Toxoplasma gondii attenuated live vaccine with deficiency of OPRT and LDH1 genes and preparation method of toxoplasma gondii attenuated live vaccine

A live attenuated vaccine, the technology of Toxoplasma gondii, applied in the field of genetic engineering, can solve the problems of reducing pathogenicity, loss of Toxoplasma gondii replication, inability to replicate in the body, etc., achieving the effects of weak virulence, improving immunity, and preventing infection

Active Publication Date: 2018-08-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Insufficient uridine acid content in Toxoplasma gondii will cause severe genetic damage, making Toxoplasma lose the ability to replicate, and then affect its virulence to the host
In addition, the research team previous...

Method used

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  • Toxoplasma gondii attenuated live vaccine with deficiency of OPRT and LDH1 genes and preparation method of toxoplasma gondii attenuated live vaccine
  • Toxoplasma gondii attenuated live vaccine with deficiency of OPRT and LDH1 genes and preparation method of toxoplasma gondii attenuated live vaccine
  • Toxoplasma gondii attenuated live vaccine with deficiency of OPRT and LDH1 genes and preparation method of toxoplasma gondii attenuated live vaccine

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Experimental program
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Effect test

Embodiment 1

[0039] Example 1: Construction of Toxoplasma gondii auxotrophic strain △oprt△ldh1

[0040] (1) Starting strain ME49

[0041] ME49 is a type II worm strain of the genus Toxoplasma of the family Toxoplasma of the order Coccidia, and has an orotate phosphoribosyltransferase gene (oprt) and a lactate dehydrogenase 1 gene (ldh1), and the orotate phosphoribosyltransferase gene and the nucleotide sequences of the lactate dehydrogenase 1 gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0042] (2) Construction of pSAG1-Cas9-TgU6-sgoprt plasmid

[0043] The pSAG1-Cas9-TgU6-sgoprt plasmid is the CRISPR plasmid of the CRISPR / Cas9 system. The plasmid was constructed using the pSAG1-Cas9-TgU6-ccdb-sgMIC3 plasmid as a template, using the NEB company Q5 point mutation kit ( Site-Directed Mutagenesis Kit) to replace the MIC3 target-specific gRNA with the gene OPRT target-specific gRNA, the specific operation steps are as follows:

[0044] ① Use the gRNA online design websit...

Embodiment 2

[0136] Embodiment 2 Toxoplasma gondii △ oprt △ ldh1 worm strain purposes

[0137] 2.1 Gene knockout strain △oprt△ldh1 dilution injection

[0138] (1) Dilute injection formula

[0139]

[0140]

[0141] (2) Preparation method of diluted injection

[0142] ① Mix the above-mentioned mixed solution with a fixed volume for 10 minutes with a magnetic stirrer;

[0143] ②Use a filter with a pore size of 0.22 μm to filter and sterilize in the ultra-clean bench.

[0144] 2.2 Mice toxicity test of △oprt△ldh1 double knockout strain

[0145] 1) Use HFF cells to culture the tachyzoites of Toxoplasma gondii △oprt△ldh1 double knockout strain in vitro. After 20-30% of the parasites have escaped from the host cells, discard the medium in the original culture flask and transfer it to the culture flask Add PBS to wash away residual escaping worms and medium, wash twice, and add the diluted injection prepared in step (1);

[0146] 2) Scrape off the cells with a disposable cell scraper, ...

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Abstract

The invention discloses an attenuated live vaccine capable of preventing toxoplasma gondii infection. The vaccine contains a toxoplasma gondii vaccine strain with deficiency of both the orotate phosphoribosyltransferase (OPRT) gene and the lactate dehydrogenase 1 (LDH1) gene, wherein the nucleotide sequence of the OPRT gene is as shown in SEQ ID NO:1, and the nucleotide sequence of the LDH1 gene is as shown in SEQ ID NO:2. According to the attenuated toxoplasma gondii live vaccine used for preventing the animal toxoplasmosis provided by the invention, for the vaccine strain, the OPRT and LDH1genes are knocked out, thus the vaccine strain has the advantages that the virulence is weak and basically no pathogenicity is caused, and the vaccine strain can be used for preventing toxoplasma gondii infection of animals, and has the potential of being prepared into a genetic engineering vaccine for resisting toxoplasma gondii.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a live attenuated vaccine for preventing Toxoplasma gondii infection, especially a live attenuated vaccine for Toxoplasma gondii lacking orotate phosphoribosyltransferase (OPRT) and lactate dehydrogenase 1 (LDH1) genes. The invention also relates to a preparation method of the vaccine. Background technique [0002] Toxoplasma gondii can infect almost all warm-blooded animals including humans, causing zoonotic toxoplasmosis. Humans and animals are mainly infected by eating meat contaminated by Toxoplasma gondii and its oocysts. About 1 / 3 of the world's people are infected with Toxoplasma gondii. When human immunity is normal, Toxoplasma gondii infection generally does not show obvious clinical symptoms. In immunocompromised individuals, Toxoplasma gondii infection can lead to fever, blindness, encephalitis, etc., which seriously threaten human life and health. In animal husbandr...

Claims

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Application Information

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IPC IPC(8): A61K39/002A61P33/02C12N1/11C12N15/113C12N15/90
CPCA61K39/002A61K2039/522A61P33/02C12N9/0006C12N9/1077C12N15/113C12N15/902C12N2310/10C12N2310/20C12Y204/0201
Inventor 申邦周太芳夏宁波赵俊龙周艳琴方瑞贺兰潘明崔建敏杨旭柯
Owner HUAZHONG AGRI UNIV
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