Fusion protein, fluorescent probe responding to glycollic acid and application of fluorescent probe

Abstract

The invention relates to the field of biological detection, in particular to fusion protein, a fluorescent probe and application thereof. The invention provides a fusion protein. The fusion protein comprises an acid-sensitive polypeptide, a circulating rearranged fluorescent protein and a connecting peptide, the amino acid sequence of the connecting peptide comprises one or more of LE, TR, SAG and GT. The fluorescent probe disclosed by the invention breaks through limitation of a traditional detection method, can realize rapid real-time quantitative glycollic acid detection, has good detection characteristics of high cost performance, simplicity and convenience in operation, rapid reaction, high specificity and the like, and is a user-friendly glycollic acid quantitative detection tool.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to fusion proteins, fluorescent probes and applications thereof. Background Art

[0002] Glycolic acid, also known as glycolic acid, is the smallest α-hydroxy acid. The molecular structure of glycolic acid has both hydroxyl and carboxyl groups, and has the dual properties of acid and alcohol. Glycolic acid is an important organic chemical that is widely used in a variety of chemical processes. Due to the wide range of applications, continuous economic development and increased global efforts to control plastic pollution, the market demand for glycolic acid is growing. It is estimated that by 2027, the global market size of glycolic acid will reach US$469.6 million. Improving the output and quality of glycolic acid and reducing production costs are the keys to improving market competitiveness and meeting market demand.

[0003] There are many chemical production processes for glycolic acid, among which the mainstream processes include chloroacetic acid hydrolysis, formaldehyde cyanidation, formaldehyde hydrogen carboxylation, and dimethyl oxalate (DMO) hydrogenation hydrolysis. The production of glycolic acid by chemical synthesis has always been plagued by problems such as low production efficiency, high production costs, and non-renewable properties. Using microorganisms to produce chemicals is currently the most promising alternative to chemical synthesis.

[0004] Since the research on the production of glycolic acid by microbial fermentation began, a variety of engineered strains have been used, including Saccharomyces cerevisiae, Escherichia coli, Kluyveromyces lactis, Corynebacterium glutamicum, etc. Escherichia coli has gradually become the main host of glycolic acid biosynthesis because of its clear genetic background, fast growth and reproduction, and low price.

[0005] Although satisfactory results have been achieved in the production of glycolic acid by E. coli, there are still problems such as the high price of inducers and the decline in performance of modified strains. Therefore, it is necessary to use metabolic modification engineering to screen excellent glycolic acid production strains. In order to screen an engineered strain with excellent glycolic acid production performance and stable genetic traits, a large number of combinatorial optimizations and even mutation libraries are often generated. Screening high-yield strains in E. coli mutation libraries in a short period of time is a technical difficulty in the current modification engineering. The rate-limiting step to solve this problem is the rapid quantification of glycolic acid produced by the strain.

[0006] The concentration detection of glycolic acid greatly limits the efficiency of screening. The commonly used detection technologies such as HPLC, LC-MS, and Western blot are not only cumbersome but also time-consuming. Therefore, it is urgent to develop a new glycolic acid detection method to achieve simple and rapid glycolic acid detection. Summary of the invention

[0007] In view of this, the present invention provides a fusion protein, a fluorescent probe and applications thereof. In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0008] The first technical solution of the present invention is to provide a fusion protein, including: an acid-sensitive polypeptide, a cyclically rearranged fluorescent protein, a connecting peptide 1 and a connecting peptide 2.

[0009] The amino acid sequence of the connecting peptide 1 includes: one of LE and SAG;

[0010] The amino acid sequence of the connecting peptide 2 includes one of TR and GT.

[0011] Furthermore, in the above fusion protein, the amino acid sequence combination of the connecting peptide 1 and the connecting peptide 2 includes: any one of LE / TR and SAG / GT.

[0012] Furthermore, in the above fusion protein, the amino acid sequences of the connecting peptide 1 and the connecting peptide 2 are LE and TR, respectively.

[0013] Furthermore, in the above fusion protein, the acid-sensitive polypeptide is: glycolic acid-sensitive polypeptide.

[0014] Further, in some embodiments of the present invention, in the above fusion protein, the amino acid sequence of the acid-sensitive polypeptide is as shown in SEQ ID NO: 17:

[0015] MKDERRPICEVVAESIERLIIDGVLKVGQPLPSERRLCEKLGFSRSALREGLTVLRGRGIIETAQGRDSRVARLNRVQDTSPLIHLFSTQPRTLYDLLDVRALLEGESARLAATLGTQADFVVITRCY EKMLAASENNKEISLIEHAQLDHAFHLAICQASHNQVLVFTLQSLTDLMFNSVFASVNNLYHRPQQKKQIDRQHARIYNAVLQRLPHVAQRAARDHVRTVKKNLHDIELEGHHLIRSAVPLEMNLS*.

[0016] Further, in some embodiments of the present invention, in the above-mentioned fusion protein, the amino acid sequence of the circularly rearranged fluorescent protein is as shown in SEQ ID NO:18: NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN.

[0017] Furthermore, in the above fusion protein, the cyclically rearranged fluorescent protein is located at any one of positions 89 / 91, 115 / 116, 142 / 143, 163 / 164, 188 / 189 and 211 / 212 of the acid-sensitive polypeptide.

[0018] Preferably, in the above fusion protein, the cyclically rearranged fluorescent protein is located at position 89 / 91 of the acid-sensitive polypeptide.

[0019] Further, in the present invention, the above fusion protein has: an amino acid sequence as shown in any of SEQ ID NO: 1 to SEQ ID NO: 7; further, in the above fusion protein, the sequence of SEQ ID NO: 1 is:

[0020] MKDERRPICEVVAESIERLIIDGVLKVGQPLPSERRLCEKLGFSRSALREGLTVLRGRGIIETAQGRDSRVARLNRVQDTSPLIHLFST

[0021] LENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDE

[0022] LYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQ

[0023] HDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNTRPRTLYDLLDVRALLEGESARLAA

[0024] TLGTQADFVVITRCYEKMLAASENNKEISLIEHAQLDHAFHLAICQASHNQVLVFTLQSLTDLMFNSVFASVNNLYHRPQQKKQIDRQHARIYNAVLQRLPHVAQRAARDHVRTVKKNLHDIELEGHHLIRSAVPLEMNLS*. (89 / 91, the amino acid sequence combination of the connecting peptide is LE / TR)

[0025] Furthermore, in the above fusion protein, the sequence of SEQ ID NO:2 is:

[0026] MKDERRPICEVVAESIERLIIDGVLKVGQPLPSERRLCEKLGFSRSALREGLTVLRGRGIIETAQGRDSRVARLNRVQDTSPLIHLFST

[0027] QPRTLYDLLDVRALLEGESARLAATLLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSK

[0028] DPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL

[0029] PVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLE

[0030] YNTRGTQADFVVITRCYEKMLAASENNKEISLIEHAQLDHAFHLAICQASHNQVLVFTLQSLTDLMFNSVFASVNNLYHRPQQKKQIDRQHARIYNAVLQRLPHVAQRAARDHVRTVKKNLHDIELEGHHLIRSAVPLEMNLS*. (115 / 116 position, the amino acid sequence combination of the connecting peptide is LE / TR)

[0031] Furthermore, in the above fusion protein, the sequence of SEQ ID NO:3 is:

[0032] MKDERRPICEVVAESIERLIIDGVLKVGQPLPSERRLCEKLGFSRSALREGLTVLRGRGIIETAQGRDSRVARLNRVQDTSPLIHLFST

[0033] QPRTLYDLLDVRALLEGESARLAATLGTQADFVVITRCYEKMLAASENNKEISLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHY

[0034] QQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHK

[0035] FSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFE

[0036] GDTLVNRIELKGIDFKEDGNILGHKLEYNTRLIEHAQLDHAFHLAICQASHNQVLVFTLQSLTDLMFNSVFASVNNLYHRPQQKKQIDRQHARIYNAVLQRLPHVAQRAARDHVRTVKKNLHDIELEGHHLIRSAVPLEMNLS*. (142 / 143, the amino acid sequence combination of the connecting peptide is LE / TR)

[0037] Further, in some embodiments of the present invention, in the above fusion protein, the sequence of SEQ ID NO: 4 is: MKDERRPICEVVAESIERLIIDGVLKVGQPLPSERRLCEKLGFSRSALREGLTVLRGRGIIETAQGRDSRVARLNRVQDTSPLIHLFSTQPRTLYDLLDVRALLEGESARLAATLGTQADFVVITRCYEKMLAASENNKEISLIEHAQLDHAFHLAICQASHNLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGI TLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAE VKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNTRQVLVFTLQSLTDLMFNSVFASVNNLYHRPQQKKQIDRQHARIYNAVLQRLPHVAQRAARDHVRTVKKNLHDIELEGHHLIRSAVPLEMNLS*. (Position 163 / 164, the connecting peptide amino acid sequence combination is LE / TR)

[0038] Further, in some embodiments of the present invention, in the above fusion protein, the sequence of SEQ ID NO: 5 is: MKDERRPICEVVAESIERLIIDGVLKVGQPLPSERRLCEKLGFSRSALREGLTVLRGRGIIETAQGRDSRVARLNRVQDTSPLIHLFSTQPRTLYDLLDVRALLEGESARLAATLGTQADFVVITRCYEKMLAASENNKEISLIEHAQLDHAFHLAICQASHNQVLVFTLQSLTDLMFNSVFASVNNLLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQ SKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKS AMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNTRYHRPQQKKQIDRQHARIYNAVLQRLPHVAQRAARDHVRTVKKNLHDIELEGHHLIRSAVPLEMNLS*. (Position 188 / 189, the connecting peptide amino acid sequence combination is LE / TR)

[0039] Further, in some embodiments of the present invention, in the above fusion protein, the sequence of SEQ ID NO: 6 is: MKDERRPICEVVAESIERLIIDGVLKVGQPLPSERRLCEKLGFSRSALREGLTVLRGRGIIETAQGRDSRVARLNRVQDTSPLIHLFSTQPRTLYDLLDVRALLEGESARLAATLGTQADFVVITRCYEKMLAASENNKEISLIEHAQLDHAFHLAICQASHNQVLVFTLQSLTDLMFNSVFASVNNLYHRPQQKKQIDRQLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDG PVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSR YPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNTRHARIYNAVLQRLPHVAQRAARDHVRTVKKNLHDIELEGHHLIRSAVPLEMNLS*. (Position 211 / 212, the connecting peptide amino acid sequence combination is LE / TR)

[0040] Further, in some embodiments of the present invention, in the above fusion protein, the sequence of SEQ ID NO:7 is: MKDERRPICEVVAESIERLIIDGVLKVGQPLPSERRLCEKLGFSRSALREGLTVLRGRGIIETAQGRDSRVARLNRVQDTSPLIHLFSTSAGNVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPW PTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNGTPRTLYDLLDVRALLEGESARLAATLGTQADFVVITRCY EKMLAASENNKEISLIEHAQLDHAFHLAICQASHNQVLVFTLQSLTDLMFNSVFASVNNLYHRPQQKKQIDRQHARIYNAVLQRLPHVAQRAARDHVRTVKKNLHDIELEGHHLIRSAVPLEMNLS*. (Position 89 / 91, the connecting peptide amino acid sequence combination is SAG / GT)

[0041] The second technical solution of the present invention is to further provide a nucleic acid molecule encoding the above fusion protein, which has: a nucleotide sequence as shown in any of SEQ ID NO: 8 to SEQ ID NO: 14;

[0042] Further, in some embodiments of the present invention, in the above nucleic acid molecule, the sequence of SEQ ID NO:8 is:

[0043] ATGAAAGATGAACGTCGTCCGATCTGCGAAGTTGTTGCTGAATCTATCGAACGTCTGATCATCGATGGCGTTCTGAAAGTTGGCCAGCC

[0044] ACTTCCAAGCGAACGTCGTCTGTGCGAGAAACTGGGTTTCTCTCGTTCTGCTCTGCGTGAAGGTCTGACCGTGTTGCGTGGTCGTGGTA

[0045] TCATCGAAACTGCTCAGGGTCGTGATAGCCGTGTAGCTCGTCTGAACCGTGTTCAAGACACCAGCCCGCTGATCCACCTGTTCTCCACT

[0046] CTCGAAAACGTCTATATCAAGGCCGACAAGCAGAAAAACGGCATTAAGGCTAACTTCAAGATCAGACACAACATCGAGGATGGTGGCGT

[0047] GCAGCTGGCCTACCATTATCAGCAGAACACACCAATCGGAGACGGACCAGTGCTGCTCCCAGATAATCACTACCTGAGCGTCCAGTCCA

[0048] AGCTGTCTAAAGACCCTAACGAGAAGCGGGATCATATGGTGCTGCTCGAATTTGTCACAGCCGCTGGGATCACTCTGGGTATGGACGAG

[0049] CTCTATAAAGGAGGGACCGGTGGCAGTATGGTGTCAAAGGGCGAGGAACTGTTCACAGGAGTGGTCCCCATTCTGGTGGAGCTCGACGG

[0050] CGATGTCAATGGACACAAATTTTCCGTGTCTGGCGAGGGCGAAGGAGATGCTACCTACGGGAAGCTGACACTCAAATTCATCTGCACCA

[0051] CAGGCAAGCTGCCAGTGCCCTGGCCTACTCTGGTCACTACCCTCACCTACGGGGTGCAGTGTTTCTCCAGATATCCCGACCACATGAAG

[0052] CAGCATGATTTCTTTAAATCTGCTATGCCTGAGGGGTACATCCAGGAACGGACAATTTTCTTTAAGGACGATGGTAACTACAAAACACG

[0053] CGCAGAGGTGAAGTTCGAAGGCGACACTCTGGTCAATCGAATCGAGCTGAAGGGAATTGACTTTAAAGAAGATGGGAACATCCTGGGTC

[0054] ACAAGCTGGAGTACAATACTAGGCCGCGTACCTTGTACGACCTGCTGGACGTTCGTGCTCTGCTGGAAGGCGAATCTGCTCGTCTGGCA

[0055] GCTACTCTGGGTACTCAGGCGGACTTCGTAGTTATCACTCGTTGCTACGAGAAGATGCTGGCAGCAAGCGAGAACAACAAAGAAATCTC

[0056] TCTGATCGAACACGCGCAGCTGGACCACGCATTCCACCTGGCAATCTGCCAGGCGAGCCACAACCAGGTTCTGGTATTCACTCTGCAGT

[0057] CTCTGACCGACCTGATGTTCAACTCCGTGTTCGCTTCTGTTAACAACCTGTACCACCGTCCGCAGCAGAAGAAACAGATCGACCGTCAG

[0058] CACGCACGTATCTACAACGCTGTTCTGCAGCGTCTGCCACACGTAGCACAGCGTGCTGCTCGTGATCACGTTCGTACCGTGAAGAAGAACCTGCACGACATCGAACTGGAAGGTCATCACCTGATCCGTTCTGCGGTTCCGTTGGAAATGAACCTGTCTTAA。

[0059] Further, in some embodiments of the present invention, in the above nucleic acid molecule,The sequence of SEQ ID NO:9 is: ATGAAAGATGAACGTCGTCCGATCTGCGAAGTTGTTGCTGAATCTATCGAACGTCTGATCATCGATGGCGTTCTGAAAGTTGGCCAGCCACTTCCAAGCGAACGTCGTCTGTGCGAGAAACTGGGTTTCTCTCGTTCTGCTCTGCGTGAAGGTCTGACCGTGTTGCGTGGTCGTGGTATCATCGAAACTGCTCAGGGTCGTGATAGCCGTGTAGCTCGTCTGAACCGTGTTCAAGACACCAGCCCGCTGATCCACCTGTTCTCCACTCAGCCGCGTACCTTGTACGACCTGCTGGACGTTCGTGCTCTGCTGGAAGGCGAATCTGCTCGTCTGGCAGCTACTCTGCTCGAAAACGTCTATATCAAGGCCGACAAGCAGAAAAACGGCATTAAGGCTAACTTCAAGATCAGACACAACATCGAGGATGGTGGCGTGCAGCTGGCCTACCATTATCAGCAGAACACACCAATCGGAGACGGACCAGTGCTGCTCCCAGATAATCACTACCTGAGCGTCCAGTCCAAGCTGTCTAAAGACCCTAACGAGAAGCGGGATCATATGGTGCTGCTCGAATTTGTCACAGCCGCTGGGATCACTCTGGGTATGGACGAGCTCTATAAAGGAGGGACCGGTGGCAGTATGGTGTCAAAGGGCGAGGAACTGTTCACAGGAGTGGTCCCCATTCTGGTGGAGCTCGACGGCGATGTCAATGGACACAAATTTTCCGTGTCTGGCGAGGGCGAAGGAGATGCTACCTACGGGAAGCTGACACTCAAATTCATCTGCACCACAGGCAAGCTGCCAGTGCCCTGGCCTACTCTGGTCACTACCCTCACCTACGGGGTGCAGTGTTTCTCCAGATATCCCGACCACATGAAGCAGCATGATTTCTTTAAATCTGCTATGCCTGAGGGGTACATCCAGGAACGGACAATTTTCTTTAAGGACGATGGTAACTACAAAACACGCGCAGAGGTGA,

[0060] AGTTCGAAGGCGACACTCTGGTCAATCGAATCGAGCTGAAGGGAATTGACTTTAAAGAAGATGGGA

[0061] ACATCCTGGGTCACAAGCTGGAGTACAATACTAGGGGTACTCAGGCGGACTTCGTAGTTATCACTCGTTGCTACGAGAAGATGCTGGCA

[0062] GCAAGCGAGAACAACAAAGAAATCTCTCTGATCGAACACGCGCAGCTGGACCACGCATTCCACCTGGCAATCTGCCAGGCGAGCCACAA

[0063] CCAGGTTCTGGTATTCACTCTGCAGTCTCTGACCGACCTGATGTTCAACTCCGTGTTCGCTTCTGTTAACAACCTGTACCACCGTCCGC

[0064] AGCAGAAGAAACAGATCGACCGTCAGCACGCACGTATCTACAACGCTGTTCTGCAGCGTCTGCCACACGTAGCACAGCGTGCTGCTCGT

[0065] GATCACGTTCGTACCGTGAAGAAGAACCTGCACGACATCGAACTGGAAGGTCATCACCTGATCCGTTCTGCGGTTCCGTTGGAAATGAACCTGTCTTAA。

[0066]

[0067]

[0068]

[0069] Furthermore, in some embodiments of the present invention, in the above nucleic acid molecule, the sequence of SEQ ID NO:13 is as follows:

[0070] ATGAAAGATGAACGTCGTCCGATCTGCGAAGTTGTTGCTGAATCTATCGAACGTCTGATCATCGATGGCGTTCTGAAAGTTGGCCAGCC

[0071] ACTTCCAAGCGAACGTCGTCTGTGCGAGAAACTGGGTTTCTCTCGTTCTGCTCTGCGTGAAGGTCTGACCGTGTTGCGTGGTCGTGGTA

[0072] TCATCGAAACTGCTCAGGGTCGTGATAGCCGTGTAGCTCGTCTGAACCGTGTTCAAGACACCAGCCCGCTGATCCACCTGTTCTCCACT

[0073] CAGCCGCGTACCTTGTACGACCTGCTGGACGTTCGTGCTCTGCTGGAAGGCGAATCTGCTCGTCTGGCAGCTACTCTGGGTACTCAGGC

[0074] GGACTTCGTAGTTATCACTCGTTGCTACGAGAAGATGCTGGCAGCAAGCGAGAACAACAAAGAAATCTCTCTGATCGAACACGCGCAGC

[0075] TGGACCACGCATTCCACCTGGCAATCTGCCAGGCGAGCCACAACCAGGTTCTGGTATTCACTCTGCAGTCTCTGACCGACCTGATGTTC

[0076] AACTCCGTGTTCGCTTCTGTTAACAACCTGTACCACCGTCCGCAGCAGAAGAAACAGATCGACCGTCAGCTCGAAAACGTCTATATCAA

[0077] GGCCGACAAGCAGAAAAACGGCATTAAGGCTAACTTCAAGATCAGACACAACATCGAGGATGGTGGCGTGCAGCTGGCCTACCATTATC

[0078] AGCAGAACACACCAATCGGAGACGGACCAGTGCTGCTCCCAGATAATCACTACCTGAGCGTCCAGTCCAAGCTGTCTAAAGACCCTAAC

[0079] GAGAAGCGGGATCATATGGTGCTGCTCGAATTTGTCACAGCCGCTGGGATCACTCTGGGTATGGACGAGCTCTATAAAGGAGGGACCGG

[0080] TGGCAGTATGGTGTCAAAGGGCGAGGAACTGTTCACAGGAGTGGTCCCCATTCTGGTGGAGCTCGACGGCGATGTCAATGGACACAAAT

[0081] TTTCCGTGTCTGGCGAGGGCGAAGGAGATGCTACCTACGGGAAGCTGACACTCAAATTCATCTGCACCACAGGCAAGCTGCCAGTGCCC

[0082] TGGCCTACTCTGGTCACTACCCTCACCTACGGGGTGCAGTGTTTCTCCAGATATCCCGACCACATGAAGCAGCATGATTTCTTTAAATC

[0083] TGCTATGCCTGAGGGGTACATCCAGGAACGGACAATTTTCTTTAAGGACGATGGTAACTACAAAACACGCGCAGAGGTGAAGTTCGAAG

[0084] GCGACACTCTGGTCAATCGAATCGAGCTGAAGGGAATTGACTTTAAAGAAGATGGGAACATCCTGGGTCACAAGCTGGAGTACAATACT

[0085] AGGCACGCACGTATCTACAACGCTGTTCTGCAGCGTCTGCCACACGTAGCACAGCGTGCTGCTCGTGATCACGTTCGTACCGTGAAGAAGAACCTGCACGACATCGAACTGGAAGGTCATCACCTGATCCGTTCTGCGGTTCCGTTGGAAATGAACCTGTCTTAA。

[0086] Furthermore, in some embodiments of the present invention, in the above nucleic acid molecule, the sequence of SEQ ID NO:14 is:

[0087] ATGAAAGATGAACGTCGTCCGATCTGCGAAGTTGTTGCTGAATCTATCGAACGTCTGATCATCGATGGCGTTCTGAAAGTTGGCCAGCC

[0088] ACTTCCAAGCGAACGTCGTCTGTGCGAGAAACTGGGTTTCTCTCGTTCTGCTCTGCGTGAAGGTCTGACCGTGTTGCGTGGTCGTGGTA

[0089] TCATCGAAACTGCTCAGGGTCGTGATAGCCGTGTAGCTCGTCTGAACCGTGTTCAAGACACCAGCCCGCTGATCCACCTGTTCTCCACT

[0090] AGCGCGGGCAACGTCTATATCAAGGCCGACAAGCAGAAAAACGGCATTAAGGCTAACTTCAAGATCAGACACAACATCGAGGATGGTGG

[0091] CGTGCAGCTGGCCTACCATTATCAGCAGAACACACCAATCGGAGACGGACCAGTGCTGCTCCCAGATAATCACTACCTGAGCGTCCAGT

[0092] CCAAGCTGTCTAAAGACCCTAACGAGAAGCGGGATCATATGGTGCTGCTCGAATTTGTCACAGCCGCTGGGATCACTCTGGGTATGGAC

[0093] GAGCTCTATAAAGGAGGGACCGGTGGCAGTATGGTGTCAAAGGGCGAGGAACTGTTCACAGGAGTGGTCCCCATTCTGGTGGAGCTCGA

[0094] CGGCGATGTCAATGGACACAAATTTTCCGTGTCTGGCGAGGGCGAAGGAGATGCTACCTACGGGAAGCTGACACTCAAATTCATCTGCA

[0095] CCACAGGCAAGCTGCCAGTGCCCTGGCCTACTCTGGTCACTACCCTCACCTACGGGGTGCAGTGTTTCTCCAGATATCCCGACCACATG

[0096] AAGCAGCATGATTTCTTTAAATCTGCTATGCCTGAGGGGTACATCCAGGAACGGACAATTTTCTTTAAGGACGATGGTAACTACAAAAC

[0097] ACGCGCAGAGGTGAAGTTCGAAGGCGACACTCTGGTCAATCGAATCGAGCTGAAGGGAATTGACTTTAAAGAAGATGGGAACATCCTGG

[0098] GTCACAAGCTGGAGTACAATGGCACCCCGCGTACCTTGTACGACCTGCTGGACGTTCGTGCTCTGCTGGAAGGCGAATCTGCTCGTCTG

[0099] GCAGCTACTCTGGGTACTCAGGCGGACTTCGTAGTTATCACTCGTTGCTACGAGAAGATGCTGGCAGCAAGCGAGAACAACAAAGAAAT

[0100] CTCTCTGATCGAACACGCGCAGCTGGACCACGCATTCCACCTGGCAATCTGCCAGGCGAGCCACAACCAGGTTCTGGTATTCACTCTGC

[0101] AGTCTCTGACCGACCTGATGTTCAACTCCGTGTTCGCTTCTGTTAACAACCTGTACCACCGTCCGCAGCAGAAGAAACAGATCGACCGT

[0102] CAGCACGCACGTATCTACAACGCTGTTCTGCAGCGTCTGCCACACGTAGCACAGCGTGCTGCTCGTGATCACGTTCGTACCGTGAAGAAGAACCTGCACGACATCGAACTGGAAGGTCATCACCTGATCCGTTCTGCGGTTCCGTTGGAAATGAACCTGTCTTAA. (The amino acid sequence combination of the linker peptide is SAG / GT)

[0103]

[0104]

[0105] (EcGCL TM )

[0106] The third technical solution of the present invention is to provide a recombinant vector, including: the above-mentioned nucleic acid molecule.

[0107] The fourth technical solution of the present invention is to further provide a host for transforming and / or transfecting the above-mentioned recombinant vector.

[0108] The fifth technical solution of the present invention is to further provide a fluorescent probe, including the above-mentioned fusion protein.

[0109] The sixth technical solution of the present invention is to provide a method for preparing the fluorescent probe, culturing the host, expressing and purifying the fluorescent probe to obtain the fluorescent probe.

[0110] The seventh technical solution of the present invention is to further provide at least one of the following applications in the quantitative detection of glycolic acid: the above-mentioned fusion protein, the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector, the above-mentioned host, the above-mentioned fluorescent probe and / or the fluorescent probe obtained by the above-mentioned preparation method.

[0111] The present invention also provides an eighth technical solution, which is to provide at least one of the following products:

[0112] The above-mentioned fusion protein, the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector, the above-mentioned host, the above-mentioned fluorescent probe and / or the fluorescent probe obtained by the above-mentioned preparation method.

[0113] Beneficial effects:

[0114] The fluorescent probe of the present invention breaks through the limitations of traditional detection methods and can achieve rapid and accurate quantitative detection of glycolic acid. It also has good detection characteristics such as simple operation, rapid response, high sensitivity, and high specificity. It is a user-friendly tool for quantitative detection of glycolic acid.

[0115] The present invention provides a genetically encoded glycolic acid fluorescent probe, comprising a glycolic acid sensitive polypeptide and a cyclically rearranged fluorescent protein, wherein the cyclically rearranged fluorescent protein is located between the sequences of the glycolic acid sensitive polypeptide. The present invention also relates to the application of the fluorescent probe in detecting glycolic acid. The glycolic acid optical probe provided by the present invention can rapidly and quantitatively detect glycolic acid in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0117] Figure 1 The SDS-PAGE diagram of the fluorescent probe integrated into the site 89 / 91 is shown; wherein: M represents protein marker;

[0118] Figure 2 The fluorescence intensity changes of the fluorescent probes whose integration sites are 89 / 91, 115 / 116, 142 / 143, 163 / 164, 188 / 189 and 211 / 212 respectively in response to glycolic acid are shown;

[0119] Figure 3 The change of fluorescence intensity of the fluorescent probe with LE / TR, SAG / GT and no connecting peptide at both ends of the cyclically rearranged fluorescent protein in response to glycolic acid is shown;

[0120] Figure 4 Schematic diagram showing the composition of the fluorescent probe with the integration site being 89 / 91 and the connecting peptide being LE / TR respectively;

[0121] Figure 5 The fluorescence spectrum property diagram of the fluorescent probe with the integration site of 89 / 91 is shown;

[0122] Figure 6 The titration curves of the fluorescent probe with the fusion site of 89 / 91 to different concentrations of glycolic acid are shown;

[0123] Figure 7 A bar graph showing the specific detection of the fluorescent probe with the integration site being 89 / 91;

[0124] Figure 8 The titration curves of the fluorescent probe with 89 / 91 fusion sites responding to glycolic acid at different pH values ​​are shown;

[0125] Fig. 9 The time curve of the fluorescent probe integrated into site 89 / 91 in response to glycolic acid is shown;

[0126] Fig.10 The bar graph shows the response of the fluorescent probe with 89 / 91 fusion site to glycolic acid at different temperatures;

[0127] Fig.11 Glycolic acid concentration changes. DETAILED DESCRIPTION

[0128] The invention discloses a fusion protein, a fluorescent probe and applications thereof.

[0129] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0130] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0131] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0132] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0133] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0134] The purpose of the present invention is to provide a genetically encoded fluorescent probe and method for quantitatively detecting glycolic acid outside cells.

[0135] A method for quantitatively detecting glycolic acid in a sample is provided.

[0136] In a first aspect, the present invention provides a fluorescent probe comprising a glycolic acid-sensitive polypeptide and a cyclically rearranged fluorescent protein, wherein the cyclically rearranged fluorescent protein is located between the sequences of the glycolic acid-sensitive polypeptide.

[0137] The second aspect of the present invention provides a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding the fluorescent probe described in the first aspect of the present invention.

[0138] The third aspect of the present invention provides a vector, wherein the vector contains the nucleic acid molecule described in the second aspect of the present invention.

[0139] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the third aspect of the present invention.

[0140] As used herein, the term "glycolic acid-sensitive polypeptide" refers to a polypeptide that responds to glycolic acid, including any response of a chemical, biological, electrical or physiological parameter of the polypeptide associated with the interaction of the glycolic acid-sensitive polypeptide. Responses include small changes, such as changes in the orientation of amino acids or peptide fragments of the polypeptide and changes in, for example, the primary, secondary or tertiary structure of the polypeptide, including, for example, changes in protonation, electrochemical potential and / or conformation. It is understood that the detectable change need not be a conformational change as long as the fluorescence of the fluorescent protein moiety is altered.

[0141] The term "fluorescent protein" as used herein refers to a protein that fluoresces when illuminated with excitation light.

[0142] The term "circular permutated FP (cpFP)" as used herein is well known to those skilled in the art and refers to connecting the ends of the original fluorescent protein molecule together and then disconnecting the protein from any site to produce a new N-terminus and C-terminus, such as green fluorescent protein GFP and its derived circular permutated green fluorescent protein (cpGFP). Fluorescent proteins and their sequences that can be used in the present invention are known in the art. Exemplarily, the amino acid sequence of cpGFP is shown in SEQ ID NO: 18.

[0143] The term "connector peptide" as used herein refers to an amino acid or nucleotide sequence that connects two parts in a polypeptide, protein or nucleic acid of the present invention. Exemplarily, the number of amino acids selected at the amino terminus of the connection region between the glycolic acid-sensitive polypeptide and the cyclically rearranged fluorescent protein of the present invention is 2, the sequence is LE, and the number of amino acids selected at the carboxyl terminus is 2, the sequence is TR.

[0144] The glycolic acid fluorescent probe described in the present invention includes a glycolic acid-sensitive polypeptide and a cyclically rearranged fluorescent protein, wherein the glycolic acid-sensitive polypeptide refers to the glycolic acid-binding protein GlcC, and the cyclically rearranged fluorescent protein refers to the cyclically rearranged green fluorescent protein cpGFP, cpGFP is inserted between the GlcC sequence, and the interaction between GlcC and glycolic acid causes the change of the fluorescence intensity of cpGFP.

[0145] The "change in fluorescence intensity" of the present invention refers to the change in fluorescence intensity ΔF / F0 (%) of the glycolic acid fluorescent probe after binding to glycolic acid. The change can be an increase in fluorescence intensity or a decrease in fluorescence intensity. The greater the fluorescence change, the better the properties of the probe.

[0146] In the fluorescent probe of the present invention, cpGFP can be located at or fused to any position of the glycolic acid-sensitive polypeptide. Specifically, cpGFP is located in the flexible region of the glycolic acid-sensitive polypeptide, and the flexible region refers to some specific structures such as ring domains in the higher-order structure of the protein, which have higher mobility and flexibility than other higher-order structures of the protein, and the region can dynamically change the spatial structural conformation after the protein binds to the ligand. The flexible region described in the present invention mainly refers to the region where the fusion site of GlcC in the glycolic acid-binding protein is located. Exemplarily, cpGFP is located at the following sites selected from the amino acid sequence of the glycolic acid-binding protein: 89 / 91, 115 / 116, 142 / 143, 163 / 164, 188 / 189, 211 / 212, and its amino acid sequence is shown in SEQ ID NO: 1 to SEQ ID NO: 6. Exemplarily, the amino acid sequences of the connecting peptides at both ends of cpGFP are: LE / TR, SAG / GT, and its complete amino acid sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 7. The full-length sequence or a fragment thereof of the fluorescent probe of the present invention can usually be obtained by PCR amplification, artificial synthesis or homologous recombination.

[0147] After obtaining the nucleotide sequence encoding the fluorescent probe, the present invention transforms the nucleotide sequence encoding the fluorescent probe into an expression vector to obtain a recombinant expression vector. In one embodiment, the expression vector can use a commercially available pET30a vector without other special requirements. Exemplarily, the expression vector is double-digested with NdeI and XhoI, and then the digested product of the expression vector and the nucleotide sequence encoding the fluorescent probe obtained by PCR amplification are homologously recombined to obtain a recombinant expression vector.

[0148] The present invention also provides application of the glycolic acid fluorescent probe in the quantitative detection of glycolic acid.

[0149] Conventional genetic engineering molecular biology cloning methods are mainly used in the embodiments of the present invention, and these methods are well known to those of ordinary skill in the art.

[0150] The pET30a-GlcC and pET30a-cpGFP plasmids used in the examples of the present invention were purchased from Qingke Biotechnology Co., Ltd. All primers used for PCR were synthesized, purified and verified to be correct by mass spectrometry by Qingke Biotechnology Co., Ltd.

[0151] The expression plasmids constructed in the examples of the present invention were sequenced by Qingke Biotechnology Co., Ltd. and Jinweizhi Biotechnology Co., Ltd. The PrimeSTAR DNA polymerase, NdeI and XhoI restriction endonucleases used in each example were purchased from TaKaRa Co., Ltd. MultiS.

[0152] The One Step Cloning Kit used in the examples of the present invention was purchased from Novazon Biotech, Inc. Sodium glycolate was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0153] The agarose gel DNA recovery kit (enhanced) and plasmid mini-extraction kit used in the examples of the present invention were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0154] The Escherichia coli DH5α, Escherichia coli K12 and Escherichia coli BL21 used in the examples of the present invention were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0155] The Tris-HCl, inorganic salts, etc. used in the examples of the present invention were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0156] The buffer involved in the following examples is as follows:

[0157] Buffer 1: 50 mM Tris-HCl (pH 7.5), 500 mM NaCl;

[0158] Washing buffer 1: 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 20 mM imidazole;

[0159] Washing buffer 2: 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 40 mM imidazole;

[0160] Elution buffer: 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 300 mM imidazole;

[0161] Reaction buffer: 50 mM potassium phosphate buffer, pH 7.0, 0.5 mM thiamine pyrophosphate (ThDp), 5 mM magnesium chloride, 50 mM formaldehyde.

[0162] The culture medium involved in the following examples is as follows:

[0163] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, and the balance is water.

[0164] LB solid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 2%, and the balance is water.

[0165] In Examples 1 to 4 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0166] The present invention will be further described below in conjunction with embodiments:

[0167] Example 1 Construction of Glycolic Acid Fluorescent Probe Expression Vector

[0168] 1. PCR amplification of target fragment

[0169] The method is mainly used for gene fragment amplification and colony PCR identification of positive clones. The reaction system of the PCR amplification is shown in Table 1, and the amplification program is shown in Table 2. The full-length GlcC gene from Escherichia coli was amplified by PCR.

[0170] Table 1 PCR amplification reaction system

[0171]

[0172]

[0173] Table 2 PCR amplification program

[0174]

[0175] 2. Endonuclease double digestion reaction

[0176] The vector pET30a was digested with NdeI and XhoI, and the system for double digestion of the vector plasmid is shown in Table 3.

[0177] Table 3 Double enzyme digestion reaction system

[0178]

[0179] 3. Homologous recombination reaction between target fragment and vector

[0180] After agarose gel electrophoresis, the PCR products and double-enzyme digestion products were recovered using the gel recovery kit (enhanced) of Tiangen Biotechnology Co., Ltd. The recovered products were used for homologous recombination reaction. The homologous recombination reaction system is shown in Table 4.

[0181] Table 4 Homologous recombination reaction system

[0182]

[0183] 4. Take 10 μL of homologous recombination product and transform it into 100 μL of E. coli DH5α competent cells, and grow them overnight on LB plates containing the antibiotic kanamycin (50 μg / mL).

[0184] 5. Pick a single colony and inoculate it into 2 mL LB liquid culture medium. Culture it at 37°C and 220 rpm / min for 4 h. Take 1 mL of the bacterial solution and send it to Qingke Biotechnology Co., Ltd. for sequencing verification.

[0185] 2. Experimental Results

[0186] In this example, the following sites were selected to insert cpGFP based on the structure of glycolic acid sensitive polypeptide GlcC, and the corresponding plasmid was successfully obtained by molecular cloning. The amino acid sequence of the exemplary fluorescent probe is shown in Table 5.

[0187] Table 5 Amino acid sequences of fluorescent probes at different fusion sites

[0188]

[0189] In this example, the connecting peptides at both ends of cpGFP were changed based on the plasmid in which cpGFP was integrated into the 89 / 91 site of GlcC, and the corresponding plasmid was successfully obtained by molecular cloning. The amino acid sequence of the exemplary fluorescent probe is shown in Table 6.

[0190] Table 6 Amino acid sequences of fluorescent probes with different connecting peptides

[0191]

[0192]

[0193] Example 2 Expression and purification of glycolic acid fluorescent probe

[0194] 1. Expression and purification of glycolic acid fluorescent probe

[0195] 1. Transform the expression vector (e.g., glycolic acid fluorescent probe expression vector based on pET30a) into E. coli BL21 (DE3) cells and grow them overnight on LB plates containing the antibiotic kanamycin (50 μg / mL).

[0196] 2. Pick a single colony from the plate and inoculate it into 10 mL LB liquid medium. Cultivate it at 37°C and 220 rpm / min for 13 h to obtain seed solution. Inoculate 10 mL of seed solution into 1 L LB medium with antibiotics at a ratio of 1:100. Shake the culture at 37°C and 220 rpm / min for about 2.5 h until the OD reaches 0. 600 When the pH reaches 0.6-0.8, add 1 mL of 1 M IPTG, the final concentration of IPTG is 1 mM, and continue inducing culture at 16°C for 24 h.

[0197] 3. After induction, centrifuge at 4°C, 4000rpm for 20min to collect the bacterial solution. Remove the supernatant, add 20mL buffer 1, and resuspend the bacterial cells. Ultrasonicate the cells, and calculate the ultrasonic time based on 10min for 1L of cells. After ultrasonication, centrifuge at 4°C, 18000rpm for 30min to collect the supernatant.

[0198] 4. The supernatant collected by centrifugation was passed through a nickel column affinity chromatography column. After repeated flow-through three times, 8 times the column volume was washed with wash buffer 1 and wash buffer 2 respectively, and finally the purified protein was eluted with elution buffer.

[0199] 2. Experimental Results

[0200] The SDS-PAGE image of the fusion protein composed of glycolic acid sensitive peptide GlcC and cpGFP is shown in Figure 1 , Figure 1 The M represents a protein marker.

[0201] Example 3 Fluorescence detection and performance of glycolic acid fluorescent probe

[0202] 1. Experimental steps

[0203] 1. The protein identified by SDS-PAGE was measured by Nanodrop and diluted to a 2 μM protein solution using buffer 1. Sodium glycolate, sodium acetate, ethanol, glyoxylic acid, sodium chloroacetate, sodium lactate, sodium pyruvate, sodium oxalate, sodium isocitrate, sodium fumarate and sodium citrate were prepared into a stock solution with a final concentration of 100 mM using buffer 1.

[0204] 2. Use buffer 1 to dilute sodium glycolate to 2mM, take 100μL of 2μM protein solution and add it to a black 96-well plate, add 100μL of 2 mM sodium lactate solution to make the final concentration 1mM, and add 100μL of buffer 1 to the parallel wells as a control group. Use a multifunctional fluorescence microplate reader to measure the fluorescence intensity of the protein emitted at 515nm after 480nm fluorescence excitation. The calculation method for the change in fluorescence intensity is as follows:

[0205] ΔF / F0(%)=(sample fluorescence intensity-control fluorescence intensity) / control fluorescence intensity×100

[0206] 3. Take 100 μL of 2 μM protein solution of the fluorescent probe with fusion site 89 / 91 and add it to a black 96-well plate. Add 100 μL of 2 mM sodium glycolate solution to make the final concentration 1 mM. Use a multifunctional fluorescence microplate reader to measure the emission spectrum of the protein at 495-600 nm after fluorescence excitation at 480 nm, and read once every 1 nm.

[0207] 4. Take 100 μL of 2 μM protein solution of the fluorescent probe with fusion site 89 / 91 and add it to a black 96-well plate, add 100 μL of sodium glycolate solution to make the final concentration in the range of 10 nM to 100 mM, and use a multifunctional fluorescence microplate reader to measure the fluorescence intensity of the protein emitted at 515 nm after fluorescence excitation at 480 nm.

[0208] 5. Use buffer 1 to dilute sodium acetate, ethanol, glyoxylic acid, sodium chloroacetate, sodium lactate, sodium pyruvate, sodium oxalate, sodium isocitrate, sodium fumarate and sodium citrate to 2 mM respectively, take 100 μL of 2 μM protein solution of the fluorescent probe with fusion site 89 / 91 into a black 96-well plate, add 100 μL of 2 mM sodium acetate, ethanol, glyoxylic acid, sodium chloroacetate, sodium lactate, sodium pyruvate, sodium oxalate, sodium isocitrate, sodium fumarate and sodium citrate solution respectively to make the final concentration of 1 mM, and use a multifunctional fluorescence microplate reader to measure the fluorescence intensity of the protein emitted at 515 nm after fluorescence excitation at 480 nm.

[0209] 6. Dilute sodium glycolate to 2 mM using buffer 1 with pH values ​​of 7.0, 7.2, 7.4, 7.6, 7.8 and 8.0, respectively, elute the fluorescent probe protein solution with fusion site 89 / 91 using the above buffer 1 with different pH values, take 100 μL of 2 μM protein solution of fluorescent probe with fusion site 89 / 91 and add it to a black 96-well plate, add 100 μL of 2 mM sodium glycolate solution with the same pH value to make the final concentration 1 mM. Use a multifunctional fluorescence microplate reader to measure the fluorescence intensity of the protein at 515 nm after fluorescence excitation at 480 nm.

[0210] 7. Dilute sodium glycolate to 2 mM using buffer 1, take 100 μL of 2 μM protein solution of fluorescent probe with fusion site 89 / 91 and add it to a black 96-well plate, and add 100 μL of 2 mM sodium glycolate solution to make the final concentration 1 mM. Measure the fluorescence intensity of the protein emitted at 515 nm after 480 nm fluorescence excitation at 0 min, 1 min, 2 min, 3 min, 5 min, 10 min and 20 min after adding sodium glycolate.

[0211] 8. Dilute sodium glycolate to 2 mM using buffer 1, add 100 μL of 2 μM protein solution of fluorescent probe with fusion site 89 / 91 to a black 96-well plate, and add 100 μL of 2 mM sodium glycolate solution to make the final concentration 1 mM. Use a multifunctional fluorescence microplate reader to measure the fluorescence intensity of the protein at 515 nm after 480 nm fluorescence excitation at temperatures of 25°C, 28°C, 30°C, and 35°C.

[0212] 2. Experimental Results

[0213] The fluorescence intensity changes of the fluorescent probes with fusion sites of 89 / 91, 115 / 116, 142 / 143, 163 / 164, 188 / 189 and 211 / 212 are shown in Figure 2 As shown in Table 7, the fluorescent probe with fusion site 89 / 91 has the greatest response to glycolic acid.

[0214] Table 7 Response of fluorescent probes at different fusion sites to glycolic acid

[0215]

[0216] The fluorescence intensity changes of the fluorescent probes with LE / TR, SAG / GT and no connecting peptide at both ends of the cyclically rearranged fluorescent protein are shown in Figure 2. Figure 3 As shown in Table 8. Among them, the fluorescent probe with the amino acid sequence of the connecting peptide at both ends of cpGFP being LE / TR has the greatest response to glycolic acid. The schematic diagram of the composition of the fluorescent probe is shown in Figure 4 shown.

[0217] Table 8 Response of fluorescent probes with different linked peptides to glycolic acid

[0218]

[0219] The emission spectrum of the fluorescent probe with fusion site 89 / 91 excited at 480 nm is as follows Figure 5 As shown, the results show that after adding glycolic acid, the fluorescence intensity of the probe is higher than that of the control group as a whole, and reaches the maximum change in fluorescence intensity at 515 nm, which is about 93.25%.

[0220] Figure 6 The results show that the Kd (binding constant) of the fluorescent probe with fusion site 89 / 91 is 101 μM, and the fluorescence intensity varies from -11.80% to 90.47%. Table 9 shows the responses of the fluorescent probe with fusion site 89 / 91 to different concentrations of glycolic acid.

[0221] Table 9 Response of fluorescent probe to different concentrations of glycolic acid

[0222]

[0223] Figure 7 This indicates that the fluorescent probe with fusion site 89 / 91 has good specificity. Its fluorescence response to different substrate molecules is shown in Table 10.

[0224] Table 10 Response of fluorescent probes to different substrate molecules

[0225]

[0226]

[0227] Figure 8 This indicates that the response affinity of the fluorescent probe fused with 89 / 91 dots to glycolic acid at different pH values ​​will not be affected. Its Kd values ​​for glycolic acid at different pH values ​​are shown in Table 11.

[0228] Table 11 Kd of fluorescent probes for glycolic acid at different pH values

[0229]

[0230] Fig. 9 This indicates that the fluorescent probe with fusion site 89 / 91 reacts faster to glycolic acid, reaching the maximum value of fluorescence intensity change at 3 minutes. Its response to glycolic acid at different time points is shown in Table 12.

[0231] Table 12 Response of fluorescent probe to glycolic acid at different time points

[0232]

[0233] Fig.10 This indicates that the response of the fluorescent probe with fusion site 89 / 91 to glycolic acid is not affected by temperature within the range of 25°C-35°C. Its response to glycolic acid at different temperatures is shown in Table 13.

[0234] Table 13 Response of fluorescent probe to glycolic acid at different temperatures

[0235]

[0236] Example 4 Quantitative detection of glycolic acid concentration produced by recombinant Escherichia coli using a glycolic acid fluorescent probe

[0237] 1. Experimental steps

[0238] 1. pETDuet-1-EcGCL TM Plasmids were purchased from Genewise Biotechnology Co., Ltd., among which EcGCL TM Located between NdeI and XhoI in the multiple cloning site 2 of pETDuet-1. EcGCL TM The nucleotide sequence is SEQ ID NO:16.

[0239] 2. Using the molecular cloning method in Example 1, the AldA gene was amplified from the genomic DNA of the Escherichia coli K12 strain, and the AldA gene fragment was constructed into the vector pETDuet-1-EcGCL TM The nucleotide sequence of AldA is shown in SEQ ID NO:15.

[0240] 3. Insert the expression vector (e.g. AldA and EcGCL based on pETDuet-1) TM The expression vector was transformed into E. coli BL21 (DE3) cells and grown overnight on LB plates containing the antibiotic ampicillin (100 μg / mL).

[0241] 4. Pick a single colony from the plate and inoculate it into LB liquid culture medium. Cultivate it at 37°C and 220 rpm / min for 13 h to obtain seed solution. Inoculate the seed solution into liquid LB culture medium with antibiotics at a ratio of 1:100. Shake the culture at 37°C and 220 rpm / min for about 2.5 h until the OD 600 When the pH reaches 0.6-0.8, add 1M IPTG, the final concentration of IPTG is 0.05mM, and continue inducing culture at 16℃ for 24h.

[0242] 5. Centrifuge the induced bacterial solution at 4000 rpm for 10 min. Resuspend the colonies in reaction buffer at a ratio of 11 g / L and react at 40°C and 250 rpm.

[0243] 6. Take the reaction solution at different time points and centrifuge it at 4000 rpm for 10 min. Detect the pH value and record it.

[0244] 7. Take 5 μL of the supernatant after centrifugation and add it to a black 96-well plate, add 195 μL of a protein solution with a final concentration of 1 μM and a fluorescent probe with a fusion site of 89 / 91, and use a multifunctional fluorescence microplate reader to measure the fluorescence intensity of the protein at 515 nm after fluorescence excitation at 480 nm.

[0245] 2. Experimental Results

[0246] The results of the test are as follows Fig.11 As shown in Table 14, as time increases, the glycolic acid production of recombinant E. coli gradually increases compared to wild-type E. coli. Within the first hour after the reaction, the glycolic acid production of recombinant E. coli shows a rapid upward trend and eventually reaches a maximum value (10.25 mM) in the second hour. As the reaction time increases, the glycolic acid concentrations produced by recombinant and wild-type E. coli change as shown in Table 14.

[0247] Table 14 Glycolic acid concentration produced by two E. coli at different time points

[0248]

[0249] It can be seen from the above examples that the fluorescent probe provided by the present invention exhibits good characteristics such as high specificity, stable properties and convenient detection when quantifying glycolic acid in vitro, and can quantify the concentration of glycolic acid produced during the recombinant Escherichia coli reaction.

[0250] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fusion protein, characterized in that The N-terminus of the cyclically rearranged fluorescent protein is connected to the acid-sensitive polypeptide via a connecting peptide 1, and the C-terminus is connected to the acid-sensitive polypeptide via a connecting peptide 2; The amino acid sequence of the connecting peptide 1 includes: one or more of LE and SAG; The amino acid sequence of the connecting peptide 2 includes one or more of TR and GT.

2. The fusion protein according to claim 1, characterized in that The cyclically rearranged fluorescent protein is located at any one of the positions 89 / 91, 115 / 116, 142 / 143, 163 / 164, 188 / 189 and 211 / 212 of the acid-sensitive polypeptide.

3. The fusion protein according to claim 1 or 2, characterized in that It has an amino acid sequence shown in any of SEQ ID NO: 1 to SEQ ID NO:

7.

4. A nucleic acid molecule encoding a fusion protein according to any one of claims 1 to 3, characterized in that: Having a nucleotide sequence shown in any of SEQ ID NO: 8 to SEQ ID NO:

14.

5. A recombinant vector, characterized in that: include: The nucleic acid molecule according to claim 4.

6. A host, characterized in that Transform and / or transfect the recombinant vector as claimed in claim 5.

7. A fluorescent probe, characterized in that The invention comprises the fusion protein according to any one of claims 1 to 3.

8. The method for preparing a fluorescent probe according to claim 7, characterized in that: The host as claimed in claim 6 is cultured, expressed and purified to obtain the fluorescent probe.

9. As follows① ～ At least one of the following is used in the quantitative detection of glycolic acid: ① The fusion protein according to any one of claims 1 to 2; ② The fusion protein according to claim 3; ③ The nucleic acid molecule according to claim 4; ④ The recombinant vector according to claim 5; 5. The host according to claim 6; 6. The fluorescent probe as claimed in claim 7; ⑦ The fluorescent probe obtained by the preparation method as described in claim 8.

10. As follows① ～ At least one product from ⑦: ① The fusion protein according to any one of claims 1 to 2; ② The fusion protein according to claim 3; ③ The nucleic acid molecule according to claim 4; ④ The recombinant vector according to claim 5; 5. The host according to claim 6; 6. The fluorescent probe as claimed in claim 7; ⑦ The fluorescent probe obtained by the preparation method as described in claim 8.

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