Toxoplasma gondii strain with ddi1, rad23 and dsk2 genes deleted, and construction method and application of toxoplasma gondii strain

A technology of gene deletion and construction method, applied in the field of genetic engineering, can solve problems such as no research reports

Inactive Publication Date: 2020-05-15
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] DNA damage-induced protein 1 (ddi1), ultraviolet shear repair protein 23 (rad23) and ubiquitin family protein 2 (dsk2) all belong to the UBL-UBA shuttle protein family, which are important components of the ubiquitin-protein system. The main function is to identify and transport ubiquitinated proteins

Method used

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  • Toxoplasma gondii strain with ddi1, rad23 and dsk2 genes deleted, and construction method and application of toxoplasma gondii strain
  • Toxoplasma gondii strain with ddi1, rad23 and dsk2 genes deleted, and construction method and application of toxoplasma gondii strain
  • Toxoplasma gondii strain with ddi1, rad23 and dsk2 genes deleted, and construction method and application of toxoplasma gondii strain

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Experimental program
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Effect test

Embodiment 1

[0043] The preparation of the ddi1, rad23 and dsk2 gene-deleted Toxoplasma gondii strain of the present invention is achieved through the following steps.

[0044] 1. Basic strain RHΔku80

[0045] RHΔku80 strain is a Toxoplasma strain with DNA damage-induced protein 1 gene (ddi1), ultraviolet shear repair protein 23 gene (rad23) and ubiquitin family protein gene (dsk2), which can be continuously passaged on HFF cells.

[0046] 2. Construction of Toxoplasma three-gene deletion strain (ΔΔΔddi1rad23dsk2)

[0047] 1. Construction of pSAG1-CAS9-U6gRNA(ddi1) plasmid

[0048] Specific steps are as follows:

[0049] 1) Use E-CRISP (http: / / www.e-crisp.org / E-CRISP / ) to design a specific sgRNA sequence for ddi1 gene knockout

[0050] 2) Using the pSAG1-CAS9-U6gRNA (UPRT) plasmid as a template, design primers to amplify fragment 1, fragment 2, and fragment 3 respectively, and add ddi1-specific sgRNA to the upstream primer of fragment 2 and the downstream primer of fragment 3. The prime...

Embodiment 2

[0168] The proliferation rate and virulence of Toxoplasma ΔΔΔddi1rad23dsk2 strain were significantly slowed down: Plaque test can comprehensively evaluate the invasion, proliferation and release ability of Toxoplasma gondii. Collect the newly released tachyzoites of ΔΔΔddi1rad23dsk2 and RHΔku80 strains, count and inoculate 500 tachyzoites into a 6-well cell culture plate covered with HFF cells, culture for 7 days, fix with 4% paraformaldehyde at room temperature for 10 min, and crystal violet Stain for 20 minutes, wash with PBS, and observe the size of the plaque area. The results showed that the plaques formed by ΔΔΔddi1rad23dsk2 were almost invisible to the naked eye, and tiny plaques could be seen under the microscope (see Figure 10 ), indicating that the growth and reproduction of Toxoplasma gondii were significantly inhibited after deletion of ddi1, rad23 and dsk2.

[0169] 200 newly released tachyzoites of ΔΔΔddi1rad23dsk2 and RHΔku80 were inoculated intraperitoneally ...

Embodiment 3

[0171] The ΔΔΔddi1rad23dsk2 strain has a good immune protection effect on mice: 6 groups of mice were set up, with 5 6-week-old female BALB / c mice in each group, among which 200 ΔΔΔddi1rad23dsk2 tachyzoites were immunized intraperitoneally in four groups, and one group was inoculated with A group of 200 RHΔku80 tachyzoites was intraperitoneally injected with PBS. The mice in the RHΔku80 inoculation group all died within 8 days. After 12 days, the mice in the ΔΔΔddi1rad23dsk2 immunization group were challenged again with 500 and 1000 tachyzoites of the strong virus strain RHΔku80 and 1000 and 2000 tachyzoites of the attenuated Pru strain. The PBS injection group was inoculated with RHΔku80 tachyzoites 100 offspring were reproduced, and the clinical symptoms and mortality were recorded. The mice in the PBS injection group all died within 8 days after being inoculated with RHΔku80 tachyzoites, and the mice immunized with the ΔΔΔddi1rad23dsk2 knockout strain survived within 30 day...

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Abstract

The invention discloses a Toxoplasma gondii strain with ddi1, rad23 and dsk2 genes deleted and a construction method and application of the Toxoplasma gondii strain. Three shuttle protein genes, namely the ddi1, rad23 and dsk2 genes, of a ubiquitin-proteasome system are simultaneously deleted in the Toxoplasma gondii strain, so the delta delta delta delta ddi1rad23dsk2 three-gene-deleted strain isobtained. The fecundity of the strain is reduced, the toxicity of the strain to mice is reduced, and the strain has a good immune protection effect on Toxoplasma gondii infection and has the potential of becoming an attenuated vaccine against toxoplasmosis.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a ddi1, rad23 and dsk2 gene-deleted toxoplasma strain and its construction method and application. Background technique [0002] Toxoplasma gondii (Toxoplasma gondii for short) is an obligate intracellular parasitic protozoa, belonging to the apicomplexan subphylum, which infects almost all warm-blooded animals and humans, and is a serious zoonosis Original. Toxoplasma gondii is widely infecting, and it can harm hosts in various forms, and the degree of harm to different hosts, different growth or physiological stages is also very different. In the case of normal host immunity, it is mostly recessive infection; infants and immunosuppressed patients infected with Toxoplasma gondii may cause serious or fatal diseases, and Toxoplasma gondii infection can also cause abortion, stillbirth and other reproductive disorders in pregnant animals and pregnant women The important reason is...

Claims

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Application Information

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IPC IPC(8): C12N1/11C12N15/113C12N15/30C12N15/90A61K39/002A61P33/02C12R1/10
CPCC12N15/113C12N15/902C07K14/45A61K39/002A61P33/02C12N2310/20A61K2039/522
Inventor 刘群张恒毋亚运刘晶薛阳飞应铸
Owner CHINA AGRI UNIV
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