CN102861327B - Cell inactivated vaccine, egg yoke antibody and injection and freeze-dried powder containing same - Google Patents
Cell inactivated vaccine, egg yoke antibody and injection and freeze-dried powder containing same Download PDFInfo
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Abstract
The invention discloses cell inactivated vaccine which simultaneously contains porcine circovirus epitopes and goose circovirus epitopes. The cell inactivated vaccine is prepared by the method including screening high-pathogenicity strains from porcine circoviruses; selecting a plurality of virus epitopes; connecting the epitopes with one another; then inserting the connected epitopes into genomes of goose circoviruses to proliferate; and adding Freund adjuvants to formaldehyde for inactivation so as to obtain the cell inactivated vaccine. The invention simultaneously discloses an egg yolk antibody prepared from the cell inactivated vaccine, injection and freeze-dried powder containing the egg yolk antibody. Genes of the porcine circovirus are inserted into the genomes of the goose circoviruses, the immunogenicity of the goose circoviruses is strengthened, immune systems of goose bodies are excited, so that the titer of generated antibodies is higher than that of egg yolk antibodies generated by porcine circovirus vaccine used singularly, a clinical application effect is good, and generated circovirus antibodies have an auxiliary treatment effect for porcine circovirus diseases.
Description
Technical field
The present invention relates to injection and the lyophilized powder of a kind of cell inactivation vaccine, yolk antibody and yolk antibody, belong to disease immune field.
Background technology
Porcine circovirus 2 type (PVC2) is the main pathogen of postweaning multisystemic wasting syndrome and nephropathy dermatitis syndrome, this disease also can cause immunosuppressant simultaneously, easily cause secondary or the accompanying infection of other diseases, there is the feature such as high rate, high popular, highly pathogenic, high death, cause huge economic loss to pig industry.The mechanism of causing a disease of PVC2 it be unclear that, and it is considered to the immune destruction of pig the main cause causing relevant swine diseases.Due to immunity degradation, pig is stress easily suffer other pathogen invasion with during pathogen infection, and its clinical symptoms is mainly progressive emaciation, anemia and jaundice.Present means of prevention has antiviral agents, Chinese medicine etc., but poor effect.
Chicken yolk antibody IgY is a kind of Mitochondrion IgG of chicken, and chicken IgY is functionally equivalent to mammal IgG, but different in structure, has the space conformation of typical immunoglobulin.Chicken yolk antibody has lot of superiority: (1), due to the gap of birds and mammal phylogenetics, birds are more suitable for the specific antibody of producing resisting mammal antigen.Yolk antibody can not excite complement system, does not react with the antibody of resisting mammal antigen, and not with the Fc receptors bind of mammal and antibacterial, this is significant in immunologic diagnosis.(2) yolk antibody has bioactive immunoglobulin as one, is storing, produces, processes, ingests and ensureing in digestion process that its stability is very crucial.Multitest shows, chicken IgY has good stability, acidproof, alkaline-resisting, heat-resisting.Yolk antibody is more easily preserved, and deposits 6 months antagonist activity and have no significant effect under 4 DEG C of conditions under depositing 5 years or room temperature condition.(3) yolk antibody amount is large, and cost is low, is convenient to large-scale production.Antibody from yolk concentration energy long term maintenance is being equivalent to even exceed in the level of Serum antibody concentrations.1 laying hen is (on average to lay eggs weekly 5 ~ 6 pieces, on average the about 15mL of every piece of egg yolk calculates) the lay eggs antibody amount of production of institute is equivalent to the amount of 90 ~ 100mL serum or 180 ~ 200mL antibodies in blood, such as 1 rabbit can be extracted IgG for 1 month and is about 200mg, and 1 month 1 laying hen can extract more than IgY2000mg from egg.In addition, in actual production, the blood sampling of animal not only to animal itself be greatly stress, and time-consuming, take a lot of work, large-scale production is unpractical, and it is many easily to utilize laying hen to produce antibody.Generally, yolk antibody does not have toxic and side effects to animal, the shortcoming that hyper-immune serum is expensive, yield poorly not only is avoided by high immunity yolk antibody treatment Animal diseases, and the side effect that hyper-immune serum can be avoided to cause over the course for the treatment of, be a kind of up-and-coming novel immunoglobulin preparation.The preparation of current yolk antibody and production technology are ripe, the production that can guarantee both quality and quantity, and yolk antibody is diagnosed at diseases of bird and livestock, treat and the application of prevention gets more and more.But the treatment of current resisting porcine circovirus 2 type vaccine is more single, and yolk antibody preparation method is loaded down with trivial details, and the loss of antibody is higher.
Summary of the invention
The object of this invention is to provide a kind of cell inactivation vaccine.
In order to realize above object, the technical solution adopted in the present invention provides a kind of cell inactivation vaccine, is prepared by following methods:
1) get pathological material of disease and make homogenate, with the centrifugal 15-20min of 10000-12000rpm after multigelation, get supernatant, filter, inoculation sensitive single-layer PK-15 cell;
2) by the continuous passage of PK-15 cell, cell freeze thawing, extract the DNA of PCV2 virus, for PCV2 virus O RF1 gene, the two pairs of Specific PCR primers be made up of the nucleotide sequence of the nucleotide sequence and SEQ ID NO:3 and SEQ ID NO:4 with SEQ ID NO:1 and SEQ ID NO:2 in sequence table increase to genes of interest;
3) extension amplification outcome enters PMD-18T carrier and checks order, qualification;
4) screen multiple virus epitopes with antigen immune activity, the protein immunization through specific primer PCR, product electrophoresis, order-checking, clone, purification, each epi-position of detection is active, selects 5 epitope points;
5) connected by 5 epitope points, be inserted in goose porcine circovirus standard strain gene, then virus goes down to posterity on sensitivity cell PK-15 monolayer, the strain of adaptive immune originality;
6) expand propagation, measure virus titer by IPMA method;
7) show stabilized cell pathological changes occurrence law when virus goes down to posterity in PK-15 cell, the malicious valency of planting poison is stabilized in 10
3.9tCID
50/ 1.0ml, collecting cell, formalin-inactivated Jia Fushi adjuvant obtains cell inactivation vaccine.
Cell multigelation 3 times in described step 1).
Employing 0.22 μm of filter membrane is filtered in described step 1).
Described step 2) middle PK-15 cell continuous passage 6 generation, step 5) virus went down to posterity for 45 generations on cell PK-15 monolayer.
The present invention also aims to the yolk antibody providing a kind of cell inactivation vaccine to prepare.
Technical program of the present invention also lies in the yolk antibody providing a kind of cell inactivation vaccine to prepare, prepared by following methods:
1) bacteria inactivation vaccine is used, at interval of 7-10 days, three immunity of fundamental immunity, booster immunization and reinforced immunological are carried out to healthy laying hen group, start after booster immunization to detect antibody titer, when pig circular ring virus antibody titer reach 1:64, goose circovirus antibody tire and reach more than 1:64 time, collect high-immunity egg;
2) cleaning, sterilization high-immunity egg, dry, asepsis gets yolk, obtains yolk stock solution;
3) in yolk stock solution, add isopyknic sterilized water, stirring and evenly mixing, to color whitens, obtains yolk solution;
4) yolk solution is put into 60-65 DEG C of water-bath homogeneous heating 30min, then add the acidified aqueous solution of pH4.9-5.2 temperature 4-6 DEG C, addition is 6 times of yolk solution volume, mixing, staticly settles, gets supernatant, the centrifugal 20min of 15000rpm, gets supernatant;
5) add sad in supernatant, sad addition is the 0.8-1.0% of liquor capacity, mixing, and leave standstill, discard impurity floating thing, the centrifugal 10min of 20000rpm, gets supernatant, leaves standstill;
6) with the filter membrane of 0.45 μm to step 5) gained supernatant liquid filtering, obtain yolk antibody solution, then through the membrane filtration of MCW 300KD, obtain the solution containing yolk antibody refined.
In described step 1), the amount of basic immunity bacteria inactivation vaccine is 1.5ml, booster immunization and reinforced immunological inoculation dosage 1 times.
In described step 4), acidified aqueous solution is the acidifying water of hydrochloric acid preparation, and pre-coo time is 1-2h.
The present invention also aims to provide the injection containing yolk antibody.
Technical program of the present invention also lies in providing the injection containing yolk antibody, this egg yolk antibody injection comprises yolk antibody solution 1 part, formaldehyde 0.002-0.003 part and thimerosal 0.0001-0.0002 part.
The present invention also aims to provide the lyophilized powder containing yolk antibody.
Technical program of the present invention also lies in providing the lyophilized powder containing yolk antibody, this yolk antibody lyophilized powder comprises yolk antibody solution 1 part, formaldehyde 0.002-0.003 part, thimerosal 0.0001-0.0002 part, glucose 0.03-0.04 part, sorbitol 0.01-0.02 part, glycine 0.02-0.03 part and mannitol 0.030.04 part.
Present invention employs up-to-date cold and hot acidification yolk liquid technology, sterilization and acidification are synchronously carried out, decrease production stage, also the loss of antibody is relatively reduced, this product is that a kind of poultry can biological preparation simultaneously, compare and produce corresponding serum product, reduce cost and labor intensity.The vaccine that this product adopts gene integration technology, clone etc. to select advanced technology to make increases its immunogenicity and Antybody therapy scope, the porcine circovirus gene of pig is inserted in goose porcine circovirus genome, strengthen the immunogenicity to goose porcine circovirus and the immune stimulation to goose body, make it produce antibody to tire height than the yolk antibody that single use pig circular ring virus vaccine produces, clinical application effect is good, and the annulus antibody simultaneously producing goose has adjuvant treatment effect to pig annulus disease.
Detailed description of the invention
The pig farm of serious pig circular ring virus 2 is there is in pathological material of disease collection of the present invention from Shandong District.
Embodiment 1
The cell inactivation vaccine that the present embodiment provides, is prepared by following methods:
1) get pathological material of disease and make homogenate, after multigelation 3 times, the centrifugal 15-20min of 10000-12000rpm, gets supernatant, through 0.22 μm of membrane filtration, and inoculation sensitive single-layer PK-15 cell;
2) by the continuous passage of PK-15 cell, cell freeze thawing, extract the DNA of PCV2 virus, for PCV2 virus O RF1 gene, the two pairs of Specific PCR primers be made up of the nucleotide sequence of the nucleotide sequence and SEQ ID NO:3 and SEQ ID NO:4 with SEQ ID NO:1 and SEQ ID NO:2 in sequence table increase to genes of interest, wherein
Primer 1PCV2.1:5'-TTCGGTACCAGCTATGACGTATCCAAG-3'(SEQ ID NO:1),
PCV2.2：5'-GCCAAGCTTTCACTTCGTAATGGTTTT-3'（SEQ ID NO：2）；
Primer 2 PCV2.1:5'-TTGGCCTAAGCTATGACGTATCCAA-3'(SEQ ID NO:3),
PCV2.2：5'-CCAAGCTTTCACTTCGTTGAAGTTT-3'（SEQ ID NO：4）；
3) extension amplification outcome enters PMD-18T carrier and checks order, qualification;
4) screen multiple virus epitopes with antigen immune activity, the protein immunization through specific primer PCR, product electrophoresis, order-checking, clone, purification, each epi-position of detection is active, selects 5 epitope points;
5) connected by 5 epitope points with splicing, be inserted in goose porcine circovirus standard strain gene, then virus went down to posterity for 45 generations on sensitivity cell PK-15 monolayer, the strain of adaptive immune originality;
6) expand propagation, measure virus titer by IPMA method;
7) show stabilized cell pathological changes occurrence law when virus goes down to posterity in PK-15 cell, the malicious valency of planting poison is stabilized in 10
3.9tCID
50/ 1.0ml, collecting cell, the obtained cell inactivation vaccine of formalin-inactivated Jia Fushi adjuvant mixing.
Embodiment 2
The yolk antibody that the present embodiment provides, is prepared by following methods:
1) bacteria inactivation vaccine is used, at interval of 7 days, three immunity of fundamental immunity, booster immunization and reinforced immunological are carried out to healthy laying hen group, basic immunity vaccine 1.5ml, booster immunization and reinforced immunological inoculation dosage 1 times, start after booster immunization to detect antibody titer, when pig circular ring virus antibody titer reach 1:64, goose circovirus antibody tire and reach more than 1:64 time, collect high-immunity egg;
2) flowing water cleaning, 84 disinfectant solution sterilization high-immunity eggs, dry, asepsis gets yolk, obtains yolk stock solution;
3) in yolk stock solution, add isopyknic sterilized water, stirring and evenly mixing, to color whitens, obtains yolk solution;
4) yolk solution is put into 60 DEG C of water-bath homogeneous heating 30min, then add the aqueous hydrochloric acid solution of pH4.9 temperature 4 DEG C, addition is 6 times of yolk solution volume, mixing, and staticly settle, get supernatant, the centrifugal 20min of 15000rpm, gets supernatant;
5) add sad in supernatant, sad addition is 0.8% of liquor capacity, mixing, and leave standstill, discard impurity floating thing, the centrifugal 10min of 20000rpm, gets supernatant, leaves standstill;
6) with the filter membrane of 0.45 μm to step 5) gained supernatant liquid filtering, obtain yolk antibody solution, then through the membrane filtration of MCW 300KD, obtain the solution containing yolk antibody refined.
Embodiment 3
The yolk antibody that the present embodiment provides, is prepared by following methods:
1) bacteria inactivation vaccine is used, at interval of 10 days, three immunity of fundamental immunity, booster immunization and reinforced immunological are carried out to healthy laying hen group, basic immunity vaccine 1.5ml, booster immunization and reinforced immunological inoculation dosage 1 times, start after booster immunization to detect antibody titer, when pig circular ring virus antibody titer reach 1:64, goose circovirus antibody tire and reach more than 1:64 time, collect high-immunity egg;
2) flowing water cleaning, bromo geramine sterilization high-immunity egg, dry, asepsis gets yolk, obtains yolk stock solution;
3) in yolk stock solution, add isopyknic sterilized water, stirring and evenly mixing, to color whitens, obtains yolk solution;
4) yolk solution is put into 65 DEG C of water-bath homogeneous heating 30min, then add the aqueous hydrochloric acid solution of pH5.2 temperature 6 DEG C, addition is 6 times of yolk solution volume, mixing, and staticly settle, get supernatant, the centrifugal 20min of 15000rpm, gets supernatant;
5) add sad in supernatant, sad addition is 1.0% of liquor capacity, mixing, and leave standstill, discard impurity floating thing, the centrifugal 10min of 20000rpm, gets supernatant, leaves standstill;
6) with the filter membrane of 0.45 μm to step 5) gained supernatant liquid filtering, obtain yolk antibody solution, then through the membrane filtration of MCW 300KD, obtain the solution containing yolk antibody refined.
Embodiment 4
The yolk antibody that the present embodiment provides, is prepared by following methods:
1) bacteria inactivation vaccine is used, at interval of 8 days, three immunity of fundamental immunity, booster immunization and reinforced immunological are carried out to healthy laying hen group, basic immunity vaccine 1.5ml, booster immunization and reinforced immunological inoculation dosage 1 times, start after booster immunization to detect antibody titer, when pig circular ring virus antibody titer reach 1:64, goose circovirus antibody tire and reach more than 1:64 time, collect high-immunity egg;
2) flowing water cleaning, bromo geramine sterilization high-immunity egg, dry, asepsis gets yolk, obtains yolk stock solution;
3) in yolk stock solution, add isopyknic sterilized water, stirring and evenly mixing, to color whitens, obtains yolk solution;
4) yolk solution is put into 63 DEG C of water-bath homogeneous heating 30min, then add the aqueous hydrochloric acid solution of pH5.0 temperature 5 DEG C, addition is 6 times of yolk solution volume, mixing, and staticly settle, get supernatant, the centrifugal 20min of 15000rpm, gets supernatant;
5) add sad in supernatant, sad addition is 0.9% of liquor capacity, mixing, and leave standstill, discard impurity floating thing, the centrifugal 10min of 20000rpm, gets supernatant, leaves standstill;
6) with the filter membrane of 0.45 μm to step 5) gained supernatant liquid filtering, obtain yolk antibody solution, then through the membrane filtration of MCW 300KD, obtain the solution containing yolk antibody refined.
Embodiment 5
The injection containing embodiment 2 yolk antibody that the present embodiment provides, comprises embodiment 2 yolk antibody solution 1 part, 0.002 part, formaldehyde, thimerosal 0.0001 part.
The preparation method of egg yolk antibody injection, comprises the following steps: by yolk antibody solution 1 part refining for embodiment 2, through degerming membrane filtration, get filtrate, add 0.002 part of formaldehyde and 0.0001 part of thimerosal, obtain egg yolk antibody injection.
Antibody titer change during in order to detect the placement at normal temperatures of the present embodiment egg yolk antibody injection, the shelf-life of this product is determined with this, do following test: this product be kept in 37 DEG C of incubators, preserve 24 months, 1 week, 2 weeks respectively, January, February, April, August, December, after 16 months, 20 months, 24 months, by agar diffusion (AGP) method, the yolk antibody measuring each point in time sampling product is tired change, the results are shown in Table 1.
Table 1 room temperature places the change that injection different time sections is tired
Quality standard:
[character] this product is faint yellow, in fine and close agglomerate.
[steriling test] is undertaken by " Chinese veterinary pharmacopoeia ", asepsis growth.
[mycoplasma inspection] is undertaken by " Chinese veterinary pharmacopoeia ", grows without mycoplasma.
[exogenous virus inspection] is undertaken by " Chinese veterinary pharmacopoeia ", conforms with the regulations.
[safety verification] with 12 age in days SPF chicken 5, each intramuscular injection plumage part, observes 2 weeks, and all should be good for and live, injection site does not have pathological changes.
[shelf-life], according to room temperature bioactivity experimental result, can deposit 2 years.
[efficacy test] is undertaken being diluted to 1 part/mL by the specification of producing.
1) protection detects: with nursery pig 30, is divided into 3 groups, often organizes 10, is respectively test group, matched group and blank group.To 10 pig muscle injection 0.1mL/kg egg yolk antibody injection of test group, matched group and the isolated rearing of blank group.After 24h, get test group and matched group nursery pig intramuscular injection inoculation diluting cells virus liquid, record 96h death condition, the protective rate of test group is more than 96%, and counteracting toxic substances matched group mortality rate is more than 82%, and blank group pig is not any change.
2) healing power measures: with nursery pig 30, as negative control group, inoculates the cytopathy venom after diluting for 20, after infecting 12h for 10, wherein 10 as treatment group, every intramuscular injection this product 0.1mL/kg, every day 1 time, continuous 3 days, another 10 as positive controls, isolated rearing.72h after treatment, record each group of death condition, counteracting toxic substances positive controls mortality rate more than 83%, treatment group survival rate is more than 90%, and healthy negative control group 10 is not only any change.
Embodiment 6
The injection containing embodiment 3 yolk antibody that the present embodiment provides, comprises embodiment 3 yolk antibody solution 1 part, 0.003 part, formaldehyde, thimerosal 0.0002 part.
The preparation method of egg yolk antibody injection, comprises the following steps: by yolk antibody solution 1 part refining for embodiment 3, through degerming membrane filtration, get filtrate, add 0.003 part of formaldehyde and 0.0002 part of thimerosal, obtain egg yolk antibody injection.
Antibody titer change during in order to detect the placement at normal temperatures of the present embodiment egg yolk antibody injection, the shelf-life of this product is determined with this, do following test: this product be kept in 37 DEG C of incubators, preserve 24 months, 1 week, 2 weeks respectively, January, February, April, August, December, after 16 months, 20 months, 24 months, by agar diffusion (AGP) method, the yolk antibody measuring each point in time sampling product is tired change, the results are shown in Table 2.
Table 2 room temperature places the change that injection different time sections is tired
Quality standard:
[character] this product is faint yellow, in fine and close agglomerate.
[steriling test] is undertaken by " Chinese veterinary pharmacopoeia ", asepsis growth.
[mycoplasma inspection] is undertaken by " Chinese veterinary pharmacopoeia ", and mycoplasma grows.
[exogenous virus inspection] is undertaken by " Chinese veterinary pharmacopoeia ", should conform with the regulations.
[safety verification] with 12 age in days SPF chicken 5, each intramuscular injection plumage part, observes 2 weeks, and all should be good for and live, injection site does not have pathological changes.
[shelf-life], according to room temperature bioactivity experimental result, can deposit 2 years.
[efficacy test] is undertaken being diluted to 1 part/mL by the specification of producing.
1) protection detects: with nursery pig 30, is divided into 3 groups, often organizes 10, is respectively test group, matched group and blank group.To 10 pig muscle injection 0.1mL/kg egg yolk antibody injection of test group, matched group and the isolated rearing of blank group.After 24h, get test group and matched group nursery pig intramuscular injection inoculation diluting cells virus liquid, record 96h death condition, the protective rate of test group is more than 95%, and counteracting toxic substances matched group mortality rate is more than 86%, and blank group pig is not any change.
2) healing power measures: with nursery pig 30, as negative control group, inoculates the cytopathy venom after diluting for 20, after infecting 12h for 10, wherein 10 as treatment group, every intramuscular injection this product 0.1mL/kg, every day 1 time, continuous 3 days, another 10 as positive controls, isolated rearing.72h after treatment, record each group of death condition, counteracting toxic substances positive controls mortality rate more than 80%, treatment group survival rate is more than 90%, and healthy negative control group 10 is not only any change.
Embodiment 7
The injection containing embodiment 4 yolk antibody that the present embodiment provides, comprises embodiment 4 yolk antibody solution 1 part, 0.003 part, formaldehyde, thimerosal 0.0001 part.
The preparation method of egg yolk antibody injection, comprises the following steps: by yolk antibody solution 1 part refining for embodiment 4, through degerming membrane filtration, get filtrate, add 0.003 part of formaldehyde and 0.0001 part of thimerosal, obtain egg yolk antibody injection.
Antibody titer change during in order to detect the placement at normal temperatures of the present embodiment egg yolk antibody injection, the shelf-life of this product is determined with this, do following test: this product be kept in 37 DEG C of incubators, preserve 24 months, 1 week, 2 weeks respectively, January, February, April, August, December, after 16 months, 20 months, 24 months, by agar diffusion (AGP) method, the yolk antibody measuring each point in time sampling product is tired change, the results are shown in Table 3.
Table 3 room temperature places the change that injection different time sections is tired
Quality standard:
[character] this product is faint yellow, in fine and close agglomerate.
[steriling test] is undertaken by " Chinese veterinary pharmacopoeia ", asepsis growth.
[mycoplasma inspection] is undertaken by " Chinese veterinary pharmacopoeia ", and mycoplasma grows.
[exogenous virus inspection] is undertaken by " Chinese veterinary pharmacopoeia ", should conform with the regulations.
[safety verification] with 12 age in days SPF chicken 5, each intramuscular injection plumage part, observes 2 weeks, and all should be good for and live, injection site does not have pathological changes.
[shelf-life], according to room temperature bioactivity experimental result, can deposit 2 years.
[efficacy test] is undertaken being diluted to 1 part/mL by the specification of producing.
1) protection detects: with nursery pig 30, is divided into 3 groups, often organizes 10, is respectively test group, matched group and blank group.To 10 pig muscle injection 0.1mL/kg egg yolk antibody injection of test group, matched group and the isolated rearing of blank group.After 24h, get test group and matched group nursery pig intramuscular injection inoculation diluting cells virus liquid, record 96h death condition, the protective rate of test group is more than 95%, and counteracting toxic substances matched group mortality rate is more than 85%, and blank group pig is not any change.
2) healing power measures: with nursery pig 30, as negative control group, inoculates the cytopathy venom after diluting for 20, after infecting 12h for 10, wherein 10 as treatment group, every intramuscular injection this product 0.1mL/kg, every day 1 time, continuous 3 days, another 10 as positive controls, isolated rearing.72h after treatment, record each group of death condition, counteracting toxic substances positive controls mortality rate is more than 85%, and treatment group survival rate is more than 95%, and healthy negative control group 10 is not only any change.
Embodiment 8
What the present embodiment provided contains embodiment 2 yolk antibody lyophilized powder, comprises embodiment 2 yolk antibody solution 1 part, 0.002 part, formaldehyde, thimerosal 0.0001 part, glucose 0.04 part, sorbitol 0.01 part, glycine 0.03 part, 0.03 part, mannitol.
The preparation method of yolk antibody lyophilized powder, comprise the following steps: by yolk antibody solution 1 part refining for embodiment 2, through degerming membrane filtration, get filtrate, add 0.002 part of formaldehyde, 0.0001 part of thimerosal, glucose 0.04 part, sorbitol 0.01 part, glycine 0.03 part, 0.03 part, mannitol, prepares yolk antibody lyophilized powder through lyophilizing program.Lyophilizing program is that ﹣ 40 DEG C starts distillation, is raised to ﹣ 35 DEG C and keeps 14h after 2h.
Embodiment 9
What the present embodiment provided contains embodiment 3 yolk antibody lyophilized powder, comprises embodiment 3 yolk antibody solution 1 part, 0.003 part, formaldehyde, thimerosal 0.0002 part, glucose 0.04 part, sorbitol 0.01 part, glycine 0.03 part, 0.03 part, mannitol.
The preparation method of yolk antibody lyophilized powder, comprise the following steps: by yolk antibody solution 1 part refining for embodiment 3, through degerming membrane filtration, get filtrate, add 0.003 part of formaldehyde, 0.0002 part of thimerosal, glucose 0.04 part, sorbitol 0.01 part, glycine 0.03 part, 0.03 part, mannitol, prepares yolk antibody lyophilized powder through lyophilizing program.Lyophilizing program is that ﹣ 40 DEG C starts distillation, is raised to ﹣ 35 DEG C and keeps 14h after 2h.
Embodiment 10
What the present embodiment provided contains embodiment 4 yolk antibody lyophilized powder, comprises embodiment 4 yolk antibody solution 1 part, 0.003 part, formaldehyde, thimerosal 0.0001 part, glucose 0.04 part, sorbitol 0.01 part, glycine 0.03 part, 0.03 part, mannitol.
The preparation method of yolk antibody lyophilized powder, comprise the following steps: by yolk antibody solution 1 part refining for embodiment 4, through degerming membrane filtration, get filtrate, add 0.003 part of formaldehyde, 0.0001 part of thimerosal, glucose 0.04 part, sorbitol 0.01 part, glycine 0.03 part, 0.03 part, mannitol, prepares yolk antibody lyophilized powder through lyophilizing program.Lyophilizing program is that ﹣ 40 DEG C starts distillation, is raised to ﹣ 35 DEG C and keeps 14h after 2h.
The commercially available prod Performance comparision of egg yolk antibody injection of the present invention and emergence sales company:
With nursery pig 60, make this product group for first group 30, second group 30 only as commercially available prod group, isolated rearing, cytopathy venom after subcutaneous vaccination dilution, when showing clinical symptoms, starts treatment, first group every intramuscular injection this product 0.1mL/kg, every day 1 time, continuous 3 days, second group of intramuscular injection commercially available prod 0.1mL/kg, every day 1 time, continuous 3 days.Treatment after 72h, record each group morbidity and treatment situation as shown in table 4.
Table 4 is group morbidity and treatment situation respectively
Sequence table
SEQUENCE LISTING
<110> Zhengzhou Houyi Pharmaceutical Co., Ltd.
The injection of <120> a cell inactivation vaccine, yolk antibody and yolk antibody and lyophilized powder
<130>
<160>4
<210>1
<211>27
<212>DNA
<213> artificial sequence
<400>1
ttcggtaccagctatgacgtatccaag 27
<210>2
<211>27
<212>DNA
<213> artificial sequence
<400>2
gccaagctttcacttcgtaatggtttt 27
<210>3
<211>25
<212>DNA
<213> artificial sequence
<400>3
ttggcctaagctatgacgtatccaa 25
<210>4
<211>25
<212>DNA
<213> artificial sequence
<400>4
ccaagctttcacttcgttgaagttt 25
Claims (9)
1. a cell inactivation vaccine, is characterized in that, is prepared by following methods:
1) get pathological material of disease and make homogenate, centrifugal after multigelation, the centrifugal 15-20min of 10000-12000rpm, gets supernatant, filters, inoculation sensitive single-layer PK-15 cell;
2) by the continuous passage of PK-15 cell, cell freeze thawing, extract the DNA of PCV2 virus, for PCV2 virus O RF1 gene, the two pairs of Specific PCR primers be made up of the nucleotide sequence of the nucleotide sequence and SEQ ID NO:3 and SEQ ID NO:4 with SEQ ID NO:1 and SEQ ID NO:2 in sequence table increase to genes of interest;
3) extension amplification outcome enters PMD-18T carrier and checks order, qualification;
4) screen multiple virus epitopes with antigen immune activity, the protein immunization through specific primer PCR, product electrophoresis, order-checking, clone, purification, each epi-position of detection is active, selects 5 epitope points;
5) connected by 5 epitope points, be inserted in goose porcine circovirus standard strain gene, then virus goes down to posterity on sensitivity cell PK-15 monolayer, the strain of adaptive immune originality;
6) expand propagation, measure virus titer by IPMA method;
7) show stabilized cell pathological changes occurrence law when virus goes down to posterity in PK-15 cell, the malicious valency of planting poison is stabilized in 10
3.9tCID
50/ 1.0ml, collecting cell, formalin-inactivated Jia Fushi adjuvant obtains cell inactivation vaccine.
2. a kind of cell inactivation vaccine according to claim 1, is characterized in that, cell multigelation 3 times in described step 1).
3. a kind of cell inactivation vaccine according to claim 1, is characterized in that, filters employing 0.22 μm of filter membrane in described step 1).
4. a kind of cell inactivation vaccine according to claim 1, is characterized in that, described step 2) middle PK-15 cell continuous passage 6 generation, step 5) virus went down to posterity for 45 generations on cell PK-15 monolayer.
5. the yolk antibody prepared of cell inactivation vaccine as claimed in claim 1, is characterized in that, prepared by following methods:
1) with cell inactivation vaccine according to claim 1, at interval of 7-10 days, three immunity of fundamental immunity, booster immunization and reinforced immunological are carried out to healthy laying hen group, start after booster immunization to detect antibody titer, when pig circular ring virus antibody titer reach 1:64, goose circovirus antibody tire and reach more than 1:64 time, collect high-immunity egg;
2) cleaning, sterilization high-immunity egg, dry, asepsis gets yolk, obtains yolk stock solution;
3) in yolk stock solution, add isopyknic sterilized water, stirring and evenly mixing, to color whitens, obtains yolk solution;
4) yolk solution is put into 60-65 DEG C of water-bath homogeneous heating 30min, then add the acidified aqueous solution of pH4.9-5.2 temperature 4-6 DEG C, addition is 6 times of yolk solution volume, mixing, staticly settles, gets supernatant, the centrifugal 20min of 15000rpm, gets supernatant;
5) add sad in supernatant, sad addition is the 0.8-1.0% of liquor capacity, mixing, and leave standstill, discard impurity floating thing, the centrifugal 10min of 20000rpm, gets supernatant, leaves standstill;
6) with the filter membrane of 0.45mm to step 5) gained supernatant liquid filtering, obtain yolk antibody solution, then through the membrane filtration of model MCW 300KD, obtain the solution containing yolk antibody refined.
6. a kind of yolk antibody according to claim 5, is characterized in that, in described step 1), the amount of basic immunity cell inactivation vaccine is 1.5ml, booster immunization and reinforced immunological inoculation dosage 1 times.
7. a kind of yolk antibody according to claim 5, is characterized in that, in described step 4), acidified aqueous solution is the acidifying water of hydrochloric acid preparation, and pre-coo time is 1-2h.
8. containing, for example an injection for yolk antibody according to claim 5, it is characterized in that, comprise yolk antibody solution 1 part, formaldehyde 0.002-0.003 part and thimerosal 0.0001-0.0002 part.
9. the lyophilized powder containing, for example yolk antibody according to claim 5, it is characterized in that, comprise yolk antibody solution 1 part, formaldehyde 0.002-0.003 part, thimerosal 0.0001-0.0002 part, glucose 0.03-0.04 part, sorbitol 0.01-0.02 part, glycine 0.02-0.03 part and mannitol 0.02-0.03 part.
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| CN104161180B (en) * | 2013-07-18 | 2016-09-21 | 河南联合英伟饲料有限公司 | The feed additive of a kind of anti-porcine viral diseases, preparation method and application |
| CN104161181B (en) * | 2013-07-18 | 2016-06-08 | 河南联合英伟饲料有限公司 | The feed additive of a kind of anti-pig bacterial disease, preparation method and application |
| CN104161182B (en) * | 2013-07-18 | 2016-07-13 | 河南联合英伟饲料有限公司 | The feed additive of a kind of anti-goose viral disease, preparation method and application |
| CN104208679B (en) * | 2013-09-30 | 2016-08-17 | 郑州后羿制药有限公司 | Compositions, freeze-dried mixed powder and the preparation method that a kind of resisting porcine circovirus is sick |
| CN104208674B (en) * | 2013-09-30 | 2016-08-17 | 郑州后羿制药有限公司 | A kind of freeze-dried mixed powder of anti-porcine viral diseases and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6497883B1 (en) * | 1999-06-10 | 2002-12-24 | Merial | Porcine circovirus recombinant poxvirus vaccine |
| CN1749397B (en) * | 2004-08-18 | 2010-12-08 | 金宁一 | Bi-recombinant active carrier vaccine of co-expression pig gyrate virus and foot and mouth disease virus |
| KR20100135903A (en) * | 2008-04-16 | 2010-12-27 | 버지니아 테크 인터렉추얼 프라퍼티스, 인크. | Chimeric porcine circovirus pcv2gen-1rep and uses thereof |
| CN101942528B (en) * | 2010-10-20 | 2012-07-04 | 福建省农业科学院畜牧兽医研究所 | Primer and probe for detecting goose circovirus |
| CN102716476B (en) * | 2012-05-31 | 2015-04-22 | 郑州后羿制药有限公司 | Cell inactivated vaccine, egg yolk antibody injection, and preparation method of cell inactivated vaccine |
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Effective date of registration: 20160302 Address after: 450061 Xingang Avenue, Xinzheng port, Henan, Zhengzhou Patentee after: Henan Hou Yi bioengineering Limited by Share Ltd Address before: 451162 Xingang, Henan, Zhengzhou, Hong Kong airport on the eastern side of the road on the eastern side of Zhengzhou Patentee before: Zhengzhou Houyi Pharmaceutical Co., Ltd. |
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