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Production of hscarb2 Gene Transgenic Mice and Its Application as Animal Model of Enterovirus Infection

A gene and genotype technology, applied in the field of preparation of enterovirus-infected animal models, can solve problems such as not being EV71

Inactive Publication Date: 2015-09-16
NAT INST OF HEALTH REPRESENTED BY THE SEC OF THE DEPT OF HEALTH & HUMAN SERVICES NAT INST OF HEALTH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Recent studies have found that human scavenger receptor class B, membrane protein type 2 (human scavenger receptor class B, member 2; hSCARB2) is the receptor for entry of enterovirus type 71 and coxsackie virus type 16 (Coxsackie A16) into cells ( Yamayoshi et al. Nat Med 15(7):798-801, 2009), although mouse SCARB2 has 85.8% homology with human SCARB2 (hSCARB2), it was shown that it does not act as a receptor for EV71

Method used

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  • Production of hscarb2 Gene Transgenic Mice and Its Application as Animal Model of Enterovirus Infection
  • Production of hscarb2 Gene Transgenic Mice and Its Application as Animal Model of Enterovirus Infection
  • Production of hscarb2 Gene Transgenic Mice and Its Application as Animal Model of Enterovirus Infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Preparation of SCARB2 gene transgenic mice

[0023] The synthesized codon-optimized human SCARB2 gene (SEQ ID NO.1) fragment was inserted into the pEF-1α plasmid (the plasmid was based on the pcDNA3.1 (-) vector (Invitrogen) using EcoRI and BamHI restriction enzymes. ) is a modified vector in which the original cytomegalovirus enhancer / promoter is replaced by the promoter of growth factor 1α (EF-1α), located in the promoter and bovine growth hormone (BGH) Multiple cloning sites between poly A tails were used to obtain the pEF-1α-hSCARB2 construct ( figure 1 ).

[0024]Generation of transgenic animals was performed according to the protocol described by Brinster et al. (Brinster et al., 1985). The C57BL / 6 mice used were purchased from National Applied Research Laboratories-Laboratory Animal Center (National Applied Research Laboratories-Laboratory Animal Center, Taiwan). Plastids were linearized with restriction enzymes prior to microinjection and recovered...

Embodiment 2

[0030] Example 2: Symptom analysis of viremia, hand-foot-mouth disease (HFMD) and nervous system lesions in SCARB2 gene transfected mice after enterovirus infection

[0031] 1-day or 4-day-old mice were subcutaneously injected into enterovirus EV71E59 or 5746-tw98 isolates (1x10 6 or 1x10 7 pfu, in 10 μL RPMI medium), to simulate the observation of the division of enterovirus in the main organs of the mouse after the newborn mouse was infected with the virus, and the external symptoms. The control group was given VP-SFM medium. For the detection of enterovirus EV71, the infected mice were sacrificed after a certain number of days, blood or organ tissue samples were collected, and specific real-time RT-PCR was performed for the VP1 gene.

[0032] Total RNA extracted from blood samples or tissue homogenates is converted into cDNA by reverse transcription. The resulting cDNA was then used with EV71 VP1 specific primer pair, forward primer: 5'-AGAGAGTCACTTGCTTGGCAGACA-3' (SEQ I...

Embodiment 3

[0037] Example 3: Using SCARB2 gene transfected mice to detect the prevention and treatment effect of anti-EV-71 antibody on enterovirus infection

[0038] In this example, the anti-EV-71 monoclonal antibody known to have enterovirus neutralizing activity was administered to SCARB2 gene-transformed mice previously infected with the natural EV71 5746-TW98 enteropathy isolate, and the monoclonal antibody was detected The effect of the antibody on protecting mice against enterovirus infection is to evaluate and prove the practicability of the SCARB2 gene transgenic mice of the present invention as a platform for screening anti-enterovirus drugs.

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Abstract

The present invention concerns the manufacture of EV71 acceptor SCARB 2 transgenic mouse. The manufactured SCARB 2 transgenic mouse can be used as an animal model infected with the hand-foot-and-mouth disease, such as infected with enterovirus 71 or Coxsackies virus A16, so as to allow for estimation of enterovirus viral vaccine immune protection efficacies and application of enterovirus infection in pathological study.

Description

technical field [0001] The present invention relates to the preparation of enterovirus-infected animal models, more particularly to the manufacture of SCARB2 gene transfected mice, and its application as an animal model of hand, foot and mouth disease. Background technique [0002] Enterovirus 71 (EV71) has been confirmed to cause hand, foot and mouth disease (HFMD), herpetic angina, and neurological lesions such as aseptic meningitis, encephalitis, and polio-like syndrome, even in infants and young children. deadly disease, and severe outbreaks worldwide (AbuBakar et al., 1999, Virus Res 61(1), 1-9; Chang, Huang, and Lin, 1998, Lancet 352(9125), 367-8; Bible et al., 2007, Rev Med Virol 17(6), 371-9). Up to now, the control and medical care of the EV71 epidemic through public health are still insufficient. There is currently no vaccine available for clinical use. Therefore, it is very important to study the mechanism of enterovirus 71 infection for the development of effe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85A01K67/027A61D19/04C12Q1/68
Inventor 周彦宏庄再成林怡玟
Owner NAT INST OF HEALTH REPRESENTED BY THE SEC OF THE DEPT OF HEALTH & HUMAN SERVICES NAT INST OF HEALTH
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