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Construction method for coccidia expressing exogenous fluorescent protein

A construction method and protein coccidia technology, applied in the field of genetic engineering, can solve the problems of limited expression of fluorescent reporter genes, poor fluorescent signal, and poor fluorescent labeling effect, etc.

Active Publication Date: 2021-03-26
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

In the prior art, homologous recombination technology is usually used to knock the fluorescent reporter gene into the target gene. This method is easy to operate, but the expression of the fluorescent reporter gene is limited, the fluorescent signal is poor, and the fluorescent labeling effect is not good.

Method used

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  • Construction method for coccidia expressing exogenous fluorescent protein
  • Construction method for coccidia expressing exogenous fluorescent protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Construction method of gene editing plasmid pCRISPR::EtA plasmid containing gRNA

[0056] 1. According to the Eimeria tenella (E.tenella) gene sequence (ETH_00017055) information, design gRNA, construct sgRNA vector, and the designed primer is CRISPR-EtA-F (containing EtA target-specific gRNA sequence) (SEQ ID NO: 2) and CRISPR-R (GOI-gRNA-Rv) (SEQ ID NO: 3).

[0057] 2. Using CRISPR-EtA-F and CRISPR-R as primers, pSAG1::Cas9-U6::sgUPRT plasmid as a template, use the Q5 point mutation kit to perform PCR reaction, then perform KLD reaction on the PCR product, and then KLD mixture Transform Stbl3 competent cells, and then identify positive clones to obtain pCRISPR::EtA plasmid.

[0058] The sequencing result of the pCRISPR::EtA plasmid is shown in SEQ ID NO: 4, and the sequencing result shows that the gRNA has been integrated into the plasmid. The results of sequencing indicated that a suitable pCRISPR::EtA plasmid was obtained.

Embodiment 2

[0060] Construction method of homologous recombination plasmid

[0061] 1. Amplify the gene fragment of the downstream 5'-homology arm of the EtA gene

[0062] Primers EtA 5'-ARM-F (SEQ ID NO: 5, containing the HindIII restriction site) and EtA 5'-ARM-R (SEQ ID NO: 6) were designed to target the genome DNA of Eimeria tenella Guangdong strain As a template, the 5'-ARM sequence of the EtA gene was amplified as the downstream homology arm sequence. After the PCR product was sequenced, the nucleotide sequence of the downstream EtA 5'-ARM was obtained as shown in SEQ ID NO:1.

[0063] 2. Amplification of screening genes and fluorescent genes

[0064] Construction of EtA-DHFR-mCherry-3UTRDHFR Sequence Fragment Containing Upstream EtA 5' Homology Arm

[0065] (1) Amplify the upstream regulatory sequence of DHFR and the open reading frame sequence of DHFR: design primers DHFR-5'-F (SEQ ID NO: 7) and DHFR-R (SEQ ID NO: 8), and use them as upstream and downstream primers , with the ...

Embodiment 3

[0074] The gene fragment of the downstream 5'-homology arm obtained in Example 2, the EtA-DHFR-mCherry-3'UTRDHFR homology arm sequence containing the upstream EtA 5' homology arm sequence and the gene fragment of the downstream 3'-homology arm Ligation is performed to obtain a long fragment containing 5'-homology arm-DHFR-mCherry-3'UTRDHFR homology arm sequence-3'-homology arm and the pcDNA 3.1(+) vector after digestion with BamHI and Hind III The sequences were ligated sequentially through the ClonExpress MultiS One Step Cloning Kit to obtain the successful targeting DHFR resistance plasmid pEtA::DHFR-mCherry (SEQ ID NO: 25).

[0075] Use CRISPR / Cas9-mediated integration at specific sites to insert resistance screening elements and fluorescent genes, use the upstream homology arm of the EtA gene to connect the resistance screening element and the fluorescent gene to be knocked in, and the downstream homology arm of the EtA gene. Construct recombinant plasmids as targeting pla...

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Abstract

The invention provides a construction method for coccidia expressing exogenous fluorescent protein, and belongs to the technical field of gene engineering. The construction method for coccidia expressing exogenous fluorescent protein comprises the following steps: designing gRNA for a target gene of the coccidiosis, and constructing a gene editing plasmid containing the gRNA; constructing a homologous recombinant plasmid containing a target gene and an mCherry gene; co-transfecting the gene editing plasmid and the homologous recombinant plasmid into sporozoites of coccidia; and infecting animals with the transfected sporozoites, and performing screening to obtain the coccidia expressing exogenous fluorescent protein. A fluorescent protein mCherry gene and a screening gene are knocked intoa coccidia genome by combining a gene editing technology and a homologous recombination technology, and the constructed coccidia can stably and efficiently express a fluorescent signal. According to the construction method, not only can the coccidia expressing the exogenous fluorescent protein be successfully constructed, but also fluorescent protein expression is stable and high in efficiency, and the construction method is suitable for research on growth, genetic engineering or drug screening of coccidia.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a construction method for expressing exogenous fluorescent protein coccidian. Background technique [0002] mCherry is a red fluorescent dye used for tracking, such as molecular labeling and cellular component localization. mCherry is a protein isolated from coral (Discosoma), and its maximum excitation light and emission light are 587nm and 610nm, respectively. mCherry has advantages over other fluorescent protein tags due to its color and photostability of monomeric molecules. [0003] mCherry has high sensitivity, is easy to detect, and is used for in vivo detection, and is not destructive to cells, organelles, etc.; it is not limited by different developmental stages of insects in vivo; it does not require any substrate and exogenous cofactors to participate; in vivo Color changes are also sometimes observed under natural light. [0004] In the stud...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/11C12N15/65C12N15/90C12N15/64C12Q1/25C12R1/90
CPCC12N15/65C12N15/902C12Q1/025
Inventor 曲自刚
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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