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Yeast-expressed Coxsackievirus A10 virus-like particles and applications thereof

A Coxsackie virus, A10 technology, applied in the field of biomedicine, can solve the problems of no vaccine and little research.

Active Publication Date: 2018-10-09
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the HFMD caused by CA10 is gradually increasing, most of the current research is still on EV71 and CA16, among which the inactivated vaccine of EV71 is already on the market, and a variety of candidate vaccines are also available for CA16, but there are still few studies on CA10, and no vaccine is available yet

Method used

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  • Yeast-expressed Coxsackievirus A10 virus-like particles and applications thereof
  • Yeast-expressed Coxsackievirus A10 virus-like particles and applications thereof
  • Yeast-expressed Coxsackievirus A10 virus-like particles and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] Expression and purification of embodiment 1 VLP

[0118] In order to express CA10-VLP, the 3CD and P1 genes were inserted into the pPink-HC vector together to construct plasmid YCA10-003 ( figure 1 A), and then use the plasmid to transform Pichia pastoris competent, small expression to obtain cell lysate centrifuged supernatant 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 YCA10-003 showed obvious antigen-antibody reactions ( figure 1 B). The two most responsive transformants were selected from the yeast clones for Western blotting analysis, and the experimental results are shown in the figure ( figure 1 C), using anti-CA10VP0, anti-CA10VP3, and anti-CA10VP1 as detection antibodies, corresponding bands appeared in the three yeast lysate samples, with sizes of 39kDa, 29kDa, and 37kDa, resp...

Embodiment 2

[0123] Example 2. Immunogenicity of CA10-VLP in mice

[0124] Before immunizing mice, CA10-VLP and control antigen were prepared and quantified ( image 3 A), followed by the addition of aluminum adjuvants, respectively. Two groups (6 / group) of ICR mice were injected intraperitoneally with CA10-VLP vaccine and control antigen at 0, 2, and 4 weeks, and blood was collected at 6 weeks for ELISA analysis and neutralization experiments. The results of ELISA with CA10-VLP coated plates showed that the mice in the vaccine group had obvious antigen-antibody responses ( image 3 B), and the geometric mean titer reached 45254.8 ( image 3 C); while the control group had no obvious response. In the virus microneutralization experiment, the serum of mice in the control group failed to show neutralization ability even at the lowest dilution (1:16); while the serum of mice in the vaccine group was resistant to homologous strain CA10 / S0273b and heterologous strains Strains CA10 / kowalik a...

Embodiment 3

[0125] Example 3.CA10-VLP vaccine antiserum has protective effect in vivo

[0126] Firstly, the in vivo protective effect of anti-VLP antibody was evaluated by virus challenge test. 6-day-old neonatal mice were intraperitoneally injected with 75 μl of anti-VLP serum or control serum, and 24 hours later, intraperitoneally injected with a lethal dose of CA10 / Kowalik or CA10 / S0148b virus, and then observed the clinical symptoms and death of virus-challenged mice . The results showed that mice injected with control serum gradually developed severe clinical symptoms after CA10 / Kowalik virus challenge, including slow movement, ataxia and paralysis, and all died within 9 days after challenge; The mice with VLP serum did not appear obvious clinical symptoms in the observation period of 15 days ( Figure 4 A-B). Similarly, after CA10 / S0148b virus challenge, all mice in the control group died within 7 days, and all mice injected with anti-VLP serum survived without obvious clinical s...

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Abstract

The present invention provides yeast-expressed Coxsackievirus A10 virus-like particles and applications thereof. According to the present invention, specifically the gene sequences of the P1 protein and the 3CD protein of Coxsackievirus A10 are transformed into yeast cells, and expression is performed to obtain the novel Coxsackievirus A10 virus-like particles; and the yeast-expressed Coxsackievirus A10 virus-like particles have characteristics of high expression, strong immunogenicity and good specificity.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, the invention relates to yeast-expressed Coxsackievirus A10 virus-like particles and applications thereof. Background technique [0002] Hand, foot and mouth disease (HFMD) is an infectious disease caused by enterovirus, mainly infecting children under 5 years old. HFMD mainly causes some mild symptoms, such as fever and herpes or ulcers on the hands, feet, and mouth, which usually heal spontaneously; however, HFMD can also cause some serious complications, such as aseptic meningitis, pulmonary edema, etc. If not treated in time, it can even lead to death. [0003] Traditionally, the pathogens causing HFMD are mainly EV71 and CA16. In recent years, the cases caused by CA6 and CA10 have gradually increased. In 2008, the co-circulation of CA6 and CA10 caused an outbreak of HFMD in Finland. In the same year, one of the main pathogens of the HFMD outbreak in Singapore was confirmed to be C...

Claims

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Application Information

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IPC IPC(8): C12N15/41C07K14/085C12N7/04A61K39/125A61P31/14
CPCA61K39/12C12N7/00C07K14/005C12N2770/32051C12N2770/32034C12N2770/32022C12N2770/32023Y02A50/30
Inventor 黄忠周瑜刘庆伟
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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