CN102626514B - Soft-shelled turtle systemic septicemia spherical virus inactivated vaccine and its preparation method - Google Patents
Soft-shelled turtle systemic septicemia spherical virus inactivated vaccine and its preparation method Download PDFInfo
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- CN102626514B CN102626514B CN 201210018723 CN201210018723A CN102626514B CN 102626514 B CN102626514 B CN 102626514B CN 201210018723 CN201210018723 CN 201210018723 CN 201210018723 A CN201210018723 A CN 201210018723A CN 102626514 B CN102626514 B CN 102626514B
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Abstract
The invention discloses a soft-shelled turtle systemic septicemia spherical virus inactivated vaccine and its preparation method, and is characterized in that the vaccine contains the inactivated soft-shelled turtle systemic septicemia spherical virus with the collection number being CGMCCNo.5378. The preparation method comprises the following steps of: cutting internal organ of sick soft-shelledturtles into pieces, adding into a TNE buffer, centrifuging, taking a supernatant, centrifuging, taking a white precipitate, suspending in the TNE buffer, centrifuging the precipitate, taking the supernatant, and centrifuging to obtain a white precipitate, namely the purified virus; diluting the purified virus by the use of aseptic normal saline until the final concentration of viral protein reaches 0.5-1mg/ml, and adding 0.5% (final volume) of formalin for inactivation in a thermostat water bath cauldron; and completely emulsifying inactivated virus and a complete Freund's adjuvant accordingto the volume ratio of 1:1 to obtain the inactivated vaccine. The soft-shelled turtle systemic septicemia spherical virus inactivated vaccine proposed for the first time is safe and effective and hasstrong immunity, and the preparation method is simple and easy to operate.
Description
Technical field
The present invention relates to technical field of vaccines, especially relate to a kind of Trionyx sinensis Wiegmann systematicness septicemia spherical viruses inactivated vaccine and preparation method thereof.
Background technology
Trionyx sinensis Wiegmann, the formal name used at school Trionyx sinensis (Wiegmann) always is medicated diet and excellent tonic product.The eighties in 20th century, the rise of greenhouse cultivation significantly improves the output of regarding as rare and precious Trionyx sinensis Wiegmann originally, and what rise is the popular wantonly of disease thereupon.The anti-Zhiduo County of tradition uses antibiotic and chemical drugs to carry out, and for viral disease, not only does not play therapeutic effect, on the contrary Trionyx sinensis Wiegmann is caused murder by poisoning, contaminated environment, and cause drug residue, hit the consumption confidence of consumer.Since 2007, in Zhejiang area, especially break out a kind of systemic septicemia with Hangzhou, Trionyx sinensis Wiegmann ecologic breeding field, area, Ningbo, cardinal symptom is systemic hemorrhagic deteriorated blood, research worker is through Epidemiological study, tissue slice and researchs such as ultrathin section observation and artificial challenge's test, determine that a kind of novel globular virus is its constitutional virulence factor, this novel globular virus is Trionyx sinensis Wiegmann systematicness septicemia spherical viruses.A kind of new virus that Trionyx sinensis Wiegmann systematicness septicemia spherical viruses formula is separated from the morbidity Trionyx sinensis Wiegmann.This virus can cause hemorrhage, the deteriorated blood of Trionyx sinensis Wiegmann systematicness, and the oncoming force is violent, and cumulative mortality can reach 90-100% in 7-10 days.
Vaccine refers to for the generation that prevents, controls infectious disease, popular; be used for the premunitive vaccine preventive biological products of human body; it can effectively control the generation of disease; use the back not pollute; do not have any residual; can preserve the ecological environment effectively and the quality of consumable products, and life-time service can not produce drug resistance yet.Vaccine mainly comprises new generation vaccines such as inactivated vaccine and subunit vaccine, recombinant vaccine, wherein the inactivated vaccine antibacterial that refers to select for use immunogenicity good, virus, rickettsia, spiral shell time body etc., through artificial culture, with physics or chemical method it is killed again and make.This kind vaccine loses fertility, but keeps immunogenicity.Killed vaccine enters behind the human body can not growth and breeding, short to the body stimulation time, obtain long-lasting immunity and need repeatedly repeated inoculation.With respect to new generation vaccines such as subunit vaccine, recombinant vaccines, inactivated vaccine advantage such as is convenient to make, simple with it and is had the advantage that can not be substituted.At Trionyx sinensis Wiegmann systematicness septicemia disease, all there is not the relevant research of method that prevents and treats at present both at home and abroad, more there is not the correlational study report about Trionyx sinensis Wiegmann systematicness septicemia spherical viruses inactivated vaccine and preparation method thereof.
Summary of the invention
Technical problem to be solved by this invention provides a kind of inventor of utilization and separate the systemic septicemia spherical viruses of the Trionyx sinensis Wiegmann that obtains as the Strain of producing vaccine in the Trionyx sinensis Wiegmann body of suffering from systemic septicemia, prepare safety, the systemic septicemia spherical viruses inactivated vaccine of Trionyx sinensis Wiegmann effective, that immunity is strong and its preparation method is provided, simple, the easy row of the preparation method of this inactivated vaccine.
The present invention solves the problems of the technologies described above the technical scheme that adopts:
1, virus characteristic
Trionyx sinensis Wiegmann systematicness septicemia spherical viruses (Soft-shelled turtle systemic septicemia sphericalvirus, STSSSV), strain is the p1015 strain, preserving number was CGMCC No.5378, was deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 21st, 2011.This virus does not have cyst membrane, diameter 23-40nm, and nucleus is inside and outside all can breed.
2, a kind of Trionyx sinensis Wiegmann systematicness septicemia spherical viruses inactivated vaccine, this vaccine contain the Trionyx sinensis Wiegmann systematicness septicemia spherical viruses through deactivation that preserving number is CGMCCNo.5378.
3, a kind of preparation method of Trionyx sinensis Wiegmann systematicness septicemia spherical viruses inactivated vaccine, step is as follows:
(1) viral purification
Get the Trionyx sinensis Wiegmann that suffers from systemic septicemia, the Trionyx sinensis Wiegmann viscera tissue of will falling ill shreds, and in mass volume ratio 1g: the 10ml ratio joins the Trionyx sinensis Wiegmann internal organs in the TNE buffer, under 4 ℃, behind the centrifugal 20min of 8000r/min, gets supernatant under 4 ℃, the centrifugal 1h of 35000g; Get core position, bottom white precipitate then, be resuspended in the 5mlTNE buffer, 4 ℃ of refrigerator ice baths spend the night, the precipitation that to spend the night is blown and beaten evenly gently, under 4 ℃, and the centrifugal 20min of 8000r/min, get supernatant under 4 ℃, the centrifugal 1h of 35000g, the white precipitate that obtains is purified virus;
(2) antigen deactivation
The purified virus that step (1) is obtained is diluted to virus protein final concentration 0.5-1mg/ml with 0.9% physiological saline solution, add the formalin of final volume 0.5% then in 37 ℃ of thermostat water bath deactivation 24h, every 2-3h mixing that vibrates obtains aseptic inactivation of viruses;
(3) emulsifying
The inactivation of viruses that step (2) is obtained obtains Trionyx sinensis Wiegmann systematicness septicemia spherical viruses inactivated vaccine with after not the formula Freund's complete adjuvant mixed in 1: 1 by volume in 4 ℃ of intermittent oscillation emulsifying 6-8h.
Described TNE buffer compound method: Tris 3.0289g, NaCl 6.00g, EDTA 0.9306g supply distilled water to 500ml, adjust pH 7.6, autoclaving, and 4 ℃ of cold preservations are standby.
Compared with prior art, the invention has the advantages that: a kind of Trionyx sinensis Wiegmann systematicness of the present invention septicemia spherical viruses inactivated vaccine and preparation method thereof has following advantage:
(1) the present invention has proposed Trionyx sinensis Wiegmann systematicness septicemia spherical viruses inactivated vaccine and preparation method thereof first, and the present invention is unique, production technology efficiently, and itself has primacy.
(2) it is less demanding to working condition to produce Trionyx sinensis Wiegmann systematicness septicemia spherical viruses inactivated vaccine by the present invention, is convenient to operation and enforcement, and preparation process is simple, easy to be realized.
(3) under the natural conditions, virus is mainly by eat disease Trionyx sinensis Wiegmann and be with malicious Chi Shuijinpao to infect of Trionyx sinensis Wiegmann, and (injected dose is by body weight＜100g, injection 0.1ml not reach the viral dosage injected in the efficacy test of the present invention; 100g＜body weight＜200g, injection 0.2ml; 200g＜body weight＜300g, injection 0.3ml; Body weight＞300g, injection 0.4ml), so the protective rate in the natural environment may be higher.
In sum, a kind of Trionyx sinensis Wiegmann of the present invention systematicness septicemia spherical viruses inactivated vaccine have safety, effectively, immunity is strong, the preparation method of this inactivated vaccine is simple, easy row.
Trionyx sinensis Wiegmann systematicness septicemia spherical viruses (Soft-shelled turtle systemic septicemia sphericalvirus, STSSSV), strain is the P1015 strain, preserving number is CGMCC No.5378, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 21st, 2011, the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Description of drawings
Fig. 1 is the electron microscopic observation result of the Trionyx sinensis Wiegmann systematicness septicemia spherical viruses particle of purification.
The specific embodiment
Describe in further detail below in conjunction with the present invention of accompanying drawing embodiment.
One, specific embodiment
A kind of Trionyx sinensis Wiegmann systematicness of the present invention septicemia spherical viruses inactivated vaccine, this vaccine contain the Trionyx sinensis Wiegmann systematicness septicemia spherical viruses through deactivation that preserving number is CGMCC No.5378.This Trionyx sinensis Wiegmann systematicness septicemia spherical viruses (Soft-shelled turtle systemic septicemia spherical virus, STSSSV), strain is the P1015 strain, preserving number was CGMCC No.5378, was deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 21st, 2011.This virus does not have cyst membrane, diameter 23-40nm, and nucleus is inside and outside all can breed.Concrete preparation method is as follows:
(1) viral purification
Get the Trionyx sinensis Wiegmann that suffers from systemic septicemia, the Trionyx sinensis Wiegmann viscera tissue of will falling ill shreds, and in mass volume ratio 1g: the 10ml ratio joins the Trionyx sinensis Wiegmann internal organs in the TNE buffer, under 4 ℃, behind the centrifugal 20min of 8000r/min, gets supernatant under 4 ℃, the centrifugal 1h of 35000g; Get core position, bottom white precipitate then, be resuspended in the 5mlTNE buffer, 4 ℃ of refrigerator ice baths spend the night, the precipitation that to spend the night is blown and beaten evenly gently, under 4 ℃, and the centrifugal 20min of 8000r/min, get supernatant under 4 ℃, the centrifugal 1h of 35000g, the white precipitate that obtains is purified virus;
(2) antigen deactivation
The purified virus that step (1) is obtained is diluted to virus protein final concentration 0.5mg/ml with 0.9% physiological saline solution, add the formalin of final volume 0.5% then in 37 ℃ of thermostat water bath deactivation 24h, every 2-3h mixing that vibrates obtains aseptic inactivation of viruses;
(3) emulsifying
Inactivation of viruses with after not the formula Freund's complete adjuvant mixed in 1: 1 by volume, is obtained Trionyx sinensis Wiegmann systematicness septicemia spherical viruses inactivated vaccine in 4 ℃ of intermittent oscillation emulsifying 6h.
TNE buffer compound method: Tris 3.0289g, NaCl 6.00g, EDTA 0.9306g supply distilled water to 500ml, adjust pH 7.6, autoclaving, and 4 ℃ of cold preservations are standby.
In addition to the implementation, the virus protein final concentration can also be 1mg/ml or 0.75mg/ml; The vibration emulsification times of having a rest can also be 7h or 8h.
Two, the method for inspection
1, product inspection: adequately emulsified inactivated vaccine is the milky oil emulsion, get one after another drop ofly not scatter in 5min waterborne, 4 ℃ place 15 days not stratified.
2, aseptic detection: get 100ul Trionyx sinensis Wiegmann systematicness septicemia spherical viruses inactivated vaccine, coating beef agar plate is inverted for 28 ℃ and is cultivated 24-48h, observes to have or not bacterial growth.Asepsis growth is qualified.
3, safety detection:
A. get the inactivated vaccine inoculated into chick embryo, every embryo 0.15ml cultivated 10 days for 34 ℃, observed the chicken embryo death situation.
B. get inactivated vaccine inoculation Trionyx sinensis Wiegmann, every 0.3ml observes death condition in the Trionyx sinensis Wiegmann 10 days.
It is qualified not having death in egg inoculation 3-10d and the Trionyx sinensis Wiegmann inoculation 15d.
4, efficacy test
(1) immunity
The different size Trionyx sinensis Wiegmann is by back leg intramuscular injection vaccine, and other establishes matched group by body weight injection corresponding dosage normal saline.
Immunity is carried out to Trionyx sinensis Wiegmann in fully emulsified back, and injected dose is injected 0.1ml by body weight＜100g; 100g＜body weight＜200g, injection 0.2ml; 200g＜body weight＜300g, injection 0.3ml; Body weight＞300g, injection 0.4ml.Other establishes matched group, by body weight injection corresponding dosage normal saline.
(2) slightly improve concentration virus liquid as counteracting toxic substances liquid by Trionyx sinensis Wiegmann incidence tissue
A. get the Trionyx sinensis Wiegmann that suffers from systemic septicemia typical case disease symptom and dissect, get internal organs and put into the beaker that is embedded in the ice;
B. viscera tissue is shredded by m/v=1g: 10ml adds the homogenate of TM buffer, and in 4 ℃, under the 6000g condition, centrifugal 20min gets supernatant;
C. centrifugal gained precipitation is resuspended with a small amount of TM buffer, homogenate again, in 4 ℃, the centrifugal 20min of 6000g merges two times centrifugal gained supernatant;
After the membrane filtration that d. will merge gained supernatant via hole diameter 0.45 μ m is removed broken tissue, again with filtrate through 0.22 μ m pin type filter filtration sterilization, by volume every milliliter of filtrate adds 1000IU penicillin and 1000 μ g streptomycins;
E. putting into 4 ℃ of refrigerator ice baths spends the night and namely gets counteracting toxic substances liquid;
(3) Trionyx sinensis Wiegmann of getting immune 15 days carries out the counteracting toxic substances experiment, and the experimental group injection is through the counteracting toxic substances liquid of method for preparing, and the same immunizing dose of dosage, is observed death condition in 3-10 day by 50 every group.
Mortality rate is 100% behind the matched group counteracting toxic substances of injecting normal saline as a result; Injection inactivated vaccine group all is lower than 50% with mortality rate behind the counteracting toxic substances liquid injection counteracting toxic substances, and the result shows that vaccine has obvious protection to render a service to Trionyx sinensis Wiegmann.
Satisfy above four kinds of examination requirementses be considered as qualified.
Claims (3)
1. Trionyx sinensis Wiegmann systematicness septicemia spherical viruses inactivated vaccine, it is characterized in that: this vaccine contains the Trionyx sinensis Wiegmann systematicness septicemia spherical viruses through deactivation that preserving number is CGMCC No.5378, and concrete preparation method is as follows:
(1) viral purification
Get the Trionyx sinensis Wiegmann that suffers from systemic septicemia, the Trionyx sinensis Wiegmann viscera tissue of will falling ill shreds, and in mass volume ratio 1g:10ml ratio the Trionyx sinensis Wiegmann internal organs is joined in the TNE buffer, under 4 ℃, behind the centrifugal 20min of 8000r/min, gets supernatant under 4 ℃, the centrifugal 1h of 35000g; Get core position, bottom white precipitate then, be resuspended in the 5mlTNE buffer, 4 ℃ of refrigerator ice baths spend the night, the precipitation that to spend the night is blown and beaten evenly gently, under 4 ℃, and the centrifugal 20min of 8000r/min, get supernatant under 4 ℃, the centrifugal 1h of 35000g, the white precipitate that obtains is purified virus;
(2) antigen deactivation
The purified virus that step (1) is obtained is diluted to virus protein final concentration 0.5-1mg/ml with 0.9% physiological saline solution, add the formalin of final volume 0.5% then in 37 ℃ of thermostat water bath deactivation 24h, every 2-3h mixing that vibrates obtains aseptic inactivation of viruses;
(3) emulsifying
The inactivation of viruses that step (2) is obtained obtains Trionyx sinensis Wiegmann systematicness septicemia spherical viruses inactivated vaccine in 4 ℃ of intermittent oscillation emulsifying 6-8h after 1:1 mixes by volume with formula Freund's complete adjuvant not.
2. the preparation method of a Trionyx sinensis Wiegmann according to claim 1 systematicness septicemia spherical viruses inactivated vaccine is characterized in that step is as follows:
(1) viral purification
Get the Trionyx sinensis Wiegmann that suffers from systemic septicemia, the Trionyx sinensis Wiegmann viscera tissue of will falling ill shreds, and in mass volume ratio 1g:10ml ratio the Trionyx sinensis Wiegmann internal organs is joined in the TNE buffer, under 4 ℃, behind the centrifugal 20min of 8000r/min, gets supernatant under 4 ℃, the centrifugal 1h of 35000g; Get core position, bottom white precipitate then, be resuspended in the 5mlTNE buffer, 4 ℃ of refrigerator ice baths spend the night, the precipitation that to spend the night is blown and beaten evenly gently, under 4 ℃, and the centrifugal 20min of 8000r/min, get supernatant under 4 ℃, the centrifugal 1h of 35000g, the white precipitate that obtains is purified virus;
(2) antigen deactivation
The purified virus that step (1) is obtained is diluted to virus protein final concentration 0.5-1mg/ml with 0.9% physiological saline solution, add the formalin of final volume 0.5% then in 37 ℃ of thermostat water bath deactivation 24h, every 2-3h mixing that vibrates obtains aseptic inactivation of viruses;
(3) emulsifying
The inactivation of viruses that step (2) is obtained obtains Trionyx sinensis Wiegmann systematicness septicemia spherical viruses inactivated vaccine in 4 ℃ of intermittent oscillation emulsifying 6-8h after 1:1 mixes by volume with formula Freund's complete adjuvant not.
3. the preparation method of a kind of Trionyx sinensis Wiegmann systematicness septicemia spherical viruses inactivated vaccine according to claim 2, it is characterized in that: described TNE buffer compound method: Tris 3.0289g, NaCl 6.00g, EDTA 0.9306g supply distilled water to 500ml, adjust pH 7.6, autoclaving, 4 ℃ of cold preservations are standby.
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| CN103013932B (en) * | 2012-12-26 | 2014-11-05 | 宁波大学 | Quick purifying method of portunus trituberculatus reovirus |
| CN104459119B (en) * | 2014-11-14 | 2016-04-13 | 宁波大学 | A kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips and preparation method thereof |
| CN106377502A (en) * | 2016-10-12 | 2017-02-08 | 中崇信诺生物科技泰州有限公司 | Emulsifying method for animal inactivated vaccine |
| CN109096389B (en) * | 2017-06-21 | 2022-09-23 | 宁波大学 | Soft-shelled turtle systemic sepsis spherical virus STSSV blocking agent and preparation method and application thereof |
Citations (2)
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| CN1188673A (en) * | 1997-01-22 | 1998-07-29 | 王卯光 | Soft-shelled turtle tetra vaccinum and preparation method |
| CN101926994A (en) * | 2010-08-19 | 2010-12-29 | 中国医学科学院医学生物学研究所 | Turtle shell immunoadjuvant and influenza vaccine containing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1188673A (en) * | 1997-01-22 | 1998-07-29 | 王卯光 | Soft-shelled turtle tetra vaccinum and preparation method |
| CN101926994A (en) * | 2010-08-19 | 2010-12-29 | 中国医学科学院医学生物学研究所 | Turtle shell immunoadjuvant and influenza vaccine containing the same |
Non-Patent Citations (4)
| Title |
|---|
| 中华鳖细菌疫苗免疫佐剂的筛选及其初步应用;郝贵杰等;《大连水产学院学报》;20100228;第25卷(第1期);19-23 * |
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| 郝贵杰等.中华鳖细菌疫苗免疫佐剂的筛选及其初步应用.《大连水产学院学报》.2010,第25卷(第1期),19-23. |
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