CN102978167A - Method for preparing nephropathogenic avian infectious bronchitis viruses and live vaccines by using cell lines - Google Patents
Method for preparing nephropathogenic avian infectious bronchitis viruses and live vaccines by using cell lines Download PDFInfo
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Abstract
The present invention provides a method for preparing nephropathogenic avian infectious bronchitis viruses W93 and vaccines thereof by using cell lines, wherein the method comprises: adopting cell lines such as a Vero cell line, a BHK-21 cell line, a PK-15 cell line, an Marc145 cell line, a ST cell line and an IBRS-2 cell line; inoculating nephropathogenic avian infectious bronchitis viruses after the cell lines form a well-growing cell monolayer, wherein the nephropathogenic avian infectious bronchitis viruses absorb the cell lines; changing a cell maintaining liquid to culture, adding an incubation agent to incubate the cells for 20-40 min to carry out nephropathogenic avian infectious bronchitis virus proliferation; and harvesting the nephropathogenic avian infectious bronchitis viruses when CPE achieves more than 75%. According to the method for preparing the nephropathogenic avian infectious bronchitis virus vaccines by using the cell lines, a purpose of safe and efficient nephropathogenic avian infectious bronchitis virus and vaccine production can be achieved.
Description
Technical field
The present invention relates to a kind of chicken avian infectious bronchitis virus virus with clone production and chicken avian infectious bronchitis virus living vaccine and preparation method thereof, belong to the live vaccine field.
Background technology
Chicken infectious bronchitis (Avian Infectious Bronchitis, IB) is the viral infectious of a kind of height contact of being caused by chicken avian infectious bronchitis virus virus (Infectious Bronchitis Virus, IBV).By the pathogenic symptom that occurs behind the virus infection, namely pathotype can be divided into breathing pattern, have a liking for kidney type etc.Avian infectious bronchitis virus disorder of genitourinary system also occurs take renal lesions as main, with respiratory symptom, shows as the pale enlargement of kidney, uriniferous tubules and ureterectasia and is full of urate, discharges white loose stool etc.The anti-disease of effecting a permanent cure adopts the weak malicious seedlings of chicken embryo tissue of low virulent strain W93 more at present.Although this seedling security is good at present, immune effect is desirable, but the production of chicken embryo seedling, large except labour intensity, output is on the low side, it is in short supply also to be faced with China's present SPF chicken embryo, and adopts the seedling of general chicken embryo, may be subjected to not only that maternal antibody disturbs in the chicken embryo, make vaccine valence unstable, and the embryo borne causal agent is very easily along with the dispersal by man who causes some epidemic disease is used in development, production and the granting of vaccine.
Summary of the invention
In view of this, main purpose of the present invention is to provide a kind of chicken avian infectious bronchitis virus virus of producing with clone and method and the product of chicken avian infectious bronchitis virus living vaccine, with realize with safer, more effectively produce the purpose of chicken avian infectious bronchitis virus virus and vaccine.
Technical scheme
The invention provides a kind of method with clone production chicken avian infectious bronchitis virus virus, may further comprise the steps:
1) with clone through had digestive transfer culture, cultivate with cell growth medium, form well-grown cell monolayer;
2) with chicken avian infectious bronchitis virus virus inoculation in above-mentioned cell monolayer, carry out the absorption of chicken avian infectious bronchitis virus virus;
3) with the cell of above-mentioned viral adsorption, use cell maintenance medium instead and cultivate, carry out the propagation of chicken avian infectious bronchitis virus virus;
4) reach 75% when above, results chicken avian infectious bronchitis virus virus as CPE.
Preferably, cell of the present invention is Vero clone, BHK-21 cells, PK-15 clone, Marc145 clone, ST clone, any one or a few clone of IBRS-2 clone.
Preferably, cell of the present invention is Vero clone.
Preferably, step 2 of the present invention) behind virus inoculation, also comprises adding the process that agent was hatched cell 20-40 minute of hatching in.
It is preferably, of the present invention that to hatch agent be D-glucosamine, Interferon, rabbit or lipopolysaccharides.
It is more preferably, of the present invention that to hatch agent be D-glucosamine.
Preferably, incubation time of the present invention is 30 minutes.
Preferably, chicken avian infectious bronchitis virus virus of the present invention is chicken avian infectious bronchitis virus virus W93 strain.
Preferably, the chicken avian infectious bronchitis virus virus of inoculation usefulness is the chicken avian infectious bronchitis virus virus that goes down to posterity in the chicken embryo and obtain step 2 of the present invention).
Therefore the present invention can obtain a kind of chicken avian infectious bronchitis virus virus according to method preparation described above, the chicken avian infectious bronchitis virus virus that described virus is produced for using clone.
Another purpose of the present invention is to provide a kind of method of producing chicken avian infectious bronchitis virus living vaccine with clone; comprise the chicken avian infectious bronchitis virus virus of using aforesaid method to obtain; add lyophilized vaccine, be prepared as chicken avian infectious bronchitis virus living vaccine.
Therefore the present invention can obtain a kind of chicken avian infectious bronchitis virus living vaccine, it is characterized in that, uses clone to produce chicken avian infectious bronchitis virus living vaccine.
Can be found out by top technical scheme and the follow-up embodiment of the present invention, the contriver is surprised to find that when chicken avian infectious bronchitis virus viral proliferation cell hatched and can helps chicken avian infectious bronchitis virus virus to adapt to clone, so that become possibility with clone breeding chicken avian infectious bronchitis virus virus, thereby realized producing chicken avian infectious bronchitis virus vaccine with clone.And in the prior art, no matter use which kind of cell or the clones such as Vero cell, BHK-21 cell, PK-15 cell, Marc145 cell, ST cell, IBRS-2 cell both at home and abroad, perhaps use the methods such as synchronized culture, band poison go down to posterity, do not realize that all chicken avian infectious bronchitis virus virus obtains effectively propagation in clone (cell), and reach the degree of utilizing clone to produce chicken avian infectious bronchitis virus virus or vaccine.
Secondly, the contriver also finds can go down to posterity at the chicken embryo first before the infectious bronchitis virus inoculating cell, can help the easier clone that is adapted to of infectious bronchitis virus, promote virus in the propagation of clone and titre and the quality accelerating to produce cytopathic speed and improve the cells produce infectious bronchitis virus.
Embodiment
Having used common business-like easy cultured cells in the embodiment of the invention is such as cell or clone production chicken avian infectious bronchitis virus virus and vaccines such as Vero cell (African green monkey kidney cell), BHK-21 cell (young hamster kidney passage cell), PK-15 cell (porcine kidney cell), Marc145 cell (epithelioid cell derives from monkey-kidney cells), ST cell (pig testis cell), IBRS-2 cells (porcine kidney cell).
The viral preferred chicken avian infectious bronchitis virus virus of using chicken embryo adaptation of virus of the chicken avian infectious bronchitis virus of inoculation usefulness in the embodiment of the invention, or preferably use clone to produce the chicken avian infectious bronchitis virus virus of acquisition; But use chicken avian infectious bronchitis virus virus chicken embryo adaptation of virus or that clone is produced to be used for inoculation, its purpose is only in order to improve the tiring of chicken avian infectious bronchitis virus virus of inoculation usefulness, and so that the chicken avian infectious bronchitis virus virus of inoculation usefulness more adapt in cell and grow, and making chicken avian infectious bronchitis virus virus on cell, cytopathy can occur faster, the titre of the chicken avian infectious bronchitis virus virus of acquisition effectiveness higher, chicken avian infectious bronchitis virus vaccine is better.Therefore, inoculation of the present invention is not limited to use through the chicken avian infectious bronchitis virus of chicken embryo adaptation of virus and the chicken avian infectious bronchitis virus virus of cultivating through clone with chicken avian infectious bronchitis virus virus, and the chicken avian infectious bronchitis virus virus in other sources as tissue-derived chicken avian infectious bronchitis virus virus liquid also can be used for inoculating cell system and produce chicken avian infectious bronchitis virus virus and vaccine.
The vaccine that the embodiment of the invention uses clone to produce the chicken avian infectious bronchitis virus is freeze-dried live vaccine, can also utilize the chicken avian infectious bronchitis virus virus of the method acquisition of the embodiment of the invention, add inactivator, add beastly pharmaceutically acceptable vaccine adjuvant after the deactivation, obtain chicken avian infectious bronchitis virus inactivated vaccine.
Serum is bovine serum in the cell culture fluid of the present invention, preferred foetal calf serum, and other animality serum such as rabbit anteserum, sheep blood serum etc. all can be used as cell culture fluid.
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention that NM concrete experimental technique in the following example carries out according to the normal experiment method usually.
Embodiment 1 Vero cells produce chicken avian infectious bronchitis virus virus and chicken avian infectious bronchitis virus living vaccine
In the present embodiment, select African green monkey kidney cell line Vero cell (available from Shanghai Inst. of Life Science, CAS cell resource center) as producing the clone of chicken avian infectious bronchitis virus virus with the chicken infectious bronchitis living vaccine, chicken avian infectious bronchitis virus virus W93 strain seed culture of viruses is available from China Veterinery Drug Inspection Office.The prescription of used Growth of Cells nutrient solution is: 94%v/v DMEM (production of Invitrogen company) liquid, and 6%v/v foetal calf serum (production of PAA company), the pH value is adjusted into 7.2; The prescription of used cell maintenance medium is: 99%v/v DMEM (production of Invitrogen company) liquid, and 1%v/v foetal calf serum (production of PAA company), the pH value is adjusted into 7.2.Below for using the concrete steps of Vero cells produce chicken avian infectious bronchitis virus virus and vaccine:
1. produce the preparation of planting with chicken avian infectious bronchitis virus virus: at first can be first with chicken avian infectious bronchitis virus virus W93 strain with times dilution in 1: 100 by volume of aseptic pH7.2 phosphate buffered saline buffer after, inoculate 10 age in days SPF chicken embryos through allantoic cavity, every embryo 0.1ml, putting 37 ℃ continues to hatch, discard the front dead germ of 24h, take out all embryos behind the 36h and put into 4 ℃ of refrigerator overnight, collect allantoic fluid, passed continuously for 10 generations with SPF chicken embryo, aseptic collection allantoic fluid and take 3000rpm at a high speed 4 ℃ of centrifugal 50min get supernatant liquor and be required chicken avian infectious bronchitis virus virus (to embryo toxicity power as 10
8.7EID
50/ 0.1ml), for subsequent use.
Vero clone through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, is continued to cultivate in 37 ℃ with cell growth medium, when forming good individual layer, be used for continuing to go down to posterity or virus inoculation; The chicken avian infectious bronchitis virus virus of the above-mentioned steps preparation of 100 times of dilutions of inoculation, 37 ℃ adsorbed 1-2 hour, and then added cell maintenance medium and cultivate in 37 ℃.Add D-glucosamine (concentration is 300mmol/L) after 6 hours and hatch half an hour, rear with pH7.2 phosphate buffered saline buffer flushing three times, continue to add cell maintenance medium and cultivate.Day by day observed and recorded meets behind the poison 48-72 hour CPE greater than 75% time when more round shrinking born of the same parents occurring (or cell monolayer) and receives poison, and is centrifugal in-20 ℃ of preservations, as the production seed culture of viruses after the freeze thawing 3 times.
2. seedling is with the preparation of chicken avian infectious bronchitis virus virus: use the cell growth medium culturing cell to forming good cell monolayer, inoculate above-mentioned production with chicken avian infectious bronchitis virus virus seed culture of viruses and after carrying out viruses adsorption, using cell maintenance medium instead cultivates, adding is hatched agent and is carried out hatching of cell, with the above-mentioned agent flush away of hatching, again add the cell maintenance medium culturing cell, the results virus liquid, it is viral with the chicken avian infectious bronchitis virus namely to obtain seedling, and concrete steps are:
Above-mentioned cell growth medium is cultivated the well-grown Vero cell monolayer that obtains, add the cell maintenance medium that seed culture of viruses is used in the production that contains the acquisition of 3%v/v above-mentioned steps, put 37 ℃ of absorption 1-2 hour; Behind the viral adsorption, add cell maintenance medium in Vero clone, in 37 ℃, cultivate.Discard cell maintenance medium after 6 hours, add D-glucosamine (concentration is 300mmol/L) and hatch half an hour.With pH7.2 phosphate buffered saline buffer flushing three times, with the above-mentioned agent flush away of hatching, again add cell maintenance medium culturing cell system, and observe day by day.Meet behind the poison 48-72 hour CPE greater than 75% time when more round shrinking born of the same parents occurring (or cell monolayer), can gather in the crops the nutrient solution that contains chicken avian infectious bronchitis virus virus.Centrifugally after the freeze thawing 3 times can obtain chicken avian infectious bronchitis virus virus liquid.
Seedling is with the check of chicken avian infectious bronchitis virus virus liquid: test and EID by the Pharmacopoeia of the People's Republic of China (version in 2005) appendix
50Mensuration, the viral level of every 0.1ml virus liquid is 10
8.9EID
50, and to 1% chicken red blood cell without aggegation.Place-20 ℃ to preserve the chicken avian infectious bronchitis virus virus liquid that obtains.
3. the preparation of vaccine: after steriling test and viral level are measured qualified virus liquid and are mixed again with sucrose gelatin protective material by 9: 1 (virus liquids: protective material) join seedling; lyophilized vaccine is with 40 ℃-50 ℃ be advisable (8% gelatin, 40% sucrose protective materials; through 115 ℃ of autoclavings 40 minutes; put 4 ℃ of preservations, be finished in 72 hours).In the process of adding, should shake virus liquid, after fully mixing, be vaccinogen liquid.With the aseptic quantitative separating of vaccinogen liquid, vacuum freezedrying seals and can obtain the chicken infectious bronchitis freeze-dried live vaccine rapidly.Prepare three batches, lot number is 2010501,2010502,2010503, and wherein viral level is 10 in every plumage part
4EID
50
4. the security of chicken infectious bronchitis living vaccine and efficacy test
Safety testing
With each 60 of 1 age in days, 7 ages in days and 14 age in days SPF chick, every batch of vaccine uses each 10, set up simultaneously with criticizing with each 10 of age in days control group chickens, above-mentioned freeze-dried live vaccine 10 plumage parts are diluted to the dosage of 0.1ml, the good vaccine 0.1ml of experimental group collunarium inoculation dilution, the physiological saline of control group collunarium equivalent, under the same conditions feeding and management was observed 28.The result shows that this vaccine safety is reliable, and all chick physiological activities are normal, without reactions such as death and adnormal respiration, nervous symptoms.Observe through cuing open inspection, experimental group and control group chick are all without pathological changes such as kidney enlargement and urate deposition.The evidence vaccine safety, result such as table 1.
Table 1 chicken infectious bronchitis living vaccine safety verification result
2). efficacy test
With each 60 of 1 age in days, 7 ages in days and 14 age in days SPF chick, every batch of vaccine uses each 10, sets up simultaneously with criticizing with each 10 of age in days control group chickens.The vaccine of a using dosage of every collunarium, control group collunarium physiological saline 1-2 drips, and 10-14 is after day, together with 10 of each batch contrast chickens, with the strong malicious collunarium of 104EID50 avian infectious bronchitis virus X strain (available from China Veterinery Drug Inspection Office), every chicken 1-2 drips, and observes 28.According to dead and the avian infectious bronchitis virus clinical symptom is arranged or observe the summation of pathological change, calculate control group sickness rate and immune group protection ratio.Detailed results sees Table 2.
Table 2 chicken infectious bronchitis living vaccine efficacy test result
Embodiment 2 BHK-21 cells produce chicken avian infectious bronchitis virus virus and chicken infectious bronchitis living vaccines
In the present embodiment, selecting baby hamster kidney cell is the clone that chicken avian infectious bronchitis virus virus and chicken infectious bronchitis living vaccine are produced in BHK-21 cell (available from China Veterinery Drug Inspection Office) conduct, and chicken avian infectious bronchitis virus virus W93 strain seed culture of viruses is available from China Veterinery Drug Inspection Office.The prescription of used Growth of Cells nutrient solution is: 95%v/v DMEM (production of Invitrogen company) liquid, and 5%v/v foetal calf serum (production of PAA company), the pH value is adjusted into 7.2; The prescription of used cell maintenance medium is: 99%v/v DMEM (production of Invitrogen company) liquid, and 1%v/v foetal calf serum (production of PAA company), the pH value is adjusted into 7.2.
Below for using the concrete steps of BHK-21 cells produce chicken avian infectious bronchitis virus virus and vaccine:
1. produce the preparation of planting with chicken avian infectious bronchitis virus virus: at first can be first with chicken avian infectious bronchitis virus virus W93 strain with times dilution in 1: 100 by volume of aseptic pH7.2 phosphate buffered saline buffer after, inoculate 10 age in days SPF chicken embryos through allantoic cavity, every embryo 0.1ml, putting 37 ℃ continues to hatch, discard the front dead germ of 24h, take out all embryos behind the 36h and put into 4 ℃ of refrigerator overnight, collect allantoic fluid, passed continuously for 10 generations with SPF chicken embryo, aseptic collection allantoic fluid and take 3000rpm at a high speed 4 ℃ of centrifugal 50min get supernatant liquor and be required chicken avian infectious bronchitis virus virus (to embryo toxicity power as 10
8.7EID
50/ 0.1ml), for subsequent use.
BHK-21 cells through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, is continued to cultivate in 37 ℃ with cell growth medium, when forming good individual layer, be used for continuing to go down to posterity or virus inoculation; The chicken avian infectious bronchitis virus virus of the above-mentioned steps preparation of 100 times of dilutions of inoculation, 37 ℃ adsorbed 1-2 hour, and then added cell maintenance medium and cultivate in 37 ℃.Add D-glucosamine (concentration is 300mmol/L) after 6 hours and hatch half an hour, rear with pH7.2 phosphate buffered saline buffer flushing three times, continue to add cell maintenance medium and cultivate.Day by day observed and recorded meets behind the poison 48-72 hour CPE greater than 75% time when more round shrinking born of the same parents occurring (or cell monolayer) and receives poison, and is centrifugal in-20 ℃ of preservations, as the production seed culture of viruses after the freeze thawing 3 times.
2. seedling is with the preparation of chicken avian infectious bronchitis virus virus: use the cell growth medium culturing cell to forming good cell monolayer, inoculate above-mentioned production with chicken avian infectious bronchitis virus virus seed culture of viruses and after carrying out viruses adsorption, using cell maintenance medium instead cultivates, adding is hatched agent and is carried out hatching of cell, with the above-mentioned agent flush away of hatching, again add the cell maintenance medium culturing cell, the results virus liquid, it is viral with the chicken avian infectious bronchitis virus namely to obtain seedling, and concrete steps are:
Above-mentioned cell growth medium is cultivated the well-grown BHK-21 cell monolayer that obtains, add the cell maintenance medium that seed culture of viruses is used in the production that contains the acquisition of 3%v/v above-mentioned steps, put 37 ℃ of absorption 1-2 hour; Behind the viral adsorption, add cell maintenance medium at BHK-21 cells, in 37 ℃, cultivate.Discard cell maintenance medium after 6 hours, add D-glucosamine (concentration is 300mmol/L) and hatch half an hour.With pH7.2 phosphate buffered saline buffer flushing three times, with the above-mentioned agent flush away of hatching, again add cell maintenance medium culturing cell system, and observe day by day.Meet behind the poison 48-72 hour CPE greater than 75% time when more round shrinking born of the same parents occurring (or cell monolayer), can gather in the crops the nutrient solution that contains chicken avian infectious bronchitis virus virus.Centrifugally after the freeze thawing 3 times can obtain chicken avian infectious bronchitis virus virus liquid.
Seedling is with the check of chicken avian infectious bronchitis virus virus liquid: test and EID by the Pharmacopoeia of the People's Republic of China (version in 2005) appendix
50Mensuration, the viral level of every 0.1ml virus liquid is 10
8.9EID
50, and to 1% chicken red blood cell without aggegation.Place-20 ℃ to preserve the chicken avian infectious bronchitis virus virus liquid that obtains.
3. the preparation of vaccine: after steriling test and viral level are measured qualified virus liquid and are mixed again with sucrose gelatin protective material by 9: 1 (virus liquids: protective material) join seedling; lyophilized vaccine is with 40 ℃-50 ℃ be advisable (8% gelatin, 40% sucrose protective materials; through 115 ℃ of autoclavings 40 minutes; put 4 ℃ of preservations, be finished in 72 hours).In the process of adding, should shake virus liquid, after fully mixing, be vaccinogen liquid.With the aseptic quantitative separating of vaccinogen liquid, vacuum freezedrying seals and can obtain the chicken infectious bronchitis freeze-dried live vaccine rapidly.Prepare three batches, lot number is 2010511,2010512,2010513, and wherein viral level is 10 in every plumage part
4EID
50
4. the security of chicken infectious bronchitis living vaccine and efficacy test
Safety testing
With each 60 of 1 age in days, 7 ages in days and 14 age in days SPF chick, every batch of vaccine uses each 10, set up simultaneously with criticizing with each 10 of age in days control group chickens, above-mentioned 10 plumage part freeze-dried live vaccines are diluted to the dosage of 0.1ml, the good vaccine 0.1ml of experimental group collunarium inoculation dilution, the physiological saline of control group collunarium equivalent, under the same conditions feeding and management was observed 28.The result shows that this vaccine safety is reliable, and all chick physiological activities are normal, without reactions such as death and adnormal respiration, nervous symptoms.Observe through cuing open inspection, experimental group and control group chick are all without pathological changes such as kidney enlargement and urate deposition.The evidence vaccine safety, result such as table 3.
Table 3 chicken infectious bronchitis living vaccine safety verification result
Annotate: "-" expression is reactionless.
Efficacy test
With each 60 of 1 age in days, 7 ages in days and 14 age in days SPF chick, every batch of vaccine uses each 10, sets up simultaneously with criticizing with each 10 of age in days control group chickens.The vaccine of a using dosage of every collunarium, control group collunarium physiological saline 1-2 drips, and 10-14 is after day, together with 10 of each batch contrast chickens, with 10
4EID
50The strong malicious collunarium of avian infectious bronchitis virus X strain (available from China Veterinery Drug Inspection Office), every chicken 1-2 drips, and observes 28.According to dead and the avian infectious bronchitis virus clinical symptom is arranged or observe the summation of pathological change, calculate control group sickness rate and immune group protection ratio.Detailed results sees Table 4.
Table 4 chicken infectious bronchitis living vaccine efficacy test result
Embodiment 3 PK-15 cells produce chicken avian infectious bronchitis virus virus and chicken infectious bronchitis living vaccines
In the present embodiment, select porcine kidney cell line PK-15 cell (available from China Veterinery Drug Inspection Office) as producing the clone of chicken avian infectious bronchitis virus virus with the chicken infectious bronchitis living vaccine, chicken avian infectious bronchitis virus virus W93 strain seed culture of viruses is available from China Veterinery Drug Inspection Office.The prescription of used Growth of Cells nutrient solution is: 94%v/v DMEM (production of Invitrogen company) liquid, and 6%v/v foetal calf serum (production of PAA company), the pH value is adjusted into 7.2; The prescription of used cell maintenance medium is: 99%v/v DMEM (production of Invitrogen company) liquid, and 1%v/v foetal calf serum (production of PAA company), the pH value is adjusted into 7.2.
Below for using the concrete steps of PK-15 cells produce chicken avian infectious bronchitis virus virus and vaccine:
1. produce the preparation of planting with chicken avian infectious bronchitis virus virus: at first can be first with chicken avian infectious bronchitis virus virus W93 strain with times dilution in 1: 100 by volume of aseptic pH7.2 phosphate buffered saline buffer after, inoculate 10 age in days SPF chicken embryos through allantoic cavity, every embryo 0.1ml, putting 37 ℃ continues to hatch, discard the front dead germ of 24h, take out all embryos behind the 36h and put into 4 ℃ of refrigerator overnight, collect allantoic fluid, passed continuously for 10 generations with SPF chicken embryo, aseptic collection allantoic fluid and take 3000rpm at a high speed 4 ℃ of centrifugal 50min get supernatant liquor and be required chicken avian infectious bronchitis virus virus (to embryo toxicity power as 10
8.7EID
50/ 0.1ml), for subsequent use.
PK-15 clone through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, is continued to cultivate in 37 ℃ with cell growth medium, when forming good individual layer, be used for continuing to go down to posterity or virus inoculation; The chicken avian infectious bronchitis virus virus of the above-mentioned steps preparation of 100 times of dilutions of inoculation, 37 ℃ adsorbed 1-2 hour, and then added cell maintenance medium and cultivate in 37 ℃.Add D-glucosamine (concentration is 300mmol/L) after 6 hours and hatch half an hour, rear with pH7.2 phosphate buffered saline buffer flushing three times, continue to add cell maintenance medium and cultivate.Day by day observed and recorded meets behind the poison 48-72 hour CPE greater than 75% time when more round shrinking born of the same parents occurring (or cell monolayer) and receives poison, and is centrifugal in-20 ℃ of preservations, as the production seed culture of viruses after the freeze thawing 3 times.
2. seedling is with the preparation of chicken avian infectious bronchitis virus virus: use the cell growth medium culturing cell to forming good cell monolayer, inoculate above-mentioned production with chicken avian infectious bronchitis virus virus seed culture of viruses and after carrying out viruses adsorption, using cell maintenance medium instead cultivates, adding is hatched agent and is carried out hatching of cell, with the above-mentioned agent flush away of hatching, again add the cell maintenance medium culturing cell, the results virus liquid, it is viral with the chicken avian infectious bronchitis virus namely to obtain seedling, and concrete steps are:
Above-mentioned cell growth medium is cultivated the well-grown PK-15 cell monolayer that obtains, add the cell maintenance medium that seed culture of viruses is used in the production that contains the acquisition of 3%v/v above-mentioned steps, put 37 ℃ of absorption 1-2 hour; Behind the viral adsorption, add cell maintenance medium in PK-15 clone, in 37 ℃, cultivate.Discard cell maintenance medium after 6 hours, add D-glucosamine (concentration is 300mmol/L) and hatch half an hour.With pH7.2 phosphate buffered saline buffer flushing three times, with the above-mentioned agent flush away of hatching, again add cell maintenance medium culturing cell system, and observe day by day.Meet behind the poison 48-72 hour CPE greater than 75% time when more round shrinking born of the same parents occurring (or cell monolayer), can gather in the crops the nutrient solution that contains chicken avian infectious bronchitis virus virus.Centrifugally after the freeze thawing 3 times can obtain chicken avian infectious bronchitis virus virus liquid.
Seedling is with the check of chicken avian infectious bronchitis virus virus liquid: test and EID by the Pharmacopoeia of the People's Republic of China (version in 2005) appendix
50Mensuration, the viral level of every 0.1ml virus liquid is 10
8.9EID
50, and to 1% chicken red blood cell without aggegation.Place-20 ℃ to preserve the chicken avian infectious bronchitis virus virus liquid that obtains.
3. the preparation of vaccine: after steriling test and viral level are measured qualified virus liquid and are mixed again with sucrose gelatin protective material by 9: 1 (virus liquids: protective material) join seedling; lyophilized vaccine is with 40 ℃-50 ℃ be advisable (8% gelatin, 40% sucrose protective materials; through 115 ℃ of autoclavings 40 minutes; put 4 ℃ of preservations, be finished in 72 hours).In the process of adding, should shake virus liquid, after fully mixing, be vaccinogen liquid.With the aseptic quantitative separating of vaccinogen liquid, vacuum freezedrying seals and can obtain the chicken infectious bronchitis freeze-dried live vaccine rapidly.Prepare three batches, lot number is 2010521,2010522,2010523, and wherein viral level is 10 in every plumage part
4EID
50
4. the security of chicken infectious bronchitis living vaccine and efficacy test
Safety testing
With each 60 of 1 age in days, 7 ages in days and 14 age in days SPF chick, every batch of vaccine uses each 10, set up simultaneously with criticizing with each 10 of age in days control group chickens, above-mentioned 10 plumage part freeze-dried live vaccines are diluted to the dosage of 0.1ml, the good vaccine 0.1ml of experimental group collunarium inoculation dilution, the physiological saline of control group collunarium equivalent, under the same conditions feeding and management was observed 28.The result shows that this vaccine safety is reliable, and all chick physiological activities are normal, without reactions such as death and adnormal respiration, nervous symptoms.Observe through cuing open inspection, experimental group and control group chick are all without pathological changes such as kidney enlargement and urate deposition.The evidence vaccine safety, result such as table 5.
Table 5 chicken infectious bronchitis living vaccine safety verification result
Annotate: "-" expression is reactionless.
Efficacy test
With each 60 of 1 age in days, 7 ages in days and 14 age in days SPF chick, every batch of vaccine uses each 10, sets up simultaneously with criticizing with each 10 of age in days control group chickens.The vaccine of a using dosage of every collunarium, control group collunarium physiological saline 1-2 drips, and 10-14 is after day, together with 10 of each batch contrast chickens, with 10
4EID
50The strong malicious collunarium of avian infectious bronchitis virus X strain (available from China Veterinery Drug Inspection Office), every chicken 1-2 drips, and observes 28.According to dead and the avian infectious bronchitis virus clinical symptom is arranged or observe the summation of pathological change, calculate control group sickness rate and immune group protection ratio.Detailed results sees Table 6.
Table 6 chicken infectious bronchitis living vaccine efficacy test result
Embodiment 4 Marc 145 cells produce chicken avian infectious bronchitis virus virus and chicken infectious bronchitis living vaccines
In the present embodiment, select Marc 145 cell (epithelioid cells, derive from monkey-kidney cells, available from China Veterinery Drug Inspection Office) as producing the clone of chicken avian infectious bronchitis virus virus with the chicken infectious bronchitis living vaccine, chicken avian infectious bronchitis virus virus W93 strain seed culture of viruses is available from China Veterinery Drug Inspection Office.The prescription of used Growth of Cells nutrient solution is: 94%v/v DMEM (production of Invitrogen company) liquid, and 6%v/v foetal calf serum (production of PAA company), the pH value is adjusted into 7.2; The prescription of used cell maintenance medium is: 98%v/v DMEM (production of Invitrogen company) liquid, and 2%v/v foetal calf serum (production of PAA company), the pH value is adjusted into 7.2.
Below for using the concrete steps of Marc 145 cells produce chicken avian infectious bronchitis virus virus and vaccine:
1. produce the preparation of planting with chicken avian infectious bronchitis virus virus: at first can be first with chicken avian infectious bronchitis virus virus W93 strain with times dilution in 1: 100 by volume of aseptic pH7.2 phosphate buffered saline buffer after, inoculate 10 age in days SPF chicken embryos through allantoic cavity, every embryo 0.1ml, putting 37 ℃ continues to hatch, discard the front dead germ of 24h, take out all embryos behind the 36h and put into 4 ℃ of refrigerator overnight, collect allantoic fluid, passed continuously for 10 generations with SPF chicken embryo, aseptic collection allantoic fluid and take 3000rpm at a high speed 4 ℃ of centrifugal 50min get supernatant liquor and be required chicken avian infectious bronchitis virus virus (to embryo toxicity power as 10
8.7EID
50/ 0.1ml), for subsequent use.
Marc 145 clones through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, are continued to cultivate in 37 ℃ with cell growth medium, when forming good individual layer, be used for continuing to go down to posterity or virus inoculation; The chicken avian infectious bronchitis virus virus of the above-mentioned steps preparation of 100 times of dilutions of inoculation, 37 ℃ adsorbed 1-2 hour, and then added cell maintenance medium and cultivate in 37 ℃.Add D-glucosamine (concentration is 300mmol/L) after 6 hours and hatch half an hour, rear with pH7.2 phosphate buffered saline buffer flushing three times, continue to add cell maintenance medium and cultivate.Day by day observed and recorded meets behind the poison 48-72 hour CPE greater than 75% time when more round shrinking born of the same parents occurring (or cell monolayer) and receives poison, and is centrifugal in-20 ℃ of preservations, as the production seed culture of viruses after the freeze thawing 3 times.
2. seedling is with the preparation of chicken avian infectious bronchitis virus virus: use the cell growth medium culturing cell to forming good cell monolayer, inoculate above-mentioned production with chicken avian infectious bronchitis virus virus seed culture of viruses and after carrying out viruses adsorption, using cell maintenance medium instead cultivates, adding is hatched agent and is carried out hatching of cell, with the above-mentioned agent flush away of hatching, again add the cell maintenance medium culturing cell, the results virus liquid, it is viral with the chicken avian infectious bronchitis virus namely to obtain seedling, and concrete steps are:
Above-mentioned cell growth medium is cultivated well-grown Marc 145 cell monolayers that obtain, add the cell maintenance medium that seed culture of viruses is used in the production that contains the acquisition of 3%v/v above-mentioned steps, put 37 ℃ of absorption 1-2 hour; Behind the viral adsorption, add cell maintenance medium in Marc 145 clones, in 37 ℃, cultivate.Discard cell maintenance medium after 6 hours, add D-glucosamine (concentration is 300mmol/L) and hatch half an hour.With pH7.2 phosphate buffered saline buffer flushing three times, with the above-mentioned agent flush away of hatching, again add cell maintenance medium culturing cell system, and observe day by day.Meet behind the poison 48-72 hour CPE greater than 75% time when more round shrinking born of the same parents occurring (or cell monolayer), can gather in the crops the nutrient solution that contains chicken avian infectious bronchitis virus virus.Centrifugally after the freeze thawing 3 times can obtain chicken avian infectious bronchitis virus virus liquid.
Seedling is with the check of chicken avian infectious bronchitis virus virus liquid: test and EID by the Pharmacopoeia of the People's Republic of China (version in 2005) appendix
50Mensuration, the viral level of every 0.1ml virus liquid is 10
8.9EID
50, and to 1% chicken red blood cell without aggegation.Place-20 ℃ to preserve the chicken avian infectious bronchitis virus virus liquid that obtains.
3. the preparation of vaccine: after steriling test and viral level are measured qualified virus liquid and are mixed again with sucrose gelatin protective material by 9: 1 (virus liquids: protective material) join seedling; lyophilized vaccine is with 40 ℃-50 ℃ be advisable (8% gelatin, 40% sucrose protective materials; through 115 ℃ of autoclavings 40 minutes; put 4 ℃ of preservations, be finished in 72 hours).In the process of adding, should shake virus liquid, after fully mixing, be vaccinogen liquid.With the aseptic quantitative separating of vaccinogen liquid, vacuum freezedrying seals and can obtain the chicken infectious bronchitis freeze-dried live vaccine rapidly.Prepare three batches, lot number is 2010531,2010532,2010533, and wherein viral level is 10 in every plumage part
4EID
50
4. the security of chicken infectious bronchitis living vaccine and efficacy test
Safety testing
With each 60 of 1 age in days, 7 ages in days and 14 age in days SPF chick, every batch of vaccine uses each 10, set up simultaneously with criticizing with each 10 of age in days control group chickens, above-mentioned 10 plumage part freeze-dried live vaccines are diluted to the dosage of 0.1ml, the good vaccine 0.1ml of experimental group collunarium inoculation dilution, the physiological saline of control group collunarium equivalent, under the same conditions feeding and management was observed 28.The result shows that this vaccine safety is reliable, and all chick physiological activities are normal, without reactions such as death and adnormal respiration, nervous symptoms.Observe through cuing open inspection, experimental group and control group chick are all without pathological changes such as kidney enlargement and urate deposition.The evidence vaccine safety, result such as table 7.
Table 7 chicken infectious bronchitis living vaccine safety verification result
Annotate: "-" expression is reactionless.
Efficacy test
With each 60 of 1 age in days, 7 ages in days and 14 age in days SPF chick, every batch of vaccine uses each 10, sets up simultaneously with criticizing with each 10 of age in days control group chickens.The vaccine of a using dosage of every collunarium, control group collunarium physiological saline 1-2 drips, and 10-14 is after day, together with 10 of each batch contrast chickens, with 10
4EID
50The strong malicious collunarium of avian infectious bronchitis virus X strain (available from China Veterinery Drug Inspection Office), every chicken 1-2 drips, and observes 28.According to dead and the avian infectious bronchitis virus clinical symptom is arranged or observe the summation of pathological change, calculate control group sickness rate and immune group protection ratio.Detailed results sees Table 8.
Table 8 chicken infectious bronchitis living vaccine efficacy test result
Embodiment 5 ST cells produce chicken avian infectious bronchitis virus virus and chicken infectious bronchitis living vaccines
In the present embodiment, select pig testis clone ST cell (available from Chinese Typical Representative culture collection center) as producing the clone of chicken avian infectious bronchitis virus virus with the chicken infectious bronchitis living vaccine, chicken avian infectious bronchitis virus virus W93 strain seed culture of viruses is available from China Veterinery Drug Inspection Office.The prescription of used Growth of Cells nutrient solution is: 95%v/v α-MEM (production of Invitrogen company) liquid, and 5%v/v foetal calf serum (production of PAA company), the pH value is adjusted into 7.2; The prescription of used cell maintenance medium is: 99%v/v α-MEM (production of Invitrogen company) liquid, and 1%v/v foetal calf serum (production of PAA company), the pH value is adjusted into 7.2.
Below for using the concrete steps of ST cells produce chicken avian infectious bronchitis virus virus and living vaccine:
1. produce the preparation of planting with chicken avian infectious bronchitis virus virus: at first can be first with chicken avian infectious bronchitis virus virus W93 strain with times dilution in 1: 100 by volume of aseptic pH7.2 phosphate buffered saline buffer after, inoculate 10 age in days SPF chicken embryos through allantoic cavity, every embryo 0.1ml, putting 37 ℃ continues to hatch, discard the front dead germ of 24h, take out all embryos behind the 36h and put into 4 ℃ of refrigerator overnight, collect allantoic fluid, passed continuously for 10 generations with SPF chicken embryo, aseptic collection allantoic fluid and take 3000rpm at a high speed 4 ℃ of centrifugal 50min get supernatant liquor and be required chicken avian infectious bronchitis virus virus (to embryo toxicity power as 10
8.7EID
50/ 0.1ml), for subsequent use.
ST clone through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, is continued to cultivate in 37 ℃ with cell growth medium, when forming good individual layer, be used for continuing to go down to posterity or virus inoculation; The chicken avian infectious bronchitis virus virus of the above-mentioned steps preparation of 100 times of dilutions of inoculation, 37 ℃ adsorbed 1-2 hour, and then added cell maintenance medium and cultivate in 37 ℃.Add D-glucosamine (concentration is 300mmol/L) after 6 hours and hatch half an hour, rear with pH7.2 phosphate buffered saline buffer flushing three times, continue to add cell maintenance medium and cultivate.Day by day observed and recorded meets behind the poison 48-72 hour CPE greater than 75% time when more round shrinking born of the same parents occurring (or cell monolayer) and receives poison, and is centrifugal in-20 ℃ of preservations, as the production seed culture of viruses after the freeze thawing 3 times.
2. seedling is with the preparation of chicken avian infectious bronchitis virus virus: use the cell growth medium culturing cell to forming good cell monolayer, inoculate above-mentioned production with chicken avian infectious bronchitis virus virus seed culture of viruses and after carrying out viruses adsorption, using cell maintenance medium instead cultivates, adding is hatched agent and is carried out hatching of cell, with the above-mentioned agent flush away of hatching, again add the cell maintenance medium culturing cell, the results virus liquid, it is viral with the chicken avian infectious bronchitis virus namely to obtain seedling, and concrete steps are:
Above-mentioned cell growth medium is cultivated the well-grown ST cell monolayer that obtains, add the cell maintenance medium that seed culture of viruses is used in the production that contains the acquisition of 3%v/v above-mentioned steps, put 37 ℃ of absorption 1-2 hour; Behind the viral adsorption, add cell maintenance medium in ST clone, in 37 ℃, cultivate.Discard cell maintenance medium after 6 hours, add D-glucosamine (concentration is 300mmol/L) and hatch half an hour.With pH7.2 phosphate buffered saline buffer flushing three times, with the above-mentioned agent flush away of hatching, again add cell maintenance medium culturing cell system, and observe day by day.Meet behind the poison 48-72 hour CPE greater than 75% time when more round shrinking born of the same parents occurring (or cell monolayer), can gather in the crops the nutrient solution that contains chicken avian infectious bronchitis virus virus.Centrifugally after the freeze thawing 3 times can obtain chicken avian infectious bronchitis virus virus liquid.
Seedling is with the check of chicken avian infectious bronchitis virus virus liquid: test and EID by the Pharmacopoeia of the People's Republic of China (version in 2005) appendix
50Mensuration, the viral level of every 0.1ml virus liquid is 10
8.9EID
50, and to 1% chicken red blood cell without aggegation.Place-20 ℃ to preserve the chicken avian infectious bronchitis virus virus liquid that obtains.
3. the preparation of vaccine: after steriling test and viral level are measured qualified virus liquid and are mixed again with sucrose gelatin protective material by 9: 1 (virus liquids: protective material) join seedling; lyophilized vaccine is with 40 ℃-50 ℃ be advisable (8% gelatin, 40% sucrose protective materials; through 115 ℃ of autoclavings 40 minutes; put 4 ℃ of preservations, be finished in 72 hours).In the process of adding, should shake virus liquid, after fully mixing, be vaccinogen liquid.With the aseptic quantitative separating of vaccinogen liquid, vacuum freezedrying seals and can obtain the chicken infectious bronchitis freeze-dried live vaccine rapidly.Prepare three batches, lot number is 2010541,2010542,2010543, and wherein viral level is 10 in every plumage part
4EID
50
4. the security of chicken infectious bronchitis living vaccine and efficacy test
Safety testing
With each 60 of 1 age in days, 7 ages in days and 14 age in days SPF chick, every batch of vaccine uses each 10, set up simultaneously with criticizing with each 10 of age in days control group chickens, above-mentioned 10 plumage part freeze-dried live vaccines are diluted to the dosage of 0.1ml, the good vaccine 0.1ml of experimental group collunarium inoculation dilution, the physiological saline of control group collunarium equivalent, under the same conditions feeding and management was observed 28.The result shows that this vaccine safety is reliable, and all chick physiological activities are normal, without reactions such as death and adnormal respiration, nervous symptoms.Observe through cuing open inspection, experimental group and control group chick are all without pathological changes such as kidney enlargement and urate deposition.The evidence vaccine safety, result such as table 9.
Table 9 chicken infectious bronchitis living vaccine safety verification result
Annotate: "-" expression is reactionless.
Efficacy test
With each 60 of 1 age in days, 7 ages in days and 14 age in days SPF chick, every batch of vaccine uses each 10, sets up simultaneously with criticizing with each 10 of age in days control group chickens.The vaccine of a using dosage of every collunarium, control group collunarium physiological saline 1-2 drips, and 10-14 is after day, together with 10 of each batch contrast chickens, with 10
4EID
50The strong malicious collunarium of avian infectious bronchitis virus X strain (available from China Veterinery Drug Inspection Office), every chicken 1-2 drips, and observes 28.According to dead and the avian infectious bronchitis virus clinical symptom is arranged or observe the summation of pathological change, calculate control group sickness rate and immune group protection ratio.Detailed results sees Table 10.
Table 10 chicken infectious bronchitis living vaccine efficacy test result
Embodiment 6 IBRS-2 cells produce chicken avian infectious bronchitis virus virus and chicken infectious bronchitis living vaccines
In the present embodiment, select porcine kidney cell line IBRS-2 cell (available from China Veterinery Drug Inspection Office) as producing the clone of chicken avian infectious bronchitis virus virus with the chicken infectious bronchitis living vaccine, chicken avian infectious bronchitis virus virus W93 strain seed culture of viruses is available from China Veterinery Drug Inspection Office.The prescription of used Growth of Cells nutrient solution is: 95%v/v MEM41500 (production of Invitrogen company) liquid, and 5%v/v foetal calf serum (production of PAA company), the pH value is adjusted into 7.2; The prescription of used cell maintenance medium is: 99%v/v MEM41500 (production of Invitrogen company) liquid, and 1%v/v foetal calf serum (production of PAA company), the pH value is adjusted into 7.2.
Below for using the concrete steps of IBRS-2 cells produce chicken avian infectious bronchitis virus virus and vaccine:
1. produce the preparation of planting with chicken avian infectious bronchitis virus virus: at first can be first with chicken avian infectious bronchitis virus virus W93 strain with times dilution in 1: 100 by volume of aseptic pH7.2 phosphate buffered saline buffer after, inoculate 10 age in days SPF chicken embryos through allantoic cavity, every embryo 0.1ml, putting 37 ℃ continues to hatch, discard the front dead germ of 24h, take out all embryos behind the 36h and put into 4 ℃ of refrigerator overnight, collect allantoic fluid, passed continuously for 10 generations with SPF chicken embryo, aseptic collection allantoic fluid and take 3000rpm at a high speed 4 ℃ of centrifugal 50min get supernatant liquor and be required chicken avian infectious bronchitis virus virus (to embryo toxicity power as 10
8.7EID
50/ 0.1ml), for subsequent use.
IBRS-2 clone through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, is continued to cultivate in 37 ℃ with cell growth medium, when forming good individual layer, be used for continuing to go down to posterity or virus inoculation; The chicken avian infectious bronchitis virus virus of the above-mentioned steps preparation of 100 times of dilutions of inoculation, 37 ℃ adsorbed 1-2 hour, and then added cell maintenance medium and cultivate in 37 ℃.Add D-glucosamine (concentration is 300mmol/L) after 6 hours and hatch half an hour, rear with pH7.2 phosphate buffered saline buffer flushing three times, continue to add cell maintenance medium and cultivate.Day by day observed and recorded meets behind the poison 48-72 hour CPE greater than 75% time when more round shrinking born of the same parents occurring (or cell monolayer) and receives poison, and is centrifugal in-20 ℃ of preservations, as the production seed culture of viruses after the freeze thawing 3 times.
2. seedling is with the preparation of chicken avian infectious bronchitis virus virus: use the cell growth medium culturing cell to forming good cell monolayer, inoculate above-mentioned production with chicken avian infectious bronchitis virus virus seed culture of viruses and after carrying out viruses adsorption, using cell maintenance medium instead cultivates, adding is hatched agent and is carried out hatching of cell, with the above-mentioned agent flush away of hatching, again add the cell maintenance medium culturing cell, the results virus liquid, it is viral with the chicken avian infectious bronchitis virus namely to obtain seedling, and concrete steps are:
Above-mentioned cell growth medium is cultivated the well-grown IBRS-2 cell monolayer that obtains, add the cell maintenance medium that seed culture of viruses is used in the production that contains the acquisition of 3%v/v above-mentioned steps, put 37 ℃ of absorption 1-2 hour; Behind the viral adsorption, add cell maintenance medium in IBRS-2 clone, in 37 ℃, cultivate.Discard cell maintenance medium after 6 hours, add D-glucosamine (concentration is 300mmol/L) and hatch half an hour.With pH7.2 phosphate buffered saline buffer flushing three times, with the above-mentioned agent flush away of hatching, again add cell maintenance medium culturing cell system, and observe day by day.Meet behind the poison 48-72 hour CPE greater than 75% time when more round shrinking born of the same parents occurring (or cell monolayer), can gather in the crops the nutrient solution that contains chicken avian infectious bronchitis virus virus.Centrifugally after the freeze thawing 3 times can obtain chicken avian infectious bronchitis virus virus liquid.
Seedling is with the check of chicken avian infectious bronchitis virus virus liquid: test and EID by the Pharmacopoeia of the People's Republic of China (version in 2005) appendix
50Mensuration, the viral level of every 0.1ml virus liquid is 10
8.9EID
50, and to 1% chicken red blood cell without aggegation.Place-20 ℃ to preserve the chicken avian infectious bronchitis virus virus liquid that obtains.
3. the preparation of vaccine: after steriling test and viral level are measured qualified virus liquid and are mixed again with sucrose gelatin protective material by 9: 1 (virus liquids: protective material) join seedling; lyophilized vaccine is with 40 ℃-50 ℃ be advisable (8% gelatin, 40% sucrose protective materials; through 115 ℃ of autoclavings 40 minutes; put 4 ℃ of preservations, be finished in 72 hours).In the process of adding, should shake virus liquid, after fully mixing, be vaccinogen liquid.With the aseptic quantitative separating of vaccinogen liquid, vacuum freezedrying seals and can obtain the chicken infectious bronchitis freeze-dried live vaccine rapidly.Prepare three batches, lot number is 2010551,2010552,2010553, and wherein viral level is 10 in every plumage part
4EID
50
4. the security of chicken infectious bronchitis living vaccine and efficacy test
Safety testing
With each 60 of 1 age in days, 7 ages in days and 14 age in days SPF chick, every batch of vaccine uses each 10, set up simultaneously with criticizing with each 10 of age in days control group chickens, above-mentioned 10 plumage part freeze-dried live vaccines are diluted to the dosage of 0.1ml, the good vaccine 0.1ml of experimental group collunarium inoculation dilution, the physiological saline of control group collunarium equivalent, under the same conditions feeding and management was observed 28.The result shows that this vaccine safety is reliable, and all chick physiological activities are normal, without reactions such as death and adnormal respiration, nervous symptoms.Observe through cuing open inspection, experimental group and control group chick are all without pathological changes such as kidney enlargement and urate deposition.The evidence vaccine safety, result such as table 11.
Table 11 chicken infectious bronchitis living vaccine safety verification result
Annotate: "-" expression is reactionless.
Efficacy test
With each 60 of 1 age in days, 7 ages in days and 14 age in days SPF chick, every batch of vaccine uses each 10, sets up simultaneously with criticizing with each 10 of age in days control group chickens.The vaccine of a using dosage of every collunarium, control group collunarium physiological saline 1-2 drips, and 10-14 is after day, together with 10 of each batch contrast chickens, with 10
4EID
50The strong malicious collunarium of avian infectious bronchitis virus X strain (available from China Veterinery Drug Inspection Office), every chicken 1-2 drips, and observes 28.According to dead and the avian infectious bronchitis virus clinical symptom is arranged or observe the summation of pathological change, calculate control group sickness rate and immune group protection ratio.Detailed results sees Table 12.
Table 12 chicken infectious bronchitis living vaccine efficacy test result
The above only is preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. the method with clone production chicken avian infectious bronchitis virus virus is characterized in that, may further comprise the steps:
1) with clone through had digestive transfer culture, cultivate with cell growth medium, form well-grown cell monolayer;
2) with chicken avian infectious bronchitis virus virus inoculation in above-mentioned cell monolayer, carry out the absorption of chicken avian infectious bronchitis virus virus;
3) with the cell of above-mentioned viral adsorption, use cell maintenance medium instead and cultivate, carry out the propagation of chicken avian infectious bronchitis virus virus;
4) reach 75% when above, results chicken avian infectious bronchitis virus virus as CPE.
2. method according to claim 1 is characterized in that, described cell is Vero clone, BHK-21 cells, PK-15 clone, Marc145 clone, ST clone, any one or a few clone of IBRS-2 clone.
3. method according to claim 1 is characterized in that step 2) in behind virus inoculation, also comprise adding the process that agent was hatched cell 20-40 minute of hatching.
4. method according to claim 3 is characterized in that, described to hatch agent be D-glucosamine, Interferon, rabbit or lipopolysaccharides.
5. method according to claim 3 is characterized in that, described incubation time is 30 minutes.
6. method according to claim 3 is characterized in that, described chicken avian infectious bronchitis virus virus is chicken avian infectious bronchitis virus virus W93 strain.
7. described method is characterized in that step 2 according to claim 1～6) in the chicken avian infectious bronchitis virus virus of inoculation usefulness be the chicken avian infectious bronchitis virus virus that in the chicken embryo, goes down to posterity and obtain.
8. the chicken avian infectious bronchitis virus virus of the described method of any one preparation according to claim 1～7 is characterized in that, uses clone to produce chicken avian infectious bronchitis virus virus.
9. method of producing chicken avian infectious bronchitis virus living vaccine with clone; it is characterized in that; right to use requires 8 described chicken avian infectious bronchitis virus viruses, adds lyophilized vaccine, is prepared as chicken avian infectious bronchitis virus living vaccine.
10. the chicken avian infectious bronchitis virus living vaccine of a method preparation according to claim 9 is characterized in that, uses clone to produce chicken avian infectious bronchitis virus living vaccine.
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| CN104845941A (en) * | 2014-11-18 | 2015-08-19 | 天津瑞普生物技术股份有限公司 | Avian infectious bronchitis virus IBV-K136, monoclonal antibody cell line 3D5 prepared by using avian infectious bronchitis virus IBV-K136, monoclonal antibodies, and applications of avian infectious bronchitis virus IBV-K136 and monoclonal antibodies |
| CN105267961A (en) * | 2015-11-16 | 2016-01-27 | 河北远征药业有限公司 | Method of avian infectious laryngotracheitis live vaccine using cell line |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103599531A (en) * | 2013-09-23 | 2014-02-26 | 天津瑞普生物技术股份有限公司 | Chicken infectious bronchitis live vaccine, heat-resisting protective agent and preparation method of the vaccine |
| CN104845941A (en) * | 2014-11-18 | 2015-08-19 | 天津瑞普生物技术股份有限公司 | Avian infectious bronchitis virus IBV-K136, monoclonal antibody cell line 3D5 prepared by using avian infectious bronchitis virus IBV-K136, monoclonal antibodies, and applications of avian infectious bronchitis virus IBV-K136 and monoclonal antibodies |
| CN104845941B (en) * | 2014-11-18 | 2018-03-30 | 天津瑞普生物技术股份有限公司 | A kind of avian infectious bronchitis virus IBV K136, cell strain of monoclonal antibody 3D5, monoclonal antibody and its application using its preparation |
| CN105267961A (en) * | 2015-11-16 | 2016-01-27 | 河北远征药业有限公司 | Method of avian infectious laryngotracheitis live vaccine using cell line |
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