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Coxsackievirus A16-type virus strain and use thereof

A Coxsackie virus and virus strain technology, applied in the fields of molecular biology and virology, to achieve stable titer, good immunogenicity, and efficient proliferation

Active Publication Date: 2014-05-21
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, the isolation of CA16 virus strains suitable for vaccine production has huge economic and social benefits

Method used

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  • Coxsackievirus A16-type virus strain and use thereof
  • Coxsackievirus A16-type virus strain and use thereof
  • Coxsackievirus A16-type virus strain and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Isolation, cultivation and identification of embodiment 1CA16 virus strain

[0024] (1) Virus isolation and culture

[0025] 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.

[0026] (2) Virus identification

[0027] 1. VP1 full-length sequence analysis results

[0028] The VP1 protein produced by the strain was analyzed for the full-length sequence of VP1, and its amino acid sequence is shown in SEQ ID NO.2, and the gene sequence encoding the VP1 protein is shown in SEQ ID NO.1.

[0029] 2. Electron microscope inspection results

[0030] The strain CA16 virus was observed under an ele...

Embodiment 2

[0039] Embodiment 2 detects the primer and the kit of CA16 virus strain

[0040] 1. Primers for detecting the CA16 virus strain, including:

[0041] CA16 upstream primer: 5'-TTGCAGACATGATTGACCAG-3' and

[0042] CA16 downstream primer: 5'-GAGTGATGGTTCAACACACA-3'.

[0043] 2. A detection kit for detecting the CA16 virus strain containing the above primers.

Embodiment 3

[0044] The preparation of embodiment 3CA16 vaccine

[0045] 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.

[0046] (1) Establish cell master seed and working seed bank (Vero cell)

[0047] To resuscitate the 120th generation African green monkey kidney cells (Vero cells) seeds 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, thaw the cells within one minute, without Bacteria aspirated the suspension, centrifuged at 1000rpm for 3min, discarded the supernatant, added MEM cell growth medium containing 10% calf serum, mixed gently by blowing, and inoculated 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...

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Abstract

The invention provides a coxsackievirus A16-type virus strain and a use thereof. The coxsackievirus A16-type virus strain has the preservation number of CGMCC No. 5371. The full-length sequence analysis and the mass spectrometry analysis on a VP1 protein produced by the coxsackievirus A16-type virus strain prove that the oxsackievirus A16-type virus strain is a good CA16 virus strain which is not polluted by allothigenes and has good immunogenicity. The CA16 virus strain can efficiently proliferate in Vero cells and has virus titer of 7.41g CCID 50 / ml. The CA16 virus strain or a vaccine prepared from the CA16 virus strain can be used for preventing diseases caused by CA16 viruses, and has the characteristics of stable titer, good immunogenicity and less immunizing dose.

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 (Hand foot mouth disease, HFMD) is a global infectious disease, which has been reported in most parts of the world. It is common in infants and young children and is caused by enteroviruses. Clinically, the main manifestations are fever, rashes and ulcers on the hands, feet, mouth and other parts, which are related to comprehensive diseases such as myocarditis, pericarditis and refractory shock. [0003] Coxsackievirus A16 type (Cox A16, CA16) is one of the main pathogens of hand, foot and mouth disease. It is a single-stranded positive-sense RNA virus of the Picornaviridae family. It is mainly transmitted through fecal-oral, respiratory and close contact, and can be transmitted to the fetus through the placenta to cause intrauterine infec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C07K14/085C12N9/12C12N15/41C12N15/54C12N15/63C12N5/10G01N33/68A61K39/125A61P31/14C07K16/10C07K16/06C12N5/16C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 高强李雅静王巍巍尹卫东
Owner SINOVAC BIOTECH
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