Preparation of chicken eimeria tenella strain with EYFP knocked into Et.Actin gene

An Eimeria, gene technology, used in genetic engineering, plant genetic improvement, microorganism-based methods, etc.

Pending Publication Date: 2021-11-02
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method can solve the transgenic problem in the existing gene editing methods, and provide an effective technical means for the functional analysis of unknown coccidia genes and the development of new anticoccidial drugs and vaccines

Method used

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  • Preparation of chicken eimeria tenella strain with EYFP knocked into Et.Actin gene
  • Preparation of chicken eimeria tenella strain with EYFP knocked into Et.Actin gene

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1 Using FnCas12a / crRNA-mediated gene editing technology to knock EYFP into Eimeria tenena Et. Actin Gene.

[0036] 1. Expression and purification of FnCas12a protein.

[0037] The expression and purification process of FnCas12a protein is similar to that of SaCas9 (https: / / pubmed.ncbi.nlm.nih.gov / 30927491 / ). The sequence information of the plasmid expressing FnCas12a is shown in SEQ ID NO.1. Use BeyoGold TM His-tag Purification Resin (P2210, Beyont) kit was used to purify FnCas12a protein, and the eluent was 200 mM imidazole buffer (200 mM imidazole, 50 mM NaH 2 PO 4 , 300 mM NaCl, pH 8.0). The purified protein was preserved in 50% glycerol and stored at -80°C for later use. For the expression and purification process of FnCas12a, see figure 1 .

[0038] 2. Synthesis of crRNA in vitro.

[0039] According to the crRNA design principle of FnCas12a, design Et. Actin The crRNA sequence (SEQID NO.2) of the gene editing site was synthesized in vitro using ...

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Abstract

The invention discloses preparation of a chicken eimeria tenella strain with EYFP knocked into an Et.Actin gene. According to the invention, an FnCas12a protein, crRNA synthesized in vitro and a homologous recombination fragment (an Et.Actin gene containing a P2A-DHFR-EYFP expression cassette) are introduced into chicken eimeria tenella sporozoite by utilizing a Lonza 4D-Nucleofector nuclear transformation instrument, so that the editing of the chicken eimeria tenella is realized. The edited insect strain is screened through pyrimethamine in a chicken body to obtain a high-purity gene edited insect strain. Then, the gene edited insect strain is verified by utilizing PCR and Sanger sequencing technologies, and it is proved that the EYFP successfully marks the Et.Actin gene. After sporozoites of the strain are subjected to cell culture and a chicken is infected, cecum mucosa is taken and smeared to show that the EYFP is expressed in the whole life history of the gene edited strain. The gene editing method can be used for performing fixed-point marking on a chicken coccidiosis gene, and is beneficial to analyzing the function of the coccidiosis gene. The invention also has important significance for research and development of novel anticoccidial drugs and vaccines.

Description

technical field [0001] The invention relates to the field of molecular editing, in particular to the preparation of an Eimeria tenella strain in which EYFP is knocked into the Et. Et. Actin A method for preparing a gene-genetic Eimeria tenella strain and its application, the method utilizes FnCas12a-mediated gene editing technology for Eimeria chicken tenella Et. Actin Genes are marked. The method can perform site-specific labeling on the Eimeria tenella gene, which is helpful for analyzing the function of the coccidia gene and developing new anticoccidial drugs and vaccines. Background technique [0002] Chicken coccidiosis is an intestinal parasitic disease caused by one or more apicomplexan protozoa, characterized by diarrhea, bloody stools, low feed conversion, growth retardation and death. It is prone to outbreaks in intensive farms and brings huge economic losses to the poultry farming industry. Global losses due to chicken coccidiosis are as high as US$ 3 billion p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/11C12N15/30C12N15/90C12R1/90
CPCC12N15/902C07K14/455
Inventor 谷峰程培培
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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