CN102268410A - Newcastle disease virus heat-resistant attenuated strain adapted to baby hamster kidney passage cell and preparation method and application thereof - Google Patents
Newcastle disease virus heat-resistant attenuated strain adapted to baby hamster kidney passage cell and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a newcastle disease virus heat-resistant attenuated strain adapted to baby hamster kidney passage cells and a preparation method and an application thereof, and the microbial preservation number is CCTCC V201113; the classification term is newcastle disease virus TS09-C strain; the preservation time is May 9th, 2011; the preservation unit is China center for type culture collection; the preservation address is Wuhan university, Wuhan, China; the strain has the following advantages: 1. the strain is fully adapted to BHK21 cells, and has a proliferation titer of up to 108.0TCID50/ml; 2. the strain has good heat resistance, and the HA titer does not decrease obviously after the strain is treated at 56 degree for 1 hour; 3. the strain has an attenuated characteristic, has chicken embryo average lethal time of more than 90 hours, and an intracerebral pathogenicity index of 0.00; 4. a newcastle disease heat-resistant live vaccine (passage cell source) prepared by the attenuated strain has the advantages of heat resistance, long storage life, good protective efficacy, prevention of chicken embryos for laying from carrying heterogenous virus, and the like.
Description
Technical field
The invention belongs to the microbial virus field, relate to the new strain of a kind of virus, be specifically related to a strain and adapt to heat-resisting low virulent strain of Avian pneumo-encephalitis virus of young hamster kidney passage cell and its production and application, its microbial preservation number is: CCTCC V201113; Classification name: Avian pneumo-encephalitis virus TS09-C strain; The preservation time is: on May 9th, 2011; Depositary institution: Chinese typical culture collection center; Preservation address: China. Wuhan. Wuhan University.
Background technology
(Newcastle disease virus NDV) is Paramyxoviridae Rubulavirus member to Avian pneumo-encephalitis virus, is the minus-stranded rna virus that cyst membrane is arranged.(Newcastle disease is a kind of very easily infectious crushing disease ND) to the newcastle disease that is caused by NDV, and mortality ratio can reach more than 90%, is regarded as one of two kinds of category-A poultry dieases (another kind of is bird flu) by International Office of Epizootics.The prevention and control of ND are puted prevention first with immunity.The current ND vaccine of producing in the world and using has two big classes, i.e. living vaccine and inactivated vaccine, living vaccine comprise low virulence and mesogenic living vaccine again.Mesogenic living vaccine immune effect is good, the antibody high conformity, but have stronger toxic side effects, the poultry of this type of vaccine immunity not to meet export requirement to chick, dove and quail etc.; Safe in utilization, the good immune effect of inactivated vaccine, but need intramuscular injection, time-consuming, immune cost height, and influence Quality of poultry meatan issue of growing importance, make its application be subjected to certain restriction; Low virulence living vaccine is most widely used in China with advantages such as it are easy to use (can by multiple mode such as spice, drinking-water, spraying, collunarium, eye droppings or injection to seedling) and toxic side effect is less.But low virulence live-vaccine heat-proof poor-performing and refrigerated condition require high, often influence immune effect of vaccine because of preservation or improper use, are unfavorable for carrying out large scale application in general higher southern area and the relatively poor rural area of refrigerated condition of China's temperature.Therefore, the low heat-resisting living vaccine of virulence of ND has comparatively wide application prospect in China.
Current, the ND production of vaccine that comprises heat-resisting living vaccine mainly is to use the chicken embryo, and with common chicken embryo produce vaccine just may make vaccine pollute some in process of production can be by the eqpidemic disease cause of disease of chicken embryo vertical transmission, cause these cause of diseases along with the use of vaccine wide-scale distribution.Using the allos passage cell to prepare the ND vaccine can avoid the chicken embryo to propagate the risk of other eqpidemic diseases.About the proliferation research of NDV strain in various cells report is arranged all both at home and abroad.Studies show that: NDV can duplicate and produce infective virus particle in chick embryo allantoic liquid, and the NDV virulent strain can duplicate in multiple cultured cell in vitro, but low virulent strain can only duplicate in containing tryptic cell.But do not see up to now, the research report of the heat-resisting low virulent strain of NDV that adapts to passage cell yet.
Existing technical literature about newcastle disease virus strain, mainly contain following several: as number of patent application is that the document " newcastle disease virus D 90 strain and application thereof " of CN200710003046.9 discloses newcastle disease virus D 90 strain, also disclose the application of this strain in antitumor, but the D90 strain do not possess the passage cell of adaptation and heat-stable characteristic; Number of patent application is that the document recombinant new castle disease LaSota attenuated vaccine strain of infectious bursal disease virus VP 2 gene " express " of CN200610001693.1 and document that number of patent application is CN200610075781.6 " are expressed the recombinant new castle disease LaSota attenuated vaccine strain of avian influenza virus H5 subtype HA protein " and disclosed the two kinds of Avian pneumo-encephalitis virus Lasota strains that can express the exogenous virus gene, and these two kinds of strains do not possess adaptation passage cell and heat-stable characteristic yet equally.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, and a kind of heat-resisting low virulent strain of Avian pneumo-encephalitis virus new, that adapt to passage cell is provided.
A further object of the invention provides described low virulent strain and is applied to prepare the heat-resisting living vaccine (subculture cell source) that prevents newcastle disease, and that described heat-resisting living vaccine has is heat-resisting, long preservative period, protection is good and can avoid producing and carry advantage such as allos virus with the chicken embryo.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a strain adapts to the heat-resisting low virulent strain of Avian pneumo-encephalitis virus (NDV) of young hamster kidney passage cell (BHK21), and its microbial preservation number is: CCTCC V201113; Classification name: Avian pneumo-encephalitis virus TS09-C strain; The preservation time is: on May 9th, 2011; Depositary institution: Chinese typical culture collection center; Preservation address: China. Wuhan. Wuhan University.
The preparation method of the heat-resisting low virulent strain of Avian pneumo-encephalitis virus of described adaptation young hamster kidney passage cell, be under the culture condition that contains 0.2 μ g/mlTPCK-pancreatin, Avian pneumo-encephalitis virus TS09 chicken embryo adapted strain is carried out 17 continuous passages in the BHK21 cell cultivate, wherein having 3 times is Method of Limited Dilution method purifying, reach the heat-resisting low virulent strain of Avian pneumo-encephalitis virus that the 17th generation promptly obtained to adapt to young hamster kidney passage cell, i.e. Avian pneumo-encephalitis virus TS09-C strain.
Press such scheme, the propagation of the heat-resisting low virulent strain of described Avian pneumo-encephalitis virus in the BHK21 cell is tired and is reached 10
8.0TCID
50/ ml.
Press such scheme, hemagglutinin-neuraminidase (HN) gene order of the heat-resisting low virulent strain of described Avian pneumo-encephalitis virus is SEQID NO:1.
Press such scheme, the nucleotide homology of the HN gene order of heat-resisting low virulent strain of described Avian pneumo-encephalitis virus and prototype-strain TS09 strain is 88.2%.
The present invention adapts to the cell proliferation titration of the heat-resisting low virulent strain of Avian pneumo-encephalitis virus of young hamster kidney passage cell: after the virocyte culture is centrifugal, gets supernatant liquor and carry out half cell infection dosage (TCID
50) mensuration, supernatant liquor is carried out 10 times of gradient dilutions, get 10
-5, 10
-6, 10
-7With 10
-8The inoculation of extent of dilution sample grows up to the BHK21 cell of fine and close individual layer, and inoculum size is the 0.1ml/ hole, and each extent of dilution is inoculated 5 porocytes.The observation of cell pathology then is judged to be the positive as cytopathy occurring in 120 hours, as cytopathy do not occur and then be judged to be feminine gender, calculates TCID with the Reed-Muench method
50It is 10 that the cell proliferation of this strain is as a result tired
8.0TCID
50/ ml.
The present invention adapts to the cytopathic effect of the heat-resisting low virulent strain of Avian pneumo-encephalitis virus of young hamster kidney passage cell and observes: viral liquid is with 10
-3After the dilution, inoculation grows up to the BHK21 cell of fine and close individual layer, observation of cell pathology situation.Cell is at virus inoculation about 48 hours as a result, begins to occur differing in size, oval-shaped cavity, cytogamy, in 72～96 hours, a large amount of cytoclasises, death occur, is suspended in the nutritive medium.
Hemagglutinin-neuraminidase (HN) gene that the present invention is adapted to the heat-resisting low virulent strain of Avian pneumo-encephalitis virus of young hamster kidney passage cell carries out RT-PCR amplification and sequencing, the HN gene order of finding this attenuated vaccine strain is SEQ ID NO:1, and the sequence total length is 1851bp.
The HN gene order that the present invention is adapted to the heat-resisting low virulent strain of Avian pneumo-encephalitis virus of young hamster kidney passage cell is analyzed: carry out the comparison of HN gene order by MegAlign program (DNAstar software package), confirm that the HN gene nucleotide homology of this heat-resisting low virulent strain and its prototype-strain TS09 strain is 88.2%.
The heat-resisting low virulent strain of Avian pneumo-encephalitis virus that the present invention adapts to young hamster kidney passage cell can be used for preparing the heat-resisting living vaccine of newcastle disease (subculture cell source), is used for the prevention of newcastle disease.
The heat-resisting low virulent strain of Avian pneumo-encephalitis virus that the present invention adapts to young hamster kidney passage cell is prepared into the method for vaccine: select the BHK21 cell that growth conditions is good, grow up to fine and close individual layer for use, behind the tipping nutrient solution, change serum-free DMEM nutritive medium (containing 0.2 μ g/ml TPCK-pancreatin) to contain 0.1% viral seed liquor, place 37 degree to cultivate, every day the observation of cell state.The cell of discovery growth failure, pollution should be discarded.Cultivate after 2～3 days, have the cell more than 80% pathology to occur approximately, and control cells is in good condition, can gather in the crops, behind three multigelations, put-20 degree and preserve.Take a morsel and carry out steriling test and virus titer check.The viral liquid that is up to the standards and protective material by 1: 1 mixed, are fully shaken up the back packing, carry out vacuum freezedrying rapidly.Carry out then physical behavior, pure, tire, checks such as safety and effectiveness.The product that is up to the standards is the heat-resisting living vaccine of newcastle disease (subculture cell source).
The present invention adapts to the heat-resisting low virulent strain of NDV of BHK21 cell and compares with existing NDV low virulent strain, has the following advantages:
1, fully adapted to the BHK21 cell, its propagation titre can reach 10
8.0TCID
50/ ml;
2, have the better heat-resisting characteristic, 1 hour HA of 56 degree processing tires not have obviously and descends;
3, have weak malicious characteristic, the average lethal time of chicken embryo was greater than 90 hours, and intracerbral pathogenicity index is 0.00;
4, with the heat-resisting living vaccine of newcastle disease (subculture cell source) of this strain preparation have heat-resisting, long preservative period, protection good, can avoid producing and carry advantage such as allos virus with the chicken embryo, have broad application prospects.
Description of drawings
Fig. 1 is that the present invention adapts to the cytopathy figure after the heat-resisting low virulent strain of the Avian pneumo-encephalitis virus of young hamster kidney passage cell infects the BHK21 cell, (A) infects 48 hours BHK21 cell; (B) 96 hours BHK21 cell of infection; (C) normal control cell;
Fig. 2 is that the present invention adapts to the heat-resisting low virulent strain of Avian pneumo-encephalitis virus of young hamster kidney passage cell and the HN gene order comparative result of prototype-strain TS09 strain;
Fig. 3 is that the present invention adapts to the heat-resisting low virulent strain of Avian pneumo-encephalitis virus of young hamster kidney passage cell and the HN gene nucleotide homology analysis result of prototype-strain TS09 strain.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be appreciated that these examples only to be used to the present invention is described and be not used in restriction the scope of protection of present invention.
The present invention adapts to the preparation of the heat-resisting low virulent strain of Avian pneumo-encephalitis virus of young hamster kidney passage cell
With the DMEM nutritive medium (serum-free) that contains 0.2 μ g/ml TPCK-pancreatin is inoculation liquid and cell maintenance medium, NDVTS09 chicken embryo adapted strain is carried out 17 continuous passages in the BHK21 cell, except that the 11st, 13 and 15 on behalf of (extent of dilution is by the high dilution decision that CPE occurs) the Method of Limited Dilution method purified virus, all the other are 10
-2The dilution inoculation.The virus that goes down to posterity is carried out biological characteristics mensuration, obtained table 1.
The go down to posterity cultivation results of table 1.NDV TS09 chicken embryo adapted strain on bhk cell
Annotate :-expression cell does not have pathology; +～++ ++ expression cytopathy variability (being respectively 25%～100%);
aRepresent that this time propagating method is for carrying out the purifying of virus with the Method of Limited Dilution method;
bThe Method of Limited Dilution multiple of expression Method of Limited Dilution method.
Can draw by table 1, the 2nd, 3 generation virus do not detect the HA activity, do not have typical CPE yet and occur, in the 4th, 5 generations, engendered typical CPE, and HA tires and also rises to 2
3, show that this stage is the adaptive phase of TS09 strain in the BHK21 cell; In the 6th～10 generation process, the HA of virus tires and is stabilized in 2
5About, and CPE is comparatively obvious, shows that this stage is the stationary phase of TS09 strain in the BHK21 cell; In the 11st～17 generation, through behind 3 Method of Limited Dilution method purified viruses, the HA of virus tires and is stabilized in 2
6About, and virus titer is by 10 of the 10th generation
6.8TCID
50/ ml rises to 10
8.0TCID
50/ ml.Therefore, through 17 continuous passages (comprising Method of Limited Dilution method purifying 3 times) in the BHK21 cell, the TS09 strain has adapted to the BHK21 cell fully, thus obtained a strain new, the purified new-castle disease virus cell adapted strain, called after TS09-C strain.Its microbial preservation number is: CCTCC V201113; Classification name: Avian pneumo-encephalitis virus TS09-C strain; The preservation time is: on May 9th, 2011; Depositary institution: Chinese typical culture collection center; Preservation address: China. Wuhan. Wuhan University.
Compare with prototype-strain TS09 strain, the cell proliferation titre of TS09-C strain is by 10
3.3TCID
50/ ml rises to 10
8.0TCID
50/ ml, its chicken embryo propagation titre is by 10
8.6EID
50/ ml drops to 10
5.7EID
50/ ml shows that the TS09-C strain changes the passage cell adapted strain into by chicken embryo adapted strain.In addition, heat-resistant quality, MDT and ICPI measurement result show that the TS09-C strain has kept the characteristic of the heat-resisting and weak poison of its maternal strain.
The present invention adapts to the cytopathic effect of the heat-resisting low virulent strain of NDV of BHK21 cell
With TS09-C strain virus liquid of the present invention with 10
-3After the dilution, inoculation grows up to the BHK21 cell of fine and close individual layer, and observation of cell pathology situation obtains Fig. 1.
As seen from Figure 1, cell is at virus inoculation about 48 hours, begins to occur differing in size, oval-shaped cavity, cytogamy; In 72～96 hours, a large amount of cytoclasises, death occur, be suspended in the nutritive medium; Normal control cell then growth conditions is good.The result shows: the TS09-C strain has cytopathic effect to the BHK21 cell.
The present invention adapts to the HN gene sequencing and the analysis of the heat-resisting low virulent strain of NDV of BHK21 cell
The Avian pneumo-encephalitis virus HB92 strain of having delivered according to GenBank (the GenBnak accession number: HN gene order AY225110), design a pair of primer, the HN gene of amplification total length, it is synthetic to give birth to worker's biotechnology company limited by Shanghai.Concrete sequence is as follows: NDV-F2:5 '-CGGTTGTAGATGACCAAAGG-3 '; NDV-R2:5 '-AATCTAATCACATTAGCACTAGCTG-3 ';
The extraction reference reagent box specification sheets of virus genome RNA carries out, and the RNA of extraction is dissolved in 17ul DEPC water, adds 1 μ l reverse transcriptase primer, 75 ℃ of 5min, ice bath adds reverse transcription reaction liquid: 5 * RT Buffer, 5 μ l, dNTPs (10mmol/L) 1 μ l and M-MLV 1 μ l then immediately.42 ℃ of 30min, 95 ℃ of 5min preserve pending PCR reaction for-20 ℃.
PCR reaction system: 10 * Buffer 5 μ l, MgCl
2(25mmol/L) 3 μ l, dNTPs (10mmol/L) 1.5 μ l, each 2 μ l of upstream and downstream primer (10 μ mol/L), Taq enzyme 5U, RT product 5 μ l, remaining water is mended to 50 μ l.Reaction conditions: 95 ℃ of 5min; 30 * (94 ℃ of 30sec, 55 ℃ of 1min, 72 ℃ of 1min); 72 ℃ of 10min.1% agarose gel electrophoresis testing goal band.Special positive band is carried out purifying with the DNA purification kit reclaim, deliver to Shanghai living worker's biotechnology company limited and carry out sequencing.
Sequencing result is analyzed: carry out the Blast comparison with other NDV strains, remove the sequence that does not belong to the HN gene, the HN gene order of the TS09-C strain that finally draws is SEQ ID NO:1, and the sequence total length is 1851bp.Adopt MegAlign program (DNAstar software package), the HN gene of TS09-C strain and prototype-strain TS09 strain is carried out sequence alignment and homology analysis, obtain Fig. 2 and Fig. 3 respectively.
Can be drawn by Fig. 2, the TS09-C strain is compared with the TS09 strain, and the maximum characteristic variation of TS09-C strain HN gene is from the 1735th to 1851, has inserted 117 Nucleotide altogether.
Can be drawn by Fig. 3, the HN gene nucleotide homology of TS09-C strain and TS09 strain is 88.2%; The highest with the homology of V4 strain, be 99.6%; Minimum with the homology of HB92 strain, be 83.7%.
The heat-resisting low virulent strain of Avian pneumo-encephalitis virus that utilizes the present invention to adapt to young hamster kidney passage cell prepares the heat-resisting living vaccine of ND (subculture cell source)
Below sketch the preparation process of the heat-resisting living vaccine of ND (subculture cell source):
1.BHK21 cell is preserved by our unit, cell frozen, the recovery with the cultivation carry out according to ordinary method.
2. seed culture of viruses production seed culture of viruses is TS09-C strain of the present invention, and this strain is identified, preserved and supply by the applicant.Imitating inspection is Beijing strain (CVCC AV1611 strain) with strong poison, identifies, takes care of and supply by China Veterinery Drug Inspection Office.
3. the production of vaccine and the inspection of semifinished product
3.1 the BHK21 cell that growth conditions is good, grow up to fine and close individual layer is selected in the production of vaccine for use, behind the tipping nutrient solution, change serum-free DMEM nutritive medium (containing 0.2 μ g/ml TPCK-pancreatin), place 37 degree to cultivate to contain 0.1% viral seed liquor, every day the observation of cell state.The cell of discovery growth failure, pollution should be discarded.Cultivate after 2～3 days, have the cell more than 80% pathology to occur approximately, and control cells is in good condition, can gather in the crops, after three freeze thawing repeatedly, low-speed centrifugal is got supernatant, obtains viral liquid, puts-20 degree and preserves.The viral liquid that takes a morsel carries out the inspection of semifinished product.
3.2 the inspection of semifinished product is carried out steriling test by existing " Chinese veterinary drug allusion quotation " appendix, answers asepsis growth; Work in-process are made 10 times of serial dilutions with sterile saline, get 10
-5, 10
-6, 10
-7Three extent of dilution are respectively inoculated BHK21 cell 5 holes, and every hole 0.1ml puts 37 ℃ and continues to cultivate.The observation of cell pathology cytopathy person occurs in 120 hours and is judged to infection, calculates TCID
50, every 0.1ml viral level should be not less than 10
6.0TCID
50, otherwise be judged to defective.
3.3 join seedling and packing the work in-process that are up to the standards are mixed in the same container, plumage part adds a certain proportion of 5% sucrose skimmed milk (quality and volume ratio) used as stabilizers in accordance with regulations, add suitable microbiotic simultaneously, fully shake up, quantitatively packing, every plumage part viral level should be not less than 10
5.0TCID
50Carry out vacuum freezedrying (being undertaken) after the packing rapidly by " People's Republic of China's veterinary biologics rules " version appendix in 2000.
4. inspection after construction is with reference to existing " Chinese veterinary drug allusion quotation " appendix, the finished product vaccine carried out checks such as physical behavior, aseptic, mycoplasma, exogenous virus, safety and effectiveness respectively, all should be qualified.
5. effect is used to prevent newcastle disease with purposes.Also can be used for preventing the newcastle disease of dove, quail.
6. usage and consumption are pressed label and are indicated plumage part, with sterile saline or suitable diluted.Collunarium or eye droppings immunity, every 0.05ml; The drinking-water immunity, dosage doubles.
7. storage and validity period-15 ℃ preservation down, validity period is 24 months; 2～8 ℃ of preservations, validity period are 12 months.
8. specification 250 plumages part/bottle.
Claims (8)
1. a strain adapts to the heat-resisting low virulent strain of Avian pneumo-encephalitis virus of young hamster kidney passage cell, and its microbial preservation number is: CCTCCV201113; Classification name: Avian pneumo-encephalitis virus TS09-C strain; The preservation time is: on May 9th, 2011; Depositary institution: Chinese typical culture collection center; Preservation address: China. Wuhan. Wuhan University.
2. press the preparation method of the heat-resisting low virulent strain of Avian pneumo-encephalitis virus of the described adaptation young hamster of claim 1 kidney passage cell, it is characterized in that: the propagation of the heat-resisting low virulent strain of described Avian pneumo-encephalitis virus in young hamster kidney passage cell is tired and is reached 10
8.0TCID
50/ ml.
3. press the preparation method of the heat-resisting low virulent strain of Avian pneumo-encephalitis virus of the described adaptation young hamster of claim 1 kidney passage cell, it is characterized in that: the hemagglutinin of the heat-resisting low virulent strain of described Avian pneumo-encephalitis virus-Neuraminidase Gene sequence is SEQ ID NO:1, and the sequence total length is 1851bp.
4. press the preparation method of the heat-resisting low virulent strain of Avian pneumo-encephalitis virus of the described adaptation young hamster of claim 1 kidney passage cell, it is characterized in that: the nucleotide homology of the HN gene order of heat-resisting low virulent strain of described Avian pneumo-encephalitis virus and prototype-strain TS09 strain is 88.2%.
5. the preparation method of the heat-resisting low virulent strain of Avian pneumo-encephalitis virus of the described adaptation young hamster of claim 1 kidney passage cell, it is characterized in that: under the culture condition that contains 0.2 μ g/ml TPCK-pancreatin, Avian pneumo-encephalitis virus TS09 chicken embryo adapted strain is carried out 17 continuous passages in young hamster kidney passage cell cultivate, comprising there being 3 times is Method of Limited Dilution method purifying, reach the heat-resisting low virulent strain of Avian pneumo-encephalitis virus that the 17th generation promptly obtained to adapt to young hamster kidney passage cell, i.e. Avian pneumo-encephalitis virus TS09-C strain.
6. heat-resisting living vaccine that prevents newcastle disease is characterized in that being made up of the heat-resisting low virulent strain of Avian pneumo-encephalitis virus of the adaptation young hamster kidney passage cell of the described significant quantity of claim 1 and pharmaceutically acceptable carrier and auxiliary material.
7. by the heat-resisting living vaccine of the described prevention newcastle disease of claim 6, it is characterized in that described newcastle disease is newcastle disease, dove newcastle disease or quail newcastle disease.
8. the preparation method of the heat-resisting living vaccine of the described prevention newcastle disease of claim 6; it is characterized in that including following steps: select young hamster kidney passage cell for use; behind the tipping nutrient solution; change serum-free DMEM nutritive medium with the Avian pneumo-encephalitis virus TS09-C strain virus seed liquor that contains volume ratio 0.1%; place 37 degree to cultivate after 2～3 days; through three multigelations; the centrifuging and taking supernatant; promptly obtain viral liquid, put-20 degree and preserve, get viral liquid and carry out steriling test and virus titer check; viral liquid through being up to the standards mixes with the skimmed milk that protective material contains sucrose; fully shake up the back packing, carry out vacuum freezedrying rapidly, promptly get the heat-resisting living vaccine that prevents newcastle disease.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420261A (en) * | 2015-10-09 | 2016-03-23 | 湖北省农业科学院畜牧兽医研究所 | Newcastle disease virus heat-resistant transformation method and application |
| CN105749271A (en) * | 2016-02-23 | 2016-07-13 | 湖北省农业科学院畜牧兽医研究所 | Preparation method and application of Newcastle disease chicken embryo inoculation vaccine |
| CN108379575A (en) * | 2018-04-24 | 2018-08-10 | 湖北省农业科学院畜牧兽医研究所 | A kind of inactivated vaccine and preparation method thereof prepared with newcastle disease virus thermostabilization strain |
| CN112126629A (en) * | 2020-08-19 | 2020-12-25 | 湖北省农业科学院畜牧兽医研究所 | Heat-resistant Newcastle disease virus mutant strain and preparation method and application thereof |
| CN118222521A (en) * | 2024-04-25 | 2024-06-21 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Preparation and application of newcastle disease virus recombinant attenuated strain and vaccine |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA962593A (en) * | 1969-11-29 | 1975-02-11 | Homu Ito | Highly attenuated newcastle disease virus vaccine and production thereof |
| CN101182494A (en) * | 2007-09-05 | 2008-05-21 | 扬州大学 | Gene VII type new castle disease virus weakening strain A-NDV-VII and construction method thereof |
-
2011
- 2011-06-17 CN CN 201110163109 patent/CN102268410B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA962593A (en) * | 1969-11-29 | 1975-02-11 | Homu Ito | Highly attenuated newcastle disease virus vaccine and production thereof |
| CN101182494A (en) * | 2007-09-05 | 2008-05-21 | 扬州大学 | Gene VII type new castle disease virus weakening strain A-NDV-VII and construction method thereof |
Non-Patent Citations (4)
| Title |
|---|
| 《Avian Pathology》 20071112 Jarra Jagne 等 Vaccination of village chickens in The Gambia against Newcastle disease using the heat-resistant, food-pelleted V4 vaccine 721-724 第20卷, 第4期 * |
| JARRA JAGNE 等: "Vaccination of village chickens in The Gambia against Newcastle disease using the heat‐resistant, food‐pelleted V4 vaccine", 《AVIAN PATHOLOGY》 * |
| 罗青平等: "新城疫V4耐热弱毒株的研究进展", 《中国家禽》 * |
| 董伟等: "新城疫不同弱毒株疫苗对鸭免疫效果比较", 《防检技术》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420261A (en) * | 2015-10-09 | 2016-03-23 | 湖北省农业科学院畜牧兽医研究所 | Newcastle disease virus heat-resistant transformation method and application |
| CN105749271A (en) * | 2016-02-23 | 2016-07-13 | 湖北省农业科学院畜牧兽医研究所 | Preparation method and application of Newcastle disease chicken embryo inoculation vaccine |
| CN108379575A (en) * | 2018-04-24 | 2018-08-10 | 湖北省农业科学院畜牧兽医研究所 | A kind of inactivated vaccine and preparation method thereof prepared with newcastle disease virus thermostabilization strain |
| CN112126629A (en) * | 2020-08-19 | 2020-12-25 | 湖北省农业科学院畜牧兽医研究所 | Heat-resistant Newcastle disease virus mutant strain and preparation method and application thereof |
| CN118222521A (en) * | 2024-04-25 | 2024-06-21 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Preparation and application of newcastle disease virus recombinant attenuated strain and vaccine |
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