A protein fluorescent probe for specific detection of polysulfides and its preparation method and application
A technology of fluorescent probes and polysulfides, applied in the direction of microorganism-based methods, biochemical equipment and methods, chemical instruments and methods, etc. Low detection limit, strong anti-interference ability, and simple preparation method have been achieved due to problems such as sulfide levels and slow progress in polysulfide research.
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Embodiment 1
[0058] Example 1: Preparation of protein fluorescent probes for specific detection of polysulfides
[0059] A method for preparing a protein fluorescent probe for specifically detecting polysulfides, the steps are as follows:
[0060] (1) Using the green fluorescent protein roGFP gene (GeneBank accession number: AJ002682.1) as a template, the gene sequence of the protein fluorescent probe was constructed by site-directed mutation. The site-directed mutation was to mutate the 202nd serine in the roGFP amino acid sequence to Cysteine, the cysteine at position 204 is mutated to serine; remove the start codon atg and insert it into the multiple cloning site of the expression vector pET30 to obtain a protein fluorescent probe with a 6-his tag at the N-terminal The expression vector pET30-psGFP ( figure 2 ); the nucleotide sequence of the protein fluorescent probe is shown in SEQ ID NO.1;
[0061] (2) Transform the expression vector pET30-psGFP obtained in step (1) into Escheri...
Embodiment 2
[0064] Embodiment 2: psGFP detects the concentration of polysulfides in different samples
[0065] The psGFP prepared in Example 1 and dimercaptothreitol (DTT) were reacted on ice for 30 minutes to make the psGFP reaction complete. After measurement, the concentration of psGFP prepared in Example 1 was 3 mg / mL, and the above-mentioned dimercaptothreitol The concentration of alcohol was 1 mM; the reacted psGFP was desalted with a desalting column to elute unbound small molecules, and the protein was collected again to obtain the reduced protein fluorescent probe.
[0066] The above-mentioned reduced protein fluorescent probes were combined with HSSH, H 2 o 2 Sample ice bath reaction 30min, HSSH, H 2 o 2 The concentration of the samples was 100 μM, and the reaction product was obtained after desalting by the desalting column, and the reaction product was dissolved in 50 mM Tris-HCl buffer solution to obtain the sample to be tested, so that the concentration of psGFP in the sa...
Embodiment 3
[0068] Example 3: Detection of polysulfide concentration in Saccharomyces cerevisiae by psGFP
[0069] Prepare the competent state of Saccharomyces cerevisiae: Pick a single colony of Saccharomyces cerevisiae and add it to 5mL YPD medium (peptone 20g / L, yeast powder 10g / L, glucose 20g / L) at 30°C and 220rpm for overnight culture. solution, transfer the culture solution to 50mL fresh YPD medium, wait for OD 600 0.8, 3000rpm, 5min to collect the cells, wash with ice-bathed sterile water, add 1mL sterile water to resuspend, and prepare the competent cells of Saccharomyces cerevisiae.
[0070] i. The concentration detection of polysulfide in the cytoplasm, the steps are as follows:
[0071] The gene sequence (SEQ ID NO.1) of the protein fluorescent probe constructed in Example 1 was inserted into the expression vector YEPlac195, the promoter of the protein fluorescent probe was TEF1, and the Saccharomyces cerevisiae expression vector YEPlac195-psGFP ( Figure 5 ); the Saccharomyc...
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