CN110368490A - Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine and preparation method thereof - Google Patents

Abstract

The present invention relates to Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine and preparation method thereof.The bivalent inactivated vaccine effective component includes the Raccoon dog parvovirus VP2 albumen and canine distemper virus inactivation antigen that RDPV-SN1 plants of recombinant baculovirus transfection Sf9 cell culture obtain；There are virulence to return high wind danger for racoon dog canine distemper, parvovirus bigeminal live vaccine by the present invention, and the lower problem of immune efficacy after the generally existing Raccoon dog parvovirus inactivation of bivalent inactivated vaccine used in the market, provide a kind of bivalent inactivated vaccine by Raccoon dog parvovirus VP2 albumen and the higher canine distemper virus inactivation antigen preparation of viral level.The Combined vaccine can be applied to the prevention of two kinds of diseases of Raccoon dog parvovirus enteritis and canine distemper simultaneously;, controllability good, highly-safe with immunogenicity be strong, culture efficiency and high degree of automation, the advantages such as be produced on a large scale; it can reach " needle two is anti-" purpose, have broad application prospects.

Description

Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine and preparation method thereof

Technical field

The present invention relates to a kind of Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine and preparation method thereof, belong to beast Use field of biological product.

Background technique

Raccoon dog parvovirus enteritis is the acute, high degree in contact as caused by Parvoviridae parvovirus category Raccoon dog parvovirus Sexually transmitted disease.Raccoon dog parvovirus enteritis is widely present in world's every country at present, which mainly causes the enteritis of racoon dog, dead Rate is up to 30%~60%.

Canine distemper is that one kind as caused by paramyxovirus section Morbillivirus canine distemper virus is acute, hot, high degree in contact Sexually transmitted disease, young animal is mostly acute, lethal is passed through, and adults can be at chronic, persistent infection, and clinical symptoms are with double Phase body temperature increases, is main feature with catarrhal pneumonia, gastroenteritis, occasionally has nervous symptoms appearance, is easy other secondary bacteriums The mixed infection and superinfection, the death rate of virus may be up to 80%, be the most serious for endangering China's fur animal farming One of epidemic disease.Racoon dog canine distemper annual any season can occur, and especially young racoon dog is before and after ablactation and finishing period is quicker Sense has very high morbidity and mortality.

Since racoon dog canine distemper and Canine Parvovirus Enteritis have the characteristics that morbidity is anxious, lacks specific treatment method, at present still Using vaccine inoculation as main preventions.

It is sold currently on the market without the dedicated canine distemper of racoon dog, Canine Parvovirus Enteritis vaccine, many raisers use substitution Vaccine can not obtain preferable immune protective effect, therefore, generally require to carry out secondary immunity to racoon dog, not only improve cost, but also The workload of raiser is increased, while causing the biggish stress reaction of recoon dog.And canine distemper vaccine is mostly live seedling, there are virulence Return strong risk；The vaccine of Canine Parvovirus Enteritis preparation is mainly inactivated virus vaccine or live vaccine, and inactivated vaccine is immune Originality is poor, and live vaccine returns strong risk there are virulence.

Therefore, Raccoon dog parvovirus baculovirus vector, the development of canine distemper bivalent inactivated vaccine are extremely urgent, can effectively control Racoon dog canine distemper and Canine Parvovirus Enteritis processed realize " needle two is anti-" purpose.

Summary of the invention

We isolate one plant of Raccoon dog parvovirus from the clinical doubtful tiny pathological material of disease of racoon dog, and Successful amplification goes out the tiny disease of the racoon dog Malicious VP2 gene, we use insect cell baculoviral --- Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus expression system construction The recombinant baculovirus of expression Raccoon dog parvovirus VP2 albumen, transfects SF9 cell, cultivates and obtain virus-like particle, the virus Sample particle has good immunogenicity, safety；CDV-11 the plant of canine distemper Natural Avirulent Strain of my company separation, through the training that suspends It supports, viral level is 10^7.5TCID₅₀/ ml or more still has preferable immunogenicity to recoon dog after inactivating, safety after inactivation Property it is high.

The object of the present invention is to provide a kind of Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine and its preparation sides Method, to cope with current Raccoon dog parvovirus enteritis and canine distemper prevalence situation.

Technical solution of the present invention

1. a kind of Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine, it is characterised in that the effective component of the vaccine Including RDPV-SN1 plants of recombinant baculovirus express Raccoon dog parvovirus VP2 albumen, CDV-11 plants of inactivation antigens of canine distemper virus, Aluminium hydroxide gel adjuvant；The described vaccine is for preventing racoon dog canine distemper and Canine Parvovirus Enteritis.

2. Raccoon dog parvovirus enteritis of the present invention, canine distemper bivalent inactivated vaccine, it is characterised in that the racoon dog is thin Small virus VP2 albumen is RDPV-SN1 plants of transfection Sf9 cells of recombinant baculovirus by expression Raccoon dog parvovirus VP2 albumen, training It supports to cell and infection acquisition occurs, which is named as Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus recombinant baculovirus RDPV-SN1 plants, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microorganism was delivered on May 16th, 2019 The preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center, research institute, deposit number are CGMCC No.17685.

3. Raccoon dog parvovirus enteritis of the present invention, canine distemper bivalent inactivated vaccine, it is characterised in that the canine distemper Viral CDV-11 plants of inactivation antigen is obtained after BEI is inactivated by CDV-11 plants of canine distemper virus, and the strain is in 2009 07 Deliver within 15th Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Chinese microorganism strain guarantor the moon The preservation of administration committee's common micro-organisms center is hidden, deposit number is CGMCC No.3201.

4. Raccoon dog parvovirus enteritis of the present invention, canine distemper bivalent inactivated vaccine, it is characterised in that expression racoon dog it is thin RDPV-SN1 plants of recombinant baculovirus of building of small virus VP2 albumen comprises the following steps:

(1) melittin signal peptide (HBM) and beta-actin the building of transfer vector: are added on pVL1393 carrier Insulator (HS4) sequence constructs transfer vector pVL-HBM-HS4；

(2) building of recombinant transfer vector: Raccoon dog parvovirus RDPV-SN1 plants of VP2 gene is extracted, by the VP2 gene of extraction (SEQ ID No:3) is cloned into transfer vector pVL-HBM-HS4, obtains recombinant transfer vector pVL-HBM-HS4-VP2；

(3) recombinant baculovirus RDPV-SN1 plants of building: by recombinant transfer vector pVL-HBM-HS4-VP2 and linearisation Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus (AcMNPV) genomic DNA cotransfection Sf9 cell obtains expression Raccoon dog parvovirus The recombinant baculovirus of VP2 albumen is named as RDPV-SN1 plants of Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus recombinant baculovirus.

5. Raccoon dog parvovirus enteritis of the present invention, canine distemper bivalent inactivated vaccine, it is characterised in that the system of the vaccine Preparation Method the following steps are included:

(1) Raccoon dog parvovirus VP2 albumen: eugonic Sf9 cell inoculation bioreactor is selected to carry out the full training that suspends It supports, inoculum density is 1.5 × 10⁶~2.0 × 10⁶A cell/ml, 27 DEG C of cultures.When Sf9 cell density reaches in bioreactor To 1.5 × 10⁶~3.0 × 10⁶When a cell/ml, recombinant baculovirus RDPV-SN1 plants, 27 DEG C is inoculated with by MOI=0.2~2.0 72~96h is cultivated, cell culture is harvested, 2%BEI solution is added thereto, makes BEI final concentration of 0.1%, 37 DEG C after mixing 48h is inactivated, to 1% swine erythrocyte hemagglutinative titer >=1:2048,50% sodium thiosulfate is then added into cell culture Solution, make its final concentration of 0.2%, stir 1h after terminate inactivation；Through sterile and inactivation after the assay was approved to get Raccoon dog parvovirus VP2 albumen；

(2) canine distemper virus CDV-11 plants of inactivation antigens: select eugonic Vero cell inoculation bioreactor into Row microcarrier suspension culture, when Vero cell density reaches 2.0 × 10 in bioreactor⁶~2.5 × 10⁶When a cell/ml, CDV-11 plants of canine distemper virus are inoculated with by MOI=0.1, are fallen off on a small quantity to 80% or so cell rounding protrusion of micro-carrier surface and having When, 10~15min is quickly stirred, microcarrier is filtered out with 75 μm or so filter plates, harvests virus liquid, with 20 μm and 5 μm of filter core removals Cell fragment, harvests cell culture, and canine distemper virus content should be not less than 10^7.5TCID₅₀/ml；To the cell culture of harvest Middle addition 2%BEI solution, makes BEI final concentration of 0.1%, and 37 DEG C of inactivation 48h, are then added into cell culture after mixing 50% hypo solution, make its final concentration of 0.2%, stir 1h after terminate inactivation；It is qualified to examine through sterile and inactivation Afterwards to get CDV-11 plants of inactivation antigens of canine distemper virus；

(3) the Raccoon dog parvovirus VP2 albumen that step (1) obtains is diluted to and 1% swine erythrocyte hemagglutinative titer is not less than 1:256, the CDV-11 plants of inactivation antigens of canine distemper virus obtained with step (2) are uniformly mixed by proper proportion, the hydrogen with sterilizing Oxidation aluminium glue is carried out adding 1% thimerosal solution with seedling in the ratio of 9:1, make its final concentration of 0.01%, after being sufficiently stirred ?.

Beneficial effect of the present invention

A kind of Raccoon dog parvovirus enteritis of the present invention, canine distemper bivalent inactivated vaccine and preparation method thereof.The present invention relates to A kind of Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine preparation, key is using the rod-shaped disease of insect cell Poison --- the recombination of Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus expression system construction expression Raccoon dog parvovirus VP2 albumen is rod-shaped Virus, and the recombinant baculovirus and canine distemper of the production technology production expression Raccoon dog parvovirus VP2 albumen using the culture that suspends Viral (Natural Avirulent Strain) is mixed with Combined vaccine with aluminium hydroxide gel after inactivating.It is tiny that the Combined vaccine can be used for simultaneously racoon dog The prevention of two kinds of diseases of viral enteritis and canine distemper has safety and immunogenicity good, is suitble to large-scale industrialized production The advantages of.

Raccoon dog parvovirus enteritis prepared by the present invention, canine distemper bivalent inactivated vaccine using the culture that suspends production work Skill, with cultivating, viral level is high, be not necessarily to mixing, differences between batches are small, training method automates, is not necessarily to mass production personnel, training The advantages that demand area is small, pollution is small, at low cost is supported, successfully solves that spinner culture production efficiency is low, labor intensity is high, product The problems such as uniformity is poor is production technology preferred in vaccine preparation.

Raccoon dog parvovirus enteritis of the invention, canine distemper bivalent inactivated vaccine through safety testing the result shows that, to 2 months The age above recoon dog single dose inoculation (1ml/ is only), single dose repeated inoculation and overdose inoculation (3.0ml/ is only), are observed after inoculation 21, recoon dog is strong to live, and spirit, diet, excrement are normal, without other abnormal clinical symptoms；Dissect, each internal organs are normal, Without lesion, i.e., the bivalent inactivated vaccine is to recoon dog safety.After potency test shows single dose vaccine inoculation, racoon dog on the 14th after being immunized Parvovirus HI antibody reaches protectiveness level；21 days after immune, racoon dog canine distemper virus neutralizing antibody reaches protectiveness level More than；And it is immune 6 months latter, immune recoon dog still has the resistance to canine distemper virus, parvovirus 100%.Vaccine is exempted from The epidemic disease phase is set to 6 months, storage life be 2~8 DEG C 18 months.

Microbial resources information of the present invention

A kind of Raccoon dog parvovirus enteritis of the invention, canine distemper bivalent inactivated vaccine, preparation are expression racoon dog with strain RDPV-SN1 plants of the recombinant baculovirus of parvovirus VP2 albumen is named as Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus weight Baculoviral RDPV-SN1 plants of group, which delivered Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 on May 16th, 2019 The preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center, Institute of Microorganism, Academia Sinica, deposit number are CGMCC No.17685；CDV-11 plants of canine distemper virus delivered BeiChen West Road, Chaoyang District, BeiJing City 1 on 07 15th, 2009 Number No. 3 China Committee for Culture Collection of Microorganisms's common micro-organisms center, Institute of Microorganism, Academia Sinica preservations of institute, Deposit number is CGMCC No.3201 (referring to CN101612396B).

Detailed description of the invention

Fig. 1 is in Raccoon dog parvovirus VP2 gene PCR amplification figure: 1 is Raccoon dog parvovirus VP2 gene amplification product；2 For blank control；M is DL2000Marker.

Fig. 2 is that bacterium colony PCR verifying VP2 gene is connected in pVL-HBM-HS4 carrier figure: 1-3 is 3 different bacterium colony PCR Electrophoresis result；4 be blank control；M is DL2000Marker.

Specific embodiment

1. RDPV-SN1 plants of recombinant baculovirus buildings

(1) Raccoon dog parvovirus RDPV-SN1 plants of separation take morbidity racoon dog intestinal contents that suitable physiological saline is added, and are added about The chloroform of total amount 1/3, sufficiently oscillation 15min, 3000r/min are centrifuged 30min, take supernatant, filtration sterilization, according to MOI be 0.1~ On 0.4 inoculation F81 cell, the MEM culture solution containing 2% newborn bovine serum, 37 DEG C of 96~120h of culture, observation are changed after cell is adherent Cytopathy, the harvest when the typical parvovirus cytopathy for seine occur, falling off, freezes in -20 DEG C.

Isolated RDPV-SN1 plants of Raccoon dog parvovirus are inoculated in the F81 cell for growing up to 80% single layer, 37 DEG C of cultures 72 ~120h, after 75% or more lesion, freeze thawing harvest.

(2) RDPV-SN1 plants of DNA of Raccoon dog parvovirus RDPV-SN1 plants of VP2 gene magnification Raccoon dog parvovirus are template, with VP2-F, VP2-R carry out PCR amplification as primer, according to the RDPV VP2 gene order design primer announced on GenBank:

VP2-F:5 '-aatgaattca gtgatggagc agttcaacc (EcoR I) -3 ' 29 (sequence 1)

VP2-R:5 '-atgggtacct taatataatt ttctaggtg (Kpn I) -3 ' 29 (sequence 2)

As a result the purpose band for amplifying 1755bp size, is consistent with expected results, and gel electrophoresis result is shown in Fig. 1.Amplification Segment is following (sequence 3):

(3) PCR product after purification is connect by transfer vector pMD19-VP2 building with identification with pMD19-T carrier, is connected Product converts DH5 α competent cell, will be coated on the LB solid medium containing Amp after culture bacterium solution concentration, 37 DEG C of cultures 16h carries out bacterium colony PCR identification after the bacterium colony culture on picking plate, as a result amplifies the purpose band of 1.7kb size.Sequencing, It as a result is 1755bp, sequence is as shown in SEQ ID NO:3.Show that RDPV-SN1 plants of VP2 gene successful clones of Raccoon dog parvovirus arrive On pMD19-T carrier.There is the pMD19-T plasmid of RDPV-SN1 plants of VP2 genes of Raccoon dog parvovirus to be named as pMD-VP2 clone.

(4) transfer vector pVL-HBM building and identification

Using pMD19-HBM plasmid as template, HBM-F, HBM-R carry out the amplification of HBM gene PCR as primer.Primer sequence It is classified as:

HBM-F:5 '-agcggatcca aaccatgaaa ttc-3 ' (BamH I) 23 (sequence 4)

HBM-R:5 '-ataagatctg catgcggtac ccc-3 ' (Pst I) 23 (sequence 5)

HBM gene magnification PCR product is obtained, purpose band size is 170bp.It is right with restriction enzyme BamH I/Pst I HBM gene amplification product and plasmid pVL1393 carry out digestion, and purifying is separately recovered after gel electrophoresis, uses T₄DNA ligase Connection converts DH5 α competent cell.Converted product is inoculated with the LB liquid medium containing Amp, picking single colonie carries out bacterium Fall PCR identification.It will identify that correct bacterium solution send sequencing company to be sequenced, selection is sequenced correct bacterium solution and saves.

The HBM gene PCR amplification system are as follows: 25 μ l, consisting of: 1 μ l, 2 × Pfu PCR of pMD19-HBM plasmid 10 μ l, 2 × Taq PCR MasterMix of MasterMix 2.5 μ l, upstream and downstream primer 1 μ l, ddH₂O 9.5μl；The PCR expands Increase reaction condition are as follows: 94 DEG C of 5min；95 DEG C of 45min, 60 DEG C of 45min, 72 DEG C of 30min, 30 circulations；72 DEG C of 10min, 4 DEG C of guarantors Hiding.

The HBM gene endonuclease reaction system are as follows: 50 μ l, consisting of: the DNA fragmentation of HBM gene PCR product glue recycling 10 μ l react 5 I enzyme of μ l, BamH of Buffer, 2 I enzyme of μ l, Pst 2 μ l, ddH₂O 31μl。

The pVL1393 plasmid enzyme restriction reaction system are as follows: 50 μ l, consisting of: 5 μ l of pVL1393 plasmid, reacts Buffer 5 I enzyme of μ l, BamH, 2 I enzyme of μ l, Pst 2 μ l, ddH₂O 36μl。

The HBM gene and pVL1393 plasmid linked system are as follows: digestion HBM gene PCR product 6 μ l, digestion pVL1393 2 μ l of plasmid reacts Buffer 1 μ l, T₄1 μ l of DNA ligase.

(5) transfer vector pVL-HBM-HS4 building and identification

Using pMD19-HS4 plasmid as template, HS4-F, HS4-R carry out the amplification of HS4 gene PCR as primer.Primer sequence It is classified as:

HS4-F:5 '-atatacgtag agctcacggg gacagccccc ccccaaag (SnaB I) -3 ' 38 (sequence 6)

HS4-R:5 '-acatacgtaa atattctcac tgactccgtc ctggagtt (SnaB I) -3 ' 38 (sequence 7)

Obtain HS4 gene magnification PCR product, purpose band size about 1.3kb.With restriction enzyme SnaB I to HS4 base Gene-amplification product and plasmid pVL-HBM carry out digestion, and agarose gel electrophoresis after purification connects target fragment and pVL-HBM carrier It connects, converts DH5 α competent cell.Converted product is inoculated with the LB liquid medium containing Amp, picking single colonie carries out bacterium colony PCR identification.It will identify that correct bacterium solution send sequencing company to be sequenced, selection is sequenced correct bacterium solution and saves.

The HS4 gene PCR amplification system are as follows: 25 μ l, consisting of: 1 μ l, 2 × Pfu PCR of pMD19-HS4 plasmid 10 μ l, 2 × Taq PCR MasterMix of MasterMix 2.5 μ l, upstream and downstream primer 1 μ l, ddH₂O 9.5μl；The PCR expands Increase reaction condition are as follows: 94 DEG C of 5min；95 DEG C of 45min, 60 DEG C of 45min, 72 DEG C of 2min, 30 circulations；72 DEG C of 10min, 4 DEG C of guarantors Hiding.

The HS4 gene endonuclease reaction system are as follows: 50 μ l, consisting of: the DNA fragmentation of HS4 gene PCR product glue recycling 10 μ l react 5 I enzyme of μ l, SnaB of Buffer, 2 μ l, alkaline phosphatase 1 μ l, ddH₂O 32μl。

The pVL-HBM plasmid enzyme restriction reaction system are as follows: 50 μ l, consisting of: 5 μ l of pVL-HBM plasmid, reaction I enzyme of Buffer5 μ l, SnaB, 2 μ l, alkaline phosphatase 1 μ l, ddH₂O 32μl。

The HS4 gene and pVL-HBM plasmid linked system are as follows: digestion HS4 gene PCR product 6 μ l, digestion pVL-HBM 2 μ l of plasmid reacts Buffer 1 μ l, T₄1 μ l of DNA ligase.

(6) transfer vector pVL-HBM-HS4-VP2 building and identification

Transfer vector pVL-HBM-HS4 and pMD-VP2 plasmid are used into I/Kpn of EcoR, I 37 DEG C of digestion 3h, digestion products respectively It recycles, purify after gel electrophoresis.The VP2 gene of the pVL-HBM-HS4 plasmid of digestion purifying and digestion purifying is used into T₄DNA connects Enzyme connection is connect, DH5 α competent cell is converted.By converted product be inoculated with the LB liquid medium containing Amp, picking single colonie, into Row bacterium colony PCR identifies purpose band occur at about 1.7kb.Concrete outcome is shown in Fig. 2.To identify correct bacterium solution send sequencing company into Row sequencing, selection are sequenced correct bacterium solution and save.

The plasmid pVL-HBM-HS4 and pMD-VP2 endonuclease reaction system is equal are as follows: 50 μ l, consisting of: 5 μ l of plasmid, instead Answer 5 I enzyme of μ l, EcoR of Buffer 2 μ l, Kpn I 2 μ l, ddH₂O 36μl。

The pVL-HBM-HS4 plasmid and pMD-VP2 plasmid linked system are as follows: 6 μ l of digestion products VP2 gene, digestion matter Grain 2 μ l of pVL-HBM-HS4, reacts Buffer 1 μ l, T₄1 μ l of DNA ligase.

(7) transfection and purifying of recombinant baculovirus

1) it transfects the transfer vector pVL-HBM-HS4-VP2 for constructing success and linearisation Autographa californica multicapsid nucleopolyhedrosisvirus caryogram is polygonal Precursor virus genomic DNA cotransfection Sf9 cell, 27 DEG C of cultures to the 3rd day started cytopathy occur, and the 5th day when harvests cell Culture supernatant.

2) recombinant baculovirus of harvest is carried out plaque purification by plaque purification.In the Tissue Culture Dish that diameter is 6cm Inoculation 2.0 × 10⁶~2.5 × 10⁶A Sf9 cell, places 5min at room temperature.Gradient dilution (dilution: 10^-4~10^-7) above-mentioned Cotransfection viral supernatants are inoculated with the virus liquid of 1.0ml difference dilution in each culture dish, are placed at room temperature for 1h, fill virus Divide infection cell.Sterile water prepares 2% agarose, adds 2 × Grace culture medium of 1 volume, is uniformly mixed.Abandon cell culture Culture solution supernatant in ware covers 1% agarose of 4.0ml in cell surface, culture dish is placed on wet ring after agarose solidification In border, 27 DEG C are cultivated 7, observe plaque.Single plaque agar block inoculation SF-SFM serum free medium on picking culture dish, 4 It DEG C rocks overnight, harvests culture, be inoculated with Sf9 cell, 27 DEG C of 72~96h of culture, harvest cell culture as recombinates rod-shaped For viral RDPV-SN1 plants of F0 for virus liquid, which is named as the recombination of Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus RDPV-SN1 plants of baculoviral, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese science was delivered on May 16th, 2019 The preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center, institute of microbiology, institute, deposit number CGMCC No.17685。

2. RDPV-SN1 plants of recombinant baculovirus identifications

(1) Sf9 cell density is diluted to 2.0 × 10 by viral subculture⁶A cell/ml is inoculated with F0 for recombinant baculovirus RDPV-SN1 plants, 27 DEG C of constant-temperature table cultures are set, revolving speed is 100~105r/min.72~96h is cultivated, cell culture is harvested, As F1 generation recombinant baculovirus RDPV-SN1 strain virus liquid.

(2) PAGE gel electrophoresis carries out RDPV-SN1 plants of cell cultures of F1 generation recombinant baculovirus of harvest SDS-PAGE detection, while using the Sf9 cell for infecting empty baculoviral (no foreign gene baculoviral) as negative control. Concrete operations are as follows: 5 × Loading Buffer, the boiling water bath 5min of 10 μ l is added in the cell culture for taking 40 μ l to harvest, 12000r/min is centrifuged 1min, takes supernatant to carry out PAGE gel (12%) electrophoresis, takes gel to be dyed, decolourized after electrophoresis Afterwards, as a result there is obvious purpose band near 65kD in F1 generation recombinant baculovirus RDPV-SN1 plants of cell culture, and infection is empty The Sf9 cell culture of baculoviral does not occur respective strap.

(3) Western Blot identification carries out RDPV-SN1 plants of cell cultures of F1 generation recombinant baculovirus of harvest Western Blot detection, while using the Sf9 cell culture for infecting empty baculoviral and Sf9 cell culture as feminine gender Control.Concrete operations are as follows: 5 × Loading Buffer of 20 μ l, boiling water bath is added in the cell culture for taking 80 μ l to harvest 5min, sampling carry out PAGE gel (12%) electrophoresis.Product after SDS-PAGE electrophoresis is transferred to PVDF and (gathers inclined difluoro Vinyl film) on film, 2h is closed with 5% skim milk, the anti-recoon dog enteritis virus positive blood in the diluted rabbit source 1:100 is added in rinsing Clear to be incubated for 2h, rinsing is added the goat anti-rabbit igg antibody that the diluted horseradish peroxidase of 1:2000 (HRP) is marked and is incubated for 2h, drift It washes, enhanced chemical luminous fluorescent substrate is then added dropwise, is exposed using X-ray film tabletting.

As a result there is apparent purpose band, table at 65kD in F1 generation recombinant baculovirus RDPV-SN1 plants of cell culture Bright VP2 albumen successful expression in Sf9 cell.

(4) the F1 generation recombinant baculovirus RDPV-SN1 strain virus liquid of harvest is pressed MTT relative effectivenes by viral level measurement Method (progress [J] microbiology immunology that Huang Shihe, complementary modul pine measure baculovirus titers method is in progress, and 2007 (02): 79-83. viral level) is measured, result is 6.0 × 10^7.0PFU/ml。

(5) RDPV-SN1 plants of cell cultures of F1 generation recombinant baculovirus of harvest are pressed in expression albumen blood clotting titration Expression albumen hemagglutinative titer, result 1:2048 are measured according to Raccoon dog parvovirus blood clotting (HA) test method.

(6) expression albumen VP2 determining the protein quantity carries out determining the protein quantity with double antibody sandwich ELISA.As a result F1 It is 72.5mg/L for RDPV-SN1 plants of expression protein contents of recombinant baculovirus.

(7) observation of expression albumen Electronic Speculum surpasses RDPV-SN1 plants of cell cultures of recombinant baculovirus through sucrose density gradient Speed centrifugation, sucrose solution concentration is respectively 40%, 50%, 60%, centrifugal speed 29000r/min, is centrifuged 2h, then collects Positioned at the band of 50% and 60% concentration intersection.Virus-like particle sample after a small amount of purifying concentration is added drop-wise to carbon film On copper mesh, after air-drying, 2% sodium phosphotungstate solution is added dropwise and carries out negative staining, Electronic Speculum is observed, as a result F1 generation recombinant baculovirus RDPV- The Raccoon dog parvovirus sample particle of SN1 plants of expression is suitable with Raccoon dog parvovirus particle size, morphology is close.

(8) RDPV-SN1 plants of cell culture legs of F1 generation recombinant baculovirus are inoculated 2~12 by safety testing Susceptible recoon dog 5 of monthly age health, 3.0ml/ only, are observed 10, and as a result all inoculation recoon dogs are strong lives.

(9) seed culture of viruses is inoculated with well-grown Sf9 cell, 27 DEG C of 72~96h of culture, harvest cell training by Study On Immunogenicity Support object.After binary ethylenimine (BEI) inactivation completely, by cell culture and the aluminium hydroxide gel of sterilizing in the ratio system of 9:1 Standby vaccine, leg inoculate 5 2~12 monthly ages susceptible recoon dogs of health (RDPV HI antibody titer is not higher than 1:4), 1.0ml/ Only, while the identical recoon dog of 5 conditions being taken not to be inoculated with as control.14 days after immune, it is equal that recoon dog RDPV HI antibody titer is immunized Not less than 1:32, compares recoon dog RDPV HI antibody titer and be not higher than 1:4；Way is gavaged through esophagus respectively to all recoon dogs simultaneously Diameter is inoculated with Raccoon dog parvovirus RDPV-SN1 strain virus liquid 15ml and (contains 15 × 10^7.0TCID₅₀), it is observed continuously 10, racoon dog is as a result immunized Sub all strong work, control recoon dog all fall ill.

3. the preparation and product inspection of Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine

(1) vaccine preparation

1) preparation of Raccoon dog parvovirus VP2 albumen: eugonic Sf9 cell inoculation bioreactor is selected to be hanged entirely Floating culture, inoculum density are 1.5 × 10⁶~2.0 × 10⁶A cell/ml, 27 DEG C of cultures, when Sf9 cell is close in bioreactor Degree reaches 1.5 × 10⁶~3.0 × 10⁶When a cell/ml, it is inoculated with recombinant baculovirus RDPV-SN1 plants by MOI=0.2~2.0, 27 DEG C of 72~96h of culture harvest cell culture, and 2%BEI solution is added thereto, makes BEI final concentration of 0.1%, after mixing 37 DEG C of inactivation 48h, then into cell culture addition 50% hypo solution, make its final concentration of 0.2%, stirring Inactivation is terminated after 1h；It examines through sterile and inactivation to get Raccoon dog parvovirus VP2 albumen, to 1% swine erythrocyte hemagglutinative titer >=1: 2048,2~8 DEG C save backup；

2) preparation of canine distemper virus CDV-11 plants of inactivation antigens: eugonic Vero cell inoculation cell culture is selected Tank carries out microcarrier suspension culture, when Vero cell density reaches 2.0 × 10 in bioreactor⁶~2.5 × 10⁶A cell/ml When, it is inoculated with canine distemper virus CDV-11 plants by MOI=0.1, using the DMEM cell culture for containing 2% newborn bovine serum when connecing poison Liquid, temperature are 34 DEG C, connect different time sampling observation after poison, to 80% or so cell rounding protrusion of micro-carrier surface and have a small amount of When falling off, 10~15min is quickly stirred, microcarrier is filtered out with 75 μm or so filter plates, virus liquid is harvested, with 20 μm and 5 μm of filter cores Cell fragment is removed, cell culture is harvested, canine distemper virus content should be not less than 10^7.5TCID₅₀/ml；It is trained to the cell of harvest It supports and 2%BEI solution is added in object, make BEI final concentration of 0.1%, 37 DEG C of inactivation 48h after mixing, then into cell culture Addition 50% hypo solution, make its final concentration of 0.2%, stir 1h after terminate inactivation, through it is sterile and inactivation examine To get CDV-11 plants of inactivation antigens of canine distemper virus after qualification, 2~8 DEG C are saved backup:

3) the Raccoon dog parvovirus VP2 albumen that step (1) obtains is diluted to 1% swine erythrocyte hemagglutinative titer not less than 1: 256, the CDV-11 plants of inactivation antigens of canine distemper virus obtained with step 2) are uniformly mixed by proper proportion, the hydroxide with sterilizing Aluminium glue is carried out adding 1% thimerosal solution with seedling in the ratio of 9:1, make its final concentration of 0.01%, it is quantitative after being sufficiently stirred Packing covers, labeling.

(2) Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine product inspection

1) after character is stood, upper layer is supernatant liquid, and it is in homogenous suspension after shaking that lower layer, which is a small amount of precipitating,.

2) steriling test, loading amount, the antiseptic mercurials determination of residual amount are tested by existing " Chinese veterinary pharmacopoeia " annex, are answered Asepsis growth.

3) safety verification 2~12 monthly ages susceptible recoon dog of health (RDPV HI antibody titer is not higher than 1:4) 5, each leg Vaccine 3.0ml is subcutaneously injected, after inoculation, periodically observes daily and measures recoon dog body temperature 1 time (with preceding thermometric time consistency is inoculated with), It is observed continuously 21.Be inoculated with that recoon dog is all strong to live, spirit, diet, excrement, body temperature compared with inoculation is preceding without significant change, without it Its abnormal clinical symptom；Or body temperature is increased more than 1 DEG C, but is delaied and should be no more than 2.

4) efficacy test

1. serological method 2~12 monthly ages susceptible recoon dog of health (RDPV HI antibody titer is not higher than 1:4) 5, each leg Vaccine 1.0ml is subcutaneously injected in portion, while the identical recoon dog of 5 conditions being taken not to be inoculated with as control.14 days after immune, adopt respectively Blood separates serum, measures RDPV HI antibody titer and canine distemper virus neutralize antibody titers, and recoon dog RDPV HI antibody effect is immunized Valence should all be not less than 1:32, and control recoon dog RDPV HI antibody titer should all be not higher than 1:4；It 21 days after immune, takes a blood sample, surveys respectively Determine canine distemper virus neutralize antibody titers, recoon dog canine distemper virus neutralize antibody titers, which are immunized, should all be not less than 1:50, compare racoon dog Sub- canine distemper virus neutralize antibody titers should all be not higher than 1:4.

2. Immunization method is with 2~12 monthly ages susceptible recoon dog of health (RDPV HI antibody titer is not higher than 1:4) 10, respectively Vaccine 1.0ml is subcutaneously injected in leg, while the identical recoon dog of 10 conditions being taken not to be inoculated with as control.14 days after immune, at random It chooses 5 immune recoon dogs and 5 control recoon dogs and gavages approach inoculation Raccoon dog parvovirus RDPV-SN1 strain virus liquid through esophagus respectively 15ml (contains 15 × 10^7.0TCID₅₀), it is observed continuously 10, control recoon dog answers 100% morbidity, and immune recoon dog answers 100% protection；Exempt from 21 days after epidemic disease, remaining 5 immune recoon dogs and 5 control recoon dog difference collunarium inoculation 2.0ml, intraperitoneal injection 8.0ml are (altogether 10.0ml contains 1.0 × 10^5.5TCID₅₀) canine distemper virus ZC strain virus liquid, it is observed continuously 21 after attacking poison, control recoon dog answers 100% morbidity, immune recoon dog answer 100% protection.

Embodiment

Following embodiment combination attached drawing further illustrates the present invention.But it is not construed as limiting the scope of the present invention.Ability Field technique people should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and Form is modified or is replaced, but these modifications or substitutions are fallen within the protection scope of the present invention.

The RDPV-SN1 plants of suspension culture process researchs of embodiment 1 --- recombinant baculovirus

1. RDPV-SN1 plants of triangular flask culture process researchs of recombinant baculovirus

(1) influence of the cell density to expression albumen hemagglutinative titer

When Sf9 cell density is 1.5 × 10⁶A cell/ml, 2.0 × 10⁶A cell/ml, 2.5 × 10⁶A cell/ml, 3.0×10⁶When a cell/ml, be inoculated with recombinant baculovirus RDPV-SN1 plants by MOI=0.5,60h, 72h after inoculation, 84h, 96h, 108h, 120h sampling, measurement expression albumen hemagglutinative titer.

As a result by the Sf9 cell of the different cell densities of RDPV-SN1 plants of recombinant baculovirus inoculations, the Sf9 cell when connecing poison Density is 1.5 × 10⁶、2.0×10⁶、2.5×10⁶、3.0×10⁶When a cell/ml, expression albumen hemagglutinative titer is without obvious poor Different, 72h expression albumen hemagglutinative titer peaks after connecing poison, is not less than 1:2048, and maintain to 96h.Concrete outcome is shown in Table 1。

The different cell density samples of table 1 express albumen hemagglutinative titer

(2) influence of the virus inoculation amount to expression albumen hemagglutinative titer

When Sf9 cell density is 1.5 × 10⁶When a cell/ml, by MOI=0.05,0.1,0.2,0.5,1.0,2.0, 3.0 recombinant baculovirus RDPV-SN1 plants of inoculations, 60h, 72h, 84h, 96h, 108h, 120h are sampled after inoculation, measurement expression egg White hemagglutinative titer.

As a result when MOI is 0.2,0.5,1.0,2.0, albumen hemagglutinative titer no significant difference is expressed.72h expresses egg after connecing poison White hemagglutinative titer peaks, and is not less than 1:2048, and maintain to 96h；Proteinemina is expressed when MOI is 0.05,0.1,3.0 Solidifying potency is lower.Concrete outcome is shown in Table 2.

The different infection multiplicity samples of table 2 express albumen hemagglutinative titer

(3) influence of the different harvest times to expression albumen hemagglutinative titer

When Sf9 cell density is 1.5 × 10⁶When a cell/ml, recombinant baculovirus RDPV-SN1 is inoculated with by MOI=0.2 Strain, 60h, 72h, 84h, 96h, 108h, 120h are sampled after inoculation, measurement expression albumen hemagglutinative titer.

As a result 72h expression albumen hemagglutinative titer peaks after connecing poison, and maintains to 96h, and it is equal to express albumen hemagglutinative titer Not less than 1:2048；120h expression albumen hemagglutinative titer is begun to decline after connecing poison.Concrete outcome is shown in Table 3.

The different harvest time samples of table 3 express albumen hemagglutinative titer

To sum up test result, it is contemplated that the factors such as production, we determine RDPV-SN1 plants of production technologies of recombinant baculovirus Are as follows: inoculation initial cell density is 1.5 × 10⁶~3.0 × 10⁶A cell/ml, MOI are 0.2~2.0, meet 72~96h after poison, Harvest cell culture.

2. RDPV-SN1 plants of bioreactor inoculating process tests of recombinant baculovirus

(1) cell inoculation and amplification are cultivated the well-grown Sf9 cell inoculation 30L bioreactor of triangular flask culture, Inoculum density is set to 1.5 × 10⁶~2.0 × 10⁶A cell/ml, cell culture temperature are 27 DEG C, dissolved oxygen 45%, during culture Timing sampling observes cell state and records cell density and vigor；Reach 3.0 × 10 to cell density⁶A cell/ml or more When, supplemented medium continues culture to cell density and is higher than 3.0 × 10⁶Sf9 cell suspension is transferred to 90L by a cell/ml It is cultivated in bioreactor, inoculum density is 1.5 × 10⁶~2.0 × 10⁶A cell/ml, timing sampling during culture are seen It examines cell state and records viable cell density and vigor.Reach 3.0 × 10 to cell density⁶When a cell/ml or more, training is added Base is supported, continues culture to cell density and reaches 3.0 × 10⁶Sf9 cell suspension is transferred to 350L cell by a cell/ml or more It is cultivated in culture tank.It is amplified to 1000L bioreactor culture in the same way.

(2) 1000L bioreactor, which connects poison and tests, reaches 1.5 × 10 to Sf9 cell density in 1000L bioreactor⁶ A cell/ml, 3.0 × 10⁶When a cell/ml, respectively according to MOI=0.2,2.0 recombinant baculovirus RDPV-SN1 plants of inoculations Seed culture of viruses is sampled respectively at 72h, 96h, measurement expression albumen hemagglutinative titer.As a result 72h, 96h after poison, expression albumen blood clotting effect are connect Valence no significant difference is not less than 1:2048.Concrete outcome is shown in Table 4.

4 1000L bioreactor of table connects poison test expression albumen blood clotting titration result

We conducted RDPV-SN1 plants of triangular flask culture process researchs of recombinant baculovirus, are carried out according to test result Bioreactor process certification test has finally determined that RDPV-SN1 plants of bioreactors of recombinant baculovirus suspend and has cultivated work Skill: when Sf9 cell density reaches 1.5 × 10 in bioreactor⁶~3.0 × 10⁶When a cell/ml, by MOI=0.2~2.0 Recombinant baculovirus RDPV-SN1 plants, 27 DEG C of 72~96h of culture of inoculation harvests cell culture.

The CDV-11 plants of suspension culture process researchs of embodiment 2 --- canine distemper virus

1. CDV-11 plants of 7L bioreactor culture process researchs of canine distemper virus

(1) after microcarrier preparation and sterilizing weigh I type microcarrier of Cytodex every time, with no Ca²⁺、Mg²⁺PBS (0.1mol/L, pH value 7.2, similarly hereinafter) cleaning 2~3 times, 121 DEG C of sterilizing 30min of PBS are added.PBS is discarded after sterilizing, The MEM cell culture fluid containing 8% newborn bovine serum is added, at 36.5 DEG C, containing 5%CO₂4h is placed in incubator.

(2) Vero cell microcarrier suspension culture selection form is good, eugonic Vero rolling bottle cell, uses EDTA- Trypsase dispersion liquid digestion prepares cell suspension, is inoculated with stirring-type Tissue Culture Flask, revolving speed 30r/min, and cell density is 4.5×10⁵A cell/ml, microcarrier concentration 4g/L, cultivation temperature are 36.5 DEG C~37.5 DEG C.

(3) influence Vero cell inoculation stirring-type Tissue Culture Flask culture of the cultivation temperature to viral level, when cell is close Degree reaches 2.0 × 10⁶When a cell/ml, the DMEM cell culture fluid containing 2% newborn bovine serum is changed, is inoculated with dog by MOI=0.1 CDV-11 plants of distemper virus, 31 DEG C, 34 DEG C, 37 DEG C of cultures are set respectively.48h, 72h, 96h, 120h are separately sampled after connecing poison, measurement Canine distemper virus content.

As a result canine distemper virus cultivates viral level highest under the conditions of 34 DEG C, and the viral level cultivated under the conditions of 37 DEG C is slightly Viral level under the conditions of lower than 34 DEG C, culture viral level is minimum under the conditions of 31 DEG C.Concrete outcome is shown in Table 5.

The different cultivation temperature sample virus contents of table 5

(4) influence Vero cell inoculation stirring-type Tissue Culture Flask culture of the cell density to viral level when connecing poison, when Cell density reaches 1.5 × 10⁶A cell/ml, 2.0 × 10⁶A cell/ml, 2.5 × 10⁶A cell/ml, 3.0 × 10⁶It is a thin When born of the same parents/ml, the DMEM cell culture fluid containing 2% newborn bovine serum is changed, is inoculated with canine distemper virus CDV-11 by MOI=0.1 respectively Strain, cultivation temperature are 34 DEG C.48h, 72h, 96h, 120h are separately sampled after connecing poison, measure canine distemper virus content.

Cell density is 2.0 × 10 when as a result connecing poison⁶A cell/ml and 2.5 × 10⁶Viral level is most when a cell/ml Height, cell density is 1.5 × 10 when connecing poison⁶A cell/ml and 3.0 × 10⁶Viral level is slightly lower when a cell/ml.According to test As a result cell density is 2.0 × 10 when production connects poison⁶~2.5 × 10⁶A cell/ml.Concrete outcome is shown in Table 6.

The different cell density sample virus contents of table 6

(5) influence Vero cell inoculation stirring-type Tissue Culture Flask culture of the poison amount to viral level is connect, cell density is worked as Reach 2.0 × 10⁶When a cell/ml, change the DMEM cell culture fluid containing 2% newborn bovine serum, respectively by MOI=0.05, 0.1,0.15 canine distemper virus CDV-11 plants of inoculation, cultivation temperature are 34 DEG C.48h, 72h, 96h, 120h are separately sampled after connecing poison, Measure canine distemper virus content.

Viral level highest when being as a result inoculated with canine distemper virus CDV-11 plants by MOI=0.1, is connect by MOI=0.05,0.15 Viral level, which is below, at canine distemper virus CDV-11 plants of kind is inoculated with canine distemper virus CDV-11 strain virus content by MOI=0.1. Canine distemper virus when producing is connect into poison amount according to test result and is determined as MOI=0.1.Concrete outcome is shown in Table 7.

7 difference of table connects poison amount sample virus content

(6) influence Vero cell inoculation stirring-type Tissue Culture Flask culture of the serum-concentration to viral level, when cell is close Degree reaches 2.0 × 10⁶When a cell/ml, the DMEM cell culture fluid containing 0,1%, 2%, 3% newborn bovine serum is changed respectively, is pressed MOI=0.1 is inoculated with canine distemper virus CDV-11 plants, and cultivation temperature is 34 DEG C.48h, 72h, 96h, 120h are separately sampled after connecing poison, Measure canine distemper virus content.

As a result with the DMEM cell culture fluid culture virus containing 2%, 3% newborn bovine serum, virus multiplication is very fast, and virus contains It measures higher；DMEM cell culture fluid culture disease with the DMEM cell culture fluid containing serum-free and containing 1% newborn bovine serum concentration Viral level is lower when malicious.According to test result, when production culture medium select concentration containing newborn bovine serum for 2% DMEM cell Culture solution.Concrete outcome is shown in Table 8.

The different serum-concentration sample virus contents of table 8

(7) influence Vero cell inoculation stirring-type Tissue Culture Flask culture of the different harvest times to viral level, when thin Born of the same parents' density reaches 2.0 × 10⁶When a cell/ml, the DMEM cell culture fluid containing 2% newborn bovine serum is changed, is connect by MOI=0.1 Canine distemper virus CDV-11 plants of kind, cultivation temperature are 34 DEG C.48h, 72h, 96h, 120h are sampled after connecing poison, observe cytopathy feelings Condition, and measure canine distemper virus content.

48h after poison is met, for cell without obvious lesion, viral level is lower；72h after poison is met, 70%~80% is thin on microcarrier Born of the same parents are rounded protrusion and lesion occur, and a small amount of cell falls off from microcarrier, and viral level peaks value；Connect 96h after poison, cytopathy It becomes apparent, 80% or so cell falls off from microcarrier, and dissociate mortality cell in culture medium, and viral level maintains peak Value；120h after poison is met, almost without normal cell, cell falls off from microcarrier, and almost cell-free on microcarrier, viral level has Declined.It, can when 80% or so cell rounding protrusion of micro-carrier surface and having falls off on a small quantity the result shows that meet 72~96h after poison Harvest virus liquid.Concrete outcome is shown in Table 9.

The different harvest times of table 9 influence viral level

According to the above experimental study, it is determined that processing parameter is cultivated in CDV-11 plants of suspensions of canine distemper virus: Vero is thin It is 2.0 × 10 that born of the same parents, which cultivate to cell density,⁶~2.5 × 10⁶When a cell/ml, canine distemper virus CDV-11 is inoculated with by MOI=0.1 Strain, culture medium uses the DMEM cell culture fluid containing 2% newborn bovine serum when connecing poison, and cultivation temperature is 34 DEG C, connects difference after poison Time sampling, observe cell state, when 80% or so cell rounding protrusion of micro-carrier surface and have on a small quantity fall off when, harvest virus Liquid.

2. bioreactor verification test

(1) bioreactor washing and sterilizing and microcarrier balance clean up bioreactor, 121 DEG C of sterilizing 30min, After sterilizing, 100% point of correction of slack tank dissolved oxygen electrode is completed.Sterilized microcarrier is squeezed into bioreactor, is added Equilibrated Medium to minimum volume of culture, equilibration time is no less than 4h.

(2) 42L bioreactor connects poison test Vero cell inoculation 7L bioreactor, when cell density reaches 2.0 × 10⁶When a cell/ml or more, digestion transfer is carried out, 7L bioreactor cell is transferred to 42L bioreactor culture, inoculation Density is not less than 4.5 × 10⁵A cell/ml.For cell in 42L bioreactor culture 2~3 days, cell density reached 2.0 × 10⁶ ~2.5 × 10⁶When a cell/ml, the DMEM cell culture fluid of 2% newborn bovine serum is changed, is inoculated with hundstaupe pyreticosis by MOI=0.1 CDV-11 plants malicious, cultivation temperature is 34 DEG C, and different time samples after connecing poison, observes cell state, left to micro-carrier surface 80% Right cell rounding protrusion and have on a small quantity when falling off, quickly stirs 10~15min, filter out microcarrier with 75 μm or so filter plates, harvest Virus liquid successively removes cell fragment with 20 μm and 5 μm of filter cores, canine distemper virus content is measured by sampling.Canine distemper as the result is shown Viral level is 10^7.5TCID₅₀/ ml or more.

We when cultivation temperature, connecing poison cell density, connect malicious amount, serum-concentration, have studied in terms of harvest time five Canine distemper virus, which suspends, cultivates production technology, and carries out bioreactor process certification test, and dog has been determined according to test result Distemper virus, which suspends, cultivates production technology are as follows: Vero cell culture to cell density is 2.0 × 10⁶~2.5 × 10⁶A cell/ml When, it is inoculated with canine distemper virus CDV-11 plants by MOI=0.1, culture medium uses the DMEM cell containing 2% newborn bovine serum when connecing poison Culture solution, cultivation temperature are 34 DEG C, observe cell state after connecing poison, connect 72~96h after poison, 80% or so cell of micro-carrier surface When being rounded protrusion and having a small amount of fall off, 10~15min is quickly stirred, microcarrier is filtered out with 75 μm or so filter plates, harvests virus liquid, Cell fragment is removed with 20 μm and 5 μm of filter cores.

The RDPV-SN1 plants of BEI inactivation technology researchs of embodiment 3 --- recombinant baculovirus

Take RDPV-SN1 plants of each 300ml of cell culture of 001,002,003 batch of recombinant baculovirus, quantitative separating, 100ml/ bottles.2%BEI solution is added in every bottle of cell culture, making its final concentration is respectively 0.05%, 0.1%, 0.2%, is set 37 DEG C inactivation, during which every 4~8h mix 1 time, respectively at for 24 hours, 32h, 40h, 48h sampling, 50% sodium thiosulfate is added Solution makes 2 times of its final concentration of BEI, and stirring 1h terminates inactivation, and sampling carries out inactivation inspection.

As a result 0.05%BEI solution, 37 DEG C of inactivation 48h, 0.1%BEI solution, 37 DEG C of inactivation 40h, 0.2%BEI solution, 37 DEG C of inactivation 40h, can be RDPV-SN1 plants with complete inactivation recombinant baculovirus, and concrete outcome is shown in Table 10.To guarantee that recombination is rod-shaped Viral RDPV-SN1 plants of inactivation thoroughly, while considering production cost, determine used in production final concentration of 0.1% BEI, 37 DEG C Inactivate 48h.

RDPV-SN1 plants of various concentration BEI solution inactivation tests of 10 recombinant baculovirus of table

Note: "+" represents inactivation completely, and "-" representative does not inactivate completely.

The CDV-11 plants of BEI inactivation technology researchs of embodiment 4 --- canine distemper virus

Take CDV-11 plants of each 300ml of cell culture of 001,002,003 batch of canine distemper virus, quantitative separating, 100ml/ bottles. 2%BEI solution is added in every bottle of cell culture, making its final concentration is respectively 0.05%, 0.1%, 0.2%, 37 DEG C of inactivations are set, Period every 4~8h mix 1 time, respectively at for 24 hours, 32h, 40h, 48h sampling, 50% hypo solution, which is added, makes its end Concentration is 2 times of BEI, and stirring 1h terminates inactivation, and sampling carries out inactivation inspection.

As a result 0.05%BEI solution, 37 DEG C of inactivation 48h, 0.1%BEI solution, 37 DEG C of inactivation 40h, 0.2%BEI solution, 37 DEG C of inactivation 40h, can be CDV-11 plants with complete inactivation canine distemper virus, and concrete outcome is shown in Table 11.To guarantee canine distemper virus CDV-11 plants of inactivations thoroughly, while considering production cost, determines the BEI in production using final concentration of 0.1%, 37 DEG C of inactivations 48h。

CDV-11 plants of various concentration BEI solution inactivation tests of 11 canine distemper virus of table

Note: "+" represents inactivation completely, and "-" representative does not inactivate completely.

Embodiment 5 --- the preparation of Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine

1. prepared by Raccoon dog parvovirus VP2 albumen

(1) production is prepared with seed culture of viruses

1) it is 1.5 × 10 that seed culture of viruses is inoculated with well-grown, cell density by MOI=0.2~2.0 by seed culture of viruses breeding⁶~2.0 × 10⁶The Sf9 cell of a cell/ml, 27 DEG C of 72~96h of culture harvest cell culture, quantitative separating.Indicate title, harvest day Phase, generation etc..

2) seed culture of viruses is examined

1. viral level is measured by MTT relative effectivenes method, every milliliter of viral level answers >=1.0 × 10^7.0PFU。

2. pure property is tested by existing " Chinese veterinary pharmacopoeia " annex, should be without bacterium, mould, mycoplasma and exogenous virus Pollution.

3. specificity

Seed culture of viruses PCR detection is detected with PCR method, the band of about 1.7kb should occurs.

Protein-specific is expressed by recombinant baculovirus RDPV-SN1 plants of cell cultures PBS (0.015mol/L, pH value It is 6.4, similarly hereinafter) it is diluted to the suspension of 8 blood coagulation units, it mixes well, makees with the Raccoon dog parvovirus specific antisera of equivalent For neutralization group, while setting up positive controls (RDPV-SN1 plants of cell cultures of recombinant baculovirus are diluted to 8 blood with PBS The suspension of solidifying unit and the PBS of equivalent are mixed well) and negative control group (PBS), 37 DEG C of neutralization 60min, shaking 2~3 times.In With after, 96 hole Microhemagglutination plate of V-type is respectively added by above 3 groups, and every group of at least 10 holes, every hole 0.025ml, then every hole is again 1% swine erythrocyte 0.025ml, shake well is added, 2~8 DEG C of 40~50min of effect observe result.Neutralization group and negative control Group should all not be aggregated, and positive controls should be all aggregated.

3) virus seed subculture should be no more than for 3 generations.

4) 2~8 DEG C of virus seed conservation preservations, storage life are 9 months.

(2) prepared by cell

1) cell recovery takes the Sf9 cell of Liquid nitrogen storage, puts into 37 DEG C of warm water immediately and is quickly dissolved, by Sf9 cell Suspension 620r/min is centrifuged 5min, abandons supernatant, and cell is resuspended with SF-SFM serum free medium, is placed in the triangle of suitable capacity Bottle, 27 DEG C of shaking table cultures, revolving speed are 100~105r/min.

2) cell expansion culture works as Sf9 cell density and reaches 3.0 × 10⁶It when a cell/ml or more, is passed on, is inoculated with Cell density is 1.0 × 10⁶~1.5 × 10⁶A cell/ml, cell are passaged to certain amount, inoculating cell culture tank, 27 DEG C after Continuous culture.

(3) prepared by antigen for vaccine

1) bioreactor cleaning, sterilizing clean up bioreactor, 121 DEG C of sterilizing 30min, after sterilizing, Correct dissolved oxygen electrode.

2) cell inoculation and amplification culture select eugonic Sf9 cell, inoculating cell culture tank, and inoculum density is 1.5×10⁶~2.0 × 10⁶A cell/ml, 27 DEG C of cultures are higher than 3.0 × 10 to cell density⁶When a cell/ml, training is added Base is supported, continues culture to cell density and is higher than 3.0 × 10⁶When a cell/ml, bioreactor inner cell suspension is transferred to newly Bioreactor culture, inoculum density are 1.5 × 10⁶~2.0 × 10⁶A cell/ml.

3) poison and harvest are connect when Sf9 cell density reaches 1.5 × 10⁶~3.0 × 10⁶When a cell/ml, by MOI=0.2 ~2.0 recombinant baculovirus RDPV-SN1 plants, 27 DEG C of 72~96h of culture of inoculations, harvest cell culture, indicate title, harvest Date, lot number etc. set -15 DEG C or less preservations, to be checked spare.

(4) inactivate into cell culture addition 2%BEI solution, make its final concentration of 0.1%, 37 DEG C of inactivation 48h；It goes out After work, into cell culture add 50% hypo solution, make its final concentration of 0.2%, stir 1h after eventually It only inactivates, 2~8 DEG C of preservations are to be checked spare.

2. CDV-11 plants of semi-finished product preparations of canine distemper virus and inspection

(1) cell prepare recovery Vero cell, growth-promoting media be the cell culture fluid containing 10% newborn bovine serum, set 37 DEG C, Containing 5%CO₂It is cultivated in incubator, when growing up to fine and close single layer, with EDTA- trypsase dispersion liquid vitellophag, passage proliferation Culture.

(2) preparation of production seed culture of viruses and inspection

1) preparation of production seed culture of viruses takes well-grown Vero cell monolayer, discards growth-promoting media (containing 10% newborn bovine serum MEM cell culture fluid), seed culture of viruses is added in the ratio of maintaining liquid (the MEM cell culture fluid containing 2% newborn bovine serum) amount 2% Into maintaining liquid, it is then dispensed into cell bottle, sets 37 DEG C, containing 5%CO₂Continue to cultivate in incubator, to 80% or so cell Occur harvesting when exemplary fusion type cytopathy, freeze thawing 1 time, quantitative separating, freezen protective indicates harvest date, Virus passages Deng.

2) seed culture of viruses is examined

It is serially diluted, takes 1. seed culture of viruses is carried out 10 times with the MEM cell culture fluid containing 2% newborn bovine serum by viral level 10^-4、10^-5、10^-6、10^-74 dilutions are inoculated with 96 porocyte culture plates of Vero cell for having grown up to single layer, each dilution respectively 8 holes of degree inoculation, every 100 μ l of hole, while setting and not connecing 8 holes of poison control.Add the MEM cell culture containing 2% newborn bovine serum in every hole 100 μ l of liquid sets 37 DEG C, containing 5%CO₂It is cultivated in incubator, observes 96~120h, recorded the hole count of cytopathy (CPE), press Reed-Muench method calculates TCID₅₀.Every milliliter of viral level answers >=10^5.5TCID₅₀。

2. pure property carries out sterile, mycoplasma by existing " Chinese veterinary pharmacopoeia " annex and exogenous virus is examined, sterile, nothing is answered Mycoplasma growth, also pollutes without exogenous virus.

3. seed culture of viruses is diluted to 10 with sterile saline by specificity^3.0TCID₅₀/ ml, with 10 times of diluted canine distemper virus Specific antisera mixed in equal amounts as neutralization group, while setting and not neutralizing control group and (be diluted to seed culture of viruses with sterile saline 10^3.0TCID₅₀/ ml is mixed with equivalent MEM cell culture fluid) and cell controls group, 37 DEG C of neutralization 30min.After neutralization respectively Inoculation has grown up to 24 porocyte culture plates of Vero cell of single layer, and neutralization group is inoculated with 12 holes, the hole 0.5ml/, do not neutralize control group and Cell controls group is respectively inoculated with 6 holes, the hole 0.5ml/, and every hole adds the MEM cell culture fluid 0.5ml containing 2% newborn bovine serum, sets 37 DEG C, contain 5%CO₂It is cultivated in incubator, observes 96~120h.Cytopathy should all not occur in neutralization group and cell controls group, no Cytopathy should all be occurred by neutralizing control group.

(3) preparation of seedling virus liquid

1) bioreactor cleaning, sterilizing and microcarrier balance clean up bioreactor, 121 DEG C of sterilizing 30min. The complete microcarrier that will sterilize after sterilizing squeezes into bioreactor, and the MEM cell culture fluid for containing 8% newborn bovine serum is added It is balanced to minimum volume of culture, equilibration time is no less than 4h.100% point of dissolved oxygen electrode of school is completed during BALANCE CARRIERS Just.

2) cell inoculation selects well-grown Vero cell monolayer, after EDTA- trypsin digestion, inoculating cell training Tank is supported, inoculum density is not less than 4.5 × 10⁵A cell/ml, culture medium are the MEM cell culture fluid containing 8% newborn bovine serum, Continuous culture 2~3 days, when cell density is higher than 2.0 × 10⁶Digestion transfer is carried out when a cell/ml.

3) cell suspension is transferred in digester by cell dissociation transfer, abandons supernatant, PBS is added into digester (0.01mol/L, pH7.2) is cleaned 2 times, abandons PBS, and trypsase is added and is digested, and is added after digesting completely and is contained 8% new born bovine The MEM cell culture fluid of serum terminates digestion.Cell suspension is transferred to new bioreactor culture, inoculum density is not less than 4.5×10⁵A cell/ml.

4) it connects poison and harvest cell suspension is transferred to new bioreactor culture 2~3 days, when cell density reaches 2.0 ×10⁶~2.5 × 10⁶When a cell/ml, it is inoculated with canine distemper virus CDV-11 plant, using containing 2% newborn bovine serum when connecing poison DMEM cell culture fluid, temperature are 34 DEG C, and connecing poison amount is MOI=0.1.Different time samples after connecing poison, observes cell state, to 80% or so cell rounding protrusion of micro-carrier surface and have on a small quantity when falling off, 10~15min is quickly stirred, with 75 μm or so filter plates Microcarrier is filtered out, virus liquid is harvested, successively removes cell fragment with 20 μm and 5 μm of filter cores, title is indicated, the harvest date, criticizes Number, -15 DEG C or less preservations are to be checked spare.

(4) inspection of semifinished product

1) steriling test is tested by existing " Chinese veterinary pharmacopoeia " annex, answers asepsis growth.

2) viral level is measured by 1.2.1.1, and every milliliter of viral level answers >=10^7.5TCID₅₀。

(5) inactivate into cell culture addition 2%BEI solution, make its final concentration of 0.1%, 37 DEG C of inactivation 48h；It goes out After work, into cell culture add 50% hypo solution, make its final concentration of 0.2%, stir 1h after eventually It only inactivates, 2~8 DEG C of preservations are to be checked spare.

3. inactivation to be examined to qualified RDPV-SN1 plants of cell cultures of recombinant baculovirus and canine distemper with seedling and packing CDV-11 plants of inactivation antigens are uniformly mixed by proper proportion, are carried out in the ratio of 9:1 with seedling with the aluminium hydroxide gel of sterilizing, then plus The thimerosal solution for entering 1%, make its final concentration of 0.01%, be sufficiently stirred, quantitative separating, cover, labeling, prepare the tiny disease of racoon dog Viral enteritis, canine distemper bivalent inactivated vaccine.

Embodiment 6 --- Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine product inspection

1. after character is stood, upper layer is supernatant liquid, it is in homogenous suspension after shaking that lower layer, which is a small amount of precipitating,.

2. steriling test is tested by existing " Chinese veterinary pharmacopoeia " annex, asepsis growth is answered.Inspection result is shown in Table 12.

3. loading quantity inspection is checked by existing " Chinese veterinary pharmacopoeia " annex, regulation should be met.Inspection result is shown in Table 12.

12 loading quantity inspection result of table

4. Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine difference leg were inoculated for 2 monthly ages by safety verification The susceptible recoon dog of health (RDPV HI antibody titer is not higher than 1:4) 5,3.0ml/, as immune group；Separately take 5 recoon dogs unavoidably Epidemic disease, as a control group.After inoculation, periodically observes daily and measure recoon dog body temperature 1 time (with preceding thermometric time consistency is inoculated with), continuously Observation 21 days, as a result immune group recoon dog is all strong lives, and spirit, diet, excrement, body temperature are compared with before inoculation without significant change, nothing Other abnormal clinical symptoms, locally and systemically adverse reaction caused by not occurring because of vaccine.With control group no significant difference.Specifically It the results are shown in Table 13.

13 vaccine safety inspection result of table

5. efficacy test

(1) the serological method 2 monthly ages susceptible recoon dog of health (RDPV HI antibody titer is not higher than 1:4) 5, each leg Vaccine 1.0ml is subcutaneously injected, while the identical recoon dog of 5 conditions being taken not to be inoculated with as control.14 days after immune, to all recoon dogs It takes a blood sample respectively, carries out the measurement of RDPV HI antibody titer, recoon dog RDPV HI antibody titer is as a result immunized and is not less than 1:32 (protection Property value for antibody), control recoon dog RDPV HI antibody titer be not higher than 1:4；21 days after immune, take a blood sample respectively to all recoon dogs, into As a result the measurement of row canine distemper virus neutralize antibody titers is immunized recoon dog canine distemper virus neutralize antibody titers and is not less than 1:50 (protection antibody value), control recoon dog canine distemper virus neutralize antibody titers are not higher than 1:4.Concrete outcome is shown in Table 14.

14 serological method efficacy test result of table

(2) the Immunization method 2 monthly ages susceptible recoon dog of health (RDPV HI antibody titer is not higher than 1:4) 10, each leg Vaccine 1.0ml is subcutaneously injected, while the identical recoon dog of 10 conditions being taken not to be inoculated with as control.14 days after immune, 5 are randomly selected Only immune recoon dog and 5 control recoon dogs gavage approach inoculation Raccoon dog parvovirus RDPV-SN1 strain virus liquid 15ml through esophagus respectively (contain 15 × 10^7.0TCID₅₀), it is observed continuously 10, control recoon dog 100% falls ill, and recoon dog 100% is immunized and protects；21 after immune Day, remaining 5 immune recoon dogs and 5 control recoon dog difference collunarium inoculation 2.0ml, (total 10ml contains 1.0 to intraperitoneal injection 8.0ml ×10^5.5TCID₅₀) canine distemper virus ZC strain virus liquid, it is observed continuously 21 after attacking poison, control recoon dog 100% falls ill, and racoon dog is immunized Son 100% is protected.Concrete outcome is shown in Table 15, table 16.

15 Raccoon dog parvovirus of table attacks clinical observation result after poison

16 canine distemper virus of table attacks clinical observation result after poison

6. the antiseptic mercurials determination of residual amount is measured by existing " Chinese veterinary pharmacopoeia " annex, regulation should be met.3 batches of epidemic diseases Seedling result meets regulation, and concrete outcome is shown in Table 17.

17 antiseptic mercurials of table remain measurement

Sequence table

<110>Qilu Animal Health Products Co., Ltd.

<120>Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine and preparation method thereof

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 29

<212> DNA

<213>artificial synthesized (VP2-F)

<400> 1

aatgaattca gtgatggagc agttcaacc 29

<210> 2

<211> 29

<212> DNA

<213>artificial synthesized (VP2-R)

<400> 2

atgggtacct taatataatt ttctaggtg 29

<210> 3

<211> 1755

<212> DNA

<213>Raccoon dog parvovirus VP2 gene (VP2)

<400> 3

atgagtgatg gagcagttca accagacggt ggtcaacctg ctgtcagaaa tgaaagagct 60

acaggatctg ggaacgggac tggaggcggg ggtggtggtg gttctggtgg tgtggggatt 120

tctacgggta ctttcaataa tcagacggaa tttaaatttt tggaaaacgg atgggtggaa 180

atcacagcaa actcaagcag acttgtacat ttaaatatgc cagaaagtga aaattataga 240

agagtggttg taaataatat ggataaaact gcagttaatg gaaacatggc tttagatgat 300

attcatgcac aaattgtaac accttggtca ttggttgatg caaatgcttg gggagtttgg 360

tttaatccag gagattggca actaattgtt aatactatga gtgaattgca tttagttagt 420

tttgaacaag aaatttttaa tgttgtttta aagactgttt cagaatctgc tactcagcca 480

ccaactaaag tttataataa tgatttaact gcatcattga tggttgcatt agatagtaat 540

aatactatgc catttactcc agcagctatg agatctgaga cattgggttt ttatccatgg 600

aaaccaacca taccaactcc atggagatat tatttccaat gggatagaac attaatacca 660

tctcatactg gaactagtgg cacaccaaca aatatatacc atggtacaga tccagatgat 720

gttcaatttt atactattga aaattctgtg ccagtacact tactaagaac aggtgatgaa 780

tttgctacag gaacattttt ttttgattgt aaaccatgta gactaacaca tacatggcaa 840

acaaatagag cattgggctt accaccattt ctaaattctt tgcctcaagc tgaaggagct 900

actaactttg gtgatatagg agttcaacaa gataaaagac gtggtgtaac tcaaatggga 960

aatacaaact atattactga agctactatt atgagaccag ctgaggttgg ttatagtgca 1020

ccatattatt cttttgaggc gtctacacaa gggccattta aaacacctat tgcagcagga 1080

cgggggggag cgcaaacaga tgaaaatcaa gcagcagatg gtgatccaag atatgcattt 1140

ggtagacaac atggtcaaaa aactaccaca acaggagaaa cacctgagag atttacatat 1200

atagcacatc aagatacagg aagatatcca gaaggagatt ggattcaaaa tactaacttt 1260

aaccttcctg taacaaatga taatgtattg ctaccaacag atccaattgg aggtaaaaca 1320

ggaattaact atactaatat atttaatact tatggtcctt taactgcatt aaataatgta 1380

ccaccagttt atccaaatgg tcaaatttgg gataaagaat ttgatactga cttaaaacca 1440

agacttcatg taaatgcacc atttgtttgt caaaataatt gtcctggtca attatttgta 1500

aaagttgcgc ctaatttaac aaatgaatat gatcctgatg catctgctaa tatgtcaaga 1560

attgtaactt actcagattt ttggtggaaa ggtaaattag tatttaaagc taaactaaga 1620

gcctctcata cttggaatcc aattcaacaa atgagtatta atgtagataa ccaatttaac 1680

tatctaccaa gtaatattgg aggtatgaaa attgtatttg aaaaatctca actagcacct 1740

agaaaattat attaa 1755

<210> 4

<211> 23

<212> DNA

<213>artificial synthesized (BM-F2)

<400> 4

agcggatcca aaccatgaaa ttc 23

<210> 5

<211> 23

<212> DNA

<213>artificial synthesized (HBM-R)

<400> 5

ataagatctg catgcggtac ccc 23

<210> 6

<211> 38

<212> DNA

<213>artificial synthesized (HS4-F)

<400> 6

atatacgtag agctcacggg gacagccccc ccccaaag 38

<210> 7

<211> 38

<212> DNA

<213>artificial synthesized (HS4-R)

<400> 7

acatacgtaa atattctcac tgactccgtc ctggagtt 38

Claims (5)

1. a kind of Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine, it is characterised in that the effective component of the vaccine includes Raccoon dog parvovirus VP2 albumen, canine distemper virus CDV-11 plants of inactivation antigens, hydrogen-oxygens of RDPV-SN1 plants of recombinant baculovirus expression Change aluminium glue adjuvant；

The described vaccine is for preventing racoon dog canine distemper and Canine Parvovirus Enteritis.

2. Raccoon dog parvovirus enteritis as described in claim 1, canine distemper bivalent inactivated vaccine, it is characterised in that the racoon dog is thin Small virus VP2 albumen is RDPV-SN1 plants of transfection Sf9 cells of recombinant baculovirus by expression Raccoon dog parvovirus VP2 albumen, training It supports to cell and infection acquisition occurs, which is named as Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus recombinant baculovirus RDPV-SN1 plants, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microorganism was delivered on May 16th, 2019 The preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center, research institute, deposit number are CGMCC No.17685.

3. Raccoon dog parvovirus enteritis as described in claim 1, canine distemper bivalent inactivated vaccine, it is characterised in that the canine distemper Viral CDV-11 plants of inactivation antigen is obtained after BEI is inactivated by CDV-11 plants of canine distemper virus, and the strain is in 2009 07 Deliver within 15th Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Chinese microorganism strain guarantor the moon The preservation of administration committee's common micro-organisms center is hidden, deposit number is CGMCC No.3201.

4. such as claim 1, the 2 Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine, it is characterised in that expression racoon dog RDPV-SN1 plants of recombinant baculovirus of building of parvovirus VP2 albumen comprises the following steps:

(1) melittin signal peptide (HBM) and beta-actin insulation the building of transfer vector: are added on pVL1393 carrier Sub (HS4) sequence, constructs transfer vector pVL-HBM-HS4；

(2) building of recombinant transfer vector: Raccoon dog parvovirus RDPV-SN1 plants of VP2 gene is extracted, by the VP2 gene (SEQ of extraction ID No:3) it is cloned into transfer vector pVL-HBM-HS4, obtain recombinant transfer vector pVL-HBM-HS4-VP2；

(3) recombinant baculovirus RDPV-SN1 plants of building: by recombinant transfer vector pVL-HBM-HS4-VP2 and linearisation clover Gamma nuclear polyhedrosis virus (AcMNPV) genomic DNA cotransfection Sf9 cell obtains expression Raccoon dog parvovirus VP2 egg White recombinant baculovirus is named as RDPV-SN1 plants of Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus recombinant baculovirus.

5. Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine according to claim 1, it is characterised in that the vaccine Preparation method the following steps are included:

(1) it Raccoon dog parvovirus VP2 albumen: selects eugonic Sf9 cell inoculation bioreactor to carry out the full culture that suspends, connects Kind density is 1.5 × 10⁶~2.0 × 10⁶A cell/ml, 27 DEG C of cultures.When Sf9 cell density reaches 1.5 in bioreactor ×10⁶~3.0 × 10⁶When a cell/ml, it is inoculated with recombinant baculovirus RDPV-SN1 plants by MOI=0.2~2.0,27 DEG C of cultures 72~96h harvests cell culture, and 2%BEI solution is added thereto, makes BEI final concentration of 0.1%, 37 DEG C of inactivations after mixing Then 48h adds 50% hypo solution to 1% swine erythrocyte hemagglutinative titer >=1:2048 into cell culture, Make its final concentration of 0.2%, stir 1h after terminate inactivation；Through sterile and inactivation after the assay was approved to get Raccoon dog parvovirus VP2 egg It is white；

(2) canine distemper virus CDV-11 plants of inactivation antigens: eugonic Vero cell inoculation bioreactor is selected to carry out micro- Carrier, which suspends, to be cultivated, when Vero cell density reaches 2.0 × 10 in bioreactor⁶~2.5 × 10⁶When a cell/ml, by MOI =0.1 CDV-11 plant canine distemper virus of inoculation, when 80% or so cell rounding protrusion of micro-carrier surface and when having a small amount of fall off, fastly Speed 10~15min of stirring, filters out microcarrier with 75 μm or so filter plates, harvests virus liquid, broken with 20 μm and 5 μm of filter core removal cells Piece, harvests cell culture, and canine distemper virus content should be not less than 10^7.5TCID₅₀/ml；It is added into the cell culture of harvest 2%BEI solution makes BEI final concentration of 0.1%, 37 DEG C of inactivation 48h after mixing, the then addition 50% into cell culture Hypo solution, make its final concentration of 0.2%, stir 1h after terminate inactivation；Through it is sterile and inactivation after the assay was approved to get CDV-11 plants of inactivation antigens of canine distemper virus；

(3) the Raccoon dog parvovirus VP2 albumen that step (1) obtains is diluted to 1% swine erythrocyte hemagglutinative titer not less than 1: 256, the CDV-11 plants of inactivation antigens of canine distemper virus obtained with step (2) are uniformly mixed by proper proportion, the hydrogen-oxygen with sterilizing Change aluminium glue to carry out adding 1% thimerosal solution with seedling in the ratio of 9:1, make its final concentration of 0.01%, after being sufficiently stirred i.e. At.