KR101835989B1 - Recombinant influenza virus to form cross-protection against multiple subtypes h3 and h7 of influenza viruses and vaccine comprising the same - Google Patents

Recombinant influenza virus to form cross-protection against multiple subtypes h3 and h7 of influenza viruses and vaccine comprising the same Download PDF

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KR101835989B1
KR101835989B1 KR1020160111653A KR20160111653A KR101835989B1 KR 101835989 B1 KR101835989 B1 KR 101835989B1 KR 1020160111653 A KR1020160111653 A KR 1020160111653A KR 20160111653 A KR20160111653 A KR 20160111653A KR 101835989 B1 KR101835989 B1 KR 101835989B1
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influenza virus
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influenza
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최영기
김세미
최여정
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충북대학교 산학협력단
아이디바이오 주식회사
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
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    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus

Abstract

The present invention relates to a novel influenza virus effective for a crossing-over immunity reaction against different subtypes H3 and H7, respectively, to a vaccine comprising the influenza virus as active ingredient, and to a method for preventing or treating an influenza virus infection disease by administering the vaccine to an individual. The recombinant influenza viruses of the present invention comprises a chimeric hemagglutinin gene produced by mixing a hemagglutinin HA1 region and an HA2 region derived from two different subtypes of H3 and H7 to show an effect of the crossing-over immunity reaction against viruses with various subtypes and clades, thereby being very useful as a viral vaccine having a wide range of neutralizing antibody effects.

Description

인플루엔자 바이러스의 다중 아형 H3 및 H7 에 대한 다중 교차 면역반응을 형성하는 신규한 재조합 인플루엔자 바이러스 및 이를 포함하는 백신{RECOMBINANT INFLUENZA VIRUS TO FORM CROSS-PROTECTION AGAINST MULTIPLE SUBTYPES H3 AND H7 OF INFLUENZA VIRUSES AND VACCINE COMPRISING THE SAME}FIELD OF THE INVENTION The present invention relates to novel recombinant influenza viruses which form multiple cross-over immune responses against multiple subtypes H3 and H7 of influenza viruses and vaccines comprising the same. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to recombinant influenza viruses }

본 발명은 각기 다른 H3 및 H7 아형의 인플루엔자 바이러스에 대한 교차 면역반응에 효과가 있는 신규한 인플루엔자 바이러스, 이를 유효성분으로 함유하는 백신, 상기 백신을 개체에 투여하여 H3 및 H7 인플루엔자 바이러스 감염 질환을 예방 또는 치료하는 방법, 상기 바이러스 또는 이의 항원을 포함하는 인플루엔자 바이러스의 진단용 조성물 또는 상기 바이러스의 검출 방법에 관한 것이다.The present invention relates to a novel influenza virus which is effective for cross-reacting with influenza viruses of H3 and H7 subtypes of different H3 and H7 subtypes, a vaccine containing the influenza virus as an active ingredient, and a method for preventing and treating H3 and H7 influenza virus infectious diseases Or a composition for the diagnosis of influenza virus comprising the virus or an antigen thereof, or a method for detecting the virus.

인플루엔자 바이러스는 직경이 약 125nm인 입자 크기를 갖는 RNA 외피 바이러스이다. 바이러스는 기본적으로 리피드 이중층 구조 및 외부 글리코단백질을 갖는 바이러스 외피에 의해 둘러 쌓여진 내부 뉴클레오캡시드 또는 핵단백질과 결합된 리보핵산(RNA)의 코어로 이루어져 있다. 바이러스 외피의 내부층은 주로 매트릭스 단백질로 이루어지며, 외부층은 주로 숙주-유래 리피드 물질로 이루어진다.Influenza viruses are RNA envelope viruses with a particle size of about 125 nm in diameter. The virus consists essentially of a core of ribonucleic acid (RNA) bound to an internal nucleocapsid or nucleoprotein surrounded by a viral envelope with a lipid bilayer structure and an exogenous glycoprotein. The inner layer of the viral envelope consists mainly of matrix proteins and the outer layer consists mainly of host-derived lipid material.

인플루엔자 바이러스 게놈은 11개의 단백질 (HA, NA, NP, M1, M2, NS1, NEP, PA, PB1, PB1-F2, PB2)를 코드화시키는 8개의 단일 RNA 가닥 상에 함유된다. 게놈의 분절화 성질은 세포 공동서식 동안 상이한 바이러스 균주 사이에서 전체 유전자의 교환을 허용한다. 8개의 RNA 분절은 혈구응집소를 인코딩하는 HA; 뉴라미니다아제를 인코딩하는 NA; 핵단백질을 인코딩하는 NP; 동일한 RNA 분절로부터 상이한 해독 구조를 사용함으로써 2개의 매트릭스 단백질 (M1 및 M2)을 인코딩하는 M; 동일한 RNA 분절로부터 상이한 해독 구조를 사용함으로써 2개의 독특한 비구조 단백질 (NS1 및 NEP)을 인코딩하는 NS; RNA 폴리머라아제를 인코딩하는 PA; 동일한 RNA 분절로부터 상이한 해독 구조를 사용함으로써 RNA 폴리머라아제 및 PB1-F2 단백질 (아팝토시스를 유도함)을 인코딩하는 PB1; 및 RNA 폴리머라아제를 인코딩하는 PB2로 이루어져 있다.The influenza virus genome is contained on eight single strands of RNA encoding 11 proteins (HA, NA, NP, M1, M2, NS1, NEP, PA, PB1, PB1-F2 and PB2). The segmentation nature of the genome allows the exchange of whole genes between different viral strains during cell co-culture. Eight RNA segments are HA that encode hemagglutinin; NA encoding neuraminidase; An NP encoding a nuclear protein; M encoding two matrix proteins (M1 and M2) by using different decoding structures from the same RNA segment; NS encoding two unique nonstructural proteins (NS1 and NEP) by using different decoding structures from the same RNA segment; PAs encoding RNA polymerases; PB1 encoding RNA polymerase and PB1-F2 protein (inducing apoptosis) by using different detoxification structures from the same RNA segment; And PB2 encoding an RNA polymerase.

이러한 인플루엔자 바이러스는 두 개의 표면 당단백질인, 헤마글루티닌(Hemagglutinin: HA) 및 뉴라미니다아제(Neuraminidase: NA)로 구성된 고도의 다형성 입자로서, 헤마글루티닌은 바이러스의 숙주 세포에의 부착과 바이러스의 세포 침투 동안 바이러스-세포막의 융합을 매개하며, 이러한 표면 단백질, 특히 헤마글루티닌은 인플루엔자 서브타입의 항원 특이성을 결정하는 것으로 알려져 있다.These influenza viruses are highly polymorphic particles consisting of two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), and hemagglutinin is a highly polymorphic particle composed of two And the fusion of virus-cell membranes during cell infiltration of the virus, and these surface proteins, in particular hemagglutinin, are known to determine the antigen specificity of the influenza subtype.

인플루엔자 바이러스는 항원의 차이에 기초하여 A형, B형 및 C형으로 분류한다. 인플루엔자 A 바이러스는 서브타입 또는 타입, 지리적 기원, 균주 번호 및 단리 연도를 포함하는 명명법에 의하여, 예를 들면 A/베이징/353/89로 기술된다. 적어도 16개의 HA 서브타입(H1-H16)과 9개의 NA 서브타입(N1-N9)이 있다. 모든 서브타입은 조류에서 발견되나, H1-H3 및 N1-N2는 인간, 돼지 및 말에서 발견된다[참고 문헌 : Murphy and Webster, orthomyxoviruses, in Virology, ed. Fields, B.N., Knipe, D.M., Chanock,R.M., 1091-1152(Raven Press, New York, (1990)]. 최근 HA서브타입의 경우 H18, NA N11까지도 발견되었다는 보고가 있다[참고: Suxiang Tong et al,"New World Bats Harbor Diverse Influenza A Viruses" PLoS Pathogens,(October 2013)].Influenza viruses are classified as type A, type B, and type C based on differences in antigen. Influenza A viruses are described by nomenclature including subtype or type, geographic origin, strain number and isolation date, for example, A / Beijing / 353/89. There are at least 16 HA subtypes (H1-H16) and 9 NA subtypes (N1-N9). All subtypes are found in algae, but H1-H3 and N1-N2 are found in humans, pigs and horses (Reference: Murphy and Webster, Orthomyxoviruses, in Virology, ed. Recently, it has been reported that H18 and NA N11 have also been found in recent HA subtypes (Suxiang Tong et al., (1999)). , "New World Bats Harbor Diverse Influenza A Viruses" PLoS Pathogens, (October 2013)].

인플루엔자는 인수 공통 전염병으로서, 인플루엔자 바이러스는 변이성이 크며 한 종에서 다른 종으로 바로 전파될 수 있는 가능성을 가지고 있어, 고병원성 인플루엔자의 세계적인 전파를 해결하는 일이 큰 과제로 떠오르고 있다. 수의분야와 관련하여 인플루엔자 바이러스는 거의 모든 포유류에 감염을 일으키므로 숙주 범위가 넓은 질병에 속한다. 돼지, 닭과 같은 산업동물의 경우 단일 감염으로 소모성 질병 혹은 다른 바이러스 및 세균과 혼합 감염됨으로써 농가에 막대한 경제적 피해를 주기 때문에, 지속적인 생독 및 사독백신을 통한 주기적인 백신접종을 실시하고 있다.As influenza is a common infectious disease, influenza viruses are highly variable and have the potential to spread from one species to another, thus addressing the global spread of highly pathogenic influenza is becoming a big challenge. With regard to the field of veterinary medicine, influenza viruses infect almost all mammals and thus belong to a wide range of host diseases. Pigs, and chickens are infected with consumable diseases or other viruses and germs to cause a great economic damage to farmers. Therefore, they are regularly vaccinated with viruses and sadox vaccines.

본 발명자들은 다양한 아형을 가지고 있는 A형 인플루엔자를 보다 효과적으로 예방 및 치료할 수 있는 백신을 개발하기 위하여 노력하였으며, 그 결과 각기 다른 H3 및 H7의 두 가지 아형(subtype)에서 유래된 헤마글루티닌 HA1 영역과 HA2 영역을 융합하여 제조한 키메릭 헤마글루티닌 유전자를 포함하는 재조합 인플루엔자 바이러스의 경우 H3N2 혈청형, H7N9 혈청형을 가진 A형 인플루엔자 바이러스에 대해 교차 면역반응을 효과적으로 형성하는 것을 확인함으로써 본 발명을 완성하게 되었다.The present inventors have sought to develop a vaccine capable of more effectively preventing and treating influenza A having various subtypes, and as a result, the hemagglutinin HA1 region derived from two different subtypes of H3 and H7 And HA2 region, it was found that the recombinant influenza virus containing the chimeric hemagglutinin gene efficiently cross-reacts with influenza A virus having H3N2 serotype and H7N9 serotype, .

한국공개특허 제10-2010-0102593호Korean Patent Laid-Open No. 10-2010-0102593 한국공개특허 제10-2008-0113217호Korean Patent Laid-Open No. 10-2008-0113217

따라서 본 발명의 목적은, 각기 다른 H3 및 H7의 두 가지 아형(subtype)의 키메릭 헤마글루티닌 유전자를 포함하는 재조합 인플루엔자 바이러스를 제공하는 것이다.It is therefore an object of the present invention to provide a recombinant influenza virus comprising two chimeric hemagglutinin genes of two different subtypes of H3 and H7.

본 발명의 다른 목적은, H3 및 H7의 다중 아형에 대한 교차 면역반응을 형성하는 신규한 재조합 바이러스 백신을 제공하는 것이다.It is another object of the present invention to provide a novel recombinant virus vaccine which forms a cross-immune response to multiple subtypes of H3 and H7.

본 발명의 또 다른 목적은, 상기 조성물을 이용하여 인플루엔자 바이러스 감염 질환을 예방 또는 치료하는 방법을 제공하는 것이다.It is still another object of the present invention to provide a method for preventing or treating an influenza virus infection disease using the composition.

본 발명의 또 다른 목적은, 상기 바이러스 또는 항원을 포함하는 인플루엔자 바이러스의 진단용 조성물을 제공하는 것이다. It is still another object of the present invention to provide a composition for the diagnosis of influenza virus containing the virus or antigen.

나아가 본 발명의 목적은 상기 바이러스 또는 이의 항원을 이용한 항원-항체 반응을 통해 감염될 또는 감염된 세포 내에서 인플루엔자 바이러스(기탁번호: KCTC 13007BP)를 검출하는 것을 특징으로 하는, 인플루엔자 바이러스(기탁번호: KCTC 13007BP)의 검출 방법을 제공하는 것이다.Further, the object of the present invention is to provide an influenza virus (accession number: KCTC (KCTC)), which is characterized by detecting an influenza virus (accession number: KCTC 13007BP) in an infected or infected cell through an antigen-antibody reaction using the virus or antigen thereof 13007BP). ≪ / RTI >

상기와 같은 본 발명의 목적을 달성하기 위해서, 본 발명은 각기 다른 두 가지 아형(subtype)의 키메릭 헤마글루티닌 유전자를 포함하는 재조합 인플루엔자 바이러스(기탁번호: KCTC 13007BP)를 제공한다.In order to achieve the above object, the present invention provides a recombinant influenza virus (accession number: KCTC 13007BP) comprising two different subtypes of chimeric hemagglutinin gene.

본 발명의 일실시예에 있어서, 상기 키메릭 헤마글루티닌 유전자는 A/Perth/16/2009 (H3N2) 유래의 HA1 및 융합펩티드 유전자 영역과; A/Anhui/1/2013 (H7N9) 유래의 HA2 유전자 영역이 융합된 형태일 수 있다. In one embodiment of the present invention, the chimeric hemagglutinin gene comprises HA1 and a fusion peptide gene region derived from A / Perth / 16/2009 (H3N2); A / Anhui / 1/2013 (H7N9) -induced HA2 gene region may be fused.

본 발명의 일실시예에 있어서, 상기 키메릭 헤마글루티닌 유전자는 서열번호 3으로 표시되는 폴리뉴클레오티드로 이루어질 수 있다.In one embodiment of the present invention, the chimeric hemagglutinin gene may be a polynucleotide represented by SEQ ID NO: 3.

또한, 본 발명은 상기 바이러스를 유효성분으로 포함하는 인플루엔자 바이러스 백신을 제공한다. The present invention also provides an influenza virus vaccine comprising the virus as an active ingredient.

본 발명의 일실시예에 있어서, 상기 백신은 바이러스를 약독화된 생독 백신, 사독 백신 또는 서브유닛 백신(면역원성 단편)일 수 있다.In one embodiment of the present invention, the vaccine may be a live attenuated vaccine, a sadox vaccine or a subunit vaccine (immunogenic fragment).

본 발명의 일실시예에 있어서, 상기 바이러스는 H3N2 혈청형 및 H7N9 혈청형을 가진 A형 인플루엔자 바이러스 모두에 대해 교차 면역반응을 형성할 수 있다. In one embodiment of the invention, the virus is capable of cross-reacting against all influenza A viruses with H3N2 serotype and H7N9 serotype.

또한, 본 발명은 상기 백신을 인간을 제외한 개체에 투여하여 인플루엔자 바이러스 감염 질환을 예방 또는 치료하는 방법을 제공한다.The present invention also provides a method for preventing or treating an influenza virus infection disease by administering the vaccine to an individual other than a human.

또한, 본 발명은 상기 바이러스 또는 이의 항원을 포함하는 인플루엔자 바이러스의 진단용 조성물을 제공한다. The present invention also provides a composition for the diagnosis of influenza virus comprising the virus or antigen thereof.

나아가, 본 발명은 상기 바이러스 또는 이의 항원을 이용한 항원-항체 반응을 통해 감염될 또는 감염된 세포 내에서 인플루엔자 바이러스(기탁번호: KCTC 13007BP)를 검출하는 것을 특징으로 하는, 인플루엔자 바이러스(기탁번호: KCTC 13007BP)의 검출 방법을 제공한다.Furthermore, the present invention relates to an influenza virus (accession number: KCTC 13007BP), which is characterized by detecting an influenza virus (Accession No .: KCTC 13007BP) in the infected or infected cells through an antigen-antibody reaction using the virus or its antigen ). ≪ / RTI >

본 발명의 재조합 인플루엔자 바이러스(기탁번호: KCTC 13007BP)는 각기 다른 두 가지 아형(subtype)에서 유래된 헤마글루티닌 HA1 및 융합 펩타이드 영역과 HA2 영역을 융합하여 제조한 키메릭 헤마글루티닌 유전자를 포함함으로써 다양한 종류의 아형(subtype) 및 계통(clade)을 가진 바이러스에 대해 교차 면역반응 효과를 나타냄에 따라, 광범위한 중화 항체 효과를 가진 바이러스 백신의 용도로서 매우 유용하게 사용될 수 있다.The recombinant influenza virus of the present invention (accession number: KCTC 13007BP) comprises hemagglutinin HA1 derived from two different subtypes and a chimeric hemagglutinin gene prepared by fusing the fusion peptide region and the HA2 region , It exhibits a cross-immune response effect against viruses having various subtypes and clades and thus can be very usefully used as a virus vaccine having a wide range of neutralizing antibody effects.

도 1A는 일반적인 인플루엔자 바이러스의 헤마글루티닌 유전자 구조를 모식화하여 나타낸 것이며, 도 1B는 본 발명의 chimeric H3/H7 헤마글루티닌 유전자 구조를 모식화하여 나타낸 것이다.
도 2는 역유전학(Reverse Genetics) 시스템으로 본 발명의 재조합 인플루엔자 바이러스를 제조하는 과정을 간략하게 나타낸 모식도이다.
도 3은 본 발명의 재조합 인플루엔자 바이러스의 효능을 확인하기 위하여 실험동물인 마우스에 백신 스케쥴을 도식화하여 나타낸 것이다.
도 4는 본 발명의 재조합 인플루엔자 바이러스의 중화 항체에 대한 능력 검증을 위해, 재조합 인플루엔자 바이러스 백신을 마우스 1차 또는 2차 접종한 다음 동종 바이러스 마우스 적응(mouse-adapted) PERTH(H3N2)를 100MLD50의 양으로 감염한 뒤 14일 동안 마우스의 체중변화를 측정하여 나타낸 그래프이다.
도 5는 본 발명의 재조합 인플루엔자 바이러스의 중화 항체에 대한 능력 검증을 위해, 재조합 인플루엔자 바이러스 백신을 마우스 1차 또는 2차 접종한 다음 이종 바이러스 A/Anhui/1/2013(H7N9)를 100MLD50의 양으로 감염한 뒤 14일 동안 마우스의 체중변화를 측정하여 나타낸 그래프이다.1A is a schematic representation of the hemagglutinin gene structure of a common influenza virus, and FIG. 1B is a schematic representation of the chimeric H3 / H7 hemagglutinin gene structure of the present invention.
FIG. 2 is a schematic diagram showing a process of producing the recombinant influenza virus of the present invention by a reverse genetics system. FIG.
FIG. 3 is a diagram illustrating a vaccine schedule in a mouse as an experimental animal to confirm the efficacy of the recombinant influenza virus of the present invention.
Of Figure 4 is 100MLD to, recombinant virus vaccines Mouse primary or secondary inoculation following homologous virus mouse adaptation (mouse-adapted) PERTH (H3N2 ) for the ability to verify the neutralizing antibody of the recombinant virus of the present invention 50 Lt; RTI ID = 0.0 > 14 < / RTI > days after infection.
5 is for the ability to verify the neutralizing antibody of the recombinant virus of the present invention, a recombinant virus vaccine mouse primary or secondary inoculation, and then the amount of the heterologous virus A / Anhui / 1 / 100MLD the 2013 (H7N9) 50 And the mice were weighed for 14 days.

하나의 양태로서, 본 발명은 키메릭 헤마글루티닌 유전자를 포함하는 재조합 인플루엔자 바이러스(기탁번호: KCTC 13007BP)에 관한 것이다.In one embodiment, the present invention relates to a recombinant influenza virus (Accession Number: KCTC 13007BP) comprising a chimeric hemagglutinin gene.

"헤마글루티닌(hemagglutinin)"은 인플루엔자 바이러스의 표면 당단백질 중 하나로서 바이러스의 숙주 세포에의 부착과 바이러스의 세포 침투 동안 바이러스-세포막의 융합을 매개하며, 인플루엔자 서브타입의 항원 특이성을 결정하는 인플루엔자 바이러스의 표면 항원단백질이다."Hemagglutinin" is one of the surface glycoproteins of influenza virus that mediates the attachment of the virus to the host cell and the fusion of the virus-cell membrane during the infiltration of the virus into the cell, and determines the antigen specificity of the influenza subtype It is surface antigen protein of influenza virus.

본 발명에서 상기 키메릭 헤마글루티닌 유전자는 A/Perth/16/2009 (H3N2) 유래의 HA1 및 융합펩티드 유전자 영역과; A/Anhui/1/2013 (H7N9) 유래의 HA2 유전자 영역이 융합된 형태이다. HA2 부분은 엑토도메인과 transmembrane cytopalsmic domain으로 구성되어 있으며, 엑토도메인은 fusion peptide, a helix loop, 그리고 coiled coiled part 등으로 구성되어 있다.In the present invention, the chimeric hemagglutinin gene comprises HA1 and a fusion peptide gene region derived from A / Perth / 16/2009 (H3N2); A / Anhui / 1/2013 (H7N9). The HA2 region consists of an ecto domain and a transmembrane cytopathic domain. The ecto domain consists of fusion peptide, a helix loop, and coiled coiled part.

본 발명의 상기 키메릭 헤마글루티닌 유전자는 서열번호 3의 폴리뉴클레오티드 서열 또는 서열번호 3의 뉴클레오타이드 서열과 적어도 95%, 적어도 98%, 적어도 98.5%, 적어도 99%, 적어도 99.2%, 적어도 99.4%, 적어도 99.6%, 적어도 99.8% 또는 적어도 99.9%의 서열 동일성을 갖는 뉴클레오타이드 서열을 포함할 수 있다.The chimeric hemagglutinin gene of the present invention comprises at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4% , At least 99.6%, at least 99.8%, or at least 99.9% sequence identity to the nucleotide sequence of SEQ ID NO: 1.

또한, 본 발명의 재조합 인플루엔자 바이러스는 상기 키메릭 헤마글루티닌 유전자 외의 6개 혹은 7개의 다른 유전자는 현재 인체백신의 백본(backbone) 바이러스로 이용되는 A/Puerto Rico/08/34 (H1N1)로부터 유래된 것을 이용할 수 있다.In addition, the recombinant influenza virus of the present invention can be obtained from A / Puerto Rico / 08/34 (H1N1), which is currently used as a backbone virus of human vaccine, from 6 or 7 other genes other than the chimeric hemagglutinin gene Quot; can be used.

본 발명의 일실시예에 있어서, 본 발명자들은 상기한 키메릭 헤마글루티닌 유전자와 이외 7개의 유전자를 A/Puerto Rico/08/34 (H1N1)로부터 제공받아 역유전학(Reverse Genetics) 시스템을 이용하여 본 발명의 재조합 인플루엔자 바이러스를 제조하였으며, 이를 'cHA H3/H7N1'로 명명하였다. 또한, 본 바이러스를 2016년 4월 15일자로 한국생명공학연구원 미생물자원센터(KCTC)에 기탁하였으며, 기탁번호 KCTC 13007BP를 부여받았다.In one embodiment of the present invention, the present inventors have provided the above-mentioned chimeric hemagglutinin gene and seven other genes from A / Puerto Rico / 08/34 (H1N1) and used reverse genetics system To produce the recombinant influenza virus of the present invention, which was named 'cHA H3 / H7N1'. On April 15, 2016, the virus was deposited with KCTC (Korea Research Institute of Bioscience and Biotechnology) and received the deposit number KCTC 13007BP.

또 다른 양태로서, 본 발명은 감수성 숙주 동물을 감염시키고 질병을 일으킬 수 있는 인플루엔자 바이러스에 대한 면역 반응을 유도하는 백신에 관한 것이다. 바람직하게, 본 발명의 백신은 상기 cHA H3/H7N1 바이러스(기탁번호 KCTC 13007BP)를 유효성분으로 포함할 수 있다.In another aspect, the present invention relates to a vaccine that induces an immune response against influenza viruses that can infect a susceptible host animal and cause disease. Preferably, the vaccine of the present invention may comprise the cHA H3 / H7N1 virus (Accession No. KCTC 13007BP) as an active ingredient.

본 발명의 백신이 면역 반응을 일으킬 수 있는 숙주 동물은 인간, 개, 고양이, 돼지, 말, 닭, 오리, 칠면조, 페럿 등을 포함할 수 있으며, 바람직하게는 인간이다.The host animal in which the vaccine of the present invention can cause an immune response may include a human, a dog, a cat, a pig, a horse, a chicken, a duck, a turkey, a pearl and the like, preferably a human.

본 발명의 백신은 약독화된 생독 백신 또는 사독 백신, 서브유닛 백신(subunit vaccine), 합성 백신(synthetic vaccine) 또는 유전공학 백신(genetic engineering vaccine)일 수 있으나, 효과적인 면역 반응을 유도하는 생독 백신이 바람직하다.The vaccine of the present invention may be an attenuated live vaccine or a sadox vaccine, a subunit vaccine, a synthetic vaccine or a genetic engineering vaccine, but a live vaccine that induces an effective immune response desirable.

본 발명에서 사용된 용어 "생독 백신"이란 살아있는 바이러스 활성성분을 포함하는 백신을 의미한다. 또한 용어 "약독화된(attenuation)"이란 살아있는 병원체의 독성을 인공적으로 약하게 한 것으로, 병원체의 필수 대사에 관여하는 유전자를 변이시켜 체내에서 질병을 일으키지 못하고 면역 체계만을 자극해서 면역성을 유도하는 것을 의미한다. 바이러스의 약독화는 자외선(UV) 조사, 약품처리 또는 시험관 내 고차 연속 계대배양에 의해 달성될 수 있다. 약독화는 또한 명확한 유전 변화를 만듦으로써, 예를 들어 독성을 제공하는 것으로 알려진 바이러스 서열의 특정 결실 또는 바이러스 게놈 내로의 서열의 삽입에 의해 달성될 수 있다.As used herein, the term " virulence vaccine "means a vaccine comprising live viral active ingredients. The term "attenuation" refers to artificially weakening the toxicity of living pathogens, mutating the genes involved in essential metabolism of pathogens, inducing immune system stimulating the immune system without causing disease in the body do. The attenuation of the virus can be achieved by ultraviolet (UV) irradiation, drug treatment, or in vitro high-order serial passaging. Inactivation can also be accomplished by making clear genetic changes, for example by insertion of sequences into viral genomes or specific deletions of viral sequences known to provide toxicity.

용어 "사독 백신"이란 불활화 백신 또는 사균 백신이라고도 하며, 죽은 바이러스를 포함한 백신이다. 이의 예로는 전 바이러스 백신(whole-virus vaccine)과 분할 백신(split vaccine)이 있으며, 이들은 공지된 방법으로 용이하게 제조가능하다. 예컨대 전 바이러스 백신은 바이러스 전체에 포르말린을 처리하여 제조할 수 있으며, 분할 백신은 바이러스에 에테르를 처리하여 외피만을 분쇄, 수득하여 제조할 수 있다.The term " Sadox vaccine "is also referred to as an inactivated vaccine or an inactivated vaccine, and is a vaccine containing a dead virus. Examples thereof include whole-virus vaccines and split vaccines, which are readily manufacturable by known methods. For example, the whole virus vaccine can be prepared by treating the whole virus with formalin, and the divided vaccine can be prepared by treating the virus with ether to crush and obtain only the envelope.

용어 "서브유닛 백신"은 바이러스의 구성성분 중 면역기능을 일으킬 수 있는 항원 성분만을 추출하여 제조한 백신으로, 바이러스 방어에 필요한 항원 부위에 대해서면 면역형성을 유도함으로써 부작용을 최소화할 수 있다. 예컨대, 본 발명의 재조합 인플루엔자 바이러스의 키메릭 헤마글루티닌(HA) 단백질을 추출하여 사용할 수 있다.The term "subunit vaccine" is a vaccine prepared by extracting only an antigen component that can cause an immune function among components of a virus, thereby minimizing adverse effects by inducing the immune formation against the antigen site necessary for viral defense. For example, the chimeric hemagglutinin (HA) protein of the recombinant influenza virus of the present invention can be extracted and used.

"유전공학 백신"은 바이러스의 병원성을 일으키는 특이 유전자를 변형하거나 제거한 것일 수 있다.A "genetic engineering vaccine" may be a modification or removal of a specific gene that causes virulence of the virus.

또한 본 발명의 백신으로 사용되는 바이러스 폴리펩티드(키메릭 헤마글루티닌) 또는 이의 단편들은 업계에 공지된 방법을 이용하여 제조할 수 있다. 예를 들어, 바이러스 폴리펩티드는 고체상 합성 방법을 이용하여 합성될 수 있으며, 재조합 DNA 기술을 이용하여 생성될 수도 있다.The virus polypeptides (chimeric hemagglutinin) or fragments thereof used as the vaccine of the present invention can also be produced using methods known in the art. For example, viral polypeptides may be synthesized using solid phase synthesis methods and may be generated using recombinant DNA techniques.

본 발명의 백신은 나출된(naked) 핵산 조성물들도 포함할 수 있다. 본 발명의 일실시예에서, 핵산은 본 발명의 재조합 인플루엔자 바이러스의 키메릭 헤마글루티닌(HA) 단백질을 인코딩하는 뉴클레오티드 서열을 포함할 수 있다. 핵산 백신접종 방법들은 업계에 공지되어 있는 방법을 통해 실시할 수 있다. 예를 들어, 미국등록특허 제6,063,385호 및 제6,472,375호에 설명되어 있다. 상기 핵산은 플라스미드 또는 유전자 발현 카세트(expression cassette)의 형태일 수 있다. 일실시예에서, 상기 핵산은 동물에 투여되는 리포좀에 둘러싸여진 채로 제공된다.The vaccine of the present invention may also include naked nucleic acid compositions. In one embodiment of the invention, the nucleic acid may comprise a nucleotide sequence encoding the chimeric hemagglutinin (HA) protein of the recombinant influenza virus of the invention. Methods of nucleic acid vaccination can be carried out by methods known in the art. For example, in U.S. Patent Nos. 6,063,385 and 6,472,375. The nucleic acid may be in the form of a plasmid or gene expression cassette. In one embodiment, the nucleic acid is provided surrounded by liposomes administered to the animal.

또한, 본 발명의 백신은 추가적으로 용매, 면역증강제(adjuvant) 및 부형제로 이루어진 군으로부터 선택된 1종 이상을 더 포함할 수 있다. 상기 용매로는 생리식염수 또는 증류수가 있으며, 면역증강제로는 프레운즈(Freund's) 불완전체 또는 완전체 어쥬번트, 알루미늄 하이드록사이드 겔과 식물성 및 광물성 오일 등이 있으며, 부형제로는 알루미늄 포스페이트, 알루미늄 하이드록사이드 또는 알루미늄 포타슘 설페이트가 있으나, 이에 한정되는 것은 아니며, 당해 분야의 통상의 지식을 가진 자가 기술자에게 잘 알려진 백신 제조에 사용되는 물질을 더 포함할 수 있다.In addition, the vaccine of the present invention may further comprise at least one member selected from the group consisting of a solvent, an adjuvant, and an excipient. Examples of the solvent include physiological saline or distilled water. Examples of the immunity enhancer include Freund's incomplete or complete adjuvant, aluminum hydroxide gel, vegetable and mineral oil, etc. Examples of excipients include aluminum phosphate, aluminum hydroxide Side or aluminum potassium sulfate, but are not limited thereto and may further comprise materials used in the manufacture of vaccines well known to those skilled in the art.

본 발명의 백신은 키메릭 헤마글루티닌 유전자를 포함하는 재조합 인플루엔자 바이러스를 23~28 HAU(hemagglutination unit, 혈구응집 단위)로 포함할 수 있으나, 이에 한정되는 것은 아니다. 다만, 백신의 혈구응집 단위가 23 HAU 미만인 경우, 투여 대상 개체에 효과적으로 항체형성을 유도하지 못할 수 있으며, 28 HAU을 초과하는 경우, 효율대비 비경제적일 수 있다.The vaccine of the present invention may include, but is not limited to, a recombinant influenza virus comprising a chimeric hemagglutinin gene in a hemagglutination unit (HAU) of 2 3 to 2 8 HAU. However, if less than the hemagglutination units of vaccine 2 3 HAU, and may not be able to induce antibody formation for effective administration of the target object may be a non-economic efficiency when compared to in excess of 2 8 HAU.

본 발명의 백신은 경구형 또는 비경구형 제제로 제조할 수 있으며, 바람직하게는 비경구형 제제인 주사액제로 제조하며, 진피내, 근육내, 복막내, 비강 또는 경막외(eidural) 경로로 투여할 수 있다.The vaccine of the present invention may be prepared in oral or parenteral formulations, preferably in the form of a parenteral injection, and may be administered intradermally, intramuscularly, intraperitoneally, nasally or via an eidural route have.

또 다른 양태로서, 본 발명은 상기 백신을 인플루엔자 바이러스 감염이 의심되는 개체에 투여하여 인플루엔자 바이러스 감염 질환을 예방 또는 치료하는 방법에 관한 것이다.In another aspect, the present invention relates to a method for preventing or treating an influenza virus infectious disease by administering the vaccine to a subject suspected of influenza virus infection.

본 발명에서 용어 "인플루엔자 바이러스 감염 질환"이란 인플루엔자 바이러스의 감염으로 유발되는 질환으로서, 부비강염, 발작적 천식, 중이염, 낭성 섬유종, 기관지염, 폐렴, 설사 등을 예시할 수 있으나(Pitkaranta와 Hayden, 1998. Ann. Med.), 본 발명은 이들에 제한되지 않는다.The term "influenza virus infection disease" in the present invention refers to diseases caused by influenza virus infection such as sinusitis, seizure asthma, otitis media, cystic fibrosis, bronchitis, pneumonia and diarrhea (Pitkaranta and Hayden, 1998. Ann Med.), The present invention is not limited thereto.

본 발명에서 용어, "개체"란 인플루엔자 바이러스에 이미 감염되었거나 감염될 수 있는 인간을 포함한 모든 동물을 의미한다. 본 발명의 추출물을 포함하는 조성물을 개체에 투여함으로써, 상기 질환을 효율적으로 예방 및 치료할 수 있다. 예를 들어, 본 발명의 조성물로 다양한 인플루엔자 바이러스 아형 또는 변이형의 인플루엔자 바이러스로 감염된 인간을 치료할 수 있다. 또한, 본 발명의 조성물로 다양한 인플루엔자 바이러스 아형 또는 변이형의 조류 인플루엔자로 감염된 닭 또는 돼지를 치료할 수 있다. 본 발명의 조성물은 기존의 인플루엔자 바이러스 감염 질환 치료제와 병행하여 투여할 수 있다.As used herein, the term "individual" refers to all animals, including humans, who are already infected or susceptible to influenza virus. By administering to a subject a composition comprising the extract of the present invention, the disease can be effectively prevented and treated. For example, a composition of the present invention can treat humans infected with various influenza virus subtypes or variant influenza viruses. In addition, the composition of the present invention can treat chickens or pigs infected with various influenza virus subtypes or variant avian influenza viruses. The composition of the present invention can be administered in combination with a conventional therapeutic agent for influenza virus infection.

본 발명에서 용어 "예방"이란 조성물의 투여에 의해 인플루엔자 바이러스 감염을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다. 본 발명에서 용어 "치료"란 조성물의 투여에 의해 인플루엔자 바이러스 감염에 의한 증세가 호전되거나 이롭게 변경하는 모든 행위를 의미한다.The term "prophylactic" in the present invention means all actions that inhibit or delay the onset of an influenza virus infection by administration of the composition. The term "treatment" in the present invention means any action that improves or alleviates symptoms due to influenza virus infection by the administration of the composition.

본 발명의 조성물은 약제학적으로 유효한 양으로 투여한다. 용어 "약제학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체의 종류 및 중증도, 연령, 성별, 감염된 바이러스 종류, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로, 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and the effective dose level will depend on the species and severity of the subject, age, sex, The activity of the compound, the sensitivity to the drug, the time of administration, the route of administration, the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 상세하게 후술되어 있는 실시예들을 참조하면 명확해질 것이다. 이하, 본 발명을 실시예에 의해 상세히 설명하기로 한다. 그러나 이들 실시예들은 본 발명을 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are for illustrating the present invention specifically, and the scope of the present invention is not limited to these examples.

<실시예 1>&Lt; Example 1 >

실험에 필요한 바이러스 준비Prepare the virus for the experiment

<1-1> A/Perth/16/2009 (<1-1> A / Perth / 16/2009 ( H3N2H3N2 ), A/), A / AnhuiAnhui /1/2013 (/ 1/2013 ( H7N9H7N9 ) 및 A/Puerto Rico/08/34 () And A / Puerto Rico / 08/34 ( H1N1H1N1 ) 바이러스 준비) Virus preparation

A/Perth/16/2009 (H3N2) 바이러스는 녹십자(경기도 용인, 한국)로부터 수득하였고, A/Puerto Rico/08/34 (H1N1) 바이러스는 Dr. Robert G. Webster(Division of Virology, Department of Infectious Diseases, St. Jude Childrens Research Hospital, Memphis, TN)로부터 수득하였다. A/Anhui/1/2013 (H7N9) 바이러스 또한 Dr. Robert G. Webster로부터 수득하였다. A / Perth / 16/2009 (H3N2) virus was obtained from Green Cross (Yongin, Korea) and A / Puerto Rico / 08/34 (H1N1) From Robert G. Webster (Division of Virology, Department of Infectious Diseases, St. Jude Childrens Research Hospital, Memphis, TN). A / Anhui / 1/2013 (H7N9) Obtained from Robert G. Webster.

<실시예 2>&Lt; Example 2 >

본 발명의 키메릭 헤마글루티닌 유전자를 포함하는 재조합 바이러스 제조Production of recombinant viruses containing the chimeric hemagglutinin gene of the present invention

본 발명자들은 A형 인플루엔자 바이러스의 각기 다른 아형(subtype) 및 각기 다른 그룹형(clade)의 인플루엔자에 대한 교차 면역반응(cross-protection) 효과를 가지는 범 중화 항체를 개발하기 위하여, 먼저 헤마글루티닌의 HA1(globular head domain)과 HA2(stalk region) 유전자간 영역을 각각 두 가지 아형(혈청형)으로부터 분리 융합하여 키메릭 헤마글루타닌 유전자를 제조하였으며, 이를 현재 인체백신의 백본 바이러스(A/Puerto Rico/08/34 (H1N1))에 포함시켜 재조합 바이러스를 제조하였다. In order to develop a cross-neutralizing antibody having a cross-protection effect on different subtypes of influenza A virus and different influenza strains of different types of clones, the present inventors firstly applied hemagglutinin The chimeric hemagglutanin gene was isolated from two subtypes (serotype) of HA1 (globular head domain) and HA2 (stalk region) Rico / 08/34 (H1N1)) to prepare a recombinant virus.

<2-1> 키메릭 헤마글루티닌 유전자 제조<2-1> Generation of chimeric hemagglutinin gene

먼저, 본 발명자들은 키메릭 헤마글루티닌 유전자를 제조하기 위하여, A/Perth/16/2009 (H3N2)의 HA1 및 fusion peptide 유전자 영역(서열번호 1)과; A/Anhui/1/2013 (H7N9)의 HA2 Ectodomain 유전자 영역(서열번호 2)을 융합하여 하나의 키메릭 H3/H7 헤마글루티민 유전자를 제조하였다.First, the present inventors prepared HA1 and fusion peptide gene regions (SEQ ID NO: 1) of A / Perth / 16/2009 (H3N2) A chimeric H3 / H7 hemaglutimine gene was prepared by fusing the HA2 Ectodomain gene region (SEQ ID NO: 2) of A / Anhui / 1/2013 (H7N9).

A/Perth/16/2009 (H3N2)는 Bm HA 1F (5-TAT TCG TCT CAG GGA GCA AAA GCA GGG G- 3: 서열번호 11)와 HA1을 특정하게 인식하는 프라이머(5-CAT TCC CTG CCA TCC CCC TTC AAT GAA ACC- 3: 서열번호 12)를 사용하여 A/Perth/16/2009 (H3N2)의 HA1 및 fusion peptide 유전자 영역(서열번호 1)을 만들고, HA2를 특정하게 인식하는 프라이머(5-GGT TTC ATT GAA GGG GGA TGG CAG GGA ATG- 3: 서열번호 13)와 Bm NS 890R(5-ATA TCG TCT CGT ATT AGT AGA AAC AAG GGT GTT TT- 3: 서열번호 14), A/Anhui/1/2013 (H7N9)의 HA2 Ectodomain 유전자 영역(서열번호 2)을 사용하여 각각의 유전자 단편을 만들고, Bm HA 1F(5‘TAT TCG TCT CAG GGA GCA AAA GCA GGG G3‘: 서열번호 15), Bm NS 890R 프라이머(5’-ATA TCG TCT CGT ATT AGT AGA AAC AAG GGT GTT TT3‘: 서열번호 16)를 사용하여 fusion PCR 과정을 통하여 융합, 증폭과정을 거쳐 본 발명의 키메릭 유전자(서열번호 3)를 제조하였다. (5-CAT TCC CTG CCA TCC (SEQ ID NO: 11) and HA1 specifically recognize Bm HA 1F (5-TAT TCG TCG CAG GGA GCA AAA GCA GGG G- HA1 and a fusion peptide gene region (SEQ ID NO: 1) of A / Perth / 16/2009 (H3N2) were constructed using CCC TTC AAT GAA ACC- 1) and Bm NS 890R (5-ATA TCG TCT CGT ATT AGT AGA AAC AAG GGT GTT TT-3: SEQ ID NO: 14), A / Anhui / 1 / Each of the gene fragments was prepared using the HA2 Ectodomain gene region (SEQ ID NO: 2) of 2013 (H7N9), and Bm HA 1F (5'TAT TCG TCT CAG GGA GCA AAA GCA GGG G3 ' (SEQ ID NO: 3) of the present invention by fusion and amplification through a fusion PCR process using primers (5'-ATA TCG TCT CGT ATT AGT AGA AAC AAG GGT GTT TT3 ': SEQ ID NO: 16) Respectively.

<2-2> 키메릭 헤마글루티닌 유전자를 포함하는 재조합 바이러스 제조&Lt; 2-2 > Production of Recombinant Virus Containing Chimeric Hemagglutinin Gene

상기 실시예<2-1>에서 준비한 본 발명의 키메릭 헤마글루티닌 유전자(서열번호 3) 및 A/Puerto Rico/08/34의 NA(서열번호 4), M(서열번호 5), NP(서열번호 6), NS(서열번호 7), PA(서열번호 8), PB1(서열번호 9), PB2(서열번호 10) 유전자를 각각 발현벡터 vPHW2000(사용논문:J Gen Virol. 2013 Jun;94(Pt 6):1230-5. doi: 10.1099/vir.0.051284-0. Epub 2013 Mar 13.)에 삽입하여 준비하였다. 상기 서열번호 3~10의 유전자가 각각 삽입되어 있는 발현벡터들을 형질감염 시약 TransIT-LT1 transfection reagent (Mirus Bio)를 이용하여 함께 모두 혼합한 후 40분간 실온에 놓아두었다. (SEQ ID NO: 4), M (SEQ ID NO: 5), and NP (SEQ ID NO: 5) of A / Puerto Rico / 08/34 of the present invention prepared in Example <2-1> (SEQ ID NO: 6), NS (SEQ ID NO: 7), PA (SEQ ID NO: 8), PB1 (SEQ ID NO: 9) and PB2 (SEQ ID NO: 10) were used as expression vectors vPHW2000 (using J Gen Virol. 94 (Pt 6): 1230-5. Doi: 10.1099 / vir.0.051284-0. Epub 2013 Mar. 13). The expression vectors in which the genes of SEQ ID NOS: 3 to 10 were respectively inserted were mixed together using transfection reagent TransIT-LT1 transfection reagent (Mirus Bio), and then placed at room temperature for 40 minutes.

24시간 전에 준비해 둔 Vero 세포(ATCCCCL-81™)에 벡터 혼합액을 조심스럽게 넣은 다음 6시간 후에 배양액을 무혈청 배양액(GIBCO™ Opti-MEM I Reduced-Serum Medium (1X) liquid)으로 교체해 주었다. 형질감염 후 36시간이 지나면 0.2μg/ml의 L-1-tosylamido-2-phenylehtyl chlorometyl ketone(TPCK)-트립신(Sigma-Aldrich)를 함유하는 Opti-MEM I 1ml를 형질감염된 세포에 첨가해주고, 48시간이 지난 후부터 10일령 SPF 유정란을 이용하여 상층액을 접종하였으며, 48시간 후 상기 유정란에서 본 발명의 재조합 바이러스를 분리하였으며, 이렇게 분리된 재조합 바이러스는 cHA H3/H7N1라 명명하였으며, 불활성화하여 하기 실험에서 사용하였다. The vector mixture was carefully placed in Vero cells (ATCCCCL-81 ™) prepared 24 hours before and after 6 hours, the culture medium was replaced with GIBCO ™ Opti-MEM I Reduced-Serum Medium (1X) liquid. After 36 h of transfection, 1 ml of Opti-MEM I containing 0.2 μg / ml L-1-tosylamido-2-phenylehtyl chlorometyl ketone (TPCK) -trypsin (Sigma-Aldrich) was added to the transfected cells, After the passage of time, the supernatant was inoculated using 10-day-old SPF fertilized eggs. After 48 hours, the recombinant viruses of the present invention were isolated from the fertilized eggs. The thus isolated recombinant virus was named cHA H3 / H7N1, Was used in the experiment.

상기 방법에 따라 제조한 본 발명의 재조합 바이러스는 2016년 4월 15일자로 한국생명공학연구원 미생물자원센터(KCTC)에 기탁하였으며, 기탁번호 KCTC 13007BP를 부여받았다.The recombinant virus of the present invention prepared according to the above method was deposited with the KCTC of the Korea Research Institute of Bioscience and Biotechnology as of April 15, 2016 and received the deposit number KCTC 13007BP.

<실시예 3>&Lt; Example 3 >

본 발명의 The 키메릭Chimeric 헤마글루티닌Hemagglutinin 유전자를 포함하는 재조합 바이러스의 마우스 동물실험을 통한 항원성 평가 Antigenicity evaluation of recombinant viruses containing genes by mouse animal test

본 발명에서 개발된 재조합 백신 후보주 바이러스 cHA H3/H7N1의 항원성을 평가화하기 위하여, 실시예 2에서 제조한 재조합 바이러스(기탁번호:KCTC 13007BP)를 10일령의 SPF 유정란에 10개씩 접종하고 48시간 후에 요수를 검출하여 0.5% 칠면조 RBC로 헤마글루티닌 테스트를 통해 역가를 확인하였다. 분리된 요수를 0.025% 농도로 포르말린을 처리하고 72시간 동안 4℃에서 보관한 후 완전히 불활화되어 증식 가능한 바이러스가 없는 것을 10일령 SPF 유정란에 접종하여 확인하였다.In order to evaluate the antigenicity of the recombinant vaccine candidate main virus cHA H3 / H7N1 developed in the present invention, 10 recombinant viruses (accession number: KCTC 13007BP) prepared in Example 2 were inoculated into 10 days-old SPF fertilized eggs and 48 Afterwards, urine was detected and titers were confirmed by hemagglutinin test with 0.5% turkey RBC. Separated rats were inoculated with formalin at 0.025% concentration, stored at 4 ° C for 72 hours, and completely inactivated, and no proliferative virus was confirmed by inoculation on 10 - day - old SPF fertilized eggs.

20% 수크로오스 쿠션을 넣은 SW28 스윙로터 버켓(Beckman, USA)에 불활화된 바이러스 요수를 넣고 25,000rpm에서 3시간동안 침강시켜 바이러스만 순수하게 분리하였다. 분리된 바이러스는 단백질량을 측정한 후 헤마글루티닌 단백질의 농도가 1.7μg/1ml 및 3.5μg/1ml가 되도록 만들어 백신으로 이용하였다.The inactivated virus strain was placed in a SW28 swing rotor bucket (Beckman, USA) containing 20% sucrose cushion and settled at 25,000 rpm for 3 hours to isolate the virus purely. The isolated virus was used as a vaccine to measure the amount of protein and make hemagglutinin protein concentration 1.7 μg / 1 ml and 3.5 μg / 1 ml.

원하는 농도의 바이러스 용액을 4주령의 BALB/C 마우스에 헤마글루티닌 단백질의 함량(1.7μg/0.1ml, 3.5μg/0.1ml)에 따라 1차 근육접종 함으로써 항원성을 평가하였으며, 이후 2주가 경과한 시점에 동일한 방법으로 2차 접종을 실시하였다.Antigenicity was evaluated by primary intramuscular injection of the desired concentration of virus solution according to the content of hemagglutinin protein (1.7 μg / 0.1 ml, 3.5 μg / 0.1 ml) in 4-week-old BALB / C mice, The second vaccination was carried out in the same way at the time of elapse.

상기 1차 및 2차 접종 후 2주가 경과한 시점에 마우스의 혈청을 채취하여 중화항체에 대한 능력 검증을 실시하였으며, 백신 효과가 입증되는지 확인하기 위하여 동종 및 이종, 이형 바이러스에 감염시킨 후 몸무게 변화와 생존율을 측정하였다. 모든 실험에서는 대조군으로 A/Perth/16/2009 (H3N2) 바이러스만을 접종한 그룹과 PBS로 접종한 두 개의 그룹을 포함시켰다. 또한 감염 3, 5, 7일 후 마우스의 폐 조직을 채취하여 바이러스의 역가를 측정하였다. 본 실험에서 사용된 마우스는 각 그룹당(실험군 및 대조군의 마우스 사용량은 동일함) 폐조직 채취용 3마리씩(3, 5, 7일간 1일 3마리씩: 총 9마리의 마우스 사용)과 몸무게 변화 및 생존율에 5마리 사용하였다. Two weeks after the first and second inoculation, the serum of the mouse was collected to test the ability of the neutralizing antibody. To confirm whether the vaccine effect was proved, the mice were infected with homologous, heterologous, And survival rate were measured. All experiments included two groups inoculated with PBS / A / Perth / 16/2009 (H3N2) as a control group. After 3, 5, and 7 days of infection, the lung tissue of the mice was collected to measure the titer of the virus. The mice used in this experiment were divided into three groups (3 mice per day for 3, 5, and 7 days: total of 9 mice were used) for collecting lung tissue for each group (the mouse and control groups were the same) Were used.

Figure 112016084887714-pat00001
Figure 112016084887714-pat00001

<3-1> 백신접종 후 동종 바이러스 감염에 따른 마우스의 몸무게 및 생존율 측정<3-1> Measurement of the weight and survival rate of mice after vaccination with allogeneic virus infections

본 발명의 키메릭 헤마글루티닌 유전자를 포함하는 재조합 바이러스 백신의 범 인플루엔자 중화 능력효력을 검증하기 알아보기 위하여, 상기 과정을 통해 제조된 본 발명의 재조합 바이러스 백신을 마우스에 접종한 다음 동종 바이러스인 maPerth(H3N2)를 100MLD50의 양으로 감염한 뒤 14일 동안 마우스의 체중변화 및 생존율을 측정하였다.In order to examine the effect of the recombinant virus vaccine containing the chimeric hemagglutinin gene of the present invention on the ability to neutralize influenza virus, the recombinant virus vaccine of the present invention prepared above was inoculated into a mouse, MaPerth (H3N2) was infected at a dose of 100MLD 50 and the weight change and survival rate of mice were measured for 14 days.

그 결과 도 4A에서 나타낸 바와 같이, 본 발명의 재조합 바이러스로 1차 및 2차 백신 실시한 실험군에서 동종 바이러스에 대하여 감염 후 7일까지 약간의 체중변화는 발생하였으나, 14일이 경과한 시점에는 대부분의 마우스에서 몸무게가 회복되는 것을 확인할 수 있었다. 또한, 도 4B에서 나타낸 바와 같이, 생존율을 100%인 것을 모니터링할 수 있었다.As a result, as shown in FIG. 4A, in the experimental group in which the first and second vaccines were performed with the recombinant virus of the present invention, slight weight change occurred until day 7 after infection with the homologous virus, but at 14 days, We were able to confirm that weight recovery was achieved in mice. In addition, as shown in FIG. 4B, it was possible to monitor the survival rate of 100%.

<3-2> 백신접종 후 이종 바이러스 감염에 따른 마우스의 몸무게 및 생존율 측정<3-2> Measurement of weight and survival rate of mice after vaccination with heterologous virus infection

본 발명의 키메릭 헤마글루티닌 유전자를 포함하는 재조합 바이러스 백신의 범 인플루엔자 중화 능력효력을 검증하기 알아보기 위하여, 상기 과정을 통해 제조된 본 발명의 재조합 바이러스 백신을 마우스에 접종한 다음, 헤마글루티닌 및 뉴라미다아제 유전자가 모두 다른 이종 바이러스인 A/Perth/16/09(H3N2)을 100MLD50의 양으로 감염한 뒤 14일 동안 마우스의 체중변화 및 생존율을 측정하였다.In order to examine the effect of the recombinant virus vaccine containing the chimeric hemagglutinin gene of the present invention on the ability to neutralize influenza virus, the recombinant virus vaccine of the present invention prepared in the above procedure was inoculated into a mouse, A / Perth / 16/09 (H3N2), which is a heterologous virus with a different luteinin and neuramidase gene, was infected at a dose of 100MLD 50 , and the weight change and survival rate of the mice were measured for 14 days.

그 결과, 1차 백신 후 바이러스 감염이 일어나면 2일째 경과 후부터 체중이 감소하기 시작하여, H3N2로 단독 백신한 그룹의 결과 6 ~ 7일째, PBS 그룹은 8일 째 모두 죽는 것을 확인할 수 있었다(도 5A 참조). 또한, 백신 후 바이러스 감염 시에는 HA 함량에 관계없이 생존율이 100%임을 알 수 있었다(도 5B 참조). As a result, when the virus infection occurred after the first vaccination, the body weight began to decrease from the second day after the first vaccination. As a result, it was confirmed that the group of H3N2 alone died at 6-7th day and the PBS group died at 8th day Reference). In addition, it was found that the survival rate was 100% regardless of the HA content at the time of virus infection after vaccination (see FIG. 5B).

<3-3> 백신접종 후 다양한 바이러스 감염에 따른 바이러스의 <3-3> Vaccination of various virus infections after vaccination 역가Potency 측정 Measure

본 발명의 재조합 바이러스 백신을 마우스에 접종한 다음 maCA04(H1N1), W150(H5N1;clade 2.2), VN/1203(H5N1;clade 1) 및 ma81(H5N2) 바이러스 각각으로 마우스를 감염시킨 후 3, 5, 7일 후 마우스 폐조직을 채취하여 바이러스의 역가를 측정하였다. 마우스는 마취제를 이용하여 마우스 체중 ㎏당 30~40㎎으로 복강 마취를 실시한 후 비강 내 접종(intranasal inoculation)을 통하여 100MLD50 30ul를 감염시켰다. MLD50 는 마우스가 50% 치사할 수 있는 바이러스의 역가를 나타내며, 본 실험에서는 MLD50의 100배에 해당하는 양을 감염시켰다. 이에 따른 바이러스 역가를 측정하기 위하여 폐조직을 채취하기 위한 마우스는 바이러스 감염 후 3, 5, 7일 째에 대조군 및 실험군에서 3마리씩 폐 조직을 채취하였다. The mice were inoculated with maCA04 (H1N1), W150 (H5N1; clade 2.2), VN / 1203 (H5N1; clade 1) and ma81 (H5N2) , And 7 days later, mouse lung tissue was collected to measure the titer of the virus. Mice were anesthetized by intraperitoneal anesthesia with 30 ~ 40 mg / kg body weight per mouse and then injected with 100MLD 50 Lt; / RTI &gt; MLD 50 represents the titer of a virus that can kill 50% of the mice, and in this experiment infected 100 times the amount of MLD 50 . In order to measure virus titers, 3 mice were collected from control and experimental groups at 3, 5, and 7 days after viral infection.

측정된 바이러스 역가는 하기 표 2에서 나타내었다.The measured virus titers are shown in Table 2 below.

Figure 112016084887714-pat00002
Figure 112016084887714-pat00002

이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

한국생명공학연구원Korea Biotechnology Research Institute KCTC13007BPKCTC13007BP 2013041520130415

<110> Chungbuk National University Industry-Academic Cooperation Foundation I.D.Bio. <120> RECOMBINANT INFLUENZA VIRUS TO FORM CROSS-PROTECTION AGAINST H3 and H7 SUBTYPES OF INFLUENZA VIRUSES AND VACCINE COMPRISING THE SAME <130> PN1603-106 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 1035 <212> DNA <213> Influenza A virus <400> 1 atgaagacta tcattgcttt gagctacatt ctatgtctgg ttttcgctca aaaacttcct 60 ggaaatgaca acagcacggc aacgctgtgc cttgggcacc atgcagtacc aaacggaacg 120 atagtgaaaa caatcacgaa tgaccaaatt gaagttacta atgctactga gctggttcag 180 agttcctcaa caggtgaaat atgcgacagt cctcatcaga tccttgatgg aaaaaactgc 240 acactaatag atgctctatt gggagaccct cagtgtgatg gcttccaaaa taagaaatgg 300 gacctttttg ttgaacgcag caaagcctac agcaactgtt acccttatga tgtgccggat 360 tatgcctccc ttaggtcact agttgcctca tccggcacac tggagtttaa caatgaaagc 420 ttcaattgga ctggagtcac tcaaaacgga acaagctctg cttgcataag gagatctaaa 480 aacagtttct ttagtagatt gaattggttg acccacttaa acttcaaata cccagcattg 540 aacgtgacta tgccaaacaa tgaacaattt gacaaattgt acatttgggg ggttcaccac 600 ccgggtacgg acaaagacca aatcttcctg tatgctcaag catcaggaag aatcacagtc 660 tctaccaaaa gaagccaaca aaccgtaagc ccgaatatcg gatctagacc cagagtaagg 720 aatatcccta gcagaataag catctattgg acaatagtaa aaccgggaga catacttttg 780 attaacagca cagggaatct aattgctcct aggggttact tcaaaatacg aagtgggaaa 840 agctcaataa tgagatcaga tgcacccatt ggcaaatgca attctgaatg catcactcca 900 aatggaagca ttcccaatga caaaccattc caaaatgtaa acaggatcac atacggggcc 960 tgtcccagat atgttaagca aaacactctg aaattggcaa cagggatgcg aaatgtacca 1020 gagaaacaaa ctaga 1035 <210> 2 <211> 666 <212> DNA <213> Influenza A virus <400> 2 ggcctatttg gtgctatagc gggtttcatt gaaaatggat gggaaggcct aattgatggt 60 tggtatggtt tcagacacca gaatgcacag ggagagggaa ctgctgcaga ttacaaaagc 120 actcaatcgg caattgatca aataacagga aaattaaacc ggcttataga aaaaaccaac 180 caacaatttg agttgataga caatgaattc aatgaggtag agaagcaaat cggtaatgtg 240 ataaattgga ccagagattc tataacagaa gtgtggtcat acaatgctga actcttggta 300 gcaatggaga accagcatac aattgatctg gctgattcag aaatggacaa actgtacgaa 360 cgagtgaaaa gacagctgag agagaatgct gaagaagatg gcactggttg ctttgaaata 420 tttcacaagt gtgatgatga ctgtatggcc agtattagaa ataacaccta tgatcacagc 480 aaatacaggg aagaggcaat gcaaaataga atacagattg acccagtcaa actaagcagc 540 ggctacaaag atgtgatact ttggtttagc ttcggggcat catgtttcat acttctagcc 600 attgtaatgg gccttgtctt catatgtgta aagaatggaa acatgcggtg cactatttgt 660 atataa 666 <210> 3 <211> 1701 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 3 atgaagacta tcattgcttt gagctacatt ctatgtctgg ttttcgctca aaaacttcct 60 ggaaatgaca acagcacggc aacgctgtgc cttgggcacc atgcagtacc aaacggaacg 120 atagtgaaaa caatcacgaa tgaccaaatt gaagttacta atgctactga gctggttcag 180 agttcctcaa caggtgaaat atgcgacagt cctcatcaga tccttgatgg aaaaaactgc 240 acactaatag atgctctatt gggagaccct cagtgtgatg gcttccaaaa taagaaatgg 300 gacctttttg ttgaacgcag caaagcctac agcaactgtt acccttatga tgtgccggat 360 tatgcctccc ttaggtcact agttgcctca tccggcacac tggagtttaa caatgaaagc 420 ttcaattgga ctggagtcac tcaaaacgga acaagctctg cttgcataag gagatctaaa 480 aacagtttct ttagtagatt gaattggttg acccacttaa acttcaaata cccagcattg 540 aacgtgacta tgccaaacaa tgaacaattt gacaaattgt acatttgggg ggttctccac 600 ccgggtacgg acaaagacca aatcttcctg tatgctcaag catcaggaag aatcacagtc 660 tctaccaaaa gaagccaaca aaccgtaagc ccgaatatcg gatctagacc cagagtaagg 720 aatatcccta gcagaataag catctattgg acaatagtaa aaccgggaga catacttttg 780 attaacagca cagggaatct aattgctcct aggggttact tcaaaatacg aagtgggaaa 840 agctcaataa tgagatcaga tgcacccatt ggcaaatgca attctgaatg catcactcca 900 aatggaagca ttcccaatga caaaccattc caaaatgtaa acaggatcac atacggggcc 960 tgtcccagat atgttaagca aaacactctg aaattggcaa cagggatgcg aaatgtacca 1020 gagaaacaaa ctagaggcat atttggcgca atcgcgggtt tcattgaaaa tggatgggaa 1080 ggcctaattg atggttggta tggtttcaga caccagaatg cacagggaga gggaactgct 1140 gcagattaca aaagcactca atcggcaatt gatcaaataa caggaaaatt aaaccggctt 1200 atagaaaaaa ccaaccaaca atttgagttg atagacaatg aattcaatga ggtagagaag 1260 caaatcggta atgtgataaa ttggaccaga gattctataa cagaagtgtg gtcatacaat 1320 gctgaactct tggtagcaat ggagaaccag catacaattg atctggctga ttcagaaatg 1380 gacaaactgt acgaacgagt gaaaagacag ctgagagaga atgctgaaga agatggcact 1440 ggttgctttg aaatatttca caagtgtgat gatgactgta tggccagtat tagaaataac 1500 acctatgatc acagcaaata cagggaagag gcaatgcaaa atagaataca gattgaccca 1560 gtcaaactaa gcagcggcta caaagatgtg atactttggt ttagcttcgg ggcatcatgt 1620 ttcatacttc tagccattgt aatgggcctt gtcttcatat gtgtaaagaa tggaaacatg 1680 cggtgcacta tttgtatata a 1701 <210> 4 <211> 1444 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 4 agcaaaagca ggagtttaaa atgaatccaa atcagaaaat aataaccatt ggatcaatct 60 gtctggtagt cggactaatt agcctaatat tgcaaatagg gaatataatc tcaatatgga 120 ttagccattc aattcaaact ggaagtcaaa accatactgg aatatgcaac caaaacatca 180 ttacctataa aaatagcacc tgggtaaagg acacaacttc agtgatatta accggcaatt 240 catctctttg tcccatccgt gggtgggcta tatacagcaa agacaatagc ataagaattg 300 gttccaaagg agacgttttt gtcataagag agccctttat ttcatgttct cacttggaat 360 gcaggacctt ttttctgacc caaggtgcct tactgaatga caagcattca agtgggactg 420 ttaaggacag aagcccttat agggccttaa tgagctgccc tgtcggtgaa gctccgtccc 480 cgtacaattc aagatttgaa tcggttgctt ggtcagcaag tgcatgtcat gatggcatgg 540 gctggctaac aatcggaatt tcaggtccag ataatggagc agtggctgta ttaaaataca 600 acggcataat aactgaaacc ataaaaagtt ggaggaagaa aatattgagg acacaagagt 660 ctgaatgtgc ctgtgtaaat ggttcatgtt ttactataat gactgatggc ccgagtgatg 720 ggctggcctc gtacaaaatt ttcaagatcg aaaaggggaa ggttactaaa tcaatagagt 780 tgaatgcacc taattctcac tatgaggaat gttcctgtta ccctgatacc ggcaaagtga 840 tgtgtgtgtg cagagacaat tggcatggtt cgaaccggcc atgggtgtct ttcgatcaaa 900 acctggatta tcaaatagga tacatctgca gtggggtttt cggtgacaac ccgcgtcccg 960 aagatggaac aggcagctgt ggtccagtgt atgttgatgg agcaaacgga gtaaagggat 1020 tttcatatag gtatggtaat ggtgtttgga taggaaggac caaaggtcac agttccagac 1080 atgggtttga gatgatttgg gatcctaatg gatggacaga gactgatagt aagttctctg 1140 tgaggcaaga tgttgtggca atgactgatt ggtcagggta tagcggaagt ttcgttcaac 1200 atcctgagct gacagggcta gactgtatga ggccgtgctt ctgggttgaa ttaatcaggg 1260 gacgacctaa agaaaaaaca atctggacta gtgcgagcag catttctttt tgtggcgtga 1320 atagtgatac tgtagattgg tcttggccag acggtgctga gttgccattc agcattgaca 1380 agtagtctgt tcaaaaaact ccttgtttct actaataacc cggcggccca aaatgccgac 1440 tcgg 1444 <210> 5 <211> 1100 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 5 actggtcgac ctccgaagtt gggggggagc aaaagcaggt agatattgaa agatgagtct 60 tctaaccgag gtcgaaacgt acgtactctc tatcatcccg tcaggccccc tcaaagccga 120 gatcgcacag agacttgaag atgtctttgc agggaagaac accgatcttg aggttctcat 180 ggaatggcta aagacaagac caatcctgtc acctctgact aaggggattt taggatttgt 240 gttcacgctc accgtgccca gtgagcgagg actgcagcgt agacgctttg tccaaaatgc 300 ccttaatggg aacggggatc caaataacat ggacaaagca gttaaactgt ataggaagct 360 caagagggag ataacattcc atggggccaa agaaatctca ctcagttatt ctgctggtgc 420 acttgccagt tgtatgggcc tcatatacaa caggatgggg gctgtgacca ctgaagtggc 480 atttggcctg gtatgtgcaa cctgtgaaca gattgctgac tcccagcatc ggtctcatag 540 gcaaatggtg acaacaacca atccactaat cagacatgag aacagaatgg ttttagccag 600 cactacagct aaggctatgg agcaaatggc tggatcgagt gagcaagcag cagaggccat 660 ggaggttgct agtcaggcta gacaaatggt gcaagcgatg agaaccattg ggactcatcc 720 tagctccagt gctggtctga aaaatgatct tcttgaaaat ttgcaggcct atcagaaacg 780 aatgggggtg cagatgcaac ggttcaagtg atcctctcac tattgccgca aatatcattg 840 ggatcttgca cttgacattg tggattcttg atcgtctttt tttcaaatgc atttaccgtc 900 gctttaaata cggactgaaa ggagggcctt ctacggaagg agtgccaaag tctatgaggg 960 aagaatatcg aaaggaacag cagagtgctg tggatgctga cgatggtcat tttgtcagca 1020 tagagctgga gtaaaaaact accttgtttc tactaataac ccggcggccc aaaatgccga 1080 ctcggagcga aagatatacc 1100 <210> 6 <211> 1559 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 6 agcagggtag ataatcactc actgagtgac atcaaaatca tggcgtctca aggcaccaaa 60 cgatcttacg aacagatgga gactgatgga gaacgccaga atgccactga aatcagagca 120 tccgtcggaa aaatgattgg tggaattgga cgattctaca tccaaatgtg caccgaactc 180 aaactcagtg attatgaggg acggttgatc caaaacagct taacaataga gagaatggtg 240 ctctctgctt ttgacgaaag gagaaataaa taccttgaag aacatcccag tgcggggaaa 300 gatcctaaga aaactggagg acctatatac aggagagtaa acggaaagtg gatgagagaa 360 ctcatccttt atgacaaaga agaaataagg cgaatctggc gccaagctaa taatggtgac 420 gatgcaacgg ctggtctgac tcacatgatg atctggcatt ccaatttgaa tgatgcaact 480 tatcagagga caagagctct tgttcgcacc ggaatggatc ccaggatgtg ctctctgatg 540 caaggttcaa ctctccctag gaggtctgga gccgcaggtg ctgcagtcaa aggagttgga 600 acaatggtga tggaattggt cagaatgatc aaacgtggga tcaatgatcg gaacttctgg 660 aggggtgaga atggacgaaa aacaagaatt gcttatgaaa gaatgtgcaa cattctcaaa 720 gggaaatttc aaactgctgc acaaaaagca atgatggatc aagtgagaga gagccggaac 780 ccagggaatg ctgagttcga agatctcact tttctagcac ggtctgcact catattgaga 840 gggtcggttg ctcacaagtc ctgcctgcct gcctgtgtgt atggacctgc cgtagccagt 900 gggtacgact ttgaaaggga gggatactct ctagtcggaa tagacccttt cagactgctt 960 caaaacagcc aagtgtacag cctaatcaga ccaaatgaga atccagcaca caagagtcaa 1020 ctggtgtgga tggcatgcca ttctgccgca tttgaagatc taagagtatt aagcttcatc 1080 aaagggacga aggtgctccc aagagggaag ctttccacta gaggagttca aattgcttcc 1140 aatgaaaata tggagactat ggaatcaagt acacttgaac tgagaagcag gtactgggcc 1200 ataaggacca gaagtggagg aaacaccaat caacagaggg catctgcggg ccaaatcagc 1260 atacaaccta cgttctcagt acagagaaat ctcccttttg acagaacaac cattatggca 1320 gcattcaatg ggaatacaga ggggagaaca tctgacatga ggaccgaaat cataaggatg 1380 atggaaagtg caagaccaga agatgtgtct ttccaggggc ggggagtctt cgagctctcg 1440 gacgaaaagg cagcgagccc gatcgtgcct tcctttgaca tgagtaatga aggatcttat 1500 ttcttcggag acaatgcaga ggagtacgac aattaaagaa aaataccctt gtttctact 1559 <210> 7 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 7 actggtcgac ctccgaagtt gggggggagc aaaagcaggg tgacaaaaac ataatggatc 60 caaacactgt gtcaagcttt caggtagatt gctttctttg gcatgtccgc aaacgagttg 120 cagaccaaga actaggcgat gccccattcc ttgatcggct tcgccgagat cagaaatccc 180 taagaggaag gggcagtact ctcggtctgg acatcaagac agccacacgt gctggaaagc 240 agatagtgga gcggattctg aaagaagaat ccgatgaggc acttaaaatg accatggcct 300 ctgtacctgc gtcgcgttac ctaactgaca tgactcttga ggaaatgtca agggactggt 360 ccatgctcat acccaagcag aaagtggcag gccctctttg tatcagaatg gaccaggcga 420 tcatggataa gaacatcata ctgaaagcga acttcagtgt gatttttgac cggctggaga 480 ctctaatatt gctaagggct ttcaccgaag agggagcaat tgttggcgaa atttcaccat 540 tgccttctct tccaggacat actgctgagg atgtcaaaaa tgcagttgga gtcctcatcg 600 gaggacttga atggaatgat aacacagttc gagtctctga aactctacag agattcgctt 660 ggagaagcag taatgagaat gggagacctc cactcactcc aaaacagaaa cgagaaatgg 720 cgggaacaat taggtcagaa gtttgaagaa ataagatggt tgattgaaga agtgagacac 780 aaactgaaga taacagagaa tagttttgag caaataacat ttatgcaagc cttacatcta 840 ttgcttgaag tggagcaaga gataagaact ttctcgtttc agcttattta gtactaaaaa 900 acacccttgt ttctactaat aacccggcgg cccaaaatgc cgactcggag c 951 <210> 8 <211> 2233 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 8 agcgaaagca ggtactgatc caaaatggaa gattttgtgc gacaatgctt caatccgatg 60 attgtcgagc ttgcggaaaa aacaatgaaa gagtatgggg aggacctgaa aatcgaaaca 120 aacaaatttg cagcaatatg cactcacttg gaagtatgct tcatgtattc agattttcac 180 ttcatcaatg agcaaggcga gtcaataatc gtagaacttg gtgatccaaa tgcacttttg 240 aagcacagat ttgaaataat cgagggaaga gatcgcacaa tggcctggac agtagtaaac 300 agtatttgca acactacagg ggctgagaaa ccaaagtttc taccagattt gtatgattac 360 aaggagaata gattcatcga aattggagta acaaggagag aagttcacat atactatctg 420 gaaaaggcca ataaaattaa atctgagaaa acacacatcc acattttctc gttcactggg 480 gaagaaatgg ccacaaaggc agactacact ctcgatgaag aaagcagggc taggatcaaa 540 accagactat tcaccataag acaagaaatg gccagcagag gcctctggga ttcctttcgt 600 cagtccgaga gaggagaaga gacaattgaa gaaaggtttg aaatcacagg aacaatgcgt 660 aagcttgccg accaaagtct cccgccgaac ttctccagcc ttgaaaattt tagagcctat 720 gtggatggat tcgaaccgaa cggctacatt gagggcaagc tgtctcaaat gtccaaagaa 780 gtaaatgcta gaattgaacc ttttttgaaa acaacaccac gaccacttag acttccgaat 840 gggcctccct gttctcagcg gtccaaattc ctgctgatgg atgccttaaa attaagcatt 900 gaggacccaa gtcatgaagg agagggaata ccgctatatg atgcaatcaa atgcatgaga 960 acattctttg gatggaagga acccaatgtt gttaaaccac acgaaaaggg aataaatcca 1020 aattatcttc tgtcatggaa gcaagtactg gcagaactgc aggacattga gaatgaggag 1080 aaaattccaa agactaaaaa tatgaagaaa acaagtcagc taaagtgggc acttggtgag 1140 aacatggcac cagaaaaggt agactttgac gactgtaaag atgtaggtga tttgaagcaa 1200 tatgatagtg atgaaccaga attgaggtcg cttgcaagtt ggattcagaa tgagtttaac 1260 aaggcatgcg aactgacaga ttcaagctgg atagagctcg atgagattgg agaagatgtg 1320 gctccaattg aacacattgc aagcatgaga aggaattatt tcacatcaga ggtgtctcac 1380 tgcagagcca cagaatacat aatgaaggga gtgtacatca atactgcctt gcttaatgca 1440 tcttgtgcag caatggatga tttccaatta attccaatga taagcaagtg tagaactaag 1500 gagggaaggc gaaagaccaa cttgtatggt ttcatcataa aaggaagatc ccacttaagg 1560 aatgacaccg acgtggtaaa ctttgtgagc atggagtttt ctctcactga cccaagactt 1620 gaaccacata aatgggagaa gtactgtgtt cttgagatag gagatatgct tataagaagt 1680 gccataggcc aggtttcaag gcccatgttc ttgtatgtga gaacaaatgg aacctcaaaa 1740 attaaaatga aatggggaat ggagatgagg cgttgcctcc tccagtcact tcaacaaatt 1800 gagagtatga ttgaagctga gtcctctgtc aaagagaaag acatgaccaa agagttcttt 1860 gagaacaaat cagaaacatg gcccattgga gagtccccca aaggagtgga ggaaagttcc 1920 attgggaagg tctgcaggac tttattagca aagtcggtat tcaacagctt gtatgcatct 1980 ccacaactag aaggattttc agctgaatca agaaaactgc ttcttatcgt tcaggctctt 2040 agggacaacc tggaacctgg gacctttgat cttggggggc tatatgaagc aattgaggag 2100 tgcctgatta atgatccctg ggttttgctt aatgcttctt ggttcaactc cttccttaca 2160 catgcattga gttagttgtg gcagtgctac tatttgctat ccatactgtc caaaaaagta 2220 ccttgtttct act 2233 <210> 9 <211> 2341 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 9 agcgaaagca ggcaaaccat ttgaatggat gtcaatccga ccttactttt cttaaaagtg 60 ccagcacaaa atgctataag cacaactttc ccttatactg gagaccctcc ttacagccat 120 gggacaggaa caggatacac catggatact gtcaacagga cacatcagta ctcagaaaag 180 ggaagatgga caacaaacac cgaaactgga gcaccgcaac tcaacccgat tgatgggcca 240 ctgccagaag acaatgaacc aagtggttat gcccaaacag attgtgtatt ggaggcgatg 300 gctttccttg aggaatccca tcctggtatt tttgaaaact cgtgtattga aacgatggag 360 gttgttcagc aaacacgagt agacaagctg acacaaggcc gacagaccta tgactggact 420 ctaaatagaa accaacctgc tgcaacagca ttggccaaca caatagaagt gttcagatca 480 gatggcctca cggccaatga gtctggaagg ctcatagact tccttaagga tgtaatggag 540 tcaatgaaca aagaagaaat ggggatcaca actcattttc agagaaagag acgggtgaga 600 gacaatatga ctaagaaaat gataacacag agaacaatgg gtaaaaagaa gcagagattg 660 aacaaaagga gttatctaat tagagcattg accctgaaca caatgaccaa agatgctgag 720 agagggaagc taaaacggag agcaattgca accccaggga tgcaaataag ggggtttgta 780 tactttgttg agacactggc aaggagtata tgtgagaaac ttgaacaatc agggttgcca 840 gttggaggca atgagaagaa agcaaagttg gcaaatgttg taaggaagat gatgaccaat 900 tctcaggaca ccgaactttc tttcaccatc actggagata acaccaaatg gaacgaaaat 960 cagaatcctc ggatgttttt ggccatgatc acatatatga ccagaaatca gcccgaatgg 1020 ttcagaaatg ttctaagtat tgctccaata atgttctcaa acaaaatggc gagactggga 1080 aaagggtata tgtttgagag caagagtatg aaacttagaa ctcaaatacc tgcagaaatg 1140 ctagcaagca tcgatttgaa atatttcaat gattcaacaa gaaagaagat tgaaaaaatc 1200 cgaccgctct taatagaggg gactgcatca ttgagccctg gaatgatgat gggcatgttc 1260 aatatgttaa gcactgtatt aggcgtctcc atcctgaatc ttggacaaaa gagatacacc 1320 aagactactt actggtggga tggtcttcaa tcctctgacg attttgctct gattgtgaat 1380 gcacccaatc atgaagggat tcaagccgga gtcgacaggt tttatcgaac ctgtaagcta 1440 cttggaatca atatgagcaa gaaaaagtct tacataaaca gaacaggtac atttgaattc 1500 acaagttttt tctatcgtta tgggtttgtt gccaatttca gcatggagct tcccagtttt 1560 ggggtgtctg ggatcaacga gtcagcggac atgagtattg gagttactgt catcaaaaac 1620 aatatgataa acaatgatct tggtccagca acagctcaaa tggcccttca gttgttcatc 1680 aaagattaca ggtacacgta ccgatgccat ataggtgaca cacaaataca aacccgaaga 1740 tcatttgaaa taaagaaact gtgggagcaa acccgttcca aagctggact gctggtctcc 1800 gacggaggcc caaatttata caacattaga aatctccaca ttcctgaagt ctgcctaaaa 1860 tgggaattga tggatgagga ttaccagggg cgtttatgca acccactgaa cccatttgtc 1920 agccataaag aaattgaatc aatgaacaat gcagtgatga tgccagcaca tggtccagcc 1980 aaaaacatgg agtatgatgc tgttgcaaca acacactcct ggatccccaa aagaaatcga 2040 tccatcttga atacaagtca aagaggagta cttgaggatg aacaaatgta ccaaaggtgc 2100 tgcaatttat ttgaaaaatt cttccccagc agttcataca gaagaccagt cgggatatcc 2160 agtatggtgg aggctatggt ttccagagcc cgaattgatg cacggattga tttcgaatct 2220 ggaaggataa agaaagaaga gttcactgag atcatgaaga tctgttccac cattgaagag 2280 ctcagacggc aaaaatagtg aatttagctt gtccttcatg aaaaaatgcc ttgtttctac 2340 t 2341 <210> 10 <211> 2341 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 10 agcgaaagca ggtcaattat attcaatatg gaaagaataa aagaactacg aaatctaatg 60 tcgcagtctc gcacccgcga gatactcaca aaaaccaccg tggaccatat ggccataatc 120 aagaagtaca catcaggaag acaggagaag aacccagcac ttaggatgaa atggatgatg 180 gcaatgaaat atccaattac agcagacaag aggataacgg aaatgattcc tgagagaaat 240 gagcaaggac aaactttatg gagtaaaatg aatgatgccg gatcagaccg agtgatggta 300 tcacctctgg ctgtgacatg gtggaatagg aatggaccaa taacaaatac agttcattat 360 ccaaaaatct acaaaactta ttttgaaaga gtcgaaaggc taaagcatgg aacctttggc 420 cctgtccatt ttagaaacca agtcaaaata cgtcggagag ttgacataaa tcctggtcat 480 gcagatctca gtgccaagga ggcacaggat gtaatcatgg aagttgtttt ccctaacgaa 540 gtgggagcca ggatactaac atcggaatcg caactaacga taaccaaaga gaagaaagaa 600 gaactccagg attgcaaaat ttctcctttg atggttgcat acatgttgga gagagaactg 660 gtccgcaaaa cgagattcct cccagtggct ggtggaacaa gcagtgtgta cattgaagtg 720 ttgcatttga ctcaaggaac atgctgggaa cagatgtata ctccaggagg ggaagtgagg 780 aatgatgatg ttgatcaaag cttgattatt gctgctagga acatagtgag aagagctgca 840 gtatcagcag atccactagc atctttattg gagatgtgcc acagcacaca gattggtgga 900 attaggatgg tagacatcct taggcagaac ccaacagaag agcaagccgt ggatatatgc 960 aaggctgcaa tgggactgag aattagctca tccttcagtt ttggtggatt cacatttaag 1020 agaacaagcg gatcatcagt caagagagag gaagaggtgc ttacgggcaa tcttcaaaca 1080 ttgaagataa gagtgcatga gggatatgaa gagttcacaa tggttgggag aagagcaaca 1140 gccatactca gaaaagcaac caggagattg attcagctga tagtgagtgg gagagacgaa 1200 cagtcgattg ccgaagcaat aattgtggcc atggtatttt cacaagagga ttgtatgata 1260 aaagcagtca gaggtgatct gaatttcgtc aatagggcga atcaacgatt gaatcctatg 1320 catcaacttt taagacattt tcagaaggat gcgaaagtgc tttttcaaaa ttggggagtt 1380 gaacctatcg acaatgtgat gggaatgatt gggatattgc ccgacatgac tccaagcatc 1440 gagatgtcaa tgagaggagt gagaatcagc aaaatgggtg tagatgagta ctccagcacg 1500 gagagggtag tggtgagcat tgaccgtttt ttgagaatcc gggaccaacg aggaaatgta 1560 ctactgtctc ccgaggaggt cagtgaaaca cagggaacag agaaactgac aataacttac 1620 tcatcgtcaa tgatgtggga gattaatggt cctgaatcag tgttggtcaa tacctatcaa 1680 tggatcatca gaaactggga aactgttaaa attcagtggt cccagaaccc tacaatgcta 1740 tacaataaaa tggaatttga accatttcag tctttagtac ctaaggccat tagaggccaa 1800 tacagtgggt ttgtaagaac tctgttccaa caaatgaggg atgtgcttgg gacatttgat 1860 accgcacaga taataaaact tcttcccttc gcagccgctc caccaaagca aagtagaatg 1920 cagttctcct catttactgt gaatgtgagg ggatcaggaa tgagaatact tgtaaggggc 1980 aattctcctg tattcaacta taacaaggcc acgaagagac tcacagttct cggaaaggat 2040 gctggcactt taactgaaga cccagatgaa ggcacagctg gagtggagtc cgctgttctg 2100 aggggattcc tcattctggg caaagaagac aagagatatg ggccagcact aagcatcaat 2160 gaactgagca accttgcgaa aggagagaag gctaatgtgc taattgggca aggagacgtg 2220 gtgttggtaa tgaaacggaa acgggactct agcatactta ctgacagcca gacagcgacc 2280 aaaagaattc ggatggccat caattagtgt cgaatagttt aaaaacgacc ttgtttctac 2340 t 2341 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tattcgtctc agggagcaaa agcagggg 28 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cattccctgc catccccctt caatgaaacc 30 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ggtttcattg aagggggatg gcagggaatg 30 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 atatcgtctc gtattagtag aaacaagggt gtttt 35 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 attcgtctca gggagcaaaa gcagggg 27 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 atatcgtctc gtattagtag aaacaagggt gtttt 35 <110> Chungbuk National University Industry-Academic Cooperation Foundation          I.D.Bio. <120> RECOMBINANT INFLUENZA VIRUS TO FORM CROSS-PROTECTION AGAINST          H3 and H7 SUBTYPES OF INFLUENZA VIRUSES AND VACCINE COMPRISING THE          SAME <130> PN1603-106 <160> 16 <170> KoPatentin 3.0 <210> 1 <211> 1035 <212> DNA <213> Influenzae virus <400> 1 atgaagacta tcattgcttt gagctacatt ctatgtctgg ttttcgctca aaaacttcct 60 ggaaatgaca acagcacggc aacgctgtgc cttgggcacc atgcagtacc aaacggaacg 120 atagtgaaaa caatcacgaa tgaccaaatt gaagttacta atgctactga gctggttcag 180 agttcctcaa caggtgaaat atgcgacagt cctcatcaga tccttgatgg aaaaaactgc 240 acactaatag atgctctatt gggagaccct cagtgtgatg gcttccaaaa taagaaatgg 300 gacctttttg ttgaacgcag caaagcctac agcaactgtt acccttatga tgtgccggat 360 tatgcctccc ttaggtcact agttgcctca tccggcacac tggagtttaa caatgaaagc 420 ttcaattgga ctggagtcac tcaaaacgga acaagctctg cttgcataag gagatctaaa 480 aacagtttct ttagtagatt gaattggttg acccacttaa acttcaaata cccagcattg 540 aacgtgacta tgccaaacaa tgaacaattt gacaaattgt acatttgggg ggttcaccac 600 ccgggtacgg acaaagacca aatcttcctg tatgctcaag catcaggaag aatcacagtc 660 tctaccaaaa gaagccaaca aaccgtaagc ccgaatatcg gatctagacc cagagtaagg 720 aatatcccta gcagaataag catctattgg acaatagtaa aaccgggaga catacttttg 780 attaacagca cagggaatct aattgctcct aggggttact tcaaaatacg aagtgggaaa 840 agctcaataa tgagatcaga tgcacccatt ggcaaatgca attctgaatg catcactcca 900 aatggaagca ttcccaatga caaaccattc caaaatgtaa acaggatcac atacggggcc 960 tgtcccagat atgttaagca aaacactctg aaattggcaa cagggatgcg aaatgtacca 1020 gagaaacaaa ctaga 1035 <210> 2 <211> 666 <212> DNA <213> Influenzae virus <400> 2 ggcctatttg gtgctatagc gggtttcatt gaaatggat gggaaggcct aattgatggt 60 tggtatggtt tcagacacca gaatgcacag ggagagggaa ctgctgcaga ttacaaaagc 120 actcaatcgg caattgatca aataacagga aaattaaacc ggcttataga aaaaaccaac 180 caacaatttg agttgataga caatgaattc aatgaggtag agaagcaaat cggtaatgtg 240 ataaattgga ccagagattc tataacagaa gtgtggtcat acaatgctga actcttggta 300 gcaatggaga accagcatac aattgatctg gctgattcag aaatggacaa actgtacgaa 360 cgagtgaaaa gacagctgag agagaatgct gaagaagatg gcactggttg ctttgaaata 420 tttcacaagt gtgatgatga ctgtatggcc agtattagaa ataacaccta tgatcacagc 480 aaatacaggg aagaggcaat gcaaaataga atacagattg acccagtcaa actaagcagc 540 ggctacaaag atgtgatact ttggtttagc ttcggggcat catgtttcat acttctagcc 600 attgtaatgg gccttgtctt catatgtgta aagaatggaa acatgcggtg cactatttgt 660 atataa 666 <210> 3 <211> 1701 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 3 atgaagacta tcattgcttt gagctacatt ctatgtctgg ttttcgctca aaaacttcct 60 ggaaatgaca acagcacggc aacgctgtgc cttgggcacc atgcagtacc aaacggaacg 120 atagtgaaaa caatcacgaa tgaccaaatt gaagttacta atgctactga gctggttcag 180 agttcctcaa caggtgaaat atgcgacagt cctcatcaga tccttgatgg aaaaaactgc 240 acactaatag atgctctatt gggagaccct cagtgtgatg gcttccaaaa taagaaatgg 300 gacctttttg ttgaacgcag caaagcctac agcaactgtt acccttatga tgtgccggat 360 tatgcctccc ttaggtcact agttgcctca tccggcacac tggagtttaa caatgaaagc 420 ttcaattgga ctggagtcac tcaaaacgga acaagctctg cttgcataag gagatctaaa 480 aacagtttct ttagtagatt gaattggttg acccacttaa acttcaaata cccagcattg 540 aacgtgacta tgccaaacaa tgaacaattt gacaaattgt acatttgggg ggttctccac 600 ccgggtacgg acaaagacca aatcttcctg tatgctcaag catcaggaag aatcacagtc 660 tctaccaaaa gaagccaaca aaccgtaagc ccgaatatcg gatctagacc cagagtaagg 720 aatatcccta gcagaataag catctattgg acaatagtaa aaccgggaga catacttttg 780 attaacagca cagggaatct aattgctcct aggggttact tcaaaatacg aagtgggaaa 840 agctcaataa tgagatcaga tgcacccatt ggcaaatgca attctgaatg catcactcca 900 aatggaagca ttcccaatga caaaccattc caaaatgtaa acaggatcac atacggggcc 960 tgtcccagat atgttaagca aaacactctg aaattggcaa cagggatgcg aaatgtacca 1020 gagaaacaaa ctagaggcat atttggcgca atcgcgggtt tcattgaaaa tggatgggaa 1080 ggcctaattg atggttggta tggtttcaga caccagaatg cacagggaga gggaactgct 1140 gcagattaca aaagcactca atcggcaatt gatcaaataa caggaaaatt aaaccggctt 1200 atagaaaaaa ccaaccaaca atttgagttg atagacaatg aattcaatga ggtagagaag 1260 caaatcggta atgtgataaa ttggaccaga gattctataa cagaagtgtg gtcatacaat 1320 gctgaactct tggtagcaat ggagaaccag catacaattg atctggctga ttcagaaatg 1380 gacaaactgt acgaacgagt gaaaagacag ctgagagaga atgctgaaga agatggcact 1440 ggttgctttg aaatatttca caagtgtgat gatgactgta tggccagtat tagaaataac 1500 acctatgatc acagcaaata cagggaagag gcaatgcaaa atagaataca gattgaccca 1560 gtcaaactaa gcagcggcta caaagatgtg atactttggt ttagcttcgg ggcatcatgt 1620 ttcatacttc tagccattgt aatgggcctt gtcttcatat gtgtaaagaa tggaaacatg 1680 cggtgcacta tttgtatata a 1701 <210> 4 <211> 1444 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 4 agcaaaagca ggagtttaaa atgaatccaa atcagaaaat aataaccatt ggatcaatct 60 gtctggtagt cggactaatt agcctaatat tgcaaatagg gaatataatc tcaatatgga 120 ttagccattc aattcaaact ggaagtcaaa accatactgg aatatgcaac caaaacatca 180 ttacctataa aaatagcacc tgggtaaagg acacaacttc agtgatatta accggcaatt 240 catctctttg tcccatccgt gggtgggcta tatacagcaa agacaatagc ataagaattg 300 gttccaaagg agacgttttt gtcataagag agccctttat ttcatgttct cacttggaat 360 gcaggacctt ttttctgacc caaggtgcct tactgaatga caagcattca agtgggactg 420 ttaaggacag aagcccttat agggccttaa tgagctgccc tgtcggtgaa gctccgtccc 480 cgtacaattc aagatttgaa tcggttgctt ggtcagcaag tgcatgtcat gatggcatgg 540 gctggctaac aatcggaatt tcaggtccag ataatggagc agtggctgta ttaaaataca 600 acggcataat aactgaaacc ataaaaagtt ggaggaagaa aatattgagg acacaagagt 660 ctgaatgtgc ctgtgtaaat ggttcatgtt ttactataat gactgatggc ccgagtgatg 720 ggctggcctc gtacaaaatt ttcaagatcg aaaaggggaa ggttactaaa tcaatagagt 780 tgaatgcacc taattctcac tatgaggaat gttcctgtta ccctgatacc ggcaaagtga 840 tgtgtgtgtg cagagacaat tggcatggtt cgaaccggcc atgggtgtct ttcgatcaaa 900 acctggatta tcaaatagga tacatctgca gtggggtttt cggtgacaac ccgcgtcccg 960 aagatggaac aggcagctgt ggtccagtgt atgttgatgg agcaaacgga gtaaagggat 1020 tttcatatag gtatggtaat ggtgtttgga taggaaggac caaaggtcac agttccagac 1080 atgggtttga gatgatttgg gatcctaatg gatggacaga gactgatagt aagttctctg 1140 tgaggcaaga tgttgtggca atgactgatt ggtcagggta tagcggaagt ttcgttcaac 1200 atcctgagct gacagggcta gactgtatga ggccgtgctt ctgggttgaa ttaatcaggg 1260 gacgacctaa agaaaaaaca atctggacta gtgcgagcag catttctttt tgtggcgtga 1320 atagtgatac tgtagattgg tcttggccag acggtgctga gttgccattc agcattgaca 1380 agtagtctgt tcaaaaaact ccttgtttct actaataacc cggcggccca aaatgccgac 1440 tcgg 1444 <210> 5 <211> 1100 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 5 actggtcgac ctccgaagtt gggggggagc aaaagcaggt agatattgaa agatgagtct 60 tctaaccgag gtcgaaacgt acgtactctc tatcatcccg tcaggccccc tcaaagccga 120 gatcgcacag agacttgaag atgtctttgc agggaagaac accgatcttg aggttctcat 180 ggaatggcta aagacaagac caatcctgtc acctctgact aaggggattt taggatttgt 240 gttcacgctc accgtgccca gtgagcgagg actgcagcgt agacgctttg tccaaaatgc 300 ccttaatggg aacggggatc caaataacat ggacaaagca gttaaactgt ataggaagct 360 caagagggag ataacattcc atggggccaa agaaatctca ctcagttatt ctgctggtgc 420 acttgccagt tgtatgggcc tcatatacaa caggatgggg gctgtgacca ctgaagtggc 480 atttggcctg gtatgtgcaa cctgtgaaca gattgctgac tcccagcatc ggtctcatag 540 gcaaatggtg acaacaacca atccactaat cagacatgag aacagaatgg ttttagccag 600 cactacagct aaggctatgg agcaaatggc tggatcgagt gagcaagcag cagaggccat 660 ggaggttgct agtcaggcta gacaaatggt gcaagcgatg agaaccattg ggactcatcc 720 tagctccagt gctggtctga aaaatgatct tcttgaaaat ttgcaggcct atcagaaacg 780 aatgggggtg cagatgcaac ggttcaagtg atcctctcac tattgccgca aatatcattg 840 ggatcttgca cttgacattg tggattcttg atcgtctttt tttcaaatgc atttaccgtc 900 gctttaaata cggactgaaa ggagggcctt ctacggaagg agtgccaaag tctatgaggg 960 aagaatatcg aaaggaacag cagagtgctg tggatgctga cgatggtcat tttgtcagca 1020 tagagctgga gtaaaaaact accttgtttc tactaataac ccggcggccc aaaatgccga 1080 ctcggagcga aagatatacc 1100 <210> 6 <211> 1559 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 6 agcagggtag ataatcactc actgagtgac atcaaaatca tggcgtctca aggcaccaaa 60 cgatcttacg aacagatgga gactgatgga gaacgccaga atgccactga aatcagagca 120 tccgtcggaa aaatgattgg tggaattgga cgattctaca tccaaatgtg caccgaactc 180 aaactcagtg attatgaggg acggttgatc caaaacagct taacaataga gagaatggtg 240 ctctctgctt ttgacgaaag gagaaataaa taccttgaag aacatcccag tgcggggaaa 300 gatcctaaga aaactggagg acctatatac aggagagtaa acggaaagtg gatgagagaa 360 ctcatccttt atgacaaaga agaaataagg cgaatctggc gccaagctaa taatggtgac 420 gatgcaacgg ctggtctgac tcacatgatg atctggcatt ccaatttgaa tgatgcaact 480 tatcagagga caagagctct tgttcgcacc ggaatggatc ccaggatgtg ctctctgatg 540 caaggttcaa ctctccctag gaggtctgga gccgcaggtg ctgcagtcaa aggagttgga 600 acaatggtga tggaattggt cagaatgatc aaacgtggga tcaatgatcg gaacttctgg 660 aggggtgaga atggacgaaa aacaagaatt gcttatgaaa gaatgtgcaa cattctcaaa 720 gggaaatttc aaactgctgc acaaaaagca atgatggatc aagtgagaga gagccggaac 780 ccagggaatg ctgagttcga agatctcact tttctagcac ggtctgcact catattgaga 840 gggtcggttg ctcacaagtc ctgcctgcct gcctgtgtgt atggacctgc cgtagccagt 900 gggtacgact ttgaaaggga gggatactct ctagtcggaa tagacccttt cagactgctt 960 caaaacagcc aagtgtacaga cctaatcaga ccaaatgaga atccagcaca caagagtcaa 1020 ctggtgtgga tggcatgcca ttctgccgca tttgaagatc taagagtatt aagcttcatc 1080 aaagggacga aggtgctccc aagagggaag ctttccacta gaggagttca aattgcttcc 1140 aatgaaaata tggagactat ggaatcaagt acacttgaac tgagaagcag gtactgggcc 1200 ataaggacca gaagtggagg aaacaccaat caacagaggg catctgcggg ccaaatcagc 1260 atacaaccta cgttctcagt acagagaaat ctcccttttg acagaacaac cattatggca 1320 gcattcaatg ggaatacaga ggggagaaca tctgacatga ggaccgaaat cataaggatg 1380 atggaaagtg caagaccaga agatgtgtct ttccaggggc ggggagtctt cgagctctcg 1440 gacgaaaagg cagcgagccc gatcgtgcct tcctttgaca tgagtaatga aggatcttat 1500 ttcttcggag acaatgcaga ggagtacgac aattaaagaa aaataccctt gtttctact 1559 <210> 7 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 7 actggtcgac ctccgaagtt gggggggagc aaaagcaggg tgacaaaaac ataatggatc 60 caaacactgt gtcaagcttt caggtagatt gctttctttg gcatgtccgc aaacgagttg 120 cagaccaaga actaggcgat gccccattcc ttgatcggct tcgccgagat cagaaatccc 180 taagaggaag gggcagtact ctcggtctgg acatcaagac agccacacgt gctggaaagc 240 agatagtgga gcggattctg aaagaagaat ccgatgaggc acttaaaatg accatggcct 300 ctgtacctgc gtcgcgttac ctaactgaca tgactcttga ggaaatgtca agggactggt 360 ccatgctcat acccaagcag aaagtggcag gccctctttg tatcagaatg gaccaggcga 420 tcatggataa gaacatcata ctgaaagcga acttcagtgt gatttttgac cggctggaga 480 ctctaatatt gctaagggct ttcaccgaag agggagcaat tgttggcgaa atttcaccat 540 tgccttctct tccaggacat actgctgagg atgtcaaaaa tgcagttgga gtcctcatcg 600 gaggacttga atggaatgat aacacagttc gagtctctga aactctacag agattcgctt 660 ggagaagcag taatgagaat gggagacctc cactcactcc aaaacagaaa cgagaaatgg 720 cgggaacaat taggtcagaa gtttgaagaa ataagatggt tgattgaaga agtgagacac 780 aaactgaaga taacagagaa tagttttgag caaataacat ttatgcaagc cttacatcta 840 ttgcttgaag tggagcaaga gataagaact ttctcgtttc agcttattta gtactaaaaa 900 acacccttgt ttctactaat aacccggcgg cccaaaatgc cgactcggag c 951 <210> 8 <211> 2233 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 8 agcgaaagca ggtactgatc caaaatggaa gattttgtgc gacaatgctt caatccgatg 60 attgtcgagc ttgcggaaaa aacaatgaaa gagtatgggg aggacctgaa aatcgaaaca 120 aacaaatttg cagcaatatg cactcacttg gaagtatgct tcatgtattc agattttcac 180 ttcatcaatg agcaaggcga gtcaataatc gtagaacttg gtgatccaaa tgcacttttg 240 aagcacagat ttgaaataat cgagggaaga gatcgcacaa tggcctggac agtagtaaac 300 agtatttgca acactacagg ggctgagaaa ccaaagtttc taccagattt gtatgattac 360 aaggagaata gattcatcga aattggagta acaaggagag aagttcacat atactatctg 420 gaaaaggcca ataaaattaa atctgagaaa acacacatcc acattttctc gttcactggg 480 gaagaaatgg ccacaaaggc agactacact ctcgatgaag aaagcagggc taggatcaaa 540 accagactat tcaccataag acaagaaatg gccagcagag gcctctggga ttcctttcgt 600 cagtccgaga gaggagaaga gacaattgaa gaaaggtttg aaatcacagg aacaatgcgt 660 aagcttgccg accaaagtct cccgccgaac ttctccagcc ttgaaaattt tagagcctat 720 gtggatggat tcgaaccgaa cggctacatt gagggcaagc tgtctcaaat gtccaaagaa 780 gtaaatgcta gaattgaacc ttttttgaaa acaacaccac gaccacttag acttccgaat 840 gggcctccct gttctcagcg gtccaaattc ctgctgatgg atgccttaaa attaagcatt 900 gaggacccaa gtcatgaagg agagggaata ccgctatatg atgcaatcaa atgcatgaga 960 acattctttg gatggaagga acccaatgtt gttaaaccac acgaaaaggg aataaatcca 1020 aattatcttc tgtcatggaa gcaagtactg gcagaactgc aggacattga gaatgaggag 1080 aaaattccaa agactaaaaa tatgaagaaa acaagtcagc taaagtgggc acttggtgag 1140 aacatggcac cagaaaaggt agactttgac gactgtaaag atgtaggtga tttgaagcaa 1200 tatgatagtg atgaaccaga attgaggtcg cttgcaagtt ggattcagaa tgagtttaac 1260 aaggcatgcg aactgacaga ttcaagctgg atagagctcg atgagattgg agaagatgtg 1320 gctccaattg aacacattgc aagcatgaga aggaattatt tcacatcaga ggtgtctcac 1380 tgcagagcca cagaatacat aatgaaggga gtgtacatca atactgcctt gcttaatgca 1440 tcttgtgcag caatggatga tttccaatta attccaatga taagcaagtg tagaactaag 1500 gagggaaggc gaaagaccaa cttgtatggt ttcatcataa aaggaagatc ccacttaagg 1560 aatgacaccg acgtggtaaa ctttgtgagc atggagtttt ctctcactga cccaagactt 1620 gaaccacata aatgggagaa gtactgtgtt cttgagatag gagatatgct tataagaagt 1680 gccataggcc aggtttcaag gcccatgttc ttgtatgtga gaacaaatgg aacctcaaaa 1740 attaaaatga aatggggaat ggagatgagg cgttgcctcc tccagtcact tcaacaaatt 1800 gagagtatga ttgaagctga gtcctctgtc aaagagaaag acatgaccaa agagttcttt 1860 gagaacaaat cagaaacatg gcccattgga gagtccccca aaggagtgga ggaaagttcc 1920 attgggaagg tctgcaggac tttattagca aagtcggtat tcaacagctt gtatgcatct 1980 ccacaactag aaggattttc agctgaatca agaaaactgc ttcttatcgt tcaggctctt 2040 agggacaacc tggaacctgg gacctttgat cttggggggc tatatgaagc aattgaggag 2100 tgcctgatta atgatccctg ggttttgctt aatgcttctt ggttcaactc cttccttaca 2160 catgcattga gttagttgtg gcagtgctac tatttgctat ccatactgtc caaaaaagta 2220 ccttgtttct act 2233 <210> 9 <211> 2341 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 9 agcgaaagca ggcaaaccat ttgaatggat gtcaatccga ccttactttt cttaaaagtg 60 ccagcacaaa atgctataag cacaactttc ccttatactg gagaccctcc ttacagccat 120 gggacaggaa caggatacac catggatact gtcaacagga cacatcagta ctcagaaaag 180 ggaagatgga caacaaacac cgaaactgga gcaccgcaac tcaacccgat tgatgggcca 240 ctgccagaag acaatgaacc aagtggttat gcccaaacag attgtgtatt ggaggcgatg 300 gctttccttg aggaatccca tcctggtatt tttgaaaact cgtgtattga aacgatggag 360 gttgttcagc aaacacgagt agacaagctg acacaaggcc gacagaccta tgactggact 420 ctaaatagaa accaacctgc tgcaacagca ttggccaaca caatagaagt gttcagatca 480 gatggcctca cggccaatga gtctggaagg ctcatagact tccttaagga tgtaatggag 540 tcaatgaaca aagaagaaat ggggatcaca actcattttc agagaaagag acgggtgaga 600 gacaatatga ctaagaaaat gataacacag agaacaatgg gtaaaaagaa gcagagattg 660 aacaaaagga gttatctaat tagagcattg accctgaaca caatgaccaa agatgctgag 720 agagggaagc taaaacggag agcaattgca accccaggga tgcaaataag ggggtttgta 780 tactttgttg agacactggc aaggagtata tgtgagaaac ttgaacaatc agggttgcca 840 gtggaggca atgagaagaa agcaaagttg gcaaatgttg taaggaagat gatgaccaat 900 tctcaggaca ccgaactttc tttcaccatc actggagata acaccaaatg gaacgaaaat 960 cagaatcctc ggatgttttt ggccatgatc acatatatga ccagaaatca gcccgaatgg 1020 ttcagaaatg ttctaagtat tgctccaata atgttctcaa acaaaatggc gagactggga 1080 aaagggtata tgtttgagag caagagtatg aaacttagaa ctcaaatacc tgcagaaatg 1140 ctagcaagca tcgatttgaa atatttcaat gattcaacaa gaaagaagat tgaaaaaatc 1200 cgaccgctct taatagaggg gactgcatca ttgagccctg gaatgatgat gggcatgttc 1260 aatatgttaa gcactgtatt aggcgtctcc atcctgaatc ttggacaaaa gagatacacc 1320 aagactactt actggtggga tggtcttcaa tcctctgacg attttgctct gattgtgaat 1380 gcacccaatc atgaagggat tcaagccgga gtcgacaggt tttatcgaac ctgtaagcta 1440 cttggaatca atatgagcaa gaaaaagtct tacataaaca gaacaggtac atttgaattc 1500 acaagttttt tctatcgtta tgggtttgtt gccaatttca gcatggagct tcccagtttt 1560 ggggtgtctg ggatcaacga gtcagcggac atgagtattg gagttactgt catcaaaaac 1620 aatatgataa acaatgatct tggtccagca acagctcaaa tggcccttca gttgttcatc 1680 aaagattaca ggtacacgta ccgatgccat ataggtgaca cacaaataca aacccgaaga 1740 tcatttgaaa taaagaaact gtgggagcaa acccgttcca aagctggact gctggtctcc 1800 gacggaggcc caaatttata caacattaga aatctccaca ttcctgaagt ctgcctaaaa 1860 tgggaattga tggatgagga ttaccagggg cgtttatgca acccactgaa cccatttgtc 1920 agccataaag aaattgaatc aatgaacaat gcagtgatga tgccagcaca tggtccagcc 1980 aaaaacatgg agtatgatgc tgttgcaaca acacactcct ggatccccaa aagaaatcga 2040 tccatcttga atacaagtca aagaggagta cttgaggatg aacaaatgta ccaaaggtgc 2100 tgcaatttat ttgaaaaatt cttccccagc agttcataca gaagaccagt cgggatatcc 2160 agtatggtgg aggctatggt ttccagagcc cgaattgatg cacggattga tttcgaatct 2220 ggaaggataa agaaagaaga gttcactgag atcatgaaga tctgttccac cattgaagag 2280 ctcagacggc aaaaatagtg aatttagctt gtccttcatg aaaaaatgcc ttgtttctac 2340 t 2341 <210> 10 <211> 2341 <212> DNA <213> Artificial Sequence <220> <223> chimeric gene <400> 10 agcgaaagca ggtcaattat attcaatatg gaaagaataa aagaactacg aaatctaatg 60 tcgcagtctc gcacccgcga gatactcaca aaaaccaccg tggaccatat ggccataatc 120 aagaagtaca catcaggaag acaggagaag aacccagcac ttaggatgaa atggatgatg 180 gcaatgaaat atccaattac agcagacaag aggataacgg aaatgattcc tgagagaaat 240 gagcaaggac aaactttatg gagtaaaatg aatgatgccg gatcagaccg agtgatggta 300 tcacctctgg ctgtgacatg gtggaatagg aatggaccaa taacaaatac agttcattat 360 ccaaaaatct acaaaactta ttttgaaaga gtcgaaaggc taaagcatgg aacctttggc 420 cctgtccatt ttagaaacca agtcaaaata cgtcggagag ttgacataaa tcctggtcat 480 gcagatctca gtgccaagga ggcacaggat gtaatcatgg aagttgtttt ccctaacgaa 540 gtgggagcca ggatactaac atcggaatcg caactaacga taaccaaaga gaagaaagaa 600 gaactccagg attgcaaaat ttctcctttg atggttgcat acatgttgga gagagaactg 660 gtccgcaaaa cgagattcct cccagtggct ggtggaacaa gcagtgtgta cattgaagtg 720 ttgcatttga ctcaaggaac atgctgggaa cagatgtata ctccaggagg ggaagtgagg 780 aatgatgatg ttgatcaaag cttgattatt gctgctagga acatagtgag aagagctgca 840 gtatcagcag atccactagc atctttattg gagatgtgcc acagcacaca gattggtgga 900 attaggatgg tagacatcct taggcagaac ccaacagaag agcaagccgt ggatatatgc 960 aaggctgcaa tgggactgag aattagctca tccttcagtt ttggtggatt cacatttaag 1020 agaacaagcg gatcatcagt caagagagag gaagaggtgc ttacgggcaa tcttcaaaca 1080 ttgaagataa gagtgcatga gggatatgaa gagttcacaa tggttgggag aagagcaaca 1140 gccatactca gaaaagcaac caggagattg attcagctga tagtgagtgg gagagacgaa 1200 cagtcgattg ccgaagcaat aattgtggcc atggtatttt cacaagagga ttgtatgata 1260 aaagcagtca gaggtgatct gaatttcgtc aatagggcga atcaacgatt gaatcctatg 1320 catcaacttt taagacattt tcagaaggat gcgaaagtgc tttttcaaaa ttggggagtt 1380 gaacctatcg acaatgtgat gggaatgatt gggatattgc ccgacatgac tccaagcatc 1440 gagatgtcaa tgagaggagt gagaatcagc aaaatgggtg tagatgagta ctccagcacg 1500 gagagggtag tggtgagcat tgaccgtttt ttgagaatcc gggaccaacg aggaaatgta 1560 ctactgtctc ccgaggaggt cagtgaaaca cagggaacag agaaactgac aataacttac 1620 tcatcgtcaa tgatgtggga gattaatggt cctgaatcag tgttggtcaa tacctatcaa 1680 tggatcatca gaaactggga aactgttaaa attcagtggt cccagaaccc tacaatgcta 1740 tacaataaaa tggaatttga accatttcag tctttagtac ctaaggccat tagaggccaa 1800 tacagtgggt ttgtaagaac tctgttccaa caaatgaggg atgtgcttgg gacatttgat 1860 accgcacaga taataaaact tcttcccttc gcagccgctc caccaaagca aagtagaatg 1920 cagttctcct catttactgt gaatgtgagg ggatcaggaa tgagaatact tgtaaggggc 1980 aattctcctg tattcaacta taacaaggcc acgaagagac tcacagttct cggaaaggat 2040 gctggcactt taactgaaga cccagatgaa ggcacagctg gagtggagtc cgctgttctg 2100 aggggattcc tcattctggg caaagaagac aagagatatg ggccagcact aagcatcaat 2160 gaactgagca accttgcgaa aggagagaag gctaatgtgc taattgggca aggagacgtg 2220 gtgttggtaa tgaaacggaa acgggactct agcatactta ctgacagcca gacagcgacc 2280 aaaagaattc ggatggccat caattagtgt cgaatagttt aaaaacgacc ttgtttctac 2340 t 2341 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tattcgtctc agggagcaaa agcagggg 28 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cattccctgc catccccctt caatgaaacc 30 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ggtttcattg aagggggatg gcagggaatg 30 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 atatcgtctc gtattagtag aaacaagggt gtttt 35 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 attcgtctca gggagcaaaa gcagggg 27 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 atatcgtctc gtattagtag aaacaagggt gtttt 35

Claims (9)

H3 및 H7의 두 가지 아형(subtype)의 키메릭 헤마글루티닌 유전자를 포함하는 재조합 인플루엔자 바이러스(기탁번호: KCTC 13007BP).Recombinant influenza virus (accession number: KCTC 13007BP) comprising two subtypes of the chimeric hemagglutinin gene of H3 and H7. 제1항에 있어서,
상기 키메릭 헤마글루티닌 유전자는 A/Perth/16/2009 (H3N2) 유래의 HA1 및 융합펩티드 유전자와; A/Anhui/1/2013 (H7N9) 유래의 HA2 유전자가 융합된 형태인 것을 특징으로 하는 재조합 인플루엔자 바이러스.The method according to claim 1,
The chimeric hemagglutinin gene comprises HA1 and a fusion peptide gene derived from A / Perth / 16/2009 (H3N2); A / Anhui / 1/2013 (H7N9) -infected recombinant influenza virus. 제1항에 있어서,
상기 키메릭 헤마글루티닌 유전자는 서열번호 3으로 표시되는 폴리뉴클레오티드로 이루어진 것을 특징으로 하는 재조합 인플루엔자 바이러스.The method according to claim 1,
Wherein the chimeric hemagglutinin gene comprises the polynucleotide of SEQ ID NO: 3. 제1항의 바이러스를 유효성분으로 포함하는 인플루엔자 바이러스 백신.An influenza virus vaccine comprising the virus of claim 1 as an active ingredient. 제4항에 있어서,
상기 백신은 바이러스를 약독화된 생독 백신, 사독 백신 또는 서브유닛 백신(면역원성 단편)인 것을 특징으로 하는 인플루엔자 바이러스 백신.5. The method of claim 4,
Wherein the vaccine is an influenza virus vaccine characterized in that the virus is an attenuated virulent vaccine, a sadox vaccine or a subunit vaccine (immunogenic fragment). 제4항에 있어서,
상기 바이러스가 H3N2 혈청형 및 H7N9 혈청형을 가진 A형 인플루엔자 바이러스 모두에 대해 교차 면역반응을 형성하는 것을 특징으로 하는 인플루엔자 바이러스 백신.5. The method of claim 4,
Wherein said virus forms a cross-immune response against both influenza A viruses having H3N2 serotype and H7N9 serotype. 제4항의 백신을 인간을 제외한 개체에 투여하여 인플루엔자 바이러스 감염 질환을 예방 또는 치료하는 방법.A method for preventing or treating an influenza virus infection disease by administering the vaccine of claim 4 to an individual other than a human. 제1항의 바이러스 또는 이의 항원을 포함하는 인플루엔자 바이러스의 진단용 조성물.A diagnostic composition for influenza virus comprising the virus of claim 1 or an antigen thereof. 제1항의 바이러스 또는 이의 항원을 이용한 항원-항체 반응을 통해 감염될 또는 감염된 세포 내에서 인플루엔자 바이러스(기탁번호: KCTC 13007BP)를 검출하는 것을 특징으로 하는, 인플루엔자 바이러스(기탁번호: KCTC 13007BP)의 검출 방법.(Accession No .: KCTC 13007BP), characterized in that it detects an influenza virus (Accession No: KCTC 13007BP) in the infected or infected cells through an antigen-antibody reaction using the virus of claim 1 or an antigen thereof Way.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101964044B1 (en) * 2018-03-14 2019-04-02 인제대학교 산학협력단 Multi-valent live-attenuated influenza vaccine platform using recombinant adenovirus
CN109666767A (en) * 2019-02-01 2019-04-23 扬州大学 Detect and distinguish one-step method real-time fluorescent quantification PCR primer, probe and the kit of the strong and weak poison of H7 subtype avian influenza virus
WO2021055679A1 (en) * 2019-09-20 2021-03-25 Academia Sinica Chimeric hemagglutinin protein and a vaccine composition comprising the same

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101964044B1 (en) * 2018-03-14 2019-04-02 인제대학교 산학협력단 Multi-valent live-attenuated influenza vaccine platform using recombinant adenovirus
WO2019177256A1 (en) * 2018-03-14 2019-09-19 인제대학교 산학협력단 Multivalent live influenza vaccine platform using recombinant adenovirus
US11672854B2 (en) 2018-03-14 2023-06-13 Inje University Industry-Academic Cooperation Foundation Multivalent live influenza vaccine platform using recombinant adenovirus
CN109666767A (en) * 2019-02-01 2019-04-23 扬州大学 Detect and distinguish one-step method real-time fluorescent quantification PCR primer, probe and the kit of the strong and weak poison of H7 subtype avian influenza virus
WO2021055679A1 (en) * 2019-09-20 2021-03-25 Academia Sinica Chimeric hemagglutinin protein and a vaccine composition comprising the same

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