TWI580786B - Vaccine of avian VII genotype new disease and its preparation method - Google Patents

Description

Vaccine of avian VII genotype Newcastle disease and preparation method thereof

The invention relates to a vaccine suitable for poultry and a preparation method thereof, in particular to a vaccine for avian VII genotype Newcastle Disease (ND) and a preparation method thereof.

According to the results of Taiwan (Ke et al., 2008) and Korea (Jeon et al., 2008), it is pointed out that the main cause of vaccine immune failure and ND persistent outbreak in Asian ND is the antigenic variation of genotype VII (Jeon et Al., 2008; Boven et. al., 2008). Among the various means used to control pandemics including, for example, Newcastle disease in poultry, vaccines often play an indispensable role. In particular, in the case where a specific virus strain of such an epidemic causes a large number of infections and a major outbreak occurs in a short period of time, it is generally impossible to use physical means such as controlling environmental sanitation or using chemical agents such as antibiotics or antiviral agents. To fight against it, at this time, it is usually only possible to rely on vaccination to suppress the extent and extent of infection for epidemic prevention.

Secondly, avian Newcastle disease is an acute infectious disease caused by the Newcastle disease virus strain (NDV) of the animal paramyxovirus. However, as of now, there is no reliable and effective treatment that can be trusted. Therefore, in the conventional technology, the prevention means used to prevent the occurrence of such infectious diseases is usually a method of using a plan to immunize chickens of different ages, in particular, a Newcastle disease to activate the vaccine.

Furthermore, in terms of a method for producing a live vaccine for avian Newcastle disease, a method conventionally adopted is to firstly use a low toxicity virulence strain HB1 strain, F strain, LaSota strain, LaSota-clone 30 strain or N79 strain. The virus strain is inoculated into the chicken embryo allantoic cavity without accidental antigen and antibody at 10 days of age, and is incubated at 37 ° C; the qualified chicken embryo allantoic fluid is regularly collected as an immunogen, followed by an appropriate amount of chilled drying additive. The mixture is well mixed, and then quantitatively dispensed into a sterile drug delivery bottle, and further vacuum-dried and dried to prepare a vaccine.

However, when the vaccine produced by the above conventional method is used for epidemic prevention, there are still several major problems as described below; for example, even if the vaccinated flock is not completely effective in preventing infection, the ND epidemic often occurs. Occurs, and spreads between chickens. Therefore, how to increase the average antibody price distribution of chickens after vaccination, so as to stop the spread of Newcastle disease virus (NDV) in breeding chickens and effectively prevent the spread of ND epidemic has become a major issue.

In addition, recent studies have also pointed out that some NDVs isolated from vaccinated chickens in China, followed by VII genotype isolates made of ND deactivated vaccines applied to chickens to provide resistance VII The protective ability of the genotype ND virus strain to prevent the death or disease of the breeding chicken caused by the disease. However, although the application of such a vaccine can protect the chicken and partially exempt the attack of the NDV strain, it is still unsatisfactory in effect. Therefore, it is urgent to develop a live vaccination vaccine or a deactivated vaccine which can be applied to poultry to effectively suppress Newcastle disease.

In view of the above, the present inventors reviewed the preparation method of the vaccine, and found out that the use of a poultry Newcastle disease to activate the vaccine not only solves the problem of the conventional technology, but also can be applied to birds to effectively suppress VII. Genotype Newcastle disease, thus completing the present invention.

In short, the present invention provides a VII genotype Newcastle disease deactivated vaccine capable of effectively preventing and reducing symptoms such as avian respiratory and nervous system damage caused by infection of avian VII genotype Newcastle virus strain; and providing a preparation The method of deactivating a vaccine for the VII genotype Newcastle disease.

Specifically, according to one aspect of the present invention, the present invention provides a live vaccine of avian VII genotype Newcastle disease, which comprises at least a live toxic antigen solution and a diluent; wherein the live toxic antigen liquid system is The live toxic antigen solution obtained by infecting the SPF chicken embryo allantoic sac and/or BHK-21 cells by the virus strain of Xincheng diseased chicken; the virus strain is deposited in the Food Industry Development Research Institute of the consortium, and the registration number is BCRC 970070, BCRC. 970071, BCRC 970072, date of registration of the Republic of China on March 24, 105; the dilution is at least one of phosphate buffered saline (PBS), purified water, 5% dextrose, and mixtures thereof. In particular, the live vaccination vaccine of the invention is particularly suitable for administration to chickens, in particular for raising chickens.

Secondly, according to another aspect of the present invention, the present invention further provides a live vaccine, which is dried, wherein the content of the viable antigen solution is in the range of 95% by volume to 65% by volume; The content of the liquid is in the range of 5 vol% to 35 vol%.

Furthermore, according to other aspects of certain embodiments, the present invention also provides a method for preparing a live vaccination vaccine for avian VII genotype Newcastle disease, comprising: (a) screening a virus strain from a field Newcastle disease chicken; the virus The company is deposited in the Food Industry Development Research Institute, and the registration numbers are BCRC 970070, BCRC 970071, BCRC 970072, and the date of registration is March 24, 105, the Republic of China; (b) the virus strain obtained in (a) above. The viable antigen solution containing the avian VII genotype Newcastle virus strain is obtained by culturing in the SPF chicken embryo allantoic sac and/or BHK-21 cells; (c) the viable antigen solution obtained in the above (b) The diluent is thoroughly mixed to form a live vaccine of avian VII genotype Newcastle disease.

In addition, according to still another aspect of certain embodiments, the present invention also provides a method for preparing a live vaccine of avian VII genotype Newcastle disease, which further comprises the live vaccine obtained in the above (b) in a vacuum refrigeration Drying is carried out to obtain a refrigerated vacuum drying live vaccine of avian VII genotype Newcastle disease.

In particular, in the above method for producing a viable antigen solution according to the present invention, the SPF chicken embryo allantoic capsule of the above (b) is cultured at 37 ° C in a chicken embryo allantoic sac of a specific pathogen at 9 days old.

Further, in the above method for producing a viable antigen solution of the present invention, the BHK-21 cell line of the above (b) is cultured in a cell culture incubator at 37 ° C and a concentration of 4.5% to 5.5% of carbon dioxide.

Specifically, according to one of the embodiments, the present invention provides a deactivated vaccine for avian VII genotype Newcastle disease, which comprises at least a deactivated antigen solution, an oily adjuvant; wherein the deactivated antigen solution The deactivated antigen solution obtained by infecting the SPF chicken embryo allantoic sac and/or BHK-21 cells by the virus strain of the Newcastle disease chicken; the virus strain is deposited in the Food Industry Development Research Institute of the consortium, and the storage number is BCRC 970070 BCRC 970071, BCRC 970072, date of registration of the Republic of China on March 24, 105; the oily adjuvant is at least one of a mineral white oil, an emulsifier, pure water, and a mixture thereof. In particular, the deactivated vaccine of the invention is particularly suitable for administration to chickens, especially chickens.

Secondly, according to another aspect of the present invention, the present invention also provides a deactivated vaccine for avian VII genotype Newcastle disease, which is dried, wherein the content of the deactivated antigen solution is from 95% by volume to 65%. The range of the volume %; the content of the oily adjuvant is in the range of 5 vol% to 35 vol%.

Further, according to other aspects of certain embodiments, the present invention also provides a method for preparing a deactivated vaccine for avian VII genotype Newcastle disease, comprising: (a) screening a virus strain from a field Newcastle disease chicken; the virus strain Deposited in the Food Industry Development Research Institute, the registration number is BCRC 970070, BCRC 970071, BCRC 970072, the date of registration is March 24, 105, the Republic of China; (b) the virus strain obtained in (a) above, The deactivated antigen solution containing the avian VII genotype Newcastle disease virus strain is obtained by culturing in SPF chicken embryo allantoic sac and/or BHK-21 cells; (c) for the antigen liquid obtained in the above (b), at 37 Deactivated treatment in the presence of °C and formalin (100 ppm to 300 ppm) for 12 to 24 hours to obtain a deactivated antigen solution; (d) Deactivated antigen solution and oil adjuvant obtained in (c) above Mixed to form a deactivated vaccine for avian VII genotype Newcastle disease.

In addition, according to still another aspect of certain embodiments, the present invention also provides a method for preparing a deactivated vaccine for avian VII genotype Newcastle disease, which further comprises the deactivated vaccine obtained in the above (b). In order to obtain a refrigerated deactivated vaccine of avian VII genotype Newcastle disease.

In particular, in the above method for producing a deactivated antigen solution of the present invention, the SPF chicken embryo allantoic capsule of the above (b) is cultured at 37 ° C in a chicken embryo allantoic sac of a specific pathogen at 9 days old.

Further, in the above method for producing a deactivated antigen solution of the present invention, the BHK-21 cell line of the above (b) is cultured in a cell culture incubator at 37 ° C, 4.5% to 5.5% carbon dioxide concentration. Detailed Description of the Invention

First, the specific terms or nouns used in the specification are described in the specification. However, the following description is merely illustrative, and is not intended to limit the scope of the invention. The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein.

In this article, the prevention of avian VII genotypes in Newcastle disease infection refers to inhibition of avian VII genotype Newcastle virus strain replication, inhibition of avian VII genotype Newcastle virus strain transmission, or prevention of avian VII genotype Newcastle virus strain It grows in its host and alleviates the symptoms of a disease or condition caused by infection with the avian VII genotype Newcastle virus strain. The method of the present invention, for example, can effectively prevent and reduce damage to the respiratory and nervous systems of birds.

The term avian VII genotype Newcastle disease vaccine and its preparation method herein refers to a vaccine for preventing a disease or disease caused by avian VII-type Newcastle disease virus strain. The avian VII genotype Newcastle disease vaccine and its preparation method may include any vaccine effective for treating or preventing avian VII genotype Newcastle disease infection. The preferred avian VII genotype Newcastle disease vaccine used in the method of the present invention and the preparation method thereof are virus strain vaccines.

The term "animal" as used herein refers to all non-human animals, including mammals.

The term "bird" in this article means a member of chicken, chicken, turkey, duck, hen, hen, pheasant, chicken, cock, pheasant and avian.

Preferably, the method of the invention is applied to a non-human mammal; the best is avian.

The term "viral strain vaccine" as used herein means a strain of avian VII genotype Newcastle virus suitable for use as a deactivation of a vaccine, and may be a chicken embryo allantoic sac and/or a cell preparation.

The term "effective amount" as used herein refers to the amount of avian VII genotype Newcastle disease vaccine and its preparation method which can sufficiently cause an avian immune response after administration. The immune response involves, but is not limited to, an innately induced, cellular and/or humoral immune response.

In this context, the numerical values and parameters used to define the scope of the invention intrinsically inevitably contain standard deviations due to individual test methods, and are therefore mostly expressed in approximate numerical values, although in specific embodiments Relevant values that are presented as accurately as possible. As used herein, "about" generally refers to the consideration of those of ordinary skill in the art to which the invention pertains, and generally means that the actual value falls within an acceptable standard error of the average value, for example, the actual value is in one Within ±10%, ±5%, ±1%, or ±0.5% of a particular value or range.

The object of the present invention is to provide an avian VII genotype Newcastle disease vaccine and a preparation method thereof, which are obtained by avian Newcastle disease virus strain collected from a field breeding house, and the virus strain is isolated from avian Newcastle disease chickens The NDV is deposited with the Food Industry Development Research Institute. The registration numbers are BCRC 970070, BCRC 970071, BCRC 970072, and the registration date is March 24, 105, the Republic of China.

The object of the present invention is to provide an avian VII genotype Newcastle disease vaccine and a preparation method thereof, which are produced by infecting chicken embryo culture, animal inoculation, or tissue culture, thereby producing a stable and immune vaccine. The antigen, the infectious virus strain culture expression system is simple, the yield is large, the production speed is fast, the safety is high, and the economic benefit is obtained.

According to one aspect of the present invention, the infectious virus strain culture expression system for use in the avian VII genotype Newcastle disease vaccine of the present invention and the preparation method thereof is not particularly limited, and for example, may be chicken embryo culture, Animal inoculation, or tissue culture. According to some embodiments of the present invention, the vaccine antigen source may be, for example, at least one of chicken embryo culture, animal inoculation, or tissue culture; the vaccine antigen suitable for use in the present invention is preferably a chicken embryo culture, animal. Inoculation, or tissue culture; better use sources are chicken embryo culture, or tissue culture; the best source of use is chicken embryo culture.

The object of the present invention is to provide an avian VII genotype Newcastle disease vaccine and a preparation method thereof, which are produced by infecting cell culture to obtain a cell supernatant containing a virus strain, wherein the cell strain includes all pairs of NDV. A mammalian kidney cell strain with good susceptibility, such as African Green monkey kidney (Vero cell), Madin-Darby canine kidney (MDK cell) and other cell lines. The infected cell culture method can rapidly expand the production scale and the quality is stable and stable, and has economic benefits.

According to one aspect of the present invention, the cell strain of the avian VII genotype Newcastle disease vaccine and the preparation method thereof for use in the present invention is not particularly limited, and for example, may be Vero cells, BHK- 21 cells, or MDCK cells. According to some embodiments of the present invention, the cell strain of the infected cell culture may be, for example, at least one of Vero cells, BHK-21 cells, or MDCK cells; and the cell strain suitable for the infected cell culture of the present invention is compared. The source of use is Vero cells, BHK-21 cells, or MDCK cells; the more preferred source is Vero cells, or BHK-21 cells; the preferred source is BHK-21 cells.

The object of the present invention is to provide an avian VII genotype Newcastle disease vaccine and a preparation method thereof, which are vaccinated by a live vaccine, an attenuated vaccine or a deactivated vaccine, and the vaccination type is simple and antigen Stable and easy to save.

According to one aspect of the present invention, the vaccination type of the avian VII genotype Newcastle disease vaccine and the preparation method thereof for use in the present invention is not particularly limited, and for example, may be a live vaccine or an attenuated vaccine. Or to activate the vaccine. According to some embodiments of the present invention, the vaccination species source may be, for example, at least one of a live vaccination vaccine, an attenuated vaccine, or a deactivated vaccine; and the vaccination type suitable for use in the present invention is preferably a live venom Vaccines, attenuated vaccines, or deactivated vaccines; better use of attenuated vaccines, or deactivated vaccines; a particularly good type of deactivated vaccine.

The object of the present invention is to provide an avian VII genotype Newcastle disease vaccine and a preparation method thereof, which are deactivated or chemically modified to modify an antigenic epitope (epitopes) to remove a virus. The activity of the strain. Commonly used dereactive treatments are chemical or physical, and the choice of method depends on the structure of the molecule and the extent of infection by each pathogen. By deactivated reaction treatment, a vaccine antigen with inactivity is obtained, and the residual toxicity is extremely low, achieving a safe and stable effect.

According to one aspect of the present invention, the vaccine for use in the vaccine of the avian VII genotype Newcastle disease of the present invention and the preparation method thereof are not particularly limited, and for example, may be an alkylating agent, Ethylene oxide, beta-propiolactone, alcohol, or formalin. According to some embodiments of the present invention, the deactivation reaction treatment may be, for example, at least one of an alkylating agent, an ethylene oxide, a β-propiolactone, an alcohol, or a formalin, and the reaction is carried out at 37 ° C. -16 hours; preferably used in the deactivation treatment of the present invention as an alkylating agent, ethylene oxide, β-propiolactone, alcohol, or formalin, at a reaction temperature of 30-40 ° C for 1-20 hours; Preferably, the reaction is carried out as an alkylating agent, alcohol or fumarin at 33-39 ° C for 8-20 hours; particularly preferably alcohol or formalin, the reaction is carried out at 36-37 ° C for 10-12 hours.

The object of the present invention is to provide a vaccine for avian VII genotype Newcastle disease and a preparation method thereof, which may comprise additional ingredients such as an adjuvant. Because the antigen has a very high safety after being deactivated, it can cause an adverse immune response and often needs to be used in combination with an adjuvant. The adjuvant allows the antigen at the injection site to be slowly released to stimulate the immune response to activate the cells. As the adjuvant, various adjuvants known in the art can be used as an adjuvant vaccine effect.

According to one aspect of the present invention, the adjuvant for use in the vaccine of the avian VII genotype Newcastle disease of the present invention and the preparation method thereof are not particularly limited, and for example, may be an inorganic adjuvant or an organic adjuvant. , synthetic adjuvants, oils; inorganic adjuvants, such as aluminum hydroxide, alum, etc.; organic adjuvants, microorganisms and their products such as mycobacteria (Mycobacterium tuberculosis, BCG), Bacillus brevis, Bordetella, endotoxin, bacterial extraction (cell wall acyl dipeptide), etc.; synthetic adjuvants, such as synthetic double-stranded polynucleotides (double-stranded polyadenylation, uridine), levamisole, isoproterenol, etc.; Such as Freund's adjuvant, peanut oil emulsion adjuvant, mineral oil, vegetable oil and the like.

According to the avian VII genotype Newcastle disease vaccine according to the present invention and the preparation method thereof, the adjuvant may be, for example, a self-aqueous aluminum hydroxide gel, an immunostimulant, an alum, Freund's incomplete adjuvant, At least one of an oily adjuvant, a water-soluble adjuvant, or a water-in-oil-in-water (W/O/W); an adjuvant suitable for use in the present invention Good use as aqueous aluminum hydroxide gel, immunostimulant, alum, Freund's incomplete adjuvant, oily adjuvant, water-soluble adjuvant, or water-in-oil-in-oil adjuvant -water, W/O/W); better for aqueous aluminum hydroxide gel, immunostimulant, oil adjuvant, water soluble adjuvant, or water-in-water double phase adjuvant (water-in-oil) -in-water, W/O/W); especially good for oily adjuvants.

The object of the present invention is to provide a vaccine for avian VII genotype Newcastle disease, a preparation method thereof and a live vaccine. The vaccine is stored in a cryopreservation manner, and the vaccine is first stored in a refrigerated manner. Refrigerate to 2~8 °C. This preservation method can maintain the efficacy and safety of the drug itself and increase the shelf life of the drug.

The vaccine for chilled and dried poultry VII genotype Newcastle disease of the present invention is mixed with the diluent before immunization of the animal. Dilutions for this purpose can be known to those skilled in the art. Preferably, the diluent used in the present invention is phosphate buffered saline (PBS), mineral oil (OE), pure water or 5% dextrose. More preferably, the diluent is a phosphate buffer, a mineral oil or pure water. Most preferably, the diluent used in the present invention is a phosphate buffer. In the present invention, about 40 ml of a dilution solution containing about 2 ml of a live virus solution of avian VII genotype Newcastle disease can be added to a live virus vaccine (about 20 doses/bottle) of a refrigerated dry poultry VII genotype Newcastle disease tissue culture culture solution (about 20 doses/bottle). For animal vaccination.

According to one aspect of the present invention, the preservation method of the avian VII genotype Newcastle disease live vaccine for use in the present invention is not particularly limited. For example, it may be refrigerated vacuum drying, sublimation drying, desorption, Or the method of storage such as heating and drying. According to some embodiments of the present invention, the storage mode source may be, for example, at least one of refrigerating vacuum drying, sublimation drying, desorption, or heat drying; and the storage method suitable for the present invention is preferably a refrigerating vacuum. Drying, sublimation drying, desorption, or heat drying; better use is refrigerated vacuum drying, desorption, or heat drying; the best use is vacuum vacuum drying.

According to certain specific embodiments, the avian VII genotype Newcastle disease vaccine of the present invention can be administered by subcutaneous injection or intramuscular injection by drinking water, orally, spraying, eye-dropping, nodding, and deactivation of the vaccine system. The vaccine is inoculated by the method of inoculation, and thus the vaccine of the present invention has an excellent effect of easy and rapid inoculation, high safety, and good economic efficiency.

According to one aspect of the present invention, the method of inoculation which can be used in the live vaccination vaccine or deactivated vaccine of the avian VII genotype of the present invention is not particularly limited. For example, the live vaccination vaccine can be used for drinking water. Oral, spray, eye, nose, etc.; the deactivated vaccine can be inoculated by subcutaneous injection or intramuscular injection. According to some embodiments of the present invention, the source of the inoculation method may be, for example, at least one of oral, spray, eye, nose, subcutaneous, or intramuscular injection; the inoculation method suitable for use in the present invention is preferably used. The method is oral, spray, eye, nose, subcutaneous, or intramuscular injection; better use is subcutaneous or intramuscular; the best use of live vaccine is oral, and deactivated vaccine for intramuscular injection .

The invention will be further described below in conjunction with specific embodiments.

In the following, the embodiments of the present invention will be described in more detail in the detailed description of the embodiments of the present invention so that the spirit and content of the present invention are more complete and easy to understand; however, those of ordinary skill in the art should understand The invention is of course not limited to these examples, and other similar or equivalent functions and order of steps may be utilized to achieve the invention.

In addition, the scope of the invention may be further exemplified by the following specific examples, which are not intended to limit the scope of the invention. Preparation of Live Vaccine "Preparation Example 1 to Preparation Example 3"

First, one of the VII genotypes of Newcastle disease virus strains obtained from the screening of the Newcastle disease chickens in the field (stored in the Food Industry Development Research Institute of the consortium, the registration number is BCRC 970070, BCRC 970071, BCRC 970072, and the registration date is 105 Republic of China. March 24, 2014) As a source of infection, inoculated in a 9-day-old specific-pathogen-free (SPF) chicken embryo allantoic cavity, or BHK-21 cells (C-13). ) (ATCC CCL-10) host, then cultured in a 37 ° C incubator (Alex Alex Electric Processing Factory, AI-528) or incubator (Thermo Scientific Forma, Cat. No. 311) Then, after culturing for 24 to 32 hours, the culture solution was extracted from the above-mentioned chicken embryo allantoic cavity and BHK-21 cells, and centrifuged to obtain VII genotype Newcastle disease antigen liquids M1 to M3. Next, the VII genotype Newcastle disease antigen liquids M1 to M3 were mixed with the diluent, and the live vaccination vaccines were designated as VL1, VL2, and VL3, and the ratios and amounts were recorded in Table 1.

"Table 1"         <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> Live Vaccine</td><td> Preparation 1 </td><td> Preparation Example 2 </td><td> Preparation Example 3 </td></tr><tr><td> Source of infection (registered number) </td><td> TW-CB-ND02 (BCRC 970070) </ Td><td> 0.5 mL </td><td> - </td><td> - </td></tr><tr><td> TW-CB-ND04 (BCRC 970071) </td> <td> - </td><td> 0.5 mL </td><td> - </td></tr><tr><td> TW-CB-ND06 (BCRC 970072) </td><td > - </td><td> - </td><td> 0.5 mL </td></tr><tr><td> host</td><td> chicken embryo allantoic sac</td>< Td> BHK-21 cells</td><td> BHK-21 cells</td></tr><tr><td> Culture conditions</td><td> Temperature (°C)/time (hr) < /td><td> 37/24 </td><td> 37/28 </td><td> 37/32 </td></tr><tr><td> Immune antigen solution</td> <td> Immune antigen (10⁸ TCID₅₀/mL) </td><td> 5.5 mL </td><td> 7 mL </td>< Td> 9.5 mL </td></tr><tr><td> No. VII genotype Newcastle disease antigen solution</td><td> M1 </td><td> M2 </td><td> M3 </td></tr><tr><td> Diluent</td><td> Potassium Phosphate Buffer (pH=7.4, 0.2 mol/L) </td><td> 1.5 mL </td> <td> 1 mL </td><td > 0.2 mL </td></tr><tr><td> pure water</td><td> 1.5 mL </td><td> 1 mL </td><td> 0.1 mL </td> </tr><tr><td> 5% (w/w) glucose</td><td> 1.5 mL </td><td> 1 mL </td><td> 0.2 mL </td>< /tr><tr><td> Live Vaccine</td><td> VL1 </td><td> VL2 </td><td> VL3 </td></tr></TBODY></ TABLE> "Preparation of Deactivated Vaccine" "Preparation Example 4 to Preparation Example 6"       

First, one of the VII genotypes of Newcastle disease virus strains obtained from the screening of the Newcastle disease chickens in the field (stored in the Food Industry Development Research Institute of the consortium, the registration number is BCRC 970070, BCRC 970071, BCRC 970072, and the registration date is 105 Republic of China. March 24, 2014) As a source of infection, inoculated in a 9-day-old specific-pathogen-free (SPF) chicken embryo allantoic cavity, or BHK-21 cells (C-13). ) (ATCC CCL-10) host, then cultured in a 37 ° C incubator (Alex Alex Electric Processing Factory, AI-528) or incubator (Thermo Scientific Forma, Cat. No. 311) Then, after culturing for 48 hours, the culture solution was extracted from the above-mentioned chicken embryo allantoic cavity and BHK-21 cells, and centrifuged to obtain VII genotype Newcastle disease antigen solution M4 to M6.

Then, the above-mentioned VII genotype Newcastle disease antigen solution M4~M6 and 20% solution of formalin (HT501128-4L, manufactured by Sigma) are thoroughly mixed at a ratio of the antigen solution to the formalin volume ratio of 4000:1; Deactivation treatment was carried out for 12 hours at 22 ° C and 37 ° C to obtain deactivated antigen solutions X1 to X3.

The deactivated antigen liquids X1 to X3 obtained above in accordance with the contents shown in Table 2 and the adjuvant were thoroughly mixed to form a deactivated vaccine VD4 to VD6 of avian VII genotype Newcastle disease.

"Table 2"         <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> Deactivated vaccine</td><td> Preparation 4 </td><td> Preparation Example 5 </td><td> Preparation Example 6 </td></tr><tr><td> Source of infection (registered number) </td><td> TW-CB-ND02 (BCRC 970070) </ Td><td> 0.5 ml </td><td> - </td><td> - </td></tr><tr><td> TW-CB-ND04 (BCRC 970071) </td> <td> - </td><td> 0.5 ml </td><td> - </td></tr><tr><td> TW-CB-ND06 (BCRC 970072) </td><td > - </td><td> - </td><td> 0.5 ml </td></tr><tr><td> host</td><td> chicken embryo allantoic sac</td>< Td> BHK-21 cells</td><td> BHK-21 cells</td></tr><tr><td> Culture conditions</td><td> Temperature (°C)/time (hr) < /td><td> 37/18 </td><td> 37/28 </td><td> 37/32 </td></tr><tr><td> Immune antigen solution</td> <td> Immune antigen (10⁸ TCID₅₀/mL) </td><td> 5.5 mL </td><td> 7 mL </td>< Td> 9.5 mL </td></tr><tr><td> No. VII genotype Newcastle disease antigen solution</td><td> M4 </td><td> M5 </td><td> M6 </td></tr><tr><td> Deactivation reaction</td><td> Formalin (20%): antigen solution (V/V) </td><td> 1/3500 </ Td><td> 1/4000 </td>< Td> 1/4500 </td></tr><tr><td> Reaction conditions</td><td> Temperature (°C)/time (hr) </td><td> 20/4.2 </td ><td> 22/2.8 </td><td> 24/1.1 </td></tr><tr><td> Deactivate the antigen solution</td><td> X1 </td><td> X2 </td><td> X3 </td></tr><tr><td> oily adjuvant </td><td> mineral white oil</td><td> 3.5 mL </td ><td> 1.5 mL </td><td> 0.3 mL </td></tr><tr><td> Potassium Phosphate Buffer (pH=7.4, 0.2 mol/L) </td><td> 1 mL </td><td> 1 mL </td><td> 0.1 mL </td></tr><tr><td> Montanide 80 </td><td> 0.5 mL </td>< Td> 0.5 mL </td><td> 0.1 mL </td></tr><tr><td> Reaction Conditions </td><td> Temperature (°C)/Time (hr)/Speed (rpm) </td><td> 20/0.2/1800 </td><td> 22/0.4/2000 </td><td> 24/0.6/2500 </td></tr><tr><td> Deactivated vaccine</td><td> VD4 </td><td> VD5 </td><td> VD6 </td></tr></TBODY></TABLE> Verification "Embodiment 1 - Example 3"       

The live vaccines VL1 to VL3 obtained above were administered by subcutaneous injection to chickens aged 2 to 3 weeks, and each of the examples was administered with 6 vaccines. The dose of each vaccine was 0.2 ml. After 3 weeks, blood was collected to separate serum S1~S30.

Next, a hemagglutination (HA) test was performed on a 96-well U-shaped plate, and 50 μl of HI buffer (3.25 g KH ₂ P0 ₄ , 10.8 g Na ₂ HP0 _{4) was} added from the 1st to the 10th row. 170 g of NaCl, three times of deionized pure water was added to 2,000 mL), and 50 μl of the VII genotype Newcastle disease antigen solution was added to the first column.

After fully mixing with a micropipette, take 50 μl of the first row of VII genotype Newcastle disease antigen solution into the second row, and then take 50 μl to the third row, so that the sequence is diluted to the 10th row, and the antigen is doubled. Dilute to 1024 times. In the 11th row as the control group, the VII genotype Newcastle disease antigen solution was replaced with 100 μL of HI buffer, and each row was repeatedly tested with the same sample of 3 wells.

Finally, add 50 μL of 1% chicken red blood cell solution (cRBC) to each well, and then gently shake the well plate with your hand, then seal the well plate with a film, and let it stand at room temperature or 4 ° C for 30 minutes, then read it; The highest dilution factor of the VII genotype Newcastle disease antigen solution is still the hemoglobin agglutination effect of its HA, which is the highest dilution ratio of 1 unit (1HAU) antigen, and the blood coagulation effect is the price of HA. When performing the HI test, 8HAU is usually taken to obtain the HA price of the NDV virus strain.

Before performing the hemagglutination inhibition test (HI), the VII genotype Newcastle disease antigen solution must be diluted with HI buffer to contain 8 HAU of the VII genotype Newcastle disease antigen solution per 25 μl of the dilution.

Next, add 25 μL of HI buffer to the 96-well U-shaped plate, and add 25 μL of the above-mentioned test serum S1~S30 to the plate in sequence, then mix thoroughly with a micropipette, row 1 Mix 25 μl of the serum into the second row, mix well and then take 25 μl and add to the third row. Dilute the sequence to the 10th row. The 11th row is the control group without antiserum. This row only adds 25 The amount of μL 8 HAU antigen was allowed to act at room temperature for 30 minutes. After the antigen and the serum to be tested were combined, 25 μL of 1% cRBC was added to each well. After 30 minutes at room temperature, the blood cells were agglutinated. The highest dilution factor is still the blood cell agglutination effect, and the average value of the viral power of the test results of each group of 6 chickens in each example is recorded in Table 3.

Then, after 6 weeks from the vaccination, the survival rates of the six inoculated chickens in each group in each example were observed, and the average values of the test results of the survival rates of the respective examples were recorded in Table 3, respectively.

In addition, the same test was repeated by using the same test as the control group without using the avian VII genotype Newcastle disease vaccine and the preparation method thereof, and measuring the viral power in the blood of each control group and observing each control group. The survival rate of chickens.

"table 3"         <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> Control example</td><td> Example 1 </ Td><td> Example 2 </td><td> Example 3 </td></tr><tr><td> Shit group </td><td> PBS </td><td > VL1 </td><td> VL2 </td><td> VL3 </td></tr><tr><td> How to Apply</td><td> Subcutaneous Injection</td><td > Point nose</td><td> Subcutaneous injection</td><td> Intramuscular injection</td></tr><tr><td> Application dose (μl) </td><td> 25 < /td><td> 20 </td><td> 25 </td><td> 30 </td></tr><tr><td> hit time (week) </td><td> 2 </td><td> 2 </td><td> 2 </td><td> 2 </td></tr><tr><td> number of hits (only) </td> <td> 6 </td><td> 6 </td><td> 6 </td><td> 6 </td></tr><tr><td> Survival rate (%) </td ><td> 15 ± 9.3 </td><td> 99 ± 1.3 </td><td> 100 ± 0.3 </td><td> 95 ± 6.3 </td></tr><tr><td > Antibody price</td><td> 16X </td><td> 512X </td><td> 1024X </td><td> 1024X </td></tr></TBODY></ TABLE>

As shown in Table 3 above, in the control group, the chickens which did not apply the avian VII genotype Newcastle disease live vaccine of the present invention showed only a weak antibody titer of less than 16X; in contrast, in Example 1 In Example 3, the chickens of the avian VII genotype Newcastle disease vaccine of the present invention and the preparation method thereof showed a high-strength antibody titer of at least 512X or more.

In addition, the results of the survival test also showed that the survival rate of the chickens in the control group was less than 20% after infection for 6 weeks, and the survival rate of the chickens decreased to 0 after 8 weeks of infection; on the other hand, in the examples In 1 to 3, the chickens of the avian VII genotype Newcastle disease vaccine of the present invention and the preparation method thereof were all survived even after infection for more than 8 weeks, showing a survival rate of up to 95%. "Validation Validation of Deactivated Vaccines" "Examples 4 to 6"

The live vaccines VD4~VD6 obtained above were administered by subcutaneous injection to chickens of 2 to 3 weeks of immunization, and 6 animals were administered in each example. The dose of each vaccine was 0.2 ml. Three weeks later, blood was collected to separate serum S31~S60.

Next, a hemagglutination (HA) test was performed on a 96-well U-shaped plate, and 50 μl of HI buffer (3.25 g KH ₂ P0 ₄ , 10.8 g Na ₂ HP0 _{4) was} added from the 1st to the 10th row. 170 g of NaCl, three times of deionized pure water was added to 2,000 mL), and 50 μl of the VII genotype Newcastle disease antigen solution was added to the first column.

After fully mixing with a micropipette, take 50 μl of the first row of VII genotype Newcastle disease antigen solution into the second row, and then take 50 μl to the third row, so that the sequence is diluted to the 10th row, and the antigen is doubled. Dilute to 1024 times. In the 11th row as the control group, the VII genotype Newcastle disease antigen solution was replaced with 100 μL of HI buffer, and each row was repeatedly tested with the same sample of 3 wells.

Finally, add 50 μL of 1% chicken red blood cell solution (cRBC) to each well, and then gently shake the well plate with your hand, then seal the well plate with a film, and let it stand at room temperature or 4 ° C for 30 minutes, then read it; The highest dilution factor of the VII genotype Newcastle disease antigen solution is still the hemoglobin agglutination effect of its HA, which is the highest dilution ratio of 1 unit (1HAU) antigen, and the blood coagulation effect is the price of HA. When performing the HI test, 8HAU is usually taken to obtain the HA price of the NDV virus strain.

Before performing the hemagglutination inhibition test (HI), the VII genotype Newcastle disease antigen solution must be diluted with HI buffer to contain 8 HAU of the VII genotype Newcastle disease antigen solution per 25 μl of the dilution.

Next, add 25 μL of HI buffer to the 96-well U-shaped plate, and add 25 μL of the above-mentioned test serum S1~S30 to the plate in sequence, then mix thoroughly with a micropipette, row 1 Mix 25 μl of the serum into the second row, mix well and then take 25 μl and add to the third row. Dilute the sequence to the 10th row. The 11th row is the control group without antiserum. This row only adds 25 The amount of μL 8 HAU antigen was allowed to act at room temperature for 30 minutes. After the antigen and the serum to be tested were combined, 25 μL of 1% cRBC was added to each well. After 30 minutes at room temperature, the blood cells were agglutinated. The highest dilution factor is still the blood cell agglutination effect, and the average value of the viral power of the test results of each group of 6 chickens in each example is recorded in Table 4.

Then, after 6 weeks of the vaccine administration, the survival rates of the 6 inoculated chickens in each group in each example were observed, and the average values of the test results of the survival rates of the respective examples were recorded in Table 4, respectively.

In addition, the same test was repeated by using the same test as the control group without using the avian VII genotype Newcastle disease vaccine and the preparation method thereof, and measuring the viral power in the blood of each control group and observing each control group. The survival rate of chickens.

"Table 4"         <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> Control example</td><td> Example 4 </ Td><td> Example 5 </td><td> Example 6 </td></tr><tr><td> Shit Group </td><td> PBS </td><td > VD4 </td><td> VD5 </td><td> VD6 </td></tr><tr><td> How to Apply</td><td> Subcutaneous Injection</td><td > Point nose</td><td> Subcutaneous injection</td><td> Intramuscular injection</td></tr><tr><td> Application dose (μl) </td><td> 25 < /td><td> 20 </td><td> 25 </td><td> 30 </td></tr><tr><td> hit time (week) </td><td> 2 </td><td> 2 </td><td> 2 </td><td> 2 </td></tr><tr><td> number of hits (only) </td> <td> 6 </td><td> 6 </td><td> 6 </td><td> 6 </td></tr><tr><td> Survival rate (%) </td ><td> 15 ± 9.3 </td><td> 99 ± 1.3 </td><td> 100 ± 0.3 </td><td> 95 ± 6.3 </td></tr><tr><td > Antibody price</td><td> 16X </td><td> 512X </td><td> 1024X </td><td> 1024X </td></tr></TBODY></ TABLE>

As shown in Table 4 above, in the control group, the chickens which did not apply the avian VII genotype Newcastle disease live vaccine of the present invention showed only a weak antibody titer of less than 16X; in contrast, in Example 4 In Example 6, the chicken of the avian VII genotype Newcastle disease vaccine of the present invention and the preparation method thereof showed a high-strength antibody titer of at least 512X or more.

In addition, the results of the survival test also showed that the survival rate of the chickens in the control group was less than 20% after infection for 6 weeks, and the survival rate of the chickens decreased to 0 after 8 weeks of infection; on the other hand, in the examples In 4 to 6, the birds of the avian VII genotype Newcastle disease vaccine of the present invention and the preparation method thereof were all survived even after infection for more than 8 weeks, showing a survival rate of up to 95%.

Thus, it was confirmed that the chicken which was administered with the deactivated vaccine was able to increase the immunoprotective ability, and thus had an excellent effect against the infection of the VII genotype Newcastle disease. Therefore, the avian VII genotype Newcastle disease active vaccine and the deactivated vaccine of the present invention can significantly increase the viral power of the chicken and enhance the immunity and survival rate of the birds and the like.

In summary, the content of the present invention has been exemplified by the above embodiments, but the present invention is not limited to the embodiments. A person skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention; for example, combining or modifying the technical contents exemplified in the foregoing embodiments. As a new embodiment, these embodiments are of course considered as belonging to the present invention. Therefore, the scope of protection to be covered in this case also includes the scope of the patent application described below and the scope defined by it.

no.

TW Republic of China, Bioresource Collection and Research Center (BCRC), the registration date is March 24, 105, the registration number is BCRC 970070, BCRC 970071, BCRC 970072.

no.

Claims (5)

A live vaccination vaccine for avian VII genotype Newcastle disease, which comprises at least a live toxic antigen solution and a diluent; wherein the live vaccination vaccine is a non-recombinant vaccine; the live toxic antigen liquid system is for utilizing chickens from Newcastle disease The virus strain is infected with SPF chicken embryo allantoic sac and/or BHK-21 cells; the virus strain is deposited in the Food Industry Development Research Institute, and the registration number is BCRC 970070, BCRC 970071, or BCRC 970072, The date is March 24, 105, the Republic of China; wherein the content of the live antigen solution is in the range of 95% by volume to 50% by volume; and the content of the diluent is in the range of 5% by volume to 50% by volume. The dilution was prepared by mixing pure water, phosphate buffered saline (PBS), and 5% dextrose. A method for preparing a live vaccination vaccine for avian VII genotype Newcastle disease, which is a live vaccination vaccine for avian VII genotype Newcastle disease described in claim 1, which comprises: (a) screening from a field Newcastle disease chicken The virus strain is deposited in the Food Industry Development Research Institute, and the registration number is BCRC 970070, BCRC 970071, or BCRC 970072, and the registration date is March 24, 105, the Republic of China; (b) will be in the above steps ( a) the obtained virus strain is cultured in SPF chicken embryo allantoic sac and/or BHK-21 cells to obtain a live toxic antigen liquid containing avian VII genotype Newcastle disease virus strain; wherein the virus strain is in the SPF chicken The embryonic allantoic sac was cultured at 37 ° C in a 9-day-old chicken embryo allantoic sac without specific pathogen; the virus strain was in the cell culture incubator at 37 ° C, 4.5% to 5.5% carbon dioxide concentration of the BHK-21 cell line. to cultivate; (c) The live toxic antigen solution and the diluted solution obtained in the above step (b) are thoroughly mixed to form a live vaccination vaccine for avian VII genotype Newcastle disease. A deactivated vaccine for avian VII genotype Newcastle disease, which comprises at least a deactivated antigen solution and an oily adjuvant; wherein the deactivated vaccine is a non-recombinant vaccine; the deactivated antigenic liquid system is utilized for the chicken The virulent antigen solution obtained by infecting the SPF chicken embryo allantoic sac and/or BHK-21 cells is obtained by deactivation treatment; the virus strain is deposited in the Food Industry Development Research Institute, and the registration number is BCRC. 970070, BCRC 970071, or BCRC 970072, date of registration of the Republic of China on March 24, 105; the oily adjuvant is at least one of a mineral white oil, an emulsifier, pure water, and a mixture thereof. The deactivated vaccine for avian VII genotype Newcastle disease according to claim 3, wherein the content of the deactivated antigen solution is in the range of 95% by volume to 50% by volume; and the content of the oily adjuvant is 5% by volume to 50% by volume range. A method for preparing a deactivated vaccine for avian VII genotype Newcastle disease, which is a live vaccination vaccine for avian VII genotype Newcastle disease described in claim 3 or 4, which comprises: (a) from the field Xincheng disease chicken Only the virus strain is screened; the virus strain is deposited in the Food Industry Development Research Institute, and the registration number is BCRC 970070, BCRC 970071, or BCRC 970072, and the storage date is March 24, 105, the Republic of China; (b) The virus strain obtained in the step (a) is cultured in an SPF chicken embryo allantoic sac and/or BHK-21 cell to obtain a live toxic antigen liquid containing the avian VII genotype Newcastle disease virus strain; wherein the virus strain is The culture conditions in the SPF chicken embryo allantoic sac were cultured at 37 ° C in the 9-day-old chicken embryo allantoic sac without specific pathogen; The culture condition of the virus strain in the BHK-21 cells was cultured in a cell culture incubator at 37 ° C and a carbon dioxide concentration of 4.5% to 5.5%. (c) deactivating the antigen solution obtained in the above step (b) at 37 ° C in the presence of formalin (100 ppm to 300 ppm) for 12 to 24 hours to obtain a deactivated antigen solution; (d) The deactivated antigen solution obtained in the step (c) and the oily adjuvant are thoroughly mixed to form a deactivated vaccine of avian VII genotype Newcastle disease.