CN110368490B - Raccoon dog parvovirus enteritis and canine distemper bivalent inactivated vaccine and preparation method thereof - Google Patents

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

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CN110368490B
CN110368490B CN201910659547.5A CN201910659547A CN110368490B CN 110368490 B CN110368490 B CN 110368490B CN 201910659547 A CN201910659547 A CN 201910659547A CN 110368490 B CN110368490 B CN 110368490B
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raccoon
canine distemper
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CN110368490A (en
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董海曼
李雯
宋晓飞
葛平萍
秦旭伟
李营
贾爱琴
王蕾
徐龙涛
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Qilu Animal Health Products Co ltd
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    • C12N2750/14011—Parvoviridae
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    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
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Abstract

The invention relates to a raccoon dog parvovirus enteritis and canine distemper bigeminal inactivated vaccine and a preparation method thereof. The active ingredients of the bivalent inactivated vaccine comprise a raccoon parvovirus VP2 protein obtained by culturing a recombinant baculovirus RDPV-SN1 strain transfected Sf9 cell and a canine distemper virus inactivated antigen. The invention provides a bivalent inactivated vaccine prepared from a raccoon parvovirus VP2 protein and a canine parvovirus inactivated antigen with higher virus content, aiming at the problem that the bivalent inactivated vaccine used in the market generally has the risk of returning strong toxicity in the presence of canine distemper and parvovirus, and has the problem of low immune efficacy after the raccoon parvovirus is inactivated. The combined vaccine can be simultaneously applied to prevention of two diseases of raccoon dog parvovirus enteritis and canine distemper, has the advantages of good immunogenicity, high safety, strong controllability, high culture efficiency and automation degree, large-scale production and the like, can achieve the purpose of preventing two diseases by one needle, and has wide application prospect.

Description

Raccoon dog parvovirus enteritis and canine distemper bigeminal inactivated vaccine and preparation method thereof
Technical Field
The invention relates to a raccoon dog parvovirus enteritis and canine distemper bigeminal inactivated vaccine and a preparation method thereof, belonging to the field of biological products for livestock.
Background
Raccoon parvovirus enteritis is an acute and highly contact infectious disease caused by raccoon parvovirus of parvoviridae. At present, raccoon dog parvovirus enteritis is widely existed in various countries of the world, the raccoon dog parvovirus enteritis is mainly caused by the disease, and the mortality rate can reach 30-60%.
Canine Distemper is an acute, thermal and high-contact infectious disease caused by Canine Distemper Virus (CDV) in the measles virus of paramyxoviridae, young animals mostly pass through the acute and lethal way, adult animals can be infected chronically and persistently, clinical symptoms are mainly characterized by biphasic temperature rise and accompanied by catarrhal pneumonia and gastroenteritis, occasionally neurological symptoms occur, mixed infection and secondary infection of other bacteria and viruses are easy to carry out, the death rate can reach 80 percent, and the Canine Distemper is one of the most serious epidemic diseases harming the fur animal breeding industry in China. Raccoon dog distemper can occur in any season all the year around, and especially, raccoon dogs are more sensitive before and after lactation interruption and during the breeding period, and have very high morbidity and mortality.
Because raccoon dog canine distemper and parvovirus enteritis have the characteristics of acute morbidity and lack of a specific treatment method, vaccination is still used as a main prevention and control means at present.
At present, no raccoon dog dedicated canine distemper and parvovirus enteritis vaccine is sold in the market, many farmers use the substitute vaccine and cannot obtain a good immune protection effect, and therefore, the raccoon dog needs to be immunized secondarily, the cost is increased, the workload of the farmers is increased, and meanwhile, a large stress reaction of the raccoon dog is caused. Most canine distemper vaccines are live vaccines, and the risk of strong toxicity exists; the vaccine prepared by parvoviral enteritis is mainly a whole virus inactivated vaccine or a live vaccine, the immunogenicity of the inactivated vaccine is poor, and the live vaccine has the risk of strong virulence.
Therefore, the development of raccoon parvovirus baculovirus vectors and canine distemper bivalent inactivated vaccines is urgent, raccoon canine distemper and parvovirus enteritis can be effectively controlled, and the purpose of preventing both canine distemper and parvovirus enteritis by one needle is realized.
Disclosure of Invention
A raccoon parvovirus is separated from clinical suspected raccoon parvovirus materials, and a gene VP2 of the raccoon parvovirus is successfully amplified, an insect cell baculovirus, namely alfalfa armyworm nucleopolyhedrosis virus expression system is adopted to construct a recombinant baculovirus expressing a protein VP2 of the raccoon parvovirus, SF9 cells are transfected, and virus-like particles are cultured and obtained and have good immunogenicity and safety; the CDV-11 strain of the canine distemper natural low virulent strain separated by the company is cultured in a suspension way, and the virus content is 10 7.5 TCID 50 More than ml, still has better immunogenicity to the raccoon dog after inactivation, and has high safety after inactivation.
The invention aims to provide a raccoon dog parvovirus enteritis and canine distemper bigeminal inactivated vaccine and a preparation method thereof, so as to cope with the current raccoon dog parvovirus enteritis and canine distemper epidemic situation.
Technical scheme of the invention
1. A raccoon parvovirus enteritis and canine distemper bigeminal inactivated vaccine is characterized in that the effective components of the vaccine comprise a raccoon parvovirus VP2 protein expressed by a recombinant baculovirus RDPV-SN1 strain, a canine distemper virus CDV-11 strain inactivated antigen and an aluminum hydroxide gel adjuvant; the vaccine is used for preventing raccoon dog distemper and parvovirus enteritis.
2. The raccoon parvovirus enteritis and canine distemper bigeminal inactivated vaccine is characterized in that the raccoon parvovirus VP2 protein is obtained by transfecting Sf9 cells by recombinant baculovirus RDPV-SN1 strain expressing the raccoon parvovirus VP2 protein, culturing until the cells are infected, wherein the strain is named as alfalfa Thinleaus nudus nuclear polyhedrosis virus recombinant baculovirus RDPV-SN1 strain, and is delivered to China microorganism culture preservation management committee general microbiological culture collection of China institute of microbiology, Ministry of China, Ministry of sciences, Ministry of China, No.3, Kyoho, No.1, Kyoho, in Beijing, 05 and 16 days in 2019, and the preservation number is CGMCC No. 17685.
3. The raccoon dog parvovirus enteritis and Canine Distemper bivalent inactivated vaccine is characterized in that the inactivated antigen of the Canine Distemper virus CDV-11 strain is obtained by inactivating a Canine Distemper Virus (CDV) CDV-11 strain by BEI, the strain is delivered to the general microorganism center of China Committee for culture Collection of microorganisms of Ministry of China academy of sciences No.3 of Ministry of microbiology of Navy, No.1 of Beijing, Injaward-Yang district, Beijing City, at 07-15 days in 2009, and the collection number is CGMCC No. 3201.
4. The raccoon parvovirus enteritis and canine distemper bigeminy inactivated vaccine is characterized in that the construction of a recombinant baculovirus RDPV-SN1 strain expressing the raccoon parvovirus VP2 protein comprises the following steps:
(1) construction of transfer vector: adding melittin signal peptide (HBM) and beta-actin insulator (HS4) sequences to the pVL1393 vector to construct a transfer vector pVL-HBM-HS 4;
(2) construction of recombinant transfer vectors: extracting a VP2 gene of raccoon parvovirus RDPV-SN1 strain, cloning the extracted VP2 gene (SEQ ID No:3) into a transfer vector pVL-HBM-HS4 to obtain a recombinant transfer vector pVL-HBM-HS4-VP 2;
(3) construction of recombinant baculovirus RDPV-SN1 strain: the recombinant transfer vector pVL-HBM-HS4-VP2 and the genome DNA of the linearized Autographa californica nuclear polyhedrosis virus (AcMNPV) are cotransfected with Sf9 cells to obtain the recombinant baculovirus expressing the protein of the raccoon dog parvovirus VP2, which is named as the Autographa californica nuclear polyhedrosis virus recombinant baculovirus RDPV-SN1 strain.
5. The raccoon dog parvovirus enteritis and canine distemper bigeminal inactivated vaccine is characterized in that the preparation method of the vaccine comprises the following steps:
(1) raccoon parvovirus VP2 protein: selecting Sf9 cells with vigorous growth, inoculating the cells into a cell culture tank for full suspension culture, wherein the inoculation density is 1.5 multiplied by 10 6 ～2.0×10 6 Cells/ml, cultured at 27 ℃. When the cell density of Sf9 in the cell culture tank reaches 1.5X 10 6 ～3.0×10 6 Inoculating a recombinant baculovirus RDPV-SN1 strain at the MOI of 0.2-2.0 when each cell/ml is cultured at 27 ℃ for 72-96 h, harvesting a cell culture, adding a 2% BEI solution into the cell culture to ensure that the BEI final concentration is 0.1%, uniformly mixing, inactivating the cell culture at 37 ℃ for 48h, and ensuring that the hemagglutination titer of 1% pig red blood cells is more than or equal to 1:2048, adding 50% sodium thiosulfate solution to the cell culture to make the final concentration 0.2%, stirring for 1h, and stopping inactivation; after being sterilized and inactivated and tested to be qualified, the raccoon parvovirus VP2 protein is obtained;
(2) inactivated antigen of canine distemper virus CDV-11 strain: inoculating Vero cells with vigorous growth into a cell culture tank for microcarrier suspension culture, and culturing the Vero cells in the cell culture tank until the Vero cell density reaches 2.0 × 10 6 ～2.5×10 6 Inoculating CDV-11 canine distemper virus at MOI of 0.1 per ml, rapidly stirring for 10-15 min when about 80% of cells on the surface of the microcarrier become round and have small amount of shedding, filtering the microcarrier with a filter plate of about 75 μm, collecting virus solution, removing cell debris with filter elements of 20 μm and 5 μm, and collecting cell culture product, wherein the content of canine distemper virus is not less than 10 7.5 TCID 50 Per ml; adding 2% BEI solution into the harvested cell culture to make BEI final concentration be 0.1%, mixing uniformly, inactivating at 37 deg.C for 48h, adding 50% sodium thiosulfate solution into the cell culture to make its final concentration be 0.2%, stirring for 1h, and terminating inactivation; after the canine distemper virus CDV-11 strain inactivated antigen is obtained after the canine distemper virus CDV-11 strain is subjected to sterility and inactivation inspection and is qualified;
(3) diluting the protein VP2 of the raccoon parvovirus obtained in the step (1) until the hemagglutination titer of 1% of pig red blood cells is not less than 1:256, uniformly mixing the protein with the inactivated antigen of the CDV-11 strain of the canine distemper virus obtained in the step (2) according to a proper proportion, carrying out seedling preparation with sterilized aluminum hydroxide gel according to a proportion of 9:1, adding 1% thimerosal solution to enable the final concentration to be 0.01%, and fully stirring to obtain the vaccine.
The invention has the beneficial effects
The invention relates to a raccoon dog parvovirus enteritis and canine distemper bigeminal inactivated vaccine and a preparation method thereof. The invention relates to preparation of a raccoon parvovirus enteritis and canine distemper bivalent inactivated vaccine, which is characterized in that an insect cell baculovirus-alfalfa Spodoptera litura nucleopolyhedrovirus expression system is utilized to construct a recombinant baculovirus expressing the protein VP2 of raccoon parvovirus, a production process of suspension culture is utilized to produce the recombinant baculovirus expressing the protein VP2 of raccoon parvovirus and canine distemper virus (natural low virulent strain), and the inactivated raccoon parvovirus and aluminum hydroxide gel are mixed to prepare the bivalent vaccine. The bigeminal vaccine can be used for preventing two diseases of raccoon dog parvovirus enteritis and canine distemper, and has the advantages of good safety and immunogenicity, and suitability for large-scale industrial production.
The raccoon dog parvovirus enteritis and canine distemper bivalent inactivated vaccine prepared by the invention adopts a production process of suspension culture, has the advantages of high virus culture content, no need of mixing, small batch difference, automatic culture mode, no need of mass production personnel, small culture required area, small pollution, low cost and the like, successfully solves the problems of low production efficiency, high labor intensity, poor product uniformity and the like of spinner bottle culture, and is a preferred production process in vaccine preparation.
The safety test result of the raccoon dog parvovirus enteritis and canine distemper bivalent inactivated vaccine shows that after 21 days of inoculation, raccoon dogs are healthy and alive, the spirit, diet and feces are normal, and other abnormal clinical symptoms do not exist when the raccoon dogs are inoculated with a single dose (1 ml/raccoon dog), a single dose is repeatedly inoculated with the raccoon dogs and an overdose is inoculated with the raccoon dogs (3.0 ml/raccoon dog), and after the raccoon dogs are inoculated with the virus enteritis and the canine distemper bivalent inactivated vaccine; and after the autopsy, all organs are normal and have no pathological changes, namely the bivalent inactivated vaccine is safe for the raccoon dog. The efficacy test shows that after the vaccine is inoculated in a single dose, the raccoon parvovirus HI antibody reaches the protective level 14 days after immunization; 21 days after immunization, neutralizing antibodies of raccoon canine distemper viruses reach a protective level or above; and 6 months after immunization, the immunized raccoon dog still has 100% resistance to canine distemper virus and parvovirus. The immunization period of the vaccine is 6 months, and the storage period is 2-8 ℃ and 18 months.
The invention relates to microbial strain resource information
The invention relates to a raccoon parvovirus enteritis and canine distemper bigeminal inactivated vaccine, which is prepared by using a recombinant baculovirus RDPV-SN1 strain with a strain expressing the protein VP2 of raccoon parvovirus, named as alfalfa Spodoptera litura nuclear polyhedrosis virus recombinant baculovirus RDPV-SN1 strain, wherein the strain is delivered to China general microbiological culture Collection center of China microbiology institute, institute of microbiology, No.3, China academy of sciences, North West Lu 1, at the sunward area, Beijing, within 05 and 16 days of 2019, and the preservation number is CGMCC No. 17685; the CDV-11 strain of Canine Distemper Virus (CDV) has been deposited in the general microbiological culture Collection center of the institute of microbiology, China academy of sciences, China Committee for culture Collection of microorganisms, China, institute of microbiology, No.3, West Lu 1, of the south-facing-Yang district, Beijing, 2009, 07, 15 days, with the deposit number of CGMCC No.3201 (see CN 101612396B).
Drawings
FIG. 1 is a diagram showing the PCR amplification result of the gene VP2 of raccoon parvovirus: 1 is an amplification product of a raccoon parvovirus VP2 gene; 2 is blank control; m is DL2000 Marker.
FIG. 2 shows the colony PCR verification of the connection of VP2 gene to pVL-HBM-HS4 vector: 1-3 are PCR electrophoresis results of 3 different colonies; 4 is blank control; m is DL2000 Marker.
Detailed Description
1. Construction of recombinant baculovirus RDPV-SN1 strain
(1) Separating raccoon parvovirus RDPV-SN1 strain, taking the content of the diseased raccoon intestine, adding a proper amount of normal saline, adding chloroform with the total amount of about 1/3, fully oscillating for 15min, centrifuging for 30min at 3000r/min, taking supernatant, filtering for sterilization, inoculating F81 cells according to the MOI of 0.1-0.4, changing MEM culture solution containing 2% newborn bovine serum after the cells are attached to the wall, culturing for 96-120 h at 37 ℃, observing cytopathic effect, harvesting when the typical parvovirus cytopathic effect of netting and shedding occurs, and freezing and storing at-20 ℃.
And (3) inoculating the separated raccoon parvovirus RDPV-SN1 strain into F81 cells growing into a 80% monolayer, culturing at 37 ℃ for 72-120 h, freezing and thawing after more than 75% of lesions are obtained.
(2) The gene amplification of the raccoon parvovirus RDPV-SN1 strain VP2 is carried out by taking the DNA of the raccoon parvovirus RDPV-SN1 strain as a template and taking VP2-F and VP2-R as primers to carry out PCR amplification, and the primers are designed according to the RDPVVP2 gene sequence published on GenBank: VP 2-F: 5 '-aatgaattca gtgatggagc agttcaacc (EcoRI) -3' 29 (SEQ ID NO: 1) VP 2-R: 5 '-atgggtacct taatataatt ttctaggtg (KpnI) -3' 29 (SEQ ID NO: 2)
As a result, a 1755bp band was amplified, which is consistent with the expected result, and the results of gel electrophoresis are shown in FIG. 1. The amplified fragments were as follows (sequence 3):
atgagtgatg gagcagttca accagacggt ggtcaacctg ctgtcagaaa tgaaagagct 060acaggatctg ggaacgggac tggaggcggg ggtggtggtg gttctggtgg tgtggggatt 120tctacgggta ctttcaataa tcagacggaa tttaaatttt tggaaaacgg atgggtggaa 180atcacagcaa actcaagcag acttgtacat ttaaatatgc cagaaagtga aaattataga 240agagtggttg taaataatat ggataaaact gcagttaatg gaaacatggc tttagatgat 300attcatgcac aaattgtaac accttggtca ttggttgatg caaatgcttg gggagtttgg 360tttaatccag gagattggca actaattgtt aatactatga gtgaattgca tttagttagt 420tttgaacaag aaatttttaa tgttgtttta aagactgttt cagaatctgc tactcagcca 480ccaactaaag tttataataa tgatttaact gcatcattga tggttgcatt agatagtaat 540aatactatgc catttactcc agcagctatg agatctgaga cattgggttt ttatccatgg 600aaaccaacca taccaactcc atggagatat tatttccaat gggatagaac attaatacca 660tctcatactg gaactagtgg cacaccaaca aatatatacc atggtacaga tccagatgat 720gttcaatttt atactattga aaattctgtg ccagtacact tactaagaac aggtgatgaa 780tttgctacag gaacattttt ttttgattgt aaaccatgta gactaacaca tacatggcaa 840acaaatagag cattgggctt accaccattt ctaaattctt tgcctcaagc tgaaggagct 900actaactttg gtgatatagg agttcaacaa gataaaagac gtggtgtaac tcaaatggga 960aatacaaact atattactga agctactatt atgagaccag ctgaggttgg ttatagtgca 1020ccatattatt cttttgaggc gtctacacaa gggccattta aaacacctat tgcagcagga 1080cgggggggag cgcaaacaga tgaaaatcaa gcagcagatg gtgatccaag atatgcattt 1140ggtagacaac atggtcaaaa aactaccaca acaggagaaa cacctgagag atttacatat 1200atagcacatc aagatacagg aagatatcca gaaggagatt ggattcaaaa tactaacttt 1260aaccttcctg taacaaatga taatgtattg ctaccaacag atccaattgg aggtaaaaca 1320ggaattaact atactaatat atttaatact tatggtcctt taactgcatt aaataatgta 1380ccaccagttt atccaaatgg tcaaatttgg gataaagaat ttgatactga cttaaaacca 1440agacttcatg taaatgcacc atttgtttgt caaaataatt gtcctggtca attatttgta 1500aaagttgcgc ctaatttaac aaatgaatat gatcctgatg catctgctaa tatgtcaaga 1560attgtaactt actcagattt ttggtggaaa ggtaaattag tatttaaagc taaactaaga 1620gcctctcata cttggaatcc aattcaacaa atgagtatta atgtagataa ccaatttaac 1680tatctaccaa gtaatattgg aggtatgaaa attgtatttg aaaaatctca actagcacct 1740agaaaattat attaa 1755
(3) construction and identification of transfer vector pMD19-VP2 purified PCR product is connected with pMD19-T vector, the ligation product transforms DH5 alpha competent cells, culture broth is concentrated and then spread on LB solid culture medium containing Amp, culture is carried out for 16h at 37 ℃, colony on a plate is selected for culture, colony PCR identification is carried out, and the result is that a target band with the size of 1.7kb is amplified. The sequencing result is 1755bp, and the sequence is shown in SEQ ID NO. 3. The result shows that the gene of the raccoon parvovirus RDPV-SN1 strain VP2 is successfully cloned to a pMD19-T vector. The pMD19-T plasmid cloned with the VP2 gene of the raccoon parvovirus RDPV-SN1 strain is named as pMD-VP 2.
(4) Construction and identification of transfer vector pVL-HBM
HBM gene PCR amplification is carried out by taking pMD19-HBM plasmid as a template and HBM-F, HBM-R as a primer. The primer sequence is as follows:
HBM-F: 5'-agcggatcca aaccatgaaa ttc-3' (BamHI) 23 (SEQ ID NO: 4)
HBM-R: 5'-ataagatctg catgcggtac ccc-3' (Pst I) 23 (SEQ ID NO: 5)
Obtaining HBM gene amplification PCR product, wherein the target band size is 170 bp. The HBM gene amplification product and the plasmid pVL1393 are subjected to enzyme digestion by using restriction enzyme BamHI/Pst I, and are respectively recovered and purified after gel electrophoresis, and T is used 4 DNA ligase ligation transformed DH5 alpha competent cells. Inoculating the transformation product into LB liquid culture medium containing Amp, picking single colony, and carrying out colony PCR identification. And (4) sending the bacteria liquid with correct identification to a sequencing company for sequencing, and selecting the bacteria liquid with correct sequencing for storage.
The HBM gene PCR amplification system is as follows: 25 μ l, consisting of: pMD19-HBM plasmid 1. mu.l, 2 XPFu PCR MasterMix 10. mu.l, 2 XPAQQ PCR MasterMix 2.5. mu.l, upstream and downstream primers 1. mu.l, ddH 2 O9.5 μ l; the PCR amplification reaction conditions are as follows: 5min at 94 ℃; 45min at 95 ℃, 45min at 60 ℃, 30min at 72 ℃ and 30 cycles; preserving at 72 ℃ for 10min and 4 ℃.
The HBM gene enzyme digestion reaction system is as follows: 50 μ l, consisting of: HBM gene PCR product gel recovered DNA fragment 10. mu.l, reaction Buffer 5. mu.l, BamHI enzyme 2. mu.l, pstI enzyme 2. mu.l, ddH 2 O 31μl。
The pVL1393 plasmid enzyme digestion reaction system is as follows: 50 μ l, consisting of: pVL1393 plasmid 5. mu.l, Buffer 5. mu.ll, 2. mu.l of BamHI enzyme, 2. mu.l of pstI enzyme, ddH 2 O 36μl。
The HBM gene and pVL1393 plasmid connecting system is as follows: mu.l of HBM gene PCR product, 2. mu.l of pVL1393 plasmid, 1. mu.l of Buffer, T 4 DNA ligase 1. mu.l.
(5) Construction and identification of transfer vector pVL-HBM-HS4
HS4 gene PCR amplification is carried out by taking pMD19-HS4 plasmid as a template and HS4-F and HS4-R as primers. The primer sequence is as follows:
HS 4-F: 5 '-atatacgtag agctcacggg gacagccccc ccccaaag (SnaBI) -3' 38 (SEQ ID NO: 6)
HS 4-R: 5 '-acatacgtaa atattctcac tgactccgtc ctggagtt (SnaBI) -3' 38 (SEQ ID NO: 7)
The HS4 gene amplification PCR product was obtained, and the target band size was about 1.3 kb. The HS4 gene amplification product and plasmid pVL-HBM are cut by restriction endonuclease SnaB I, and after agarose gel electrophoresis purification, the target fragment is connected with pVL-HBM vector to transform DH5 alpha competent cells. Inoculating the transformation product into LB liquid culture medium containing Amp, picking single colony, and carrying out colony PCR identification. And (4) sending the bacteria liquid with correct identification to a sequencing company for sequencing, and selecting the bacteria liquid with correct sequencing for storage.
The HS4 gene PCR amplification system is as follows: 25 μ l, consisting of: pMD19-HS4 plasmid 1. mu.l, 2 XPFu PCR MasterMix 10. mu.l, 2 XPAQQ PCR MasterMix 2.5. mu.l, upstream and downstream primers 1. mu.l, ddH 2 O9.5 μ l; the PCR amplification reaction conditions are as follows: 5min at 94 ℃; 45min at 95 ℃, 45min at 60 ℃, 2min at 72 ℃ and 30 cycles; preserving at 72 ℃ for 10min and 4 ℃.
The enzyme digestion reaction system of the HS4 gene is as follows: 50 μ l, consisting of: 10. mu.l of DNA fragment recovered from HS4 gene PCR product gel, 5. mu.l of reaction Buffer, 2. mu.l of SnaBI enzyme, 1. mu.l of alkaline phosphatase, ddH 2 O 32μl。
The pVL-HBM plasmid enzyme digestion reaction system is as follows: 50 μ l, consisting of: pVL-HBM plasmid 5. mu.l, reaction Buffer 5. mu.l, SnaBI enzyme 2. mu.l, alkaline phosphatase 1. mu.l, ddH 2 O 32μl。
The connecting system of the HS4 gene and the pVL-HBM plasmid is as follows: enzyme-cleaved HS4 gene PCRMu.l of the product, 2. mu.l of the pVL-HBM plasmid, 1. mu.l of Buffer, and T 4 DNA ligase 1. mu.l.
(6) Construction and identification of transfer vector pVL-HBM-HS4-VP2
The transfer vector pVL-HBM-HS4 and the plasmid pMD-VP2 are respectively cut by EcoRI/Kpn I for 3h at 37 ℃, and the cut products are recovered and purified after gel electrophoresis. The enzyme-cleaved and purified pVL-HBM-HS4 plasmid and the enzyme-cleaved and purified VP2 gene were ligated using T 4 DNA ligase ligation transformed DH5 alpha competent cells. The transformation product is inoculated into LB liquid culture medium containing Amp, a single colony is picked up and is subjected to colony PCR identification, and a target band appears at the position of about 1.7 kb. The specific results are shown in FIG. 2. And (4) sending the bacteria liquid with correct identification to a sequencing company for sequencing, and selecting the bacteria liquid with correct sequencing for storage.
The restriction enzyme reaction systems of the plasmid pVL-HBM-HS4 and pMD-VP2 are as follows: 50 μ l, consisting of: mu.l of plasmid, 5. mu.l of reaction Buffer, 2. mu.l of EcoRI enzyme, 2. mu.l of KpnI, ddH 2 O 36μl。
The pVL-HBM-HS4 plasmid and pMD-VP2 plasmid connecting system is as follows: enzyme digestion product VP2 gene 6. mu.l, enzyme digestion plasmid pVL-HBM-HS 42. mu.l, reaction Buffer 1. mu.l, T 4 DNA ligase 1. mu.l.
(7) Transfection and purification of recombinant baculovirus
1) Transfection the successfully constructed transfer vector pVL-HBM-HS4-VP2 and linearized Autographa californica nuclear polyhedrosis virus genomic DNA were co-transfected into Sf9 cells, cultured at 27 ℃ until cytopathic effect began to appear on day 3, and cell culture supernatant was harvested on day 5.
2) Plaque purification the harvested recombinant baculovirus was plaque purified. Seeding of 2.0X 10 in cell culture dishes 6cm in diameter 6 ～2.5×10 6 Sf9 cells were allowed to stand at room temperature for 5 min. Gradient dilution (dilution: 10) -4 ～10 -7 ) 1.0ml of virus solution with different dilutions was inoculated into each culture dish and left at room temperature for 1h to allow the virus to fully infect the cells. Sterile water was prepared into 2% agarose, 1 volume of 2 × Grace medium was added, and mixed well. Discarding the culture supernatant from the cell culture dish, covering the cell surface with 4.0ml of 1% agarose, coagulating the agarose, and collecting the supernatantThe culture dish was placed in a humid environment and incubated at 27 ℃ for 7 days, and the plaque was observed. Selecting a single plaque agar block on a culture dish, inoculating an SF-SFM serum-free culture medium, shaking overnight at 4 ℃, harvesting a culture, inoculating Sf9 cells, culturing at 27 ℃ for 72-96 h, and harvesting a cell culture, namely recombinant baculovirus RDPV-SN1 strain F0-generation virus liquid, wherein the recombinant baculovirus is named as Autographa californica nuclear polyhedrosis virus recombinant baculovirus RDPV-SN1 strain, and is delivered to China microorganism culture collection management committee general microbiological culture collection of institute of microbiology, China institute of academy of sciences, No.1, North West Lu, 3, of the sunward region, Beijing city, on 05 and 16 days in 2019, and the preservation number is CGMCC No. 17685.
2. Identification of recombinant baculovirus RDPV-SN1 strain
(1) Virus subculture Sf9 cells were diluted to a density of 2.0X 10 6 Inoculating an F0 generation recombinant baculovirus RDPV-SN1 strain per cell/ml, and culturing in a shaking table at the constant temperature of 27 ℃ at the rotating speed of 100-105 r/min. Culturing for 72-96 h, and harvesting a cell culture, namely F1 generation recombinant baculovirus RDPV-SN1 strain virus liquid.
(2) SDS-PAGE gel electrophoresis the harvested cell culture of the F1 recombinant baculovirus strain RDPV-SN1 was subjected to SDS-PAGE detection, while Sf9 cells infected with empty baculovirus (without exogenous gene) were used as negative controls. The specific operation is as follows: and (3) adding 10 mu l of 5 Xloading Buffer into 40 mu l of the harvested cell culture, centrifuging for 1min in a boiling water bath for 5min at 12000r/min, taking the supernatant, carrying out SDS-PAGE gel (12%) electrophoresis, and taking the gel after the electrophoresis, wherein after the electrophoresis, a remarkable target band appears in the cell culture of the F1 generation recombinant baculovirus RDPV-SN1 strain near 65kD, and a corresponding band does not appear in the Sf9 cell culture infected with the empty baculovirus.
(3) Western Blot identification Western Blot assay was performed on harvested cell cultures of the F1-generation recombinant baculovirus RDPV-SN1 strain while using Sf9 cell cultures infected with empty baculovirus and Sf9 cell cultures as negative controls. The specific operation is as follows: 80 μ l of the harvested cell culture was taken, 20 μ l of 5 × Loading Buffer was added, and a sample was taken for 5min in a boiling water bath and subjected to SDS-PAGE gel (12%) electrophoresis. Transferring the product after SDS-PAGE electrophoresis to a PVDF (polyvinylidene fluoride membrane) membrane, sealing for 2h by using 5% skimmed milk, rinsing, adding rabbit-derived anti-raccoon enteritis virus positive serum diluted by 1:100, incubating for 2h, rinsing, adding Horse Radish Peroxidase (HRP) -labeled goat anti-rabbit IgG antibody diluted by 1:2000, incubating for 2h, rinsing, then dripping an enhanced chemiluminescence fluorescent substrate, and performing exposure by using an X-ray film.
As a result, a remarkable band appears at 65kD in the F1 generation recombinant baculovirus RDPV-SN1 strain cell culture, and the VP2 protein is successfully expressed in Sf9 cells.
(4) Virus content determination the harvested F1 generation recombinant baculovirus RDPV-SN1 strain virus fluid was studied according to the MTT relative potency method (Huangshi and Yumei pine. determination of baculovirus titre method [ J]Microbiological immunological progress, 2007(02):79-83.) assay of virus content, result was 6.0 × 10 7.0 PFU/ml。
(5) Measurement of hemagglutination potency of expressed protein harvested cell cultures of F1 generation recombinant baculovirus RDPV-SN1 strain were tested for hemagglutination potency of expressed protein according to the raccoon parvovirus Hemagglutination (HA) test method, and the result was 1: 2048.
(6) Protein content determination of expression protein VP2 protein content determination was performed by double antibody sandwich ELISA. As a result, the protein content of the F1 generation recombinant baculovirus RDPV-SN1 strain expression protein is 72.5 mg/L.
(7) Expression protein electron microscopy observation is carried out on cell cultures of the recombinant baculovirus RDPV-SN1 strain through sucrose density gradient ultracentrifugation, the concentration of sucrose solution is 40%, 50% and 60% respectively, the centrifugation speed is 29000r/min, centrifugation is carried out for 2h, and then strips located at the junction of the concentration of 50% and the concentration of 60% are collected. And (3) dripping a small amount of purified and concentrated virus-like particle samples on a copper net with a carbon film, air-drying, dripping 2% sodium phosphotungstate solution for negative staining, and observing by an electron microscope, wherein the raccoon parvovirus-like particles expressed by the F1 generation recombinant baculovirus RDPV-SN1 strain are equivalent in size and morphology to the raccoon parvovirus particles.
(8) Safety test 5 healthy and susceptible raccoon dogs of 2-12 months age are subcutaneously inoculated with 3.0 ml/raccoon dog in 3.0ml in the leg of the cell culture of the F1-generation recombinant baculovirus RDPV-SN1 strain, and observation is carried out for 10 days, so that all the inoculated raccoon dogs are healthy and alive.
(9)Immunogenicity test Sf9 cells growing well are inoculated to the virus seeds, cultured for 72-96 h at 27 ℃, and cell cultures are harvested. After complete inactivation by Binary Ethyleneimine (BEI), preparing a vaccine by using a cell culture and sterilized aluminum hydroxide gel according to a ratio of 9:1, subcutaneously inoculating 5 healthy and susceptible raccoon dogs (RDPVHI antibody titer is not higher than 1:4) with 1.0 ml/raccoon dog with 2-12 months age on the leg, and simultaneously taking 5 raccoon dogs with the same condition as a control without inoculation. The titer of the RDPVHI antibody of the immunized raccoon dog is not lower than 1:32 14 after the immunization, and the titer of the RDPVHI antibody of the control raccoon dog is not higher than 1: 4; simultaneously, all raccoon dogs are respectively inoculated with 15ml of raccoon dog parvovirus RDPV-SN1 strain virus liquid (containing 15 multiplied by 10) through the esophagus drenching way 7.0 TCID 50 ) And continuously observing for 10 days, so that the immunized raccoon dog is all healthy and alive, and the control raccoon dog is all attacked.
3. Preparation and finished product inspection of raccoon dog parvovirus enteritis and canine distemper bivalent inactivated vaccine
(1) Vaccine preparation
1) Preparation of raccoon parvovirus VP2 protein: selecting Sf9 cells with vigorous growth, inoculating the cells into a cell culture tank for full suspension culture, wherein the inoculation density is 1.5 multiplied by 10 6 ～2.0×10 6 Culturing at 27 deg.C when the cell density of Sf9 in the cell culture tank reaches 1.5 × 10 6 ～3.0×10 6 Inoculating a recombinant baculovirus RDPV-SN1 strain at the MOI of 0.2-2.0 when each cell/ml is cultured at 27 ℃ for 72-96 h, harvesting a cell culture, adding a 2% BEI solution into the cell culture to enable the BEI final concentration to be 0.1%, uniformly mixing, inactivating the cell culture at 37 ℃ for 48h, adding a 50% sodium thiosulfate solution into the cell culture to enable the final concentration to be 0.2%, stirring for 1h, and stopping inactivation; performing sterility and inactivation tests to obtain the raccoon parvovirus VP2 protein, wherein the hemagglutination titer of 1% of pig red blood cells is more than or equal to 1:2048, and the raccoon parvovirus VP2 protein is stored at 2-8 ℃ for later use;
2) preparation of inactivated antigen of canine distemper virus CDV-11 strain: inoculating Vero cells with vigorous growth into a cell culture tank for microcarrier suspension culture, and culturing the Vero cells in the cell culture tank until the Vero cell density reaches 2.0 × 10 6 ～2.5×10 6 Inoculating CDV-11 strain at MOI of 0.1 per cell/ml, inoculating DMEM cell culture solution containing 2% newborn calf serum at 34 deg.C, and inoculatingSimultaneously sampling and observing, rapidly stirring for 10-15 min when about 80% of cells on the surface of the microcarrier become round and convex and have a small amount of shedding, filtering the microcarrier by using a filter plate of about 75 microns to obtain virus liquid, removing cell fragments by using filter elements of 20 microns and 5 microns to obtain cell culture, wherein the content of canine distemper virus is not less than 10 7.5 TCID 50 Per ml; adding a 2% BEI solution into the harvested cell culture to enable the final concentration of BEI to be 0.1%, uniformly mixing, inactivating at 37 ℃ for 48h, then adding a 50% sodium thiosulfate solution into the cell culture to enable the final concentration to be 0.2%, stirring for 1h, stopping inactivation, obtaining the inactivated antigen of the CDV-11 strain of the canine distemper virus after the inactivation is qualified through sterility and inactivation inspection, and storing at 2-8 ℃ for later use:
3) diluting the protein VP2 of the raccoon parvovirus obtained in the step (1) until the hemagglutination titer of 1% pig red blood cells is not less than 1:256, uniformly mixing the protein with the inactivated antigen of CDV-11 strain of canine distemper virus obtained in the step 2) in a proper proportion, preparing seedlings with sterilized aluminum hydroxide gel in a proportion of 9:1, adding 1% thimerosal solution to enable the final concentration to be 0.01%, fully stirring, quantitatively packaging, covering and labeling.
(2) Inspection of raccoon dog parvovirus enteritis and canine distemper bigeminal inactivated vaccine finished product
1) After standing, the upper layer is clear liquid, the lower layer is a small amount of precipitate, and the mixture is shaken to form a uniform suspension.
2) And (4) carrying out sterile inspection, loading and mercury preservative residual quantity measurement according to the appendix of the current Chinese veterinary pharmacopoeia, and carrying out sterile growth.
3) 5 healthy susceptible raccoon dogs (RDPV HI antibody titer is not higher than 1:4) with 2-12 months of age are used for safety inspection, 3.0ml of vaccine is injected subcutaneously into each leg, after inoculation, the body temperature of the raccoon dogs is observed and measured for 1 time (consistent with the temperature measurement time before inoculation) at regular time every day, and the raccoon dogs are continuously observed for 21 days. The inoculated raccoon dogs are all healthy and alive, and have no obvious changes in spirit, diet, excrement and body temperature compared with those before inoculation, and no other abnormal clinical symptoms; or the body temperature rises above 1 deg.C, but the remaining time should not exceed 2 days.
4) Efficacy test
Firstly, a serology method comprises the steps of injecting 1.0ml of vaccine subcutaneously into 5 healthy and susceptible raccoon dogs (RDPV HI antibody titer is not higher than 1:4) with the age of 2-12 months, and simultaneously taking 5 raccoon dogs with the same condition as a control without inoculation. Respectively taking blood 14 days after immunization, separating serum, and determining the RDPV HI antibody titer and the canine distemper virus neutralizing antibody titer, wherein the RDPV HI antibody titer of the immunized raccoon dog is not lower than 1:32, and the RDPV HI antibody titer of the control raccoon dog is not higher than 1: 4; and (3) respectively collecting blood 21 days after immunization, and determining the titer of the neutralizing antibody of the canine distemper virus of the immunized raccoon dog, wherein the titer of the neutralizing antibody of the canine distemper virus is not lower than 1:50, and the titer of the neutralizing antibody of the canine distemper virus of the control raccoon dog is not higher than 1: 4.
② an immunization challenge method uses 10 healthy and susceptible raccoon dogs (RDPV HI antibody titer is not higher than 1:4) with 2-12 months of age, 1.0ml of vaccine is injected subcutaneously on each leg, and simultaneously, 10 raccoon dogs with the same condition are not inoculated as a control. After 14 days of immunization, 5 immunized raccoon dogs and 5 control raccoon dogs are randomly selected to be respectively inoculated with 15ml of virus liquid (containing 15 multiplied by 10) of raccoon parvovirus RDPV-SN1 strains through the esophagus drenching way 7.0 TCID 50 ) Continuously observing for 10 days, wherein the control raccoon dog needs 100% of diseases, and the immune raccoon dog needs 100% of protection; 21 days after immunization, the rest 5 immunized raccoon dogs and 5 control raccoon dogs are respectively inoculated with 2.0ml through nasal drip and 8.0ml through intraperitoneal injection (10.0 ml in total, containing 1.0 multiplied by 10) 5.5 TCID 50 ) The virus liquid of the canine distemper virus ZC strain is continuously observed for 21 days after counteracting the virus, the control raccoon dog is required to be 100% attacked, and the immune raccoon dog is required to be 100% protected.
Examples
The following examples further illustrate the invention in conjunction with the accompanying drawings. But should not be construed to limit the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1 study of recombinant baculovirus RDPV-SN1 Strain suspension culture Process
1. Research on recombinant baculovirus RDPV-SN1 strain triangular flask culture process
(1) Effect of cell Density on hemagglutination Titers of expressed proteins
When the cell density of Sf9 is 1.5X 10 6 2.0X 10 cells/ml 6 2.5X 10 cells/ml 6 Individual cell/ml, 3.0X 10 6 At each cell/ml, the recombinant baculovirus RDPV-SN1 strain was inoculated at an MOI of 0.5, and samples were taken 60h, 72h, 84h, 96h, 108h, and 120h after inoculation to determine the hemagglutination titer of the expressed protein.
As a result, the recombinant baculovirus RDPV-SN1 strain was inoculated with Sf9 cells at different cell densities, and the Sf9 cell density was 1.5X 10 when inoculated 6 、2.0×10 6 、2.5×10 6 、3.0×10 6 The hemagglutination titer of the expressed protein has no obvious difference when each cell/ml, and the hemagglutination titer of the expressed protein reaches the peak 72 hours after virus inoculation, which is not lower than 1:2048 and is maintained for 96 hours. Specific results are shown in table 1.
TABLE 1 hemagglutination titers of expressed proteins from samples of different cell densities
Figure GDA0002203556800000111
(2) Effect of virus inoculum size on hemagglutination titer of expressed proteins
When the cell density of Sf9 is 1.5X 10 6 The individual cells/ml were inoculated with recombinant baculovirus RDPV-SN1 strain at MOI 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, and sampled at 60h, 72h, 84h, 96h, 108h, and 120h after inoculation to determine the hemagglutination titer of the expressed protein.
As a result, there was no significant difference in the hemagglutination titers of the expressed proteins at MOI's of 0.2, 0.5, 1.0 and 2.0. After inoculation, the hemagglutination titer of the expressed protein reaches a peak within 72 hours, which is not lower than 1:2048, and is maintained for 96 hours; the expressed protein has lower hemagglutination titer when the MOI is 0.05, 0.1 and 3.0. The specific results are shown in Table 2.
TABLE 2 expression of protein hemagglutination titers in samples of different multiplicity of infection
Figure GDA0002203556800000112
Figure GDA0002203556800000121
(3) Effect of different harvest times on the hemagglutination Titers of expressed proteins
When the cell density of Sf9 is 1.5X 10 6 At each cell/ml, the recombinant baculovirus RDPV-SN1 strain was inoculated at an MOI of 0.2, and samples were taken 60h, 72h, 84h, 96h, 108h, and 120h after inoculation to determine the hemagglutination titer of the expressed protein.
The result shows that the hemagglutination titer of the expressed protein reaches the peak 72h after the inoculation, and is maintained to 96h, and the hemagglutination titer of the expressed protein is not lower than 1: 2048; the hemagglutination titer of the expressed protein begins to decline 120 hours after the inoculation. The specific results are shown in Table 3.
TABLE 3 expression of protein hemagglutination titers in samples at different harvest times
Figure GDA0002203556800000122
In conclusion, considering the factors of production, we defined the production process of the recombinant baculovirus RDPV-SN1 strain as follows: initial cell density of 1.5X 10 for seeding 6 ～3.0×10 6 And (3) each cell/ml, wherein the MOI is 0.2-2.0, and the cell culture is harvested 72-96 hours after inoculation.
2. Inoculation process test of recombinant baculovirus RDPV-SN1 strain cell culture tank
(1) Cell inoculation and amplification culture Sf9 cells growing well in a triangular flask were inoculated into a 30L cell culture pot at an inoculation density of 1.5X 10 6 ～2.0×10 6 Each cell/ml, the cell culture temperature is 27 ℃, the dissolved oxygen is 45%, sampling is carried out at regular time during the culture period, the cell state is observed, and the cell density and the cell activity are recorded; when the cell density reaches 3.0X 10 6 When the cell density is more than 3.0X 10, the culture medium is supplemented, and the culture is continued until the cell density is higher than 3.0X 10 6 Each cell/ml, Sf9 cell suspension was transferred to a 90L cell culture tank for culture at a seeding density of 1.5X 10 6 ～2.0×10 6 Individual cells/ml, samples were taken periodically during the culture, cell status was observed and viable cell density and viability were recorded. When the cell density reaches 3.0 multiplied by 10 6 When the cell density is more than one cell/ml, the culture medium is supplemented, and the culture is continued until the cell density reaches 3.0 multiplied by 10 6 The Sf9 cell suspension was transferred to a 350L cell culture tank for culture at a cell/ml or more. The cells were cultured in 1000L cell culture pots in the same manner.
(2)1000L cell culture jar detoxification test when the cell density of Sf9 in 1000L cell culture jar reached 1.5X 10 6 Individual cells/ml, 3.0X 10 6 At each cell/ml, recombinant baculovirus RDPV-SN1 strain was inoculated at MOI of 0.2 and 2.0, respectively, and samples were taken at 72h and 96h, respectively, to determine the hemagglutination titer of the expressed protein. The results show that the hemagglutination titer of the expressed protein is not obviously different at 72h and 96h after inoculation, and is not lower than 1: 2048. The specific results are shown in Table 4.
TABLE 41000L measurement results of hemagglutination titer of expressed protein in cell culture tank virus inoculation test
Figure GDA0002203556800000131
We have conducted the research on the culture process of the recombinant baculovirus RDPV-SN1 strain triangular flask, and have conducted the cell culture tank process verification test according to the test results, and finally determined the suspension culture process of the recombinant baculovirus RDPV-SN1 strain cell culture tank: when the cell density of Sf9 in the cell culture tank reaches 1.5X 10 6 ～3.0×10 6 When the number of cells per ml is one, a recombinant baculovirus RDPV-SN1 strain is inoculated according to the MOI of 0.2-2.0, and the cells are cultured for 72-96 h at the temperature of 27 ℃, so that cell cultures are obtained.
Example 2 study of suspension culture Process of CDV-11 Strain of Canine distemper Virus
1. Study on culture process of 7L cell culture tank of canine distemper virus CDV-11 strain
(1) Microcarrier preparation and Sterilization A Cytodex type I microcarrier was weighed each time and treated with Ca-free medium 2+ 、Mg 2+ The PBS (0.1mol/L, pH7.2, the same below) is washed for 2-3 times, and then the PBS is added for sterilization at 121 ℃ for 30 min. After sterilization, PBS was discarded, and MEM cell culture medium containing 8% newborn calf serum and containing 5% CO at 36.5 deg.C 2 The incubator is placed for 4 hours.
(2) Vero cell microcarrier suspension culture is adopted to select Vero spinner flask cells with good shape and vigorous growth, and EDTA-trypsin dispersion is used for eliminatingPreparing cell suspension, inoculating into stirring cell culture bottle at rotation speed of 30r/min and cell density of 4.5 × 10 5 Each cell/ml, the concentration of the microcarrier is 4g/L, and the culture temperature is 36.5-37.5 ℃.
(3) Influence of culture temperature on virus content Vero cell inoculation stirring type cell culture flask culture is carried out when cell density reaches 2.0 x 10 6 When the cells are cultured per ml, DMEM cell culture solution containing 2% newborn calf serum is replaced, and canine distemper virus CDV-11 strain is inoculated according to MOI of 0.1, and cultured at 31 ℃, 34 ℃ and 37 ℃ respectively. And sampling 48h, 72h, 96h and 120h after inoculation, and determining the content of canine distemper virus.
As a result, the canine distemper virus has the highest virus content when cultured at 34 ℃, the virus content when cultured at 37 ℃ is slightly lower than that of the canine distemper virus when cultured at 34 ℃, and the virus content when cultured at 31 ℃ is the lowest. The specific results are shown in Table 5.
TABLE 5 Virus content of samples at different culture temperatures
Figure GDA0002203556800000141
(4) Influence of cell density on virus content during virus inoculation Vero cell inoculation stirring type cell culture flask culture is carried out, and when the cell density reaches 1.5 multiplied by 10 6 2.0X 10 cells/ml 6 2.5X 10 cells/ml 6 Individual cell/ml, 3.0X 10 6 When each cell is per ml, DMEM cell culture solution containing 2% newborn calf serum is replaced, and canine distemper virus CDV-11 strain is inoculated according to MOI of 0.1 respectively, and the culture temperature is 34 ℃. And sampling 48h, 72h, 96h and 120h after inoculation, and determining the content of canine distemper virus.
As a result, the cell density at the time of inoculation was 2.0X 10 6 Individual cells/ml and 2.5X 10 6 The virus content is highest when each cell/ml, and the cell density is 1.5 multiplied by 10 when receiving virus 6 Individual cells/ml and 3.0X 10 6 The virus content was slightly lower at individual cells/ml. According to the test results, the cell density at the time of virus inoculation is 2.0X 10 6 ～2.5×10 6 Individual cells/ml. The specific results are shown in Table 6.
TABLE 6 Virus content of samples of different cell densities
Figure GDA0002203556800000142
(5) Influence of virus inoculation amount on virus content Vero cell inoculation stirring type cell culture flask culture is carried out when cell density reaches 2.0 multiplied by 10 6 When each cell/ml, DMEM cell culture medium containing 2% newborn bovine serum was replaced, and canine distemper virus CDV-11 strain was inoculated at an MOI of 0.05, 0.1, and 0.15, respectively, at a culture temperature of 34 ℃. And sampling 48h, 72h, 96h and 120h after inoculation, and determining the content of canine distemper virus.
As a result, the virus content was highest when the strain CDV-11 was inoculated at an MOI of 0.1, and was lower than that when the strain CDV-11 was inoculated at an MOI of 0.05 or 0.15. According to the test results, the inoculation amount of the canine distemper virus during production is determined as MOI 0.1. The specific results are shown in Table 7.
TABLE 7 Virus content of samples with different virus receiving amounts
Figure GDA0002203556800000143
Figure GDA0002203556800000151
(6) Influence of serum concentration on virus content Vero cell inoculation stirring type cell culture flask culture is carried out when cell density reaches 2.0 x 10 6 When each cell is cultured per ml, DMEM cell culture solution containing 0%, 1%, 2% and 3% newborn bovine serum is respectively replaced, and canine distemper virus CDV-11 strain is inoculated according to MOI of 0.1, and the culture temperature is 34 ℃. And sampling 48h, 72h, 96h and 120h after inoculation, and determining the content of canine distemper virus.
As a result, the virus is cultured by DMEM cell culture solution containing 2% and 3% of newborn bovine serum, the virus is proliferated faster, and the virus content is higher; the virus content was lower when the virus was cultured in serum-free DMEM cell culture medium and 1% newborn bovine serum-containing DMEM cell culture medium. According to the test results, the culture medium was DMEM cell culture medium containing newborn bovine serum at a concentration of 2%. The specific results are shown in Table 8.
TABLE 8 Virus content of samples at different serum concentrations
Figure GDA0002203556800000152
(7) Influence of different harvest time on virus content Vero cell inoculation stirring type cell culture bottle culture, when the cell density reaches 2.0 x 10 6 When each cell is per ml, DMEM cell culture solution containing 2% newborn calf serum is replaced, and canine distemper virus CDV-11 strain is inoculated according to MOI of 0.1, and the culture temperature is 34 ℃. Sampling 48h, 72h, 96h and 120h after inoculation, observing cytopathic condition, and determining the content of canine distemper virus.
48 hours after inoculation, cells have no obvious lesion and the virus content is lower; after 72h of inoculation, 70-80% of cells on the microcarrier become round and protrude to present pathological changes, a small amount of cells fall off from the microcarrier, and the virus content reaches a high peak value; 96 hours after inoculation, cytopathic effect is obvious, about 80% of cells fall off from the microcarrier, a large number of dead cells are separated from the culture medium, and the virus content is maintained at a high peak value; 120h after inoculation, almost no normal cells exist, cells fall off from the microcarriers, almost no cells exist on the microcarriers, and the virus content is reduced. The results show that virus liquid can be obtained when about 80% of cells on the surface of the microcarrier become round and protuberant and have a small amount of shedding after 72-96 hours after virus inoculation. Specific results are shown in Table 9.
TABLE 9 Effect of different harvest times on Virus content
Figure GDA0002203556800000153
Figure GDA0002203556800000161
According to the experimental research, the suspension culture production process parameters of the canine distemper virus CDV-11 strain are determined as follows: vero cellCulturing until the cell density is 2.0 × 10 6 ～2.5×10 6 Inoculating CDV-11 strain of canine distemper virus at MOI of 0.1 per ml, inoculating DMEM cell culture solution containing 2% newborn bovine serum at 34 deg.C, sampling at different time, observing cell state, and collecting virus solution when 80% of cells on the surface of the microcarrier become round and have small amount of shedding.
2. Cell culture tank validation test
(1) Cleaning and sterilizing the cell culture tank and balancing the microcarrier, cleaning the cell culture tank, sterilizing for 30min at 121 ℃, and finishing 100% point correction of the empty tank dissolved oxygen electrode after the sterilization is finished. And (3) pumping the sterilized microcarrier into a cell culture tank, and supplementing an equilibrium culture medium to a minimum culture volume, wherein the equilibrium time is not less than 4 h.
(2)42L cell culture tank inoculation test Vero cell inoculation 7L cell culture tank when cell density reaches 2.0 x 10 6 When the cell/ml is more than one, performing digestion transfer, transferring cells in a 7L cell culture tank to a 42L cell culture tank, and culturing at an inoculation density of not less than 4.5 × 10 5 Individual cells/ml. The cells are cultured in a 42L cell culture tank for 2-3 days, and the cell density reaches 2.0 multiplied by 10 6 ～2.5×10 6 When each cell/ml is detected, 2% of newborn bovine serum DMEM cell culture solution is replaced, a canine distemper virus CDV-11 strain is inoculated according to the MOI of 0.1, the culture temperature is 34 ℃, sampling is carried out at different time after inoculation, the cell state is observed, when about 80% of cells on the surface of a microcarrier become round and protuberants and a small amount of cells fall off, rapid stirring is carried out for 10-15 min, the microcarrier is filtered by a filter plate of about 75 mu m, a virus solution is obtained, cell fragments are sequentially removed by filter elements of 20 mu m and 5 mu m, and the canine distemper virus content is measured by sampling. The results show that the content of the canine distemper virus is 10 7.5 TCID 50 More than ml.
The canine distemper virus suspension culture production process is researched from five aspects of culture temperature, cell density during virus inoculation, virus inoculation amount, serum concentration and harvest time, a cell culture tank process verification test is carried out, and the canine distemper virus suspension culture production process is determined to be as follows according to the test result: vero cell is cultured until the cell density is 2.0 multiplied by 10 6 ～2.5×10 6 When each cell is per mlInoculating CDV-11 strain of canine distemper virus at MOI of 0.1, inoculating DMEM cell culture solution containing 2% newborn bovine serum to a culture medium during inoculation, culturing at 34 ℃, observing the cell state after inoculation, rapidly stirring for 10-15 min after inoculation for 72-96 h when about 80% of cells on the surface of the microcarrier become round and protrude and a small amount of cells fall off, filtering the microcarrier by using a filter plate of about 75 mu m, harvesting virus liquid, and removing cell fragments by using filter elements of 20 mu m and 5 mu m.
Example 3 inactivation study of the recombinant baculovirus RDPV-SN1 Strain BEI
Collecting cell cultures of 001, 002 and 003 batches of recombinant baculovirus RDPV-SN1 strain 300ml respectively, quantitatively subpackaging, and packaging 100 ml/bottle. Adding 2% BEI solution into each bottle of cell culture to enable the final concentration to be 0.05%, 0.1% and 0.2% respectively, inactivating at 37 ℃, uniformly mixing for 1 time every 4-8 h during the period, sampling for 24h, 32h, 40h and 48h respectively, adding 50% sodium thiosulfate solution to enable the final concentration to be 2 times of BEI, stirring for 1h to terminate inactivation, and sampling for inactivation inspection.
Results of 0.05% BEI solution, inactivation at 37 ℃ for 48h, 0.1% BEI solution, inactivation at 37 ℃ for 40h, 0.2% BEI solution, and inactivation at 37 ℃ for 40h can completely inactivate the recombinant baculovirus RDPV-SN1 strain, and specific results are shown in Table 10. In order to ensure complete inactivation of the recombinant baculovirus RDPV-SN1 strain and consider the production cost, BEI with the final concentration of 0.1% is determined to be adopted in the production, and inactivation is carried out for 48 hours at 37 ℃.
TABLE 10 inactivation test of the recombinant baculovirus RDPV-SN1 strain in BEI solutions at different concentrations
Figure GDA0002203556800000171
Note: "+" indicates complete inactivation and "-" indicates incomplete inactivation.
Example 4 inactivation of CDV-11 BEI in Canine distemper Virus
Collecting 001, 002 and 003 batches of CDV-11 cell cultures of canine distemper virus strain in 300ml each, quantitatively packaging, and packaging in 100 ml/bottle. Adding 2% BEI solution into each bottle of cell culture to enable the final concentration to be 0.05%, 0.1% and 0.2% respectively, inactivating at 37 ℃, uniformly mixing for 1 time every 4-8 h during the period, sampling for 24h, 32h, 40h and 48h respectively, adding 50% sodium thiosulfate solution to enable the final concentration to be 2 times of BEI, stirring for 1h to terminate inactivation, and sampling for inactivation inspection.
As a result, the CDV-11 strain of the canine distemper virus can be completely inactivated by 0.05% BEI solution, inactivation at 37 ℃ for 48h, 0.1% BEI solution, inactivation at 37 ℃ for 40h, 0.2% BEI solution and inactivation at 37 ℃ for 40h, and the specific results are shown in Table 11. In order to ensure thorough inactivation of the canine distemper virus CDV-11 strain and simultaneously consider the production cost, BEI with the final concentration of 0.1% is determined to be adopted in production, and inactivation is carried out for 48 hours at 37 ℃.
TABLE 11 inactivation test of BEI solutions of CDV-11 strains of Canine distemper Virus at different concentrations
Figure GDA0002203556800000172
Figure GDA0002203556800000181
Note: "+" indicates complete inactivation and "-" indicates incomplete inactivation.
Example 5 preparation of raccoon dog parvovirus enteritis and Canine distemper bivalent inactivated vaccine
1. Preparation of raccoon parvovirus VP2 protein
(1) Preparation of seed poison for production
1) Seed inoculation and good growth are carried out on the seeds according to the MOI of 0.2-2.0 and the cell density of 1.5 multiplied by 10 6 ～2.0×10 6 Culturing Sf9 cells per ml at 27 ℃ for 72-96 h, harvesting cell culture, and quantitatively packaging. Name, date harvested, generation number, etc. are noted.
2) Virus seed test
Firstly, the virus content is measured according to the MTT relative efficiency method, and the virus content per ml is more than or equal to 1.0 multiplied by 10 7.0 PFU。
Secondly, the purity is tested according to the appendix of the traditional Chinese beast pharmacopoeia, and bacteria, mould, mycoplasma and exogenous virus pollution do not exist.
③ specificity
The virus PCR detection is carried out by the PCR method, and a band of about 1.7kb should appear.
Expressing protein specificity, diluting a recombinant baculovirus RDPV-SN1 strain cell culture into a suspension with 8 hemagglutination units by PBS (0.015mol/L, the pH value is 6.4, the same below), fully mixing the suspension with an equal amount of raccoon parvovirus specific antiserum to serve as a neutralization group, simultaneously setting a positive control group (the suspension of the recombinant baculovirus RDPV-SN1 strain cell culture diluted into 8 hemagglutination units by PBS is fully mixed with an equal amount of PBS) and a negative control group (PBS), neutralizing for 60min at 37 ℃, and shaking for 2-3 times. And after neutralization, adding the 3 groups into V-shaped 96-hole micro hemagglutination plates, wherein each group has at least 10 holes and each hole has 0.025ml, then adding 0.025ml of 1% pig red blood cells into each hole, fully shaking, acting at 2-8 ℃ for 40-50 min, and observing the result. Neither the neutralization group nor the negative control group should be agglutinated, and the positive control group should be agglutinated completely.
3) The virus generation should not exceed 3 generations.
4) The virus seeds are preserved at the temperature of 2-8 ℃ and the preservation period is 9 months.
(2) Cell preparation
1) And (3) recovering the cells, taking Sf9 cells preserved by liquid nitrogen, immediately putting warm water at 37 ℃ for rapid dissolution, centrifuging Sf9 cell suspension at 620r/min for 5min, removing supernatant, resuspending the cells by using SF-SFM serum-free culture medium, placing the cells in a triangular flask with proper volume, and carrying out shake culture at 27 ℃ at the rotating speed of 100-105 r/min.
2) When the cell density of Sf9 reaches 3.0X 10, the cells are expanded and cultured 6 When the cell density is more than one cell/ml, passage is carried out, and the density of inoculated cells is 1.0X 10 6 ～1.5×10 6 And (4) each cell/ml, carrying out cell passage to a certain number, inoculating a cell culture tank, and continuously culturing at 27 ℃.
(3) Antigen preparation for vaccine production
1) Cleaning and sterilizing the cell culture tank, cleaning the cell culture tank, sterilizing at 121 ℃ for 30min, and correcting the dissolved oxygen electrode after the sterilization is finished.
2) Inoculating and amplifying to select Sf9 cells with vigorous growth, inoculating to cell culture tank at density of 1.5 × 10 6 ～2.0×10 6 Culturing at 27 deg.C until cell density is higher than 3.0 × 10 6 When each cell/ml, the culture medium is supplemented, and the culture is continued until the cell density is higher than 3.0X 10 6 When the cell is per ml, transferring the cell suspension in the cell culture tank to a new cell culture tank for culture, wherein the inoculation density is 1.5 multiplied by 10 6 ～2.0×10 6 Individual cells/ml.
3) Inoculation and harvesting when Sf9 cell density reached 1.5X 10 6 ～3.0×10 6 Inoculating recombinant baculovirus RDPV-SN1 strain at MOI of 0.2-2.0 per ml, culturing at 27 deg.C for 72-96 hr, collecting cell culture, storing at-15 deg.C or below with name, date, lot number, etc. noted, and checking.
(4) Inactivation A2% BEI solution was added to the cell culture to a final concentration of 0.1%, and inactivation was carried out at 37 ℃ for 48 h; and after inactivation is finished, adding a 50% sodium thiosulfate solution into the cell culture to enable the final concentration of the solution to be 0.2%, stirring for 1 hour, then stopping inactivation, and storing at 2-8 ℃ for later use.
2. Preparation and inspection of canine distemper virus CDV-11 strain semi-finished product
(1) Preparing and recovering Vero cells, wherein the growth solution is a cell culture solution containing 10% newborn calf serum, placing at 37 ℃ and containing 5% CO 2 Culturing in an incubator, digesting the cells with EDTA-trypsin dispersion liquid when the cells grow into a compact monolayer, and carrying out subculture proliferation.
(2) Preparation and inspection of production virus seed
1) Preparation of seed Virus for production Vero cell monolayer with good growth, discarding growth medium (MEM cell culture medium containing 10% newborn calf serum), adding seed Virus into maintenance medium at a ratio of 2% of maintenance medium (MEM cell culture medium containing 2% newborn calf serum), subpackaging into cell bottles, standing at 37 deg.C and 5% CO 2 Continuously culturing in an incubator, harvesting when about 80% of cells have typical confluent cytopathy, freeze thawing for 1 time, quantitatively packaging, freezing and storing, and noting harvest date, toxin generation and the like.
2) Virus seed test
(virus content) virus seeds were serially diluted 10-fold with MEM cell culture containing 2% newborn calf serum, and 10 cells were collected -4 、10 -5 、10 -6 、10 -7 And 4 dilutions are respectively inoculated to Vero cell 96-well cell culture plates which have grown into a monolayer, 8 dilutions are inoculated to each well, 100 mu l of each well, and 8 wells of non-inoculated control are arranged. Each well was supplemented with 100. mu.l of MEM cell culture containing 2% newborn calf serum, and the mixture was left at 37 ℃ with 5% CO 2 Culturing in an incubator, observing for 96-120 h, recording the number of holes of cytopathic effect (CPE), and calculating TCID according to Reed-Muench method 50 . The virus content per ml should be more than or equal to 10 5.5 TCID 50 。
Secondly, the purity is tested according to the appendix of the current Chinese veterinary pharmacopoeia, and the virus is aseptic, mycoplasma and exogenous virus, and the virus is aseptic, mycoplasma-free and exogenous virus-free.
③ the virus seeds are specifically diluted to 10 by sterile normal saline 3.0 TCID 50 The mixture was mixed with 10-fold diluted antiserum specific for canine distemper virus to give a neutralization group, and a neutralization control group (prepared by diluting the seed virus with 10 times of sterile physiological saline) was added 3.0 TCID 50 Each ml was mixed with an equal amount of MEM cell culture medium) and cell control, neutralized at 37 ℃ for 30 min. After neutralization, 24-well Vero cell culture plates with grown monolayers were inoculated, the neutralized group was inoculated with 12 wells at 0.5 ml/well, the non-neutralized control group and the cell control group were inoculated with 6 wells at 0.5 ml/well, each well was supplemented with 0.5ml of MEM cell culture medium containing 2% newborn bovine serum, the mixture was left at 37 ℃ and 5% CO 2 Culturing in an incubator, and observing for 96-120 h. The neutralization group and the cell control group should have no cytopathy, and the neutralization control group should have cytopathy.
(3) Preparation of virus liquid for preparing vaccine
1) Cleaning and sterilizing the cell culture tank, balancing the microcarrier, cleaning the cell culture tank, and sterilizing for 30min at 121 ℃. And (3) after the sterilization is finished, pumping the completely sterilized microcarrier into a cell culture tank, adding MEM cell culture solution containing 8% newborn calf serum to the minimum culture volume for balancing, wherein the balancing time is not less than 4 h. The calibration of the 100% point of the dissolved oxygen electrode was done during equilibration of the support.
2) Inoculating Vero cell to select well grown Vero cell monolayer, digesting with EDTA-trypsin, inoculatingThe seeding density of the seed cell culture tank is not lower than 4.5 multiplied by 10 5 Each cell/ml, the culture medium is MEM cell culture solution containing 8% newborn calf serum, and the continuous culture is carried out for 2-3 days when the cell density is higher than 2.0 multiplied by 10 6 The digestion transfer was performed at individual cells/ml.
3) Cell digestion transfer the cell suspension was transferred to a digester, the supernatant was discarded, PBS (0.01mol/L, pH7.2) was added to the digester and washed 2 times, PBS was discarded, trypsin was added for digestion, and MEM cell culture containing 8% newborn calf serum was added to stop digestion after digestion was completed. Transferring the cell suspension into a new cell culture tank for culture, wherein the inoculation density is not lower than 4.5 × 10 5 Individual cells/ml.
4) Inoculating virus and harvesting cell suspension, transferring to a new cell culture tank, and culturing for 2-3 days until cell density reaches 2.0 × 10 6 ～2.5×10 6 Inoculating CDV-11 strain of canine distemper virus at a cell/ml, and inoculating DMEM cell culture solution containing 2% newborn calf serum at 34 deg.C with MOI of 0.1. Sampling at different time after virus inoculation, observing cell state, rapidly stirring for 10-15 min when about 80% of cells on the surface of the microcarrier become round and convex and a small amount of cells fall off, filtering the microcarrier by using a filter plate of about 75 microns, harvesting virus liquid, sequentially removing cell fragments by using filter elements of 20 microns and 5 microns, marking name, harvesting date and batch number, storing at-15 ℃ and waiting for standby.
(4) Inspection of semi-finished product
1) The sterility test is carried out according to the appendix of the traditional Chinese veterinary pharmacopoeia, and the bacteria can grow aseptically.
2) The virus content is measured according to item 1.2.1.1, and the virus content per ml is more than or equal to 10 7.5 TCID 50 。
(5) Inactivation A2% BEI solution was added to the cell culture to a final concentration of 0.1%, and inactivation was carried out at 37 ℃ for 48 h; and after inactivation is finished, adding a 50% sodium thiosulfate solution into the cell culture to enable the final concentration of the cell culture to be 0.2%, stirring for 1h, stopping inactivation, and storing at 2-8 ℃ to be ready for use.
3. Preparing and subpackaging, namely uniformly mixing a cell culture of a recombinant baculovirus RDPV-SN1 strain qualified in inactivation test and an inactivated antigen of a canine distemper CDV-11 strain in a proper proportion, preparing seedlings with sterilized aluminum hydroxide gel in a ratio of 9:1, adding a 1% thimerosal solution to enable the final concentration to be 0.01%, fully stirring, quantitatively subpackaging, covering and labeling to prepare the raccoon parvovirus enteritis and canine distemper bigeminal inactivated vaccine.
Example 6 detection of raccoon dog parvovirus enteritis and Canine distemper bigeminal inactivated vaccine finished product
1. After standing, the upper layer is clear liquid, the lower layer is a small amount of precipitate, and the mixture is shaken to form a uniform suspension.
2. The sterility test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the growth is carried out aseptically. The results of the test are shown in Table 12.
3. The loading inspection is carried out according to the appendix of the current Chinese animal pharmacopoeia and is in line with the regulations. The results of the test are shown in Table 12.
TABLE 12 results of the load inspection
Figure GDA0002203556800000211
4. Safety inspection 5 healthy and susceptible raccoon dogs (RDPV HI antibody titer is not higher than 1:4) with 3.0ml per dog parvovirus enteritis and canine distemper bivalent inactivated vaccine are respectively subcutaneously inoculated on legs to serve as an immunization group; another 5 raccoon dogs were not immunized and served as control groups. After inoculation, the body temperature of the raccoon is regularly observed and measured for 1 time every day (the body temperature is consistent with the temperature measurement time before inoculation), and the raccoon is continuously observed for 21 days, so that the raccoon in the immunization group is completely healthy and alive, and the spirit, diet, excrement and body temperature have no obvious change compared with those before inoculation, and have no other abnormal clinical symptoms, and no local and systemic adverse reactions caused by the vaccine. No obvious difference from the control group. The results are shown in Table 13.
TABLE 13 vaccine safety test results
Figure GDA0002203556800000212
Figure GDA0002203556800000221
5. Efficacy test
(1) Serological method 5 healthy susceptible raccoon dogs (RDPV HI antibody titer is not higher than 1:4) with 2 months of age are injected with 1.0ml of vaccine subcutaneously on each leg, and 5 raccoon dogs with the same conditions are not inoculated as a control. After 14 days of immunization, blood is respectively collected from all raccoon dogs, and RDPV HI antibody titer determination is carried out, so that the RDPV HI antibody titer of the immunized raccoon dogs is not lower than 1:32 (protective antibody value), and the RDPV HI antibody titer of the control raccoon dogs is not higher than 1: 4; and (3) taking blood from all raccoons 21 days after immunization, and performing canine distemper virus neutralization antibody titer determination, wherein the results show that the neutralizing antibody titer of the immunized raccoon canine distemper virus is not lower than 1:50 (protective antibody value), and the neutralizing antibody titer of the control raccoon canine distemper virus is not higher than 1: 4. The specific results are shown in Table 14.
TABLE 14 serological method potency test results
Figure GDA0002203556800000222
(2) The immune challenge method comprises the steps of injecting 1.0ml of vaccine subcutaneously into 10 healthy and susceptible raccoon dogs (RDPV HI antibody titer is not higher than 1:4) with 2 months of age, and simultaneously taking 10 raccoon dogs with the same condition without inoculation as a control. After 14 days of immunization, 5 immunized raccoon dogs and 5 control raccoon dogs are randomly selected and inoculated with 15ml of virus solution (containing 15 multiplied by 10) of raccoon parvovirus RDPV-SN1 strain through the esophagus drenching way respectively 7.0 TCID 50 ) Continuously observing for 10 days, controlling the raccoon dog to have 100 percent of disease, and protecting the immunized raccoon dog by 100 percent; 21 days after immunization, the rest 5 immunized raccoon dogs and 5 control raccoon dogs are respectively inoculated with 2.0ml through nasal drip and 8.0ml through intraperitoneal injection (10 ml in total, containing 1.0 multiplied by 10) 5.5 TCID 50 ) The virus liquid of the canine distemper virus ZC strain is continuously observed for 21 days after counteracting the virus, and compared with 100 percent of the developed raccoon dogs, the immunized raccoon dogs are 100 percent protected. The results are shown in tables 15 and 16.
Watch 15 clinical observation result of raccoon dog parvovirus after virus challenge
Figure GDA0002203556800000231
TABLE 16 clinical observation results of canine distemper virus after challenge
Figure GDA0002203556800000241
6. The determination of the residual quantity of the mercury preservative is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the determination is in accordance with the regulations. The results of 3 batches of vaccine are in accordance with the regulations, and the specific results are shown in Table 17.
TABLE 17 mercury based preservative residue measurement results
Figure GDA0002203556800000242
Figure GDA0002203556800000251
Sequence listing
<110> Qilu animal health products Co., Ltd
<120> raccoon dog parvovirus enteritis and canine distemper bigeminal inactivated vaccine and preparation method thereof
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aatacaaact atattactga agctactatt atgagaccag ctgaggttgg ttatagtgca 1020
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Claims (1)

1. A raccoon parvovirus enteritis and canine distemper bigeminal inactivated vaccine is characterized in that the effective components of the vaccine are raccoon parvovirus VP2 protein expressed by a recombinant baculovirus RDPV-SN1 strain, canine distemper virus CDV-11 strain inactivated antigen and an aluminum hydroxide gel adjuvant;
the vaccine is used for preventing canine distemper and raccoon dog parvovirus enteritis;
the racoon dog parvovirus VP2 protein is obtained by transfecting Sf9 cells with a recombinant baculovirus RDPV-SN1 strain expressing racoon dog parvovirus VP2 protein, culturing until the cells are infected, wherein the strain of virus is named as an alfalfa Spodoptera litura nucleopolyhedrovirus recombinant baculovirus RDPV-SN1 strain, and is delivered to the general microbiological center of China microbiological culture collection management Committee of Microbiol research institute of China academy of sciences No.3 of North West Lu 1 of the Yangyo city in Beijing on the 05 and 16 days of 2019, and the preservation number is CGMCC No. 17685;
the inactivated antigen of the canine distemper virus CDV-11 strain is obtained by inactivating the canine distemper virus CDV-11 strain by BEI, and the strain is preserved by the China general microbiological culture Collection center of the microbiological research institute of China academy of sciences, China national institute of sciences, No.1 West way, 3 North Cheng, the Beijing city, on 07-15 th of 2009 with the preservation number of CGMCC number 3201;
the construction of the recombinant baculovirus RDPV-SN1 strain for expressing the protein of the raccoon parvovirus VP2 comprises the following steps:
(1) construction of transfer vector: adding a melittin signal peptide HBM and a beta-actin insulator HS4 sequence to the pVL1393 vector to construct a transfer vector pVL-HBM-HS 4;
(2) construction of recombinant transfer vectors: extracting a VP2 gene of raccoon parvovirus RDPV-SN1 strain, cloning the extracted VP2 gene into a transfer vector pVL-HBM-HS4 to obtain a recombinant transfer vector pVL-HBM-HS4-VP2, wherein the VP2 gene sequence is shown as SEQ ID No. 3;
(3) construction of recombinant baculovirus RDPV-SN1 strain: the recombinant transfer vector pVL-HBM-HS4-VP2 and linearized Autographa californica nuclear polyhedrosis virus AcMNPV genome DNA are subjected to cotransfection with Sf9 cells to obtain recombinant baculovirus expressing raccoon dog parvovirus VP2 protein, and the recombinant baculovirus is named as Autographa californica nuclear polyhedrosis virus recombinant baculovirus RDPV-SN1 strain;
the preparation method of the vaccine comprises the following steps:
(1) preparing raccoon parvovirus VP2 protein: selecting Sf9 cells with vigorous growth, inoculating the cells into a cell culture tank for full suspension culture, wherein the inoculation density is 1.5 multiplied by 10 6 ～2.0×10 6 Cells/ml, cultured at 27 ℃. When the cell density of Sf9 in the cell culture tank reaches 1.5X 10 6 ～3.0×10 6 Inoculating a recombinant baculovirus RDPV-SN1 strain at MOI = 0.2-2.0 when each cell/ml, culturing at 27 ℃ for 72-96 h, harvesting a cell culture, adding a 2% BEI solution into the cell culture to enable the BEI final concentration to be 0.1%, uniformly mixing, inactivating at 37 ℃ for 48h, enabling the hemagglutination titer of 1% pig red blood cells to be more than or equal to 1:2048, adding a 50% sodium thiosulfate solution into the cell culture to enable the final concentration to be 0.2%, stirring for 1h, and stopping inactivation; after being sterilized and inactivated and tested to be qualified, the raccoon parvovirus VP2 protein is obtained;
(2) preparing inactivated antigen of canine distemper virus CDV-11 strain: inoculating Vero cells with vigorous growth into a cell culture tank for microcarrier suspension culture, and culturing the Vero cells in the cell culture tank until the Vero cell density reaches 2.0 × 10 6 ～2.5×10 6 Inoculating CDV-11 canine distemper virus at MOI =0.1 per cell/ml, rapidly stirring for 10-15 min when 80% of cells on the surface of the microcarrier become round and have a small amount of shedding, filtering the microcarrier with a 75 μm filter plate, harvesting virus liquid, removing cell debris with 20 μm and 5 μm filter elements, and harvesting cell culture product with canine distemper virus content not lower than 10 7.5 TCID 50 Per ml; adding 2% BEI solution into the harvested cell culture to make BEI final concentration 0.1%, mixing, inactivating at 37 deg.C for 48 hr, adding 50% sodium thiosulfate solution into the cell culture,the final concentration is 0.2%, and the inactivation is stopped after stirring for 1 h; after the canine distemper virus CDV-11 strain inactivated antigen is obtained after the canine distemper virus CDV-11 strain is subjected to sterility and inactivation inspection and is qualified;
(3) diluting the protein VP2 of the raccoon parvovirus obtained in the step (1) until the hemagglutination titer of 1% of pig red blood cells is not less than 1:256, uniformly mixing the protein with the inactivated antigen of the CDV-11 strain of the canine distemper virus obtained in the step (2) according to a proper proportion, carrying out seedling preparation with sterilized aluminum hydroxide gel according to a proportion of 9:1, adding 1% thimerosal solution to enable the final concentration to be 0.01%, and fully stirring to obtain the vaccine.
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CN104258386A (en) * 2014-09-12 2015-01-07 齐鲁动物保健品有限公司 Combination of mink viral enteritis inactivated vaccine and canine distemper live vaccine

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