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Enterovirus type 71 and use thereof

A virus strain, strain technique, applied to a virus that can be used for prophylaxis, force, monoclonal virus strain, and/or purification. ,The field of virus strains for treating or resisting hand, foot and mouth disease can solve the problem of not found HFMD (hand foot and mouth disease, etc.

Active Publication Date: 2010-06-02
SINOVAC BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no cases of HFMD (hand, foot and mouth disease) were found in this outbreak

Method used

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  • Enterovirus type 71 and use thereof
  • Enterovirus type 71 and use thereof
  • Enterovirus type 71 and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: the cultivation of Vero cell (ATCC CCL81)

[0049] Cell resuscitation method: take the cell seeds out of the liquid nitrogen, centrifuge at 2000 rpm for 5 min, and resuspend the cells with resuscitation solution (MEM medium containing 20% ​​calf serum). The resuspended cells were approximately 1.0 x 10 5 Cell / CM 2 The amount of was inoculated into an empty cell culture flask and incubated overnight (about 16 hr) at 37°C. Then, the growth medium (MEM medium containing 5% calf serum) was replaced, and the cells were continued to be cultured to a monolayer and then passaged.

[0050] The cell subculture method is as follows: the monolayer cells are digested with digestive solution (0.25% trypsin (containing 0.03% EDTA)), and inoculated according to the split ratio of 1:5 (the number of cells is about 1.5-2.0×10 5 ), cultured at 37°C for 2 days for virus subculture, and cultured for 4 days at 37°C for cell subculture. The pH was controlled at 7.2-7.4 durin...

Embodiment 2

[0051] Example 2: Virus Culture

[0052] In the HFMD epidemic area (Fuyang, Anhui) in 2008, throat swab specimens from clinically confirmed typical HFMD patients were collected, frozen at -20°C immediately, and transported to the laboratory for isolation of EV71 virus.

[0053] Thoroughly agitate the throat swab in the specimen preservation solution (at least 40 times) to wash off the virus and virus-containing cells attached to the swab, then centrifuge at 10,000rpm for 20min at 4°C, and filter it out with a 0.22um filter. After bacteria, it was inoculated into the Vero cells cultured in the above-mentioned Example 1 for 2 days to inoculate the virus, and the cell density reached about 80%. Harvest cultures when cytopathic changes (CPE) reach +++ (1+, <25%; 2+, 25%-50%; 3+, 50%-75%; 4+, 75%-100%) , Freezing at -20°C, and then thawing at room temperature, thereby destroying the cells and releasing the virus particles from the cells. Afterwards, the virus particles were harve...

Embodiment 3

[0054] Example 3: Plaque Purification

[0055] The virus seed obtained in embodiment 2 is diluted to 10CCID 50 . The diluted virus seeds were then inoculated onto 6-well plates overgrown with Vero cells for plaque purification.

[0056] Specifically, wash the cell plate twice with PBS, inoculate 1.0ml of virus in each well, absorb at 35°C for 120min, shake the 6-well plate once every 30min, and wash the plate twice with PBS; Mix and prepare nutrient agar, control the temperature at about 45°C, then add nutrient agar to 6-well plate, add 3.0ml to each well. Next, place the 6-well plate at 35°C, 5% CO 2 Cultured in the incubator for 96hr.

[0057] Then, mix equal volumes of liquid A and liquid C, control the temperature at about 45°C, add 3.0ml of staining to each well, and then place the 6-well plate at 35°C, 5% CO 2 Cultured in the incubator for 96hr. Turn the 6-well plate upside down and observe white spots on the bottom of the culture plate. Circle the single circular ...

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Abstract

The invention relates to an enterovirus type 71 (abbreviated as EV71) and use thereof, in particular to a monoclonal virus strain which is separated by a plaque assay method. The progeny viruses of the monoclonal virus strain show genetic stability. The monoclonal virus strain can be used in the production of a vaccine (particularly the production of a vaccine for infant). The virus strain or the vaccine produced by using the same can be used in the prevention of diseases (such as hand-foot-and-mouth diseases, particularly hand-foot-and-mouth diseases in children) caused by the EV71 and has the characteristics of stable titer, high immunogenicity and small immunizing dose.

Description

[0001] The invention relates to a strain of enterovirus 71 (hereinafter referred to as EV71) and its application. Specifically, the present invention relates to a monoclonal virus strain isolated by a plaque method, whose progeny virus is genetically stable and can be used in the production of vaccines for humans (especially vaccine production for infants). The virus strain of the present invention or the vaccine produced by it can be used to prevent diseases caused by EV71 virus (such as hand, foot and mouth disease, especially children's hand, foot and mouth disease), and has stable titer, strong immunogenicity, and small immune dose. features. Background technique [0002] EV71 is a member of the genus Enterovirus in the Picornaradae family and belongs to human Enterovirus A. The particles of the virus are icosahedral three-dimensionally symmetrical spherical structures, without envelopes and protrusions, with a diameter of about 24-30 nm, and the nucleic acid is single-st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/125A61K35/76A61P31/14C07K16/10C12N15/02C12N5/12C12R1/93A61K35/765
Inventor 高正伦张小梅高强刘玉芬惠增弟张卉公雪杰张建三尹卫东
Owner SINOVAC BIOTECH
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