WO2002036618A1 - Nouveaux genes codant pour les proteines g et m1 d'un virus coreen isole de la necrose hematopoietique infectieuse-scs (nhi-scs) et vaccin permettant de lutter contre le virus de la necrose hematopoietique infectieuse chez un salmonide - Google Patents

Nouveaux genes codant pour les proteines g et m1 d'un virus coreen isole de la necrose hematopoietique infectieuse-scs (nhi-scs) et vaccin permettant de lutter contre le virus de la necrose hematopoietique infectieuse chez un salmonide Download PDF

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WO2002036618A1
WO2002036618A1 PCT/KR2001/001871 KR0101871W WO0236618A1 WO 2002036618 A1 WO2002036618 A1 WO 2002036618A1 KR 0101871 W KR0101871 W KR 0101871W WO 0236618 A1 WO0236618 A1 WO 0236618A1
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ihnv
protein
scs
gene
proteins
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PCT/KR2001/001871
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Jeong-Woo Park
Chang-Hoon Moon
Won-Joon Yoon
Kyung-Won Park
Han-Geun Kim
Tae-Kwang Oh
Hyung-Kwoun Kim
Dae-Kyun Chung
Wha-Ja Cho
Jeong-Min Park
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Rna Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/145—Rhabdoviridae, e.g. rabies virus, Duvenhage virus, Mokola virus or vesicular stomatitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/205—Rhabdoviridae, e.g. rabies virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a novel protein isolated from infectious hematopoietic necrosis virus (IHNV), a gene encoding the protein and a vaccine using the protein or the gene and more particularly, the present invention relates to a novel protein isolated from Korean type infectious hematopoietic necrosis virus; a gene encoding the protein; and a vaccine against hematopoietic necrosis symptoms in fish containing the protein or the gene.
  • IHNV infectious hematopoietic necrosis virus
  • Salmonid fish is one of the most expensive pisces and there is great demand for this pisces. Therefore, it is mass cultivated in many regions over the world, including North America, North Europe, Japan, and etc. In case of Norway, industries related to salmon cultivation comprises more than 15% of total GNP. Thus, the cultivating industry can greatly contribute to national economy and moreover, as the exclusive economic zones of the respective countries are enlarged its importance increases. Particularly, Korea is surrounded by the sea on three sides and the whole area of the East Sea has a water temperature and water quality suitable for cultivation of salmon. Therefore, if complete control of diseases can be achieved, there are ample possibilities for success of salmon cultivation in this area. However, viral diseases which cause catastrophic death of fmgerlings is a severe problem in salmon cultivation.
  • IHNV is a fish virus belonging to rhabdovirus, which affects spleen, kidneys, brain or digestive tract of various salmons including Pacific salmons, Atlantic salmons, rainbow trout and sockeye salmon, and leads to abrupt and mass death of fry and juveniles of those fishes. It was initially isolated in natural fishes, but has spread widely and finally has been found in the cultivated rainbow trout and salmons. It is now found that IHNV is one of causes of fish death and researches and studies are conducted to develop a vaccine against the fish virus (Park M.A. et al, J. Fish Dis. 16:471-478, 1993; and Hah Y.C. et al, Kor. J. Microbiol 22(2):85-90, 1984).
  • IPNV As for IPNV, it has been reported that it includes four structural proteins: VP1, VP2, VP3 and VP4, and a neutralizing epitope exists in VP2, which is the most outwardly exposed protein on the native virus (Nagy et al, International Congress of Virology, Edmonton, Canada, OP 25.2, p311, 1987). On the basis of this, a recombinant vaccine using VP2 has been developed (Lawrence et al, Gene, 79: 369-374, 1989; and Christie, Dev. Biol. Stand. 90:1-9, 1997).
  • IHNV immunodeficiency virus
  • G protein includes five structural proteins: L(polymerase), G(glycoprotein), N(nucleocapsid), Ml (matrix proteinl), M2(matrix protein2), and a neutralizing epitope exists in G protein, an outer coat protein (Engelking et al, Virus Research 13:213-230, 1989).
  • a recombinant vaccine using G protein has been developed (Gilmore et al, Bio/Technology 6(3):85-90, 1988; d Xu et al., J. Virol. 65(3):1611-1615, 1991).
  • the majority of the IHNV vaccines are produced in only several countries such as USA, Norway, etc. Furthermore, the vaccines are developed with targeting G protein of IHNV of certain serotypes found in the corresponding countries. Therefore, it is expected that these vaccines targeting G protein, particularly known to have many mutants, would not be effective to prevent the mass death of salmonid in Korea. In addition, these vaccines show very weak neutralizing effect against actual viral infection and thus fail to be effectively utilized. Inactivated virus which has been widely used until now as a preventive vaccine costs a great deal for its preparation. Thus, from an economic point of view, such vaccines are not suitable for applying to fish which is cheaper than the price required to prepare the vaccines.
  • DNA vaccines or protein vaccines against some structural proteins have been developed.
  • these vaccines also fail to provide satisfactory effects.
  • the cause of the failure is partially attributable to the fact that a target gene for production of such vaccines is focused on only VP2 gene for IPNV and only G protein gene for IHNV and an expression system with higher efficiency has not been developed.
  • IHNV shows different virulences according to regions from which the viruses are isolated and proteins of the viruses are different according to various serotypes (Picher et al., Crit. Rev. Microbiol. 7:287-364, 1980). Therefore, a vaccine against a certain serotype is not effective against a virus of another serotype.
  • IHNV In case of IHNV, although there is no standard serotype officially approved, there are many approaches to classifying the serotypes of IHNV. One of them is to classify IHNV according to the differences in molecular weights of the five structural proteins, L, G, N, Ml and M2. Representatively, Hsu et al. classify IHNV into five types (electropherotypes) on the basis of the difference between molecular weights of G and N proteins (Hsu et al, Appl. Environ. Microbiol. 52:1353-1361, 1986). Also, Winton et al. classify IHNVs of twelve types isolated in various parts of the world into four groups using three kinds of monoclonal antibodies (Winton et al, Dis. Aquat. Org. 4:199-204, 1988).
  • IHNV isolated from the Korean type is different in serological characteristics and sizes of structural proteins from IHNVs isolated in foreign countries (Park M.A. et al, J. Fish Dis. 16:471-478, 1993)
  • IHNV vaccines developed in the foreign countries also are not effective to prevent the mass death of fish caused by Korean type IHNV. Indeed, it has been reported that a neutralizing antibody against G protein produced in a foreign country fails to neutralize Korean type IHNV-PRT (Park M.A. et al, J. Fish Dis. 16:471-478, 1993).
  • the present inventors have searched for a novel target protein against a Korean type IHNV serotype to develop a vaccine which has superior ability to induce immunity to Korean type IHNV serum as well as higher neutralizing efficiency as compared to conventional vaccines against IHNV, and have made intensive research to develop a method for mass-producing the protein.
  • the present inventors discovered genes encoding G protein and Ml protein from IHNV-SCS, a Korean type isolated strain, and confirmed that G protein and Ml protein stimulate production of a neutralizing antibody.
  • the inventors optimized conditions under which G and Ml proteins can be mass-produced in an E. coli strain transformed with an expression vector containing the gene and showed that the gene is useful as an effective vaccine against
  • G protein and Ml protein isolated from IHNV-SCS of Korean type IHNV serum and genes encoding the proteins are provided.
  • an antibody using IHNV-SCS G protein and IHNV-SCS Ml protein as antigens there is provided an antibody using IHNV-SCS G protein and IHNV-SCS Ml protein as antigens.
  • a vaccine against infectious hematopoietic necrosis virus in salmon containing the IHNV-SCS G and/or Ml gene(s), or protein(s).
  • Fig. 1 shows a cleavage map of an expression vector p ⁇ T28a/IHNV-SCS-G comprising the IHNV G gene
  • Fig. 2 shows a cleavage map of an expression vector pET28a/IHNV-SCS-Ml comprising the IHNV Ml gene
  • Fig. 3 shows a photograph of a gel.
  • pET28a/IHNV-SCS-Ml cut with restriction enzyme Nde I was subjected to electrophoresis.
  • band shift was observed. Therefore, it was noted that the size was increased, indicating that the Ml gene is inserted into pET28a vector;
  • pET28a vector 2 pET28a/IHNV-SCS-Ml;
  • Fig. 4a shows a result of SDS-PAGE.
  • E. coli transformed with ⁇ ET28a/IHNV- SCS-M1 during culturing, was treated with IPTG as an expression inducer at various concentrations. After treatment, the strain was further cultured for 4 hours and expression levels of Ml protein were measured and compared; M: size marker
  • Fig. 4b shows a result of SDS-PAGE.
  • E. coli transformed with pET28a/IHNV- SCS-M1 during culturing, was treated with 0.3 mM of IPTG as an expression inducer.
  • M size marker 1: non-transformed E.coli BL21(DE3)
  • Fig. 5a shows a result of SDS-PAGE.
  • E. coli transformed with pET28a/IHNV- SCS-M1 during culturing, was treated with lactose as an expression inducer at various concentrations. After treatment, the strain was further cultured, for 4 hours and expression levels of Ml protein were measured and compared;
  • SCS-M1 during culturing, was treated with 5 mM of lactose as an expression inducer. After treatment, the strain was examined for its expression levels of Ml protein according to time; M: size marker
  • Fig. 6a (Fractions 1-9) and Fig. 6b (Fractions 10-18) show results of SDS-PAGE, wherein Ml protein expressed in fractions separated by a Ni-NTA column was observed at about 31 kDa;
  • FigJ shows a Western blot analysis of reaction between Ml protein and anti- serum (RT-1, RT-2, RT-3 and RT-4) isolated from the rainbow trout surviving after infection by IHNV-SCS;
  • RT-NS serum isolated from healthy rainbow trout not infected by IHNV-SCS
  • Fig. 8 is a graph showing a result of a neutralization assay, which reveals that the anti-serum isolated from the rainbow trout surviving after infection by IHNV-SCS has a neutralizing activity against IHNV;
  • Fig. 9 is a graph showing inhibitive effects of virus multiplication of egg liquid extracted from rainbow trouts which were treated with a IHNV-G vaccine, and IHNV-G + IHNV-M1 vaccines, respectively;
  • control 2 treated with IHNV-G
  • FIG. 10 is a graph showing preventive effects of mass death by the DNA vaccine of fry grown from eggs of mother rainbow trouts which were treated with DNA vaccines having the G gene and Ml gene of IHNV-SCS introduced therein.
  • D not treated with DNA vaccine
  • the Korean type IHNV-SCS used in the present invention was isolated from fry of rainbow trout which perished from infection while being cultivated in Korea.
  • the strain has characteristics serologically different from strains isolated from IHNV in foreign countries (Park M. A. et al, J, Fish Dis. 16:471-478, 1993).
  • the G gene of IHNV-SCS according to the present invention is consisted of 1458 base pairs having the nucleotide sequence represented by SEQ ID NO: 5 and encodes G protein consisted of 485 amino acids represented by SEQ ID NO: 6.
  • the G protein has a calculated molecular weight of about 54.4 kDa.
  • the Ml gene of IHNV-SCS according to the present invention is consisted of 693 base pairs having the nucleotide sequence represented by SEQ ID NO: 7 and encodes Ml protein consisted of 230 amino acids represented by SEQ ID NO: 8.
  • the Ml protein has a calculated molecular weight of about 29.5 kDa.
  • the IHNV-SCS G gene and Ml gene according to the present invention were deposited in Korea Culture Center of Microorganisms on August 2, 2000 as transformed E. coli (Deposition Access Nos.: KCCM- 10207 and KCCM- 10208) which are yielded by transformation of E. coli BL21(DE3) with plasmids produced by inserting the IHNV-SCS G and Ml genes into pET28a, a vector for prokaryote expression, respectively. Also, the present invention provides a method for mass-producing G protein and
  • the expression control sequence includes preferably T7 promoter, although it is not limited thereto.
  • the medium may preferably be treated with IPTG (isopropyl- ⁇ -D-thio-galactopyranoside) or lactose as expression inducers.
  • IPTG isopropyl- ⁇ -D-thio-galactopyranoside
  • lactose isopropyl- ⁇ -D-thio-galactopyranoside
  • the medium may be preferably treated with lactose in a concentration of 1 to 20 mM for 4 to 6 hours, in order to achieve a desirable result in terms of protein production yield. More preferably, the medium may be treated with IPTG in a concentration of 0.3 mM for about 6 hours or with lactose in a concentration of 5 mM for about 5 hours. In the examples discussed later, by using the above conditions, about 1.4 mg/m of protein is obtained. In another embodiment of the present invention, in order to determine that IHNV-
  • SCS Ml protein shows immunogenecity against IHNV-SCS
  • reaction of anti-serum against IHNV-SCS with Ml is examined using a Western blot analysis. Further, whether an antibody against Ml protein has a neutralizing activity against IHNV-SCS is tested using a neutralization assay.
  • G protein is already known as a structural protein having high immunogenecity.
  • G protein itself or the gene of IHNV-SCS can be used as a prophylactic vaccine against IHNV infection.
  • G protein is also known as a protein having relatively many variations among serotypes of IHNV. The reason the existing vaccines do not succeed in preventing infections is that the vaccines are produced targeting mainly the G protein. Therefore, in order to enhance efficiency of vaccines using the IHNV-SCS G protein or gene according to the present invention, the vaccines may be produced by combinedly employing the Ml protein or gene, or a neutralizing epitope of IPNV which is another main cause of viral infection in salmonid fish.
  • the neutralizing epitope of IPNV may be selected from VP2 and VP3 known for their neutralizing activities.
  • the present invention provides a DNA vaccine comprising an eukaryote expression vector containing G gene and/or Ml gene of IHNV-SCS as an active component. Further, the present invention provides a protein vaccine comprising G and/or Ml protein mass-produced in a cell culture system such as E. coli as an active component.
  • the DNA vaccine is mostly applied to the mother fish for the purpose of enhancing immunogenecity of the mother fish and preventing vertical infection of virus through transfer of antibodies produced in the mother fish to the fry, thereby preventing mass death at a fry stage. Also, since G and/or Ml protein(s) is(are) produced continuously after administration of the vaccine, it is not necessary to repeatedly administer the vaccine.
  • Administration of the vaccine is effected by one of the methods of directly injecting the vaccine to fry or the mother fish, immersing the subjects into a pool containing the vaccine dissolved in water, or orally administering the vaccine in a mixture with feed, in case of protein vaccine.
  • At least one of the three methods can be selected according to type of vaccine, and size and age offish.
  • a DNA vaccine it is preferable to administer to the mother fish by direct injection.
  • cultivated fish are anesthetized with about 0.04 % of 2-phenoxyethanol and administered with 10 to 100 ⁇ g of a DNA vaccine via intramuscular injection.
  • the vaccine may be administered along with a adjuvant such as ⁇ - glucan, chitosan, levamisol, etc, GM-CSF, TGF- ⁇ or a hormone to enhance the effect of the vaccine.
  • Administration via injection is known to be the most effective administration methods. There are advantages that it can induce immunity in the mother fish and antibodies produced in the mother fish can be transmitted to fry, thereby providing immunity to virus in a fry stage after hatching.
  • a protein vaccine it is orally administered after combining with feed.
  • Fry in an early stage after hatching does not take feed.
  • an immersion method is used. Fry are immersed in a solution of a protein vaccine dissolved in water for 1 to 2 minutes.
  • effective amounts of the gene/protein are 0.1 to 2 ⁇ gl l, preferably 0.5 to 1 ⁇ gl i.
  • the antibodies may be a polyclonal antibody or a monoclonal antibody.
  • the monoclonal antibody can be isolated by the conventional methods known to the art, such as cell fusion, using a hybridoma.
  • cDNA library of CHSE-214 cell lines (ATCC CRL 1681) infected by IHNV-SCS, a strain isolated in Korea, which is one of viruses causing infectious hematopoietic necrosis in salmonid fish such as rainbow trout (Salmo girdnerii), was constructed.
  • CHSE-214 Chonook salmon embryos cell lines as host cells of IHNV- SCS were cultured in an EMEM (Eagles' Minimum Essential Medium; Sigma, U.S.A.) containing 10%o fetal bovine serum (FBS), 50 mg/4. of streptomycin (GibcoBRL,
  • CHSE-214 cells grown to a single layer were infected with Korean type IHNV-SCS (Park M.A. et al, J. Fish, Dis. 16:471-478, 1993). At 25 hours after infection, the medium was removed and the cells were washed with phosphate buffered saline (PBS). A denaturing solution (4M guanidium thiocyanate, 0.5% SDS, 25mM sodium citrate, pH 7.0, 0.1 M 2- mercaptoethanol) was added to the washed medium to dissolve the cells.
  • PBS phosphate buffered saline
  • cDNA was synthesized with the isolated mRNA as a template using a ZAP- cDNA synthesis kit (Stratagene, U.S.A.), according to the instructions of the manufacturer.
  • the synthesis of cDNA was carried out using hybrid oligo (dT) linker- primer containing a Xho I site and MMLV-RT.
  • dsDNA was synthesized using DNA polymerase I and linked with an EcoR I adaptor. The DNA was cut at both ends with Xho I to form cohesive ends for EcoR I and Xho I. And the DNA was connected to a EcoR l-Xho I site of a ⁇ -ZAP vector.
  • the recombinant ⁇ ZAP DNA was packaged into ⁇ phage.
  • a E. coli XLl-Blue MRF strain (Stratagene, U.S.A.) was infected with the recombinant vector to construct a cDNA library.
  • a PCR primer was prepared to be used as a probe to screen G and Ml genes of IHNV-SCS in the cDNA library.
  • a primer having the nucleotide sequence represented by S ⁇ Q ID NO: 1 forward
  • a primer having the nucleotide sequence represented by S ⁇ Q ID NO: 2 reverse
  • a primer having the nucleotide sequence represented by S ⁇ Q ID NO: 2 reverse
  • a primer having the nucleotide sequence represented by S ⁇ Q ID NO: 3 forward
  • a primer having the nucleotide sequence represented by S ⁇ Q ID NO: 4 reverse
  • [ ⁇ - 32 P]-dCTP was added for obtaining DNA radiolabeled with 32 P.
  • the PCR products included 442 bp for the pair of primers [SEQ ID NO: 1 + SEQ ID NO: 2] and 453 bp for the pair of primers [SEQ ID NO: 3 + SEQ ID NO: 4], respectively.
  • the nucleotide sequences of both products were determined and compared with the nucleotide sequences of G and Ml genes recorded in GenBank to examine homology. As a result, it was confirmed that the two sequences have homology to G gene and Ml gene of IHNV - WRAC, respectively. Therefore, the cDNA library constructed in Example 1-1 was screened using the products as probes. After screening was performed by three times, two clones containing full-length G or Ml gene of IHNV-SCS, respectively, were selected.
  • Example 1-1) was analyzed by means of an automated sequencer (Applied Biosystemics, U.S.A.). As a result, it was found that the G gene has an open reading frame of 1458 bp
  • the G protein of IHNV-SCS is consisted of 487 amino acids (SEQ ID NO: 6) with a calculated molecular weight of 54.4 kDa and the Ml protein of IEINV-SCS is consisted of 230 amino acids (SEQ ID NO: 8) with a calculated molecular weight of 25.9 kDa.
  • the G gene of IHNV-SCS identified in Example 2 was cloned into pET 28a (Novagen, U.S.A.) which is a vector for expression of prokaryotes.
  • the G gene of IHNV-SCS cloned in Example 1 was amplified with the primers represented by SEQ ID NOs: 1 and 2 by PCR using a Taq-polymerase.
  • the amplified PCR product was cut with restriction enzyme Sacl-Xhol and inserted between
  • the plasmid vector containing the G gene was excised with Nde I and its size was compared with pET28a, a negative control. In the comparison, it was shown that the size was increased and thus the G gene was duly cloned.
  • E. coli BL21 (D ⁇ 3) (Novagen, U.S.A.) transformed with the produced pET28a IHNV-SCS G was deposited in Korea Culture Center of Microorganisms on August 2, 2000 (Deposition Access No.: KCCM- 10207).
  • coli BL21 (DE3) (Novagen, U.S.A.) transformed with the produced pET28a/IHNV-SCS Ml was deposited in Korea Culture Center of Microorganisms on August 2, 2000 (Deposition Access No.: KCCM-10208).
  • E. coli BL21 (DE3)/pET28a/IHNV-SCS Ml (herein after referred to as "Ml cell") was cultured in 5 ml of LB medium containing 50 mgimi of kanamycin for 10 hours. The medium was diluted to 1/100 and inoculated into 100 in?, of LB medium containing 50 mgimi of kanamycin. When OD 600 reached to 0.6, IPTG
  • IPTG isopropyl- ⁇ -D-thio-galactopyranoside
  • concentrations of added IPTG were 0.3 mM, 0.5 mM, 0J mM, 1 mM and 2 mM.
  • expression levels for respective concentrations were measured by SDS-PAGE analysis.
  • expression patterns of the Ml protein were examined at time points after addition of IPTG(0 hours); 2 hours, 4 hours, 6 hours and 8 hours after treatment with 0.3 mM of IPTG.
  • coli transformed with only pET28a, and pET28a/IHNV-SCS-Ml transformant non-treated with IPTG were used.
  • the expression levels of the induced Ml cells were examined by SDS-PAGE analysis. Concretely, 1 mi aliquots were taken from respective treatments and controls. Each sample was then centrifuged at 14,000 x g for 30 seconds. The supernatant was removed to obtain pellets, which were resuspended into 500 ⁇ i of distilled water. A 1.5 mi test tube containing the suspended sample was subjected to ultrasonic treatment while kept in an ice bath, to disrupt cells. The ultrasonic treatment was performed by 3 cycles of ultrasonication for 15 seconds and cooling for 10 seconds.
  • the disrupted cells were centrifuged at 14,000 x g for 5 minutes. 20 ⁇ i of the supernatant was taken and mixed with 40 ⁇ i of 5 x SDS-sample buffer, followed by boiling at 110°C for 5 minutes. The product was again centrifuged at 14,000 x g for 5 minutes. 200 ⁇ i of the supernatant was taken and loaded on a SDS-polyacrylamide gel.
  • the gel was stained with a Coomassie staining solution (0.1 %> coomassie blue R 250, 42% acetic acid and 42 % methylalcohol) for 30 minutes and destained with a destaining solution (1.5% acetic acid and 7.5 % methylalcohol) for 30 minutes. From the SDS analysis, as shown in Fig. 4a and Fig. 4b, it can be seen that the expression level of IHNV-SCS Ml protein is highest when cells were treated with about 0.3 mM of IPTG for 6 to 8 hours.
  • Ml cells were cultured in 100 ml of LB medium containing 50 mgimi of kanamycin. When OD 600 reached to 0.8, lactose was added to the medium at various concentrations and times. The concentration of added lactose were 0.3 mM, 0.5 mM, 0J mM, 1 mM and 2 mM. At 4 hours after the treatment with the inducer, expression patterns for respective concentrations were measured by SDS-PAGE analysis. Also, in order to determine expression patterns of the Ml protein according to time treated with lactose, expression patterns of the Mlprotein were examined at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours after treatment with 5 mM lactose.
  • Ml protein 5-1) Purification and quantitative analysis of IHNV-SCS Ml protein 5-1) Purification of Ml protein The Ml protein expressed in Example 4 was purified on a Ni-NTA agarose column (QIAGEN, U.S.A.) using 6 x His tags positioned at N-terminal and C-terminal of a pET28a vector.
  • the Ml cells were cultured in 5 ml of LB medium containing 50 mgimi of kanamycin for 10 hours.
  • the medium was diluted to 1/100 and inoculated into 1 I of LB medium containing 50 mgimi of kanamycin.
  • IPTG isopropyl- ⁇ -D-thio-galactopyranoside
  • the medium was cultured for a further 6 hours.
  • 1 mi of the medium was taken before the addition of IPTG, to serve as a negative control.
  • 1 mi of each sample was taken and centrifuged at 4,000 x g for 20 minutes to obtain Ml cell pellets.
  • the pellets were resuspended in 50 mi of lysis buffer (50 mM NaH 2 PO 4 , 300 mM sodium chloride, 10 mM imidazole). The suspension was added with Imglmi of lysozyme and kept in an ice bath for 30 minutes. After cell disruption by ultrasonic treatment, the disrupted cells were centrifuged at 9,000 x g for 20 minutes and the supernatant was removed.
  • lysis buffer 50 mM NaH 2 PO 4 , 300 mM sodium chloride, 10 mM imidazole.
  • Total protein amount (mg ) (OD 595 x R dll x M - 0.047)/0.0567 in which the R dll is a dilution rate and M is an added amount of sample. The results are shown in Table 1 below.
  • the G protein contains a neutralizing epitope (Engelking et al, Virus Research 13:213-230, 1989).
  • the Ml protein of IHNV-SCS was examined for its immunogenecity by a Western blot analysis and neutralization assay to determine the utility of the Ml protein as a vaccine.
  • anti-serum was collected from rainbow trout fry naturally infected by IHNV-SCS but surviving from the infection.
  • Two types of anti-serum, RT-1 and RT-2 were obtained from fry surviving from the infection of IHNV-SCS and captured at Jungseon in Korea; and two types of anti-serum, RT-3 and RT-4 were obtained from fry surviving from the infection of IHNV-SCS and captured at Dongbang and Inje in Korea, respectively.
  • serum RT-NS obtained from fry not infected by IHNV-SCS was used. All anti-sera were kept at -20 ° C until used.
  • Proteins of IHNV-SCS was subjected to SDS-PAGE on a 10% separation gel under reducing conditions according to the method of Laemmli (Laemmli, Nature 227:680-685, 1970).
  • the separated proteins were electrophoresed using a transfer buffer (25 mM Tris, 192 mM glycine, 20 % methaol, pH 8.3) at 40 mA for 16 hours and transferred onto a nitrocellulose (NC) membrane.
  • the NC membrane was washed with phosphate-buffered saline (PBS) and soaked into PBS containing 1 % bovine serum albumin (BSA) for 1 hour to block the reaction.
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • the anti-sera obtained from fry were diluted with PBS-Tween 20 (0.05 %) and reacted with the NC membrane at room temperature for 2 hours. Then, the NC membrane was reacted with mouse antibody against rainbow trout immunoglobulin and finally with goat anti-mouse IgG antibody conjugated with alkaline phosphatase.
  • the membrane was soaked in a carbonate buffer (0.1 M NaHCO 3 , 1 mM MgCl 2 , pH 9.8) containing BCIP-NBT (5-bromo-4-chloro-3- indolyl phophate sodium salt 0.15 mgimi, p-nitro blue tetrazolium chloride 0.3 mgimi) as a coloring substrate at room temperature for 15 minutes to develop the color reaction.
  • An in vitro neutralization assay was performed according to the method of Okamoto et al. (Okamoto et al, J. fish Dis. 6:19-25, 1983) to determine whether the antibodies against the IHNV-SCS Ml protein have neutralizing activity against IHNV infection.
  • the four anti-sera (RT-1, RT-2, RT-3 and RT-4) were serially diluted by halves with an EMEM medium. The respective dilutions were placed into a 96-well plate.
  • Freshly prepared IHNV-SCS was diluted 100 TCID 60 /50 ⁇ i, and added to each well, followed by incubation at room temperature for 1 hour with continuous shaking.
  • CHSE-214 cells 100 ⁇ i, 1 x 10 5 cellssM were added to each well, followed by incubation at 18 ° C for 10 days.
  • the neutralization titer (ND 50 /50 ⁇ i) was expressed as the reciprocal of the maximum dilution of the anti serum, by which 50% of inoculated cells can be protected from virus infection.
  • ND 50 /50 ⁇ i was expressed as the reciprocal of the maximum dilution of the anti serum, by which 50% of inoculated cells can be protected from virus infection.
  • the G gene of IHNV-SCS was amplified with a forward primer of the nucleotide sequence represented by SEQ ID NO: 9 containing Nbel and a reverse primer of the nucleotide sequence represented by SEQ ID NO: 10 containing EcoRI.
  • the amplified DNA was cut with Nhel and EcoRI and inserted between N zel-EcoRI of MCS (multi cloning site) A of pIR ⁇ S (hereinafter referred to as "IHNV-G vaccine").
  • the Ml gene of IHNV-SCS was amplified with a forward primer of the nucleotide sequence represented by SEQ ID NO: 11 containing Bam ⁇ and a reverse primer of the nucleotide sequence represented by SEQ ID NO: 12 containing Notl.
  • the amplified DNA was cut with Bam ⁇ I and Notl and inserted between BmdHl - Notl of MCS B of pIRES (hereinafter referred to as "IH ⁇ V-M1 vaccine").
  • the virus titer was 103.3/m ⁇ .
  • the group treated with the egg liquid treated with the vaccine had a reduced virus titer.
  • the egg liquid treated with only the IHNV-G vaccine showed a IHNV titer reduced to 102.2/m£ and the egg liquid treated with the IHNV-G and Ml vaccines showed a IHNV titer reduced to lOlJ/in From these results, it can be seen that substances capable of inhibiting multiplication of virus are present in the egg liquids of mother rainbow trout juveniles treated with the DNA vaccines of IHNV-SCS according to the present invention and also that the amount of the substances upon co- treatment with the IHNV-G and Ml vaccines is greater than treatment with only the IHNV-G vaccine.
  • fry hatched from the mother rainbow trout treated with the DNA vaccines in Example 7 was examined for their survival rate to confirm that resistance of mother fish to IHNV-SCS is transmitted to their fry.
  • Eggs were harvested from the mother fish treated with the DNA vaccine comprising the IHNV G gene and the IHV Ml gene prepared in Example 7 and cultivated.
  • the fry of the control group began to die from 15 days after hatching and at 30 days, 90 % of fry were dead and only 10% survived.
  • the perished fry were subjected to RT-PCR against IHNV-SCS G protein and were found to have been infected by IHNV-SCS.
  • the fry from mother fish treated with the vaccines did not die until 30 days after hatching, however, from 30 days they began to die and at 40 days, 20 % of the fry were dead. Further, after 45 days, the death rate did not increase and 80 % of the fry survived the end of the trial. From this, it was demonstrated that fry hatched from mother fish treated with the vaccines according to the present invention have a high resistance to IHNV-SCS infection.
  • novel G and Ml proteins of IHNV-SCS which is isolated from the Korean type IHNV-SCS can be used effectively as a prophylactic vaccine against IHNV infection causing abrupt and mass death in salmonid fish.
  • genes encoding the proteins can be directly used to prepare DNA vaccines.
  • the vaccines using the IHNV-SCS G and/or Ml genes, and the IHNV-SCS G and/or Ml proteins have a superior ability to induce immune response to the conventional IHNV vaccines and are especially able to prevent the Korean type IHNV-SCS infection, which was not controlled by the conventional IHNV vaccines.
  • Tlie microo ⁇ ao ⁇ aro identified under I above was -urompa ⁇ ird by:

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Abstract

La présente invention concerne des nouvelles protéines G et M1 isolées à partir d'un virus coréen de la nécrose hématopoïétique infectieuse-SCS, un des virus de la nécrose hématopoïétique infectieuse (NHI); des nouveaux gènes codant pour ces protéines; des vaccins utilisant ces gènes et/ou ces protéines; et un procédé permettant de produire ces protéines à l'échelle industrielle. Les protéines G et M1 et/ou les gènes codant pour ces protéines peuvent être utilisés de manière efficace comme vaccins pour lutter contre le virus de la nécrose hématopoïétique infectieuse.
PCT/KR2001/001871 2000-11-04 2001-11-05 Nouveaux genes codant pour les proteines g et m1 d'un virus coreen isole de la necrose hematopoietique infectieuse-scs (nhi-scs) et vaccin permettant de lutter contre le virus de la necrose hematopoietique infectieuse chez un salmonide WO2002036618A1 (fr)

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CN106890327A (zh) * 2017-02-28 2017-06-27 兰州威特森生物科技有限公司 一种防治虹鳟鱼传染性造血器官坏死症的活载体疫苗及其制备方法和应用
CN109136200A (zh) * 2018-09-20 2019-01-04 中国水产科学研究院黑龙江水产研究所 一种重组传染性造血器官坏死病毒及其构建方法与应用

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US7249294B2 (en) 2002-06-24 2007-07-24 Hynix Semiconductor Inc. Semiconductor memory device with reduced package test time

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106890327A (zh) * 2017-02-28 2017-06-27 兰州威特森生物科技有限公司 一种防治虹鳟鱼传染性造血器官坏死症的活载体疫苗及其制备方法和应用
CN109136200A (zh) * 2018-09-20 2019-01-04 中国水产科学研究院黑龙江水产研究所 一种重组传染性造血器官坏死病毒及其构建方法与应用

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