CN101985610A - Salmonella typhi ghost, preparation method and application thereof serving as delivery system - Google Patents
Salmonella typhi ghost, preparation method and application thereof serving as delivery system Download PDFInfo
- Publication number
- CN101985610A CN101985610A CN2010105195099A CN201010519509A CN101985610A CN 101985610 A CN101985610 A CN 101985610A CN 2010105195099 A CN2010105195099 A CN 2010105195099A CN 201010519509 A CN201010519509 A CN 201010519509A CN 101985610 A CN101985610 A CN 101985610A
- Authority
- CN
- China
- Prior art keywords
- ghost
- salmonella typhi
- ty21a
- plasmid
- cell
- 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
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a salmonella typhi ghost, a preparation method and application thereof serving as a delivery system. The ghost is prepared by cracking salmonella typhi thalli with cracking genes E, so that target modules can be delivered to targets by the ghost serving as a delivery vector.
Description
Technical field
The present invention relates to a kind of salmonella typhi ghost, its preparation method with and as the application of delivery system, belong to molecular biology and microbial technology field.
Background technology
(Bacterial Ghost is the E genetic expression of inducible phage PhiX174 in Gram-negative bacteria BG) to the bacterium ghost, the complete bacterium ghost that the host cell cracking is formed.Hydrophobic protein of being made up of 91 amino acid of PhiX174E genes encoding, this albumen itself be without any enzymic activity, but can play the effect that connects inside and outside film by oligomerization.Show that through electron photomicrograph interior adventitia is realized at the passage place merging, and forms the periplasmic space of a sealing.The diameter range of cracking passage is between 40nm and 200nm.The size of cracking passage is subjected to the influence of cell individual difference, relates generally to the degree that aqtocytolysis takes place, the variation that cytolemma took place in the size cases of sieve aperture and the cracking process on the peptidoglycan layer.In a single day passage forms, and under the ordering about of high osmotic pressure, intracellular most contents thing comprises that nucleic acid, rrna and other components are excluded out, have formed a complete ghost in cell.
The preparation of bacterium ghost is to realize by the strict expression regulation to lysis genes E.According to the study, the lysis genes E of PhiX174 can be controlled by a plurality of expression systems, as the control of transcribing of thermally sensitized λ pL/pR-cI857 promotor, or some chemical induced promoter L acPO-LacI
q, or expression system such as tol.
Experiment confirm E gene also can effectively cracking pleuritis pneumonia actinobacillus, Klebsiella pneumonia, haemolysis mannheim bacterium, p pestic, helicobacter pylori, Salmonella typhimurium, vibrio cholerae, Edwardsiella, Vibrio anguillarum etc. except that the cleavable intestinal bacteria.Infer that thus E cracking system can mediate all Gram-negative bacteria generation cracking.
Because preparation process is gentle and need not Denaturation, so cell walls is got off by complete preservation to a great extent.Bacterium ghost itself can be directly as a kind of good vaccine because it has kept coating structure and associated antigen protein under the viable bacteria state, as lipopolysaccharides, CPG and pili etc., thus can be effectively by antigen presenting cell (APCs) engulf, processing and submission.Present known ghost can and have been obtained good immune effect by animal models such as approach immune mouse, rabbit, pigs such as subcutaneous, vein, collunarium.The pleuropneumonia actinomycetes (A.pleuropneumoniae, APP) ghost has been induced the provide protection at APP through the intramuscular injection immune swine; While is in APP aerosol infection model, but the respiratory mucosa immunne response of APP ghost inducing producing specificity,, safety more effective than whole-cell vaccines.(V.cholerae Ghost VCG) through mode immune rabbits such as subcutaneous or intramuscular injection, can induce body fluid and the cellullar immunologic response of generation at the cell envelope composition to the vibrio cholerae ghost.
In addition, the bacterium ghost still is a kind of fabulous delivery system, utilizes genetic engineering means, can easily other compositions such as exotic antigen albumen, nucleic acid or medicine be imported in the bacterium ghost.External target antigen can anchor to adventitia (OM) or inner membrance (IM), or is filled in periplasmic space (PPS) or endochylema pericentral siphon (CPS).This reorganization ghost has complete, natural outer membrane structure, can be targeted to antigen presenting cell, can excite body fluid and cellullar immunologic response, and mucosal immune response.People such as Eko at first make gene grappling in vibrio cholerae (V.cholerae) inner membrance of chlamydia trachomatis major outer membrane albumen (OMP) express, respectively in the nose and the intramuscular injection immune mouse, the result induces stratum mucosum to produce the intensive Th1 immune response that continues with the vibrio cholerae ghost of expressing OMP1.This vaccine delivery system is developed human interior infection of born of the same parents of avoiding the Powdery Mildew mushroom of protection in the future and has been brought hope, has now set about entering the clinical experiment stage.Experiment confirm directly is suspended in cryodesiccated ghost in the relevant damping fluid that contains plasmid, and plasmid can enter ghost by the cracking passage, and with ghost inner membrance generation non-specific binding.Experiment shows, still mannheim bacterium ghost (Mannheimia haemolytica Ghosts) of intestinal bacteria ghost (E.coli Ghost) no matter, and a ghost loads the plasmid (pEGFP-N1) of about 4000-6000 copy, average 2000 copies.Studies show that the intestinal bacteria ghost up to 96% successfully loads DNA.People such as Paukner S confirm that the intestinal bacteria ghost that is mounted with pEGFP-N1 can be efficiently by mouse macrophage (RAW264.7) picked-up, and transfection efficiency reaches 58%, far above non-virus carriers such as liposome or polymers.People such as Ebensen T find that the mannheim bacterium ghost that is mounted with pEGFP-N1 can not only be absorbed by mouse macrophage, can also be efficiently by human dendritic cell (DC) picked-ups, and transfection efficiency is all near 52%.On this basis, people such as Kudela P confirms that (maturation mix, the transfection efficiency that associating MM) can not only effectively improve ghost reach 85% to ghost, and this efficient is near adenovirus with facilitating ripe medium; Can also effectively promote the DCs maturation.Comprehensively it seems, no matter the intestinal bacteria ghost still be mannheim bacterium ghost all effectively mediated dna be cell (APCs) to antigen delivery, and two kinds of ghosts no significant difference on the transfection efficiency of mediated dna.The reorganization mannheim bacterium ghost that is mounted with plasmid is compared with naked DNA through intracutaneous or intramuscular injection path immune mouse, and reorganization mannheim bacterium ghost can stimulate body to produce more effective specificity humoral and cell (CD4
+And CD8
+) immune response, and change the reaction of Th2 type into by Th1/Th2 mixed type reaction and preponderate.
The intestinal mucosa immunity plays a very important role in the immune response of body, and the immunity of numerous disease is that the approach by mucosal immunity obtains, and this immunization route often can just can start by oral vaccine, has simple and easy to do advantage.Above-mentioned research does not all relate to the research of this respect.Also lack the application of ghost in the intestinal mucosa immunity in the prior art at present, also do not have in the intestinal mucosa immunity, to take on the ghost of delivery vector.
Summary of the invention
An object of the present invention is to provide a kind of delivery vector that can be used as, safe, genetic background is ghost clearly, with satisfy can be effective, safe in intestine immunity use.
A further object of the present invention provides the preparation method of described ghost.
Another purpose of the present invention provides the application of described ghost as molecular vehicle.
Based on above purpose, the present invention at first provides a kind of salmonella typhi ghost, and described ghost is to cause the host cell cracking by the abduction delivering product of lysis genes E in salmonella typhi, and the intact bacterial ghost that forms.
In an optimized technical scheme, described salmonella typhi is Ty21a.Ty21a is a kind of typhoid fever Ty21a less-virulent strain of work, is that Germanier induced non-rite-directed mutagenesis to obtain in 1975 with chemical mutagen nitrosoguanidine mutagenesis and uviolizing.Ty21a genetic background is clear, and security is good, is the oral attenuated live vaccine that early gets the Green Light, and its mode such as gastrointestinal tract infection with natural infection enters human body, is effective bacteria carrier of intestine immunity.
In an optimized technical scheme, described gene E is that carrier is exercised the cracking function with plasmid pMB.Described lysis genes E is directed and is cloned into plasmid pMB acquisition recombinant plasmid pMBE.The temperature control expression system of described pMB is a λ pL/pR-cI857 Controlling System.Described pMB plasmid is on the basis of commercialization plasmid pBV220 (brilliant bio tech ltd is dodged in Shanghai), and directed RepA gene (the GenBank accession number Z49272.1) structure that inserts the helicobacter pylori of synthetic forms at distance initiating terminal 1300bp place.
The present invention also provides the preparation method of described ghost, said method comprising the steps of:
(1) cultivates described salmonella typhi;
(2) the carrier electricity that will contain lysis genes E is transformed into described salmonella typhi;
(3) 42 ℃ of expression of inducing described gene E;
(4) collect the thalline that step (3) obtains.
In an optimized technical scheme, described salmonella typhi is Ty21a.
In another optimized technical scheme, described expression plasmid is pMBE.The present invention adopts electric method for transformation that cracking plasmid pMBE (containing lysis genes E) is imported in the people Salmonella typhi Ty21a vaccine strain.Used electric method for transformation adopts the electricity commentaries on classics buffering system of optimizing (to contain the 0.5M sorbyl alcohol, 0.5M N.F,USP MANNITOL, the phosphate buffered saline buffer of 10% glucose) and the electricity of optimizing transform voltage (250-500V), electric capacity (10-30 μ F) and resistance (100-300 Ω), greatly improved transformation efficiency.And the traditional chemical method for transformation can not be successfully with recombinant plasmid transformed to Corynebacterium diphtheriae Ty21a.Reorganization Ty21a (pMBE) engineering bacteria is identified through colony PCR amplification E gene.Through identifying that correct reorganization Ty21a (pMBE) engineering bacteria is cultured to OD at 28 ℃
600During for 0.3-0.4, culture temperature rises to 42 ℃ rapidly and induces the E expression of gene, realizes the cracking of bacterium.After cracking is finished, centrifugal collection thalline, PBS washing back freeze-drying is preserved standby.
The present invention also provides the application of described ghost as the molecule delivery vector, and described application comprises:
(1) makes and sent molecule and contact, so that described molecule is loaded in the described ghost with described ghost;
(2) will load the ghost transduction of being sent molecule or be transfected into target cell.
In an optimized technical scheme, the described molecule of being sent is a nucleic acid molecule.
In the ghost of preparation, inner membrance of bacterium and adventitia form the cavity with intact cell membrane structure, and described loading imports to dna molecular in this cavity exactly.Being about to freeze dried Ty21aBG is resuspended in and contains the finite concentration plasmid and (as the egfp expression plasmid, in HBS damping fluid pEGFP-N1), add CaCl then
2To 25mM, hatch certain hour for 37 ℃, centrifugal collection.
In an optimized technical scheme, described target cell is the antigen presenting cell that comprises scavenger cell.
12h is inoculated in 6 orifice plates as scavenger cell RAW264.7 with finite concentration with antigen presenting cell (APCs) before the transfection, adds the Ty21a ghost that is mounted with plasmid then by a certain percentage, hatches 2h for 37 ℃.
The present invention utilizes cracking plasmid pMBE successfully to prepare the Ty21a ghost, and lysis efficiency is up to 99%.Carry out the loading of Ty21a ghost on this basis, result's confirmation is sent through the Ty21a ghost, but the carrier for expression of eukaryon efficient loading is approximately loaded the plasmid of 2000-5000 copy in each ghost in the Ty21a ghost; Because Corynebacterium diphtheriae Ty21a is engulfed by antigen presenting cell such as scavenger cell etc. easily, itself compares with independent plasmid, improve recombinant plasmid greatly and entered into the efficient of antigen presenting cell, efficiently express intracellular thereby mediate eucaryon plasmid.Experiment showed, with mouse macrophage RAW264.7 to be the antigen presenting cell of representative, can effectively absorb eukaryon expression plasmid, and at cell inner expression.
Ty21a genetic background is clear, and security is good, and the preparation of Ty21a ghost need not violent physics, chemical treatment, thereby kept its outer membrane structure effectively, had natural adjuvant characteristic, simultaneously, most of intracellular organic matter of bacterium, especially nucleic acid is released, and its security is improved greatly.And traditional ablation method physical method (as irradiation) and chemical process (as the formalin deactivation etc.) in the deactivation bacterium, natural structure and some antigen of bacterium have also been destroyed, and endobacillary endochylema composition and nucleic acid component also are retained, and security is relatively poor.With the delivery system of Ty21a ghost as materials such as recombinant protein, nucleic acid or medicines, have following advantage: 1. salmonella typhi Ty21a is born of the same parents' endophyte, and easier in antigen presenting cell (APCs) picked-up, target is good; 2. compare with the intestinal bacteria ghost, because cracking process control is suitable, the after birth of the Ty21a ghost that present technique is prepared is more complete, is fit to loading active substance; 3. the security of Ty21a ghost is good.
Description of drawings
Fig. 1 expresses the reorganization Ty21a PCR qualification result of E gene;
The lysis efficiency of Fig. 2 Ty21a ghost;
The scanning electron microscope and the transmission electron microscope results of Fig. 3 Ty21a ghost;
The expression of Fig. 4 fluorescence microscope green fluorescent protein in scavenger cell RAW264.7;
Fig. 5 fluidic cell detects the expression of green fluorescent protein in scavenger cell RAW264.7.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment and replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
Test materials
1 bacterial strain and plasmid
People's typhoid fever Ty21a vaccine strain (available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute).
2 main agents and instrument
Key instrument has:
PCR instrument (Thermocyler, Biometra), horizontal strip electrophoresis instrument (Liuyi Instruments Plant, Beijing), enzyme connection instrument (Thermo, Multiskan MK3), supercentrifuge (HITACHI, CR22GIII), electric shock instrument (BIO-RAD Gene X cell), scanning electronic microscope (HITACHI, S-2400) and transmission electron microscope (PHILIPS, Megaview II), flow cytometer (BDBioscience).
Main agents has:
Tryptones, yeast extract are available from Oxoid company, and plasmid extraction kit is available from TIANGEN company, and primer, restriction enzyme, PCR test kit are available from TAKARA company.
Preparation, the evaluation of embodiment 1 salmonella typhi Ty21a ghost
1.1 the preparation of salmonella typhi Ty21a ghost:
Use the method (forward primer 5 '-GAATTCGGCATGGTACGCTG-3 ' of PCR, reverse primer 5 '-GGATCCGGCTCACTCCTTCC-3 '), obtain lysis genes E from phage PhiX174 (available from U.S. typical case culture center ATCC13706-B1), then lysis genes E orientation is connected among the temperature control expression plasmid pMB, obtains cracking plasmid pMBE.The method that adopts electricity to transform is transformed into cracking plasmid pMBE among the people Salmonella typhi Ty21a, used electric method for transformation adopts the electricity commentaries on classics buffering system of optimizing (to contain the 0.5M sorbyl alcohol, 0.5M N.F,USP MANNITOL, the phosphate buffered saline buffer of 10% glucose) and the electricity of optimizing transform voltage (250-500V), electric capacity (10-30 μ F) and resistance (100-300 Ω).Pcr amplification E gene is identified (see Fig. 1, swimming lane 1 is molecular weight contrasting marking DL2000, and swimming lane 2-3 is an E gene amplification thing, swimming lane 4 positive contrasts, swimming lane 5 negative contrasts).Through identifying that correct reorganization Ty21a (pMBE) engineering bacteria is cultured to OD at 28 ℃
600When being 0.3 left and right sides, culture temperature rises to 42 ℃ rapidly and induces cracking.After cracking is finished, centrifugal collection thalline, PBS washing back freeze-drying is preserved standby.
Test-results: reorganization Ty21a (pMBE) engineering bacteria after will transforming is through pcr amplification, and the band of 276bp appears in 1% agarose electrophoresis, illustrates that cracking plasmid pMBE successfully changes in the Ty21a bacterium.
1。The evaluation of 2 salmonella typhi Ty21a ghosts:
Measure the lysis efficiency (see figure 2) of reorganization Ty21a (pMBE) engineering bacteria by method for counting colonies; By scanning electron microscopic observation, methods such as transmission electron microscope observing are carried out preliminary evaluation (see Fig. 3, A is the scanning electron microscope result, and B is a transmission electron microscope results) to the form of Ty21a ghost.
Test-results: PCR is identified that correct reorganization Ty21a (pMBE) engineering bacteria is cultured to the logarithmic growth cycle in 28 ℃ of 200r/min.Culture is divided into 2 groups, and one group is elevated to 42 ℃ rapidly with culture temperature and induces E genetic expression, and another is organized in 28 ℃ and continues to cultivate, and enumeration is carried out in the different time points sampling.The result shows that Ty21a bacterium (pMBE) is the cracking fullest when inducing the back at 4h, and lysis efficiency reaches more than 99%.
Scanning electron microscope and transmission electron microscope Electronic Speculum result show that most bacteriums are cavity shape structure, and can see the bacteriolyze duct inducing the back to form ghost, and diameter is between 100-200nm.Ghost has kept complete bacterial outer membrane, but because intracellular organic matter is released out, shrinkage (referring to Fig. 3) takes place bacterium.
The loading and the in-vitro transfection of embodiment 2 salmonella typhi Ty21a ghosts
2.1 the evaluation of plasmid pEGFP-N1 and a large amount of the extraction:
At first egfp expression plasmid pEGFP-N1 (available from CLONTECH company) is carried out enzyme and cut evaluation; Identify that correct plasmid extracts in a large number and concentrate standby.
2.2Ty21a the loading of ghost:
In the ghost of preparation, inner membrance of bacterium and adventitia formation-individual cavity with intact cell membrane structure, its loading imports to DNA in this cavity exactly.
Loading process: freeze dried Ty21a BG is resuspended in the pEGFP-N1HBS damping fluid that concentration is 2-20mg/ml, and the ratio of ghost and plasmid is loaded the plasmid of the above-mentioned concentration of 100-500uL for the 10mg ghost, and then the adding final concentration is the CaCl of 20-30mM
2Solution is hatched 10-20min for 28-37 ℃, centrifugal collection, and PBS (PH7.4) washing back is preserved standby.
Efficiency of loading: in the Ty21a ghost of above-mentioned loading plasmid pEGFP-N1, use the plasmid that each ghost of quantitative PCR detection approximately loads the 2000-5000 copy.
2.3 the in-vitro transfection of salmonella typhi Ty21a ghost
2.3.1 the cultivation of mouse macrophage RAW264.7:
Mouse macrophage RAW264.7 (available from BJ Union Hospital's cell centre), with containing 10% foetal calf serum, the DMEM substratum of 1%L-glutamine and 1% mycillin (above reagent is all available from GIBCO company), at 37 ℃, 5%CO
2Condition under cultivate.
2.3.2Ty21a the in-vitro transfection of ghost:
The transfection conditions optimizing process: 12h is with cell (10 before the transfection
5-10
6) be inoculated in 6 orifice plates, then by a different ratios (cell: ghost=1: 100-2000) add Ty21a ghost, Ty21a ghost or the plasmid that is mounted with plasmid.Hatch 2h for 37 ℃.The ghost cell of not engulfing is removed in no thermal source PBS (PH7.4) washing 2 times.Add 2ml 5%FBS or 10%FBS DMEM substratum, hatch for 37 ℃.Respectively at 24h, 36h after the transfection and 48h collecting cell, fluorescence microscope egfp expression situation.Determine best transfection process.
Standard transfection process: the 12h mouse macrophage RAW264.7 cell (10 that growth conditions is good before the transfection
5-10
6) be inoculated in 6 orifice plates, then so that (cell: ghost=1: 100-2000) adding is mounted with the Ty21a ghost of plasmid, sets up sky ghost and plasmid pEGFP-N1 contrast simultaneously by different ratios.Hatch 2h for 37 ℃, the ghost cell of not engulfing is removed in no thermal source PBS (PH7.4) washing 2 times.Adding 2ml contains the DMEM substratum of 5-10%FBS, 37 ℃ are continued to hatch, different time 24h, 36h and 48h collecting cell after transfection, observe the egfp expression situation with fluorescent microscope and fluidic cell respectively, Fig. 4 and Fig. 5 are respectively after the transfection 48 hours fluorescent microscopes (Fig. 4) and fluidic cell (Fig. 5) observations.
Experimental result: egfp expression plasmid pEGFP-N1 is loaded into the Ty21a ghost for preparing, and transfection is to mouse macrophage RAW264.7, use the expression of fluorescence microscope green fluorescent protein, the result as shown in Figure 4, A is the RAW264.7 negative control, do not observe the expression of fluorescin, B is the RAW264.7 cell that contains the Ty21a ghost that has transformed plasmid pEGFP-N1, can be observed the expression of fluorescin.The fluidic cell detected result has also confirmed the expression of green fluorescent protein, and the result as shown in Figure 5.The result of two kinds of methods shows that the Ty21a ghost that loads the pEGFP-N1 plasmid has obtained effective expression in RAW264.7.
Claims (10)
1. people salmonella typhi ghost, thus described ghost is to induce lysis genes E to express in the people salmonella typhi by temperature control to cause the bacterium cracking, and the complete bacterium ghost that forms.
2. ghost according to claim 1 is characterized in that, described people salmonella typhi is Ty21a.
3. ghost according to claim 2 is characterized in that, described gene E is that carrier is exercised the cracking function with plasmid pMB.
4. method for preparing people salmonella typhi ghost said method comprising the steps of:
(1) cultivates described salmonella typhi;
(2) the recombinant plasmid pMBE that will contain lysis genes E is transformed into described salmonella typhi with electric method for transformation, obtains the recombinant human salmonella typhi of stable conversion pMBE;
(3) 28 ℃ of recombinant human salmonella typhis of cultivating above-mentioned conversion pMBE, 42 ℃ of expression of inducing described gene E;
(4) ghost of 2-4 hour collection step (3) acquisition behind abduction delivering.
5. method according to claim 4 is characterized in that, described people salmonella typhi is Ty21a.
6. method according to claim 4 is characterized in that, the buffering system of described electric method for transformation is for containing the 0.5M sorbyl alcohol, 0.5M N.F,USP MANNITOL, the phosphate buffered saline buffer of 10% glucose.
7. method according to claim 4 is characterized in that, it is 250-500V, electric capacity 10-30 μ F that the electricity of described electric method for transformation transforms voltage, resistance 100-300 Ω.
8. according to arbitrary described ghost is as the application of molecule delivery vector among the claim 1-3, described application comprises:
(1) will be sent molecule is loaded in the described ghost;
(2) will load the ghost transduction of being sent molecule or be transfected into target cell.
9. application according to claim 8 is characterized in that, the described molecule of being sent is a nucleic acid molecule.
10. application according to claim 8 is characterized in that, described target cell is the antigen presenting cell that comprises scavenger cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105195099A CN101985610A (en) | 2010-10-26 | 2010-10-26 | Salmonella typhi ghost, preparation method and application thereof serving as delivery system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105195099A CN101985610A (en) | 2010-10-26 | 2010-10-26 | Salmonella typhi ghost, preparation method and application thereof serving as delivery system |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101985610A true CN101985610A (en) | 2011-03-16 |
Family
ID=43710012
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010105195099A Pending CN101985610A (en) | 2010-10-26 | 2010-10-26 | Salmonella typhi ghost, preparation method and application thereof serving as delivery system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101985610A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102352338A (en) * | 2011-09-30 | 2012-02-15 | 中国人民解放军军事医学科学院军事兽医研究所 | Brucella shell as well as preparation method and application thereof |
| CN103275914A (en) * | 2013-06-03 | 2013-09-04 | 中国人民解放军军事医学科学院微生物流行病研究所 | Bacterial ghost presenting protective antigens and application thereof |
| CN104066834A (en) * | 2011-12-22 | 2014-09-24 | 万科斯蒙股份有限公司 | Method for producing high yield attenuated salmonella strains |
| CN104353072A (en) * | 2014-10-10 | 2015-02-18 | 中国农业科学院哈尔滨兽医研究所 | Application of bacterial ghost in preparation of viral vaccine adjuvant |
| CN104498524A (en) * | 2014-12-31 | 2015-04-08 | 山东农业大学 | Edwardsiella tarda ghost of recombinant vibrio vulnificus gene vvhA and construction |
| CN112111438A (en) * | 2020-09-22 | 2020-12-22 | 江苏省家禽科学研究所 | Preparation method of Salmonella indiana ghost |
| CN112410360A (en) * | 2021-01-18 | 2021-02-26 | 西南大学 | Chicken pathogenic bacterium ghost and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996028551A1 (en) * | 1995-03-13 | 1996-09-19 | The Secretary Of State For Defence | Vaccines for plague |
| CN101503694A (en) * | 2009-03-02 | 2009-08-12 | 中国农业科学院哈尔滨兽医研究所 | Rearranged bacterial virus bacteriolysis gene, perforating vector thereof and use in vaccine preparation |
-
2010
- 2010-10-26 CN CN2010105195099A patent/CN101985610A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996028551A1 (en) * | 1995-03-13 | 1996-09-19 | The Secretary Of State For Defence | Vaccines for plague |
| CN101503694A (en) * | 2009-03-02 | 2009-08-12 | 中国农业科学院哈尔滨兽医研究所 | Rearranged bacterial virus bacteriolysis gene, perforating vector thereof and use in vaccine preparation |
Non-Patent Citations (2)
| Title |
|---|
| 李小妹等: "禽致病性大肠杆菌(APEC)菌蜕的制备研究", 《中国预防兽医学报》 * |
| 蔡昆等: "细菌菌蜕的研究进展", 《中国生物制品学杂志》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102352338A (en) * | 2011-09-30 | 2012-02-15 | 中国人民解放军军事医学科学院军事兽医研究所 | Brucella shell as well as preparation method and application thereof |
| CN104066834A (en) * | 2011-12-22 | 2014-09-24 | 万科斯蒙股份有限公司 | Method for producing high yield attenuated salmonella strains |
| US9493738B2 (en) | 2011-12-22 | 2016-11-15 | Vaximm Ag | Method for producing high yield attenuated Salmonella strains |
| CN103275914A (en) * | 2013-06-03 | 2013-09-04 | 中国人民解放军军事医学科学院微生物流行病研究所 | Bacterial ghost presenting protective antigens and application thereof |
| CN104353072A (en) * | 2014-10-10 | 2015-02-18 | 中国农业科学院哈尔滨兽医研究所 | Application of bacterial ghost in preparation of viral vaccine adjuvant |
| CN104353072B (en) * | 2014-10-10 | 2018-04-27 | 中国农业科学院哈尔滨兽医研究所 | Application of the bacterium ghost in viral vaccine adjuvant is prepared |
| CN104498524A (en) * | 2014-12-31 | 2015-04-08 | 山东农业大学 | Edwardsiella tarda ghost of recombinant vibrio vulnificus gene vvhA and construction |
| CN112111438A (en) * | 2020-09-22 | 2020-12-22 | 江苏省家禽科学研究所 | Preparation method of Salmonella indiana ghost |
| CN112410360A (en) * | 2021-01-18 | 2021-02-26 | 西南大学 | Chicken pathogenic bacterium ghost and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101985610A (en) | Salmonella typhi ghost, preparation method and application thereof serving as delivery system | |
| Cao et al. | Identification of fish source Vibrio alginolyticus and evaluation of its bacterial ghosts vaccine immune effects | |
| Lubitz et al. | Applications of bacterial ghosts in biomedicine | |
| Zhu et al. | Generation of biotechnology‐derived flavobacterium columnare ghosts by PhiX174 gene E‐mediated inactivation and the potential as vaccine candidates against infection in grass carp | |
| Liang et al. | Evaluation of efficacy, biodistribution and safety of antibiotic-free plasmid encoding somatostatin genes delivered by attenuated Salmonella enterica serovar Choleraesuis | |
| Tu et al. | Effect of oral immunization with Aeromonas hydrophila ghosts on protection against experimental fish infection | |
| Kahieshesfandiari et al. | Streptococcosis in Oreochromis sp.: is feed-based biofilm vaccine of Streptococcus agalactiae effective? | |
| Hajam et al. | Incorporation of membrane-anchored flagellin into Salmonella Gallinarum bacterial ghosts induces early immune responses and protection against fowl typhoid in young layer chickens | |
| Zhang et al. | Bacterial ghost as delivery vehicles loaded with DNA vaccine induce significant and specific immune responses in common carp against spring viremia of carp virus | |
| Cao et al. | Construction of Vibrio mimicus ghosts as a novel inactivated vaccine candidate and its protective efficacy against ascites disease in grass carps (Ctenopharyngodon idella) | |
| Thwaite et al. | Nanostructured recombinant protein particles raise specific antibodies against the nodavirus NNV coat protein in sole | |
| Cai et al. | Oral immunization with surface immunogenic protein from Streptococcus agalactiae expressed in Lactococcus lactis induces protective immune responses of tilapia (Oreochromis niloticus) | |
| Zhao et al. | Oral vaccination with recombinant Lactobacillus casei expressing Aeromonas hydrophila Aha1 against A. hydrophila infections in common carps | |
| Chaudhari et al. | Construction of an attenuated Salmonella delivery system harboring genes encoding various virulence factors of avian pathogenic Escherichia coli and its potential as a candidate vaccine for chicken colibacillosis | |
| Li et al. | An oral vaccine against spring viremia of carp virus induces protective immunity in common carp (Cyprinus carpio L.) | |
| Jawale et al. | Development of a biosafety enhanced and immunogenic Salmonella enteritidis ghost using an antibiotic resistance gene free plasmid carrying a bacteriophage lysis system | |
| CN102274496A (en) | O/Asia I type foot and mouth disease virus bivalent genetic engineering polypeptide vaccine, its preparation method and its purpose | |
| Li et al. | Oral vaccination with recombinant Lactobacillus casei with surface displayed OmpK fused to CTB as an adjuvant against Vibrio mimicus infection in Carassius auratus | |
| CN101979504A (en) | Yersinia pestis ghost, preparation method thereof, and application of Yersinia pestis ghost serving as vaccine | |
| CN105154377B (en) | Recombinant salmonella pullorum, preparation method and application | |
| CN101991863B (en) | Double-target DNA vaccine and constructing method thereof | |
| US20180153945A1 (en) | Oral e. coli vector-based vaccine for prevention of coccidiosis in poultry | |
| Chaudhari et al. | Safety evaluation and immunogenicity of arabinose-based conditional lethal Salmonella Gallinarum mutant unable to survive ex vivo as a vaccine candidate for protection against fowl typhoid | |
| CN109402035A (en) | A kind of parenteral enteropathogenic E. Coli recombinant bacterial strain and its application | |
| CN104232502A (en) | Bacterial ghost of aeromonas veronii as well as 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 | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20110316 |