CN102716479A - Pig breeding and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus disease duplex inactivated vaccine and preparation method and application thereof - Google Patents
Pig breeding and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus disease duplex inactivated vaccine and preparation method and application thereof Download PDFInfo
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Abstract
The invention particularly relates to a pig breeding and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus disease duplex inactivated vaccine and a preparation method and application thereof. The pig breeding and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus disease duplex inactivated vaccine is prepared by enabling pig breeding and respiratory syndrome virus NVDC-JXA1 strain to inoculate with a Marc-145 cell, enabling a porcine parvovirus virulent strain to inoculate with an ST cell, respectively obtaining infection cell culture fluid to be inactivated by using formaldehyde solution, and conducting ultrafiltration, concentration and mixing to obtain mixed virus liquid to be mixed and emulsified with oil adjuvant. The inactivated vaccine comprises, by weight, 1-2 parts of the oil adjuvant and 1 part of mixed virus liquid aqueous phase, the inactivated vaccine is prepared by preparing pig breeding and respiratory syndrome virus liquid, porcine parvovirus liquid, an oil phase, the aqueous phase and emulsifying, and NVDC-JXA1 strain pig breeding and respiratory syndrome-porcine parvovirus disease duplex inactivated vaccine quality standards are built.
Description
Technical field
The present invention relates to the veterinary biologics technical field, be specifically related to a kind of Porcine reproductive and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus bivalent inactivated vaccine.
Background technology
Porcine reproductive and respiratory syndrome (PRRS); Claim reproductive and respiratory syndrome again; Be a kind of height contagious disease of the pig that caused by porcine reproductive and respiratory syndrome virus (PRRSV), the pig of all ages and classes, kind and sex all can infect, but with in-pig and 1 monthly age with the susceptible of interior piglet.This disease is a principal character with sow miscarriage, stillborn fetus, weak tire, mummy tire and piglet dyspnea, septicemia, high mortality etc.Owing to still do not have this sick specific medicament of treatment at present, therefore use specificity vaccine to become the major measure of prevention and control PRRS.Developed the vaccine of two types of inactivated vaccine and attenuated live vaccines at present both at home and abroad, it is generally acknowledged the attenuated live vaccines good immune effect, and the inactivated vaccine safety has been better.Inactivated vaccine is the vaccine of early developing, and uses the main pig body generation humoral immune reaction that stimulates in back.Because of it is dead Seedling, so biggest advantage is safety, diffusing poison, is convenient to store and transportation.Bayer company has released inactivated vaccine PRRomiSetM first in 1997; The existing 20 tame enterprises of China are produced the PRRS inactivated vaccine by Ministry of Agriculture's approval at present: CH-1a strain (biotechnology development company of Harbin dimension section) and NVDC-JXA1 strain (19 families such as Guangdong Dahuanong Animal Health Products Co., Ltd.); Be mainly used in the immunity on the negative pig farm of PRRS, control PRRS is played a role with the reduction economic loss.
Porcine parvovirus is that the breeding difficulty of the boar that caused by pig parvoviral (PPV) is sick; With infected the sow particularly negative multiparity sow of first farrowing sow and serology output stillborn fetus, monster, mummy tire, weak son, and the sow non-evident sympton is a characteristic.Pig parvoviral is a kind of very obstinate cause of disease, is distributed widely in all over the world, and swinery infects the back and is difficult to eradicate, and causes enormous economic loss to pig industry.At present; This disease is polluted very serious in China, positive rate reaches more than 90%, almost is difficult to the pig farm of finding a PPV negative; And should disease normal and breeding difficulty disease mixed infections such as pig circular ring virus 2 viral disease, pig blue-ear disease, porcine pseudorabies have become one of more common swine diseases of China at present.Prove that through blood clotting inhibition test and serum neutralization test the PPV that is separated to of various places belongs to a serotype together so far.Primary disease does not still have the efficacious therapy method at present, but because the serotype of PPV is single and immunogenicity is high, so vaccination remains generally acknowledged effective prevention and control measure, can to boar particularly the reserve boar carry out vaccination and prevent primary disease.The vaccine of at present having succeeded in developing also large scale investment use has inactivated vaccine and attenuated live vaccines, and main use at present is the PPV inactivated vaccine.From beginning in 1976, external just report relevant for the PPV Inactivated Vaccine, and in the eighties in 20th century in national widespread usage such as the U.S., Australia, France.China Pan Xue pearl (1987) has at first successfully been developed the PPV inactivated vaccine, and after this Wu Jie, Han Xiaocheng, Xiao Chi, leaf priority such as face south is successfully developed the PPV inactivated vaccine.The pig parvoviral vaccine that present China has obtained new veterinary drug certificate has: the porcine parvovirus inactivated vaccines (wH-1 strain) of the pig parvoviral oil emulsion inactivated vaccine of academy of agricultural sciences, Shanghai animal and veterinary Research Institute, Wuhan Zhongbo Biochemical Co., Ltd's porcine parvovirus inactivated vaccines (CP-99 strain), Hua Zhong Agriculture University, Wuhan Keqian Animal Biological Products Co., Ltd., Zhongmu Industry Co.,Ltd's joint research and development.Important function has been brought into play in the prevention and control that are applied as China's porcine parvovirus of these vaccines.
In order effectively to prevent above-mentioned 2 kinds of eqpidemic diseases, in the routine immunization program is all listed the vaccine of these 2 kinds of infectious disease by each enterprise of raising pigs greatly.Because of only being arranged at present on the market, sells single Seedling; To prevent this 2 kinds of eqpidemic diseases simultaneously; The same age bracket pig only often needs immune 2 pins; This makes the immune density of each age group pig increase, and the expense of vaccine, the manpower when annotating Seedling and to pig cause stress and the short time high density annotate Seedling and cause the phase mutual interference between each vaccine, the pig farm operator is suffered untold misery.Along with the development of China's intensive pig production industry, traditional univalent vaccine can't satisfy immune needs, presses for to prevent many sick vaccines by a pin; Therefore, in order to satisfy the epidemic prevention demand of pig industry, develop a kind of Porcine reproductive and respiratory syndrome-porcine parvovirus bivalent inactivated vaccine safe, stable and can " pin prevent two diseases " and seem particularly urgent and important.
Summary of the invention
For overcoming the defective of prior art; The object of the present invention is to provide a kind of Porcine reproductive and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus bivalent inactivated vaccine; Make each antigenic component in the vaccine all reach the immune effect of corresponding single Seedling, can prevent sick and these the two kinds of eqpidemic diseases of porcine parvovirus of Porcine reproductive and respiratory syndrome simultaneously.
Another object of the present invention is to provide the method for preparing of a kind of Porcine reproductive and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus bivalent inactivated vaccine.
Another purpose of the present invention is to set up Porcine reproductive and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus bivalent inactivated vaccine quality standard with Porcine reproductive and respiratory syndrome-porcine parvovirus bivalent inactivated vaccine.
The technical scheme that is adopted for the present invention of realization above-mentioned purpose is following:
A kind of Porcine reproductive and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus bivalent inactivated vaccine; It is by porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain inoculation Marc-145 cell; Pig parvoviral virulent strain inoculation ST cell is gathered in the crops the infection cell culture fluid respectively and behind formalin deactivation, ultrafiltration and concentration, is mixed the hybrid virus liquid that obtains and process with oily adjuvant mixing and emulsifying; It is made up of 1-2 part oil adjuvant and 1 part of hybrid virus liquid water in weight portion.
Oily adjuvant described in the present invention is by the injection white oil in weight portion: 90-95 part, Si Ben-80:5-8 part, aluminium stearate 1-3 part are formed.
The liquid of hybrid virus described in the present invention water is made up of 95-98 part hybrid virus liquid, 2-5 part tween 80 in weight portion.
Hybrid virus liquid of the present invention is that spissated porcine reproductive and respiratory syndrome virus infection cell culture fluid and spissated pig parvoviral infection cell culture fluid are the proportioning concentration of 1:1 according to volume ratio.
Wherein before the deactivation, the PRRSV viral level is 2 * 10 in the porcine reproductive and respiratory syndrome virus infection cell culture fluid
6-7TCID
50/ ml, the PPV viral level is 2 * 10 in the pig parvoviral infection cell culture fluid
6-7TCID
50/ ml.
The method for preparing of a kind of Porcine reproductive and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus bivalent inactivated vaccine, it may further comprise the steps:
1) preparation of porcine reproductive and respiratory syndrome virus liquid: the Marc-145 cell that porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain 5% amount inoculation has by volume been grown up to monolayer; Rotating and culturing; When cytopathy appears in 70% above cell, results porcine reproductive and respiratory syndrome virus infection cell culture fluid, freeze thawing; Through centrifugal or remove by filter cell debris; Reuse formalin is carried out inactivation treatment, is concentrated into the half the of original volume through ultrafiltration and concentration more afterwards, obtains concentrating porcine reproductive and respiratory syndrome virus liquid;
2) preparation of pig parvoviral liquid: adopting synchronous inoculation method, when the ST cell divides bottle to go down to posterity, is that 1～2% amount is inoculated pig parvoviral virulent strain synchronously by volume; When being cultured to 70% above cell and cytopathy occurring, results pig parvoviral infection cell culture fluid, freeze thawing; Through centrifugal or remove by filter cell debris; Reuse formalin is carried out inactivation treatment, is concentrated into the half the of original volume through ultrafiltration and concentration more afterwards, obtains concentrating pig parvoviral liquid;
3) oil phase preparation: with the injection white oil: 90-95 part, Si Ben-80:5-8 part are mixed post-heating, are added aluminium stearate 1-3 part again, with add be stirred to transparent till, make oil phase after the sterilization;
4) the concentrated pig parvoviral liquid water preparation: the concentrated porcine reproductive and respiratory syndrome virus liquid and the step 2 of getting step 1)) is 1: 1 mixed by volume; Stir, obtain hybrid virus liquid, get hybrid virus liquid 95-98 part then; The cooled tween 80 2-5 part of sterilizing; Fully stir, dissolve fully, make water until tween 80;
5) emulsifying: get oil phase 1-2 part and put stirring in the emulsifying filling, slowly add 1 part of water again, stirring makes mixture fully emulsified, processes Porcine reproductive and respiratory syndrome-porcine parvovirus bivalent inactivated vaccine.
The temperature of rotating and culturing is 37 ℃ in the step 1), rotary speed be 8～9 change/hour, used culture fluid is to contain the DMEM cell culture fluid that mass concentration is 2% hyclone; Using final concentration is that 0.1% formalin is carried out 37 ℃ of inactivation treatment, and inactivation time is 16-20 hour.
Temperature is 37 ℃ when cultivating step 2), and incubation time is 72～96 hours; Using final concentration is that 0.1% formalin was in 37 ℃ of inactivation treatment 15-20 hours.
Mixing speed is 3000r/min in the step 5), and mixing time is 30-60 minute.
Porcine reproductive and respiratory syndrome NVDC-JXA1 strain of the present invention-porcine parvovirus bivalent inactivated vaccine is applied to set up Porcine reproductive and respiratory syndrome-porcine parvovirus bivalent inactivated vaccine quality standard.
The method for using of bivalent inactivated vaccine of the present invention is: 4～6 monthly ages of first farrowing sow immunity 1 time, and booster immunization is 1 time after 2～3 weeks; The multiparity sow is in breeding immunity in preceding 1 month 1 time; Breeding boar is exempted from 6 monthly age head, immunity in later per 6 months 1 time.Each intramuscular injection, the 2ml/ head.
Beneficial effect of the present invention is:
1. Porcine reproductive and respiratory syndrome NVDC-JXA1 strain of the present invention-porcine parvovirus bivalent inactivated vaccine is a kind of Seedling that joins more; Each antigenic component in the vaccine all reaches the immune effect of corresponding single Seedling; Can prevent sick and these the two kinds of eqpidemic diseases of porcine parvovirus of Porcine reproductive and respiratory syndrome simultaneously; Reduce the immune density of each age group pig and the expense of vaccine; Manpower when having avoided annotating Seedling and to pig cause stress and the short time high density annotate Seedling and cause the phase mutual interference between each vaccine, accomplish " pin is prevented two diseases ";
2. method for preparing of the present invention is simple to operation, is easy to be extended and applied;
3. the present invention has set up the quality standard of Porcine reproductive and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus bivalent inactivated vaccine in China first.
Embodiment below in conjunction with concrete is done further explain to the present invention.
The specific embodiment
The method for preparing of a kind of Porcine reproductive and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus bivalent inactivated vaccine, it may further comprise the steps:
1) preparation of porcine reproductive and respiratory syndrome virus liquid: the Marc-145 cell that porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain 5% amount inoculation has by volume been grown up to monolayer; Rotating and culturing; When cytopathy appears in 70% above cell, results porcine reproductive and respiratory syndrome virus infection cell culture fluid, freeze thawing; Through centrifugal or remove by filter cell debris; Reuse formalin is carried out inactivation treatment, is concentrated into the half the of original volume through ultrafiltration and concentration more afterwards, obtains concentrating porcine reproductive and respiratory syndrome virus liquid;
2) preparation of pig parvoviral liquid: adopting synchronous inoculation method, when the ST cell divides bottle to go down to posterity, is that 1～2% amount is inoculated pig parvoviral virulent strain synchronously by volume; When being cultured to 70% above cell and cytopathy occurring, results pig parvoviral infection cell culture fluid, freeze thawing; Through centrifugal or remove by filter cell debris; Reuse formalin is carried out inactivation treatment, is concentrated into the half the of original volume through ultrafiltration and concentration more afterwards, obtains concentrating pig parvoviral liquid;
3) oil phase preparation: with the injection white oil: 90-95 part, Si Ben-80:5-8 part are mixed post-heating, are added aluminium stearate 1-3 part again, with add be stirred to transparent till, make oil phase after the sterilization;
4) the concentrated pig parvoviral liquid water preparation: the concentrated porcine reproductive and respiratory syndrome virus liquid and the step 2 of getting step 1)) is 1: 1 mixed by volume; Stir, obtain hybrid virus liquid, get hybrid virus liquid 95-98 part then; The cooled tween 80 2-5 part of sterilizing; Fully stir, dissolve fully, make water until tween 80;
5) emulsifying: get oil phase 1-2 part and put stirring in the emulsifying filling, slowly add 1 part of water again, stirring makes mixture fully emulsified, processes Porcine reproductive and respiratory syndrome-porcine parvovirus bivalent inactivated vaccine.
It below is the preferred embodiment of the invention.
Embodiment 1
The preparation of a kind of Porcine reproductive and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus bivalent inactivated vaccine; Carry out according to following steps: the Marc-145 cell that 1) porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain 5% amount inoculation has by volume been grown up to monolayer; In 37 ℃ with 8 change/hour the rotating speed rotating and culturing; When cytopathy appears in 70% above cell, results porcine reproductive and respiratory syndrome virus infection cell culture fluid, freeze thawing 2 times; Through centrifugal or remove by filter cell debris; The reuse final concentration be 0.1% formalin in 37 ℃ of deactivations 18 hours, ultrafiltration and concentration to original volume half the again obtains concentrating porcine reproductive and respiratory syndrome virus liquid afterwards; 2) adopt synchronous inoculation method; When the ST cell divides bottle to go down to posterity, be that 2% amount is inoculated pig parvoviral CG03 strain strain synchronously by volume, when being cultured to 70% above cell and cytopathy occurring; Results pig parvoviral infection cell culture fluid; Freeze thawing 2 times, through centrifugal or remove by filter cell debris, the reuse final concentration is that 0.1% formalin was in 37 ℃ of deactivations 16 hours; Be concentrated into the half the of original volume through ultrafiltration and concentration more afterwards, obtain concentrating pig parvoviral liquid; 3) with the injection white oil: 94 parts, Si Ben-80:6 part is mixed post-heating, is added 2 parts of aluminium stearate again, with add be stirred to transparent till, make oil phase after the sterilization; 4) get that to concentrate porcine reproductive and respiratory syndrome virus liquid and concentrated pig parvoviral liquid be 1: 1 mixed by volume; Stir, obtain hybrid virus liquid, get 96 parts of hybrid virus liquid then; The cooled 4 parts of tween 80s of sterilizing; Fully stir, dissolve fully, make water until tween 80; 5) get 1.5 parts of oil phases and put stirring in the emulsifying filling, slowly add 1 part of water again, 3000r/min stirred 30 minutes, made mixture fully emulsified, processed Porcine reproductive and respiratory syndrome-porcine parvovirus bivalent inactivated vaccine.
Embodiment 2
The preparation of a kind of Porcine reproductive and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus bivalent inactivated vaccine; Carry out according to following steps: the Marc-145 cell that 1) porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain 5% amount inoculation has by volume been grown up to monolayer; In 37 ℃ with 9 change/hour the rotating speed rotating and culturing; When cytopathy appears in 70% above cell, results porcine reproductive and respiratory syndrome virus infection cell culture fluid, freeze thawing 2 times; Through centrifugal or remove by filter cell debris; The reuse final concentration be 0.1% formalin in 37 ℃ of deactivations 18 hours, ultrafiltration and concentration to original volume half the again obtains concentrating porcine reproductive and respiratory syndrome virus liquid afterwards; 2) adopt synchronous inoculation method; When the ST cell divides bottle to go down to posterity, be that 2% amount is inoculated pig parvoviral CG03 strain strain synchronously by volume, when being cultured to 70% above cell and cytopathy occurring; Results pig parvoviral infection cell culture fluid; Freeze thawing 2 times, through centrifugal or remove by filter cell debris, the reuse final concentration is that 0.1% formalin was in 37 ℃ of deactivations 16 hours; Be concentrated into the half the of original volume through ultrafiltration and concentration more afterwards, obtain concentrating pig parvoviral liquid; 3) with the injection white oil: 90 parts, Si Ben-80:8 part is mixed post-heating, is added 1 part of aluminium stearate again, with add be stirred to transparent till, make oil phase after the sterilization; 4) get that to concentrate porcine reproductive and respiratory syndrome virus liquid and concentrated pig parvoviral liquid be 1: 1 mixed by volume; Stir, obtain hybrid virus liquid, get 98 parts of hybrid virus liquid then; The cooled 2 parts of tween 80s of sterilizing; Fully stir, dissolve fully, make water until tween 80; 5) get 1 part of oil phase and put stirring in the emulsifying filling, slowly add 1 part of water again, 3000r/min stirred 30 minutes, made mixture fully emulsified, processed Porcine reproductive and respiratory syndrome-porcine parvovirus bivalent inactivated vaccine.
Embodiment 3
The preparation of a kind of Porcine reproductive and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus bivalent inactivated vaccine; Carry out according to following steps: the Marc-145 cell that 1) porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain 5% amount inoculation has by volume been grown up to monolayer; In 37 ℃ with 9 change/hour the rotating speed rotating and culturing; When cytopathy appears in 70% above cell, results porcine reproductive and respiratory syndrome virus infection cell culture fluid, freeze thawing 2 times; Through centrifugal or remove by filter cell debris; The reuse final concentration be 0.1% formalin in 37 ℃ of deactivations 18 hours, ultrafiltration and concentration to original volume half the again obtains concentrating porcine reproductive and respiratory syndrome virus liquid afterwards; 2) adopt synchronous inoculation method; When the ST cell divides bottle to go down to posterity, be that 2% amount is inoculated pig parvoviral CG03 strain strain synchronously by volume, when being cultured to 70% above cell and cytopathy occurring; Results pig parvoviral infection cell culture fluid; Freeze thawing 2 times, through centrifugal or remove by filter cell debris, the reuse final concentration is that 0.1% formalin was in 37 ℃ of deactivations 16 hours; Be concentrated into the half the of original volume through ultrafiltration and concentration more afterwards, obtain concentrating pig parvoviral liquid; 3) with the injection white oil: 95 parts, Si Ben-80:5 part is mixed post-heating, is added 3 parts of aluminium stearate again, with add be stirred to transparent till, make oil phase after the sterilization; 4) get that to concentrate porcine reproductive and respiratory syndrome virus liquid and concentrated pig parvoviral liquid be 1: 1 mixed by volume; Stir, obtain hybrid virus liquid, get 95 parts of hybrid virus liquid then; The cooled 5 parts of tween 80s of sterilizing; Fully stir, dissolve fully, make water until tween 80; 5) get 2 parts of oil phases and put stirring in the emulsifying filling, slowly add 1 part of water again, 3000r/min stirred 30 minutes, made mixture fully emulsified, processed Porcine reproductive and respiratory syndrome-porcine parvovirus bivalent inactivated vaccine.
Porcine reproductive and respiratory syndrome NVDC-JXA1 strain of the present invention-porcine parvovirus bivalent inactivated vaccine is applied to set up Porcine reproductive and respiratory syndrome-porcine parvovirus bivalent inactivated vaccine quality standard, and step is following:
1) character (comprising outward appearance, dosage form, stability, viscosity), steriling test and formaldehyde, antiseptic mercurials residual quantity: measure by " Chinese veterinary drug allusion quotation " appendix, should meet corresponding standard;
2) safety verification: get 3～6 age in week PRRSV antigen, negative antibody and 5 of negative (the HI antibody titer is not higher than 1: 8) piglets of PPV antigen-antibody, every intramuscular injection vaccine 4ml observed 21, the part and the systemic adverse reactions that are caused by vaccine should not occur; With 5 of 3～5 age in days neonatal rats, each subcutaneous injection vaccine 0.1ml observed 10, all should be good for work;
3) efficacy test:
The Porcine reproductive and respiratory syndrome part: with 3～6 age in week 10 of PRRSV antigen, negative antibody pigs, 5 intramuscular injection vaccine 2ml wherein, 5 as contrast, isolated rearing under the equal conditions in addition.After 28 days, all pigs (contain 10 to the strong malicious 3ml of rear portion intramuscular injection PRRSV NVDC-JXA1 strain that picks up the ears
5TCID
50), every day thermometric and observing 21.5 contrast pigs should all fall ill, and at least 2 death, and immune swine should at least 4 strong living;
The pig parvoviral part: following method is appointed and is selected one of which
With 5 of PPV antigen, negative antibody (the HI antibody titer is not higher than 1: the 8) healthy guinea pigs of body weight 350～400g, every vaccinate 0.5ml respectively, after 28 days, together with 4 of the contrast Cavia porcelluss the same terms under, blood sampling, separation of serum, mensuration HI antibody; At least 4 antibody positives of immune guinea pig, its HI antibody titer geometrical mean should be not less than 1: 64, and contrast Cavia porcellus HI antibody titer all is not higher than 1: 8;
With 10 of negative (the HI antibody titer is not higher than 1: 8) pigs of 4～6 PPV antigen-antibodies in age in week, 5 intramuscular injection vaccine 2ml wherein, 5 as contrast in addition, isolated rearing under the equal conditions, after 28 days, all pigs blood samplings, separation of serum is measured HI antibody.At least 4 antibody positives of immune swine, its HI antibody titer geometrical mean should be not less than 1: 64, and contrast pig HI antibody titer all is not higher than 1: 8.
According to above-mentioned quality standard, the Porcine reproductive and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus bivalent inactivated vaccine prepared to embodiments of the invention 1-3 carries out the check of safety and immune efficacy.
The vaccine safety check:
3 batches of vaccines of embodiment of the invention preparation have been carried out safety testing respectively.Content of the test comprise to 3～5 age in days neonatal rats, 3～6 age in week ablactational baby pig, 4～6 monthly age replacement gilts, breeding boar and conceived 30～50 days in-pig carry out the safety testing of 1 single dose inoculation, overdose inoculation, single dose repeated inoculation respectively.Result of the test shows, behind 3 batches of vaccination animals, local responses such as redness, pruritus, ulceration all do not occur; It is normal with the mental status to search for food; Nonsystemic reaction, in-pig are observed childbirth always, the performance of clinical symptoms such as no miscarriage, premature labor, stillborn fetus, weak son; From the blood of its whole fetuses that produce or internal organs, all can not isolate PRRSV and PPV, visible vaccine safety is reliable.
Vaccine immunity is renderd a service:
Get 30 of 6 monthly ages primiparity replacement gilts (PRRSV and PPV antigen, antibody are all negative), be divided into three groups at random, 10 every group; Exempt from the vaccine in the embodiment of the invention 1 in 6 monthly age head for first group, 2～3 week back booster immunizations 1 time; Second group of vaccine in 1 embodiment of the invention 1 of breeding immunity in previous month; The 3rd group not immune as contrast; Each immunity is intramuscular injection, the 2ml/ head; The blood sampling regularly of inoculation back, separation of serum is measured PRRSV antibody horizontal and PPV HI antibody horizontal in the serum; First group of test pig (being first farrowing sow secondary immunity group) exempts from can detect in back 14 days PRRSV antibody and PPV HI antibody in head as a result; Raise gradually subsequently; Immunity was set up fully in 21～28 days; Exempt from the back and the antibody peak occurred in 2～4 months, its antibody horizontal begins to descend after 5 months, reduces to 28 days left and right horizontal to 6 months antibody titers; And the antibody Changing Pattern of second group of test pig (being immune group of first farrowing sow) and first group are basic identical, but the antibody peak value is lower than first group, and level when antibody titer has been reduced to 28 days during to 4 months.It is thus clear that, for the primiparity replacement gilt, only immune 1 time effect before 2 its immune effects of immunity are superior to breeding before breeding.
Learn from else's experience and produce 35 of sows, wherein 20 in this vaccine of breeding immunity in previous month, intramuscular injection, and the 2ml/ head, 15 as contrast, isolated rearing under the equal conditions in addition; The blood sampling regularly of immunity back; Separation of serum is measured PRRSV antibody horizontal and PPV HI antibody horizontal in the serum, and in the time of pregnant 30～50 days; Respectively extract above-mentioned immune swine and contrast pig out 10 at random; Be divided into 2 groups, 5 every group, one group of strong malicious 3ml of ear rear portion intramuscular injection PRRSV NVDC-JXA1 strain (contains 10
5TCID
50), another group musculi colli injection pig parvoviral PPV7909 standard virulent strain 2ml (contains 10
5TCID
50), observe to childbirth; As a result the multiparity sow 1 its immune effect of immunity is with above-mentioned first farrowing sow secondary immunity group before breeding, the antibody peak appears at exempts from afterwards 2～4 months, level when antibody titer is reduced to 28 days during to 6 months; The counteracting toxic substances protection is the result show, all morbidities behind the contrast pig counteracting toxic substances PRRSV, and 2 death, and 4 breeding difficulty symptoms such as miscarriage, stillborn fetus, mummy tire or fetus heavily absorb occur behind the counteracting toxic substances PPV, and equal 5/5 protection of immune swine; Pig that immune swine is farrowed birth back can detect PRRSV antibody and PPV HI antibody at the 3rd day, peaked in the 10th～14 day, and lasting decline of 3～4 week back PRRSV antibody, 6～7 week back PPV HI antibody also begin to continue decline.
Get 6 monthly age 15 of boars (PRRSV and PPV antigen, antibody are all negative); Wherein 10 with the vaccine among 2ml/ the dosage intramuscular injection embodiment 1; 5 conduct contrasts in addition, isolated rearing under the equal conditions, regularly take a blood sample in the immunity back; Separation of serum is measured PRRSV antibody horizontal and PPV HI antibody horizontal in the serum; Boar immunity as a result 1 time antibody Changing Pattern and multiparity sow, booster immunization first farrowing sow basic identical; It is all positive to inoculate back 28 days immune swine PRRSV antibody, and PPV HI tires all>=and 1: 128, and contrast pig PRRSV antibody is all negative; PPV HI antibody all≤1: 8; Exempt from the back and the antibody peak occurred in 2～3 months, but the antibody peak value is lower than the first farrowing sow of multiparity sow and booster immunization, level when antibody titer is reduced to 28 days during to 4 months.
The immune duration that shows vaccine according to above-mentioned result of the test is decided to be 4 months.
Field test:
Field test is carried out on 3 batches of Porcine reproductive and respiratory syndrome-porcine parvovirus bivalent inactivated vaccine subordinate's of Wen Shi group in Guangdong that the embodiment of the invention is prepared pig farm; The test pig kind comprises multiparity sow, replacement gilt, breeding boar; Through observation to the searching for food of swinery after the immunity, drinking-water, body temperature situation; And regular antibody test, the vaccine evaluation immune effect.The result shows, mine massively food, drinking-water, body temperature etc. of immune swine are all no abnormal; The multiparity sow is exempted from the back and can produce strong immunity on 21st～28 in preceding 1 month this vaccine of dosage immunity with the 2ml/ head of breeding, and duration of immunity is 6 months, sustainable 3～4 weeks of the PRRSV maternal antibody of its cub pig, sustainable 6～7 weeks of PPV maternal antibody; Replacement gilt is in 4～6 monthly ages immunity, 1 deuterzooid vaccine, booster immunization 1 time again after 2～3 weeks, and each 2ml/ head, the immune effect of generation is with the multiparity sow; Breeding boar is exempted from this vaccine in 6 monthly age head, the 2ml/ head, and duration of immunity can reach 4 months, immunity in later per 6 months 1 time, duration of immunity reaches 6 months.Through the tracking to the whole production phase, immune swinery does not all have the generation of high-pathogenicity porcine reproductive and respiration syndrome and porcine parvovirus.Show Porcine reproductive and respiratory syndrome-porcine parvovirus bivalent inactivated vaccine that the present invention prepares to immune swine safety, effectively.
Above-mentioned embodiment is merely preferred implementation of the present invention; Can not limit the scope of the present invention's protection with this, the variation of any unsubstantiality that those skilled in the art is done on basis of the present invention and replacement all belong to the present invention's scope required for protection.
Claims (10)
1. Porcine reproductive and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus bivalent inactivated vaccine; It is characterized in that: it is by porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain inoculation Marc-145 cell; Pig parvoviral virulent strain inoculation ST cell is gathered in the crops the infection cell culture fluid respectively and behind formalin deactivation, ultrafiltration and concentration, is mixed the hybrid virus liquid that obtains and process with oily adjuvant mixing and emulsifying; It is made up of 1-2 part oil adjuvant and 1 part of hybrid virus liquid water in weight portion.
2. Porcine reproductive and respiratory syndrome NVDC-JXA1 strain according to claim 1-porcine parvovirus bivalent inactivated vaccine is characterized in that: said oily adjuvant is by the injection white oil in weight portion: 90-95 part, Si Ben-80:5-8 part, aluminium stearate 1-3 part are formed.
3. Porcine reproductive and respiratory syndrome NVDC-JXA1 strain according to claim 2-porcine parvovirus bivalent inactivated vaccine is characterized in that: said hybrid virus liquid water is made up of 95-98 part hybrid virus liquid, 2-5 part tween 80 in weight portion.
4. Porcine reproductive and respiratory syndrome NVDC-JXA1 strain according to claim 3-porcine parvovirus bivalent inactivated vaccine is characterized in that: said hybrid virus liquid is that spissated porcine reproductive and respiratory syndrome virus infection cell culture fluid and spissated pig parvoviral infection cell culture fluid are the proportioning concentration of 1:1 according to volume ratio.
5. Porcine reproductive and respiratory syndrome NVDC-JXA1 strain according to claim 1-porcine parvovirus bivalent inactivated vaccine is characterized in that: wherein before the deactivation, the PRRSV viral level is 2 * 10 in the porcine reproductive and respiratory syndrome virus infection cell culture fluid
6-7TCID
50/ ml, the PPV viral level is 2 * 10 in the pig parvoviral infection cell culture fluid
6-7TCID
50/ ml.
6. the method for preparing of Porcine reproductive and respiratory syndrome NVDC-JXA1 strain-porcine parvovirus bivalent inactivated vaccine, it is characterized in that: it may further comprise the steps
1) preparation of porcine reproductive and respiratory syndrome virus liquid: the Marc-145 cell that porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain 5% amount inoculation has by volume been grown up to monolayer; Rotating and culturing; When cytopathy appears in 70% above cell, results porcine reproductive and respiratory syndrome virus infection cell culture fluid, freeze thawing; Through centrifugal or remove by filter cell debris; Reuse formalin is carried out inactivation treatment, is concentrated into the half the of original volume through ultrafiltration and concentration more afterwards, obtains concentrating porcine reproductive and respiratory syndrome virus liquid;
2) preparation of pig parvoviral liquid: adopt synchronous inoculation method; When the ST cell divides bottle to go down to posterity, be that 1～2% amount is inoculated pig parvoviral virulent strain synchronously by volume, when being cultured to 70% above cell and cytopathy occurring; Results pig parvoviral infection cell culture fluid; Freeze thawing, through centrifugal or remove by filter cell debris, reuse formalin is carried out inactivation treatment; Be concentrated into the half the of original volume through ultrafiltration and concentration more afterwards, obtain concentrating pig parvoviral liquid;
3) oil phase preparation: with the injection white oil: 90-95 part, Si Ben-80:5-8 part are mixed post-heating, are added aluminium stearate 1-3 part again, with add be stirred to transparent till, make oil phase after the sterilization;
4) the concentrated pig parvoviral liquid water preparation: the concentrated porcine reproductive and respiratory syndrome virus liquid and the step 2 of getting step 1)) is 1: 1 mixed by volume; Stir, obtain hybrid virus liquid, get hybrid virus liquid 95-98 part then; The cooled tween 80 2-5 part of sterilizing; Fully stir, dissolve fully, make water until tween 80;
5) emulsifying: get oil phase 1-2 part and put stirring in the emulsifying filling, slowly add 1 part of water again, stirring makes mixture fully emulsified, processes Porcine reproductive and respiratory syndrome-porcine parvovirus bivalent inactivated vaccine.
7. the method for preparing of Porcine reproductive and respiratory syndrome NVDC-JXA1 strain according to claim 6-porcine parvovirus bivalent inactivated vaccine; It is characterized in that: the temperature of rotating and culturing is 37 ℃ in the step 1); Rotary speed be 8～9 change/hour, used culture fluid is to contain the DMEM cell culture fluid that mass concentration is 2% hyclone; Using final concentration is that 0.1% formalin is carried out 37 ℃ of inactivation treatment, and inactivation time is 16-20 hour.
8. the method for preparing of Porcine reproductive and respiratory syndrome NVDC-JXA1 strain according to claim 6-porcine parvovirus bivalent inactivated vaccine is characterized in that: step 2) in when cultivating temperature be 37 ℃, incubation time is 72～96 hours; Using final concentration is that 0.1% formalin was in 37 ℃ of inactivation treatment 15-20 hours.
9. the method for preparing of Porcine reproductive and respiratory syndrome NVDC-JXA1 strain according to claim 6-porcine parvovirus bivalent inactivated vaccine is characterized in that: mixing speed is 3000r/min in the step 5), and mixing time is 30-60 minute.
10. Porcine reproductive and respiratory syndrome NVDC-JXA1 strain as claimed in claim 1-porcine parvovirus bivalent inactivated vaccine is applied to set up Porcine reproductive and respiratory syndrome-porcine parvovirus bivalent inactivated vaccine quality standard.
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