CN106085965B - A kind of duck tembusu virus, the vaccine and preparation method thereof prepared by the virus - Google Patents
A kind of duck tembusu virus, the vaccine and preparation method thereof prepared by the virus Download PDFInfo
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Abstract
The present invention discloses a kind of duck tembusu virus, by vaccine of virus preparation and preparation method thereof, which is duck tembusu virus GDHD2014 3；The virus is preserved in China typical culture collection center on April 11st, 2016, and preserving number is CCTCC No：V201621；The preparation method of the vaccine of the treatment duck tembusu virus, comprises the following steps：1) Virus culture：Duck tembusu virus GDHD2014 3 is seeded in Virus culture cell, then harvest virus liquid and is filtered；2) inactivation of virus：24~48h is inactivated by formalin is added in virus liquid；3) emulsify：It by vaccinogen liquid, mixes and is emulsified with white-oil adjuvant, obtain vaccine；Antigen immunogenicity in vaccine is good, and vaccine, which is made, using low concentration virus to play a protective role, and protective rate is up to more than 90%.
Description
Technical field
The present invention relates to biological technical fields, and in particular to a kind of duck tembusu virus and its application.
Background technology
Duck tembusu virus disease (Duck Tembusu Virus Strain), also referred to as egg Duckling flavivirus disease is by flavivirus
A kind of acute infectious disease for encroaching on laying duck ovary caused by section's duck tembusu virus.Under mainly causing high laying ducks egg production drastically
Drop, feed intake drastically decline.If laying duck suffers from this disease, egg production can rapidly decline from peak in one week even to stop production, and adopts
Appetite is also reduced to normal 20% or so from normal level.Through clinical analysis of diagnosis, diseased laying duck necrosis of ovary, spleen swells
Greatly, the clinical pathologic characteristics such as the liquefaction and with unshaped ovum.This disease is propagated extensively at home, and each province kind laying duck culture zone is annual
There is generation.Since the disease in 2010 is found, present irregular hair has been developed to from initial summer egg-laying peak
Raw, which presents a kind of irregular variation, very harmful to laying duck aquaculture.This disease is temporarily listed without vaccine, therefore is sent out
Now for the strain with preferable immunogenicity for the vaccine development of egg Duckling flavivirus disease, preventing the disease has great meaning
Justice.
The content of the invention
For overcome the deficiencies in the prior art, of the invention first is designed to provide a kind of duck tembusu virus and (draws
Fourth literature name：Duck Tembusu Virus, DTMUV), for the virus to the adaptable of cell, virus production is efficient.
Achieving the object of the present invention can be reached by adopting the following technical scheme that：A kind of duck tembusu virus, the virus
It is named as duck tembusu virus GDHD2014-3；The virus was preserved in China typical culture collection on April 11st, 2016
Center, address：In the Bayi Road Wuhan University of Wuhan City, Hubei Province Hongshan District, preserving number is CCTCC No：V201621, classification life
Entitled duck tembusu virus GDHD2014-3 (Latin names：Duck Tembusu vi rus strain GDHD2014-3),
Its sequence is as shown in sequence table SEQ No.1.
Second object of the present invention is to provide for a kind of vaccine for treating duck tembusu virus, and the antigen in vaccine is exempted from
Epidemic focus is good, and low dose can have effect.
Achieving the object of the present invention can be reached by adopting the following technical scheme that：A kind of epidemic disease for treating duck tembusu virus
Seedling is prepared by above-mentioned duck tembusu virus GDHD2014-3.
Third object of the present invention is to provide for a kind of preparation method for the vaccine for treating duck tembusu virus, can support
It is anti-morbidity dosage duck tembusu virus attack and laying rate is kept to maintain more than 92.4%.
Achieving the object of the present invention can be reached by adopting the following technical scheme that：Treatment duck Tan Busu diseases as described above
The preparation method of the vaccine of poison, vaccine preparation comprise the following steps：
1) Virus culture：
Duck tembusu virus GDHD2014-3 is seeded in the culture solution of the cell containing Virus culture, then harvest virus
Liquid simultaneously filters, and -20 DEG C save backup；
2) inactivation of virus：
The formalin inactivation 24 of virus liquid 1000~10000 times of volumes of volume is added in the virus liquid that step 1) is obtained
~48h；Then ammonium sulfate is added in virus liquid, precipitates 6~8h, 5000rpm centrifugations after stirring evenly under conditions of temperature is 4 DEG C
40min abandons supernatant, and precipitation is suspended with sterilizing distilled water, and loaded in bag filter, 6~8h of stirring dialysis, is purified in PBS
Duck tembusu virus GDHD2014-3；Viral 10000rpm is centrifuged into 5~10min, it is vaccinogen liquid to take supernatant；
3) emulsify：
The vaccinogen liquid that step 2) is obtained, mixes with white-oil adjuvant and is emulsified, and obtains duck tembusu virus
GDHD2014-3 vaccines.
Preferably, in step 1), the Virus culture with cell be ST cells, Vero cells or PK15 cells wherein
One of.
Preferably, in step 1), when the sugar of culture solution is 1~10mmol, and pH value is 6.5~7.5, harvest virus
Liquid.
Preferably, in step 2), the addition quality of the ammonium sulfate and the volume ratio of virus liquid are：300~400g/L.
Preferably, in step 1), the method for the Virus culture is half perfusion type cultural method.
Preferably, the half perfusion type cultural method comprises the following steps：
I) virus multiplication is carried out using both arms cell culture apparatus：Both arms cell culture apparatus equipped with carrier is carried out high
Pressure sterilizing, then adds in culture solution, and 12~36h is cultivated under conditions of 4~25 DEG C and is placed on 20~30min in 37 DEG C of water-baths；
Sterile access Virus culture cell, at 37 DEG C, 5%CO2Under conditions of carry out cell amplification cultivation 36~120h；
II) culture solution is replaced：Glucose content and pH value in culture solution are measured daily, when sugar is 1~10mmol,
When pH value is 6.5~7.5, culture fluid exchange is carried out；
III) virus inoculation：When Virus culture cell quantity reaches 108~1010When a, tembusu virus containing duck is accessed
The allantoic fluid of GDHD2014-3, at 37 DEG C, 5%CO2Under conditions of carry out virus amplification culture；
IV) culture solution is supplemented：Glucose content and pH value monitoring are carried out daily after connecing poison, when sugar is 1 in culture solution
During~10mmol, when pH value is 6.5~7.5, the duck tembusu virus GDHD2014-3 virus liquids of collected volume 10~90%, so
The fresh medium for filling into equivalent afterwards continues to cultivate；
V) preserve：By the virus liquid of step IV) harvest in -100~-20 DEG C, multigelation 1~5 time centrifuges after freeze thawing
Cell fragment, filtering are taken, obtains the duck tembusu virus GDHD2014-3 virus liquids for inoculation, -20 DEG C of preservations.
Preferably, step I) in, the culture solution includes the hyclone of volume ratio 1~10%, volume ratio 1~5%
Mycillin, the Hepes liquid of volume ratio 1~5%.
Preferably, repeating step IV) 4~6 times, each duck tembusu virus GDHD2014-3 virus liquids are collected mixed
It closes.
Compared with prior art, the beneficial effects of the present invention are：
1st, duck tembusu virus GDHD2014-3 provided by the invention is strong to cell adaptation, and virus production is efficient；
2nd, the antigen immunogenicity in vaccine of the present invention is good, and vaccine, which is made, using low concentration virus to play
Protective effect, protective rate is up to more than 90%；
3rd, after duck tembusu virus GDHD2014-3 was cultivated for 4~6 generations by half perfusion type cultural method of the present invention, disease
Malicious titre reaches 107.5ELD50/ mL, and general duck tembusu virus averagely needs passage at least 30 generations that can just reach；
4th, half perfusion type cultural method of the present invention reduces production cost, improves virus production efficiency, often cultivates one
4~6 duck tembusu virus GDHD2014-3 virus liquids can continuously be harvested by criticizing cell, and harvest virus quantity is Nostoc commune Vanch mode
2.5 times or more, greatly reduce virus production cost.
Description of the drawings
Fig. 1 is the electron microscope of duck tembusu virus GDHD2014-3.
Duck tembusu virus GDHD2014-3 of the present invention was preserved in Chinese Typical Representative culture on April 11st, 2016
Object collection, address：In the Bayi Road Wuhan University of Wuhan City, Hubei Province Hongshan District, preserving number is CCTCC No：V201621,
Its sequence is as shown in sequence table SEQ No.1.
Specific embodiment
In the following, with reference to attached drawing and specific embodiment, the present invention is described further：
A kind of duck tembusu virus, the viral nomenclature are duck tembusu virus GDHD2014-3；The virus was in 2016 4
It is preserved within 11st China typical culture collection center the moon, preserving number is CCTCC No：V201621, sequence such as sequence table SEQ
Shown in No.1.
Duck tembusu virus GDHD2014-3 is used for the preparation of vaccine, is comprised the following steps：
First, Virus culture：
Virus is cultivated using half perfusion type cultural method,
I) virus multiplication is carried out using both arms cell culture apparatus：Both arms cell culture apparatus equipped with carrier is carried out high
Pressure sterilizing, then adds in culture solution, and 12~36h is cultivated under conditions of 4~25 DEG C and is placed on 20~30min in 37 DEG C of water-baths；
Sterile access Vero cells, at 37 DEG C, 5%CO2Under conditions of carry out cell amplification cultivation 36~120h；
II) culture solution is replaced：Glucose content and pH value in culture solution are measured daily, when sugar is 1~10mmol,
When pH value is 6.5~7.5, culture fluid exchange is carried out；
III) virus inoculation：When Vero cell quantities reach 108~1010When a, tembusu virus containing duck is accessed
The allantoic fluid of GDHD2014-3, at 37 DEG C, 5%CO2Under conditions of carry out virus amplification culture；
IV) culture solution is supplemented：Glucose content and pH value monitoring are carried out daily after connecing poison, when sugar is 1 in culture solution
During~10mmol, when pH value is 6.5~7.5, the duck tembusu virus GDHD2014-3 virus liquids of collected volume 10~90%, so
The fresh medium for filling into equivalent afterwards continues to cultivate；Step IV) is repeated 4~6 times, by each duck tembusu virus
GDHD2014-3 virus liquids collect mixing；
V) preserve：By the virus liquid of step IV) harvest in -100~-20 DEG C, multigelation 1~5 time centrifuges after freeze thawing
Cell fragment, filtering are taken, obtains the duck tembusu virus GDHD2014-3 virus liquids for inoculation, -20 DEG C of preservations；
The culture solution includes the hyclone of volume ratio 1~10%, 1~5% mycillin of volume ratio, and volume ratio 1~
5% Hepes liquid.
2nd, inactivation of virus：
Virus liquid volume 1000~10000 is added in the duck tembusu virus GDHD2014-3 virus liquids that step 1 is obtained
The formalin of times volume inactivates 24~48h；Then quality is added in virus liquid and the volume ratio of virus liquid is：300~
The ammonium sulfate of 400g/L, precipitates 6~8h under conditions of temperature is 4 DEG C after stirring evenly, 5000rpm centrifugation 40min abandon supernatant, sink
It forms sediment and is suspended with sterilizing distilled water, loaded in bag filter, 6~8h of dialysis, the duck tembusu virus purified are stirred in PBS
GDHD2014-3；Viral 10000rpm is centrifuged into 5~10min, it is vaccinogen liquid to take supernatant；
3rd, emulsify：
The vaccinogen liquid that step 2 is obtained, mixes with white-oil adjuvant and is emulsified, and obtains duck tembusu virus
GDHD2014-3 vaccines.
Embodiment 1
The present embodiment is used for the screening and identification for illustrating duck tembusu virus GDHD2014-3：
The sample of the morbidity laying duck ovary RT-PCR positives was collected in laying duck farm of difference city of Guangdong Province from 2014, through place
It is seeded to after reason in the SPF chicken embryos of 7-9 ages in days, after cultivating 2 days, dead chicken embryo was collected in 7 days, allantoic fluid is carried out after precooling
It collects, obtains duck tembusu virus GDHD2014-3.
To this, viral genome sequence is analyzed, complete genome sequence as shown in sequence table SEQ ID No.1, with
The sequence of existing duck tembusu virus is compared in GenBank, and the homology of nucleotide is more than 98%.
In electric Microscopic observation duck tembusu virus GDHD2014-3, as shown in Figure 1, virion diameter range is in 50-
There is cyst membrane on 150nm, surface, have fibre prominent on cyst membrane, in surrounding increased radioactivity.
According to《Chinese veterinary pharmacopoeia》Method carries out sterile, mycoplasma inspection to the virus, the results showed that without mycoplasma and its
Its microbial contamination.
Embodiment 2
The present embodiment is used to illustrate that half perfusion type cultural method cultivates virus：
I) virus multiplication is carried out using both arms cell culture apparatus：Both arms cell culture apparatus equipped with carrier is carried out high
Pressure sterilizing, then adds in culture solution, is cultivated under conditions of 25 DEG C and be placed on 30min in 37 DEG C of water-baths for 24 hours；Sterile access Vero
Cell, at 37 DEG C, 5%CO2Under conditions of carry out cell amplification cultivation 36~120h；
II) culture solution is replaced：Glucose content and pH value in culture solution are measured daily, when sugar is 1~10mmol,
When pH value is 6.5~7.5, culture fluid exchange is carried out；
III) virus inoculation：When Vero cell quantities reach 108~1010When a, tembusu virus containing duck is accessed
The allantoic fluid of GDHD2014-3, at 37 DEG C, 5%CO2Under conditions of carry out virus amplification culture；
IV) culture solution is supplemented：Glucose content and pH value monitoring are carried out daily after connecing poison, when sugar is 1 in culture solution
During~10mmol, when pH value is 6.5~7.5, then the duck tembusu virus GDHD2014-3 virus liquids of collected volume 40% are mended
The fresh medium for entering equivalent continues to cultivate；Step IV) is repeated 6 times, by each duck tembusu virus GDHD2014-3 viruses
Liquid collects mixing；
V) preserve：By the virus liquid of step IV) harvest in -20 DEG C, multigelation 5 times, centrifuging and taking cell is broken after freeze thawing
Piece, filtering obtain the duck tembusu virus GDHD2014-3 virus liquids for inoculation, -20 DEG C of preservations；
The culture solution includes the hyclone of volume ratio 10%, 5% mycillin of volume ratio, the Hepes of volume ratio 5%
Liquid.
Chicken embryo median lethal rate method is used to measure the virus of the strain virus to the duck tembusu virus GDHD2014-3 of acquisition
Concentration, method are as follows：By SPF hatchings to 7~9 days, by virus with cell maintenance medium by gradient with 10 times of series from 10-1Times
Than being diluted to 10-8Times, respectively by 100uL/ pieces of the poison disease vaccination of each dilution factor, each dilution factor is inoculated with 6 pieces of eggs, sets simultaneously
Blanc cell maintaining liquid control group.It puts and chicken embryo is cultivated in 37 DEG C of incubators, 2~6 days observation cell infection virus situations, record is extremely
Chicken embryo quantity is died, calculates virus concentration, virus concentration is 10 after measured7.5ELD50/mL。
Embodiment 3
The present embodiment is used for the preparation for illustrating vaccine：
1000 times of virus liquid volume will be added in the duck tembusu virus GDHD2014-3 virus liquids obtained in embodiment 2
The formalin inactivation of volume is for 24 hours；Then the volume ratio that quality and virus liquid are added in virus liquid is the ammonium sulfate of 300g/L,
It stirs evenly to be placed under conditions of temperature is 4 DEG C and precipitates 8h, 5000rpm centrifugation 40min abandon supernatant, precipitation is hanged with sterilizing distilled water
It is floating, loaded in bag filter, dialysis 8h, the duck tembusu virus GDHD2014-3 purified are stirred in PBS；By virus
10000rpm centrifuges 10min, and it is vaccinogen liquid to take supernatant；
It by vaccinogen liquid, mixes and is emulsified with white-oil adjuvant, obtain duck tembusu virus GDHD2014-3 vaccines.
Obtained duck tembusu virus GDHD2014-3 vaccines are subjected to laying ducks immuning effect test.200 are laid eggs
The laying duck that rate is 90% is divided into 2 groups at random, and every group 100, immune group every injects 0.5mL duck tembusu viruses
GDHD2014-3 vaccines, another set injecting normal saline is as blank control group.Two groups are recorded in 20 days after vaccine injection
Daily laying rate and feed intake, based on laying rate and basic feed intake compare, the results are shown in Table 1.After 20 days, experiment
Every laying duck injects the duck tembusu virus GDHD2014-3 progress artificial onsets of equivalent in group and control group, records two respectively
Group attacks after poison situation of laying eggs daily in 15 days, and the results are shown in Table 2.
Table 1 vaccinate after laying rate
Table 2 carries out the laying rate after artificial onset
| Immunization time | Immune group laying rate (%) | Control group laying rate (%) |
| 1st day | 90 | 75 |
| 2nd day | 92 | 80 |
| 3rd day | 89 | 69 |
| 4th day | 90 | 43 |
| 5th day | 94 | 46 |
| 6th day | 93 | 30 |
| 7th day | 96 | 32 |
| 8th day | 90 | 15 |
| 9th day | 94 | 16 |
| 10th day | 90 | 10 |
| 11st day | 95 | 9 |
| 12nd day | 96 | 12 |
| 13rd day | 92 | 10 |
| 14th day | 93 | 14 |
| 15th day | 95 | 11 |
From table 2 it can be seen that morbidity agent can be resisted using the duck tembusu virus GDHD2014-3 of the present invention vaccines prepared
It measures the attack of duck flavivirus and laying rate is kept to maintain more than 92.4%.This result shows that, use duck tembusu virus
Vaccine prepared by GDHD2014-3 has preferable protecting effect for laying ducks, can resist the infection of duck flavivirus.
For those skilled in the art, technical solution that can be as described above and design are made other each
Kind is corresponding to be changed and deforms, and all these change and deform the protection model that should all belong to the claims in the present invention
Within enclosing.
Claims (7)
1. a kind of duck tembusu virus, it is characterised in that：The viral nomenclature is duck tembusu virus（Duck Tembusu Virus, DTMUV）GDHD2014-3；The virus was preserved in China typical culture collection center on April 11st, 2016, protected
Tibetan number is CCTCC V201621；For the virion diameter range in 50-150nm, there is cyst membrane on surface, has fibre prominent on cyst membrane, in four
All increased radioactivities.
2. a kind of vaccine for treating duck tembusu virus, it is characterised in that：By duck tembusu virus described in claim 1
GDHD2014-3 is prepared.
3. it is a kind of it is as claimed in claim 2 treatment duck tembusu virus vaccine preparation method, it is characterised in that including with
Lower step：
1）Virus culture：
Duck tembusu virus GDHD2014-3 is seeded in the culture solution of the cell containing Virus culture, then harvests virus liquid simultaneously
Filtering, -20 DEG C save backup；
2）Inactivation of virus：
By step 1）The formalin that virus liquid 1000 ~ 10000 times of volumes of volume are added in the virus liquid of acquisition inactivates 24 ~ 48h；
Then ammonium sulfate is added in virus liquid, precipitates 6 ~ 8h, 5000rpm centrifugation 40min after stirring evenly under conditions of temperature is 4 DEG C,
Supernatant is abandoned, precipitation is suspended with sterilizing distilled water, and loaded in bag filter, 6 ~ 8h of dialysis, the smooth cloth of duck purified are stirred in PBS
Virus of reviving GDHD2014-3；Viral 10000rpm is centrifuged into 5 ~ 10min, it is vaccinogen liquid to take supernatant；
3）Emulsification：
By step 2）The vaccinogen liquid of acquisition, mixes with white-oil adjuvant and is emulsified, and obtains duck tembusu virus GDHD2014-3
Vaccine.
4. the preparation method of the vaccine for the treatment of duck tembusu virus as claimed in claim 3, it is characterised in that：Step 1）In,
The Virus culture is one of ST cells, Vero cells or PK15 cells with cell.
5. the preparation method of the vaccine for the treatment of duck tembusu virus as claimed in claim 3, it is characterised in that：Step 1）In,
When the sugar of culture solution is 1 ~ 10mmol, and pH value is 6.5~7.5, virus liquid is harvested.
6. the preparation method of the vaccine for the treatment of duck tembusu virus as claimed in claim 3, it is characterised in that：Step 2）In,
The addition quality of the ammonium sulfate and the volume ratio of virus liquid are：300~400g/L.
7. the preparation method of the vaccine for the treatment of duck tembusu virus as claimed in claim 3, it is characterised in that：Step 1）In,
The method of the Virus culture is half perfusion type cultural method.
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