CN101948809A - Inactivated vaccine for preventing duck viral hepatitis and preparation method thereof - Google Patents
Inactivated vaccine for preventing duck viral hepatitis and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an inactivated vaccine for preventing duck viral hepatitis and a preparation method thereof. The invention provides a Duck hepatitis A virus type 1 DHAV-SD strain with the CGMCC No.3746. The strain provided by the invention is a virulent duck hepatitis virus strain with good immunogenicity. The strain is inoculated onto a sensitive cell, cytochylema is obtained, and after the cytochylema is inactivated and emulsified, and the safe, the effective and controllable duck viral hepatitis inactivated vaccine (cell vaccine) can be prepared and is favorable for controlling the duck viral hepatitis. The inactivated vaccine can prevent and control the infection and outburst of the duck viral hepatitis pertinently; meanwhile, the production process is simplified; and the inactivated vaccine can be produced in a large scale and is applied clinically.
Description
Technical field
The invention belongs to the veterinary biological product technical field, be specifically related to prevent inactivated vaccine (cell vaccine) of duck viral hepatitis and preparation method thereof.
Background technology
China is the first aquatic bird big producing country in the world, has advantageous waters, geographical resource advantage.Foster duck already is the important component part of China's aquatic bird aquaculture; in the rural economy of China, be seized of important status; yet wreaking havoc of duck disease not only restricting China's aquaculture striding forward to mass-producing, intensification direction; and affect the development of internationalization such as the import and export multilateral trade of China's bird product, can bring huge social, economy and human life's property damage to countries and regions simultaneously.Duck viral hepatitis is duck group's a common disease, and duckling is had bigger harm.The prevention and control of duck viral hepatitis directly affect the development that the duck industry is supported by China, all are that the most important thing of being paid close attention to is produced in China's livestock industry all the time.
Duck viral hepatitis (Duck virus hepatitis, DVH) be by DHAV (Duck hepatitis A virus, DHAV) the acute height lethality transmissible disease of a kind of duckling that causes, main 4 weeks of infringement are with interior duckling, the easy infection of following duckling of 1 week particularly, mortality ratio is higher.Duck viral hepatitis is a Picornaviridae Avihepatovirus Tobamovirus, the positive chain RNA virus of no cyst membrane non-segmented negative, and no blood clotting has strong resistibility to acid, heat.This viral RNA total length 7729～7799 has only an open reading frame, and translation produces a polyprotein that contains 2254aa.Because of the DHAV antigenic specificity, DHAV can be divided into three genotype, is respectively DHAV-1, DHAV-2 (Taiwan) and DHAV-3 (Korea), and serum-free is learned cross reaction between three hypotypes, is mainly DHAV-1 and DHAV-2 at China's popular at present.
All there is generation and popular in most duck countries of supporting to duck viral hepatitis in the world at present, and all there be the popular of this disease in the provinces and cities of the many foster ducks of China.In the prevention and control to duck viral hepatitis, oneself is developed into three kinds present vaccine: the weak malicious seedling (tissue seedling) of aluminium hydroxide DHV chicken embryoization, the weak malicious deactivation vaccine (tissue seedling) of aluminium hydroxide DHV chicken embryoization and the strong malicious deactivation vaccine of aluminium hydroxide DHV chicken embryoization (tissue seedling).Start with from cause of disease, further study molecule pathogenesis and virus variation mechanism, develop corresponding vaccine and be the key that prevention and control should disease.
Summary of the invention
The purpose of this invention is to provide a kind of inactivated vaccine that prevents duck viral hepatitis and preparation method thereof.
The invention provides DHAV 1 type (Duck hepatitis A virus type 1) DHAV-SD strain, CGMCC No.3746.DHAV 1 type DHAV-SD strain CGMCC No.3746 is in April, 2009, Liu Wenjun, Zhou Yuancheng separate a strain duck viral hepatitis I C-type virus C (DHAV-1) that obtains in the morbidity duck body of duck field, Weifang City, Shandong Province, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 21st, 2010 and (be called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCCNo.3746.
DHAV 1 type claims duck hepatitis virus 1 type again.
The present invention protects the application of DHAV 1 type DHAV-SD strain CGMCC No.3746 in preparation 1 type duck virus hepatitis vaccine (cell vaccine).
The present invention also protects the 1 type duck virus hepatitis vaccine (cell vaccine) that DHAV 1 type DHAV-SD strain CGMCC No.3746 deactivation is obtained.
The present invention also protects a kind of method of the DHAV-SD of preparation strain virus liquid, comprises the steps: DHAV 1 type DHAV-SD strain CGMCC No.3746 is cultivated the back smudge cells in host cell, and the supernatant that obtains is viral liquid; Described host cell is BHK21 cell or DF-1 cell.
Described DHAV 1 type DHAV-SD strain CGMCC No.3746 is cultivated the back smudge cells in host cell, the method for collecting supernatant liquor can comprise the steps:
1. DHAV 1 type DHAV-SD strain CGMCC No.3746 is inoculated described host cell, cultivate the back, collect supernatant, be and produce with kind of a poison by the freeze thawing smudge cells; 2. will produce with planting the described host cell of poison inoculation, cultivate the back, collect supernatant, be seedling with planting poison by the freeze thawing smudge cells; 3. seedling is inoculated a described host cell with kind of a poison, cultivate the back, collect supernatant liquor by the freeze thawing smudge cells.
Described step 1., step 2. with step 3. in: MOI all can be 0.001 when inoculating described host cell, and incubation time all can be 48～72 hours; Described step 1., step 2. with step 3. in: incubation time is preferably 48 hours; Described step 3. in, can pass through the centrifugal 30～40min of 5000rpm (Thermo legend RT whizzer, angle rotor #3332) and collect supernatant liquor.
When described host cell is the BHK21 cell, before described inoculation, can carry out following amplification cultivation earlier:
(1) 37 ℃ of water-baths of frozen BHK21 cell are melted, the centrifugal 5min of 800rpm gets precipitation; With cell culture fluid suspension precipitation, obtain 2 * 10
5The cell suspension of cell/ml, 37 ℃, 5%CO
2Leave standstill and be cultured to every milliliter and contain 1 * 10
6Cell; (2) peptic cell on the basis of step (1) is resuspended in the cell culture fluid then, makes every milliliter to contain 2 * 10
5Cell is cultured to every milliliter under the following conditions then and contains 1 * 10
6Cell: pH7.4, saturation dissolved oxygen are 50%, 37 ℃, 100 commentaries on classics/min (Wheaton company
Magnetic stirring apparatus, agitator article No.: 356907; Agitator arm article No. 356886).
Described cell culture fluid specifically can be: the foetal calf serum of the DMEM of 95 parts by volume and 5 parts by volume mixes, and adds two anti-(penicillin 100U/ml, Streptomycin sulphate 100U/ml).
The present invention also protects a kind of method of preparation 1 type duck virus hepatitis vaccine (cell vaccine), is the DHAV-SD strain virus liquid that above arbitrary described method prepares is carried out deactivation and emulsification as seedling successively with viral liquid, obtains 1 type duck virus hepatitis vaccine.
The method of described deactivation is specific as follows: add formaldehyde in described DHAV-SD strain virus liquid, the volumn concentration that makes formaldehyde is 0.1%, 37 ℃ of deactivation 24 hours, obtains viral liquid after the deactivation.Described emulsive method is specific as follows: with 3 parts by volume oil phases, 1 parts by volume water and 1% (quality percentage composition) Thiomersalate aqueous solution, the final concentration that makes Thiomersalate is 0.01% (quality percentage composition), obtains duck virus hepatitis vaccine; The preparation method of described oil phase is as follows: 94 parts by volume white oils and 6 parts by volume Si Ben-80 are mixed, obtain mixed solution, add 1.6～1.8 gram aluminum stearates in per 100 milliliters of mixed solutions; The preparation method of described water is as follows: with the viral liquid mixing after 4 parts by volume tween-80s and the 96 parts by volume deactivations.
During vaccine immunity duckling provided by the invention, adopt the mode of single immunization, the recommendation immunizing dose is 0.2ml.During vaccine immunity kind duck provided by the invention, adopt the mode of single immunization, the recommendation immunizing dose is 1ml.
The invention provides a strain good immunogenic duck hepatitis virus virulent strain is arranged.This strain is inoculated on the sensitive cells, harvested cell liquid, emulsification after the deactivation can obtain a kind of safe, effective, controlled duck virus hepatitis inactivated vaccine (cell vaccine), helps the prevention and control of duck viral hepatitis.The present invention can prevent and treat the infection and the outburst of duck viral hepatitis targetedly, has simplified production technique simultaneously, is produced on a large scale and is applied to clinical.
Description of drawings
Fig. 1 is a DHAV PCR detected result; Swimming lane 1 negative contrast, 2 is the DHAV positive control, and 3 is DHAV SD strain, and 4 is Marker5000.
Fig. 2 is DHAV virus particle electron microscopic observation result.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Test method among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.% among the following embodiment if no special instructions, is the quality percentage composition.Quantitative test in following examples all is provided with revision test three times, results averaged.
The discovery of embodiment 1, duck hepatitis virus SD strain and evaluation
One, the discovery of duck hepatitis virus SD strain
The present inventor is from open-air certain duck field (in April, 2009, duck field in the Weifang City, Shandong Province, find by Liu Wenjun, Zhou Yuancheng) morbidity duck body in separate and obtained a strain duck viral hepatitis I C-type virus C (DHAV-1), with its called after DHAV-SD strain, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 21st, 2010 and (be called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCCNo.3746.
Two, the evaluation of duck hepatitis virus SD strain
1, morphology of virus
The virus particle of DHAV-SD strain is the icosahedron symmetry, the about 40nm of diameter.Electron microscopic observation the results are shown in Figure 2.
2, molecular biology identification
Extract the RNA of DHAV-SD strain, after reverse transcription, a pair of Auele Specific Primer (P1 and P2) that the DHAV complete genome sequence of delivering according to GENBANK (FJ496340) designs carries out the RT-PCR amplification, and amplified production is the purpose band of 348bp.The electrophorogram of PCR product is seen Fig. 1.Sequencing result is shown in the sequence 1 of sequence table.The result shows that the DHAV-SD strain is the DHAV positive.
Upstream primer P1:5 '-CAAAGCCCATCCACTGTTTT-3 ';
Downstream primer P2:5 '-CACTAGGGCTGGGTCATTGT-3 '.
Extract the RNA of DHAV-SD strain, after reverse transcription, carry out complete sequence determination.The complete sequence of strain is shown in the sequence 2 of sequence table, and the DHAV complete genome sequence of delivering with GENBANK has sequence difference.
3, blood clotting
The DHAV-SD strain is to chicken, rabbit, pig, all not aggegations of guinea-pig red blood cell.
4, the median infective dose (TCID of pair cell
50)
The cell toxicant in the 16th generation of DHAV-SD strain is 10
7.1TCID
50The cell toxicant in/ml, 27 generations is 10
7.29TCID
50/ ml.
5, to the median infective dose (EID of chicken embryo
50)
The titre in the 13rd generation of DHAV-SD strain is 10
7.25EID
50/ 0.2ml.
6, the virulence of duckling is measured
In 3 generations of DHAV-SD strain, measured the virulence of duckling, and titre is 10
4.45ID
50/ ml, 10
3.9LD
50/ ml.ID
50Be median infective dose, LD
50Be medium lethal dose.
7, stability
The DHAV-SD strain was passed for 32 generations at cell, and every 0.1ml viral level is 10
7TCID
50More than, the DHAV-SD strain 32 generations with interior be stable, can be used as production of vaccine with kind the poison.
8, immunogenicity
DHAV-SD strain F
1～F
32All have good immunogenicity, be beneficial to the research of vaccine.
9, specificity
The DHAV-SD strain is diluted to 10
4TCID
50/ ml mixes with the anti-duck hepatitis virus specific serum of equivalent, and room temperature was placed after 1 hour, and 10 of inoculation SPF chicken embryos were observed 120 hours.In 24～120 hours, do not cause specificity death, and survive 8 at least.
The preparation of embodiment 2, duck virus hepatitis inactivated vaccine (cell vaccine)
One, chick embryo fibroblast is the cultivation of DF-1
Chick embryo fibroblast is DF-1: (ATCC clone is numbered: CRL-12203) available from Central Plains, Beijing company.
Cell culture fluid: the FBS of the DMEM of 9 parts by volume and 1 parts by volume mixes, and adds two anti-(penicillin 100U/ml, Streptomycin sulphate 100U/ml).
1, cell recovery
DF-1 cell cryopreservation pipe is taken out from liquid nitrogen, in 37 ℃ of water-baths, heat rapidly, enchylema is melted.With the centrifugal 5min of frozen pipe 800rpm in desk centrifuge, abandon supernatant.Cell precipitation is blown and beaten evenly (in the 6cm diameter culture dish) in the 3ml substratum, put into CO
2In the constant incubator, 37 ℃, 5%CO
2Leave standstill cultivation.
2, passage
Use electric suction apparatus, inhale along the culture dish wall and remove waste liquid.PBS cleans 1-2 time, inhales and removes waste liquid.Add pancreatin, 37 ℃ of digestion along the culture dish wall.By the time cellular form becomes circle, inhale and remove pancreatin, with DMEM substratum piping and druming cell, cell evenly is suspended in the substratum, obtain cell suspension.Cell suspension evenly is divided into several parts, joins in the new culture dish, add new DMEM substratum, place 37 ℃ of cultivations.
3, cell cryopreservation
Cell culture fluid is placed centrifuge tube, the centrifugal 5min of 800rpm.Carefully abandon supernatant, use the cells frozen storing liquid re-suspended cell.With resuspended good cell packing, every frozen pipe is no more than 1.5ml.Frozen pipe places 4 ℃, 30min earlier, then-20 ℃, 2h, ℃ spends the night then-80, puts into liquid nitrogen container then and preserves.
Two, the preparation of viral liquid and TCID
50Mensuration
1, the preparation of viral liquid
DHAV-SD strain inoculation DF-1 cell; Cytopathy reaches 80% when above (about 48 hours), harvested cell liquid, and multigelation 3 times is collected supernatant (viral liquid).
2, the TCID of viral liquid
50Measure
(1) prepares cell
Get well-grown DF-1 cell, the digestion back prepares cell suspension with 10%FBS DMEM; Adjusting cell concn is 5 * 10
5Individual/ml, add 96 porocyte culture plates then, the 0.1ml/ hole.37 ℃, 5%CO
2Cultivation makes it be paved with individual layer.
(2) prepare virus
The viral liquid of step 1 preparation is carried out 10 times of gradient dilutions.
(3) TCID of viral liquid
50Measure
After cell covers with individual layer in 96 orifice plates,, add viral dilution liquid, each gradient 8-16 hole, every hole 0.1ml, 37 ℃, 5%CO with the nutrient solution sucking-off
2Cultivate and observe pathology hole count and record after 96 hours.Each dilution CPE the results are shown in Table 1.
Each dilution CPE result of table 1
| The viral dilution degree | The hole count that CPE occurs |
| 10 3 | 16/16 |
| 10 4 | 16/16 |
| 10 5 | 16/16 |
| 10 6 | 13/16 |
| 10 7 | 7/16 |
| 10 8 | 1/16 |
| 10 9 | 0/16 |
| 10 10 |
(4) calculate according to the Reed-MuenchShi method
Press the Reed-MuenchShi method and calculate TCID
50, intermediate data sees Table 2.
Table 2 is pressed the Reed-MuenchShi method and is calculated TCID
50Intermediate data
Annotate: A, B and C item are raw data; The D item deducts the value (promptly not having CPE hole=cell hole count-CPE hole count) of C item for the B item; The E item is that (reason is 10 to the value that adds from bottom to top of C item
8If have this cell hole virus inoculation amount of CPE bigger 10
7Also necessarily have CPE, and the like); The F item is the value (reason 10 that the D item adds from top to bottom
6If be these three cell hole virus inoculation amounts of no CPE littler 10
7Necessarily there is not CPE yet, and the like); The G item is E item and F item sum (promptly each group accumulation cell hole count sum equals the accumulation CPE hole count and the no CPE hole count sum of accumulation of this group); The H item is E item and G item sum (promptly accumulate the CPE percentage and equal to accumulate CPE hole count and the ratio of accumulating cell hole count sum).As seen from Table 2, the extent of dilution of CPE to occur (be TCID to the half cell
50) between 10
-6With 10
-7Between.
Distance is than=(being higher than 50% percentage ratio-50)/(be higher than 50% percentage ratio-be lower than 50% percentage ratio)=(87.5-50)/(87.5-40)=0.73.
TCID
50=be higher than 50% viral dilution degree negative logarithm+distance than=6+0.73=6.73.
Admittedly can make the extent of dilution of 50% cell generation pathology is 10
-6.73, when viral liquid carries out 10
6.73When doubly diluting, contain 1 TCID among the 0.1ml
50, the viral level in every milliliter of viral liquid is 10
7.73TCID50.
Three, the preparation of duck virus hepatitis inactivated vaccine (cell vaccine)
1, produces with kind of the preparation of poison
DHAV-SD strain inoculation DF-1 cell, MOI are 0.001, and (MOI is the virus quantity that is used to infect and the ratio of cell number; The unit of virus quantity is TCID
50, the unit of cell number is individual); When pathology appears in 80% above cell (inoculating back 48 hours) observed in inoculation back, harvested cell liquid, and multigelation 3 times is collected supernatant (sampling Detection TCID
50, viral level 〉=10
6.5TCID
50The conduct of/0.1ml is produced with kind of a poison).Supernatant places 2～8 ℃ of preservations.
Produce with kind of the pure property check of poison:
(1) steriling test
T.G, G.P tubule and G.A slant medium are not all observed bacterium colony.
(2) mycoplasma check
In the observation period, do not find that considerable change appears in bottle and tubule culture color, the liquid culture of transplanting does not have " fried egg " shape mycoplasma bacterium colony on solid medium.
(3) exogenous virus check
All survivals in two groups of chicken embryos 7 days, it is normal to cut open the inspection fetation, and chorioallantoic membrane does not have pathology, and the chicken blastochyle does not have blood clotting.Do not see bacterium, mould and mycoplasma growth in the virus, no exogenous virus pollutes, and proves that it is purified.
2, the seedling preparation of venom
Get and produce with kind of a malicious inoculating cell, MOI is 0.001; Inoculation back cytopathy reaches 80% when above (inoculating back 48 hours), harvested cell liquid, and multigelation 3 times is collected supernatant (sampling Detection TCID
50, viral level 〉=10
6.5TCID
50/ 0.1ml's is malicious with planting as seedling).Supernatant places 2～8 ℃ of preservations.
3, the preparation of vaccine
(1) preparation of viral liquid and deactivation
Seedling is inoculated a DF-1 cell with kind of a poison, and MOI is 0.001, observes once every 12 hours; Cytopathy (CPE) reaches 80% when above (inoculating back 48 hours), collects enchylema, multigelation 3 times, 5000rpm (Thermo legend RT whizzer, angle rotor #3332) centrifugal 30min collects supernatant (sampling Detection is answered asepsis growth), and supernatant (viral liquid) places 2～8 ℃ of preservations.
Add formaldehyde in supernatant, the volumn concentration that makes formaldehyde is 0.1%, 37 ℃ of deactivation 24 hours (reaching 37 ℃ with temperature in the bottle picks up counting), during jolting 3～4 times, 2～8 ℃ of preservations (inactivation of viruses liquid) after the deactivation.
(2) emulsification
Oil phase preparation: by injection white oil 94 parts by volume, Jia Siben-806 parts by volume after the mixing, adds stearic acid aluminium (per 100 milliliters of mixed solutions add 1.75 gram aluminum stearates) then, be stirred to gradually transparent till, autoclaving is standby.
The water preparation: get sterilization tween-80 4 parts by volume, add inactivation of viruses liquid 96 parts by volume, shake well dissolves tween-80 fully.
Emulsification: get oil phase 3 parts by volume and be put in the emulsion tank, starting the motor slow rotation stirs, slowly add water 1 parts by volume simultaneously, stirred 2～5 minutes with 10000r/min again after adding, before stopping stirring, add the 1% Thiomersalate aqueous solution, making its final concentration is 0.01%, obtains duck virus hepatitis inactivated vaccine (lot number is 200912001).
(7) packing
Quantitatively packing seals 2～8 ℃ of preservations (lot number is 200912001).
The preparation of embodiment 3, duck virus hepatitis inactivated vaccine (cell vaccine)
Adopt the DHAV-SD strain to prepare vaccine, method obtains preventing duck virus hepatitis inactivated vaccine (lot number is 200912002) with embodiment 2.
The preparation of embodiment 4, duck virus hepatitis inactivated vaccine (cell vaccine)
Adopt the DHAV-SD strain to prepare vaccine, method obtains preventing duck virus hepatitis inactivated vaccine (lot number is 2009120023) with embodiment 2.
The preparation of embodiment 5, duck virus hepatitis inactivated vaccine (cell vaccine)
One, the cultivation of immature hamster nephrocyte BHK21
Immature hamster nephrocyte BHK21: available from China Veterinery Drug Inspection Office (clone is numbered CL6).
Cell culture fluid: the foetal calf serum of the DMEM of 95 parts by volume and 5 parts by volume mixes, and adds two anti-(penicillin 100U/ml, Streptomycin sulphate 100U/ml).
1, cell recovery
BHK21 cell cryopreservation pipe is taken out from liquid nitrogen, in 37 ℃ of water-baths, heat rapidly, enchylema is melted.With the centrifugal 5min of frozen pipe 800rpm in desk centrifuge, abandon supernatant.With cell precipitation piping and druming evenly, cell suspension is according to 2 * 10
5The concentration inoculation culture ware (containing cell culture fluid) of cell/ml is put into CO
2In the constant incubator, 37 ℃, 5%CO
2Leave standstill cultivation.
2, passage and suspension culture
After treating that cell covers with culture dish, cell concn reaches 1 * 10
6During cell/ml, peptic cell is resuspended in the fresh cell culture fluid, carries out cell counting, is 1 * 10 according to cell concn
5The amount inoculating cell of cell/ml is cultivated reactor (containing cell culture fluid) and is carried out suspension culture, suspension culture stage significant parameter is set is: pH7.4, saturation dissolved oxygen are 30%[saturation dissolved oxygen=(the saturated content of dissolved oxygen under dissolved oxygen actual measurement content/actual measurement condition) * %; Oxygen saturated solubleness in water is 6.7mg/L in the time of 37 ℃], 37 ℃ of temperature, rotating speed 100 commentaries on classics/min (Wheaton company
Magnetic stirring apparatus, agitator article No.: 356907; Agitator arm article No. 356886).
3, cell harvesting
Every day, cell counting was carried out in sampling, treated that cell concn reaches 1 * 10
6Go down to posterity during cell/ml or gather in the crops the freezing preservation in back.
Two, the preparation of viral liquid and TCID
50Mensuration
Replace the DF-1 cell with the BHK21 cell, other is with the step 2 of embodiment 2.
The median infective dose that is viral liquid is 10
7.73TCID
50/ ml.
Three, the preparation of duck virus hepatitis inactivated vaccine (cell vaccine)
Replace the DF-1 cell with the BHK21 cell, other is with the step 3 of embodiment 2.
To duck virus hepatitis inactivated vaccine (lot number is 200912004).
The preparation of embodiment 6, duck virus hepatitis inactivated vaccine (cell vaccine)
Adopt the DHAV-SD strain to prepare vaccine, method obtains preventing duck virus hepatitis inactivated vaccine (lot number is 200912005) with embodiment 5.
The preparation of embodiment 7, duck virus hepatitis inactivated vaccine (cell vaccine)
Adopt the DHAV-SD strain to prepare vaccine, method obtains preventing duck virus hepatitis inactivated vaccine (lot number is 2009120026) with embodiment 5.
The quality inspection of embodiment 8, duck virus hepatitis inactivated vaccine
One, the inspection after construction of inactivated vaccine
Vaccine with six lot numbers of embodiment 2 to embodiment 7 preparation carries out following detection:
(1) physical behavior
Outward appearance: should be milky white emulsion.
Formulation: be water-in-oil-type (W/O).Get a clean suction pipe, draw a small amount of vaccine and drip in cold water, should be the oil droplet shape, indiffusion.
Viscosity: with 1ml suction pipe (the outlet internal diameter is 1.2mm), draw about 25 ℃ vaccine, make its vertical outflow naturally, record flows out the 0.4ml required time, should be no more than 5 seconds.
Stability: under 37 ℃ of left and right sides conditions, placed 21 or vaccine 5ml is added centrifuge tube, centrifugal through 3000r/min, 15 minutes, breakdown of emulsion not.
Against regulation person is judged to defective.
(2) steriling test is undertaken by " Chinese veterinary drug allusion quotation " regulation, should be asepsis growth.
(3) safety verification is with 10 of the Beijing ducks of 3～7 ages in days, and each neck subcutaneous injection vaccine 1ml observed 14, any part and the systemic adverse reactions that are caused by vaccination should not occur.
(4) Beijing duck of efficacy test about with 1 age in days is 10, and each subcutaneous injection vaccine 0.3ml is in the time of 21 days, together with 10 of the identical contrast ducks of raising condition, each chest muscle injection 10
7TCID
50Strong poison was observed 7, should all be good for and live.
(5) formaldehyde, Thiomersalate assay are measured by the prescriptive procedure of " Chinese veterinary drug allusion quotation ", and its formaldehyde solution amount (40% formaldehyde) should not surpass 0.1%, and the antiseptic mercurials residual quantity should be no more than 0.01%.
Two, the safety examination of duck virus hepatitis inactivated vaccine
Vaccine with six lot numbers of embodiment 2 to embodiment 7 preparation carries out following detection:
(1) various dosage are to the safety testing of different days Beijing duck duckling
With vaccine neck subcutaneous vaccination 1 age in days, 3 ages in days, 5 age in days Beijing duck ducklings, 10 every group, 0.2ml/ only.The inoculation back was observed 14 days, and all duckling inoculation positions are not seen swelling, necrosis, do not see systemic adverse reactions.
(2) the reusable safety testing of single dose
With 10 3 age in days ducklings of vaccine neck inoculated with subcutaneous injections, weekly, each 0.2ml/ only repeats 3 times; 10 of neck subcutaneous vaccination kind ducks in 30 age in week, 1 time weekly, each 0.5ml/ only repeats 3 times.The inoculation back was observed 14 days, and the duck inoculation position is not seen swelling, necrosis, does not see systemic adverse reactions.
The safety testing of (3) overdose inoculations
With 10 of vaccine neck subcutaneous vaccination 3 age in days ducklings, 1ml/ only.10 ducklings of overdose inoculation, the inoculation back was observed 14 days, and all duckling inoculation positions are not seen swelling, necrosis, do not see systemic adverse reactions.
(4) vaccine is to the influence test of laying ducks production performance
Get 20 of the laying duckses of egg-laying peak in age in vaccine immunity 28 week, be divided into 2 groups, every group 10, difference subcutaneous injection vaccine, immunizing dose is 0.5ml and 1ml, sets up negative control simultaneously, every subcutaneous injection 0.5ml physiological saline, raised 14 days simultaneously with condition, observations, 200912001 batches of vaccines the results are shown in Table 3.
Table 3 vaccine is to the influence of laying ducks
In sum, security verification is the result show, the vaccine of 6 different lot numbers all is safe, does not cause any untoward reaction.
The efficacy test of embodiment 9, duck virus hepatitis inactivated vaccine
Vaccine with six lot numbers of embodiment 2 to embodiment 7 preparation detects respectively.
One, to the effectiveness of duckling immunity
Duckling (kind is a Beijing duck, available from experimentation on animals base, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences Changping) is divided into 19 groups, 10 every group, carries out following processing respectively:
First group: inject the vaccine of 200912001 lot numbers, every duckling subcutaneous injection 0.1ml;
Second group: inject the vaccine of 200912001 lot numbers, every duckling subcutaneous injection 0.2ml;
The 3rd group: inject the vaccine of 200912001 lot numbers, every duckling subcutaneous injection 0.4ml;
The 4th group: inject the vaccine of 200912002 lot numbers, every duckling subcutaneous injection 0.1ml;
The 5th group: inject the vaccine of 200912002 lot numbers, every duckling subcutaneous injection 0.2ml;
The 6th group: inject the vaccine of 200912002 lot numbers, every duckling subcutaneous injection 0.4ml;
The 7th group: inject the vaccine of 200912003 lot numbers, every duckling subcutaneous injection 0.1ml;
The 8th group: inject the vaccine of 200912003 lot numbers, every duckling subcutaneous injection 0.2ml;
The 9th group: inject the vaccine of 200912003 lot numbers, every duckling subcutaneous injection 0.4ml;
The tenth group: inject the vaccine of 200912004 lot numbers, every duckling subcutaneous injection 0.1ml;
The 11 group: inject the vaccine of 200912004 lot numbers, every duckling subcutaneous injection 0.2ml;
The 12 group: inject the vaccine of 200912004 lot numbers, every duckling subcutaneous injection 0.4ml;
The 13 group: inject the vaccine of 200912005 lot numbers, every duckling subcutaneous injection 0.1ml;
The 14 group: inject the vaccine of 200912005 lot numbers, every duckling subcutaneous injection 0.2ml;
The 15 group: inject the vaccine of 200912005 lot numbers, every duckling subcutaneous injection 0.4ml;
The 16 group: inject the vaccine of 200912006 lot numbers, every duckling subcutaneous injection 0.1ml;
The 17 group: inject the vaccine of 200912006 lot numbers, every duckling subcutaneous injection 0.2ml;
The 18 group: inject the vaccine of 200912006 lot numbers, every duckling subcutaneous injection 0.4ml;
The 19 group (blank group): injecting normal saline, each every duckling subcutaneous injection 0.2ml.
Adopt single immunization in the test, immunity back the 14th day, each is organized the equal intramuscular injection duck viral hepatitis of duckling virulent strain and (available from China Veterinery Drug Inspection Office, AV2112), attacks the toxic agent amount and be 10
7TCID
50, observed continuously again 14 days afterwards.Attack the 14th day statistics in poison back and attack poison protection number, the results are shown in Table 4.
The potency test result of table 4 duckling virus hepatitis inactivated vaccine
The duckling potency test shows: with 0.1ml dosage group immunity duckling, attack malicious protection ratio more than 80%; Carry out immunity with 0.2ml dosage group and 0.4ml dosage group, attack malicious protection ratio 100%; The duck of blank group is all morbidities or dead all, and protection ratio is 0%.
Two, to kind of the effectiveness of duck immunity
Kind of the duck (kind is a Beijing duck, available from experimentation on animals base, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences Changping) of will laying eggs is divided into 19 groups, 10 every group, carries out following processing respectively:
First group: inject the vaccine of 200912001 lot numbers, every duck subcutaneous injection 0.5ml;
Second group: inject the vaccine of 200912001 lot numbers, every duck subcutaneous injection 1ml;
The 3rd group: inject the vaccine of 200912001 lot numbers, every duck subcutaneous injection 1.5ml;
The 4th group: inject the vaccine of 200912002 lot numbers, every duck subcutaneous injection 0.5ml;
The 5th group: inject the vaccine of 200912002 lot numbers, every duck subcutaneous injection 1ml;
The 6th group: inject the vaccine of 200912002 lot numbers, every duck subcutaneous injection 1.5ml;
The 7th group: inject the vaccine of 200912003 lot numbers, every duck subcutaneous injection 0.5ml;
The 8th group: inject the vaccine of 200912003 lot numbers, every duck subcutaneous injection 1ml;
The 9th group: inject the vaccine of 200912003 lot numbers, every duck subcutaneous injection 1.5ml;
The tenth group: inject the vaccine of 200912004 lot numbers, every duck subcutaneous injection 0.5ml;
The 11 group: inject the vaccine of 200912004 lot numbers, every duck subcutaneous injection 1ml;
The 12 group: inject the vaccine of 200912004 lot numbers, every duck subcutaneous injection 1.5ml;
The 13 group: inject the vaccine of 200912005 lot numbers, every duck subcutaneous injection 0.5ml;
The 14 group: inject the vaccine of 200912005 lot numbers, every duck subcutaneous injection 1ml;
The 15 group: inject the vaccine of 200912005 lot numbers, every duck subcutaneous injection 1.5ml;
The 16 group: inject the vaccine of 200912006 lot numbers, every duck subcutaneous injection 0.5ml;
The 17 group: inject the vaccine of 200912006 lot numbers, every duck subcutaneous injection 1ml;
The 18 group: inject the vaccine of 200912006 lot numbers, every duck subcutaneous injection 1.5ml;
The 19 group (blank group): injecting normal saline, every duckling subcutaneous injection 1ml.
Adopt single immunization in the test, immunity back was collected and is respectively organized duck's egg that laying ducks produces (every group picked at random 20) on the the 30th to 35 day, hatched; Duckling went out behind the shell 7 days, and each organizes equal intramuscular injection duck hepatitis SD strain virulent virus, attacks the toxic agent amount and is 10
7TCID
50, observed continuously again 14 days.Attack the 14th day statistics in poison back and attack poison protection number, the results are shown in Table 5.
The potency test result of table 5 kind of duck virus hepatitis inactivated vaccine
Planting the duck potency test shows: plant duck with the immunity of 0.5ml dosage group, 7 age in days ducklings are attacked malicious protection ratio more than 80%; Carry out immunity with 1ml dosage group and 1.5ml dosage group, attack malicious protection ratio 100%; The duck of blank group is all morbidities or dead all, and protection ratio is 0%.
Claims (10)
1. DHAV 1 type (Duck hepatitis A virus type 1) DHAV-SD strain, its preserving number is CGMCC No.3746.
2. the application of DHAV 1 type DHAV-SD strain CGMCC No.3746 in preparation 1 type duck virus hepatitis vaccine.
3. the 1 type duck virus hepatitis vaccine that DHAV 1 type DHAV-SD strain CGMCC No.3746 deactivation is obtained.
4. the method for preparing DHAV-SD strain virus liquid comprises the steps: DHAV 1 type DHAV-SD strain CGMCC No.3746 is cultivated the back smudge cells in host cell, collects supernatant liquor, and this supernatant liquor is DHAV-SD strain virus liquid; Described host cell is BHK21 cell or DF-1 cell.
5. method as claimed in claim 4 is characterized in that: described DHAV 1 type DHAV-SD strain CGMCC No.3746 is cultivated the back smudge cells in host cell, the method for collecting supernatant liquor comprises the steps:
1. DHAV 1 type DHAV-SD strain CGMCC No.3746 is inoculated described host cell, cultivate the back, collect supernatant, be and produce with kind of a poison by the freeze thawing smudge cells;
2. will produce with planting the described host cell of poison inoculation, cultivate the back, collect supernatant, be seedling with planting poison by the freeze thawing smudge cells;
3. seedling is inoculated a described host cell with kind of a poison, cultivate the back, collect supernatant liquor by the freeze thawing smudge cells.
6. method as claimed in claim 5 is characterized in that: described step 1., step 2. with step 3. in: MOI is 0.001 when inoculating described host cell, and incubation time is 48～72 hours; Described step 1., step 2. with step 3. in: incubation time is preferably 48 hours; Described step 3. in, collect supernatant liquor by the centrifugal 30～40min of 5000rpm.
7. as arbitrary described method in the claim 4 to 6, it is characterized in that: described host cell is the BHK21 cell; Described host cell carries out following amplification cultivation earlier before described inoculation:
(1) 37 ℃ of water-baths of frozen BHK21 cell are melted, the centrifugal 5min of 800rpm gets precipitation; With cell culture fluid suspension precipitation, obtain 2 * 10
5The cell suspension of cell/ml, 37 ℃, 5%CO
2Leave standstill and be cultured to every milliliter and contain 1 * 10
6Cell;
(2) peptic cell on the basis of step (1) is resuspended in the cell culture fluid then, makes every milliliter to contain 2 * 10
5Cell is cultured to every milliliter under the following conditions then and contains 1 * 10
6Cell: pH7.4, saturation dissolved oxygen are 50%, 37 ℃, 100 commentaries on classics/min.
8. a method for preparing 1 type duck virus hepatitis vaccine is that the DHAV-SD strain virus liquid that arbitrary described method in the claim 4 to 7 prepares is carried out deactivation and emulsification as seedling successively with viral liquid, obtains 1 type duck virus hepatitis vaccine.
9. method as claimed in claim 8 is characterized in that: the method for described deactivation is as follows: add formaldehyde in described DHAV-SD strain virus liquid, the volumn concentration that makes formaldehyde is 0.1%, 37 ℃ of deactivation 24 hours, obtains viral liquid after the deactivation.
10. method as claimed in claim 8 or 9, it is characterized in that: described emulsive method is as follows: with 3 parts by volume oil phases, 1 parts by volume water and 1% (quality percentage composition) Thiomersalate aqueous solution, the final concentration that makes Thiomersalate is 0.01% (quality percentage composition), obtains duck virus hepatitis vaccine; The preparation method of described oil phase is as follows: 94 parts by volume white oils and 6 parts by volume Si Ben-80 are mixed, obtain mixed solution, add 1.6～1.8 gram aluminum stearates in per 100 milliliters of mixed solutions; The preparation method of described water is as follows: with the viral liquid mixing after 4 parts by volume tween-80s and the 96 parts by volume deactivations.
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2010
- 2010-08-06 CN CN2010102473069A patent/CN101948809B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| 《广西畜牧兽医》 20090228 李婵等 一株I型鸭肝炎病毒的分离与鉴定 18-19 1-10 第25卷, 第1期 2 * |
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