CN109790522B - Attenuated swine fever virus-marked live vaccine strain and vaccine composition for oral administration using the same - Google Patents
Attenuated swine fever virus-marked live vaccine strain and vaccine composition for oral administration using the same Download PDFInfo
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Abstract
The present invention relates to an attenuated swine fever virus-marked live vaccine strain and a vaccine composition for oral administration using the same, and more particularly, to an attenuated swine fever virus-marked live vaccine strain (CSFV BErns attenuated C-P50), a vaccine composition for oral administration comprising the same, and a serological identification method of a swine infected with swine fever virus in the wild and a swine vaccinated with the attenuated swine fever virus-marked live vaccine strain. The vaccine composition for oral administration of the present invention is formulated into a bait formulation, so that pigs can easily ingest the vaccine composition, and the problem that the vaccine is difficult to administer to breeding wild pigs and black pigs which are fiercely used as injection only is solved. The serological identification method can identify antigens and antibodies of pigs infected with the swine fever virus in the wild and pigs inoculated with the attenuated swine fever virus marked live vaccine strain, so that the DIVA has excellent effect.
Description
Technical Field
The present invention relates to an attenuated swine fever virus-marked live vaccine strain (attenuated swine fever virus live marker vaccine strain) and a vaccine composition for oral administration using the same, and more particularly, to an attenuated swine fever virus-marked live vaccine strain (CSFV BErns attenuated C-P50) obtained by subjecting an existing swine fever virus vaccine strain Flc-LOM-BErns to an alternate continuous culture between cells, a vaccine composition for oral administration comprising the same, and a serological identification method of swine infected with swine fever virus in the wild and swine vaccinated with the attenuated swine fever virus-marked live vaccine strain.
Background
Swine fever Virus (Classical Swine Fever Virus: CSFV) is an RNA virus of about 40-50 nm in size and belongs to the Flaviviridae family. Furthermore, it is a virus belonging to the genus Pestivirus (Pestivirus), consisting of a 12.3kb gene consisting of a sense single strand RNA (positive sense single strand RNA) and a protein synthesized from about 3,900 amino acids.
The symptoms of swine infected with swine fever virus are various according to the sensitivity, age and virus type of swine, but the symptoms of inappetence, diarrhea or constipation, skin cyanosis, abnormal use of hind legs or the carrier, etc. generally occur while hyperpyrexia occurs. In addition, it is a very important disease, since the infectious agent is very strong, the infected pigs are almost all dead regardless of age, so that it is prescribed as a first type of livestock infectious disease of korean livestock infectious disease prevention method, and the world animal health Organization (OIE) also regards swine fever as an eradication subject, taking measures to prohibit import, export, etc. in epidemic situation-occurring countries.
Swine fever virus is transmitted to other individuals by the infection of swine feces, urine, tears, nasal discharge, etc., but may also be transmitted through agents such as human shoes, clothing, and instruments. The virus mainly invaded by digestive tract and respiratory tract proliferates in tonsils, lymph nodes and cells of solid organs to cause toxemia, and under the condition of acute infection, symptoms such as hyperpyrexia, redness of skin, lack of appetite, constipation, diarrhea, leukopenia, paralysis and death can appear. In addition, in the case of pregnant pigs, the virus infects the fetus through the placenta and symptoms such as resorption, abortion, stillbirth and weak piglets are present depending on the age of the fetus. Furthermore, a large amount of virus is discharged through the feces of the sick pigs, so that secondary infection may also occur.
The incubation period of classical swine fever virus is mainly 6 to 11 days, but may also be 20 to 30 days, so that there is a relatively high risk of spreading when moving before clinical symptoms occur, and thus it is important to diagnose and prevent spreading at an early stage.
Although swine fever virus does not occur in the united states and japan, infection of farmed pigs by wild boars frequently occurs in a plurality of countries in europe. The southeast asia or northeast asia, china, etc. are continuously occurring, and the world animal health Organization (OIE) only acknowledges that countries that do not use vaccines and that do not have an epidemic situation for more than 1 year are clean countries. However, if vaccines and diagnostic methods containing hog cholera signatures are commercialized in this country and their efficacy is prominent, OIE will recognize that this country is a clean country and encourage its use worldwide.
The swine fever virus vaccine in use in korea was an attenuated live vaccine (live attenuated vaccine) LOM strain used since 1976, which is the only vaccine prepared by korean manufacturers and used nationwide in a manner of full delivery, and the use of other vaccine strains was prohibited according to national policies. In order to commercialize a novel swine fever vaccine strain, it is necessary to make a decision by a swine fever extermination committee composed of civil authorities, to perform a thorough field clinical experiment by a small committee composed of technical experts, and to uniformly replace the vaccine strain by a government after policy advice. The swine fever LOM vaccine strain currently in use after this system shows excellent immunogenicity during the use for 40 years, thereby taking the responsibility of eradicating korean swine fever. However, experts and first-line pig raising personnel are continually raising problems with pathogenicity and safety of LOM vaccines.
As a representative event that raises the safety problem of LOM vaccine strain, there is an event that korean has been given a non-vaccine policy after the declaration of swine fever cleaning in 2002, but swine fever has occurred again in 2002 and 2003 nationwide, so LOM vaccination of all pigs nationwide has been restarted since 2004, and many cases of abortion and stillbirth have occurred in vaccination of pregnant sows, and this has become the focus. In addition, the blood meal preparation products used as feed additives in the blood meal preparation product of the swine fever non-vaccine area since 1999 were unintentionally contaminated and circulated with the swine fever LOM vaccine strain, whereby very similar symptoms to swine fever occurred in 150 farmed vaccine strains among 300 farmers of the swine fever island, and specific lesion symptoms occurred in the parenchymal organs when the swine was necropsied, so the pathogenicity disputes for the LOM vaccine strain were continuously expanded until the infected swine of the swine fever island disappeared in 2009. Recently in 2014, the island of Jizhou developed improper circulation of swine fever LOM vaccine strain again and inoculated to pregnant sows, thereby confirming abortion, stillbirth and vertical infection of fetal LOM vaccine strain, and thus the problem of vaccine safety became the focus again.
In addition, LOM vaccine strains have a problem that it is difficult to identify serologically, that is, it is difficult to distinguish field strain-infected antibodies from LOM vaccine strains-infected antibodies in pigs, and attempts have been made to produce swine fever-marked live vaccine strains in the form of DIVA (distinguishing infected animals from vaccinated animals, differentiating infected in vaccinated animals) vaccine in order to solve the problem.
Thus, the present inventors developed a more attenuated swine fever marker live vaccine, and expected that the purification of swine fever in korea was advanced thereby, and that the excellent efficacy of the attenuated swine fever marker live vaccine was approved from OIE, thereby being popularized and used worldwide, contributing to eradication of swine fever. Moreover, a series of efforts have been made to prepare vaccine compositions in the form of baits for oral administration of vaccines to farmed wild or black pigs that are harsher in nature and difficult to handle and difficult to inject.
As a result, the present inventors confirmed that when a swine fever virus vaccine strain named Flc-LOM-BErns (refer to korean laid-open patent No. 2010-012688) in which the Erns gene of CSFV (swine fever virus, classical Swine Fever Virus) was replaced with the Erns gene of BVDV (bovine viral diarrhea virus, bovine Viral Disease Virus) was proliferated by alternate continuous culture between different types of specific cells, the virus titer was improved, and thus not only was excellent efficacy as a vaccine, but also attenuation was obtained as compared with the existing Flc-LOM-BErns vaccine strain, and thus the occurrence probability of side effects such as abortion and stillbirth was reduced in pregnant sows. Furthermore, it was found that serological identification of pigs inoculated with the attenuated vaccine strain and pigs infected in the wild was enabled, thereby completing the present invention.
Disclosure of Invention
Technical problem to be solved
The invention aims to provide attenuated CSFV BErns attenuated C-P50, which is obtained by culturing a CSFV vaccine strain Flc-LOM-BErns alternately and continuously.
It is another object of the present invention to provide a vaccine composition for oral administration comprising the attenuated swine fever virus-tagged live vaccine strain and a pharmaceutically acceptable carrier.
Another object of the present invention is to provide a method for preparing attenuated swine fever virus-marked live vaccine strain, wherein Flc-LOM-Berns vaccine strain is sequentially subjected to successive subculture in CPK cells (cloned porcine kidney cells, cloned porcine kidney cell), BK cells (bovine kidney cells, bovine kidney cell), PK cells (porcine kidney cells, porcine kidney cell) and CPK cells to proliferate to a final titer of 1X 10 6.0 ～1×10 7.0 TCID 50 /ml。
It is another object of the present invention to provide a method for identifying pigs infected with swine fever virus in the field and pigs vaccinated with said attenuated swine fever virus marker live vaccine strain, wherein competition enzyme-linked immunosorbent assay (competitive enzyme-linked immunosorbent assay, C-ELISA) is used.
Technical proposal
In order to achieve the above object, the present invention provides an attenuated swine fever virus-marked live vaccine strain obtained by subjecting swine fever virus vaccine strain Flc-LOM-Berns to an alternate continuous culture between cells.
Furthermore, the present invention provides a vaccine composition for oral administration comprising the attenuated swine fever virus-tagged live vaccine strain and a pharmaceutically acceptable carrier.
Furthermore, the present invention provides a method for preparing an attenuated swine fever virus-tagged live vaccine strain comprising the steps of: (a) Flc-LOM-Berns vaccine strains were inoculated into CPK cells (cloned porcine kidney cells, cloned porcine kidney cell) and then subjected to successive subcultures to proliferate to have a size of 1X 10 3.0 ～1×10 4.0 TCID 50 Titers of/ml; (b) Inoculating the virus propagated in the step (a) again into BK cells (bovine kidney cells, bovine kidney cell) and then performing successive subculture to propagate to have 1×10 4.0 ～1×10 5.0 TCID 50 Titers of/ml; (c) Re-inoculating the virus propagated in step (b) to PK cells (porcine kidney cells, porcine kidney ce)ll) and then carrying out successive subcultures to proliferate to have a length of 1X 10 5.0 ～1×10 6.0 TCID 50 Titers of/ml; and (d) inoculating the virus propagated in the step (c) again into CPK cells (cloned porcine kidney cells, cloned porcine kidney cell) and then performing successive subculture to proliferate to have 1X 10 6.0 ～1×10 7.0 TCID 50 Titers per ml.
In addition, the present invention provides a method for identifying swine infected with swine fever virus in the field and swine inoculated with an attenuated swine fever virus-tagged live vaccine strain obtained by alternately and continuously culturing swine fever virus vaccine strain Flc-LOM-Berns among cells, comprising the steps of: (a) Simultaneously adding a detection sample and an enzyme-labeled antibody into a swine fever virus antigen adsorption plate to enable the antibody in the detection sample and the enzyme-labeled antibody to react competitively; (b) Adding a color developing agent to the plate of step (a) to induce an enzymatic reaction with the enzyme-labeled antibody; (c) Measuring absorbance after the enzymatic reaction of step (b); (d) Calculating a% PC value using the absorbance measured in said step (c); and (e) when the% PC value calculated in the step (d) is equal to or greater than a predetermined threshold (cut-off), determining that the swine is wild-infected with a swine fever virus, and when the% PC value calculated in the step (d) is less than the threshold, determining that the swine is vaccinated with the attenuated swine fever virus-marker live vaccine strain.
In addition, the invention provides the use of the attenuated swine fever virus-marked live vaccine strain in preparation of attenuated live vaccine and/or marked live vaccine of swine fever virus as immune response inducer for field swine fever virus and attenuated swine fever virus.
In addition, the invention provides application of the attenuated swine fever virus marked live vaccine strain in preparation of attenuated live vaccine and/or marked live vaccine of swine fever virus as a preparation for defending infection of field swine fever virus.
In addition, the invention provides the use of attenuated swine fever virus-marked live vaccine strains in the preparation of attenuated live vaccines and/or marked live vaccines for swine fever virus as agents for preventing, preventing or defending against abortion, stillbirth or fetal vertical infection caused by wild swine fever virus in pregnant sows.
In addition, the invention provides the use of the attenuated swine fever virus-marked live vaccine strain in the preparation of a swine fever virus attenuated live vaccine and/or a marked live vaccine as a preparation for identifying pigs infected with swine fever virus in the wild and pigs vaccinated with the attenuated swine fever virus-marked live vaccine strain.
Effects of the invention
As for the attenuated swine fever virus marked live vaccine strain CSFV BErns attenuated C-P50, the safety is enhanced because the problems of the existing swine fever virus vaccine strain, namely the problems of abortion, stillbirth and other side effects of pregnant sows are relieved.
In addition, the vaccine composition for oral administration of the present invention is formulated into a bait formulation so that pigs can easily ingest it, thus solving the problem of the existing swine fever virus vaccine that it is difficult to administer the vaccine to the cultivated wild pigs and black pigs, which are harsher in nature, due to the use thereof as an injection only.
In addition, the serological identification method can identify antigens and antibodies of pigs infected with the swine fever virus in the wild and pigs vaccinated with the attenuated swine fever virus marker live vaccine strain CSFV BErns attenuated C-P50, so that DIVA (distinguishing infected animals from vaccinated animals, differentiating infected in vaccinated animals) has excellent effect.
Drawings
FIG. 1 is a photograph showing whether or not Flc-LOM-Berns virus was proliferated (A: CPK-15 cells (normal cells), B: reaction to antibodies specific to Classical Swine Fever Virus (CSFV) and BVDV Erns) confirmed by PLA (Peroxidase-linked assay).
FIG. 2 is a photograph showing whether or not Flc-LOM-Berns virus was proliferated (A: DNA size marker, B: marker live vaccine strain positive) confirmed by RT-PCR (reverse transcription-polymerase chain reaction).
FIG. 3 is a graph showing viral titers when Flc-LOM-Berns viruses were serially subcultured in CPK cells, BK cells, PK cells and CPK cells.
FIG. 4 is a graph showing virus titers when Flc-LOM-Berns viruses were continuously subcultured in single cells (FIG. 4A: continuous subcultured to 50 generations in CPK cells, FIG. 4B: continuous subcultured to 20 generations in CPK cells, 21 to 50 generations in BK cells, FIG. 4C: continuous subcultured to 20 generations in CPK cells, 21 to 50 generations in PK cells, FIG. 4D: continuous subcultured to 20 generations in CPK cells, 21 to 30 generations in BK cells, 31 to 30 generations in PK cells).
FIG. 5 is a diagram showing a patterned method of identifying swine infected with swine fever virus in the field and swine vaccinated with the attenuated swine fever virus marker live vaccine strain of the present invention (CSFV BErns attenuated C-P50) using C-ELISA.
PREFERRED EMBODIMENTS FOR CARRYING OUT THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used in this specification is well known and commonly employed in the art.
In one embodiment of the present invention, in order to prepare a novel swine fever virus-tagged live vaccine strain ensuring safety and high titer, the existing Flc-LOM-Berns virus strain (Korean laid-open patent publication No. 2010-012688, accession No. KCTC12304 BP) was sequentially subjected to successive subculture in CPK cells (cloned porcine kidney cells, cloned porcine kidney cell), BK cells (bovine kidney cells, bovine kidney cell), PK cells (porcine kidney cells, porcine kidney cell), and CPK cells, resulting in about 1X 10 6.5 TCID 50 Titers per ml, and the attenuated swine fever virus-tagged live vaccine strain thus obtained was designated as CSFV BErns attenuated C-P50. In addition, it was confirmed that CSFV BErns attenuated C-P50 produced antibodies well in vaccinated pigs to effectively protect against field infection, thus not only functioning well as a swine fever virus vaccine, but also significantly lower in terms of the miscibility, stillbirth and fetal vertical infection rate of pregnant sows than the existing vaccine 。
Accordingly, in one aspect the present invention relates to an attenuated swine fever virus marker live vaccine strain (CSFV BErns attenuated C-P50) obtained by subjecting swine fever virus vaccine strain Flc-LOM-Berns to an alternate continuous culture between cells.
The attenuated swine fever virus-marked live vaccine strain of the present invention was designated as CSFV Berns attenuated C-P50, and was deposited at the Korean institute of life and engineering, and accepted at 12.08 of 2015, to obtain accession No. KCTC18431P.
In the present invention, the Flc-LOM-BErns vaccine strain used in preparing the CSFV BErns attenuated C-P50 vaccine strain refers not only to the vaccine strain deposited with accession number KCTC12304B, but also to the swine fever virus-marked live vaccine strain prepared by the method disclosed and described in korean laid-open patent No. 2010-01261288, i.e., the swine fever virus-marked live vaccine strain prepared by substituting the Erns gene of BVDV (bovine viral diarrhea virus, bovine Viral Disease Virus) for the Erns gene of CSFV (swine fever virus, classical Swine Fever Virus). The Flc-LOM-Berns vaccine strain prepared in this way has the problem of low potency and insufficient attenuation.
In the present invention, the inter-cell alternate continuous culture is preferably performed by continuous subculture in the CPK cells (cloned porcine kidney cells, cloned porcine kidney cell), BK cells (bovine kidney cells, bovine kidney cell), PK cells (porcine kidney cells, porcine kidney cell) and CPK cells (cloned porcine kidney cells, cloned porcine kidney cell) in this order, but is not limited to this order. The vaccine strain subjected to alternate continuous culture between cells is characterized by having a structure of 1X 10 6.0 ～1×10 7.0 TCID 50 The titer per ml is preferably 1X 10 6.5 TCID 50 Titers per ml.
In another aspect the invention relates to a vaccine composition for oral administration comprising said attenuated classical swine fever virus marker live vaccine strain CSFV BErns attenuated C-P50 and a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier is not particularly limited, and solid carriers generally used for oral administration of pharmaceuticals can be used, and for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like can be used. In particular, when the vaccine composition for oral administration is formulated into a bait formulation capable of being easily ingested by pigs, a pig feed powder may be used as the carrier, to which a binder (gelatinized starch), a perfume, or the like may be further added.
When the attenuated swine fever virus-marked live vaccine strain of the present invention is administered in a usual injection form, it is necessary to administer the attenuated swine fever virus-marked live vaccine strain to a site adjacent to or in the muscle of a subject swine to a virus content of at least 1X 10 3.0 TCID 50 In view of the weight and health status of the swine to be administered, it is preferable that the swine to be administered should be administered once within a predetermined administration period (even once administration induces an immune response against the attenuated swine fever virus-labeled live vaccine strain and forms antibodies).
When the attenuated swine fever virus-marked live vaccine strain of the present invention is administered in a bait formulation, it is necessary to administer a vaccine comprising at least 1X 10 before feeding the administration subject pig 5.0 TCID 50 A bait formulation having a virus content of 2ml or more, but is preferably administered once within a prescribed administration period (even if administered (ingested) once, an immune response to an attenuated swine fever virus-labeled live vaccine strain is induced to form an antibody) in view of the weight and health status of the administration subject pigs.
In another aspect the invention relates to a method for preparing an attenuated classical swine fever virus marked live vaccine strain CSFV berts attenuated C-P50, preferably the vaccine strain deposited under accession number KCTC18431P, comprising the steps of:
(a) Flc-LOM-Berns vaccine strain (preferably the vaccine strain deposited under accession number KCTC12304 BP) is inoculated into CPK cells (cloned porcine kidney cells, cloned porcine kidney cell) and then subjected to successive subculture for proliferation to have a length of 1X 10 3.0 ～1×10 4.0 TCID 50 The titer per ml is preferably increased to 1X 10 3.5 TCID 50 Titers of/ml; (b) Re-inoculating the virus propagated in the step (a) to BK cells (bovine kidneyCells, bovine kidney cell) and then subjected to successive subcultures to proliferate to have a cell size of 1X 10 4.0 ～1×10 5.0 TCID 50 The titer per ml is preferably increased to 1X 10 4.5 TCID 50 Titers of/ml; (c) Inoculating the virus propagated in the step (b) again into PK cells (pig kidney cells, porcine kidney cell), and then performing continuous subculture to proliferate to have a size of 1×10 5.0 ～1×10 6.0 TCID 50 The titer per ml is preferably increased to 1X 10 5.5 TCID 50 Titers of/ml; and (d) inoculating the virus propagated in the step (c) again into CPK cells (cloned porcine kidney cells, cloned porcine kidney cell) and then performing successive subculture to proliferate to have 1X 10 6.0 ～1×10 7.0 TCID 50 The titer per ml is preferably increased to 1X 10 6.5 TCID 50 Titers per ml.
In order to further effectively increase the titer of the vaccine strain, the present invention is characterized by further comprising a step of confirming the presence of a mixed bacterium, preferably a mycoplasma, or a mixed virus, which may be canine parainfluenza virus, before the step (a), before the step (b), before the step (c), before the step (d), or a mixture thereof, and removing the mixed bacterium, preferably the mixed bacterium. Preferably, the mycorrhizal examination and the canine parainfluenza virus examination are performed on all the cells used in the continuous subculture, and the mycorrhizal examination and the canine parainfluenza virus examination are also performed on the vaccine strain of each step.
The present invention is also characterized in that the continuous subculture of the step (a) is performed at least until the proliferation is performed for 20 generations (preferably 20 generations), the continuous subculture of the step (b) is performed at least until the proliferation is performed for 10 generations (preferably 10 generations), the continuous subculture of the step (c) is performed at least until the proliferation is performed for 10 generations (preferably 10 generations), and the continuous subculture of the step (d) is performed at least until the proliferation is performed for 10 generations (preferably 10 generations).
In one embodiment of the invention, the swine fever Erns recombinant protein and monoclonal antibodies thereof are utilized to perform C-ELISA on swine serum samples, absorbance is measured, then a% PC value is calculated, and compared with a critical value, so that the serum samples of pigs infected with swine fever virus in the wild show positive, and the serum samples of pigs inoculated with the CSFV BErns attenuated C-P50 vaccine strain of the invention show negative, thereby confirming that the serological identification of pigs infected with swine fever virus in the wild and pigs inoculated with the CSFV BErns attenuated C-P50 vaccine strain of the invention can be performed.
Accordingly, in a further aspect the present invention relates to a method for identifying pigs infected with swine fever virus in the wild and pigs vaccinated with attenuated swine fever virus marker live vaccine strain CSFV berts attenuated C-P50, preferably the vaccine strain deposited under accession number KCTC18431P, comprising the steps of:
(a) Simultaneously adding a detection sample and an enzyme-labeled antibody into a swine fever virus antigen adsorption plate to enable the antibody in the detection sample and the enzyme-labeled antibody to react competitively;
(b) Adding a color developing agent to the plate of step (a) to induce an enzymatic reaction with the enzyme-labeled antibody;
(c) Measuring absorbance after the enzymatic reaction of step (b);
(d) Calculating a% PC value using the absorbance measured in said step (c); and
(e) And (c) determining pigs infected with swine fever virus in the wild when the% PC value calculated in the step (d) is equal to or greater than a predetermined threshold (cut-off) value, and determining pigs vaccinated with the attenuated swine fever virus-marker live vaccine strain when the% PC value calculated in the step (d) is less than the threshold value.
The present invention is characterized in that the% PC value of the step (d) can be calculated using the following equation 1, and the predetermined critical value of the step (e) is 20.
[ mathematics 1]
Competition ratio of positive control (% PC) = [ (average absorbance of negative control solution (NC) -absorbance of test sample (OD))/average absorbance of negative control solution (NC) ]100
In addition, as the enzyme of the enzyme-labeled antibody, for example, beta-glucuronidase, beta-D-glucosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, invertase, preferably peroxidase, may be used for realizing the qualitative or quantitative measurement of the formation of antigen-antibody complex, but is not limited thereto. The enzyme may be conjugated to the antibody by methods known in the art. The color former is a substrate of an enzyme bound to an antibody, and may be different depending on the type of enzyme, and since an antibody bound to HPR (horseradish peroxidase ) is used in the present invention, TMB (Tetramethylbenzidine) which is a substrate for HPR is used.
Detailed Description
The present invention will be described in more detail with reference to examples. These examples are merely for illustrating the present invention, and the scope of the present invention is not limited to these examples, as will be apparent to those skilled in the art. The substantial scope of the present invention is therefore defined according to the appended claims and equivalents thereof.
EXAMPLE 1 preparation of attenuated classical swine fever Virus marker live vaccine strain CSFV BErns attenuated C-P50
EXAMPLE 1-1 cultivation of Flc-LOM-Berns Virus in CPK cells
The Flc-LOM-Berns virus strain (accession number: KCTC12304 BP) was first prepared by the method described in Korean laid-open patent publication No. 2010-012688. In order to increase the titer of the Flc-LOM-Berns virus and to attenuate it, cells for subculturing the virus were explored. As a result of conducting a plurality of experiments, it was possible to obtain a swine fever virus-labeled live virus more efficiently in the case of transfection and subculture of CPK cells (cloned porcine kidney cells, cloned porcine kidney cell) than PK cells (porcine kidney cells, porcine kidney cell) and BK cells (bovine kidney cells, bovine kidney cell), and thus CPK cells were selected as cells for the first subculture.
In order to further increase the titer of the Flc-LOM-Berns virus, first, the mycoplasma that contaminated the prepared Flc-LOM-Berns virus were removed by the following method. FirstMRA (mycoplasma remover (Mycoplasma removal agent), ann Xuezhi medical (MPbio), product code (Cat No.) 3050044, unit size (Unit size)) was added to cells and viruses contaminated with mycoplasma at a concentration of 0.5. Mu.g/ml, and cultured. I.e. at 25cm 2 When 10ml of the culture solution was used, 0.1ml of MRA stock solution (stock) was added. MRA was added to the culture broth at a concentration of 0.5. Mu.g/ml each time the broth was replaced or subcultured and cultured. Contamination of the mycoplasma was confirmed by PCR every 4 passages of continuous culture. After 4 consecutive generations, MRA was not added and the culture was continued until 4 to 5 generations, and then contamination with the Mycobacterium was confirmed by PCR, thereby confirming whether contamination with the Mycobacterium was again caused.
Canine parainfluenza virus contaminated with Flc-LOM-BErns virus was also removed using hyperimmune antisera. To prepare a hyperimmune antiserum, a purified canine parainfluenza virus was mixed with Freund's incomplete adjuvant (IFA: incomplete Freund's adjuvant) mixed with mineral oil, and rabbits were inoculated with 2ml of the mixture at one week intervals for 6 weeks, and inoculated 6 times in total, to prepare a hyperimmune antiserum against canine parainfluenza virus. Thereafter, the hyperimmune antiserum was mixed with the contaminated virus at 1:1 and cultured in an incubator (incubator) at 37℃for 1 hour. After that, the prepared cells were washed twice with PBS, and then sensitized with a mixture of virus and antisera in an incubator (incubator) at 37℃for 2 hours, and then the sensitized solution was removed. The cells are continuously sensitized to 4 passages by adding an appropriate culture medium to the cells and culturing for three to 5 days. Antisera were re-mixed for priming at each priming, and virus supernatant was recovered at each subculture and canine parainfluenza virus genes were confirmed.
CPK is transfected, and needs to be removed from the swine fever marked live virus within 10 generations, and also needs to be used after necessary examination when the CPK is used for PK and BK cells.
After that, the culture was performed for about 24 hours to give a monolayer culture of about 80% of CPK cells, the cell culture supernatant was removed and washed three times with PBS, and then the Flc-LOM-Berns virus strain was inoculated (protectionTibetan number: KCTC12304 BP) and CO at 37 °c 2 Sensitization was performed in the incubator for 1 hour. After that, the sensitization solution was removed and CO was added to the medium at 37℃C 2 The virus was cultured in the incubator for 72 hours, and then the supernatant was recovered.
As for the confirmation of virus proliferation, no cytopathic effect (CPE) was observed, and thus, the confirmation was carried out by performing fluorescent antibody diagnosis (FA; fluorescent assay), PLA (Peroxidase-linked assay) and RT-PCR (reverse transcription-polymerase chain reaction) as described below.
[ confirmation by fluorescent antibody diagnostic method (fluorescent assay) ]
As reagents, there were prepared a swine fever E2-specific monoclonal antibody, a fluorescein isothiocyanate-labeled (FITC-labeled) anti-mouse IgG and a swine fever-specific polyclonal antibody (derived from rabbits, goats), a fluorescein isothiocyanate-labeled (FITC-labeled) anti-rabbit IgG, a fluorescein isothiocyanate-labeled (FITC-labeled) anti-goat IgG and an Erns-specific monoclonal antibody of BVDV (bovine viral diarrhea virus).
First, a swine fever-specific monoclonal antibody or a specific polyclonal antibody, an Erns-specific monoclonal antibody of BVDV (bovine viral diarrhea virus) was diluted to a use concentration in PBS and reacted with immobilized PK-15 cells at 37 ℃ for 40 minutes, washed 3 to 5 times with PBS, and then an anti-mouse FITC (monoclonal antibody) or an anti-rabbit FITC (polyclonal antibody), an anti-goat FITC (polyclonal antibody) was diluted 200-fold and reacted at 37 ℃ for 40 minutes. After that, the Flc-LOM-Berns virus reacted specifically with the anti-CSFV E2 monoclonal antibody and the anti-BVDV Erns monoclonal antibody, as a result of observation with a fluorescence microscope after washing 3 to 5 times with PBS, whereby bright green fluorescence was observed in cytoplasm.
[ confirmation by PLA (Peroxidase-linked assay) ]
ABC Kit (ABC Kit, vector PK-4000), swine fever specific monoclonal antibody, erns specific monoclonal antibody of BVDV (bovine viral diarrhea virus), biotinylated anti-mouse IgG, DAB (3, 3'-diaminobenzidine, 3' -diaminobenzodine) and the like were prepared as reagents.
First, swine fever specific monoclonal antibodies and Erns specific monoclonal antibodies of BVDV (bovine viral diarrhea virus) were diluted to the use concentration in PBS and reacted with immobilized PK-15 cells at 37 ℃ for 40 min, washed 3-5 times with PBS, then Biotinylated anti-mouse IgG was diluted and reacted at 37 ℃ for 40 min (Biotinylated) anti-mouse IgG in an ABC Kit (ABC Kit) as 10mL of pbs+1 drop solution (solution 1 drop) Biotinylated (biotinated) anti-mouse IgG (other product) was diluted 200-fold).
After the reaction, washing 3 to 5 times with PBS and reacting 10mL of AB solution (AB solution) as 10mL of PBS+2 drop of A solution (A solution 2 drop) +2 drop of B solution (B solution 2 drop) at 37℃for about 20 to 40 minutes at room temperature, thus preparing in advance), washing 3 to 5 times with PBS, then separating DAB substrate solution (DAB substrate solution) and reacting 10 minutes at room temperature (10X DAB is 0.6g DAB+100mL DW+DAB substrate solution (substrate solution) is 1mL 10X DAB+9mL PBS+10. Mu.L H) 2 O 2 (1000X))。
After that, the cells were stained brown by washing with distilled water three times and observing the results by a microscope (FIG. 1), it was observed that the Flc-LOM-Berns virus reacted specifically with the anti-CSFV E2 monoclonal antibody and the anti-BVDV Erns monoclonal antibody.
[ confirmation by RT-PCR ]
As a reagent, a swine fever antigen diagnostic kit (RT-PCR premix (premix), median Diagnostics company) and an RNA isolation kit were prepared.
Subsequently, virus RNA (viral RNA) was isolated using an RNA isolation kit, and after RNA was added to an RT-PCR premix (premix), RT-PCR was performed (primer was included in the premix (premix), and the 5' NCR site was amplified to 421 bp), and then the mixture was loaded on a gel, and the size of the band (band) was confirmed (FIG. 2).
EXAMPLE 1-2 continuous subculture in BK cells, PK cells and CPK cells
To enhance culture in CPK cells of example 1-1The virus titer of the virus propagated to 20 generations was again cultured continuously from 21 to 30 generations in BK cells under the same culture conditions as in example 1-1, and the resulting titer was 1X 10 3.5 TCID 50 /ml is increased by 1X 10 1.0 TCID 50 Per ml to 1X 10 4.5 TCID 50 /ml. Thereafter, the cells were again proliferated in PK cells from 31 to 40 generations under the same culture conditions as in example 1-1, and the resulting titers were 1X 10 4.5 TCID 50 /ml is increased by 1X 10 1.0 TCID 50 Per ml to 1X 10 5.5 TCID 50 /ml. Finally, the cells were returned to CPK again and proliferated from 41 to 50 generations under the same culture conditions as in example 1-1, resulting in titers of 1X 10 5.5 TCID 50 /ml is increased by 1X 10 1.0 TCID 50 Per ml to 1X 10 6.5 TCID 50 /ml. In addition, as in example 1-1, the examination of the mycoplasma and canine parainfluenza virus was performed every time the cells were cultured, and only negative cases were used. From this, it was confirmed that a high titer (1X 10) was achieved at a level sufficient for use as a vaccine 6.5 TCID 50 /ml) (fig. 3).
Thus, the result of increasing the virus titer in the alternate continuous culture between cells different from each other was shown to be compared with the case of continuous culture in a single cell type.
For the comparative example, continuous subculture with CPK cells alone was carried out until the highest titer at 50 passages remained at 1X 10 4.5 TCID 50 Per ml (FIG. 4A), the highest titer at 21 to 50 successive generations of subculture with BK cells was also maintained at 1X 10 4.5 TCID 50 Per ml (FIG. 4B), the titers of successive subcultures of 21 to 50 generations with PK cells remained at 1X 10 4.5 TCID 50 /ml (FIG. 4C). In addition, when viruses cultured in CPK cells to 20 generations and in BK cells to 30 generations were again subjected to successive subculture of 31 to 50 generations in PK cells, the highest titer was shown to be 1X 10 5.5 TCID 50 Per ml (FIG. 4D), but not up to 1X 10 6.5 TCID 50 /ml。
Therefore, it was confirmed that the increase in the viral titer to 1X 10 was possible in particular when the intercellular continuous culture was sequentially performed in CPK cells, BK cells, PK cells and CPK cells 6.5 TCID 50 Each ml, and the thus-increased classical swine fever virus vaccine strain was designated as CSFV BErns attenuated C-P50 (accession number: KCTC 18431P).
Formulation example composition of bait formulation
The titer generated during the preparation of the CSFV Berns attenuated C-P50 vaccine strain of example 1 was 1X 10 3 ～1×10 6 TCID 50 The vaccine strains/ml are formulated into bait formulations which can be orally administered to pigs at different titers, respectively. In the case of the composition of the bait dosage form, the ingredients of the bait dosage form were selected as ingredients of 85% by weight of pig feed powder, 14% by weight of binder (gelatinized starch), 1% by weight of apple flavor, and were prepared by coating the dosage form surface with palm oil (coating) through three palatability investigations. The dosage form was dried at 56 ℃ for 5 hours and at room temperature for one day. To prepare the bait formulation, a square bait formulation was prepared by placing pig feed powder into a compression molding die, and the labeled live vaccine strain was filled into each plastic packaging container in an amount of 2mL and sealed. Therefore, the plastic of wild boars or cultured boars, black pigs or ordinary cultured pigs can be torn when the wild boars or the cultured boars, black pigs or ordinary cultured pigs are chewed, so that bait formulations and vaccines can be ingested simultaneously.
EXAMPLE 2 efficacy test of attenuated classical swine fever Virus marker live vaccine strain CSFV BErns attenuated C-P50
EXAMPLE 2-1 implementation of neutralization assay for measuring antibody titers of classical swine fever virus produced in pigs
The attenuated CSFV BErns attenuated C-P50 (accession No. KCTC 18431P) vaccine strain obtained in example 1 was subjected to muscle inoculation with a syringe, or formulated as a bait and orally administered as described in the formulation examples, and a neutralization test (neutralization peroxidase-linked assay; NPLA) was used in order to investigate the antibody formation rate of swine to the swine fever vaccine strain.
First, an ABC kit (Vector PK-4000), a swine fever-specific monoclonal antibody, biotinylated anti-mouse IgG, DAB, etc. were prepared as reagents.
Next, 50. Mu.l of minimum essential medium α (Alpha-minimum essential medium, a-medium) to which only antibiotics were added was used, and negative control pig serum without vaccinated strain and pig serum with vaccinated strain were subjected to 2-fold gradient dilution (2 fold) in 96 microwell plates (microplates), respectively. After dilution, the swine fever virus used in the neutralization test was 1×10 2 TCID 50 Per ml and added to wells of diluted porcine serum in an amount of 50 μl, respectively. The PK cells were added to the wells at 37℃for a sensitization time of 60 min and at 5% CO 2 Incubated at 37℃for 3 days, followed by PLA staining. The swine fever specific monoclonal antibody was diluted to a working concentration in PBS and reacted with immobilized PK-15 cells at 37℃for 40 minutes, washed 3 to 5 times with PBS, and then Biotinylated anti-mouse IgG was diluted and reacted at 37℃for 40 minutes. Biotinylated (Biotinylated) anti-mouse IgG (other product) in 10mL of pbs+1 drop solution (solution 1 drop) in ABC Kit (ABC Kit) 200 fold dilution.
After the reaction, washing 3-5 times with PBS and reacting AB solution (AB solution) at 37 ℃ for 40 minutes (10 mL of AB solution (AB solution) pbs+2 drop) +2 drop of B solution (B solution 2 drop) +2 drop) is prepared beforehand because it needs to react for 20-40 minutes at room temperature), washing 3-5 times with PBS, then separating DAB substrate solution (DAB substrate solution) and reacting DAB substrate solution (substrate solution) at room temperature for 10 minutes (10X DAB is 0.6g dab+100mL dw+10 μl H of pbs+10 mL 10X DAB substrate solution (substrate solution) 1 mL) 2 O 2 (1000X))。
After that, the cell periphery of the cells growing the wells of swine fever virus was specifically stained brown in the result of washing with distilled water three times and observing by a microscope, and the antibody titer was calculated after reading also the wells not stained. The world animal health Organization (OIE) prescribes that when the swine fever antibody titer in the swine body is 10 times or more by the neutralization test method, it is judged that the swine fever antibody is positive, and when the swine fever antibody titer in the swine body is 32 times or more by the neutralization test method, the swine fever virus can be defended even if it is infected.
As a result of experiments, in individuals who had undergone muscle vaccination of piglets with attenuated swine fever-marked live vaccine strains at different titers, the antibodies before vaccination were negative, and after one week of vaccination, the following results were investigated, namely, at 1X 10 1.0 TCID 50 About 2 to 8 times per ml, 1X 10 2.0 TCID 50 About 4 to 16 times per ml, 1X 10 3.0 TCID 50 And/ml of about 16-32 times. Two weeks after vaccination, the following results were investigated, respectively, at 1×10 1.0 TCID 50 About 128 times per ml at 1X 10 2.0 TCID 50 About 128-256 times per ml, 1X 10 3.0 TCID 50 At/ml, about 256-512 times (Table 1).
In addition, attenuated swine fever-marked live vaccine strains were formulated as baits at different titers and orally administered to individuals of piglets, antibodies were negative prior to vaccination, and one week, two weeks, three weeks, four weeks after vaccination were investigated and shown at 1×10 3.0 TCID 50 Ml and 1X 10 4.0 TCID 50 Antibody titers at/ml were negative. In individuals who were orally administered, the dose was 1X 10 5.0 TCID 50 At/ml, the antibodies at one and two weeks of vaccination were negative, but of 7 pigs at three weeks post-vaccination, 2 and 5 were investigated and at four weeks of vaccination, 7 pigs all formed 16-64 fold of antibodies. In addition, in the case of individuals who were orally administered, the dose was 1X 10 6.0 TCID 50 At/ml, 7 pigs each showed 16-64 fold antibody titers at three weeks of vaccination (table 2).
EXAMPLE 2-2 implementation of challenge vaccination
Challenge vaccination was performed in order to see whether antibodies formed in pigs by muscle vaccination or oral administration of vaccine strains as in example 2-1 would normally exert their efficacy.
Attack inoculation of classical swine fever virusFor YC11WC (Yonchon strain), 1X 10 was used 5.0 TCID 50 2ml intramuscular injection and 2ml nasal inoculation were performed per ml. The Lian Chuan strain was a virus isolated from wild boars in the last 2011, which was a virus belonging to genotype 2.1b similar to classical swine fever virus epidemic in Korea since 2000, and was found to be very pathogenic.
As a result of clinical observation after challenge inoculation, in individuals who have vaccinated piglets with attenuated swine fever-marked live vaccine strains at different titers, 1X 10 strains were observed at 21 days after challenge inoculation, although no death occurred during the observation period 1.0 TCID 50 Ml and 1X 10 2.0 TCID 50 In the group inoculated/ml, heat reaction, inappetence, leucopenia and the like were observed, and complete defense was not performed. Furthermore, 1×10 3.0 TCID 50 In the group vaccinated with/ml or more, it was found that not only death but also specific clinical symptoms did not occur after challenge vaccination (Table 1).
Therefore, it was confirmed that the ratio was 1×10 1.0 TCID 50 In the case of inoculation in/ml, although antibody titers are established, the minimum injection dose that can be defended against field viruses should be 1X 10 3.0 TCID 50 And/ml or more.
In addition, 3 pigs of the negative control group developed clinical symptoms (neurological symptoms, thermal reactions, leukopenia, anorexia, postamble paralysis, body surface erythema, etc.) specifically after challenge inoculation, and died 12 days and 14 days, 18 days after challenge inoculation, respectively. Spleen congestion and bleeding, punctate bleeding of the kidneys, mesenteric lymph node congestion, hypertrophy, and the like also occurred in 3 pigs of the negative control group after necropsy. The world animal health Organization (OIE) prescribes that swine fever antibodies in pigs have a potency of 10 times or more by neutralization test, and that they can be protected even when they are infected with swine fever virus.
In contrast, attenuated swine fever-marked live vaccine strains were formulated as baits at different titers and administered orally to individuals of piglets, and when observed 21 days after challenge inoculation, they were seen to be administered at 1×10 5.0 TCID 50 In the group inoculated per ml, clinical symptoms and death did not appear and swine fever virus was protected. However, at 1X 10 3.0 TCID 50 Ml and 1X 10 4.0 TCID 50 In the group inoculated/ml, there were developed clinical symptoms peculiar to swine fever such as hyperthermia, inappetence, leukopenia, postparalytic paralysis, neurological symptoms, diarrhea, body surface erythema, etc., and at 1X 10 3.0 TCID 50 Of 7 pigs vaccinated/ml, 4 had died, at 1X 10 4.0 TCID 50 Of the 7 pigs vaccinated/ml, 2 deaths were observed (Table 2).
The final results of the correlation between the antibody formation rate and the challenge vaccination defense rate and the serological discrimination of pigs subjected to muscle vaccination or oral administration with the attenuated swine fever virus-marked live vaccine strain (CSFV BErns attenuated C-P50) of the present invention are shown in Table 3 below.
TABLE 1
After the attenuated swine fever marked live vaccine strains are used for respectively carrying out muscle inoculation on piglets at different titers, the minimum concentration antibody titers are formed and the attack inoculation defense efficiency is investigated
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F: high heat reaction at 40 ℃, a: inappetence, L: leukopenia, R: postamble paralysis, C: neurological symptoms, D: diarrhea, S: body surface erythema
TABLE 2
After attenuated swine fever marked live vaccine strains are respectively formulated into baits with different titers and then orally applied to piglets, investigation on the formation of minimum concentration antibody titers and the attack inoculation defense efficiency
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F: high heat reaction at 40 ℃, a: inappetence, L: leukopenia, R: postamble paralysis, C: neurological symptoms, D: diarrhea, S: body surface erythema
TABLE 3
EXAMPLE 3 serological identification
C-ELISA conditions for identifying sera from wild-type infection with swine fever were set using the swine fever Erns recombinant protein and monoclonal antibodies. The swine fever Erns ELISA is an ELISA kit for detecting wild-type infected serum, which uses the principle that since the Erns site of a swine fever marker live vaccine strain is replaced with the BVDV Erns site, swine fever Erns antibodies will not appear in individuals vaccinated with the marker live vaccine strain, but swine fever Erns antibodies will be produced in individuals infected with wild-type swine fever virus, and thus, when using the swine fever marker live vaccine strain, swine fever Erns ELISA detects only wild-type infected serum as positive.
The serum of wild infection swine fever and the serum and negative serum of inoculated marked live vaccine are used to set the recombinant protein coating concentration, the serum dilution times, the secondary antibody concentration and the critical (cut-off) value.
The swine fever Erns ELISA was performed as follows (fig. 5). First, 100. Mu.l of the Erns recombinant protein of swine fever was coated on a "polysorb plate" as ELISA plate at a concentration of 0.0125. Mu.g/well, respectively, and blocked at room temperature for 2 hours. A blocking solution was prepared by including 5% horse serum (horse serum) in PBST (0.1% Tween (Tween) 20). Blocked ELISA plates were washed three times with PBST (0.1% Tween (Tween) 20), and porcine test serum was diluted 10-fold and separately split at 100. Mu.l and reacted in an incubator at 37℃for 1 hour. After the reaction, PBST (0.1% Tween (Tween) 20) was washed three times, and a swine fever Erns monoclonal antibody (C-Erns 1) bound to HPR was subjected to strain isolation at a concentration of 5. Mu.g/well and an amount of 100. Mu.l per well, and reacted in an incubator at 37℃for 1 hour.
After the reaction, the reaction mixture was washed three times with PBST (0.1% Tween (Tween) 20) and a color developing agent (TMB) was added, and then it was confirmed whether or not color development was performed for 10 minutes, and the OD value was measured by an enzyme-labeled instrument (ELISA reader) at 450 nm. The positive and negative determinations were calculated by "% pc= (NC-OD)/nc×100" when the% PC value was calculated, and were determined to be positive when the% PC value was 20 or more.
As a result, all pigs showed negative results for 84 pigs negative for the classical swine fever virus antibody, and all pigs also showed negative results for 31 pigs vaccinated with the CSFV BErns attenuated C-P50 strain of the present invention (Table 4). In contrast, the investigation of all pigs was positive for 75 pigs vaccinated with LOM vaccine strain and 43 pigs vaccinated with field swine fever virus (table 4), so that it was possible to confirm the DIVA (differentiating infected in vaccinated animals) effect which was distinguishable from pigs vaccinated with CSFV BErns attenuated C-P50 strain of the invention.
TABLE 4
Serological identification using swine fever Erns C-ELISA kit
EXAMPLE 4 RT-PCR was performed in fetal vertically infected organs
The following experiment was conducted in order to see whether the virus vertically infects a fetus from a pregnant sow when the CSFV BErns attenuated C-P50 vaccine strain (accession number: KCTC 18431P) and the existing LOM vaccine strain and the existing Flc-Lom-BErns vaccine strain (accession number: KCTC12304 BP) of the present invention are inoculated to a sow in the early or middle of pregnancy.
First, pregnant sows inoculated at the early and middle stages of pregnancy were randomly selected from groups inoculated with each vaccine strain, and the lactating piglets and abortions and stillbirth fetuses produced by these sows were necropsied, and amplified by RT-PCR in order to confirm whether or not viruses were present in the important solid organs spleen, kidney, heart, tonsil, mesenteric lymph node, lung and brain tissues collected.
First, a swine fever antigen diagnostic kit (RT-PCR premix (premix), median Diagnostics company) and an RNA isolation kit were prepared as reagents. Next, various organs were emulsified (emulisife), and in order to isolate the total RNA (total RNA) in an emulsion (emulison liquid), virus RNA (viral RNA) was isolated using RNA isolation kit. Thereafter, RNA was added to RT-PCR premix (premix) for RT-PCR (primer included in premix) to amplify the 5' NCR site to 421 bp), and then the size of the band was confirmed by gel loading (gel loading).
As a result, it was confirmed that the yield of fluid and dead time during pregnancy caused by the LOM vaccine was 50%, and that 10% of fluid and dead time were also exhibited in the group vaccinated with the Flc-Lom-Berns vaccine strain, whereby insufficient progress of attenuation was confirmed. In contrast, the group vaccinated with the CSFV Berns attenuated C-P50 vaccine strain of the invention showed no yield, no dead yield, and thus it was confirmed that the attenuation was obtained (tables 5 and 6).
In addition, regarding the vertical infection rate to the fetus in pregnant sows, 50% in the group vaccinated with the LOM vaccine strain, 20% in the group vaccinated with the Flc-Lom-BErns vaccine strain, and 10% in the group vaccinated with the CSFV BErns attenuated C-P50 vaccine strain of the present invention were investigated, thereby exhibiting more excellent safety (tables 5, 6).
TABLE 5
TABLE 6
The specific portions of the present invention are described in detail above, and these are merely preferred embodiments, and the scope of the present invention is not limited thereto, as will be clear to those having ordinary skill in the art. Therefore, the true scope of the invention should be defined according to the following claims and their equivalents.
Claims (14)
1. The attenuated swine fever virus marked live vaccine strain is characterized by being obtained by alternately and continuously culturing swine fever virus vaccine strain Flc-LOM-Berns among cells, wherein the alternately and continuously culturing among cells is sequentially continuous subculturing in cloned pig kidney cells, cattle kidney cells, pig kidney cells and cloned pig kidney cells;
wherein, the method for preparing the attenuated swine fever virus marked live vaccine strain comprises the following steps:
(a) Flc-LOM-Berns vaccine strains were inoculated into cloned porcine kidney cells and then subjected to successive subculture for proliferation to have a length of 1X 10 3.0 ～1×10 4.0 TCID 50 Titers of/ml;
(b) Inoculating again the virus propagated in the step (a) into bovine kidney cells, and then performing continuous subculture to propagate to 1×10 4.0 ～1×10 5.0 TCID 50 Titers of/ml;
(c) Inoculating the virus propagated in step (b) into pig kidney cells again, and then performing continuous subculture to propagate to 1×10 5.0 ～1×10 6.0 TCID 50 Titers of/ml; and
(d) Inoculating the virus propagated in step (c) again into cloned pig kidney cells, and then performing continuous subculture to proliferate to have a ratio of 1×10 6.0 ～1×10 7.0 TCID 50 Titers of/ml;
wherein the continuous subculture of step (a) is carried out at least until proliferation is carried out for 20 generations, the continuous subculture of step (b) is carried out at least until proliferation is carried out for 10 generations, the continuous subculture of step (c) is carried out at least until proliferation is carried out for 10 generations, and the continuous subculture of step (d) is carried out at least until proliferation is carried out for 10 generations.
2. The attenuated swine fever virus marker live vaccine strain of claim 1, wherein the swine fever virus vaccine strain Flc-LOM-BErns is a vaccine strain deposited under accession number KCTC12304 BP.
3. The attenuated swine fever virus marker live vaccine strain of claim 1, wherein the marker live vaccine strain has a length of 1 x 10 6.0 ～1×10 7.0 TCID 50 Titers per ml.
4. A swine fever virus vaccine composition for oral administration comprising the attenuated swine fever virus-tagged live vaccine strain of any one of claims 1-3 and a pharmaceutically acceptable carrier.
5. The swine fever virus vaccine composition for oral administration of claim 4, wherein the composition is formulated as a bait formulation.
6. A method for preparing an attenuated swine fever virus-tagged live vaccine strain, comprising the steps of:
(a) Flc-LOM-Berns vaccine strains were inoculated into cloned porcine kidney cells and then subjected to successive subculture for proliferation to have a length of 1X 10 3.0 ～1×10 4.0 TCID 50 Titers of/ml;
(b) Inoculating again the virus propagated in the step (a) into bovine kidney cells, and then performing continuous subculture to propagate to 1×10 4.0 ～1×10 5.0 TCID 50 Titers of/ml;
(c) Inoculating the virus propagated in the step (b) into pig kidney cells again, then carrying out continuous subculture,to proliferate to have 1X 10 5.0 ～1×10 6.0 TCID 50 Titers of/ml; and
(d) Inoculating the virus propagated in step (c) again into cloned pig kidney cells, and then performing continuous subculture to proliferate to have a ratio of 1×10 6.0 ～1×10 7.0 TCID 50 Titers of/ml;
wherein the continuous subculture of step (a) is carried out at least until proliferation is carried out for 20 generations, the continuous subculture of step (b) is carried out at least until proliferation is carried out for 10 generations, the continuous subculture of step (c) is carried out at least until proliferation is carried out for 10 generations, and the continuous subculture of step (d) is carried out at least until proliferation is carried out for 10 generations.
7. The method of producing an attenuated swine fever virus marker live vaccine strain of claim 6, further comprising the step of identifying and removing mixed bacteria, mixed viruses or a mixture thereof, prior to performing each of the steps (a) - (d).
8. The method of preparing an attenuated swine fever virus marker live vaccine strain of claim 7, wherein the mixed bacterium is a mycoplasma and the mixed virus is canine parainfluenza virus.
9. The method of preparing an attenuated swine fever virus-marked live vaccine strain of claim 6, wherein the Flc-LOM-BErns vaccine strain of step (a) is a vaccine strain deposited under accession number KCTC12304 BP.
10. The attenuated swine fever virus marked live vaccine strain is used as an immune response inducer for field swine fever viruses in the preparation of attenuated live vaccine or marked live vaccine of swine fever viruses,
Wherein the attenuated swine fever virus marked live vaccine strain is obtained by alternately and continuously culturing swine fever virus vaccine strain Flc-LOM-Berns among cells,
the alternate continuous culture among cells is continuous subculture among cloned pig kidney cells, cattle kidney cells, pig kidney cells and cloned pig kidney cells in sequence;
wherein, the method for preparing the attenuated swine fever virus marked live vaccine strain comprises the following steps:
(a) Flc-LOM-Berns vaccine strains were inoculated into cloned porcine kidney cells and then subjected to successive subculture for proliferation to have a length of 1X 10 3.0 ～1×10 4.0 TCID 50 Titers of/ml;
(b) Inoculating again the virus propagated in the step (a) into bovine kidney cells, and then performing continuous subculture to propagate to 1×10 4.0 ～1×10 5.0 TCID 50 Titers of/ml;
(c) Inoculating the virus propagated in step (b) into pig kidney cells again, and then performing continuous subculture to propagate to 1×10 5.0 ～1×10 6.0 TCID 50 Titers of/ml; and
(d) Inoculating the virus propagated in step (c) again into cloned pig kidney cells, and then performing continuous subculture to proliferate to have a ratio of 1×10 6.0 ～1×10 7.0 TCID 50 Titers of/ml;
wherein the continuous subculture of step (a) is carried out at least until proliferation is carried out for 20 generations, the continuous subculture of step (b) is carried out at least until proliferation is carried out for 10 generations, the continuous subculture of step (c) is carried out at least until proliferation is carried out for 10 generations, and the continuous subculture of step (d) is carried out at least until proliferation is carried out for 10 generations.
11. The attenuated swine fever virus marked live vaccine strain is used as a preparation for defending infection of field swine fever virus in preparation of attenuated live vaccine or marked live vaccine of swine fever virus,
wherein the attenuated swine fever virus marked live vaccine strain is obtained by alternately and continuously culturing swine fever virus vaccine strain Flc-LOM-Berns among cells,
the alternate continuous culture among cells is continuous subculture among cloned pig kidney cells, cattle kidney cells, pig kidney cells and cloned pig kidney cells in sequence;
wherein, the method for preparing the attenuated swine fever virus marked live vaccine strain comprises the following steps:
(a) Flc-LOM-Berns vaccine strains were inoculated into cloned porcine kidney cells and then subjected to successive subculture for proliferation to have a length of 1X 10 3.0 ～1×10 4.0 TCID 50 Titers of/ml;
(b) Inoculating again the virus propagated in the step (a) into bovine kidney cells, and then performing continuous subculture to propagate to 1×10 4.0 ～1×10 5.0 TCID 50 Titers of/ml;
(c) Inoculating the virus propagated in step (b) into pig kidney cells again, and then performing continuous subculture to propagate to 1×10 5.0 ～1×10 6.0 TCID 50 Titers of/ml; and
(d) Inoculating the virus propagated in step (c) again into cloned pig kidney cells, and then performing continuous subculture to proliferate to have a ratio of 1×10 6.0 ～1×10 7.0 TCID 50 Titers of/ml;
wherein the continuous subculture of step (a) is carried out at least until proliferation is carried out for 20 generations, the continuous subculture of step (b) is carried out at least until proliferation is carried out for 10 generations, the continuous subculture of step (c) is carried out at least until proliferation is carried out for 10 generations, and the continuous subculture of step (d) is carried out at least until proliferation is carried out for 10 generations.
12. The use of attenuated swine fever virus-marked live vaccine strains in the preparation of attenuated live vaccines or marked live vaccines for swine fever virus as a preparation for preventing, preventing or defending against abortion, stillbirth or vertical infection of a fetus caused by field swine fever virus in pregnant sows,
wherein the attenuated swine fever virus marked live vaccine strain is obtained by alternately and continuously culturing swine fever virus vaccine strain Flc-LOM-Berns among cells,
the alternate continuous culture among cells is continuous subculture among cloned pig kidney cells, cattle kidney cells, pig kidney cells and cloned pig kidney cells in sequence;
wherein, the method for preparing the attenuated swine fever virus marked live vaccine strain comprises the following steps:
(a) Flc-LOM-Berns vaccine strains were inoculated into cloned porcine kidney cells and then subjected to successive subculture for proliferation to have a length of 1X 10 3.0 ～1×10 4.0 TCID 50 Titers of/ml;
(b) Inoculating again the virus propagated in the step (a) into bovine kidney cells, and then performing continuous subculture to propagate to 1×10 4.0 ～1×10 5.0 TCID 50 Titers of/ml;
(c) Inoculating the virus propagated in step (b) into pig kidney cells again, and then performing continuous subculture to propagate to 1×10 5.0 ～1×10 6.0 TCID 50 Titers of/ml; and
(d) Inoculating the virus propagated in step (c) again into cloned pig kidney cells, and then performing continuous subculture to proliferate to have a ratio of 1×10 6.0 ～1×10 7.0 TCID 50 Titers of/ml;
wherein the continuous subculture of step (a) is carried out at least until proliferation is carried out for 20 generations, the continuous subculture of step (b) is carried out at least until proliferation is carried out for 10 generations, the continuous subculture of step (c) is carried out at least until proliferation is carried out for 10 generations, and the continuous subculture of step (d) is carried out at least until proliferation is carried out for 10 generations.
13. The use according to any one of claims 10 to 12, wherein the Flc-LOM-BErns vaccine strain is a vaccine strain deposited under accession number KCTC12304 BP.
14. The use according to any one of claims 10 to 12, wherein the attenuated swine fever virus-marker live vaccine strain is for muscle vaccination or oral administration.
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