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Eimeria tenella calcium-dependent protein kinase gene and application thereof

A technology of Eimeria coccidiosis and genes, applied in the field of bioengineering, can solve the problems of drug resistance of coccidiosis and achieve good immunogenicity

Inactive Publication Date: 2012-07-04
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 will solve the technical problem of urgently needing to develop novel vaccines and new drugs for the problem that drug resistance against coccidiosis is very easy to produce drug resistance, and provides a kind of Eimeria tenella calcium-dependent protein kinase (EtCDPK) gene, which EtCDPK can be used in the Eimeria sporozoites play a special role in recognizing and binding to the invasion site of chicken intestinal epithelial cells, and have high potential for the development of new vaccines or new drugs that inhibit the invasion of chicken intestinal epithelial cells by sporozoites and block the life cycle of coccidia. Value

Method used

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  • Eimeria tenella calcium-dependent protein kinase gene and application thereof
  • Eimeria tenella calcium-dependent protein kinase gene and application thereof
  • Eimeria tenella calcium-dependent protein kinase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Cloning and analysis of the full-length cDNA of the Eimeria tenella EtCDPK gene

[0054] In the early stage of the laboratory, the differentially expressed genes of sporozoites and sporulated oocysts were screened on a large scale using suppression subtractive hybridization technology and cDNA microarray technology, and a large number of differentially expressed gene ESTs were obtained. ESTs of highly expressed genes (gene clone number: ZB1-H07), Blast homology comparison analysis found that ZB1-H07 is highly homologous to Plasmodium falciparum calcium-dependent protein kinase (CDPK), which is speculated to be Eimeria tenella Calcium-dependent protein kinase, named EtCDPK.

[0055] The full-length cDNA of the EtCDPK gene was amplified by RACE technology, and the specific steps were as follows:

[0056] 1. RACE primer design

[0057] The ESTs sequence of ZB1-H07 was first compared with the E.tenella genome sequencing website ( http: / / www.Sanger.ac.uk. / pr...

Embodiment 2

[0091] Example 2 Analysis of expression differences of EtCDPK gene in different developmental stages of Eimeria tenella

[0092] 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 were used as templates, and real-time fluorescent quantitative PCR was used to select 18s rRNA as an internal reference to verify that the EtCDPK gene was expressed in different developmental stages of Eimeria tenella. expression in the body. Table 3 is the real-time fluorescence quantitative PCR amplification primer sequence. The results showed that the EtCDPK gene was highly expressed in the sporozoite development stage (see figure 2 ).

[0093] Table 3 real-time quantitative PCR amplification primer sequence

[0094]

Embodiment 3

[0095] Example 3 Prokaryotic expression and immunogenicity analysis of EtCDPK gene

[0096] 1. Construction of prokaryotic expression plasmids

[0097] The EtCDPK gene was connected to the prokaryotic expression vector pET28a(+), and the recombinant expression plasmid pET(28a)-EtCDPK was constructed. After the pET(28a)-EtCDPK recombinant plasmid was transformed into DH5α competent cells, the positive clones were sequenced and BamHI, Hind III double enzyme digestion identification. image 3 Shown is the enzyme digestion identification diagram of the recombinant prokaryotic expression plasmid pET(28a)-EtCDPK, in image 3 Among them, "1" represents the BamHI and HindIII double digestion product of the recombinant expression plasmid pET(28a)-EtCDPK.

[0098] The identified correct recombinant expression plasmid (pET28a-EtCDPK) was transformed into Escherichia coli BL21(DE3), and the expression was induced by 1mmol / L IPTG. The predicted molecular weight of EtCDPK is 49.3KDa. Usi...

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Abstract

The invention discloses an eimeria tenella gene. The nucleotide sequence of the gene coded calcium-dependent protein kinase comprises a DNA sequence for coding an SEQ ID NO.1 amino acid sequence or a DNA sequence for coding an amino acid sequence with calcium-dependent protein kinase activity obtained by deleting, adding, inserting or replacing one or a plurality of amino acids in the SEQ ID NO.1amino acid sequence. The invention also discloses a protein coded by the gene. The eimeria tenella gene is a new gene highly expressed in a sporozoite phase of the eimeria tenella, and is related to the invasion of a to a host cell. The gene has high application value in developing new vaccines or new medicines for inhibiting the invasion of the sporozoite to the host cell and blocking the life cycle of the eimeria tenella.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an Eimeria tenella calcium-dependent protein kinase 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. [0003] In view of the hazards of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/12C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12Q1/68C07K16/40C07K16/06A61K39/012A61K48/00A61P33/02
Inventor 董辉韩红玉黄兵李洋姜连连赵其平朱顺海马卫娇程军曾艳波
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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