CN1737153A - A kind of recombinant BCG and its preparation method - Google Patents
A kind of recombinant BCG and its preparation method Download PDFInfo
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- CN1737153A CN1737153A CN 200510027967 CN200510027967A CN1737153A CN 1737153 A CN1737153 A CN 1737153A CN 200510027967 CN200510027967 CN 200510027967 CN 200510027967 A CN200510027967 A CN 200510027967A CN 1737153 A CN1737153 A CN 1737153A
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Abstract
The invention relates to a recombinant bacillus Calmette-Guerin vaccine and its preparation method, wherein tuberculosis mycobacterium ag85b, esat-6 and IFN-gamma gene sequences are inserted into colibacillus-tuberculosis mycobacterium shuttle plasmids to form recombinant plasmid, and are transformed into BCG to form recombinant tuberculosis vaccine. The recombinant vaccine can be used for prevention and treatment of tuberculosis with better immunological effects than BCG.
Description
Technical field
The invention belongs to the recombinant vaccine field, specifically, the invention provides the recombiant vaccine of a kind of novel tubercule bacillus, BCG is gone in the IFN-γ gene clone that is about to tubercule bacillus ag85b, esat-6 and mouse/people.
Background technology
Tuberculosis still constitutes huge threat to the mankind.The global at present population near 32% has infected tubercule bacillus.Have every year 2700000 people to die from tuberculosis approximately, average every day is dead above 7000 people.The World Health Organization announces that in 1993 tuberculosis is the global healthy emergency state.
China's tuberculosis epidemic situation is more serious, is one of the high burden of 22 tuberculosis in whole world country, and the tuberculosis number occupies the second place of the world.The 4th national tuberculosis epidemiological random sampling survey interpretation of result of China in 2000, infected number surpasses 400,000,000.In 10 years of future, have 3,000 ten thousand people tuberculosis takes place.Investigation shows that China's tuberculosis epidemic situation is still quite serious, and there is spreading trend some areas.
Tuberculosis is popular be difficult to containment main increase with the multiple-drug resistance tuberculosis bacterial strain and acquired immune deficiency syndrome (AIDS) popular relevant.
Bacille Calmette-Guerin vaccine (BCG) comes out year surplus in the of 80, and global most countries is with its immunization project on the schedule.Practice shows that bacille Calmette-Guerin vaccine is a kind of safe vaccine, and its side effect is slight, and human body can be stood.The existing at present people more than 3,000,000,000 has inoculated BCG, and have the newborn infant about 100,000,000 to inoculate this vaccine every year.It can stop the generation of year child's miliary tuberculosis and tuberculous meningitis preferably.Yet it can not protect the phthisical generation of modal adult.There is data to show that BCG has 0% of South India not wait to 80% of Britain to adult's protection effect.Although deliver the documentation integrators virtual value is 50%, and its preventive effect to tuberculosis death only accounts for 5% according to estimates, therefore, is necessary to develop more effective vaccine and replaces existing BCG vaccine.
In the ascendant to the research of novel tuberculosis vaccine both at home and abroad.The vaccine of setting about at present studying is nothing more than nucleic acid vaccine, protein subunit vaccine, attenuated live vaccine, recombinant bacillus Calmette-Guerin vaccine etc.The research of recombinant bacillus Calmette-Guerin vaccine at present mainly concentrates following three aspects: 1. recombinant cytokine is as IFN-γ, IL-2, and IL-12 and GM-CSF can stimulate stronger potential immune response; 2. the protective antigen of recombinant tubercle bacillus increases the antigenicity protective number as Ag85B; 3. the protective antigen of recombinant bacillus Calmette-Guerin vaccine disappearance is as ESAT-6, CFP-10.Also there is report that Ag85B and ESAT-6 are merged the back reorganization recently and advances bacille Calmette-Guerin vaccine, and to good than the term single gene reorganization of the protection effect of tubercule bacillus.But the IFN-γ gene clone of tubercule bacillus ag85b, esat-6 and mouse (people) is gone into BCG through looking into new no relevant report.
Summary of the invention
An object of the present invention is to provide a kind of new recombinant plasmid.
Another object of the present invention provides a kind of vaccine by above-mentioned recombinant plasmid preparation.
A further object of the present invention provides a kind of preparation method of recombiant vaccine.
An object of the present invention is to provide a kind of new recombinant plasmid, be about to ag85b, esat-6 and IFN-gamma gene sequences and insert in same intestinal bacteria-mycobacterium tuberculosis shuttle plasmid sequence, thereby form recombinant shuttle plasmid.
In the above-mentioned recombinant plasmid, the common multiple clone site place that ag85b, esat-6 and IFN-γ are inserted same intestinal bacteria-mycobacterium tuberculosis shuttle plasmid is beneficial to insert the stably express of gene.
In one embodiment of the invention, the multiple clone site place that ag85b, esat-6 and IFN-γ are inserted same intestinal bacteria one mycobacterium tuberculosis shuttle plasmid successively.
Among the present invention, ag85b, esat-6 are from mycobacterium tuberculosis (H37RV), and IFN-γ can be the people source, can also be inhuman source.In one embodiment of the invention, IFN-γ is the mouse source.
In the recombinant plasmid preparation process of the present invention, adoptable intestinal bacteria-mycobacterium tuberculosis shuttle plasmid has pWV261, pWV361, pSD7 etc.In one embodiment of the invention, used intestinal bacteria-mycobacterium tuberculosis shuttle plasmid is pWV261.
On the other hand, the invention provides a kind of novel tubercule bacillus recombiant vaccine, the recombinant plasmid transformed that is about to the above-mentioned ag85b of containing, esat-6 and IFN-γ gene is gone into bacille Calmette-Guerin vaccine, thereby obtains rBCG ∷ AEI recombiant vaccine.
Ag85B is an integral part of Ag85 complex body.The Ag85 complex body is chief component and the protective antigen in BCG and the tubercule bacillus nutrient solution, is easy to be incorporated into people's fibronectin, in conjunction with the formation of back involved in diseases.It is synthetic relevant with the mycobacterium cell walls also.Ag85B is the strongest a kind of of antigenicity.Ag85B can induce the intensive delayed type hypersensitivity in the cavy of sensitization, very similar to PPD.Plasmid to mouse injection coding Ag85B can induce the intensive Th1 sample at the Ag85 complex body to react, but specificity rising IL-2, the level of IFN-γ and IFN-α.
Going down to posterity at Mtuberculosis.bovis forms in the process of bacille Calmette-Guerin vaccine, virulence occurs and reduces, and follows incomplete antigen to lose.Promptly comprise EAST-6 in the antigen of losing.Tubercule bacillus secretory protein ESAT-6 is a kind of important T cell antigen, is the major antigen target position that the T lymphocyte is discerned.Can be infected the animal model of Mycobacterium tuberculosis and the protectiveness T lymphocyte of tuberculosis patient discern.Also can induce the intensive t cell responses at mouse model; In natural infection mycobacterium tuberculosis case, experiment in vitro finds that the T cell has intensive at this antigenic IFN-gamma reaction.
IFN can be used for treating non-viral to be infected, and majority is an intracellular bacterial parasite infection curing.Its mechanism is not direct pathogen kill, but works by strengthening cellular immune function.The mechanism of IFN-γ treatment mycobacterium tuberculosis infection: 1. activated lymphocyte non-specific cell toxic action: IFN can activate natural killer cell (NK cell), and the NK cell is therefore one of the prerequisite of activated killer cell (LAK cell) of lymph, and gamma-interferon is to stimulate the NK cell, the lymphokine of activation LAK cell, these two kinds of cells all have the non-specific cell cytotoxic activity.2. activating macrophage: IFN-γ is macrophage activating factor (MAF) (MAF), plays a role in eliminating tubercule bacillus by activating macrophage.Its function has: (1) strengthens adhesivity, phagocytic function and the pinocytosis activity of scavenger cell; (2) promote the picked-up of scavenger cell to glucosamine; (3) by strengthening the active antibacterial or germicidal action of oxyradical in the born of the same parents.3. regulate and control main histocompatibility (MHC) antigen presentation: α, interferon-can just be regulated I class MHC antigen (HLA-A, B, C) expression, expression to II class (HLA-DR) does not have regulating effect, and IFN-γ can regulate two classes (I, II) antigen presentation of some cell, these cells comprise scavenger cell, star spongiocyte and inoblast have consequently strengthened T cellullar immunologic response function greatly, and then produce IFN-γ again, so just form the positivity feedback, progressively raise immunity.Interferon, rabbit also can be regulated other cytokine receptor expression on the cytolemma, IL-2, TNF-α and scavenger cell Fc acceptor, raise immunity indirectly.Therefore, for tuberculosis patient, especially the low patient of body's immunity uses interferon therapy, is a kind of effective means.
With the immune C57BL/c mouse of rBCG ∷ AEI recombiant vaccine (BCG is a bacille Calmette-Guerin vaccine, and " ∷ " represents reorganization, and A is Ag85B, and E is ESAT-6, and I is IFN-γ), use bacille Calmette-Guerin vaccine (BCG) and PBS simultaneously respectively as contrast.The result shows that the inductive serum Ag85B of institute antibody horizontal, ESAT-6 antibody horizontal and splenocyte IFN-γ express all to have and increase.This explanation rBCG ∷ AEI recombiant vaccine can be induced body fluid and the cellular immune level high than BCG, has better immune effect.
On the other hand, the invention provides the preparation method of above-mentioned rBCG ∷ AEI recombiant vaccine.Mainly may further comprise the steps:
(1) preparation of mycobacterium tuberculosis strain gene group DNA;
(2) ag85b, the amplification of esat-6 and IFN-γ gene;
(3) ag85b, sat-6 and IFN-γ gene insert same carrier respectively;
(4) be transformed into bacille Calmette-Guerin vaccine with the recon that contains ag85b, sat-6 and IFN-γ gene.
Various experiment parameters are operation selection routinely all, is decided by concrete experiment condition.
This vaccine has following advantage:
1. spectrotype is near tubercule bacillus: be cloned into the esat-6 gene in the recombinant BCG, and antigen expressed ESAT-6, wherein ESAT-6 is the protective antigen that bacille Calmette-Guerin vaccine is lost.
2. strengthened the protective antigen amount: be cloned into the ag85b gene in the recombinant BCG.
3. effect is better than BCG: because recombinant BCG is living vaccine, secretion antigen lastingly is so can keep longer immune effect.Dna vaccination and protein subunit vaccine effect all are not better than BCG.And this vaccine newly developed has increased important T cell antigen and the antigen of losing thereof again owing to both kept the antigen of former BCG, has also merged the gene of IFN-γ, can express IFN-γ.IFN can bring into play anti-microbial effect indirectly, so effect is better than BCG.
Description of drawings
Fig. 1 cuts evaluation figure for the PAEI enzyme.1, dna molecular amount standard: 2, recombinant plasmid; 3, the Ag85B double digestion; 4, the Esat-6 double digestion; 5, IFN-γ double digestion.
Fig. 2 is that rBCG ∷ AEI PCR identifies figure.6, dna molecular amount standard; 7, the Esat-6PCR band; 8, IFN-γ PCR band.
Fig. 3 is rBCG ∷ AEI Western blotting (protein imprinted hybridization) figure.9, the molecular weight of albumen standard; 10, the BCG band; 11, rBCG ∷ AEI band.
Embodiment
The structure of embodiment 1 recon PAEI
(1) preparation of mycobacterium tuberculosis H37Rv strain gene group DNA
Mycobacterium tuberculosis (H37Rv) 37 ℃, leaves standstill and cultivated for 2 weeks in the 7H9Broth liquid nutrient medium.80 ℃, deactivation in 2 hours.With DNA of bacteria (in a small amount) extraction agent box extracting genomic dna.Wherein because of the wall thickness of tubercule bacillus, the digestion time of bacterium extends to 3-5 hour.
(2) mycobacterium tuberculosis H37Rv bacterial strain ag85b, the amplification of the IFN-γ gene of esat-6 and mouse
According to ag85b, IFN-γ gene of esat-6 and mouse and carrier multiple clone site design primer:
Ag85B:5 ' end primer P1:ATATGGCCAATGACAGACGTGAGCC
3 ' end primer P2:TATGAATTCGCCGGCGCCTAACG
Esat-6 5 ' end primer P1:TATTGGCCAGAATTCATGACAGAGCAGCAGTGG
3 ' end primer P2:TTAGTCGACTGCGAACATCCCAGTG
The IFN-γ gene of mouse
5 ' end primer P1:AATGTCGACATGCAGGACCCGTAC
3 ' end primer P2:ATTGTTAACCTATTACTGAGAAGCACG
PCR reaction system: 10 * buffer 5 μ l, dNTPs 5 μ l, dna profiling 1 μ l, each 1 μ l of primer, Tag enzyme 0.25 μ l, water 36.75 μ l.Reaction conditions: 94 ℃ of 1min, 66 ℃ of 1min, 72 ℃ of 3min, totally 30 circulations, 72 ℃ are extended 10min then.Product is identified with 1% agarose gel electrophoresis.
(3) enzyme of carrier pWV261, ag85b is cut, is connected and transforms
The PCR product of carrier pWV261 plasmid, ag85b uses BalI and EcorI double digestion, 37 ℃, 4 hours.Reclaim the test kit purifying enzyme with glue and cut product.The T4DNA ligase enzyme connects, and 16 ℃, spends the night.
Preparation E.coliDH5 α competent cell: get the single bacterium colony of DH5 α in the 3mlLB nutrient solution, 37 ℃ of shaking culture are spent the night, transferred in 30mlLB nutrient solution in by 1: 100 next day, 37 ℃ of shaking culture 2 hours, 10min is placed in 0 ℃ of cooling, 4 ℃ of centrifugal 5min of 5kr/min collect thalline, are resuspended in 10ml0.1mol/LCaCl
2In.Ice bath 20min, 4 ℃ of centrifugal 5min of 5kr/min collect thalline.Add precooling 0.1mlmol/LCaCl
2Ice bath is placed standby more than 1 hour.
Transform: 10ml is connected product add in the 200mlE.coliDH5 α competent cell ice bath 30min, 37 ℃ of heat-shocked 5min, ice bath 2min immediately, add LB nutrient solution 800ml, 37 ℃ of shaking culture 1 hour are coated the dull and stereotyped last 37 ℃ of overnight incubation of the LB that contains 50 μ g/ml Kana then.Select single bacterium colony and be inoculated in 3ml respectively and contain in the LB nutrient solution of 50 μ g/ml Kana, 37 ℃ of shaking culture are spent the night, centrifugal receipts bacterium, extracting plasmid DNA.Identify with BalI and EcorI double digestion.Choose correct recon (being called for short PA) and carry out next step experiment.
(4) enzyme of recon (PA), esat-6 is cut, is connected and transforms
The PCR product of recon (PA), esat-6 uses EcorI and SalI double digestion.Connect then, transform (the same).Choose correct recon (being called for short PAE) and carry out next step experiment.
(5) enzyme of the IFN-γ gene of recon (PAE), mouse is cut, is connected and transforms
The PCR product of the IFN-γ of recon (PAE), mouse uses SalI and HpaI double digestion.Connect then, transform (the same).Choose correct recon (being called for short PAEI) and carry out next step experiment.Final recon (PAEI) has been carried out enzyme cut evaluation (as Fig. 1), the evaluation of also checking order shows that institute's insertion sequence is consistent with the full genome corresponding gene sequences of tuberculosis among the Gene Bank.
(6) recon (PAEI) electricity is transformed into bacille Calmette-Guerin vaccine
The preparation of BCG competent cell: BCG 37 ℃, leaves standstill and cultivated for 2 weeks in the 100ml7H9Broth liquid nutrient medium.Then with its ice bath 2 hours, 8000g, 4 ℃, the centrifugal receipts of 15min bacterium; 10% the glycerine 5ml/ pipe that adds precooling, 8000g, 4 ℃, the centrifugal receipts of 15min bacterium; Repeat above-mentioned steps 5 times; Add 10% the glycerine 500 μ l/ pipe of precooling at last, ice bath is standby more than 1 hour.
Get recon (PAEI) 10 μ l and add in the 400 μ l BCG competent cells, ice bath 30min changes electricity over to and transforms in the cup, carries out electricity and changes, and parameter is: voltage: 2500V, and resistance: 1000 Ω, electric capacity:: 25 μ F.Add 7H9Broth liquid nutrient medium 800ml, 37 ℃ of shaking culture 1 hour are coated on the flat board that contains 50 μ g/ml Kana on the 7H10 substratum then, cultivate 3-4 weeks, have bacterium colony to grow for 37 ℃.Select single bacterium colony and be inoculated in 5ml respectively and contain in the 7H9Broth liquid nutrient medium of 50 μ g/ml Kana, 37 ℃ leave standstill and cultivate 2-3 week, get bacterium and protect kind.
The evaluation of embodiment 2 recombinant BCGs (rBCG ∷ AEI) gene level
Get the centrifugal receipts of the bacterium 200ml bacterium of above-mentioned cultivation, add 20 μ l water boiling water and boil 30min, the centrifuging and taking supernatant carries out PCR as template.
The result shows, the band that pcr amplification obtains conform to IFN-γ gene (467bp) stripe size of esat-6 (306bp) and mouse (as Fig. 2).Because of BCG itself contains the ag85b gene, so, also can't determine wherein whether to contain the ag85b gene of external source even pcr amplification obtains the ag85b band.
The evaluation of embodiment 3 recombinant BCGs (rBCG ∷ AEI) protein level
The bacterium of getting recombinant BCG (rBCG ∷ AEI) is inoculated in the 100ml modified Russell medium, 37 ℃, leaves standstill and cultivates 4-6 week.8000g, 4 ℃, the centrifugal receipts supernatant of 30min.Freezing then draining, dialysis, freezing again draining.
Western-blot result shows that the band of gained is about 60KD, conforms to the size of prediction.(as Fig. 3).
Claims (5)
1. a recombinant plasmid is characterized in that, ag85b, esat-6 and IFN-gamma gene sequences are inserted in same intestinal bacteria-mycobacterium tuberculosis shuttle plasmid sequence, forms recombinant shuttle plasmid.
2. recombinant plasmid according to claim 1 is characterized in that ag85b, esat-6 and IFN-γ insert the multiple clone site place of same intestinal bacteria-mycobacterium tuberculosis shuttle plasmid.
3. recombinant plasmid according to claim 1 is characterized in that used intestinal bacteria-mycobacterium tuberculosis shuttle plasmid is pWV261.
4. the recombiant vaccine of a tubercule bacillus is characterized in that, recombinant plasmid transformed is gone into bacille Calmette-Guerin vaccine according to claim 1, thereby obtains rBCG ∷ AEI recombiant vaccine.
5. recombinate the according to claim 1 preparation method of rBCG ∷ AEI recombiant vaccine is characterized in that this method may further comprise the steps:
(1) preparation of mycobacterium tuberculosis strain gene group DNA;
(2) ag85b, the amplification of esat-6 and IFN-γ gene;
(3) ag85b, sat-6 and IFN-γ gene insert same carrier respectively;
(4) be transformed into bacille Calmette-Guerin vaccine with the recon that contains ag85b, sat-6 and IFN-γ gene.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010127492A1 (en) * | 2009-05-07 | 2010-11-11 | 华中科技大学 | Recombinant bcg vaccine rbcg::xb |
| US8173773B2 (en) | 2006-01-10 | 2012-05-08 | The 309Th Hospital, The People's Liberation Army | Mycobacterium tuberculosis fusion protein and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| DE4116249A1 (en) * | 1991-05-17 | 1992-11-19 | Biotechnolog Forschung Gmbh | HYBRID PLASMIDE FOR M. TUBERCULOSIS ANTIGEN, E. COLI AS A HOST AND ANTIGEN |
| WO2003018053A1 (en) * | 2001-08-22 | 2003-03-06 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Vaccine against mycobacterial-induced diseases |
| KR100597699B1 (en) * | 2003-01-30 | 2006-07-07 | 학교법인 포항공과대학교 | DNA vaccine composition with enhanced immunogenicity |
| CN100374548C (en) * | 2004-07-22 | 2008-03-12 | 天津市泌尿外科研究所 | BCG vaccine strain of recombined interferon and its preparation process |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US8173773B2 (en) | 2006-01-10 | 2012-05-08 | The 309Th Hospital, The People's Liberation Army | Mycobacterium tuberculosis fusion protein and uses thereof |
| EP2574632A1 (en) | 2006-01-10 | 2013-04-03 | The Second Affiliated Hospital of General Hospital, PLA | A mycobacterium tuberculosis fusion protein and uses thereof |
| US8445662B2 (en) | 2006-01-10 | 2013-05-21 | The 309Th Hospital, The People's Liberation Army | Mycobacterium tuberculosis fusion protein and uses thereof |
| WO2010127492A1 (en) * | 2009-05-07 | 2010-11-11 | 华中科技大学 | Recombinant bcg vaccine rbcg::xb |
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