Protein composition for diagnosis or auxiliary diagnosis of far-eastern spotted fever
A technology for spotted fever and auxiliary diagnosis, which is applied in the biological field and can solve problems such as cumbersome and time-consuming steps, high cost, and complicated processes
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Embodiment 1
[0071] The preparation of embodiment 1, 5 kinds of antigenic 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
seque...
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 protein RplA, made by protein RplY, made by protein OmpA, made by protein OmpB, made by 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 (purchased from Zhon...
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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