly concentrates on the peak of 42mmol/L NaCl elution peak and before the peak of 55mmol/L NaCl elution peak.After testing, sample target protein purity >=98% under Q-FF chromatography.Be 3 ~ 4mg/mL by purifying protein ultrafiltration and concentration to protein concentration, 4 DEG C save backup.
Gel permeation chromatography (G-50 chromatography)
Use 5 times of column volume 1mol/L NaOH process G-50 chromatography columns, and connect Ultraviolet Detector and Portable type recorder.Adopt the 20 mmol/L NaHCO of pH 9.5
3balance liquid carry out Balance Treatment, utilize balance liquid to carry out wash-out after end of the sample.Desalination is carried out and except after urea, SDS-PAGE result shows, target protein purity >=98% in sample under G-50 chromatography, and molecular weight is about 75kD ± 10% through G-50 chromatography; HPLC testing goal purity of protein >=98%, and target protein collection of illustrative plates repeatability is good; Isoelectric focusing electrophoresis shows, and the iso-electric point of target protein is between 5.0 ~ 7.0; Lowry method detects protein concentration >=3mg/mL.Oral immunity BALB/c mouse, finds that IgA and the IgG level in experimental group serum, gastric juice is significantly higher than control group, proves that target protein can produce higher immunne response by effective stimulus body; After oral immunity mouse, adopt
h.pylorisS1 strain infection, oral administration recombinant helicobacterpylori vaccine resists
h.pyloriprotection ratio>=80% infected.
Embodiment two
Bacterial cell disruption
Take engineering bacteria 2000g, add 10mmol/L pH 6.0Tris-HCl damping fluid in the ratio of 1:10, with high-pressure homogeneous crusher machine thalline after fully mixing, bacterium is broken 2 times in 600bar, adopt tubular-bowl centrifuge 8000rpm centrifugal, flow velocity 1L/min, precipitation is thick inclusion body.Microscopy bacterium breakdown ratio >=99%.
The washing of inclusion body and cracking
(1) take 300g inclusion body, add washings I in the ratio of 1:10, after high speed disperser disperses completely, the centrifugal 20min of mix and blend 15min, 4000rpm, collecting precipitation.
(2) in precipitation, washings II is added, after high speed disperser disperses completely, mixing and stirring, the centrifugal 20min of 4000rpm, collecting precipitation.
(3) in precipitation, add inclusion body lysate in the ratio of 1:10, be placed in 10 DEG C of stirrings and spend the night, the centrifugal 45min of 6000rpm, collect supernatant, for Q-FF chromatography.
After secondary washing, the foreign protein in inclusion body significantly reduces, cracking spend the night after inclusion body exist with the form of soluble proteins, through SDS-PAGE testing goal purity of protein > 40%.
Anion exchange chromatography (Q-FF chromatography)
(1) adopt 1 times of column volume 1 mol/L NaCl, 1mol/L NaOH solution process Q-FF chromatography column, and connect Ultraviolet Detector and portable record instrument.Utilize the balance liquid balance Q-FF chromatography column of 3 times of column volumes.
(2), after loading, the balance liquid containing 30mmol/L, 42mmol/L, 55mmol/L NaCl is adopted to carry out gradient elution, under ultraviolet detection condition, Fractional Collections protein sample.The balance liquid Main Function of 30mmol/L NaCl is removal of impurities albumen, after target protein LTB-UreB mainly concentrates on the peak of 42mmol/L NaCl elution peak and before the peak of 55mmol/L NaCl elution peak.After testing, sample target protein purity >=98% under Q-FF chromatography.Be 3 ~ 4mg/mL by purifying protein ultrafiltration and concentration to protein concentration, 4 DEG C save backup.
Gel permeation chromatography (G-50 chromatography)
Use 5 times of column volume 1mol/L NaOH process G-50 chromatography columns, and connect Ultraviolet Detector and Portable type recorder.Adopt the 20 mmol/L NaHCO of pH 9.0
3balance liquid carry out Balance Treatment, utilize balance liquid to carry out wash-out after end of the sample.Desalination is carried out and except after urea, SDS-PAGE result shows, target protein purity >=98% in sample under G-50 chromatography, and molecular weight is about 75kD ± 10% through G-50 chromatography; HPLC testing goal purity of protein >=98%, and target protein collection of illustrative plates repeatability is good; Isoelectric focusing electrophoresis shows, and the iso-electric point of target protein is between 5.0 ~ 7.0; Lowry method detects protein concentration >=3mg/mL.Oral immunity BALB/c mouse, finds that IgA and the IgG level in experimental group serum, gastric juice is significantly higher than control group, proves that target protein can produce higher immunne response by effective stimulus body; After oral immunity mouse, adopt
h.pylorisS1 strain infection, oral administration recombinant helicobacterpylori vaccine resists
h.pyloriprotection ratio>=80% infected.
Embodiment three
Bacterial cell disruption
Take engineering bacteria 3000g, add 10mmol/L pH 9.0Tris-HCl damping fluid in the ratio of 1:10, with high-pressure homogeneous crusher machine thalline after fully mixing, bacterium is broken 3 times in 800bar, adopt tubular-bowl centrifuge 10000rpm centrifugal, flow velocity 2L/min, precipitation is thick inclusion body.Microscopy bacterium breakdown ratio >=99%.
The washing of inclusion body and cracking
(1) take 400g inclusion body, add washings I in the ratio of 1:10, after high speed disperser disperses completely, the centrifugal 40min of mix and blend 30min, 6000rpm, collecting precipitation.
(2) in precipitation, washings II is added, after high speed disperser disperses completely, mixing and stirring, the centrifugal 40min of 6000rpm, collecting precipitation.
(3) in precipitation, add inclusion body lysate in the ratio of 1:10, be placed in 18 DEG C of stirrings and spend the night, the centrifugal 60min of 8000rpm, collect supernatant, for Q-FF chromatography.
After secondary washing, the foreign protein in inclusion body significantly reduces, cracking spend the night after inclusion body exist with the form of soluble proteins, through SDS-PAGE testing goal purity of protein > 40%.
Anion exchange chromatography (Q-FF chromatography)
(1) adopt 1 times of column volume 1 mol/L NaCl, 1mol/L NaOH solution process Q-FF chromatography column, and connect Ultraviolet Detector and portable record instrument.Utilize the balance liquid balance Q-FF chromatography column of 3 times of column volumes.
(2), after loading, the balance liquid containing 30mmol/L, 42mmol/L, 55mmol/L NaCl is adopted to carry out gradient elution, under ultraviolet detection condition, Fractional Collections protein sample.The balance liquid Main Function of 30mmol/L NaCl is removal of impurities albumen, after target protein LTB-UreB mainly concentrates on the peak of 42mmol/L NaCl elution peak and before the peak of 55mmol/L NaCl elution peak.After testing, sample target protein purity >=98% under Q-FF chromatography.Be 3 ~ 4mg/mL by purifying protein ultrafiltration and concentration to protein concentration, 4 DEG C save backup.
Gel permeation chromatography (G-50 chromatography)
Use 5 times of column volume 1mol/L NaOH process G-50 chromatography columns, and connect Ultraviolet Detector and Portable type recorder.Adopt the 20 mmol/L NaHCO of pH 11.0
3balance liquid carry out Balance Treatment, utilize balance liquid to carry out wash-out after end of the sample.Desalination is carried out and except after urea, SDS-PAGE result shows, target protein purity >=98% in sample under G-50 chromatography, and molecular weight is about 75kD ± 10% through G-50 chromatography; HPLC testing goal purity of protein >=98%, and target protein collection of illustrative plates repeatability is good; Isoelectric focusing electrophoresis shows, and the iso-electric point of target protein is between 5.0 ~ 7.0; Lowry method detects protein concentration >=3mg/mL.Oral immunity BALB/c mouse, finds that IgA and the IgG level in experimental group serum, gastric juice is significantly higher than control group, proves that target protein can produce higher immunne response by effective stimulus body; After oral immunity mouse, adopt
h.pylorisS1 strain infection, oral administration recombinant helicobacterpylori vaccine resists
h.pyloriprotection ratio>=80% infected.
Claims (8)
1. the purifying process of an oral administration recombinant helicobacterpylori vaccine, it is characterized in that: after fermentation mode High Cell Density And High Expression LTB-UreB, adopt and highly crush bacterium, washing, cracking, anion exchange chromatography, ultrafiltration, gel filtration chromatography, obtain highly purified LTB-UreB vaccine protein.
2. the purifying process of oral administration recombinant helicobacterpylori vaccine according to claim 1, is characterized in that: concrete operation step is as follows:
(1) height crushes bacterium: suspended by coli somatic Tutofusin tris (Tris-HCl) damping fluid of high expression LTB-UreB fusion rotein, after fully mixing, high pressure homogenizer is adopted to break bacterium, collecting precipitation after high speed centrifugation, i.e. inclusion body;
(2) wash: step (1) gained inclusion body employing washings I (10mmol/L Tris-HCl, 1% Tween 80, pH 6.0 ~ 9.0), washings II (10mmol/L Tris-HCl, 2 ~ 3mol/L Urea, pH 6.0 ~ 9.0) are washed, high speed centrifugation, collecting precipitation;
(3) cracking: the precipitation that step (2) obtains utilized lysate (50mmol/L Tris-HCl, 6 ~ 8mol/L Urea, pH 6.0 ~ 9.0) denatured lysis to spend the night;
(4) anionresin column purification: select Q Sepharose Fast Flow anion-exchange chromatography (being called for short Q FF chromatography) to carry out preliminary purification, balance liquid (50mmol/L Tris-HCl, 6 ~ 8mol/L Urea, pH 6.0 ~ 9.0) carry out Q-FF chromatography column balance, utilize containing different concns NaCl(30 ~ 55mmol/L) balance liquid carry out gradient elution;
(5) ultrafiltration: protein sample step (4) obtained uses ultra-filtration membrane bag to concentrate;
(6) gel filtration chromatography: adopted by the protein sample of above-mentioned gained Sepherdex G-50 gel permeation chromatography (being called for short G-50 chromatography) to carry out purifying, utilize NaHCO
3damping fluid carries out balancing and wash-out.
3. according to the purifying process of the oral administration recombinant helicobacterpylori vaccine described in claim 2, it is characterized in that: described step (1) uses that 600 ~ 800bar is high crushes bacterium technology, and broken bacterium rate >=99%, high speed centrifugation obtains inclusion body.
4. according to the purifying process of the oral administration recombinant helicobacterpylori vaccine described in claim 2, it is characterized in that: the described employing washings I of described step (2), washings II wash inclusion body secondary.
5. according to the purifying process of the oral administration recombinant helicobacterpylori vaccine described in claim 2, it is characterized in that: described step (3) adopts the lysate of pH 6.0 ~ 9.0 to carry out denatured lysis, spends the night in 10 ~ 18 DEG C of stirrings.
6. according to the purifying process of the oral administration recombinant helicobacterpylori vaccine described in claim 2, it is characterized in that: the filler that the described anion-exchange chromatography of step (4) uses is Q Sepharose Fast Flow, adopt Q-FF chromatography to carry out the purifying of target protein LTB-UreB.
7. according to the purifying process of the oral administration recombinant helicobacterpylori vaccine described in claim 2, it is characterized in that: target protein ultrafiltration and concentration to the protein concentration of step (5) purifying is 3 ~ 4 mg/mL.
8., according to the purifying process of the oral administration recombinant helicobacterpylori vaccine described in claim 2, it is characterized in that step (6) adopts G-50 chromatography to carry out desalination and except urea, makes protein renaturation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510144744.5A CN104962541A (en) | 2015-03-31 | 2015-03-31 | Purifying process for oral recombinant helicobacter pylori vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510144744.5A CN104962541A (en) | 2015-03-31 | 2015-03-31 | Purifying process for oral recombinant helicobacter pylori vaccine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104962541A true CN104962541A (en) | 2015-10-07 |
Family
ID=54216632
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510144744.5A Pending CN104962541A (en) | 2015-03-31 | 2015-03-31 | Purifying process for oral recombinant helicobacter pylori vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104962541A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105462997A (en) * | 2015-12-23 | 2016-04-06 | 江南大学 | New urease prosthetic group gene and application thereof |
| CN106480003A (en) * | 2016-10-31 | 2017-03-08 | 中国人民解放军第三军医大学 | Helicobacter pylori dominant antigen combination based on CD4+T cellular immunization and screening technique |
| CN111467368A (en) * | 2020-04-13 | 2020-07-31 | 江南大学 | Application of oligosaccharide compound containing heptasaccharide chain in preparation of helicobacter pylori vaccine |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5986051A (en) * | 1991-10-03 | 1999-11-16 | Institut Pasteur | Genes of Helicobacter pylori necessary for the regulation and maturation of urease and their use |
| CN1563388A (en) * | 2004-03-29 | 2005-01-12 | 中国人民解放军第三军医大学 | Constructing genetic engineering Vaccine of adhesin of confluent Helicobacter pylor and preparation method |
| CN1563406A (en) * | 2004-04-20 | 2005-01-12 | 中国人民解放军第三军医大学 | Purification technique in preparing genetic engineering vaccine of heat shock protein A of recombined Helicobacter pylori |
| CN1887349A (en) * | 2006-07-20 | 2007-01-03 | 中国人民解放军第三军医大学 | Helicobacter pylori vaccine based on urease B subunit active segment and its prepn process |
| CN1973903A (en) * | 2006-12-04 | 2007-06-06 | 严杰 | Oral vaccine of recombinant gene for preventing Helicobacter pylori infection and its prepn process |
| CN102838680A (en) * | 2012-09-07 | 2012-12-26 | 中国人民解放军第三军医大学 | Helicobacter pylori multiple-epitope fusion protein and multiple-epitope vaccine prepared by helicobacter pylori multiple-epitope fusion protein |
| CN103990121A (en) * | 2013-12-06 | 2014-08-20 | 上海联合赛尔生物工程有限公司 | Antigen chimera, antigen composition, vaccine and preparation method and kit thereof |
-
2015
- 2015-03-31 CN CN201510144744.5A patent/CN104962541A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5986051A (en) * | 1991-10-03 | 1999-11-16 | Institut Pasteur | Genes of Helicobacter pylori necessary for the regulation and maturation of urease and their use |
| CN1563388A (en) * | 2004-03-29 | 2005-01-12 | 中国人民解放军第三军医大学 | Constructing genetic engineering Vaccine of adhesin of confluent Helicobacter pylor and preparation method |
| CN1563406A (en) * | 2004-04-20 | 2005-01-12 | 中国人民解放军第三军医大学 | Purification technique in preparing genetic engineering vaccine of heat shock protein A of recombined Helicobacter pylori |
| CN1887349A (en) * | 2006-07-20 | 2007-01-03 | 中国人民解放军第三军医大学 | Helicobacter pylori vaccine based on urease B subunit active segment and its prepn process |
| CN1973903A (en) * | 2006-12-04 | 2007-06-06 | 严杰 | Oral vaccine of recombinant gene for preventing Helicobacter pylori infection and its prepn process |
| CN102838680A (en) * | 2012-09-07 | 2012-12-26 | 中国人民解放军第三军医大学 | Helicobacter pylori multiple-epitope fusion protein and multiple-epitope vaccine prepared by helicobacter pylori multiple-epitope fusion protein |
| CN103990121A (en) * | 2013-12-06 | 2014-08-20 | 上海联合赛尔生物工程有限公司 | Antigen chimera, antigen composition, vaccine and preparation method and kit thereof |
Non-Patent Citations (4)
| Title |
|---|
| JIE YAN ET AL.: ""Construction of prokaryotic expression system of ltB-ureBfusion gene and identification of the recombinant protein immunity and adjuvanticity"", 《WORLD JOURNAL OF GASTROENTEROLOGY》 * |
| JIE YAN ET AL.: ""Effects of lactose as an inducer on expression of Helicobacter pylori rUreB and rHpaA, and Escherichia coli rLTKA63 and rLTB"", 《WORLD JOURNAL OF GASTROENTEROLOGY》 * |
| WEI-YING ZHOU ET AL.: ""Therapeutic efficacy of a multi-epitope vaccine againstHelicobacter pylori infection in BALB/c mice model"", 《VACCINE》 * |
| 张卫军: ""幽门螺杆菌外膜蛋白双价亚单位融合疫苗的实验研究"", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105462997A (en) * | 2015-12-23 | 2016-04-06 | 江南大学 | New urease prosthetic group gene and application thereof |
| CN106480003A (en) * | 2016-10-31 | 2017-03-08 | 中国人民解放军第三军医大学 | Helicobacter pylori dominant antigen combination based on CD4+T cellular immunization and screening technique |
| CN106480003B (en) * | 2016-10-31 | 2019-09-10 | 中国人民解放军第三军医大学 | The combination of helicobacter pylori dominant antigen and screening technique based on CD4+T cellular immunity |
| CN111467368A (en) * | 2020-04-13 | 2020-07-31 | 江南大学 | Application of oligosaccharide compound containing heptasaccharide chain in preparation of helicobacter pylori vaccine |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103656632B (en) | Multivalent pneumococcal capsular polysaccharide composition, its preparation method and application | |
| CN103656631B (en) | multivalent pneumococcal capsular polysaccharide-protein conjugate composition and preparation method thereof | |
| CN104004806B (en) | One kind has anticoagulation and thrombus dissolving earthworm polypeptide and its enzymolysis preparation and application | |
| CN101081296B (en) | Method for preparing b type haemophilus influenzae capsular polysaccharide and united vaccines thereof | |
| CN104491855B (en) | Method of the aftosa whole virus particles marker vaccine of a kind of extensive preparation high yield, high-purity, high safety and products thereof | |
| CN102154325B (en) | Vaccine against human papillomavirus (HPV) as well as preparation method and application thereof | |
| CN104962541A (en) | Purifying process for oral recombinant helicobacter pylori vaccine | |
| CN102526116B (en) | Method for refining bee venom | |
| CN115969967B (en) | Triple mRNA vaccine for preventing cat rhinotracheitis, cat calicivirus disease and cat panleukopenia and preparation method thereof | |
| CN101024079B (en) | Pneumo-streptococcal-polysaccharide adventitia jointed vaccine and preparing method | |
| CN100579578C (en) | Pyloric spiral bacillus antigen recombinant vaccine | |
| WO2009127130A1 (en) | Achyranthes bidentata polypeptide and production methods and uses thereof | |
| CN102286100B (en) | SEB (staphylococcal enterotoxin B) resisting immune globulin F(ab')2 and preparation method thereof | |
| CN102078604A (en) | Meningococcus capsular polysaccharide polyvalent multivalent conjugate vaccine, preparation method and application thereof | |
| CN105535963A (en) | EV71 subunit vaccine of mixed adjuvant and preparation method thereof | |
| CN103990121B (en) | Antigen chimera, antigen composition, vaccine and preparation method and kit thereof | |
| CN101985467A (en) | Analgesic active peptide VGG and preparation method and applications thereof | |
| CN109438585B (en) | Purification process of type b haemophilus polysaccharide | |
| CN102898537B (en) | Purification method of lipopolysaccharide | |
| CN100540675C (en) | A kind of Sepharose of use 4FF gel carries out the method for purifying to bacterial polysaccharides | |
| CN101307314B (en) | Method for constructing and preparing vaccine against type 2 diabetes mellitus | |
| CN105420174A (en) | Establishment of genetically engineered bacterium expressing recombined VEGF fusion protein | |
| CN106177934B (en) | A kind of haemophilus parasuis subunit vaccine and preparation method thereof | |
| CN104592408A (en) | Method for purifying capsular polysaccharide | |
| CN102888422A (en) | DNA (Deoxyribose Nucleic Acid) vaccine vector based on B2L gene of contagious pustular dermatitis virus and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151007 |
|
| RJ01 | Rejection of invention patent application after publication |