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High throughput screening of important schistosoma japonicum antigens and application of antigens in schistosomiasis diagnosis

A schistosomiasis and antigen technology, applied in the biological field, can solve the problems of time-consuming and laborious, polluting the environment, and low sensitivity of pathogenic detection.

Active Publication Date: 2013-04-24
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The above three methods have their own advantages and disadvantages: although the results of pathogenic detection are still the gold standard for the diagnosis of schistosomiasis, the sensitivity of pathogenic detection is low, time-consuming and labor-intensive, and there is a risk of environmental pollution
The key is that there is no effective, highly sensitive, and highly specific antigen molecule used in the field of ELISA to detect schistosomiasis

Method used

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  • High throughput screening of important schistosoma japonicum antigens and application of antigens in schistosomiasis diagnosis
  • High throughput screening of important schistosoma japonicum antigens and application of antigens in schistosomiasis diagnosis
  • High throughput screening of important schistosoma japonicum antigens and application of antigens in schistosomiasis diagnosis

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preparation example Construction

[0078] The preparation method of the antigen of the present invention has the following steps:

[0079] (1). Transform or transduce a suitable host cell with a polynucleotide (or variant) encoding the positive antigen of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0080] (2). Host cells cultured in a suitable medium;

[0081] (3). Isolate and purify the antigen of the present invention from culture medium or cells.

[0082] In the present invention, polynucleotide sequences can be inserted into recombinant expression vectors. Methods well known to those skilled in the art can be used to construct expression vectors containing the antigen-encoding DNA sequence and appropriate transcriptional / translational control signals. A vector comprising the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.

[008...

Embodiment 1

[0098] Example 1 High-throughput screening of important antigens of Schistosoma japonicum

[0099] 1. Predict the secreted protein of Schistosoma japonicum: Download the Schistosoma japonicum EST database and the corresponding protein sequence from the National Human Genome South Research Center and NCBI; import the protein sequence into the signal peptide online prediction software SignalP 3.0 (http: / / www.cbs. dtu.dk / services / SignalP / ) to predict; according to the prediction results, according to the p value greater than 0.1, select the sequence predicted to be the signal peptide protein.

[0100] 2. Gene cloning: extract the total RNA of Schistosoma japonicum; amplify the target gene by RT-PCR, recover the target fragment, digest it, and clone the target fragment into the commercially available expression vector pGEX-4T-1; transform the commercially available Escherichia coli Top10 , The sequence of the positive clone was confirmed to be correct by sequencing.

[0101] 3. I...

Embodiment 2

[0112] Example 2 Comparison of diagnostic sensitivity of strong positive clones

[0113]Select 8 strong positive antigens with R≥10 (sequences are SEQ ID NO: 1-8), respectively adsorbed on the GSH-microwell plate, and set GST (glutathione-S-transferase) expressing bacteria 200 μl 5% milk powder / PBST, blocked at room temperature for 2 hours; washed five times with PBST; the absorbed serum was diluted with 5% milk powder according to 1:20, and the final serum dilution 1:1000; 100μl / well, combine for 1h at 37°C; wash five times with PBST; dilute HRP enzyme-labeled mouse anti-human IgG (H+L) antibody at 1:20,000, 100μl / well, combine for 1h at 37°C; PBST Wash 5 times; add 100 μl commercially available Super Signal ELISA Femto for detection, read the fluorescence intensity value at 425 nm; calculate the R value. And compared with the number of liver eggs per gram (EPG) of the sample, the sensitivity of the antigen was detected. The comparison results are shown in Table 2.

[0114...

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Abstract

The invention discloses high throughput screening of important schistosoma japonicum antigens and application of the antigens in schistosomiasis diagnosis. Specifically, the invention provides important schistosoma antigens and application thereof in schistosomiasis diagnosis and vaccine preparation. Based on a high throughput functional protein screening technology of glutathione S transferase (GST) fusion expression, 24 positive antigens capable of being identified by the serum of a schistosomiasis patient are acquired, wherein 8 antigens represent strong positive reaction. Schistosomiasis serologic detection shows that the positive antigens for detecting schistosoma have high sensitivity and specificity. The invention also provides a corresponding method and a kit for diagnosing and monitoring the schistosomiasis.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention discloses high-throughput screening of important antigens of Schistosoma japonicum and its application in the diagnosis of schistosomiasis. Applications in disease diagnosis and vaccine preparation. Background technique [0002] Schistosomiasis is a zoonotic disease that seriously threatens human health. The number of deaths due to schistosomiasis is about 280,000 worldwide every year. After decades of hard work, the prevention and control of schistosomiasis in my country has achieved world-renowned achievements. However, there are still about 500,000 people who are currently infected in my country. The situation of schistosomiasis prevention and control is not optimistic, and the task of prevention and control is still arduous. [0003] The diagnosis of schistosomiasis is one of the important prerequisites and means for the control of schistosomiasis. Accurate diag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435G01N33/68C12N15/12C12N15/63C12N1/21C12N1/15C12N1/19C12N5/10C12P21/02C07K16/18C07K1/14
Inventor 潘卫庆徐新东
Owner TONGJI UNIV
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