[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2002032453A1 - Synthetic peptide hog cholera vaccine and method producing it - Google Patents

Synthetic peptide hog cholera vaccine and method producing it Download PDF

Info

Publication number
WO2002032453A1
WO2002032453A1 PCT/CN2001/001188 CN0101188W WO0232453A1 WO 2002032453 A1 WO2002032453 A1 WO 2002032453A1 CN 0101188 W CN0101188 W CN 0101188W WO 0232453 A1 WO0232453 A1 WO 0232453A1
Authority
WO
WIPO (PCT)
Prior art keywords
swine fever
synthetic peptide
epitope
vaccine
polypeptide
Prior art date
Application number
PCT/CN2001/001188
Other languages
French (fr)
Chinese (zh)
Inventor
Yinghua Chen
Xiaonan Dong
Yi Xiao
Original Assignee
Tsinghua University
Beijing Feikai Biotech Co. Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tsinghua University, Beijing Feikai Biotech Co. Ltd. filed Critical Tsinghua University
Priority to AU2002223384A priority Critical patent/AU2002223384A1/en
Publication of WO2002032453A1 publication Critical patent/WO2002032453A1/en

Links

Classifications

    • 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
    • A61K39/187—Hog cholera virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
    • C12N2770/24322—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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
    • C12N2770/24334—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 vaccine prepared by bioengineering technology and a preparation method thereof, and particularly to a swine fever virus vaccine and a preparation method thereof.
  • Swine fever is a malignant infectious disease caused by classical swine fever virus ( ⁇ Classical Swine fever virus, CSFV or Hog Cholera virus, HCV). Once an outbreak, it often causes huge economic losses within a short period of time.
  • CSFV Classical Swine fever virus
  • HCV Hog Cholera virus
  • the swine fever virus subunit vaccine is a swine fever vaccine currently being developed at home and abroad.
  • the vaccine uses genetically-recombined viral envelope proteins to induce immune protection.
  • this vaccine can cope with the mutation of classical swine fever virus to a certain extent, the vaccine contains only one genetic information of the virus. When the virus mutates, the vaccine will lose its effectiveness, and the production cost of the vaccine is still relatively low.
  • the antigenic region sequence of envelope protein E2 of swine fever virus (amino acid residues 690-866) has been shown to induce immune protection, that is, to protect swine fever virus from infection (Vaccine 2000, 18: 2351; Vaccine 1999, 17: 433; J. Virology 1995, 69: 6479; Vaccine 2000, 18: 1374; J. Virology 1993, 67: 5435).
  • the object of the present invention is to provide a synthetic peptide classical swine fever virus vaccine capable of coping with mutations of classical swine fever virus.
  • Another object of the present invention is to provide a method for producing the above-mentioned synthetic peptide swine fever virus vaccine.
  • a synthetic peptide swine fever vaccine comprising at least one of the swine fever virus envelope protein E2 coupled to an IJ carrier protein or carrier polypeptide to form a conjugate.
  • the neutralizing epitope or variant epitope on the swine fever virus envelope protein E2 is located in the antigenic region on the swine fever virus envelope protein E2.
  • the antigenic region is amino acids 690-866 on the swine fever virus-coated phosphin protein E2. Acid residues.
  • neutralizing epitope and variant epitope polypeptides can be selected from the following 14 polypeptide sequences and their variant sequences:
  • the above 14 polypeptides and their conjugates formed by the mutated sequence polypeptide and the carrier can be added to the vaccine.
  • the vaccine should also include acceptable pharmaceutical adjuvants.
  • a method for preparing the above-mentioned synthetic peptide swine fever vaccine basically includes the following steps:
  • Example 1 Preparation of polyclonal swine fever polypeptide (synthetic peptide) based on classical protein of swine fever virus E2 1. Synthesize 11 synthetic peptides (polypeptides) that contain one of the neutralizing epitopes of the antigen region of the classical swine fever virus E2 protein (amino acid residues at positions 690-866). The sequences of these 11 peptides partially overlap each other. 11 The sequence of each polypeptide is:
  • MBS nrmaleimidobenzoyl-N-hydroxy succinimide ester
  • One or more of the above 11 conjugates are respectively formulated with an adjuvant (such as oil adjuvant, etc.) and mixed to prepare a multiple swine fever vaccine.
  • an adjuvant such as oil adjuvant, etc.
  • the fraction ratio of various conjugates can be adjusted according to the probability of variation of each point obtained by statistical processing in different years.
  • Example 2 Preparation of anti-variation-multiple-one swine fever polypeptide based on E2 protein of classical swine fever virus
  • MBS nrmaleimidobenzoyl- N-hydroxy succinimide ester
  • the above 3 conjugates were formulated with Shan adjuvant to prepare a multiple swine fever polypeptide vaccine. Its In the three conjugates, the proportions of the conjugates can be adjusted according to the variation probability of each point obtained by statistical processing in different years.
  • a synthetic peptide containing the neutralizing epitope of the antigenic region of the classical swine fever virus E2 protein the sequence is:
  • the above conjugate is formulated with an oil adjuvant to prepare a swine fever polypeptide vaccine.
  • MBS m-maleimidobenzoyl-hydroxy succinimide ester
  • the present invention utilizes the neutralizing epitope on envelope protein E2 of classical swine fever virus to induce pigs to produce stronger, longer-lasting immune protection characteristics.
  • a multiple vaccine is used to produce pigs.
  • a variety of antibodies to classical swine fever virus that is, when one or several epitopes of classical swine fever virus are mutated, the vaccine-injected pigs are still sufficient to deal with the mutant classical swine fever virus corresponding to other unmutated epitopes Antibodies.
  • the vaccine of the present invention has the following advantages: 1.
  • the vaccine is a highly efficient multiple vaccine that can stimulate the production of a variety of neutralizing antibodies, which can effectively deal with the mutation of the virus, thereby overcoming the shortcomings of the existing swine fever vaccines, which are poor or even ineffective. .
  • the active ingredient of the vaccine is an epitope polypeptide comprising a neutralizing epitope or a variant epitope on envelope protein E2 of swine fever virus coupled to a carrier protein or carrier polypeptide to form a conjugate.
  • epitope polypeptides Does not require attenuated or inactivated pathogens or natural proteins of pathogens, nor does it require genetically engineered protein antigens and pathogen nucleic acids, which can directly induce a predetermined, multiple neutralizing epitope and variant epitope-specific immune response, No genetic material and activity of classical swine fever virus, no side effects induced by genetic material of classical swine fever virus, no danger of reactivation of inactivated or attenuated vaccines, and the use of attenuated classical swine fever vaccine (such as Swine fever rabbit attenuated attenuated vaccine) infection and replication of live swine fever attenuated live virus induced in breeding pigs, sows and pigs, thereby overcoming the potential, current Unknown hazard.
  • attenuated classical swine fever vaccine such as Swine fever rabbit attenuated attenuated vaccine

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention is concerned with a synthetic peptide hog cholera vaccine which comprise at least a conjugate containing neutralizing epitope peptide present on swine fever envelope protein E2, i.e. amino acid residue 690-866, the said conjugate is generated by coupling the epitope peptide to a carrier protein or a carrier polypeptide. The method producing the synthetic peptide comprise: (1) artificially synthesis of peptide containing neutralizing epitope present on antigenic region; (2) coupling the said peptides to a carrier protein or a carrier polypeptide individually to produce hog cholera vaccine to form the conjugate; (3) formulating the said conjugate with acceptable adjuvant to obtain hog cholera vaccine.

Description

一种合成肽猪瘟疫苗及其制备方法  Synthetic peptide swine fever vaccine and preparation method thereof
技术领域  Technical field
本发明涉及用生物工程技术制备的疫苗及其制备方法， 特别是涉及一 种猪瘟病毒疫苗及其制备方法。  The present invention relates to a vaccine prepared by bioengineering technology and a preparation method thereof, and particularly to a swine fever virus vaccine and a preparation method thereof.
背景技术  Background technique
猪瘟是由猪瘟病毒 (^ Classical Swine fever virus, CSFV或 Hog Cholera virus, HCV) 引发的恶性传染疾病， 一旦爆发往往在短时间内带 来巨大的经济损失。 早在 50年代， 我国便研制出了猪瘟兔化弱毒疫苗， 该 疫苗是减毒活疫苗的一种， 利用减毒后的活猪瘟病毒来诱导免疫防护， 有 效地控制了猪瘟在我国的大流行和散发， 因其安全性和预防效果很好， 许 多国家也使用此疫苗。 进入 70年代后期， 猪瘟的流行以散发和慢性感染为 特征， 并且普遍出现免疫失败现象。 事实证明， 现有的猪瘟兔化弱毒疫苗 在预防已发生变异的猪瘟病毒引发的猪瘟流行时， 存在着很大的困难。 猪 瘟病毒亚单位疫苗是目前国内外正在研制的一种猪瘟疫苗， 该疫苗是利用 基因重组的病毒包膜蛋白来诱导免疫防护。 这种疫苗虽然在一定程度上能 够对付猪瘟病毒的变异， 但疫苗中只包含病毒的一个遗传信息， 当病毒发 生变异时， 该疫苗即会失去效力， 并且， 该疫苗的生产成本目前仍然较 另一方面， 猪瘟病毒包膜蛋白 E2上的抗原区序列 （第 690- 866位氨基 酸残基） 已被证实能诱导免疫防护， 即能保护猪预防猪瘟病毒的感染 (Vaccine 2000, 18:2351 ; Vaccine 1999, 17:433; J. Virology 1995, 69: 6479; Vaccine 2000, 18: 1374; J. Virology 1993, 67: 5435)。  Swine fever is a malignant infectious disease caused by classical swine fever virus (^ Classical Swine fever virus, CSFV or Hog Cholera virus, HCV). Once an outbreak, it often causes huge economic losses within a short period of time. As early as the 1950s, China developed a swine fever rabbit attenuated attenuated vaccine, which is a live attenuated vaccine that uses attenuated live swine fever virus to induce immune protection and effectively control swine fever in China. The pandemic and its distribution have been used in many countries because of its safety and prevention. In the late 1970s, the swine fever epidemic was characterized by sporadic and chronic infections, and widespread immune failure. Facts have proved that the existing rabbit swine fever attenuated attenuated vaccine has great difficulties in preventing the swine fever epidemic caused by the mutated swine fever virus. The swine fever virus subunit vaccine is a swine fever vaccine currently being developed at home and abroad. The vaccine uses genetically-recombined viral envelope proteins to induce immune protection. Although this vaccine can cope with the mutation of classical swine fever virus to a certain extent, the vaccine contains only one genetic information of the virus. When the virus mutates, the vaccine will lose its effectiveness, and the production cost of the vaccine is still relatively low. On the other hand, the antigenic region sequence of envelope protein E2 of swine fever virus (amino acid residues 690-866) has been shown to induce immune protection, that is, to protect swine fever virus from infection (Vaccine 2000, 18: 2351; Vaccine 1999, 17: 433; J. Virology 1995, 69: 6479; Vaccine 2000, 18: 1374; J. Virology 1993, 67: 5435).
发明公开 Invention Disclosure
本发明的目的是提供一种能对付猪瘟病毒变异的合成肽猪瘟病毒疫 苗。  The object of the present invention is to provide a synthetic peptide classical swine fever virus vaccine capable of coping with mutations of classical swine fever virus.
本发明的另一个目的是提供一种生产上述合成肽猪瘟病毒疫苗的方 法。  Another object of the present invention is to provide a method for producing the above-mentioned synthetic peptide swine fever virus vaccine.
为实现上述目的， 本发明采取以下设计方案： 一种合成肽猪瘟疫苗 ， 它包括至少一个偶联至 IJ载体蛋白或载体多肽上形成偶联物的包含有猪瘟病 毒包膜蛋白 E2上的中和表位或变异表位的多肽。  In order to achieve the above object, the present invention adopts the following design scheme: A synthetic peptide swine fever vaccine, comprising at least one of the swine fever virus envelope protein E2 coupled to an IJ carrier protein or carrier polypeptide to form a conjugate. A polypeptide that neutralizes an epitope or a variant epitope.
所述猪瘟病毒包膜蛋白 E2上的中和表位或变异表位位于猪瘟病毒包膜 蛋白 E2上的抗原区。  The neutralizing epitope or variant epitope on the swine fever virus envelope protein E2 is located in the antigenic region on the swine fever virus envelope protein E2.
更具体地说， 所述抗原区为猪瘟病毒包膦蛋白 E2上的第 690— 866位氨 基酸残基。 More specifically, the antigenic region is amino acids 690-866 on the swine fever virus-coated phosphin protein E2. Acid residues.
上述中和表位和变异表位的多肽可以选自于下列 14条多肽序列以及它 们的变异序列：  The above-mentioned neutralizing epitope and variant epitope polypeptides can be selected from the following 14 polypeptide sequences and their variant sequences:
CKEDYRYAISSTNEIGLLGAGGLT CAGGLTTTWKEYSHDLQ CKEDYRYAISSTNEIGLLGAGGLT CAGGLTTTWKEYSHDLQ
CSHDLQLNDGTVKAICVAGSFKVTCSHDLQLNDGTVKAICVAGSFKVT
CSFKVTALNVVSRRYLASLHKCSFKVTALNVVSRRYLASLHK
CSLHKGALLTSVTFELLFDGTNCSLHKGALLTSVTFELLFDGTN
CFDGTNPSTEEMGDDFRFGLCPFDT CPFDTSPVVKGKYNTTLLNGSAFEECFDGTNPSTEEMGDDFRFGLCPFDT CPFDTSPVVKGKYNTTLLNGSAFEE
CEEEENGSAFYLVCPIGWTEEECEEEENGSAFYLVCPIGWTEEE
CEEEPIGWTGVIECTAVSEEECEEEPIGWTGVIECTAVSEEE
CTAVSPTTLRTEVVKTFRREKCTAVSPTTLRTEVVKTFRREK
CFRREKPFPHRMDCVTTTVENED CKEDHRYAISTTNEIGLHGAGGLTCFRREKPFPHRMDCVTTTVENED CKEDHRYAISTTNEIGLHGAGGLT
CKEDHRYAISTTNEIGLHGAEGLTCKEDHRYAISTTNEIGLHGAEGLT
CFRREKPFPHRRDCVTTTVENED CFRREKPFPHRRDCVTTTVENED
为了使疫苗能更好地抵御突变的发生， 可以将上述 14条多肽以及它们 的变异序列多肽与载体形成的偶联物均加入到疫苗中。  In order to make the vaccine better able to resist the occurrence of mutations, the above 14 polypeptides and their conjugates formed by the mutated sequence polypeptide and the carrier can be added to the vaccine.
为了适于注射， 疫苗中还应该包括可接受的药用佐剂。  To be suitable for injection, the vaccine should also include acceptable pharmaceutical adjuvants.
一种制备上述合成肽猪瘟疫苗的方法， 基本上包括以下步骤：  A method for preparing the above-mentioned synthetic peptide swine fever vaccine basically includes the following steps:
( 1 ) 、 人工合成至少一条分别含有猪瘟病毒包膜蛋白 E2上抗原区中 和表位和 /或变异表位的多肽；  (1) artificially synthesizing at least one polypeptide containing a neutralizing epitope and / or a variant epitope on the antigenic region of envelope protein E2 of classical swine fever virus, respectively;
( 2 ) 、 将上述多肽分别偶联到载体蛋白或载体多肽上形成偶联物； ( 3 ) 、 将上述偶联物配以可接受的佐剂制备成合成肽猎瘟疫苗。 实施发明的最佳方式  (2) Coupling the above-mentioned polypeptide to a carrier protein or a carrier polypeptide, respectively, to form a conjugate; (3), preparing the synthetic peptide hunting plague vaccine by combining the above-mentioned conjugate with an acceptable adjuvant. The best way to implement the invention
实施例 1、 制备以猪瘟病毒 E2蛋白为基础的多联一猪瘟多肽（合成肽）疫 田 1. 人工合成分别含有猪瘟病毒 E2蛋白抗原区 （第 690- 866位氨基酸残 基） 中中和表位之一的 11个合成肽（多肽）， 这 11个多肽的序列相互部分重 叠， 11个多肽的序列为： Example 1. Preparation of polyclonal swine fever polypeptide (synthetic peptide) based on classical protein of swine fever virus E2 1. Synthesize 11 synthetic peptides (polypeptides) that contain one of the neutralizing epitopes of the antigen region of the classical swine fever virus E2 protein (amino acid residues at positions 690-866). The sequences of these 11 peptides partially overlap each other. 11 The sequence of each polypeptide is:
CKEDYRYAISSTNEIGLLGAGGLT CAGGLTTTWKEYSHDLQ CKEDYRYAISSTNEIGLLGAGGLT CAGGLTTTWKEYSHDLQ
CSHDLQLNDGTV AICVAGSFKVTCSHDLQLNDGTV AICVAGSFKVT
CSFKVTALNVVSRRYLASLHKCSFKVTALNVVSRRYLASLHK
CSLHKGALLTSVTFELLFDGTNCSLHKGALLTSVTFELLFDGTN
CFDGTNPSTEEMGDDFRFGLCPFDT CPFDTSPVVKGKYNTTLLNGSAFEECFDGTNPSTEEMGDDFRFGLCPFDT CPFDTSPVVKGKYNTTLLNGSAFEE
CEEEENGSAFYLVCPIGWTEEECEEEENGSAFYLVCPIGWTEEE
CEEEPIGWTGVIECTAVSEEECEEEPIGWTGVIECTAVSEEE
CTAVSPTTLRTEVVKTFRREKCTAVSPTTLRTEVVKTFRREK
CFRREKPFP圆 DGVTTTVENED CFRREKPFP round DGVTTTVENED
2. 利用 MBS (nrmaleimidobenzoyl - N- hydroxy succinimide ester将上述多 肽分别与载体蛋白 BSA偶联；  2. MBS (nrmaleimidobenzoyl-N-hydroxy succinimide ester) was used to couple the above peptides to the carrier protein BSA, respectively;
3. 将上述 11种偶联物中的一种或几种分别配以佐剂 （如油佐剂等） 并 混合制备成多联一猪瘟多肽疫苗。 其中， 各种偶联物的份数比可以根据不 同年份统计学处理得出的各位点的变异机率进行调整。  3. One or more of the above 11 conjugates are respectively formulated with an adjuvant (such as oil adjuvant, etc.) and mixed to prepare a multiple swine fever vaccine. Among them, the fraction ratio of various conjugates can be adjusted according to the probability of variation of each point obtained by statistical processing in different years.
实施例 2、 制备以猪瘟病毒 E2蛋白为基础的抗变异一多联一猪瘟多肽 Example 2.Preparation of anti-variation-multiple-one swine fever polypeptide based on E2 protein of classical swine fever virus
(合成肽）疫苗 (Synthetic peptide) vaccine
1. 人工合成分别含有猪瘟病毒 E2蛋白抗原区中和表位及变异表位的 3 个合成肽（多肽）， 其序列为：  1. Three synthetic peptides (polypeptides) containing the neutralizing epitope and variant epitope of the antigenic region of classical swine fever virus E2 protein were artificially synthesized, and their sequences are:
CKEDYRYAISSTNEIGLLGAGGLT G EDHRYAISTTNEIGLHGAGGLT CKEDHRYAISTTNEIGLHGAEGLT  CKEDYRYAISSTNEIGLLGAGGLT G EDHRYAISTTNEIGLHGAGGLT CKEDHRYAISTTNEIGLHGAEGLT
2. 利用 MBS (nrmaleimidobenzoyl- N- hydroxy succinimide ester将上述多 肽分别与载体蛋白牛血清白蛋白偶联；  2. MBS (nrmaleimidobenzoyl- N-hydroxy succinimide ester) was used to couple the above peptides to the carrier protein bovine serum albumin;
3. 将上述 3种偶联物分别配以汕佐剂制备成多联一猪瘟多肽疫苗。 其 中， 3种偶联物的份数比可以根据不同年份统计学处理得出的各位点的变异 机率进行调整。 3. The above 3 conjugates were formulated with Shan adjuvant to prepare a multiple swine fever polypeptide vaccine. Its In the three conjugates, the proportions of the conjugates can be adjusted according to the variation probability of each point obtained by statistical processing in different years.
实施例 3、 制备以猪瘟病毒 E2蛋白为基础的猪瘟多肽（合成肽）疫苗 Example 3. Preparation of classical swine fever polypeptide (synthetic peptide) vaccine based on E2 protein of classical swine fever virus
1. 人工合成含有猪瘟病毒 E2蛋白抗原区中和表位的 1个合成肽， 其序 列为： 1. A synthetic peptide containing the neutralizing epitope of the antigenic region of the classical swine fever virus E2 protein, the sequence is:
CKEDYRYAISSTNEIGLLGAGGLT  CKEDYRYAISSTNEIGLLGAGGLT
2. 利用戊二醛将上述多肽与载体蛋白鸡卵白蛋白偶联；  2. Coupling the above polypeptide with carrier protein chicken ovalbumin using glutaraldehyde;
3. 将上述偶联物配以油佐剂制备成猪瘟多肽疫苗。  3. The above conjugate is formulated with an oil adjuvant to prepare a swine fever polypeptide vaccine.
实施例 4、 制备以猪瘟病毒 E2蛋白为基础的抗变异一多联一猪瘟多肽 (合成肽）疫苗  Example 4.Preparation of anti-variation-multiple-one swine fever polypeptide (synthetic peptide) vaccine based on E2 protein of classical swine fever virus
1. 人工合成分别含有猪瘟病毒 E2蛋白抗原区中和表位及变异表位的 14 个合成肽（多肽）， 其序列为- 1. Synthesize 14 synthetic peptides (polypeptides) containing neutralizing epitopes and variant epitopes of the E2 protein region of classical swine fever virus, the sequence of which is-
CKEDYRYAISSTNEIGLLGAGGLTCKEDYRYAISSTNEIGLLGAGGLT
CAGGLTTTWKEYSHDLQ CSHDLQLNDGTVKAICVAGSFKVTCAGGLTTTWKEYSHDLQ CSHDLQLNDGTVKAICVAGSFKVT
CSFKVTALNVVSRRYLASLHKCSFKVTALNVVSRRYLASLHK
CSLHKGALLTSVTFELLFDGTNCSLHKGALLTSVTFELLFDGTN
CFDGTNPSTEEMGDDFRFGLCPFDTCFDGTNPSTEEMGDDFRFGLCPFDT
CPFDTSPVVKGKYNTTLLNGSAFEE CEEEENGSAFYLVCPIGWTEEECPFDTSPVVKGKYNTTLLNGSAFEE CEEEENGSAFYLVCPIGWTEEE
CEEEPIGWTGVIECTAVSEEECEEEPIGWTGVIECTAVSEEE
CTAVSPTTLRTEVVKTFRREKCTAVSPTTLRTEVVKTFRREK
CFRREKPFPHRMDCVTTTVENEDCFRREKPFPHRMDCVTTTVENED
CKEDHRYAISTT EIGLHGAGGLT CKEDHRYAISTTNEIGLHGAEGLTCKEDHRYAISTT EIGLHGAGGLT CKEDHRYAISTTNEIGLHGAEGLT
CFR EKPFPHRRDCVTTTVENED CFR EKPFPHRRDCVTTTVENED
2. 利用 MBS (m- maleimidobenzoyl- hydroxy succinimide ester 将上述多 肽分别与载体蛋白牛血清白蛋白偶联；  2. MBS (m-maleimidobenzoyl-hydroxy succinimide ester) was used to couple the above peptides to the carrier protein bovine serum albumin;
3. 将上述 14.种偶联物中的几种或全部分别配以油佐剂制备成抗变异一 多联一猪瘟多肽疫苗。 其中， 各种偶联物的份数比可以根据不同年份统计 学处理得出的各位点的变异机率进行调整。 3. Prepare several or all of the above 14. conjugates with oil adjuvant to prepare anti-mutation one Multi-unit swine fever polypeptide vaccine. Among them, the fraction ratio of various conjugates can be adjusted according to the probability of variation of each point obtained by statistical processing in different years.
工业应用 Industrial applications
本发明利用猪瘟病毒包膜蛋白 E2上的中和表位能够诱导猪产生较强， 而且时间较长的免疫防护的特性， 针对猪瘟病毒变异较多的特点， 运用多 联疫苗使猪产生多种对猪瘟病毒的抗体， 也就是说， 当猪瘟病毒的一个或 几个表位发生变异时， 注射了疫苗的猪还有足以对付该变异猪瘟病毒的对 应于其它未变异表位的抗体。 本发明的疫苗具有以下优点： 1、 该疫苗是一 种能刺激产生多种中和抗体的高效多联疫苗， 能有效对付病毒的变异， 进 而克服已有猪瘟疫苗预防效果差甚至无效的缺点。 2、 该疫苗的有效成分是 偶联到载体蛋白或载体多肽上形成偶联物的包含有猪瘟病毒包膜蛋白 E2上 的中和表位或变异表位的表位多肽， 这些表位多肽不需要减毒或灭活的病 原体或病原体的天然蛋白， 也不需要基因重组蛋白抗原以及病原体的核 酸， 能够直接诱导预先确定的、 多个中和表位及变异表位特异性的免疫应 答， 没有猪瘟病毒的遗传物质及活性， 不会产生因猪瘟病毒的遗传物质诱 发的副作用， 也没有灭活疫苗或减毒疫苗可能复性的危险， 同时也可排除 使用猪瘟弱毒疫苗 (如猪瘟兔化弱毒疫苗）在种猪、 母猪和肉猪体内诱导产 生的猪瘟减毒活病毒的感染和复制， 从而克服猪瘟减毒活病毒感染肉猪给 人类带来的潜在的、 目前未知的危害。 3、 能诱导很强的针对特定中和表位 的免疫应答， 是相应的亚单位疫苗诱导的特定中和抗体水平的 10— 20倍， 针对特定中和表位的抗体量为 10— 240微克 /毫升血清。 4、 裉据猪瘟病毒的 变异， 可以很快生产出其相应类型的疫苗， 不需进行长时间的试验， 降低 生产成本。 该技术将对世界预防医学研究产生重大影响， 将使猪瘟病毒疫 苗的制备技术产生革命性变化， 并且将带来巨大的经济效益和社会效益。  The present invention utilizes the neutralizing epitope on envelope protein E2 of classical swine fever virus to induce pigs to produce stronger, longer-lasting immune protection characteristics. In view of the characteristics of more classical swine fever virus mutations, a multiple vaccine is used to produce pigs. A variety of antibodies to classical swine fever virus, that is, when one or several epitopes of classical swine fever virus are mutated, the vaccine-injected pigs are still sufficient to deal with the mutant classical swine fever virus corresponding to other unmutated epitopes Antibodies. The vaccine of the present invention has the following advantages: 1. The vaccine is a highly efficient multiple vaccine that can stimulate the production of a variety of neutralizing antibodies, which can effectively deal with the mutation of the virus, thereby overcoming the shortcomings of the existing swine fever vaccines, which are poor or even ineffective. . 2. The active ingredient of the vaccine is an epitope polypeptide comprising a neutralizing epitope or a variant epitope on envelope protein E2 of swine fever virus coupled to a carrier protein or carrier polypeptide to form a conjugate. These epitope polypeptides Does not require attenuated or inactivated pathogens or natural proteins of pathogens, nor does it require genetically engineered protein antigens and pathogen nucleic acids, which can directly induce a predetermined, multiple neutralizing epitope and variant epitope-specific immune response, No genetic material and activity of classical swine fever virus, no side effects induced by genetic material of classical swine fever virus, no danger of reactivation of inactivated or attenuated vaccines, and the use of attenuated classical swine fever vaccine (such as Swine fever rabbit attenuated attenuated vaccine) infection and replication of live swine fever attenuated live virus induced in breeding pigs, sows and pigs, thereby overcoming the potential, current Unknown hazard. 3. Can induce a strong immune response against specific neutralizing epitopes, which is 10-20 times the level of specific neutralizing antibodies induced by corresponding subunit vaccines, and the amount of antibodies directed against specific neutralizing epitopes is 10-240 micrograms. / Ml of serum. 4. According to the mutation of swine fever virus, the corresponding type of vaccine can be produced quickly, without long-term tests, which reduces the production cost. This technology will have a major impact on world preventive medicine research, it will revolutionize the technology for preparing swine fever virus vaccine, and it will bring huge economic and social benefits.

Claims

权利要求书 Claim
1、 一种合成肽猪瘟疫苗 ， 它包括至少一个偶联到载体蛋白或载体多 肽上形成偶联物的包含有猪瘟病毒包膜蛋白 E2上的中和表位或变异表位的 多肽。 1. A synthetic peptide swine fever vaccine comprising at least one polypeptide comprising a neutralization epitope or a variant epitope on an envelope protein E2 of classical swine fever virus coupled to a carrier protein or a carrier polypeptide to form a conjugate.
2、 根据权利要求 1所述的合成肽猪瘟疫苗， 其特征在于： 所述猪瘟病 毒包膜蛋白 E2上的中和表位或变异表位位于猪瘟病毒包膜蛋白 E2上的抗原 区。  2. The synthetic peptide swine fever vaccine according to claim 1, characterized in that: the neutralization epitope or the variant epitope on the envelope protein E2 of the swine fever virus is located in the antigen region on the envelope protein E2 of the swine fever virus .
3、 裉据权利要求 2所述的合成肽猪瘟疫苗， 其特征在于： 所述抗原区 为猪瘟病毒包膜蛋白 E2上的第 690— 866位氨基酸残基。  3. The synthetic peptide classical swine fever vaccine according to claim 2, wherein the antigen region is amino acid residues 690-866 on the envelope protein E2 of classical swine fever virus.
4、 根据权利要求 1或 2或 3所述的合成肽猪瘟疫苗， 其特征在于： 所述 中和表位和变异表位的多肽选自于：  4. The synthetic peptide swine fever vaccine according to claim 1 or 2 or 3, characterized in that the polypeptide that neutralizes the epitope and the variant epitope is selected from:
CKEDYRYAISSTNEIGLLGAGGLT CKEDYRYAISSTNEIGLLGAGGLT
CAGGLTTTWKEYSHDLQ CSHDLQLNDGTVKAICVAGSFKVTCAGGLTTTWKEYSHDLQ CSHDLQLNDGTVKAICVAGSFKVT
CSFKVTALNVVSRRYLASLHKCSFKVTALNVVSRRYLASLHK
CSLHKGALLTSVTFELLFDGTNCSLHKGALLTSVTFELLFDGTN
CFDGTNPSTEEMGDDFRFGLCPFDTCFDGTNPSTEEMGDDFRFGLCPFDT
CPFDTSPVVKGKYNTTLLNGSAFEE CEEEENGSAFYLVCPIGWTEEECPFDTSPVVKGKYNTTLLNGSAFEE CEEEENGSAFYLVCPIGWTEEE
CEEEPIGWTGVIECTAVSEEECEEEPIGWTGVIECTAVSEEE
CTAVSPTTLRTEVVKTFRREKCTAVSPTTLRTEVVKTFRREK
CFRREKPFPHRMDCVTTTVENEDCFRREKPFPHRMDCVTTTVENED
CKEDHRYAISTTNEIGLHGAGGLT CKEDHRYAISTT EIGLHGAEGLTCKEDHRYAISTTNEIGLHGAGGLT CKEDHRYAISTT EIGLHGAEGLT
CFR EKPFPHRRDCVTTTVENED CFR EKPFPHRRDCVTTTVENED
5、 根据权利要求 4所述的合成肽猎瘟疫苗， 其特征在于： 所述疫苗包 括分别偶联到载体蛋白或载体多肽上形成偶联物的下述 14条多肽：  5. The synthetic peptide hunting plague vaccine according to claim 4, characterized in that: the vaccine comprises the following 14 polypeptides respectively coupled to a carrier protein or a carrier polypeptide to form a conjugate:
CKEDYRYAISSTNEIGLLGAGGLT CKEDYRYAISSTNEIGLLGAGGLT
CAGGLTTTWKEYSHDLQ CSHDLQLNDGTV AICVAGSFKVT CAGGLTTTWKEYSHDLQ CSHDLQLNDGTV AICVAGSFKVT
CSFKVTALNVVSRRYLASLH CSLH GALLTSVTFELLFDGTN CFDGTNPSTEEMGDDFRFGLCPFDT CPFDTSPVVKGKYNTTLLNGSAFEE CEEEENGSAFYLVCPIGWTEEE CEEEPIGWTGVIECTAVSEEE CTAVSPTTLRTEVVKTFRRE CFRREKPFPHRMDCVTTTVENED CKEDHRYAISTTNEIGLHGAGGLT C EDHRYAISTTNEIGLHGAEGLT CFRREKPFPHRRDCVTTTVENED  CSFKVTALNVVSRRYLASLH CSLH GALLTSVTFELLFDGTN CFDGTNPSTEEMGDDFRFGLCPFDT CPFDTSPVVKGKYNTTLLNGSAFEE CEEEENGSAFYLVCPIGWTEEE CEEEPIGWTGVIECTAVSEEE CTAVSPTTLRTEVEVKTFRRE CFRREKPFPHRMDCGAVTHRTTVTEDEDVTCTTTTED
6、 根据权利要求 1或 2或 3或 5所述的合成肽猪瘟疫苗， 其特征在于： 疫 苗中还包括可接受的药用佐剂。  6. The synthetic peptide swine fever vaccine according to claim 1 or 2 or 3 or 5, characterized in that the vaccine further comprises an acceptable medicinal adjuvant.
7、 根据权利要求 4所述的合成肽猪瘟疫苗， 其特征在于： 疫苗中还包 括可接受的药用佐剂。  7. The synthetic peptide swine fever vaccine according to claim 4, wherein the vaccine further comprises an acceptable medicinal adjuvant.
8、 一种制备上述合成肽猪瘟疫亩的方法， 基本上包括以下步骤： 8. A method for preparing the above-mentioned synthetic peptide swine plague mu, which basically includes the following steps:
( 1 ) 、 人工合成至少一条分别含有猪瘟病毒包膜蛋白 E2上抗原区中 和表位和 /或变异表位的多肽； (1) artificially synthesizing at least one polypeptide containing a neutralizing epitope and / or a variant epitope on the antigenic region of envelope protein E2 of classical swine fever virus, respectively;
( 2 ) 、 将上述多肽分别偶联到载体蛋白或载体多肽上形成偶联物； (2) coupling the above polypeptide to a carrier protein or a carrier polypeptide, respectively, to form a conjugate;
( 3 ) 、 将上述偶联物配以可接受的佐剂制备成合成肽猪瘟疫苗。(3) preparing the synthetic peptide swine fever vaccine by combining the above conjugate with an acceptable adjuvant.
9、 根据权利要求 8所述的一种制备合成肽猪瘟疫苗的方法， 其特征在 于： 所述人工合成的含有猪瘟病毒包膜蛋白 E2上抗原区中和表位的多肽选 自： 9. A method for preparing a synthetic peptide swine fever vaccine according to claim 8, characterized in that: said artificially synthesized polypeptide containing an epitope neutralizing epitope on envelope protein E2 of swine fever virus is selected from:
CKEDYRYAISSTNEIGLLGAGGLT CKEDYRYAISSTNEIGLLGAGGLT
CAGGLTTTWKEYSHDLQCAGGLTTTWKEYSHDLQ
CSHDLQLNDGTVKAICVAGSFKVTCSHDLQLNDGTVKAICVAGSFKVT
CSFKVTALNVVSRRYLASLHKCSFKVTALNVVSRRYLASLHK
CSLHKGALLTSVTFELLFDGTN CFDGTNPSTEEMGDDFRFGLCPFDTCSLHKGALLTSVTFELLFDGTN CFDGTNPSTEEMGDDFRFGLCPFDT
CPFDTSPVVKGKYNTTLLNGSAFEE i:/〕 88llz>ldi i esn ¾。ΙΛ≥ν3應Ί 33s Η33CPFDTSPVVKGKYNTTLLNGSAFEE i: /] 88llz> ldi i esn ¾. ΙΛ≥ν3 should Ί 33s Η33
3a3I00 33SAJJICS333 l333AllACS aNHECS3Hy¾  3a3I00 33SAJJICS333 l333AllACS aNHECS3Hy¾
PCT/CN2001/001188 2000-08-10 2001-07-20 Synthetic peptide hog cholera vaccine and method producing it WO2002032453A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002223384A AU2002223384A1 (en) 2000-08-10 2001-07-20 Synthetic peptide hog cholera vaccine and method producing it

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN00121291.5 2000-08-10
CN00121291A CN1338309A (en) 2000-08-10 2000-08-10 Synthetic peptide vaccine of hog cholera and its preparing process

Publications (1)

Publication Number Publication Date
WO2002032453A1 true WO2002032453A1 (en) 2002-04-25

Family

ID=4588704

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2001/001188 WO2002032453A1 (en) 2000-08-10 2001-07-20 Synthetic peptide hog cholera vaccine and method producing it

Country Status (3)

Country Link
CN (1) CN1338309A (en)
AU (1) AU2002223384A1 (en)
WO (1) WO2002032453A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007098717A2 (en) 2006-02-28 2007-09-07 Centro De Ingeniería Genética Y Biotecnología Chimeric vaccine antigens against classical swine fever virus
CN110652583A (en) * 2018-06-29 2020-01-07 西北民族大学 Bivalent inactivated antigen of swine foot-and-mouth disease and swine fever low virulent strain bigeminy vaccine

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101144818B (en) * 2007-10-19 2011-04-27 清华大学 Method for detecting pig plague virus specific antibody and its ELISA reagent kit
CN101152570B (en) * 2007-10-19 2010-11-24 清华大学 Epitope vaccine of hog cholera virus and its preparing process
CN102485749B (en) * 2009-10-09 2014-06-04 中牧实业股份有限公司 Swine fever synthesized peptide vaccine and preparation method thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0236977A2 (en) * 1986-03-07 1987-09-16 Biotechnology Research Partners, Ltd. Bovine virus diarrhea and hog cholera vaccines
EP0389034A1 (en) * 1989-03-19 1990-09-26 Akzo Nobel N.V. Hog cholera virus vaccine and diagnostic
WO1995035380A1 (en) * 1994-06-17 1995-12-28 Instituut Voor Dierhouderij En Diergezondheid Nucleotide sequences of pestivirus strains, polypeptides encoded by these sequences and use thereof for diagnosis and prevention of pestivirus infections
WO1996019498A2 (en) * 1994-12-20 1996-06-27 Akzo Nobel N.V. T cell stimulating protein of pestivirus
EP0982402A1 (en) * 1998-08-14 2000-03-01 Stichting Instituut voor Dierhouderij en Diergezondheid (ID-DLO) Pestivirus vaccination

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0236977A2 (en) * 1986-03-07 1987-09-16 Biotechnology Research Partners, Ltd. Bovine virus diarrhea and hog cholera vaccines
EP0389034A1 (en) * 1989-03-19 1990-09-26 Akzo Nobel N.V. Hog cholera virus vaccine and diagnostic
WO1995035380A1 (en) * 1994-06-17 1995-12-28 Instituut Voor Dierhouderij En Diergezondheid Nucleotide sequences of pestivirus strains, polypeptides encoded by these sequences and use thereof for diagnosis and prevention of pestivirus infections
WO1996019498A2 (en) * 1994-12-20 1996-06-27 Akzo Nobel N.V. T cell stimulating protein of pestivirus
EP0982402A1 (en) * 1998-08-14 2000-03-01 Stichting Instituut voor Dierhouderij en Diergezondheid (ID-DLO) Pestivirus vaccination

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MOORMANN R.J. ET AL., VIROLOGY, vol. 177, no. 1, July 1990 (1990-07-01), pages 184 - 198 *
VAN RIJN P.A. ET AL., VIROLOGY, vol. 237, no. 2, 27 October 1997 (1997-10-27), pages 337 - 348 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007098717A2 (en) 2006-02-28 2007-09-07 Centro De Ingeniería Genética Y Biotecnología Chimeric vaccine antigens against classical swine fever virus
CN110652583A (en) * 2018-06-29 2020-01-07 西北民族大学 Bivalent inactivated antigen of swine foot-and-mouth disease and swine fever low virulent strain bigeminy vaccine

Also Published As

Publication number Publication date
CN1338309A (en) 2002-03-06
AU2002223384A1 (en) 2002-04-29

Similar Documents

Publication Publication Date Title
Denis et al. Immunogenicity of papaya mosaic virus-like particles fused to a hepatitis C virus epitope: evidence for the critical function of multimerization
EP1218031B1 (en) Pharmaceutical composition comprising an antigen
TWI620574B (en) Synthetic peptide-based emergency vaccine against foot and mouth disease (fmd)
EP0090581A2 (en) Small peptides with the specificity of foot and mouth disease viral antigens
JP2005162761A (en) Artificial t helper cell epitope as immune stimulator for synthetic peptide immunogen
JP2006504644A5 (en)
Mohan et al. Nasal delivery of PLG microparticle encapsulated defensin peptides adjuvanted gp41 antigen confers strong and long-lasting immunoprotective response against HIV-1
Verma et al. Norovirus (NoV) specific protective immune responses induced by recombinant P dimer vaccine are enhanced by the mucosal adjuvant FlaB
WO2023064708A1 (en) Vaccine compositions against sars-cov-2 variants of concern to prevent infection and treat long-haul covid
CA2901888C (en) Influenza nucleoprotein vaccines
Chua et al. Opinion: making inactivated and subunit-based vaccines work
Zamorano et al. Recognition of B and T cell epitopes by cattle immunized with a synthetic peptide containing the major immunogenic site of VP1 FMDV 01 Campos
McCLUSKIE et al. Immunization against hepatitis B virus by mucosal administration of antigen–antibody complexes
WO2002020048A1 (en) A classical swine fever virus epitope vaccine and its producing method
EP2482844B1 (en) Adjuvanted vaccine formulations
JP2020534874A (en) Fusion peptide of CD4 helper T cell epitope and its vaccine
KR102165358B1 (en) Vaccine composition for foot and mouth disease virus a serotype
WO2002032453A1 (en) Synthetic peptide hog cholera vaccine and method producing it
Osorio et al. Immune responses to hepatitis B surface antigen following epidermal powder immunization
Kim et al. DNA vaccination against foot‐and‐mouth disease via electroporation: study of molecular approaches for enhancing VP1 antigenicity
US8691234B2 (en) Vaccine formulation potentiated by the combination of DNA and an antigen
Schulze et al. A prime-boost vaccination protocol optimizes immune responses against the nucleocapsid protein of the SARS coronavirus
KR102365464B1 (en) Development of recombinant subunit Zika virus vaccine and preparing method thereof
Venugopal et al. Immunity to St. Louis encephalitis virus by sequential immunization with recombinant vaccinia and baculovirus derived PrM/E proteins
Ge et al. Fusion expression of major antigenic segment of JEV E protein-hsp70 and the identification of domain acting as adjuvant in hsp70

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP