CN105709218A - Method for preparing proteus mirabilis-staphylococcus aureus-pseudomonas aeruginosa adsorption combined vaccine - Google Patents
Method for preparing proteus mirabilis-staphylococcus aureus-pseudomonas aeruginosa adsorption combined vaccine Download PDFInfo
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- CN105709218A CN105709218A CN201610154756.0A CN201610154756A CN105709218A CN 105709218 A CN105709218 A CN 105709218A CN 201610154756 A CN201610154756 A CN 201610154756A CN 105709218 A CN105709218 A CN 105709218A
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- staphylococcus aureus
- proteus mirabilis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/085—Staphylococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/104—Pseudomonadales, e.g. Pseudomonas
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
Abstract
The invention discloses a method for preparing a proteus mirabilis-staphylococcus aureus-pseudomonas aeruginosa adsorption combined vaccine. The method includes the steps that firstly, a proteus mirabilis culture solution is subjected to in-situ digestion, high-speed centrifugation, pre-filtering, ultrafiltration and precipitation, and a proper proteus mirabilis cell membrane soluble antigen raw solution is obtained; secondly, a staphylococcus aureus bacterium suspension is subjected to centrifugation, smashing, re-centrifugation, filtering, precipitation, enzymolysis and dialysis, and a proper staphylococcus aureus cytoplast antigen raw solution is obtained; thirdly, a staphylococcus aureus and pseudomonas aeruginosa culture solution is subjected to centrifugation and pre-filtering, formalin is added for detoxification, then purification is conducted, and a proper inactivated staphylococcus aureus and pseudomonas aeruginosa toxin raw solution is obtained. The vaccine is used for preventing burn, scalding and infection caused by one or more of conditioned pathogens including proteus mirabilis, staphylococcus aureus and pseudomonas aeruginosa before and after operations in an intramuscular deep injection mode.
Description
Technical field
The present invention relates to the preparation method of a kind of vaccine, particularly relate to a kind of proteus mirabilis-Staphylococcus aureus
Bacterium-Pseudomonas aeruginosa absorption combined vaccine (PSP adsorbs combined vaccine) preparation method, is especially for prevention, treatment,
The absorption combined vaccine preparation method that conditioned pathogen burn, preoperative and postoperative etc. infects, can including proteus mirabilis after birth
Dissolubility antigen, staphylococcus aureus cytoplasmic antigen, staphylococcus aureus toxoid, P. aeruginosa toxoid and hydrogen-oxygen
Change the preparation method of alumina gel absorption combined vaccine, belong to field of medicaments.
Background technology
In the last few years, abuse phenomenon due to domestic antibiotic and be on the rise, proteus mirabilis, staphylococcus aureus and
Pseudomonas aeruginosas etc. have become serious Nosocomial Infection Pathogens at present, are also the important causes of wound surface of burn patients infection simultaneously
Pathogenic bacteria.Serious symptom proteus mirabilis, staphylococcus aureus and Pseudomonas aeruginosa is often caused when patient immune function is bad
Infect, especially Cystic fibrosis, serious burn, surgical wound and or the patient such as operation transplantation, cancer, urinary tract illness deep
By its evil.Infect and cause different diseases, serious from slight skin infection to bacteremia, osteomyelitis, intimitis, poison pus disease etc.
Harm broad masses' is healthy.
There is presently no both at home and abroad and report about proteus mirabilis vaccine, and for pseudomonas aeruginosa vaccine research be not
A lot, research to the proper composition of Pseudomonas aeruginosa candidate vaccine at present concentrates on pseudomonas thalline and adventitia is expressed as
Point, such as formalin-inactivated thalline, exotoxin A (PEA), elastoser and alkaline protease, cilium, leukocidin etc. are logical
Cross collection of data, mainly include PseudostatTM Vaccine, Pseudomonas aeruginosa-mannose sensitive haemagglutination (PA-MSHA) pili
Strain vaccine and IC43 vaccine.Great mass of data show staphylococcus aureus candidate vaccine mainly include capsular polysaccharide (CP5, CP8 and
CP336 etc.) and toxin
(α-Hemolysin, PVL, TSST-1 etc.), glycosamine (PNAG, PNSG) and surface protein (IsdA, IsdB, SdrD and
SdrE), DNA vaccination FnBPA-CifA and Peptidoglycan (Peptidoglycan, PGN) etc..In the face of infection of staphylococcus aureus
Complexity, staphylococcus aureus contains multiple pathogenicity factors, and these factors are at bacterial growth different phase sequentially table
Reach.In the last few years, research in terms of domestic and international related vaccines company actively develops staphylococcus aureus candidate vaccine and facing
Bed test, but most of S. aureus vaccines candidate vaccine clinical research is not reaching to clinical target.And
Bacterial strain virulence factor is a lot, and content is relatively low, though and whole cell vaccine, attenuation have certain immunogenicity, but side reaction
Relatively strong, directly isolated and purified from whole cell have the antigen component technology difficulty of protectiveness relatively greatly, and method is complicated, the response rate
Low, it is unfavorable for prepared by the industrialization of vaccine, utilizes gene recombination technology to obtain related activity component, its active component natural structure
Imperfect, immunogenicity is the strongest.
Summary of the invention
It is an object of the invention to solve the problems referred to above that prior art exists, be given and a kind of novel prepare unusual deformed rod
The method of bacterium-staphylococcus aureus-Pseudomonas aeruginosa absorption combined vaccine (PSP adsorbs combined vaccine).The present invention uses
Proteus mirabilis, staphylococcus aureus and Pseudomonas aeruginosa carry out by large scale fermentation, use enzymatic digest, from
The heart, ultrafiltration, ethanol precipitation etc. obtain proteus mirabilis after birth soluble component, and employing is centrifugal, Mechanical Crushing, isoelectric point, IP are heavy
Shallow lake, enzymolysis etc. obtain staphylococcus aureus kytoplasm soluble component, use centrifugal, pre-filtering, ultrafiltration, detoxification etc. to obtain golden yellow
Then four kinds of stock solutions, adjuvants etc. are carried out by color staphylococcus and P. aeruginosa toxoid active component according to a certain percentage
Compatibility, it is mixed to form absorption combined vaccine.This vaccine includes 6 strain proteus mirabilis after birth soluble antigens, golden yellow Fructus Vitis viniferae
Coccus cytoplasmic antigen, staphylococcus aureus toxoid and P. aeruginosa toxoid, be adsorbed in after above-mentioned 9 kinds of antigens mixing
Gel aluminum hydroxide (Al(OH)3).This method technique is simple, and acquired active antigens composition kind is many, wide coverage, it is possible to have
The raising immunogenicity of effect and protectiveness.
The technical scheme that the present invention is given is: a kind of proteus mirabilis-staphylococcus aureus-Pseudomonas aeruginosa is inhaled
The preparation method of attached combined vaccine (PSP adsorbs combined vaccine), it is characterised in that have following steps:
(1) prepared by proteus mirabilis after birth soluble antigen stock solution；
(2) prepared by staphylococcus aureus cytoplasmic antigen stock solution；
(3) prepared by staphylococcus aureus toxoid stock solution；
(4) prepared by P. aeruginosa toxoid stock solution；
(5) prepared by proteus mirabilis 6 valency antigen mixed liquor；
(6) proteus mirabilis-staphylococcus aureus-Pseudomonas aeruginosa absorption combined vaccine (PSP adsorbs combined vaccine)
Preparation.
Prepared by described step (1) proteus mirabilis after birth soluble antigen stock solution: by the unusual deformation of High Density Cultivation
Bacillus Proteus mirabilis 13(PM13) prepared by strain.The culture fluid of generation is digested in place, high speed centrifugation, pre-filtering,
Ultrafiltration and precipitation, obtain suitable proteus mirabilis after birth soluble antigen stock solution.Remaining 5 strain Proteus mirabilis
28(PM28), Proteus mirabilis 43(PM43), Proteus mirabilis 62(PM62) Proteus
Mirabilis 92(PM92), Proteus mirabilis 104(PM104) cultivate and purification condition identical.Can also use
It is solvable that other proteus mirabilis bacterial strains other than the above and cultivation and purification process obtain suitable proteus mirabilis after birth
Property antigen stock.
Prepared by described step (2) staphylococcus aureus cytoplasmic antigen stock solution: by High Density Cultivation Staphylococcus aureus
Bacterium Staphylococus aureus79(SA79) prepared by strain, by the bacteria suspension produced through centrifugal, broken, centrifugal, filter,
Precipitation, enzymolysis, dialysis obtain suitable staphylococcus aureus cytoplasmic antigen stock solution.Other gold other than the above can also be used
Staphylococcus aureus bacterial strain and cultivation and purification process obtain suitable staphylococcus aureus cytoplasmic antigen stock solution.
Prepared by described step (3) staphylococcus aureus toxoid stock solution: be through High Density Cultivation Staphylococcus aureus
Bacterium Staphylococus aureus26002(CMCC26002) prepared by strain.By the medium centrifugal produced, pre-filtering, addition good fortune
Purification after your Malin's detoxification detoxification, obtains suitable inactivation staphylococcus aureus toxoid stock solution.Can also use above-mentioned with
It is former that other outer staphylococcus aureus strains and cultivation and purification process obtain suitable staphylococcus aureus toxoid antigen
Liquid.
Prepared by described step (4) P. aeruginosa toxoid stock solution: through High Density Cultivation Pseudomonas aeruginosa
Pseudomonas aeruginosa(PA101) prepared by strain.The medium centrifugal of generation, pre-filtering, addition formalin are taken off
Purification after poison detoxification, obtains suitable inactivation P. aeruginosa toxoid stock solution.Other copper other than the above can also be used
Green pseudomonas strains and cultivation and purification process obtain suitable Pseudomonas aeruginosa toxoid antigen stock solution.
Prepared by described step (5) proteus mirabilis 6 valency antigen mixed liquor: select 6 strain proteus mirabilis after births solvable
Property antigen, through lyophilization, it is desirable to freeze-dried antigen residual moisture control 3.0～8.0%, measure single antigen dry weight, it is desirable to
Single antigen dry weight is not less than 2mg/ml, becomes concentration to be 9ug/ml or its multiple each strain antigen addition normal saline dilution,
Then 6 strain antigens mix according to proportioning 1:1:1:1:1:1, and remaining is supplied with normal saline, shake up regulation pH6.8～7.2,
It is prepared as proteus mirabilis 6 valency antigen mixed liquor.
Described step (6) proteus mirabilis-staphylococcus aureus-(PSP inhales Pseudomonas aeruginosa absorption combined vaccine
Attached combined vaccine) to prepare: the effective dose preparing 1ml absorption united vaccine formulation needs proteus mirabilis antigen mixed liquor 48
～60ug, staphylococcus aureus cytoplasmic antigen fraction 0.8～1.2mg, staphylococcus aureus toxoid antigen component 3～
8EC, P. aeruginosa toxoid component 20～50ug, 0.8～1.2mg aluminium adjuvant.
The suitable adsorbent that the present invention uses includes aluminium hydroxide, aluminum phosphate and alumina gel.Aluminium hydroxide, phosphoric acid
Aluminum and alumina gel can be bought or prepare with known method.
The strain that the present invention uses is.
Proteus mirabilis: Proteus mirabilis 13(PM13), Proteus mirabilis 28(PM28),
Proteus mirabilis 43(PM43), Proteus mirabilis 62(PM62), Proteus mirabilis 92
(PM92), Proteus mirabilis 104(PM104).
Staphylococcus aureus: Staphylococus aureus79(SA79), Staphylococus aureus26002
(CMCC26002).
Pseudomonas aeruginosa: pseudomonas aeruginosa(PA101).
Above 6 strain proteus mirabilis Proteus mirabilis 13(PM13), Proteus mirabilis 28
(PM28), Proteus mirabilis 43(PM43), Proteus mirabilis 62(PM62), Proteus
Mirabilis 92(PM92), Proteus mirabilis 104(PM104).1 strain staphylococcus aureus
Staphylococus aureus79(SA79), 1 Pseudomonas aeruginosa strain pseudomonas aeruginosa(PA101) all come
From in attached Shengjing city hospital of Chinese Medical Sciences University.Our company is by proteus mirabilis, staphylococcus aureus, Pseudomonas aeruginosa
Collection and the Preliminary screening working delegation of bacterial strain complete to attached Shengjing city hospital of Chinese Medical Sciences University.Chinese Medical Sciences University is attached
Shengjing city hospital is responsible for gathering and screening from In South China, the southeast, northwest, the Grade A hospital clinic of five regions in southwest and northeast
The related strain separated, according to " for the coupling bacterial strain acquisition protocols of PSP vaccine ", attached Shengjing city hospital of Chinese Medical Sciences University is every
Plant bacterium and 125 strain original strains are provided.According to project research requirement, our company filters out 6 strain proteus mirabilises, 1 strain golden yellow
Staphylococcus, 1 Pseudomonas aeruginosa strain study original strain as project.
Staphylococcus aureus Staphylococus aureus26002(CMCC26002) by Chinese food drug assay
Academy provides.
Compared with prior art, the invention has the beneficial effects as follows: use proteus mirabilis, staphylococcus aureus and
Pseudomonas aeruginosa carries out by large scale fermentation, uses enzymatic digest, centrifugal, ultrafiltration, ethanol precipitation etc. to obtain unusual deformation
Bacillus after birth soluble component, uses centrifugal, Mechanical Crushing, isoelectric precipitation, enzymolysis etc. to obtain staphylococcus aureus kytoplasm
Soluble component, uses centrifugal, pre-filtering, ultrafiltration, detoxification etc. to obtain staphylococcus aureus and P. aeruginosa toxoid
Then four kinds of stock solutions, adjuvants etc. are carried out compatibility, are mixed to form absorption combined vaccine by active component according to a certain percentage.This method
Technique is simple, and acquired active antigens composition kind is many, wide coverage, it is possible to effective raising immunogenicity and protectiveness.
Avoiding the side reaction that prior art causes big, the response rate is low, is unfavorable for drawback prepared by the industrialization of vaccine.
Accompanying drawing explanation
Fig. 1. proteus mirabilis after birth soluble antigen stock solution preparation technology flow process.
Fig. 2. staphylococcus aureus cytoplasmic antigen stock solution preparation technology flow process.
Fig. 3. staphylococcus aureus toxoid stock solution preparation technology flow process.
Fig. 4. P. aeruginosa toxoid stock solution preparation technology flow process.
Fig. 5. proteus mirabilis-staphylococcus aureus-(PSP adsorbs associating to Pseudomonas aeruginosa absorption combined vaccine
Vaccine) technological process.
Detailed description of the invention
Embodiment 1: proteus mirabilis after birth soluble antigen stock solution.
6 strain bacterial strain PM13, PM28, PM43, PM62, PM92 and PM104 are carried out fermentation tank High Density Cultivation, collects bacterium
Body.By enzymolysis, somatic cells film carried out enzymolysis, centrifugal, pre-filtering, ultrafiltration, precipitation solvable to proteus mirabilis cell membrane
Property component carries out isolated and purified, obtains proteus mirabilis antigen stock (6 strain strain fermentations and the purification condition phase of high protectiveness
With), technological process is as shown in Figure 1.
Described purification step is as follows.
(1) enzymolysis: in the phage solution pH6.0 that fermentation tank High Density Cultivation is obtained～8.0 aseptic 0.9%NaCl solution
Mixing, then according to 4.0～6.0 × 109Individual/ml bacterial concentration adds 1～2g pancreatin, pH7.8～8.5,50～65 DEG C of digestion
4.5 hour.
(2) centrifugal: by 10000～12000rpm, 4 DEG C, 10～15min freezing high speed centrifugations, collect supernatant.
(3) pre-filtering: use 0.2+0.45um filter element that centrifuged supernatant carries out pre-filtering, obtains pre-filtering clarification
Liquid.
(4) ultrafiltration: select 10～30KD ultrafilter membrane bags that the filter analysis of pre-filtering supernatant carried out remaining medium removal, 100
～1000 times, then concentrate 10～15 times.
(5) precipitation: liquid is concentrated by ultrafiltration and adds ethanol solution according to 1:1.0～3.0, selects 0.5～1.0MHCl regulation pH extremely
4.0～6.5, place 3.0 ± 0.5 hours for 4 DEG C, 4000～5000rpm, 4 DEG C centrifugal 10～15min, collect precipitation solution aseptic
In water for injection, with 0.5～1.0MNaOH solution regulation pH7.8 ± 0.5, aseptic filtration preserves.
Embodiment 2 staphylococcus aureus cytoplasmic antigen stock solution.
SA79 bacterial strain is carried out fermentation tank High Density Cultivation, collects thalline.By bead mill method thalline carried out Mechanical Crushing,
Staphylococcus aureus kytoplasm soluble component is carried out point by pre-filtering, isoelectric precipitation 1, isoelectric precipitation 2, enzymolysis, dialysis
From purification, obtaining the staphylococcus aureus cytoplasmic antigen stock solution of high protectiveness, technological process is as shown in Figure 2.
Described purification step is as follows.
(1) Mechanical Crushing: the phage solution pH6.0～the 8.0 aseptic 0.9%NaCl that fermentation tank High Density Cultivation are obtained are molten
Liquid mixes, then by peristaltic pump bacteria suspension is pumped into and disintegrating machine carries out continuous crushing, bead particle size range 0.45-
1.25mm, percentage of damage is not less than 80%, and freezing high speed centrifugation collects supernatant.
(2) pre-filtering: use 0.2+0.45um filter element that centrifuged supernatant carries out pre-filtering, obtains pre-filtering clarification
Liquid.
(3) isoelectric precipitation 1: select 0.5～1.0MHCl regulation pre-filtering clear liquor pH to 3.0～4.5, place 2 for 4 DEG C
～3 hours, 4000～5000rpm, 4 DEG C centrifugal 10～15min, collect in precipitation solution aseptic 0.9%NaCl solution, with 0.5～
1.0MNaOH solution regulation pH to 7.0～7.2.
(4) isoelectric precipitation 2: select 0.5～1.0MHCl regulation pre-filtering clear liquor pH to 4.5～6.5, place 2 for 4 DEG C
～3 hours, 4000～5000rpm, 4 DEG C centrifugal 10～15min, collect in precipitation solution aseptic 0.9%NaCl solution, with 0.5～
1.0MNaOH solution regulation pH to 7.0～7.2.
(5) enzymolysis: use G-250 or Lorry method to measure redissolution liquid total nitrogen content, according to
60～100mg total nitrogens corresponding 1～10mgTrypsin, after adding Trypsin vibration dissolving, regulate pH to 8.0～8.2,
Hatch 2～3 hours for 30-37 DEG C, then take out placement room temperature, regulate pH to 6.5 ± 0.5.
(6) dialysis: using 1～10KD dialyzer to dialyse enzymolysis solution 24～48 hours, aseptic filtration preserves.
Embodiment 3 staphylococcus aureus toxoid stock solution.
Staphylococcus aureus CMCC26002 is carried out fermentation tank High Density Cultivation, collects bacteria suspension.By centrifugal, de-
Poison, pre-filtering, ultrafiltration, Acid precipitation, centrifugal staphylococcus aureus toxoid component is carried out isolated and purified, obtain high protectiveness
Staphylococcus aureus toxoid antigen stock solution, technological process is as shown in Figure 3.
Described purification step is as follows.
(1) centrifugal: by 10000～12000rpm, 4 DEG C, 10～15min freezing high speed centrifugations, collect supernatant.
(2) detoxification: add 0.4～1.0% formalin or glutaraldehyde solution, 30～37 DEG C, pH6.8～7.2 detoxifications 5～
7 days.
(3) pre-filtering: use 0.2+0.45um filter element that centrifuged supernatant carries out pre-filtering, obtains pre-filtering clarification
Liquid.
(4) ultrafiltration: select 10～30KD ultrafilter membrane bags to pre-filtering supernatant filter analysis 100～1000 times, be then concentrated into
20～30 times.
(5) Acid precipitation: add NaCl crystal, makes final concentration of 10～30%, selects 0.5～1.0MHCl solution regulation ultrafiltration
Liquid pH is 3.0～5.0, and 4 DEG C precipitate 2～3 hours.
(6) centrifugal: by 4000～5000rpm, 4 DEG C, 10～15min are centrifuged, and collect precipitation, add normal saline solution
Redissolving, regulation pH is 6.8～7.2, and aseptic filtration preserves.
Embodiment 4 P. aeruginosa toxoid stock solution.
PA101 is carried out fermentation tank High Density Cultivation, centrifugal collection supernatant.By adding detoxification, pre-filtering, ultrafiltration pair
P. aeruginosa toxoid carries out isolated and purified, obtains the Pseudomonas aeruginosa toxoid antigen stock solution of high protectiveness, technique
Flow process is as shown in Figure 4.
Described purification step is as follows.
(1) centrifugal: excessively 10000～12000rpm, 4 DEG C, 10～15min freezing high speed centrifugations, collect supernatant.
(2) detoxification: addition 0.4～1.0% formalin or glutaraldehyde solution, 30～37 DEG C, pH6.8～7.2.
Detoxification 12～15 days.
(3) pre-filtering: use 0.2+0.45um filter element that centrifuged supernatant carries out pre-filtering, obtains pre-filtering clarification
Liquid.
(4) ultrafiltration 1: select 300～1000KD ultrafilter membrane bags to pre-filtering supernatant filter analysis 100～1000 times, collect super
Filtrate.
(5) ultrafiltration 2: select 10～30KD ultrafilter membrane bags to pre-filtering supernatant filter analysis 100～1000 times, be then concentrated into
10～15 times, aseptic filtration preserves.
Prepared by embodiment 5 proteus mirabilis 6 valency antigen mixed liquor.
Select 6 strain proteus mirabilis after birth soluble antigens, through lyophilization, it is desirable to freeze-dried antigen residual moisture content control
System, 3.0～8.0%, measures single antigen dry weight, it is desirable to single antigen dry weight is not less than 2mg/ml, is added by each strain antigen
Normal saline dilution becomes concentration to be 9ug/ml or its multiple, and then 6 strain antigens mix according to proportioning 1:1:1:1:1:1, its
Remaining normal saline is supplied, and shakes up regulation pH6.8～7.2, is prepared as proteus mirabilis 6 valency antigen mixed liquor.
Embodiment 6 proteus mirabilises-staphylococcus aureus-(PSP adsorbs connection to Pseudomonas aeruginosa absorption combined vaccine
Close vaccine) prepare.
Proteus mirabilis-staphylococcus aureus-Pseudomonas aeruginosa absorption combined vaccine (PSP adsorbs combined vaccine)
Preparation process is as follows: prepare 1ml absorption united vaccine formulation effective dose need proteus mirabilis antigen mixed liquor 48～
60ug, staphylococcus aureus cytoplasmic antigen fraction 0.8～1.2mg, staphylococcus aureus toxoid antigen component 3～8EC,
P. aeruginosa toxoid component 20～50ug, 0.8～1.2mg aluminium adjuvant.Preparation flow represents in Figure 5.
Claims (7)
1. proteus mirabilis-staphylococcus aureus-(PSP adsorbs associating epidemic disease to Pseudomonas aeruginosa absorption combined vaccine
Seedling) preparation method, use many strains proteus mirabilis, staphylococcus aureus, Pseudomonas aeruginosa to be prepared and form, it is special
Levy and be there are following steps:
(1) prepared by proteus mirabilis after birth soluble antigen stock solution；
(2) prepared by staphylococcus aureus cytoplasmic antigen stock solution；
(3) prepared by staphylococcus aureus toxoid stock solution；
(4) prepared by P. aeruginosa toxoid stock solution；
(5) prepared by proteus mirabilis 6 valency antigen mixed liquor；
(6) proteus mirabilis-staphylococcus aureus-Pseudomonas aeruginosa absorption combined vaccine (PSP adsorbs combined vaccine)
Preparation.
A kind of proteus mirabilis-staphylococcus aureus the most according to claim 1-Pseudomonas aeruginosa absorption associating
Vaccine (PSP adsorbs combined vaccine) preparation method, it is characterised in that step (1) proteus mirabilis after birth soluble antigen stock solution
Preparation: refer to that proteus mirabilis is through High Density Cultivation, digestion in place, centrifugal, pre-filtering, ultrafiltration, ethanol precipitation, degerming mistake
Filter is prepared from, and digestion in place is obtained by trypsinization, in digestion process in place, and 4.0～6.0 × 109Individual/ml antibacterial
Concentration adds 1～2g pancreatin, and the ultrafilter membrane bag aperture that ultrafiltration is used is 10～30KD.
A kind of proteus mirabilis-staphylococcus aureus the most according to claim 1-Pseudomonas aeruginosa absorption associating
Vaccine (PSP adsorbs combined vaccine) preparation method, it is characterised in that step (2) staphylococcus aureus cytoplasmic antigen stock solution system
Standby: to refer to be sunk through High Density Cultivation, centrifugal, Mechanical Crushing, centrifugal, isoelectric precipitation 1, isoelectric point, IP by staphylococcus aureus
Shallow lake 2, enzymolysis, dialysis, aseptic filtration step obtain, and the bead particle diameter that Mechanical Crushing uses is 0.45～0.6mm, percentage of damage
Being not less than 80%, isoelectric precipitation 1 requires that pH is 3.0～4.5, and isoelectric precipitation 2 requires that pH is 4.5～6.0, and enzymolysis uses pancreas
Protease content is: 1L, 60～the 100mg/L corresponding 1～10mg trypsin of total nitrogens, dialysis use bag filter aperture be 1～
10KD。
A kind of proteus mirabilis-staphylococcus aureus the most according to claim 1-Pseudomonas aeruginosa absorption associating
Vaccine (PSP adsorbs combined vaccine) preparation method, it is characterised in that prepared by step (3) staphylococcus aureus toxoid stock solution:
Refer to suddenly be obtained through High Density Cultivation, centrifugal, detoxification, ultrafiltration, Acid precipitation, centrifugal, aseptic filtration by staphylococcus aureus
, detoxification uses 0.4%～1.0% formaldehyde or glutaraldehyde, pH6.8～7.2,30～37 DEG C of detoxifications 5～7 days, and ultrafiltration uses ultrafiltration
Pustule aperture is 10～30KD, in Acid precipitation containing sodium chloride concentration be 0-30%, pH be 3.0～5.0.
A kind of proteus mirabilis-staphylococcus aureus the most according to claim 1-Pseudomonas aeruginosa absorption associating
Vaccine (PSP adsorbs combined vaccine) preparation method, it is characterised in that prepared by step (4) P. aeruginosa toxoid stock solution: be
Refer to be obtained through High Density Cultivation, centrifugal, detoxification, ultrafiltration 1, ultrafiltration 2, aseptic filtration step by Pseudomonas aeruginosa, detoxification
Use 0.4～1.0% formaldehyde or glutaraldehyde, pH6.8～7.2,30～37 DEG C of detoxifications 12～15 days, ultrafiltration 1 uses ultrafiltration pustule
Aperture is 300～1000KD, and ultrafiltration 2 uses ultrafiltration pustule aperture to be 10～30KD.
A kind of proteus mirabilis-staphylococcus aureus the most according to claim 1-Pseudomonas aeruginosa absorption associating
Vaccine (PSP adsorbs combined vaccine) preparation method, it is characterised in that prepared by step (5) proteus mirabilis 6 valency antigen mixed liquor:
Refer to that 6 strain proteus mirabilis after birth soluble antigens are carried out compatibility mixing according to antigen concentration according to 1:1:1:1:1:1 and
Become.
A kind of proteus mirabilis-staphylococcus aureus the most according to claim 1-Pseudomonas aeruginosa absorption associating
Vaccine (PSP adsorbs combined vaccine) preparation method, it is characterised in that step (6) proteus mirabilis-staphylococcus aureus-copper
Prepared by green pseudomonas absorption combined vaccine (PSP adsorbs combined vaccine): refer to resist 6 valency proteus mirabilis after birth solubilities
Former, staphylococcus aureus cytoplasmic antigen, staphylococcus aureus toxoid, P. aeruginosa toxoid, alumina gel and nothing
Machine salt composition mixes according to a certain percentage, and 6 valency proteus mirabilis after birth soluble antigens concentration in absorption combined vaccine is
48 μ g/ml～60 μ g/ml, the single concentration of each of which strain proteus mirabilis is 8～10ug/ml, staphylococcus aureus
Cytoplasmic antigen concentration in absorption combined vaccine is 0.8mg～1.2mg/ml；Staphylococcus aureus toxoid is in absorption associating
In vaccine, concentration is 3 EC/ml～8EC/ml, P. aeruginosa toxoid absorption combined vaccine in concentration be 20 μ g/ml～
50 μ g/ml, gel aluminum hydroxide concentration in absorption combined vaccine is 0.8mg～1.2mg/ml；Absorption combined vaccine pH is 6.8
～7.5, in vaccine, adjuvant is alumina gel, refers to gel aluminum hydroxide, Fosfalugel (Yamanouchi) or alumina gel, or according to formula from
System meets the alumina gel of standards of pharmacopoeia.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021032179A1 (en) * | 2019-08-22 | 2021-02-25 | 四川大学 | Application of pseudomonas aeruginosa vaccine in treating infection associated with burn or scald injury |
| CN113354719A (en) * | 2021-05-11 | 2021-09-07 | 重庆市畜牧科学院 | Proteus mirabilis antigen identification and application thereof in detection of proteus infection |
| CN114364396A (en) * | 2019-08-22 | 2022-04-15 | 四川大学 | Application of pseudomonas aeruginosa vaccine in resisting burn and scald infection |
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| CN114364396A (en) * | 2019-08-22 | 2022-04-15 | 四川大学 | Application of pseudomonas aeruginosa vaccine in resisting burn and scald infection |
| CN114364396B (en) * | 2019-08-22 | 2024-01-23 | 四川大学 | Application of pseudomonas aeruginosa vaccine in burn and scald infection resistance |
| CN113354719A (en) * | 2021-05-11 | 2021-09-07 | 重庆市畜牧科学院 | Proteus mirabilis antigen identification and application thereof in detection of proteus infection |
| CN113354719B (en) * | 2021-05-11 | 2023-05-30 | 重庆市畜牧科学院 | Proteus mirabilis antigen identification and application thereof in detection of Proteus infection |
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