WO2020138761A1

WO2020138761A1 - Chimeric virus of porcine reproductive and respiratory syndrome virus, and vaccine using same

Info

Publication number: WO2020138761A1
Application number: PCT/KR2019/017319
Authority: WO
Inventors: 조선희; 박창훈; 백종혁; 차상호; 강석진; 유수화; 조인수
Original assignee: 주식회사 바이오포아; 대한민국(농림축산식품부 농림축산검역본부장)
Priority date: 2018-12-27
Filing date: 2019-12-09
Publication date: 2020-07-02

Abstract

The present invention relates to a chimeric virus of porcine reproductive and respiratory syndrome (PRRS) virus, the chimeric virus being usable as a vaccine. A PRRSV chimeric virus of the present invention is more attenuated than the parent strain thereof, and thus promotes the secretion of neutralizing antibodies while having low pathogenicity and high stability, thereby being usable as a vaccine in the effective prevention and treatment of PRRS diseases.

Description

Chimeric virus of swine genital and respiratory syndrome virus and vaccine using same

The present invention relates to a chimeric virus of porcine genital and respiratory syndrome virus (PRRSV) that can be used as a vaccine, and the chimeric virus of the present invention has lower pathogenicity and higher safety than the parent strain. The PRRSV chimeric virus of the present invention improves the secretion of neutralizing antibodies related to cross-immunity, and can provide a vaccine that can effectively protect porcine genital and respiratory syndrome.

Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most damaging infectious diseases in the domestic pig industry along with swine circovirus infection and foot and mouth disease. PRRS causes reproductive inability of pregnant sows, miscarriage or premature birth, reproductive disorders in stillbirths, and respiratory symptoms such as sneezing and fever in suckling pigs and piglets. In general, after infection with the virus, severe respiratory symptoms are caused by secondary infection such as bacteria, but in case of chronic infection, a decrease in body weight and an increase in mortality are observed without characteristic clinical symptoms.

The viral disease was first discovered in the United States in 1987, then in Europe, and was identified in Asia in the early 1990s. So far, PRRS has been characterized by endemic disease in swine countries and has spread worldwide, causing huge economic losses every year.

The pathogens responsible for PRRS are the PRRS virus belonging to the genus Arterivirus, Arteriviridae, and Nidovirales. The PRRS virus has a positive-sense single stranded RNA genome and is about 15.4 kilobase in size. The genome of the PRRS virus has 9 ORFs (Conzelmann et al., 1993; Meulenberg et al., 1993). Among them, ORF1a and ORF1b encoding non-structural proteins (NSPs) account for about 80% of viral genomes (Bautista et al., 2002; Meulenberg et al., 1993; Snijder and Meulenberg, 1998, 2001) . It is known that NSP1-alpha, NSP-1 beta, NSP2 to NSP8 are located in ORF1a, and NSP9 to NSP12 are located in ORF1b among the non-structural proteins. The glycosylated structural proteins GP2, GP3, GP4, GP5, non-glycosylated membrane (Membrane, M) protein, and nucleocapsid (N) protein are encoded by ORF2-7, which accounts for the remaining 20%. The minor structural proteins GP2, GP3, and GP4 form heterotrimers, which act when viruses enter the host cell. The major structural proteins, GP5, M, form heterodimers to increase the infectivity of the virus.

The PRRS virus is highly mutated due to the nature of the RNA virus, so there are many differences between the viruses. PRRS virus is largely divided into North American and European. Type I representing the European type (Lelystad virus, LV) and Type II representing the North American strain ATCC VR2332 (for genome sequence of VR2332, refer to GenBank accession number AY150564) (Murtaugh et al., Arch Virol. 1995; 140:1451-1460).

It is known that there is a genetic difference of up to 40% between the North American type and the European type, so that they do not cross-protect each other. Also, cross-defense is often not achieved even among mutants belonging to the same type (Meng, X. J. et al., 2000). For this reason, a standard mutant-based vaccine is produced for each, but it is not effectively preventing PRRS due to poor cross-defense. In order to overcome this, various attempts have been made to produce a vaccine effectively equipped with safety, immunogenicity, and defense ability.

Because of the severity of this disease, after discovering the porcine reproductive and respiratory syndrome virus (PRRSV), the cause of porcine reproductive and respiratory syndrome (PRRS), it has still been effective even though much effort has been invested to develop preventive measures against the virus for about 20 years. Prevention and management methods have not been developed. Various vaccines such as inactivated vaccines and live attenuated vaccines have been developed to control porcine reproductive and respiratory syndrome virus (PRRSV), but only attenuated vaccines with infectious properties have been found to induce satisfactory protective effects. However, because the swine respiratory syndrome virus (PRRSV) is genetically diverse, there is no cross-immunity between various virus types, so it is difficult to protect various swine respiratory syndrome virus (PRRSV) with one vaccine. In addition, the current attenuated vaccine can be produced only through successive passages of 100 to 200 or more passages in cell lines of other animal species, so there is a problem that the development period is long and the efficacy and safety are difficult to be guaranteed. Because of this, a standard mutant-based vaccine is produced for each, but it is not effectively preventing PRRS because of its poor cross-defense ability.

In order to overcome this, various attempts have been made to produce a vaccine effectively equipped with safety, immunogenicity, and defense ability (Application No. 10-2011-7004020, title of the invention: highly pathogenic porcine genital and respiratory syndrome (HP PRRS Vaccine against )).

The present invention relates to a chimeric virus of porcine genital and respiratory syndrome virus (PRRSV) that has low pathogenicity, high stability, and improved secretion of neutralizing antibodies, a vaccine comprising the same, and a method for manufacturing the same.

In one example, the chimeric virus is

(1) The nucleic acid sequence of ORF1a and ORF1b derived from the LMY ver2 mutant strain of accession number KCTC 13394BP or a nucleic acid sequence having 70% or more sequence homology with the nucleic acid sequence while maintaining functional equivalence thereto, and

(2) The nucleic acid sequence of the ORF2 to ORF7 region of the BP2017-2 isolate with accession number KCTC 13393BP or a nucleic acid sequence having 70% or more sequence homology with the nucleic acid sequence while maintaining functional equivalence thereto

It may be to include.

The present invention relates to a chimera virus of porcine reproductive and respiratory syndrome (PRRS) virus that can be used as a vaccine. The PRRSV chimeric virus of the present invention is attenuated more than the parent strain, and thus has low pathogenicity and high stability, and significantly improves the immunity of pigs by improving the secretion of neutralizing antibodies capable of cross-immunization. Therefore, it can be used as a vaccine for effective prevention and treatment of PRRS disease.

Hereinafter, the present invention will be described in more detail.

As used herein, “attenuated virus” refers to a non-toxic virus that is capable of eliciting an immune response in a target mammal without causing clinical signs of PRRS disease, and is also an attenuated virus infected and attenuated virus It may also mean that the incidence of clinical signs is lowered in animals that have not been dosed with, or that the severity of the signs is reduced compared to "control" animals infected with the non-attenuated PRRS virus. In this situation, the term “reduced/reduced” refers to a reduction of at least 10%, preferably 25%, more preferably 50%, most preferably 100% or more compared to the control group defined above.

“Vaccine composition” as used herein may be a PRRS chimeric virus or any immunogenic fragment or fraction thereof, preferably an attenuated PRRS chimeric virus, such as the PRRS chimeric virus of the present invention. This causes the host's “immunological response” to be a cellular and/or antibody-mediated immune response to PRRSV. It is preferred that the vaccine composition is capable of conferring preventive immunity against PRRSV infection and clinical signs associated therewith.

As used herein, "immune response" refers to any cell- and/or antibody-mediated immune response to a chimeric virus or vaccine administered to an animal receiving the PRRSV chimeric virus of the present invention, or a vaccine composition comprising the same. Means Usually, an “immune response” includes, but is not limited to, one or more of the following effects: antibodies, B cells, helper T cells specifically directed against antigens or antigens included in the composition or vaccine. , Production or activation of inhibitor T cells and/or cytotoxic T cells and/or γδ T cells. It is preferred that the host exhibit a therapeutic or prophylactic immunological response such that the resistance to new infection is improved and/or the clinical severity of the disease is reduced compared to a control group that has not been administered an immunogenic composition or vaccine. This prevention will be evidenced by the lack of symptoms associated with the host infections described above, as well as a decrease in frequency or severity, as well.

As used herein, "pigs", "pigs" and "pigs" may be used interchangeably.

“Vaccinating” means administering the PRRSV chimeric virus described herein or a vaccine comprising it before exposure to PRRS disease.

“Prevent” or “prophylaxis” means that as a result of receiving the PRRSV virus of the present invention or a vaccine composition comprising the same, the clinical incidence of PRRS, the severity or frequency of symptoms decreases. The decrease in severity or frequency is a result of comparing the PRRSV chimeric virus of the present invention or a vaccine composition comprising the same with an animal or group of animals that has not been administered. The animal is preferably a pig.

For convenience, the nucleotide sequence of the present specification has been described based on DNA nucleotides, and when the type of polynucleotide is RNA, a sequence in which all or some thymine (T) in the nucleotide sequence is substituted with uracil (U) it means.

In the present specification, a specific nucleotide sequence and/or amino acid "consisting of a sequence" includes the sequence, essentially comprising the sequence, and/or comprising all of the sequence, and may be used by appropriate substitution as necessary. Can.

The present invention provides a chimeric virus attenuated against swine genital and respiratory syndrome (PRRSV), a viral disease affecting pigs.

In order to achieve the above object, the present invention provides a polynucleotide comprising the structure of structural formula 1:

[Structural Formula 1]

In the above formula, [X] is a nucleic acid sequence of the NSP1 gene (NSP1-alpha gene and NSP1-beta gene) of the LMY ver2 mutant strain having accession number KCTC 13394BP, or a sequence of 70% or more within a range in which functions equivalent to the nucleic acid sequence are maintained. Containing a nucleic acid sequence having homology, including, for example, a nucleic acid sequence of the NSP1 gene of the LMY ver2 variant strain or a nucleic acid sequence having 70% or more sequence homology within a range in which functions equivalent to the nucleic acid sequence are maintained The nucleic acid sequence of the gene of the ORF1a and ORF1b regions of the LMY ver2 variant strain having the number 13394BP or the nucleic acid sequence of the gene of the genes of the ORF1a and ORF1b regions is 70% or more sequence homology, and [Y] is accession number KCTC 13393BP It is a nucleic acid sequence having a sequence homology of 70% or more within a range in which the gene nucleic acid sequence of the ORF2 to ORF7 region of the BP2017-2 mutant strain having or a function equivalent to the nucleic acid sequence is maintained. As used herein, the "equivalent function" may mean the same or similar function in qualitative (active) and/or quantitative (level) with the desired function. For example, the amino acid sequence of the protein encoded by the gene may be the same as the amino acid sequence of the protein encoded by the wild type gene.

In one example, the [X] in the nucleic acid sequence of the NSP1 gene consisting of the nucleotide sequence of SEQ ID NO: 11, all kinds of points that occur within the range in which the amino acid sequence of the NSP1 protein encoded by the NSP1 gene remains the same It may include a mutation (silent mutation), for example, can be freely selected and used within a target range for lowering the CBP level of the NSP1 gene.

For example, [X] in the nucleic acid sequence of the NSP1 gene consisting of the nucleotide sequence of SEQ ID NO: 11, position 222, position 225, position 237, position 240, position 252, position 306, position 309 Position, Position 312, Position 315, Position 324, Position 327, Position 330, Position 333, Position 336, Position 339, Position 342, Position 345, Position 357, Position 363, Position 366, Position 378, Position 379, Position 381, Position 393, Position 396, Position 543, Position 546, Position 549, Position 555, Position 558, Position 561, 573 Position, Position 579, Position 582, Position 588, Position 612, Position 618, Position 621, Position 627, Position 633, Position 639, Position 654, Position 673, Position 675, Position 678, Position 681, Position 684, Position 705, Position 708, Position 729, Position 735, Position 738, Position 741, Position 744, Position 747, Position 771, Position 786 Position, Position 789, Position 792, Position 810, Position 825, Position 828, Position 838, Position 840, Position 846, Position 849, Position 858, Position 867, Position 879, Position 882, Position 885, Position 891, Position 900, Position 903, Position 906, Position 924, Position 936, Position 939, Position 948, Position 954, Position 963, Position 966 One or more selected from the group consisting of position, 1026 position, 1029 position, 1038 position, 1047 position, 1053 position, 1066 position, 1068 position, 1086 position, and 1110 position, 25 or more, It may be a variation in which bases of 66 or more, 80 or more, or 91 whole positions are substituted.

In one example, in the nucleic acid sequence of the NSP1 gene consisting of the nucleotide sequence of SEQ ID NO: 11, G is substituted at position 222 by C, and C is substituted at position 225 by A, 237 Variation in which position T is substituted by C, mutation in position 240 is replaced by T, mutation in position 252 is substituted by C, mutation in position 306 is substituted by C, and mutation in position 309 T is substituted with C, G at position 312 is substituted with A, C at position 315 is substituted with A, T at position 324 is substituted with C, C at position 327 is G substitution at position 330, G at position 330, A substitution, C at position 333, T substitution, C at position 336 at G, G substitution, T at position 339 as C Substituted mutation, mutation in which A at position 342 is substituted by T, mutation in which T at position 345 is substituted by A, mutation in which A at position 357 is substituted by G, substitution at T at position 363 is replaced by A Variation in which T at position 366 is substituted for C, V for C at position 378, T for substitution at C at position 379, V at C at position 381 is substituted by G, Variation in which T at position 393 is substituted by C, mutation in which T at position 396 is substituted by A, mutation at which T at position 543 is substituted by C, mutation at which T at position 546 is substituted by C, 549 Variation in which position C is substituted by A, mutation in position 555 is substituted by C, mutation in position 558 is substituted by C, mutation in position 561 is substituted by A, position 573 C is substituted with T, 579 is replaced by T, C is 582, G is substituted by T, 588 is replaced by C, C is substituted, T is 612 C substitution, G at position 618, C substitution, T at position 621, C substitution, A at position 627, T substitution, T at position 633, C Substituted mutation, mutation in which T at position 639 is substituted by G, 65 C-position at position 4 is substituted with T, C-position at position 673 is substituted with T, C-position at position 675 is substituted with A, C-position at position 678 is substituted with T, 681 Variation in which position C is substituted by G, C in position 684 is substituted by G, V in position 705 is substituted by C, V in position 708 is substituted by T, Variation in position 729 A substitution with C, T at position 735, C with substitution, G at 738 with T substitution, T at 741 with C substitution, T at 744 C substitution, T at position 747, A substitution, T at position 771, C substitution, T at position 786, C substitution, C at position 789, C is T Substituted mutation, T at position 792, G substitution, G at position 810, T substitution, T at position 825, C substitution, C at position 828, C is substituted Variation, the substitution of T at position 838 by C, the variation of G at position 840 by C, the variation of C at position 846 by G, the variation at position 849 by G by A, A substitution at position 858 with G, substitution at T at position 867 with C, mutation at position T with C at position 879, substitution at C with position 882, transition at position 885 Variation in which position C is substituted by T, mutation in position 891 is substituted by T, mutation in position 900 is substituted by C, mutation in position 903 is substituted by A, position 906 T is substituted by C, G at position 924 is replaced by T, 936 at position T is substituted by C, 939 at position 939 is substituted by C, A is at position 948 Variation substituted by C, A substitution at C at position 954, V substitution at A at position 963, T substitution at C at position 966, A at position 1026 as G Substituted mutation, G at position 1029 is substituted with C, C at position 1038 Is a mutation substituted with T, a T substitution with C at 1047, a T substitution with C at 1053, a substitution with C at position 1066, a C at position 1068 1 or more, 25 or more, 66 or more, 80 or more, or 91 selected from the group consisting of a mutation substituted with C at position 1086, a substitution at C with position 1110, and a substitution with C at position 1110. Variations can include all.

For example, the [X] is the nucleic acid sequence of the NSP1 gene (SEQ ID NO: 12) and/or NSP1-beta (SEQ ID NO: 1; 5'terminal 609 base site of SEQ ID NO: 12) with the LMY ver2 variant having accession number 13394BP, Or 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99 within the range of maintaining the function equivalent to the nucleic acid sequence % Or greater than 99.5% sequence homology.

In addition, the [Y] is 70% or more, 75% or more, 80% or more within the range of maintaining the gene nucleic acid sequence of the ORF2 to ORF7 region of the BP2017-2 mutant strain having accession number KCTC 13393BP or equivalent function to the sequence, It may be a nucleic acid sequence having a sequence homology of 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more.

According to an example of the present invention, [A]n may be further included at the 3'end of [Y] of Structural Formula 1. The n is the number of nucleotides containing the base A, may be an integer of 10 to 100. Preferably 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 15 to 30 , 20 to 30, may be an integer of 20 to 26.

The polynucleotide may be RNA, reverse transcripts of the RNA (DNA), or a combination thereof. The polynucleotide may function as a genome of the PRRSV chimeric virus.

Accordingly, another example of the present invention provides a chimeric virus of porcine genital respiratory syndrome virus (PRRSV) comprising a polynucleotide having the structure of Structural Formula 1. The genome of the PRRSV chimeric virus may be DNA or RNA, and preferably RNA.

The [X] corresponds to the NSP1 gene region, including NSP1-beta of the LMY ver2 mutant with accession number 13394BP, and may be a gene fragment obtained by treating the genome of the LMY ver2 mutant with restriction enzymes AscI and PacI. For example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95 within a range that maintains the function equivalent to the nucleic acid sequence of SEQ ID NO: 1 and/or SEQ ID NO: 12, or the base sequence % Or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more.

The [Y] corresponds to the ORF2 to ORF7 region of the BP2017-2 mutant strain having accession number KCTC 13393BP, and may be a gene fragment obtained by treating the genome of the BP2017-2 mutant strain with restriction enzymes AscI and PacI, for example, a sequence 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98 within a range that maintains the nucleic acid sequence of No. 2 or equivalent function to the sequence % Or more, 99% or more, or 99.5% or more.

In one embodiment, the structural formula 1 is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more within a range maintaining the function equivalent to the nucleic acid sequence of SEQ ID NO: 4 or the nucleic acid sequence , 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more. For example, it may be a nucleic acid sequence of SEQ ID NO: 4.

In Table 1, the bolded portion of the base sequence of the NSP1 gene of LMY ver2 indicates the NSP1-beta region.

(In the above table, the polynucleotide (RNA) provided herein may include a nucleic acid sequence in which T is substituted by U among the sequences set forth in SEQ ID NOs: 1 to 4.)

The present invention provides a PRRSV chimeric virus comprising the polynucleotide of structural formula 1 as a genome. The PRRSV chimeric virus is 1 to 80 passages, 1 to 70 passages, 1 to 60 passages, 1 to 50 passages, 1 to 40 passages, 1 to 30 passages, 1 to 20 passages, or 1 to 10 passage cultured progeny viruses It may be included.

[Structural Formula 1]

In the above formula, [X] is the nucleic acid sequence of the NSP1 gene and/or NSP1-beta of the LMY ver2 variant with accession number 13394BP or a nucleic acid sequence having 70% or more sequence homology with the nucleic acid sequence, and [Y] is the accession The gene nucleic acid sequence of the ORF2 to ORF7 region of the BP2017-2 variant strain having the number KCTC 13393BP or a nucleic acid sequence having 70% or more sequence homology with the nucleic acid sequence.

For example, the [X] is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the nucleic acid sequence of the NSP1 gene and/or NSP1-beta of the LMY ver2 mutant with accession number 13394BP or the nucleic acid sequence. Or more, 95%, 96%, 97%, 98%, 99%, or 99.5% or more nucleic acid sequences.

In addition, the [Y] is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more with the gene nucleic acid sequence or the nucleic acid sequence of the ORF2 to ORF7 region of the BP2017-2 variant strain having accession number KCTC 13393BP , 95%, 96%, 97%, 98%, 99%, or 99.5% or more nucleic acid sequences.

According to an example of the present invention, [A]n may be further included at the 3'end of [Y] of Structural Formula 1. The n is the number of nucleotides containing the base A, may be an integer of 10 to 100, for example, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 15 to 80 , 15 to 70, 15 to 60, 15 to 50, 15 to 40, 15 to 30, 20 to 30, may be an integer of 20 to 26.

The polynucleotide may be RNA, reverse transcripts of the RNA (DNA), or a combination thereof. The polynucleotide may function as a genome of the PRRSV chimeric virus. Accordingly, another example of the present invention provides a chimeric virus of porcine genital respiratory syndrome virus (PRRSV) comprising a polynucleotide having the structure of Structural Formula 1. The genome of the PRRSV chimeric virus may be DNA or RNA, and preferably RNA.

The [X] corresponds to the NSP1 gene region, including NSP1-beta of the LMY ver2 mutant with accession number 13394BP, and may be a gene fragment obtained by treating the genome of the LMY ver2 mutant with restriction enzymes AscI and PacI. For example, the nucleic acid sequence of SEQ ID NO: 1 or the nucleic acid sequence and 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, It may be a nucleic acid sequence having a sequence homology of 99% or more, or 99.5% or more.

The [Y] corresponds to the ORF2 to ORF7 region of the BP2017-2 mutant strain having accession number KCTC 13393BP, and may be a gene fragment obtained by treating the genome of the BP2017-2 mutant strain with restriction enzymes AscI and PacI, for example, a sequence The nucleic acid sequence of No. 2 or 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more with the nucleic acid sequence, Or it may include a nucleic acid sequence having a sequence homology of 99.5% or more.

In one example, the structural formula 1 is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more of the nucleic acid sequence of SEQ ID NO: 4 or the nucleic acid sequence , 98% or more, 99% or more, or 99.5% or more. For example, it may be a nucleic acid sequence of SEQ ID NO: 4.

One embodiment provides the PRRSV chimeric virus comprising a nucleic acid sequence comprising the polynucleotide of SEQ ID NO: 4 as a genome, which was named LMY+BP2017. The LMY+BP2017 virus may be a virus having accession number KCTC 13394BP.

At least one base contained in the nonstructural protein 1 (NSP1) of SEQ ID NO: 5 of the LMY parent strain (Accession No. GenBank accession no.DQ473474.1.), which forms the skeleton of the LMY+BP2017 chimeric virus, The substituted variant may be a PRRSV variant strain comprising NSP1-beta.

The nucleotide sequence substituted in the nucleotide sequence of the gene encoding NSP1 and/or NSP1-beta of the LMY strain may be selected using a known SAVE (Synthetic Attenuated Virus Engineering) program, preferably, the present inventors It may be selected using the developed SAVE program. Specifically, the genetically safe NSP1 region in the genome of the parent strain LMY was analyzed by the SAVE program developed by the present inventors, and selected or substituted for a part or all of the nucleotide sequence showing a relatively high Codon Pair Bias (CPB). It may be a base sequence deoptimized. The deoptimization of the base sequence, a mutation in which the G at position 222 of the gene encoding the NSP1 protein of the LMY strain consisting of the base sequence of SEQ ID NO: 11 is substituted with C, and the mutation at C at position 225 is substituted with A, 237 A mutation in position T replaced by C, a mutation in position 240 replaced by T, a mutation in position 252 replaced by C, a mutation in position 306 replaced by C, a position 309 Of T is substituted by C, G of 312 is substituted by A, C of 315 is substituted by A, 324 of T is substituted by C, C of 327 Is a G substitution, G at position 330 is substituted with A, C at position 333 is substituted with T, C at position 336 is substituted with G, T at position 339 is C , A substitution at position 342, T substitution, T substitution at position 345, A substitution, substitution at position A at 357, G substitution, T at position 363, substitution at A Mutation, mutation in which T at position 366 is replaced by C, mutation in which C at position 378 is substituted by T, mutation in which C at position 379 is substituted by A, mutation at position 381 where C is substituted by G , Mutation in which T at position 393 is substituted by C, mutation in which T at position 396 is substituted by A, mutation at which T at position 543 is substituted by C, mutation at which T at position 546 is substituted by C, 549 C substitution at position A is substituted by A, T at position 555 is substituted by C, T at position 558 is substituted by C, T at position 561 is substituted by A, Variation at position 573 Of C is substituted with T, 579 of T is substituted with C, 582 of G is substituted with T, 588 of T is substituted with C, T of 612 Is a mutation substituted by C, a G substitution at position 618, a substitution at C at position 621, a substitution at C at position 621, a substitution at position A at position 627 by T, and a T at position 633 is C. Substituted with 63 Variation where T at position 9 is replaced by G, V substitution at C at position 654, T substitution at C at position 673, V substitution at C at position 675, 678 Variation in which position C is substituted by T, mutation in position 681 is substituted by G, mutation in position 684 is substituted by G, mutation in position 705 is substituted by C, position 708 C is a substitution with T, A at position 729 is substituted with C, a 735 position is substituted with C, a 738 position is replaced with G, and a T is substituted with T. C substitution, T at position 744, C substitution, T at position 747, A substitution, T at position 771, C substitution, C at position 786, C Substituted mutation, C substitution at position 789, T substitution at position 792, G substitution at position 792, C substitution at position 810, substitution at T, 825 position at T substitution at C Variation, the substitution of C at position 828 with T, the variation of T at position 838 with C, the variation of G at position 840 with C, the variation of C at position 846 with G, Variation in which G at position 849 is substituted by A, mutation in which A is replaced by G at position 858, mutation in which T at position 867 is substituted for C, mutation at which T at position 879 is substituted by C, 882 Variation in which position C is substituted with G, mutation in position 885 is substituted by T, mutation in position 891 is substituted by T, mutation in position 900 is substituted by C, position at 903 C is substituted with A, T at position 906 is substituted with C, G at position 924 is substituted with T, T at position 936 is substituted with C, T at position 939 Variation substituted with C, A substituted with C at position 948, A substituted with C at position 954, V substituted with A at position 963 with T, T at position 966 with C Substituted mutation, A at position 1026 is substituted with G, G at position 1029 is C substitution, C substitution at position 1038, T substitution, C substitution at position 1047, C substitution, C substitution at position 1053, C substitution, C at position 1066, C 1 or more, 25 or more selected from the group consisting of a substituted mutation, a mutation in which A at position 1068 is substituted by C, a mutation in which C at position 1086 is substituted by T, and a mutation in which T at position 1110 is substituted by C. , 66 or more, 80 or more, or may include all of the 91 variations, but is not limited thereto.

The SAVE program can be used to quantify CPB, which is a bias caused by interaction when viral gene codons are arranged in pairs, using a computer algorithm, and the proliferation of the virus is reduced when the CPB value is deoptimized, and attenuated. (Virus Attenuation by Genome-Scale Changes in Codon Pair Bias, Science, 2008, J. Robert Coleman et al.), to replace some sequences with high CPB levels with sequences with low CPB levels, preferably Codon Silent mutation according to the Pair Deoptimization principle can produce an attenuated LMY ver2 mutant used in the preparation of the chimeric virus of the present invention. The LMY ver2 mutant may include a gene encoding the NSP1-beta protein consisting of the nucleotide sequence of SEQ ID NO: 6, or a gene encoding the NSP1 protein consisting of the nucleotide sequence of SEQ ID NO: 12.

In addition, the present invention can provide cells comprising the genome of the PRRSV chimeric virus of the present invention. The cell refers to a cell in which the chimeric virus genome (DNA, RNA, or a vector containing the same) or the chimeric virus containing the genome is transfected in order to manufacture the chimeric virus in large quantities. Inside, the type of cells is not particularly limited.

According to an example of the present invention, it is possible to provide a porcine genital respiratory syndrome virus vaccine composition comprising the PRRSV chimeric virus or a passaged cultured progeny thereof.

The passaged cultured progeny is a progeny virus cultured from 1 to 80 passages, 1 to 70 passages, 1 to 60 passages, 1 to 50 passages, 1 to 40 passages, 1 to 30 passages, 1 to 20 passages, or 1 to 10 passages It may be to include.

According to an example of the present invention, the vaccine may be a live vaccine or a four vaccine, but is preferably a live vaccine. Specifically, the attenuated PRRS chimeric virus described herein may be a modified live vaccine containing, in a surviving state, one or more virus strains described above in a pharmaceutically acceptable carrier. Alternatively, or alternatively, an inactivated virus can be used to prepare a vaccine.

The vaccine may further include one or more selected from the group consisting of carriers, diluents, excipients, and adhuvants. The pharmaceutically acceptable carrier is not particularly limited in its kind, and may include any and all solvents, dispersion media, coatings, stabilizers, preservatives, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like.

On the other hand, the effective amount of the attenuated chimeric virus of the present invention included in the vaccine composition may be the amount of the virus that can attract or induce an immune response in animals to which the effective dose of the virus has been administered. The effective amount may depend on the vaccine components and dosing schedule. The dosage of the vaccine composition is in the range of TCID ₅₀ 2 to 6, preferably 3 to 4, but may vary depending on the type of individual, but is not limited thereto.

The genome of the LMY-BP2017 chimeric virus of the present invention, and a vaccine composition comprising the same, can be used to prevent swine from the effects of PRRS disease. In addition, subunits, including immunogenic fragments or fractions of the PRRS chimeric virus, can also be used to prevent swine from the effects of PRRS disease. The attenuated chimeric virus of the present invention or a vaccine composition comprising the same can be administered prophylactically before the pig is exposed to the PRRS-inducing PRRS virus strain, and can be administered to the pig at the same time the pig is exposed to the virus strain. And can be administered therapeutically after exposure of the target pig to the virus strain.

The vaccine composition provided by the present invention may be used for prevention of porcine genital respiratory syndrome (PRRS), for example, PRRS caused by North American PRRSV. In one example, the North American PRRSV may be a type II type VR2332 virus strain.

The attenuated PRRSV chimeric virus of the present invention, or a vaccine composition comprising the same, is oral, parenteral, subcutaneous, intramuscular, intradermal, sublingual, transdermal, rectal, transmucosal, surface area via inhalation, buccal administration, or its It can be administered in combination. In addition, the attenuated PRRS chimeric virus can be administered in the form of an implant capable of allowing sustained release of the attenuated virus.

The attenuated PRRSV chimeric virus of the present invention or a vaccine composition comprising the same can be administered via injection, inhalation or transplantation, with injection being particularly preferred. Depending on the desired duration and effectiveness of vaccination or treatment, the attenuated PRRSV chimeric virus or vaccine composition comprising the same may be administered once or several times, and also intermittently at different dosages daily, such as for days, weeks or months. . The injection can be given in the desired amount or by subcutaneous or nasal spraying, or alternatively continuous injection.

According to an example of the present invention, the PRRSV chimera virus of the present invention is the TCID ₅₀ of the virus measured by inoculating the chimeric virus and its parent strain (Accession No. GenBank accession no.DQ473474.1.) in pig lung macrophages. The value of can be a virus having a TCID ₅₀ value of 0.01 to 0.1 times compared to the TCID ₅₀ value of the parent strain. Preferably, the measurement period is 2 days later, and the TCID ₅₀ value may be 0.05 to 0.1 times. The PRRSV chimeric virus of the present invention has a TCID ₅₀ value lower than that of the parent strain, and is less attenuated and proliferative than the parent strain.

In addition, the PRRSV chimeric virus of the present invention and its parent strain (Accession No. GenBank accession no.DQ473474.1.) were inoculated in pigs and the neutralizing antibody content of the mutant strain measured was 2 to 8 times, preferably Provides a virus with 2 to 4 fold. Preferably, the measurement period is 28 days later. The term neutralizing antibody means an antibody having a neutralizing ability against VR2332, a representative North American PRRSV strain. Therefore, when the attenuated chimera virus of the present invention or a vaccine composition containing the same is inoculated into a pig, the titer of the neutralizing antibody of the pig increases and the immune effect on PRRS is significantly increased by increasing the expression of the pig's immune factor. I can do it.

According to one embodiment, the CPB value of the PRRSV chimeric virus LMY+BP2017 of the present invention described above represents a lower value than the LMY parent strain (Accession No. GenBank accession no.DQ473474.1.), preferably -0.39 To 0, more preferably -0.35 to -0.20, suitably -0.35 to -0.26, for example -0.2393184052058128. When the CPB level is less than -0.39, it is difficult to produce a virus, and when it exceeds 0, the operation is possible, but there is a problem in that the proliferation does not decrease and thus is not attenuated. Therefore, the attenuated LMY+BP2017 chimeric virus of the present invention preferably has the CPB level.

In addition, the present invention provides an LMY+BP2017 chimeric virus having a CpG, UpA value of 1.0 to 3.0 times that of a parent strain (Accession No. GenBank accession no.DQ473474.1.). Changes in the ratio of CpG and UpA inevitably occur during the deoptimization process. When the CpG and UpA levels of the eukaryotic gene are increased, cell proliferation is reduced and the virus is attenuated, thereby attenuating the virus. The LMY+BP2017 chimeric virus of the present invention has higher CpG and UpA levels than the parent strain, preferably 1.0 to 3.0 times higher than the parent strain, for example, CpG of the attenuated chimeric virus of the present invention. May be 1.1753, and UpA may be 1.03.

According to an example of the present invention, the genome of the LMY ver2 mutant with accession number 13394BP is treated with restriction enzymes AscI and PacI to prepare a polynucleotide fragment, and the genome of BP2017-2 mutation having accession number KCTC 13393BP is restricted. A step of preparing a polynucleotide fragment by treating with AscI and PacI, and recombining the polynucleotide fragment of LMY ver 2 with the polynucleotide fragment of BP2017-2 to prepare an infectious clone, a pig genital organ A method of producing the chimeric virus of the respiratory syndrome virus can be provided.

The polynucleotide fragment prepared by treating the genome of the LMY ver2 mutant with restriction enzymes AscI and PacI may include a region encoding NSP1-beta.

The polynucleotide fragment prepared by treating the genome of the BP2017-2 mutant with restriction enzymes AscI and PacI may include ORF2 to ORF7 sites.

In the step of recombining the fragments, ligase may be used, and the infectious clone prepared above may be inoculated into cells to prepare a chimeric virus.

The cell refers to a cell in which a chimeric virus DNA or RNA, or a vector containing it, an infectious clone, or a chimeric virus is transfected, in order to prepare a large amount of the chimeric virus, and does not specifically limit the type of cell.

In addition, an embodiment of the present invention may provide a composition for preventing or treating porcine genital respiratory syndrome, including the vaccine composition.

In a preferred form, the composition for preventing or treating porcine reproductive and respiratory syndrome of the present invention may include additional components known to those skilled in the art, and further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. Can.

In addition, it can be formulated and used in the form of oral formulations, external preparations, suppositories, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. according to a conventional method. Suitable formulations known in the art are preferred to use those disclosed in Remington's Pharmaceutical Science, recently Mack Publishing Company, Easton PA.

Carriers, excipients and diluents that may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. When formulating the composition, it is usually prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. in the composition. Is prepared. Also, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral administration include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used as diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, Witepsol, Macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The preferred dosage of the composition of the present invention depends on the condition and weight of the individual, the severity of the disease, the drug form, the route and duration of administration, but can be appropriately selected by those skilled in the art. For example, for the desired effect, the composition of the present invention may be administered in an amount of 0.0001 to 1,000 mg/kg (body weight) per day. The composition may be administered once a day or divided several times.

The composition of the present invention can be administered to a subject by various routes. Any mode of administration can be expected.

Another example of the present invention can provide a method for preventing or treating porcine genital respiratory syndrome, comprising administering a vaccine composition of the present invention to a pig.

By administering the vaccine composition comprising the chimeric virus genome of the present invention to pigs, it is possible to induce that the pig's immune response to the PRRSV antigen is enhanced, and to induce the pig's immune response to the PRRSV antigen to be enhanced, porcine genital respiratory syndrome It can provide a way to prevent or treat. Preferably it can be prevented.

Specifically, the method includes subcutaneous injection, intravenous injection, intradermal injection, parenteral injection, intramuscular injection, needle free injection, electroporation, oral delivery, intranasal delivery, oronasal delivery, Or any combination thereof.

The present invention can provide a kit for performing any of the methods described above. The kit reduces the clinical signs or effects of the container, preferably a vaccine composition containing the attenuated PRRS chimeric virus of the invention, a pharmaceutically acceptable carrier, adjuvant and PRRS infection, preferably PRRS frequency or severity Instructions for administering the immunogenic composition to an animal in need thereof may be included. The kit can also include injection means and/or other forms of administration. Also, the kit may include a solvent. The attenuated vaccine can be lyophilized and restored to a solvent to be a solution for injection and/or inhalation. The solvent can be water, physiological saline, buffer or reinforcing solvent. The kit can include a separation container containing the attenuated virus, solvent and/or pharmaceutically acceptable carrier. The instructions for use may be labels and/or printed materials attached to one or more containers.

The PRRSV virus chimeric virus according to the present invention can be used to effectively defend PRRS by significantly increasing the secretion of neutralizing antibodies when inoculated into pigs, and can be usefully used as a vaccine for the treatment of PRRS.

1 shows a general genomic arrangement of PRRS virus.

Figure 2 is a schematic diagram showing the genome sequence of the LMY + BP2017 chimeric virus synthesized by cutting and connecting a part of clones of the LMY ver2 virus and the BP2017-2 virus, respectively.

Figure 3 shows the phylogenetic tree of the PRRSV LMY ver2 virus and the LMY+BP2017 chimera virus.

FIG. 4 shows the proliferative difference between PRRSV LMY ver2 virus and LMY+BP2017 chimeric virus in Porcine Alveolar Macrophage cells (PAM cells).

Figure 5 is a PRRSV LMY, LMY+BP2017 chimera virus in pigs, comparing the secretion of neutralizing antibodies after PBS inoculation.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, the present invention is not limited by the following examples.

Since RNA is easily destroyed, the cells were transfected by synthesizing RNA from the DNA after converting to DNA.

실시예 1: 키메라 바이러스의 제조Example 1: Preparation of chimeric virus

1-1. Chimera virus design

PRRS chimera virus is a non-structural protein (NON structural Protein 1, NSP1; SEQ ID NO: 11) of the variant strain of LMY strain and ORF2, ORF3, ORF4, ORF5, ORF6 of BP2017-2 isolated in 2017 from Namsan Farm, Namsan-ri, Gongju-si, Chungnam , And ORF7 sites.

1-2. Manufacturing attenuated LMY mutants

An LMY ver2 mutant was prepared by substituting 91 bases of the nucleotide sequence of the gene at the NSP1 site using the PRRS strain, LMY strain (GenBank accession no.DQ473474.1.), isolated from the existing quarantine headquarters. The NSP1 site gene was replaced with 25 bases at the NSP1-alpha site and 66 bases at the NSP1-beta site.

Specifically, the recombinant strain LMY ver2 is a base sequence according to the Codon Pair Deoptimization Principle (Table 3) using a generally known Synthetic Attenuated Virus Engineering (SAVE) program or SAVE (Synthetic Attenuated Virus Engineering) program developed by the present inventors. A portion of the was prepared by silent mutation (silent mutation). First, using the SAVE program, the codon pair base (CPB) value, which is a bias caused by the interaction of the gene codons of the LMY virus in pairs, was quantified using a computer algorithm. CPB levels fluctuate when some nucleotide sequences of the LMY virus gene are replaced with other nucleotide sequences, and are closely related to the proliferation of the virus. Viral Attenuation by Genome-Scale Changes in Codon Pair Bias, Science, 2008, J. Robert Coleman et al. ). The present inventors selected NSP1 regions (NSP1-alphe and NSP1-beta, SEQ ID NO: 11) with high genetic stability in the genome of the LNY parent strain and analyzed them with the SAVE program, and NSP1-alpha among base regions with relatively high CPB values LMY virus mutants were prepared by deoptimization by selecting 25 bases at the site, 66 bases at the NSP1-beta site, and 91 bases, replacing them with other bases. The LMY virus mutant strain in which 91 base sequences of the NSP1 prepared by the above method were mutated (SEQ ID NO: 12) was referred to as LMY ver2, and the base sequences are shown in Table 3 below. The LMY ver2 has accession number 13394BP.

(In Table 3, the bolded and underlined portion of the nucleic acid sequence of the LMY ver2 NSP1 gene is the base portion where the mutation occurred in the LMY NSP1 gene or LMY NSP1-beta gene.)

Next, to confirm the degree of attenuation of the LMY ver2 mutant strain, CPB values were measured. The NSP1 CPB value of the LMY parent strain was measured to be about 0.0139 and the NSP1-beta CBP value to about 0.016, but the CPB value of the LMY ver2 variant strain of the present invention was measured to be about -0.2393 to NSP1 and about -0.33 to NSP1-beta. From this, it was found that the proliferative potential of the LMY ver2 mutant strain of the present invention is reduced compared to the existing parent strain, and is an attenuated strain.

Next, an infectious clone of LMY ver2 was prepared.

First, the entire gene sequence (GenBank accession no.DQ473474.1.) of the LMY strain was divided into 7 fragments and synthesized. Fragment 1 of the 7 fragments was an NPS-1 site, and the site was synthesized as a DNA fragment (SEQ ID NO: 12) in which 91 bases among the gene base sequences of the NSP1 site of the LMY strain were substituted. The synthesized fragment gene was cut with restriction enzymes in Table 5 in order, and then linked with ligase to prepare one infectious clone.

1-3. Construction of chimeric virus clones

As shown in the schematic diagram of Fig. 1, the PRRS virus contains a total of 8 ORFs, namely ORF1a, ORF1b, and ORF2 to 7.

In the genome region containing the entire structural gene of the LMY ver2 mutant amplified in Example 1-1, the regions of ORF2 to ORF7 were cleaved using restriction enzymes AscI and PacI. Subsequently, among the genomic regions of the BP2017 strain, the portions corresponding to the regions of ORF2 to ORF7 of the LMY ver2 mutant were cut with the same AscI and PacI restriction enzymes, and then to the ORF1a and ORF1b regions of the LMY ver2 mutant and the ORF2 to ORF7 regions of the BP2017 strain. One recombinant infectious clone was prepared by linking the corresponding site with a ligase, which was named LMY+BP2017. The restriction enzymes used are shown in Table 6 below.

After inserting the completed infectious clone into a high copy vector loaded with CMV promoter and ampicillin resistance gene, transfected into BHK cells (Korea Cell Line Bank) using lipofectamine, and finally LMY+BP2017 of the present invention. The chimeric virus was generated.

The LMY+BP2017 chimera virus was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on October 24, 2018, and received the accession number KCTC13675BP.

실시예 2. LMY+BP2017 키메라 바이러스의 CpG 및 UpA 수치 측정Example 2 Measurement of CpG and UpA levels of LMY+BP2017 chimeric virus

To evaluate the proliferation of the LMY+BP2017 chimera virus prepared in Example 1, the ratio of CpG and UpA in the NSP1 gene of the LMY+BP2017 chimera virus and the CpG and UpA values in the NSP1 beta gene of the parent strain LMY were measured. .

Changes in the ratio of CpG and UpA of the viral gene inevitably occur during the deoptimization process. When the levels of CpG and UpA of the gene increase, cell stress is induced and the virus's proliferation is reduced. The ratio of CpG and UpA in NSP1 beta gene of LMY+BP2017 chimeric virus and LMY virus was measured using SSE program (version 1.2), and the results are shown in Table 7 below.

As shown in Table 7, it was confirmed that the levels of CpG and UpA of the LMY+BP2017 chimera virus increased compared to the parent strain LMY, from which it was confirmed that the proliferation of the LMY+BP2017 chimera virus was significantly reduced compared to the LMY parent strain. Could.

실시예 3. LMY+BP2017 키메라 균주의 동정Example 3. Identification of LMY+BP2017 chimeric strain

3-1. Identification of LMY+BP2017 chimeric strain

The LMY+BP2017 chimeric strain was identified by sequencing the LMY+BP2017 chimeric strain synthesized in Example 1 and the NSP1 region of the LMY strain (GenBank accession no.DQ473474.1.).

Specifically, PCR was performed using the primer set of Table 8 and one step RT PCR kit (Intron) that can be detected from 1 to 1654nt, including the NSP1 region of the LMY virus, specifically, at 45°C for 30 minutes and at 95°C 5 After the initial RT process for a minute, the reaction was carried out for 2 minutes at 94°C for 30 seconds and 61°C for 30 seconds at 72°C, and the process was repeated 38 times. Next, sequencing of the amplification products by PCR results was completed to identify each strain.

3-2. Comparison of homology with other strains and phylogenetic tree

To confirm the genetic difference between the recombinant virus strain LMY ver2 of the present invention prepared in Example 1 and a known PRRS virus strain, the sequence identity of NSP1 regions of each strain was measured using a bioedit program, and the comparison results of homology are shown in the table. It is shown in 9.

As shown in Table 9, the homology comparison result showed that the LMY+BP chimera virus possesses a unique sequence different from other PRRS strains.

Based on the homology comparison result, phylogenetic tree was analyzed using bioedit program and Bootstrap was repeated 1000 times. Based on this, approximate LMY+BP2017 chimera virus and LMY phylogenetic tree were prepared and are shown in FIG. 3. From the phylogenetic tree of Fig. 3, the strain of the present virus was found.

3-3. 세포 계대 연구3-3. Cell passage research

When the LMY+BP2017 chimera virus prepared in Example 1 was passaged, it was confirmed how long the modified site was maintained. In MARC-145 cells known as PRRS virus soluble cell line, the LMY+BP2017 chimera virus of the present invention was stabilized after 30 passages, and the same method as in Example 3-1 was used to confirm gene mutation every 10 passages. Sequencing was performed.

As a result of sequencing, it was confirmed that the gene replacement site of the virus was maintained even after the 30th passage stabilization. Table 10 shows the number of nucleotide changes (nt change), mutation site (mutataion site), and amino acid mutation (a.a change) according to each passage number.

As can be seen from Table 10, even when the passage was performed 30 times or more, the mutation on the NSP1a or NSP1b gene of ORF1a was maintained, and only the mutation on the NSP2 protein or ORF2a was confirmed. Therefore, it was confirmed that even after the passage 40 times, the variation of the gene encoding the NSP1 protein of the virus produced in Example 1 was maintained.

실시예 4: LMY와 LMY+BP chimera의 증식성 비교Example 4: Comparison of proliferative properties of LMY and LMY+BP chimera

In order to confirm the proliferation of the LMY+BP2017 chimera virus prepared in Example 1, swine lung macrophages (PAM, Porcine Alveolar Macrophage) inoculated with the LMY+BP2017 chimera virus and its parent strain, LMY, and then the virus produced It was confirmed by measuring the amount of TCID ₅₀ .

Specifically, PAM cells were dispensed at a rate of 2 X 10 ⁶ cells/well per 6 well plates containing 2 ml of RPMI medium (including 10% FBS, 1% penicillin, and streptomycin). Next, LMY and LMY+BP2017 chimera viruses were inoculated into different wells at a rate of 0.01 of MOI, and after 1 hour of inoculation, all the supernatant was removed, and maintenance fluid RPMI medium (including 10% FBS, 1% penicillin, streptomycin) 2 ml was dispensed. Two days after dispensing, the supernatant of each well was collected to measure TCID ₅₀ (Tissue culture infective dose ₅₀ ). The cells used in the subsequent experiments were prepared by adding 2X10 ⁵ cell/well of MARC-145 cells to 96 wells on the day before the measurement of TCID ₅₀ in DMEM medium (10% (v/v) FBS, 1% (w/v) penicillin, streptomycin) 100ul. It was used by dispensing with.

Viruses to be inoculated were prepared as follows. The supernatant collected above was dispensed in a row in 200uL to the leftmost well of a 96-well plate, and DMEM medium (FBS, no antibiotic added) was dispensed in 180µl to the remaining wells. Next, 20 μl was collected using a multi-pipet one after the other from the left and diluted 10-fold while dispensing into the right well. The tips were diluted one after the other and the last 12th well was maintained as a negative control. The prepared virus dilution was inoculated on a plate to which MARC-145 cells were attached. At this time, all of the culture solution of the MARC-145 cell was removed, and 200 µl of PBS was dispensed into each well and discarded 3 times to perform a washing process. Thereafter, the prepared virus dilution was inoculated by dispensing 100 μl per well, and after 2 hours of inoculation, all of the inoculum was removed and 100 μl of fresh maintenance solution (DMEM medium, 10% FBS, 1% penicillin, streptomycin) was dispensed.

After dispensing, it was confirmed whether Clumping and apoptosis, a type of cytopathogenic effect (CPE), occurred in the cells for about 7 days, and finally, 4 of the 8 virus inoculation solutions were based on the dilution factor immediately before the well without CPE. Dilution multiples in which dog wells can show CPE were determined. The final value was calculated by multiplying 10 by 1 ml.

The LMY+BP2017 chimeric strain and the parent strain LMY strain were inoculated into porcine lung macrophages (PAM cells) and the TCID ₅₀ values measured after 1 to 2 days are shown in Tables 11 and 4 below.

As can be seen from Table 11 and Figure 4, the value of TCID ₅₀ measured on the 2nd day when the strain was inoculated and the virus began to be harvested was LMY+BP2017 chimera, showing a TCID ₅₀ value 100 times lower than LMY. From this, it was found that the LMY+BP2017 chimera virus of the present invention in PAM (Porcine Alveolar Macrophage) cell, which is the main infecting cell of PRRS, has at least 10-fold reduction in proliferative properties compared to LMY, and the LMY+BP2017 chimera virus of the present invention. It was confirmed that the proliferation was reduced and attenuated more than the parent strain LMY.

실시예 4: 중화항체 분비 양상 확인Example 4: Neutralizing antibody secretion pattern confirmation

After inoculation with the chimeric virus of the present invention, in order to confirm the secretion pattern of neutralizing antibodies, 12 pigs, 3 weeks old, were divided into 3 groups of 4 animals, each of which were LMY (10 ⁵ TCID ₅₀ ), LMY+BP2017 chimeric virus (10 ⁵ TCID) ₅₀ ), 2 ml of PBS was inoculated. After 4 weeks, blood was drawn from the veins of each group of pigs to separate 2 ml of serum, and the amount of neutralizing antibody in the serum was measured.

Specifically, the inspection method was conducted in accordance with a known method. All serum samples were heat inactivated at 56°C for 45 minutes before starting the test. Inactivated serum was placed in RPMI 1640 medium (10% FCS, 20mM L-glutamine, antibiotic-antimycotic mixture-100IU/ml penicillin, 100μg/ml streptomycin, 50μg/ml gentamycin, 0.25 mg/ml amphotericin B) and doubled in sequence. Diluted. Subsequently, 100 μl of each diluted serum was collected, mixed with 100 μl of the previously prepared VR2332 strain 200 TCID ₅₀ /ml and incubated at 37° C. for one hour, and then inoculated with the monolayer Marc-145 cells previously cultured the previous day. . Subsequently, after incubation at 37° C. for 1 hour, all inoculum was removed, and this was replaced with 200 μl of RPMI culture. Thereafter, the cytopathic effect (CPE) was confirmed daily while culturing at 37°C, and CPE of each well was confirmed after culturing for 5 days. In the well without CPE, virus growth was confirmed by staining with SDOW 17 antibody. The neutralizing antibody titer was measured by calculating the reciprocal of the highest dilution factor without any signs of virus growth, and the measurement results are shown in Tables 11 and 5 below.

As shown in Table 11 and FIG. 5, it was confirmed that the LMY+BP2017 chimeric virus secreted a significantly higher neutralizing antibody in cells compared to the LMY parent strain.

Claims

A polynucleotide composed of the structure of Structural Formula 1, which is a genome of the chimeric virus of porcine genital respiratory syndrome virus (PRRSV), polynucleotide:

[Structural Formula 1]

The [X] is the nucleic acid sequence of the gene of the NSP1 gene ORF1a and ORF1b region of the LMY ver2 mutant with accession number KCTC 13394BP or a nucleic acid sequence having 70% or more sequence homology with the nucleic acid sequence

The [Y] is a nucleic acid sequence of the ORF2 to ORF7 region of the BP2017-2 isolate with accession number KCTC 13393BP or a nucleic acid sequence having 70% or more sequence homology with the nucleic acid sequence.
According to claim 1, [A] n further includes a 3'end of [Y] of Structural Formula 1,

The n is the number of nucleotides of the base adenine (Adenine, A), an integer of 1 to 100, polynucleotide.
The polynucleotide of claim 1, wherein the polynucleotide of the PRRSV chimeric virus is DNA or RNA.
The polynucleotide according to claim 1, wherein [X] is a gene fragment obtained by treating the genome of the LMY ver2 mutant with accession number 13394BP with restriction enzymes AscI and PacI.
The polynucleotide according to claim 1, wherein [Y] is a gene fragment obtained by treating the genome of the BP2017-2 mutant strain having accession number KCTC 13393BP with restriction enzymes AscI and PacI.
According to claim 1, wherein [X] is, in the nucleic acid sequence of the NSP1 gene consisting of the nucleotide sequence of SEQ ID NO: 11, position 222, position 225, position 237, position 240, position 252, position 306 , Position 309, Position 312, Position 315, Position 324, Position 327, Position 330, Position 333, Position 336, Position 339, Position 342, Position 345, Position 357, 363 Position 3, Position 366, Position 378, Position 379, Position 381, Position 393, Position 396, Position 543, Position 546, Position 549, Position 555, Position 558, Position 561 , Position 573, Position 579, Position 582, Position 588, Position 612, Position 618, Position 621, Position 627, Position 633, Position 639, Position 654, Position 673, 675 Position #678, Position #678, Position #81, Position #684, Position #705, Position #708, Position #729, Position #735, Position #738, Position #731, #744, Position #747, Position #711 , Position 786, Position 789, Position 792, Position 810, Position 825, Position 828, Position 838, Position 840, Position 846, Position 849, Position 858, Position 867, 879 Position, 882 position, 885 position, 891 position, 900 position, 903 position, 906 position, 924 position, 936 position, 939 position, 948 position, 954 position, 963 position , One or more positions selected from the group consisting of position 966, position 1026, position 1029, position 1038, position 1047, position 1053, position 1066, position 1068, position 1086, and position 1110 A polynucleotide, wherein the base contains a substituted NSP1 gene.
The polynucleotide of claim 6, wherein the variant NSP1 gene is represented by a nucleic acid sequence of SEQ ID NO: 12 or a nucleic acid sequence having 70% or more homology to it.
The polynucleotide according to claim 1, wherein [Y] is represented by a nucleic acid sequence of SEQ ID NO: 2 or a nuclear sequence having 70% or more homology thereto.
The polynucleotide according to claim 1, wherein the structural formula 1 is represented by a nucleic acid sequence of SEQ ID NO: 4 or a nucleic acid sequence having 70% or more homology thereto.
A PRRSV chimeric virus comprising the polynucleotide of any one of claims 1 to 9 as a genome.
The virus according to claim 10, wherein the chimeric virus has accession number KCTC13675BP.
Porcine genital respiratory syndrome virus vaccine composition comprising the chimeric virus of PRRSV of claim 10 or a passaged cultured progeny thereof.
The vaccine composition of claim 12, wherein the chimeric virus has accession number KCTC13675BP.
The vaccine composition of claim 11, wherein the vaccine is a live vaccine.
The vaccine composition of claim 12, wherein the vaccine further comprises one or more selected from the group consisting of carriers, diluents, excipients, and adhuvants.
The method of claim 12, after inoculating the vaccine composition and its parent strain (Accession No. GenBank accession no.DQ473474.1.) into the lung macrophages of pigs, the value of the measured TCID 50 of the chimeric virus, Vaccine composition having a TCID 50 value of 0.01 to 0.1 times compared to the TCID 50 value of the strain.
According to claim 12, After the vaccine composition and its parent strain (Accession No. GenBank accession no.DQ473474.1.) inoculated into pigs, the measured neutralizing antibody value of the chimeric virus, the neutralizing antibody value of the parent strain Compared to 2 to 8 times, the vaccine composition.
Preparing a polynucleotide fragment by treating the genome of the LMY ver2 mutant with accession number 13394BP with restriction enzymes AscI and PacI;

Preparing a polynucleotide fragment by treating the genome of the BP2017-2 variant having accession number KCTC 13393BP with restriction enzymes AscI and PacI; And

A method of producing a chimeric virus of porcine genital respiratory syndrome virus, comprising the step of preparing an infectious clone by recombining the polynucleotide fragment of LMY ver 2 and the polynucleotide fragment of BP2017-2.
The method according to claim 18, wherein the polynucleotide fragment prepared by treating the genome of the LMY ver2 mutant with restriction enzymes AscI and PacI comprises a site encoding an NSP1 protein.
The method according to claim 18, wherein the polynucleotide fragment prepared by treating the genome of the BP2017-2 mutant with restriction enzymes AscI and PacI comprises ORF2 to ORF7 sites.
A pharmaceutical composition for the prevention or treatment of porcine genital respiratory syndrome, comprising the vaccine composition of claim 12.
A method of preventing or treating porcine genital respiratory syndrome, comprising administering the vaccine composition of claim 12 to a pig.