A fluorescent biosensor capable of detecting phenylalanine concentration in blood and its application

Abstract

The present invention relates to a phenylalanine fluorescent biosensor and its use in detecting phenylalanine in blood. The phenylalanine fluorescent biosensor of the present invention can accurately and rapidly detect the phenylalanine concentration in a sample. Compared with existing phenylalanine detection methods, the phenylalanine fluorescent biosensor of the present invention has the advantages of simple operation, short detection cycle, high sensitivity, strong specificity, low cost, no need for expensive and complex equipment, and amenability to high-throughput, real-time, rapid detection.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and medical detection, and particularly relates to a phenylalanine fluorescence biosensor and an application thereof in detecting the concentration of phenylalanine in blood. Background Art

[0002] Phenylalanine (Phe), an aromatic amino acid, is a crucial precursor for the synthesis of various proteins, hormones, and neurotransmitters. Factors such as genetics, liver and kidney function, and immunity can lead to Phe metabolic disorders, leading to various diseases such as phenylketonuria and hyperphenylalaninemia. Therefore, measuring Phe concentration in the blood is crucial for studying the pathophysiology and clinical diagnosis of related diseases.

[0003] Phenylketonuria (PKU) is the most common inborn error in amino acid metabolism. PKU is an autosomal recessive disorder caused by an impaired conversion of L-phenylalanine (Phe) to L-tyrosine, typically due to insufficient L-phenylalanine hydroxylase activity in the liver and kidneys. The disease is characterized by elevated concentrations of phenylalanine and its metabolites in body fluids. Typical PKU patients have very high blood L-phenylalanine concentrations, often exceeding 20 mg / dL (1.2 mmol / L). If left undiagnosed and untreated, PKU can lead to mental retardation, microcephaly, speech delay, seizures, eczema, behavioral abnormalities, and other adverse clinical symptoms. The current incidence of PKU in my country is approximately 1 in 14,000. Early diagnosis and restriction of phenylalanine-containing foods offer a good prognosis. Otherwise, half of patients live before the age of 20, and 70% die before the age of 30. Therefore, regular monitoring of L-phenylalanine levels is essential.

[0004] Currently, methods for detecting L-phenylalanine concentration include bacterial inhibition method, fluorescence spectrometry, phenylalanine dehydrogenase method, high performance liquid chromatography (HPLC), and tandem mass spectrometry (MS / MS).

[0005] The Guthrie bacterial inhibition assay was developed by Professor Guthrie in the United States in 1961. Its principle is that the Bacillus subtilis variant strain ATCC6633 requires phenylalanine for spore germination. Adding the phenylalanine antagonist β-2-thienylalanine to the culture medium inhibits spore germination, while phenylalanine relieves this inhibitory effect. If the phenylalanine content in the test sample increases, the Bacillus subtilis growth ring increases; conversely, the growth ring decreases. By comparing the growth ring size with that of a standard sample with known phenylalanine content, the phenylalanine content in the sample can be determined, allowing for diagnosis of the sample. The Guthrie bacterial inhibition assay is a semiquantitative method for measuring Phe. It is simple, easy to use, and low-cost. However, its accuracy is affected by low blood Phe concentrations, and taking antibiotics can easily lead to false negative results.

[0006] The principle of phenylalanine determination by fluorescence spectrophotometry is that phenylalanine reacts with ninhydrin in an acidic solution of a dipeptide (such as leucine and alanine) to produce a fluorescent product. The resulting fluorescence, after being enhanced and stabilized by divalent copper ions, can be measured using a fluorescence spectrophotometer. While fluorescence spectrophotometry can accurately quantify Phe, it involves numerous reagents, is complex to prepare, requires a long operation time, is susceptible to interference from other fluorescent substances, and suffers from poor stability.

[0007] The phenylalanine dehydrogenase method works by converting phenylalanine to phenylpyruvate by phenylalanine dehydrogenase. This reaction is accompanied by a reduction in the coenzyme NAD+ in the reaction mixture, generating NADH. This redox reaction converts the added yellow tetrazolium salt into formazane, a yellow substance. The amount of formazane is proportional to the amount of phenylalanine in the sample. The optical density is measured using a microplate reader and converted to the corresponding phenylalanine concentration. While the phenylalanine dehydrogenase method can accurately quantify Phe, the assay is quite cumbersome and the enzyme reagent is expensive.

[0008] High-performance liquid chromatography (HPLC) requires UV derivatization pretreatment of specimens and often requires complex dual-pump gradient elution, which is time-consuming and unsuitable for the clinical detection of serum Phe. Tandem mass spectrometry, on the other hand, is difficult to popularize due to the high cost of MS equipment, limiting their clinical application.

[0009] Of the above methods, the Guthrie bacterial inhibition method is simple, easy, and low-cost, but it cannot accurately quantify phenylalanine. While the remaining four methods can accurately quantify phenylalanine, the fluorescence spectrophotometry and phenylalanine dehydrogenation methods are cumbersome and time-consuming, while the high-performance liquid chromatography and tandem mass spectrometry methods require expensive instrumentation, making them difficult to commercialize. Summary of the Invention

[0010] The present invention relates to a phenylalanine fluorescent biosensor. The technical principle is to utilize the different protein conformations of GrsA A and PCP domain at different stages of catalysis, insert a fluorescent protein sensitive to conformational changes into a specific position of the GrsA A and PCP domain, and generate a specific phenylalanine fluorescent biosensor. The fluorescence intensity of the fluorescent protein in the sensor changes with the concentration of phenylalanine. Therefore, the phenylalanine fluorescent biosensor involved in the present invention utilizes and modifies the adenosine domain and acyl intermediate carrier domain (GrsA A and PCP domain) that bind to phenylalanine, so that they bind to phenylalanine and convert the concentration of phenylalanine into a fluorescent signal that is easy to measure. This can achieve simple, accurate, highly sensitive, low-cost, real-time and rapid detection of phenylalanine in a sample.

[0011] The present invention first provides a phenylalanine fluorescent biosensor fusion gene, characterized in that a fluorescent protein DNA sequence with a random connecting peptide is inserted into the connecting peptide connecting the C-terminal small subunit and the acyl intermediate carrier domain, or is inserted into the C-terminal small subunit of the sensor protein to form the sensor protein gene, and the phenylalanine-binding sensor protein gene is a DNA sequence of the adenosine domain and the acyl intermediate carrier domain of a non-ribosomal peptide synthetase; preferably, the amino acid sequence encoded by the DNA sequence of the adenosine domain and the acyl intermediate carrier domain of the non-ribosomal peptide synthetase is as shown in SEQ ID NO.1, and preferably, its nucleotide sequence is as shown in SEQ ID NO.2.

[0012] In a specific embodiment, the length of the connecting peptide is within 20 amino acids, preferably within 15 amino acids, more preferably within 10 amino acids, further preferably within 5 amino acids, for example, 1 to 4 amino acids, and most preferably 2 amino acids; the fluorescent protein is green fluorescent protein, yellow fluorescent protein, or red fluorescent protein. More specifically, the amino acid sequence of the fluorescent protein encoded by its fluorescent protein DNA is as shown in SEQ ID NO.3, and preferably its nucleotide sequence is as shown in SEQ ID NO.4.

[0013] In a preferred embodiment, the fluorescent protein DNA sequence with random connecting peptides is inserted between the coding sequences of proline 524 and glutamate 525 of the amino acid sequence shown in SEQ ID NO. 1 and after the coding sequence of proline 524 of GrsA Phe A domain.

[0014] The present invention also provides an expression vector containing the fluorescent sensor fusion gene according to any one of claims 1 to 3, preferably, it is included in an expression plasmid suitable for inducible expression in chassis cells, more preferably, the starting vector of the expression plasmid is a pET expression system such as pET28a, pET24a, pET17b, or a pBAD expression system such as pBAD / His, pBAD / gIII.

[0015] The present invention further provides a phenylalanine fluorescent biosensor, which is a fusion protein encoded by the above-mentioned fusion gene.

[0016] The present invention particularly provides the use of the phenylalanine fluorescent biosensor in preparing a kit for detecting phenylalanine in blood.

[0017] Accordingly, the present invention provides a kit for detecting phenylalanine in blood, which comprises the aforementioned fusion protein, preferably dissolved in a buffer solution, preferably a buffer solution of Tris salt, more specifically 20mM Tris, 500mM Nacl, 10% glycerol, pH 7.9; further preferably, it also comprises ATP and MgCl2; more preferably, it uses 100mM ATP as the mother solution and 250mM MgCl2 as the mother solution, and most preferably comprises 100mM Tris at pH 7.5, 1mM ATP, 2.5mM MgCl2, and a 3μM phenylalanine fluorescent biosensor. More specifically, the kit of the present invention includes phenylalanine fluorescent biosensor protein (preserved in 20 mM Tris, 500 mM Nacl, 10% glycerol, pH 7.9, stored at -20°C), ATP powder (stored at 4°C), MgCl2 (stored at room temperature), L-phenylalanine standard (stored at room temperature), and reaction buffer (specifically 100 mM Tris, pH 7.5).

[0018] Preferably, it further comprises a phenylalanine standard solution; further, the concentration of phenylalanine in the blood is detected by detecting the serum of the subject to be tested.

[0019] In addition, the present invention also provides a method for detecting phenylalanine in blood for non-diagnostic purposes, characterized in that the fusion protein is allowed to react with the serum to be tested, and then fluorescence detection is performed to calculate the concentration of phenylalanine in the blood; preferably, the reaction uses a buffer solution of 100mM Tris pH7.5, the reaction system is 200μL, wherein the final concentrations of each substance are ATP 1mM, MgCl2 2.5mM, and phenylalanine sensor 3μM, and 4μL of the serum sample to be tested is added.

[0020] The advantages and beneficial effects of the present invention include: Using the present invention's phenylalanine fluorescent biosensor method for detecting phenylalanine in blood, the phenylalanine concentration in a sample can be accurately and rapidly measured. Compared with existing phenylalanine detection methods, the phenylalanine fluorescent biosensor of the present invention offers advantages such as simple operation, short detection cycle, high sensitivity, strong specificity, low cost, no need for expensive and complex instrumentation, and ease of high-throughput, real-time, rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the standard curve of phenylalanine determined by HPLC;

[0022] Figure 2 is the HPLC spectrum of serum sample;

[0023] Figure 3 This is the relationship between the phenylalanine concentration and the fluorescence signal intensity of the phenylalanine fluorescent biosensor GrsA Phe A and PCP domain-cpEGFP;

[0024] Figure 4 This is the relationship between the phenylalanine concentration and the fluorescence signal intensity of the phenylalanine fluorescent biosensor GrsA Phe A domain-cpEGFP;

[0025] Figure 5 This is the standard curve of phenylalanine concentration and sensor fluorescence signal intensity of phenylalanine fluorescence biosensor; DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention are further described below with reference to specific embodiments.

[0027] Example 1 HPLC detection of phenylalanine concentration in human blood

[0028] Step 1: Prepare 2.5% and 5% perchloric acid solutions (v / v, based on commercially available concentrated perchloric acid as 100%). Chromatographic conditions were: Waters C18 column (Atlantis T3 5μm 4.6*250mm), mobile phase: acetonitrile-water (volume ratio 10:90), flow rate 0.5 ml / min, detection wavelength 190 nm, column temperature 25°C, injection volume 20 μL.

[0029] Step 2: Prepare 12.2μM, 30.5μM, 61.0μM, and 122.0μM phenylalanine standard solutions with 2.5% perchloric acid solution, and perform high performance liquid chromatography detection. Inject each concentration three times, take the mean, and perform regression analysis using the least squares method. Draw a standard curve with peak area Y as the ordinate and concentration X (μM) as the abscissa. Figure 1 ). After calculation, Y=76.87*X+87.30, and R square is 0.999.

[0030] Step 3: Draw 1 ml of fasting venous blood from the subjects into a clean centrifuge tube and centrifuge at 12000 rpm / min to separate the serum.

[0031] Step 4: Take 100 μL of serum and add an equal amount of 5% perchloric acid solution into a centrifuge tube. Vortex mix and place at room temperature for 10 minutes to allow serum protein to fully precipitate. Then centrifuge at 12000 rpm / min for 15 minutes. Take the supernatant for high performance liquid chromatography detection ( Figure 2 ), the concentration of phenylalanine in the serum sample to be tested was determined to be 69.94 μM, with an error value of 4.35 (Table 1).

[0032] Table 1 Results of HPLC detection of phenylalanine concentration in serum samples in Example 1

[0033]

[0034] Example 2 Design and Construction of Phenylalanine Fluorescent Biosensor

[0035] Step 1: Research and find the adenosine domain and PCP domain of the non-ribosomal peptide synthetase that binds phenylalanine, GrsA Phe A and PCP domain, whose amino acid sequence is SEQ ID NO.1.

[0036] Step 2: The GrsA Phe A and PCP domain nucleotide sequence (SEQ ID NO. 2) and the GrsA Phe A domain sequence from Step 1 were obtained by PCR using primer sequences of SEQ ID NOs 5-7. The nucleotide sequences were then cloned into the E. coli protein expression vector PET28a by homologous recombination to construct the plasmids PET28a-GrsA PheA and PCP domain and PET28a-GrsA Phe A domain.

[0037] Step 3: Determine the fluorescent protein insertion sites in the plasmids PET28a-GrsA Phe A and PCP domain and PET28a-GrsAPhe A domain constructed in step 2: between proline 524 and glutamate 525 in the GrsA Phe A and PCP domain, and after proline 524 in the GrsA Phe A domain, respectively. Primers include a 20-30 bp GrsA Phe A and PCP domain nucleotide fragment or a PET28a backbone fragment for PCR amplification, a 6-9 bp fluorescent protein linker peptide sequence, and a 17-20 bp homology arm of the fluorescent protein sequence (SEQ ID NOs 8-10). Using the expression vectors PET28a-GrsA Phe A and PCP domain and PET28a-GrsA Phe A domain constructed in step 2 as templates, linearize the sensor DNA fragment by PCR.

[0038] Step 4: The fluorescent protein cpEGFP DNA fragment (amino acid sequence is SEQ ID NO. 3, nucleotide sequence is SEQ ID NO. 4) was cloned into the linearized sensor DNA fragment obtained in step 3 by homologous recombination to obtain complete fluorescent biosensor plasmids PET28a-GrsA Phe A and PCP domain-cpEGFP and PET28a-GrsAPhe A domain-cpEGFP.

[0039] Step 5: Take 100 μL of two tubes of E. coli BAPⅠ competent cells from a -80 refrigerator and thaw them on ice for later use.

[0040] Step 6: Add the sensor plasmids PET28a-GrsA Phe A and PCP domain-cpEGFP and PET28a-GrsA Phe A domain-cpEGFP constructed in Step 4 to the E. coli BAPⅠ competent cells prepared in Step 5. Incubate on ice for 5 minutes, then heat shock in a 42°C water bath for 45 seconds. After heat shock, add 900 μL of antibiotic-free LB medium and recover at 37°C at 220 rpm for 1 hour. After recovery, plate the plates and incubate them upside down in a 37°C incubator overnight.

[0041] Step 7: The single clones on the plate in step 6 were inoculated into liquid LB medium for culture and sequencing to obtain the phenylalanine fluorescent biosensor strains GrsA Phe A and PCP domain-cpEGFP and GrsA Phe A domain-cpEGFP.

[0042] Example 3 Purification and Characterization of Phenylalanine Fluorescent Biosensor

[0043] Step 1: The phenylalanine fluorescent biosensor strains GrsA Phe A and PCP domain-cpEGFP and GrsA Phe A domain-cpEGFP were re-inoculated into the culture medium and cultured overnight in a shaking incubator at 37°C and 220 rpm.

[0044] Step 2: Transfer the strain in step 1 into a shake flask containing 50 ml of kanamycin-resistant LB medium at a 2% inoculum volume, and culture in a shaker at 37°C and 220 rpm for 1-2 hours until the OD600 of the bacterial solution reaches 0.6-0.8.

[0045] Step 3: Add IPTG to the bacterial solution in step 2 to make the final concentration of IPTG 0.2-0.4 mM.

[0046] Step 4: The strain obtained in step 3 was cultured in a shaker at 16°C and 220 rpm for 48 hours.

[0047] Step 5: Place the strain obtained in step 4 in a 4°C centrifuge and centrifuge at 7500 rpm for 5 minutes to precipitate the bacteria, and discard the supernatant.

[0048] Step 6: Resuspend the strain from step 5 in 20mM Tris 500mM NaCl pH 7.9 buffer. Centrifuge at 7500 rpm for 5 minutes at 4°C to pellet the cells. Discard the supernatant. Repeat this step 2-3 times.

[0049] Step 7: Resuspend the strain prepared in step 6 in 20 mM Tris 500 mM NaCl pH 7.9 buffer and lyse the cells by ultrasonication.

[0050] Step 8: Centrifuge the strain obtained in step 7 at 10,000 rpm in a 4°C centrifuge for 45 minutes to precipitate the insoluble cell debris.

[0051] Step 9: Purify the protein using the His tag in the sensor, wash with 20mM Tris 500mM Nacl 20mM imidazole pH 7.9 buffer, and elute the target protein with 20mM Tris 500mM Nacl 500mM imidazole pH 7.9 buffer.

[0052] Step 10: Use ultrafiltration tube centrifugation to reduce the imidazole concentration in the target protein to below 0.1 mM.

[0053] Step 11: Prepare the reaction system with a buffer solution of 100 mM Tris pH 7.5. The final concentrations of the substances in the reaction system are 1 mM ATP, 2.5 mM MgCl2, 0.1 μM-1 mM phenylalanine solution, and 3 μM phenylalanine fluorescent biosensor.

[0054] Step 12: Transfer the reaction system in step 11 to a 96-well fluorescence microplate. Measure the relationship between the fluorescence intensity of the phenylalanine fluorescent biosensor and the phenylalanine concentration using a fluorescence microplate reader with excitation light at 460 nm and emission light at 510 nm.

[0055] Step 13: Draw a curve with the concentration of phenylalanine as the horizontal axis and the fluorescence intensity of the phenylalanine fluorescent biosensor as the vertical axis.

[0056] The relationship between phenylalanine and the fluorescence intensity of the fluorescent biosensor GrsA Phe A and PCP domain-cpEGFP obtained by experiment ( Figure 3 ), the relationship between phenylalanine and the fluorescence intensity of the fluorescent biosensor GrsA Phe A domain-cpEGFP ( Figure 4 ).like Figure 3 As shown in the figure, when the concentration of phenylalanine is 0.1-50 μM, the fluorescence intensity of the sensor GrsA Phe A and PCP domain-cpEGFP increases with the increase of phenylalanine concentration, and the increase in fluorescence intensity is 290%. Figure 4 As shown, when the phenylalanine concentration of the phenylalanine fluorescent biosensor GrsA Phe Adomain-cpEGFP is 0.1-50 μM, the fluorescence intensity of the sensor increases with the increase of L-phenylalanine concentration, and the increase in fluorescence intensity is 44.6%.

[0057] Example 4: Detection of Phenylalanine Concentration in Human Blood by Phenylalanine Fluorescence Biosensor

[0058] Step 1: Prepare 0.1mM, 0.5mM, 1mM, 2.5mM, 5mM, and 10mM phenylalanine standard solutions in ultrapure water.

[0059] Step 2: Prepare a solution of 100 mM Tris pH 7.5, 100 mM ATP stock solution, and 250 mM MgCl2 stock solution.

[0060] Step 3: The buffer solution is 100mM Tris pH7.5, and the reaction system is 200μL, wherein the final concentrations of each substance are ATP 1mM, MgCl2 2.5mM, phenylalanine fluorescent biosensor GrsA Phe A and PCP domain-cpEGFP 3μM, and phenylalanine standard solution is added to make the final concentration 0.1μM-10μM. The mixture is allowed to stand at room temperature for 15min, and fluorescence detection is performed with excitation light 460nm and emission light 510nm. The concentration of small molecule X (μM) is used as the horizontal axis, and the fluorescence intensity of the fluorescent small molecule biosensor Y is used as the vertical axis to draw a standard curve ( Figure 5 The standard curve is drawn as Figure 5 As shown, Y=16258.11+7710.40*x, and the R square is 0.996.

[0061] Step 4: The buffer solution was 100 mM Tris pH 7.5, and the reaction system was 200 μL, with the final concentrations of each substance being 1 mM ATP, 2.5 mM MgCl2, and 3 μM of the phenylalanine sensor GrsA Phe A and PCP domain-cpEGFP. 4 μL of the serum sample to be tested was added, mixed, and allowed to stand at room temperature for 15 minutes. Fluorescence detection was performed with excitation light at 460 nm and emission light at 510 nm. The calculated phenylalanine concentration in the serum sample to be tested was 65.62 μM, with an error value of 2.22 (see Table 2).

[0062] Table 2 Results of phenylalanine fluorescence biosensor detection of phenylalanine concentration in serum samples in Example 4

[0063]

[0064] In this example, there was no statistically significant difference in phenylalanine concentration in human blood measured using the phenylalanine fluorescent biosensor of the present invention compared to the phenylalanine concentration in the same human blood sample measured using a standard HPLC method. This demonstrates that the phenylalanine fluorescent biosensor of the present invention is accurate and reliable in measuring phenylalanine concentration in human blood, and is faster, simpler to operate, and less expensive than existing methods. Sequence Listing Sequence Listing <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> A fluorescent biosensor capable of detecting phenylalanine concentration in blood and its application <160> 10 <170> PatentIn Version 3.1 <210> 1 <211> 617 <212> PRT <213> Synthetic sequence <400> 1 MLNSSKSILI HAQNKNGTHE EEQYLFAVNN TKAEYPRDKT IHQLFEEQVS KRPNNVAIVC 60 ENEQLTYHEL NVKANQLARI FIEKGIGKDT LVGIMMEKSI DLFIGILAVL KAGGAYVPID 120 IEYPKERIQY ILDDSQARML LTQKHLVHLI HNIQFNGQVE IFEEDTIKIR EGTNLHVPSK 180 STDLAYVIYT SGTTGNPKGT MLEHKGISNL KVFFENSLNV TEKDRIGQFA SISFDASVWE 240 MFMALLTGAS LYIILKDTIN DFVKFEQYIN QKEITVITLP PTYVVHLDPE RILSIQTLIT 300 AGSATSPSLV NKWKEKVTYI NAYGPTETTI CATTWVATKE TIGHSVPIGA PIQNTQIYIV 360 DENLQLKSVG EAGELCIGGE GLARGYWKRP ELTSQKFVDN PFVPGEKLYK TGDQARWLSD 420 GNIEYLGRID NQVKIRGHRV ELEEVESILL KHMYISETAV SVHKDHQEQP YLCAYFVSEK 480 HIPLEQLRQF SSEELPTYMI PSYFIQLDKM PLTSNGKIDR KQLPEPDLTF GMRVDYEAPR 540 NEIEETLVTI WQDVLGIEKI GIKDNFYALG GDSIKAIQVA ARLHSYQLKL ETKDLLKYPT 600 IDQLVHYIKD SKRRSEQ 617 <210> 2 <211> 1851 <212> DNA <213> Synthetic sequence <400> 2 atgttaaaca gttctaaaag tatattgatt catgctcaaa ataaaaatgg aacgcatgaa 60 gaggagcagt atctctttgc tgtgaacaac accaaagcgg agtatccacg tgataagacg 120 atccatcagt tatttgaaga gcaggttagt aagagaccaa acaatgtagc cattgtatgt 180 gaaaatgagc aacttaccta ccatgagctt aatgtgaaag ccaatcaact agcacggatt 240 tttatagaaa aagggattgg aaaagacact cttgttggaa ttatgatgga gaaatctatc 300 gatttatta taggcatatt agccgtttta aaagcaggtg gagcatatgt tccgattgat 360 attgaatatc ctaaggaaag aattcaatat attcttgatg atagtcaggc aagaatgcta 420 cttacccaga agcatttggt tcatttaatt cataatattc aatttaatgg gcaagtggaa 480 atttttgaag aagatactat caaaattaga gaaggaacta atctacatgt accaagtaaa 540 tcaaccgatc ttgcttatgt tatttatact tctggtacaa caggcaatcc aaaaggtaca 600 atgctggagc ataaaggaat aagtaatcta aaggtatttt tcgaaaatag tcttaacgtg 660 actgaaaagg atagaattgg tcaatttgcc agcatctctt ttgatgcatc tgtatgggag 720 atgtttatgg ctttgttaac gggggctagc ctgtatatta tcctgaagga tacaatcaat 780 gattttgtga agtttgaaca atacattaac caaaaggaaa tcactgttat tacgttacca 840 cctacctatg tagttcatct tgatccagaa cgtattttat cgatacaaac gttaattaca 900 gcaggctcag ctacctcgcc ttccttagta aacaagtgga aggagaaagt aacttacata 960 aatgcctatg gccctacgga aacaactatt tgtgcgacta catgggtagc caccaaagaa 1020 acaataggtc attcagttcc aatcggagca ccaattcaaa atacacaaat ttatattgtc 1080 gatgaaaatc ttcaattaaa atcggttggt gaagctggtg aattgtgtat tggtggagaa 1140 gggttagcaa ggggatattg gaagcgaccg gaattaactt cccagaagtt cgttgataac 1200 ccgtttgttc caggagagaa gttgtataaa acaggagatc aggcaagatg gctatctgat 1260 ggaaatattg aatatctcgg aagaatagat aaccaggtaa agattagagg tcaccgagtt 1320 gaactagaag aagttgagtc tattcttcta aagcatatgt atattagcga aactgcagta 1380 agtgtgcata aagatcacca agaacagccg tatttgtgcg cttattttgt atcggaaaag 1440 catataccac tagaacagtt aagacaattc tcatcagaag aactgccaac gtatatgatc 1500 ccttcttatt ttatccagtt agacaaaatg ccgcttacat caaatgggaa gattgatcga 1560 aagcagttgc cggaacctga tttaactttc gggatgaggg tagactatga agcgccgcga 1620 aatgaaatcg aggaaacgct tgttactatc tggcaggatg tattaggtat tgagaaaatc 1680 ggtattaaag ataatttcta tgcattaggt ggagattcta ttaaagcaat acaggttgct 1740 gctcgcctgc attcctacca attaaagcta gaaacaaaag atttattaaa gtatccaaca 1800 atcgatcaac tcgttcatta tataaaagat agtaaaagaa gaagtgagca a 1851 <210> 3 <211> 241 <212> PRT <213> Synthetic sequence <400> 3 NVYIMADKQK NGIKANFKIR HNIEDGGVQL AYHYQQNTPI GDGPVLLPDN HYLSTQSKLS 60 KDPNEKRDHM VLLEFVTAAG ITLGMDELYK GGTGGSMVSK GEELFTGVVP ILVELDGDVN 120 GHKFSVSGEG EGDATYGKLT LKFICTTGKL PVPWPTLVTT LTYGVQCFSR YPDHMKQHDF 180 FKSAMPEGYI QERTIFFKDD GNYKTRAEVK FEGDTLVNRI ELKGIDFKED GNILGHKLEY 240 N 241 <210> 4 <211> 735 <212> DNA <213> Synthetic sequence <400> 4 gtgtttaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggc aaacttcaag 60 atccgccaca acatcgagga cggcggcgtc cagctcgcct accactacca gcagaacacc 120 cccatcggcg acggccccgt cctgctgccc gacaaccact acctgagcac ccagtccaaa 180 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtaaccgcc 240 gccgggatca ctctcggcat ggacgagctg tacaagggcg gaaccggcgg aagcatggtc 300 agcaagggcg aggagctgtt caccggggtc gtacccatcc tggtcgagct ggacggcgac 360 gtaaacggcc acaagttcag cgtctccggc gagggcgagg gcgatgccac ctacggcaag 420 ctgaccctga agttcatctg caccaccggc aagctgcccg taccctggcc caccctcgtc 480 accaccctga cctacggcgt ccagtgcttc agccgctacc ccgaccacat gaagcagcac 540 gacttcttca agtccgccat gcccgaaggc tacatccagg agcgcaccat cttcttcaag 600 gacgacggca actacaagac ccgcgccgag gtcaagttcg agggcgacac cctggtcaac 660 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg 720 gagtacaacc agtcg 735 <210> 5 <211> 40 <212> DNA <213> Artificially synthesized sequences <400> 5 atataccatg ggcagcagca tgttaaacag ttctaaaagt 40 <210> 6 <211> 47 <212> DNA <213> Artificially synthesized sequences <400> 6 tcagtggtgg tggtggtggt gctcgagttg ctcacttctt cttttac 47 <210> 7 <211> 40 <212> DNA <213> Artificially synthesized sequences <400> 7 tggtggtggt ggtgctcgag caactgcttt cgatcaatct 40 <210> 8 <211> 47 <212> DNA <213> Artificially synthesized sequences <400> 8 gtcggccatg atatagacgt taaacaccgg caactgcttt cgatcaa 47 <210> 9 <211> 46 <212> DNA <213> Artificially synthesized sequences <400> 9 ggcacaagct ggagtacaac bcagtcgaac ctgatttaac tttcgg 46 <210> 10 <211> 53 <212> DNA <213> Synthetic sequence <400> 10 ggcacaagct ggagtacaac ctgctcgagc accaccacca ccaccactga gat 53

Claims

1. Use of a phenylalanine fluorescent biosensor in preparing a kit for detecting phenylalanine in blood, characterized in that: The coding gene of the phenylalanine fluorescent biosensor is obtained by inserting the DNA sequence shown in SEQ ID NO.4 between the coding sequences of proline at position 524 and glutamate at position 525 of the amino acid sequence shown in SEQ ID NO.1, or after the coding sequence of proline at position 524 of the GrsAPhe A domain; the amino acid sequence of the GrsAPhe A domain is positions 1 to 524 of the amino acid sequence shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.

2.

3. A kit for detecting phenylalanine in blood, characterized in that: The invention comprises the phenylalanine fluorescent biosensor for use as claimed in claim 1 or 2 dissolved in a buffer solution, and further comprises ATP powder, MgCl2 and a reaction buffer.

4. The kit according to claim 3, wherein The buffer solution is a Tris salt buffer solution; the reaction buffer is 100 mM Tris, pH 7.

5.

5. The kit according to claim 3, wherein The buffer solution is 20 mM Tris, 500 mM NaCl, 10% glycerol, pH 7.

9.

6. The kit according to any one of claims 3 to 5, wherein Further included is a phenylalanine standard solution.

7. A kit for detecting phenylalanine in blood, characterized in that: The invention comprises the phenylalanine fluorescent biosensor for use as claimed in claim 1 or 2, 100 mM Tris at pH 7.5, 1 mM ATP, and 2.5 mM MgCl2 dissolved in a buffer solution, wherein the concentration of the phenylalanine fluorescent biosensor is 3 μM.

8. The kit according to claim 7, wherein Further included is a phenylalanine standard solution.

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