A16 type strain of Coxsackie virus and application of the strain

A technology of coxsackie virus and virus strain, applied in the direction of antiviral agent, virus/phage, antiviral immunoglobulin, etc., to achieve the effect of small immune dose, stable titer and good immunogenicity

Active Publication Date: 2012-07-11
SINOVAC BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The outbreak and prevalence of hand, foot and mouth disease have seriously affected people's daily life, causing huge economic losses and social burdens. The use of virus vaccines to fundamentally cut off the spread of t

Method used

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  • A16 type strain of Coxsackie virus and application of the strain
  • A16 type strain of Coxsackie virus and application of the strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Isolation, cultivation and identification of embodiment 1 CA16 virus strain

[0019] (1) Virus isolation and culture

[0020] The stool samples of patients with positive CA16 virus PCR results were collected from the HFMD epidemic area in Zhejiang Province in 2010 (the cumulative number of HFMD cases reached 111,783). After treatment, they were inoculated onto African green monkey kidney passage cells (Vero), and cultured for three generations for virus isolation. , after the CA16 virus was obtained, the CA16 virus strain was obtained by the plaque method.

[0021] (2) Virus identification

[0022] 1. Sequence analysis results of VP1 conserved region

[0023] The VP1 conserved region sequence of the strain was analyzed, and compared with the sequence of the reference strain (human Coxsackievirus A16 strain shzh00-1, complete genome sequence GenBank: AY790926.1), the nucleotide homology was 94.8 %above.

[0024] 2. Electron microscope inspection results

[0025] The...

Embodiment 2

[0034] The preparation of embodiment 2 CA16 vaccine

[0035] After the CA16 virus strain infects Vero cells, cultures, harvests the virus liquid, inactivates and purifies, a vaccine stock solution is obtained for further preparation of the CA16 vaccine.

[0036] (1) Establish cell master seed and working seed bank (Vero cell)

[0037] Resuscitate the seeds of 120-generation African green monkey kidney cells (Vero cells) from ATCC in the United States. The specific operation is: take out the cell cryopreservation tube from liquid nitrogen, place it in sterile water at 39-40°C, and thaw the cells within one minute , aseptically suck out the suspension, centrifuge at 1000rpm for 3min, discard the supernatant, add MEM cell growth medium containing 10% calf serum, mix gently by blowing, inoculate the mixed cell suspension in 25cm 2 in a cell culture flask at 37°C, 5% CO 2 Cultivate in an incubator, change the medium after it adheres to the wall, and then place it at 37°C, 5% CO ...

Embodiment 3

[0050] Example 3 CA16 strain immunogenicity test

[0051] Purify the stock solution of the CA16 strain vaccine prepared in Example 2, and after inactivation, immunize mice, New Zealand rabbits, and sheep to obtain better protective effects.

[0052] Stock solution preparation method is described with embodiment 2.

[0053] Among them, the immunogenicity test on sheep is as follows:

[0054] After absorbing the vaccine stock solution and the aluminum hydroxide adjuvant in equal proportions, the sheep were immunized on the 0th day, 7th day, 14th day, and 21st day, 2ml / head / time, and the health status, Behavioral changes, etc., are documented in detail. Animals should be observed for half an hour on the day of immunization. Animals were observed twice daily for mortality. Blood was collected on day 0, day 7, day 14, day 21 and day 28. A small amount of blood was collected venously, centrifuged at 3000rpm for 10min, and the serum was separated. Determination of anti-CA16 neu...

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Abstract

The invention provides an A16 type strain of Coxsackie virus, which has the preservation number of CGMCC No. 5373. When observed with an electronic microscope, the virus is in the shape of an icosahedral three-dimensional symmetrical sphere with the diameter of 23-30nm. VP1 conserved region sequence analysis and mass spectrum analysis are respectively conducted for the strain, and the results indicate that the strain is CA 16 virus which can be efficiently proliferated in Vero cells, and the virus titer can reach 7.01g CCID50/ml, and furthermore, the strain is free of extraneous contamination, and has good immunogenicity and excellent effects.

Description

technical field [0001] The invention relates to the fields of virology and molecular biology technology, in particular to a new Coxsackievirus A16 virus strain and application thereof. Background technique [0002] Hand, foot and mouth disease is a global infectious disease, and there are reports of the prevalence of this disease in most parts of the world. It was first reported in New Zealand in 1957, Coxsackie virus was isolated in 1958, and HFMD was named in 1959. The pathogen of HFMD found in the early stage is mainly Cox A16 type, and the report related to HFMD and EV 71 infection began in the early 1970s, and EV 71 was confirmed for the first time in the United States in 1972. Since then, EV 71 infection and Cox A16 infection have appeared alternately, becoming the main pathogen of HFMD. It mostly occurs in children under 5 years old and can cause herpes on the hands, feet, mouth and other parts. A small number of children can cause complications such as pulmonary ed...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/125A61P31/14C07K16/10C07K16/06C12N5/16G01N33/569C12R1/93
Inventor 高强李雅静王巍巍尹卫东
Owner SINOVAC BIOTECH
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