Conservative neutralizing epitope polypeptide of Coxsackievirus A16 and application thereof

A Coxsackie virus, A16 technology, applied in antiviral agents, antiviral immunoglobulins, medical preparations containing active ingredients, etc., can solve the problem of unidentified neutralizing epitopes

Inactive Publication Date: 2014-06-04
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the neutralizing epitope of

Method used

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  • Conservative neutralizing epitope polypeptide of Coxsackievirus A16 and application thereof
  • Conservative neutralizing epitope polypeptide of Coxsackievirus A16 and application thereof
  • Conservative neutralizing epitope polypeptide of Coxsackievirus A16 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1 Materials and methods

[0041] 1.1 Cells, viruses and serum

[0042] For the cultivation of RD cells and VERO cells, please refer to: Liu Q, Ku Z, Cai Y, Sun B, Leng Q, Huang Z. Detection, characterization and quantitation of coxsackievirus A16 using polyclonal antibodies against recombinant capsid subunit proteins. J Virol Methods 2011 Apr;173(1):115-20.

[0043] For the strain CA16-SZ05 (GeneBank ID: EU262658), please refer to: (1) Wu Z, Gao Y, Sun L, Tien P, Jin Q. Quick identification of effective small interfering RNAs that inhibit the replication of coxsackievirus A16. Antiviral Res 2008 Dec;80(3):295-301 and (2) Yang F, Jin Q, He Y, Li L, Hou Y. The complete genome of Enterovirus 71 China strain. Sci China C Life Sci 2001 Apr;44( 2): 178-83.

[0044] Strain CA16-G08 was isolated from a fecal sample of a HFMD patient. For the isolation procedure, please refer to the literature: Li L, He Y, Yang H, Zhu J, Xu X, Dong J, Zhu Y, Jin Q. Genetic characterization of...

Embodiment 2

[0085] 1. Antigen: KLH-coupled synthetic polypeptide, the amino acid sequence of which is as described in SEQ ID NO.1-6.

[0086] 2. Immunization of mice: purebred BALB / C mice, aged 6-8 weeks. 50 μg KLH-conjugated synthetic peptide was injected intraperitoneally, and Freund's complete adjuvant was used for initial immunization. The same antigen dose was immunized with Freund's incomplete adjuvant every two weeks, and immunized three times in total. Mice were sacrificed two weeks after the last immunization for blood collection. All animal operations complied with the IACUC regulations of the Shanghai Pasteur Institute. Blood was collected from the mice during the immunization process, and ELISA was used to detect the antibody expression in the mouse serum, so as to judge the immune effect of the mice.

[0087] 3. Fusion with SP2 / 0 myeloma cell line: kill the immunized mice, and take splenocytes. Myeloma cells SP2 / 0 and mouse splenocytes were mixed at a ratio of 1:10, and ...

Embodiment 3

[0092] 1. Selection and acquisition of antigen: Synthesize the polypeptide with amino acid sequence as described in SEQ ID NO.1-6, add His tag to the nucleic acid sequence corresponding to the polypeptide, and construct it into the prokaryotic expression vector Pet28a. The expression vector of the polypeptide was transformed into Escherichia coli BL21 for expression, and the soluble protein supernatant was purified with a nickel column. The concentration of the peptide obtained after purification was measured, and the peptide was detected by western detection with His antibody. The western test is positive, ready to immunize animals;

[0093] 2. Animals to be immunized: Two New Zealand rabbits were selected, each time immunized with 200 μg of immunogen, Freund's complete adjuvant was used in the initial stage, and incomplete adjuvant was used in the later stage. Immunization was carried out at 0, 2, 4, 5, 6, 7, 8, and 9 weeks respectively, and from the 5th week, venous blood ...

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Abstract

The invention relates to a conservative neutralizing epitope polypeptide of Coxsackievirus A16 and application thereof. Specifically, the invention provides the neutralizing epitope polypeptide of Coxsackievirus A16, and also provides a binding molecule able to specifically bind with the neutralizing epitope polypeptide, antiserum containing the binding molecule, a kit for detecting the Coxsackievirus A16, a detection method and a composition inhibiting the Coxsackievirus A16. The invention also provides application of the neutralizing epitope polypeptide, the binding molecule and the antiserum in preparation of drugs for preventing and treating hand, foot and mouth disease and diagnostic agents for diagnosing hand, foot and mouth disease. According to the invention, 6 linear neutralizing epitopes on CA16VP1 are identified for the first time, the comparison result of different genotype CA16 sequences shows that the epitopes are very conservative, and the serum brought about by immunizing mice with the neutralizing epitope polypeptide can neutralize homologous and heterogenous CA16 strains. The results have important guiding significance on development of Coxsackievirus A16 vaccines and diagnostic kits.

Description

technical field [0001] The present invention relates to the epitope of antigen and its application, specifically, the conserved neutralizing epitope polypeptide of Coxsackie virus A16 type and its application. Background technique [0002] Coxsackievirus A16 (Coxsackievirus A16, CA16, CVA16) belongs to the Enterovirus genus of the picornaviridae family. The viral genome is a single-stranded positive-strand RNA of about 7410bp, which contains an open reading frame encoding a long peptide chain. The long peptide chain Divided into structural protein P1 and non-structural protein P2 and P3 regions, P1 is cleaved into viral capsid proteins VP1, VP0 and VP3 by viral protease; part of VP0 is further self-cleaved into VP2 and VP4. [0003] CA16 is a major causative agent of hand, foot and mouth disease (HFMD), which is common in children. Most patients with CA16 infection have mild clinical symptoms, and most of them can recover by themselves, including fever, oral ulcers, and red...

Claims

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Application Information

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IPC IPC(8): C07K7/08A61K39/125A61P31/14G01N33/68C07K16/10A61K39/42G01N33/569C07K16/06
Inventor 黄忠石金平
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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