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Application of anaplasma phagocytophilum protein APH1384

一种吞噬细胞、无形体的技术，应用在免疫学领域，能够解决检测成本高、操作时间长、价格昂贵抗原片等问题，达到提高灵敏度、快速精准诊断的效果

Active Publication Date: 2017-08-29
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the diagnostic antigens used in these three serological detection methods are phagocytic anaplasmic outer membrane protein P44, and the detection method using P44 as a single diagnostic antigen has the defect of missing detection, and its detection sensitivity is 80-90%
In short, the IFA method is expensive to detect, requires expensive antigen sheets, and the judgment result is subjective, and the operation time is relatively long, so it is not suitable for the detection of a large number of clinical samples; while the WB, DB and ELISA methods are simple to operate and low in cost, but currently They all use P44 as a single diagnostic antigen, which has the defect of missed detection, and the detection sensitivity needs to be improved

Method used

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  • Application of anaplasma phagocytophilum protein APH1384
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  • Application of anaplasma phagocytophilum protein APH1384

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 Preparation of APH1384 protein (Anaplasma phagocytophilum species-specific protein)

[0017] Using bioinformatics technology, 610 unknown functional proteins encoded by the genome of Anaplasma phagocytophilum were targeted to screen out the species-specific proteins of Anaplasma phagocytophilum. Some of the proteins (including APH1384) were cloned, expressed and identified for their antigenicity. The results of the study found that APH1384 has strong antigenicity. The following are the detailed cloning, expression and antigenic identification steps of APH1384. Specific primers containing restriction endonuclease sites were designed according to the aph1384 gene sequence in Gene Bank: aph1384-F-TCCGAATTCATGTTTGATATATTTAATGATGCTG; aph1384-R-GCACTCGAGTTATTCGGACGCAGAGGCT. The DNA fragment of the aph1384 gene was amplified by PCR using the whole genome DNA of Anaplasma phagocytophilum as a template. The amplified DNA fragment of the target gene and the prokaryot...

Embodiment 2

[0019] Example 2 Preparation of APH1384 Polypeptide

[0020] The antigenic epitope of APH1384 was predicted by bioinformatics method, and its amino acid sequence is shown in SEQ ID No.2. Chemically synthesize the APH1384 epitope polypeptide, and use DB technology to verify the antigenicity of the APH1384 polypeptide. The specific operations are as follows:

[0021] Add APH1384 polypeptide antigen dilution (1 μg / μl, 0.1 μg / μl, 0.01 μg / μl respectively) to the nitrocellulose membrane, dry at 37°C for 20-30 minutes, and block for 30 minutes. The primary antibody is the human positive serum (1: 2000) for 1 h, secondary antibody for 1 h (1:5000), and develop. see results image 3 , image 3 N in the table represents a negative serum, and P1-P3 each represent a positive serum for Anaplasma phagophilum. The results showed that the negative serum did not react with APH1384, while the three positive sera all combined with the APH1384 polypeptide, proving that the APH1384 polypeptide ...

Embodiment 3

[0022] Example 3 Application of APH1384 Polypeptide

[0023] Using the synthetic APH1384 polypeptide as the coating antigen, the ELISA method is used to detect the specific anti-APH1384 antibody in human serum. The specific operation steps are as follows:

[0024] (1) Coating: Dilute the APH1384 polypeptide synthesized in Example 2 to 20 μg / ml with PBS, and wrap it overnight at 4°C or at room temperature for 2 hours (96-well plate, 50 μl per well);

[0025] (2) Blocking: wash the 96-well plate three times with PBS, add blocking solution of bovine serum albumin (concentration: 1%), 200 μl per well, 37°C, 2h;

[0026] (3) Primary antibody incubation: Dilute the serum to be tested at a ratio of 1:1000 with blocking solution, add 100 μl of the dilution solution to each well, and incubate at 37°C for 1 hour;

[0027] (4) Secondary antibody incubation: wash four times with PBS (200 μl per well), add secondary antibody (HRP-labeled goat anti-human IgG, diluted 1:5000), and incubate ...

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Abstract

The invention relates to application of anaplasma phagocytophilum protein APH1384 as a granulocytic anaplasmosis diagnosis antigen. The protein can overcome the defect of inspection missing of the existing anaplasmosis diagnosis antigen P44; the sensitivity of the anaplasmosis detection is improved; the occurrence of the anaplasmosis can be favorably, fast and precisely diagnosed in clinics.

Description

technical field [0001] The invention relates to the field of immunology, in particular to the application of anaplasmin protein APH1384 of phagocytic cells. Background technique [0002] Anaplasma phagocytophilum is an obligate intracellular parasitic tick-vectored Gram-negative bacterium that causes granulocytic anaplasmosis, such as human and canine granulocytic anaplasmosis occur. According to data from the US CDC, the number of cases of human granulocytic anaplasmosis infection increased from 348 to 2389 from 2000 to 2012, showing a rapid growth trend. In addition, granulocytic anaplasmosis is localized in humans and livestock in Australia, many European countries, Japan and Korea. In China, the serological epidemiological survey organized by the Chinese Center for Disease Control and Prevention showed that the seropositive rate of neutrophil anaplasmosis among agricultural practitioners in my country was as high as 13.9%. Compared with patients with granulocytic anap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569G01N33/561
CPCG01N33/561G01N33/56966G01N33/68G01N2333/43552G01N2333/195C07K14/29G01N33/56911G01N2469/20G01N2333/43556G01N2333/29
Inventor 牛华贺美玲吴淑燕
Owner SUZHOU UNIV
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