CN109718370B - A kind of duck reovirus vaccine and preparation method thereof - Google Patents
A kind of duck reovirus vaccine and preparation method thereof Download PDFInfo
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Abstract
The present invention discloses a kind of duck reovirus vaccine, is the inactivated vaccine for being inoculated with duck reovirus seed culture of viruses on continuous cell line BHK-21 cell and obtaining.10 can be stably reached by measuring its virus titer using Reed-Muench method7.0‑107.5TCID50/0.1mL.The steady production and high titre of antigen are to prepare vaccine most critical factor, 10 times or more that duck reovirus is proliferated that its virus titer is conventional duck embryos method and primary cell method are carried out with BHK-21 cell, virus multiplication is such as carried out using bioreactor, virus titer can achieve 100 times of levels.Duck reovirus inactivated vaccine disclosed in this patent, yield is high, stay in grade, and immune duck can generate high-level serum neutralizing antibody, and good immune effect has huge application prospect.
Description
Technical field
The present invention relates to a kind of duck reovirus vaccines and preparation method thereof.
Background technique
China from 1997, the provinces such as Guangdong and Guangxi Provinces, Zhejiang, Fujian, Henan and Hubei Province broken out with duck liver it is dirty on there is the white necrosis point to be
The disease of feature.This disease encounter mixed infection or stress when the death rate it is very high.In general, this disease takes place mostly in 7~45 ages in days
Kind duck, pathogenicity rate are up to 20%~90%, and the death rate is 10~50%.Infected duck shows as abdomen sea, weak foot, attached joint or toe more
The different degrees of swelling in joint, resistance to duck excessively become stiff duck；The necrosis of dissect visible liver, spleen, pancreas, kidney and intestines.This is irregular with liver
Necrosis, blood spots/and cardiac muscle, bursa of Fabricius bleeding be main feature new epidemic disease, be commonly called as " the new hepatopathy of duck ", " duck gangrenosum acne liver
Scorching disease " etc., identify that its cause of disease is Reoviridae Orthoreovirus duck reovirus through separation.
It is still listed at present without duck reovirus vaccine, also controls disease caused by the virus without relevant drug,
The generation of duck reovirus infection disease often causes heavy economic losses to duck culturing industry.
Summary of the invention
The technical problem to be solved by the present invention is to effectively prevent duck reovirus.
In order to solve the above technical problems, the present invention discloses a kind of duck reovirus vaccine, it is in continuous cell line
The inactivated vaccine that duck reovirus seed culture of viruses obtains is inoculated on BHK-21 cell.
The preparation method of the duck reovirus vaccine comprising following steps:
(1) passage and culture of BHK-21 cell: BHK-21 cell through EDTA- pancreatin cell dispersion liquid had digestive transfer culture, with
Cell growth medium continues to cultivate, when forming cell monolayer, for continuing passage or virus inoculation；
(2) viral inoculation and culture: taking the above-mentioned cell for having formed single layer, discards cell growth medium, inoculating cell dimension
Liquid is held, reovirus seed culture of viruses is inoculated into the cell of preparation by final volume 1:100~1:1000, is inhaled after absorption and abandons virus liquid,
The harvest when there is lesion with culture solution culture to cell；
(3) viral collection, concentration, purifying: by the sick cell freeze thawing of above-mentioned inoculation, supernatant is collected after centrifugation and obtains
Virus stock solution used；Being concentrated by ultrafiltration above-mentioned virus liquid is seedling virus liquid；
(4) inactivation of virus: using 0.1% Formalin inactivation virus, as vaccine antigen；
(5) vaccine preparation is configured using vaccine antigen.
It is preferred that the cell growth medium in the step (1) is the DMEM culture solution containing 5~10% newborn bovine serum, it is described thin
It can contain in intracellular growth liquid dual anti-.The step (2) cell maintenance medium is the DMEM culture solution of serum-free, and seed culture of viruses is inoculated into carefully
Adsorb at 37 DEG C 30 after born of the same parents~it inhales abandon virus liquid after sixty minutes, connect after poison that condition of culture is 37 DEG C, 5%CO2, institute in culture solution
Stating culture solution is the DMEM culture solution containing 1~2% newborn bovine serum, and the glutamine containing 2mM in cell maintenance medium is more excellent
Select the N-N- diethyl -1-4- phenylenediamine containing 0.2mM.The every 0.1mL virus of seedling virus liquid described in the step (3) is greater than
Content 107.0TCID50.BHK-21 cell is cultivated in rolling bottle or in microcarrier reactor in the step (1).
Step (5) the configuration vaccine preparation includes the following steps:
A) prepared by water phase: 4 parts of Tween-80 for examining qualified 96 parts of vaccine antigen to be added after sterilizing are taken, start to stir,
Until being completely dissolved Tween-80, water phase is made；
B) oil is mutually prepared: 94 parts of high-quality injection white oil is taken, it is 2 parts of aluminum stearate, 4 parts of Span-80, first that white oil is slow
Heating is heated by 4% and 2% addition Span-80 and aluminum stearate when stirring, and transparent is until aluminum stearate is sufficiently dissolved to
Only, high pressure sterilization is spare；
C) it emulsifies: being emulsified using IKA mulser, 16000rpm, emulsified 5 minutes；
D) it dispenses: the vaccine of preparation is dispensed according to every bottle of 250mL.
Presently disclosed duck reovirus vaccine using viral high-adaptability continuous cell line BHK-21 cell into
Row production of vaccine, the technique have following advantage:
1. cellular context understands, no exogenous pathogen, easy proliferative, it is applicable in very much bioreactor and carries out large-scale culture,
This meets future vaccines Production trend, thus the present invention have height technique it is perspective.
2. preparing vaccine using BHK-21 continuous cell line, the clarification of haptens height is easy to carry out concentration, very suitable
Close the high-quality inactivated vaccine production for carrying out high antigen titre.
Duck reovirus proliferation is carried out with BHK-21 passage cell, its virus is measured using Reed-Muench method and drips
Degree can stably reach 107.0-107.5TCID50/0.1mL.The steady production and high titre of antigen be prepare vaccine most critical because
Element carries out 10 times that duck reovirus is proliferated that its virus titer is conventional duck embryos method and primary cell method with BHK-21 cell
More than, virus multiplication is such as carried out using bioreactor, virus titer can achieve 100 times of levels.Duck disclosed in this patent
Reovirus inactivated vaccine, yield are high, stay in grade, and immune duck can generate high-level serum neutralizing antibody, good immune effect,
With huge application prospect.
Specific embodiment
Above content of the invention is described in further detail again below by way of specific embodiment.But this should not be managed
Solution is limited only to embodiment below for the range of the above-mentioned theme of the present invention.The case where not departing from above-mentioned technical idea of the invention
Under, the various replacements or change made according to ordinary skill knowledge and customary means, should all include in model of the invention
In enclosing.
Duck reovirus used in following embodiment is voluntarily separation identification: being expanded using RT-PCR, sequencing carries out
Sequence ratio, while carrying out animal recurrence and being further determined as duck reovirus with duck embryos compatibility test.
Embodiment 1
Preparation method:
(1) it by BHK-21 cell, is digested and is dispersed with pancreatin, the DMEM culture solution of 5% newborn bovine serum is added in rolling bottle
37 DEG C, 5%CO2After culture to cell monolayer, original fluid is discarded, is cleaned cell 3 times, is used for serum-free DMEM culture solution
The inoculation of duck reovirus.
(2) duck reovirus seed culture of viruses viral inoculation and culture: is inoculated into what 1. step was prepared by final volume 1:100
Cell, and 37 DEG C adsorb 30 minutes after inhale abandon virus liquid, with containing 1% newborn bovine serum DMEM culture solution in 37 DEG C,
5%CO2There is termination when lesion to cell in culture.
(3) collection virus, concentration and purifying and viral level measurement: the cytopathy venom of collection is frozen repeatedly in -20 DEG C
After melting twice, it is centrifuged 10min in 4 DEG C, 5000rpm, takes centrifuged supernatant for being concentrated by ultrafiltration.By the supernatant after above-mentioned centrifugation
After liquid is concentrated 10 times with 30K hollow fiber column, it is dilute that the virus liquid after taking concentration with DMEM cell culture fluid does 10 times of series
It releases, takes 10-5、10-6、10-7、10-8、10-95 dilutions are inoculated with 48 hole confluent monolayers BHK-21 tissue culture plates, each dilution
Degree repeats 5 holes, while setting up negative control cell；Every hole 0.2mL, 5%CO2,37 DEG C cultivate 120 hours, observe cytopathy
(CPE), TCID50, every 0.1mL viral level 10 are calculated7.0TCID50 can be used for seedling.
(4) inactivation of virus: using 0.1% Formalin inactivation virus, as vaccine antigen.
Embodiment 2
Preparation method:
(1) it by BHK-21 cell, is digested and is dispersed with pancreatin, the DMEM culture solution of 10% newborn bovine serum is added in microcarrier
37 DEG C, 5%CO in reactor2After culture to cell monolayer, original fluid is discarded, cleans cell 3 with serum-free DMEM culture solution
It is secondary, it is inoculated with for duck reovirus.
(2) viral inoculation and culture: duck reovirus seed culture of viruses is inoculated into step by final volume 1:1000 and is 1. prepared
Cell, and inhaled after sixty minutes in 37 DEG C of absorption and abandon virus liquid, with the DMEM culture solution of 2% newborn bovine serum in 37 DEG C,
5%CO2There is termination when lesion to cell in culture.
(3) collection virus, concentration and purifying: by the sick cell freeze thawing of above-mentioned inoculation, supernatant is collected after centrifugation and obtains
Virus stock solution used；Being concentrated by ultrafiltration above-mentioned virus liquid is seedling virus liquid.Method is with embodiment 1 and carries out viral level survey
It is fixed: every 0.1mL viral level 107.5TCID50 can be used for seedling.
(4) inactivation of virus: using 0.1% Formalin inactivation virus, as vaccine antigen.
Embodiment 3
Preparation method:
(1) it by BHK-21 cell, is digested and is dispersed with pancreatin, the DMEM culture solution of 7% newborn bovine serum is added in microcarrier
37 DEG C, 5%CO in reactor2After culture to cell monolayer, original fluid is discarded, then with the glutamine containing 2mM without blood
Clear DMEM culture solution cleans cell 3 times, is inoculated with for duck reovirus.
(2) duck reovirus seed culture of viruses viral inoculation and culture: is inoculated into what 1. step was prepared by final volume 1:500
Cell, and inhaled after being adsorbed 45 minutes at 37 DEG C and abandon virus liquid, with the DMEM culture solution of 1.5% newborn bovine serum in 37 DEG C,
5%CO2There is termination when lesion to cell in culture.
(3) collection virus, concentration and purifying: by the sick cell freeze thawing of above-mentioned inoculation, supernatant is collected after centrifugation and obtains
Virus stock solution used；Being concentrated by ultrafiltration above-mentioned virus liquid is seedling virus liquid.Method is with embodiment 1 and carries out viral level survey
It is fixed: every 0.1mL viral level 108.0TCID50 can be used for seedling.
(4) inactivation of virus: using 0.1% Formalin inactivation virus, as vaccine antigen.
Embodiment 4
Preparation method:
(1) it by BHK-21 cell, is digested and is dispersed with pancreatin, the DMEM culture solution containing 6% dual anti-newborn bovine serum is added
37 DEG C, 5%CO in microcarrier reactor2Culture is to after cell monolayer, discarding original fluid, then with the glutamy containing 2mM
Amine, 0.2mM N-N- diethyl -1-4- phenylenediamine serum-free DMEM culture solution clean cell 3 times, be used for duck reovirus
Inoculation.
(2) duck reovirus seed culture of viruses viral inoculation and culture: is inoculated into what 1. step was prepared by final volume 1:500
Cell, and inhaled after being adsorbed 45 minutes at 37 DEG C and abandon virus liquid, with the DMEM culture solution of 1.5% newborn bovine serum in 37 DEG C,
5%CO2There is termination when lesion to cell in culture.
(3) collection virus, concentration and purifying: by the sick cell freeze thawing of above-mentioned inoculation, supernatant is collected after centrifugation and obtains
Virus stock solution used；Being concentrated by ultrafiltration above-mentioned virus liquid is seedling virus liquid.Method is with embodiment 1 and carries out viral level survey
It is fixed: every 0.1mL viral level 107.0TCID50 can be used for seedling.
(4) inactivation of virus: using 0.1% Formalin inactivation virus, as vaccine antigen.
The vaccine antigen of above embodiments all passes through following steps and is formulated as vaccine preparation:
A) prepared by water phase: 4 parts of Tween-80 for examining qualified 96 parts of vaccine antigen to be added after sterilizing are taken, start to stir,
Until being completely dissolved Tween-80, water phase is made；
B) oil is mutually prepared: 94 parts of high-quality injection white oil is taken, it is 2 parts of aluminum stearate, 4 parts of Span-80, first that white oil is slow
Heating is heated by 4% and 2% addition Span-80 and aluminum stearate when stirring, and transparent is until aluminum stearate is sufficiently dissolved to
Only, high pressure sterilization is spare；
C) it emulsifies: being emulsified using IKA mulser, 16000rpm, emulsified 5 minutes；
D) it dispenses: the vaccine of preparation is dispensed according to every bottle of 250mL.
The experimental test result of duck reovirus prepared by above embodiments of the present invention is as follows:
1) the duck reovirus prepared by above embodiments quality control: according to existing " Chinese veterinary pharmacopoeia " to its into
Row stability, viscosity, steriling test, content of formaldehyde measurement, as a result meet national standard, and it is qualified to examine.
2) inactivation of the duck reovirus prepared by above embodiments is examined: Example 1-4, using DMEM nutrition respectively
Liquid is with 10-1、10-2、10-3Three dilution proportion measuring samples, while the inactivation provirus sample for setting up same dilution ratio is used
In positive control, it is inoculated in 48 hole BHK-21 cell monolayer cells respectively, every group of 5 hole of repeated inoculation, 37 DEG C of every hole 0.4mL,
5%CO2Culture 96 hours.The results show that not occurring cytopathy, positive controls in inactivated samples each group dilution hole
Cytopathy is obvious in each dilution hole, up to 80% or more.It is qualified that embodiment 1-4 inactivation is examined.
3) safety testing of the duck reovirus vaccine prepared by above embodiments: the susceptible duck of 7 ages in days health is selected
5 groups are classified as, every group 10, embodiment 1-4 vaccine 1mL, remaining one group of conduct is subcutaneously injected in every muscle or neck respectively
Blank control group is observed 14, the strong work of duck is as a result tested, without any locally and systemically adverse reaction；It was solved at the 15th day
Observation is cutd open, viscera tissue is without apparent lesion.Show the duck reovirus vaccine safety of 1-4 of embodiment of the present invention preparation.
4) Vaccine potency test of the duck reovirus vaccine prepared by above embodiments: the susceptible duck of 7 ages in days health is taken
50, every group 10, duck reovirus vaccine prepared by embodiment 1-4 is injected respectively with 0.3mL/ chest muscle, residue one
Group is used as blank control group, 14,28,42 and 56 days after being immunized, takes a blood sample respectively, measures its serum neutralize antibody titers.As a result it shows
Show, the duck reovirus vaccine of embodiment 1-4 preparation can generate high-level serum neutralizing antibody, specifically be shown in Table 1.
Table 1
5) Immunoprotection test of the duck reovirus vaccine prepared by above embodiments: the susceptible duck of 7 ages in days health is taken
100, it is divided into 5 groups, every group 20.Each group is synchronous to carry out strong virus attack, and attacking strain is duck reovirus, and attacking toxic dose is respectively
Only, intramuscular injection in two times is spaced 1 day 60 cfu/, the next day chest muscle drug administration by injection embodiment 1-4 0.3mL/ only, control group 1
Inject same volume physiological saline.It is observed continuously 50 days, counts the protective rate of each group duck reovirus vaccine, implement as the result is shown
Example sample immune protective effect is good, is specifically shown in Table 2:
Table 2
。
Claims (1)
1. a kind of preparation method of duck reovirus vaccine, is characterized in that,
Include the following steps:
(1) passage and culture of BHK-21 cell: by BHK-21 cell, being digested with pancreatin and dispersed, and is added newborn containing dual anti-6%
The DMEM culture solution of cow's serum is 37 DEG C in microcarrier reactor, 5%CO2 is cultivated to cell monolayer, discards original fluid, then
Cell is cleaned with the serum-free DMEM culture solution of the glutamine containing 2mM, the N-N- diethyl -1-4- phenylenediamine of 0.2mM, is used
It is inoculated in duck reovirus；
(2) viral inoculation and culture: by duck reovirus seed culture of viruses by final volume 1:500 be inoculated into step 1. prepare it is thin
Born of the same parents, and 37 DEG C adsorb 45 minutes after inhale abandon virus liquid, with the DMEM culture solution of 1.5% newborn bovine serum in 37 DEG C, 5%CO2
Culture occurs terminating when obvious lesion to cell；
(3) viral collection virus, concentration, purifying: by the sick cell freeze thawing of above-mentioned inoculation, supernatant is collected after centrifugation and obtains
Virus stock solution used；Being concentrated by ultrafiltration above-mentioned virus liquid is seedling virus liquid；
(4) inactivation of virus: using 0.1% Formalin inactivation virus, as vaccine antigen；
(5) vaccine preparation is configured using vaccine antigen.
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| CN110354259B (en) * | 2019-06-25 | 2023-05-09 | 广东渔跃生物技术有限公司 | Preparation method of duck reovirus inactivated vaccine |
| CN111000992A (en) * | 2019-12-19 | 2020-04-14 | 广州渔跃生物技术有限公司 | Triple inactivated vaccine for duck reovirus, duck tembusu virus and duck adenovirus and preparation method thereof |
| CN111184859A (en) * | 2019-12-19 | 2020-05-22 | 广州渔跃生物技术有限公司 | Duck reovirus and duck tembusu virus combined inactivated vaccine and preparation method thereof |
| CN111053897A (en) * | 2019-12-19 | 2020-04-24 | 广州渔跃生物技术有限公司 | Duck reovirus and duck adenovirus bivalent inactivated vaccine and preparation method thereof |
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