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In-vivo efficacy evaluation method of EV71 vaccines and antiviral drug screening method

A technology of EV71 and vaccines, applied in biochemical equipment and methods, measurement/inspection of microorganisms, cells modified by introducing foreign genetic material, etc., can solve problems such as limiting research work

Active Publication Date: 2017-04-05
NAT INST FOR FOOD & DRUG CONTROL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] This characteristic limits the research work related to the virus

Method used

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  • In-vivo efficacy evaluation method of EV71 vaccines and antiviral drug screening method
  • In-vivo efficacy evaluation method of EV71 vaccines and antiviral drug screening method
  • In-vivo efficacy evaluation method of EV71 vaccines and antiviral drug screening method

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Embodiment approach

[0091] The present invention also specifically relates to the following embodiments:

[0092] In one embodiment of the present invention, the present invention relates to the method for constructing R26-hSCARB2 mouse, and it mainly comprises the following steps:

[0093] 1) Using embryonic stem cells to knock in the hSCARB2 gene

[0094]Targeting vectors capable of homologous recombination with gene sites in the genome (mutated target genes, homology arms, resistance genes, etc.) were obtained through molecular carrier construction technology; targeting vectors were introduced into embryonic stem cells by electroporation technology, and challenged The drug-resistant embryonic stem cells were taken, and 4 correctly targeted embryonic stem cell clones were screened out by PCR (see Table 1 for primers) and Southern blotting.

[0095] 2) Microinjection of embryonic stem cells into blastocysts and germline inheritance

[0096] Two of the positive embryonic stem cell clones were s...

Embodiment 1

[0127] Example 1: Construction of hSCARB2 targeting vector

[0128] Rosa26 gene can encode a non-essential nuclear RNA in almost all tissues, and can make the inserted exogenous gene express systemically without affecting the expression of other genes. We inserted the cDNA of hSCARB2 gene (SEQ ID NO:1) into the modified Ai3 targeting vector (Addgene, Madisen L, et al. A robust and high-throughput Crereporting and characterization system for the whole mouse brain. Nat Neurosci13,133-140 , 2010). The Ai3 vector in turn contains a strong CAG promoter, a termination signal flanked by loxP, and a WPRE element that enhances mRNA transcriptional stability. And insert tdTomato reporter gene (SEQ ID NO:11) at the back of hSACARB2, connect with hSCARB2 through 2A short peptide (coded by SEQ ID NO:10) ( figure 1 ). Under the action of Cre recombinase, the transcription termination signal with loxP is cut off, hSCARB2 and tdTomato can be expressed simultaneously, and the 22nd amino aci...

Embodiment 2

[0129] Example 2: Construction of hSCARB2 conditional knock-in mice

[0130] The targeting vector obtained in Example 1 was transduced into C57BL / 6ES cells by electroporation, and the clones selected by G418 were detected by PCR and southern hybridization to detect 4 correct hSCARB2 gene insertion positive clones. In order to obtain hSCARB2 knock-in mice, we selected one of the positive clones for blastocyst injection. Using BALB / c blastocysts as recipients, 98 blastocysts were injected and transplanted into ICR pseudopregnant mother mice to obtain 8 chimeric mice. Then the chimeric mice were mated with C57BL / 6 mice, and 10 C57BL / 6 background heterozygous gene knock-in mice were obtained completely derived from targeted ES cells through PCR identification. Furthermore, heterozygous mice were mated to obtain homozygous knock-in mice, which were named B6-Gt(Rosa)26Sortm1(CAG-hSCARB2-tdTomato), abbreviated as R26-STOP-hSCARB2 mice.

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Abstract

The invention relates to an in-vivo efficacy evaluation method of EV71 vaccines and an antiviral drug screening method, in particular to the biological field. More particularly, a sensitive, safe and convenience EV71 vaccine efficacy in-vivo evaluation system composed of animal models, pseudotyped viruses and living imaging is established, and a new method for quality control and evaluation of existing vaccines and development of novel vaccines is provided. The method can also be used for screening antiviral drugs relevant to the EV71 vaccines.

Description

[0001] Technical field: [0002] The invention relates to a vaccine evaluation method based on hSCARB2 gene knock-in mice and EV71-Fluc pseudovirus enterovirus 71, belonging to the field of biotechnology. The model system constructed by the invention can be used for in vivo evaluation of EV71 virus vaccine efficacy and antiviral drug screening. Background technique [0003] Human enterovirus 71 (EV71) is one of the main pathogens of hand, foot and mouth disease (HFMD), mainly infecting children under 5 years old, and can cause nervous system infection leading to aseptic meningitis, Brainstem encephalitis, myocarditis, pulmonary edema, etc. EV71 has caused sporadic outbreaks worldwide, especially in the Asia-Pacific Rim, including Japan, Malaysia, Singapore, inland China, and Taiwan, causing serious social harm to these countries and regions. In my country, since the outbreak of HFMD in 2008, the incidence of HFMD has been on the rise in recent years. Most of the severe cases...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A01K67/027C12N5/10C12Q1/70
Inventor 范昌发周舒雅刘强吴星陈盼吴曦王佑春毛群颖刘甦苏左琴黄维金李保文梁争论李文辉贺争鸣
Owner NAT INST FOR FOOD & DRUG CONTROL
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