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Eimeria tenella calcium-dependent protein kinases 4 gene, and applications thereof

A technology of Eimeria calcium and Eimeria, which is applied to the Eimeria tenella calcium-dependent protein kinase 4 gene and its application field, and can solve the problem of easy drug resistance of coccidiosis

Inactive Publication Date: 2017-06-16
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

[0006] The present invention will solve the technical problem of urgently needing to develop new vaccines and new drugs for the problem that drug resistance to coccidiosis is very easy to produce, and provides a kind of Eimeria tenella calcium-dependent protein kinase 4 gene (Et CDPK4), the Et CDPK4 plays an important role in the process of Eimeria tenella sporozoites invading host cells, and has high application value for the development of new vaccines or new drugs for the prevention and treatment of chicken coccidiosis

Method used

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  • Eimeria tenella calcium-dependent protein kinases 4 gene, and applications thereof
  • Eimeria tenella calcium-dependent protein kinases 4 gene, and applications thereof
  • Eimeria tenella calcium-dependent protein kinases 4 gene, and applications thereof

Examples

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Effect test

Embodiment 1

[0035] Example 1 Cloning and analysis of full-length cDNA of Eimeria tenella Et CDPK4 gene

[0036] 1. Materials and Methods

[0037] 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. The EST sequence of the highly expressed gene was compared with the Eimeria tenella genome database (http: / / www.genedb.org / Homepage / Etenella), and it was found that the EST and the calcium-dependent protein kinase family predicted in the coccidia genome database Calmodulin kinase (ETH_00010685) is 100% homologous. Therefore, 5'RACE and 3'RACE primers were designed and synthesized according to the sequence, and the full-length cDNA sequence containing 5'UTR, 3'UTR, and ORF was amplified using RACE technology. Follow GeneRacer TM Kit (Invitrogen Cor...

Embodiment 2

[0043] Example 2 Construction of EtCDPK4 gene prokaryotic expression system

[0044] 1. Materials and Methods

[0045] According to the full-length 1803bp of the ORF of the gene, a coding region sequence of it was selected for cloning and analysis. The size of the selected coding region is 1660bp, the number of encoded amino acids is 550aa, and the size is 62.6KDa.

[0046] Instructions for use Use Trizol reagent (TaKaRa) to extract the total RNA of pure sporozoites, and use M-MLV Reverse Transcriptase Kit (Invitrogen, USA) to reverse-transcribe all of them into cDNA as PCR amplification after 1% agarose gel electrophoresis identification. The increased template was PCR amplified using the following specific primers with restriction endonuclease sites: EtCDPK4UP: 5'-CGGGATCCAGCAGGTGATGGGTGGGCGGGAGGT-3' (SEQ ID NO.7, containing BamHI site), EtCDPK4LOW: 5'-GCGTCGACAATTCGTCCCAGTCAATCTGCCCAT-3' (SEQ ID NO.8, containing the SalI restriction site), the final target gene EtCDPK4 am...

Embodiment 3

[0049] Example 3 Obtaining and Identification of Anti-EtCDPK4 Recombinant Fusion Protein Serum

[0050] 1. Materials and Methods

[0051] Transform the pCold-I-EtCDPK4 recombinant plasmid with correct sequencing into Escherichia coli BL21 strain, use 16°C, 1mMIPTG to induce expression for 24 hours, and use the affinity resin that can specifically bind His-Tag to obtain the soluble recombinant protein Column chromatography, and then quantify the obtained protein with BCA, then immunize 2 New Zealand white rabbits and 5 Balb / c mice. (FCA) for emulsification (1:1), and a second immunization after an interval of 2 weeks. At this time, the recombinant soluble protein should be emulsified with Freund's incomplete adjuvant (1:1), and then immunized once every other week until the second immunization. After five times of immunization, blood was collected from New Zealand white rabbits and Balb / c mice for detection (WB), and the titer of immune rabbit serum was determined by indirect ...

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Abstract

The invention discloses an eimeria tenella gene, and more specifically discloses eimeria tenella calcium-dependent protein kinases 4 gene. The nucleotide sequence is disclosed in SEQ ID NO.1. The eimeria tenella calcium-dependent protein kinases 4 gene is a high expression gene at eimeria tenella spore period, is related to invading of sporozoite into host cells, and possesses relatively high application value in development of novel vaccines or novel drugs used for inhibiting invading of sporozoite into host cells and blocking eimeria tenella life history.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an Eimeria tenella calcium-dependent protein kinase 4 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. In severe cases, it can cause high 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/63C12N9/12C07K16/18A61K48/00A61K39/395A61P33/02
Inventor 韩红玉黄兵王自文董辉赵其平朱顺海李聪朱雪龙夏伟丽门启斐唐敏
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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