CN101361971B - Preparation technique of Vibrio vulnificus whole-cell immunity stimulated compound vaccine and use thereof in aquatic animal - Google Patents
Preparation technique of Vibrio vulnificus whole-cell immunity stimulated compound vaccine and use thereof in aquatic animal Download PDFInfo
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- CN101361971B CN101361971B CN2008100719137A CN200810071913A CN101361971B CN 101361971 B CN101361971 B CN 101361971B CN 2008100719137 A CN2008100719137 A CN 2008100719137A CN 200810071913 A CN200810071913 A CN 200810071913A CN 101361971 B CN101361971 B CN 101361971B
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Abstract
The invention relates to a preparation technology of vibrio vulnificus whole-bacteria immunity stimulated compound vaccine and the application thereof in aquatic animals. The technology comprises the following steps: (1) pre-amplification and expansion cultivation of the vibrio vulnificus is carried out, (2) blanching is imposed on the vibrio vulnificus in zymogen liquid cultivated, (3) non-viable bacteria inspection is carried out on the bacteria liquid blanched, (4) the inactivated vibrio vulnificus is collected by a centrifuge, (5) an ultrasonic crushing is taken on the inactivated vibrio vulnificus collected, (6) immunity stimulated compound ingredients are prepared, and (7) the immunity stimulated compound is prepared by ultrasonic treatment. The virulence of the vibrio vulnificus selected is quite strong (LD50 is about 200cfu/g), the cultivation condition and optimized culture medium can double the biological content of viable bacteria in the fermentation broth, and the biomass of the vibrio vulnificus cultivated is more than 50mg/ml, thus providing energetic technological support for the industrialized fermentation production of the vibrio vulnificus.
Description
Technical field
The present invention relates to a kind of preparation and application thereof of pathogenic bacterium inactivated vaccine.
Background technology
Vibrio vulnificus (vibrio vulnificus) is a kind of low halophagia vibrio, belongs to mermaid ill cause of disease altogether, can cause people and anguilla japonica to produce septicemia and serious wound infection.Be broadly divided into biological I, biological II, three types of biological III, wherein biological I type main harm people's health, can cause the people infect the back fatality rate up to more than 60%; Biological II type then is one of main bacteria sexually transmitted disease source of disease of anguilla japonica, in the eighties of last century 70-80 age, once allows anguilla japonica industries such as Japan, America and Europe suffer enormous economic loss; Over nearly 5 years, China's Coastal Areas is cultured anguilla japonica because of Vibrio vulnificus causes that morbidity is also very serious, presents acute clinically and two kinds of processes of delay property, and plant's anguilla japonica Vibrio vulnificus disease can be shown effect repeatedly, causes serious economic loss.Tradition adopts chemicals control Vibrio vulnificus diseases such as antibiotic and disinfectant usually, has easily caused problems such as environmental pollution, drug residue and Drug resistance, influences product water outlet, even threatens the health of consumer.
At present, study Vibrio vulnificus both at home and abroad and mainly stress epidemiology and pathogenesis etc., and the development and the exploitation of fewer concern Aquatic product vaccine.Relevant Study on Fermentation mainly concentrates on industrial microorganisms such as escherichia coli, does not appear in the newspapers so far in vibrio fermentation technology aspect; The research report of relevant aquatic animal cause of disease ISCOMs is rarely seen breadboard Aeromonas hydrophila ISCOMs research also, and the Study on Preparation of Vibrio vulnificus ISCOM vaccine is not appeared in the newspapers at home as yet.
Summary of the invention
The effect that the object of the present invention is to provide a kind of fermentation culture that can optimize the Vibrio vulnificus vaccine, improves biological content of vaccine viable bacteria in the fermentation liquid and the vaccine preparation technology of Vibrio vulnificus whole-cell immunity stimulated compound vaccine and the application of aquatic animal thereof preferably.
Purpose of the present invention technical scheme as follows realizes: it comprises the steps: that (1) increase Vibrio vulnificus and amplification culture in advance, (2) Vibrio vulnificus in the zymocyte liquid after cultivating is carried out deactivation, (3) the bacterium liquid to deactivation does not have the Vibrio vulnificus that viable bacteria is checked (4) centrifugal collection deactivation, (5) then the deactivation Vibrio vulnificus of collecting is carried out ultrasonic disruption, (6) preparation of immunostimulating complex component, (7) ultrasonic Treatment prepares immunostimulating complex.The method that Vibrio vulnificus is increased in advance with amplification culture is: the TSB culture medium of selecting TSB culture medium and improvement; To detect in the 250ml triangular flask of 50mlTSB that qualified Vibrio vulnificus strain is inoculated into the bacterial cultures that contains TSB through routine, under the condition of 25-31 ℃ of constant temperature, 100-200 rev/min shaken cultivation 14-18 hour, with this culture fluid as seed liquor; Be inoculated in the fresh autoclaved improvement TSB culture fluid in the fermentation tank large-scale culture 36-60 hour with the culture of the pre-amplification of 0.5-10%; The composition of the TSB culture fluid volume weight percentage ratio W/V of described improvement is: glucose 0.18%-0.22%, tryptone 1.2%-1.8%, soya peptone 0.2%-0.6%, Semen Maydis pulp 0.2%-0.6%, dipotassium hydrogen phosphate 0.15%-0.25%, sodium chloride 1.0%-2.0%, iron phosphate 0.0005%-0.002% and yeast extract 0.05%-0.2%; The fermentation culture conditions parameter is: inoculum concentration is 0.5-10%, temperature 25-31 ℃ of culture volume, rotating speed 100-200 rev/min; Air mass flow is 35-151/min; The pH value 6.5-7.5 of culture medium; Ferment after 20-28 hour by interval 3-10 minute, the 15-20 that works mends concentrate feed 500ml altogether second automatically, the ratio of weight and number of described concentrate feed is 3 parts of 1 part of glucose and soya peptones, is zymocyte liquid after the collection.
Strong (the LD of the Vibrio vulnificus virulence that the present invention selects for use
50Be about 200cfu/g) and the culture medium of condition of culture and optimization can make the biological content of viable bacteria of fermentation liquid realize being doubled, the Vibrio vulnificus Biomass of turning out is up to more than the 50mg/ml; For the industrialization fermenting and producing of Vibrio vulnificus vaccine provides powerful technical support.
The specific embodiment:
Concrete processing step of the present invention is as follows:
(1) Vibrio vulnificus is increased and amplification culture in advance
Select to generally acknowledge the TSB culture medium of TSB culture medium and improvement; To detect in the 250ml triangular flask of 50mlTSB that qualified Vibrio vulnificus strain is inoculated into the bacterial cultures that contains TSB through routine, under the condition of 25-31 ℃ of constant temperature, 100-200 rev/min shaken cultivation 14-18 hour, with this culture fluid as seed liquor; Be inoculated in the fresh autoclaved improvement TSB culture fluid in the fermentation tank large-scale culture 36-60 hour with the culture of the pre-amplification of 0.5-10%; The composition of the TSB culture fluid of described improvement is: (W/V is the volume weight percentage ratio of water to glucose 0.18%-0.22%, and is as follows.), tryptone 1.2%-1.8% (W/V), soya peptone 0.2%-0.6% (W/V), Semen Maydis pulp 0.2%-0.6% (V/V), dipotassium hydrogen phosphate 0.15%-0.25% (W/V), sodium chloride 1.0%-2.0% (W/V), iron phosphate 0.0005%-0.002% (W/V) and yeast extract 0.05%-0.2% (W/V); The fermentation culture conditions parameter is: inoculum concentration is 0.5-10%, temperature 25-31 ℃, rotating speed 100-200 rev/min; Air mass flow is 35-15L/min; PH value 6.5-7.5; Ferment after 20-28 hour by interval 3-10 minute, the 15-20 that works mends concentrate feed 500ml altogether second automatically, the ratio of weight and number of described concentrate feed is 3 parts of 1 part of glucose and soya peptones, is zymocyte liquid after the collection.
(2) Vibrio vulnificus in the zymocyte liquid after cultivating being carried out deactivation is collected in bacterium liquid in the sterilization tank, the interpolation final concentration is the formalin of 0.1-0.5% (V/V percent by volume), concussion roll in the room temperature jar after 3-5 hour, place 3-5 ℃ of refrigerator overnight or through 48 hours, thoroughly deactivation.Need carry out steriling test in order to ensure thorough deactivation to the bacterium liquid (being inactivated bacterial liquid) in the back sterilization tank of sterilizing, the method of steriling test is: fully shake up the inactivated bacterial liquid after the sterilization, sterilization rifle head is drawn the inactivated bacterial liquid of 0.2-1.0ml, superclean bench upper berth flat board, placing 26-30 ℃ of constant incubator cultivated 48 hours, the culture of sterilizing completely should not have bacterium colony to occur, and blank dull and stereotyped contrast is established in test.
(3) Vibrio vulnificus of centrifugal collection deactivation: under 8000-12000 rev/min rotating speed, centrifugal 20-30 minute, collect, concentrate antibacterial; The Biomass of fermentation culture antibacterial (weight in wet base) should be 25-50g.l
-1The antibacterial of described centrifugal collection is resuspended with sterile saline, is settled to Biomass 125-175g.l behind every liter of vaccine centrifugal
-1
The cultivation in a large number of above-mentioned process, deactivation and the centrifugal deactivation antibacterial that concentrates, collects are the semi-finished product of vaccine.
(4) the deactivation Vibrio vulnificus of collecting is carried out ultrasonic disruption, the preparation of thalline crude protein (antibacterial cracking, albumen discharges):
Adopt commercially available JY98-3D ultrasonic disruption instrument, the diameter of hyperacoustic ultrasonic probe (being called horn again) is 20mm, and ultrasonic power is made as 800 watts; The ultrasonic disruption instrument is carried out the cleaning sterilization with 70% ethanol; The concentrated bacterium liquid of the 200-400ml of deactivation is put into special-purpose ultrasonic cup, wait to concentrate bacterium liquid pre-freeze or pre-moltenly carry out the low temperature ultrasonication when leaving a small amount of ice crystal; Broken action routine is: 10 seconds blanking times, 5 seconds working times, it is 6 minutes/wheel that circulation like this acts on total time repeatedly.Act on 3 altogether and take turns, the omnidistance time is 18 minutes, and the practical function time is 6 minutes.The output initial power that pointer shows is about 720 watts, and the power of final stage can be near 800 watts of the values of setting.Can also adopt other bacteria breaking instrument among the present invention, the antibacterial after the deactivation be carried out broken purpose as long as can reach.
The Plondrel phthalate buffer (PBS) of using behind the ultrasonic treatment is adjusted bacterioprotein concentration to 8-12mg.ml for the first time
-1
(5) preparation of immunostimulating complex (ISCOMs) component
In the thalline crude protein of ultrasonic degradation, in every 100ml bacterium liquid, add saponin (QuilA) (the source ACCURATE CHEMICAL﹠amp of the 0.05-0.2% (W/V) of 0.05-0.2 gram; SCIENTIFIC CORPORATION) and in every ml bacterium liquid add 100-150 μ g.ml
-1Lecithin and cholesterol (final concentration).Because lecithin and cholesterol all are Organic substances not soluble in water, earlier two kinds of substance dissolves are made into 100-150mg.ml in chloroform when preparation lecithin and cholesterol
-1Storage liquid, now with the current or-20 ℃ frozen standby.
(6) ultrasonic Treatment prepares immunostimulating complex
The thalline crude protein is joined after the Seedling ratio adds saponin, lecithin and cholesterol and constitute the substrate of immunostimulating complex in above-mentioned, treat pre-freeze or pre-molten when leaving a small amount of ice crystal, place and carry out in the ultrasonic disruption instrument that temperature is not higher than 35 ℃ in low temperature ultrasonication, the control ultrasonication storehouse.Program is set to: 10 seconds blanking times, and ultrasonication 5 seconds, the omnidistance time is 18 minutes.Temperature is not higher than 60 ℃.Under cleaning condition, the immunostimulating complex of supersound process is collected in the 500ml sterilization vaccine special plastic bottle behind the mixing, measure the immunostimulating complex protein concentration, adjust the protein concentration of immunostimulating complex to 8-12mg.ml with sterilization PBS
-1Obtain goods after twice ultrasonic is handled and be Vibrio vulnificus immunostimulating complex vaccine; The immunostimulating complex vaccine that makes is frozen standby at-20 ℃.
(7) packing: Vibrio vulnificus ISCOM vaccine is sub-packed in 100ml through 150-170 ℃ of dry heat treatment 1.5-2.5 hour vaccine special glass bottle, adds a cover, label ,-20 ℃ frozen.
(8) product inspection: the finished product after the packing is carried out character check and pure check; Goods are the even dense thick liquid of pale brown color before frozen, and slight sulfur stink is arranged; Frozen state is pale brown color ice cube; Described character verifies as: goods dilute with 0.01M PBS at 1: 100, and differential centrifugation is removed cell debris, and ultracentrifugation concentrates ISCOMs again, the observation down of the rearmounted transmission electron microscope of saturated phosphotungstic acid negative staining, and can be observed diameter is 35nm-40nm cage grating texture; Described sampling observation 2/10 vaccine of packing that verifies as purely with the ISCOMs0.1-0.5ml of pipettor absorption mixing, evenly is coated with flat board under the aseptic condition, and 26-30 ℃ of calorstat cultivated to observe in 48 hours should not have bacterial growth.
The existence of contamination-free in the culture that obtains in order to guarantee, above-mentioned steps (1) afterwards step (2) the pollution bacterium colony that carries out culture before detect; Concrete grammar is as follows: all establishes with batch culture medium blank when pre-amplification of each batch and amplification culture, and culture medium blank polluter is discarded.Stochastic sampling from the antibacterial of each batch production (2/10) is drawn plate and is cultivated, and identifies colonial morphology, color and luster, observes to have or not varied bacteria growing; Carry out the antigenicity of serum agglutination test assessment antibacterial, discarded after the deactivation of substandard product high pressure.
In order to ensure in the process of step (4), pollution condition not taking place in step (2), step (4) afterwards and step (5) preferably pollute bacterium colony check and antigenicity check before, it is identical with above-mentioned steriling test method to pollute the bacterium colony method of inspection, the antigenicity detection method is for after the vaccine after concentrating makes 10-20 and doubly dilute with sterile saline (or PBS), its antigenicity of somatic agglutination test assessment.
Step (6) is before afterwards in step (5), can also carry out Protein Detection to bacterium liquid through broken process, this detection method belongs to existing known method, detection method is: the bovine serum albumin (BSA) with fresh configuration is made curve, and the Bradford method is measured the protein concentration behind the primary fragmentation.
In step (7) afterwards, can also be through the observation under the transmission electron microscope and the step of evaluation, the detailed process of this step is as follows: the structure of typical ISCOMs is the graininess of diameter 35nm-40nm, the centre is a hydrophobic region, there are 6 symmetric point-like things relatively to be evenly distributed in around it, constitute the typical substrate of immunostimulating complex (ISCOM) structure.The full bacterium ISCOMs of Vibrio vulnificus goods are not done further ultracentrifugation because of complicated component and are handled, and the rearmounted transmission electron microscope of saturated phosphotungstic acid negative staining is observed down, is difficult for observing typical ISCOMs structure.Sample is through 100 times of dilutions, 8000-12000 rev/min centrifugal 30 minutes, remove cell debris, after the 34000-36000 rev/min of centrifugal concentration, can under Electronic Speculum, observe the cage grating texture that diameter is 35nm-40nm.
Will be as follows through the result of safety detection and efficacy test through the Vibrio vulnificus ISCOM vaccine that the inventive method makes.
Described safety examination is divided into following steps:
(a) experiment fish and raising condition thereof
Selection is going strong, does not have the anguilla japonica of disease, no ectoparasitic infection, puts 50 * 30 * 40cm
3The glass aquarium is filled to 1/2 dark place of case, and each aquarium is put anguilla japonica 10-25 tail in a suitable place to breed, place indoor, 24 ℃ ± 1 ℃ of temperature-controlling air-conditioning control room temperature.The water source is sudden and violent gas tap water, changes water 1/3 every day.Common small-sized inflator continuous charge is up to off-test; Anguilla japonica is supported more than 7 days before test temporarily, checks no parasitic infection, and duration of test stops feeding.
(b) to the safety testing of Anguillar japonica
Dipping bath: is 200 μ g.ml with the sterilization tap water with the dilution of Vibrio vulnificus ISCOM vaccine
-1, dipping bath European eel 10-25 tail changed water after 24 hours; Same volume sterilization tap water dipping bath is contrast with batch anguilla japonica 10-25 tail; Experimental group is all established with matched group and is repeated parallel group, observes continuously 7-14 days, and anguilla japonica does not have the pathology reaction, and a decidable batch vaccine is safe and reliable.
Injection: sterilization PBS (0.01mol) is 2000 μ g.ml with the dilution of Vibrio vulnificus ISCOM vaccine
-1, disposable syringe is with the dosage of 0.1ml/ tail injection anguilla japonica 10-25 tail, and contrast anguilla japonica 10-25 endnote is penetrated and is waited the dosage PBS that sterilizes; Experimental group is all established with matched group and is repeated parallel group, observes continuously 7-14 days, and anguilla japonica does not have the pathology reaction, and a decidable batch vaccine is safe and reliable.
Irritate stomach: sterilization PBS (0.01mol) is 4000 μ g.ml with the dilution of Vibrio vulnificus ISCOM vaccine
-1, disposable syringe (joining silica gel tube) is irritated stomach anguilla japonica 10-25 tail with the dosage of 0.2ml/ tail, and contrast anguilla japonica 10-25 tail is irritated dosage sterilization PBS such as stomach; Experimental group is all established with matched group and is repeated parallel group, observes continuously 7-14 days, and anguilla japonica does not have the pathology reaction, and a decidable batch vaccine is safe and reliable.
Described efficacy test step is as follows:
(a) test fish standard and raising condition
Specification: 5-50g/ tail (being convenient to blood sampling).Test fish preimmune serum antibody test: select the above anguilla japonica of 3 tails to gather serum at random, the serum antibody titer before serum coagulation or the ELISA method assessment immunity.Background antibody titer agglutination titer should not be used fish greater than 22 (significantly coagulations), ELISA antibody titer as test greater than 1: 200.Health standards and the same safety examination of raising condition (a) of test fish.
(b) injecting immune, blood sampling and counteracting toxic substances
With sterile saline Vibrio vulnificus ISCOM vaccine is adjusted into 350-450 μ g.ml
-1The suspension of concentration.Anguilla japonica behind the fish neuroleptanesthesia, the above-mentioned immune suspension 0.1ml of every tail lumbar injection, every group of 10-25 tail, the isodose sterile saline of matched group anguilla japonica lumbar injection; Experimental group is all established with matched group and is repeated parallel group.Counteracting toxic substances: after immunity 25-40 days, with the bacterium liquid (2 * 10 of the Vibrio vulnificus bacterial strain of prepared fresh
4-5 * 10
5Cfu) lumbar injection counteracting toxic substances is observed in 7-14 days and the statistics death condition.
Challenge test result of the test after the laboratory vaccine injection immunity (seeing Table 1), the immune protective efficiency of immune group be 100% and the survival rate of matched group be 0%.
The survival rate that table 1 contrast and immune European eel are attacked the FJ03-X2 bacterial strain compares
(c) oral immunity, blood sampling and counteracting toxic substances
Vibrio vulnificus ISCOM vaccine carries out the oral immunity test in the eel field to anguilla japonica.By fish body weight spice every day 50ml vaccine per ton, continuous oral 7 days, obeyed again 7 days after the week at interval; With the conventional feed that does not the add vaccine anguilla japonica of throwing something and feeding is matched group.Counteracting toxic substances: carried out challenge test in 25-60 days after the 2nd immunity, test divides 2 groups, immune group and matched group, every group of 10-25 tail; Immune group is all established with matched group and is repeated parallel group.Bacterium liquid (2 * 10 with the Vibrio vulnificus bacterial strain of prepared fresh
4-5 * 10
5Cfu) lumbar injection counteracting toxic substances is observed in 7-14 days and the statistics death condition.
The immune protective efficiency of challenge test immune group is 100% behind the vaccine oral immunity of field, and the survival rate of matched group is 23.1%; Result of the test sees Table 2
The survival rate that table 2 contrast and immune Japanese eel are attacked the FJ03-X2 bacterial strain compares
The result shows: Vibrio vulnificus ISCOM vaccine safety, effective, practical.
The Vibrio vulnificus whole-cell immunity stimulated compound vaccine that makes is applied to prepare the immunoprophylaxis preparation of the Vibrio vulnificus of aquatic animal, the Vibrio vulnificus whole-cell immunity stimulated compound vaccine that makes is applied to the immunoprophylaxis of the Vibrio vulnificus of aquatic animal.Adopt the mode of drug administration by injection or adopt the mode of oral administration to carry out immunoprophylaxis.
Claims (10)
1. the preparation technology of a Vibrio vulnificus whole-cell immunity stimulated compound vaccine, it is characterized in that, it comprises the steps: that (1) increase Vibrio vulnificus and amplification culture in advance, (2) Vibrio vulnificus in the zymocyte liquid after cultivating is carried out deactivation, (3) the bacterium liquid to deactivation does not have the Vibrio vulnificus that viable bacteria is checked (4) centrifugal collection deactivation, (5) then the deactivation Vibrio vulnificus of collecting is carried out ultrasonic disruption, (6) preparation of immunostimulating complex component, (7) ultrasonic Treatment prepares immunostimulating complex; The method that Vibrio vulnificus is increased in advance with amplification culture is: the TSB culture medium of selecting TSB culture medium and improvement; To detect in the 250ml triangular flask of 50mlTSB that qualified Vibrio vulnificus strain is inoculated into the bacterial cultures that contains TSB through routine, under the condition of 25-31 ℃ of constant temperature, 100-200 rev/min shaken cultivation 14-18 hour, with this culture fluid as seed liquor; Be inoculated in the fresh autoclaved improvement TSB culture fluid in the fermentation tank large-scale culture 36-60 hour with the culture of the pre-amplification of 0.5-10%; The composition of the TSB culture fluid volume weight percentage ratio W/V of described improvement is: glucose 0.18%-0.22%, tryptone 1.2%-1.8%, soya peptone 0.2%-0.6%, Semen Maydis pulp 0.2%-0.6%, dipotassium hydrogen phosphate 0.15%-0.25%, sodium chloride 1.0%-2.0%, iron phosphate 0.0005%-0.002% and yeast extract 0.05%-0.2%; The fermentation culture conditions parameter is: inoculum concentration is 0.5-10%, temperature 25-31 ℃ of culture volume, rotating speed 100-200 rev/min; Air mass flow is 35-151/min; The pH value 6.5-7.5 of culture medium; Ferment after 20-28 hour by interval 3-10 minute, the 15-20 that works mends concentrate feed 500ml altogether second automatically, the ratio of weight and number of described concentrate feed is 3 parts of 1 part of glucose and soya peptones, is zymocyte liquid after the collection.
2. the preparation technology of Vibrio vulnificus whole-cell immunity stimulated compound vaccine according to claim 1, it is characterized in that: the described method that Vibrio vulnificus in the zymocyte liquid after cultivating is carried out deactivation is: bacterium liquid is collected in the sterilization tank, adding final concentration V/V percent by volume is the formalin of 0.1-0.5%, concussion roll in the room temperature jar after 3-5 hour, place 3-5 ℃ of refrigerator overnight or through 48 hours, thoroughly deactivation; Described steriling test is as follows: fully shake up antibacterial, sterilization rifle head is drawn the inactivated bacterial liquid of 0.2-1.0ml, and superclean bench upper berth flat board is placed 26-30 ℃ of constant incubator and cultivated 48 hours, the culture of sterilizing completely should not have bacterium colony to occur, and blank dull and stereotyped contrast is established in test.
3. the preparation technology of Vibrio vulnificus whole-cell immunity stimulated compound vaccine according to claim 1, it is characterized in that: the method for the Vibrio vulnificus of described centrifugal collection deactivation is: under 8000-12000 rev/min rotating speed, centrifugal 20-30 minute, collect, concentrate antibacterial; The weight in wet base of the Biomass of fermentation culture antibacterial should be 25-50g.l
-1The antibacterial of described centrifugal collection is resuspended with sterile saline, is settled to Biomass 125-175g.l behind every liter of vaccine centrifugal
-1
4. the preparation technology of Vibrio vulnificus whole-cell immunity stimulated compound vaccine according to claim 1, it is characterized in that: the method that described deactivation Vibrio vulnificus to collection carries out ultrasonic disruption is: adopt commercially available JY98-3D ultrasonic disruption instrument, the diameter of hyperacoustic ultrasonic probe is 20mm, and ultrasonic power is made as 800 watts; The ultrasonic disruption instrument is carried out the cleaning sterilization with 70% ethanol; The concentrated bacterium liquid of the 200-400ml of deactivation is put into special-purpose ultrasonic cup, wait to concentrate bacterium liquid pre-freeze or pre-moltenly carry out that temperature is not higher than 35 ℃ in low temperature ultrasonication, the control ultrasonication storehouse when leaving a small amount of ice crystal; Broken action routine is: blanking time 10s, working time 5s, so to act on total time repeatedly be 6 minutes/wheel in circulation; Act on 3 altogether and take turns, the omnidistance time is 18 minutes, and the practical function time is 6 minutes; The output initial power that pointer shows is about 720 watts, and the power of final stage can be near 800 watts of the values of setting.
The Plondrel phthalate buffer of using behind the ultrasonic treatment is adjusted bacterioprotein concentration to 8-12mg.ml for the first time
-1
5. the preparation technology of Vibrio vulnificus whole-cell immunity stimulated compound vaccine according to claim 1, it is characterized in that: the compound method of described immunostimulating complex component is as follows: add in the thalline crude protein of ultrasonic degradation, in every 100ml bacterium liquid in the saponin of 0.05-0.2%W/V of 0.05-0.2 gram and the every ml bacterium liquid and add final concentration 100-150 μ g.ml
-1Lecithin and cholesterol.
6. the preparation technology of Vibrio vulnificus whole-cell immunity stimulated compound vaccine according to claim 5 is characterized in that: earlier two kinds of substance dissolves are made into 100-150mg.ml in chloroform when preparation lecithin and cholesterol
-1Storage liquid, now with the current or-20 ℃ frozen standby.
7. the preparation technology of Vibrio vulnificus whole-cell immunity stimulated compound vaccine according to claim 6, it is characterized in that: the method that described ultrasonic Treatment prepares immunostimulating complex is: the thalline crude protein is joined after the Seedling ratio adds saponin, lecithin and cholesterol and constitute the substrate of immunostimulating complex in above-mentioned, waits to concentrate bacterium liquid pre-freeze or pre-moltenly carries out that temperature is not higher than 35 ℃ in low temperature ultrasonication, the control ultrasonication storehouse when leaving a small amount of ice crystal; Program is set to: 10 seconds blanking times, and ultrasonication 5 seconds, the omnidistance time is 18 minutes; Temperature is not higher than 60 ℃; Under cleaning condition, the immunostimulating complex of supersound process is collected in the 500ml sterilization vaccine special plastic bottle behind the mixing, measure the immunostimulating complex protein concentration, adjust the protein concentration of immunostimulating complex to 8-12mg.ml with sterilization PBS
-1Obtain goods after twice ultrasonic is handled and be Vibrio vulnificus immunostimulating complex vaccine; The immunostimulating complex vaccine that makes is frozen standby at-20 ℃.
8. the preparation technology of Vibrio vulnificus whole-cell immunity stimulated thing vaccine according to claim 1 is characterized in that, it also comprises the steps: packing and product inspection step; Described packing step is added a cover, is labelled for Vibrio vulnificus ISCOM vaccine being sub-packed in 100ml through 150-170 ℃ of dry heat treatment 1.5-2.5 hour vaccine special glass bottle, and-20 ℃ frozen; Described product inspection step is: the finished product after the packing is carried out character check and pure check; Goods are the even dense thick liquid of pale brown color before frozen, and slight sulfur stink is arranged; Frozen state is pale brown color ice cube; Described character verifies as: goods dilute with 0.01M PBS at 1: 100, differential centrifugation is removed cell debris, ultracentrifugation concentrates ISCOMs again, and the rearmounted transmission electron microscope of saturated phosphotungstic acid negative staining is observed down, and can be observed typical diameter is 35nm-40nm cage grating texture; Described sampling observation 2/10 vaccine of packing that verifies as purely with the ISCOMs 0.1-0.5ml of pipettor absorption mixing, evenly is coated with flat board under the aseptic condition, and 26-30 ℃ of calorstat cultivated to observe in 48 hours should not have bacterial growth.
9. the preparation technology of Vibrio vulnificus whole-cell immunity stimulated compound vaccine according to claim 1 is characterized in that, above-mentioned steps (1) afterwards step (2) the pollution bacterium colony that carries out culture before detect; Concrete grammar is as follows: all establishes with batch culture medium blank when pre-amplification of each batch and amplification culture, and culture medium blank polluter is discarded; 2/10 stroke of plate of stochastic sampling is cultivated from the antibacterial of each batch production, identifies colonial morphology, color and luster, observes to have or not varied bacteria growing; Carry out the antigenicity of serum agglutination test assessment antibacterial, discarded after the deactivation of substandard product high pressure.
10. described Vibrio vulnificus whole-cell immunity stimulated multiple according to any claim among the claim 1-9
The compound vaccine is applied to prepare the application of immunoprophylaxis preparation of the Vibrio vulnificus of aquatic animal.
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|---|---|---|---|---|
| CN101020051A (en) * | 2006-12-22 | 2007-08-22 | 广东海洋大学 | Prepn and usage of outer membrane protein subunit vaccine of seawater fish morbid vibrio |
| CN101028513A (en) * | 2006-12-22 | 2007-09-05 | 广东海洋大学 | Production and use for beach vibrio piscium efficient vaccine |
| CN100398151C (en) * | 2005-02-18 | 2008-07-02 | 浙江大学 | Ginsenoside nano microparticle and its preparing method and use |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100398151C (en) * | 2005-02-18 | 2008-07-02 | 浙江大学 | Ginsenoside nano microparticle and its preparing method and use |
| CN101020051A (en) * | 2006-12-22 | 2007-08-22 | 广东海洋大学 | Prepn and usage of outer membrane protein subunit vaccine of seawater fish morbid vibrio |
| CN101028513A (en) * | 2006-12-22 | 2007-09-05 | 广东海洋大学 | Production and use for beach vibrio piscium efficient vaccine |
Non-Patent Citations (8)
| Title |
|---|
| .创伤弧菌ISCOM疫苗的免疫学效应研究.《中国人兽共患病学报》.2007,第23卷(第11期),1075-1078. |
| 万美贞 |
| 俞伏松 |
| 林天龙 |
| 许斌福 |
| 许斌福;陈秀锦;俞伏松;陈强;万美贞;林天龙;.创伤弧菌ISCOM疫苗的免疫学效应研究.《中国人兽共患病学报》.2007,第23卷(第11期),1075-1078. * |
| 陈强 |
| 陈秀锦 |
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