CN105296439B - One breeder c-type fowl metapneumovirus strain aMPV/C-JCX and its application - Google Patents
One breeder c-type fowl metapneumovirus strain aMPV/C-JCX and its application Download PDFInfo
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Abstract
The present invention provides a breeder c-type fowl metapneumovirus strain aMPV/C-JCX and its application, and the deposit number of the virus stain is CGMCC No.10900.The virus is to collect the domestic chicken c-type fowl metapneumovirus infection diseased chicken tissue grinder liquid being separated to for the first time, it is inoculated with the single layer African green monkey kidney cell Vero of adherent growth, the inoculum that cell culture is passed on as next round, successively continuous passage obtains one plant of cell culture and adapts to strain.In adaptation process, early generation cultivation cycle is 7-9 days, is foreshortened to 3-5 days after reaching for 60 generations it can be observed that apparent cytopathy.Confirm that the cell can effectively cultivate c-type fowl metapneumovirus by transmission electron microscope, indirect immunofluorescence and immunoblotting.The titre lgTCID50=10 of virus‑4.2/0.1ml.It confirms that the virus virulence is weaker by SPF chicken, there is good immunogenicity and protectiveness, which is the ideal candidates strain for researching and producing viral vaccine.
Description
Technical field
The invention belongs to microorganism animal vaccine fields, and in particular to one plant of chicken c-type fowl metapneumovirus and its related application.
Background technique
Fowl metapneumovirus (aMPV) is a kind of cause of disease of newfound important fowl upper disease, in turkey and chicken etc.
Caused acute, the highly infectious infection of the upper respiratory tract on birds, causes breeder and chicken egg productivity of laying eggs sharply declines, and shadow
Ring the quality of eggshell.The virus belongs to paramyxovirus section metapneumovirus category, the inclined lung of the fowl observed under negative-contrast electron microscopy
Virus is polygon, ellipse, and typical diameters 80-200nm has the form presence of multiplicity of filaments shape once in a while, and length is reachable
1000nm.Fowl metapneumovirus has tetra- hypotypes of A, B, C, D.We in China southeast have found the inclined lung of chicken c-type fowl for the first time within 2012
Virus infection.Show that the white feather chicken of China's either local varieties or overseas introduction, aMPV infect all in epidemiological survey
It is generally existing.
AMPV has high infectiousness, and spread speed is fast, can also immunosupress be caused to react, the normal secondary sexuality of microbe satellite
Dye can make the infected poultry death rate increase 25%-40%, cause serious economic loss to aquaculture.China is not yet at present
For the vaccine of fowl metapneumovirus infection.During we investigate, Shuo Jia enterprise is made due to the infection of c-type fowl metapneumovirus
Decline to a great extent at Breeder hens and above the average age for marriage performance in layers, it has to it is carried out it is superseded, lose it is huge.Since chicken c-type fowl is inclined
Pneumovirinae is that China reports for the first time, we are mainly by being separately cultured and identifying to SPF chicken or chicken embryo at present, but this
The demand for carrying out the correlative studys such as viral pathogenesis mechanism and vaccine can not be fully met.The present invention utilizes African green monkey kidney cell
The Cell culture invitro characteristic discovery of Vero further investigation virus, initial in vitro culture does not generate obvious lesion, by continuous
Passage and domestication, obtain one plant of cell adapted virus.It confirms to separate through indirect immunofluorescence, transmission electron microscope and immunoblotting
Strain is chicken c-type fowl metapneumovirus aMPV/C-JCX, carries out aMPV vaccine research in a deep going way for next step and provides material.
Summary of the invention
The purpose of the present invention is to provide the chicken C hypotype fowl metapneumovirus of one plant of cell adaptation, and the purpose of the present invention is also
It is to provide the application of the fowl metapneumovirus.
To achieve the above object, the present invention is continuous on Vero cell by the chicken c-type fowl metapneumovirus for separating field
Passage, obtains the cell adapted strain aMPV/C-JCX of one plant of virulence attenuation of, the virus is on June 24th, 2015 in China
Microbiological Culture Collection administration committee common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Chinese section
Institute of microbiology of institute, postcode 100101) preservation, classification naming is fowl metapneumovirus (Avain metapneumovirus),
Deposit number is CGMCC No.10900.
Virus of the present invention is to collect the domestic chicken c-type fowl metapneumovirus infection diseased chicken tissue grinder liquid being separated to for the first time, is connect
The single layer African green monkey kidney cell Vero of kind adherent growth, the inoculum that cell culture is passed on as next round, successively continuously
Passage obtains one plant of cell culture and adapts to strain.In adaptation process, early generation cultivation cycle is 7-9 days, reaches 60 generation retractions
It is as short as 3-5 days it can be observed that apparent cytopathy.It is confirmed by transmission electron microscope, indirect immunofluorescence and immunoblotting
The cell can effectively cultivate c-type fowl metapneumovirus.Observe that virion is rounded under viral transmission electron microscope, directly
Diameter about 170nm.The titre lgTCID50=10 of virus-4.2/0.1ml.It confirms that the virus virulence is weaker by SPF chicken, has good
Good immunogenicity and protectiveness, the cell adapted virus stain are production viral vaccine ideal candidates strains.
In turn, the present invention also provides the chicken c-type fowl metapneumovirus strain aMPV/C-JCX prevents and treats the inclined lung of fowl in preparation
Application in the vaccine of disease caused by virus infection.Disease caused by the fowl metapneumovirus infects can be fowl metapneumovirus
Infection causes upper respiratory disease or-egg drop reduction etc..The chicken c-type fowl metapneumovirus strain aMPV/C-JCX system can be used
Standby production of vaccine seed culture of viruses.
The beneficial effects of the invention are as follows one plant of cell adapted strain aMPV/C-JCX is provided, which can be used in preparation
Production of vaccine seed culture of viruses, prepares vaccine, for preventing and treating disease caused by fowl metapneumovirus infects.
Detailed description of the invention
Fig. 1 is the testing result figure of immunofluorescence, it has been observed that the Vero of infection aMPV/C-JCX under fluorescence microscope
Visible apparent interior fluorescent staining in cell (figure A), and normal non-inoculating cell (figure B) unstressed configuration occurs.
Fig. 2 is Western Blot test experience result figure, uses the virus protein of specificity as primary antibody, Western
Blot detects the expression discovery of different vaccination time virus protein, and connect poison cell has apparent protein band at 48KD.
Fig. 3 morphology of virus figure, the virion observed under transmission electron microscope is rounded, diameter about 170nm.
Chick embryo development is not caused to be obstructed after Fig. 4 aMPV-JCX inoculation, figure A indicates that inoculation aMPV-JCX, B indicate inoculation
PBS (control group).
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
The acquisition and identification of 1 aMPV/C-JCX strain of embodiment
1. set out viral aMPV/C-JCZ
Virus of setting out is aMPV/C-JCZ, and the virus is on June 24th, 2015 in Chinese microorganism strain preservation management
Committee's common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postal
100101) preservation is compiled, classification naming is fowl metapneumovirus (Avain metapneumovirus), deposit number CGMCC
No.10889。
The EID50, lgEID50=10 of virus are calculated according to Reed-Muench method-4.6/0.2ml.By returning chicken test card
Real individually infected by aMPV/C-JCZ can cause the serious respiratory symptom of commercial generation yellow-feather broiler；The pathology of lung and tracheae
Check discovery, virus infection causes serious inflammatory and venereal injury.
Virus total RNA is extracted using Qiagen RNeasy Mini Kit, uses III Kit and M base of SuperScriptTM
Because of specific primer, the fowl metapneumovirus cDNA of synthesis is that template carries out RT-PCR amplification, and amplified production is with Ago-Gel electricity
Swimming detection, is connected in carrier T after recycling, is sequenced after cloning successfully by Shanghai Sheng Gong biotechnology Co., Ltd.M
A, B, C the hypotype fowl metapneumovirus registered on gene (SEQ ID No.1) and GenBank, according to MEGA 5.10 and DNAstar
Software progress sequence homology compares and phylogenetic tree construction, it is found that aMPV-JCZ is with c-type fowl metapneumovirus homology
96.0%-96.7%, and there was only the homology of 70.6%-71.7% with A and Type B.
2, viral sample is obtained
Diseased chicken turbinate, tracheae, larynx and the lung tissue sample for collecting respiratory symptom, are resuspended in PBS after grinding,
3500rpm/min is centrifuged 15 minutes, and supernatant is using being stored in -80 DEG C after 0.22 μm of filter degerming.
3, viral blind passage and adaptation on Vero cell
Vero cell is at 37 DEG C, 5%CO2In incubator, it is trained using the DMEM complete culture solution containing 5% calf serum
After single layer, after the digestion of 0.1% pancreatin, according to 2*105Cell/ml is inoculated with 25cm2Small square vase will after 37 DEG C of overnight incubations
Viral sample dilution 1ml is inoculated on cell monolayer, is adsorbed 1-2 hours, and the maintenance containing 2% calf serum is then added
Liquid, 37 DEG C of cultures and every generation observe cytopathy, and cell culture was collected cell culture, and frozen repeatedly in -40 DEG C to 7 days
Melt three times afterwards as inoculum, continue to be inoculated with Vero cell with method or freezes spare.
In adaptation process, in preceding 4 generation, does not see cytopathy, and the 5th generation 3 days after connecing poison started cytopathy occur, connects poison
It harvests within 7 days afterwards, by repeatedly passing on, cytopathy is stablized, and is detected and is found with Westen-bloting, virus protein is after connecing poison
It peaks within 5th day, then begins to reduce, this is followed by 5 days harvest virus after poison, it is more than generation to continue continuous passage 60, and carry out
Identification and immunoprotection related experiment.
4, the identification of aMPV/C-JCX strain:
(1) culture solution is discarded by after virus inoculation cell 48h according to the method described above, is carried out using 4% paraformaldehyde
After 15min is fixed, paraformaldehyde is removed, is washed 3 times using PBST, each 5min.5% skimmed milk power is prepared, after closing 2h, is made
Use aMPV N protein immune rabbit prepare the rabbit anteserum for aMPV N protein as primary antibody N protein specific antibody at room temperature
After being incubated for 2h, primary antibody is removed, PBST is added and washes 3 times, each 10min.Diluted FITC goat-anti rabbit secondary antibody is added, is protected from light incubation 45
It after minute, washes 3 times, microscopy is carried out under fluorescence microscope can see, the clearly visible green fluorescence of virus inoculation cell, control group
Then unstressed configuration (Fig. 1) in cell.
(2) culture solution is discarded after virus inoculation cell 12,24,36,48,72,96,120h according to the method described above, cracks egg
It is white, SDS-PAGE electrophoresis is carried out, the gel after SDS-PAGE electrophoresis is subjected to transferring film.After by NC film in TBST washing lotion
In wash 3 times, close 2h in 5% defatted milk at room temperature.After diluted virus protein specific antibody is incubated for 2h at room temperature, TBST is washed
3 times, each 10min.After diluted secondary antibody is incubated for 50min at room temperature, TBST is washed 3 times, and result is aobvious after each 10min, ECL colour developing
Show: virus protein is continuously increased (Fig. 2) with inoculation time.
(3) by the 60th generation cytopathy venom multigelation 3 times, 3500rpm is centrifuged 15min, discards supernatants after precipitation warp
It crosses 40000rpm ultracentrifugation 3h, abandons supernatant, be resuspended to precipitate and collect with PBS and carry out transmission electron microscope observing, the results show that thoroughly
It is rounded to virion to penetrate observed under electron microscope, diameter about 170nm (Fig. 3).
Virocyte adaptation strain is named as aMPV/C-JCX, the virus is micro- in China on June 23rd, 2015
Biological inoculum preservation administration committee common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Chinese science
Institute of microbiology of institute, postcode 100101) preservation, classification naming is fowl metapneumovirus (Avain metapneumovirus), is protected
Hiding number is CGMCC No.10900.
The virus titer TCID50 of 2 aMPV/C-JCX strain of embodiment is measured
It saves virus and carries out continuous 10 times of dilutions, be inoculated with 96 orifice plate Vero cells, each viral dilution gradient at least eight is flat
Row, setting feminine gender and positive control, 5-7 days observation cytopathies.The TCID50 of virus is calculated according to Reed-Muench method,
LgTCID50=10-4.2/0.1ml。
3 aMPV/C-JCX strain of embodiment is to SPF chicken embryo toxicity test
The cell virus of preservation directly can be inoculated with SPF chicken embryo by allantoic cavity and detect virus virulence.The virus of preservation is connect
5 pieces of SPF chicken embryos of kind, 200 microlitres of every embryonic breeding kind.It is incubated in 38 DEG C, the incubator that humidity is 40%, sees whether occur on time
Chicken embryo death situation.Chicken embryo is placed in 4 DEG C of refrigerators coolings after a week by inoculation, and tweezers break up chorion after disinfection, tear after removing shell membrane
Broken chorioallantoic membrane, while chicken embryo is taken out, observe chick embryo development.As a result, it has been found that aMPV-JCX does not cause chick embryo development to be obstructed
Or it is dead, illustrate that aMPV-JCX passes through and stablize passage on Vero cell, the strain virulence is weaker, may be used as production chicken C
The candidate strain of type fowl metapneumovirus attenuated live vaccines.
Measurement of the 4 aMPV-JCX F60 of embodiment for immune protective effect
21 age in days SPF chickens are randomly divided into 5 groups, every group 10.It is set to: healthy control group, aMPV-JCX2000
TCID50 immune group, 5000TCID50 immune group, 10000TCID50 immune group, and non-Immunization group.After 3 weeks immune, remove
Outside healthy control group, remaining four groups pass through collunarium eye droppings approach inoculation 500EID50 aMPV-JCZ and are attacked, and observe day by day
Clinical symptoms.As a result, it has been found that attacking all morbidities of poison group, there is apparent respiratory symptom.2000 TCID50 and 5000
TCID50 group can play part protection, and protective rate is 50% and 70% respectively.10000TCID50 immune group is protected completely (protects
Shield rate 100%).As a result illustrate, respiratory tract disease caused by capable of being infected chicken c-type fowl metapneumovirus using aMPV-JCX cell toxicant
Shape plays good protecting effect, may be used as preparation chicken c-type fowl metapneumovirus infection attenuated live vaccines.
Malicious SPF chicken clinical onset situation is attacked after table 1.aMPV-JCX cell toxicant is immune to summarize
Claims (4)
1. chicken c-type fowl metapneumovirus strain aMPV/C-JCX, deposit number is CGMCC No.10900.
2. chicken c-type fowl metapneumovirus strain aMPV/C-JCX described in claim 1 draws in preparation prevention and treatment fowl metapneumovirus infection
Application in the vaccine of the disease risen.
3. application according to claim 2, which is characterized in that disease caused by the fowl metapneumovirus infects is the inclined lung of fowl
Virus infection causes upper respiratory disease or egg drop reduction.
4. application of the chicken c-type fowl metapneumovirus strain aMPV/C-JCX described in claim 1 in preparation production of vaccine seed culture of viruses.
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| CN107904326B (en) * | 2017-12-12 | 2021-06-22 | 北京市农林科学院 | Nucleic acid for RT-PCR rapid typing detection of avian metapneumovirus subtype and application |
| CN109402065B (en) * | 2018-08-08 | 2021-07-30 | 温氏食品集团股份有限公司 | Muscovy duck subtype C avian metapneumovirus attenuated strain and preparation and application thereof |
| CN109628413A (en) * | 2018-12-29 | 2019-04-16 | 广东和信健康科技有限公司 | The cultural method and its product of a kind of human metapneumovirus and application |
| CN112881682A (en) * | 2021-01-11 | 2021-06-01 | 广西大学 | Indirect ELISA (enzyme-linked immunosorbent assay) detection kit for avian metapneumovirus antibody |
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| WO2012140032A1 (en) * | 2011-04-12 | 2012-10-18 | Intervet International B.V. | Avian metapneumovirus in oncolysis |
| CN103360472A (en) * | 2013-07-30 | 2013-10-23 | 北京市农林科学院 | Acian Metapneumovirus antibody ELISA detection kit |
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