Phycoerythrin PE-1 with fluorescence characteristics, fusion protein thereof and application

Abstract

The application discloses a phycobiliprotein PE-1 with fluorescence characteristics, a fusion protein thereof and application, and belongs to the technical field of genetic engineering. The application provides a novel phycobiliprotein, and the amino acid sequence of the phycobiliprotein is shown as SEQ ID NO. 1. The phycobiliprotein has high fluorescence quantum yield and fluorescence emission capacity. The application further provides a fusion protein constructed by fusing the phycobiliprotein and streptavidin through a connecting peptide, a recombinant expression vector containing the coding sequence of the fusion protein and a recombinant host cell capable of expressing the fusion protein. The fusion protein provided by the application can be used in solid-phase immunodetection systems such as ELISA and immunochromatography, and sensitive detection of target objects can be realized through a fluorescence detection mode of 540 / 580 nm. The fusion protein provided by the application has significant advantages in signal-to-noise ratio and detection stability, and can be widely applied to the fields of biomedical detection and diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a phycoerythrin PE-1 with fluorescent properties, its fusion protein, and its applications. Background Technology

[0002] Phycobiliproteins (PBPs) are a class of photosynthetic auxiliary pigment proteins found in red algae, cyanobacteria, and other algae. They mainly include R-phycoerythrin (R-PE), phycocyanin (PC), and allophycocyanin (APC). Among them, R-phycoerythrin (R-PE) has broad application prospects in bioanalysis, flow cytometry, immunodiagnostics, FRET probes, food coloring, and cosmetics due to its high molar absorption coefficient, strong fluorescence quantum yield, and good water solubility.

[0003] Currently, natural phycoerythrin is mainly derived from red algae, but its extraction process is affected by season, algal growth cycle, and environmental conditions, resulting in low yield, high cost, and difficulty in purification, as well as insufficient batch stability. These factors limit its further promotion and application in the biomedical and industrial fields.

[0004] With the development of synthetic biology and protein engineering technologies, the production of recombinant phycoerythrin through microbial expression systems has become a new solution. Although some studies have obtained several variants with improved performance through site-directed mutagenesis and regional modification, novel, unreported phycoerythrin sequences remain very limited. Due to the high conservatism and complex structure of natural sequences, existing engineering strategies struggle to break through the original sequence framework, thus limiting their application in regulating spectral properties, enhancing thermal stability, and increasing Stokes shifts.

[0005] With the development of protein engineering and computational design technologies, it has become possible to construct phycoerythrins with unique structures and functions by designing amino acid sequences independently. These artificial sequences can overcome the limitations of natural proteins, achieving functions such as spectral tunability and high stability, and represent an important development direction for future high-performance fluorescent materials and biological probes. However, there is currently a lack of novel artificial sequence design schemes for phycoerythrins and related structural and functional verification. Summary of the Invention

[0006] The purpose of this invention is to provide a phycoerythrin PE-1 with fluorescent properties, its fusion protein, and its applications. The sequence of the phycoerythrin provided by this invention has not been reported in existing literature and databases. Furthermore, experiments have shown that phycoerythrin PE-1 has the strongest fluorescence emission ability, which improves the accuracy of detection. It also has the advantage of storage stability and has good industrial application value.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] The present invention provides a phycoerythrin PE-1 with fluorescent properties, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] Furthermore, the Stokes shift of the phycoerythrin PE-1 is 20 nm.

[0010] The present invention also provides the encoding gene of the phycoerythrin PE-1, the nucleotide sequence of which is shown in SEQ ID NO.6.

[0011] The present invention also provides a recombinant expression vector containing the encoding gene of the phycoerythrin PE-1.

[0012] The present invention also provides a recombinant host cell containing the encoding gene of the phycoerythrin PE-1, wherein the recombinant host cell contains the recombinant expression vector and is capable of expressing the fusion protein.

[0013] The present invention also provides a fusion protein, the fusion protein being SA-PE, which is composed of phycoerythrin PE-1 and streptavidin linked by a linker peptide, the linker peptide being 5 to 30 amino acid residues in length.

[0014] Furthermore, the purified fusion protein SA-PE exists in tetrameric form.

[0015] Furthermore, the linker peptide has a (G4S)3 sequence.

[0016] The present invention also provides the application of the phycoerythrin PE-1 or the fusion protein SA-PE in solid-phase immunoassay.

[0017] Furthermore, the fusion protein SA-PE is able to bind to biotin.

[0018] Furthermore, the solid-phase immunoassay method includes the following steps:

[0019] (1) The target analyte is added to a solid-phase carrier pre-coated with capture antibodies for an immune reaction;

[0020] (2) Add biotinylated detection antibodies that specifically bind to the target analyte to form an immune complex;

[0021] (3) Add the fusion protein SA-PE as a fluorescent probe to bind it to the biotinylated detection antibody;

[0022] (4) After removing the unbound material, the solid-phase carrier is detected by a fluorescence microplate reader to obtain the fluorescence signal, and the target analyte is quantitatively analyzed according to the fluorescence signal intensity.

[0023] Furthermore, in the solid-phase immunoassay, the excitation wavelength of the phycoerythrin PE-1 or the fusion protein SA-PE is 530~555 nm, and the emission wavelength of the phycoerythrin PE-1 or the fusion protein SA-PE is 550~595 nm.

[0024] Furthermore, the fusion protein SA-PE exhibits the strongest fluorescence signal when used in solid-phase immunoassay with an excitation wavelength of 540 nm and an emission wavelength of 580 nm.

[0025] Compared with the prior art, the present invention has the following significant advantages:

[0026] 1. The amino acid sequence of phycoerythrin PE-1 shown in SEQ ID NO.1 provided by this invention is obtained through rational design and is different from previously disclosed phycoerythrin sequences. Furthermore, this invention fuses phycoerythrin with streptavidin, enabling it to directly participate in the biotin-avidin recognition system, achieving efficient solid-phase capture and fluorescence amplification, and reducing additional labeling steps. Compared with traditional fluorescent labels, the fusion protein provided by this invention has a high fluorescence quantum yield and strong fluorescence emission capability, improving the signal-to-noise ratio by at least 1.5 times and reducing background noise in solid-phase immunoassay. The fusion protein or phycoerythrin provided by this invention can be widely used in ELISA, immunochromatography, rapid diagnostic test strips, and other platforms, possessing significant industrial application value.

[0027] 2. Compared with the other four phycoerythrins, the phycoerythrin PE-1 provided by this invention has the strongest fluorescence emission ability at the same protein concentration; moreover, it has the largest Stokes shift, which reduces the overlap between absorption peak and emission peak, thereby improving the accuracy of detection.

[0028] 3. The phycoerythrin PE-1 provided by this invention exhibits less fluorescence decay and superior thermal stability at higher temperatures, thus having a significant advantage in storage stability and being more suitable for large-scale engineering production and application.

[0029] 4. The phycoerythrin and its fusion protein provided by this invention are structurally unique and have advantages in spectral performance, thus overcoming the problems of limited excitation / emission wavelengths, insufficient signal-to-noise ratio, and low photostability of existing phycoerythrin in immunoassays. Simultaneously, this invention aims to construct a functionalized protein fused with streptavidin, enabling its direct use in solid-phase immunoassay systems, improving detection sensitivity and ease of operation, and making it suitable for detection modes of 540 nm excitation and 580 nm emission. This provides a new fluorescent labeling tool for biomedical detection, and the novel phycoerythrin and its fusion protein exhibit superior application effects in immunoassay technology. Attached Figure Description

[0030] Figure 1 The images show the nucleic acid electrophoresis results after PCR of the target gene sequence. From left to right, they are PE-1, PE-2, PE-3, PE-4, PE-5, Marker, and cpcS.

[0031] Figure 2 The image shows the nucleic acid electrophoresis results after PCR of the target gene sequence. From left to right, these are ho1, Marker, and pebS.

[0032] Figure 3 The images are SAS-PAGE images of proteins purified by affinity chromatography. From left to right, they are PE-1, PE-2, PE-3, PE-4, PE-5, and Marker. A is zinc staining, and B is Coomassie Brilliant Blue staining.

[0033] Figure 4 This is a visible spectrum scan.

[0034] Figure 5 The image shows a fluorescence spectrum scan, with the solid line representing the excitation spectrum and the dashed line representing the emission spectrum.

[0035] Figure 6 The diagram shows the temperature stability, with A representing stability at 50℃, B representing stability at 55℃, C representing stability at 60℃, and D representing stability at 65℃.

[0036] Figure 7 The image shows the SAS-PAGE of the fusion protein SA-PE after affinity chromatography purification. From left to right, the images show SA-PE and the marker, with A being zinc stain and B being Coomassie brilliant blue stain.

[0037] Figure 8 The image shows the Western blotting results of the fusion protein SA-PE after affinity chromatography purification. From left to right, these are SA-PE and the marker.

[0038] Figure 9A standard curve for detecting IL-1 in the fusion protein SA-PE. The x-axis represents the concentration of IL-1 (pg / mL), and the y-axis represents the fluorescence value. Detailed Implementation

[0039] The following embodiments are for illustrative purposes only and are not intended to limit the technical solutions described in this invention. Although this specification has described the invention in detail with reference to the above embodiments, those skilled in the art should understand that they can still make modifications or equivalent substitutions to the invention, and all technical solutions and improvements that do not depart from the spirit and scope of the invention should be covered within the scope of the claims of this invention.

[0040] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0041] Example 1: Rational Design and Analysis of Phycoerythrin Sequence

[0042] To screen for phycobiliproteins with strong fluorescence emission and high thermal stability, this invention analyzes the sequences of phycobiliproteins in the NCBI database to determine the protein backbone, and then uses LigandMPNN for sequence filling to obtain new protein sequences. Five amino acid sequences were manually selected as shown in SEQ ID NO.1-SEQ ID NO.5, and the nucleotide sequences of their corresponding coding genes, after codon optimization, are shown in SEQ ID NO.6-SEQ ID NO.10. BLAST sequence analysis showed that the amino acid sequences shown in SEQ ID NO.1-SEQ ID NO.5 provided by this invention are significantly different from previously published amino acid sequences, with the highest similarity being 48.41% (Genbank accession number: MEO0948496.1), 41.40% (Genbank accession number: MEB3312071.1), 42.97% (Genbank accession number: WP_190389521.1), 44.44% (Genbank accession number: MFQ3679211.1), and 44.03% (Genbank accession number: WP_166278809.1), respectively. All five amino acid sequences provided by this invention are novel proteins, and their genes are all novel coding genes.

[0043] Example 2: Construction of recombinant plasmids

[0044] The nucleotide sequences shown in SEQ ID NO. 6-SEQ ID NO. 10 after codon optimization were synthesized artificially by Beijing Qingke Biotechnology Co., Ltd. Specific primer pairs PE-1-F / PE-1-R, PE-2-F / PE-2-R, PE-3-F / PE-3-R, PE-4-F / PE-4-R, and PE-5-F / PE-5-R (Table 1) were designed based on the target gene sequence. Gene PCR amplification was performed according to the instructions of Phanta Max Super-Fidelity DNA Polymerase. After amplification, the PCR products were detected by nucleic acid electrophoresis. Figure 1 After purification, the lengths of all 5 gene sequences were consistent with the theoretical lengths.

[0045] Table 1 Primer Sequences

[0046] ,

[0047] The MCS1 of plasmid pRSFDuet-1 was double-digested with restriction endonucleases BamH I and Not I, and the target band was recovered according to the gel extraction kit instructions. The gel-extracted plasmid and purified PCR product were ligated according to the ClonExpress II One Step Cloning Kit instructions, followed by heat shock transformation to... E. coli DH5α competent cells were incubated at 37°C for 30 min and then plated onto LB agar plates containing kanamycin. After culturing at 37°C for 12 h, single colonies were selected, and positive clones were screened by PCR. Positive clones were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. Clones that were verified by sequencing were preserved, and recombinant plasmids were extracted and labeled as pR-pe-1, pR-pe-2, pR-pe-3, pR-pe-4, and pR-pe-5, respectively.

[0048] The cpcS gene was obtained from the NCBI database (Genbank accession number: WP_322877014.1), and codon optimization was performed. It was then synthesized artificially by Beijing Qingke Biotechnology Co., Ltd. The cpcS gene was amplified using primer pairs cpcS-F / cpcS-R (Table 1), and the PCR products were detected by nucleic acid electrophoresis. Figure 1The recombinant plasmids pR-pe-1, pR-pe-2, pR-pe-3, pR-pe-4, and pR-pe-5 were double-digested with restriction endonucleases Nde I and Kpn I, respectively, and the target bands were recovered according to the gel extraction kit instructions. The gel-recovered recombinant plasmids and the purified cpcS gene were ligated according to the ClonExpress II One Step Cloning Kit instructions, and then heat-shocked transformed into... E. coli DH5α competent cells were incubated at 37°C for 30 min and then plated onto LB agar plates containing kanamycin. After culturing at 37°C for 12 h, single colonies were selected, and positive clones were screened by PCR. Positive clones were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. Clones that were verified by sequencing were preserved, and recombinant plasmids were extracted and labeled as pR-pe-cpcS-1, pR-pe-cpcS-2, pR-pe-cpcS-3, pR-pe-cpcS-4, and pR-pe-cpcS-5, respectively.

[0049] The ho1 gene (Genbank accession number: BAA16864.1) and pebS gene (Genbank accession number: QFG06437.1) were obtained from the NCBI database and their codons were optimized before being synthesized by Beijing Qingke Biotechnology Co., Ltd. The ho1 and pebS genes were amplified using primer pairs ho1-F / ho1-R and pebS-F / pebS-R (Table 1), respectively, and the PCR products were detected by nucleic acid electrophoresis. Figure 2 The plasmid pACYCDuet-1 was double-digested with restriction endonucleases BamH I and Not I, and the target band was recovered according to the gel extraction kit instructions. The gel-extracted plasmid and the purified ho1 gene were ligated according to the ClonExpress II One Step Cloning Kit instructions, and then heat-shock transformed into... E. coliDH5α competent cells were incubated at 37°C for 30 min and then plated onto LB agar plates containing chloramphenicol. After culturing at 37°C for 12 h, single colonies were selected, and positive clones were screened by PCR. Positive clones were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. Clones that were verified by sequencing were preserved, and recombinant plasmids were extracted and labeled pA-ho1. The MCS2 of the recombinant plasmid pA-ho1 was double-digested with restriction endonucleases Nde I and Kpn I, and the target band was recovered according to the gel extraction kit instructions. The gel-recovered recombinant plasmid and the purified pebS gene were ligated according to the ClonExpress II One Step Cloning Kit instructions, and then heat-shocked transformed into... E. coli DH5α competent cells were incubated at 37°C for 30 min and then plated onto LB agar plates containing chloramphenicol. After culturing at 37°C for 12 h, single colonies were selected, and positive clones were screened by PCR. Positive clones were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. Clones that were verified by sequencing were preserved, and recombinant plasmids were extracted and labeled pA-ho1-pebS.

[0050] Example 3: Expression and purification of recombinant phycoerythrin

[0051] Recombinant plasmids pR-pe-cpcS-1, pR-pe-cpcS-2, pR-pe-cpcS-3, pR-pe-cpcS-4, and pR-pe-cpcS-5 were co-transformed with pA-ho1-pebS to [various locations]. E. coli After BL21(DE3) competent cells were incubated upside down on LB agar plates containing kanamycin and chloramphenicol at 37°C for 12 h, single colonies were selected for PCR screening of positive bacteria. The positive bacteria were labeled BL21-1, BL21-2, BL21-3, BL21-4, and BL21-5. The five positive strains were inoculated into LB liquid medium and cultured at 37°C and 160 rpm until OD500. 600 When the concentration is 0.6~0.8, add IPTG to a final concentration of 10 mM, and continue culturing at 20℃ and 160 rpm for 18 h.

[0052] Centrifuge (5000 rpm, 20 min, 4℃) to collect bacterial cells. Wash the cells twice with PBS, then lyse the cells (10 min, sonication power 40%, 2 s on, 2 s off). After lysing, centrifuge (12000 rpm, 30 min, 4℃) and collect the supernatant. Filter the supernatant through a 0.22 μm sterile filter and purify the five proteins according to the nickel affinity chromatography column purification instructions. Elute the recombinant phycoerythrins (PE-1, PE-2, PE-3, PE-4, and PE-5) with 100 mM imidazole. SDS-PAGE electrophoresis results showed that all five proteins were purified as single protein bands. Figure 3 The molecular mass is approximately 17.5 kDa, which is consistent with the theoretical molecular mass.

[0053] Example 4: Determination of the properties of recombinant phycoerythrin

[0054] (1) Spectral scanning

[0055] The five recombinant phycoerythrins purified in Example 3 were diluted to 10 μM with PBS and their spectra were scanned using a UV-Vis spectrophotometer. Figure 4 The results showed that, among the five recombinant phycoerythrins provided by this invention, PE-1 had the strongest absorption at the same protein concentration, being 2 times, 1.4 times, 1.8 times, and 2.2 times that of PE-2, PE-3, PE-4, and PE-5, respectively.

[0056] The five recombinant phycoerythrins purified in Example 3 were diluted to 1 μM with PBS, and the excitation and emission spectra were further scanned using a fluorescence spectrophotometer. Figure 5 The results showed that the maximum excitation wavelengths of the recombinant phycoerythrins PE-1, PE-2, PE-3, PE-4, and PE-5 provided by this invention were 547 nm, 548 nm, 548 nm, 548 nm, and 548 nm, respectively, and the maximum emission wavelengths were 567 nm, 563 nm, 566 nm, 565 nm, and 564 nm, respectively. At the same protein concentration, PE-1 exhibited the strongest fluorescence emission.

[0057] The recombinant phycoerythrin PE-1 provided by this invention has the largest Stokes shift, at 20 nm. A larger Stokes shift reduces the overlap between absorption and emission peaks, thereby improving detection accuracy.

[0058] (2) Determination of fluorescence quantum yield

[0059] The fluorescence quantum yield was determined using a reference method. Using rhodamine as a reference, the fluorescence quantum yields of PE-1, PE-2, PE-3, PE-4, and PE-5 purified in Example 3 were measured to be 0.97, 0.90, 0.87, 0.96, and 0.91, respectively. Fluorescence quantum yield is generally defined as the ratio of the number of fluorescence photons emitted by a sample after light absorption to the number of photons of the absorbed excitation light; a higher value indicates stronger fluorescence. The recombinant phycoerythrin PE-1 provided by this invention exhibits a high fluorescence quantum yield, indicating that PE-1 better stabilizes the structure of phycoerythrin, which is beneficial for photon transmission and results in less energy loss.

[0060] (3) Temperature stability

[0061] The residual fluorescence intensity of the five recombinant phycoerythrins purified in Example 3 was measured after being placed at 50℃, 55℃, 60℃ and 65℃ for different times, with the initial fluorescence intensity set at 100%. Figure 6 The results show that the recombinant phycoerythrin PE-1 provided by the present invention has good thermal stability (relative fluorescence intensity ≥80%) in the temperature range of 50℃~60℃.

[0062] Plotting the natural logarithm of the percentage of residual fluorescence intensity against time, the slope of the straight line represents the inactivation rate constant. k Half-life t 1 / 2 =ln(2) / k =0.693 / k The half-lives of five recombinant phycoerythrins were calculated at 55℃, 60℃, and 65℃, and the results are shown in Table 2. The half-life of the recombinant phycoerythrin PE-1 provided by this invention is 16.3 h at 65℃.

[0063] Table 2. Half-life of recombinant phycoerythrin at different temperatures

[0064]

[0065] The half-life of recombinant phycoerythrin PE-1 at 70℃ and 75℃ was further determined. The half-life of recombinant phycoerythrin PE-1 at 70℃ and 75℃... k The values ​​were 0.0603 and 0.0721, respectively, yielding half-lives of 11.5 h and 9.6 h. PE-1 exhibited less fluorescence decay at higher temperatures and demonstrated superior thermal stability. The recombinant phycoerythrin PE-1 provided by this invention exhibits significant advantages in storage stability, making it more suitable for large-scale engineering production and application.

[0066] Example 5: Preparation of fusion protein (SA-PE)

[0067] The streptavidin (SA) gene (Genbank accession number: CAA46210.1) was obtained from the NCBI database and its codons were optimized before being synthesized by Beijing Qingke Biotechnology Co., Ltd. The SA gene was amplified using primers SA-F / SA-R (Table 1), and the recombinant plasmid pR-pe-cpcS-1 was amplified using primers F-1 / R-1 (Table 1). The PCR products were detected and purified by nucleic acid electrophoresis, ligated according to the instructions of the ClonExpress II One Step Cloning Kit, and then heat-shock transformed into... E. coli DH5α competent cells were incubated at 37°C for 30 min and then plated onto LB agar plates containing kanamycin. After culturing at 37°C for 12 h, single colonies were selected, and positive clones were screened by PCR. Positive clones were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. Clones that were verified by sequencing were preserved, and recombinant plasmids were extracted and labeled as pR-sa-pe-cpcS.

[0068] Recombinant plasmids pR-sa-pe-cpcS and pA-ho1-pebS were co-transformed into E. coli BL21(DE3) competent cells were incubated upside down on LB agar plates containing kanamycin and chloramphenicol at 37°C for 12 h. Single colonies were selected for PCR screening of positive bacteria. The obtained positive strains were inoculated into LB liquid medium and cultured at 37°C and 160 rpm until OD500. 600 When the concentration is 0.6~0.8, add IPTG to a final concentration of 10 mM, and continue culturing at 20℃ and 160 rpm for 18 h.

[0069] Centrifuge (5000 rpm, 20 min, 4℃) to collect bacterial cells. Wash the cells twice with PBS, then lyse the cells (10 min, sonication power 40%, 2 s on, 2 s off). After lysing, centrifuge (12000 rpm, 30 min, 4℃) and collect the supernatant. Filter the supernatant through a 0.22 μm sterile filter and purify the protein according to the nickel affinity chromatography instructions to obtain the fusion protein SA-PE. SDS-PAGE electrophoresis results showed that the purified SA-PE had a molecular weight of approximately 33.2 kDa and was a single protein band. Figure 7 This is consistent with the predicted molecular weight.

[0070] Example 6: Detection of biotin-binding activity of fusion protein SA-PE

[0071] Following the instructions of the Native PAGE kit, a 5% stacking gel and a 12.5% ​​separating gel were prepared. The SA-PE purified in Example 5 was analyzed by non-denaturing electrophoresis at a loading volume of 20 μL, a voltage of 150 V, and a time of 1 h. After electrophoresis, the sample was transferred to a PVDF membrane and incubated at a constant current (400 mA) for 2 h. After transfer, the membrane was blocked with 5% BSA at room temperature (2 h), and then washed three times with TBST for 10 min each time.

[0072] The secondary antibody (biotin-horseradish peroxidase) was diluted 1:5000 with secondary antibody dilution buffer. The PVDF membrane was then immersed in the secondary antibody solution and incubated at room temperature for 2 h. After incubation, the secondary antibody was recovered, and the PVDF membrane was washed three times with TBST for 10 min each time.

[0073] SA in the fusion protein SA-PE can bind to biotin, which in turn cross-links with horseradish peroxidase. The luminescent solution was uniformly applied to the PVDF membrane according to the kit instructions, and the membrane was exposed and images were acquired using an ECL luminescence analyzer. Figure 8 The results show that the SA-PE provided by this invention, after expression and purification, exists in the form of a tetramer (molecular weight of approximately 133 kDa) and can bind correctly to biotin, thus it can be applied to immunofluorescence detection.

[0074] Example 7: Wavelength Selection of Fusion Protein SA-PE in Solid-Phase Immunoassay

[0075] Dilute biotin-BSA 1:5000 with coating buffer and add 100 μL to each well of a fluorescent microplate. Seal the plate and incubate overnight at 4°C. Remove the plate, shake off the liquid from the wells and blot dry. Add 200 μL of TBST to each well, shake off the liquid and blot dry. Repeat 3 times. Add 100 μL of blocking buffer to each well, seal the plate, and incubate at 37°C for 2 hours. Remove the plate, shake off the liquid from the wells and blot dry. Finally, add 200 μL of TBST to each well, shake off the liquid and blot dry. Repeat 3 times.

[0076] The purified fusion protein SA-PE from Example 5 was diluted to 10 μg / mL with TBST. 100 μL was added to an ELISA plate and incubated at 37°C for 1 h. The liquid in the wells was then removed and the plate was patted dry. The plate was then washed three times with TBST. Finally, 100 μL of TBST was added to each well, and fluorescence intensity was detected using different excitation and emission wavelengths. The results are shown in Table 3. The strongest fluorescence signal was observed when 540 nm was used as the excitation wavelength and 580 nm as the emission wavelength.

[0077] Table 3. Fluorescence intensity of fusion protein SA-PE at different excitation and emission wavelengths.

[0078] ,

[0079] Example 8: Application of fusion protein SA-PE in solid-phase immunoassay

[0080] Dilute IL-1 capture antibody 1:500 with coating buffer and add 100 μL to each well of an ELISA plate. Seal the plate and incubate overnight at 4°C. Remove the plate, shake off the liquid from the wells and blot dry. Add 200 μL of TBST to each well, shake off the liquid and blot dry. Repeat this process three times. Add 100 μL of blocking buffer to each well, seal the plate, and incubate at 37°C for 2 hours. Remove the plate, shake off the liquid from the wells and blot dry. Finally, add 200 μL of TBST to each well, shake off the liquid and blot dry. Repeat this process three times. Serially dilute IL-1 standards with TBST to concentrations of 0, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 pg / mL. Add 100 μL to each well, setting up 3 replicates. After sealing the ELISA plate, incubate at 37°C for 2 h. Discard the liquid in the wells and pat dry. Add 200 μL of TBST to each well and wash 3 times. Dilute the biotinylated detection antibody 1:500 with TBST and add 100 μL to each well of the ELISA plate. After sealing the plate, incubate at 37°C for 1 h and wash 3 times with TBST.

[0081] The purified fusion protein SA-PE from Example 5 was diluted to 5 μg / mL with TBST and added to each well of an ELISA plate (100 μL). After incubation at 37°C for 1 h, the liquid in the wells was removed and the plate was patted dry. The plate was then washed three times with TBST. Finally, 100 μL of TBST was added to each well, and the fluorescence signal at 580 nm was measured using 540 nm as the excitation wavelength. The quantitative results are shown below. Figure 9 The detection limit of SA-PE provided by this invention for IL-1 is 0.128 pg / mL.

[0082] The above embodiments are merely illustrative of the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A phycoerythrin PE-1 having fluorescent properties, characterized in that, The amino acid sequence of the phycoerythrin is shown as SEQ ID NO.

1.

2. The phycoerythrin PE-1 according to claim 1, characterized in that, The Stokes shift of the phycoerythrin PE-1 is 20 nm.

3. The gene encoding phycoerythrin PE-1 according to claim 1, characterized in that, The nucleotide sequence of the coding gene is shown as SEQ ID NO.

6.

4. A recombinant expression vector or a recombinant host cell, characterized in that, The recombinant expression vector or the recombinant host cell contains the coding gene of the phycoerythrin PE-1 according to claim 3.

5. A fusion protein, characterized in that, The fusion protein is SA-PE, which is connected by a linker peptide with a length of 5-30 amino acid residues between the phycoerythrin PE-1 according to claim 1 and streptavidin.

6. The fusion protein of claim 5, wherein, The fusion protein SA-PE exists in a tetramer form after purification.

7. Use of the phycoerythrin PE-1 according to claim 1 or the fusion protein according to claim 5 in the preparation of a reagent for solid-phase immunoassay.

8. Use according to claim 7, characterized in that, The fusion protein SA-PE can bind to biotin.

9. Use according to claim 7, characterized in that, The phycoerythrin PE-1 has good thermal stability in a temperature range of 50-70°C.

10. Use according to claim 7, characterized in that, In solid-phase immunoassay, the excitation wavelength of the phycoerythrin PE-1 or the fusion protein SA-PE is 530-555 nm, and the emission wavelength is 550-595 nm.

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