Babesia gibsoni recombinant antigen, diagnostic reagent kit thereof and application of diagnostic reagent kit

A technology of Babesia gibsoni and recombinant antigens, applied in the field of bioengineering, can solve the problems of low recombinant expression yield and difficult purification of BgTRAP complete protein, and achieve good diagnostic specificity and high sensitivity

Active Publication Date: 2014-07-02
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The present invention aims to solve the technical problems of low yield and difficult purification of BgTRAP complete protein recombinant expression, and provides a Babesia gibsoni recombinant antigen, which contains 411 amino acids of the BgTRAP protein TSP1 functional region and antigenic region, and is a soluble Truncated antigen, the truncated antigen can not only achieve high-efficiency soluble expression and separation and purification, but also has good antigenicity, and is suitable as a diagnostic antigen for canine Babesiosis gibzii

Method used

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  • Babesia gibsoni recombinant antigen, diagnostic reagent kit thereof and application of diagnostic reagent kit
  • Babesia gibsoni recombinant antigen, diagnostic reagent kit thereof and application of diagnostic reagent kit
  • Babesia gibsoni recombinant antigen, diagnostic reagent kit thereof and application of diagnostic reagent kit

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Embodiment 1

[0028] Example 1 Bioinformatics analysis of Babesia gibsoni BgTRAP gene and encoded protein

[0029] Zhou Jinlin of Shanghai Veterinary Research Institute reported the identification of BgTRAP molecule of Babesia gibsoni for the first time in 2006 (Zhou J, Fukumoto S, Jia H, et al.Characterization of the Babesia gibsoni P18 as a homologue of thrombospondin related adhesive protein. Mol Biochem Parasitol, 2006, 148: 190-198), this molecule is a functional molecule with strong immunogenicity secreted by the apicomplex, and is closely related to the biological process of Babesia gibsoni invading red blood cells. The full-length cDNA sequence of BgTRAP has been registered in Genbank, and the sequence number is AB053292. figure 1 It is the primary structure of BgTRAP protein and the schematic diagram of the position of the truncated antigenic protein of the present invention, such as figure 1 As shown, the full length of the BgTRAP gene is 2,373bp, containing an open reading frame...

Embodiment 2

[0030] Cloning and expression of embodiment 2 Babesia gibsoni BgTRAP truncated gene

[0031] 1. Primers

[0032] According to the structural analysis of the reported Babesia gibsonia BgTRAP gene sequence and encoded protein, before the C-terminal transmembrane region, the 1233bp protein encoded by 411 amino acids (SEQ ID NO.1) containing the TSP1 functional region and antigenic region was selected. Gene fragments, design PCR amplification primers: TRAP-F: 5'-GCGAATTCAGTGCACTGGCCCTTGAAGAG-3'(SEQ ID NO.3, including EcoR I restriction site, GAATTC); TRAP-R: 5'-GACTCGAGCTTGTACTCCCAGAAAAGAG-3'( SEQ ID NO.4, including Xhol I restriction site, CTCGAA), the primers used were synthesized by Saibaisheng Company.

[0033] 2. Cloning and expression of selective fragments of Babesia gibsoni BgTRAP gene

[0034] The recombinant plasmid pBluescriptSK(+) / BgTRAP containing the full-length TRAP gene was used as a template, and the truncated antigen gene-specific primers TRAP-F and TRAP-R were...

Embodiment 3

[0037] Preparation and immunofluorescence test of embodiment 3 recombinant protein antiserum

[0038] 1. Method steps

[0039] The recombinant truncated protein purified in Example 2 and the control GST protein were used to immunize mice according to conventional methods, and the titer was determined to be above 1:16 by agar diffusion test, and blood was collected from eyeballs to prepare antiserum. The antiserum was serially diluted for immunofluorescence experiment, and the immunofluorescence experiment was carried out according to the method of Zhou et al (2006), as follows: (1) Preparation of erythrocyte antigen sheets infected with worms, high-quality slides were selected, washed and soaked in absolute ethanol and Diethyl ether equal volume mixture. When in use, take it out and wipe it clean with a silk cloth. After washing with PBS, the canine-infected Babesia gibsoni-infected erythrocytes were dropped on glass slides. Fix with methanol containing 2.5% acetone for 30 ...

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Abstract

The invention discloses a babesia gibsoni recombinant antigen, which contains a thrombospondin 1 (TSP1) functional region of babesia gibsoni apical complex secretory protein (BgTRAP) and an amino acid sequence of a glutamic acid-enriched region, wherein the amino acid sequence is shown as SEQ ID No.1. The invention also discloses a reagent kit for diagnosing canine gibsoni babesiosis and application of the reagent kit. By the babesia gibsoni recombinant antigen, high-efficiency soluble expression and separation purification can be realized, and the antigenicity is high; and the babesia gibsoni recombinant antigen serving as a diagnostic antigen for the canine gibsoni babesiosis has high specificity and sensibility.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a Babesia gibsoni recombinant antigen and its diagnostic kit and application. Background technique [0002] Babesia gibsoni (Babesia gibsoni) is a tick-borne protozoa that parasitizes in the red blood cells of dogs. emaciation, and even death. At present, the diagnosis of Babesia gibsoni mainly relies on red blood cell staining microscopy, but it is difficult to diagnose non-acute infections with low red blood cell staining rate. Therefore, it is extremely important to establish a serological diagnostic method for Babesia gibsoni to detect antibodies. In the early stage of serological diagnosis using parasite antigens, the sensitivity and specificity were limited to a certain extent. In recent years, great progress has been made in the identification of important parasite antigen molecules and the application of recombinant proteins in serological diagnosis. Among them, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/44C12N15/30C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10G01N33/569G01N33/543
Inventor 周金林杨其清周勇志曹杰张厚双
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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