CN1090602A - Pseudomonos aeruginosa MSHA fimbria strain and foundation thereof - Google Patents
Pseudomonos aeruginosa MSHA fimbria strain and foundation thereof Download PDFInfo
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- CN1090602A CN1090602A CN 93100114 CN93100114A CN1090602A CN 1090602 A CN1090602 A CN 1090602A CN 93100114 CN93100114 CN 93100114 CN 93100114 A CN93100114 A CN 93100114A CN 1090602 A CN1090602 A CN 1090602A
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Abstract
Pseudomonos aeruginosa MSHA fimbria strain is a kind of Pseudomonas aeruginosa with all pili MSHA pili, and many very thin and upright and outspoken pili are promptly arranged around thalline.The MSHA test is strong positive, and flat-plate bacterial colony adheres to the red corpuscle test and is strong positive, and saccharomycetic direct agglutination test is the MS blood clotting.The foundation of this Pseudomonos aeruginosa MSHA fimbria strain is to be sample through the reduction virulence that goes down to posterity more than the hundreds of time with natural non-all pili, the strong malicious Pseudomonas aeruginosa virulent strain of non-MSHA, and then adopt biotechnology to make it to be with whole body property MSHA pili, it has the immunogenicity that the broad-spectrum high efficacy valency is crossed over Pseudomonas, and virulence only is 1/80 of an original pilus strain.
Description
The present invention relates to a kind of microorganism, particularly Pseudomonas aeruginosa.
Pseudomonos aeruginosa MSHA fimbria strain full name of the present invention is pseudomonas aeruginosa (Ps.aeruginosa) mannose sensitive haemagglutination pilus strain, it is a kind of all pili MSHA(Mannose Sensitive Hemagglu tination that has) Pseudomonas aeruginosa of pili, this pilus strain has the wide spectrum immunogenicity of striding Pseudomonas.
At InfectImmun 1980; Reported this inferior dead thalline of Pseudomonas aeruginosa of once finding of Japanese scholar in 1975 at 40: 670 immunoregulation effect has been arranged, its dead bacterium is expelled in the animal body and can excites body fluid and cellular immunity, induce and generate the interference element and have the mitogen effect, can also induced animal the growth of cancer cells of opposing inoculation, but because of this bacterium thalline toxicity too big, immunizing potency is low, does not have all pili MSHA pili again and strides Pseudomonas high-efficiency broad spectrum immunogenicity feature, so can't practical application.Israel scholar Gilboa-Garber professor can not express the phenotypic characteristic of all pili MSHA pilus strains at In Adhesion and micrcorganismpathogenicity cida foundation symposium 80.Pitman Medical p119～141.1981 chromogenes of further illustrating Pseudomonas aeruginosa in 1980, reason may be that Pseudomonas aeruginosa is structurally different with other Gram-negative bacteria, shortage is hooked into the lip-deep gene of bacterial body with its MSHA lectin, thereby the positive strain of MSHA of green pus bacterium culture alive can not occur.
The object of the present invention is to provide the positive Pseudomonas aeruginosa pilus strain of a kind of MSHA and build the method for strain.
Pseudomonos aeruginosa MSHA fimbria strain is characterized in that:
A, many very thin and upright and outspoken pili are arranged around the thalline,
B, MSHA test is strong positive,
C, flat-plate bacterial colony adhere to red corpuscle test strong positive,
D, saccharomycetic direct agglutination test are the MS blood clotting.
2. the foundation of Pseudomonos aeruginosa MSHA fimbria strain is characterized in that:
1. reduce the virulence of Pseudomonas aeruginosa
A, strong malicious Pseudomonas aeruginosa virulent strain is a sample with natural non-all pili, non-MSHA,
Cultivate 5～20 days through going down to posterity more than 200 times at 37～41 ℃ in b, the mixed culture medium formed with ordinary culture medium and soup,
2. the expression of whole body property MSHA pili
A, extract cyto-chromatin DNA: adopt repeatedly freezing and SDS synthetic method to work in coordination with and destroy thalline, deproteinize then,
B, acquisition DNA small segment: the chromosomal DNA of getting above-mentioned attenuated strain adopts Pst I, EcoR I and the compound restriction endonuclease of HPa II to cut 1～2 hour at 65 ℃ of enzymes,
C, the DNA small segment is engaged again at random: get the liquid that contains the DNA small segment and add 0.3%～0.5% ligase enzyme, 0～12 ℃ of effect 1～12 day,
D, DNA separate double-stranded and degerming: add Pst I, EcoR I and the compound desmolase of HPa II in the liquid that long-chain DNA is arranged and cut about 5 minutes to above-mentioned the joint again, adopt the usual manner sterilization then at 65 ℃ of enzymes,
E, the DNA of strand is combined with the attenuation thalline: separate double-stranded DNA liquid and join and contain CaCl above-mentioned
2The attenuated strain inherence be added with in the ordinary culture medium of 2% agar in 37～41 ℃ of common cultivations.
The present invention carries out continuous passage to Pseudomonas aeruginosa to make it the virulence reduction, and the using gene engineering method realizes the expression of whole body property MSHA pili again.
The foundation of Pseudomonos aeruginosa MSHA fimbria strain of the present invention
One, reduces the virulence of Pseudomonas aeruginosa
The present invention is with natural non-all pili, the strong malicious Pseudomonas aeruginosa virulent strain (LD of non-MSHA
30=10
7Individual viable bacteria) is sample, in ordinary culture medium as in the product of substratum (PH=7.4～7.6) behind autoclaving that adopts the sodium-chlor by 0.3% meat extract, beef infusion broth, 1% peptone and 0.5% to constitute, adds soup again.This soup can be antibiotic medicine such as gentamicin, the polymyxin etc. in the Western medicine, also can be clearing heat and detoxicating medicine such as Acalypha australis, Philippine Violet Herb, the root bark of tree peony, Herba Bidentis Bipinnatae, bitter dish and the Radix Astragali etc. in the Chinese medicine.Above-mentioned soup is added to makes it concentration in the ordinary culture medium and be lower than Mlc.The bacteriocidal concentration of these medicines has nothing in common with each other, get its bacteriocidal concentration 1/10th as Mlc, make it to develop immunity to drugs to lower virulence.Under 37～41 ℃ temperature condition, cultivate and went down to posterity once in 5～20 days, repeatedly repeat aforesaid operations through 200 generations after its virulence ease down to can for the preparation vaccine usefulness.Though the virulence of the more original bacterium sample of its toxicity strain has descended, but still keeps original complete immunogenicity, still can form enough specific antibodies and cellular immunization after promptly injecting the non-lethal quantity of small white mouse.
Two, the expression of whole body property MSHA pili
The extraction of bacterium chromatin dna: freezing and SDS(sulfonic acid dodecane sodium salt repeatedly) the collaborative thalline that destroys of synthetic method, the Pseudomonas aeruginosa that is about to above-mentioned attenuation places the distilled water cooling to make it glaciation afterwards again with its thawing, meanwhile also needing to add the SDS medicine in distilled water corrodes, to quicken the destruction of bacteria cell wall, carry out repeatedly repeatedly just can destroying thalline.Deproteinize afterwards: add chloroform one primary isoamyl alcohol in ruined thalline, then protein is flocks and separates out.Centrifugation is removed protein and is left and taken supernatant liquor.Preferably use the diluted hydrochloric acid dissolution particulate matter again, make the DNA that is contained in it dissociate out, to increase the yield of DNA.The supernatant liquor that will contain DNA washs repeatedly with aqueous ethanolic solution, removes the chromosomal DNA that salt can obtain the attenuated strain of purifying.Available electron microscope (* 100 ten thousand times) inspection has or not the DNA gene.
The acquisition of DNA small segment: the chromosomal DNA to above-mentioned attenuated strain adopts Pst I, EcoR I and the compound restriction endonuclease of HPa II to cut 1～2 hour at 65 ℃ of enzymes, makes it become small segment.Get the liquid that contains the DNA small segment and add 0.3～0.5% ligase enzyme, 0～12 ℃ of effect 1～12 day, make various tiny dna fragmentations be bonded into long-chain DNA(two strands again at random) engage situation with submicroscopy, if do not connect, change ligase enzyme and repeat again.Add desmolase to above-mentioned the joint in the liquid that long-chain DNA is arranged again, promptly above-mentioned restriction endonuclease such as Pst I, EcoR I and the compound restriction endonuclease of HPa II were cut about 5 minutes at 65 ℃ of enzymes, made long-chain DNA become strand, went so that enter in the viable bacteria body.Adopt conventional sterilization method to remove any thalline then as adopting filter membrane or filter.The above-mentioned DNA liquid of separating double-stranded remaining strand is added to the CaCl that contains conventional concentration
2Attenuated strain in, in ordinary culture medium, promptly add 2% agar again in the mixture of 0.3% meat extract, beef infusion broth, 1% peptone and 0.5% sodium-chlor, cultivate clone repeatedly jointly in 37～41 ℃, can obtain Pseudomonos aeruginosa MSHA fimbria strain.
This bacterium is with after biotechnology is modified and has gone up whole body property MSHA pili, has the immunogenicity that the broad-spectrum high efficacy valency is crossed over Pseudomonas.Its virulence only is 1/80 of an original non-MSHA pilus strain.Use the dead bacterium of this pilus strain to can be made into hypotoxic wide spectrum immunity metabolism regulators, be injected in human body, when helping the human body activating immune system to resist multiple pathogenic microorganism infection, can also make human body recover immune metabolic balance state, can be antibiotic also can anti-virus infection; Can regulate immunocompromised state control repeated relapsing urinary tract and respiratory tract disease and malignant tumour also can regulate immune hyperfunction state and prevent and treat supersensitivity illness (as asthma, psoriatic, scleroderma, chronic persistent hepatitis B).
Drawing is described as follows:
Fig. 1 is the electron micrograph (29000 *) of Pseudomonos aeruginosa MSHA fimbria strain of the present invention
Fig. 2 is the photo of Pseudomonas aeruginosa semi-solid form of the present invention.
Fig. 3 is the oxidase test comparing result photo of Pseudomonas aeruginosa of the present invention and other bacterium.
Fig. 4 is the pigment comparing result photo of Pseudomonas aeruginosa of the present invention.
Fig. 5 is the electron micrograph (29000 *) of the MSHA pilus strain of Pseudomonas aeruginosa of the present invention
Fig. 6 is that the MSHA slide aggegation direct viewing of the MSHA pilus strain of Pseudomonas aeruginosa of the present invention is the positive photo of MSHA.
Fig. 7 presents MSHA negative findings photo after the MSHA pilus strain MSHA of Pseudomonas aeruginosa of the present invention is suppressed by seminose.
Fig. 8 is the direct Saccharomyces congloberatus of MSHA pilus strain of Pseudomonas aeruginosa of the present invention and the optical microscope photograph (1000 *) of surface adhesion test
Fig. 9 is the optical microscope photograph (1000 *) of direct Saccharomyces congloberatus bacterium of the non-MSHA pilus strain of Pseudomonas aeruginosa of the present invention and surface adhesion test
Embodiment
Get natural non-all pili, the strong malicious Pseudomonas aeruginosa virulent strain (LD=10 of non-MSHA
7Individual viable bacteria) be sample, the substratum (PH=7.5) that constitutes at the sodium-chlor by 0.3% meat extract, beef infusion broth, 1% peptone and 0.5% adds soup again behind autoclaving.This soup is that the mixed solution of the 2-8% Radix Astragali and 1% Herba Bidentis Bipinnatae is cultivated under 37～41 ℃ temperature condition and gone down to posterity once in 5～20 days.Virulence through the Pseudomonas aeruginosa of going down to posterity for 462 times adopts kind 14～18 gram small white mouses in Kunming to record LD
50=9 * 10
8Individual viable bacteria.Place the distilled water cooling to make it glaciation the Pseudomonas aeruginosa of above-mentioned attenuation, meanwhile also need in distilled water, to add the SDS medicine, thalline is destroyed through repeated multiple times afterwards again with its thawing.Add chloroform-primary isoamyl alcohol then in ruined thalline, then protein is flocks and separates out.Centrifugation is removed protein and is left and taken supernatant liquor, uses the diluted hydrochloric acid dissolution particulate matter again.The supernatant liquor that will contain DNA repeatedly washs with aqueous ethanolic solution removes the chromosomal DNA that salt can obtain the attenuated strain of purifying.Have or not the DNA gene with electron microscope (* 100 ten thousand times) inspection.Chromosomal DNA to above-mentioned attenuated strain adopts Pst I, EcoR I and the compound restriction endonuclease of HPa II, cuts 1～2 hour at 65 ℃ of enzymes, makes it become small segment.Get the liquid that contains the DNA small segment and add 0.3～0.5% ligase enzyme,, make various tiny dna fragmentations be bonded into long-chain DNA(two strands again at random) 0～12 ℃ of effect 1～12 day.Engage situation with submicroscopy.Have to above-mentioned recombine to add Pst I, EcoR I and the compound restriction endonuclease of HPa II in the liquid of long-chain DNA, cut about 5 minutes at 65 ℃ of enzymes.Use membrane filtration then, filtrate being added to contained in the attenuated strain of calcium chloride of 5% concentration, in substratum, clone the MSHA pilus strain that can obtain Pseudomonas aeruginosa repeatedly in 37～41 ℃ of co-cultivation by 0.3% meat extract, beef infusion broth, 1% peptone, 0.5% sodium-chlor and 2% agar.
It is as shown in the table that the Pseudomonas aeruginosa of the present invention that obtains above has whole mycology features of Pseudomonas aeruginosa
Pseudomonos aeruginosa MSHA fimbria strain of the present invention has the mycology feature different with the non-MSHA pilus strain of green bacillus again and sees the following form:
Application of the present invention: make 15,000,000,000 dead thalline of vaccine or the safe and reliable toxicological harmless vaccine of immunomodulator preparation injection small white mouse with this pilus strain and do not see any toxic reaction, the maximum that human body uses is 1,600,000,000 as 1/10 of small white mouse only, fool proof, case is not seen an example poisoning and an allergy sufferers surplus the clinical treatment ten thousand, contains multiple material such as vitamins B, vitamin-E, trace element, zinc, copper, iron, calcium and phosphorus etc. to the favourable easy absorption of human body in above-mentioned vaccine or the immunomodulator.This vaccine immunization animal capable excites cell and the immunity system of animal comprehensively; Can not only resist charrin's disease and also can cross over the attack of a plurality of Pseudomonas resistance to deformation bacillus, intestinal bacteria, pneumobacillus and Corynebacterium diphtheriae etc.Its wide spectrum antibody titer 100% of vaccine immunization animal and volunteers improves more than 4 times.Improve 30～256 times than tiring of wide spectrum antibody before the vaccine immunization.The equal tool positive effect of immunoregulatory activity and antitumor action.This vaccine is applied to clinical as control respiratory repeated infection and asthma inverse amplification factor reach more than 80%.Treatment repeatability chronic uropoiesis road feel dyes that treatment rate reaches 75～98%, treatment psoriatic curative ratio is 60.3% higher by 53% than international conventional curative ratio.Also evident in efficacy to cholecystitis, delay property pancreatitis, large-area burns and chronic repeatability colitis in addition.
Pseudomonos aeruginosa MSHA fimbria strain of the present invention has the mycology feature different with the non-MSHA pilus strain of green bacillus again and sees the following form:
Application of the present invention: make 15,000,000,000 dead thalline of vaccine or the safe and reliable toxicological harmless vaccine of immunomodulator preparation injection small white mouse with this pilus strain and do not see any toxic reaction, the maximum that human body uses is 1,600,000,000 as 1/10 of small white mouse only, fool proof, case is not seen an example poisoning and an allergy sufferers surplus the clinical treatment ten thousand, contains multiple material such as vitamins B, vitamin-E, trace element, zinc, copper, iron, calcium and phosphorus etc. to the favourable easy absorption of human body in above-mentioned vaccine or the immunomodulator.This vaccine immunization animal capable excites cell and the immunity system of animal comprehensively; Can not only resist charrin's disease and also can cross over the attack of a plurality of Pseudomonas resistance to deformation bacillus, intestinal bacteria, pneumobacillus and Corynebacterium diphtheriae etc.Its wide spectrum antibody titer 100% of vaccine immunization animal and volunteers improves more than 4 times.Improve 30～256 times than tiring of wide spectrum antibody before the vaccine immunization.The equal tool positive effect of immunoregulatory activity and antitumor action.This vaccine is applied to clinical as control respiratory repeated infection and asthma inverse amplification factor reach more than 80%.Treatment repeatability chronic uropoiesis road feel dyes that treatment rate reaches 75～98%, treatment psoriatic curative ratio is 60.3% higher by 53% than international conventional curative ratio.Also evident in efficacy to cholecystitis, delay property pancreatitis, large-area burns and chronic repeatability colitis in addition.
Annotate: the per-cent among the present invention all is weight percentage except that special indicating.
Claims (2)
1, Pseudomonos aeruginosa MSHA fimbria strain is characterized in that:
A, many very thin and upright and outspoken pili are arranged around the thalline,
B, MSHA test is strong positive,
C, flat-plate bacterial colony adhere to red corpuscle test strong positive,
D, saccharomycetic direct agglutination test are the MS blood clotting.
2, the foundation of Pseudomonos aeruginosa MSHA fimbria strain is characterized in that:
1. reduce the virulence of Pseudomonas aeruginosa
A, strong malicious Pseudomonas aeruginosa virulent strain is a sample with natural non-all pili, non-MSHA,
Cultivate 5～20 days through going down to posterity more than 200 times at 37～41 ℃ in b, the mixed culture medium formed with ordinary culture medium and soup,
2. the expression of whole body property MSHA pili
A, extract cyto-chromatin DNA: adopt repeatedly freezing and SDS synthetic method to work in coordination with and destroy thalline, deproteinize then,
B, acquisition DNA small segment: the chromosomal DNA of getting above-mentioned attenuated strain adopts Pst I, EcoR I and the compound restriction endonuclease of HPa II to cut 1～2 hour at 65 ℃ of enzymes,
C, the DNA small segment is engaged again at random: get the liquid that contains the DNA small segment and add 0.3%～0.5% ligase enzyme, 0～12 ℃ of effect 1～12 day,
D, DNA separate double-stranded and degerming: add Pst I, EcoR I and the compound desmolase of HPa II in the liquid that long-chain DNA is arranged and cut about 5 minutes to above-mentioned the joint again, adopt the usual manner sterilization then at 65 ℃ of enzymes,
E, the DNA of strand is combined with the attenuation thalline: separate double-stranded DNA liquid and join and contain CaCl above-mentioned
2The attenuated strain inherence be added with in the ordinary culture medium of 2% agar in 37～41 ℃ of co-cultivation.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 93100114 CN1090602A (en) | 1993-02-06 | 1993-02-06 | Pseudomonos aeruginosa MSHA fimbria strain and foundation thereof |
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| CN 93100114 CN1090602A (en) | 1993-02-06 | 1993-02-06 | Pseudomonos aeruginosa MSHA fimbria strain and foundation thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1101404C (en) * | 1998-07-29 | 2003-02-12 | 中德联合研究院 | Anti Bacillus pyocyaneu Flugge vitelline immunoglobulin products and use thereof |
| CN1297657C (en) * | 2005-03-31 | 2007-01-31 | 牟希亚 | Pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain |
| CN101914463B (en) * | 2010-02-09 | 2012-09-05 | 北京万特尔生物制药有限公司 | Proteus vulgaris (mannose-sensitive hemagglutination) piliated strain |
| WO2013004020A1 (en) * | 2011-07-07 | 2013-01-10 | 深圳市宝舜泰生物医药股份有限公司 | Pseudomonas aeruginosa (pa-msha) strain igy and preparation method and use thereof |
| CN101880640B (en) * | 2010-02-09 | 2013-05-29 | 北京万特尔生物制药有限公司 | Klebsiella pneumoniae (Mannose-sensitive hemagglutination) pilus strain |
| CN103690941A (en) * | 2013-12-30 | 2014-04-02 | 北京万特尔生物制药有限公司 | Pseudomonas aeruginosa MSHA (Mannose-Sensitive Hemagglutinin) strain tumor vaccine and preparation method thereof |
-
1993
- 1993-02-06 CN CN 93100114 patent/CN1090602A/en not_active Withdrawn
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1101404C (en) * | 1998-07-29 | 2003-02-12 | 中德联合研究院 | Anti Bacillus pyocyaneu Flugge vitelline immunoglobulin products and use thereof |
| CN1297657C (en) * | 2005-03-31 | 2007-01-31 | 牟希亚 | Pseudomonas aeruginosa mannose sensitive haemagglutination pilus strain |
| CN101914463B (en) * | 2010-02-09 | 2012-09-05 | 北京万特尔生物制药有限公司 | Proteus vulgaris (mannose-sensitive hemagglutination) piliated strain |
| CN101880640B (en) * | 2010-02-09 | 2013-05-29 | 北京万特尔生物制药有限公司 | Klebsiella pneumoniae (Mannose-sensitive hemagglutination) pilus strain |
| WO2013004020A1 (en) * | 2011-07-07 | 2013-01-10 | 深圳市宝舜泰生物医药股份有限公司 | Pseudomonas aeruginosa (pa-msha) strain igy and preparation method and use thereof |
| CN103690941A (en) * | 2013-12-30 | 2014-04-02 | 北京万特尔生物制药有限公司 | Pseudomonas aeruginosa MSHA (Mannose-Sensitive Hemagglutinin) strain tumor vaccine and preparation method thereof |
| CN103690941B (en) * | 2013-12-30 | 2016-03-23 | 北京万特尔生物制药有限公司 | A kind of Pseudomonas aeruginosa MSHA pilus strain tumor vaccine and preparation method thereof |
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Open date: 19940810 |