CN105695540A - Preparing method of gram-negative bacterium cell wall antigen - Google Patents
Preparing method of gram-negative bacterium cell wall antigen Download PDFInfo
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- CN105695540A CN105695540A CN201610154758.XA CN201610154758A CN105695540A CN 105695540 A CN105695540 A CN 105695540A CN 201610154758 A CN201610154758 A CN 201610154758A CN 105695540 A CN105695540 A CN 105695540A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12P19/00—Preparation of compounds containing saccharide radicals
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Abstract
The invention relates to a preparing method of a gram-negative bacterium cell wall antigen. The preparing method is characterized by comprising the steps of strain rejuvenation, passage and amplification, fermentation medium forming, bacterium fermenting tank culture and antigen separation and purification. The method of cutting Asn-Gly amino acid loci through hydroxylamine hydrochloride specificity is adopted for obtaining the antigen, absolute ethyl alcohol is utilized as a chemical reagent for extracting the antigen, and therefore toxicity and risk performance are small compared with those of phenol. According to the method, damage to protein components when phenol is used for extracting the polysaccharide antigen is avoided, the protein antigen components are retained to the greater degree, and therefore the mixed antigen can activate the immune response more effectively.
Description
Technical field
The preparation method that the present invention relates to a kind of gram negative bacteria cell wall antigen, particularly relates to the cell wall composition polysaccharide of a kind of proteus mirabilis and the preparation method of albumen hybrid antigen。Belong to biological technical field。
Background technology
Bacillus proteus is widely present in water, the Organic substance of soil corruption and the intestinal of humans and animals, for conditioned pathogen, can causing chronic otitis media, traumatic infection, cystitis, infantile diarrhea, alimentary toxicosis, some bacterial strains can cause septicemia, alimentary toxicosis, peritonitis, meningitis etc.。
Antigen is the immune system that a class can stimulate body, so as to there is immunne response, produces antibody and primed lymphocyte etc., and can with corresponding antibodies or primed lymphocyte in vivo or the material of the specific binding reaction of external generation。Antigen has two kinds of performances: stimulate body to produce the immunogenicity of immunne response；Specific binding immunoreactivity is there is with the product of corresponding immunne response (antibody or primed lymphocyte etc.)。The kind of antigen is a lot, can be divided into following several big class: according to antigenic substance role, be divided into complete antigen and hapten。If most protein, antibacterial, virus, bacterial exotoxin etc. are all complete antigens。Hapten is only to have immunoreactivity, and the material of non-immunogenicity, therefore also known as incomplete antigen。Hapten can be divided into again complex hapten and simple hapten。Complex hapten does not have immunogenicity, only has immunoreactivity, such as most polysaccharide and all of lipoids etc.；Simple hapten neither has immunogenicity, does not have again immunoreactivity, but antibody can be stoped to be combined with corresponding antigens or complex hapten。Hydrolyzate etc. such as pneumococcal capsular polysaccharide。Assist the need of T cell according further to when producing antibody, also can be divided into thymus dependent antigen and thymus independent antigen。
The extraction of gram negative bacilli cell wall antigen conventional at present has three kinds of methods。
1 hot phenol method: after bacterium solution multigelation, add hot phenol solution, reheat water-bath, this processing procedure mainly retains lipopolysaccharide components, and the antigenic components such as albumen can be destroyed, and phenol has strong carcinogenic and corrosiveness, and needs harmless treatment after use, processing cost is high, pollutes the environment if dealing with improperly。
2 sonioation methods: this kind of method is difficult to hold operating condition, by whole bacterial cell disruption in operation, bacterial cell contents all can be released, difficulty is improved to subsequent purification work, and effective for antigen epi-position may be destroyed by the ultrasonic instantaneous high-temperature caused, causing that immunogenicity reduces, additionally ultrasonic method is difficult to be applicable to large-scale production operation。
3 boiling methods: this kind of method is to be boiled for a long time by thalline, the problem that there is also the destroyed degeneration of effective ingredient in processing procedure, immunogenicity cannot ensure。
Summary of the invention
It is an object of the invention to solve the problems referred to above that prior art exists, the preparation method providing a kind of novel gram negative bacteria cell wall antigen。The present invention adopts the method for oxammonium hydrochloride. special cutting Asn-Gly amino acid sites to obtain antigen, and recycling dehydrated alcohol is little compared with phenol as the chemical reagent extracting antigen, its toxicity and danger。And destruction to protein component when this method avoids extract with phenol polysaccharide antigen, remain proteantigen composition greatly, so that the significantly more efficient activation immunne response of antigen of mixing。
The technical scheme that the present invention provides is: the preparation method of a kind of gram negative bacteria cell wall antigen, it is characterised in that have following steps:
(1) strain rejuvenation, go down to posterity and expand；
(2) composition of fermentation medium；
(3) bacterial fermentation tank is cultivated；
(4) separation of antigen and purification。
The rejuvenation of described step (1) strain, go down to posterity and expand。
Lyophilizing proteus mirabilis strain is recovered: by lyophilizing bacterial strain, with alcohol wipe cillin bottle aluminium lid, after removing aluminium lid, with alcohol swab wiping plug and cillin bottle surface。
Draw 0.5 ~ 1mlNB culture medium with pipettor to add in bacterial strain cillin bottle, rock and be mixed into bacteria suspension, bacteria suspension is added in the test tube containing NB culture medium, is positioned over after slight oscillatory in incubator, 37 DEG C of static gas wave refrigerator 18 ~ 24hr。
Strain passage: with after calcination inoculating loop dip the bacterial cultures in NB culture medium, be inoculated on aseptic NA inclined-plane drawing in the way of serpentine, be put in constant incubator, cultivate 18 ~ 24 hours for 37 DEG C。
Bacterial strain expands: with the culture in step on the inoculating loop picking after calcination, culture is rinsed with aseptic 0.9%NaCl, blowing and beating uniformly after bacteria suspension, with pipettor, bacteria suspension is laid in NA agar square vase, the seed liquor bacterium amount obtained with spectrophotometer method detection is close to 600 × 1010Individual, for fermentor cultivation after gram stain microscopy is qualified。
The composition of described step (2) fermentation medium。
Casein hydrolysate 20 ~ 30g/L, yeast leaching powder 8 ~ 17g/L, CH3COONa4~12g/L、MgSO40.15~0.35g/L、Na2HPO40.1~0.5g/L、NaH2PO40.1~0.4g/L、C6H12O62 ~ 6g/L, above-mentioned medium component adds in proportion and is configured to fermentation liquid, after degerming standby through 0.1+0.2 μm of aperture sleeve frit of high pressure steam sterilization。
Described step (3) bacterial fermentation tank is cultivated。
Fermentation condition is set, ventilation 8 ~ 10L/min, agitation revolution 300 ~ 400rpm, ph7.2 ~ 7.4, temperature 37 DEG C, after fermentation conditional stability, seed liquor is seeded in fermentation tank, is cultured to logarithmic growth after date, sampling and measuring OD value determines that bacterium is dense。
After mensuration bacterium is dense, by 109/ ml bacterium is dense corresponding adds 53 ~ 72ug oxammonium hydrochloride., and calculating need to add oxammonium hydrochloride. total amount, arranges condition ventilation 2 ~ 4L/min, and agitation revolution 100 ~ 200rpm, pH7.2 ~ 7.4, digest temperature 45 ~ 65 DEG C, duration 24 ~ 72hr, blowing fermentation liquid after digestion terminates。
The separation of described step (4) antigen and purification。
By fermentation liquor 8000 ~ 10000rpm centrifugal 10 ~ 20min under 4 DEG C of conditions, results supernatant, supernatant is clarified through 0.45+0.2um aperture sleeve frit, and then through 10kd ultrafilter membrane bag, clear liquor is concentrated 5 ~ 10 times, add purified water to original volume, dialyse 500 ~ 1000 times through 10kd ultrafilter membrane bag。
With sample: the ratio of dehydrated alcohol=1:2.5 ~ 1:1 adds dehydrated alcohol, regulate ph4.5 ~ 6.5, be placed in 2 ~ 8 DEG C of conditions 0.5 ~ 2.5 hour。Being shaken up by liquid through the centrifugal 10 ~ 30min of 3000 ~ 6000rpm, abandon supernatant, precipitation dehydrated alcohol is resuspended, with step centrifugal condition recentrifuge, abandons supernatant, and precipitation adjusts ph7.2 ~ 7.4 after dissolving with ultra-pure water, it is thus achieved that the antigen of extraction。
Compared with the prior art applying the chemical reagent extraction cell wall antigens such as phenol, the invention has the beneficial effects as follows。
Utilizing the method for oxammonium hydrochloride. special cutting Asn-Gly amino acid sites to obtain antigen, recycling dehydrated alcohol is little compared with phenol as the chemical reagent extracting antigen, its toxicity and danger。And destruction to protein component when this method avoids extract with phenol polysaccharide antigen, remain proteantigen composition greatly, so that the significantly more efficient activation immunne response of antigen of mixing。
Detailed description of the invention
The lyophilizing proteus mirabilis bacterium numbering that present patent application provides is Proteusmirabilis13(PM13), Proteusmirabilis28(PM28), Proteusmirabilis43(PM43), Proteusmirabilis62(PM62), Proteusmirabilis92(PM92), Proteusmirabilis104(PM104)。Above bacterial strain is both from attached Shengjing city hospital of Chinese Medical Sciences University。Collection and the Preliminary screening working delegation of proteus mirabilis bacterial strain are completed by our company to attached Shengjing city hospital of Chinese Medical Sciences University。Attached Shengjing city hospital of Chinese Medical Sciences University be responsible for gathering and screening from In South China, the southeast, northwest, five regions in southwest and northeast the Bacillus proteus (proteus vulgaris and proteus mirabilis) that is clinically separated of Grade A hospital, according to " the coupling bacterial strain acquisition protocols for PSP vaccine ", attached Shengjing city hospital of Chinese Medical Sciences University provides 125 strain Bacillus proteuss。According to project research requirement, our company filters out 6 strain proteus mirabilises and studies original strain as project。
Embodiment 1。
The rejuvenation of strain, go down to posterity and expand: lyophilizing proteus mirabilis strain being recovered: by lyophilizing bacterial strain, with alcohol wipe cillin bottle aluminium lid, after removing aluminium lid, with alcohol swab wiping plug and cillin bottle surface。Draw 0.5 ~ 1mlNB culture medium with pipettor to add in bacterial strain cillin bottle, rock and be mixed into bacteria suspension, bacteria suspension is added in the test tube containing NB culture medium, is positioned over after slight oscillatory in incubator, 37 DEG C of static gas wave refrigerator 18 ~ 24hr。Strain passage: with after calcination inoculating loop dip the bacterial cultures in NB culture medium, be inoculated on aseptic NA inclined-plane drawing in the way of serpentine, be put in constant incubator, cultivate 18 ~ 24 hours for 37 DEG C。Bacterial strain expands: with the culture in step on the inoculating loop picking after calcination, culture is rinsed with aseptic 0.9%NaCl, blowing and beating uniformly after bacteria suspension, with pipettor, bacteria suspension is laid in NA agar square vase, the seed liquor bacterium amount obtained with spectrophotometer method detection is close to 600 × 1010Individual, for fermentor cultivation after gram stain microscopy is qualified。
Embodiment 2。
The composition of fermentation medium: casein hydrolysate 20 ~ 30g/L, yeast leaching powder 8 ~ 17g/L, CH3COONa4~12g/L、MgSO40.15~0.35g/L、Na2HPO40.1~0.5g/L、NaH2PO40.1~0.4g/L、C6H12O62 ~ 6g/L, above-mentioned medium component adds in proportion and is configured to fermentation liquid, after degerming standby through 0.1+0.2 μm of aperture sleeve frit of high pressure steam sterilization。
Embodiment 3。
Bacterial fermentation is cultivated: arrange fermentation condition, ventilation 8 ~ 10L/min, agitation revolution 300 ~ 400rpm, ph7.2 ~ 7.4, temperature 37 DEG C, after fermentation conditional stability, being seeded in fermentation tank by seed liquor, be cultured to logarithmic growth after date, sampling and measuring OD value determines that bacterium is dense。After mensuration bacterium is dense。By 109/ ml bacterium is dense corresponding adds 53 ~ 72ug oxammonium hydrochloride., and calculating need to add oxammonium hydrochloride. total amount, arranges condition ventilation 2 ~ 4L/min, and agitation revolution 100 ~ 200rpm, pH7.2 ~ 7.4, digest temperature 45 ~ 65 DEG C, duration 24 ~ 72hr, blowing fermentation liquid after digestion terminates。
Embodiment 4。
The separation of antigen and purification: by fermentation liquor 8000 ~ 10000rpm centrifugal 10 ~ 20min under 4 DEG C of conditions, results supernatant, supernatant is clarified through 0.45+0.2um aperture sleeve frit, clear liquor is concentrated 5 ~ 10 times then through 10kd ultrafilter membrane bag, add purified water to original volume, dialyse 500 ~ 1000 times through 10kd ultrafilter membrane bag。With sample: the ratio of dehydrated alcohol=1:2.5 ~ 1:1 adds dehydrated alcohol, regulate ph4.5 ~ 6.5, be placed in 2 ~ 8 DEG C of conditions 0.5 ~ 2.5 hour。Being shaken up by liquid through the centrifugal 10 ~ 30min of 3000 ~ 6000rpm, abandon supernatant, precipitation dehydrated alcohol is resuspended, with step centrifugal condition recentrifuge, abandons supernatant, and precipitation adjusts ph7.2 ~ 7.4 after dissolving with ultra-pure water, it is thus achieved that the antigen of extraction。
Claims (5)
1. the preparation method of a gram negative bacteria cell wall antigen, it is characterised in that have following steps:
(1) strain rejuvenation, go down to posterity and expand；
(2) composition of fermentation medium；
(3) bacterial fermentation tank is cultivated；
(4) separation of antigen and purification。
2. the preparation method of gram negative bacteria cell wall antigen according to claim 1, it is characterised in that: the rejuvenation of step (1) strain, going down to posterity and expanding refers to:
The rejuvenation of strain: by lyophilizing bacterial strain, with alcohol wipe cillin bottle aluminium lid, after removing aluminium lid, with alcohol swab wiping plug and cillin bottle surface；
Draw 0.5 ~ 1mlNB culture medium with pipettor to add in bacterial strain cillin bottle, rock and be mixed into bacteria suspension, bacteria suspension is added in the test tube containing NB culture medium, is positioned over after slight oscillatory in incubator, 37 DEG C of static gas wave refrigerator 18 ~ 24hr；
Strain passage: with after calcination inoculating loop dip the bacterial cultures in NB culture medium, be inoculated on aseptic NA inclined-plane drawing in the way of serpentine, be put in constant incubator, cultivate 18 ~ 24 hours for 37 DEG C；
Bacterial strain expands: with the culture in the inoculating loop picking above-mentioned steps after calcination, culture is rinsed with aseptic 0.9%NaCl, blow and beat uniformly after bacteria suspension, with pipettor, bacteria suspension is laid in NA agar square vase, the seed liquor bacterium amount obtained with spectrophotometer method detection, for fermentor cultivation after gram stain microscopy is qualified。
3. the preparation method of gram negative bacteria cell wall antigen according to claim 1, it is characterised in that: the composition of step (2) fermentation medium refers to:
Casein hydrolysate 20 ~ 30g/L, yeast leaching powder 8 ~ 17g/L, CH3COONa4~12g/L、MgSO40.15~0.35g/L、Na2HPO40.1~0.5g/L、NaH2PO40.1~0.4g/L、C6H12O62 ~ 6g/L, above-mentioned medium component adds in proportion and is configured to fermentation liquid, after degerming standby through 0.1+0.2 μm of aperture sleeve frit of high pressure steam sterilization。
4. the preparation method of gram negative bacteria cell wall antigen according to claim 1, it is characterised in that: the cultivation of step (3) bacterial fermentation tank refers to:
Fermentation condition parameter, ventilation 8 ~ 10L/min, agitation revolution 300 ~ 400rpm are set, ph7.2 ~ 7.4, temperature 37 DEG C, after fermentation conditional stability, being seeded in fermentation tank by gram stain microscopy seed liquor after qualified, be cultured to logarithmic growth after date, sampling and measuring OD value determines that bacterium is dense；
After mensuration bacterium is dense, by 109The dense corresponding ratio adding 53 ~ 72ug oxammonium hydrochloride. of/ml bacterium adds oxammonium hydrochloride., resets fermentation tank parameter, ventilation 2 ~ 4L/min, agitation revolution 100 ~ 200rpm, pH7.2 ~ 7.4, digest temperature 45 ~ 65 DEG C, duration 24 ~ 72hr, blowing fermentation liquid after digestion terminates。
5. the preparation method of gram negative bacteria cell wall antigen according to claim 1, it is characterised in that: separation and the purification of step (4) antigen refer to:
By fermentation liquor 8000 ~ 10000rpm centrifugal 10 ~ 20min under 4 DEG C of conditions, results supernatant, supernatant is clarified through 0.45+0.2um aperture sleeve frit, and then through 10kd ultrafilter membrane bag, clear liquor is concentrated 5 ~ 10 times, add purified water to original volume, dialyse 500 ~ 1000 times through 10kd ultrafilter membrane bag；
With sample: the ratio of dehydrated alcohol=1:2.5 ~ 1:1 adds dehydrated alcohol, regulate ph4.5 ~ 6.5, it is placed in 2 ~ 8 DEG C of conditions 0.5 ~ 2.5 hour, being shaken up by liquid through the centrifugal 10 ~ 30min of 3000 ~ 6000rpm, abandon supernatant, precipitation dehydrated alcohol is resuspended, with step centrifugal condition recentrifuge, abandoning supernatant, precipitation adjusts ph7.2 ~ 7.4 after dissolving with ultra-pure water, it is thus achieved that the antigen of extraction。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106978469A (en) * | 2017-06-08 | 2017-07-25 | 太仓优活生物技术有限公司 | A kind of improved bacterium viable count method |
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| CN101070203A (en) * | 2007-06-18 | 2007-11-14 | 同济大学 | Method for preparing biological flocculant using strange deformation bacilli |
| CN101203605A (en) * | 2004-12-17 | 2008-06-18 | Nvi荷兰疫苗研究所 | Deacylation of lps in gram negative bacteria |
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- 2016-03-18 CN CN201610154758.XA patent/CN105695540A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101203605A (en) * | 2004-12-17 | 2008-06-18 | Nvi荷兰疫苗研究所 | Deacylation of lps in gram negative bacteria |
| CN101070203A (en) * | 2007-06-18 | 2007-11-14 | 同济大学 | Method for preparing biological flocculant using strange deformation bacilli |
Non-Patent Citations (3)
| Title |
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| FC MCINTIRE等: "Chemical, Physical, and Biological Properties of a Lipopolysaccharide from Escherichia coli K-235", 《BIOCHEMISTRY》 * |
| 夏小成等: "布鲁氏杆菌LPS抗原制备及其免疫原性测定", 《特产研究》 * |
| 邓功成等: "《微生物与人类》", 31 December 2015, 重庆大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106978469A (en) * | 2017-06-08 | 2017-07-25 | 太仓优活生物技术有限公司 | A kind of improved bacterium viable count method |
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Application publication date: 20160622 |