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Coxsackie virus a16 type virus strain, application, vaccine and preparation method thereof

A Coxsackie virus and virus strain technology, applied in the direction of antiviral immunoglobulin, biochemical equipment and methods, antiviral agents, etc., can solve the problem of large gaps, differences in antigenicity and immunogenicity, and cross-protection of different strains Level differences and other issues, to achieve the effect of strong toxicity and effective immune activity

Active Publication Date: 2020-02-18
NAT VACCINE & SERUM INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reasons are as follows: First, 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, resulting in different strains There may be differences in the level of cross-protection of the vaccine, which poses a serious challenge to the selection of vaccine strains
Secondly, although CA16 infection has been prevalent for many years, there have been many outbreaks of CA16 infection in recent years, and its pathogenic mechanism is still unclear; these all make the development of CA16 vaccine difficult.
Again, although the current patent literature discloses that the CA16 virus strain has a high serum antibody titer when detected by the neutralization test, the neutralization test uses RD cells that are not sensitive to the CA16 virus as neutralization cells, and the neutralization test The neutralization time is 3 days, which is quite different from the conventionally recognized neutralization time of 6-7 days. The result is judged when the virus has not fully replicated and grown. The reliability of the serum neutralizing antibody results through three immunizations is questionable

Method used

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  • Coxsackie virus a16 type virus strain, application, vaccine and preparation method thereof
  • Coxsackie virus a16 type virus strain, application, vaccine and preparation method thereof
  • Coxsackie virus a16 type virus strain, application, vaccine and preparation method thereof

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preparation example Construction

[0051] The fourth aspect of the present invention provides the preparation method of the vaccine, which includes: cultivating the A16 strain of Coxsackievirus, inactivating and purifying it, so as to prepare the vaccine. Preferably, said purification is performed before said inactivation and / or after said inactivation.

[0052] Preferably, the preparation method of the present invention comprises:

[0053] 1) Provide Vero cells or human diploid cells;

[0054] 2) In the Vero cells or human diploid cells obtained in step 1), the Coxsackievirus A16 strain is cultured to obtain a virus suspension;

[0055] 3) purifying and inactivating the virus suspension obtained in step 2), so as to obtain the vaccine stock solution; and

[0056] 4) Diluting the vaccine stock solution obtained in step 3) to obtain the vaccine.

[0057] Preferably, those skilled in the art can prepare the Coxsackie virus A16 type inactivated vaccine by conventional methods as required, wherein the routine pr...

Embodiment 1

[0079] Embodiment 1: Isolation of CA16 virus strain

[0080]Collect throat swab specimens from patients with HFMD (isolated from Feng XX, a 3-year-old child with severe HFMD in Shijiazhuang City, Hebei Province), centrifuge at 2000-4000rpm / min for 30min at 4°C, and take the supernatant with 0.2 Sterilize with a μm filter, and inoculate Vero cells or human diploid cells with the filtered supernatant. Before inoculating the specimen, pour off the growth solution, and each bottle of cells (8×10 6 Each / bottle) inoculated with 0.2-1ml of sample suspension, cultured at 36±1°C; after 1-2 hours of adsorption, replaced with 10ml of DMEM medium containing 2% (v / v) bovine serum to continue the culture. Use an inverted microscope to observe the cytopathic changes day by day. If there is no cytopathic effect (CPE) in 7 days, continue to observe for 7 days until 75% to 90% of the cells change, and then store them at -20°C for secondary use. Subpassaging. When suspicious cell lesions are ...

Embodiment 2

[0081] Embodiment 2: the identification method of CA16 virus strain

[0082] (1) Morphological observation: The lesions of the CA16 virus strain of Example 1 on the Vreo cells were observed under an optical microscope, and the cells were rounded and dispersed, and the particles in the cytoplasm increased. After concentration by ultrafiltration and negative staining with 2% phosphotungstic acid solution, spherical virus particles with a diameter of about 30nm can be observed under an electron microscope.

[0083] (2) Immunological test identification: After neutralizing the virus culture fluid of CA16 virus strain and the same amount of anti-CA16 immune serum at 36±1°C for 1 to 2 hours, inoculate it on a cell culture plate, continue culturing for 7 days, and observe the lesions The number of cell wells, set serum and cell controls at the same time, the titer of the virus control should not be lower than 500CCID 50 / ml. The results showed that all the wells of the virus contro...

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Abstract

The invention provides a Coxsackie virus A16 virus strain, uses of the strain, a vaccine and a preparation method of the vaccine. The preservation number of the Coxsackie virus A16 virus strain is CGMCC No.6954. The CA16 virus strain has strong virulence, can be used for evaluating the CA16 vaccine, and can also be used for researching the CA16 virus infection mechanism. A method for establishing a Coxsackie virus A16 infected animal model provided by the invention can provide a stable animal model, and provides a foundation for the development of the Coxsackie virus A16 vaccine, the screening of antiviral drugs and the researches of the CA16 virus infection mechanism. The vaccine prepared by using the CA16 virus has effective immune activity.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, relates to a Coxsackie virus A16 strain and its use, a vaccine for preventing and / or treating hand, foot and mouth disease and its preparation method, antibody or hybridoma cell or anti- The serum and its preparation method, and the establishment method of an animal model infected by Coxsackie virus A16. Background technique [0002] Hand, foot and mouth disease (HFMD) is an acute infectious disease caused by enterovirus infection. Rash, herpes, and ulcers are typical manifestations, and a small number of patients may cause complications such as myocarditis, pulmonary edema, aseptic meningitis, and encephalitis. There are more than 20 enteroviruses (types) that cause HFMD, among which Coxsackievirus A16 (Coxsackievirus A16, CA16) and enterovirus 71 (Enterovirus71, EV71) are the most common. HFMD was first reported in New Zealand in 1957, and the Coxsackie virus was isolated in 1958. Th...

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 李秀玲郝春生李懿王潇潇杨永娟郭会杰张冲陈明张晨
Owner NAT VACCINE & SERUM INST
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