Neospora caninum attenuated strain with double deletion of grx S16 and grx C5 genes, and construction method and application thereof

A technology of Neospora and a construction method, which is applied in the field of genetic engineering to achieve the effects of reducing pathogenicity and preventing reinfection

Active Publication Date: 2020-05-22
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To date, there are no effective vaccines or drugs for the control of neosporosis in animals

Method used

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  • Neospora caninum attenuated strain with double deletion of grx S16 and grx C5 genes, and construction method and application thereof
  • Neospora caninum attenuated strain with double deletion of grx S16 and grx C5 genes, and construction method and application thereof
  • Neospora caninum attenuated strain with double deletion of grx S16 and grx C5 genes, and construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1: Construction of Neospora Δgrx S16Δgrx C5 strain

[0033] (1) Basic strain Nc1: The Nc1 strain is an international standard strain with glutaredoxin S16 and glutaredoxin C5 genes, the nucleotide sequences of the glutaredoxin S16 and glutaredoxin C5 genes are sequentially are SEQ ID NO.1 and SEQ ID NO.2.

[0034] (2) Construction of pSAG1-Cas9-NcU6-sggrx S16 plasmid

[0035] The specific steps of pSAG1-Cas9-NcU6-sggrx S16 plasmid construction are as follows:

[0036] ① Use the EuPaGDT library in ToxoDB to design the targeting site for glutaredoxin S16 gene knockout, and design primers according to the target sequence to amplify the Cas9 fragment (No. 1 fragment), Neospora U6 promoter (No. 2 fragment) ), the T vector fragment (fragment No. 3), the grx S16-specific gRNA is added to the downstream primer of the U6 promoter and the upstream primer of the T vector backbone, and the plasmid map is shown in figure 1 .

[0037] Fragment Primer No. 1 (5'→3'):

[00...

Embodiment 2

[0151] Example 2: Characteristics of Neospora Δgrx S16Δgrx C5 strain

[0152] (1) In vitro invasion and reproduction characteristics of Δgrx S16Δgrx C5 strain

[0153] The plaque test can comprehensively reflect the invasion, proliferation and situation of Neospora, and the plaque area can directly reflect the invasion and reproduction ability of the parasite. Spread HFFs cells over a 12-well cell culture plate, collect tachyzoites of Δgrx S16Δgrx C5 and Nc1 strains, then inoculate 300 tachyzoites into HFF cells, culture for 7 days, fix with 4% paraformaldehyde at room temperature for 30 minutes, and wash with PBS , stained with crystal violet, after staining at room temperature for 20 min, the remaining crystal violet was washed away with PBS, and the plaque area was counted. The results showed that the plaque area of ​​Δgrx S16Δgrx C5 and Nc1 strains was significantly different (p>0.05), indicating that the invasion and proliferation ability of strains lacking grx S16 and g...

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PUM

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Abstract

The invention discloses a neospora caninum attenuated strain with the double deletion of grx S16 and grx C5 genes, and a construction method and application thereof. The strain lacks the glutaredoxinS16 gene (grx S16) and glutaredoxin C5 gene (grx C5) of neospora caninum, and nucleotide sequences of the grx S16 gene and grx C5 gene are as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The strain with the double deletion of grx S16 and grx C5 genes of neospora caninum constructed by the invention is significantly reduced in reproductive capacity and virulence, can effectively resist the reinfection of neospora caninum after inoculation of mice, and has the potential to become a neospora caninum attenuated vaccine.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an attenuated neospora strain with double deletion of grx S16 and grx C5 genes, its construction method and application. Background technique [0002] Neospora is an obligate intracellular parasitic protozoan, and neosporosis is a parasitic disease shared by many animals, which is widely prevalent in the world. The harm to cattle and dogs is particularly serious, mainly causing abortion of pregnant animals, stillbirth and nervous system dysfunction of newborn young animals. Neospora infection is one of the important reasons for abortion of dairy cows. So far, there is no effective vaccine or drug for the prevention and treatment of neosporosis in animals. At present, the most effective prevention and control measures for neosporosis are to eliminate infected cattle and purify the cattle herd on the basis of effective detection. Therefore, it is of great significance to constr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/11C12N15/85C12N15/66C12Q1/6893A61K39/002A61P33/02C12R1/90
CPCC12N9/0004C12Y120/04001C12N15/85C12N15/66C12Q1/6893A61K39/002A61P33/02C12Q2600/124A61K2039/552
Inventor 刘群宋星桔刘晶王臣荣许建海
Owner CHINA AGRI UNIV
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