CN109504664A - III type duck hepatitis A virus chicken embryo low virulent strain of one kind and its application - Google Patents
III type duck hepatitis A virus chicken embryo low virulent strain of one kind and its application Download PDFInfo
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- CN109504664A CN109504664A CN201710825604.3A CN201710825604A CN109504664A CN 109504664 A CN109504664 A CN 109504664A CN 201710825604 A CN201710825604 A CN 201710825604A CN 109504664 A CN109504664 A CN 109504664A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The present invention provides a kind of III type duck hepatitis A virus chicken embryo low virulent strain and its application, deposit numbers are as follows: CCTCC NO:V201727；Classification naming are as follows: III type duck hepatitis A virus；The preservation time is: on June 15th, 2017；Depositary institution is: China typical culture collection center, and III type live vaccine of duck virus hepatitis prepared by the present invention can effectively prevent III type duck hepatitis A.
Description
Technical field
The present invention relates to microbial virus fields, and in particular to a kind of III type duck hepatitis A virus chicken embryo low virulent strain
And its application.
Background technique
Duck virus hepatitis is the master caused by duck hepatitis A virus (DuckhepatitisAvirus, DHAV) infection
Endanger a kind of serious disease of duckling.Clinically infected duck can be caused the symptoms such as liver enlargement, bleeding, necrosis occur,
Have the characteristics that morbidity is anxious, spread speed is fast, the course of disease is short and the death rate is high, the duckling within 5 week old of main infection.
Duck hepatitis A virus (DHAV) belongs to Picornaviridae (Picornaviridae), fowl hepatitis on taxology
Poison belongs to (Avihepatovirus).Virion is spherical in shape, be in icosahedral symmetry, diameter about 22~30nm, no cyst membrane,
It is mainly made of protein coat and nucleic acid, genome is the single-stranded positive RNA of non-segmented negative, and molecular size is about 7.7kb.To mesh
Before until, it has been reported that the DHAV of 3 different serotypes, respectively serum 1 type (DHAV-1), 2 type of serum (DHAV-2) and blood
Clear III type (DHAV-3), the serum cross reaction between the DHAV of different shaped is not fully understood at present, but DHAV-1 and DHAV-
Without cross-neutralization between 3.DHAV-2 is only reported in Taiwan, and the cross reaction between DHAV-1 and DHAV-3 is current
It has no and has been reported that.
Summary of the invention
The present invention provides a kind of III type duck hepatitis A virus chicken embryo to overcome the problems of the above-mentioned prior art
III type duck A type liver can effectively be prevented by changing low virulent strain and its application, III type live vaccine of duck virus hepatitis prepared by the present invention
It is scorching.
Above-mentioned purpose of the invention is achieved through the following technical solutions:
A kind of one of technical solution provided by the invention: III type duck hepatitis A virus (DHAV-3) chicken embryo low virulent strain
HB80, deposit number are as follows: CCTCC NO:V201727；Classification naming are as follows: III type duck hepatitis A virus；The preservation time is: 2017
On June 15, in；Depositary institution is: China typical culture collection center, VP1 gene nucleotide series such as Seq ID No.1
It is shown.
Above-mentioned, III type duck hepatitis A virus (DHAV-3) chicken embryo low virulent strain HB80 is caused by duck embryos separation, passage
Low virulent strain that is weak and carrying out the adaptation acquisition of SPF chicken embryo.
Above-mentioned, III type duck hepatitis A virus (DHAV-3) chicken embryo low virulent strain HB80 is trained using SPF chicken embryo
It supports, and is proliferated in SPF chicken embryo and stablizes, is pure.
Above-mentioned, III type duck hepatitis A virus (DHAV-3) chicken embryo low virulent strain HB80 is resistant to freeze thawing, and at -20 DEG C
15 months viral levels are saved not decline.
Above-mentioned, III type duck hepatitis A virus (DHAV-3) chicken embryo low virulent strain HB80, the continuous subculture 5 in duckling body
In generation, does not occur virulence and returns by force.
The two of technical solution provided by the invention: aforementioned deposit number are as follows: the III type duck A type liver of CCTCC NO:V201727
Scorching virus chicken embryo low virulent strain is preparing the application on III type live vaccine of duck virus hepatitis.
The three of technical solution provided by the invention: a kind of vaccine composition for preventing duck virus hepatitis, including protect
Hiding number are as follows: the III type duck hepatitis A virus chicken embryo low virulent strain and veterinary biologics of CCTCC NO:V201727 is acceptable
Freeze drying protectant, chilled vacuum drying are made.
The invention has the following advantages that
1, III type live vaccine of duck virus hepatitis prepared by the present invention, can effectively prevent III type duck hepatitis A.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, the present invention and its feature, outer
Shape and advantage will become more apparent upon.Identical label indicates identical part in all the attached drawings.Not deliberately proportionally
Draw attached drawing, it is preferred that emphasis is show the gist of the present invention.
Fig. 1 is DHAV-3HB80 plants of PCR testing results, and wherein M is DNAmaker；1 is negative control；2 draw for DHAV-3
Object expands HB80 plants of results.
The chadogram that Fig. 2 is DHAV-3HB80 plants and other several plants of DHAVVP1.
Sequence table explanation:
Seq ID No.1 is that the present invention is DHAV-3HB80 plants of VP1 gene nucleotide series of the present invention, length 720bp.
Specific embodiment
In the following description, a large amount of concrete details are given so as to provide a more thorough understanding of the present invention.So
And it is obvious to the skilled person that the present invention may not need one or more of these details and be able to
Implement.In other examples, in order to avoid confusion with the present invention, for some technical characteristics well known in the art not into
Row description.
In order to thoroughly understand the present invention, detailed step and detailed structure will be proposed in following description, so as to
Illustrate technical solution of the present invention.Presently preferred embodiments of the present invention is described in detail as follows, however other than these detailed descriptions, this
Invention can also have other embodiments.
For the popularity for understanding China's duck virus hepatitis, to the main doubtful duck in duck culturing area such as Hubei, Jiangsu, Shandong
The pathological material of disease of virus hepatitis carries out RT-RCR identification, to distinguish the genotype of epidemic strain.65 parts of clinical sample of acquisition, is used respectively
DHAV-1 and DHAV-3 specific primer carries out RT-PCR detection.As a result 10 parts of DHAV-1 positive sample are detected, accounts for 15.4%；
49 parts of DHAV-3 positive sample, account for 75.4%；6 parts of negative sample, account for 9.2%.Show that DHAV-3 is that China is mainly flowed in recent years
Capable genotype.
The cultivation big country traditional as one, China, with the development of science and technology, the progress of cultural technique, via traditional
Casual household's cultivation is developing progressively as intensive, scale big farm's aquaculture model, the generation of disease also by it is fragmentary, distribute change
To concentrate outburst, therefore, the most important thing that immune prevention and control are modern farmings is carried out.Prevention is had no currently on the market
The commercialization live vaccine of DHAV-3, the country is mainly prevented using yolk antibody, but yolk antibody action period is short, is cultivated in duck
Period domestic demand Reusability, brings many problems.So exploitation DHAV-3 live vaccine research, extremely urgent.
Referring to Fig.1 shown in-Fig. 2, the present invention provides a kind of III weak poison of type duck hepatitis A virus (DHAV-3) chicken embryoization
Strain HB80, deposit number are as follows: CCTCC NO:V201727；Classification naming are as follows: III type duck hepatitis A virus；The preservation time is:
On June 15th, 2017；Depositary institution is: China typical culture collection center.
The source that 1DHAV-3HB80 plants of embodiment
1.1 virus purifications 2008, in Hubei, a kind of doubtful duck of clinical manifestation occurs for 3~20 age in days Beijing ducks of duck
The disease of virus hepatitis, dead duck liver enlargement bleeding, there is dendroid blutpunkte or blood spots.It is sterile to take duck liver of dying of illness dirty,
Add physiological saline grinding that homogenate is made, is centrifuged 10 minutes after freeze thawing, takes supernatant, filtration sterilization.Filtrate is inoculated with through allantoic cavity
10 5 pieces of age in days duck embryos, 0.1ml/ pieces, 37 DEG C of incubations.The allantoic fluid of dead duck embryos is harvested, is inoculated with duck embryos passage 2 in the same way
It is secondary.3 generations duck embryos are all dead, and the death time is consistent, the visible idiosome oedema of dissect death duck embryos, bleeding, hepatorrhagia or have white
Color necrosis point.
The identification of 1.2 neutralization tests takes dead duck embryos allantoic fluid to dilute 100 times, respectively with equivalent DHAV-1 antiserum and wait
The mixing of DHAV-3 antiserum is measured, as in serum and group；Virus control group separately is set, mixes with equivalent sterile saline, sets
37 DEG C act on 1 hour, and every group is inoculated with 10 pieces of duck embryos, 0.2ml/ pieces through allantoic cavity；10 pieces of blank control group are set simultaneously.37 DEG C are set to incubate
It educates, chicken embryo survival condition is shown in Table 1.The result shows that isolated viral is DHAV-3 virus, it is named as DHAV-3HB plants.
1 serum neutralization test qualification result of table
| Group | Inoculum/dosage | Survival number/test number |
| In DHAV-1 serum and group | Duck embryos；0.2ml | 0/10 |
| In DHAV-3 serum and group | Duck embryos；0.2ml | 10/10 |
| Virus control group | Duck embryos；0.2ml | 0/10 |
| Blank control group | Duck embryos；It is not inoculated with | 10/10 |
1.3 virus Attenuations by DHAV-3HB plant dilute 10 times through allantoic cavity inoculation 5 pieces of duck embryos, 0.1ml/ pieces, 37
It DEG C is incubated for, harvests the allantoic fluid of dead duck embryos, passed on by same procedure, until 75 generations.Harvest the 25th, 45,55 and 75 simultaneously
As tissue poison 10 2 age in days ducks respectively are subcutaneously injected through neck, 0.5ml/ is only in generation duck embryos fetus.After inoculation 3 days to duckling into
Row dissect, as a result after the 55th generation tissue poison inoculation duckling, duckling is all strong to live, and dissect is visible by naked eyes variation.Test result is shown in
Table 2.Test result show DHAV-3HB plants after 55 generation of duck embryos continuous passage to duckling safety.
The different generation duck embryos tissue poison of table 2 are inoculated with duckling safety testing
| Generation | Liver enlargement bleeding | Splenomegaly | Nephrorrhagia |
| 25th generation | Have | Have | Have |
| 45th generation | Have | Have | Have |
| 55th generation | Nothing | Nothing | Nothing |
| 75th generation | Nothing | Nothing | Nothing |
1.4 chicken embryo compatibility tests take DHAV-3HB strain the 75th generation duck embryos tissue malicious, through allantocherion vaccination SPF chicken
10 pieces of embryo, 0.2ml/ pieces.The chorioallantoic membrane and fetus, high-speed homogenization for harvesting dead chicken embryo are denoted as the 76th generation chicken embryo tissue poison；
76th generation chicken embryo tissue poison was reached into for the 80th generation according to the above method, as DHAV-3HB80 plants.
2DHAV-3HB80 plants of VP1 sequence alignment analysis of embodiment
2.1RT-PCR takes HB80 plants of chicken embryo tissue poison, carries out viral RNA according to RNA extracts kit operational manual and mentions
It takes, and carries out reverse transcription by reverse transcription reagent box operational manual, then negate transcription product and carry out PCR.Required primer sequence
As follows, theoretical amplification length is 720bp.
DHAV-3F:5 '-GGTGATTCCAATCAGCTTGGCG-3 '
DHAV-3R:5 '-TTCAATTTCTAGATGGAGCTC-3 '
Pcr amplification product is identified with 1% agarose gel electrophoresis, the result is shown in Figure 1 occurs at about 720bp
The DNA band of specificity.
The amplified production that electrophoresis goes out specific band is carried out nucleotide sequencing by 2.2 sequencings.By the sequence of acquisition
Column are compared with the VP1 gene order of Gen-Bank other DHAV strains included, and analyze genetic evolution relationship.As a result see
NO.1DHAV-3HB80 plants of VP1 sequences of SEQ ID.HB80 plants of VP1 genes and DHAV-3 represent strain in same branch, heredity
Relationship is closer.
The stability that 3DHAV-3HB80 plants of embodiment
3.1 proliferation stability are in identified good DHAV-3HB80 strain 10 generation of continuous passage in SPF chicken embryo, to the 81st
Generation, 85 generations, 90 generation viral levels, harvest yield and pure property are detected and are counted, and the results are shown in Table 3.The results show that using DHAV-
3HB80 plants of 10 generations of continuous passage, the embryo death time than more consistent, viral level stablize, show DHAV-3HB80 plants in SPF chicken embryo
Upper proliferation is stablized.
3DHAV-3HB80 plants of passage results of table
3.2 freeze-thaw stabilities set DHAV-3HB80 plants -20 DEG C of multigelations 3 times, while control group is arranged, without
Freeze thawing, while viral level is measured, it the results are shown in Table 4.The results show that DHAV-3HB80 plants -20 DEG C multigelation 3 times, virus
Content is not remarkably decreased, and shows DHAV-3HB80 plants of tolerance freeze thawing.
Table 4DHAV-3HB80 strain 3 viral level measurement results of -20 DEG C of multigelations
3.3 storage stabilities are placed in -20 DEG C of preservations for DHAV-3HB80 plants, 0 after preservation, 3,6,12,15 months difference
Viral level measurement is carried out, the results are shown in Table 5.The results show that DHAV-3HB80 plants are set -20 DEG C and saved 15 months, viral level does not have
It decreased significantly.
5DHAV-3HB80 plants of storage stabilities of table
| Holding time (moon) | 0 | 3 | 6 | 12 | 15 |
| Viral level (DELD50/0.1ml) | 107.17 | 107.32 | 107.0 | 107.0 | 107.17 |
The safety that 4DHAV-3HB80 plants of embodiment
The safety testing of 4.1 pairs of ducklings takes DHAV-3HB80 plants of seeds culture of viruses, 10 times is diluted with sterile saline, through neck
1 age in days duckling 20 is subcutaneously injected in portion, and 0.5ml/ only, separately sets blank control group 20.It being observed continuously 14, duckling is strong to live,
Spirit and appetite are normal, the results are shown in Table 6.
6 vaccine inoculation clinical observation result of table
| Grouping | Seed culture of viruses extension rate | Strong number/test number living | Clinical symptoms |
| Immune group | 10 | 20/20 | Spirit, appetite are normal |
| Blank control group | / | 20/20 | Spirit, appetite are normal |
4.2 virulence return strong test and DHAV-3HB80 plants of seeds culture of viruses are inoculated with 1 age in days duckling, and 0.5ml/ only, takes 5 in 3 days after inoculation
Dissect acquires liver mixed grinding, returns 1 age in days duckling of poison disease vaccination 5 as 1st generation, so passed for 5 generations altogether.
In 5th generation, was returned poison disease vaccination duckling 10, until dissects on the 7th and carrying out histopathology.
Test result: by DHAV-3HB80 plants of 5 generations of continuous subculture on duckling, all generation ducklings are strong to live, no clinic
Symptom；Dissect and Histopathology detection are carried out to subculture duck, the internal organs such as liver,spleen,kidney, pancreas, brain are without pathological change and pathologic group
Knit damage.Show that DHAV-3HB80 plants virulence does not occur return by force.
5DHAV-3HB80 plants of immunogenicities of embodiment are examined
DHAV-3HB80 plants of seeds culture of viruses are taken, dilute 500 times with sterile saline, inoculate 1 age in days duck 15 through neck,
0.5ml/ is only.Separately 15 are taken to attack malicious control.It after 7 days, is attacked, is observed 7, duckling survival condition is shown in Table 7 with virulent.Exempt from
Epidemic disease group is survived 14, is attacked malicious control group and is survived 2, and vaccine is 93.3% to the protective rate of duckling.
7 vaccine immunogenicity measurement result of table
| Grouping | It tests number (only) | Seed culture of viruses extension rate | Survival number/test number | Protective rate |
| Immune group | 15 | 500 | 14/15 | 93.3% |
| Attack malicious control group | 15 | / | 2/15 | 13.3% |
Presently preferred embodiments of the present invention is described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, devices and structures not described in detail herein should be understood as gives reality with the common mode in this field
It applies；Anyone skilled in the art, without departing from the scope of the technical proposal of the invention, all using the disclosure above
Methods and technical content many possible changes and modifications are made to technical solution of the present invention, or be revised as equivalent variations etc.
Embodiment is imitated, this is not affected the essence of the present invention.Therefore, anything that does not depart from the technical scheme of the invention, foundation
Technical spirit of the invention any simple modifications, equivalents, and modifications made to the above embodiment, still fall within the present invention
In the range of technical solution protection.
Sequence table
<110>Biotechnology Co., Ltd. Shanghai Chuanghong
<120>a kind of III type duck hepatitis A virus chicken embryo low virulent strain and its application
<141> 2017-09-14
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 720
<212> DNA
<213>duck hepatitis A virus (3 type)
<400> 1
ggtgattcca atcagcttgg cgatgatgaa ccagtgtgtt ttctcaactt tgagactgca 60
aatgtgccaa tacaagggga gtcacacact ttggtgaaac atctttttgg tcgtcaatgg 120
ctggttcgta ctgttcaaca tactagtgag gtacaagagt tggatttgcc agtacctgac 180
cagggtcacg catctctgtt gcgcttcttt gcctacttct ctggagaagt gattctgacc 240
attgtcaata atggaacaac accatgcatg gttgcacact cttatacaat ggacaatctc 300
acttctgaat atgctgtcac tgccatgggg ggtattctta tcccagcaaa ctctgccaag 360
aatattaata ttccatttta ttctgttaca cctttacgcc ccacacgacc catgccagca 420
tttcaggggg gtggtttgac ttttggcagg ttgtatattt ggacacaatc aggaagcgtt 480
tctgttttta tgggcctcca caagccagct ttattttttc cactgcctgc accaacttac 540
acaacacaca cacagttgaa taacattgaa accatgaatc tgcataatca atcagatcag 600
ccagactgcc acctgtgtaa gatttgcaag aaaatgaaga aatggtctcg caatcatcgc 660
ccatttcgct tctgtttgag acttaaaaca cttgcctttg agctccatct agaaattgaa 720
Claims (4)
1. a kind of III type duck hepatitis A virus chicken embryo low virulent strain, which is characterized in that deposit number are as follows: CCTCC NO:
V201727。
2. a kind of III type duck hepatitis A virus chicken embryo low virulent strain, which is characterized in that its VP1 gene nucleotide series such as Seq
Shown in ID No.1.
3. deposit number are as follows: the III type duck hepatitis A virus chicken embryo low virulent strain of CCTCC NO:V201727 is preparing duck viral
Application on III type live vaccine of hepatitis.
4. a kind of vaccine composition for preventing duck virus hepatitis, which is characterized in that including deposit number are as follows: CCTCC NO:
The III type duck hepatitis A virus chicken embryo low virulent strain and the acceptable freeze drying protectant of veterinary biologics of V201727, through cold
Freeze vacuum drying to be made.
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Cited By (1)
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| CN109797139A (en) * | 2019-01-15 | 2019-05-24 | 四川农业大学 | 3 type duck hepatitis A virus low virulent strain CH-P60 of one kind and its application |
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| CN109797139A (en) * | 2019-01-15 | 2019-05-24 | 四川农业大学 | 3 type duck hepatitis A virus low virulent strain CH-P60 of one kind and its application |
| CN109797139B (en) * | 2019-01-15 | 2021-06-08 | 四川农业大学 | 3-type duck hepatitis A virus attenuated strain CH-P60 and application thereof |
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| CN109504664B (en) | 2022-04-08 |
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