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Recombinant coccidiosis vector for expressing alpha toxin protein and fluorescent tag protein and detection method of recombinant coccidiosis vector

A technology of fluorescent label and detection method, applied in the field of coccidial vector vaccine, can solve the problem of increasing prevention and control of chicken necrotizing enteritis, achieve good application prospects, reduce workload, and achieve the effect of heritability

Pending Publication Date: 2022-03-01
FOSHAN STANDARD BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The use of antibiotic additives in feed has played a greater role in the prevention and treatment of chicken necrotic enteritis, but the resulting problems of drug resistance and drug residues cannot be ignored
At present, the addition of antibiotic additives other than anticoccidiosis to feed has been banned, which has added enormous pressure to the prevention and treatment of chicken necrotic enteritis

Method used

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  • Recombinant coccidiosis vector for expressing alpha toxin protein and fluorescent tag protein and detection method of recombinant coccidiosis vector
  • Recombinant coccidiosis vector for expressing alpha toxin protein and fluorescent tag protein and detection method of recombinant coccidiosis vector
  • Recombinant coccidiosis vector for expressing alpha toxin protein and fluorescent tag protein and detection method of recombinant coccidiosis vector

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Experimental program
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Effect test

Embodiment 1

[0078] This embodiment provides an ETH_00009555 gene editing system, the ETH_00009555 gene editing system includes sgRNA targeting the ETH_00009555 gene, Cas9 and homologous recombination fragments;

[0079] The target of the sgRNA targeting the ETH_00009555 gene is located in the 3'UTR region of the ETH_00009555 gene, and the sgRNA targeting the ETH_00009555 gene is connected to the same gene editing plasmid with the Cas9;

[0080] The homologous recombination fragment includes a 5' homology arm, a SAG13 promoter, a CPα gene, a P2A-EGFP gene and a 3' homology arm connected in sequence.

[0081] The gene editing plasmid is prepared by the following method:

[0082] (1) PCR amplification of the sgRNA targeting the ETH_00009555 gene and the coding sequence of Cas9:

[0083] Using CRISPR-ACT-F (SEQ ID NO.1) and CRISPR-R (SEQ ID NO.2) as primers, using the pSAG1::Cas9-U6::sgUPRT plasmid as a template, and using the Q5 site-directed mutagenesis kit to perform PCR React to obtain ...

Embodiment 2

[0157] This embodiment provides a recombinant coccidia vector, the recombinant coccidia vector is edited by the ETH_00009555 gene editing system described in Example 1, and the CPα gene and EGFP gene are integrated in the 3'UTR region of the ETH_00009555 gene coccidian vector.

[0158] The schematic diagram of the construction principle of the recombinant coccidian vector is as follows: figure 2 As shown, the construction method is as follows:

[0159] (1) Take 1 million fresh coccidia sporozoite suspensions, add them into the electric shock cup with a gap=4mm, and resuspend the sporozoites in the electric shock cup with electroporation buffer;

[0160] (2) Add the pSAG1::Cas9-U6::sgACT plasmid and the homologous recombination fragment in Example 1 to the electroporation solution, use the electroporation solution to adjust the volume to 800 μL, and gently pipette to mix;

[0161] (3) Place the electric shock cup in the BTX electroporator, and shock twice under the condition...

Embodiment 3

[0165] In this embodiment, the recombinant coccidial vector prepared in Example 2 is detected, and the steps are as follows:

[0166] (1) Take 1 million fresh recombinant coccidian carrier sporozoite suspensions, use the RNAeasy Animal RNA Extraction Kit, follow the steps in the instructions to extract the RNA from the strains, and then use the BeyoRT II cDNA Synthesis Kit (with gDNAEraser) to carry out cDNA synthesis;

[0167] (2) Use the obtained cDNA as a template for PCR amplification detection:

[0168] Use CPα toxin gene-F (SEQ ID NO.26), CPα toxin gene-R (SEQ ID NO.13) to amplify the α toxin gene, use EGFP-F (SEQ ID NO.27), EGFP-R1 (SEQ ID NO.16), amplify the EGFP tag protein gene, the amplification result is as follows Figure 4 shown.

[0169] It can be seen from the figure that the size of the amplified product is in line with the expectation, indicating that the α-toxin protein and EGFP-tagged protein have been successfully transcribed in the recombinant coccidio...

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Abstract

The invention provides a recombinant coccidiosis vector for expressing alpha toxin protein and fluorescent tag protein and a detection method of the recombinant coccidiosis vector, and belongs to the technical field of genetic engineering. The invention provides sgRNA (small guide ribonucleic acid) targeting an ETH00009555 gene, the target spot of the sgRNA targeting the ETH00009555 gene is located at the ETH00009555 gene, and accurate gene site-directed mutation can be carried out; a gene editing system is established by sgRNA, Cas9 and a homologous recombination fragment, so that a target fragment can be accurately subjected to homologous recombination on the ETH00009555 gene; a target gene is successfully recombined in DNA (Deoxyribose Nucleic Acid) of the recombinant coccidium vector, and the recombinant coccidium vector has an obvious fluorescence signal under a fluorescence microscope, which indicates that the recombinant coccidium vector successfully expresses the alpha toxin protein and the fluorescence tag protein.

Description

technical field [0001] The invention belongs to the field of coccidia vector vaccines, in particular to a recombinant coccidia vector expressing alpha toxin protein and fluorescent tag protein and a detection method thereof. Background technique [0002] Clostridium perfringens (Clostridium perfringens, CP) belongs to the Bacillus family Clostridium genus, is a strict anaerobic Gram-negative bacteria, of which Clostridium perfringens type A is the cause of chicken necrotic enteritis (Necrotic Enteritis, NE) the main pathogen. Chicken necrotic enteritis is often called "rotten intestine disease", which is a chicken intestinal disease with complex pathogenesis. The susceptible group of chicken necrotic enteritis is chickens aged 2 to 4 weeks. The main pathological feature is severe damage to the intestinal mucosa, which greatly reduces the production performance of animals. According to statistics, worldwide, the economic loss caused by chicken necrotic enteritis is more tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N9/22C12N15/55C12N15/65C12N15/85C12N15/90C12Q1/686G01N21/64
CPCC12N15/113C12N9/22C12N9/16C12N15/65C12N15/85C12N15/902C12Q1/686G01N21/6428C12N2310/20C12Q2521/107C12Q2563/107
Inventor 林瑞庆陆肖周德荣翁亚彪蔡晓懿谭志坚刘丽丹王新秋
Owner FOSHAN STANDARD BIO TECH
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