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Schistosoma japonicum recombinant antigen rsjmrp1 and its application

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

Active Publication Date: 2021-05-07
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 its application
  • Schistosoma japonicum recombinant antigen rsjmrp1 and its application
  • Schistosoma japonicum recombinant antigen rsjmrp1 and its application

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 instruction manual and synthesized by Invitrogen. The upstream primer sequence was 5'-GGAGCTGTCGTATTCCAGTC-3' (SEQ ID NO.4), and ...

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, which is produced by expressing a recombinant vector containing a Schistosoma japonicum MRP1 gene fragment. The sequence of the MRP1 gene fragment encodes the amino acid sequence shown in SEQ ID NO.1 Nucleotide sequence. The invention also discloses the application of the above-mentioned schistosomiasis recombinant antigen in the preparation of products for diagnosing schistosomiasis japonicum. The schistosomiasis recombinant antigen rSjMRP1 of the invention is used for diagnosing schistosomiasis, has high sensitivity and strong specificity, and is of great significance for the 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 Patents(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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