Coxsackie virus A16 type virus-like particle vaccine

A coxsackie virus, A16 technology, applied in the field of biotechnology and biomedicine, can solve problems such as an ideal vaccine without coxsackie virus

Inactive Publication Date: 2012-05-23
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

[0005] However, there is currently no effective

Method used

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  • Coxsackie virus A16 type virus-like particle vaccine
  • Coxsackie virus A16 type virus-like particle vaccine
  • Coxsackie virus A16 type virus-like particle vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] Embodiment 1, the construction of baculovirus

[0118] The gene fragments encoding P1 and 3CD of CVA16 / shzh05 were amplified and synthesized by reverse-transcribing the first-strand cDNA, and then cloned into the pFBD vector, named pFBD-CVP1 and pFBD-CV3CD, respectively. The CVP1 gene is regulated by the polyhedrin promoter in pFBD-CVP1, and the CV3CD gene is directly regulated by the p10 promoter in pFBD-CV3CD. pFBD-CVP1 / 3CD loaded with two genes at the same time ( figure 1 ) was also successfully constructed, which can co-express two proteins efficiently in the same kind of cells.

[0119] The above three structures were subsequently constructed into corresponding baculoviruses, named Bac-CVP1, Bac-3CD, and Bac-CVP1 / 3CD, respectively.

Embodiment 2

[0120] Example 2, expression of CVA16VLPs in insect cells

[0121] Sf9 insect cells were infected with Bac-CVP1, Bac-3CD and Bac-CVP1 / 3CD respectively. Firstly, the polyclonal antibody against-CVA16VPO was used to detect the expression of the target protein in infected insect cells by immunofluorescence method. The result is as figure 2 As shown in A, Bac-CVP1 and Bac-CVP1 / 3CD had bright green fluorescence; no fluorescent signal was detected in the blank group and Bac-3CD infected group. The expression of Bac-CVP1 in insect cells was also confirmed by ELISA experiments: the anti-CVA16 VP0 antibody detected the expression of Bac-CVP1 and Bac-CVP1 / 3CD cell lysis, but the blank group and Bac-3CD infection group did not detect to the signal ( figure 2 B).

[0122] Secondly, the anti-VP0 antibody Western blot analyzed the shearing process of P1 by 3CD. like figure 2 As shown in C, no bands were detected in the blank group and Bac-3CD infection group, but the lysate of Bac-...

Embodiment 3

[0125] Example 3, Immunogenicity of CVA16 virus-like particles in mice

[0126] Animal experiments were used to evaluate the immunogenicity of CVA16 virus-like particles. The crude purified virus-like particles were injected intraperitoneally at 1ug / mouse at 0, 3 and 6 weeks, with aluminum adjuvant as the adjuvant, and uninfected Sf9 cells treated in the same way as comparison. The mouse serum was collected two weeks after the last immunization, and the specific antibody was detected by recombinant antigen-coated ELISA. The results showed that the serum of the VLP immune group could react with the recombinant subunit coat protein, while the control Sf9 serum did not react ( Figure 5 ).

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Abstract

The invention relates to a coxsackie virus A16 type virus-like particle vaccine. The inventor fortuitously finds that proteins which have better spatial configurations and are suitably cut can be obtained by expressing P1 protein of CVA16 and 3CD protein of CVA16 by infecting insect cells with rhabdovirus. The proteins can be automatically assembled into a virus-like particle which has high immunogenicity.

Description

technical field [0001] The invention belongs to the fields of biotechnology and biomedicine; more specifically, the invention relates to a Coxsackievirus A16 virus-like particle vaccine. Background technique [0002] Hand, foot and mouth disease is a common disease in children, and there is still no effective vaccine and treatment. Coxsackievirus A16 (CVA16) and human enterovirus 71 (EV71) are the two most important pathogens of hand, foot and mouth disease, belonging to the family Picornaviridae Enterovirus, single-stranded positive-sense RNA virus with a genome of about 7400bp (Xu , J., Y. Qian, S. Wang, J.M. Serrano, W. Li, Z. Huang, and S. Lu. 2010. EV71: anemerging infectious disease vaccine target in the Far East Vaccine 28: 3516-21 and Zhang, Y ., D. Wang, D. Yan, S. Zhu, J. Liu, H. Wang, S. Zhao, D. Yu, L. Nan, J. An, L. Chen, H. An, A. Xu, and W. Xu. 2010. Molecular evidence of persistentepidemic and evolution of subgenotype B1 coxsackievirus A16-associated hand, ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N7/01C12N7/00A61K39/125A61P31/14
Inventor 黄忠刘庆伟蔡一村
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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