CN104162154A - Bivalent inactivated vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis, and preparation method and application thereof - Google Patents
Bivalent inactivated vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a bivalent inactivated vaccine used for prevention of bovine viral diarrhea and infectious bovine rhinotracheitis, and a preparation method and application thereof. The vaccine provided by the invention comprises the effective components--inactivated antigen of the bovine viral diarrhea virus type I and inactivated antigen of the infectious bovine rhinotracheitis virus, can prevent infection of cattle of different breeds caused by the two viruses and reduces economic losses caused by infection by the viruses. The vaccine realizes the purpose of prevention of the two viruses through one dose of the vaccine and is safe and reliable. The invention also provides a preparation method for the bivalent inactivated vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis. According to the method, the vaccine is cultured and produced by using a continuous cell line, and virus culture and preparation technology in the preparation method for the vaccine are optimized. Vaccine safety and efficacy test results show that no local and systemic adverse reaction occur after cattle is immunized with the bivalent inactivated vaccine and immune protection is realized to all the cattle; so it is proved that the vaccine is safe and reliable and can achieve the purpose of prevention of the two viruses through one dose of the vaccine.
Description
Technical field
The invention belongs to veterinary biologics technical field, more specifically relate to a kind of bivalent inactivated vaccine that prevents infectious bovine rhinotrachetis and bovine viral diarrhoea and its preparation method and application.
Technical background
Bovine viral diarrhoea (Bovine viral diarrhea, BVD) be a kind of infectious disease taking heating, mucosal erosion ulcer, leukopenia, diarrhoea, cough and the miscarriage of conceived cow or output malformation fetus as principal character being caused by bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV).Bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV) belongs to flaviviridae (Flaviviridae), pestivirus (Pestivirus).Genome is sub-thread positive chain RNA, is made up of an open reading frame (ORF) and 5 ' UTR and 3 ' UTR.Nineteen forty-six Olafson etc. finds and isolates BVDV first.At present, this disease extensively exists in the world.Li Youmin equals nineteen eighty-three and from the spleen of aborted fetus, has been separated to BVDV first, proves that Gai Bingyi China exists.Be separated to BVDV from the multiple morbidities of China cattle farm in recent years, brought huge economic loss to China's cattle-raising.Ox blood serum sample to China's part province detects, and result shows, this disease is rising situation in China.
Infectious bovine rhinotrachetis (Infectious bovine rhinotracheitis, IBR) be that the one of the cattle that caused by bovine herpes virus-1 virus (BHV-1) is acute, hot, contagious disease, clinical manifestation is the symptoms such as upper respiratory tract and tracheal mucosa inflammation, dyspnea, watery nasal discharge, also can cause pustular epivaginitis, balanitis, conjunctivitis, young cattle meningitis, mastitis, miscarriage etc.This disease sees the U.S. in twentieth century the beginning of the fifties the earliest, popular in world wide at present.China found this disease first in 1980 from New Zealand import kind cattle, all had IBRV to infect at present milch cow, cattle and the Babalus bubalis L. in most of province, caused larger economic loss, and vaccine is one of this sick major measure of prevention and control.
Commercial BVD vaccine and IBR vaccine are mainly attenuated live vaccines and inactivated vaccine, and the two respectively has its advantage.The reaction of attenuated live vaccines energy induction of immunity fast, duration of immunity is lasting and can induce local mucous membrane immunity.The sharpest edges of inactivated vaccine are its safety, do not have the risk of toxin expelling, nothing miscarriage and persistent infection, can replace in some cases attenuated vaccine to use, especially for conceived cows and the higher milk cows of economic worth.For these two kinds of infectious disease, China does not still have commercialized vaccine to put on market at present, and cattle-raising faces huge threat, epidemic situation occurs at one's wit's end, finds often mixed infection of BVDV and IBRV in clinical case research.Therefore urgent need development is a kind of can prevent the effective vaccine of above-mentioned epidemic disease simultaneously.The invention provides a kind of bivalent inactivated vaccine that can simultaneously prevent infectious bovine rhinotrachetis and bovine viral diarrhoea, carry out immunity inoculation by these goods, can effective guarantee cattle avoid the infection of BVDV and IBRV, again can " pin two be anti-", save time, laborsaving, cost-saving.
Summary of the invention
One of object of the present invention be to provide a kind of immunity high, without the bovine viral diarrhoea of immune interference and the bivalent inactivated vaccine compositions of infectious bovine rhinotrachetis, reach the object of simultaneously preventing BVD and IBR.
Two of object of the present invention is to provide the bivalent inactivated vaccine of described bovine viral diarrhoea and infectious bovine rhinotrachetis in the application in preparation prevention or treatment infectious bovine rhinotrachetis and bovine viral diarrhoea medicine.
Three of object of the present invention is to provide a kind of method of the bivalent inactivated vaccine compositions of preparing bovine viral diarrhoea and infectious bovine rhinotrachetis.
The present invention is a kind of bovine viral diarrhoea, infectious bovine rhinotrachetis bivalent inactivated vaccine, it is characterized in that effective ingredient comprises bovine viral diarrhoea I type inactivation of virus antigen and infectious bovine rhinotrachetis virus inactivation antigen.
In the present invention, preferably, the bovine viral diarrhea virus NM01 strain that described bovine viral diarrhoea I type inactivation of virus antigen is deactivation, the infectious bovine rhinotrachetis virus LN01/08 strain that described infectious bovine rhinotrachetis virus inactivation antigen is deactivation, described bovine viral diarrhoea I type virus N M01 strain, called after BVDV NM01 strain, Classification And Nomenclature is bovine viral diarrhea virus, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation is numbered: CGMCC No.6032, preservation date is on April 20th, 2012, described infectious bovine rhinotrachetis virus LN01/08 strain, called after IBRV LN01/08 strain, Classification And Nomenclature is infectious bovine rhinotrachetis virus, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation is numbered: CGMCC No.6033, preservation date is on April 20th, 2012.
The present invention seed culture of viruses IBRV used LN01/08 strain be from morbidity Nasus Bovis seu Bubali swab through MDBK cell continuous passage separating obtained through clone purification, viral titer can reach 10
8.0tCID
50more than/ml.
Further, the present invention also provides described bivalent inactivated vaccine to prevent or treat the application in infectious bovine rhinotrachetis and bovine viral diarrhoea in preparation.
Further, the present invention also provides a kind of method of preparing infectious bovine rhinotrachetis, bovine viral diarrhoea bivalent inactivated vaccine, it is characterized in that comprising: by bovine viral diarrhea virus I type virus (BVDV), infectious bovine rhinotrachetis virus (IBRV), carry out large-scale culture respectively, results virus liquid, by after virus liquid deactivation with immunological adjuvant mixing and emulsifying, be prepared into inactivated vaccine.
In method of the present invention, preferably, described bovine viral diarrhoea I type virus is bovine viral diarrhea virus NM01 strain, described infectious bovine rhinotrachetis virus is infectious bovine rhinotrachetis virus LN01/08 strain, wherein said bovine viral diarrhea virus NM01 strain, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its culture presevation is numbered: CGMCC No.6032; Described infectious bovine rhinotrachetis virus LN01/08 strain, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its culture presevation is numbered: CGMCC No.6033.
In method of the present invention, preferred, it comprises the following steps:
(1) cell line gone down to posterity and cultivate;
(2) the virus N M01 strain of bovine viral diarrhea virus I type and infectious bovine rhinotrachetis virus LN01/08 strain are inoculated respectively to the medium that covers with 90%～100% passage cell, cultivate with cell maintenance medium, kind of a poison is produced in preparation;
(3) prepared production kind poison is inoculated respectively to the medium that covers with 90%～100% passage cell, cultivate with cell maintenance medium, obtain bovine viral diarrhoea I type virus N M01 strain virus antigen liquid and infectious bovine rhinotrachetis virus LN01/08 strain virus antigen liquid;
(4) bovine viral diarrhoea I type virus N M01 strain virus antigen liquid and infectious bovine rhinotrachetis virus LN01/08 strain virus antigen liquid are added respectively to inactivator deactivation, add nertralizer neutralization, the antigen liquid after two kinds of deactivations is mixed, add immunological adjuvant, emulsifying, to obtain final product;
Wherein, preferred, described cell maintenance medium is for containing 0.5～2 μ g/ml pancreatin and 3.5%(v/v) DMEM culture fluid or the MEM culture fluid of horse serum, pH value 7.0～7.2.
Wherein, preferably, the continuous cell line described in step (1) comprises bovine kidney cells system (MDBK cell line), bull testis cell (BT cell line), Nasus Bovis seu Bubali first osteocyte system (BT cell line), Vero cell line, BHK21 cell line, cattle primary cell, mdck cell system, cattle uterus cell line (NCL-1 cell line), rabbit kidney primary cell line (CRL-1414), rabbit kidney cell system ((LLC-RK1).
Wherein, preferred, described cell line go down to posterity and cultivation comprises: cell line, through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, is continued to cultivate with cell growth medium, when cell covers with 90%～100%, goes down to posterity or virus inoculation for continuing; Wherein, described cell growth medium is for containing 6%～8%(v/v) the DMEM culture fluid of calf serum, pH value is 7.0～7.2; The cultural method of described cell line for following any one: in rolling bottle, cultivate, make its cell density reach 2 × 10
5～6 × 10
5/ ml; Or add and adhere to carrier and carry out suspension culture or do not add any carrier and carry out pure suspension culture in bioreactor, make cell density reach 2 × 10
6～5 × 10
6/ ml, the described carrier that adheres to is microcarrier or the scraps of paper.
Wherein, preferred, the cultivation temperature described in step (1), (2) or (3) is 33～37 DEG C, and cell culture environment is containing 5%CO
2the incubator of left and right or bioreactor or containing CO
2rolling bottle; The Virus culture time is 30～72 hours.
Wherein, preferred, the virus inoculation amount MOI of the bovine viral diarrhoea I type virus N M01 strain described in step (2) or (3) is 0.03～0.05, and the virus inoculation amount MOI of infectious bovine rhinotrachetis virus LN01/08 strain is 0.01～0.03.
Wherein, preferred, the inactivator described in step (4) is divinyl imines (BEI), and its final concentration is 0.002～0.003M; Described nertralizer is sodium thiosulfate, and the final concentration of sodium thiosulfate is 0.03M.
Wherein, preferred, in step (4), in every dosage vaccine, bovine viral diarrhoea 1 type virus N M01 strain virus antigen liquid deactivation provirus content is for being not less than 10
7.0tCID
50, the viral level before the deactivation of infectious bovine rhinotrachetis virus LN01/08 strain virus antigen liquid is for being not less than 10
7.5tCID
50antigen liquid after two kinds of deactivations is mixed, then the ratio that is 1-2:1-2 according to the volume ratio of antigen liquid and immunological adjuvant, add immunological adjuvant, mix, emulsifying, described emulsifying agent is 206 adjuvants or other mineral oil adjuvant, preferred, be that the hybrid antigen liquid of 54 volume parts is mixed with the immunological adjuvant of 46 volume parts.
Brief description of the drawings
Fig. 1 is the identified by immunofluorescence result of F4 for viral cell culture;
A.F4 uses the dyeing of BVDV1 type monoclonal antibody after for viral cell culture inoculating cell; B.F4 uses the dyeing of BVDV2 type monoclonal antibody after for viral cell culture inoculating cell; C. negative contrast
Fig. 2 is virus liquid the 4th generation cell culture RT-PCR amplification;
1, negative control; 2, virus liquid the 4th generation cell culture; M, Marker DL2000
Fig. 3 is BVDV NM01 strain Subtype result;
Fig. 4 is mean body temperature change curve after the safety testing vaccination first of BVD, IBR bivalent inactivated vaccine;
Fig. 5 is mean body temperature change curve after the safety testing secondary inoculation vaccine of BVD, IBR bivalent inactivated vaccine;
Fig. 6 is that after the potency test Immunization of BVD, IBR bivalent inactivated vaccine, mean body temperature changes.
Detailed description of the invention
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these amendments and replacement all fall within the scope of protection of the present invention.
The isolation identification of embodiment 1 bovine viral diarrhoea 1 type virus N M01 strain
(1) immunofluorescence test
Inventor is separated to a strain virus from clinical collection bovine blood, and this virus can produce typical cytopathic on MDBK cell, by the 4th generation virus liquid be inoculated in the 96 porocyte culture plates that cover with monolayer MDBK cell, and establish and do not inoculate blank, be placed in 37 DEG C, containing 5%CO
2incubator is cultivated 48～72 hours, after acetone is fixing, adds respectively fluorescently-labeled BVDV1 and BVDV2 monoclonal antibody, acts on after 40 minutes fluorescence microscope in 37 DEG C of wet boxes.In the hole of the visible BVDV1 of being added with monoclonal antibody, occur specific fluorescence, BVDV2 type monoclonal anti body opening and matched group are without visible fluorescence.As shown in Figure 1.
(2) RT-PCR qualification is according to the Auele Specific Primer of the synthetic BVDV of BVDV5 ' UTR gene order design, for detection of the Auele Specific Primer of BVDV virus.
Forward primer is: P324:5 '-ATGCCCTTAGTAGGACTAGCA-3 ';
Downstream primer: P326:5 '-TCAACTCCATGTGCCATGTAC-3 ',
Result shows that virus liquid the 4th generation cell culture can obtain the specificity object fragment of about 288bp through RT-PCR amplification, and negative control is without band.As shown in Figure 2.
(3) full gene sequencing is announced BVDV complete genome sequence with reference to GenBank, and the synthetic Auele Specific Primer of design, measures for BVDV whole genome sequence, and primer sequence is in table 1.Reclaim and order-checking through viral RNA preparation, viral cDNA generation, pcr amplification, fragment, result shows, this virus total length 12294bp, containing 5 ' noncoding region, ORF coding region and 3 ' noncoding region, belongs to BVDV1 type.Show separating malicious hypotype qualification result, this strain and BVDV-1a hypotype strain homology are higher, belong to BVDV1a hypotype.
Table 1BVDV complete genome sequence is measured primer table
Utilize DNAStar software to carry out the splicing of gene order sequencing result, obtain the complete genome sequence of isolated viral strain.Announce BVDV sequence with reference to GenBank, for the synthetic BVDV1 type specificity primer of BVDV5 ' UTR design, upstream BV1:5'-TCG ACG CTT TGG AGG ACA AGC-3'; Downstream BV1:5'-CCA TGT GCC ATG TAC AG-3'.Amplified fragments is sent to the handsome company in Beijing simultaneously and checks order, the BVDV1a and the BVDV1b type that log in GenBank compare, and identify for BVDV type.
Show through the sequencing results, the BVDV1a hypotype sibship that separated strain BVDV NM01 strain and GenBank log in is nearer, belongs to BVDV1a hypotype, as Fig. 3.
Above experimental result shows, this isolated strain is bovine viral diarrhea virus, called after BVDV NM01 strain, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation is numbered: CGMCC No.6032, preservation date is on April 20th, 2012.
Embodiment 2 utilizes MDBK cell line to produce bovine viral diarrhoea, infectious bovine rhinotrachetis bivalent inactivated vaccine
(1) by bovine viral diarrhoea I type NM01 strain virus and infectious bovine rhinotrachetis LN01/08 strain virus, respectively by MOI be 0.04 and MOI to be 0.01 connect poison amount is inoculated in the MDBK cell covering with, 40 hours h left and right harvesting cultures,-70 DEG C of freeze thawing 1 time, on MDBK cell, carry out breeding and plaque clone purification continuously, obtained pure kind poison.
(2) select MDBK cell line as seedling cell;
(3) above-mentioned MDBK cell line is through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, add the DMEM culture fluid of the calf serum of cell growth medium (containing 8%(v/v), pH value is 7.0) spinner culture, when cell covers with 90%～100%, goes down to posterity or virus inoculation for continuing; When microcarrier or scraps of paper suspension culture, in the backward cell culture reactor of cell dissociation with spinner culture, inoculate 1 × 10
5-2 × 10
5the cell of cell/mL, on carrier, the full cell of 80-90% remittance, realizes the continuous passage of cell or connects poison;
(4) breeding of seed culture of viruses: get well-grown above-mentioned MDBK cell, wash 2 times with PBS, bovine viral diarrhoea kind poison and infectious bovine rhinotrachetis kind poison are measured and inoculated by the poison that connects of MOI0.04 and MOI0.01 respectively; Add the DMEM culture fluid of maintenance medium (containing 1 μ g/ml pancreatin and 3.5%(v/v) horse serum, when CPE appears in pH value 7.0,30～48 hour cells 80%, receive cell culture venom as production seed culture of viruses;
Seed culture of viruses qualification: carry out the inspection of pure property according to " People's Republic of China's veterinary drug allusion quotation " annex, seed culture of viruses should be pure, and every milliliter of bovine viral diarrhoea seed culture of viruses viral level is not less than 10
7.0tCID
50, infectious bovine rhinotrachetis seed culture of viruses viral level is not less than 10
7.5tCID
50;
(5) breeding of seedling venom: in the time that above-mentioned cell has covered with 90%～100%, discard cell growth medium, wash 2 times with PBS, respectively by the inoculum concentration access seed culture of viruses that meets poison amount, infectious bovine rhinotrachetis virus MOI0.01 of bovine viral diarrhea virus MOI0.04, add the DMEM culture fluid of maintenance medium (containing 1 μ g/ml pancreatin and 3.5%(v/v) horse serum, pH value 7.0, or with pancreatin (final concentration is 0.5～2 μ g/ml) process virus 1 hour after, add DMEM, when CPE appears in 30～48 hour cells 80%, receive cell culture venom as seedling venom; The venom of results is put-20 DEG C of following preservations;
The inspection of virus liquid for seedling: test by " People's Republic of China's veterinary drug allusion quotation " annex, should be without antibacterial, mycete, mycoplasma growth.
(6) deactivation and emulsifying: 0.4mol/L BEA and 0.4mol/L NaOH are mixed in appropriate containers with volume ratio 1:1 ratio.37 ± 2 DEG C of cyclisation at least 1 hour, generate BEI, regulate pH to 7.2～7.6.By the BVDV NM01 strain being up to the standards and IBRV LN01/08 strain virus culture fluid, be placed in different vessels, adding BEI inactivator final concentration is 3mM, deactivation 36～40 hours, fully shakes up, then to add final concentration be that the sodium thiosulfate of 0.03M neutralizes.Two kinds of antigens of deactivation are mixed, make in every dosage vaccine, deactivation first two antigenic content is that BVDV NM01 strain is not less than 10
7.0tCID
50, IBRVLN01/08 strain is not less than 10
7.5tCID
50, antigen is slowly injected in 206 adjuvants, keep 30 DEG C, be 54/100 volume parts by antigen, adjuvant is that 46/100 volume parts is carried out mixing and emulsifying.Be cooled to rapidly 15 DEG C, place not shake in 24 hours for 4 DEG C, subpackage gets product.Laboratory makes 3 batches of goods altogether, is respectively BV-09001, BV-09002 and BV-09003.
Product inspection: test by " People's Republic of China's veterinary drug allusion quotation " annex, should be without antibacterial, mycete and mycoplasma growth.
Bovine viral diarrhoea, infectious bovine rhinotrachetis bivalent inactivated vaccine prepared by the present embodiment, before in every dosage, IBRV antigen contains deactivation, IBRV LN01/08 viral level is for being not less than 10
7.5tCID
50, before BVDV antigen contains deactivation, BVDV NM01 viral level is for being not less than 10
7.0tCID
50.Character inspection, safety verification, efficacy test etc. are all qualified.
Embodiment 3 utilizes suspension culture technology to produce bovine viral diarrhoea, infectious bovine rhinotrachetis bivalent inactivated vaccine
(1) MDBK cell in liquid nitrogen container is taken out, puts in 37 DEG C of water-baths and melt, 1000 revs/min centrifugal 1 minute, abandon cryopreserving liquid.With the DMEM culture medium re-suspended cell containing 6%～8% calf serum, be inoculated in ordinary cells culture bottle, be placed in 37 DEG C, 5%CO
2incubator in grow 72 hours, by the 1:5 cultivation of going down to posterity, after 72 hours, peptic cell is collected, be inoculated in 250mL triangular flask 37 DEG C, 100 revs/min vibration suspension culture; Regulate pH value with NaOH, it is controlled between 6.8-7.4; Be increased to when a certain amount of until cell volume, proceed in special-purpose suspension culture bottle and cultivate, until cell agglomerate disappears, cell concentration can be from 0.5 × 10 in 96 hours
6individual/mL grows into 2-4 × 10
6individual/mL, cell domestication finishes.
(2) by being seeded to 2L bioreactor through the MDBK cell of domestication, first allow it fully suspend with 150 revs/min; Then rotating speed is down to 50 revs/min-80 revs/min, to the defoamer that adds 0.1% in reactor, cultivates 72 hours.PH value is controlled at 7.2-7.4.When cell density reaches 2 × 10
6individual/when mL, to rise reactor from 2 liters of scaling-ups to 15.Rotating speed is down to 40-60 rev/min, makes cell density reach 2 × 10
6individual/mL.In this step, used medium is special low serum suspension medium (containing 1% calf serum).
(3) will rise reactor from 15 liters of scaling-ups to 150 through the MDBK cell of domestication.With 150 revs/min of suspension cells, add 0.1% defoamer, cultivate 48～72 hours, when cell density reaches 2 × 10
6individual/when mL.Proceed to next stage bioreactor.
(4) cell is risen to reactor from 150 liters of scaling-ups to 1000.When cell density reaches 2 × 10
6individual/when mL, to press respectively bovine viral diarrhea virus MOI0.05, infectious bovine rhinotrachetis virus MOI0.01 virus inoculation, 35 DEG C, suspension culture.Once check cellular morphology every sampling in 6 hours, approximately 14 hours results virus-culturing fluids.The special-purpose suspension medium that in this step, used medium is serum-free.
(5) adopt high pressure homogenizer by the pressure breaking of the virus-culturing fluid of results 150～300Bar, and it is tested.Method is undertaken by " People's Republic of China's veterinary drug allusion quotation " annex, should be without antibacterial, mycete, mycoplasma growth.
(6) adopt the identical method of embodiment 2 to seedling virus-culturing fluid deactivation and emulsifying, and finished product is tested, method is undertaken by " People's Republic of China's veterinary drug allusion quotation " annex, should be without antibacterial, mycete and mycoplasma growth.
Bovine viral diarrhoea, infectious bovine rhinotrachetis bivalent inactivated vaccine prepared by the present embodiment, in every dosage vaccine, bovine viral diarrhoea 1 type virus N M01 strain virus antigen liquid deactivation provirus content is for being not less than 10
7.0tCID
50, the viral level before the deactivation of infectious bovine rhinotrachetis virus LN01/08 strain virus antigen liquid is for being not less than 10
7.5tCID
50, character inspection, safety verification, efficacy test etc. are all qualified.
The overdose safety testing of experimental example 1 bovine viral diarrhoea of the present invention, infectious bovine rhinotrachetis bivalent inactivated vaccine
5-7 monthly age 20 of healthy cattle are selected in this test altogether, require the anti-BVDV of Ox blood serum and anti-IBR antibody titer to be less than 2.Test is divided into 4 groups, and 3 groups of immune group, inoculate respectively different batches vaccine (lot number: BV-09001, BV-09002 and BV-09003), every group of 5 cattle, 5 cattle of matched group.Immune group neck injection 4ml(2 head part) bovine viral diarrhoea, infectious bovine rhinotrachetis bivalent inactivated vaccine.Matched group musculi colli inoculation 4ml MDBK cell freeze thawing thing.After vaccination 21 days, same batch of every group of Niu Zaici inoculation of immune group, same dosage inactivated vaccine were exempted from as two, matched group inoculation 4ml MDBK cell freeze thawing thing.Continuous 14 days one-point measurement experimental animal rectal temperatures after each vaccination, and carry out the general reactions such as the clinical observation mental status, appetite, behavioral activity, defecation condition, eye discharge.
Result shows, after 2 multiple dose head exempt from, respectively organizes experimental animal body temperature all normal, and mean body temperature change curve as shown in Figure 4.After secondary immunity, each vaccination group and matched group experimental animal body temperature are all normal, and mean body temperature change curve as shown in Figure 5.Vaccine 2 multiple dose head exempt from and secondary immunity after, vaccination group and the matched group experimental animal mental status and appetite are good, have no untoward reaction, vaccine injection position has no the symptoms such as redness, suppuration.After getting secondary immunity, injection site muscle on the 14th is prepared pathological section, and result shows, vaccine 2 multiple doses are inoculated latter 14 days, and injection site muscle fiber recovers normally substantially, with matched group no significant difference.After 3 batches of vaccine overdose inoculation cattle, without fervescence, without visible clinical abnormal, inject latter 14 days, absorption is good, muscle fiber pathological section and matched group zero difference.
The potency test of experimental example 2 bovine viral diarrhoea of the present invention, infectious bovine rhinotrachetis bivalent inactivated vaccine
Test is used 6-9 monthly age 20 of healthy cattle altogether, requires the anti-BVDV of Ox blood serum and IBRV antibody titer to be all less than 2.Test is divided into 4 groups, every group of 5 cattle, 1 group and 3 groups of musculi colli injection 2ml(1 head parts) BVD and IBR bivalent inactivated vaccine (lot number is BV-09001), after 21 days, carry out two and exempt from, inoculation method, program and dosage and one are exempted from identical.2 groups and 4 groups is matched group, every incidence intramuscular inoculation 2ml MDBK cell freeze thawing thing.
1, BVDV efficacy test
Vaccine two is exempted from latter 21 days, and 1 group of 5 cattle of vaccine immunity group and 2 groups of 5 cattle of matched group are attacked with BVDV inspection strong malicious BVDV JL strain respectively, and every Nasus Bovis seu Bubali internal spraying inoculation 6ml(virus titer is 10
6.5-6.8tCID
50/ ml).After counteracting toxic substances every day determination test animal rectal temperature; Observe clinical symptoms; Gather nose swab and separate for virus every day; Blood sampling every other day separates with virus for numeration of leukocyte, continuous 14 days.
2, IBRV efficacy test
Vaccine two is exempted from latter 21 days, and 4 groups of every cattle of 3 groups of immune group and matched group carry out counteracting toxic substances with the strong malicious LN01/08 strain of IBRV effect inspection, and every Nasus Bovis seu Bubali internal spraying inoculation 4ml(virus titer is 10
6.4～7.0tCID
50/ ml), after counteracting toxic substances, timing in continuous 14 days is measured body temperature 1 time and is gathered nose swab and separates for virus.
Result shows; after this inactivated vaccine immunity test animal; can produce good protective effect to strong virus attack; protective rate is 100%; 1 group of immune group and immune 3 groups of relevant symptoms that all do not occur bovine viral diarrhoea or infectious bovine rhinotrachetis; and 4 groups of 2 groups of matched groups and contrasts the 3rd day equal body temperature after counteracting toxic substances obviously raises, and with symptoms such as leukopenia, diarrhoea, cough, dyspnea, watery nasal discharges.After Immunization, each group mean body temperature change curve as shown in Figure 6.
Claims (10)
1. a bovine viral diarrhoea, infectious bovine rhinotrachetis bivalent inactivated vaccine, it is characterized in that effective ingredient comprises bovine viral diarrhoea I type inactivation of virus antigen and infectious bovine rhinotrachetis virus inactivation antigen, preferably, the bovine viral diarrhea virus NM01 strain that described bovine viral diarrhoea I type inactivation of virus antigen is deactivation, the infectious bovine rhinotrachetis virus LN01/08 strain that described infectious bovine rhinotrachetis virus inactivation antigen is deactivation, wherein said bovine viral diarrhea virus NM01 strain, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its culture presevation is numbered: CGMCC No.6032, described infectious bovine rhinotrachetis virus LN01/08 strain, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its culture presevation is numbered: CGMCC No.6033.
2. the application of bivalent inactivated vaccine claimed in claim 1 in preparation prevention or treatment bovine viral diarrhoea and infectious bovine rhinotrachetis medicine.
3. prepare the method for bovine viral diarrhoea, infectious bovine rhinotrachetis bivalent inactivated vaccine for one kind, it is characterized in that comprising: bovine viral diarrhea virus I type virus, infectious bovine rhinotrachetis virus are carried out respectively to large-scale culture, results virus liquid, by after virus liquid deactivation with immunological adjuvant mixing and emulsifying, be prepared into inactivated vaccine; Preferably, described bovine viral diarrhoea I type virus is bovine viral diarrhea virus NM01 strain, described infectious bovine rhinotrachetis virus is infectious bovine rhinotrachetis virus LN01/08 strain, wherein said bovine viral diarrhea virus NM01 strain, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its culture presevation is numbered: CGMCC No.6032; Described infectious bovine rhinotrachetis virus LN01/08 strain, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its culture presevation is numbered: CGMCC No.6033.
4. method as claimed in claim 3, is characterized in that comprising the following steps:
(1) cell line gone down to posterity and cultivate;
(2) the virus N M01 strain of bovine viral diarrhea virus I type and infectious bovine rhinotrachetis virus LN01/08 strain are inoculated respectively to the medium that covers with 90%～100% passage cell, cultivate with cell maintenance medium, kind of a poison is produced in preparation;
(3) prepared production kind poison is inoculated respectively to the medium that covers with 90%～100% passage cell, cultivate with cell maintenance medium, obtain bovine viral diarrhoea I type virus N M01 strain virus antigen liquid and infectious bovine rhinotrachetis virus LN01/08 strain virus antigen liquid;
(4) bovine viral diarrhoea I type virus N M01 strain virus antigen liquid and infectious bovine rhinotrachetis virus LN01/08 strain virus antigen liquid are added respectively to inactivator deactivation, add nertralizer neutralization, the antigen liquid after two kinds of deactivations is mixed, add immunological adjuvant, emulsifying, to obtain final product;
Wherein, described cell maintenance medium is for containing 0.5～2 μ g/ml pancreatin and 3.5%(v/v) DMEM culture fluid or the MEM culture fluid of horse serum, pH value is 7.0～7.2.
5. method as claimed in claim 4, is characterized in that the cell line described in step (1) comprises bovine kidney cells system (MDBK cell line), bull testis cell (BT cell line), Nasus Bovis seu Bubali first osteocyte system (BT cell line), Vero cell line, BHK21 cell line, cattle primary cell, mdck cell system, cattle uterus cell line (NCL-1 cell line), NCL-1 cell line, rabbit kidney primary cell (CRL-1414), rabbit kidney cell ((LLC-RK1).
6. the method as described in claim 4 or 5, it is characterized in that cell line described in step (1) go down to posterity and cultivation comprises: by cell line through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, continue to cultivate with cell growth medium, when cell covers with 90%～100%, go down to posterity or virus inoculation for continuing; Wherein, described cell growth medium is for containing 6%～8%(v/v) the DMEM culture fluid of calf serum, pH value is 7.0～7.2; The cultural method of described cell line for following any one: in rolling bottle, cultivate, make its cell density reach 2 × 10
5～6 × 10
5/ ml; Or add and adhere to carrier and carry out suspension culture or do not add any carrier and carry out pure suspension culture in bioreactor, make cell density reach 2 × 10
6～5 × 10
6/ ml, the described carrier that adheres to is microcarrier or the scraps of paper.
7. method as claimed in claim 4, is characterized in that the cultivation temperature described in step (1), (2) or (3) is 33～37 DEG C, and cell culture environment is containing 5% left and right CO
2incubator or bioreactor or containing CO
2rolling bottle; The Virus culture time is 30～72 hours.
8. method as claimed in claim 4, the virus inoculation amount MOI that it is characterized in that the bovine viral diarrhoea I type virus N M01 strain described in step (2) or (3) is 0.03～0.05, and the virus inoculation amount MOI of infectious bovine rhinotrachetis virus LN01/08 strain is 0.01～0.03.
9. method as claimed in claim 4, is characterized in that the inactivator described in step (4) is divinyl imines (BEI), and its final concentration is 0.002-0.003M; Described nertralizer is sodium thiosulfate, and the final concentration of sodium thiosulfate is 0.03M.
10. method as claimed in claim 4, is characterized in that in step (4), and in every dosage vaccine, bovine viral diarrhoea 1 type virus N M01 strain virus antigen liquid deactivation provirus content is for being not less than 10
7.0tCID
50, the viral level before the deactivation of infectious bovine rhinotrachetis virus LN01/08 strain virus antigen liquid is for being not less than 10
7.5tCID
50antigen liquid after two kinds of deactivations is mixed, then the ratio that is 1-2:1-2 according to the volume ratio of antigen liquid and immunological adjuvant, add immunological adjuvant, mix, emulsifying, described emulsifying agent is 206 adjuvants or other mineral oil adjuvant, preferably, be that the hybrid antigen liquid of 54 volume parts is mixed with the immunological adjuvant of 46 volume parts.
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| CN201310184764.6A CN104162154B (en) | 2013-05-17 | 2013-05-17 | Bivalent inactivated vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis, and preparation method and application thereof |
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Denomination of invention: Preparation and application of inactivated vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis Effective date of registration: 20221008 Granted publication date: 20170208 Pledgee: Zhejiang Commercial Bank Co.,Ltd. Taizhou Branch Pledgor: SINOVET (JIANGSU) BIOLOGICAL PHARMACEUTICAL Co.,Ltd. Registration number: Y2022980017467 |