Proteins for diagnosing typhus fever, and application thereof
A typhus and protein technology, applied in the field of protein diagnosis of typhus, can solve the problems of high protection requirements for extraction and purification of rickettsia, affecting the reliability of serological diagnosis, complicated process, etc. Simple operation, accurate and reliable results
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[0059] Preparation of refolding buffer:
[0060] 6M urea refolding buffer was prepared as follows: 500mmol NaCl, 0.2L Tris-HCl buffer (0.1mol / L), 6mol urea, 0.2L glycerol and ddH 2 O mixed, adjusted to pH 7.4, with ddH 2 O was adjusted to 1 liter to obtain a refolding buffer containing 6M urea.
[0061] The urea-free refolding buffer was prepared as follows: mix 500mmol NaCl, 0.2L Tris-HCl buffer (0.1mol / L), 0.2L glycerol and ddH 2 O mixed, adjusted to pH 7.4, with ddH 2 O was adjusted to 1 liter to obtain a urea-free refolding buffer.
[0062] 5M-1M urea refolding buffer is obtained from 6M urea refolding buffer and urea-free refolding buffer according to different volume ratios.
[0063] Composition of washing buffer per 1 liter: 50mmol NaH 2 PO 4 ·H 2 O, 300mmol NaCl, 20mmol imidazole and ddH 2 O mixed, pH adjusted to 8.0 with NaOH, and ddH 2 O was adjusted to 1 L to obtain a washing buffer.
[0064] Per 1 liter of elution buffer composition: 50mmol NaH 2 PO 4 ·...
Embodiment 1
[0080] Embodiment 1, 4 kinds of antigenic proteins and preparation thereof
[0081] 1. Amino acid and nucleotide sequences of the four antigenic proteins
[0082] The amino acid sequences and nucleotide sequences of the four antigenic proteins are shown in Table 1.
[0083] Table 1 Amino Acid and Nucleotide Sequence
[0084] protein name
protein sequence
gene name
Nucleotide sequence
Sca5
SEQ ID No.1
sca5
SEQ ID No.5
FTZ
SEQ ID No.2
fts Z
SEQ ID No.6
SEQ ID No.3
SEQ ID No.7
Rp828
SEQ ID No.4
rp828
SEQ ID No.8
[0085] 2. Preparation of 4 kinds of antigenic proteins
[0086] (1) Amplification of gene fragments
[0087] Using the genomic DNA of Rickettsia prowazekii Madrid E strain as a template, the primers of sca5, ftsZ, groEL and rp828 genes (see Table 2) were used for PCR amplification respectively, and the amplification products of the four genes were obtai...
Embodiment 2
[0114] Example 2. Protein chip for diagnosing typhus and its preparation
[0115] 1. The structure of the protein chip
[0116] The protein chip is composed of an aldylated slide substrate, a detection point, a positive quality control point and a negative quality control point, wherein 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. The structure of the protein chip is as figure 2 shown.
[0117] (1) The detection points are divided into 4 types: they are made by spotting the proteins Sca5, FtsZ, GroEL and Rp828 respectively, and the above proteins are all spotted in the form of protein solution.
[0118] (2) The positive quality control point is mou...
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