CN102757940A - Duck flavivirus and preparation and application of inactivated vaccine of duck flavivirus - Google Patents
Duck flavivirus and preparation and application of inactivated vaccine of duck flavivirus Download PDFInfo
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Abstract
The invention discloses duck flavivirus and a method for preparing an oil-emulsion inactivated vaccine by using the duck flavivirus, and belongs to the field of veterinary infectious diseases. The duck flavivirus is a duck flavivirus AH-F10 strain, of which the preservation number is CCTCC V201213. The method for preparing the oil-emulsion inactivated vaccine comprises the following steps: clinically separating and purifying the duck flavivirus, inactivating the purified duck flavivirus fluid with formaldehyde, and emulsifying the inactivated duck flavivirus with an oil emulsion adjuvant to prepare the duck flavivirus inactivated oil-emulsion vaccine. The duck flavivirus inactivated vaccine prepared by the method has good immunity and less adverse effect on animals, and is suitable for the immunity and scientific experiment for ducks in a small-scale breading unit under the condition of no commodity vaccine.
Description
Technical field
The present invention relates to the preparation method of the newborn vaccine of a kind of anti-duckling inactivation of virus oil.
Background technology
Since 2010; The popular of duck egg drop reduction-death syndrome that duckling virus infection egg duck causes takes place in the East China first; And the main plant in provinces such as Zhejiang, Anhui, Fujian, Jiangsu is arrived in bamboo telegraph; Propagate into provinces and cities such as Guangdong, Shandong, Henan, Hebei afterwards, up to the present the most of main aquatic bird in the whole nation is cultured the threat that the area has all received this disease.Duck infect should virus after, main clinical shows as: high hot, depressed, apocleisis etc. appears in sick duck; The egg duck egg productivity occurs and sharply descends even stopping production; Growth retardation appears in the meat duck.This sick sickness rate is up to 100%, and mortality ratio is between 5%～10%.This virus belongs to the tembusu virus of flaviviridae, Flavivirus.
This disease belongs to reported first in China; The biological characteristics of its cause of disease; The research of aspects such as molecular epidemiology and molecule pathogenesis is all at the early-stage; And should disease in recent years China's aquatic bird culturing area be widely current with and the tremendous economic loss that causes, more should accelerate to study the research of the vaccine of duckling virus disease.The duckling virus vaccines immune effect of the present invention's preparation is better, can prevent the duckling virus disease preferably.
Summary of the invention
The objective of the invention is to prepare a kind of duckling viral inactivation vaccine, to prevent the popular of duckling virus disease.
One of the object of the invention provides a kind of duckling virus; This virus strain is deposited in Chinese typical culture collection center, the address: Chinese Wuhan Wuhan University, and the preservation name is called duckling virus of A H-F10 strain; Preservation date: on April 18th, 2012, deposit number is: CCTCC NO:V201213.
Another object of the present invention is the duckling virus vaccines of being invented to be carried out the evaluation of immune effect.
The preparation method of duckling viral inactivation vaccine of the present invention, its preparation process is following:
With adding 3 ‰ formaldehyde by volume in 37 ℃ of shaking table deactivations 36 hours in virus the 5th generation chick embryo allantoic liquid (being the above-mentioned viral allantoic fluid of gathering in the crops); After deactivation is accomplished; Inactivation of viruses liquid is 96 parts by volume; Tween-80 shakes up for 4 parts, promptly gets antigen for vaccine, by it as under go on foot the emulsive water; White-oil adjuvant as under go on foot the emulsification oil phase; The ratio of 3 parts of oil phases, 1 part of water is got oil phase, water stirring and emulsifying by volume, is prepared into duckling inactivation of virus oil emulsion vaccine, and add volumetric concentration 1% Thiomersalate solution make it in final concentration be ten thousand/, shake up subsequent use.
The separation method of duckling of the present invention virus, its step is following: its oophoropathy tissue of aseptic collection from the sick duck of infected duck flavivirus, with multigelation after its grinding homogenate three times, the centrifugal 10min of 12000r/min. Get its supernatant under the aseptic condition; Supernatant is inoculated 10 age in days SPF (being no-special pathogen) chicken embryo after the degerming of 0.22um filtering with microporous membrane, 0.2mL/ piece, hatch in 38 ℃ of incubators; Discard the dead chicken embryo of inoculation in 24 hours; Gather in the crops dead chick embryo allantoic liquid in 48 hours to 120 hours, reached for 5 generations continuously, it is subsequent use to gather in the crops viral allantoic fluid.
Compared with present technology, beneficial effect of the present invention is embodied in: the strain that the present invention prepares the duckling viral inactivation vaccine separates from the area, Anhui, and virus can stable going down to posterity in the chicken embryo, has better protection property to endemy duckling virus disease.The prepared duckling viral inactivation vaccine of the present invention adopts mature and stable making method; Has immune effect preferably; Untoward reaction to animal is less, is not having temporarily under the situation of commercially available vaccine instantly, is fit to small-scale breed unit duck crowd immunity and scientific experiment are used.
The present invention has set up tiring of fine jade expanding method check antibody, the assay good reproducibility, quick and precisely.
Description of drawings
To be antigen with the fine jade in six holes on every side expand Fig. 1 occurs six precipitation lines that link to each other clearly between the antibody
Fig. 2 is antigen and 2
0Antiserum(antisera), 2
-1Precipitation line clearly appears in the middle of the antiserum(antisera)
Embodiment
Below through specific embodiment technical scheme of the present invention is done further and to be explained.
Embodiment 1
Aseptic condition is gathered pathological tissues such as its ovary down from the sick duck of Anhui zone infected duck flavivirus, it is ground after homogenate multigelation three times, the centrifugal 10min of 12000r/min.Get its supernatant under the aseptic condition, supernatant is inoculated 10 age in days SPF (being no-special pathogen) chicken embryo, 0.2mL/ piece after the degerming of 0.22um filtering with microporous membrane; Hatch in 38 ℃ of incubators; Discard the dead chicken embryo of inoculation in 24 hours, gather in the crops the allantoic fluid of dead chicken embryo in 48 hours to 120 hours, the generation virus allantoic fluid of results is inoculated 10 age in days SPF chicken embryos; 0.2mL/ piece, hatch in 38 ℃ of incubators.Gather in the crops the allantoic fluid of dead chicken embryo in 48 hours to 120 hours, obtain s-generation virus allantoic fluid, reached for 5 generations so continuously, the results allantoic fluid is subsequent use.
In the 5th generation virus allantoic fluid of results, add 3 ‰ formaldehyde by volume in 37 ℃ of shaking table deactivations 36 hours, deactivation is pressed 96 parts of inactivation of viruses liquid after accomplishing, and tween-80 (being Tween-80) shakes up for 4 parts, be antigen for vaccine as under go on foot the emulsive water; White-oil adjuvant as under go on foot the emulsification oil phase.The ratio of 3 parts of oil phases, 1 part of water is got oil phase, water stirring and emulsifying by volume, is prepared into duckling inactivation of virus oil emulsion vaccine, and add 1% Thiomersalate solution make it final concentration and be ten thousand/, shake up subsequent use.
AH-F10 strain stability test
Back inoculation 10 age in days SPF chicken embryos, every piece of 0.2mL are handled in the duckling virus disease abrasive lapping of PCR test positive.Inoculation is placed on 38 ℃ of incubators, observes continuously 7 days.Dead chicken embryo in 24 hours to 120 hours is collected allantoic fluid, reached for 5 generations continuously, viral liquid still can obtain stable duckling virus of A H-F10 strain to 100% death of chicken embryo.
The vaccine evaluation test
Buy from non-jaundice viral disease epidemic-stricken area 50 30 age in week the health sheldrake (laying rate is 86.7%) of laying eggs, be divided into A at random, B, C, four groups of D, A, two groups 15 of B, C, two groups each 10 of D.Isolated rearing between each group.A, two groups of B experiment duck respectively only through neck subcutaneous injection flavivirus inactivated vaccine 2mL/, C, the saline water 2mL/ of two groups of experiments of D duck after neck subcutaneous injection sterilization only observed 10 days continuously, during each group experiment duck have no adverse reaction, do not have the morbidity and the phenomena of mortality.The experiment duck can produce stress response to injection, and in 3 days, egg productivity descends to some extent behind each group experiment duck vaccinate or the saline water, and each is organized rate of descent and is respectively 13.3%, 13.3%, gos up gradually after the 10%, 10%, 4th day, after a week, returns to level before the injection.
Observed for 2 weeks continuously after the vaccinate, during each group experiment duck do not have morbidity, laying rate is no abnormal.After the vaccinate the 15th day, to A, B, C respectively tests duck leg muscle injection flavivirus five generations virus allantoic fluid for three groups, every 1mL, D group experiment duck leg muscle injecting normal saline 1mL. observed for two weeks continuously.The result is for attacking the A of poison after the immunity, the B group is respectively tested duck with the control group D group of injecting normal saline and all had no adverse reaction, and laying rate descends and is respectively 13.3%, 13.3%, 10%.Attack poison group C and organize, attack poison back egg productivity and descend continuously and healthily, until week back stopping production.After the observations expiration in two weeks, each group experiment duck catched and killed cut open inspection and find A, B, D respectively tests duck for three groups does not have obvious pathology, and the D group is respectively tested duck and the ovarian follicle atrophy all occurred, is dispersed in the blutpunkte, pathologies such as enteron aisle, cloaca diffuse hemorrhage.
This shows that flavivirus inactivated vaccine of the present invention has good security, immune protective preferably.
Embodiment 2
Duckling inactivation of virus oil-emulsion antigen immune build with preparation is healthy and strong, and body weight is about the 2Kg new zealand white rabbit, operates as follows: in new zealand white rabbit neck subcutaneous injection oil emulsion vaccine 0.5mL/ only; Two weeks back neck subcutaneous injection oil emulsion vaccine 1mL/ only carries out reinforced immunological; At interval two week the back with oil-emulsion inactivated vaccinating agent once more reinforced immunological once, neck subcutaneous injection 2mL/ only, after this every around booster immunization once, each neck subcutaneous injection oil emulsion vaccine 2mL/ only, the back heart blood sampling of one of immunity week prepares antiserum(antisera) for the third time.
The reactive detection of anti-duckling virus polyclonal antibody.Add 3 ‰ formaldehyde in proportion in 37 ℃ of shaking table deactivations 36 hours in the 5th generation virus allantoic fluid with results, be prepared into agp antigen.On 1% sepharose flat board, punch with seven apertures in the human head plum blossom punch tool; Interstitial hole adds agp antigen; Six holes add the anti-duckling virus of equivalent multi-clone rabbit antiserum(antisera) on every side; In 37 ℃ of wet boxes reaction after 15 hours fine jade expand reaction and occur, interstitial hole with occur between six holes on every side that six precipitation lines that link to each other are as shown in Figure 1 clearly.
The mensuration that anti-duckling virus polyclonal antibody is tired.With the punching of seven apertures in the human head plum blossom punch tool, it is 2 that rabbit anti-serum is used the PBS dilution on 1% sepharose flat board
-1, 2
-2, 2
-3, 2
-4, 2
-5Interstitial hole adds agp antigen, and six holes add equivalent original content antiserum(antisera), 2 successively on every side
-1Antiserum(antisera), 2
-2Antiserum(antisera), 2
-3Antiserum(antisera), 2
-4Antiserum(antisera), 2
-5Antiserum(antisera), reaction is observed after 20 hours and is found out 2 in 37 ℃ of wet boxes
0Antiserum(antisera), 2
-1Antiserum(antisera), and have precipitation line to produce between the interstitial hole, promptly antibody titer is 1:2, and is as shown in Figure 2; Explain that this flavivirus inactivated vaccine has good immunity.
Claims (4)
1. a duckling virus is characterized in that, this virus strain is deposited in Chinese typical culture collection center, and its preservation name is called duckling virus of A H-F10 strain, and deposit number is: CCTCC NO:V201213.
2. the separation method of the said duckling virus of a claim 1; It is characterized in that; Said separation method is following: its oophoropathy tissue of aseptic collection from the sick duck of infected duck flavivirus, grind after homogenate multigelation three times with it, the centrifugal 10min of 12000r/min. Get its supernatant under the aseptic condition; Supernatant is inoculated 10 age in days SPF chicken embryos after the degerming of 0.22um filtering with microporous membrane, 0.2mL/ piece, hatch in 38 ℃ of incubators; Discard the dead chicken embryo of inoculation in 24 hours; Gather in the crops dead chick embryo allantoic liquid in 48 hours to 120 hours, reached for 5 generations continuously, it is subsequent use to gather in the crops viral allantoic fluid.
3. the application of the described duckling virus of claim 1 in preparation duckling viral inactivation vaccine.
4. the preparation method of the described duckling viral inactivation vaccine of claim 3 is characterized in that preparation process is following:
Get this virus the 5th generation chick embryo allantoic liquid, add 3 ‰ formaldehyde by volume, after deactivation is accomplished in 37 ℃ of shaking table deactivations 36 hours; Inactivation of viruses liquid is 96 parts by volume; Tween-80 (being Tween-80) shakes up for 4 parts, promptly gets antigen for vaccine, by it as under go on foot the emulsive water; White-oil adjuvant as under go on foot the emulsification oil phase; The ratio of 3 parts of oil phases, 1 part of water is got oil phase, water stirring and emulsifying by volume, is prepared into duckling inactivation of virus oil emulsion vaccine, and add volumetric concentration 1% Thiomersalate solution make it in final concentration be ten thousand/, shake up subsequent use.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109468281A (en) * | 2018-11-19 | 2019-03-15 | 安徽农业大学 | A kind of BHK-21 cells and its construction method that can stablize expression duck tembusu virus NS1 albumen |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220291A (en) * | 2011-05-18 | 2011-10-19 | 福建省农业科学院畜牧兽医研究所 | Duck flavivirus and inactivated vaccine thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220291A (en) * | 2011-05-18 | 2011-10-19 | 福建省农业科学院畜牧兽医研究所 | Duck flavivirus and inactivated vaccine thereof |
Non-Patent Citations (2)
| Title |
|---|
| 李玉峰等: "一种从鸭新分离的黄病毒研究初报", 《畜牧兽医学报》 * |
| 马秀丽等: "鸭黄病毒BZ株的生物学特性研究", 《中国家禽》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109468281A (en) * | 2018-11-19 | 2019-03-15 | 安徽农业大学 | A kind of BHK-21 cells and its construction method that can stablize expression duck tembusu virus NS1 albumen |
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