Eimeria tenella apical membrane antigen 1 (AMA 1) gene and application thereof

A technology of Eimeria tenella and tenderness, applied in the fields of application, gene therapy, genetic engineering, etc., which can solve the problems that there is no research report on Eimeria tenella AMA1 gene

Inactive Publication Date: 2011-11-16
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

At present, AMA1 has been found in Plasmodium, Toxoplasma gondii, and Babesia bovis, but so far there is no research report on the AMA1 gene of Eimeria tenella

Method used

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  • Eimeria tenella apical membrane antigen 1 (AMA 1) gene and application thereof
  • Eimeria tenella apical membrane antigen 1 (AMA 1) gene and application thereof
  • Eimeria tenella apical membrane antigen 1 (AMA 1) gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Cloning and analysis of full-length cDNA of Eimeria tenella AMA1 gene

[0049] Using the difference between the Eimeria tenella sporozoite gene expression library and the merozoite gene expression library, EST sequences highly expressed in the sporozoite stage and merozoite stage were obtained, and the EST sequences with high homology to the acrosomal membrane protein were selected The sequence BG589 was used to amplify the full-length gene, and the amplified full-length gene was the Eimeria tenella acrosomal membrane antigen-1 gene, named EtAMA1.

[0050] The full-length cDNA of the EtAMA1 gene was amplified by RACE technology, and the specific steps were as follows:

[0051] 1. Collection of Eimeria tenella sporozoites

[0052] 1.1 Collection and sporulation of Eimeria tenella unsporulated oocysts

[0053] Take the sporulated oocysts of Eimeria tenella stored at 4°C, centrifuge at 3000r / min for 10min, add an appropriate amount of distilled water to the pre...

Embodiment 2

[0076] Example 2 Analysis of expression differences of EtAMA1 gene in different developmental stages of Eimeria tenella

[0077]The total RNA of four developmental stages of Eimeria tenella (spored oocysts, unsporulated oocysts, sporozoites, and second-generation merozoites) were extracted, and the unsporulated eggs of Eimeria tenella The first strand of cDNA of cysts, sporulated oocysts, sporozoites, and second-generation merozoites was used as a template, and the housekeeping gene 18S rRNA was selected as an internal reference by real-time fluorescent quantitative PCR to observe the differences of EtAMA1 gene in Eimeria tenella. mRNA transcription in developmental stage worms. The experimental results show that the EtAMA1 gene is highly expressed in the sporozoite development stage of Eimeria tenella (see image 3 ). Table 3 is the real-time fluorescence quantitative PCR amplification primer sequence.

[0078] Table 3 real-time quantitative PCR amplification primer sequen...

Embodiment 3

[0080] The prokaryotic expression of embodiment 3EtAMA1 gene

[0081] 1. Extraction of prokaryotic expression vector pGEX-6P-1 plasmid

[0082] The plasmid DNA miniprep kit was used to extract the empty vector plasmid pGEX-6P-1 and the plasmid pGEM-T-BG589 containing the full-length sequence of the AMA1 gene, and stored at -20°C for future use.

[0083] 2. Cloning of the EtAMA1 gene sequence containing restriction sites

[0084] Sequence analysis was performed on the full-length sequence of the AMA1 gene. After removing the signal peptide and the transmembrane region of the AMA1 gene, primers were designed for the sequence of the extracellular region for sequence amplification. The upstream and downstream primers were SPA1 and SPA2 respectively (Table 4). BamH I and EcoRI were added to the primers, and the cloning plasmid pGEM-T-BG589 containing the full-length sequence of the AMA1 gene was used as a template to amplify the expression sequence of the AMA1 gene by PCR, thereby...

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Abstract

The invention discloses an Eimeria tenella AMA 1 gene, the nucleotide sequence of which includes a DNA sequence that codes the amino acid sequence represented by SEQ ID No.1 or a DNA sequence that codes the amino acid sequence which has AMA 1 protein activity and is obtained by omitting, adding, inserting or substituting one or more amino acid in the amino acid sequence represented by SEQ ID No.1. The invention also discloses proteins coded by the above-mentioned genes. The Eimeria tenella AMA 1 gene and proteins provided in the invention are closely related to invasion of daughter spores, escape and invasion of merozoites and escape and invasion of microgametes in the stage of sexual reproduction, and are suitable for being developed into novel vaccines or medicines for controlling coccidiosis.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an Eimeria tenella acrosomal membrane antigen-1 (Apical membrane antigen 1, AMA1) gene and its application. Background technique [0002] Chicken coccidiosis is a protozoan disease caused by coccidia of Eimeriaceae and Eimeria parasitizing in chicken intestinal epithelial cells. Among them, Eimeria tenella is the most pathogenic and harmful. It mainly parasitizes the cecum and its surrounding areas, can cause hemorrhagic enteritis, and is the most harmful to chicks. In severe cases, it can cause higher Mortality causes huge economic losses to the aquaculture industry all over the world. At present, the control of coccidiosis mainly relies on the use of anticoccidiosis drugs, but due to the long-term use of anticoccidiosis drugs, the emergence of drug resistance of chicken coccidiosis is inevitable, which makes the control of coccidiosis with drugs a major challenge. [0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/30C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C07K14/455C07K16/20C07K16/06A61K39/012A61K48/00A61P33/02
Inventor 姜连连韩红玉董辉黄兵林矫矫赵其平朱顺海马卫娇程军曾艳波李婷孔春林
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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