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A detoxification system and its application

A technology of recombining vectors and genes, which is applied in the field of genetic engineering, can solve the problems that proteins cannot be regulated, and achieve the effects of convenient regulation, good regulation effect and low background

Active Publication Date: 2022-07-15
BLUE ELEGANT BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, because the DDD regulatory system controls the expression of the target protein through ubiquitination and degradation, it cannot regulate the secreted and expressed protein.

Method used

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  • A detoxification system and its application
  • A detoxification system and its application
  • A detoxification system and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Construction of a strain of Plasmodium berghei P.bANKA using DDD to regulate the EF1g gene

[0058] In this example, the Cas9 knock-in vector pBC-DHFR-GFPm3-EF1g-Tar is constructed. The vector diagram is as follows figure 1 As shown, the vector is Amp resistant, contains a tandem expression of Cas9 protein and the Plasmodium pyrimethamine resistance gene hDHFR, using the pbeef1aa promoter, the Cas9 gene and hDHFR gene are linked by 2A peptide, using 3'Pb dhfr / ts as a terminator. In addition, the vector also contains a fusion expression cassette that uses the pbeef1aa promoter to contain EF1g homology arm 1, regulatory element DHFR, reporter protein GFP and EF1g downstream homology arm sequences in tandem, using 3'Pb dhfr / ts as a terminator, the vector also contains a P.b U6 promoter for sgRNA expression.

[0059] The EF1g gene homology arms are shown in SEQ ID NO.4-5, the sgRNA primers are shown in SEQ ID NO.6-7, and the specific sequences are shown in Tabl...

Embodiment 2

[0069] Example 2 Verifying the effect of using DDD to regulate the EF1g gene in P. berghei P.bANKA

[0070] Two Balb / c (8w, female) mice were inoculated with the P.bNAKA / pBC-DHFR-GFPm3-EF1g-Tar strain, and one mouse was inoculated with P. berghei using the CRISPR-Cas9 system to knock out the non-essential gene NT1 as a For the control, mixed administration of TMP / pyrimethamine was performed first, and the drug withdrawal TMP experiment was performed after the infection rate of Plasmodium exceeded 1%. After drug withdrawal, blood smears were taken for mice to calculate the infection rate. The results are as follows: Figure 5 shown.

[0071] from Figure 5 It can be seen that the mice in the control NT1 group died 7 days after drug withdrawal, while the infection rate of all mice in the DDD-EF1g group decreased to 0; After drug withdrawal, the infection rate continued to rise and died after 5 days, while the infection rate of 2 DDD-EF1g group mice dropped to 0 5 days after d...

Embodiment 3

[0073] Example 3 The effect of using DDD to regulate the EF1g gene in Plasmodium berghei P.bANKA

[0074] In this example, a DDD-GFP strain constructed by our company (DDD system regulates GFP expression, but does not regulate essential genes) was inoculated into Balb / c (8w, female) mice to verify whether there was TMP residue after TMP withdrawal. In addition, the DDD-EF1g strain was inoculated into 6 Balb / c (8w, female) mice, and the administration methods of the mice were shown in Table 2:

[0075] Table 2 Mice grouping and dosing table

[0076]

[0077] After the Plasmodium infection rate exceeded 1%, the G1 and G3 groups were withdrawn. Before withdrawal, the GFP fluorescence of the DDD-EF1g strain was observed, and the results were as follows Image 6 As shown, after TMP withdrawal, the G1 fluorescence was observed, and the results were as follows Figure 7 As shown, the infection rate and survival rate of mice in each group are as follows Figure 8(A)-Figure 8(B) ...

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Abstract

The present invention specifically relates to an attenuating system and application thereof, in particular to an attenuating virus and its application for attenuating Plasmodium, in particular to the use of an EF1g gene for attenuating Plasmodium. The attenuating system uses a regulatory system to regulate the expression or degradation of the EF1g gene, so as to control the growth of the malaria parasite and achieve attenuating the malaria parasite.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a virus attenuation system and its application, in particular to a virus attenuation and its application for the attenuation of Plasmodium. Background technique [0002] Malaria, AIDS and tuberculosis are the three major infectious diseases in the world. Malaria is an infectious disease caused by the single-celled protozoan Plasmodium and transmitted by Anopheles mosquitoes. The parasites that parasitize the human body are divided into four types: Plasmodium falciparum (P. .falciparum), P. malariea, P. vivax, and P. ovale. Plasmodium falciparum infection is responsible for 95% of malaria deaths, mainly in sub-Saharan Africa. At present, the animal models used for malaria research are mainly murine malaria and monkey malaria models. Murine Plasmodium can be divided into P. chaubdi, P. berghei, P. yoelii, and P. vinckei. The monkey Plasmodium is mainly P. kno...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/90C12N1/11A61K39/015A61P33/06C12R1/90
CPCA61K39/015A61P33/06C12N15/85C12N15/902C07K14/445A61K2039/522C12N2810/10Y02A50/30C12N1/36C12N9/22A01K67/0333A01K2217/072A01K2227/70C12N15/113C12N2310/20A61K35/68C12N15/79
Inventor 梁兴祥苏建华王美玲童英姚永超秦莉陈小平
Owner BLUE ELEGANT BIOTECH CO LTD
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