CN100563708C - Preparation method with Gram-negative pathogen reduction vaccine of cross protection power - Google Patents
Preparation method with Gram-negative pathogen reduction vaccine of cross protection power Download PDFInfo
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- CN100563708C CN100563708C CNB2007100347337A CN200710034733A CN100563708C CN 100563708 C CN100563708 C CN 100563708C CN B2007100347337 A CNB2007100347337 A CN B2007100347337A CN 200710034733 A CN200710034733 A CN 200710034733A CN 100563708 C CN100563708 C CN 100563708C
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Abstract
The invention discloses a kind of preparation method with Gram-negative pathogen reduction vaccine of cross protection power, may further comprise the steps: a, adopt solid medium or the fluid medium be added with iron chelating agent, carry out In vitro culture with described culture medium after getting rejuvenation of spawn; B, to the antibacterial that solid medium is cultivated, wash with the vaccine diluent of sterilizing, reach industry standard until final bacterial concentration; The bacterium liquid that perhaps above-mentioned final bacterial concentration is lower than the bacterium liquid of industry standard and cultivates with fluid medium concentrates, and the outer membrane protein that keeps thalline and come off reaches industry standard until final bacterial concentration; C, the final bacterium liquid percent by volume that step b is obtained are the formalin-inactivated of 0.1-0.3%; D, adding adjuvant are made vaccine.The inventive method technology is simple, low cost of manufacture, easy realization of industrialization.
Description
Technical field
The present invention relates to the preparation method of Gram-negative pathogen reduction vaccine.
Background technology
Many gram negative bacterias are important pathogen, as some serotype escherichia coli, Salmonella, pasteurella multocida, Riemerlla anatipestifer etc.These pathogen cause the birds M ﹠ M very high, have all brought very big harm for poultry farming and human health.Owing to antigen composition complexity, the serotype of gram negative bacteria are numerous, the inactivated vaccine for preparing through In vitro culture only has protective effect to the infection of homotype bacterium, and special-shaped bacterium infection is not produced protective effect substantially.These characteristics of gram negative bacteria have brought very big difficulty for the immunoprophylaxis of corresponding birds infectious disease.Studies show that and to express the cross protection factor (cross-protective factor when gram negative bacteria is grown in vivo; CPF); and the expression of this cross protection factor is subjected to inducing of the interior low concentration ferrum of body; and CPF can further induce body to produce cross protection antibody, thereby the infection of this each serological type strain of gram negative bacteria is all produced protective effect.This is found to be induces gram negative bacteria to express CPF when realizing In vitro culture, provide new thinking and method thereby prepare the novel gram negative bacteria inactivated vaccine with cross-protection.At present, a kind of gram negative bacteria 1 outer-membrane protein vaccine is arranged abroad, its preparation technology is: with containing iron chelating agent culture medium culturing antibacterial; Concentrate antibacterial; Abandoning supernatant is collected and the washing bacterial precipitation; Broken antibacterial; High speed centrifugation is collected the supernatant that contains outer membrane protein; Concentrated supernatant; With the outer membrane protein in the denaturant stripping adventitia; Separate the purification outer membrane protein; Make 1 outer-membrane protein vaccine.The vaccine cross immunity protection effect that this prepared goes out is better, but the extraction process complexity of outer membrane protein, and needing to use Superfreezing centrifugal, this makes that the 1 outer-membrane protein vaccine cost of preparation is too high, can't extensive use on producing.Therefore how preparing the gram negative bacteria inactivated vaccine with good cross immunity protection and energy industrialization is current problem demanding prompt solution.
Summary of the invention
The object of the present invention is to provide that a kind of technology is simple, production cost is low, can be used for the preparation method of the gram negative bacteria inactivated vaccine with cross immunity protection of large-scale production.This method may further comprise the steps: a, adopt solid medium or the fluid medium be added with iron chelating agent, carry out In vitro culture with described culture medium after getting rejuvenation of spawn; B, to the antibacterial that solid medium is cultivated, wash with the vaccine diluent of sterilizing, reach industry standard until final bacterial concentration; The bacterium liquid that perhaps above-mentioned final bacterial concentration is lower than the bacterium liquid of industry standard and cultivates with fluid medium concentrates, and the outer membrane protein that keeps thalline and come off reaches industry standard until final bacterial concentration; To the spissated method of bacterium liquid be: carry out thalline and fluid separation applications with low-speed centrifugal or microfiltration method, described liquid is concentrated with ultrafiltration, the minimum molecular cut off of used filter membrane is not more than 25kDa, be not less than 0.5kDa, with the liquid after concentrating sedimentary thalline being suspended then reaches industry standard until final bacterial concentration.C, the final bacterium liquid percent by volume that step b is obtained are the formalin-inactivated of 0.1-0.3%; D, adding adjuvant are made vaccine.This preparation method with Gram-negative pathogen reduction vaccine of cross immunity protection provided by the invention comprises the antibacterial that solid medium is cultivated, and washes with the vaccine diluent of sterilizing, and reaches industry standard until final bacterial concentration; The bacterium liquid that perhaps above-mentioned final bacterial concentration is lower than the bacterium liquid of industry standard and cultivates with fluid medium concentrates, and the outer membrane protein that keeps thalline and come off reaches industry standard until final bacterial concentration; It is characterized in that describedly being: carry out thalline and fluid separation applications with low-speed centrifugal or microfiltration method to the spissated method of bacterium liquid, described liquid is concentrated with ultrafiltration, the minimum molecular cut off of used filter membrane is not more than 25kDa, be not less than 0.5kDa, with the liquid after concentrating sedimentary thalline being suspended then reaches industry standard until final bacterial concentration.Beneficial effect of the present invention: 1) adopt the method that directly bacterium liquid is transferred to desired concn without washing, and when bacterial concentration does not reach desired concn, after carrying out thalline and fluid separation applications, collect isolating liquid, be used further to the thalline that suspends after concentrating, and make the bacterium liquid that obtains reach desired concn.Can avoid CPF in washing process, to lose like this, and keep all outer membrane protein of antibacterial, thereby guarantee that inactivated vaccine has the effect of cross immunity protection preferably.2) preparation technology of the present invention is simple, has saved the proteic separation purifying technique of external membrane, thereby greatly reduces the preparation cost of vaccine, can effectively promote the industrialization of gram negative bacteria inactivated vaccine.
The specific embodiment
Embodiment one: the present invention is used for the preparation method and the immune protective experiment thereof of fowl cholera propolis inactivated vaccine.The strong malicious freeze-drying lactobacillus of A type pasteurella multocida that present embodiment relates to is available from China Veterinery Drug Inspection Office, identified and preserved by this institute.1, rejuvenation of spawn and seedling prepare with bacterium liquid:
After freeze-drying lactobacillus dissolved with the 1ml physiological saline solution, streak inoculation on blood Martin agar culture medium was cultivated 20-22 hour for 37 ℃.The antibacterial that grows is washed, and 2 rejuvenation of continuous passage in the chicken body are got the streak inoculation of liver pathological material of disease in blood Martin agar plate, cultivate 20-22 hour for 37 ℃.Get 4-6 colonies typical streak inoculation in blood Martin agar slant, cultivated 20-22 hour for 37 ℃.Cultivating end back every pipe inclined-plane adding 2ml sterile saline washes culture, be transferred on the solid medium that contains haemachrome Martin agar, 200 μ mol/L 2 have wherein been added in the solid medium, 2 '-bipyridyl, also available 5-10 μ mol/L desferrioxamine or the lactoferrin of 0.2-0.4mg/mL as iron chelating agent.Cultivated 24 hours for 37 ℃.The antibacterial that grows after cultivating is washed with an amount of sterile saline, gained bacterium liquid is carried out count of bacteria, if bacterial concentration in 150-200 hundred million CFU/ml scopes, does not then need to concentrate again; If being higher than 20,000,000,000 CFU/ml, bacterial concentration it is diluted to 150-200 hundred million CFU/ml scopes with sterile saline; If bacterial concentration is lower than 15,000,000,000 CFU/ml, then with gained bacterium liquid centrifugal 30min under 6000rpm, again supernatant is concentrated 100 times with ultrafiltration, used ultrafilter membrane molecular cut off is advisable with 5-20kDa, maximum molecular cut off is not higher than 25kDa, to keep the outer membrane protein that comes off in the supernatant.Wash with the antibacterial of the concentrated solution of holding back, and bacterial concentration is adjusted to 150-200 hundred million CFU/ml centrifugation.Except above-mentioned method for concentration, also available polyethylene glycol precipitation, gel chromatography etc.
If adopting the haemachrome Martin nutrient broth that contains above iron chelating agent is culture medium, then the bacterium liquid centrifugal 10-30min separating thallus under 6000-8000rpm after cultivating can be concentrated certain multiple with centrifuged supernatant with above-mentioned ultrafiltration again.Wash with the antibacterial of concentrated solution, bacterial concentration is adjusted to 150-200 hundred million CFU/ml centrifugation.
2, water is proposed the preparation of propolis adjuvant:
Take by weighing a certain amount of propolis virgin rubber, it is pulverized the surfactant that the back adds distilled water and 1-2%, surfactant can adopt Tween 80, Polyethylene Glycol etc., 50 ℃ are soaked 2-5h down, supersound process 30-45min under 40KHz then, solution temperature is controlled at 50-60 ℃, after the ultrasonic end with centrifugal 20-30min under the solution 6000-8000rpm, get supernatant, 60 ℃ of 2-4h that sterilize down promptly make water and carry propolis adjuvant.Contain pure propolis 20-30mg/ml.
3, fowl cholera propolis inactivated vaccine preparation:
Above-mentioned inactivated bacterial liquid and propolis aqueous extract equal-volume are mixed, fully promptly make novel cholera propolis inactivated vaccine behind the mixing, wherein bacterial concentration is 7,500,000,000 CFU/ml, and propolis content is 12mg/ml.Draw the 0.1ml vaccine, add in the nutrient agar that is cooled to about 46 ℃, mixing rapidly solidifies and is placed on 37 ℃ of incubators and cultivates 24h, and the result does not have bacterial growth, shows that inactivated vaccine meets aseptic requirement.
4, the safety experiment of fowl cholera propolis inactivated vaccine:
With the preparation vaccination in 5 February older chicken, every chest intramuscular injection 3ml observed for 2 weeks and carries out security inspection, experimental result is not seen any untoward reaction after showing chicken inoculation, illustrates that the safety of vaccine is good.
5, fowl cholera propolis inactivated vaccine is tested the short-term cross protection of white mice:
Getting 60 body weight is the healthy fowl cholera negative antibody white mice of 15-18g, is divided into three groups at random, 20 every group, and respectively at the novel fowl cholera propolis of back subcutaneous injection 0.3ml inactivated vaccine, conventional fowl cholera propolis inactivated vaccine and normal saline.After 14 days, each group is carried out counteracting toxic substances (wherein the minimal lethal dose of fowl pasteurella multocida C48-3 strain and P1662 strain is respectively 25CFU and 20CFU) with the C48-3 strain of fowl pasteurella multocida and the P1662 strain of 2 times of minimal lethal doses respectively.10 white mice of every kind of antibacterial counteracting toxic substances were observed for 2 weeks behind the counteracting toxic substances, calculated immune protective rate.Experimental result is referring to table 1.
Table 1 fowl pasteurella multocida vaccine short-term cross protection experimental result
The result shows: with the fowl cholera propolis inactivated vaccine of the inventive method preparation to homotype bacterium (C
48-3Strain) immune protective rate reaches 100%, and the immune protective rate of special-shaped bacterium (P1662) is reached 90%; Conventional fowl cholera propolis inactivated vaccine is 100% to the immune protective rate of homotype bacterium, and only is 30% to the immune protective rate of special-shaped bacterium; The normal saline control animals is all dead.Explanation obviously is better than conventional fowl cholera propolis inactivated vaccine with the short-term cross immunity protection of the fowl cholera propolis inactivated vaccine that the inventive method makes.
6, the fowl cholera propolis inactivated vaccine that makes with the inventive method is tested the long-term cross immunity protection of chicken:
Get 60 2 the monthly age big healthy fowl cholera negative antibody chicken, be divided into three groups at random, every group 20, respectively at the novel fowl cholera propolis of chest muscle injection 1ml inactivated vaccine, conventional fowl cholera propolis inactivated vaccine and normal saline, each group is used the fowl pasteurella multocida C of 2 times of minimal lethal doses respectively after 180 days
48-3Counteracting toxic substances is carried out in strain and P1662 strain, and 10 chickens of every group of antibacterial counteracting toxic substances were observed for 2 weeks behind the counteracting toxic substances, calculated immune protective rate.Experimental result is referring to table 2.
The long-term cross protection experimental result of table 2 fowl pasteurella multocida vaccine
Experimental result shows: the fowl cholera propolis inactivated vaccine that makes with the inventive method reaches 80% to the immune protective rate of homotype bacterium (C48-3 strain), and the immune protective rate of special-shaped bacterium (P1662) is reached 70%; Conventional fowl cholera propolis inactivated vaccine is 80% to the immune protective rate of homotype bacterium, and only is 10% to the immune protective rate of special-shaped bacterium; The normal saline control animals is all dead.Explanation obviously is better than conventional fowl cholera propolis inactivated vaccine with the long-term cross immunity protection of the fowl cholera propolis inactivated vaccine that the inventive method makes.
Embodiment two: the present invention is used for the preparation method and the immune protective experiment thereof of escherichia coli inactivated vaccine
(serotype is O to the standard escherichia coli CMCC44115 strain freeze-drying lactobacillus that present embodiment relates to
2Type), available from China Veterinery Drug Inspection Office, identify and preserve by this institute.
1, rejuvenation of spawn and seedling prepare with bacterium liquid
After freeze-drying lactobacillus dissolved with the 1ml physiological saline solution, streak inoculation on blood Martin agar culture medium was cultivated 20-22 hour for 37 ℃.The antibacterial that grows is washed, and 2 rejuvenation of continuous passage in the chicken body are got the streak inoculation of liver pathological material of disease in blood Martin agar culture plate, cultivate 20-22 hour for 37 ℃.Get 4-6 colonies typical streak inoculation in blood Martin agar slant, cultivated 20-22 hour for 37 ℃.Cultivating end back every pipe inclined-plane adding 2ml physiological saline solution washes culture, be transferred in the triangular flask that contains the haemachrome martin's bouillon, 200 μ mol/L 2 have been added in the broth bouillon, 2 '-bipyridyl is as iron chelating agent, also available 5-10 μ mol/L desferrioxamine or the lactoferrin of 0.2-0.4mg/ml replaces.Cultivated 24 hours for 37 ℃.With the centrifugal 30min of bacterium liquid 6000rpm after cultivating, again supernatant is concentrated 100 times with ultrafiltration (the minimum molecular cut off of used filter membrane is 10kDa), wash with the antibacterial of the concentrated solution of holding back, directly bacterial concentration is adjusted to 50-100 hundred million CFU/ml without washing with centrifugation.The formaldehyde that adds 0.3% (percent by volume) again, 37 ℃ of following deactivation 24h, during shake up for several times, 4-8 ℃ of cold preservation is standby after aseptic detections is safe.
2, the preparation of oil emulsion
With diluent antibacterial is diluted to the bacterium liquid that contains desired concn (175CFU/ml), the tween 80 of interpolation 2% is a water; With the span-80 of No. 7 mineral oil addings 6%, it is oil phase that every then 100ml adds the 2g aluminium stearate.
3, the present invention is used for the preparation of escherichia coli inactivated vaccine
To add 1 part of water in 2.5 parts of oil phases, through emulsify at a high speed 2min, and add 1: 10000 thimerosal, make Water-In-Oil escherichia coli inactivated vaccine.
4, the cross immunity protection is measured
Get 40 the 7 non-immune broiler chicken of age in days escherichia coli, be divided into 2 groups at random, 20 every group, first group of vaccine (0.3ml/ is only) that the chest intramuscular injection prepares, the normal saline of second group of chest intramuscular injection equal volume.Use the escherichia coli O of 2 times of minimal lethal doses after 14 days respectively
2Serological type strain and O
78Serological type strain intersection counteracting toxic substances, 10 chickens of every kind of antibacterial counteracting toxic substances observed for 2 weeks, calculated immune protective rate.Experimental result is referring to table 3.
Table 3 compares with the bacillus coli vaccine cross protection experimental result that the inventive method makes
Experimental result shows: the escherichia coli inactivated vaccine that makes with the inventive method is to homotype bacterium (O
2Serological type strain) immune protective rate reaches 100%, to special-shaped bacterium (O
78Serological type strain) immune protective rate reaches 90%.The matched group chicken is all dead, illustrates that the escherichia coli inactivated vaccine that makes with the inventive method has good short-term cross immunity protection effect.
Embodiment three: the present invention is used for the preparation method and the immune protective experiment thereof of Riemerlla anatipestifer vaccine.
The Riemerlla anatipestifer freeze-drying lactobacillus that present embodiment relates to (serotype is the I type) separates in the clinical duck body of dying of illness.
1, rejuvenation of spawn and seedling prepare with bacterium liquid
Freeze-drying lactobacillus is inoculated in the serum nutrient broth after dissolving with the 1ml physiological saline solution, cultivates 16-18 hour for 37 ℃.Get culture fluid and be inoculated in the serum nutrient agar panel, cultivated 16-18 hour for 37 ℃.The antibacterial that grows is washed with sterile saline, as seed liquor.Seed liquor is inoculated in the ratio of culture fluid volume 5% makes in the broth bouillon that contains haemachrome serum that vaccine uses, 200 μ mol/L 2 have been added in the broth bouillon, 2 ' bipyridyl is as iron chelating agent, also available 5-10 μ mol/l desferrioxamine or the lactoferrin of 0.2-0.4mg/ml replaces.37 ℃ of ventilation shaking tables are cultivated 24h, with the centrifugal 30min of bacterium liquid 6000rpm after cultivating, again supernatant is concentrated 100 times with ultrafiltration (the minimum molecular cut off of used filter membrane is 10kDa), wash with the antibacterial of the concentrated solution of holding back, directly bacterial concentration is adjusted to 150-300 hundred million CFU/ml without washing with centrifugation.The formaldehyde that adds 0.3% (percent by volume) again, 37 ℃ of following deactivation 24h, during shake up for several times, 4-8 ℃ of cold preservation is standby after aseptic detections is safe.
2, the preparation of oil emulsion
With diluent antibacterial is diluted to the bacterium liquid that contains desired concn (175CFU/ml), the tween 80 of interpolation 2% is a water; With the span-80 of No. 7 mineral oil addings 6%, it is oil phase that every then 100ml adds the 2g aluminium stearate.
3, the present invention is used for the riemerella anatipestifer inactivated vaccine preparation
To add 1 part of water in 2.5 parts of oil phases, through emulsify at a high speed 2min, and add 1: 10000 thimerosal, make the Water-In-Oil riemerella anatipestifer inactivated vaccine.
4, the cross immunity protection is measured
Get 40 the 7 non-immune ducklings of age in days Riemerlla anatipestifer, be divided into 2 groups at random, 20 every group, first group of vaccine (0.3ml/ is only) that the chest intramuscular injection prepares, the normal saline of second group of chest intramuscular injection equal volume.Respectively with the Riemerlla anatipestifer I type serological type strain and the II type serological type strain intersection counteracting toxic substances of 2 times of minimal lethal doses, 10 ducks of every kind of antibacterial counteracting toxic substances observed for 2 weeks, calculated immune protective rate after 14 days.Experimental result is referring to table 4.
Table 4 cross protection experimental result relatively
Experimental result shows: the riemerella anatipestifer inactivated vaccine that makes with the inventive method reaches 100% to the immune protective rate of homotype bacterium (I type serological type strain), and the immune protective rate of special-shaped bacterium (II type serological type strain) is reached 90%.The matched group duckling is all dead, illustrates that the riemerella anatipestifer inactivated vaccine that makes with the inventive method has good short-term cross immunity protection effect.
Claims (1)
1, a kind of preparation method with Gram-negative pathogen reduction vaccine of cross protection power is characterized in that may further comprise the steps:
A, adopt solid medium or the fluid medium be added with iron chelating agent, carry out In vitro culture with described culture medium after getting rejuvenation of spawn;
B, to the antibacterial that solid medium is cultivated, wash with the vaccine diluent of sterilizing, reach industry standard until final bacterial concentration; The bacterium liquid that perhaps above-mentioned final bacterial concentration is lower than the bacterium liquid of industry standard and cultivates with fluid medium concentrates, and the outer membrane protein that keeps thalline and come off reaches industry standard until final bacterial concentration; To the spissated method of bacterium liquid be: carry out thalline and fluid separation applications with low-speed centrifugal or microfiltration method, described liquid is concentrated with ultrafiltration, the minimum molecular cut off of used filter membrane is not more than 25kDa, be not less than 0.5kDa, with the liquid after concentrating sedimentary thalline being suspended then reaches industry standard until final bacterial concentration;
C, the final bacterium liquid percent by volume that step b is obtained are the formalin-inactivated of 0.1-0.3%;
D, adding adjuvant are made vaccine.
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| CN100563708C true CN100563708C (en) | 2009-12-02 |
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| CN102614511B (en) * | 2012-04-05 | 2014-12-24 | 青岛农业大学 | Propolis-adjuvant inactivated vaccine for mink pasteurellosis |
| CN105288607A (en) * | 2015-11-13 | 2016-02-03 | 湖北省农业科学院畜牧兽医研究所 | Preparation method for inactivated vaccine against avian pasteurellosis |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5766607A (en) * | 1994-10-25 | 1998-06-16 | Kansas State University Research Foundation | Method of culturing M. bovis in low available iron media and isolation of outer membrane proteins |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5766607A (en) * | 1994-10-25 | 1998-06-16 | Kansas State University Research Foundation | Method of culturing M. bovis in low available iron media and isolation of outer membrane proteins |
Non-Patent Citations (4)
| Title |
|---|
| 兽医生物制品实用技术. 刑钊等，118，126，127，162， 166，167，172，173，中国农业大学出版社. 2000 |
| 兽医生物制品实用技术. 刑钊等，118，126，127，162， 166，167，172，173，中国农业大学出版社. 2000 * |
| 蜂胶的生物学活性及其在医学上的应用前景（上）. 李英华，朱威，胡福良.蜜蜂杂志，第2期. 2004 |
| 蜂胶的生物学活性及其在医学上的应用前景(上). 李英华，朱威，胡福良.蜜蜂杂志，第2期. 2004 * |
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