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
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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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