CN-121991806-A - Toxoplasma attenuated vaccine strain RH delta rop67, construction method and application thereof

CN121991806ACN 121991806 ACN121991806 ACN 121991806ACN-121991806-A

Abstract

The invention discloses a toxoplasma attenuated vaccine strain RH delta rop67, a construction method and application thereof, belonging to the technical field of parasitic disease prevention and control and biological product preparation. The attenuated vaccine strain is obtained by targeted knocking out of ROP67 gene construction in toxoplasma strain RH delta ku80 through CRISPR/Cas9 mediated gene editing technology. Compared with wild type insect strains, the attenuated vaccine strain shows remarkable attenuation property and good immunogenicity. Test results show that the attenuated vaccine strain can induce a host to generate a specific immune response mainly based on cellular immunity, maintain the protection effect on toxoplasma infection in a longer immune period, has the protection effect on tachyzoite infection and chronic infection stages of toxoplasma strains with different virulence, can improve the survival ability of the host on the tachyzoite infection, and reduces the formation level of encapsulation in tissues. The invention provides a technical scheme with long-term immunity potential for the development of toxoplasma attenuated vaccines.

Inventors

SONG HONGYU
CAO HUI
HUANG SHIBO
ZHENG XIAONAN
ZHU XINGQUAN

Assignees

山西农业大学

Dates

Publication Date: 20260508
Application Date: 20260212

Claims (8)

1. The toxoplasma attenuated vaccine strain RH delta ROP67 is characterized in that the RH delta ROP67 is obtained by knocking out a ROP67 gene of a toxoplasma RH delta ku80 insect strain, and the accession number of the ROP67 gene in a toxoplasma whole genome database TOXODB is TGME 49-305590.
2. Use of the toxoplasma attenuated vaccine strain RH Δrop67 according to claim 1 for the preparation of a vaccine for controlling toxoplasma infection.
3. A vaccine for controlling toxoplasma infection comprising the attenuated toxoplasma vaccine strain RH Δrop67 according to claim 1.
4. A method of constructing a toxoplasma attenuated vaccine strain RH Δrop67 according to claim 1, comprising the step of knocking out the ROP67 gene of toxoplasma RH Δku80 strain.
5. The construction method according to claim 4, wherein the ROP67 gene of the Toxoplasma gondii RH delta ku80 strain is knocked out, comprising the steps of: Constructing CRISPR/Cas9 knockout plasmids of target ROP67 genes, respectively amplifying homologous arms of 5 'ends and 3' ends of the ROP67 genes, screening a DHFR fragment and a pUC19 vector skeleton, and connecting after enzyme digestion to obtain homologous DHFR plasmids; And jointly introducing the CRISPR/Cas9 knockout plasmid and the homologous DHFR fragment into toxoplasma RH delta ku80 tachyzoites, and screening and monoclonal to obtain a toxoplasma attenuated vaccine strain RH delta rop67.
6. The construction method of claim 5, wherein the CRISPR/Cas9 knockout plasmid targeting the ROP67 gene is obtained by performing PCR amplification using pSAG a Cas9-U6-SgUPRT as a template and using a primer pair as shown in SEQ ID No.1-2 to replace SgRNA targeting UPRT in pSAG a Cas9-U6-SgUPRT with SgRNA targeting ROP 67.
7. The construction method of claim 6, wherein the nucleotide sequence of SgRNA of the targeted ROP67 is set forth in SEQ ID No. 3.
8. The construction method according to claim 5, wherein the primer sequences for amplifying the 5 'and 3' homology arms of the ROP67 gene are shown in SEQ ID NO. 4-SEQ ID NO. 7; The primer sequence of the amplified screening mark DHFR fragment is shown as SEQ ID NO. 8-SEQ ID NO. 9; The primer sequence of the pUC19 vector skeleton is shown in SEQ ID NO. 10-SEQ ID NO. 11; The primer sequence for amplifying the homologous DHFR fragment is shown as SEQ ID NO. 12-SEQ ID NO. 13.

Description

Toxoplasma attenuated vaccine strain RH delta rop67, construction method and application thereof Technical Field The invention relates to the technical field of parasitic disease prevention and control and biological product preparation, in particular to a toxoplasma attenuated vaccine strain RH delta rop67, a construction method and application thereof. Background Toxoplasma gondii (Toxoplasma gondii) is a typical obligate intracellular parasitic protozoa capable of infecting a wide variety of warm-blooded animals, including humans and a wide variety of domestic animals, and has a host adaptability, a diverse transmission pattern, and can form a long-term persistent infection in the host. Toxoplasma infection has important effects in the fields of animal husbandry production, reproductive safety and public health, and particularly in pregnant women and immunocompromised people, serious pathological consequences are more likely to be caused after infection. In most individuals with normal immune function, toxoplasma infection is often in a recessive infection state, but when the host immune regulation capacity is reduced, serious clinical manifestations such as abortion, fetal dysplasia, central nervous system injury and the like can occur, and even life is endangered. Related studies have shown that toxoplasma can attack brain tissue and remain latent for a long period of time, resulting in neurological symptoms such as headache, somnolence, cognitive dysfunction, etc., which constitute a constant threat to host health. At present, the prevention and treatment of toxoplasmosis still takes medicine treatment as the main treatment, but the existing chemical medicines mainly play a role in inhibiting tachyzoite stage, and are difficult to effectively remove tissue encapsulation formed in the chronic infection process. In addition, the long-term or repeated administration can cause toxic and side effects and drug resistance, and the application of the drug is limited in large-scale prevention and control. Therefore, the exploration of safer and durable prevention and control strategies has important practical significance. Vaccine immunization is generally considered to be one of the effective means of preventing and controlling toxoplasma infection. In recent years, research has been continued around toxoplasma vaccines, and various vaccine forms have been developed, including nucleic acid vaccines, recombinant protein vaccines, nano-delivery vaccines, vector vaccines, attenuated live vaccines, and the like. Among them, attenuated live vaccines have significant advantages in inducing systemic immune responses because of their ability to replicate in a limited manner in a host and mimic the natural course of infection. Research has reported that a variety of toxoplasma attenuated strains can produce a range of immunoprotection effects in different hosts. For example, a partially attenuated strain may induce a specific immune response in a host against toxoplasma, reduce clinical symptoms or pathogenic burden after infection, and block transmission to some extent. However, the traditional attenuated insect strains are mostly obtained through natural mutation or serial passage, the genetic background is complex, and the potential risks of insufficient stability and virulence reversion exist, so that the safety and popularization and application of the attenuated insect strains are affected. Along with the analysis of toxoplasma genome information and the maturation of CRISPR/Cas9 and other gene editing technologies, the site-directed modification of pathogenic related genes is carried out based on molecular level, so that an attenuated insect strain with clear genetic background and controllable toxicity is constructed, and the attenuated insect strain gradually becomes an important development direction of toxoplasma vaccine research. The coryneform proteins play a key role in the invasion of toxoplasma into host cells and in the regulation of host immune responses, and their loss of function may significantly affect the pathogenicity and immunogenicity of parasites. However, the effect of different coryneform protein gene deletions on vaccine safety and immunoprotection effects remains to be further systematically assessed. Therefore, based on a specific coryneform protein gene deletion strategy, the toxoplasma attenuated vaccine strain with good safety and immune protection capability is constructed, and has important research value and application prospect for preventing and controlling toxoplasma infection and researching and developing novel vaccines. Disclosure of Invention The invention aims to provide a toxoplasma attenuated vaccine strain RH delta ROP67, a construction method and application thereof, so as to solve the problems in the prior art, adopt a CRISPR/Cas9 mediated gene editing technology, target and knock out a rod protein related gene ROP67 in the toxoplasma RH delta ku80 strain, construct a