Schistosoma japonicum recombinant antigen rSjMRP1 and application thereof

A technology of recombining antigens and recombinant antigen proteins, which is applied in the field of bioengineering to achieve the effect of strong specificity and high sensitivity

Active Publication Date: 2018-03-09
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

Although there have been many reports on the research of genetically engineered diagnostic antigens, there is no genetically engineered antigen that can be applied on a large scale.

Method used

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  • Schistosoma japonicum recombinant antigen rSjMRP1 and application thereof
  • Schistosoma japonicum recombinant antigen rSjMRP1 and application thereof
  • Schistosoma japonicum recombinant antigen rSjMRP1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Livestock Positive Serum IgG Screening Schistosoma japonicum Adult Phage Display cDNA Library

[0029] 1. Materials

[0030] The phage display cDNA library of adult Schistosoma japonicum (Sj44d) was constructed and preserved by our laboratory, with a library capacity of 4.98×10 6 pfu, the recombination rate was 93.8%, and 95.6% of the inserted fragments were larger than 300bp (Sun Yi et al., 2007). The cercariae of Schistosoma japonicum were provided by the snail laboratory of this research laboratory. The positive sera of sheep, buffalo and cattle with Schistosoma japonicum (positive in stool test) were collected and preserved in our laboratory after infection with Schistosoma japonicum cercariae. T7 phage host strain BLT5403 was purchased from Novagen. The T7 vector primer sequence was provided by the T7Select 10-3b Cloning Kit manual, synthesized by Invitrogen, the upstream primer sequence was 5'-GGAGCTGTCGTATTCCAGTC-3' (SEQ ID NO.4), and the downstream ...

Embodiment 2

[0054] Example 2 Cloning and expression of candidate diagnostic antigen target gene for domestic animal schistosomiasis japonicum

[0055] 1. Materials

[0056] Adult worms of Schistosoma japonicum were collected from infected mice in our laboratory and stored in liquid nitrogen tanks. The pET-28b(+) vector was stored in our laboratory. Escherichia coli BL21(DE3) was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0057] 2. Method

[0058] 2.1 Total RNA extraction and cDNA preparation of Schistosoma japonicum

[0059] The total RNA of Schistosoma japonicum adult worms was extracted by Trizol method, and the genome was digested with a reverse transcription kit (TaKaRa) and reverse transcribed into cDNA.

[0060] 2.2 Cloning of the candidate diagnostic antigen gene of Schistosoma japonicum

[0061] Screening the phage display cDNA library of Schistosoma japonicum adult worms with positive IgG from domestic animals, the EST sequence SjMRP1 with the most repeated ti...

Embodiment 3

[0092] Example 3 Evaluation of the diagnostic effect of recombinant antigens rSjMRP, rSjOAT, and rSjHSP70HOMO

[0093] 1. Establishment of ELISA diagnostic method

[0094] 1.1 Method steps of ELISA diagnosis

[0095] After the antigen was diluted with coating solution (CB buffer pH=9.6), each well was coated with 100 μl of antigen, and left overnight at 4°C. The plate was washed three times with PBST and placed on a shaker for 5 min each time. Shake the plate and shake dry, add 1% gelatin blocking solution to each well, 37°C, 1h. The plate was washed three times with PBST and placed on a shaker for 5 min each time. Add diluted primary antiserum to each well at 37°C for 1h. The plate was washed three times with PBST and placed on a shaker for 5 min each time. Add diluted HRP-labeled secondary antibody serum to each well, 37°C, 1h. The plate was washed three times with PBST and placed on a shaker for 5 min each time. Add 100 μl of TMB color development solution to each we...

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Abstract

The invention discloses a schistosoma japonicum recombinant antigen. The recombinant antigen is prepared by expressing a recombinant vector of a schistosoma japonicum MRP1 gene segment; the sequence of the MRP1 gene segment is the nucleotide sequence of the amino acid sequence shown in SEQ ID NO.1. The invention further discloses application of the abovementioned schistosoma japonicum recombinantantigen in preparation of products for diagnosing schistosoma japonicum. The schistosoma japonicum recombinant antigen rSjMRP1 is used for diagnosing schistosoma japonicum, is high in sensitivity, high in specificity, and is of important significance on prevention and treatment of schistosomiasis.

Description

technical field [0001] The invention relates to the technical field of biological engineering, in particular to rSjMRP1, a recombinant antigen of schistosomiasis japonicum and its application in the diagnosis of schistosomiasis japonicum. Background technique [0002] Schistosomiasis occurs in the tropics and subtropics, and is the most important parasitic disease worldwide, with important socioeconomic significance. Schistosomiasis is endemic in 74 countries, with approximately 120 million people symptomatic and 20 million severely affected. Although rarely fatal at low levels of infection, its relapsing nature makes it a lifelong deranged chronic disease. Schistosomiasis japonicum is prevalent in my country, which is prevalent in 5 provinces along the middle and lower reaches of the Yangtze River (Hunan, Hubei, Jiangxi, Anhui and Jiangsu) and 2 provinces of Sichuan and Yunnan. Although remarkable achievements have been made in the prevention and control of schistosomiasi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12G01N33/68
CPCC07K14/43559G01N33/6893G01N2333/43547
Inventor 刘金明冯金涛许瑞金亚美李浩陆珂傅志强
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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