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Toxoplasma gondii attenuated live vaccine with deficiency of OMPDC and LDH1 genes

A live attenuated vaccine, technology of Toxoplasma gondii, applied in the field of genetic engineering, can solve the problems of inability to replicate in vivo, loss of replication of Toxoplasma gondii, reduce pathogenicity, etc., to prevent acute, chronic and congenital infections, and improve immunity. Effect

Active Publication Date: 2018-08-24
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 serious genetic damage, making Toxoplasma lose the ability to replicate, and then affect its virulence
In addition, the research team previously developed a Toxoplasma gondii mutant lacking lactate dehydrogenase 1 (LDH1), and found that the mutant can grow normally in vitro, but cannot replicate in vivo after infecting animals, significantly reducing pathogenicity

Method used

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  • Toxoplasma gondii attenuated live vaccine with deficiency of OMPDC and LDH1 genes
  • Toxoplasma gondii attenuated live vaccine with deficiency of OMPDC and LDH1 genes
  • Toxoplasma gondii attenuated live vaccine with deficiency of OMPDC and LDH1 genes

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Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Construction of Toxoplasma gondii auxotrophic strain △ompdc△ldh1

[0035] (1) Starting strain ME49

[0036] ME49 is a type II worm strain of the genus Toxoplasma of the family Toxoplasma in the order Coccidia, and has an orotidine-5-phosphate decarboxylase gene and a lactate dehydrogenase 1 gene, and the orotidine-5-phosphate decarboxylation The nucleotide sequences of the enzyme gene and the lactate dehydrogenase 1 gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0037] (2) Construction of pSAG1-Cas9-TgU6-sgompdc plasmid

[0038] The pSAG1-Cas9-TgU6-sgompdc 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 ompdc target-specific gRNA, the specific operation steps are as follows:

[0039] ① Use gRNA to design the web...

Embodiment 2

[0131] Embodiment 2 Toxoplasma gondii △ ompdc △ ldh1 worm strain purposes

[0132] 2.1 Gene knockout strain △ompdc△ldh1 diluted injection

[0133] (1) Dilute injection formula

[0134]

[0135] (2) Preparation method of diluted injection

[0136] ① Mix the above-mentioned mixed solution with a fixed volume for 10 minutes with a magnetic stirrer;

[0137] ②Use a filter with a pore size of 0.22 μm to filter and sterilize in the ultra-clean bench.

[0138] 2.2 Mice toxicity test of △ompdc△ldh1 double knockout strain

[0139] 1) Use HFF cells to culture Toxoplasma gondii △ompdc△ldh1 double-knockout strain tachyzoites in vitro. After 20-30% of the parasites have escaped from the host cells, discard the medium in the original culture bottle and transfer it to the culture bottle. Add PBS to wash away residual escaping worms and medium, wash twice, and add the diluted injection prepared in step (1);

[0140] 2) Scrape off the cells with a disposable cell scraper, blow the susp...

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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 simultaneous deficiency of the orotidine-5-phosphate decarboxylase gene and the lactic dehydrogenase 1 gene, wherein the nucleotide sequence of the orotidine-5-phosphate decarboxylase gene is as shown in SEQ ID NO:1, and the nucleotide sequence of the lactic dehydrogenase 1 gene is as shown in SEQ ID NO:2. The attenuated live vaccine provided by the invention has the advantages that basically no virulence exists, the immunocompetencefor toxoplasma gondii of animals can be improved, the acute and chronic as well as congenital infection of toxoplasma gondii can be prevented, the vaccine strain cuts off the drug resistance screening label, and thus the vaccine strain has the potential of being prepared into the genetically engineered vaccine for resisting toxoplasma gondii.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to an attenuated live vaccine for preventing toxoplasma infection, especially an attenuated toxoplasma gondii lacking orotidine-5-phosphate decarboxylase (OMPDC) and lactate dehydrogenase 1 (LDH1) genes live vaccines. Background technique [0002] Toxoplasma gondii can infect almost all warm-blooded animals including humans, causing a 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 the human immune function is normal, Toxoplasma gondii infection generally does not show obvious clinical symptoms, but in immunocompromised individuals, Toxoplasma gondii infection can cause fever, blindness, encephalitis, etc., seriously threatening human life and health. In animal husbandry production, Toxoplasma gondii infection leads to miscarria...

Claims

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Application Information

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IPC IPC(8): A61K39/002A61P33/02C12N15/79C12N15/90C12N15/53C12N15/60
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张丽红梁笑寒
Owner HUAZHONG AGRI UNIV
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