Application of Eimeria stiedai SAG8 protein

A technology of Eimeria and protein, applied in the application field of Eimeria stezeri SAG8 protein, to achieve good immunogenicity and reactogenicity, high sensitivity and specificity

Active Publication Date: 2020-04-10
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of reports about the method of pre-mortem diagnosis of the disease, so it is of great significance to establish a serological diagnosis method to accurately diagnose hepatic coccidiosis in rabbits.

Method used

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  • Application of Eimeria stiedai SAG8 protein
  • Application of Eimeria stiedai SAG8 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Bioinformatics analysis and cloning sequencing of EsSAG8

[0030] 1. cDNA amplification of EsSAG8

[0031] Use the open reading frame finder ORF Finder (http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html) to determine the open reading frame and deduced amino acid sequence of EsSAG8; use SignalP4.1 in the Biological Sequence Analysis Center (http: / / www.cbs.dtu.dk / services / SignalP / ) to predict whether there is a signal peptide and a transmembrane region in the protein; use the ExPASy website (http: / / web.expasy.org / ) to predict the molecular weight (MW) of the protein ), isoelectric point (pI), conserved domains and protein properties.

[0032] The sporulated oocysts of Eimeria stutzeri stored at 4°C in 2.5% potassium dichromate were washed repeatedly with normal saline for 5 times, and extracted using a total RNA extraction kit (MiniBEST Universal RNA Extraction Kit, TaKaRa, Japan). ) for the extraction of RNA, and then using the extracted total RNA of Eimeria stezer...

Embodiment 2

[0038] Example 2: Western blot analysis

[0039] Prepare a 12% SDS-PAGE gel, and perform SDS-PAGE separation on the purified rEsSAG8 protein obtained according to the method in Example 1; Device at 0.8mA / cm 2 Transfer at a constant current for 40 minutes; after the end, take out the film and wash it 3 times with TBST, 5 minutes each time; use 5% skimmed milk powder PBS solution to seal at room temperature for 2 hours; then wash the film 3 times with TBST, 5 minutes each time; Rabbit-positive serum of coccidia Meieria (diluted 1:100 with PBS buffer) as the primary antibody, incubated overnight at 4°C (about 12h); poured off the liquid, washed 3 times with TBST, each 5min; then added PBS to dilute (Dilution ratio 1:3000) with HRP-labeled rabbit anti-goat IgG secondary antibody, incubated at room temperature for 2h and soaked in TBST for 4 times, developed color with DAB, and added pure water to terminate the reaction.

[0040] Western blot analysis showed that rEsSAG8 could be...

Embodiment 3

[0041] Embodiment 3: establishment and detection verification of indirect ELISA method

[0042] 1. Establishment of indirect ELISA method

[0043] The establishment method of indirect ELISA was carried out according to previous reports (CROWTHER J R. The ELISA Guidebook [J]. Methods in Molecular Biology, 2000, 149(149): III-IV, 1-413.). Using 0.1M carbonate buffer (pH 9.6), the purified rEsSAG8 protein was used as the antigen in indirect ELISA (1:20, 1:40, 1: 80, 1:160, 1:320, 1:640) and added to a 96-well microtiter plate (Invitrogen), 100 μl per well, and coated overnight at 4°C. After washing 3 times with PBST (0.01M PBS+0.05% Tween-20), the coated ELISA plate was blocked with 5% skimmed milk powder (diluted with 0.01M PBS solution), 300 μL per well, and incubated at 37°C for 2h. Wash again with PBST for 3 times, and at the same time use 0.01M PBS solution to carry out 6 different concentrations of Eimeria stezeri negative and positive sera (1:20, 1:40, 1:80, 1:160 , 1:3...

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Abstract

The invention relates to the technical field of biology, and discloses a series of related applications of an Eimeria skrjabini SAG8 protein as a rabbit coccidiosis diagnosis antigen and the like, andrelated experimental results show that the Eimeria skrjabini SAG8 protein can be recognized by Eimeria skrjabini positive serum, and has good immunogenicity and reactogenicity; and meanwhile, the Eimeria skrjabini SAG8 protein shows extremely high sensitivity and specificity in an indirect ELISA method, and various results prove that the Eimeria skrjabini SAG8 protein can be used as a diagnosticantigen of rabbit coccidiosis and can be applied to related vaccines and detection kits.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of the Eimeria stuzeri SAG8 protein. Background technique [0002] Rabbit coccidiosis (Rabbit coccidiosis) is a kind of protozoan disease caused by a variety of coccidia of the genus Eimeria parasitic in the intestinal tract and liver of rabbits. It is a common and multiple disease of rabbits. One of the major diseases that seriously endanger domestic rabbits. Eimeria stiedai, as the most pathogenic coccidia in rabbits, mainly invades the liver and bile duct epithelial cells of rabbits, leading to liver cirrhosis and cholestasis, thus causing severe hepatic rabbit balls. Bug disease. The main clinical features of sick rabbits are diarrhea, growth retardation, weakness and emaciation, and severe infection can lead to death. Due to the high morbidity and mortality of hepatic rabbit coccidiosis, Eimeria stezeri is regarded as one of the most harmful pathogens facing t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/543A61K39/012A61P33/02
CPCG01N33/56905G01N33/54306A61K39/012A61P33/02Y02A50/30
Inventor 杨光友陶圆圆彭雪蓉
Owner SICHUAN AGRI UNIV
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