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Coxsackie virus A16-type virus strain and applications thereof

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 stable titer, good immunogenicity and efficient proliferation

Active Publication Date: 2014-03-26
SINOVAC BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 the virus is one of the more effective prevention methods at present. There is no CA16 virus vaccine yet Therefore, it is of great economic and social benefit to isolate the CA16 virus strain suitable for vaccine production

Method used

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  • Coxsackie virus A16-type virus strain and applications thereof
  • Coxsackie virus A16-type virus strain and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0019] (1) Virus isolation and culture

[0020] The fecal 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 93.8 %above.

[0024] 2. Electron microscope inspection results

[0025] The ...

Embodiment 2

[0034] The preparation of embodiment 2CA16 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, and 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% C...

Embodiment 3CA16

[0050] Embodiment 3CA16 virus 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-...

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Abstract

The invention provides a coxsackie virus A16-type virus strain. The collection number of the coxsackie virus A16-type virus strain is CGMCC No.5372, wherein CGMCC refers to China General Microbiological Culture Collection Center. The virus is a 20-face stereoscopic symmetrical sphere under observation through an electron microscope, and the diameter of the virus is 23-30nm. VP1 conserved region sequence analysis and mass spectrum analysis are respectively performed on the virus strain, and a result shows a CA16 virus. The CA16 virus can be efficiently proliferated in Vero cells (African green monkey kidney cells), and the virus titer can reach 6.61g CCID50 / ml. Moreover, the virus strain has no external pollution, better immunogenicity and a good effect.

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 mouth disease (HFMD) is an infectious disease caused by enterovirus, which mostly occurs in children under 5 years old, and can cause herpes on the hands, feet, mouth and other parts, and a small number of children can cause pulmonary edema, asymptomatic Bacterial meningoencephalitis and other complications. In some severely ill children, if the disease develops rapidly, it will lead to death. There are more than 20 types (types) of enteroviruses that cause hand, foot and mouth disease. Coxsackievirus types 16, 4, 5, 9, and 10 in group A, types 2 and 5 in group B, and enterovirus type 71 are all It is the most common pathogen of hand, foot and mouth disease. It is mainly transmitted through fecal-oral, respiratory and close contact, and can be transmi...

Claims

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Application Information

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