CN103041384B - A kind of vaccine combination and its preparation method and application - Google Patents
A kind of vaccine combination and its preparation method and application Download PDFInfo
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Abstract
The invention provides a kind of vaccine combination, this vaccine combination comprises swine fever virus antigen and pig blue-ear disease antigen, wherein, to be that swine fever rabbit is weak poison strain to this swine fever virus, this pig blue-ear disease antigen is high-pathogenicity porcine reproductive and respiratory syndrome virus, and this pig blue-ear disease antigen and swine fever virus antigenic content ratio are 100:1-100:10, and this vaccine combination can eliminate the interference effect between two kinds of antigen, reach the object that a pin prevents two kinds of infectious disease, greatly reduce immune time and Animal stress reaction.
Description
Technical field
The present invention relates to the vaccine combination of a kind of prevention and therapy swine fever, pig blue-ear disease.
Background technology
Swine fever (classicalswinefever, CSF) is the crushing infectious disease of a kind of serious harm pig industry caused by swine fever virus (classicalswinefevervirus, CSFV).This sick mortality rate is up to 80-90%, and OIE (OIE) is classified as OIE disease register, and China is also classified as a class infectious disease.The anti-system of swine fever is always the problem that whole world swine fever exists state and pays much attention to, and each state has all dropped into a large amount of human and material resources for this reason.Current vaccination is still the important means controlling swine fever.The pig testis passage cell Seedling that the hog cholera lapinised virus vaccine that China produces has spleen to drench Seedling, newborn rabbit Tissue vaccine, bull testis primary cell Seedling and grow up for nearest 2 years, vaccine immunity is still the basic means of China's prevention and control swine fever.
Porcine reproductive and respiratory syndrome (also known as the blue ear of pig) is by porcine reproductive and respiratory syndrome virus (porcinereproductiveandrespiratorysyndromevirus, PRRSV) cause with sow heating, miscarriage, piglet mortality rate before and after wean raises, and all ages and classes pig respiratory disorder etc. is the disease of Clinical symptoms.At present, this disease is one of important diseases having a strong impact on world's pig industry development.Vaccination is the most effective method of this disease of prevention, both at home and abroad existing commercial Porcine reproductive and respiratory syndrome vaccine.Existing inactivated vaccine, the also live vaccine prepared of useful low virulent strain.Due to regionality and the epidemiologic feature difference on pig farm, the Attenuate vaccine of the classical reproductive and respiratory syndrome of some pig farm immunity, the Attenuate vaccine of the immune high-pathogenicity blue ear disease had, brings a lot of puzzlement to clinically the immunity of reproductive and respiratory syndrome.
Porcine reproductive and respiratory syndrome virus (PRRSV) is a kind of positive chain RNA virus, has found two kinds of genotype at present: Europe class and american type, and order first two genotype all detects in China.PRRSV Genome Size is about 15kb, there is multiple open reading frame, the wherein sequence of first open reading frame (ORF1a and ORF1b) containing PRRSV genomic 80%, it is encoded about rna replicon and the albumen of transcribing, and comprises RNA RNA-dependent polymerase.(Strawetal，DiseasesofSwine，9thedition，chapter24(2006))。ORF1a and ORF1b is translated into a polyprotein (poly-protein), this polyprotein is cut into multiple non-structural protein by the proteinase activity region wherein contained, comprise Nsp1-Nsp12 (see, Vriesetal, SeminarsinVirology, 8:33-47 (1997); Allendeetal, JournalofGeneralVirology, 80:307-315 (1999)).According to the Genetic Variation Analysis to domestic high-pathogenicity blue ear disease strain, highly pathogenic strain is current advantage epidemic isolates, and variation is very fast.Highly pathogenic PRRS is a class animal epidemic, is a class epidemic disease of current serious harm China pig industry, shows as and all have strong pathogenicity to respiratory system, propagating system, nervous system, immune system, digestive system.Virus virulence is extremely strong, pig infects high-pathogenicity porcine reproductive and respiratory syndrome virus can produce high-caliber viremia, the economic loss that highly pathogenic PRRS causes pig farm is very big, have a lot of pig farm because this going to wreck property of disease is hit, this fact of leading strain that high-pathogenicity porcine reproductive and respiratory syndrome virus Yi Shi China present stage reproductive and respiratory syndrome is popular is unquestionable.
PRRSV variant each strain ORF5 aminopeptidase gene acid homology is 96%-100%, and NsP2 gene has 30 amino acid whose disappearances, the strain NVDC-JXA1-R of prior art, and as prevention high-pathogenicity blue ear disease epidemic situation Government bidding vaccine strain, play an important role in control high-pathogenicity blue ear disease epidemic situation.There are again two strain high-pathogenicity blue ear disease variant TJM-F92 strains and the listing of HuN4-F112 strain live vaccine subsequently, basically identical on the effect of above-mentioned seed culture of viruses and immune effect.
Classical strain reproductive and respiratory syndrome strain is mostly America separated strain, classical reproductive and respiratory syndrome is two class animal epidemics, show as and have stronger pathogenicity to piglet respiratory system and sow propagating system, virus disseminating ability is stronger, the replication capacity of classical reproductive and respiratory syndrome virus is viral lower than high-pathogenicity blue ear disease more than 10000 times, swinery is once infect high-pathogenicity porcine reproductive and respiratory syndrome virus, and classical reproductive and respiratory syndrome virus is just difficult to detect in pig body.Mainly contain Porcine reproductive and respiratory syndrome live vaccine R98 strain, pig blue-ear disease ATCCVR-2332 strain, pig blue-ear disease CH-1R strain at present.
At present, the low virulent strain that PRRS live vaccine uses is domestication's strain, and all become avirulence through Attenuation, and have good immunogenic attenuated vaccine strain, the homology between each strain of PRRSV variant is very high.Complete ORF5 and part Nsp2 Gene sequence comparison analysis discovery is carried out by the PRRSV variant be separated all parts of the country, from the PRRSV very high homology on different pig farm, the GP5 gene of new isolated strain is also very high homology each other, and homology reaches 98.5--100% (see CN101280292A Instructions Page 9 2.2.7 sequence analysis).At present, the variant strain that the PRRSV live vaccine generally used uses has NVDC-JXA1-R strain, TJM-F92 strain and HuN4-F112 strain, although different strain there are differences in virulence and biological property etc., but its immunogenicity is similar, because very high homology between PRRS live vaccine variation strain, there is immunogenic albumen and all exist.Therefore, use the live vaccine of Different Variation strain, all can obtain good immune effect.
Reproductive and respiratory syndrome attenuated vaccine strain also has classical strains except variant; the vaccine strain of current application has Porcine reproductive and respiratory syndrome R98 strain, ATCCVR-2332 strain, CH-1R strain; homology between classical strains is very high; its intersecting protective is also fine, all can resist the attack of the strong poison of classical strains well.Vaccine prepared by each strain all has good immune protective to classical reproductive and respiratory syndrome virulent strain.
Homology between porcine reproductive and respiratory syndrome virus variant and classical strains is only 69.8-86.2%; up to the present the vaccine also having classical strains to prepare can protect the successful examples of the attack of high-pathogenicity blue ear disease poison; but generally believe, the effect of classical strains to preventing and treating highly pathogenic reproductive and respiratory syndrome can not show a candle to the effective of the low virulent strain vaccine of variant.If feeding and management level is better, and it is popular that disease reproductive and respiratory syndrome does not occur within the scope of raising, vaccine prepared by classical strain and variant also has certain intersecting protective.
The infectious disease of this two boar is very serious to the harm of raising pigs, and current immunity inoculation is still the important measures of the anti-swine fever processed of China, pig blue-ear disease.And the immunity simultaneously of pig blue-ear disease vaccine and swine Fever Vaccine can make the effect of vaccine significantly reduce (Hubei animal and veterinary to have document to show, 7th phase in 2009, PRRSV immune is to the test of hog cholera immune influential effect), also have document to show the immune essentially no immunosuppressant simultaneously of two kinds of antigens, its indication " essentially no immunosuppressant " refers to the immune effect (CN102727883A Porcine reproductive and respiratory syndrome and swine fever Combined vaccine and uses thereof) that significantly can not weaken the other side.But in clinical immunization program, these two kinds of vaccines generally take another vaccine of more than seven days, interval ability immunity, and this also describes from the side under existence conditions, and this two kinds of vaccines immunity simultaneously interference phenomenon can occur.But due to two kinds of vaccines immunization time relatively, in clinical practice, spininess time, multiple dose, panimmunity program are difficult to reasonable arrangement, both loaded down with trivial details, time-consuming, effort, increase cost, and easily cause Lous kind, directly affect immune effect.
Du Xizhong etc. (" PRRSV immune is to the test of hog cholera immune influential effect ", " Hubei animal and veterinary ", 7th phase in 2009,21st page, code of post issue 38-352) disclose the immunity simultaneously of pig blue-ear disease Attenuate vaccine and live vaccines of hog cholera and can produce certain impact to hog cholera immune, cause live vaccines of hog cholera antibody titer can reduce 1-2 titre.
Summary of the invention
For solving the deficiencies in the prior art, the object of the invention is the vaccine combination providing a kind for the treatment of and prevent swine fever, pig blue-ear disease,, the problem of interference can be produced when solving the immunity simultaneously of existing vaccine, provide the optimum proportioning of two class antigens in this vaccine combination.
Main purpose of the present invention is to provide a kind of vaccine combination, described vaccine combination comprises swine fever virus antigen and PRRS virus antigen, wherein, to be that swine fever rabbit is weak poison strain to described swine fever virus, described pig blue-ear disease antigen is high-pathogenicity porcine reproductive and respiratory syndrome virus, and described pig blue-ear disease antigen and swine fever virus antigenic content ratio are 100: 1--100: 10.
Preferably, described pig blue-ear disease antigen and swine fever virus antigenic content ratio are 100: 3--100: 10.
Preferably, described pig blue-ear disease antigen comprises the virus of HuN4-F112 strain, NVDC-JXA1-R strain, TJM-F92 strain work.
Another object of the present invention is to provide a kind of vaccine combination, described vaccine combination comprises swine fever virus antigen and pig blue-ear disease antigen, wherein, to be that swine fever rabbit is weak poison strain to described swine fever virus, described pig blue-ear disease antigen is classical PRRS virus, and described pig blue-ear disease antigen and swine fever virus antigenic content ratio are 100: 1-1000: 1.
Preferably, described pig blue-ear disease antigen and swine fever virus antigenic content ratio are 100: 1-300: 1.
Preferably, described pig blue-ear disease antigen comprises the virus of ATCCVR-2332 strain, CH-1R strain, R98 strain work.
Another object of the present invention is to provide a kind of method preparing described vaccine combination, and described preparation method comprises:
Cultivate propagation swine fever virus, prepare described swine fever virus antigen;
Cultivate propagation PRRS virus, prepare described PRRS virus antigen;
Carry out proportioning according to described pig blue-ear disease antigen and swine fever virus antigenic content ratio 100: 1--100: 10, prepare described vaccine combination.
Another object of the present invention is to provide a kind of method preparing described vaccine combination, and described preparation method comprises:
Cultivate propagation swine fever virus, prepare described swine fever virus antigen;
Cultivate propagation PRRS virus, prepare described PRRS virus antigen;
Carry out proportioning according to described pig blue-ear disease antigen and swine fever virus antigenic content ratio 100: 1-1000: 1, prepare described vaccine combination.
An also object of the present invention is to provide the application of described vaccine combination preparing prevention and therapy swine fever and pig blue-ear disease medicine.
The present invention is by the proportion relation between adjustment two kinds of antigens, have found the best psma ligand ratio can eliminating interference effect between two kinds of antigens completely, really accomplish the immune interference effect eliminated between two kinds of antigens, reach the object that a pin prevents two kinds of infectious disease.
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 embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
For pig high-pathogenicity blue ear disease HuN4-F112 strain, JXA1-R strain, TJM-F92 strain, swine fever is described in the embodiment of the present invention 1 ~ 6, reproductive and respiratory syndrome totivirus antigen proportion relation affects immune effect; For pig blue-ear disease classical strains CH-1R, R98 strain, swine fever is described in embodiment 7 ~ 10, reproductive and respiratory syndrome totivirus antigen proportion relation affects immune effect.In the following example, NM specific experiment method, carries out according to experimental technique well known to those skilled in the art usually.
The preparation of embodiment 1 swine fever virus antigen liquid
1. by grow up to good monolayer to swine fever virus, (fever virus lapinized Chinese Strain is by China Veterinery Drug Inspection Office's preservation, preserving number AV1412) Digestive system of high responsive ST cell (purchased from ATCC) containing 0.125% pancreatin and 0.03%EDTA, carry out digestion dispersion, with suitable density inoculating cell culture bottle after cell counting, add the MEM cell culture fluid of 1.5-5%FBS, connecing toxic agent amount according to M.O.I.=0.01-0.6 adds kind of a poison simultaneously, preferably connecing toxic agent amount is further M.O.I.=0.1-0.6, preferably connecing toxic agent amount is further M.O.I.=0.2-0.4, be placed in 34-37 DEG C of incubator to cultivate, preferably cultivation temperature is 34-35 DEG C further.
2. cultivate and carry out first time after three days and receive poison, add the cell maintenance medium of 1.5-3%FBS after receiving poison, received poison once every 2 days later, malicious 5 times can be received continuously.After receiving poison, antigen mixing is placed in-20 DEG C of storages.
3. after the virus liquid mixed sampling gathered in the crops 5 times, carry out TCID
50measure with RID, antigen liquid viral level 10
6.5tCID
50/ ml (immunofluorescence), rabbit body infective dose should be not less than 500,000RID/ml (rabbit precursor reactant full-boiled process), detects simultaneously, conform with the regulations according to Chinese veterinary pharmacopoeia version " live vaccines of hog cholera " touchstone in 2010.
The preparation method of embodiment 2 pig blue-ear disease highly pathogenic mutant strain antigen
1. the going down to posterity and cultivation of seedling cell: Marc145 or MA104 cell line disperseed to go down to posterity through EDTA-trypsinization liquid, continues to cultivate with cell growth medium, when forming fine and close monolayer, for subsequent use, goes down to posterity or virus inoculation for continuing.
2. the breeding of cell seed culture of viruses: by reproductive and respiratory syndrome HuN4-F112 strain (commercially available) virus liquid, the cell bottle of good cell monolayer has been grown into by the dosage access of M.O.I.=0.001-0.01, preferably connecing toxic agent amount is further M.O.I.=0.005-0.01, adsorb after 1 hour, add cell maintenance medium in cell line monolayer, continue to cultivate, gather in the crops after pathological changes appears in 70-80% cell, venom freeze thawing 1-2 rearmounted less than-20 DEG C preservations of results, take a morsel and do the inspection of semifinished product, antigen liquid viral level 10
6.5tCID
50/ ml, all conforms with the regulations according to national standard inspection.
By reproductive and respiratory syndrome JXA1-R strain (commercially available) virus liquid, the cell bottle of good cell monolayer has been grown into by the dosage access of M.O.I.=0.001-0.01, preferably connecing toxic agent amount is further M.O.I.=0.005-0.01, adsorb after 1 hour, add cell maintenance medium in cell line monolayer, continue to cultivate, gather in the crops after pathological changes appears in 70-80% cell, venom freeze thawing 1-2 rearmounted less than-20 DEG C preservations of results, take a morsel and do the inspection of semifinished product, antigen liquid viral level 10
7.0tCID
50/ ml, all conforms with the regulations according to national standard inspection.
By reproductive and respiratory syndrome TJM-F92 strain (commercially available) virus liquid, the cell bottle of good cell monolayer has been grown into by the dosage access of M.O.I.=0.001-0.01, preferably connecing toxic agent amount is further M.O.I.=0.005-0.01, adsorb after 1 hour, add cell maintenance medium in cell line monolayer, continue to cultivate, gather in the crops after pathological changes appears in 70-80% cell, venom freeze thawing 1-2 rearmounted less than-20 DEG C preservations of results, take a morsel and do the inspection of semifinished product, antigen liquid viral level 10
6.5tCID
50/ ml, all conforms with the regulations according to national standard inspection.
Bivalent vaccine composition is prepared into after embodiment 3 swine fever, pig high-pathogenicity blue ear disease (HuN4-F112, JXA1-R, TJM-F92) antigen and swine fever virus antigen different ratio
Swine fever virus antigen (the viral level 10 that implementation column 1 is prepared
6.5tCID
50/ ml), three kinds of PRRS virus antigens that embodiment 2 prepares carry out proportioning according to the antigenic content of table 1 respectively, then mix with the freeze drying protectant of equivalent, and make pig blue-ear disease, swine fever bigeminal live vaccine vaccine combination, concrete condition is in table 1.
The different ratio of table 1 pig blue-ear disease antigen, swine fever antigen and grouping
Mixed successively by above-mentioned antigen, be then that the amount of 1: 1 adds the mixed liquor of 2wt% gelatin and 15wt% lactose as freeze drying protectant by volume, stirring and evenly mixing carries out aseptic subpackaged after 1 hour, and 2-8 DEG C of preservation is for subsequent use.
Embodiment 4 carries out effect evaluation (antibody detection or protest test) by after the swine fever made, pig blue-ear disease (HuN4-F112) bivalent vaccine composition immune animal
1. vaccine immunity: choose the 4-6 piglet in age in week 135 that swine fever, pig blue-ear disease antibody are double-negative, be divided into 9 groups at random, the 1st group 15, immune commercial goods swine Fever Vaccine 1 immunizing dose/head (10
4.5tCID
50/ head or 7500RID/ head part) (Pulaike Biological Engineering Co., Ltd. produces, lot number 1201004), 8th group 15, (Harbin Wei Ke biotech company produces immunity commercial goods pig blue-ear disease attenuated vaccine, product batch number 2012008), immunity 1 doses (10
4.5tCID
50/ head part), the 2nd group to the 7th group each 15, swine fever, pig blue-ear disease Combined vaccine respectively after immune different ratio; 9th group is not immune matched group 15, and observed after immunization piglet clinical response, result occurs without any untoward reaction situation.
2. antibody detection: take a blood sample for every 2 weeks after immunity, after separation of serum, the stop band restrain test kit adopting IDEXX company to produce detects hog cholera antibody, and adopt the antibody that indirect immunofluorescence method detection PRRS virus produces, concrete testing result is respectively in table 2 and table 3.
After table 2 vaccine immunity, piglet produces the dynamic change (5 meansigma methodss) of CSFV antibody
The dynamic change (5 meansigma methodss) of young PRRSV antibody after table 3 vaccine immunity
Surprisingly, when swine fever virus antigen and PRRS virus antigen are all with the immunizing dose (swine Fever Vaccine 10 that existing product specifies
4.5tCID
50/ head part, Porcine reproductive and respiratory syndrome vaccine 10
4.5tCID
50/ head part (HuN4-F112 strain)) when carrying out immunity, immune interference effect can be produced between two kinds of antigens, as what report in document, reproductive and respiratory syndrome antigen can disturb swine fever antigen to produce the speed of antibody, and the time that after showing as immunity, hog cholera antibody produces and peak value all can be postponed.And along with the increase of swine fever virus antigenic content, its immune interference phenomenon weakens gradually, when pig blue-ear disease antigen and swine fever virus antigenic content ratio are 100: 3--100: 10 time, immune interference phenomenon can be eliminated completely.When swine fever antigen content continues to strengthen subsequently, the immune effect of reproductive and respiratory syndrome also can be subject to the impact of swine fever antigen.So when two kinds of psma ligands ratios are only in particular range, immune interference phenomenon could be eliminated.
3. protest test: by the piglet after vaccine immunity, the corresponding vaccine quality standard issued according to country carries out protest test, by immune group pig and matched group pig, uses the strong poison (10 of swine fever crossdrift system respectively
5mLD), the strong poison (10 of pig blue-ear disease HuN4-F112
4.0tCID
50/ ml) to attack, after counteracting toxic substances, every day carries out body temperature measurement and clinicing symptom observation, and carries out cuing open inspection respectively at after counteracting toxic substances 16 days, 21 days, carries out each internal organs Pathologic Observation.Immunoprotection result is judged according to clinical symptoms, pathological change.
Table 4 vaccine potency inspection (counteracting toxic substances protection) test grouping and process
Find with after pig high-pathogenicity blue ear disease HuN4-F112 strong virus attack, when the ratio of pig blue-ear disease antigen and swine fever virus antigen is between 100: 0.3--100: 30, the immune protective effect that pig blue-ear disease antigen produces all can be fine.But along with the increasing of swine fever antigen amount, when ratio is less than 100: 30, the immune effect of pig blue-ear disease antigen can be interfered, the Immunization protective rate of pig blue-ear disease vaccine is caused to reduce.Specifically referring to table 5.
By the counteracting toxic substances protection result of highly pathogenic PRRS strong virus attack piglet after table 5 vaccine immunity
| Group (TCID 50Ratio) | Body temperature (>=40.5 DEG C) | Clinical symptoms | Pathological change | Protective rate |
| 2(100∶0.3) | 0/5 | 0/5 | 0/5 | 5/5 |
| 3(100∶1) | 0/5 | 0/5 | 0/5 | 5/5 |
| 4(100∶3) | 0/5 | 0/5 | 0/5 | 5/5 |
| 5(100∶10) | 0/5 | 0/5 | 0/5 | 5/5 |
| 6(100∶30) | 1/5(4) | 1/5 | 1/5 | 4/5 |
| 7(100∶100) | 3/5(5) | 3/5 | 2/5 | 2/5 |
| 8(1∶0) | 0/5 | 0/5 | 0/5 | 5/5 |
| 9 (matched group is without antigens) | 5/5(≥7) | 4/5 | 5/5 | 0/5 |
Note: the natural law that () interior numeral is had a fever continuously
Find with after swine fever crossdrift system strong virus attack; when swine fever virus antigenic content is low; the immune effect of reproductive and respiratory syndrome antigen meeting severe jamming swine fever antigen; along with swine fever antigen content increases; interference effect fades away; but after exceeding certain limit, swine fever antigen also can be subject to the interference of pig blue-ear disease antigen, thus the immune protective effect of swine fever antigen is caused to reduce.When the optimal proportion of two kinds of antigens is between 100: 3--100: 10, without any interference effect between two kinds of antigens.Specifically refer to table 6.
By the counteracting toxic substances protection result of swine fever crossdrift system strong virus attack piglet after table 6 vaccine immunity
| Group | Body temperature (>=40.5 DEG C) | Clinical symptoms | Pathological change | Protective rate |
| 1(0∶1) | 0/5 | 0/5 | 0/5 | 5/5 |
| 2(100∶0.3) | 3/5(5) | 3/5 | 3/5 | 2/5 |
| 3(100∶1) | 2/5(4) | 1/5 | 2/5 | 3/5 |
| 4(100∶3) | 1/5(5) | 0/5 | 0/5 | 5/5 |
| 5(100∶10) | 0/5 | 0/5 | 0/5 | 5/5 |
| 6(100∶30) | 2/5(2) | 1/5 | 2/5 | 3/5 |
| 7(100∶100) | 4/5(5) | 3/5 | 3/5 | 2/5 |
| 9 (matched group is without antigens) | 5/5(≥7) | 5/5 | 5/5 | 0/5 |
Note: the natural law that () interior numeral is had a fever continuously
Embodiment 5 carries out effect evaluation (protest test) by after the swine fever made, pig blue-ear disease (JXA1-R) bivalent vaccine composition immune animal
1. vaccine immunity: choose the 4-6 piglet in age in week 90 that swine fever, pig blue-ear disease antibody are double-negative, be divided into 9 groups at random, the 1st group 10, immune commercial goods swine Fever Vaccine 1 immunizing dose/head (10
4.5tCID
50/ head) (Pulaike Biological Engineering Co., Ltd. produces, lot number 1201004), 8th group 10, immunity commercial goods pig blue-ear disease attenuated vaccine (JXA1-R strain Pulaike Biological Engineering Co., Ltd., product batch number 1202020B), immunity 1 doses (10
5.0tCID
50/ head part), the 2nd group to the 7th group each 10, swine fever, pig blue-ear disease Combined vaccine compositions respectively after immune different ratio; 9th group is not immune matched group 10, and observed after immunization piglet clinical response, result occurs without any untoward reaction situation.
2. protest test: by the piglet after vaccine immunity, the corresponding vaccine quality standard issued according to country carries out protest test, by immune group pig and matched group pig, uses the strong poison (10 of swine fever crossdrift system respectively
5mLD), the strong poison (10 of pig blue-ear disease JXA1-R
4.5tCID
50/ ml) 3ml attack, after counteracting toxic substances, every day carries out body temperature measurement and clinicing symptom observation, and carries out cuing open inspection respectively at after counteracting toxic substances 16 days, 21 days, carries out each internal organs Pathologic Observation.Immunoprotection result is judged according to clinical symptoms, pathological change.
The grouping of table 7 vaccine protest test and process
Find with after pig high-pathogenicity blue ear disease NVDV-JXAI strong virus attack, when the ratio of pig blue-ear disease antigen and swine fever virus antigen is between 100: 0.3--100: 30, the immune protective effect that pig blue-ear disease antigen produces all can be fine.But along with the increasing of swine fever antigen amount, when ratio is less than 100: 30, the immune effect of pig blue-ear disease antigen can be interfered, the Immunization protective rate of pig blue-ear disease vaccine is caused to reduce.Specifically referring to table 8.
By the counteracting toxic substances protection result of highly pathogenic PRRS strong virus attack piglet after table 8 vaccine immunity
| Group (TCID 50Ratio) | Body temperature (>=40.5 DEG C) | Clinical symptoms | Pathological change | Protective rate |
| 2(100∶0.3) | 1/5(1) | 0/5 | 0/5 | 5/5 |
| 3(100∶1) | 0/5 | 0/5 | 0/5 | 5/5 |
| 4(100∶3) | 0/5 | 0/5 | 0/5 | 5/5 |
| 5(100∶10) | 0/5 | 0/5 | 0/5 | 5/5 |
| 6(100∶30) | 1/5(3) | 1/5 | 1/5 | 4/5 |
| 7(100∶100) | 3/5(5) | 3/5 | 2/5 | 2/5 |
| 8(1∶0) | 0/5 | 0/5 | 0/5 | 5/5 |
| 9 (matched group is without antigens) | 5/5(≥7) | 4/5 | 5/5 | 0/5 |
Note: the natural law that () interior numeral is had a fever continuously
Find with after swine fever crossdrift system strong virus attack; when swine fever virus antigenic content is low; the immune effect of reproductive and respiratory syndrome antigen meeting severe jamming swine fever antigen; along with swine fever antigen content increases; interference effect fades away; but after exceeding certain limit, swine fever antigen also can be subject to the interference of pig blue-ear disease antigen, thus the immune protective effect of swine fever antigen is caused to reduce.When the optimal proportion of two kinds of antigens is between 100: 3--100: 10, without any interference effect between two kinds of antigens.Specifically refer to table 9.
By the counteracting toxic substances protection result of swine fever crossdrift system strong virus attack piglet after table 9 vaccine immunity
| Group | Body temperature (>=40.5 DEG C) | Clinical symptoms | Pathological change | Protective rate |
| 1(0∶1) | 0/5 | 0/5 | 0/5 | 5/5 |
| 2(100∶0.3) | 3/5(5) | 3/5 | 3/5 | 2/5 |
| 3(100∶1) | 2/5(4) | 1/5 | 2/5 | 3/5 |
| 4(100∶3) | 1/5(3) | 0/5 | 0/5 | 5/5 |
| 5(100∶10) | 0/5 | 0/5 | 0/5 | 5/5 |
| 6(100∶30) | 2/5(3) | 1/5 | 2/5 | 3/5 |
| 7(100∶100) | 3/5(5) | 3/5 | 3/5 | 2/5 |
| 9 (matched group is without antigens) | 5/5(≥7) | 5/5 | 5/5 | 0/5 |
Note: the natural law that () interior numeral is had a fever continuously
Effect evaluation (protest test) is carried out after embodiment 6 swine fever, pig blue-ear disease (TJM-F92) bivalent vaccine composition immune animal.
1. vaccine immunity: choose the 4-6 piglet in age in week 90 that swine fever, pig blue-ear disease antibody are double-negative, be divided into 9 groups at random, the 1st group 10, immune commercial goods swine Fever Vaccine 1 immunizing dose/head (10
4.5tCID
50/ head) (Pulaike Biological Engineering Co., Ltd. produces, lot number 1201004), 8th group 10, immunity commercial goods pig blue-ear disease attenuated vaccine (TJM-F92 strain Jilin Special Research Biological Technology Co., Ltd., product batch number 2011120701), immunity 1 doses (10
5.0tCID
50/ head part), the 2nd group to the 7th group each 10, swine fever, pig blue-ear disease Combined vaccine compositions respectively after immune different ratio; 9th group is not immune matched group 10, and observed after immunization piglet clinical response, result occurs without any untoward reaction situation.
2. protest test: by the piglet after vaccine immunity, the corresponding vaccine quality standard issued according to country carries out protest test, by immune group pig and matched group pig, uses the strong poison (10 of swine fever crossdrift system respectively
5mLD), the strong poison (10 of pig blue-ear disease TJ strain
4.5tCID
50/ ml) attack of 2ml collunarium, after counteracting toxic substances, every day carries out body temperature measurement and clinicing symptom observation, and carries out cuing open inspection respectively at after counteracting toxic substances 16 days, 21 days, carries out each internal organs Pathologic Observation.Immunoprotection result is judged according to clinical symptoms, pathological change.
The grouping of table 10 vaccine protest test and process
Find with after pig high-pathogenicity blue ear disease TJ strain strong virus attack, when the ratio of pig blue-ear disease antigen and swine fever virus antigen is between 100: 0.3--100: 10, the immune protective effect that pig blue-ear disease antigen produces all can be fine.But along with the increasing of swine fever antigen amount, when ratio is less than 100: 30, the immune effect of pig blue-ear disease antigen can be interfered, the Immunization protective rate of pig blue-ear disease vaccine is caused to reduce.Specifically refer to table 11.
By the counteracting toxic substances protection result of highly pathogenic PRRS strong virus attack piglet after table 11 vaccine immunity
| Group (TCID 50Ratio) | Body temperature (>=40.5 DEG C) | Clinical symptoms | Pathological change | Protective rate |
| 2(100∶0.3) | 2/5(2) | 0/5 | 0/5 | 5/5 |
| 3(100∶1) | 0/5 | 0/5 | 0/5 | 5/5 |
| 4(100∶3) | 0/5 | 0/5 | 0/5 | 5/5 |
| 5(100∶10) | 0/5 | 0/5 | 0/5 | 5/5 |
| 6(100∶30) | 2/5(4) | 2/5 | 2/5 | 3/5 |
| 7(100∶100) | 3/5(4) | 3/5 | 2/5 | 2/5 |
| 8(1∶0) | 0/5 | 0/5 | 0/5 | 5/5 |
| 9 (matched group is without antigens) | 5/5(≥7) | 4/5 | 5/5 | 0/5 |
Note: the natural law that () interior numeral is had a fever continuously
Find with after swine fever crossdrift system strong virus attack; when swine fever virus antigenic content is low; the immune effect of reproductive and respiratory syndrome antigen meeting severe jamming swine fever antigen; along with swine fever antigen content increases; interference effect fades away; but after exceeding certain limit, swine fever antigen also can be subject to the interference of pig blue-ear disease antigen, thus the immune protective effect of swine fever antigen is caused to reduce.When the optimal proportion of two kinds of antigens is between 100: 3--100: 10, without any interference effect between two kinds of antigens.Specifically refer to table 12.
By the counteracting toxic substances protection result of swine fever crossdrift system strong virus attack piglet after table 12 vaccine immunity
| Group | Body temperature (>=40.5 DEG C) | Clinical symptoms | Pathological change | Protective rate |
| 1(0∶1) | 0/5 | 0/5 | 0/5 | 5/5 |
| 2(100∶0.3) | 3/5(5) | 3/5 | 3/5 | 2/5 |
| 3(100∶1) | 2/5(5) | 2/5 | 2/5 | 3/5 |
| 4(100∶3) | 1/5(2) | 0/5 | 0/5 | 5/5 |
| 5(100∶10) | 0/5 | 0/5 | 0/5 | 5/5 |
| 6(100∶30) | 2/5(4) | 1/5 | 2/5 | 3/5 |
| 7(100∶100) | 3/5(6) | 3/5 | 3/5 | 2/5 |
| 9 (matched group is without antigens) | 5/5(≥7) | 5/5 | 5/5 | 0/5 |
Note: the natural law that () interior numeral is had a fever continuously
By being prepared into vaccine combination to after three kinds of reproductive and respiratory syndrome variant attenuated IBDVs virus antigens and swine fever antigen different ratio; protest test all confirms in certain ratio range; namely, when swine fever, pig blue-ear disease antigen ratio are listed between 100: 3-100: 10, the phenomenon that immune interference does not occur two kinds of antigens can be reached completely.
Attached: swine fever, PRRS virus morbidity standard and counteracting toxic substances protection criterion
Piglet morbidity standard after 1.1 reproductive and respiratory syndrome virus counteracting toxic substances: 1. body temperature at least 3rd reaches more than 41 DEG C, or occurs that spiritual appetite declines, eye conjunctivitis, the respiratory symptom such as to cough, breathe heavily.2. substantially cuing open inspection, there is lamellar consolidation in pulmonary.Meet above 2, be judged to morbidity.
Counteracting toxic substances protection criterion after 1.2 PRRS virus counteracting toxic substances: contrast pig 5/5 falls ill, and at least dead 2, immune swine should at least 4/5 head protection.
After 1.3 swine fever virus counteracting toxic substances, piglet morbidity criterion is: after pig strong virus attack, body temperature is elevated to more than 41 DEG C, in delaying pattern of fever.Incubation period is 24 ~ 84 hours, and loss of appetite or absolutely useless, has loose bowels, generally dead in 5 ~ 9 days.The course of disease is the shortest 4 days, the longest 13.5 days.
Piglet counteracting toxic substances protection criterion after 1.4 swine fever virus counteracting toxic substances: will with sterile saline
often head part vaccine dilutes 3000 times, intramuscular injection without the healthy susceptible pig 2 of swine fever neutralizing antibody, every 1ml.After 10 ~ 14 days, together with contrast pig 3, injection swine fever crossdrift system blood poison 1ml (10
5minimum lethal dose), observe 16.Contrast pig all falls ill, and at least dead 2, immune swine is all strong lives or slightly body temperature reaction, but is qualified without swine fever clinical symptoms.
The preparation method of embodiment 7 pig blue-ear disease classical strains
1. the going down to posterity and cultivation of seedling cell: Marc-145 or MA104 cell line disperseed to go down to posterity through EDTA-trypsinization liquid, continues to cultivate with cell growth medium, when forming fine and close monolayer, for subsequent use, goes down to posterity or virus inoculation for continuing.
2. the breeding of the classical strain virus of pig blue-ear disease: by reproductive and respiratory syndrome CH-1R strain (commercially available) virus liquid, the cell bottle of good cell monolayer has been grown into by the dosage access of m.o.i.=0.001-0.01, preferably connecing venomous scheme amount is further M.O.I.=0.001-0.005, adsorb after 1 hour, add cell maintenance medium in cell line monolayer, continue to cultivate, gather in the crops after pathological changes appears in 70-80% cell, venom freeze thawing 1-2 rearmounted less than-20 DEG C preservations of results, take a morsel and do the inspection of semifinished product, antigen liquid viral level 10
8.0tCID
50/ ml, all conforms with the regulations according to national standard inspection.
By reproductive and respiratory syndrome R98 strain (commercially available) virus liquid, the cell bottle of good cell monolayer has been grown into by the dosage access of m.o.i.=0.001-0.01, preferably connecing venomous scheme amount is further M.O.I.=0.001-0.005, adsorb after 1 hour, add cell maintenance medium in cell line monolayer, continue to cultivate, gather in the crops after pathological changes appears in 70-80% cell, venom freeze thawing 1-2 rearmounted less than-20 DEG C preservations of results, take a morsel and do the inspection of semifinished product, antigen liquid viral level 10
7.5tCID
50/ ml, all conforms with the regulations according to national standard inspection.
Bivalent vaccine composition is prepared into after embodiment 8 swine fever, pig blue-ear disease (CH-1R) antigen and swine fever virus antigen different ratio
By the swine fever virus antigen (viral level 10 prepared
6.5tCID
50/ ml), PRRS virus antigen (viral level 10
8.0tCID
50/ ml, carries out proportioning according to different antigenic contents, then mixes with the freeze drying protectant of equivalent, and make pig blue-ear disease, swine fever bigeminal live vaccine vaccine combination, concrete condition is in table 13:
The different ratio of table 13 pig blue-ear disease antigen, swine fever antigen and grouping
Mixed successively by above-mentioned antigen, be then that the amount of 1: 1 adds the mixed liquor of 2wt% gelatin and 15wt% lactose as freeze drying protectant by volume, stirring and evenly mixing carries out aseptic subpackaged after 1 hour, and 2-8 DEG C of preservation is for subsequent use.
Embodiment 9 carries out effect evaluation (protest test) by after the vaccine combination immune animal made
1. vaccine immunity: choose the 4-6 piglet in age in week 90 that swine fever, pig blue-ear disease antibody are double-negative, be divided into 9 groups at random, 1st group 10, immunity commercial goods pig blue-ear disease attenuated vaccine 1 doses/head (Bo Linge lot number 245-D03B) 1 immunizing dose/head, 8th group 10, immune commercial goods swine Fever Vaccine 10
4.5tCID
50/ head (Pulaike Biological Engineering Co., Ltd.'s lot number 1201004), the 2nd group to the 7th group each 10, swine fever, pig blue-ear disease (CH-1R) Combined vaccine vaccine combination respectively after immune different ratio; 9th group is not immune matched group 10, and observed after immunization piglet clinical response, result occurs without any untoward reaction situation.
2. protest test: by the piglet after vaccine immunity, the corresponding vaccine quality standard issued according to country carries out protest test, by immune group pig and matched group pig, uses the strong poison (10 of swine fever crossdrift system respectively
5mLD), the strong poison (10 of the strong malicious CH-1R of pig blue-ear disease
5.0TcID
50/ ml) to attack, after counteracting toxic substances, every day carries out body temperature measurement and clinicing symptom observation, and carries out cuing open inspection respectively at after counteracting toxic substances 16 days, 21 days, carries out each internal organs Pathologic Observation.Judge immunoprotection result according to clinical symptoms, pathological change, concrete grouping situation is in table 14:
Table 14 vaccine combination efficacy test test grouping and process
Find with after pig high-pathogenicity blue ear disease CH-1R strong virus attack, when the ratio of pig blue-ear disease antigen and swine fever virus antigen is between 100: 1--10000: 1, the immune protective effect that pig blue-ear disease antigen produces all can be fine.But along with swine fever antigen measures increasing, when ratio is less than 30: 1, the immune effect of pig blue-ear disease antigen can be interfered, the Immunization protective rate of pig blue-ear disease vaccine is caused to reduce.Concrete condition refers to table 15.
Attack the counteracting toxic substances protection result of piglet with the strong poison (CH-1R) of pig blue-ear disease after table 15 vaccine combination immunity piglet
Note: the natural law that () interior numeral is had a fever continuously
Find with after swine fever crossdrift system strong virus attack; when swine fever virus antigenic content is low; the immune effect of reproductive and respiratory syndrome antigen meeting severe jamming swine fever antigen; along with swine fever antigen content increases; interference effect fades away; but after exceeding certain limit, swine fever antigen also can be subject to the interference of pig blue-ear disease antigen, thus the immune protective effect of swine fever antigen is caused to reduce.When the optimal proportion of two kinds of antigens is between 100: 1--300: 1, without any interference effect between two kinds of antigens.Specifically refer to table 16.
By the counteracting toxic substances protection result of swine fever crossdrift system strong virus attack piglet after table 16 vaccine combination immunity piglet
Note: the natural law that () interior numeral is had a fever continuously
Embodiment 10 is according to such scheme; we have also carried out counteracting toxic substances protection evaluated the swine fever prepared, pig blue-ear disease (R98 strain) bivalent vaccine composition; find with after pig high-pathogenicity blue ear disease CH-1R strong virus attack; when the ratio of pig blue-ear disease antigen and swine fever virus antigen is between 100: 1--10000: 1, the immune protective effect that pig blue-ear disease antigen produces all can be fine.But along with swine fever antigen measures increasing, when ratio is less than 30: 1, the immune effect of pig blue-ear disease antigen can be interfered, the Immunization protective rate of pig blue-ear disease vaccine is caused to reduce.Result is as table 17:
Attack the counteracting toxic substances protection result of piglet with the strong poison (CH-1R) of pig blue-ear disease after table 17 vaccine combination immunity piglet
Note: the natural law that () interior numeral is had a fever continuously
Find with after swine fever crossdrift system strong virus attack; when swine fever virus antigenic content is low; the immune effect of reproductive and respiratory syndrome antigen meeting severe jamming swine fever antigen; along with swine fever antigen content increases; interference effect fades away; but after exceeding certain limit, swine fever antigen also can be subject to the interference of pig blue-ear disease antigen, thus the immune protective effect of swine fever antigen is caused to reduce.When the optimal proportion of two kinds of antigens is between 100: 1--300: 1, without any interference effect between two kinds of antigens.Specifically refer to table 18.
By the counteracting toxic substances protection result of swine fever crossdrift system strong virus attack piglet after table 18 vaccine combination immunity piglet
Note: the natural law that () interior numeral is had a fever continuously
As what report in document, reproductive and respiratory syndrome antigen can disturb swine fever antigen to produce the speed of antibody, and the time that after showing as immunity, hog cholera antibody produces and peak value all can be postponed.The present invention is immunity after different ratio, and find, along with the continuous increase of swine fever virus antigenic content, its immune interference phenomenon weakens gradually, and when pig blue-ear disease antigen and swine fever virus antigenic content ratio are less than 30: 1, immune interference phenomenon still exists.But surprisingly, when classical strain (CH-1R) antigen of swine fever virus antigen and PRRS virus with 100: 1-300: 1 amount carry out proportioning time, immune interference phenomenon can be eliminated completely.
Beneficial effect of the present invention
1. have found the optimum proportioning that Combined vaccine immunity does not produce interference, make the real noiseless effect of the generation of immune latter two antibody;
2. greatly reduce immune time and Animal stress reaction, improve the economic benefit of animal.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (3)
1. a vaccine combination, described vaccine combination comprises swine fever virus antigen and pig blue-ear disease antigen, wherein, to be that swine fever rabbit is weak poison strain to described swine fever virus, described pig blue-ear disease antigen is the virus that CH-1R strain or R98 strain are lived, and described pig blue-ear disease antigen and swine fever virus antigenic content ratio are 100:1-300:1.
2. prepare a method for vaccine combination described in claim 1, described preparation method comprises:
Cultivate propagation swine fever virus, prepare described swine fever virus antigen, described pig blue-ear disease antigen is the virus that CH-1R strain or R98 strain are lived;
Cultivate propagation PRRS virus, prepare described PRRS virus antigen;
Carry out proportioning according to described pig blue-ear disease antigen and swine fever virus antigenic content ratio 100:1-300:1, prepare described vaccine combination.
3. vaccine combination according to claim 1 prevents the application of swine fever and pig blue-ear disease medicine in preparation.
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