Toxoplasma gondii attenuated live vaccine with deletion of AP2IV-1 gene and construction method thereof

A technology of AP2IV-1, 1. AP2IV-1 is applied in the construction method and the vaccine containing the attenuated parasite strain, the field of the attenuated parasite of Toxoplasma gondii, and can solve the problems of strong virulence, restricting the use of vaccines, etc. The effect of disease reduction, high immune protection efficacy, and reduced proliferation rate

Active Publication Date: 2020-09-01
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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  • Toxoplasma gondii attenuated live vaccine with deletion of AP2IV-1 gene and construction method thereof
  • Toxoplasma gondii attenuated live vaccine with deletion of AP2IV-1 gene and construction method thereof
  • Toxoplasma gondii attenuated live vaccine with deletion of AP2IV-1 gene and construction method thereof

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-deficient strain was constructed with ΔKu80 RH as the basic strain.

[0041] ΔKu80 RH is a Toxoplasma type I strain that loses the ability to form cysts in animals. The sequence of AP2IV-1 is conserved in different types of Toxoplasma gondii, 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-fragment seamless cloning kit from TransGen Biotech ( Seamless Cloning and Assembly Kit) to replace the gRNA of the UPRT gene with the gRNA of the AP2IV-1 gene. The specific steps are as follows:

[0044] (1) Design of gRNA

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

Embodiment 2

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

[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 RH ΔAP2IV-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 for 10 5 The tachyzoites were placed in a 12-well plate filled with HFF cells (human foreskin fibroblasts, purchased from ATCC Company) (put a sterile cell slide before laying the cells). After 1 hour of inoculation, the uninvaded insects were washed away, and the cultivation 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 for 30...

Embodiment 3

[0129] Example 3 Monitoring of Humoral Immunity and Cellular Immune Response of RH ΔAP2IV-1 Strain Immunized Host

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Abstract

The invention relates to the technical field of toxoplasma gondii vaccines, in particular to a toxoplasma gondii attenuated live vaccine with deletion of AP2IV-1 gene and a construction method thereof. The invention provides a toxoplasma gondii strain with deletion of the AP2IV-1 gene. The amino acid sequence of AP2IV-1 is shown in SEQ ID NO.1.. Compared with a starting insect strain, the AP2IV-1-gene-deleted insect strain is remarkably reduced in toxicity, and the AP2IV-1-gene-deleted insect strain hardly has pathogenicity in a mouse body and has relatively high safety to a host. The AP2IV-1-gene-deleted strain can provide long-term effective immune protection for acute and chronic reinfection of different types of high-dose toxoplasma gondii, and has the potential of becoming an attenuated live vaccine for preventing animal toxoplasmosis.

Description

technical field [0001] The invention relates to the technical field of toxoplasma gondii vaccines, in particular to an attenuated toxoplasma gondii strain lacking AP2IV-1 gene, a construction method thereof and a vaccine containing the attenuated toxoplasma strain. Background technique [0002] Toxoplasma Gondii (Toxoplasma Gondii) is a zoonotic obligate cellular parasite that can grow in almost any warm-blooded cell. Humans can be infected with Toxoplasma gondii, but most people are recessively infected. Toxoplasma gondii infection in pregnant women and immunocompromised persons can cause serious consequences. At the same time, toxoplasmosis also causes huge economic losses to animal husbandry, and infection with toxoplasma can easily cause abortion in pigs, sheep and other animals. At present, the treatment of toxoplasmosis is still based on drug therapy - the combination of hexamethazine and sulfadiazine, but this type of drug is ineffective for the treatment of toxopla...

Claims

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Application Information

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Patent Type & Authority Applications(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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