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Rapid construction method and application of ACE2 humanized mouse model

A human-derived, transgenic vector technology, applied in biochemical equipment and methods, applications, botanical equipment and methods, etc., can solve the requirements of high experimental conditions, need to go through line establishment, expression verification, etc., the preparation cycle is not less than 9 -12 months time, high failure rate issues

Active Publication Date: 2021-11-23
SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV +2
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Problems solved by technology

However, the continuous passage of the virus in mice is used to screen the SARS-CoV-2 virus strains adapted to mice, and then to establish a susceptible model. The method has a long period of time, a high failure rate, and high requirements for experimental conditions. It can be carried out, but it is not conducive to the rapid establishment and development of models; expressing human ACE2 in mice through conventional transgenic methods usually needs to go through the process of strain establishment and expression identification, which takes at least 9-12 months; through gene editing Human ACE2 is expressed in mice in the same way, because gene editing is affected by the size of the inserted fragment, it is difficult to perform complex genetic operations, and the strain also needs to go through line establishment, expression verification, etc., and the preparation period is not less than 9-12 months time

Method used

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  • Rapid construction method and application of ACE2 humanized mouse model
  • Rapid construction method and application of ACE2 humanized mouse model
  • Rapid construction method and application of ACE2 humanized mouse model

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Experimental program
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Effect test

Embodiment 1

[0015] ACE2 humanized mouse model construction.

[0016] 1) Construction of Piggybac transgene vector: PiggyBac transposon 5' inverted repeat (ITR), CAG promoter, human ACE2 coding region, ribosome access site (IRES), luciferase ), woodchuck hepatitis post-transcriptional regulatory element (WPRE), polyadenylation (polyA) site and 3' inverted repeat (ITR) sequences are SEQ ID NO: 1-8. The CAG promoter was amplified by PCR amplification using the plvct-tTR-KRAB plasmid as a template, and the remaining fragments were obtained by total gene synthesis. During the construction process, the two ITRs and restriction sites were first connected into the PBR322 vector by the In-Fusion method to obtain the PBR322-ITR backbone vector; then the CAG promoter, synthetic human ACE2 fragment, IRES-Luciferase-WPRE -polyA was connected into the PBR322-ITR backbone vector by In-Fusion to obtain the final PBR322-ITR-ACE2 transgenic vector. After the vector was digested and sequenced and verified...

Embodiment 2

[0024] Construction of ACE2 humanized mouse SARS-CoV-2 virus susceptibility model

[0025] ACE2 humanized mouse SARS-CoV-2 virus infection and detection experiments were completed in a biosafety level 3 laboratory (BSL-3), and 5 F0 mice with positive expression of Luciferase and 5 wild-type C57BL / 6 In the biological safety cabinet, 50 μL (4.15 × 10 4 PFU) SARS-CoV-2 virus solution was instilled into the nasal cavity of mice. After 4 days of infection, the lung tissues of the mice were collected, RNA was extracted and reverse-transcribed, and real-time fluorescent quantitative PCR (Realtime PCR) was performed to detect the presence of The content of SARS-CoV-2 virus, the results are as follows image 3 As shown, the results show that: 4 days after SARS-CoV-2 virus infection, the virus content in the lung tissue of ACE2 humanized mice positive for Luciferase expression is about 1000 times that of wild-type mice, indicating that ACE2 humanized mice have Novel coronavirus pneum...

Embodiment 3

[0027] Evaluation of SARS-CoV-2 neutralizing antibody protection using ACE2 humanized mice.

[0028] group number of mice Administration (2h after infection) Dose (mg / kg) Route of administration control group 6 PBS - intraperitoneal injection Neutralizing antibody low dose group 5 Neutralizing Antibody-Low dose 5 intraperitoneal injection Medium dose group of neutralizing antibody 5 Neutralizing Antibody-Medium dose 10 intraperitoneal injection Neutralizing antibody high dose group 5 Neutralizing Antibody-High dose 20 intraperitoneal injection

[0029] After infection and administration, the state and body weight of the mice were recorded every day. After 5 days of infection, the lung tissues of the surviving mice were taken, RNA was extracted and reverse-transcribed, and Realtime PCR was performed to detect the presence of SARS-CoV-2 virus in the lung tissues of different groups of mice. content. Experimental r...

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Abstract

The invention provides a transgenic vector, a method for rapidly constructing an ACE2 humanized animal model by using the transgenic vector, and application of the ACE2 humanized animal model by aiming at research and development of SARS-CoV-2 drugs. The transgenic vector comprises a PiggyBac transposon 5'end inverted repeat sequence (ITR), a CAG promoter, a human ACE2 coding region, a ribosome access site (IRES), firefly luciferase, a dial rat hepatitis post-transcriptional regulatory element (WPR), a polyA site and a 3 'end inverted repeat sequence (ITR), the transgenic vector can efficiently insert human ACE2 and luciferase gene expression cassettes into a mouse genome, and the luciferase and human ACE2 expressed transgenic mice can be rapidly screened through a luciferase living body imaging system.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a rapid construction strategy of an ACE2 humanized mouse model and its application in the research and development of anti-new coronavirus SARS-CoV-2 therapeutic drugs. Background technique [0002] The current research and development of vaccines and antiviral drugs against the new coronavirus SARS-CoV-2 still needs to be strengthened urgently. [0003] Animal models are critical for understanding viral pathogenesis, vaccine development and drug screening. Therefore, the development of animal models susceptible to the new coronavirus SARS-CoV-2 is an important part of the development of virus vaccines and antiviral drugs. Among various animal models, non-human primates (NHP) are more similar to humans and have better predictability for clinical effects. However, the application of NHP animal models is limited by high cost, small quantity, and high requirements for bree...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12N15/53C12N15/57A01K67/027A61K49/00
CPCC12N15/8509C12N9/485C12N9/0069A01K67/0278A61K49/0043C12Y304/17023C12Y113/12007C12N2800/107C12N2800/90C12N2830/001A01K2227/105A01K2267/0337
Inventor 孙瑞林王津津池骏慈磊
Owner SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV
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