A fluorescent probe for detecting squamous cell carcinoma antigen and use thereof

Abstract

The application belongs to the technical field of bioengineering, and relates to a fluorescent probe for detecting squamous cell carcinoma antigen and application thereof. The application utilizes phage display technology to screen short peptides S1 and S2 which are specific to squamous cell carcinoma antigen and bind to the squamous cell carcinoma antigen. A cyclic fluorescent protein R-GECO1 is inserted into the short peptides S1 and S2, and a fluorescent probe with the structure of S1-R-GECO1-S2 is formed. The fluorescent probe provided by the application has relatively small protein molecular weight and is easy to mature. The fluorescent probe has large dynamic change of fluorescence and good specificity, and can detect squamous cell carcinoma antigen in and out of cells.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a fluorescent probe for detecting squamous cell carcinoma antigen and its uses. Background Technology

[0002] Squamous cell carcinoma antigen (SCC-Ag) is a glycoprotein isolated from cervical squamous epithelial cells and is widely present in the cytoplasm of squamous cell carcinoma cells in the uterus, cervix, lung, head and neck, esophagus, and anal canal. Studies have shown that the expression level of SCC-Ag is related to the invasion and metastasis of squamous cell carcinoma. The concentration of SCC-Ag in the serum of cancer patients usually changes with the progression of the disease. An increase often indicates disease progression and the possibility of metastasis. Therefore, SCC-Ag is often used for treatment monitoring and prognosis.

[0003] SCC-Ag has been widely used in preoperative diagnosis, postoperative observation, monitoring of metastasis and recurrence after treatment, and assessment of patient prognosis for tumors. Currently, there are two methods for detecting serum SCC-Ag: chemiluminescence immunoassay and enzyme-linked immunosorbent assay (ELISA). However, these methods have some drawbacks: cumbersome procedures, poor selectivity, and susceptibility to environmental influences. Therefore, it is necessary to design a highly sensitive and specific method for detecting squamous cell carcinoma antigen. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention designs a fluorescent probe based on fluorescent proteins that can specifically detect squamous cell carcinoma antigens. 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 squamous cell carcinoma antigens both inside and outside cells, and can be used for clinical biomarker detection, as well as providing a tool for further scientific research.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] A fluorescent probe for detecting squamous cell carcinoma antigen includes short peptides S1 and S2 that specifically bind to squamous cell carcinoma antigen, and a cyclic fluorescent protein R-GECO1; wherein the cyclic fluorescent protein R-GECO1 is inserted between the short peptides S1 and S2 to form a fluorescent probe with an S1-R-GECO1-S2 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.

[0007] The complete amino acid sequence of the fluorescent probe for detecting squamous cell carcinoma antigen is (SEQ ID NO.1):

[0008] TLSWHQNLRLMEGVSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYK

[0009] AKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHSTGGMDELYKGGT

[0010] GGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTA

[0011] KLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVM

[0012] NFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEAGAY

[0013] SRDVVLNMWR

[0014] The complete nucleotide sequence of the fluorescent probe for detecting squamous cell carcinoma antigen is (SEQ ID NO.2): ACCCTGAGCTGGCATCAGAACCTGCGCCTGATGGAAGGCGTGAGCGAACGCATGTATCCGGAAGATGGCGCGCTGAAAAGCGAAATTAAAAAAGGCCTGCGCCTGAAAGATGGCGGCCATTATGCGGCGGAAGTGAAAACCACCTATAAAGCGAAAAAACCGGTGCAGCTGCCGGGCGCGTATATTGTGGATATTAAACTGGATATTGTGAGCCATAACGAAGATTATACCATTGTGGAACAGTGCGAACGCGCGGAAGGCCGCCATAGCACCGGCGGCATGGATGAACTGTATAAAGGCGGCACCGGCGGCAGCCTGGTGAGCAAAGGCGAAGAAGATAACATGGCGATTATTAAAGAATTTATGCGCTTTAAAGTGCATATGGAAGGCAGCGTGAAC GGCCATGAATTTGAAATTGAAGGCGAAGGCGAAGGCCGCCCGTATGAAGCGTTTCAGACCGCGAAACTGAAAGTGACCAAAGGCGGCCCGCTGCCGTTTGCGTGGGATATTCTGAGCCCGCAGTTTATGTATGGCAGCAAAGCGTATATTAAACATCCGGCGGATATTCCGGATTATTTTAAACTGAGCTTTCCGGAAGGCT TTCGCTGGGAACGCGTGATGAACTTTGAAGATGGCGGCATTATTCATGTGAACCAGGATAGCAGCCTGCAGGATGGCGTGTTTATTTATAAAGTGAAACTGCGCGGCACCAACTTTCCGCCGGATGGCCCGGTGATGCAGAAAAAAACCATGGGCTGGGAAGCGGGCGCGTATAGCCGCGATGTGGTGCTGAACATGTGGCGC

[0015] The fluorescent probe for detecting squamous cell carcinoma antigen, wherein the amino acid sequence of the short peptide S1 (SEQ ID NO. 3):

[0016] TLSWHQNLRLME

[0017] The nucleotide sequence of short peptide S1 (SEQ ID NO.4):

[0018] ACCCTGAGCTGGCATCAGAACCTGCGCCTGATGGAA

[0019] The fluorescent probe for detecting squamous cell carcinoma antigen, wherein the amino acid sequence of the short peptide S2 (SEQ ID NO. 5):

[0020] AYSRDVVLNMWR

[0021] The nucleotide sequence of short peptide S2 (SEQ ID NO.6):

[0022] GCGTATAGCCGCGATGTGGTGCTGAACATGTGGCGC

[0023] The fluorescent probe for detecting squamous cell carcinoma antigen, wherein the cyclic fluorescent protein R-GECO1 can be selected from different colored fluorescent proteins according to the color development requirements, such as using red cyclic fluorescent protein R-GECO1, whose amino acid sequence (SEQ ID NO.7):

[0024] GVSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNG HEFEIEGEGEGRPYEAFQTAKLKVTKGGGLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEAG

[0025] The nucleotide sequence of the red cyclic fluorescent protein R-GECO1 (SEQ ID NO.8):

[0026] GGCGTGAGCGAACGCATGTATCCGGAAGATGGCGCGCTGAAAAGCGAAATTAAAAAAGGCCTGCGCCTGAAAGATGGCGGCCATTATGCGGCGGAAGTGAAAACCACCTATAAAGCGAAAAAACCGGTGCAGCTGCCGGGCGCGTATATTGTGGATATTAAACTGGATATTGTGAGCCATAAC GAAGATTATACCATTGTGGAACAGTGCGAACGCGCGGAAGGCCGCCATAGCACCGGCGGCATGGATGAACTGTATAAAGGCGGCACCGGCGGCAGCCTGGTGAGCAAAGGCGAAGAAGATAACATGGCGATTATTAAAGAATTTATGCGCTTTAAAGTGCATATGGAAGGCAGCGTGAACGGC CATGAATTTGAAATTGAAGGCGAAGGCGAAGGCCGCCCGTATGAAGCGTTTCAGACCGCGAAACTGAAAGTGACCAAAGGCGGCCCGCTGCCGTTTGCGTGGGATATTCTGAGCCCGCAGTTTATGTATGGCAGCAAAGCGTATATTAAACATCCGGCGGATATTCCGGATTATTTTAAACTG AGCTTTCCGGAAGGCTTTCGCTGGGAACGCGTGATGAACTTTGAAGATGGCGGCATTATTCATGTGAACCAGGATAGCAGCCTGCAGGATGGCGTGTTTATTTATAAAGTGAAACTGCGCGGCACCAACTTTCCGCCGGATGGCCCGGTGATGCAGAAAAAAACCATGGGCTGGGAAGCGGGC

[0027] The aforementioned use of squamous cell carcinoma antigen in in vitro or live-cell detection can be achieved by packaging its DNA sequence into a prokaryotic expression system, purifying the expressed probe protein for in vitro detection, or by packaging its DNA sequence into a eukaryotic expression system, transfecting cells to express the probe protein in live cells for live-cell detection.

[0028] The beneficial effects of this invention are:

[0029] 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.

[0030] 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

[0031] Figure 1 Fluorescence spectra of fluorescent probes at different concentrations of squamous cell carcinoma antigen (0-280 ng / mL).

[0032] Figure 2 Linear relationship between fluorescence intensity and the concentration of the corresponding squamous cell carcinoma antigen. Detailed Implementation

[0033] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and technical solutions.

[0034] Example 1: Phage display technology was used to pan for squamous cell carcinoma antigen affinity peptides to obtain short peptides S1 and S2 that can specifically bind to squamous cell carcinoma antigens.

[0035] 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 that specifically bind to squamous cell carcinoma antigens are screened from a phage library, then enriched and amplified. Using the downstream primers M13-96gⅢ provided in the kit, short peptides S1 and S2 are obtained, with their amino acid sequences as shown in SEQ ID NO.3 and SEQ ID NO.5.

[0036] Example 2: Construction and preparation of the pRSETb-S1-R-GECO1-S2 fluorescent probe

[0037] The red fluorescent protein R-GECO1 was inserted between short peptides S1 and S2, and pRSETb was used as the expression vector to form a fluorescent probe with the structure pRSETb-S1-R-GECO1-S2. The interaction between short peptides S1 and S2 and squamous cell carcinoma antigen led to an increase in the fluorescence signal of the fluorescent protein R-GECO1.

[0038] The fluorescent probe pRSETb-S1-R-GECO1-S2 recombinant plasmid was transformed into BL21 competent bacteria, and clones were picked and cultured. The culture was then transferred to Erlenmeyer flasks containing 1L of ampicillin-containing LB broth 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:

[0039] After activating positive clones on agar plates (containing ampicillin), a single colony was picked and inoculated into a conical flask containing 10 mL of LB broth containing the corresponding antibiotic. The flask was then incubated overnight at 37°C with shaking at 200 rpm. The following day, the seed culture was inoculated at a 1:100 ratio into LB broth containing ampicillin (final concentration 100 mg / mL) and incubated at 37°C with shaking at 200 rpm. OD was then calculated. 600 When the concentration reaches 0.5-1.0, add IPTG to a final concentration of 0.5 mmol / L, then incubate at 16°C with shaking at 180 rpm for 3-4 hours. Centrifuge the sample taken during induction at 10,000×g for 10 min, discard the supernatant, and add 10 mL of Binding Buffer to the cells and mix well. Sonicate on ice at 300W, with a 5-second stimulation every 5 seconds for a total of 30 min. Add Triton to a final concentration of 0.5%, incubate on ice for 30 min, then centrifuge at 10,000×g for 15 min at 4°C. Take 1 mL of Ni agarose beads after shaking well, centrifuge at 1,000g for 2 min at 4°C; remove the supernatant with a pipette, wash off the alcohol with pure water, centrifuge at 1,000g for 2 min, repeat three times, wash four times with Binding Buffer, and centrifuge at 1,000g for 2 min for later use. The supernatant was mixed with Ni agarose beads and incubated at 4°C in the dark for 6 hours to ensure thorough binding of the target protein to the Ni agarose beads. After centrifugation at 10,000×g for 5 minutes at 4°C, the upper layer of cell lysis buffer was discarded. 5 mL of Binding Buffer was added to the Ni agarose beads for washing and mixing, followed by centrifugation at 10,000×g for 5 minutes at 4°C. This process was repeated three times. 1 mL of Elution Buffer was added to the Ni agarose beads and mixed. After centrifugation at 10,000×g for 5 minutes at 4°C, the supernatant was collected as the protein solution. This step was repeated until the elution buffer became colorless. Place the obtained protein solution into a dialysis bag rinsed with deionized water and seal it with a dialysis clamp. Immerse the dialysis bag in 1L of protein dialysis buffer and stir with a magnetic stirrer at 4°C in the dark for 4 hours. Discard the protein dialysis buffer, add 1L of PBS, and continue stirring at 4°C in the dark for 4 hours. After that, aspirate the protein solution into centrifuge tubes and store at -20°C, taking care to avoid light.

[0040] Example 3: Response of pRSETb-S1-R-GECO1-S2 fluorescent probe to squamous cell carcinoma antigen

[0041] This study investigated the performance of the pRSETb-S1-R-GECO1-S2 fluorescent probe in the in vitro quantitative detection of squamous cell carcinoma antigen. Figure 1 The fluorescence spectral response of pRSETb-S1-R-GECO1-S2 fluorescent probe for in vitro detection of squamous cell carcinoma antigen is shown. The results indicate that the fluorescence intensity of the pRSETb-S1-R-GECO1-S2 fluorescent probe gradually increases with increasing squamous cell carcinoma antigen concentration. The calibration curve of fluorescence intensity versus squamous cell carcinoma antigen concentration is shown below. Figure 2 As shown. In Figure 2 A linear positive correlation was observed between fluorescence intensity and squamous cell carcinoma antigen concentration. Within the range of 1-100 ng / mL, the calibration equation was F = 0.0166x + 1.0277, where F and X represent fluorescence intensity and squamous cell carcinoma antigen concentration, respectively, and R0... 2 The value is 0.9959.

[0042] 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 squamous cell carcinoma antigen. 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 squamous cell carcinoma antigen, a lung cancer marker, both intracellularly and extracellularly.

Claims

1. A fluorescent probe for detecting squamous cell carcinoma antigen, characterized in that, The probe includes short peptides S1 and S2 that can specifically bind to squamous cell carcinoma antigens, and a cyclic fluorescent protein R-GECO1; the cyclic fluorescent protein R-GECO1 is inserted between short peptides S1 and S2 to form a fluorescent probe with an S1-R-GECO1-S2 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 squamous cell carcinoma antigen as described in claim 1 in the preparation of a drug for detecting squamous cell carcinoma antigen.

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

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