Eimeria tenella invasion-related protein Et CHP559 gene and application thereof

A technology of Eimeria cocci and protein amino acids, applied in the field of bioengineering, can solve problems such as easy generation of drug resistance, and achieve the effect of good immunogenicity

Inactive Publication Date: 2015-02-04
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

[0004] The present invention aims to solve the technical problem of the urgent need to develop new vaccines and new drugs for the problem that drug resistance to coccidiosis is easily produced, and provides a Eimeria tenella invasion-

Method used

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  • Eimeria tenella invasion-related protein Et CHP559 gene and application thereof
  • Eimeria tenella invasion-related protein Et CHP559 gene and application thereof
  • Eimeria tenella invasion-related protein Et CHP559 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Cloning and analysis of full-length cDNA of Eimeria tenella Et CHP559 gene

[0037] 1. Materials

[0038] Insect strains and in vitro cultured cells: Eimeria tenella (E. tenella) was preserved and propagated by Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. DF-1 (chicken embryo fibroblast) cells were used for infection, inhibition, immunofluorescence experiments and transfection of eukaryotic expression recombinant plasmids.

[0039] 2. Method

[0040] 2.1 Total RNA extraction

[0041]Before the experiment, the clean metal extraction utensils were wrapped with tinfoil and baked in an oven at 180°C for 6 hours. The operating table and the centrifuge tube rack were sterilized with 75% alcohol to ensure no RNase contamination. The pipettes, pipette tips and centrifuge tubes used are all designed for RNA extraction.

[0042] The extraction of sporozoite total RNA was performed according to the instructions of Trizol reagent,...

Embodiment 2

[0066] Example 2 Differential expression analysis of Et CHP 559 gene in different developmental stages of Eimeria tenella

[0067] Total RNA of four developmental stages of Eimeria tenella (unsporulated oocysts, sporulated oocysts, sporozoites, and second-generation merozoites) were extracted, and Eimeria tenena unsporulated The first strand of cDNA of oocysts, sporulated oocysts, sporozoites, and second-generation merozoites was used as a template, and real-time fluorescent quantitative PCR was used to select 18s rRNA as an internal reference to verify that the EtCHP559 gene was in different developmental stages of Eimeria tenella. expression in worms. Table 3 is the real-time fluorescence quantitative PCR amplification primer sequence. The results showed that the Et CHP559 gene was highly expressed in the sporozoite development stage (see figure 2 ). figure 2 Among them, UO represents the unsporulated oocyst of Eimeria tenella; SO represents the sporulated oocyst of Eim...

Embodiment 3

[0070] Example 3 Construction of Et CHP 559 Gene Prokaryotic Expression Recombinant Plasmid and Expression of Recombinant Protein

[0071] 1. Construction of prokaryotic expression recombinant plasmid (pGEX-4T-Et CHP559) and expression of recombinant protein

[0072] The recombinant cloning plasmid pGEM-T-Et CHP559, which was sequenced correctly before, was double-digested with restriction endonucleases BamHI and Xho I, and then combined with the prokaryotic expression vector pGEX-4T which was double-digested with the same restriction endonucleases. -2 connection to construct the recombinant expression plasmid pGEX-4T-Et CHP559. After transforming Escherichia coli TOP10, it was identified by PCR and double enzyme digestion (BamH Ⅰ and Xho Ⅰ), and the target band with the expected size was obtained (see image 3 ), indicating that the prokaryotic expression plasmid containing the Et CHP559 gene was constructed successfully. image 3 Shown is the enzyme digestion identificatio...

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Abstract

The invention discloses an eimeria tenella Et CHP559 gene. The Et CHP559 gene includes an open reading frame nucleotide sequence represented as the SEQ ID No.1. The eimeria tenella conserved protein Et CHP559 gene is a new gene of the eimeria tenella and is relative to sporozoite invaded host cells. The eimeria tenella conserved protein Et CHP559 gene has quite high application value in development of a new vaccine or a new medicine capable of inhibiting the sporozoite invaded host cells and blocking a life history of the eimeria tenella.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an Eimeria tenella invasion-related protein Et CHP559 gene and application thereof. Background technique [0002] Chicken coccidiosis is a kind of extremely serious global parasitic disease caused by several Eimeria coccidiosis parasitic intestines, which seriously endangers the growth and development of chickens, and is one of the most harmful diseases in intensive chicken farming. It causes huge economic losses to the poultry industry every year. Eimeria tenella is one of the most pathogenic coccidia in chickens. It mainly parasitizes the cecum and its surrounding areas, can cause hemorrhagic enteritis, and is the most harmful to chicks. death rate. The current method of controlling coccidiosis in chickens is mainly based on adding anticoccidial drugs to the feed for prevention. However, due to the long-term use of anticoccidial drugs, the generation of chicken coccidi...

Claims

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Application Information

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IPC IPC(8): C12N15/30C07K14/455C12N15/70C12N15/85C07K16/20A61K39/012A61K48/00A61K45/00A61K39/395A61P33/02
Inventor 韩红玉黄兵翟颀董辉赵其平朱顺海梁思婷李莎杨斯涵
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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