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Enterovirus 71 type virus strain, vaccine, animal model establishment method

A technology of enteroviruses and animal models, applied in the direction of antiviral immunoglobulins, microbial-based methods, biochemical equipment and methods, etc., can solve the differences in cross-protection levels of virus strains, unclear pathogenic mechanisms, and the development of EV71 vaccines Difficulties and other problems, to achieve effective immune activity and protection against viruses, good cross-immunogenicity, and easy control of the production process

Active Publication Date: 2015-01-14
NAT VACCINE & SERUM INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the possible differences in antigenicity and immunogenicity among strains of different genotypes, between strains of the same genotype and different genotype subtypes, and even between different strains of the same genotype subtype, the cross-protection level of different strains may be different , which poses a severe challenge to the selection of vaccine strains; although EV71 infection has been popular for many years, EV71 infection has broken out many times in recent years and caused more severe cases and even death cases, and its pathogenic mechanism is still unclear; these make EV71 There are certain difficulties in the development of vaccines

Method used

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  • Enterovirus 71 type virus strain, vaccine, animal model establishment method
  • Enterovirus 71 type virus strain, vaccine, animal model establishment method
  • Enterovirus 71 type virus strain, vaccine, animal model establishment method

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preparation example Construction

[0053] Another aspect of the present invention provides a vaccine preparation method, which includes: culturing EV71 virus strain 1 or EV71 virus strain 2, inactivating and purifying, thereby preparing the vaccine. Preferably, said purification is performed before said inactivation and / or after said inactivation.

[0054] Preferably, the preparation method of the present invention comprises:

[0055] 1) Provide Vero cells or human diploid cells;

[0056] 2) culturing EV71 virus strain 1 or EV71 virus strain 2 in the Vero cells or human diploid cells obtained in step 1), thereby obtaining a virus suspension;

[0057] 3) purifying and inactivating the virus suspension obtained in step 2), so as to obtain the vaccine stock solution; and

[0058] 4) Diluting the vaccine stock solution obtained in step 3) to obtain the vaccine.

[0059] Herein, Vero cells refer to passaged African green monkey kidney cells, which can be obtained from ATCC; human diploid cells are derived from no...

Embodiment 1

[0081] Embodiment 1: the separation and identification method of EV71 virus strain 1

[0082] (1) Isolation method of EV71 virus strain 1

[0083] Candidate strains must have complete records, history, and sources. Determine the biological characteristics of candidate virus strains. The inactivated vaccines prepared by candidate virus strains should have high virus yield, strong ability to induce immune protection, broad cross-protection spectrum, and stable biological characteristics, and should be based on epidemiology and molecular epidemiology Selected virus strains with broad current and future epidemic potential. Animals are immunized with candidate viruses, serum is separated, serum neutralizing antibody titers are determined, and viruses with a broad protective spectrum and strong ability to induce immune protection are screened as inactivated vaccine virus strains.

[0084] Throat swab specimens were collected from a patient with HFMD (Huang XX, a 4-year-old HFMD ch...

Embodiment 2

[0089] Embodiment 2: Purification and passage stability analysis of EV71 virus strain 1

[0090] (1) Plaque purification

[0091] The EV71 virus suspension obtained in Example 1 was serially diluted, and after the cells were observed to grow into a single layer under a microscope, the original culture medium in the six-hole cell culture plate was discarded, and 0.1-0.5ml / hole of the virus suspension was added, 36±1 Adsorb at ℃ for 1-2 hours. After the adsorption is over, pour off the liquid in the plate, wash once with sterile PBS, add a cover, and culture in a carbon dioxide incubator at 36±1℃. 3-7 days after virus inoculation, observe the formation of plaques under a microscope, pick plaques, and further purify plaques, and then purify twice in this way.

[0092] (2) Passage stability analysis of the virus

[0093] Taking Vero cells as an example, the EV71 virus strain was selected for continuous passage on Vero cells, harvested when the lesion was 75% to 90%, and the tite...

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Abstract

The invention provides an Enterovirus 71 (EV71 for short) type virus strain, a vaccine, and an animal model establishment method. The preservation number of the virus strain is CGMCC No.5539. The EV71 type virus strain provided in the invention has the advantages that: it has strong toxicity, and can be used for evaluation of an EV71 vaccine or drug. The establishment method of the animal model for evaluation of the EV71 vaccine effect provided in the invention can provide a stable animal model, thus providing the basis for development of the EV71 vaccine and screening of antiviral drugs. The vaccine prepared from the EV71 virus strain is in accordance with the relevant requirements of ''Chinese Pharmacopoeia (three parts) 2010 edition'', and has effective immunoactivity and toxicity attack protection capability. The preparation method of the vaccine has the advantages of easily controllable production process, good repeatability, and suitability for large-scale production, and the prepared vaccine has stable quality.

Description

technical field [0001] The invention belongs to the field of vaccine production, in particular, relates to an enterovirus 71 virus strain and its application, a vaccine prepared by the enterovirus 71 virus strain and a preparation method, and a method for evaluating enterovirus 71 Establishment method of animal model of vaccine effect. Background technique [0002] Hand, foot and mouth disease (HFMD) is an acute infectious disease caused by enterovirus infection. It mostly occurs in infants under 5 years old. Herpes and ulcers are typical manifestations, and a small number of patients can cause complications such as myocarditis, pulmonary edema, aseptic meningitis, and encephalitis. There are more than 20 kinds of enteroviruses (types) that cause HFMD, among which Coxsackie virus A16 (CA16) and enterovirus 71 (hereinafter referred to as EV71) are the most common, among which EV71 not only causes HFMD, And can cause serious central nervous system complications, such as meni...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K39/125A61P31/14C07K16/10C07K16/06C12N5/16A01K67/027C12R1/93
Inventor 李秀玲郝春生王潇潇张中洋郭会杰陈蕾吴蕴怡李懿张晨刘宇
Owner NAT VACCINE & SERUM INST
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