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Protein composition for diagnosis or auxiliary diagnosis of far-eastern spotted fever

A spotted fever and auxiliary diagnosis technology, applied in the biological field, can solve the problems of high protection requirements, complicated process and high cost of rickettsia

Inactive Publication Date: 2015-03-04
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One of the reasons for misdiagnosis and missed diagnosis is that spotted fever has not been understood by most people, including medical staff; the other is laboratory diagnostic methods, including indirect immunofluorescence (IFA) technology, enzyme-linked immunosorbent assay (ELISA) Technology, etc. These detection methods have high specificity, but they require a large amount of samples, the steps are cumbersome and time-consuming, and all of them require purified whole bacteria of Rickettsia as antigens, and the protection requirements for culturing and purifying Rickettsia are high. The process is complex and expensive, so it is difficult to be widely promoted and used

Method used

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  • Protein composition for diagnosis or auxiliary diagnosis of far-eastern spotted fever
  • Protein composition for diagnosis or auxiliary diagnosis of far-eastern spotted fever
  • Protein composition for diagnosis or auxiliary diagnosis of far-eastern spotted fever

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] The preparation of embodiment 1, 5 kinds of antigen proteins

[0072] Vector pET-32a(+) was purchased from Novagen, the product catalog number is 69015

[0073] Vector pQE-30 was purchased from Qiagen, the product catalog number is 33203.

[0074] Escherichia coli M15 was purchased from Novagen, the product catalog number is 34210.

[0075] Escherichia coli BL21 (DE3) was purchased from Novagen, the product catalog number is 69450.

[0076] 1. The protein sequences and coding gene sequences of the five antigenic proteins

[0077] The amino acid sequences of the five antigenic proteins and the nucleotide sequences of the coding genes are shown in Table 1.

[0078] Table 1 is the protein and gene sequence

[0079] protein name

protein sequence

gene name

coding gene sequence

RplA

sequence 1

rplA

sequence 6

PPML

sequence 2

wxya

sequence 7

OmpA

sequence 3

ompA

sequence 8

OmpB

sequenc...

Embodiment 2

[0114] Embodiment 2, protein chip and its preparation

[0115] 1. Structure of the protein chip

[0116] The structure of the protein chip: it is composed of an aldylated slide substrate, a detection point, a positive quality control point and a negative quality control point, and the detection point, the positive quality control point and the negative quality control point are all spotted on the substrate. There were 5 replicates for each detection point, positive quality control point and negative quality control point. Detection points, positive quality control points and negative quality control points are arranged in a matrix on the substrate.

[0117] The detection points are divided into 5 types: made by spotting protein RplA, spotting protein RplY, spotting protein OmpA, spotting protein OmpB, spotting protein GroEL; It is made in the form of protein solution.

[0118] Positive quality control points (2 positive quality control points) consisted of mouse IgG (purcha...

Embodiment 3

[0127] Example 3, Application of Antigen Protein and Protein Chip in Diagnosis of Far East Spotted Fever

[0128] Cy5-labeled goat anti-mouse IgG was purchased from KPL, USA, and the product catalog number was 072-02-18-06.

[0129] FITC-labeled goat anti-mouse IgG was purchased from Beyontien Institute of Biotechnology, catalog number A0568.

[0130] Heilongjiang Rickettsia Antigen Tablets, Rickettsia Prussiana Antigen Tablets, Rickettsia Rickettsia Antigen Tablets, Coxiella Beinsoni Phase I Antigen Tablets, Orientia Tsutsugamushi Antigen Tablets; the above antigen sheets are as follows Method preparation: apply the inactivated corresponding bacteria on the glass slide, and then fix it with acetone for 15 minutes.

[0131] Prepare 1 liter of phosphate buffered saline (PBS) as follows: mix 8g NaCl, 0.2g KCl, 3.53g Na 2 HPO 4 12H 2 O, 0.24g KH 2 PO 4 Mix with water, and dilute to 1 liter with water, the pH value of the obtained solution is 7.4, that is, PBS buffer solutio...

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Abstract

The invention discloses a protein composition for diagnosis or auxiliary diagnosis of far-eastern spotted fever. The protein composition provided in the invention is composed of the following 5 proteins: RplA protein, RplY protein, OmpA protein, OmpB protein and GroEL protein. Experiments involved in the invention prove that, with the protein composition composed of the 5 proteins and a protein chip with the 5 proteins pointed on a substrate, fast and accurate serological analytical diagnosis on the serum of patients or animals suffering the far-eastern spotted fever can be realized. Diagnosis of far-eastern spotted fever by the protein composition or chip has the advantages of accurate and reliable results, simple operation, less sample consumption, time saving and labor saving, thus being able to replace the traditional tedious, tiring and time-consuming serological diagnosis method to perform serological diagnosis of far-eastern spotted fever.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a protein composition for diagnosing or assisting the diagnosis of Far Eastern spotted fever. Background technique [0002] Rickettsial disease is a zoonotic natural foci disease that seriously threatens human health. It has seriously threatened human health in history and is still one of the important causes of human morbidity and death today. A tick-borne infectious disease caused by the critters. Rickettsia heilongjiangensis is a newly discovered spotted fever group Rickettsia in my country, which causes Far-eastern tick-borne spotted fever. Its epidemic areas are mainly distributed in Northeast China and Russia. Far East and Japan. [0003] In my country, there are few reports of spotted fever, and the important reason is that misdiagnosis and missed diagnosis are quite common. Spotted fever is often misdiagnosed as upper respiratory tract infection, malaria, leptospirosis, typ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
Inventor 齐永温博海王锡乐熊小路段长松李佳明贾引军龚文平焦俊
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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