WO2005117960A1 - Vaccin adn contre le sras et procede de preparation de celui-ci, utilisation de gene glycoproteine spicule (spike) des coronavirus pour vaccin - Google Patents

Vaccin adn contre le sras et procede de preparation de celui-ci, utilisation de gene glycoproteine spicule (spike) des coronavirus pour vaccin Download PDF

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Publication number
WO2005117960A1
WO2005117960A1 PCT/CN2004/000501 CN2004000501W WO2005117960A1 WO 2005117960 A1 WO2005117960 A1 WO 2005117960A1 CN 2004000501 W CN2004000501 W CN 2004000501W WO 2005117960 A1 WO2005117960 A1 WO 2005117960A1
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WO
WIPO (PCT)
Prior art keywords
gene
sars
vaccine
vaccine according
pcdna3
Prior art date
Application number
PCT/CN2004/000501
Other languages
English (en)
Chinese (zh)
Inventor
Yixin Zeng
Wenlin Huang
Jian Wang
Haide Tan
Peng Liu
Zhigang Pan
Qisheng Feng
Jiang Li
Lixi Huang
Miaohua Zhang
Lizhen Chen
Original Assignee
Cancer Center Sun Yat-Sen University
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 Cancer Center Sun Yat-Sen University filed Critical Cancer Center Sun Yat-Sen University
Priority to PCT/CN2004/000501 priority Critical patent/WO2005117960A1/fr
Publication of WO2005117960A1 publication Critical patent/WO2005117960A1/fr

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Classifications

    • 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
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Coronavirus is a non-segmental positive-strand RNA virus with a genome of nearly 30kb that can be transmitted in humans and animals. It mainly infects the respiratory systems of humans and animals. Coronavirus particles are a kind of virus There are four types of structural proteins in viruses: spike (Sike), membrane (Mmbrance, M), envelope (Evelop, E), and nucleoprotein (N). Because coronavirus is an RNA virus, it is very unstable and is susceptible to mutations to avoid host immune surveillance and rejection. Therefore, one must look for The SARS-associated coronavirus has a stable and immunoprotective antigen for the development of related vaccines.
  • spike S is a structural protein that induces a protective immune response.
  • Some researchers have confirmed that the C-terminus of the spike protein of coronavirus is Its epitope is located.
  • SARS-associated coronavirus causes infection through the respiratory tract, and there is no vaccine to prevent SARS.
  • the content of the invention is a structural protein that induces a protective immune response.
  • N-terminal amplification primers Dl and D4
  • SARS-associated coronavirus spike protein belongs to its epitope. Therefore, the present invention is based on this finding.
  • the SARS-associated coronavirus spike protein is extracted and cloned into pcDNA3, which is amplified, purified, and formulated to effectively induce the body to produce antibodies and prevent the virus from infecting the body. Clinical application prospects.
  • Figure 1 is the primer design for DNA vaccine and protein subunit vaccine research
  • Figure 2 is an electrophoresis diagram of pcDNA3 plasmid identification of S gene transformation.
  • Figure 3 is a technical roadmap for the preparation of a SARS nucleic acid vaccine.
  • Example 1 The present invention will be further described below through specific examples.
  • Example 1
  • the PCR method was used to amplify it. After PCR, it was digested with EcoRl and digested with pcDNA3, and ligated to transform E. coli. Positive clones were screened for ampicillin (Amp resistance), and the SARS nucleic acid vaccine was obtained by culturing, purifying, and formulating.
  • the cloned into the eukaryotic expression plasmid was the Si region of the S gene of the SARS-associated coronavirus.
  • the cloned into the eukaryotic expression plasmid were the transmembrane segment of SARS-associated coronavirus S gene (base numbers: 3686 ⁇ 3648, see attached table 1) and the C-terminal fragment.
  • the S gene amplified by PCR was digested with EcoRl, and the pcDNA3 plasmid was digested at the same time, ligated, transformed into E. coli, cultured, and the positive clones were screened by ampicillin resistance to obtain the S gene pcDNA3 recombinant, which was subjected to conventional agarose gel electrophoresis The results are shown in Figure 2.
  • Rats No.1 and No.2 were intramuscularly injected with 50 ⁇ g pcDNA3-S N on each of their four legs, that is, a total of 200 ⁇ g.
  • mice 3 and 4 200 pcDNA3-S N was injected into the tail vein of each mouse to add a mixed volume of mycoplasma 2000.
  • the second group Shuttle-Sc test group
  • the third group pcDNA3 empty vector group

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Communicable Diseases (AREA)
  • Pulmonology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne le domaine des modifications génétiques biologiques, un vaccin ADN du syndrome respiratoire aigu sévère (SRAS), son procédé de préparation et l'utilisation du gène spike de coronavirus du SRAS pour un vaccin contre le SRAS. Cette invention est un type de vaccin ADN, extrait du gène spike, cloné en ADN pc3, puis amplifié, purifié et préparé pour un vaccin ADN. Par rapport au vaccin activé vivant classique de virus, cette invention est plus sûre et présente une meilleure immunogénicité, elle peut efficacement induire un anticorps dans un organisme et protéger cet organisme contre une infection virale. Cette invention possède un potentiel important d'utilisation clinique.
PCT/CN2004/000501 2004-06-04 2004-06-04 Vaccin adn contre le sras et procede de preparation de celui-ci, utilisation de gene glycoproteine spicule (spike) des coronavirus pour vaccin WO2005117960A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2004/000501 WO2005117960A1 (fr) 2004-06-04 2004-06-04 Vaccin adn contre le sras et procede de preparation de celui-ci, utilisation de gene glycoproteine spicule (spike) des coronavirus pour vaccin

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2004/000501 WO2005117960A1 (fr) 2004-06-04 2004-06-04 Vaccin adn contre le sras et procede de preparation de celui-ci, utilisation de gene glycoproteine spicule (spike) des coronavirus pour vaccin

Publications (1)

Publication Number Publication Date
WO2005117960A1 true WO2005117960A1 (fr) 2005-12-15

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2004/000501 WO2005117960A1 (fr) 2004-06-04 2004-06-04 Vaccin adn contre le sras et procede de preparation de celui-ci, utilisation de gene glycoproteine spicule (spike) des coronavirus pour vaccin

Country Status (1)

Country Link
WO (1) WO2005117960A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111505286A (zh) * 2020-04-28 2020-08-07 郑州伊美诺生物技术有限公司 一种新型冠状病毒特异性抗体双抗原夹心elisa检测试剂盒及其制备方法
CN112538105A (zh) * 2020-06-29 2021-03-23 斯克里普斯研究院 稳定的冠状病毒刺突(s)蛋白抗原和相关疫苗

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GUO Y.J. ET AL: "Cloning of SARS coronavirus S2 gene and immunization effect of its DNA vaccine", ACAD J SEC MIL MED UNIV, vol. 24, no. 7, July 2003 (2003-07-01), pages 707 - 709 *
HOU W. ET AL: "Homologous analysis of SARS coronavirus spike protein encoding gene and amino acid sequence", ACTA ACADEMIAE MEDICINAE QINGDAO UNIVERSITY, vol. 39, no. 2, June 2003 (2003-06-01), pages 114 - 117 *
YP Y. ET AL: "Contruction of DNA vaccine against severe acute respiratory syndrome", LETTERS IN BIOLOGY, vol. 14, no. 3, May 2003 (2003-05-01), pages 200 - 201 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111505286A (zh) * 2020-04-28 2020-08-07 郑州伊美诺生物技术有限公司 一种新型冠状病毒特异性抗体双抗原夹心elisa检测试剂盒及其制备方法
CN112538105A (zh) * 2020-06-29 2021-03-23 斯克里普斯研究院 稳定的冠状病毒刺突(s)蛋白抗原和相关疫苗
CN112538105B (zh) * 2020-06-29 2022-04-12 斯克里普斯研究院 稳定的冠状病毒刺突(s)蛋白抗原和相关疫苗

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