CN101940787A - Adjuvant for improving effect of porcine reproductive and respiratory syndrome inactivated vaccine, preparation method thereof and application thereof - Google Patents
Adjuvant for improving effect of porcine reproductive and respiratory syndrome inactivated vaccine, preparation method thereof and application thereof Download PDFInfo
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Abstract
The invention discloses an adjuvant for improving the effect of a porcine reproductive and respiratory syndrome (PRRSV) inactivated vaccine, a preparation method thereof and application thereof and relates to the preparation method and steps of Hemagglutinating virus of Japan envelope (HVJ-E) and application of a medicament for improving the effect of the PRRSV inactivated vaccine. The preparation method of adjuvant comprises the following steps of: breeding Hemagglutinating virus of Japan (HVJ) by using an SPF chick embryo, and then concentrating, purifying, counting and inactivating the HVJ to prepare the Hemagglutinating virus of Japan envelope (HVJ-E); immunizing PRRSV negative pigs with the HVJ-E and the PRRSV inactivated vaccine together, and setting a PBS contrast group and a PRRSV inactivated vaccine group at the same time, wherein compared with the contrast group, the HVJ-E and PRRSV inactivated vaccine group can obviously improve the response level of hunoral immunity and cellular immunity of the pigs; and after immunizing, expelling toxin by using a PRRSV virulent strain (JXA1), wherein the result shows that the clinical manifestations and the weight increasing of the HVJ-E and PRRSV inactivated vaccine group are better than those of the contrast group, and the generated viremiavirusemia rate, toxin expelling rate and virus distribution rate are obviously reduced when compared with the contrast group. The adjuvant of the invention has the advantages of convenient production and low cost and is suitable for large-scale production.
Description
Affiliated technical field
The invention belongs to field of biological pharmacy, particularly, the present invention relates to preparation and the application of Sendai virus cyst membrane (HVJ-E), refer more particularly to the Sendai virus cyst membrane as pig breeding and respiratory tract obstruction syndrome virus inactivated vaccine immunological adjuvant as immunological adjuvant.
Background technology
Pig breeding and respiratory tract obstruction syndrome (porcine reproductive and respiratory syndrome, PRRS) from 1987 since the U.S., with the respiratory tract obstruction that causes sow miscarriage, weak product, stillbirth, mummy tire and institute has age pig is cardinal symptom, brings the tremendous economic loss for world's pig industry.With China is example, and the annual economic loss that causes because of porcine respiratory and breeding difficulty syndrome reaches more than 200 hundred million yuan.The cause of disease of PRRS is pig breeding and respiratory disorder syndrome virus (PRRSV).PRRSV belongs to the coronaviridae Arterivirus in the 6th meeting report of ICTV, be the underlying stock single-stranded RNA virus, and cyst membrane is arranged, and can be divided into two subgroups, and the A subgroup is an Europe class; The B subgroup is an american type, and at present, the strain of acquisition that China separates belongs to american type mostly.
Being used at present prevent the vaccine of PRRS mainly is inactivated vaccine and weak malicious Seedling.Inactivated vaccine is wide in China's scope of application, and its advantage is that safety is good, and shortcoming is that immunizing dose is big, immune time is many, the immune generation phase is longer.Especially neutralizing antibody speed is slower because the PRRS inactivated vaccine produces antibody; and inactivated vaccine almost can not the inducing cell immunity generation; therefore usually can occur clinically producing the report that non-specific antibody quickens to fall ill because of the inoculation inactivated vaccine; this mainly is that (Antibody dependent effect ADE) causes because the antibody of PRRSV relies on enhancement effect.Therefore, the immunological adjuvant of the effective raising of research PRRS inactivated vaccine immunizing potency is extremely urgent.
Sendai virus (Hemagglutinating virus of Japan, HVJ) belong to Paramyxoviridae, Respirovirus, the sub-thread minus-stranded rna virus, genome total length 15384bp, coding nucleoprotein (NP), phosphoprotein (P), stromatin (M), fusion rotein (F), hemagglutinin neuraminidase (HN) and 6 kinds of structural protein of high molecular weight protein (L) and some non-structural proteins.HVJ is a cytoplasm type sub-thread minus-stranded rna virus, now confirmed Sendai virus cyst membrane (the Hemagglutinating virus of Japan envelope of deactivation, HVJ-E) still can be through the gene induced I receptor identification of retinoic acid pathway activation dendritic cell, activate the natural immunity and acquired immunity, stimulate DC to produce IL-12, IL-6, cytokines such as IFN-β and TNF-α, wherein IL-6 is particularly crucial, it can suppress the activation of regulatory T cells (CD4+CD25+Treg), promote the propagation of CD4+ and CD8+T cell, stop IL-10, the secretion of inhibitive ability of immunity cytokines such as TGF-β, in addition, also can induce expression and the comprehensive NK of the activation cell of Local C XCL10 in the body behind the injection HVJ-E.Therefore, HVJ-E has possessed the potential of comprehensive raising autoimmunity, and has brought into play important effect in antitumor research.
At present, the research of immunological adjuvant is the focus of immunology research, yet do not appear in the newspapers as yet as the research of immunological adjuvant with HVJ-E, based on this true present situation, the present invention adopts HVJ-E as immunologic adjuvant, observes the raising that it can not only promote the anti-PRRSV antibody horizontal of animal, efficiently the generation of the inducing cell factor, alleviate the clinical symptoms behind the animals received strong virus attack, reduce the toxemic generation of zoosis and virus is drained T/A.This technology can be used for the preparation of animal vaccine, especially the preparation and the application of pig breeding and respiratory tract obstruction syndrome inactivated vaccine.
Summary of the invention
At above defective, the invention provides a kind of immunological adjuvant that can improve PRRSV inactivated vaccine immunizing potency.
The present invention also provides a kind of preparation method that can improve the HVJ-E immunological adjuvant of PRRSV inactivated vaccine immunizing potency.
The present invention also provides the pharmaceutical composition that contains described HVJ-E.
The present invention also provides described HVJ-E purposes in improving PRRS inactivated vaccine immunizing potency.
On the one hand, the invention provides a kind of HVJ-E adjuvant that can be used for improving PRRSV inactivated vaccine immunizing potency, wherein said HVJ-E is for to inoculate 10 age in days SPF Embryo Gallus domesticus after ultraviolet inactivation forms through HVJ.
The step that the present invention prepares the HVJ-E immunological adjuvant is as follows:
The first step: HVJ breeding: with containing 1000 times of the two anti-PBS solution dilution kind poison of mycillin, chick embryo allantoic liquid behind the 72h, is collected in inoculated into chick embryo allantocherion chamber.
Second step: the HVJ concentrates: PEG6000 concentrates HVJ.
The 3rd step: HVJ calculating particles: measure tiring of Sendai virus with hemagglutination test, according to the blood clotting valency and 1HAU (the blood clotting valency unit) ≈ 3 * 10 of Sendai virus
6Formula to calculating go out the population of the Sendai virus of purification.
The 4th step: the preparation of HVJ-E: use ultraviolet inactivation HVJ, form HVJ-E.
On the other hand, the invention provides and comprise the pharmaceutical composition that can accept carrier on HVJ-E immunological adjuvant and the materia medica, said composition can be used for improving the immunizing potency of PRRSV inactivated vaccine.
Acceptable carrier can be this area compounding pharmaceutical compositions any diluent, solvent or excipient commonly used on the wherein said materia medica, include but not limited to glucose injection, sterilization redistilled water, normal saline, phosphate buffer (PBS) and sodium phosphate buffer, be preferably PBS.In a preferred embodiment of the invention, HVJ-E of the present invention is dissolved in the PBS injection.
The present invention studies improving behind the PRRSV inactivated vaccine inoculation piglet antibody horizontal and cytokine secretion amount etc. described HVJ-E.
In one embodiment of the invention, be object of study with the negative pig of PRRSV, intramuscular injection HVJ-E of the present invention and PRRS inactivated vaccine carry out the immunity inactivated vaccine of corresponding dosage (inject simultaneously in contrast) simultaneously.Collect before the immunity and the peripheral blood of immunity back different time points (for example immune preceding 0 day and immunity back 7,14,21 and 28 days), detect by peripheral blood mononuclear lymphocyte proliferation assay, ELISA, studied the negative pig of the PRRSV level of anti-PRRSV antibody and changes of cytokine situation in the serum after immunity in great detail.After the immunity 28 days, immune swine is carried out counteracting toxic substances, collect the peripheral blood and the nasal secretion of different time points before and after the counteracting toxic substances (behind preceding 0 day of counteracting toxic substances, the counteracting toxic substances 3,7,14 and 21 days), studied toxin expelling and the viremia situation of experiment pig behind strong virus attack by RT-PCR.Experimental result shows: HVJ-E of the present invention can quicken anti-PRRSV production of antibodies; Promote generation and the release of IL-6 and IFN-γ, suppress the generation of IL-4, improved the immunity of body; Behind the counteracting toxic substances; HVJ-E can significantly improve the clinical symptoms of attacking the back experiment pig; reduce the drainage that experiment pig produces viremia and reduces virus; thereby HVJ-E of the present invention has significant protective effect inducing antibody to produce in advance and improving aspects such as cytokine generation level, and can effectively alleviate the symptoms such as heating, viremia and toxin expelling that strong virus attack brings.This result of the test has confirmed HVJ-E of the present invention to the protective effect of PRRS inactivated vaccine immunostimulant, has promoted the live pig fertility performance to improve, and can effectively avoid now using always the adverse side effects such as inflammatory reaction that cause in the adjuvant.
For achieving the above object, the present invention has adopted following technical scheme: a kind of adjuvant that improves pig breeding and respiratory tract obstruction syndrome inactivated vaccine immunizing potency prepares the Sendai virus cyst membrane with Sendai virus, as immunological adjuvant.
Virus of the present invention comprises following strain:
The Fushimi strain, Aritsugn strain, MN strain, Z strain, BB1 strain, TianJin strain.
Described strain is used to prepare the animal immune adjuvant, especially the immunological adjuvant of pig breeding and respiratory disorder syndrome inactivated vaccine.
Preparation method of the present invention comprises the steps:
1) preparation contains the chick embryo allantoic liquid of Sendai virus;
2) with the allantoic fluid for preparing in the step 1) after PEG6000 concentrates, rebasing with 20% sucrose, the centrifugal 2h of 20000r/min;
3) collecting precipitation, the PBS washing precipitation, 20000r/min recentrifuge 2h removes sucrose;
4) collecting precipitation is suspended in the apyrogeneity normal saline;
5) with 99mJ/cm
2The Sendai virus 2h that collects of uitraviolet intensity deactivation;
6) the Sendai virus called after Sendai virus cyst membrane of deactivation is inoculated 9 age in days SPF Embryo Gallus domesticus with the Sendai virus cyst membrane, behind the 72h, collects chick embryo allantoic liquid, measures the blood clotting valency of allantoic fluid.
Described ablation method is 99mJ/cm
2Ultraviolet inactivation 2h.
After adopting above technical scheme, advantage part of the present invention is:
1. HVJ-E of the present invention and the pharmaceutical composition that contains HVJ-E can excite experimenter's non-specific and specific immunity, strengthen experimenter's self resistivity, thereby to Different Kinds of Pathogens microorganism has the wide spectrum resistant function, can be used for improving the immune effect of vaccine, have advantage of wide range of application.
2. compare with other adjuvants, HVJ-E of the present invention and the pharmaceutical composition that contains HVJ-E do not possess infectivity, have safe and the little characteristics of side effect.
Description of drawings
The invention will be further elaborated below in conjunction with drawings and Examples.
Fig. 1 .pCpG induces the specific PBMC reaction of PRRSV.
Fig. 2. immunity back the 14th day and the 28th day cytokine IFN-γ, the expression of IL-6.
PRRSV specific antibody level behind Fig. 3 .ELISA method detection immunity and the counteracting toxic substances.
Fig. 4. the average rectal temperature of pig behind the counteracting toxic substances.
Fig. 5. respectively organize the weight increase situation of pig behind the counteracting toxic substances.
The specific embodiment
As shown in the figure, a kind of adjuvant that improves pig breeding and respiratory tract obstruction syndrome inactivated vaccine immunizing potency prepares the Sendai virus cyst membrane with Sendai virus, as immunological adjuvant.
Virus of the present invention comprises following strain:
The Fushimi strain, Aritsugn strain, MN strain, Z strain, BB1 strain, TianJin strain.
Described strain is used to prepare the animal immune adjuvant, especially the immunological adjuvant of pig breeding and respiratory disorder syndrome inactivated vaccine.
Preparation method of the present invention comprises the steps:
1) preparation contains the chick embryo allantoic liquid of Sendai virus;
2) with the allantoic fluid for preparing in the step 1) after PEG6000 concentrates, rebasing with 20% sucrose, the centrifugal 2h of 20000r/min;
3) collecting precipitation, the PBS washing precipitation, 20000r/min recentrifuge 2h removes sucrose;
4) collecting precipitation is suspended in the apyrogeneity normal saline;
5) with 99mJ/cm
2The Sendai virus 2h that collects of uitraviolet intensity deactivation;
6) the Sendai virus called after Sendai virus cyst membrane of deactivation is inoculated 9 age in days SPF Embryo Gallus domesticus with the Sendai virus cyst membrane, behind the 72h, collects chick embryo allantoic liquid, measures the blood clotting valency of allantoic fluid.
Described ablation method is 99mJ/cm
2Ultraviolet inactivation 2h.
The preparation of embodiment 1.HVJ-E
1.HVJ breeding: from-70 ℃ of refrigerators, get a frozen HVJ seed culture of viruses (Z strain),, after waiting to dissolve,, collect supernatant with the centrifugal 10min of 2000r/min in slowly dissolving on ice.The supernatant of collecting also is inoculated in 10 age in days SPF chick chorioallantoic membrane chambeies with 1000 times of the PBS solution dilutions that contains 100U/ml penicillin and 0.1mg/ml streptomycin etc., the virus of drawing 100 μ L dilution with disposable sterilized injector.Observe the chicken embryo death situation behind the 24h, discard SPF Embryo Gallus domesticus dead in the 24h.Behind the 72h, aseptic collection chick embryo allantoic liquid.
2.HVJ concentrate: the allantoic fluid collected is removed impurity in the allantoic fluid through the centrifugal 1h of 10000rpm, the chick embryo allantoic liquid behind the impurity is removed in collection, pack into bag filter and place the beaker that contains PEG 6000 to concentrate, collect concentrated allantoic fluid and slowly be added on 20% sucrose solution surface, the centrifugal 2h of 20000rpm, supernatant discarded, collecting precipitation, the resuspended precipitation of PBS, the centrifugal 2h of 20000rpm collects virus and removes sucrose.
3.HVJ virion is calculated: measure tiring of Sendai virus with hemagglutination test.Concrete steps are:
(1) adds the 0.05ml normal saline with microsyringe to the every hole of template, 96 holes " V ", add 24 holes altogether.
(2) whenever ranked first the hole with microsyringe to " V " template and add the 0.05ml Sendai virus, do doubling dilution then, discard after being diluted to the 23rd hole, last hole does not add virus hole in contrast.
(3) in each hole, add 1% (V/V) chicken red blood cell with microsyringe.
(4) with after shaking 1-2 minute on the V-type agglutination plate earthquake device, room temperature (20-25 ℃) leaves standstill judged result after 15-30 minute.
(5) according to the blood clotting valency and the 1HAU (blood clotting valency unit)=3 * 10 of Sendai virus
6The formula to calculating of HVJ virion goes out the population of the Sendai virus of purification.
4.HVJ-E preparation: Sendai virus solution is suspended in pyrogen-free PBS, and evenly lay is in the 10cm plate and place under the ultraviolet transilluminator.With 99mJ/cm
2Ultraviolet radiation Sendai virus solution 2h.Sendai virus after the deactivation (HVJ-E) with containing 1000 times of the two anti-PBS solution dilutions of mycillin, is inoculated 100 μ L in the SPF chick chorioallantoic membrane, collect allantoic fluid behind the 72h, detect the blood clotting valency, judge the infectivity of HVJ-E.Behind the prepared HVJ-E inoculated into chick embryo, the erythrocyte that its allantoic fluid can not the coagulation chicken loses infectivity, has good biological safety.
Embodiment 2.HVJ-E strengthens pig breeding and obstacle syndrome virus inactivated vaccine Immunogenicity Study
1. reagent
Culture medium (RPMI-1640), con A (ConA), polymine (PEI, 25kDa), MTT (available from Sigma company); Hyclone (available from Hyclone); PRRSV antibody assay kit (available from IDEXX company); Pig IFN-γ immune reagent kit (available from Invitrogen International Inc.) pig IL-10 and IL-12 immune reagent kit are (available from R﹠amp; D International Inc.), the vacuum hemospasia pipe that contains the Lithium acid heparin anticoagulant is a Hunan pharmaceuticals product, 96 orifice plates (available from Corning Co.LTD.) Ficoll-Paque (available from DingGuo Biotech), DNA/RNA extraction agent box, one-step method RT-PCR test kit are all available from TAKARA company.
2. test method
Choose 15 ablactational baby pig, gather serum, in advance with pig PRRS antibody assay kit (IDEXX, the U.S.) detect, the result shows in the serum of all test pig all nonreactive PRRSV antibody, simultaneously through RT-PCR detect (forward primer: 5 '-GAGTTTCAGCGGAACAATGG-3, downstream primer: 5 '-GCCGTTGACCGTAGTGGAG-3 '), be the PRRSV feminine gender, in addition, carried out swine fever virus (forward primer: 5 '-AACATGGATGGTGTAACTGGT-3, downstream primer 5 '-TTCTCTATAGTGTTGGTCATTCC-3 '), porcine circovirus 2 type (forward primer: 5 '--CGAGAAAGCGAAAGGAACAGA-3 ', downstream primer: 5 '-GGTAACCATCCCACCACTT-3 ') and pig parvoviral (forward primer: 5 '-AGTTAGAATAGGATGCCGAGGAA-3 ', downstream primer: 5 '-AGAGTCTGTTGGTGTATTTATTGG-3 ') PCR detect, the result is all negative.Piglet is divided into 3 groups at random: full dosage PRRSV inactivated vaccine matched group, HVJ-E+ full dosage inactivated vaccine group and PBS matched group, every group of 5 pigs.
Before the immunity, the vena cava anterior blood sampling.Every pig of PBS matched group is cervical region intramuscular injection 2ml PBS (pH 7.2) behind ear; Complete every pig of dosage inactivated vaccine matched group is in the isopyknic PRRSV inactivated vaccine of same position intramuscular injection, and experimental group (the full dosage inactivated vaccine of HVJ-E+ group) injection contains and of the present inventionly contains 1 * 10
6The PBS buffer of virus like particle HVJ-E, cumulative volume are 2ml.1 week, 2 weeks, 3 weeks and 4 weeks took a blood sample separation of serum respectively at vena cava anterior after immunity then.Measure peripheral blood mononuclear lymphopoiesis situation, Cytokine of Serum content and anti-PRRSV antibody titer.
After 4 weeks of immunity, (JXA1) carries out counteracting toxic substances with PRRSV virulent strain, and every pig is in musculi colli injection 10
.4.5The virulent strain of TCID50, every pig injection 2ml.Behind the counteracting toxic substances, measure the body temperature of pig every day, observe the behavior of pig, and 3 days, 1 week, 2 weeks and 3 all vena cava anteriors blood samplings behind counteracting toxic substances, separation of serum is used for viremia and detects; Gather the nasal mucosa cotton simultaneously and wipe away, be used to detect the toxin expelling situation of PRRSV,
3. result of the test and discussion
3.1pCpG induce the specific PBMC reaction of PRRSV
Referring to Fig. 1: mtt assay detects the periphery lymphocyte propagation situation of HVJ-E mediation.The result shows that the periphery lymphocyte propagation of PRRSV inactivated vaccine group and PBS group does not have notable difference, the lymphopoiesis of the 14th day and 28 days is then apparently higher than PRRSV inactivated vaccine group and PBS group (p＜0.05) after the immunity of HVJ-E+PRRSV inactivated vaccine group, the 14th day the stimulation index (SI) in immunity back will be higher than behind the counteracting toxic substances the 28th day SI, but the two no significant difference.
3.2HVJ-E promote the generation of PRRS inactivated vaccine antibody horizontal
Referring to Fig. 2: after each experimental group and the matched group immunity, vena cava anterior blood sampling separation of serum, the result of anti-PRRSV antibody in the serum after test kit detects, as can be seen from Figure 2, piglet is after HVJ-E and the complete common immunity of dosage PRRSV inactivated vaccine, and antibody horizontal is significantly the highest, and presents ascendant trend in 28 days always, compare significant difference (p＜0.05) with full dosage inactivated vaccine group.This result of the test shows: HVJ-E of the present invention can effectively improve the immune effect of vaccine.After the immunity 28 days, attack with PRRSV virulent strain (JXA1), and behind counteracting toxic substances 3 days, 1 week, 2 weeks and the blood sampling of 3 weeks, measure anti-PRRSV antibody horizontal in the serum, the result shows, the antibody horizontal of HVJ-E and full dosage PRRS inactivated vaccine immune group is on a declining curve behind counteracting toxic substances, prompting has produced neutralizing antibody when counteracting toxic substances, neutralizing antibody neutralizes to attacking virulent strain, has caused declines of tiring of the antibody ELISA in the serum, and separately full the antibody horizontal of dosage inactivated vaccine immune group present ascendant trend, proof is when counteracting toxic substances, and the inactivated vaccine immune group does not produce neutralizing antibody separately.The PBS matched group shows as the ELISA negative antibody all the time before counteracting toxic substances, and antibody titer rises rapidly behind the counteracting toxic substances, has pointed out the self-protection after the body actute infection to begin performance.In a word, can judge the generation that HVJ-E can shift to an earlier date induction of immunity pig neutralizing antibody, protect immune animal to avoid virus infraction effectively from this result.
3.3 cytokine assay result
Participate in regulating the immune response reaction referring to Fig. 3: IL-6 and IFN-γ, can promote the circulation of IgG secretion cell in phagocyte and the blood, stimulate the generation of Th1 cytokines, promote lymphocytic cell toxicant and bactericidal action.ELISA measures the result of Cytokine of Serum.As shown in Figure 3, piglet is after HVJ-E and the complete common immunity of dosage PRRS inactivated vaccine, its Cytokine of Serum content IL-6 and IFN-γ significantly raise, compare with full dosage inactivated vaccine group, significant difference (p＜0.05), dosage inactivated vaccine group and PBS immune group generation cytokine is on close level the two no significant difference entirely.The present embodiment result of the test shows that HVJ-E of the present invention has produced high-caliber IL-6 and IFN-γ by stimulating body, play crucial effects to regulating the immunne response network, wherein IFN-γ is extraordinary anti-virus formulation, IL-6 then can suppress regulatory T cells, promote the generation of Th1 cytokines, proved absolutely that IL-6 and IFN-γ are bringing into play important effect in the process that infects at anti-PRRSV, this has also obtained confirmation from clinical effectiveness.
3.4 the clinical observation result behind the counteracting toxic substances
Behind the counteracting toxic substances, each group test pig is carried out clinical marking, marking the results are shown in Table 1, and HVJ-E therefrom of the present invention as can be seen can improve the survival rate of piglet significantly, effectively improves the clinical manifestation behind the strong virus attack.5 pigs of PBS matched group are all dead behind counteracting toxic substances, add the then all survivals of full dosage inactivated vaccine immune group that contain HVJ-E.3 of the survivals in 21 days behind counteracting toxic substances of the complete independent immune group of dosage inactivated vaccine.
Clinical manifestation marking behind table 1. counteracting toxic substances
aGive a mark s.0 according to neural and respiratory symptom, asymptomatic; 1, mild; 2, serious symptoms
3.5 the body temperature behind the counteracting toxic substances changes
Referring to Fig. 4: behind the counteracting toxic substances, occurring fervescence at first is PRRSV inactivated vaccine immune swine, and this group experiment pig presented anorexia and hyperpyrexia (41.042.0 ℃) in 4 days behind counteracting toxic substances, and subsequently, the PBS matched group also shows as the hyperpyrexia symptom.The pig of HVJ-E and the common immune group of PRRSV inactivated vaccine began to occur fervescence in 6 days behind counteracting toxic substances, but after 5 days, its body temperature is all reduced to below 41 ℃.Because the performance in 16 days behind counteracting toxic substances of the pig of PBS matched group is dying, its body temperature descends rapidly, behind counteracting toxic substances 10-18 days, and PBS matched group and PRRSV inactivated vaccine group each dead 4 pigs and 2 pigs, all the other pigs all survive.
3.6 the weightening finish of survival pig changes
Referring to Fig. 5: counteracting toxic substances is after 21 days, all survival pigs are carried out weighing, gained weight is deducted body weight before the counteracting toxic substances, calculate the weightening finish of each pig in the counteracting toxic substances 21 days, PBS survival pig weightening finish 3.6kg wherein, PRRSV inactivated vaccine immune swine survival pig average weight gain 2.5kg, the common immune swine survival of HVJ-E and PRRSV pig average weight gain 6.4kg.
3.7 viremia behind the counteracting toxic substances and toxin expelling testing result
Respectively at 3,7,14 and 21 days acquisition test Sanguis sus domestica samples behind counteracting toxic substances same day, the counteracting toxic substances and nasal cavity swab, carry out PRRSV in nasal secretion and the serum with the method for RT-PCR, the result shows, the generation of viremia and virus are drained and are seen PRRSV inactivated vaccine immune group the earliest, wherein have 3/5 to produce viremia, 1/5 beginning toxin expelling.In 1 week behind the counteracting toxic substances, except that 2 pigs of HVJ-E and the common immune group of PRRSV inactivated vaccine, all pigs have all produced viremia, and the situation of toxin expelling and viremia production are similar.Behind the counteracting toxic substances 7-14 days, each dead 1 pig of PBS matched group and inactivated vaccine matched group, in addition, viremia and toxin expelling rate begin to descend.Counteracting toxic substances 21 days, in the middle of the pig of survival, PBS matched group and inactivated vaccine matched group respectively have 1 to present toxin expelling, and all survival pigs all show as the viremia feminine gender.This result has confirmed that piglet has shown good resistance after HVJ-E and the common immunity of PRRSV inactivated vaccine, not only shows as whole survivals clinically, and viremia positive rate and toxin expelling rate also decline to a great extent.
The toxin expelling of test pig and viremia detect behind table 2. counteracting toxic substances
3.8 cardinal principle lesion examining result
After experiment finished, the pig that will survive was slaughtered and dissects, and observed pathological changes substantially, and pathological changes result is described below.HVJ-E+ inactivated vaccine group: indivedual pig spleen edges have hemorrhage, and other internal organs are normal.The inactivated vaccine matched group: pulmonary has hemorrhage, but does not have necrosis, the hilar lymph node enlargement, and spleen, kidney are normal substantially.The PBS matched group: the spleen infarction, the lungs diffuse hemorrhage, submandibular lymph nodes, hilar lymph node and inguinal lymph nodes are hemorrhage, and kidney has the point-like petechia.
3.9 the viral tissue distribution behind the counteracting toxic substances
When dissected is collected the heart, liver,spleen,kidney, tonsil and lymph node, after dismembyator is ground, carries out RT-PCR and detects.The result shows, the institute of PBS matched group and inactivated vaccine matched group nearly all presents the positive in a organized way, HVJ-E and the common immune group of inactivated vaccine organize positive rate then very low.
Survive after the 21 days tissue distribution of pig of table 3. counteracting toxic substances
aLymph is become the mixture of submandibular lymph nodes, hilar lymph node and inguinal lymph nodes
In a word, the present embodiment result of the test shows: but HVJ-E human body immunity improving function of the present invention; Promote generation and the release of IL-6 and IFN-γ, improve antibody and produce level, provide guarantee for removing cause of disease fast; HVJ-E can significantly improve the attacking ability of the anti-PRRSV virulent strain of immune piglet, significantly improves the clinical manifestation behind the strong virus attack; The RT-PCR testing result shows that HVJ-E and PRRSV inactivated vaccine immune group can reduce the generation of virus drainage and viremia; Clinical anatomic result shows that the viral tissue distribution of HVJ-E and PRRSV inactivated vaccine immune group is less, can significantly protect each internal organs to avoid damage.In sum, HVJ-E of the present invention can improve the immunoprotection function of PRRSV live vaccine, and HVJ-E of the present invention adopts administered intramuscular mode easily, thereby can promote the immunoprophylaxis that is used for large scale of pig farm.
Claims (5)
1. an adjuvant that improves pig breeding and respiratory tract obstruction syndrome inactivated vaccine immunizing potency prepares the Sendai virus cyst membrane with Sendai virus, as immunological adjuvant.
2. a kind of adjuvant that improves pig breeding and respiratory tract obstruction syndrome inactivated vaccine immunizing potency according to claim 1 is characterized in that described virus comprises following strain:
The Fushimi strain, Aritsugn strain, MN strain, Z strain, BB1 strain, TianJin strain.
3. a kind of adjuvant that improves pig breeding and respiratory tract obstruction syndrome inactivated vaccine immunizing potency according to claim 2, it is characterized in that described strain is used to prepare the animal immune adjuvant, especially the immunological adjuvant of pig breeding and respiratory disorder syndrome inactivated vaccine.
4. method for preparing the Sendai virus cyst membrane, this preparation method comprises the steps:
1) preparation contains the chick embryo allantoic liquid of Sendai virus;
2) with the allantoic fluid for preparing in the step 1) after PEG6000 concentrates, rebasing with 20% sucrose, the centrifugal 2h of 20000r/min;
3) collecting precipitation, the PBS washing precipitation, 20000r/min recentrifuge 2h removes sucrose;
4) collecting precipitation is suspended in the apyrogeneity normal saline;
5) with 99mJ/cm
2The Sendai virus 2h that collects of uitraviolet intensity deactivation;
6) the Sendai virus called after Sendai virus cyst membrane of deactivation is inoculated 9 age in days SPF Embryo Gallus domesticus with the Sendai virus cyst membrane, behind the 72h, collects chick embryo allantoic liquid, measures the blood clotting valency of allantoic fluid.
5. a kind of method for preparing the Sendai virus cyst membrane according to claim 4 is characterized in that described ablation method is 99mJ/cm
2Ultraviolet inactivation 2h.
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| CN104195114A (en) * | 2014-09-05 | 2014-12-10 | 青岛易邦生物工程有限公司 | Avian pneumovirus and application thereof |
| CN104211783A (en) * | 2013-05-22 | 2014-12-17 | 青岛红桥明勤生物科技有限公司 | Preparation of subunit vaccine for porcine reproductive and respiratory syndrome |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104211783A (en) * | 2013-05-22 | 2014-12-17 | 青岛红桥明勤生物科技有限公司 | Preparation of subunit vaccine for porcine reproductive and respiratory syndrome |
| CN104211783B (en) * | 2013-05-22 | 2017-11-07 | 青岛明勤生物科技有限公司 | A kind of preparation of blue otopathy subunit vaccine |
| CN104174018A (en) * | 2014-09-05 | 2014-12-03 | 青岛易邦生物工程有限公司 | Swollen head syndrome vaccine and preparation method thereof |
| CN104195114A (en) * | 2014-09-05 | 2014-12-10 | 青岛易邦生物工程有限公司 | Avian pneumovirus and application thereof |
| CN109701012A (en) * | 2019-03-01 | 2019-05-03 | 龙阔（苏州）生物工程有限公司 | A kind of vaccine adjuvant and its application and porcine reproductive and respiratory syndrome vaccine |
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