A fluorescent probe for detecting neuron-specific enolase and use thereof

Abstract

The application belongs to the technical field of bioengineering, and relates to a fluorescent probe for detecting neuron-specific enolase and application. The application screens a specific binding short peptide E for neuron-specific enolase by using phage display technology, inserts a circular fluorescent protein R-GECO1 into the short peptide E, divides the E peptide chain into two parts E1 and E2, and forms a probe structure in the form of E1-R-GECO1-E2. The fluorescent probe protein provided by the application has relatively small molecular weight and is easy to mature, has large fluorescence dynamic change, has good specificity, and can detect neuron-specific enolase in and outside cells.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a fluorescent probe for detecting neuron-specific enolase and its uses. Background Technology

[0002] Neuron-specific enolase (NSA) is an acidic protease specific to neuroendocrine cells. Its activity is highest in brain tissue cells, intermediate in peripheral nerves and neurosecretive tissues, and lowest in non-neural tissues, serum, and cerebrospinal fluid. Excessive NSA expression has been found in tumors of neuroendocrine origin, particularly small cell lung cancer, leading to a significant increase in serum NSA levels. NSA is a biomarker for small cell lung cancer.

[0003] Neuron-specific enolases possess many characteristics, such as large molecular weight, acidity, and stability, and are easily leaked from cells after damage, making them easy to detect. Current detection methods mainly include immunoradioassay or radioimmunoassay based on antigen-antibody immune reactions, fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), and chemiluminescent immunoassay. However, these methods share common drawbacks: poor specificity and high false positive rates; furthermore, their complex operation and long detection time prevent timely identification of pathogens, thus limiting the detection of neuron-specific enolases.

[0004] Compared to other dye probes, fluorescent protein bioprobes have better biocompatibility and lower toxicity, making them significant scientifically and practically valuable, thus laying a solid foundation for comprehensive research on tumor markers. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention designs a fluorescent probe based on fluorescent proteins to specifically detect neuron-specific enolases. The protein has a relatively small molecular weight and is easy to mature, exhibits large fluorescence dynamics, and demonstrates good specificity. It can quantitatively detect neuron-specific enolases both inside and outside cells, and can be used for clinical biomarker detection, as well as providing a tool for further scientific research.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] A fluorescent probe for detecting neuron-specific enolase comprises a short peptide E that specifically binds to neuron-specific enolase and a cyclic fluorescent protein R-GECO1; wherein the fluorescent protein R-GECO1 is inserted into the peptide chain of the short peptide E, dividing the E peptide chain into two parts, short peptide E1 and short peptide E2, forming a fluorescent probe with an E1-R-GECO1-E2 structure; the amino acid sequence of the fluorescent probe is shown in SEQ ID NO.1, and the DNA sequence of the fluorescent probe is shown in SEQ ID NO.2.

[0008] The complete amino acid sequence of the fluorescent probe for detecting neuron-specific enolase is (SEQ ID NO. 1):

[0009] FGSAKNGVSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNG HEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEAGILNCCY

[0010] The complete nucleotide sequence of the fluorescent probe for detecting neuron-specific enolase is (SEQ ID NO. 2):

[0011] TTTGGCAGCGCGAAAAACGGCGTGAGCGAACGCATGTATCCGGAAGATGGCGCGCTGAAAAGCGAAATTAAAAAAGGCCTGCGCCTGAAAGATGGCGGCCATTATGCGGCGGAAGTGAAAACCACCTATAAAGCGAAAAAACCGGTGCAGCTGCCGGGCGCGTATATTGTGGATATTAAACTGGATATTGTGAGCCATAACGAAGATTATACCATTGTGGAACAGTGCGAACGCGCGGAAGGCCGCCATAGCACCGGCGGCATGGATGAACTGTATAAAGGCGGCACCGGCGGCAGCCTGGTGAGCAAAGGCGAAGAAGATAACATGGCGATTATTAAAGAATTTATGCGCTTTAAAGTGCATATGGAAGGCAGCGTGAACGGCCATGAATTTGAAATTGAAGGCGAAGGCGAAGGCCGCCCGTATGAAGCGTTTCAGACCGCGAAACTGAAAGTGACCAAAGGCGGCCCGCTGCCGTTTGCGTGGGATATTCTGAGCCCGCAGTTTATGTATGGCAGCAAAGCGTATATTAAACATCCGGCGGATATTCCGGATTATTTTAAACTGAGCTTTCCGGAAGGCTTTCGCTGGGAACGCGTGATGAACTTTGAAGATGGCGGCATTATTCATGTGAACCAGGATAGCAGCCTGCAGGATGGCGTGTTTATTTATAAAGTGAAACTGCGCGGCACCAACTTTCCGCCGGATGGCCCGGTGATGCAGAAAAAAACCATGGGCTGGGAAGCGGGCATTCTGAACTGCTGCTA

[0012] The fluorescence probe for detecting neuron-specific enolase, wherein the amino acid sequence of peptide E (SEQ ID NO.3):

[0013] FGSAKILNCCY

[0014] The nucleotide sequence of peptide E (SEQ ID NO.4):

[0015] TTTGGCAGCGCGAAAATTCTGAACTGCTGCTAT

[0016] The fluorescent probe for detecting neuron-specific enolase can utilize various cyclic fluorescent protein R-GECO1; this invention employs red fluorescent protein R-GECO1, whose amino acid sequence (SEQ ID NO. 5):

[0017] GVSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNG HEFEIEGEGEGRPYEAFQTAKLKVTKGGGLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEAG

[0018] The nucleotide sequence of the cyclic fluorescent protein R-GECO1 (SEQ ID NO.6):

[0019] GGCGTGAGCGAACGCATGTATCCGGAAGATGGCGCGCTGAAAAGCGAAATTAAAAAAGGCCTGCGCCTGAAAGATGGCGGCCATTATGCGGCGGAAGTGAAAACCACCTATAAAGCGAAAAAACCGGTGCAGCTGCCGGGCGCGTATATTGTGGATATTAAACTGGATATTGTGAGCCATAAC GAAGATTATACCATTGTGGAACAGTGCGAACGCGCGGAAGGCCGCCATAGCACCGGCGGCATGGATGAACTGTATAAAGGCGGCACCGGCGGCAGCCTGGTGAGCAAAGGCGAAGAAGATAACATGGCGATTATTAAAGAATTTATGCGCTTTAAAGTGCATATGGAAGGCAGCGTGAACGGC CATGAATTTGAAATTGAAGGCGAAGGCGAAGGCCGCCCGTATGAAGCGTTTCAGACCGCGAAACTGAAAGTGACCAAAGGCGGCCCGCTGCCGTTTGCGTGGGATATTCTGAGCCCGCAGTTTATGTATGGCAGCAAAGCGTATATTAAACATCCGGCGGATATTCCGGATTATTTTAAACTG AGCTTTCCGGAAGGCTTTCGCTGGGAACGCGTGATGAACTTTGAAGATGGCGGCATTATTCATGTGAACCAGGATAGCAGCCTGCAGGATGGCGTGTTTATTTATAAAGTGAAACTGCGCGGCACCAACTTTCCGCCGGATGGCCCGGTGATGCAGAAAAAAACCATGGGCTGGGAAGCGGGC

[0020] The fluorescent probe for detecting neuron-specific enolase can have its DNA sequence packaged into a prokaryotic expression system, and the expressed probe protein purified for in vitro detection. Alternatively, its DNA sequence can be packaged into a eukaryotic expression system, and the probe protein expressed in living cells via transfection for in vivo detection.

[0021] The beneficial effects of this invention are:

[0022] 1. The fluorescent probe provided by this invention can replace antibodies and secondary antibodies based on immune responses, reducing the types of reagents and eliminating the need for multiple steps in sandwich ELISA, thus saving the sample processing steps.

[0023] 2. The fluorescent probe provided by this invention is easy to mature, exhibits large fluorescence dynamic changes, and has good specificity. It can be used for in vitro detection by purifying proteins through prokaryotic expression vectors, or for real-time detection in living cells for related scientific research by packaging eukaryotic expression vectors. Attached Figure Description

[0024] Figure 1 Fluorescence spectra of fluorescent probes at different concentrations of neuron-specific enolase (0-280 ng / mL).

[0025] Figure 2 Linear relationship between fluorescence intensity and the concentration of the corresponding neuron-specific enolase. Detailed Implementation

[0026] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and technical solutions.

[0027] Example 1: Phage display technology for screening neuron-specific enolase affinity peptides

[0028] The phage display technology described in this invention is based on the affinity between biomolecules and target molecules. Through a repetitive process of adsorption-elution-amplification, phages containing specific binding to neuron-specific enolases are screened from a phage library, then enriched and amplified. Using the downstream primer M13-96 gⅢ provided in the kit, a short peptide E is obtained, the amino acid sequence of which is shown in SEQ ID NO.3.

[0029] Example 2: Construction and preparation of pRSETb-E-RGECO1 fluorescent probe

[0030] This invention provides a fluorescent probe comprising a short peptide E that specifically recognizes neuron-specific enolase and a fluorescent protein R-GECO1; wherein the fluorescent protein R-GECO1 is inserted into the short peptide E, dividing E into two parts, E1 and E2, forming a probe structure of the form E1-R-GECO1-E2; the interaction between the short peptide E and neuron-specific enolase leads to an increase in the fluorescence signal of the fluorescent protein R-GECO1.

[0031] The pRSETb-E-RGECO1 recombinant plasmid of the probe was transformed into BL21 competent bacteria, and clones were picked and cultured. The cultures were then transferred to 1L Erlenmeyer flasks containing ampicillin for large-volume culture. After 3-4 hours, IPTG induction solution was added to a final concentration of 0.5 mmol / L and induced overnight. Protein was then extracted from the bacterial culture. The specific steps are as follows:

[0032] (1) Prepare beads: Shake the Ni agarose beads well and take 1 mL. Centrifuge at 1,000 g for 2 min at 4℃. Use a pipette to remove the upper liquid, add pure water to wash away the alcohol, centrifuge at 1,000 g for 2 min, repeat three times, wash 4 times with Binding Buffer, and centrifuge at 1,000 g for 2 min for later use.

[0033] (2) Bacterial preparation: Centrifuge the bacterial culture at 10,000×g for 10 min, discard the upper culture medium, and add 10mL Binding Buffer to the bacterial cells and mix well.

[0034] (3) Bacterial disruption: The bacteria were disrupted by sonication at 300W on ice, with a 5s excitation every 5s for a total of 30min. Triton was added to a final concentration of 0.5%, and the bacteria were placed on ice for 30min. Then, the bacteria were centrifuged at 10,000×g for 15min at 4℃.

[0035] (4) Protein binding: Take the supernatant from (3) and mix it with Ni agarose beads. In a 4°C refrigerator, shake it in the dark for 6 hours to allow the target protein to fully bind with the Ni agarose beads.

[0036] (5) Washing: After centrifuging at 10,000×g for 5 min at 4℃, discard the upper cell lysis buffer, add 5 mL Binding Buffer to Ni agarose beads, wash and mix well, then centrifuge at 10,000×g for 5 min at 4℃, and repeat three times.

[0037] (6) Elution: Add 1 mL of Elution Buffer to Ni agarose beads, mix well, centrifuge at 10,000×g for 5 min at 4℃, and collect the supernatant as the protein solution. Repeat this step until the elution solution becomes colorless.

[0038] (7) Dialysis: Place the protein solution obtained in (6) into a dialysis bag that has been washed with deionized water and seal it with a dialysis clamp. Immerse the dialysis bag in 1L of protein dialysis solution and stir it with a magnetic stirrer at 4°C in the dark for 4 hours. Then discard the protein dialysis solution, add 1L of PBS, and continue stirring at 4°C in the dark for 4 hours. After that, aspirate the protein solution and put it into a centrifuge tube. Store it at -20°C, taking care to avoid light.

[0039] Example 3: Response of the pRSETb-E-RGECO1 fluorescent probe to neuron-specific enolases

[0040] Under these conditions, the performance of the pRSETb-E-RGECO1 fluorescent probe in the in vitro quantitative detection of neuron-specific enolases was investigated. Figure 1The fluorescence spectral response was used for the in vitro detection of neuron-specific enolase. The results showed that the fluorescence intensity of the pRSETb-E-RGECO1 fluorescent probe gradually increased with increasing neuron-specific enolase concentration. The calibration curve of fluorescence intensity versus neuron-specific enolase concentration is shown below. Figure 2 As shown. In Figure 2 A linear positive correlation was observed between fluorescence intensity and neuron-specific enolase concentration. Within the range of 40-200 ng / mL, the calibration equation was F = 0.0278x + 0.8666, where F and X represent fluorescence intensity and neuron-specific enolase concentration, respectively, and R0... 2 The value is 0.9709.

[0041] As can be seen from the above embodiments, the fluorescent probe provided by the present invention is a gene-encoded fluorescent protein biological probe that specifically responds to neuron-specific enolase. This probe protein has a relatively small molecular weight and is easy to mature, exhibits large dynamic changes in fluorescence, and has good specificity, enabling the detection of neuron-specific enolase, a lung cancer marker, both intracellularly and extracellularly.

Claims

1. A fluorescent probe for detecting neuron-specific enolase, characterized in that, The probe comprises a short peptide E that specifically binds to neuron-specific enolases and a cyclic fluorescent protein R-GECO1; the fluorescent protein R-GECO1 is inserted into the short peptide E chain, dividing the E peptide chain into two parts, short peptide E1 and short peptide E2, forming a fluorescent probe with an E1-R-GECO1-E2 structure; the amino acid sequence of the fluorescent probe is shown in SEQ ID NO.1, and the DNA sequence of the fluorescent probe is shown in SEQ ID NO.

2.

2. The use of the fluorescent probe for detecting neuron-specific enolase as described in claim 1 in the preparation of a drug for detecting neuron-specific enolase.

3. The use according to claim 2, characterized in that, The DNA sequence of a fluorescent probe for detecting neuron-specific enolase is packaged into a prokaryotic expression system, and the expressed probe protein is purified for in vitro detection.

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