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Construction method and applications for toxoplasma adenylosuccinate lyase gene knockout strains

A technology of adenylate succinic acid and gene knockout, applied in the fields of biology and genetic engineering, can solve the problem of toxoplasmosis prevention without good vaccine and other problems

Active Publication Date: 2019-08-06
枝江市宜合众畜牧有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there is no good vaccine for the prevention of toxoplasmosis, so it is of great value to construct a vaccine against toxoplasmosis with good immunogenicity and immune protection

Method used

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  • Construction method and applications for toxoplasma adenylosuccinate lyase gene knockout strains
  • Construction method and applications for toxoplasma adenylosuccinate lyase gene knockout strains
  • Construction method and applications for toxoplasma adenylosuccinate lyase gene knockout strains

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Experimental program
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Effect test

Embodiment 1

[0013] Embodiment 1: Construction of Toxoplasma gondii ΔADSL strain

[0014] (1) Starting strain ME49

[0015] ME49 is a type II worm strain of the genus Toxoplasma of the family Toxoplasma in the order Coccidia with an adenylosuccinate lyase gene, and the nucleotide sequence of the adenylosuccinate gene is shown in SEQ ID NO:1.

[0016] (2) Construction of pSAG1-Cas9-TgU6-sgADSL plasmid

[0017] ① Use the gRNA online design website (http: / / www.e-crisp.org / E-CRISP / designcrispr.html) to design the target gene targeting site, and design the gRNA primers according to the designed target sequence:

[0018] Upstream primer gRNA-ADSL-F: 5'-GACACTGCTCAATTTCGTCGGTTTTAGAGCTAGAA ATAGC-3' (SEQ ID NO: 3)

[0019] Downstream primer gRNA-R: 5'-AACTTGACATCCCCATTTAC-3' (SEQ ID NO: 4)

[0020] ② Prepare the following reaction system in a sterilized PCR tube, in which the template DNA: pSAG1-Cas9-TgU6-sgUPRT plasmid (purchased from http: / / www.addgene.org):

[0021]

[0022] ③PCR reaction...

Embodiment 2

[0058] The purposes of embodiment 2 Toxoplasma gondii △ ADSL worm strain

[0059] 2.1 Gene knockout strain △ADSL diluted injection

[0060] 1) Dilute injection formula

[0061]

[0062] 2) Preparation method of diluted injection

[0063] ①Mix with a magnetic stirrer for 5 minutes.

[0064] ② Filter and sterilize with a 0.22um filter.

[0065] 2.2 Toxicity test of △ADSL knockout strain on mice

[0066] 1) Use HFF cells to culture Toxoplasma gondii tachyzoites in vitro, until 30-50% of the parasites have escaped from the cells; discard the medium in the original culture bottle, and wash away the escaped parasites and residual medium with PBS , add fresh FBS-free dilution.

[0067] 2) Scrape off the cells with a disposable cell scraper, repeatedly blow and beat the suspension 8-10 times with a 5ml syringe, so that the insect vesicles burst and the worms escape, and filter and purify the worms with a sterile 3μm pore-diameter filter membrane; count the cells Count and dil...

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Abstract

The invention discloses a construction method for toxoplasma gene knockout strains. The method knocks out a toxoplasma adenylosuccinate enzyme gene; the nucleotide sequence of the adenylosuccinate enzyme gene is shown as SEQ ID NO : 1; and a toxoplasma gene knockout strain has a nucleotide sequence shown as SEQ ID NO : 2 at an adenylosuccinate enzyme site. The gene knockout strain has the advantage of being small in virulence, can enhance the immunocompetence of animals to toxoplasma, and has the potential to become an anti-toxoplasma genetic engineering vaccine.

Description

technical field [0001] The present invention relates to a construction method of Toxoplasma gondii adenylate succinate lyase (abbreviated as ADSL) gene knockout strain, and the use of the adenylate succinate lyase gene deletion strain constructed by the method, which belongs to biology and field of genetic engineering. Background technique [0002] Toxoplasma gondii is a zoonotic parasitic protozoa, about 1 / 3 of the world's population may be infected with Toxoplasma gondii, because most of them are invisible after infection. Toxoplasma can parasitize in all nucleated cells of warm-blooded animals, and its hosts are extremely wide. In South America and some parts of Europe, the infection rate of humans and animals is as high as 80-90%; in China, the infection rate is generally around 10%. Toxoplasma gondii is an important opportunistic protozoan. Immunocompetent infected persons are usually asymptomatic, whereas immunocompetent infected persons may develop encephalitis, imm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/11C12N15/85C12R1/90
CPCC12N9/88C12N15/85C12Y403/02002
Inventor 方瑞王璐瑶唐定杨静李森阳杨承杭赵俊龙申邦周艳琴
Owner 枝江市宜合众畜牧有限公司
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