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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: 2020-11-13
GUANGZHOU JENNIO BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • SARS-CoV-2 pseudovirus mouse in-vivo packaging system and preparation method thereof
  • SARS-CoV-2 pseudovirus mouse in-vivo packaging system and preparation method thereof
  • 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. The method comprises the following steps: S1, constructing an SARS-CoV-2 pseudovirus packaging plasmid system based on a lentivirus packaging plasmid system and a nymphaea tetragona transposon system; and S2, mixing the SARS-CoV-2 pseudovirus packaging plasmid system obtained in the step S1 with a nymphaeatetragona transposase expression plasmid, and transfecting mouse hepatocytes in a hydrodynamic injection manner; and then integrating the sequence required by SARS-CoV-2 pseudovirus packaging into the genome of the mouse hepatocyte by the nymphaea tetragona transposon system in a shearing and pasting manner. According to the present invention, the SARS-CoV-2 pseudovirus can be continuously produced and secreted in the mouse body, and the process that the target organ is continuously invaded and attacked by the SARS-CoV-2 pseudovirus can be simulated so as to simulate the pathological characteristics of the new coronal pneumonia (COVID-19). The animal model based on the SARS-CoV-2 pseudovirus mouse in-vivo packaging system is high in safety, and research can be carried out without a P3-level laboratory. The SARS-CoV-2 pseudovirus packaging plasmid system is introduced in a hydrodynamic injection mode, which 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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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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