Attenuated live vaccine of Toxoplasma gondii lacking ap2iv-1 gene and its construction method

A technology of AP2IV-1 and Toxoplasma gondii, applied in the direction of microorganism-based methods, other methods of inserting foreign genetic materials, vaccines, etc., can solve the problems of restricting the use of vaccines, strong virulence, etc., and achieve high immune protection efficacy and pathogenicity. The effect of reducing the disease and reducing the growth rate

Active Publication Date: 2022-07-05
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, the only commercially available live vaccine against Toxoplasma gondii is the S48 strain isolated from stillborn sheep, which has been weakened after passage in the laboratory. However, it has the risk of strong virulence, which limits the use of this vaccine

Method used

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  • Attenuated live vaccine of Toxoplasma gondii lacking ap2iv-1 gene and its construction method
  • Attenuated live vaccine of Toxoplasma gondii lacking ap2iv-1 gene and its construction method
  • Attenuated live vaccine of Toxoplasma gondii lacking ap2iv-1 gene and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction of Toxoplasma gondii AP2IV-1 gene deletion strain

[0040] The AP2IV-1 gene deletion strain was constructed with ΔKu80 RH as the base strain.

[0041] ΔKu80 RH is a type I strain of Toxoplasma gondii that loses the ability to form cysts in animals. The sequence of AP2IV-1 is conserved among different types of Toxoplasma gondii, and its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2.

[0042] 1. Construction of CRISPR / CAS9 system plasmid pSAG1-CAS9-TgU6-sgAP2IV-1

[0043] Using the pSAG1-CAS9-TgU6-sgUPRT plasmid as a template, the multi-segment seamless cloning kit ( (Seamless Cloning and Assembly Kit) replace the gRNA of UPRT gene with the gRNA of AP2IV-1 gene, the specific operation steps are as follows:

[0044] (1) Design of gRNA

[0045] The AP2IV-1 gRNA was designed in the ToxoDB (http: / / gRNA.ctegd.uga.edu / ) database of Toxoplasma gondii. Since the AP2IV-1 gene sequence is less than 2000b...

Embodiment 2

[0103] Example 2 Detection of proliferation rate, virulence and immune protection of Toxoplasma gondii AP2IV-1 gene deletion strains

[0104] 1. In vitro proliferation experiment of RH ΔAP2IV-1 strain

[0105] The intracellular proliferation rate of the Toxoplasma gondii AP2IV-1 gene deletion strain RHΔAP2IV-1 constructed in Example 1 was detected, and the specific method was as follows:

[0106] Tachyzoites of freshly released ΔKu80 RH and RH ΔAP2IV-1 strains were collected and inoculated with 10 5 Tachyzoites were placed in a 12-well plate confluent with HFF cells (human foreskin fibroblasts, purchased from ATCC Company) (a sterile cell slide was placed before the cells were plated). After 1 h of inoculation, the non-invading worms were washed away, and the culture was continued in the incubator. After culturing for 24h or 48h, carry out the IFA test, the specific method is as follows:

[0107] ① Fix the cells infected with Toxoplasma gondii in 4% paraformaldehyde at 37°C...

Embodiment 3

[0129] Example 3 Humoral immunity and cellular immune response monitoring of host immunized with RHΔAP2IV-1 strain

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Abstract

The invention relates to the technical field of Toxoplasma gondii vaccines, in particular to a live attenuated Toxoplasma gondii vaccine lacking the AP2IV-1 gene and a construction method thereof. The present invention provides a Toxoplasma strain with AP2IV-1 gene deletion, and the amino acid sequence of AP2IV-1 is shown in SEQ ID NO.1. Compared with the worm strain, the virulence of the AP2IV-1 gene-deleted strain is significantly reduced, and it has almost no pathogenicity in mice, and has high safety to the host. The AP2IV-1 gene deletion strain can provide long-term and effective immune protection against acute and chronic reinfection of different types of high-dose Toxoplasma gondii, and has the potential to become a live attenuated vaccine for the prevention of toxoplasmosis in animals.

Description

technical field [0001] The invention relates to the technical field of Toxoplasma gondii vaccines, in particular to an attenuated Toxoplasma gondii strain lacking the AP2IV-1 gene, a construction method thereof and a vaccine containing the attenuated strain. Background technique [0002] Toxoplasma Gondii is a zoonotic obligate cellular parasite that can grow on almost any warm-blooded cell. Humans can be infected with Toxoplasma gondii, but most people have a latent infection. Pregnant women and immunocompromised people are infected with Toxoplasma gondii with more serious consequences. At the same time, toxoplasmosis also causes huge economic losses to animal husbandry. At present, the treatment of toxoplasmosis is still mainly based on drug therapy - the combination of hexylpyridine and sulfadiazine, but these drugs are ineffective for the treatment of Toxoplasma gondii cysts. Therefore, there is an urgent need to develop a toxoplasmosis vaccine. Studies have found th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/11C12N15/79C12N15/90C12N15/30A61K39/002A61P33/02C12R1/90
CPCC07K14/45C12N15/79C12N15/902A61K39/002A61P33/02A61K2039/522A61K2039/552
Inventor 索勋刘贤勇王超越胡丹丹张思新汤新明马辉明王思
Owner CHINA AGRI UNIV
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