TWI597363B - Suitable for Type VII Newcastle disease vaccine strains - Google Patents

Description

Virus strain suitable for type VII Newcastle disease vaccine

The present invention relates to a virus strain of type VII Newcastle disease vaccine and its gene coding, in particular to a virus strain suitable for type VII Newcastle disease vaccine, a coding gene thereof and a coding gene thereof.

According to the research report, Newcastle disease (ND) is the most common malignant infectious disease in various poultry diseases. Since the discovery of Newcastle disease in 1926, it has spread to most countries in the world. The popularity of the region is not the same.

Chicken Newcastle disease was first discovered in Indonesia in 1926 and soon discovered in the New Town of the United Kingdom. Newcastle disease (ND) is an acute infectious disease caused by Newcastle Virus (NDV), which first broke out in New Towns in Indonesia and the United Kingdom in 1926, hence the name Newtown Virus (Alexander DJ, 1982). It belongs to Paramyxoviridae , a member of the Rubulavirus family (Rima BK et al., 1995) and belongs to the first paramyxovirus of poultry. The genome of the Newcastle virus is a single-stranded negative-strand RNA containing approximately 15 kb of base and six major structural proteins: 3'-NP-PMF-HN-L-5'.

In recent years, due to the avian paramyxovirus (Avian Paramyxovirus, APMV) is often found in between the many types of poultry, pigeons and other birds; when infected with the virus, often resulting in a variety of clinical symptoms in birds, for example, caused by avian Respiratory, digestive and neurological disorders. Due to the large difference in toxicity between various types of Newcastle virus, the incidence of Newcastle disease, pathogenic symptoms, pathological signs, and mortality rate are also significantly different depending on the type of virus. Although the symptoms are not significant at the time of mildness, but death is caused in severe cases, it has recently caused special attention, attention and continuous research and investigations related to such pathogens in countries around the world.

Newcastle viruses can be classified into three categories: velogenic, mesogenic, and lentogenic depending on the severity of infection and the severity of clinical symptoms caused by infection (Alexander D J, 1997). NDV virulent strains are highly infectious and usually characterized by strong infections; while NDV attenuated strains are weakly infectious and pathogenic, generally without acute infections, in fact, multiple strains including La Sota Attenuated NDV is often used as a live vaccine in production.

However, because of the high mortality rate caused by the strong virulence infection of Newcastle disease, it is a serious hazard to the chicken industry. There are no effective treatments, and they can only rely on strict disinfection, isolation, and the use of live vaccines and deactivated vaccines to prevent vaccination. At present, vaccines against Newcastle disease mainly use LaSota and Hitchner B1 vaccines or inactivated vaccines, and these two viruses have been used as vaccine strains for decades. Both LaSota and Hitchner B1 strains belong to the gene type II, and the current strains of the Asian region are mainly gene type VIIV. Therefore, the above two vaccines can not provide strong immune protection in clinical applications, and this situation can also explain the reasons for the outbreak of Newcastle disease in some areas.

In addition, based on the existing vaccine quality problems, immunization methods, immunization programs, chicken farm management standards, and the standard of chicken antibody levels, the immune failure of Newcastle disease is not uncommon. The virus strain which can be regarded as a good vaccine candidate needs to have the characteristics of weak virulence and good genetic stability. At present, the Newcastle virus isolated at home and abroad generally has a cleavage site, low titer and easy susceptibility of virulent strains. Disadvantages such as variation. Most of the F-protein cleavage site sequences of Newcastle virus are highly toxic, but the pathogenicity to chicken is weak. It is speculated that this virulence difference may be caused by the difference in viral replication ability. In order to solve this problem, it is necessary to study the epidemic characteristics and variation characteristics of the strains popular in the region, and to screen and isolate the virus strains with suitable vaccines to make the prevention and control of Newcastle disease more targeted and effective.

Therefore, it is not expected and eagerly expected to develop a virus strain of type VII Newcastle disease vaccine which can solve the problem of the conventional technology such as low immunity response, weak infectious power, and weak anti-epidemic effect.

In view of this, the present inventors have diligently studied and searched for various possible solutions for solving the problems of the conventional technology, and have selected a problem that not only improves the above-mentioned conventional technology, but also has no harm to the breeding environment and poultry health. It has an anti-epidemic effect that can effectively improve the host's immune response to Newcastle disease virus, the host's infectivity within a safe range, and the long-lasting antibody immunity, and can be made into a vaccine with long-term efficacy against Newcastle disease. The novel virus strain suitable for type VII Newcastle disease vaccine and its coding gene have heretofore completed the present invention.

Specifically, according to one aspect of the present invention, a virus strain suitable for type VII Newcastle disease vaccine and a coding gene thereof, wherein the biological registration number of the virus strain is BCRC 970070 and the name is avian type 7 Newcastle disease virus can be provided. Genotype VII Newcastle disease virus VII NDV/TW-CB-ND02 virus strain; the biological strain of the virus strain is BCRC 970071, the name is Genotype VII Newcastle disease virus VII NDV/TW-CB a virus strain of -ND04; or a virus strain of BCRC 970072, which is named as Genotype VII Newcastle disease virus VII NDV/TW-CB-ND06, which is a virus strain of the virus strain On March 24, 2016, the company was deposited at the Bioresource Collection and Research Center (BCRC) in the Republic of China, the Food Industry Development Institute.

In some embodiments, the virus strain of the present invention is applicable to a strain of type VII Newcastle disease vaccine and a gene encoding the same, wherein the nucleotide sequence of the virus strain having the F ₀ protein is SEQ ID NO: 1 to SEQ ID NO :3, which correspond to avian VII Newcastle disease virus VII NDV/TW-0CB-ND02, avian type VII Newcastle disease virus VII NDV/TW-CB-ND04, and avian type VII Newcastle disease virus VII NDV/CB-ND06.

In some embodiments, the virus strain of the present invention is applicable to a strain of type VII Newcastle disease vaccine and a gene encoding the same, wherein the nucleotide sequence of the virus strain having the HN protein is SEQ ID NO: 4 to SEQ ID NO: 6. Corresponding to avian type VII Newcastle disease virus VII NDV/TW-0CB-ND02, avian type VII Newcastle disease virus VII NDV/TW-CB-ND04, and avian type VII Newcastle disease virus VII NDV/CB-ND06.

Specifically, according to the present invention, in some specific embodiments, the virus strain of the present invention is applicable to a Newcastle disease type VII vaccine strain and a gene encoding the same, wherein the viral strain has a nucleotide sequence of the P protein as SEQ ID. NO:7~SEQ ID NO:9, which correspond to avian VII Newcastle disease virus VII NDV/TW-0CB-ND02, avian type VII Newcastle disease virus VII NDV/TW-CB-ND04, and avian type VII Newcastle disease virus VII NDV/CB-ND06.

In addition, in some specific embodiments, the present invention is applicable to a strain of a Newcastle disease type VII vaccine and a gene encoding the same, wherein the virus strain has a cut-off amino acid sequence of F ₀ protein (112-117) as SEQ ID NO: 12, any one of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

Furthermore, in some embodiments, the present invention is applicable to a virus strain of type VII Newcastle disease vaccine and a gene encoding the same, wherein the virus strain infects SPF chicken embryos has an average lethal time of 20 to 40 hours; after weakening The average lethal time of the virus strain exceeded 60 hours.

Moreover, in some embodiments, the present invention is applicable to a strain of a Newcastle disease type VII vaccine and a gene encoding the same, wherein the virus strain infects a brain pathogenicity index of SPF chickens. The average value is 1.7~2; after weakening, the average value of the pathogenicity index of the virus strain is below 0.4.

Wherein, in some embodiments, the virus strain of the present invention for use in a Newcastle disease type VII vaccine and a gene encoding the same, wherein the virus strain is used to infect at least one of chicken embryo culture, animal inoculation, or tissue culture. .

According to some embodiments, the virus strain of the present invention for use in a Newcastle disease type VII vaccine and a gene encoding the same, wherein the virus strain is for infecting at least one of Vero cells, BHK-21 cells, or MDCK cells.

Further, according to some embodiments, the virus strain of the present invention is applicable to a strain of type VII Newcastle disease vaccine and a gene encoding the same, wherein the nucleotide sequence of the virus strain, and one or more nucleotides of the nucleotide sequence It can be further replaced by other nucleotides.

Furthermore, according to certain embodiments, the virus strain of the present invention suitable for the Newcastle disease type VII vaccine and the gene encoding the same can be further used for an immunogenic composition, the immunogenic composition system can be included in A pharmaceutically acceptable carrier.

Furthermore, according to some specific embodiments, the virus strain of the present invention suitable for the Newcastle disease type VII vaccine and the gene encoding the same can be further used for a vaccine composition, wherein the vaccine composition can be pharmaceutically acceptable. Accepted carrier.

According to some specific embodiments, the virus strain of the present invention applicable to the type VII Newcastle disease vaccine and the gene encoding the same can be further used for a vaccine composition, wherein the vaccine composition is a monovalent vaccine composition.

According to certain embodiments, the virus strain of the present invention which is suitable for the Newcastle disease type VII vaccine and its coding gene can be further used for a vaccine composition which is a multivalent vaccine composition.

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 context, the term "viral strain suitable for type VII Newcastle disease vaccine and its coding gene" refers to a specific antigen that a virus strain can be used to induce a host immune response, including but not limited to whole virus, surface hemagglutination antigen ( Hemagglutinin surface antigen, surface neuraminidase surface antigen, and internal nucleocapsid antigen, or derivative.

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 "bird type VII Newcastle disease deactivated vaccine" as used herein refers to a vaccine for preventing a condition or disease caused by infection with avian type VII Newcastle disease virus strain. The avian VII genotype Newcastle disease deactivated vaccine may include any vaccine effective to treat or prevent avian VII genotype Newcastle disease infection. The preferred avian VII genotype Newcastle disease deactivated vaccine used in the method of the present invention is a virus strain vaccine.

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 deactivated vaccine that is sufficient to 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 invention separates a virus strain suitable for type VII Newcastle disease vaccine and its coding gene from field sampling.

Specifically, according to one aspect of the present invention, it is possible to provide a virus strain suitable for type VII Newcastle disease vaccine and a coding gene thereof, wherein the biological deposit number of the virus strain is BCRC 970070, and the name is avian type 7 Genotype VII Newcastle disease virus VII NDV/TW-CB-ND02 virus strain; the biological strain of the virus strain is BCRC 970071, the name is Genotype VII Newcastle disease virus VII NDV /TW-CB-ND04 virus strain; or the virus strain of the virus strain BCRC 970072, the name is Genotype VII Newcastle disease virus VII NDV/TW-CB-ND06, which The three strains were deposited on March 24, 2016 in the Bioresource Collection and Research Center (BCRC), located in the Republic of China, the Food Industry Development Institute.

According to some specific embodiments, the virus strain suitable for type VII Newcastle disease vaccine of the present invention and the gene encoding the same, the virus strain is applied to vaccine production, and the length of the nucleotide sequence of the F ₀ protein is not particularly The definition is as long as it does not contradict or deviate from the spirit of the present invention and can achieve the effect of causing a host immune response. For example, for a virus strain suitable for type VII Newcastle disease vaccine and a gene encoding the same, the nucleotide sequence of the F ₀ protein may be, for example, from the first nucleotide to about the 900th nucleotide. In certain embodiments, preferably from about 20 nucleotides to about 800 nucleotides; more preferably from about 50 nucleotides to about 700 nucleotides; Particularly preferred is in the range of from about 60 nucleotides to about 600 nucleotides; most preferably in the range of from about 100 nucleotides to about 500 nucleotides.

According to some specific embodiments, the virus strain suitable for the type VII Newcastle disease vaccine of the present invention and the gene encoding the same, the virus strain is applied to vaccine production, and the length of the nucleotide sequence of the HN protein is not particularly limited. It suffices that it does not contradict or depart from the spirit of the present invention and can achieve the effect of causing a host immune response. For example, for a virus strain suitable for type VII Newcastle disease vaccine and a gene encoding the same, the nucleotide sequence of the HN protein may be, for example, from the first nucleotide to about the 900th nucleotide; In certain embodiments, preferably from about 20 nucleotides to about 800 nucleotides; more preferably from about 50 nucleotides to about 700 nucleotides; Preferably, it is in the range of from about 60 nucleotides to about 600 nucleotides; most preferably in the range of from about 100 nucleotides to about 500 nucleotides.

According to some specific embodiments, the virus strain suitable for the type VII Newcastle disease vaccine of the present invention and the gene encoding the same, the virus strain is applied to vaccine production, and the length of the nucleotide sequence of the P protein is not particularly limited. It suffices that it does not contradict or depart from the spirit of the present invention and can achieve the effect of causing a host immune response. For example, for a virus strain suitable for type VII Newcastle disease vaccine and a gene encoding the same, the nucleotide sequence of the P protein may be, for example, from the first nucleotide to about 1188 nucleotides; In certain embodiments, preferably from about 20 nucleotides to about 1100 nucleotides; more preferably from about 50 nucleotides to about A range of 900 nucleotides; particularly preferably in the range of from about 60 nucleotides to about 850 nucleotides; most preferably in the range of from about 100 nucleotides to about 800 nucleotides.

According to some specific embodiments, the virus strain suitable for the type VII Newcastle disease vaccine of the present invention and the coding gene thereof, the viral strain having the amino acid sequence of the cut point of the F ₀ protein is not particularly limited, as long as it is not It is possible to contradict or depart from the spirit of the present invention and to achieve the amino acid sequence of the cleavage point of the F ₀ protein which causes host virulence. For example, for a virus strain suitable for type VII Newcastle disease vaccine and a gene encoding the same, the virus strain has a cut-point amino acid sequence of F ₀ protein, which may be, for example, ¹¹² RRQKRF ¹¹⁷ , ¹¹² RRQRRF ¹¹⁷ , ¹¹² KRQKRF ¹¹⁷ Or at least one of ¹¹² KRQRRF ¹¹⁷ ; in some embodiments, preferably at least one of ¹¹² RRQKRF ¹¹⁷ , ¹¹² RRQRRF ¹¹⁷ , ¹¹² KRQKRF ¹¹⁷ , or ¹¹² KRQRRF ¹¹⁷ ; more preferably ¹¹² RRQKRF ¹¹⁷ , ¹¹² RRQRRF ¹¹⁷ Or ¹¹² KRQKRF ¹¹⁷ at least one of them; particularly preferably ¹¹² RRQKRF ¹¹⁷ , or ¹¹² RRQRRF ¹¹⁷ .

According to some specific embodiments, the effect of the pathogenicity identification of the host mean time to death (MDT) of the virus strain suitable for the type VII Newcastle disease vaccine of the present invention and its coding gene is not particularly limited, as long as it is not It is possible to violate or depart from the spirit of the present invention and to achieve a range in which the average death time of the host is caused. For example, for a virus strain suitable for type VII Newcastle disease vaccine and a gene encoding the same, the virus strain causes the average death time of the host, for example, may range from about 1 hour to about 120 hours; In the embodiment, it is preferably in the range of about 5 hours to about 110 hours; more preferably in the range of about 10 hours to about 90 hours; particularly preferably in the range of about 20 hours to about 80 hours. The best is in the range of about 30 hours to about 60 hours.

Specifically, according to one aspect of the present invention, a virus strain suitable for type VII Newcastle disease vaccine and a gene encoding the same can be provided, wherein after the virus strain is weakened, the average lethal time of the virus strain exceeds 60 hours.

According to some embodiments, the effect of the pathogenicity identification of the pathogenicity index (ICPI) of the host brain in the virus strain of the type VII Newcastle disease vaccine of the present invention and the gene encoding the same is not particularly limited, as long as It is within the scope of not departing from or departing from the spirit of the invention and capable of achieving a pathogenicity index which causes vaccination in the host brain. For example, for a virus strain suitable for type VII Newcastle disease vaccine and a gene encoding the same, the virus strain inoculates a host with a pathogenicity index, for example, may range from about 0 to about 2; In some embodiments, it is preferably in the range of from about 0.5 to about 1.99; more preferably in the range of from about 0.6 to about 1.95; more preferably in the range of from about 0.7 to about 1.9; A range of from about 0.9 to about 1.85.

Specifically, according to one aspect of the present invention, a virus strain suitable for type VII Newcastle disease vaccine and a coding gene thereof can be provided, which is an average of the pathogenicity index of the virus strain after the virus strain is weakened. The value is below 0.4.

Wherein, in some specific embodiments, the virus strain of the present invention is applicable to the VII type Newcastle disease vaccine and the gene encoding the same, wherein the virus strain is not particularly limited for use in the method of infection culture, as long as it does not violate or It is possible to leave the spirit of the invention. For example, it may be chicken embryo culture, animal inoculation, or tissue culture. According to some embodiments of the present invention, the virus strain is used for infection culture, for example, at least one of chicken embryo culture, animal vaccination, or tissue culture; and the novel VII type vaccine suitable for use in the present invention The virus strain and its coding gene line are used for infection culture. The preferred source of use is chicken embryo culture, animal inoculation, or tissue culture; the better use source is chicken embryo culture, or tissue culture; the best use source is chicken embryo culture. .

According to one aspect of the present invention, the virus strain of the present invention for use in a Newcastle disease type VII vaccine and a gene encoding the same, wherein the virus strain is used for the type of infected cells is not particularly limited as long as it does not contradict or deviate from the present invention. The spirit can be. For example, it may be Vero cells, BHK-21 cells, or MDCK cells. According to some embodiments of the invention, the The cell strain derived from the infected cell species may be, for example, at least one selected from Vero cells, BHK-21 cells, or MDCK cells; the virus cell strain suitable for the type VII Newcastle disease vaccine of the present invention and the infected cell type encoding the gene thereof Preferably, the source is Vero cells, BHK-21 cells, or MDCK cells; more preferably the source is Vero cells, or BHK-21 cells; a particularly useful source is Vero cells.

Specifically, according to one aspect of the present invention, a virus strain suitable for type VII Newcastle disease vaccine and a coding gene thereof, wherein the nucleotide sequence of the virus strain, and one or more nucleosides in the nucleotide sequence can be provided The acid can be further replaced by other nucleotides.

Further, in particular, according to one aspect of the present invention, a virus strain suitable for type VII Newcastle disease vaccine and a gene encoding the same can be provided, and further used for an immunogenic composition comprising a pharmaceutically acceptable carrier .

Further, in particular, according to one aspect of the present invention, a virus strain suitable for a Newcastle disease type VII vaccine and a gene encoding the same can be provided, and further used in a vaccine composition comprising a pharmaceutically acceptable carrier.

Furthermore, in particular, according to one aspect of the present invention, a virus strain suitable for the VII type Newcastle disease vaccine and a gene encoding the same can be provided, and further used for a vaccine composition which is a monovalent vaccine composition.

Further, in particular, according to one aspect of the present invention, a virus strain suitable for type VII Newcastle disease vaccine and a gene encoding the same can be provided, and further used for a vaccine composition which is a multivalent vaccine composition.

S1‧‧‧ steps

S2‧‧‧ steps

S3‧‧‧ steps

S4‧‧‧ steps

S5‧‧ steps

S6‧‧ steps

W‧‧‧ specimen

Y‧‧‧ virus strain

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart schematically showing the steps of collecting a type VI Newcastle disease vaccine strain in a specific embodiment of the present invention: In Fig. 1, step S1 represents a poultry farm from a Newcastle disease (NDV) infection. The step of collecting the virus strain; the step S2 is a step of culturing the virus strain collected in the step 1 in the SPF chicken embryo chorioallantoid; the step S3 is a step of observing whether the infected cells have virus spots; the step S4 is indicating The step of measuring the average lethal time of the infected chicken embryo; the step S5 is a step of measuring the pathogenicity index of the infected chicken embryo; and the step S6 is for performing the type VII Newcastle disease vaccine strain collected in the step S1. The steps of nucleotide sequencing of F ₀ protein, HN protein and P protein; W line indicates sample V1~V5; Y line indicates virus strain TW-CB-ND01, TW-CB-ND02, TW-CB-ND03, TW-CB-ND04, TW-CB-ND06.

Hereinafter, the isolation and screening methods of the virus strain of the present invention will be further described by way of examples.

<<Example 1 to Example 5>> "Field field collection virus strain"

First, according to the collection procedure of the virus strain of type VII Newcastle disease vaccine shown in Figure 1, the poultry farm that has been infected with Newcastle disease (NVD) in Taiwan, from the lungs, trachea, intestines of infected chickens, and A histopathological sample was collected from the brain, and a total of 5 samples were collected, each weighing 10 grams each. At room temperature, the five histopathological samples were placed in a modified minimal essential medium containing 2% fetal bovine serum, and thoroughly ground with a homogenizer; the suspensions were respectively taken, followed by After centrifugation at 1,500 rpm for 15 minutes, it was filtered through a 0.2 μm filter; the upper sputum was taken as a sample V1-V5.

"Chicken embryo allantoic cavity culture virus strain"

Next, 50 ml of the sample V1-V5 was inoculated into the chicken embryo anterior chamber of the 9-day-old specific pathogen free (SPF), and then placed in an incubator at 37 ° C, and culture was continued for 7 days. Observe the daily death of chicken embryos. The pathological anatomy of the dead chicken embryo was performed and the liquid in the allantoic cavity was collected as a sample of the virus strain, encoding TW-CB-ND01, TW-CB-ND02, TW-CB-ND03, TW-CB-ND04, TW-CB. -ND06.

"Infected specimens containing Newcastle disease virus have virus spots"

First, the virus strain samples were separately cultured with Vero cell strain (green monkey kidney cells, ATCC: CCL81) in a 6-well culture dish in DMEM medium containing 10% fetal bovine serum (FBS) (Dulbecco's Cultured in a culture environment of modified Eagle medium) (5 ml/well), 5% CO ₂ and 37 °C.

The above cells were grown to a cell density of about 80%, which was referred to as a monolayer of Vero cells. The monolayer of Vero cells were removed from the original medium, washed once with phosphate-buffered saline (PBS, pH=7.2), and then 0.5 ml of the culture solution containing the virus strain sample was placed in each well to carry out the culture solution. The dilutions were 10 ¹ times, 10 ² times, 10 ³ times, 10 ⁴ times, 10 ⁵ times, and ¹⁰⁶ times, respectively, and the last well was 0.5 ml of the cell culture liquid control group.

The culture plate was placed in an environment of 5% CO ₂ and 37 ° C for one hour; the plate was gently shaken every 15 minutes during the action to allow the culture solution containing the virus strain sample to uniformly cover the cell layer.

Then, the culture medium containing the virus strain sample was removed, and 4 ml of a maintenance medium containing 1% agar (agar) was added to each well, and the cells were cultured in a culture environment of 5% CO ₂ and 37 ° C, and 48 after the culture. Hours, selected cultured cells were infected with virus to produce significant CPE, and 0.5 ml of 0.1% neutral red solution was added to each well, wrapped in tin foil, placed at 5% CO ₂ and 37 ° C. The culture was carried out in a culture environment, and after culturing overnight, the state of occurrence of virus spots was observed.

The samples of the NDV strains according to Examples 1 to 5 of the present invention were subjected to CPE results after infection of the cells, and the statistics are shown in Table 1.

Identification of Newcastle Disease Virus

1. Mean death time (MDT)

At room temperature, diluted with the PBS 10 ^{6-fold, 107-fold, 108-fold, 109-fold} dilution of Example 1 to Example 5 of the embodiment samples strain TW-CB-ND01 ~ TW- CB-ND06 containing In the solution, 0.1 ml each was inoculated into 5 9-10 day old SPF chicken embryos, and then placed in an incubator at 37 ° C. The eggs were examined twice a day, and the green chicken embryos were recorded. The time of death was continuously implemented for 7 days and the minimum lethal dose (MDT) was determined.

The results of MDT identification of NDV of Examples 6 to 10 are shown in Table 3.

According to the MDT test results of the TW-CB-ND02, TW-CB-ND04, and TW-CB-ND06 Newcastle disease virus strains of Example 6 to Example 10 of Table 3, the MDT can be confirmed to be 44 hours to 48 hours. Therefore, the three strains of TW-CB-ND02, TW-CB-ND04, and TW-CB-ND06 are virulent strains, and it can be confirmed that the virus strain of the virus strain of the present invention is significantly superior to the current commercial vaccine. The virus strain is, for example, a BC virus strain (MDT: 62 hours), a LaSota virus strain (MDT: 90 hours), and the like.

On the other hand, the MDT of TW-CB-ND01 of Example 6 and TW-CB-ND03 of Example 8 exceeded 120 hours or more, and thus TW-CB-ND01 and TW-CB-ND03 were weak virus strains.

Intracerebral pathogenicity index (ICPI)

First, to measure the highest dilution factor of 1 unit (1HAU) of antigen, there is still a hemagglutination effect of HA. The first time, 50 μL of HI buffer (3.25g KH ₂ PO ₄ , 10.8g) is added to each well of U-shaped plate. Na ₂ HPO ₄ , 170 g NaCl, D ₃ W was added to 2,000 mL).

50 μl of the serum virus antigen to be tested was added to the first well, mixed well with physiological saline, and 25 μl of the solution was injected into the second well, thus performing two-fold serial dilution, and the last well was discarded with 25 μl of the mixed solution.

Finally, 50 μL of 1% chicken red blood cell solution (cRBC) was added to each well, and it was interpreted after 30 minutes at room temperature. The highest titer of the antigen still has the hemagglutination effect of the HA, which is 1 unit (1HAU). The highest dilution factor of the antigen is still the hemoglobin agglutination.

Samples of virus strains were taken and the HA price was higher than 16 and diluted 10 times with normal saline. In each group, 10 animals were inoculated in the brain of 1 day old SPF chicken, each 0.05 ml, observed every 24 hours after inoculation, continuous observation On the 8th, the normal chicken record value was 0, sick chicken 1, dead chicken 2, and ICPI was the average value of each chicken per day for all 8 days.

The results of ICPI identification of NDV of Examples 11 to 15 are shown in Table 4.

According to the results of the ICICP test of TW-CB-ND02, TW-CB-ND04, and TW-CB-ND06 Newcastle disease virus strains of Examples 11 to 15 of Table 4, it can be confirmed that the average value of ICPI is between 1.7 and 1.8. Therefore, the three strains of TW-CB-ND02, TW-CB-ND04, and TW-CB-ND06 are virulent strains, and it can be confirmed that the virus strain of the virus strain of the present invention is significantly superior to the current commercial vaccine. The virus strain is, for example, a BC virus strain (ICPI: 0.25), a LaSota virus strain (ICPI: 0.5), or the like.

On the other hand, the ICPI of TW-CB-ND01 of Example 11 and TW-CB-ND03 of Example 13 were all below 0.3, and thus TW-CB-ND01 and TW-CB-ND03 were weak virus strains.

"Sequencing of Newcastle Disease Virus"

First, RNA was extracted from the Newcastle disease virus strain of the present invention, but in order to avoid RNase contamination of the experimental apparatus and digestion of viral RNA, all the solutions used for contacting RNA were treated with diethylpyrocarbonate (DEPC). Formulated in distilled water. After autoclaving, other plastic materials should also be placed in a specific storage area dedicated to the operation of RNA viruses to avoid mixing with non-RNA materials.

Then, the above-mentioned infected specimens V1-V5 collected from the allantoic cavity were mixed with 250 μL of allantoic fluid and 750 μL of TriZole (Invitrogen) for 5 minutes, and 200 μL of chloroform was added and mixed to a chyle-like form. Stand still for 10 minutes and then at 13,000 rpm, 4 Centrifuge at °C for 10 minutes, take about 500 μL of the upper layer of water to a new centrifuge tube, add equal amount of isopropanol, mix well, centrifuge at 13,000 rpm for 10 minutes at 4 ° C, discard the supernatant, and add 1 mL of 70% alcohol. The salt was removed and centrifuged at 13,000 rpm for 10 minutes at 4 ° C. The supernatant was discarded, and the precipitate was dried in an oven at 50-60 ° C. The precipitate was suspended in 50 μL of DEPC-treated water to infect the sample (I1 to I5).

Furthermore, the infected samples (I1 to I5) were first cultured with Vero cell strain in DMEM medium containing 2% fetal calf blood, and then commercially available ribonucleotide (RNA) extraction combination kit (QIAamp) was used. Viral RNA mini kit, Qiagen Inc., Santa Clara, CA), RNA from infected samples (I1 to I5) was extracted from infected cell lines into RNA samples (R1 to R5) infected with the virus.

Using the RNA sample (R1~R5) as a template, the full-length gene of the virus was copied into several fragments by using the reverse transcription-polymerase chain reaction (RT-PCR) technique commonly used in the laboratory. The first cDNA synthesis was performed.

Subsequently, a polymerase chain reaction (PCR) was carried out, and the primer sequences SEQ ID NO: 10 and SEQ ID NO: 11 in Table 5 were used as primers for the polymerase chain reaction, and the reaction conditions were first heated at 94 ° C, 5 In minutes, the protein enzymes present in the reaction mixture were destroyed and 35 temperature cycles were performed. Each reaction was a denaturation reaction at 94 ° C for 1 minute, a refining reaction at 55 ° C for 1 minute, and a DNA synthesis reaction at 72 ° C for 1 minute per 1 Kb. After 35 cycles of reaction, the cells were replenished at 72 ° C for 10 minutes to complete the ends of the PCR synthesis product. After the reaction was completed, the product of the F protein of the target gene was obtained.

After the PCR reaction. 5 μL of the DNA was electrophoresed on a 1% agarose film, and the size of the DNA fragment in the PCR product was analyzed as "Table 6".

Next, the products of the above Examples 16 to 20 were placed on ice, and 45 μL of the PCR product was subjected to electrophoresis on a 1% agarose film. After completion of the electrophoresis, the colloid was placed in a 5 μg/mL EB (ethidium bromide) staining solution, soaked for 5 minutes, and then faded with secondary distilled water for 10 minutes.

The film of the specific DNA fragment was cut with a surgical blade under a UV lamp having a wavelength of 320 nm. Place the microcentrifuge tube that has been pre-weighed and weigh the total weight to determine the weight of the film.

DNA was recovered from the agarose gel using a VIOGENE Gel Extraction System. First, 0.5 mL buffer GEX was added to the microcentrifuge tube containing the colloid. The tube was placed in a water bath at 60 ° C for 10 minutes, and the tube was inverted every 2 minutes to uniformly suspend the GEX in the solution. The gel extraction column was placed in a collection tube, and the dissolved gel and GEX mixture was added to a concentration column at 13,000 rpm for 30 seconds to remove the filtrate; 0.5 mL of Washing Buffer was added to centrifuge at 13,000 rpm. 30 seconds, discard the filtered liquid; add 0.7mL Washing Buffer, centrifuge at 13000rpm for 30 seconds, then discard the filtrate; put the concentrated column into a new collection tube, add 30μL of treated Distilled water (DEPC), centrifuged at 13,000 rpm for 2 minutes after standing for 1 minute, collected the filtrate to achieve purification of the genetic material of the Newcastle disease virus of the present invention, and directly performed the amino acid sequence of the cut point of the F ₀ protein of the Newcastle disease virus strain, and Full-gene nucleotide sequence analysis (using a fully automated analyzer), the results are shown in Tables 7 and 8.

Table 7 shows the results of analysis of the amino acid sequence of the cleavage point of the F ₀ protein of NDV of Examples 21 to 25. According to the amino acid sequence data of the cut point of the F ₀ protein described in Table 7, it was confirmed that TW-CB-ND02 of Example 22, TW-CB-ND04 of Example 24, and TW-CB-ND06 of Example 26 were strong. The strain can also confirm that the virus strain of the present invention has the amino acid sequence of the cut point of the strong virus strain F ₀ protein, which is significantly different from the virus strain of the currently commercially available vaccine, for example, the BC virus strain ( ¹¹² GRQGRL ¹¹⁷ ) and the LaSota virus strain ( ¹¹² GRQGRL ¹¹⁷ ) and so on.

Further, it was confirmed that CB-ND01 of Example 21 and TW-CB-ND03 of Example 23 were attenuated strains.

Therefore, the Newcastle disease virulent strains of TW-CB-ND02, TW-CB-ND04, and TW-CB-ND06 have two pairs of basic amino acids separated by Q and are easily recognized by the host's protease (furin). Lysis; TW-CB-ND01, TW-CB-ND03 Attenuated strain of Newcastle disease does not have two pairs of basic amino acids separated by Q, but is transferred to the offspring in the form of inactive precursor F ₀ protein, which is not easy to be The host's protease (furin) recognizes and cleaves, and the infection activity is reduced or lost.

In summary, the virus strains of the present invention applicable to the type VII Newcastle disease vaccine and the coding genes thereof TW-CB-ND01, TW-CB-ND02, TW-CB-ND03, TW-CB, which are exemplified in Examples 1 to 25 -ND04, TW-CB-ND06, which is TW-CB-ND02, TW-CB-ND04 and TW-CB-ND06 are strong virus strains; TW-CB-ND01 and TW-CB-ND03 are weak virus strains.

Therefore, it was confirmed that the virus strain of the present invention can be compared with the virus strain of the commercially available vaccine because the infection effect of MDT is 50% stronger, and the average value of ICPI is nearly twice as high. The avian VII genotype Newcastle disease virus strain of the present invention can obviously cause an immune response in chickens, and can be developed into a vaccine having an anti-VII genotype Newcastle disease infection and related applications.

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.

【Biomaterial Storage】

TW Republic of China, Bioresource Collection and Research Center (BCRC), the storage date is March 24, 2016, the registration number is BCRC 970070, BCRC 970071, BCRC 970072.

<110> Guonian Industrial Co., Ltd.

<120> Virus strain suitable for type VII Newcastle disease vaccine and its coding gene

<160> 15

<210> SEQ ID NO: 1

<211> 529

<212> DNA

<213> Chicken Seven Newcastle Disease Virus

<400> 1

<210> SEQ ID NO: 2

<211> 389

<212> DNA

<213> Chicken Seven Newcastle Disease Virus

<400> 2

<210> SEQ ID NO: 3

<211> 374

<212> DNA

<213> Chicken Seven Newcastle Disease Virus

<400> 3

<210> SEQ ID NO: 4

<211> 426

<212> DNA

<213> Chicken Seven Newcastle Disease Virus

<400> 4

<210> SEQ ID NO: 5

<211> 1716

<212> DNA

<213> Chicken Seven Newcastle Disease Virus

<400> 5

<210> SEQ ID NO: 6

<211> 1716

<212> DNA

<213> Chicken Seven Newcastle Disease Virus

<400> 6

<210> SEQ ID NO: 7

<211> 1188

<212> DNA

<213> Chicken Seven Newcastle Disease Virus

<400> 7

<210> SEQ ID NO: 8

<211> 1188

<212> DNA

<213> Chicken Seven Newcastle Disease Virus

<400> 8

<210> SEQ ID NO: 9

<211> 1188

<212> DNA

<213> Chicken Seven Newcastle Disease Virus

<400> 9

<210> SEQ ID NO: 10

<211> 20

<212> DNA

<213> Artificial sequence

<400> 10

<210> SEQ ID NO: 11

<211> 25

<212> DNA

<213> Artificial sequence

<400> 11

<210> SEQ ID NO: 12

<211> 6

<212> PRT

<213> F ₀ protein of chicken type 7 Newcastle disease virus

<400> 12

<210> SEQ ID NO: 13

<211> 6

<212> PRT

<213> F ₀ protein of chicken type 7 Newcastle disease virus

<400> 13

<210> SEQ ID NO: 14

<211> 6

<212> PRT

<213> F ₀ protein of chicken type 7 Newcastle disease virus

<400> 14

<210> SEQ ID NO: 15

<211> 6

<212> PRT

<213> F ₀ protein of chicken type 7 Newcastle disease virus

<400> 15

Claims (8)

A virus strain suitable for the VII type Newcastle disease vaccine, which is a virus with a biological registration number of BCRC 970070 and a name of Genotype VII Newcastle disease virus VII NDV/TW-CB-ND02 The virus strain of the virus strain is BCRC 970071, the virus strain named Genotype VII Newcastle disease virus VII NDV/TW-CB-ND04: or the biological registration number of the virus strain is BCRC 970072, a virus strain named Genotype VII Newcastle disease virus VII NDV/TW-CB-ND06, which was deposited on March 24, 2016 in the Republic of China, the corporation. The Bioresource Collection and Research Center (BCRC); and the virus strain is cultured in any one of a chicken embryo process and a cell culture process. The virus strain suitable for the Newcastle disease type VII vaccine according to claim 1, wherein the nucleotide sequence of the virus strain having the F ₀ gene is any one of SEQ ID NO: 1 to SEQ ID NQ: 3. The virus strain suitable for the Newcastle disease type VII vaccine according to claim 1, wherein the virus strain has any one of the nucleotide sequences of the HN gene of SEQ ID NO: 4 to SEQ ID NO: 6. The virus strain suitable for the Newcastle disease type VII vaccine according to claim 1, wherein the virus strain has any one of the nucleotide sequences of the P gene of SEQ ID NO: 7 to SEQ ID NO: 9. The virus strain suitable for the Newcastle disease type VII vaccine according to claim 1, wherein the virus strain has the amino acid sequence of the cut point of the F ₀ protein, that is, the amino acid at positions 112 to 117 is SEQ ID NO: 12, SEQ. ID NO: 13. Any one of SEQ ID NO: 14. The virus strain suitable for the type VII Newcastle disease vaccine described in claim 1, wherein the virus strain infects SPF chicken embryos has an average lethal time of 20 to 50 hours; after weakening, the average lethal time of the virus strain exceeds 60 hours. . The virus strain suitable for the type VII Newcastle disease vaccine described in claim 1, wherein the average value of the pathogenicity index of the virus strain infected with the SPF chicken is 1.7~2; after weakening, the brain of the virus strain The average value of the internal pathogenicity index is below 0.4. The virus strain suitable for the Newcastle disease type VII vaccine according to any one of claims 2 to 4, wherein one or more nucleotides in the nucleotide sequence of the virus strain can be further replaced by other nucleotides .