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Eimeria tenella serine/threonine protein kinase (Et STK) gene and application thereof

A kind of Eimeria and gene technology, applied in the direction of anti-enzyme immunoglobulin, application, genetic engineering, etc., can solve the problems such as easy to produce drug resistance, and achieve the effect of good immunogenicity

Inactive Publication Date: 2016-08-03
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 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 serine / threonine protein kinase (EtSTK) gene, which EtSTK plays an important role in the invasion of host cells by Eimeria tenella, 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 serine/threonine protein kinase (Et STK) gene and application thereof
  • Eimeria tenella serine/threonine protein kinase (Et STK) gene and application thereof
  • Eimeria tenella serine/threonine protein kinase (Et STK) gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Cloning and analysis of the full-length eDNA of the Eimeria tenella EtSTK gene

[0044] 1. Materials

[0045] 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.

[0046] 2. Method

[0047] 2.1 Total RNA extraction

[0048] 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.

[0049] The extraction of sporozoite total RNA was performed according to the instructions of Trizol rea...

Embodiment 2

[0072] Example 2 Analysis of Expression Differences of EtSTK Gene in Different Developmental Stages of Eimeria tenella

[0073] 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 18srRNA as an internal reference to verify that the EtSTK gene was used in different developmental stages of Eimeria tenella. expression in the body. Table 3 is the real-time fluorescence quantitative PCR amplification primer sequence.

[0074] Table 3 Real-time fluorescence quantitative PCR amplification primer sequence

[0075]

[0076]The results showed that EtSTK gene mRNA was transcribed in the four developmental stages of Eimeria tenella, and the transcription le...

Embodiment 3

[0077] Example 3 Construction of EtSTK Gene Prokaryotic Expression Recombinant Plasmid and Expression of Recombinant Protein

[0078] The recombinant cloning plasmid pGEM-T-easy-EtSTK, which was sequenced correctly before, was double-digested with restriction endonucleases BamHI and EcoRI, and then connected to the prokaryotic expression vector pColdI which was double-digested with the same restriction endonucleases, The recombinant expression plasmid pColdI-EtSTK was constructed. After transforming Escherichia coli TOP10, the target band with the expected size was obtained by PCR and double enzyme digestion (see image 3 ), indicating that the prokaryotic expression plasmid containing the EtSTK gene was constructed successfully.

[0079] The successfully constructed pColdI-EtSTK recombinant expression plasmid was transformed into Escherichia coli BL21(DE3), and its expression was induced by 1mmol / LIPTG at 37°C and analyzed by SDS-PAGE electrophoresis. It was found that the ...

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Abstract

The invention discloses an Eimeria tenella gene which is Et STK gene and includes an amino acid sequence shown as SEQ ID NO.1. Moreover, the invention provides a preparation method of a vaccine for prophylaxis and treatment of coccidiosis in chicken. The method includes: connecting the mature peptide of Eimeria tenella conserved protein Et STK with an expression vector of escherichia coli or pichia pastoris cell to obtain a recombinant expression vector, transferring the recombinant expression vector into a host of escherichia coli or pichia pastoris cell to obtain a recombination strain, and cultivating the recombination strain to obtain the vaccine of coccidiosis in chicken. The method has quite high application value.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an Eimeria tenella EtSTK gene and its application. 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 (Eimeriatenella) is one of the most pathogenic coccidia in chickens. It mainly parasitizes in the cecum and its surrounding areas, can cause hemorrhagic enteritis, and is the most harmful to chicks. mortality 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 coccidiosis resi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/12C07K16/40A61K39/012A61P33/02
CPCA61K39/012A61K2039/552C07K16/40C12N9/1205C12Y207/01037
Inventor 董辉黄兵朱雪龙韩红玉赵其平朱顺海李聪王自文门启斐夏伟丽杨致远
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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