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Yeast-expressed coxsackie A6 virus-like particles and application thereof

A virus and particle technology, applied in the field of biomedicine, can solve the problem of no vaccine for hand, foot and mouth disease

Active Publication Date: 2017-11-24
中国科学院上海免疫与感染研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is currently no vaccine available against HFMD

Method used

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  • Yeast-expressed coxsackie A6 virus-like particles and application thereof
  • Yeast-expressed coxsackie A6 virus-like particles and application thereof
  • Yeast-expressed coxsackie A6 virus-like particles and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Example 1 Expression and purification of VLP

[0123] In order to express CA6 VLP, the inventors inserted the 3CD and P1 genes together on the pPink-HC vector ( figure 1 A) Plasmid YCA6-003 was constructed, and then the plasmid was used to transform Pichia pastoris into competent yeast, and a small amount of expression was used to obtain the centrifuged supernatant of the cell lysate for ELISA and Western blotting analysis. Yeast clones transformed with empty vector pPink-HC were operated in parallel as negative controls. Compared with the control group, the yeast lysate supernatant transformed with plasmid YCA6-003 showed obvious antigen-antibody reaction ( figure 1 B). Pick the three most responsive ones from the yeast clones for Western blotting analysis, and the experimental results are shown in the figure ( figure 1 C), using anti-CA6VP1, anti-CA6VP0, and anti-CA6VP3 as detection antibodies, corresponding bands appeared in the three yeast lysate samples, with si...

Embodiment 2

[0125] Embodiment 2 mouse immunization

[0126] Before immunizing mice, CA6 VLP and control antigen were prepared and quantified ( image 3 A), followed by mixing with aluminum adjuvant. Two groups (6 mice / group) of ICR mice were injected intraperitoneally with CA6 VLP vaccine and control antigen at 0, 2 and 5 weeks respectively. Blood was collected at 7 and 9 weeks for ELISA analysis. Cover the plate with VP0, VP1, and VP3 mixture in equal proportions, and the ELISA results are as follows: image 3 As shown in B, among the 6 sera collected from the mice in the vaccine group, 5 showed obvious antigen-antibody reactions, while the control group had no obvious reactions. Notably, when coated with CA6 VLP, mice in all vaccine groups showed significant antigen-antibody responses ( image 3 C), and the geometric mean titer reached 17959 ( image 3 D). The above results not only prove that CA6 VLP has high immunogenicity, but also show that the antibody induced by CA6 VLP is m...

Embodiment 3

[0127] Example 3 In vitro cross-neutralization experiment of CA6-VLP serum

[0128] The obtained anti-CA6 serum was initially diluted 2 times at 1:16, and the results of cross-neutralization experiments on CA16, CA10 and EV71 viruses confirmed that CA6 serum had no neutralizing cross-effect on the three viruses at the lowest dilution.

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Abstract

The invention provides yeast-expressed coxsackie A6 virus-like particles and an application thereof. Specifically, the method provides a method for preparing the coxsackie A6 virus-like particles. According to the method, through expression of coxsackie A6 virus P1 protein and 3CD protein coding sequences, which are optimized by codon, in yeast cells, VLP (the virus-like particles) can be formed by automatic-assembling, and the VLP is high in expression amount and easy to purify; and the purified VLP is relatively strong in immunogenicity.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, the invention relates to yeast-expressed Coxsackievirus A6 virus-like particles and applications thereof. Background technique [0002] HFMD is a contagious disease that is widespread in the Asia-Pacific region. It mainly infects children under the age of five, causing symptoms such as fever, herpes on the hands, feet, mouth and buttocks, and coughing. A small number of children may develop severe neurological symptoms and cardiopulmonary system complications in a short period of time, and even die. However, there is currently no vaccine available against HFMD. [0003] Since 2008, enterovirus type 71 (EV71) and coxsackievirus type 16 (CA16) have become the two main pathogens of hand, foot and mouth disease due to their widespread spread, and vaccine research and development are mostly aimed at these two viruses. However, in recent years, there have been increasing reports on the globa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/41C12N7/04A61K39/125A61P31/14C12R1/84
CPCA61K39/12C12N7/00C07K14/005A61K2039/5258C12N2770/32034C12N2770/32023C12N2770/32022
Inventor 黄忠周瑜刘庆伟
Owner 中国科学院上海免疫与感染研究所
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