WO1997027302A1 - Vaccine prepared using human-origin hantaan virus nucleocapsid protein expressed from escherichia coli - Google Patents

Vaccine prepared using human-origin hantaan virus nucleocapsid protein expressed from escherichia coli Download PDF

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Publication number
WO1997027302A1
WO1997027302A1 PCT/KR1997/000016 KR9700016W WO9727302A1 WO 1997027302 A1 WO1997027302 A1 WO 1997027302A1 KR 9700016 W KR9700016 W KR 9700016W WO 9727302 A1 WO9727302 A1 WO 9727302A1
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protein
virus
human
escherichia coli
gene
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PCT/KR1997/000016
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French (fr)
Japanese (ja)
Inventor
Hyun Su Kim
Wang Don Yoo
Soo Ok Kim
Kap Soo Noh
Sun Pyo Hong
Young Chol Shin
Sung Hee Lee
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Cheil Jedang Corporation
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Priority claimed from KR1019960001638A external-priority patent/KR0178110B1/en
Application filed by Cheil Jedang Corporation filed Critical Cheil Jedang Corporation
Priority to AU15591/97A priority Critical patent/AU1559197A/en
Publication of WO1997027302A1 publication Critical patent/WO1997027302A1/en

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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
    • 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
    • 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
    • C12N2760/12011—Bunyaviridae
    • C12N2760/12111—Hantavirus, e.g. Hantaan virus
    • C12N2760/12122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention comprises culturing a nucleocapsid protein gene selected from a human-derived Hunter virus strain, an expression plasmid containing the gene, an E. coli transformant transformed with the expression plasmid, and a transformant.
  • the present invention relates to a nucleoside protein obtained by expressing and purifying a protein, and a prophylactic vaccine against Hunter virus containing the protein as an active ingredient.
  • Hunter virus a member of the genus Huntervirus of the Vernier virus family, is a single-stranded ribonucleic acid genome (RNA genome) consisting of three segments (S, M, and L segments, respectively), which cause renal symptomatic hemorrhagic fever. It contains.
  • the L-section RNA encodes RNA-dependent RNA polymerase
  • the M-section RNA encodes glycoproteins G1 and G2
  • the S-section RNA encodes nucleoside N-protein. It is known that Gl, G2 and nucleocapsid N proteins are involved in the immune response.
  • the value as a vaccine substance that could be used could not be determined.
  • these proteins are not only difficult to purify but also genetically engineered in E. coli, yeast or animal cells.
  • the prophylactic tract against renal symptomatic hemorrhagic fever is currently present in the brain or animal cells of mammals, despite the numerous problems of using the virus itself as a vaccine substance. In fact, only vaccines produced by directly culturing and inactivating them with formalin are supplied.
  • the present inventors have conducted intensive research to solve the stability problem caused by the inactivated virus vaccine, and as a result, have been able to transfer a nucleoside protein that has never been used in pure isolation. It was found that large-scale production of Escherichia coli by this method and its use as a component of a vaccine for the prevention of Hunter's virus could lead to the development of a vaccine that is safer and more effective in preventing hemorrhagic fever.
  • the present inventors studied the antigenicity of various proteins constituting human-derived huntan virus for the purpose of the present invention, and as a result, The protein of the skin protein, ie, the nucleoside protein, is selected, its nucleotide sequence is analyzed, expressed in E. coli, the expressed protein is separated and purified, and the neutralized antibody form of the purified protein is purified.
  • the protein of the skin protein ie, the nucleoside protein
  • An object of the present invention is to provide a nucleocapsid protein gene of a human-derived hunter virus whose base sequence has been clarified by the present inventors.
  • the present invention also provides a recombinant expression plasmid containing a nucleocapsid protein gene as a foreign gene, an Escherichia coli transformant transformed with the expression plasmid, and culturing the transformant in an appropriate medium.
  • the present invention relates to a human-derived nucleocapsid protein of Hunter's virus obtained through a separation and purification process.
  • an object of the present invention is to provide a vaccine for preventing hemorrhagic fever with nephropathy, which contains, as an active ingredient, a nucleocabside protein of a human-derived hunter virus obtained purely by the genetic engineering method as described above. .
  • Fig. 1 shows the nucleotide sequence of a primer for synthesizing and amplifying the nucleocapsid protein gene of human-derived Hunter virus.
  • FIG. 2 schematically shows a method for producing pThNP8 and pThNP9, which are recombinant gene carriers containing the nucleocapsid protein gene of the human-derived Hunter virus.
  • Figure 3A shows the nucleotide sequence of the nucleocapsid protein gene of the human-derived Hunter virus compared to the known Hans Equine virus 76-118, and B shows the analogous amino acid sequence of the hunter virus. This is shown in comparison with virus 76-118.
  • Figure 4 shows a schematic representation of the production process for the expression plasmid pEThNP.
  • FIG. 5 shows a schematic representation of the production process of the expression plasmid pKKhNP.
  • FIG. 6 shows a method of purifying a nucleocapsid protein expressed from Escherichia coli containing pEThNP or pKKhNP, respectively.
  • FIG. 7 is a photograph of polyatarylamide gel electrophoresis showing the purity of nucleosides expressed by E. coli BL (pEThNP) in each purification step.
  • Figure 8 shows the results of ⁇ ⁇ stamp lotting using human patient blood and a monoclonal antibody to confirm the nucleocapsid protein purified from E. coli.
  • the infection source Hunter virus
  • the virus was isolated and the RNA genome was isolated from this virus.
  • the recombinant vector having the nucleocapsid protein gene was isolated and confirmed, and the single-stranded DNA was transformed using helper phage Ml3KO7. By separation, it was used as an experimental material for gene sequencing.
  • the nucleotide sequence analyzed for the entire nucleocapsid protein and the deduced amino acid sequence are as shown in FIGS. 3A and 3B. From this, the nucleocapsid gene isolated from human-derived hunter virus according to the present invention is shown. It can be seen that the nucleotide sequence of the nucleotide sequence is 95% similar to that of the known gene sequence of the known 76-11 virus, and the amino acid sequence is 98.1% similar. From the nucleocapsid protein gene having the altered nucleotide sequence, to produce an effective and safe vaccine against Hansin virus aimed at by the present invention, first, amplification and expression of a foreign gene must be carried out. Production process, culture of the transformant, and isolation and purification of the nucleocapsid protein.The invention is completed by investigating the efficacy of the finally produced actin. it can.
  • nucleocapsid protein ⁇ gene was obtained by performing the polymerase chain reaction using the five types of primers shown in FIG. 1, a sufficient amount of the nucleocapsid protein ⁇ gene to be cloned into plasmid was obtained.
  • Appropriate restriction enzyme recognition sites were added to the gene so that it could be cloned into plasmids pET-3a and pKK223-3, respectively.
  • plasmid pET-38 contains a part of the 510-derived amino acid sequence, the nucleoside protein cloned and expressed in this plasmid is converted into a fusion protein containing an additional 12 amino acids at the N-terminus.
  • plasmid pKK223-3 does not have such a sequence, it is expressed as an original protein, not as a fusion protein.
  • the process for producing the expressed plasmids pEThNP and pKKhNP desired by the present invention by cloning the nucleoside psitoprotein gene into each of the plasmids pET-3a and pKK223-3 is shown in FIG. 4 and FIG. This is shown schematically in FIG.
  • the desired nucleocapsid protein is produced in large quantities by inserting each of the produced expression plasmids into host cells E. coli BL21 (DE3) and HB101 using an electric field impact method.
  • E. c01iBL (pEThNP) and E.co] iBL (pKKhNP) which can be used for E. coli, were deposited and deposited on August 18, 995, with the Korean Society for the Oncology of Korea. , Given a deposit number (KFCC-10865 and KFCC-10868) 0
  • the obtained Escherichia coli transformant is cultured in an appropriate medium to express a large amount of protein by a conventional method used in the field of gene engineering.
  • the expressed protein was purified by performing the illustrated procedure. Poria for purified protein
  • the results of acrylamide gel electrophoresis and Western plotting are shown in Figs. 7 and 8, and it can be confirmed that the purified protein is the target nucleocapsid protein of Hunter virus.
  • the purity of the purified protein judged from the results of electrophoresis was 95% or more.
  • the molecular weight of the nucleoside protein obtained from the expression plasmid pEThNP was measured at about 49.5 kDa, while the molecular weight of the protein obtained from pKK hNP was measured at about 48 kDa.
  • this is due to the s10 leader sequence located in front of the BamHI site, which is the plasmid cloning site of pET-3a used to construct pEThNP. That is, the nucleoside protein produced from pET h NP contains an additional 12 amino acids as compared to the protein in the natural state, and such a structural difference indicates that the protein production amount in the transformant is also large. It is thought to play a much larger role.
  • nucleoforce psid protein produced from E. coli transformed with pEThNP and pKKhNP by ⁇ ⁇ stamp lotting the amount of nucleoforce psid protein derived from pEThNP was 5% less than the amount of protein derived from PKKhNP. It is supported by the fact that there are many times t ⁇ .
  • the purified nucleoside protein is used as a vaccine stock solution, and aluminum hydroxide gel can be added as an auxiliary agent in the process of producing a final vaccine product from this stock solution, in addition to thimerosal and Purified gelatin can be added.
  • Aluminum hydroxide gel is powerful for use as an immunoadjuvant, and the preferred loading is up to 0.625 mg per purified stock solution for vaccine production, and thimerosal is 0.01% (w / v) as a preservative.
  • the power of use ⁇ preferred, the power of purified gelatin is preferably ⁇ 0.02% (wZv) as a stabilizer.
  • Tween 80 can be added in an amount of 0.01% (w / v).
  • the pectin product of the present invention produced from the nucleocapsid protein of the human Hanyu virus by the method described above, it is currently commercially available.
  • Hunterbucks was used as a comparative vaccine in a plaque reduction neutralization ⁇ experiment and confirmed that the vaccine of the present invention showed superior neutralizing ability compared to comparative pectin. / 8 No special abnormal symptoms were found even when vaccinated three times at 10-day intervals.
  • the vaccine according to the invention has been shown to be inactive or to show antigenicity in order to completely eliminate the basis for the occurrence of a safety accident due to the use of the poisoned virus itself as a pectin substance.
  • nucleocapsid protein Since a specific protein component, namely nucleocapsid protein, was mass-produced and purified by a genetic engineering method and used as a vaccine substance, it completely solved the stability problems that have been continuously raised in the vaccine for the prevention of hemorrhagic fever in symptomatic patients. It is considered to be an epoch-making invention that has been solved.
  • Example 1 Isolation of Hantaan virus from infected patients
  • Vero E 6 Vero E 6
  • 5 DMEM medium was added, and the cells were cultured at 37 ° C for 10 to 12 days.
  • the first subculture consists of inoculating a new T-125 culture flask packed with Vero E6 cells, which is packed with 150 £ of the culture from the already cultured flask, and repeating the above process. went.
  • the cells that have undergone the three subcultures in this manner are treated with trypsin, an IFA (Indirect Fluorescent Assay) is performed to test for the presence or absence of antigen, and the presence of the virus is confirmed. Used as material for separation. ,
  • the supernatant obtained here was combined with the supernatant, and then extracted once with a mixture of phenol Z-cloth form (1: 1) and I-time with cro-form form.
  • 1Z10 volume of 3 M sodium acetate and 2 volumes of isopropyl alcohol were added, mixed well, and stored at ⁇ 20 ° C. for 16 hours. This was centrifuged in a microcentrifuge at 12, OOO rpm for 15 minutes to precipitate, and the precipitate was washed twice with 70% ethyl alcohol and then dried. The dried precipitate was dissolved in 10 / £ of sterile water without ribonuclease (RNase) and stored at 4. Ribonucleic acid 5 Then, cDNA was synthesized in a total of 20 volumes, and the reaction conditions at this time were as shown in Table 1 below.
  • the primary reaction solution 5 was taken and subjected to a secondary polymerization enzyme chain reaction using primers NP2 and NP3.
  • the 5'-ends of the primers NP2 and NP3 used at this time contain the restriction enzyme recognition sites of Bg1II (AGATCT) and EcoRI (GAATTC), respectively.
  • the equipment used for the polymerase chain reaction was a Perkin Elmer
  • the conditions of the polymerase chain reaction were as shown in Table 2 below, using a siribonucleic acid temperature cycler (DNA thermal cycler 480).
  • Vector-1 PT7T3 18U and PT7T3 19U DNA were cut with restriction enzymes BglII and EcoRI as shown in FIG. 2 and then purely separated by an electric extraction method. These two DNAs (vector and amplified foreign gene) were reacted overnight at 15 in the presence of T4 DNA ligase to produce recombinant plasmid DNA (pThNP 8 and pThNP 9).
  • E. coli NM522 was transformed by the CaC12 transformation method (see: Molecular Cloning; Laboratory Manual, Co Id Spring Harbor).
  • the transformed Escherichia coli is cultured in a selection medium containing an antibiotic (ampicillin 50 ug / 8), selected, separated in a small amount of DNA, and then reacted with a restriction enzyme (Bg111, EcoRI) to obtain an accurate result. Only those strains containing the recombinant plasmid DNA containing the various amplified DNA sections were selected.
  • an antibiotic ampicillin 50 ug / 8
  • a restriction enzyme Bg111, EcoRI
  • Examples pThNP8 and pThNP9 obtained in 4 is added by Uni consisting respectively inserted and 30 minutes after cultured Escherichia coli are Ml 3 KO 7 helper phage (helper phage) 1 0 8 / above, the Ganamaishin then after 1 hour The mixture was added to 50 gZ and shake-cultured overnight.
  • the nucleocapsid protein gene for cloning into the known plasmids PET-3a and pKK223-3 according to the present invention was amplified by the polymerase chain reaction as described below.
  • the nucleoside psid protein gene to be cloned into the plasmid ⁇ -3a was obtained by the same method as described in Example 3; NP1, NP2 containing the Bg1II (AGATCT) recognition site and NP2 GAATTC) was produced through three rounds of amplification from three primers consisting of NP3 containing the recognition site.
  • the protein gene for cloning into plasmid pKK223-3 is replaced by primer NP containing an EcoRI (GAATTC) recognition site instead of primers ⁇ 2 and ⁇ 3 during the secondary polymerase chain reaction. Production was carried out in the same manner as in Example 3, except that NP5 containing 4 and Sa11 (GTCGAC) recognition site was used.
  • the cloning process of the nucleocapsid protein gene is shown in FIGS.
  • the two types of double-stranded DNA amplified by the method of Example 6 were confirmed for their size on agar gel electrophoresis, and then contained a 1.3 kb nucleoside peptide gene.
  • the DNA section thus obtained was recovered using a zinc kit.
  • the recovered DNA sections were cloned into plasmid pET-3a containing a part of the s10-derived amino acid sequence and into plasmid pKK223-3 containing no derived amino acid sequence.
  • the gene to be cloned into pET-3a in the collected DNA section was double digested with restriction enzymes Bg1II and Ec0RI, and the gene to be cloned into pKK223-3 was Klenow enzyme. in after making the processing to the both ends blunt-ended (blunt end the), was cut by using only E co RI, Fuwenoru / / black port Holm mixture (1: 1) was extracted once with the final Dissolved in 20 lb of sterile distilled water.
  • 5 g of the gene carrier pET-3a was double-digested with EcoRI and BamHI, and the same amount of pKK223-3 was double-digested with EcoRI and SmaI.
  • E. coli BL21 (DE3) was used for PEThNP, and E. coli HB101 was used for pKKhNP.
  • Escherichia coli BL2] (DE3) or HB101 was inoculated into a 2 Om LB medium from an agar plate medium and cultured with shaking at 37 ° C for 18 hours. Each of these was inoculated into one fresh LB medium and shake-cultured at 37 ° C until the absorbance at a wavelength of 600 nm reached 0.5 to 0.8. The culture was left at 0 ° C for 20 minutes to recover the cells, and the cells were collected by centrifugation. The cells were recovered by washing the cells once with one cold sterilized distilled water, then with 0.5, and finally with 20% glycerol.
  • Gene-Pulser has a capacitance of 25 F, a resistance of 200 ⁇ , Electric field strength 1 2.5 kV Zon, immobilized once with electric field and immediately applied to SOC medium (2% Bacto-tryptone, 0.5% yeast extract, lOmM NaCl, 2.5m KC 1, 1 OmM M gC 1 2 , 1 OmM MgS0 4, 2 OmM glucose) was added. After shaking for 1 hour at 37 ° C, spread each of the two LB agar plates supplemented with 50 zg / ampicillin at 0.lm £ and 0.
  • the method for purifying nucleoside psid proteins expressed from E. coli, including pEThNP and pKKhNP, is shown briefly in FIG.
  • the recovered cells were treated with 5 TE buffer solution (5 OmM
  • the cells were suspended in Tris, 1 mM EDTA, pH 8.0), and the cells were disrupted using an ultrasonic sonicator or French press. The degree of disruption was Bradford. Using a quantitative (Bradford assay) method, the procedure was performed until the protein concentration of the lysate was further increased. The crushed solution was centrifuged at 8,000 ⁇ g for 1 hour, and the supernatant was collected. Ammonium sulfate was added to the supernatant to a concentration of 30% of the saturation concentration, dissolved, left at room temperature for 1 hour, centrifuged at 8,000 X g for another 30 minutes, and the supernatant was discarded. Was resuspended in 20n6 TE buffer solution.
  • the eluate received here was diluted 5-fold with a 5 OmM Tris (pH 8.0) solution and dropped onto an immunoaffinity (i-thigh unoaffinity) column to which a single monoclonal antibody against nucleocapsid protein was bound.
  • PBS buffer solution 8 g NaC 1, 0. 2 g KC 1, 1. 44 g Na 2 HP0 4. 0. 24 g KH 2? 0, / i, pH 8. 0
  • 2 M Tris buffer solution (pH 8.0) was added 0.3 times the eluate volume.
  • a hybridoma was cultured and purified from the culture using a protein A column.
  • the hybridoma was purified from the human Hanyu virus, which was cultured in a mammalian mouse, and inoculated into valve C (Balb / c) mice. From this, spleen cells and myeloma cells were obtained, and then polyethylene glycol was obtained. This was made by fusing it and screening only the fused cells producing a single group antibody against the nucleocapsid protein in the fused cells.
  • binding of the purified monoclonal antibody was carried out by using the Cephalos 4B activated by CNBr of Pamacia according to the manufacturer's recommendation method.
  • the protein expressed from Escherichia coli is the nucleocapsid protein of Hunter virus
  • the protein expressed and purified from recombinant plasmid was subjected to mm electrophoresis on a polyacrylamide gel, and the protein was electrophoresed from the gel. Transfer to the membrane, 97/27302
  • the washed membrane is diluted appropriately and mixed with a secondary antibody (goat anti-humanlgG or goat anti-mouse IgG) to which a peroxidase enzyme is attached and a PBS solution containing 5% skim milk.
  • the reaction was shaken for a while. After washing the membrane three times with PBST solution for 5 minutes each, and performing a color reaction using 4-chloro-1-naphthol as a color reagent, a band specific to the desired nucleoside psid protein was obtained at the expected position. (Approximately 5 OKd) (see Fig. 8).
  • Example 12 Investigation of the efficacy of a vaccine produced using the purified nucleoside protein
  • An aluminum hydroxide gel as an auxiliary agent was added in a mixing ratio of 0.625 mg per 0.5 of the unpurified stock solution of the vaccine purified in Example 10, and the mixture was allowed to stand at 4 days for 15 days. Thereafter, 0.01% (w / v) thimerosal and 0.02% purified gelatin were added to produce the final test vaccine.
  • test vaccine In order to investigate the neutralizing efficacy of the test vaccine produced in this way, an experiment was conducted with guinea pigs, and Hanyuichi Bax of Green Cross Co., Ltd., which is currently sold, was used as a comparative pegutin. At this time, three different concentrations of test vaccines were prepared and inoculated into guinea-big so that the antigen amount became 10 gZ0.5m 20 g / 0.5 and 40 g / 0.57 ⁇ , and the vaccine was compared with the test vaccine. The immunogenicity of the vaccine was determined by examining the blood of Guineabig obtained after inoculation of the vaccine with Guineabig by a blackout neutralization test. The plaque reduction neutralization test proceeded as described below. 1) In order to obtain antibodies for the plaque reduction neutralization test, the above test vaccine and the comparative vaccine prepared at three concentrations were subcutaneously inoculated (0. vaccination) three times at 10-day intervals.
  • Serum was collected from Guinea big and immobilized at 56 for 30 minutes. Then, using a medium for fat and oil (MEM + M199 1: 1) containing 3% fetal bovine serum, the following steps were performed: ⁇ Diluted at 0, 1:20, 1:40, 1:80 and 1: 160.
  • a primary agar overlay medium (Agarose overlay) was separated for each culture vessel.
  • the composition of the primary agar overlay medium is as follows.
  • Ml 99 was used in a culture medium containing no fenol red, and fetal calf serum used was heat-treated at 56 ° C for 30 minutes.
  • each inoculated cell was cultured at 37 for 10 to 11 days.
  • the primary agar overlay medium of 3.5 m per culture vessel is separated, and the composition of the secondary agar overlay medium is as follows.
  • the purified nucleoside peptide was used! ⁇ In the case of the test vaccine produced, 40 jg 0 0.5? ⁇ And 20 ⁇ / At a concentration of 0.5, the neutralizing ability was superior to that of the comparative vaccine, and at a concentration of 10 g / 0.5, the neutralizing ability was equivalent. Therefore, the vaccine of the present invention, which is produced by mass-producing a nucleocapsid protein, which is one of the substances showing antigenicity in human-derived Hunter virus, by a genetic engineering method, has a higher hemorrhagic syndrome than the existing vaccine.
  • a single protein with a clear component is used without using the virus itself, which has not only excellent immunogenicity against heat but also highly likely to cause side effects such as toxic substances, as a vaccine substance. By using it as a vaccine substance, it is an excellent vaccine that has significantly improved the stability of vaccine preparations.

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Abstract

A nucleocapsid protein gene obtained by screening human-origin Hantaan virus strains and sequenced; an expression plasmid containing the above gene as a foreign gene; transgenic Escherichia coli prepared by transforming with the above plasmid; a nucleocapsid protein obtained by expressing a protein by culturing the above transformant and purifying the obtained protein; and a vaccine containing the above protein as the active ingredient for preventing hemorrhagic fever with renal syndrome.

Description

明 細 書 大腸菌から発現したヒト由来ハンターンウィルスのヌクレオカプシド 蛋白質を用いたワクチン  Description Vaccine using nucleocapsid protein of human-derived Hantaan virus expressed from Escherichia coli
技術分野  Technical field
この発明はヒト由来のハンターンウィルス菌株から選別したヌクレオカプシド蛋白質遺 伝子、 この遗伝子を含有する発現プラスミ ド、 発現プラスミ ドにより形質転換された大腸 菌形質転換体、 形質転換体を培養して蛋白質を発現し、 これを精製することによって得ら れるヌクレオ力プシド蛋白質及び、 該蛋白質を有効成分として含有するハンターンウィル スに対する予防ワクチンに関するものである。  The present invention comprises culturing a nucleocapsid protein gene selected from a human-derived Hunter virus strain, an expression plasmid containing the gene, an E. coli transformant transformed with the expression plasmid, and a transformant. The present invention relates to a nucleoside protein obtained by expressing and purifying a protein, and a prophylactic vaccine against Hunter virus containing the protein as an active ingredient.
背景技術  Background art
バーニアウィルス科のハンターウィルス属に厲するハンターンウィルスは、 腎症候出血 熱を誘発させる病原菌として、 三つのセグメント （それぞれ S, M, Lセグメント） から なる一本鎖リボ核酸ゲノム （R NA genome)を含有する。 この中で L切片 R NAは R NA 依存型 RNAポリマラーゼを暗号化し、 M切片 R NAは糖蛋白質の G 1及び G 2を暗号化 し、 S切片 R NAはヌクレオ力プシド N蛋白質を暗号化する力 <、 ここで G l、 G 2及びヌ クレオカプシド N蛋白質が免疫反応に関与しているものと知られている。  Hunter virus, a member of the genus Huntervirus of the Vernier virus family, is a single-stranded ribonucleic acid genome (RNA genome) consisting of three segments (S, M, and L segments, respectively), which cause renal symptomatic hemorrhagic fever. It contains. In this, the L-section RNA encodes RNA-dependent RNA polymerase, the M-section RNA encodes glycoproteins G1 and G2, and the S-section RNA encodes nucleoside N-protein. It is known that Gl, G2 and nucleocapsid N proteins are involved in the immune response.
ハン夕一ンウィルス感染に対する効果的な予防ワクチンを開発するため、 今までウィル ス自体またはこれら免疫反応に関与する蛋白質を用いた多数の研究が行われて来た。 しか し、 ウィルス自体を薬毒化または不活化して用いるのは通常効果面において優れたものと 知られている反面、 第一に、 種類によるウィルス培養自体が容易でなく、 培養液からのゥ ィルス精製が難しく、 精製中にゥィルスが破壊され完全なゥィルスを得られなし、場合が多 く、 第二に、 精製されたウィルスをワクチンとして使用するためにはホルマリンや熱処理 を介して薬毒化または不活化させなければならないが、 この過程でワクチン物質が変性す ることがあり、 第三に、 ワクチン製造において最も重要なものと言える安全性が充分でな い等の問題がある。 Numerous studies have been conducted on viruses themselves or on proteins involved in these immune responses to develop effective preventive vaccines against Han Yun virus infection. However, it is known that the use of a virus itself after poisoning or inactivating it is generally excellent in terms of effect. On the other hand, first, it is not easy to culture the virus depending on the type, and the virus from the culture solution is used. It is difficult to purify, and the virus is destroyed during purification, and complete virus is not obtained.In many cases, the purified virus is poisoned or immobilized via formalin or heat treatment for use as a vaccine. It must be activated, but this process can lead to denaturation of the vaccine material, and thirdly, the lack of safety, which is the most important factor in vaccine production. Problems.
即ち、 例えばインシユリンまたは成長ホルモン等のように大腸菌を用いて生産された既 存の遗伝子組換え蛋白質は大きな副作用無しに医薬品として広く用いているが、 1世代ハ ンターンウィルスワクチンの場合、 生きている鼠の脳にウィルスを接種培養した後、 嵐の 脳乳液からウィルスを精製するので、 いく ら精製をよくするとしても既に副作用の素地の あるものと知られている嵐脳のベイシックミエリン蛋白質 （bas i c myel i n protein) を完 全に除去することができなく、 その他分析されない鼠脳由来の蛋白質が微量包含されてい る場合もあるので、 これら動物由来外部蛋白質によってワクチンの安定性についての問題 が発生する可能性がある。  That is, existing recombinant protein produced using Escherichia coli, for example, inulin or growth hormone, is widely used as a drug without significant side effects. After inoculating and cultivating the virus in the rat brain, the virus is purified from the storm brain milk, so that no matter how much the purification is improved, the basic myelin protein of the storm brain, which is already known to have a substrate for side effects (Basic myel in protein) cannot be completely removed, and may contain trace amounts of other rat brain-derived proteins that are not analyzed. May occur.
従って、 細胞のない生産体制 （Cel l free production system)や遗伝子操作によって発 熱反応等を起こす可能性のある有害な物質及び蛋白質を包含しないように、 免疫蛋白質の 遺伝子のみを発現しょうとする努力が進行されて来た。  Therefore, to express only immune protein genes so as not to include harmful substances and proteins that may cause a thermogenic reaction or the like due to a cell-free production system or gene manipulation. Efforts have been made.
かかる努力の一環として、 Schraal john等は原型ハンターンウィルス 7 6 - 1 1 8株の各 セグメン卜に対する塩基配列及び遺伝子配列を明らかにし、 ハンターンウィルスの外皮蛋 白質 G 1及び G 2を暗号化している Mセグメントをワクシニア遣伝子と結合させ動物 （Ha mster)に注入した後、 ハンターンウィルスに対する防禦効果を観察し、 ヌクレオ力プシド 蛋白質を暗号化する Sセグメントは、 外皮蛋白質の防禦能を増加させるものと報告したこ と; ^ある。 更に、 これらはバクロウィルス （Baculovi rus)を用いてハンターンウィルスの G l、 G 2及びヌクレオ力プシド蛋白質を昆虫細胞において発現し、 この発現細胞を破砕 して得られた細胞溶解物で動物に接種することを試したが、 ハンターンウィルスのヌクレ ォカプシド蛋白質による直接的な中和抗体形成は観察できなかった。  As part of this effort, Schraal john et al. Clarified the nucleotide sequence and gene sequence for each segment of the prototype Hunter virus 761-118, and encoded the Hunter virus coat proteins G1 and G2. After binding the M segment to the vaccinia gene and injecting it into an animal (Hamster), observe the protective effect against Hunter virus and observe that the S segment, which encodes the nucleoside psid protein, has the ability to protect the coat protein. Reported that it would increase; In addition, they express Hl, G2, and nucleopsid proteins of Hunter virus in insect cells using baculovirus (Baculovirus), and express them to animals with cell lysate obtained by disrupting the expressing cells. Inoculation was attempted, but no direct neutralizing antibody formation by the Huntan virus nucleocapsid protein was observed.
このように腎症候出血熱予防ワクチンの製造において段々関心力〈高まっているヌクレオ 力プシド蛋白質は、 ウィルスのリボ核酸中 Sセグメントから発現してそれぞれのリボ核酸 と結合して存在するウィルス構造蛋白質であって、 ウィルス蛋白質全体の大部分を占める ので、 ウィルス培養後大量で精製することが容易であるという特性がある。 し力、し、 上記 言及の通り、 ヌクレオ力プシド蛋白質の中和能に関する研究としては、 遺伝子をべキシニ ァウィルスという媒介体を用いて付与するか、 あるいは発現したヌクレオカプシド蛋白質 を包含している全体細胞溶解物を動物に投与したの力〈全部であり、 純粋分離したヌクレオ 力プシド蛋白質を中和抗体形成研究に使用したことがないので、 ヌクレオカプシド蛋白質 による中和能、 特に中和抗体を形成して肾症候出血熱を予防することのできるワクチン物 質としての価値が判別できなかった。 更に、 ハンターンウィルスまたはハン夕一ウィルス に属する他のウィルスの外皮蛋白質 （G 1及び G 2 ) の場合にはこれら蛋白質が精製しが たいだけでなく、 大腸菌、 酵母または動物細胞において遺伝工学的方法で大量生産が不可 能であるので、 ウィルス自体をワクチン物質として使用する場合の数多い問題点にもかか わらず、 現在腎症候出血熱に対する予防ヮクチンとしては哺乳マウスの脳または動物細胞 においてウィルスを直接培養した後、 これをホルマリンで不活化させ製造したワクチンの みが供給されている実状である。 As described above, the interest in the production of a vaccine for preventing hemorrhagic fever with renal syndrome is increasing. Since it occupies most of the whole virus protein, it has the property that it is easy to purify it in large quantities after culturing the virus. Power, then, above As noted, studies on the ability to neutralize nucleocapsid proteins include gene transfer using a vector called vexinia virus or administration of whole cell lysates containing expressed nucleocapsid proteins to animals. Since it has not been used for research on the formation of neutralizing antibodies, the ability to neutralize nucleocapsid proteins, especially the formation of neutralizing antibodies, prevents hemorrhagic fever with symptomatic effects. The value as a vaccine substance that could be used could not be determined. Furthermore, in the case of the coat proteins (G1 and G2) of Hunter virus or other viruses belonging to Hanyuichi virus, these proteins are not only difficult to purify but also genetically engineered in E. coli, yeast or animal cells. In spite of the numerous problems of using the virus itself as a vaccine substance, the prophylactic tract against renal symptomatic hemorrhagic fever is currently present in the brain or animal cells of mammals, despite the numerous problems of using the virus itself as a vaccine substance. In fact, only vaccines produced by directly culturing and inactivating them with formalin are supplied.
発明の開示  Disclosure of the invention
本発明者らは現在の不活化ウィルスワクチンによる安定性の問題を解決するため集中的 な研究を行った結果、 今まで純粋分離して使用されたことのないヌクレオ力プシド蛋白質 を遣伝工学的方法によつて大腸菌から大量生産し、 これをハンターンウィルス予防ワクチ ンの成分として使用すると臂症候出血熱に対しより安全であり予防効果の優れたワクチン が開発できることを見出した。  The present inventors have conducted intensive research to solve the stability problem caused by the inactivated virus vaccine, and as a result, have been able to transfer a nucleoside protein that has never been used in pure isolation. It was found that large-scale production of Escherichia coli by this method and its use as a component of a vaccine for the prevention of Hunter's virus could lead to the development of a vaccine that is safer and more effective in preventing hemorrhagic fever.
更に、 ハンターンウィルス感染に対する予防ワクチン技術分野において、 今までは主に 野鼠を宿主とした公知のハンターンウィルスをその研究対象として用いたが、 本発明では ヒトに対する免疫性の増加によりワクチンとしての効率性を高めることができるとの可能 性に基づいて、 感染された患者の血液より分離したヒト由来のハンターンウィルス （cl i n ical i solate) を出発物質として使用することによって、 優れた結果が得られた。  Furthermore, in the field of preventive vaccine against Hunter's virus infection, a well-known Hunter's virus, mainly using a wild mouse as a host, has been used as a research object. Based on the potential for increased efficiency, superior results have been obtained by using as a starting material a clinical isoleate of human origin (clinical isolate) isolated from the blood of infected patients. Obtained.
即ち、 本発明者らは、 本発明の目的によってヒト由来のハンターンウィルスを構成して いる多様な蛋白質の抗原性を研究し、 その結果、 ヒト由来ハンターンウィルス菌株から内 皮蛋白質、 即ちヌクレオ力プシド蛋白質の适伝子を選別してその塩基配列を分析し、 大腸 菌内で発現した後、 発現した蛋白質を分離、 精製し、 純粋精製された蛋白質の中和抗体形 成能を研究することによって、 ヮクチン物質としての価値を確認した。 That is, the present inventors studied the antigenicity of various proteins constituting human-derived huntan virus for the purpose of the present invention, and as a result, The protein of the skin protein, ie, the nucleoside protein, is selected, its nucleotide sequence is analyzed, expressed in E. coli, the expressed protein is separated and purified, and the neutralized antibody form of the purified protein is purified. By studying the ability to grow, we confirmed its value as a pectin substance.
本発明の目的は、 本発明者らによってその塩基配列が明らかになったヒト由来ハンター ンウィルスのヌクレオカプシド蛋白質遺伝子を提供するものである。  An object of the present invention is to provide a nucleocapsid protein gene of a human-derived hunter virus whose base sequence has been clarified by the present inventors.
本発明はまた、 ヌクレオカプシド蛋白質遺伝子を外来遗伝子として含有する組換え発現 プラスミ ド、 上記発現プラスミ ドによって形質転換された大腸菌形質転換体及び、 この形 質転換体を適当な培地で培養し、 分離及び精製過程を経て得られたヒト由来ハンターンゥ ィルスのヌクレオカプシド蛋白質に関するものである。  The present invention also provides a recombinant expression plasmid containing a nucleocapsid protein gene as a foreign gene, an Escherichia coli transformant transformed with the expression plasmid, and culturing the transformant in an appropriate medium. The present invention relates to a human-derived nucleocapsid protein of Hunter's virus obtained through a separation and purification process.
結局、 本発明は上記のような遺伝工学的方法によって純粋に得られたヒト由来ハンター ンウィルスのヌクレオカブシド蛋白質を有効成分として含有する腎症候出血熱予防ワクチ ンを提供することを目的とする。  After all, an object of the present invention is to provide a vaccine for preventing hemorrhagic fever with nephropathy, which contains, as an active ingredient, a nucleocabside protein of a human-derived hunter virus obtained purely by the genetic engineering method as described above. .
図面の簡単な説明  BRIEF DESCRIPTION OF THE FIGURES
図 1はヒト由来ハンターンウィルスのヌクレオカブシド蛋白質遗伝子を合成増幅するた めのプライマーの塩基配列を示したものである。  Fig. 1 shows the nucleotide sequence of a primer for synthesizing and amplifying the nucleocapsid protein gene of human-derived Hunter virus.
図 2はヒト由来ハンターンウィルスのヌクレオカプシド蛋白質遺伝子を包含した組換え 遺伝子運搬体である pThNP 8及び pThNP 9の製造方法を図式化して示したもので める。  FIG. 2 schematically shows a method for producing pThNP8 and pThNP9, which are recombinant gene carriers containing the nucleocapsid protein gene of the human-derived Hunter virus.
図 3は Aはヒト由来ハンターンウィルスのヌクレオカプシド蛋白質遺伝子の塩基配列を 公知のハン夕一ンウィルス 76— 1 18と比較して示したものであり、 Bは類推されたァ ミノ酸配列をハンターンウィルス 76- 1 18と比較して示したものである。  Figure 3A shows the nucleotide sequence of the nucleocapsid protein gene of the human-derived Hunter virus compared to the known Hans Equine virus 76-118, and B shows the analogous amino acid sequence of the hunter virus. This is shown in comparison with virus 76-118.
図 4は発現プラスミ ド pEThNPの製造過程を図式化して示したものである。  Figure 4 shows a schematic representation of the production process for the expression plasmid pEThNP.
図 5は発現ブラスミ ド pKKhNPの製造過程を図式化して示したものである。  FIG. 5 shows a schematic representation of the production process of the expression plasmid pKKhNP.
図 6は p EThNPまたは pKKhNPを含有する大腸菌からそれぞれ発現したヌクレ ォカプシド蛋白質の精製方法を示したものである。 図 7は大腸菌形質転換体 E. c o l i BL (pEThNP) から発現したヌクレオ力 プシド蛋白質の精製段階別純度を示すポリアタリルァミ ドゲル電気泳動写真である。 図 8は大腸菌から精製されたヌクレオカブシド蛋白質を確認するためにヒト患者血液と 単一クローン抗体を用いてゥヱスタンプロッティングを行った結果を示したものである。 発明を実施するための最良の形態 FIG. 6 shows a method of purifying a nucleocapsid protein expressed from Escherichia coli containing pEThNP or pKKhNP, respectively. FIG. 7 is a photograph of polyatarylamide gel electrophoresis showing the purity of nucleosides expressed by E. coli BL (pEThNP) in each purification step. Figure 8 shows the results of ゥ ヱ stamp lotting using human patient blood and a monoclonal antibody to confirm the nucleocapsid protein purified from E. coli. BEST MODE FOR CARRYING OUT THE INVENTION
本発明では、 まずヒト由来ハン夕一ンウィルス （clinical isolate) のヌクレオカプシ ド蛋白質をコ一ディングする遺伝子配列を明らかにするためにハンターンウィルスに感染 された患者の血液から感染源であるハンターンウイルスを分離し、 このウィルスから R N Aゲノムを分離した。  In the present invention, first, in order to elucidate the gene sequence coding for the nucleocapsid protein of human-derived Han isolate virus (clinical isolate), the infection source, Hunter virus, was determined from the blood of a patient infected with Hunter virus. The virus was isolated and the RNA genome was isolated from this virus.
逆転写酵素による相補 DN A合成と重合酵素連鎖反応 （Polymerase Chain Reaction)に よる遺伝子増幅をためには、 まず Schiraaljohnと Dalrymple (1983) によって既に明らか になった遺伝子両末端の塩基配列を基礎として 1 7個、 22個、 そして 23個からなるプ ライマー （図 1, NP 1. NP 2及び NP 3) を DNA合成器 （Applied Biosystems, In c.， Model 381A) を用いて合成した。 合成した 5 '末端プライマーには制限酵素 B g 1 I 1, 3 '末端には制限酵素 E c 0 R I認識部位を有するようにし、 バクテリアとファージ の複製オリジンを全部有したファージミ ド（phagemid)ベクターの Bg 1 I I、 E c oR I 場所に結合させた。 これを大腸菌 NM 522で形質転換させた後、 ヌクレオカプシド蛋白 質の遗伝子を有した組換えベクターを単離して確認し、 ヘルパーファージ （helper phage ) Ml 3 KO 7を用いて一本鎖 DNAを分離することによって、 遺伝子塩基配列決定の実 験材料として使用した。  In order to perform complementary DNA synthesis by reverse transcriptase and gene amplification by Polymerase Chain Reaction, the base sequence at both ends of the gene, which was already revealed by Schiraaljohn and Dalrymple (1983), was used as a basis. Primers consisting of 7, 22, and 23 primers (Fig. 1, NP 1. NP 2 and NP 3) were synthesized using a DNA synthesizer (Applied Biosystems, Inc., Model 381A). A phagemid vector containing a restriction enzyme Bg1I1 and a restriction enzyme Ec0RI recognition site at the 3 'end of the synthesized 5'-end primer and all replication origins of bacteria and phages. Bg1II, EcoRI site. After transforming this with E. coli NM522, the recombinant vector having the nucleocapsid protein gene was isolated and confirmed, and the single-stranded DNA was transformed using helper phage Ml3KO7. By separation, it was used as an experimental material for gene sequencing.
ヌクレオカプシド蛋白質全体に対して分析された塩基配列及び類推されたァミノ酸配列 は図 3の A及び Bに示した通りであり、 これから本発明によってヒ卜由来のハンターンゥ ィルスから分離したヌクレオカブシド遗伝子の塩基配列は、 公知の 76— 1 1 8ウィルス の該当遗伝子塩基配列と比較する時 95 %の類似性を示し、 ァミノ酸配列は 98. 1 %の 類似性を示すことがわかる。 このように変化された塩基配列を有するヌクレオカプシド蛋白質遺伝子から、 本発明が 目的とする効果的であり安全なハン夕一ンウィルス予防ワクチンを製造するためには、 ま ず外来遺伝子の増幅、 発現プラスミ ド及び形質転換体の製造、 形質転換体の培養及びヌク レオカプシド蛋白質の分離及び精製過程を経なければならないし、 最終的に製造されたヮ クチンの効能性を調査することによつて発明が完成できる。 The nucleotide sequence analyzed for the entire nucleocapsid protein and the deduced amino acid sequence are as shown in FIGS. 3A and 3B. From this, the nucleocapsid gene isolated from human-derived hunter virus according to the present invention is shown. It can be seen that the nucleotide sequence of the nucleotide sequence is 95% similar to that of the known gene sequence of the known 76-11 virus, and the amino acid sequence is 98.1% similar. From the nucleocapsid protein gene having the altered nucleotide sequence, to produce an effective and safe vaccine against Hansin virus aimed at by the present invention, first, amplification and expression of a foreign gene must be carried out. Production process, culture of the transformant, and isolation and purification of the nucleocapsid protein.The invention is completed by investigating the efficacy of the finally produced actin. it can.
即ち、 本発明では、 図 1に示した 5種のプライマーを使用して重合酵素連鎖反応を行う ことによって、 プラスミ ドにクローニングするに十分な量のヌクレオカブシド蛋白質遗伝 子を確保し、 これら遺伝子にそれぞれプラスミ ド pET— 3 a及び pKK 223— 3にク ローニングできるよう適切な制限酵素認識部位を添加した。 ここで、 プラスミ ド pET— 38は510誘導アミノ酸配列の一部を包含するので、 このプラスミ ドにクローニングさ れ発現したヌクレオ力プシド蛋白質は N末端に更に 12個のアミノ酸を含有した融合蛋白 質に発現する反面、 プラスミ ド pKK 223— 3にはこのような配列がないので融合蛋白 質でな 、元来の蛋白質形態に発現するようになる。  That is, in the present invention, by performing the polymerase chain reaction using the five types of primers shown in FIG. 1, a sufficient amount of the nucleocapsid protein に gene to be cloned into plasmid was obtained. Appropriate restriction enzyme recognition sites were added to the gene so that it could be cloned into plasmids pET-3a and pKK223-3, respectively. Here, since plasmid pET-38 contains a part of the 510-derived amino acid sequence, the nucleoside protein cloned and expressed in this plasmid is converted into a fusion protein containing an additional 12 amino acids at the N-terminus. On the other hand, since plasmid pKK223-3 does not have such a sequence, it is expressed as an original protein, not as a fusion protein.
プラスミ ド pET-3 a及び pKK 223 - 3それぞれにヌクレオ力プシト蛋白質遺伝 子をクロ一ニングして、 本発明が目的とする発現プラスミ ド pEThNP及び pKKhN Pを製造するための過程は、 図 4及び図 5に図式化して示した通りである。 また、 製造さ れたそれぞれの発現プラスミ ドを電気場衝撃法を使用して宿主細胞である大腸菌 BL 21 (DE 3)及び HB 101内に挿入することによって、 所望するヌクレオカプシド蛋白質 を大量で生産することのできる大腸菌形質転換体 E. c 01 i BL (pEThNP)及 び E. c o】 i BL (pKKhNP) を得、 これらを I 995年 8月 18日付で社団法 人韓国腫菌協会に寄託し、 寄託番号が与えられた （KFCC— 10865及び KFCC - 10868) 0 The process for producing the expressed plasmids pEThNP and pKKhNP desired by the present invention by cloning the nucleoside psitoprotein gene into each of the plasmids pET-3a and pKK223-3 is shown in FIG. 4 and FIG. This is shown schematically in FIG. In addition, the desired nucleocapsid protein is produced in large quantities by inserting each of the produced expression plasmids into host cells E. coli BL21 (DE3) and HB101 using an electric field impact method. E. c01iBL (pEThNP) and E.co] iBL (pKKhNP), which can be used for E. coli, were deposited and deposited on August 18, 995, with the Korean Society for the Oncology of Korea. , Given a deposit number (KFCC-10865 and KFCC-10868) 0
次の段階で、 本発明では遗伝工学分野で使用される通常の方法によって、 得られた大腸 菌形質転換体を適当な培地で培養して蛋白質を大量で発現し、 図 6に具体的に図示した手 続きを行うことによって、 発現した蛋白質を精製した。 精製された蛋白質に対するポリア クリルアミ ドゲル電気泳動及びウエスタンプロッティングを行った結果は、 図 7及び図 8 に示した通りであるが、 これから精製された蛋白質が目的とするハンターンウィルスのヌ クレオカプシド蛋白質であることが確認でき、 特に電気泳動結果から判断された精製され た蛋白質の純度は 95%以上であった。 In the next step, according to the present invention, the obtained Escherichia coli transformant is cultured in an appropriate medium to express a large amount of protein by a conventional method used in the field of gene engineering. The expressed protein was purified by performing the illustrated procedure. Poria for purified protein The results of acrylamide gel electrophoresis and Western plotting are shown in Figs. 7 and 8, and it can be confirmed that the purified protein is the target nucleocapsid protein of Hunter virus. In particular, the purity of the purified protein judged from the results of electrophoresis was 95% or more.
一方、 発現プラスミ ド pEThNPから得られたヌクレオ力プシド蛋白質の分子量は約 49. 5kDaに測定された反面、 p KK hNPから得られた蛋白質の分子量は約 48 k Daに測定されたが、 これは上記で言及した通り、 pEThNPの製作に使用された pE T- 3 aのプラスミ ドクローニング部位である B amH I部位前方に存在する s 10リー ダー （leader)配列に起因するものである。 即ち、 p E T h N Pから生産されたヌクレオ 力プシド蛋白質は天然状態の蛋白質に比べて更に 12個のアミノ酸を含有しており、 かか る構造上の差異は形質転換体においての蛋白質生産量も遙に増大させる役割をするものと 考えられる。 これは pEThNP及び pKKhNPによりそれぞれ形質転換された大腸菌 から生産されたヌクレオ力プシド蛋白質をゥヱスタンプロッテイングで分析してみた結果 、 pEThNP由来のヌクレオ力プシド蛋白質量が PKKhNPに由来の蛋白質量より 5 倍程多 tゝ事実から裏付けられる。  On the other hand, the molecular weight of the nucleoside protein obtained from the expression plasmid pEThNP was measured at about 49.5 kDa, while the molecular weight of the protein obtained from pKK hNP was measured at about 48 kDa. As mentioned above, this is due to the s10 leader sequence located in front of the BamHI site, which is the plasmid cloning site of pET-3a used to construct pEThNP. That is, the nucleoside protein produced from pET h NP contains an additional 12 amino acids as compared to the protein in the natural state, and such a structural difference indicates that the protein production amount in the transformant is also large. It is thought to play a much larger role. As a result of analyzing nucleoforce psid protein produced from E. coli transformed with pEThNP and pKKhNP by ゥ ヱ stamp lotting, the amount of nucleoforce psid protein derived from pEThNP was 5% less than the amount of protein derived from PKKhNP. It is supported by the fact that there are many times t ゝ.
精製されたヌクレオ力プシド蛋白質をワクチンの精製原液とし、 この精製原液から最終 ワクチン製品を製造するための過程において、 補助剤としてアルミニウムヒドロキサイ ド ゲルを添加することができ、 その以外にもチメロサール及び精製ゲラチンを添加すること ができる。 アルミニウムヒドロキサイドゲルは免疫補助剤用として使用する力く、 好ましい 添加量はワクチン製造用精製原液 0. 当たり最大 0. 625mgであり、 チメロサール は保存剤として 0. 01 % (w/v)量で使用するの力 <好ましく、 精製ゼラチンは安定化 剤として 0. 02% (wZv) の量で使用するの力 <好ましい。 この以外にも Tween 8 0を 0. 01% (w/v) の量で添加することができる。  The purified nucleoside protein is used as a vaccine stock solution, and aluminum hydroxide gel can be added as an auxiliary agent in the process of producing a final vaccine product from this stock solution, in addition to thimerosal and Purified gelatin can be added. Aluminum hydroxide gel is powerful for use as an immunoadjuvant, and the preferred loading is up to 0.625 mg per purified stock solution for vaccine production, and thimerosal is 0.01% (w / v) as a preservative. The power of use <preferred, the power of purified gelatin is preferably <0.02% (wZv) as a stabilizer. Besides this, Tween 80 can be added in an amount of 0.01% (w / v).
以上説明の方法によってヒト由来ハン夕一ンウィルスのヌクレオカプシド蛋白質から製 造された本発明のヮクチン製品効能性を調査するため、 現在市販して t、る株式会社緑十字 のハンターバックスを比較ワクチンとしてプラーク減少中和^法で実験した結果、 本発 明のワクチンが比較ヮクチンに比べて卓越した中和能力を示すことを確認し、 ワクチン効 能テストのためギニァビグに 40/ 8ずっ1 0日間隔で 3回接種した場合においても特別 な異常症状は発見されなかった。 更に、 本発明によるワクチンは不活化させるか、 あるい は薬毒化ゥィルス自体をヮクチン物質で使用することによる安全事故発生の素地を完全に 排除するため、 それから抗原性を示すものとして明らかになった特定の蛋白質成分、 即ち ヌクレオカプシド蛋白質を遗伝工学的方法で大量生産及び精製してワクチン物質として使 用したので、 肾症候出血熱予防ワクチンにおいて今まで絶え間なく提起されて来た安定性 問題を完全に解決した画期的な発明と考えられる。 In order to investigate the efficacy of the pectin product of the present invention produced from the nucleocapsid protein of the human Hanyu virus by the method described above, it is currently commercially available. Hunterbucks was used as a comparative vaccine in a plaque reduction neutralization ^ experiment and confirmed that the vaccine of the present invention showed superior neutralizing ability compared to comparative pectin. / 8 No special abnormal symptoms were found even when vaccinated three times at 10-day intervals. Furthermore, the vaccine according to the invention has been shown to be inactive or to show antigenicity in order to completely eliminate the basis for the occurrence of a safety accident due to the use of the poisoned virus itself as a pectin substance. Since a specific protein component, namely nucleocapsid protein, was mass-produced and purified by a genetic engineering method and used as a vaccine substance, it completely solved the stability problems that have been continuously raised in the vaccine for the prevention of hemorrhagic fever in symptomatic patients. It is considered to be an epoch-making invention that has been solved.
以下、 本発明を下記実施例により更に詳しく説明する。 しかし、 これは本発明の理解を 助けるものであり、 本発明の範囲がこれらの実施例によって制限されるものではない。 実施例  Hereinafter, the present invention will be described in more detail with reference to the following examples. However, this aids the understanding of the present invention, and the scope of the present invention is not limited by these examples. Example
実施例 1 ：感染患者からのハンターンウィルス分離  Example 1: Isolation of Hantaan virus from infected patients
背症候群出血熱症状の患者 （男性， 32歳） から採取した血液 1 00 £を T— 25培 養フラスコ一杯なるよう生長したベロ E 6 (Ve r o E 6 ) 細胞に接種した。 T— 25 フラスコ内で宿主細胞にゥィルスが良く吸着するように 2時間培養した後、 5 の D M E M培地を添加し、 1 0乃至 1 2日間 37 °Cで培養した。 一番目の継代培養は、 ベロ E 6紬 胞がぎっしりと詰まった新しい T一 25培養フラスコに既に培養したフラスコから 1 50 £の培養液を取って接種した後、 上記の過程を反復して行った。 このように三回の継代 培養を経た細胞をトリブシン処理し、 抗原存在可否試験のための I FA (Indirect Fluor escent Assay) を行ってウィルス存在を確認した後、 培養液を収集して RNAゲノム分離 の材料として使用した。 ,  One hundred pounds of blood from a patient with back syndrome hemorrhagic fever (male, 32 years old) was inoculated into Vero E 6 (Vero E 6) cells grown to fill a T-25 culture flask. After culturing for 2 hours in a T-25 flask so that the virus was well adsorbed to the host cells, 5 DMEM medium was added, and the cells were cultured at 37 ° C for 10 to 12 days. The first subculture consists of inoculating a new T-125 culture flask packed with Vero E6 cells, which is packed with 150 £ of the culture from the already cultured flask, and repeating the above process. went. The cells that have undergone the three subcultures in this manner are treated with trypsin, an IFA (Indirect Fluorescent Assay) is performed to test for the presence or absence of antigen, and the presence of the virus is confirmed. Used as material for separation. ,
実施例 2 ：ハンターンウィルス RNAゲノムの精製及び c DNAの合成  Example 2: Purification of Hantaan virus RNA genome and cDNA synthesis
患者から分離して Ve r 0 E 6細胞において増殖されたハン夕一ンウィルス培養液 1 00 を取って、 溶液 A ( 4. 2M Guanidine isot iocyanate, 25mM Tr i s— W Take Han Han virus 100 culture separated from the patient and grown in Ver0E6 cells, and add solution A (4.2 M Guanidine isot iocyanate, 25 mM Tris- W
HC 1 (pH 8. 0) , 0. 5 % Sarkosyl, 0. 7 % yS-mercaptoethanol) 400 と よく混合した。 ここに、 溶液 B (1 Tris-HCl(pH8.0), 0.1M EDTA, 10¾ SDS) 50 ^を 加えてよく混合した後、 65°Cで 5分間溶解した。 混合溶液に同一容積のフヱノール ク ロロホルム （1 : 1) 混合液を加えて 65'Cで 30分間放置した後、 遠心分離して上澄液 のみを取り、 残りの溶液に更に溶液 Aを 30 加えて 65 °Cで 5分間放置した後、 遠 心分離した。 ここで取った上澄液を先の上澄液と合わせた後、 これをフエノール Zクロ口 ホルム （1 : 1) 混合液で 1回、 クロ口ホルムで I回抽出した。 抽出した溶液に 1Z1 0 容積の 3 M酢酸ナトリウム及び 2倍容積のイソプロピルアルコールを加えてよく混合した 後、 — 20°Cで 1 6時間保管した。 これをマイクロ遠心分離器で 1 2, O O O r pmで 1 5分間遠心分離して沈殿させ、 沈殿を 70%エチルアルコールで 2回洗浄した後乾燥した 。 乾燥した沈殿をリボ核酸分解酵素 (RNa s e) のない滅菌水 1 0 /£に溶解した後 4 で保管した。 リボ核酸 5
Figure imgf000011_0001
を取って全体 20 容積において cDNAを合成したが 、 この時の反応条件は下記表 1の通りであり、 37てで 1時間反応させた。 Mix well with HC 1 (pH 8.0), 0.5% Sarkosyl, 0.7% yS-mercaptoethanol) 400. 50 B of solution B (1 Tris-HCl (pH 8.0), 0.1 M EDTA, 10 ° SDS) was added thereto, mixed well, and dissolved at 65 ° C. for 5 minutes. Add the same volume of phenol chloroform (1: 1) to the mixed solution, leave it at 65'C for 30 minutes, centrifuge it, take only the supernatant, and add 30 more solution A to the remaining solution. And left at 65 ° C for 5 minutes, followed by centrifugation. The supernatant obtained here was combined with the supernatant, and then extracted once with a mixture of phenol Z-cloth form (1: 1) and I-time with cro-form form. To the extracted solution, 1Z10 volume of 3 M sodium acetate and 2 volumes of isopropyl alcohol were added, mixed well, and stored at −20 ° C. for 16 hours. This was centrifuged in a microcentrifuge at 12, OOO rpm for 15 minutes to precipitate, and the precipitate was washed twice with 70% ethyl alcohol and then dried. The dried precipitate was dissolved in 10 / £ of sterile water without ribonuclease (RNase) and stored at 4. Ribonucleic acid 5
Figure imgf000011_0001
Then, cDNA was synthesized in a total of 20 volumes, and the reaction conditions at this time were as shown in Table 1 below.
表 1. ハンターンウィルス cDNA合成条件  Table 1. Hunter virus cDNA synthesis conditions
Tr i s - HC】 （pH8. 4) 1 OmM Tris-HC] (pH8.4) 1 OmM
KC 1 5 OmM  KC 15 OmM
MgC 12 7mM MgC 1 2 7mM
dNTP s 1 mM  dNTPs 1 mM
. RNa s e抑制剤 1 Hi  RNa se inhibitor 1 Hi
ランダムプライマー 50 pmol  50 pmol random primer
AMV逆転写酵素 12 Units 実施例 3 ：塩基配列決定用遗伝子増幅  AMV reverse transcriptase 12 Units Example 3: Gene amplification for nucleotide sequence determination
DN A重合酵素による塩基配列決定用遗伝子増幅のためには、 図 1に示した 5種類のプ ライマ中、 それぞれ 1 7、 23及び 22me r長さの NPし NP 2及び NP 3を使用し た。 即ち、 実施例 2で合成したヌクレオ力ブンドの cDNAが包含された溶液 5 を取 つてプライマ一 NP 1及び NP 3を使用してヌクレオ力プシド遺伝子を 1次増幅させた。 For amplification of DNA for DNA sequencing using DNA polymerase, the five types of primers shown in Figure 1 were used. In the lyma, NPs of lengths 17, 23 and 22 mer, respectively, NP2 and NP3 were used. That is, the solution 5 containing the cDNA of the nucleoforce band synthesized in Example 2 was used to perform primary amplification of the nucleoside gene using primers NP1 and NP3.
1次反応した溶液中には所望するヌクレオカプシド遗伝子が十分でないので、 1次反応溶 液 5 を取ってプライマ一 NP 2及び NP 3を使用して 2次重合酵素連鎖反応を行った 。 この時使用したプライマ一 NP 2及び NP 3の 5 '末端にはそれぞれ Bg 1 I I (AG ATCT) 及び Ec oR I (GAATTC) の制限酵素認識部位が包含されている。 重合酵素連鎖反応に使用された機器は、 パーキンエルマ一社 （PERKIN ELMER) のデォキ Since the desired nucleocapsid gene was not sufficient in the solution subjected to the primary reaction, the primary reaction solution 5 was taken and subjected to a secondary polymerization enzyme chain reaction using primers NP2 and NP3. The 5'-ends of the primers NP2 and NP3 used at this time contain the restriction enzyme recognition sites of Bg1II (AGATCT) and EcoRI (GAATTC), respectively. The equipment used for the polymerase chain reaction was a Perkin Elmer
O C  O C
シリボ核酸温度サイクラ— （DNA thermal cycler 480)を使用し、 重合酵素連鎖反応の 条件は下記の表 2に示した通りである。 The conditions of the polymerase chain reaction were as shown in Table 2 below, using a siribonucleic acid temperature cycler (DNA thermal cycler 480).
表 2. 重合酵素連鎖反応溶液組成及び遂行手続き  Table 2. Polymerase chain reaction solution composition and performance procedure
Tr i s-HC】 1 OmM Tr s-HC] 1 OmM
KC 1 5 OmM  KC 15 OmM
反応組成 MgC 12 2. 5mM  Reaction composition MgC 12 2.5 mM
4 dNTP s 0. 2mM  4 dNTP s 0.2mM
プライマー 50 pmol  Primer 50 pmol
Ta q重合酵素 2. 5 Units  Ta q Polymerase 2.5 Units
92°C ( 1 5 ) →46°C (2^·) →72で （3分） 20回反復 遂行手続き 92°C (1 5秒） → 46。C ( 2分） →Ί 2て (3.5分） 1 5回反復 92 ° C (15) → 46 ° C (2 ^ ·) → 72 (3 minutes) 20 repetitions Execution procedure 92 ° C (15 seconds) → 46. C (2 minutes) → Ί2 (3.5 minutes) 1 Repeat 5 times
92。C (1 5秒:) → 46°C ( 2分;) →72°C (10分） 1回 実施例 4 ：増幅された ¾伝子のクローニング  92. C (15 seconds :) → 46 ° C (2 minutes;) → 72 ° C (10 minutes) 1 time Example 4: Cloning of amplified ¾ gene
実施例 3の方法によつて増幅された一本鎖 D N Aの大きさを寒天ゲル電気泳動上で確認 した後、 電気抽出方法によりゲルから抽出した。 抽出した DNAに蛋白質分解酵素 （Prot einaseK)を 100 g/ となるように添加し 37°Cで 30分間反応させた後、 70 °C で 15分間反応させ酵素を不活性化した。 増幅された D N Aの両方末端の制限酵素認識部 位をそれぞれ Bg l I I及び E c oR Iで切断した後、 フヱノール Zクロ口ホルム抽出に よつて切断された増幅 D N Aを純粋に分離した。 Confirm the size of single-stranded DNA amplified by the method of Example 3 on agar gel electrophoresis. After that, the gel was extracted from the gel by an electric extraction method. Proteolytic enzyme (Proteinase K) was added to the extracted DNA at a concentration of 100 g / l and reacted at 37 ° C for 30 minutes, and then reacted at 70 ° C for 15 minutes to inactivate the enzyme. After restriction sites at both ends of the amplified DNA were cleaved with BglII and EcoRI, respectively, the amplified DNA which had been cleaved by extraction with phenol Z-cloth form was purely separated.
—方、 ベクタ一 PT7T3 18U及び PT7T3 1 9U DNAを図 2に示した通 り制限酵素 Bg l I I及び Ec oR Iで切断した後、 電気抽出法により純粋分離した。 こ れら 2種類の DN A (ベクタ一及び増幅された外来遺伝子） を T 4 DN Aリガーゼ存在下 に 15でで一夜反応させて組換えプラスミ ド DNA (pThNP 8及び pThNP 9) を 製造した後、 CaC 12 形質転換法 （参照： Molecular Cloning ； Laboratory Manual, Co Id Spring Harbor) によって大腸菌 NM 522に形質転換させた。 形質転換された大腸菌 を抗生剤 （アムピシリン 50 ug/ 8) を包含する選択培地で培養して選別し、 DNAを 少量分離した後、 制限酵素 (Bg 1 1 1, Ec oR I) を反応させ正確な増幅 DNA切片 が包含された組換えブラスミ ド D N Aが入っている菌株のみを選別した。  On the other hand, Vector-1 PT7T3 18U and PT7T3 19U DNA were cut with restriction enzymes BglII and EcoRI as shown in FIG. 2 and then purely separated by an electric extraction method. These two DNAs (vector and amplified foreign gene) were reacted overnight at 15 in the presence of T4 DNA ligase to produce recombinant plasmid DNA (pThNP 8 and pThNP 9). E. coli NM522 was transformed by the CaC12 transformation method (see: Molecular Cloning; Laboratory Manual, Co Id Spring Harbor). The transformed Escherichia coli is cultured in a selection medium containing an antibiotic (ampicillin 50 ug / 8), selected, separated in a small amount of DNA, and then reacted with a restriction enzyme (Bg111, EcoRI) to obtain an accurate result. Only those strains containing the recombinant plasmid DNA containing the various amplified DNA sections were selected.
実施例 5 ：遗伝子塩基配列の決定  Example 5: Determination of gene base sequence
実施例 4で得た pThNP8及び pThNP9がそれぞれ挿入されている大腸菌を培養 した 30分後に Ml 3 KO 7ヘルパーファージ （helper phage) を 1 08 / 以上なるよ うに添加し、 それから 1時間後にガナマイシンを 50 gZ となるように添加して一夜 振蕩培養した。 PEGZNaC 1 (20%PEG 6000/2M NaC】） を用いた一 本鎖 DNA分離方法 （参照： Molecular Cloning ； A Laboratory Manual, Cold Spring Ha rbor, 1982) によって分離された一本鎖 DNAを材料とし、 Dideoxy Sequencing Kit (U SB社） から提供する方法により塩基配列を決定した。 決定された塩基配列から類推され たアミノ酸配列を明らかにした後、 これを既存のハン夕一ンウィルス 76— 1 1 8菌株の ヌクレオカプシド蛋白質を暗号化する遺伝子塩基配列及びァミノ酸配列と比較し、 その結 果を図 3の A及び Bに示した。 実施例 6 ：重合酵素連鎖反応によるヌクレオカプシド蛋白質遗伝子の増幅 Examples pThNP8 and pThNP9 obtained in 4 is added by Uni consisting respectively inserted and 30 minutes after cultured Escherichia coli are Ml 3 KO 7 helper phage (helper phage) 1 0 8 / above, the Ganamaishin then after 1 hour The mixture was added to 50 gZ and shake-cultured overnight. Single-stranded DNA separated by a single-stranded DNA separation method using PEGZNaC 1 (20% PEG 6000 / 2M NaC) (see: Molecular Cloning; A Laboratory Manual, Cold Spring Harbor, 1982) The nucleotide sequence was determined by the method provided from Dideoxy Sequencing Kit (USB). After elucidating the amino acid sequence deduced from the determined nucleotide sequence, the amino acid sequence was compared with the existing nucleotide sequence encoding the nucleocapsid protein of the Hanuin virus 76-1118 strain and the amino acid sequence, The results are shown in Figs. Example 6: Amplification of nucleocapsid protein gene by polymerase chain reaction
本発明によって公知のプラスミ ド PET— 3 a及び pKK 223— 3にクロ一ニングす るためのヌクレオカプシド蛋白質遗伝子は、 次の説明の通りそれぞれ重合酵素連鎖反応に よって増幅させた。  The nucleocapsid protein gene for cloning into the known plasmids PET-3a and pKK223-3 according to the present invention was amplified by the polymerase chain reaction as described below.
プラスミ ド ρΕΤ— 3 aにクローニングするためのヌクレオ力プシド蛋白質遺伝子は、 実施例 3の記載と同様の方法で NP 1、 Bg 1 I I (AGATCT)認識部位を含有する NP 2及び E c oR I (GAATTC)認識部位を含有する NP 3からなる 3種のプライ マーから二回の増幅過程を介して製造した。 一方、 プラスミ ド pKK 223— 3にクロー ニングするための蛋白質遺伝子は、 2次重合酵素連鎖反応時プライマ一 ΝΡ 2及び ΝΡ 3 の代わりに E c oR I (GAATTC)認識部位を含有するプライマ一 NP 4及び S a 1 1 (GTCGAC)認識部位を含有する NP 5を使用する以外には実施例 3と同様の方法 で製造した。  The nucleoside psid protein gene to be cloned into the plasmid ρΕΤ-3a was obtained by the same method as described in Example 3; NP1, NP2 containing the Bg1II (AGATCT) recognition site and NP2 GAATTC) was produced through three rounds of amplification from three primers consisting of NP3 containing the recognition site. On the other hand, the protein gene for cloning into plasmid pKK223-3 is replaced by primer NP containing an EcoRI (GAATTC) recognition site instead of primers ΝΡ2 and ΝΡ3 during the secondary polymerase chain reaction. Production was carried out in the same manner as in Example 3, except that NP5 containing 4 and Sa11 (GTCGAC) recognition site was used.
実施例 7：発現プラスミ ド pEThNP及び pKKhNPの作製  Example 7: Production of expression plasmids pEThNP and pKKhNP
ヌクレオカプシド蛋白質遗伝子のクロ一ニング過程は図 4及び図 5に示した。 実施例 6 の方法によつて増幅された 2種類の二本鎖 D N Aは、 寒天ゲル電気泳動上でその大きさを 確認した後、 1. 3 kb大きさのヌクレオ力プシド蛋白質 ¾伝子を包含した DNA切片を ジンクリンキッ トを使用して回収した。 回収した DNA切片はそれぞれ s 10誘導アミノ 酸配列の一部を包含するプラスミ ド pET— 3 a及び、 誘導アミノ酸配列を全く包含しな いプラスミ ド pKK 223— 3内にクローニングした。 回収した DNA切片中 pET— 3 aにクロ-一ニングするための遺伝子は、 制限酵素 B g 1 I I及び E c 0 R Iで二重消化し 、 pKK 223— 3にクローニングするための遺伝子はクレノウ酵素で処理して両末端を ブラントエンド （blunt end)に作った後、 E c o R Iのみを使用して切断し、 フヱノール / /クロ口ホルム混合液 （1 : 1) で 1回抽出した後、 最終 20 £の滅菌蒸溜水に溶解し た。 一方、 遗伝子運搬体である pET— 3 aの 5 gを Ec oR Iと BamH Iで二重消 ィ匕し、 同量の pKK 223— 3を Ec pR Iと Sma Iで二重消化した後、 最終濃度 5 m Mの EDTA存在下に 7 (TCで 1 0分間熱処理し、 フエノール/クロ口ホルム混合液 （1 ： 1) で 1回抽出した後、 それぞれ 20 £の滅菌蒸溜水に溶解した。 切断された各遗伝 子運搬体 5； ( とヌクレオ力プシド DNA1 0 / を混合し、 T4DNAリガ一ゼを用い て 25 °Cで 3時間反応させ接合させることによって、 本発明が目的とする発現プラスミ ド pEThNP及び pKKhNPの作製を完了した。 The cloning process of the nucleocapsid protein gene is shown in FIGS. The two types of double-stranded DNA amplified by the method of Example 6 were confirmed for their size on agar gel electrophoresis, and then contained a 1.3 kb nucleoside peptide gene. The DNA section thus obtained was recovered using a zinc kit. The recovered DNA sections were cloned into plasmid pET-3a containing a part of the s10-derived amino acid sequence and into plasmid pKK223-3 containing no derived amino acid sequence. The gene to be cloned into pET-3a in the collected DNA section was double digested with restriction enzymes Bg1II and Ec0RI, and the gene to be cloned into pKK223-3 was Klenow enzyme. in after making the processing to the both ends blunt-ended (blunt end the), was cut by using only E co RI, Fuwenoru / / black port Holm mixture (1: 1) was extracted once with the final Dissolved in 20 lb of sterile distilled water. On the other hand, 5 g of the gene carrier pET-3a was double-digested with EcoRI and BamHI, and the same amount of pKK223-3 was double-digested with EcoRI and SmaI. After, final concentration 5 m Heat-treated with 7 (TC for 10 minutes) in the presence of M EDTA, extracted once with a phenol / chloroform-form mixture (1: 1), and dissolved in 20 £ of sterile distilled water, respectively.運 搬 Gene carrier 5; (and nucleoside psid DNA10 / are mixed and reacted at 25 ° C. for 3 hours using T4 DNA ligase to allow the expression plasmid pEThNP and The construction of pKKhNP was completed.
実施例 8 ：形質転換体の製造  Example 8: Production of transformant
大腸菌の形質転換は電気場衝擎法を使用し、 宿主細胞としては PEThNPは大腸菌 B L 21 (DE 3) 、 そして pKKhNPは大腸菌 HB 1 0 1をそれぞれ使用した。  Transformation of Escherichia coli used an electric field impact method. As host cells, E. coli BL21 (DE3) was used for PEThNP, and E. coli HB101 was used for pKKhNP.
形質転換用宿主細胞を多量で得るために 2 Omの LB培地に大腸菌 BL 2】 （DE 3) または HB 10 1を寒天平板培地から接種し、 37°Cで 1 8時間振蕩培養した。 これらの それぞれを 1 の新しい LB培地に接種し、 600 nmの波長において吸光度が 0. 5乃 至 0. 8に到達するまで 37 °Cで振蕩培養した。 菌体の回収のために培養液を 0°Cで 20 分間放置した後、 遠心分離で菌体を回収した。 回収しは菌体を 1 の冷滅菌蒸溜水で 1回 洗浄した後、 更に 0. 5 で洗浄し、 最終的に 20%グリセロールで洗浄した。 これを 3 の 1 0%グリセロール溶液に再懸濁させた後分株して一 70°Cで保管し、 各形質転換時 1個ずつ取り出して使用した。 電気場衝撃法に使用した機器は B i 0— R a d社が製作し た Gen e— Pu 1 s e rを使用し、 形質転換は次の通りである。 ― 70°Cで保管された 菌体 40 £とデォキシリボ核酸 2 を混合して電極間隔が 0. 2 cmのキューべッ トに 入れ、 Gene— Pu l s e rを静電容量 25 F、 抵抗 200 Ω、 電気場の強さ 1 2. 5 kVZonに固定して 1回電気場衝撃を加え、 直ちに の SOC培地 （2%バク ト トリ プトン， 0. 5%酵母エキス， l OmM NaC l, 2. 5m KC 1, 1 OmM M gC 12 , 1 OmM MgS04 , 2 OmM グルコース） を添加した。 37てで1時間 振蕩培養した後 50 zg/ のアムピシリンが添加された 2個の LB寒天平板培地にそれ ぞれ 0. l m£、 0. ずつ塗抹し、 37°Cインキュベータ一で 1 2乃至 1 8時間培養し た。 組換プラスミ ドのスクリニングは単一コロニーのクラッキング （cracking) 方法を使 用した。 即ち、 クラッキングバッファ一 （0. 05M Tr i s (pH 6. 8) , 1 % SDS, 2mM EDTA, 0. 4M ショ糖， 0. 01% ブロモフエノールブル一:） 80 £に寒天平板培地から 1ループの菌体を入れてボルテキスミキサー （vortex mi er ) で懸濁し、 マイクロ遠心分離器で 12, 000 r pmで 15分間遠心分離した後、 上澄 液を取つて寒天ゲル電気泳動で所望するヌクレオカブシド遣伝子運搬体である p E T h N Pと PKKhNPプラスミ ドを確認した。 更に、 最終的に分離した組換えプラスミ ドを制 限酵素で切断して確認することによってスクリニングを行つた。 In order to obtain a large amount of host cells for transformation, Escherichia coli BL2] (DE3) or HB101 was inoculated into a 2 Om LB medium from an agar plate medium and cultured with shaking at 37 ° C for 18 hours. Each of these was inoculated into one fresh LB medium and shake-cultured at 37 ° C until the absorbance at a wavelength of 600 nm reached 0.5 to 0.8. The culture was left at 0 ° C for 20 minutes to recover the cells, and the cells were collected by centrifugation. The cells were recovered by washing the cells once with one cold sterilized distilled water, then with 0.5, and finally with 20% glycerol. This was resuspended in 3 10% glycerol solution, separated, stored at 170 ° C., and used one at a time for each transformation. The equipment used for the electric field shock method was Gen e-Pu 1 ser manufactured by Bi0-Rad, and the transformation was as follows. -Mix 40 μg of bacterial cells stored at 70 ° C with deoxyribonucleic acid 2 and put them in a cubet with an electrode spacing of 0.2 cm.Gene-Pulser has a capacitance of 25 F, a resistance of 200 Ω, Electric field strength 1 2.5 kV Zon, immobilized once with electric field and immediately applied to SOC medium (2% Bacto-tryptone, 0.5% yeast extract, lOmM NaCl, 2.5m KC 1, 1 OmM M gC 1 2 , 1 OmM MgS0 4, 2 OmM glucose) was added. After shaking for 1 hour at 37 ° C, spread each of the two LB agar plates supplemented with 50 zg / ampicillin at 0.lm £ and 0. The cells were cultured for 8 hours. Screening of recombinant plasmids uses a single colony cracking method. Used. 1 cracking buffer (0.05 M Tris (pH 6.8), 1% SDS, 2 mM EDTA, 0.4 M sucrose, 0.01% bromophenol: 1) One loop from agar plate at 80 £ of putting cells were suspended in Volte Kiss mixer (vor t ex mi er), it was centrifuged for 15 minutes at 12, 000 r pm in micro centrifuge, optionally in connexion agar gel electrophoresis preparative supernatant PET hNP and PKKhNP plasmids, which are nucleocapsid gene carriers, were identified. Furthermore, screening was performed by confirming the finally separated recombinant plasmid by digestion with a restriction enzyme.
実施例 9 ：形質転換体の培養  Example 9: Culture of transformant
大腸菌の培養のためには LB培地 （酵母エキス 0. 5%, バク ト トリプトン l%, Na C 1 1 %, ρΗ 7. 0) に葡萄糖とェムピシリンをそれぞれ 0. 5%、 1 00 8 7^と なるように添加して使用し、 一夜培養した腫菌液を新しい培地 1 に 5%となるように接 種して 37てで 200 r pmで振蕩培養した。 培養液の吸光度 （八600)が 0. 5乃至 0. 8に到達した時 I PTGを 0. 1乃至 2mMとなるように添加し、 更に 4乃至 8時間培養 した。 培養液を遠心分雠して菌体を回収し、 0. 8%NaC 1溶液で 1回洗净した。 For cultivation of E. coli, glucose and empicillin were added to LB medium (0.5% yeast extract, 1% bactotryptone, 1% NaC, ρΗ7.0) with 0.5% glucose and 10087% respectively. The tumor cell solution cultured overnight was inoculated into a fresh medium 1 at 5% and shake-cultured at 37 rpm at 200 rpm. When the absorbance ( 600 ) of the culture solution reached 0.5 to 0.8, IPTG was added to be 0.1 to 2 mM, and the cells were further cultured for 4 to 8 hours. The culture was centrifuged to collect the cells, and the cells were washed once with a 0.8% NaCl solution.
実施例 10 ： ヌクレオカプシド蛋白質の分離及び精製  Example 10: Separation and purification of nucleocapsid protein
pEThNP及び pKKhNPを包含する大腸菌から発現したヌクレオ力プシド蛋白質 を精製する方法は図 5に簡単に示した。 回収した菌体は 5 の TE緩衝溶液 （5 OmM The method for purifying nucleoside psid proteins expressed from E. coli, including pEThNP and pKKhNP, is shown briefly in FIG. The recovered cells were treated with 5 TE buffer solution (5 OmM
Tr i s, 1 mM EDTA, pH 8. 0) に懸濁させ、 超音波機 （ultra sonicator) またはフレンチプレス （French press) を使用して菌体を破砕したが、 破砕の程度はブラ ッ ドフォード定量 (Bradford assay)方法を使用して破砕液の蛋白質濃度が更に増加しな I、時まで行つた。 破砕した溶液を 8 , 000 X gで 1時間遠心分離して上澄液を取つた。 上澄液に硫酸アンモニゥムを飽和濃度の 30%となるように添加して溶解した後常温で 1 時間放置し、 8， 000 X gで更に 30分間遠心分離して上澄液を捨てて沈殿物は 2 0n6 の T E緩衝溶液に再懸濁させた。 再懸濁させた溶液中の硫酸ァンモニゥムを除去するため 2 の TE緩衝溶液に 2時間ずつ 2回透析を行った。 透析した溶液は〗次でパマシア社の セフアクリル Sを使用してゲル據過 (filtration) を行い、 これを 2次で D E A Eセファ デックス A- 50陰イオン交換樹脂を使用して 2次精製した。 陰イオン交換樹脂に使用し た溶出液は、 50mM Tr i s (pH8. 0) に NaC lを】 . 0M濃度となるように 添加したものを使用した。 ここで受けた溶出液を 5 OmM Tr i s (pH 8. 0)溶液 で 5倍稀釈してヌクレオカブシド蛋白質に対する単一ク口一ン抗体が結合した免疫親和 （ i腿 unoaffinity) カラムに滴下し、 PBS緩衝溶液 （8 g NaC 1, 0. 2 g KC 1 , 1. 44 g Na2 HP04. 0. 24 g KH2 ?0, / i, pH 8. 0) で洗浄し た後、 10 OmMの酢酸を使用して溶出した。 溶出液の pHを調整するために 2MTr i s緩衝溶液 （pH8. 0) を溶出液容積の 0. 3倍添加した。 これを更に PBS緩衝溶液 で透析し、 蛋白質を所望する濃度で濃縮するためにアミコン社が製作したセントリコンま たはセントリフラップを使用した。 各段階での精製された蛋白質はポリアクリルアミ ドゲ ル電気泳動を介して確認したが （図 7参照） 、 これから最終的に精製された蛋白質の純度 は 95%以上であるものと判断された。 The cells were suspended in Tris, 1 mM EDTA, pH 8.0), and the cells were disrupted using an ultrasonic sonicator or French press. The degree of disruption was Bradford. Using a quantitative (Bradford assay) method, the procedure was performed until the protein concentration of the lysate was further increased. The crushed solution was centrifuged at 8,000 × g for 1 hour, and the supernatant was collected. Ammonium sulfate was added to the supernatant to a concentration of 30% of the saturation concentration, dissolved, left at room temperature for 1 hour, centrifuged at 8,000 X g for another 30 minutes, and the supernatant was discarded. Was resuspended in 20n6 TE buffer solution. To remove the ammonium sulfate in the resuspended solution, dialysis was performed twice for 2 hours each against 2 TE buffer solutions. The dialyzed solution was used by Pamacia Gel filtration was performed using Cefacryl S, and this was secondarily purified using a DEAE Sephadex A-50 anion exchange resin in a second step. The eluate used for the anion exchange resin was 50 mM Tris (pH 8.0) to which NaCl was added at a concentration of】 0.0M. The eluate received here was diluted 5-fold with a 5 OmM Tris (pH 8.0) solution and dropped onto an immunoaffinity (i-thigh unoaffinity) column to which a single monoclonal antibody against nucleocapsid protein was bound. , PBS buffer solution (8 g NaC 1, 0. 2 g KC 1, 1. 44 g Na 2 HP0 4. 0. 24 g KH 2? 0, / i, pH 8. 0) was washed with 10 Elution was performed using OmM acetic acid. To adjust the pH of the eluate, 2 M Tris buffer solution (pH 8.0) was added 0.3 times the eluate volume. This was further dialyzed against a PBS buffer solution, and Centricon or Centriflap manufactured by Amicon was used to concentrate the protein to a desired concentration. The purified protein at each step was confirmed by polyacrylamide gel electrophoresis (see Fig. 7), and from this it was determined that the purity of the finally purified protein was 95% or more.
免疫親和力ラムに使用された単一群抗体としてはハイプリ ドーマを培養し、 プロテイン Aカラムを使用して培養液から精製したものを使用した。 ここでハイプリ ドーマは哺乳マ ウスで培養したヒト由来ハン夕一ンウィルスを精製してバルブ C (Balb/c) マウスに接種 し、 これから脾臓細胞とミエロマ一 （myeloma)細胞を得た後ポリエチレングリコールを使 用してこれを融合させ、 融合された細胞中でヌクレオカプシド蛋白質に対する単一群抗体 を生産する融合細胞のみをスクリニングすることによって製作した。 また、 精製された単 —群抗体の結合はパマシア社の CNB rによって活性化されたセファロス 4 Bを使用して 製造会社の推薦方法によって行った。  As a single group antibody used in the immunoaffinity ram, a hybridoma was cultured and purified from the culture using a protein A column. Here, the hybridoma was purified from the human Hanyu virus, which was cultured in a mammalian mouse, and inoculated into valve C (Balb / c) mice. From this, spleen cells and myeloma cells were obtained, and then polyethylene glycol was obtained. This was made by fusing it and screening only the fused cells producing a single group antibody against the nucleocapsid protein in the fused cells. In addition, binding of the purified monoclonal antibody was carried out by using the Cephalos 4B activated by CNBr of Pamacia according to the manufacturer's recommendation method.
実施例 1 1 ： ヌクレオ力プシド蛋白質のウエスタンブロッテイング  Example 11 1: Western Blotting of Nucleotide Psid Protein
大腸菌から発現した蛋白質がハンターンウィルスのヌクレオカプシド蛋白質であること を 9¾するため、 組換えプラスミ ドから発現し精製された蛋白質に対しポリアクリルアミ ドゲルで mm泳動を行い、 電気泳動したゲルから蛋白質を膜に移し、 腎症候出血熱患者の 97/27302 In order to confirm that the protein expressed from Escherichia coli is the nucleocapsid protein of Hunter virus, the protein expressed and purified from recombinant plasmid was subjected to mm electrophoresis on a polyacrylamide gel, and the protein was electrophoresed from the gel. Transfer to the membrane, 97/27302
血清と抗ヌクレオカプシド蛋白質単一クローン抗体 h t 9040を用いてウエスタンブロ ッティング （Western blotting) を行った。 この時、 特異性を有する蛋白贾のみの接着の ため膜を 5%のスキムミルク （ 1<
Figure imgf000018_0001
を包含する？83溶液 （1 当たり 82 Na C 1, 0. 2 g KC 1, 1. 4 g Na2 HPCU， 0. 24 g KH2 P04 , P H 7. 4 ) と 30分間十分に反応させた後、 上記で言及した 1次抗体と室温で 1時間以上 反応させた。 付着されない抗体を除去するため PBST溶液 （PBS+0. 5%Twe e n 20) で膜を 3回それぞれ 5分間洗浄した。 洗浄完了された膜は適当に稀釈され、 ペル ォキシダーゼ (peroxidase) 酵素が付着された 2次抗体（goat anti-humanlgGまたは goat anti-mouse IgG)及び 5 %スキムミルクを包含する P B S溶液の混合液と 1時間振蕩し て反応させた。 更に PBST溶液で膜を 3回それぞれ 5分間洗浄した後、 4 -クロロ- 1 一ナフトールを発色試薬で使用して発色反応を行った結果、 予想した位置で所望するヌク レオ力プシド蛋白質特有のバンド （約 5 OKd) を確認した （図 8参照） 。 Western blotting was performed using serum and anti-nucleocapsid protein monoclonal antibody ht9040. At this time, 5% of skim milk (1 <
Figure imgf000018_0001
Embrace? 83 solution (1 per 82 Na C 1, 0. 2 g KC 1, 1. 4 g Na 2 HPCU, 0. 24 g KH 2 P0 4, PH 7. 4) and was allowed to sufficiently react for 30 minutes, the The reaction was carried out for 1 hour or more at room temperature with the primary antibody mentioned in the above. The membrane was washed three times for 5 minutes each with a PBST solution (PBS + 0.5% Tween 20) to remove unattached antibodies. The washed membrane is diluted appropriately and mixed with a secondary antibody (goat anti-humanlgG or goat anti-mouse IgG) to which a peroxidase enzyme is attached and a PBS solution containing 5% skim milk. The reaction was shaken for a while. After washing the membrane three times with PBST solution for 5 minutes each, and performing a color reaction using 4-chloro-1-naphthol as a color reagent, a band specific to the desired nucleoside psid protein was obtained at the expected position. (Approximately 5 OKd) (see Fig. 8).
実施例 1 2 ：精製されたヌクレオ力プシド蛋白質を用いて製造されたワクチンの効能性 調査  Example 12: Investigation of the efficacy of a vaccine produced using the purified nucleoside protein
実施例 1 0で精製されたワクチン精製原液 0. 5 当たり補助剤のアルミニウムヒドロ イシ一ドゲルを 0. 625mgの配合比で添加し、 4てで 1 5日間定置させた。 その後、 0 . 01 % (w/v) チメロサールと 0. 02%精製ゼラチンを加えて最終試験ワクチンを 製造した。  An aluminum hydroxide gel as an auxiliary agent was added in a mixing ratio of 0.625 mg per 0.5 of the unpurified stock solution of the vaccine purified in Example 10, and the mixture was allowed to stand at 4 days for 15 days. Thereafter, 0.01% (w / v) thimerosal and 0.02% purified gelatin were added to produce the final test vaccine.
このように製造された試験ワクチンめ中和効能を調査するためにギニァビグを対象とし て実験し、 比較ヮグチンとしては現在販売している株式会社緑十字のハン夕一バックスを 使用した。 この時、 抗原量が 1 0 gZ0. 5m 20 g/0. 及び 40 g/0 . 57 ^となるように 3種の違う濃度の試験ワクチンを製造してギニァビグに接種し、 試験 ワクチンと比較ヮクチンをギニァビグに接種した後に得られたギニァビグの血淸をブラー ク減少中和試験法で検査することによって、 ワクチンの免疫原性を判定した。 プラーク減 少中和試験は下記の記載の通り進行された。 1) プラーク減少中和試験のための抗体を得るため、 3種の濃度で製造された上記試験 ワクチンと比較ワクチンをギニァビグに 1 0日間隔で 3回皮下接種 （0. 接種） し た。 In order to investigate the neutralizing efficacy of the test vaccine produced in this way, an experiment was conducted with guinea pigs, and Hanyuichi Bax of Green Cross Co., Ltd., which is currently sold, was used as a comparative pegutin. At this time, three different concentrations of test vaccines were prepared and inoculated into guinea-big so that the antigen amount became 10 gZ0.5m 20 g / 0.5 and 40 g / 0.57 ^, and the vaccine was compared with the test vaccine. The immunogenicity of the vaccine was determined by examining the blood of Guineabig obtained after inoculation of the vaccine with Guineabig by a blackout neutralization test. The plaque reduction neutralization test proceeded as described below. 1) In order to obtain antibodies for the plaque reduction neutralization test, the above test vaccine and the comparative vaccine prepared at three concentrations were subcutaneously inoculated (0. vaccination) three times at 10-day intervals.
2 ) ギニァビグから血清を採取して 56でで 30分間非動化処理した後、 3 %牛胎児血 清を含有する油脂用培地 （MEM + M1 99= 1 ： 1) を使用してそれぞれ 1 ： 〗 0、 1 : 20、 1 : 40、 1 : 80及び 1 : 1 60で稀釈した。  2) Serum was collected from Guinea big and immobilized at 56 for 30 minutes. Then, using a medium for fat and oil (MEM + M199 = 1: 1) containing 3% fetal bovine serum, the following steps were performed:稀 Diluted at 0, 1:20, 1:40, 1:80 and 1: 160.
3)稀釈させた血清と 70PFUZ培養容器 （直径 6 cm) となるように稀釈させたハン ターンウィルス攻撃菌株 76- 1 1 8を同量ずつ試験管で混合した後、 37でで 1時間反 応させた。  3) After mixing the diluted serum and the Hunter virus-infected strain 76-118 diluted in a 70 PFUZ culture vessel (diameter 6 cm) in equal amounts in a test tube, react at 37 for 1 hour. I let it.
4 ) 予め準備した 6 cmの細胞培養容器で断層培養したベロ E 6細胞に上記混合液を 0. 2 ずつ接種した後、 37でで 90分間反応させた。  4) The above mixture was inoculated in 0.2 to each Vero E6 cells that had been subjected to fault culture in a 6 cm cell culture vessel prepared in advance, and reacted at 37 for 90 minutes.
5)反応後接種液を除去し、 各培養容器当たり ずつの 1次寒天重層培地 （Agarose overlay)を分株した。 1次寒天重層培地の組成は次の通りである。  5) After the reaction, the inoculum was removed, and a primary agar overlay medium (Agarose overlay) was separated for each culture vessel. The composition of the primary agar overlay medium is as follows.
Ml 99培地 5 Omi  Ml 99 medium 5 Omi
牛胎児血清 1 QmS  Fetal bovine serum 1 QmS
7%NaHC03 SmS 7% NaHC0 3 SmS
寒天 0. 8 g  Agar 0.8 g
蒸溜水 5  Distilled water 5
この時 Ml 99はフユノールレツ ドを含有しない培地で使用し、 牛胎児血清は 56てで 30分間熱処理したものを使用した。  At this time, Ml 99 was used in a culture medium containing no fenol red, and fetal calf serum used was heat-treated at 56 ° C for 30 minutes.
6 ) 1次寒天重層が終わるとそれぞれ接種された細胞を 37でで 1 0乃至 1 1日間培養 した。  6) Upon completion of the primary agar overlay, each inoculated cell was cultured at 37 for 10 to 11 days.
7 )培養後各培養容器当たり 3. 5 mずつの 1次寒天重層培地を分株し、 2次寒天重層 培地の組成は次の通りである。  7) After culturing, the primary agar overlay medium of 3.5 m per culture vessel is separated, and the composition of the secondary agar overlay medium is as follows.
Ml 99培地 5 Qnd 4 0 OmM ME S 1 m£ Ml 99 medium 5 Qnd 4 0 OmM ME S 1 m £
(2- (N-Morpho 1 i no) ethane sulfonic acid, monohydrate)  (2- (N-Morpho 1 i no) ethane sulfonic acid, monohydrate)
5 % BS A 5 mi  5% BS A 5 mi
0. 6 % 中性的（neutral red) \ i  0.6% neutral red \ i
I N Na OH 1. 5m6  I N Na OH 1.5m6
寒天 0. 7 g  Agar 0.7 g
蒸溜水 3 2. 5mi  Distilled water 32.5 mi
8) 2次寒天重雇が終わると 3日間培養した後直径 2IDID程度のプラーク数を観察し、 そ の結果を下記の表 3に示した。 8) At the end of secondary agar hiring, after culturing for 3 days, the number of plaques with a diameter of about 2IDID was observed, and the results are shown in Table 3 below.
W 7/ W 7 /
表 3. 精製された人間由来ハンターンウィルスヌクレオカプシド蛋白質から製造された 試験ワクチンの親和逆価試験 血清稀釈倍数 ブラーク数 （PFU) 培養容器 Table 3. Affinity reversal test of test vaccines produced from purified human hunter virus nucleocapsid protein Serum dilution factor Braak number (PFU) Culture vessel
1 : 1 0 70 1: 1 0 70
正常血清 1 : 20 72  Normal serum 1: 20 72
1 : 4 0 67  1: 4 0 67
1 : 8 0 75  1: 8 0 75
1 : 1 0 8 (40 g), 12 (20yg) , 21 (lOj g) 試験ワクチン 1 : 20 11 (40^ g), 22 (20 xg) , 32 (lOwg) 血 1 : 0 17 (40 zg), 28 (20/ g) ， 47 (10〃 g) 1: 10 8 (40 g), 12 (20 yg), 21 (lOj g) Test vaccine 1: 20 11 (40 ^ g), 22 (20 xg), 32 (lOwg) Blood 1: 0 17 (40 zg ), 28 (20 / g), 47 (10〃 g)
1 : 80 26 (40// g), 42 (20/ g) , 73 (10 ί g) 1: 80 26 (40 // g), 42 (20 / g), 73 (10 ί g)
1 : 1 6 0 28 (40 ), 45 (20 g) , 75 (lOfig) 1: 1 6 0 28 (40), 45 (20 g), 75 (lOfig)
1 : 1 0 22 1: 1 0 22
比較ワクチン 1 : 20 32  Comparative vaccine 1: 20 32
血清 1 : 0 4 3  Serum 1: 0 4 3
1 : 80 6 5 上記表 3の結果からわかるように、 精製されたヌクレオ力プシド蛋白質を用! <、て製造さ れた試験ワクチンの場合、 4 0 j gノ 0. 5?^及び20〃 /0. 5 の濃度では比較ヮ クチンに比べて卓越した中和能力を示し、 1 0 g/0. 5 の濃度では同等の中和能力 を示した。 従って、 ヒト由来ハンターンウィルスにおいて抗原性を示す物質の一つであるヌクレオ 力プシド蛋白質を遺伝工学的方法で大量生産して製造された本発明のワクチンは、 既存の ワクチンに比べて肾症候群出血熱に対し優れた免疫原性を有しているだけでなく、 特に毒 性物質含有等副作用の可能性の高いウィルス自体をワクチン物質として使用しないで、 成 分が明らかになった単一蛋白質をワクチン物質として使用することによって、 ワクチン製 剤の安定性を顕、著に改善した優れたワクチンである。 1: 80 6 5 As can be seen from the results in Table 3 above, the purified nucleoside peptide was used! <In the case of the test vaccine produced, 40 jg 0 0.5? ^ And 20〃 / At a concentration of 0.5, the neutralizing ability was superior to that of the comparative vaccine, and at a concentration of 10 g / 0.5, the neutralizing ability was equivalent. Therefore, the vaccine of the present invention, which is produced by mass-producing a nucleocapsid protein, which is one of the substances showing antigenicity in human-derived Hunter virus, by a genetic engineering method, has a higher hemorrhagic syndrome than the existing vaccine. A single protein with a clear component is used without using the virus itself, which has not only excellent immunogenicity against heat but also highly likely to cause side effects such as toxic substances, as a vaccine substance. By using it as a vaccine substance, it is an excellent vaccine that has significantly improved the stability of vaccine preparations.

Claims

Q Q
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Figure imgf000023_0001
Figure imgf000023_0001
9 9
2 . 下記に示す通りに 5 '及び 3 '末端にそれぞれ更に塩基配列を含有することを特徴と する請求項 1記載のヒト由来ハンターンウィルスのヌクレオカプシド蛋白質 c D N A遗伝 子。 2. The human huntan virus nucleocapsid protein cDNA 遗 gene according to claim 1, further comprising a nucleotide sequence at each of the 5 'and 3' ends as shown below.
TAGTAGTAGACTCCTTAAAGAGCTACTAGAACAACGATGGCAACTATGGA -50  TAGTAGTAGACTCCTTAAAGAGCTACTAGAACAACGATGGCAACTATGGA -50
し N蛋白質  N protein
GG. TTACAG.AGGG. ATC TGCCCATGAGGGTCAATTAGTCATAGCCA - 100 GGCAGAAGGTGAGGGATGCAGA^ACAGTATGAAAAGGATCCAGATGAG - 150 丁 TGAACAAGAGAACATTAACTGACCGAGAGGGTGTTGCAGTATCTATCCA -200 GGCAAAGATTGATGAGTTAAAAAGGCAACTGGCAGATAGGATTGCAACTG -250 GGAAGAACCTTGGAA GAACAAGATCCAACAGGGGTAGAGCCTGGAGAC -300 CATCTG. AG.AGAGATC TGCTCAGTTATGGTAATGTGTTGGATTTAAA -350 CCACTTGGATATTGATGAACCCACAGGACAGACAGCAGACTGGCTAAGCA -400 TTGTCGTCTATCTCACATCCTTTGTTGTCCCGACACTTCTGAAAGCTCTA -450 TATATGTTGACAACAAGGGGGAGGC. CTACCAAGGATAATAAAGGAAC -500 CCGCATTCGATTTAAGGATGATAGCTCCTTCGAGGATGHAATGGTATCC -550 GAAAACC. AAACATCTGTATGTCTCCTTGCCAAATGCACAGTCAAGTATG -600 AAGGCAGAACAGATTACACCTCGTAGATATAGAACAGCACTCTGTGCACT -650 CTACCCTGCACACATCAAGCCACGGCACATGATCAGCCCAGTCATCACTG -700 TAATTCCTTTTTTCCCATTCGCAAAAGATTCGAGTGATCGTAnGAACAA -750  .. GG TTACAG.AGGG ATC TGCCCATGAGGGTCAATTAGTCATAGCCA - 100 GGCAGAAGGTGAGGGATGCAGA ^ ACAGTATGAAAAGGATCCAGATGAG -.. 150 Ding TGAACAAGAGAACATTAACTGACCGAGAGGGTGTTGCAGTATCTATCCA -200 GGCAAAGATTGATGAGTTAAAAAGGCAACTGGCAGATAGGATTGCAACTG -250 GGAAGAACCTTGGAA GAACAAGATCCAACAGGGGTAGAGCCTGGAGAC -300 CATCTG AG.AGAGATC TGCTCAGTTATGGTAATGTGTTGGATTTAAA -350 CCACTTGGATATTGATGAACCCACAGGACAGACAGCAGACTGGCTAAGCA -400 TTGTCGTCTATCTCACATCCTTTGTTGTCCCGACACTTCTGAAAGCTCTA -450 TATATGTTGACAACAAGGGGGAGGC CTACCAAGGATAATAAAGGAAC -500 CCGCATTCGATTTAAGGATGATAGCTCCTTCGAGGATGHAATGGTATCC -550 GAAAACC. AAACATCTGTATGTCTCCTTGCCAAATGCACAGTCAAGTATG -600 AAGGCAGAACAGATTACACCTCGTAGATATAGAACAGCACTCTGTGCACT -650 CTACCCTGCACACATCAAGCCACGGCACATGATCAGCCCAGTCATCACTG -700 TAATTCCTTTTCCCATTCGCAAAAGATTCGAGTGATCGTAnGAAAAA
TGGTTAAGCGAA C^TGCAAGCTCCTTCCAGATACAACAGCACTTAGCCT -800 CCTTCGTGGTCC7CC AACAAACAGGGACTACTTACGGCAGCGGCAACTGG -850  TGGTTAAGCGAA C ^ TGCAAGCTCCTTCCAGATACAACAGCACTTAGCCT -800 CCTTCGTGGTCC7CC AACAAACAGGGACTACTTACGGCAGCGGCAACTGG -850
CATTACGTAATATGGAGACA.AAGCAGTCAAACGCTATACGCCACCATCCA -900  CATTACGTAATATGGAGACA.AAGCAGTCAAACGCTATACGCCACCATCCA -900
CAAGCAGCTGCCTGTAGCATCATTGAAGACATTCAGTCACCATCATCAAT -950  CAAGCAGCTGCCTGTAGCATCATTGAAGACATTCAGTCACCATCATCAAT -950
ATCGCTAnTCCTGCCCCACCAGACCGCTCTCCCCCAACATCTTTCTTTA - 1000 TAGCAGGTATTCCTGAGCTTGGAGCAmTTCTCCATCCTCCAGGATATG - 1050 CGAAATACAATCATGGCATCTAAGACAGTTGGAACATCTGAGGAGAAGCT - 1100 GCGAAAGAAATC.ATCATTCTATCAGTCTTACCTCACCAGGACACAATCAA -1150 TGCGGATACAACTGGATCAAAGGATTATTGTGCTCTTTATCGTTGCTTGG - 1200 CGGAAGCA.AGCTGTGGACAACTTCCACTTAGGAGATGACATGGATCCTGA - 1250 GCTAAGIACACTGGCACAGAGCTTGATTGATGTCAAAGTC- AGGAAATTT - 1300 CCAACCAAGAGCCTTTGAAACTCTAATTA - 1329 ATCGCTAnTCCTGCCCCACCAGACCGCTCTCCCCCAACATCTTTCTTTA - 1000 TAGCAGGTATTCCTGAGCTTGGAGCAmTTCTCCATCCTCCAGGATATG - 1050 CGAAATACAATCATGGCATCTAAGACAGTTGGAACATCTGAGGAGAAGCT - 1100 GCGAAAGAAATC.ATCATTCTATCAGTCTTACCTCACCAGGACACAATCAA -1150 TGCGGATACAACTGGATCAAAGGATTATTGTGCTCTTTATCGTTGCTTGG - 1200 CGGAAGCA.AGCTGTGGACAACTTCCACTTAGGAGATGACATGGATCCTGA - 1250 GCTAAGIACACTGGCACAGAGCTTGATTGATGTCAAAGTC- AGGAAATTT - 1300 CCAACCAAGAGCCTTTGAAACTCTAATTA - 1329
3. 請求項 1または 2記載のヒト由来ハンターンウィルスのヌクレオ力プシド蛋白質遗伝 子を含有する組換え発現プラスミ ド。 3. A recombinant expression plasmid containing the human hantan virus nucleoside psid protein gene according to claim 1 or 2.
4. ヒト由来ハンターンウィルスのヌクレオ力プシド蛋白質遺伝子をべクタ一 pET— 3 a内に挿入させ製造することを特徴とする請求項 3記載の発現ブラスミ ド p E T h N P。  4. The expression plasmid pEThNP according to claim 3, which is produced by inserting a nucleoside psid protein gene of a human-derived Hunter virus into vector pET-3a.
5. ヒト由来ハンターンウィルスのヌクレオ力プシド蛋白質遺伝子をベクター pKK 22 3— 3内に挿入させ製造することを特徴とする請求項 3記載の発現プラスミ ド pEThN  5. The expression plasmid pEThN according to claim 3, which is produced by inserting the nucleoside psid protein gene of the human-derived Hunter virus into the vector pKK223-3.
6. 請求項 3記載の発現プラスミ ドによって形質転換された大腸菌形質転換体。 6. An Escherichia coli transformant transformed by the expression plasmid according to claim 3.
7. 発現プラスミ ド pEThNPで大腸菌 BL 21 (D E 3 ) を形質転換させ製造される ことを特徴とする請求項 6記載の大腸菌形質転換体 E. c 01 i BL (pEThNP) (寄託番号： KFCC - 10865) 。  7. The E. coli transformant E. c01iBL (pEThNP) according to claim 6, which is produced by transforming Escherichia coli BL 21 (DE 3) with the expression plasmid pEThNP (deposit number: KFCC- 10865).
8. 発現プラスミ ド pKKhNPで大腸菌 HB 1 01を形質転換させ製造されることを特 徴とする請求項 6記載の大腸菌形質転換体 E. c 01 i BL (pKKhNP) (寄託番 号： KFCC - 10868)。  8. The Escherichia coli transformant E. c01iBL (pKKhNP) according to claim 6, which is produced by transforming Escherichia coli HB101 with the expression plasmid pKKhNP (deposit number: KFCC-10868). ).
9. 請求項 6記載の大腸菌形質転換体の培養液から分離、 精製されたヒト由来ハンターン ウィルスのヌクレオカプシド蛋白質。  9. A nucleocapsid protein of a human-derived Hantaan virus isolated and purified from a culture solution of the transformant of Escherichia coli according to claim 6.
10. 請求項 7記載の大腸菌形質転換体培養液から得られ、 ァミノ末端部位に更に s 10 リーダ一 （leader)配列を含有することを特徵とするヌクレオ力プシド融合蛋白質。 10. A nucleoside fusion protein obtained from the culture solution of the transformant of Escherichia coli according to claim 7, further comprising an s 10 leader sequence at an amino terminal site.
1 1. 硫酸アンモニゥム沈殿法、 ゲル濾過 (filtration) , 陰イオン交換樹脂カラム及び 免疫親和- (i麵 unoaffinity) カラムを用いて精製されることを特徴とする請求項 9または 10記載の蛋白質。 11. The protein according to claim 9, wherein the protein is purified using ammonium sulfate precipitation, gel filtration, anion exchange resin column, and immunoaffinity column.
12. 請求項 9から得られたヒト由来ハンターンウィルスのヌクレオカプシド蛋白質を有 効成分として含有する腎症候出血熱予防ヮクチン。  12. A prophylactic tract with renal symptomatic hemorrhagic fever, comprising as an active ingredient the nucleocapsid protein of human-derived hantan virus obtained in claim 9.
13. 薬剤学的に許容する補助剤を含有することを特徴とする請求項 12記載のワクチン 13. The vaccine according to claim 12, comprising a pharmaceutically acceptable adjuvant.
1 4 . 薬剤学的に許容する補助剤がアルミニウムヒドロキシードゲル、 チメロサール、 精 製ゼラチンから選択される 1種以上であることを特徴とする請求項 1 4記載のワクチン。 14. The vaccine according to claim 14, wherein the pharmaceutically acceptable adjuvant is at least one selected from aluminum hydroxide gel, thimerosal, and refined gelatin.
PCT/KR1997/000016 1996-01-25 1997-01-25 Vaccine prepared using human-origin hantaan virus nucleocapsid protein expressed from escherichia coli WO1997027302A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0553475B2 (en) * 1988-11-18 1993-08-10 Ryoku Juji Kk
US5298423A (en) * 1987-11-25 1994-03-29 The United States Of America As Represented By The Secretary Of The Army Nucleotide sequences encoding the expression of a Hantaan virus nucleocapsid protein and G1 and G2 glycoproteins
JPH08325291A (en) * 1995-05-30 1996-12-10 Jiro Arikawa Antigenic protein of hantavirus and monoclonal antibody

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US5298423A (en) * 1987-11-25 1994-03-29 The United States Of America As Represented By The Secretary Of The Army Nucleotide sequences encoding the expression of a Hantaan virus nucleocapsid protein and G1 and G2 glycoproteins
JPH0553475B2 (en) * 1988-11-18 1993-08-10 Ryoku Juji Kk
JPH08325291A (en) * 1995-05-30 1996-12-10 Jiro Arikawa Antigenic protein of hantavirus and monoclonal antibody

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