CN1241938C - Purification technique in preparing genetic engineering vaccine of heat shock protein A of recombined Helicobacter pylori - Google Patents
Purification technique in preparing genetic engineering vaccine of heat shock protein A of recombined Helicobacter pylori Download PDFInfo
- Publication number
- CN1241938C CN1241938C CN 200410022360 CN200410022360A CN1241938C CN 1241938 C CN1241938 C CN 1241938C CN 200410022360 CN200410022360 CN 200410022360 CN 200410022360 A CN200410022360 A CN 200410022360A CN 1241938 C CN1241938 C CN 1241938C
- Authority
- CN
- China
- Prior art keywords
- purification
- renaturation
- purifying
- nickel ion
- superdex
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention discloses purifying technology in the preparation of recombining helicobacter pylori heat shock protein A gene engineering bacterium vaccines, which uses the technology of the high-pressure bacterium breakage and the filtration of the genetic engineering bacteria, the affinity chromatography purification of nickel ions, concentrating schizolysis, gel filtrating chromatography, renaturation, etc. Thus, the recombining helicobacter pylori heat shock protein A of high purity is obtained. The purifying technology of the present invention has the advantages of simplicity, convenience, easy amplification and good repetitiveness. The obtained target protein has high purity. Animal experiments prove that the recombining helicobacter pylori heat shock protein A obtained by the purification of the present invention has good immune activity and immune protection.
Description
Technical field
The invention belongs to field of biological pharmacy, be specifically related to the purification process in the preparation of Heat Shock Protein A Gene of Helicobactor pylori engineered vaccine.
Technical background
Helicobacter pylori (Helicobacter pylori, Hp) be a kind of Gram-negative, screw shaped, microaerobe that is colonizated in the stomach, infection rate surpasses 50% in whole world crowd, be the main paathogenic factor of disease of upper digestive tract such as chronic gastritis, peptide ulceration and non-ucler dyspepsia, and closely related with the generation of stomach malignant tumour.
Known Hp bacterial strain can both produce two kinds of heat shock protein(HSP)s, i.e. HspA and HspB.HspA is made up of 118 amino acid, and 91 amino acid of N end and intestinal bacteria GroES homology are very high, can be used as molecular chaperones, plays an important role in synthesizing, assemble at Hp albumen; 27 amino acid regions of C end are peculiar by Hp, are rich in Histidine and halfcystine, are nickel ion calmodulin binding domain CaMs and relevant with urease activity.The coexpression in intestinal bacteria with hspA and urease gene, the ureaclastic ability of urease strengthens more than 4 times.In a word, HspA participates in the assembling of urease and the performance of urease activity, is one of important virulence factor of Hp.HspA can excitating organism produces one of effective antigens of protective immune response, is the important candidate composition of Hp subunit vaccine.
Helicobacter pylori HspA is the homologous protein of HSP family member GroES (Hsp10), compares with other bacterium, and the HspA of Hp has uniqueness, includes a metal ion binding site at the C of HspA art end (being the b zone of HspA).Discover that HspA and nickel ion bonded ability are far longer than bonding force (the Kansau I with cobalt, iron, zinc, cupric ion, Guillain F, et al.Nickel binding andimmunological properties of the c-terminal domain of the Helicobacterpylori GroES homologue (HspA) .Mol Microbiol, 1996,22 (5): 1013-1023).The applicant has made up the colibacillus engineering (Ran Xiangyang that efficiently expresses recombinant helicobacterpylori heat shock protein(HSP) A, Zou Quanming etc. recombinant helicobacterpylori heat shock protein(HSP) A subunit efficiently expresses and preliminary purification. cell and molecular immunology magazine [J] .2002,18 (6): 539-542).
The applicant is groping that first purification of samples to rHspA is further purified, discovery is under non-sex change condition, ion exchange column, hydrophobic chromatography post and gel permeation chromatography post, all the foreign protein of 30KD size can not be removed, and this albumen is main foreign protein in the rHspA sample behind the affinitive layer purification.Best (the Amersham Pharmacia Biotech AB of the resolving power of employed Superdex 75 gel permeation chromatography posts in protein molecular is 3～70KD scope, Gel Filtration Principles and Methods, code No.18-1022-18), in theory should be able to be with the albumen sepn of 30KD and 16KD, yet in the Superdex75 gel permeation chromatography on the affine sample of rHspA behind the sample wash-out only formed simple spike (seeing accompanying drawing 1), before SDS-PAGE result shows the peak, the peak point, the foreign protein of tail of the peak 30KD accompanies with rHspA all the time, analyzes this foreign protein and may form polymer with rHspA.
Opening the polymeric common method of albumen is to use denaturing agent such as SDS, urea, Guanidinium hydrochloride etc. and adds reductive agent such as beta-mercaptoethanol, DTT (Ling Mingsheng, Xu Xiangyu, Ding Shubiao. the purifying of the recombinant protein that exists with inclusion body and external folding. Chinese biochemical drug magazine, 1995,16 (3): 135-139), attempted the first purification of samples of rHspA is concentrated back Guanidinium hydrochloride cracking, renaturation, repurity, can observation separate the 30KD foreign protein with rHspA.The result shows, yet both is not separated.Analysis may be that 30KD foreign protein and rHspA have separated under the sex change condition, but in renaturation process, both have formed polymer again.
This shows and adopt conventional purifying process can not obtain highly purified rHspA.
Summary of the invention
Purpose of the present invention is intended to can not be directly applied for the constructed recombinant helicobacterpylori heat shock protein(HSP) A recombinant vaccine albumen of the inventor at existing purification process, and provide a kind of technology simple and direct, the purification process of the purity of protein high-recovery that obtains recombinant helicobacterpylori heat shock protein(HSP) A recombinant vaccine preparation preferably.
The present invention has adopted following steps:
(1) height crushes bacterium: the colibacillus engineering thalline 200-500g that will efficiently express solubility expression reorganization heat shock protein(HSP) A (rHspA) is floating with 1: 10 (W/V) ratio suspendible of nickel ion affinity chromatograph level pad suspension, adopt the cell homogenates machine that it is mixed after 4 ℃ of precoolings, break bacterium 4～6 times with high pressure homogenizer, high speed centrifugation is collected supernatant.
(2) filter: directly use the nickel ion affinity chromatograph post to carry out elementary purifying by filtering with microporous membrane above-mentioned supernatant liquor.
(3) nickel ion affinity chromatograph column purification: select the nickel ion affinity chromatograph post to carry out preliminary purification, use 20mmol/L Tris under pH7.0～9.0 conditions, target protein to be carried out purifying, collect the target protein peak.
(4) concentrate, cracking: with step (3) just purification of samples add Guanidinium hydrochloride or urea after concentrating, and DTT or beta-mercaptoethanol sex change cracking;
(5) Superdex gel permeation chromatography purifying:,, use gel-filtration level pad balance with gel-filtration column Superdex purifying with the metaprotein sample that step (4) is obtained;
(6) renaturation: adopt dilution to wait step (5) purification of samples and hold dialysis method or chromatography column renaturation method renaturation, the sample after the renaturation adopts Superdex gel permeation chromatography separating monomer and aggressiveness.
The height that uses in described employing production of step (1) or the pilot scale purifying crushes the bacterium technology, and broken bacterium to bacterium is cracked rate greater than 98%, and differential centrifugation obtains the inclusion body throw out.
The described centrifuged supernatant of step (2) directly goes up the chromatography column purification behind 0.45 μ m filtering with microporous membrane.
The described nickel ion purifying of step (3) filler is one of Chelating Sepharose HP, ChelatingSepharose FF.
First purification of samples ultrafiltration and concentration to the protein concentration of step (4) is 20～30g/L, and the sex change cracking is to add Guanidinium hydrochloride to 6～7mol/L, adding DTT to 50mmol/L, 4 ℃, stirs more than 30 minutes;
The gel permeation chromatography post that step (5) is used is one of Superdex 75, Superdex 200, SuperdexHR 10/30.
The chromatography column renaturation method that step (6) is used, can adopt Sephadex G-25 desalination to remove on urea renaturation and the nickel ion affinity chromatograph post and repeat to fold, the nickel ion affinity chromatograph column packing is one of Chelating SepharoseHP, Chelating Sepharose FF.
The invention effect
The rHspA that the inventor is constructed, directly sample carries out under the unfavorable situation of purification effect under natural condition behind the use nickel ion affinity chromatograph, under the sex change condition, directly the affinitive layer purification sample to rHspA is further purified, adopt the nickel ion affinity chromatograph post, from the broken bacterium liquid of large intestine engineering bacteria of expressing HspA, extract rHspA, obtained the ideal purification effect, sex change rHspA to final purifying acquisition, adopt dilution refolding or chromatography on-column refolding, thereby can obtain purity greater than 98% rHspA.(rHspA purity＞75% that preliminary purification gets, the rHsp A molecular weight that makes up acquisition through the evaluation inventor is about 15KD).
Referring to Fig. 2, show by SDS-PAGE, under 8mol/L urea condition, use Superdex 75 gel permeation chromatography posts to find, compare with non-sex change condition, occurred bimodal at 130ml～170ml place in the chromatography collection of illustrative plates, SDS-PAGE shows the 30KD foreign protein is separated with target protein rHspA, and rHspA purity＞98% behind the purifying, and the target protein yield is about 67%, referring to Fig. 3,1 is the low molecular protein standard among the figure; 2 is the protein sample before the purifying not; 3 is the sample behind the nickel ion affinity chromatograph purifying; 4 for Superdex under the sex change condition 75 foreign protein peak sample when secondarily purified, based on the 30KD foreign protein; 5 ~ 10 target protein peak samples for Superdex under the sex change condition 75 collection when secondarily purified.
RHspA by above-mentioned art breading different batches makes 15%SDS-PAGE, presents single band, and molecular mass is about 15KD.The analysis of HPLC C4 post presents single peak, and purity is more than 98%.Iso-electric point is about pH6.2.Different each peak-to-peak number average of rHspA collection of illustrative plates are consistent, and the retention time at each peak all fluctuates in ± 10s, and the peptide mapping repeatability is good.RHspA behind the purifying and mucosal adjuvant CT (Toxins,exo-, cholera) or LTB (E.coli LT B subunit) be oral immunity BalB/c mouse altogether, find that IgA and IgG level that rHspA adds in mucosal adjuvant group serum, the gastrointestinal fluid are significantly higher than control group (PBS group) (P＜0.01), but prove that HspA effective stimulus body produces higher immunne response; After rHspA and mucosal adjuvant CT (Toxins,exo-, cholera) or LTB (E.coli LT B subunit) are total to the oral immunity mongolian gerbil; adopt Hp SS1 strain (Australian Lee professor A. is so kind as to give) to infect; find that it is 21% that rHspA adds mucosal adjuvants group mongolian gerbil infection rate; control group (PBS group) infection rate is 87%, and the protection ratio that calculates the anti-Hp infection of rHspA is 70%.
Description of drawings
Fig. 1 is the Superdex 7526/60 gel permeation chromatography collection of illustrative plates of rHspA affinity purification sample under the non-sex change condition;
Fig. 2 is rHspA affinity purification sample Superdex 7526/60 a gel permeation chromatography purifying collection of illustrative plates under the sex change condition;
Fig. 3 is Superdex 7526/60 a chromatography column purifying rHspA affinity purification sample result under the sex change condition.
Embodiment
Below in conjunction with embodiment the present invention is described in detail:
Embodiment one:
With the recombinant helicobacterpylori heat shock protein(HSP) A engineering bacteria that the applicant makes up, by high density fermentation, the target protein expression rate is 23%, and centrifugal collection thalline carries out purifying according to the following steps:
(1) height crushes bacterium: the thalline 500g that efficiently expresses is floating with 1: 10 (W/V) ratio suspendible of TE nickel ion affinity chromatograph damping fluid suspension, and adopt the cell homogenates machine that it is mixed after 4 ℃ of precoolings.Adopting high pressure homogenizer is break bacterium under the condition of 75Mpa (to break bacterium 5 times altogether at pressure, crack rate greater than 98% to bacterium), after broken bacterium finishes, bacterium liquid smear staining takes a morsel, the integrity of microscope observing cell guarantees that cytoclasis is complete, with 15, the centrifugal 40min of 000g collects supernatant and abandons precipitation.
(2) filter: directly use the nickel ion affinity chromatograph post to carry out elementary purifying by 0.45 μ m filtering with microporous membrane above-mentioned supernatant liquor.
(3) nickel ion affinity chromatograph column purification: select nickel ion affinity chromatograph post Chelating SepharoseHP to carry out preliminary purification, use 20mmol/L Tris under the pH8.5 condition, target protein to be carried out purifying, nickel ion affinity chromatograph level pad: 10mmol/L imidazoles, 20mmol/L Tir, 0.5mol/L NaCl), adopt imidazoles (0.5mol/L imidazoles, 20mmol/L Tris, 0.5mol/L NaCl) gradient elution is collected the target protein peak.
(4) concentrate, cracking: step (3) just purification of samples after ultrafiltration (film bag aperture is less than 5KD) is concentrated into protein concentration and is 30g/L, add Guanidinium hydrochloride to 6mol/L, add DTT to 50mmol/L, 4 ℃, stir more than 30 minutes.
(5) Superdex gel permeation chromatography purifying: under the sex change condition, the metaprotein sample that step (4) is obtained, adopt gel-filtration column Superdex 75 purifying (level pad: 8mol/L urea, 0.15mol/L NaCl, 20mmol Tris, pH8.5), collect target peak, when ultraviolet absorption peak is zero, stop.
(6) renaturation: use nickel ion affinity chromatograph post ChelatingSepharose FF to carry out on-column refolding method renaturation the HspA of purifying under the sex change condition, method is that chromatography column is through balance liquid (8mol/L urea, 20mmolTris, pH8.5) after the balance, last sample sex change target protein sample, use renaturation solution (20mmol/L PBS, 2mmolGSH, 0.2mmol/L GSSG, 0.15mol/L NaCl) slowly gradient elution post to urea is removed fully, uses imidazole buffer (20mmol/L PBS, 0.5mol/L imidazoles, pH8.5) wash-out target protein is collected protein peak.
Embodiment two:
Sample preparation is used nickel ion affinity purification post Chelating Sepharose FF preliminary purification with example one, and purification condition is with example one; Adopt 8mol/L urea and 50mmol/L DTT sex change crack protein; Under the sex change condition, (level pad: the 6mol/L Guanidinium hydrochloride, 20mmolTris pH8.5), collects the target protein peak to adopt gel-filtration column Superdex 75 purifying.Metaprotein adopts gel-filtration column G-25 to carry out the post desalination and removes the denaturing agent renaturation, uses renaturation solution (20mmol NaHCO on the albumen behind the sample
3/ Na
2CO
3, 20mmol EDTA pH10), collects the target protein peak.
Embodiment three:
Sample preparation, purification condition are with example one; The sex change crack protein adopts 8mol/L urea and 50mmol/LDTT; Under the sex change condition, and employing gel-filtration column Superdex 200 purifying (level pad: the 6mol/L Guanidinium hydrochloride, 20mmol Tris pH8.5), collects the target protein peak; The HspA metaprotein, use nickel ion affinity chromatograph post Chelating Sepharose HP to carry out on-column refolding, method is that chromatography column is through balance liquid (8mol/L urea, 20mmol Tris, pH8.5) after the balance, last sample sex change target protein sample, use renaturation solution (20mmol/L PBS, 2mmol GSH, 0.2mmol/L GSSG, 0.15mol/L NaCl) slowly gradient elution post to urea remove fully, use imidazole buffer (20mmol/L PBS, 0.5mol/L imidazoles, pH8.5) wash-out target protein is collected protein peak.
Embodiment four:
Sample preparation is used nickel ion affinity purification post Chelating Sepharose FF preliminary purification with example one, and purification condition is with example one; The sex change crack protein adopts 6mol/L Guanidinium hydrochloride and 50mmol/L DTT; Under the sex change condition, (level pad: the 6mol/L Guanidinium hydrochloride, 20mmol Tris pH8.5), collects the target protein peak to adopt gel-filtration column Superdex HR 10/30 purifying.Metaprotein adopts gel-filtration column G-25 to carry out the post desalination and removes the denaturing agent renaturation, uses renaturation solution (20mmol NaHCO on the albumen behind the sample
3/ Na
2CO
3, 20mmolEDTA pH10), collects the target protein peak.
Result: 15%SDS-PAGE, target protein present single band, and the branch quality is about 15KD.The analysis of HPLC C4 post presents single peak, and purity is 98.2%.Iso-electric point is about pH6.2.The oral immunity BALB/c mouse can stimulate mouse to produce specificity sIgA secretion, and mongolian gerbil is attacked malicious protection test and confirmed that it can effectively induce gerbil jird to prevent and removing Hp infects, and protection ratio is 70%.
Claims (7)
1. the purification process during a recombinant helicobacterpylori heat shock protein(HSP) A recombinant vaccine prepares, it is characterized in that: after expressing rHspA with the fermentation mode high-density, adopt height to crush bacterium, filtration, the sequential combination of nickel ion affinity chromatograph, concentrated cracking, gel permeation chromatography and renaturation technology, obtain high purity rHspA, step is as follows:
(1) height crushes bacterium: the colibacillus engineering thalline that will efficiently express solubility recombinant helicobacterpylori heat shock protein(HSP) A is floating with nickel ion affinity chromatograph level pad suspendible, adopt the cell homogenates machine that it is mixed after the precooling, with the broken bacterium of high pressure homogenizer, high speed centrifugation is collected supernatant;
(2) filter: above-mentioned supernatant liquor is stand-by by filtering with microporous membrane;
(3) nickel ion affinity chromatograph column purification: select the nickel ion affinity chromatograph post to carry out preliminary purification, use Tris under pH 7.0～9.0 conditions, target protein to be carried out purifying, adopt the imidazoles gradient elution;
(4) concentrate, cracking: with step (3) just purification of samples add Guanidinium hydrochloride or urea after concentrating, and DTT or beta-mercaptoethanol sex change cracking;
(5) Superdex gel permeation chromatography purifying:,, use gel-filtration level pad balance with gel-filtration column Superdex purifying with the metaprotein sample that step (4) is obtained;
(6) renaturation: adopt dilution to wait step (5) purification of samples and hold dialysis method or chromatography column renaturation method renaturation, the sample after the renaturation adopts Superdex gel permeation chromatography separating monomer and aggressiveness.
2. purification process described in claim 1 is characterized in that the high-pressure homogeneous broken bacterium technology of the 60～80Mpa that uses in described employing production of step (1) or the pilot scale purifying, and broken bacterium to bacterium is cracked rate greater than 98%, and differential centrifugation obtains the inclusion body throw out.
3. the purification process described in claim 1 is characterized in that: the described centrifuged supernatant of step (2), directly go up the chromatography column purification behind 0.45 μ m filtering with microporous membrane.
4. purification process described in claim 1, it is characterized in that: the described nickel ion purifying of step (3) filler is one of Chelating Sepharose HP, Chelating Sepharose FF.
5. purification process described in claim 1, it is characterized in that: first purification of samples ultrafiltration and concentration to the protein concentration of step (4) is 20～30g/L, the sex change cracking is to add Guanidinium hydrochloride to 6～7mol/L or urea 6～8mol/L and add DTT or beta-mercaptoethanol to 10～50mmol/L, 4 ℃, stir more than 30 minutes.
6. purification process described in claim 1, it is characterized in that: the gel permeation chromatography post that step (5) is used is one of Superdex 75, Superdex 200, Superdex HR 10/30.
7. purifying construction method described in claim 1, it is characterized in that: the chromatography column renaturation method that step (6) is used, can adopt Sephadex G-25 desalination to remove on urea renaturation and the nickel ion affinity chromatograph post and repeat to fold, the nickel ion affinity chromatograph column packing is one of Chelating Sepharose HP, Chelating Sepharose FF.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410022360 CN1241938C (en) | 2004-04-20 | 2004-04-20 | Purification technique in preparing genetic engineering vaccine of heat shock protein A of recombined Helicobacter pylori |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410022360 CN1241938C (en) | 2004-04-20 | 2004-04-20 | Purification technique in preparing genetic engineering vaccine of heat shock protein A of recombined Helicobacter pylori |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1563406A CN1563406A (en) | 2005-01-12 |
| CN1241938C true CN1241938C (en) | 2006-02-15 |
Family
ID=34480059
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410022360 Expired - Fee Related CN1241938C (en) | 2004-04-20 | 2004-04-20 | Purification technique in preparing genetic engineering vaccine of heat shock protein A of recombined Helicobacter pylori |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1241938C (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962541A (en) * | 2015-03-31 | 2015-10-07 | 芜湖康卫生物科技有限公司 | Purifying process for oral recombinant helicobacter pylori vaccine |
| CN107459575B (en) * | 2016-06-03 | 2021-08-31 | 硕英生医股份有限公司 | Monoclonal antibody having immunosuppressive function against pathogen, antigen-binding fragment thereof, and hybridoma producing same |
| CN107417765B (en) * | 2017-09-26 | 2020-12-04 | 珠海联邦制药股份有限公司 | Method for separating and purifying recombinant protein in escherichia coli autolysis expression system |
| CN112812969A (en) * | 2021-01-12 | 2021-05-18 | 福建基诺厚普生物科技有限公司 | System purification method for recombinant expression polypeptide in genetic engineering |
-
2004
- 2004-04-20 CN CN 200410022360 patent/CN1241938C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1563406A (en) | 2005-01-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100460013C (en) | Oral administration recombinant helicobacterpylori vaccine and preparing method thereof | |
| CN106967696B (en) | Allergen of artemisia pollen and application thereof | |
| WO2013065009A1 (en) | A sortase-click reaction suite for synthesis of multivalent dendrimeric protein assembly | |
| JP2005097311A (en) | Antigen | |
| CN101062015A (en) | Urease epitope fusion peptide liposome bacterin for preventing the helicobacter pylori infecting | |
| WO1995033482A1 (en) | Treatment and prevention of helicobacter infection | |
| CN1241938C (en) | Purification technique in preparing genetic engineering vaccine of heat shock protein A of recombined Helicobacter pylori | |
| CN110642927B (en) | Application of protein in preparation of medicine for preventing cryptococcus pyogenes infection | |
| US7517533B2 (en) | Pseudomonas aeruginosa antigens | |
| CN1844398A (en) | Fusion expression method for metallothionein and use thereof | |
| CN1275981C (en) | Method of monomerizing human serum albumin polymers | |
| JPH06511154A (en) | Method for producing recombinant Borrelia protein | |
| CN109337853B (en) | Purification method of pseudomonas aeruginosa genetic engineering vaccine candidate antigen rExoU | |
| CN108251445B (en) | Large-scale preparation process and application of GCRV II S9 and S10 recombinant proteins | |
| CN112999341B (en) | Edwardsiella tarda outer membrane protein vaccine and preparation and application thereof | |
| RU2799574C1 (en) | Method of producing cholera toxin for control of production of cholera chemical vaccine | |
| Young et al. | Reassembly of a fimbrial hemagglutinin from Pseudomonas solanacearum after purification of the subunit by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis | |
| CN116217680B (en) | Immunoglobulin binding proteins with high alkali stability and uses thereof | |
| RU2478644C2 (en) | Method for preparing pharmacologically acceptable mixture of substances containing low-molecular ingredients of cell wall peptidoglycane of gram-negative bacteria and possessing immunostimulatory activity | |
| CN1563388A (en) | Constructing genetic engineering Vaccine of adhesin of confluent Helicobacter pylor and preparation method | |
| JP3693900B2 (en) | Method for producing protein having 1A5B structure | |
| CN113896775A (en) | Specific immunogenic peptide fragment of helicobacter pylori | |
| US20210308245A1 (en) | Construction and use of tandem epitope polypeptide of outer membrane protein of campylobacter jejuni, gene, recombinant plasmid and recombinant bacteria | |
| CN1648254A (en) | Goose parvo-virus VP1 and VP3 gene non-overlapping sequence pronucleus expression protein and its preparing method | |
| AU702878B2 (en) | Treatment and prevention of helicobacter infection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: CHONGQING KANGWEI BIOLOGICAL TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: MILITARY MEDICAL UNIV NO.3, P.L.A. CLINICAL MICROBIOLOGY STAFF ROOM, MEDICAL LABORATORY DEPARTMENT, THIRD MILITARY MEDICAL UNIVERSITY Effective date: 20060630 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20060630 Address after: 400038 Chongqing Shapingba city gaotanyan Garden City Yuyuan Yan building B 7-5 Patentee after: Chongqing Kangwei biotechnology Ltd. Address before: Chongqing city Shapingba street 400038 gaotanyan No. 30 Department of clinical microbiology Patentee before: Third Military Medical University, Chinese People's Liberation Army |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20060215 Termination date: 20180420 |