A kind of sars-cov-2 pseudovirus mouse in vivo packaging system and preparation method thereof

A technology of sars-cov-2 and sars-cov-2s, which is applied to the in vivo packaging system and preparation of SARS-CoV-2 pseudovirus in mice, can solve the problem that the optimal time window is difficult to determine, the time window is short, and the simulation cannot be performed. The problem of continuous invasion of animal target organs, etc., achieves the effect of low cost, simple operation and high safety

Active Publication Date: 2020-12-25
GUANGZHOU JENNIO BIOLOGICAL TECH CO LTD
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  • Abstract
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Problems solved by technology

[0004] However, when the SARS-CoV-2 pseudovirus packaged in vitro is used in animal experiments, the pseudovirus can only be injected successively (such as tail vein injection) to infect animals. The time window of the experiment is short, and it cannot simulate the impact of SARS-CoV-2 virus. Continuous invasion of animal target organs, and the optimal time window is difficult to determine, evaluating the effect of drug intervention

Method used

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  • A kind of sars-cov-2 pseudovirus mouse in vivo packaging system and preparation method thereof
  • A kind of sars-cov-2 pseudovirus mouse in vivo packaging system and preparation method thereof
  • A kind of sars-cov-2 pseudovirus mouse in vivo packaging system and preparation method thereof

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Embodiment 1

[0036] S1: construct the SARS-CoV-2 pseudovirus packaging plasmid system, the specific steps are as follows:

[0037] (1) Replace the envelope protein with SARS-CoV-2 S protein, the specific steps are as follows:

[0038] Using the pMD2.G vector as the backbone, the VSV-G sequence was excised by enzyme digestion, and then the excised sequence was replaced by the CDS region of the SARS-CoV-2 S protein in the way of a gateway. The replaced envelope protein particle pMD2 .G(SARS-CoV-2-S) map such as figure 2 shown.

[0039] (2) Construct the envelope protein particle PH-TB-SARS-CoV-2-S, the specific steps are as follows:

[0040] Using the plasmid PH-TB as the backbone, fish the sequence from CMVenhancer to SARS-CoV-2-S from the plasmid pMD2.G(SARS-CoV-2-S) by PCR, and insert the sequence by gateway Between ITR(L) and ITR(R), the final plasmid map constructed is as follows image 3 shown.

[0041](3) Construct the packaging plasmid PH-TB-GAG-POL, the specific steps are as f...

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Abstract

The invention provides a preparation method of a pseudovirus mouse in vivo packaging system, comprising the following steps: S1 constructs a SARS-CoV-2 pseudovirus packaging plasmid system based on a lentivirus packaging plasmid system and a Sleeping Beauty transposon system, and S2 combines the steps In S1, the SARS‑CoV‑2 pseudovirus packaging plasmid system was mixed with the Sleeping Beauty transposase expression plasmid to transfect mouse hepatocytes by hydrodynamic injection, and then the Sleeping Beauty transposon system packaged the SARS‑CoV‑2 pseudovirus The desired sequence was integrated into the genome of mouse hepatocytes in a cut-and-paste manner. The invention can continuously produce and secrete SARS-CoV-2 pseudoviruses in mice, and can simulate the process of target organs being continuously invaded and attacked by SARS-CoV-2 viruses, thereby simulating the pathological characteristics of new coronary pneumonia (COVID-19). The animal model based on the SARS‑CoV‑2 pseudovirus mouse in vivo packaging system is highly safe, and research can be carried out without the need for a P3 laboratory. The method of hydrodynamic injection to introduce the SARS-CoV-2 pseudovirus packaging plasmid system is simple to operate and low in cost.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a SARS-CoV-2 pseudovirus mouse in vivo packaging system and a preparation method thereof. Background technique [0002] The SARS-CoV-2 pseudovirus is a defective virus particle coated with the SARS-CoV-2 Spike protein (S protein), so it can use the same virus invasion mechanism of SARS-CoV-2 (with ACE2 as the receptor) to carry the DNA or RNA sequences are introduced into cells, but they carry sequences that cannot guide the regeneration of viruses, so they can be used to study the mechanism of a single invasion process and test the effect of drug intervention. Generally, experimental animals and operators have no substantial risk of infection . [0003] At present, pseudovirus packaging is usually completed in cell lines cultured in vitro, and the system that can be used for SARS-CoV-2 pseudovirus packaging consists of retroviruses such as human immunodeficiency...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/86C07K14/165A01K67/027
CPCC12N15/86C07K14/005A01K67/0278C12N2770/20052C12N2770/20023C12N2770/20043C12N2800/107C12N2800/90A01K2217/206A01K2227/105A01K2267/0337Y02A50/30
Inventor 谢鹏胡策叶钊成
Owner GUANGZHOU JENNIO BIOLOGICAL TECH CO LTD
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