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Molecular marker for eimeria tenella sensitive strain and drug-resistant strain and application of molecular marker

A technology of Eimeria and molecular markers, applied in the field of ATPase (EtASNA1) protein, can solve problems such as the inability to realize the correlation between protein and drug resistance of coccidia, human growth retardation, nematode growth arrest, etc.

Active Publication Date: 2020-08-11
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

Previous studies have shown that downregulation of ASNA1 leads to growth arrest and increased sensitivity to arsenite in nematodes, decreased insulin secretion, growth retardation and increased apoptosis in humans
However, due to factors such as the difficulty of screening and difficult operation of experiments, there is no report of ASNA1 in Eimeria tenella
[0004] In addition, in order to study the correlation between protein and coccidia drug resistance, it is extremely difficult to obtain a single drug-resistant strain of chicken coccidia resistant to a certain drug, although chicken coccidia are easier to resist in the process of long-term drug use in the field Coccidiostats produce drug resistance, but the use of chicken farms is complicated, and the drug-resistant strains produced are resistant to multiple drugs at the same time, that is, multi-drug resistance or cross-resistance, which is not conducive to the screening of drug-resistant genes
There is no mature method in this field to realize the screening of drug-resistant strains, so it is impossible to realize the research on the correlation between protein and drug resistance of coccidia, which has become the current stage to restrict the protein or marker of drug resistance of coccidia bottle neck

Method used

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  • Molecular marker for eimeria tenella sensitive strain and drug-resistant strain and application of molecular marker
  • Molecular marker for eimeria tenella sensitive strain and drug-resistant strain and application of molecular marker
  • Molecular marker for eimeria tenella sensitive strain and drug-resistant strain and application of molecular marker

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Et Cloning and expression of ASNA1 gene

[0033] Experimental strains and experimental animals

[0034] The experimental strains and experimental animals used in this study were all provided by the Shanghai Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0035] Extraction and cDNA preparation

[0036] Use TRIzol reagent to extract total RNA from purified Eimeria tenella sporulated oocysts, and use M-MLV reverse transcription kit to reverse transcribed it into the first strand of cDNA template for PCR amplification.

[0037] Amplification

[0038] Use the primers in Table 1 to obtain amplification using conventional PCR amplification methods Et The ORF sequence of the ASNA1 gene is shown in SEQID NO. 1.

[0039] Table 1 PCR primers

[0040] name sequence Et ASNA1

UP: 5'-ATGACAGACGACGAGTTTCCCCTTG-3' (SEQ ID NO.3) LP: 5'-TGCATCAATTCGGCGAAAGAAAGGG-3' (SEQ ID NO.4)

[0041] according to Et The ORF sequence of ASNA1 gene, the primers were de...

Embodiment 2

[0044] Example 2 Differential expression and localization of EtASNA1 gene

[0045] In order to detect Et ASNA1 mRNA transcription levels at different developmental stages of Eimeria tenella, we used unsporulated oocysts, sporulated oocysts, sporozoites and second-generation merozoites as templates, and 18S rRNA as templates. The internal control was analyzed by qPCR and Western Blot respectively Et The transcription and translation levels of ASNA1 in four different developmental stages of Eimeria tenella.

[0046] Among them, the primers for qPCR (fluorescence quantitative PCR) are: EtASNA1-AP 5'-GGCGTCGGCAAGACAACCAC -3' (SEQ ID NO. 5) and EtASNA1-SP 5'-GTGGACAGCAGCAGCACTGATTC -3' (SEQ ID NO. 6).

[0047] The result is figure 2 , image 3 As shown, the results confirmed that the mRNA transcription level of EtASNA1 was higher in the unsporulated oocyst and second-generation merozoite stages, and lowest in the sporozoite stage. by image 3 We can see that the translation level of EtA...

Embodiment 3

[0049] Example 3 Et Analysis of the difference between ASNA1 in sensitive and resistant strains of Eimeria tenella

[0050] Using the qPCR method, the first strand of cDNA of the sporulated oocysts of Eimeria tenella susceptible strains, diclazuril-resistant strains and maduramycin-resistant strains were used as templates, and 18S rRNA was used as the internal control. Et The mRNA transcription level of ASNA1 in Eimeria tenella susceptible strains, diclazuril-resistant strains and maduramycin-resistant strains. It turns out that compared with sensitive strains, Et The expression of ASNA1 was significantly up-regulated in diclazuril-resistant strains and maduramycin-resistant strains, which is consistent with the results of previous transcriptome sequencing. After that, we used the worm protein of the sporulated oocysts of Eimeria tenella-sensitive strains, diclazuril-resistant strains and maduramycin-resistant strains as templates, and rabbit anti-r Et ASNA1 polyclonal antibody is...

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Abstract

The invention provides a molecular marker for an eimeria tenella sensitive strain and a drug-resistant strain and application of the molecular marker. The molecular marker is ATPase (EtASNA1) homologous with eimeria tenella ASNA1. The invention finds that the transcription and protein expression of mRNA of a diclazuril drug-resistant strain and a maduramycin drug-resistant strain are obviously higher than that of the sensitive strain; and the influence of drug stimulation on the EtASNA1 transcription level is detected through an in-vitro culture experiment and an in-vivo experiment, and the invention finds that addition of drugs can cause up-regulation of expression. An antibody inhibition test detects that the EtASNA1 participates in invasion of host cells by polypides. Research results show that the EtASNA1 may participate in invasion, growth and development of eimeria tenella, may be closely related to formation of drug resistance of the eimeria tenella, and may play an important role in resisting drugs.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a molecular marker for Eimeria tenella-sensitive strains, diclazuril-resistant strains, and maduramycin-resistant strains, and applications thereof. The ATPase homologous to Meliococcus ASNA1 ( Et ASNA1) protein. Background technique [0002] Chicken coccidiosis is a serious intestinal parasitic disease caused by several Eimeria species. Eimeria tenella is the most severely harmful to chickens and the most common disease. Drug therapy is currently the most commonly used treatment method, and the widespread use of anticoccidial drugs has also exacerbated the problem of coccidial resistance. However, no mechanism has been found for the formation of drug resistance in coccidia, and no target genes have been found to control the development of drug resistance in coccidia. With the general emergence of drug resistance, anti-coccidial drugs have gradually decreased or even become i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14C12N15/11C07K16/40C12Q1/6893C12Q1/04A61P33/02
CPCC12N9/14C07K16/40C12Q1/6893A61P33/02C12Q2600/158Y02A50/30
Inventor 韩红玉董辉于钰黄兵赵其平朱顺海赵焕之
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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