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Method for knocking out eimeria tenella N-myristoyl transferase gene

A technology of Eimeria, myristoyl, applied in the field of microbiology

Active Publication Date: 2020-04-03
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] N-myristoyltransferase plays an important role in worms, but there is no method for knocking out the N-myristoyltransferase gene of Eimeria tenoni in the prior art

Method used

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  • Method for knocking out eimeria tenella N-myristoyl transferase gene
  • Method for knocking out eimeria tenella N-myristoyl transferase gene
  • Method for knocking out eimeria tenella N-myristoyl transferase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Acquisition of sporozoites from Eimeria tenella Guangdong strain

[0101] The Eimeria tenella strain preserved in 2.5% potassium dichromate solution (the conventional source of the worm strain is enough), was centrifuged at 3600rpm for 5min to remove the potassium dichromate solution, and then reweighted with PBS (0.01M, pH7.4) Suspended, centrifuged at 3600rpm for 5min, and repeated three times. Subsequently, the obtained precipitate was washed with saturated saline (at room temperature, the solubility of NaCl in water is 36.0 g dissolved in 100 g water).

[0102] Resuspend, centrifuge at 2000rpm for 10min to obtain the supernatant, then add the supernatant to water (the volume ratio of supernatant to water is 1:5), then centrifuge at 3600rpm for 10min to obtain purified oocysts. Resuspend the oocysts with PBS (0.01M, pH7.4), then add 200mm glass beads (the ratio of PBS to glass beads is 1:1), and beat with wrist force until 95% sporulation The oocysts were decystate...

Embodiment 2

[0104] Construction of knockout plasmid pCRISPR::EtNMT plasmid and homologous template plasmid pEtNMT::DHFR plasmid for drug screening

[0105] (1) Cloning of EtNMT gene of Eimeria tenderensis Guangdong strain:

[0106] Total RNA was extracted from oocysts of Eimeria tenella, and cDNA synthesis was performed using a one-step RT-PCR kit (Takara Bio, Japan). The keyword "glycylpetide N-tetradecanoyltransferase" was entered in the online website ToxoDB (https: / / toxodb.org / toxo / ), and the species-related glycylpetide N-tetradecanoyltransferase of Eimeria acervulina exists in the ToxoDB database, and its ToxoDB No: EAH_00054370) , while Eimeria tenella was absent. Therefore, blast the EAH_00054370 sequence as a template (parameter: database: RefSeq Representative genome (RefSeq_representative_genomes), select the organism as "Eimeria tenella (taxid: 5802)", the EtNMT gene is located in Eth_Scaff32 in the Eimeria genome, and use the same After the source sequence and related speci...

Embodiment 3

[0142] Transfection of Eimeria tenella sporozoites and screening of positive clones

[0143] Take 1×10 7 A newly extracted fresh and vigorous Eimeria tenenia sporozoite suspension was added to the electroporation cup, and electroporation buffer Cytomix (composition of Cytomix, 120mmol / L KCl, 0.15mmol / L CaCl 2 ,10mmol / LK 2 HPO 4 pH 7.6, 25mmol / L HEPES pH 7.6, 2mmol / L EGTA, 5mmol / L MgCl 2 ) to resuspend worms, then add pCRISPR::EtNMT plasmid (5 μg) and pEtNMT::DHFR homologous template plasmid (1 μg), make the volume to 800 μL, mix thoroughly, and place the electroporation cup in the Bio-Rad electroporator, Under the conditions of voltage 2000v, capacitance 25μF, and capacitance ∞, electric shocks were performed twice, and after electrotransfection, the sporozoites were 7 The density of sporozoites / bottle was inoculated onto the monolayer MDBK cells that had covered the bottom of the 80% cell bottle (before inoculation, the cells were washed twice with 0.1mol / L, pH 7.2 PBS),...

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Abstract

The invention provides a method for knocking out an eimeria tenella N-myristoyl transferase gene, which belongs to the technical field of microorganisms, and comprises the following steps: mixing sporozoites of eimeria tenella with pCRISPR: EtNMT plasmids and pEtNMT: DHFR plasmids, and carrying out electrotransformation to obtain the eimeria tenella with the knockout N-myristoyl transferase gene.By adopting the method provided by the invention, the eimeria tenella N-myristoyl transferase gene can be successfully knocked out, and a foundation is laid for researching functions of the eimeria tenella gene and researching and developing vaccines.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a method for knocking out Eimeria tenella N-myristoyltransferase gene. Background technique [0002] Eimeria is one of the most important pathogens in animal husbandry, which can cause significant economic losses in poultry, rabbit, pig, cattle, sheep and other breeding industries. According to statistics, coccidiosis causes global chicken industry losses of more than 5 billion U.S. dollars every year. In my country, the economic loss due to chicken coccidiosis is as high as 6 billion to 7 billion yuan, and the drug expenditure alone is as high as 1.2 billion yuan. . In intensive farming, the use of anticoccidiostats to control coccidia greatly reduces economic losses and promotes the healthy development of the poultry industry. However, with the passage of time, severe resistance to the commercially available coccidiostats has emerged. The prevention and con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N1/13C12R1/90
CPCC12N15/113C12N15/902C12N9/1025C12N2310/20C12N9/22C12N15/102C12N15/1137C12N9/1029C12Y203/01097C12N15/1031C12N15/79
Inventor 蔡建平曲自刚许笑汪靖龚振兴王恒何雪阳王文青
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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