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Applications of rabbit encephalitozoon cuniculi spore wall protein SWP1 to preparation of reagent for diagnosing or detecting rabbit encephalitozoon cuniculi infection

A technology of microsporidia and sporozoite protein, which is applied in the field of veterinary disease diagnosis, and can solve the problems of untested specificity and sensitivity of recombinant PTP2 protein, slow growth of parasites, laborious process of purifying parasites and antigen preparation, etc.

Inactive Publication Date: 2014-04-16
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cost of parasite culture is very high, and the parasites grow slowly, and the process of purifying parasites and preparing antigens is laborious
[0007] There are also literature reports using recombinant PTP2 protein or PTP3 protein expressed in Escherichia coli as a diagnostic antigen, but PTP2 protein is the polar tube protein of the parasite, and the specificity and sensitivity of using recombinant PTP2 protein remains to be tested

Method used

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  • Applications of rabbit encephalitozoon cuniculi spore wall protein SWP1 to preparation of reagent for diagnosing or detecting rabbit encephalitozoon cuniculi infection
  • Applications of rabbit encephalitozoon cuniculi spore wall protein SWP1 to preparation of reagent for diagnosing or detecting rabbit encephalitozoon cuniculi infection
  • Applications of rabbit encephalitozoon cuniculi spore wall protein SWP1 to preparation of reagent for diagnosing or detecting rabbit encephalitozoon cuniculi infection

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1 Preparation of Recombinant Rabbit Encephalitic Microsporidia Sporophyte Protein SWP1-GST

[0025] 1. Amplification of the nucleotide sequence encoding the recombinant rabbit encephalitic microsporidia sporoderm protein SWP1

[0026] The present invention designs specific primers according to the Microsporidia SWP1 gene sequence published in the NCBI database, amplifies the partial sequence of the SWP1 gene (amino acid 21 to the stop codon) in the fox kidney sample infected with Microsporidia, and passes Sequencing analyzes the sequence of a gene. Compared with the SWP1 sequence of GB-M1, the SWP1 sequence amplified by the present invention has a deletion of a repeated sequence (the sequence has been submitted to Genebank, the sequence number is KF169728), and the nucleotide sequence of the amplified SWP1 sequence is As shown in SEQ ID NO.2, the encoded amino acid sequence is shown in SEQ ID NO.1.

[0027] 2. The amplified sequence was inserted between the Xh...

Embodiment 2

[0029] Example 2 Application of Recombinant Microsporidium Rabbit Encephalitis Sporosporidium SWP1 in Diagnosis or Detection of Rabbit Encephalitis Infection in Fox Serum

[0030] 1. Samples to be tested: 200 fox serum samples suspected to be infected by Encephalitozoon cuniculi, 3 positive fox serum samples infected by Encephalitozoon cuniculi, positive foxes infected by Toxoplasma gondii Serum samples, positive fox serum samples for Neospora Caninum infection, positive fox serum samples for Cryptosporidium parvum infection.

[0031] 2. Method

[0032] (1) Measure the concentration of the purified fusion protein SWP1-GST prepared in Example 1 with a BCA protein concentration kit (Pierce) and coat it on an ELISA plate (Jite), with 0.1ug per well, and coat at 4°C overnight. After blocking with 5% skim milk at 37°C for 2h, serum samples diluted 100 times with 5% skim milk were added and incubated at 37°C for 1h. After washing 6 times with PBST, add HRP-labeled goat anti-dog I...

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Abstract

The invention discloses application of rabbit encephalitozoon cuniculi spore wall protein SWP1 to preparation of a reagent for diagnosing or detecting rabbit encephalitozoon cuniculi infection. A specific primer is designed according to the gene sequence of the rabbit encephalitozoon cuniculi spore wall protein SWP1, published by a database of NCBI (National Center of Biotechnology Information), and is amplified in a fox kidney sample infected with encephalitozoon cuniculi to obtain the SWP1nucleotide sequence shown as SEQ ID NO.2. The fused-expressed SWP1-GST is used for the detection of the sample, the result shows that the fused-expressed SWP1-GST protein has good specificity on the detection of fox serum infected with rabbit encephalitozoo cuniculi, and has no cross reaction with positive serum infected with toxoplasma gondii, neospora caninum and cryptosporidium. In addition, the research result shows that the sensitivity of the SWP1 protein, which is taken as a detection antigen, is obviously higher than that of the PTP2 (Protein-tyrosine-phosphatase 2) protein as a detection antigen. Therefore, the method for diagnosing or detecting the rabbit encephalitozoonosis cuniculi is high in sensitivity, and the diagnosis and prevention of the rabbit encephalitozoonosis cuniculi are further promoted.

Description

technical field [0001] The present invention relates to a method for diagnosing or detecting the infection of Microsporidia encephalitis in rabbits, in particular to a method for diagnosing or detecting Microsporidium rabbit encephalitidis infection by using ELISA method with the sporozoite protein SWP1 of Microsporidium rabbit encephalitidis as antigen , in order to improve the sensitivity of diagnosis, the invention belongs to the technical field of veterinary disease diagnosis. Background technique [0002] Rabbit encephalitis microsporidiosis is a chronic, recessive or subclinical zoonotic protozoan disease caused by rabbit encephalitis microsporidia (also known as rabbit encephalitis protozoa, Encephalitozoon cuniculi). Rabbit encephalitis protozoa has a wide range of hosts, including invertebrates, rodents, lagomorphs, herbivores, carnivores and primates, etc. It has been reported that rabbits, rats, mice, guinea pigs, voles, Domestic dogs, cattle, foxes, pigs, minks ...

Claims

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Application Information

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IPC IPC(8): G01N33/68C12N15/30C12N15/70C07K19/00
CPCG01N33/56905G01N33/68
Inventor 贾洪林张彦龙
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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