A biological probe for identifying velvet antler polypeptide and its recombinant plasmid

Abstract

The present invention belongs to the technical field of cell biology and molecular biology, and relates to a biological probe for identifying antler polypeptide and a recombinant plasmid thereof. The biological probe is designed and prepared based on the cyclized rearranged fluorescent protein cpFP technology and biological subcloning technology. The probe includes cyclized rearranged Mcherry fluorescent protein and interactive proteins TβRI-R and TβRI-F for identifying antler polypeptide respectively connected at both ends thereof, and constitutes a recombinant plasmid with an expression vector plasmid. After the probe is transfected into living cells, it can be self-expressed in the living cells. When the probe expressed in the cells interacts with the antler polypeptide, the probe shows red fluorescence, which can reflect whether the antler polypeptide enters the cell by dynamically and real-time detecting the presence or absence of the fluorescent signal, thereby realizing visual detection of the antler polypeptide; the probe fusion protein expression can also be realized through a prokaryotic expression system, and then the antler polypeptide is detected by scanning the intensity of the fluorescence emission wavelength, thereby realizing the detection of the antler polypeptide in the liquid.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology and molecular biology, and relates to a biological probe for identifying pilose antler polypeptide and a recombinant plasmid thereof, and specifically to a biological probe for detecting pilose antler polypeptide and a recombinant plasmid thereof based on circularly permuted fluorescent proteins (cpFP) technology. Background Art

[0002] Deer antler is a traditional precious Chinese medicine, and deer antler peptide is a natural peptide with a molecular weight of 3.2k extracted by biochemical techniques such as gel filtration and ion exchange chromatography. It is one of the main active ingredients of deer antler, with anti-inflammatory, anti-apoptotic and chondrocyte proliferation effects. Previous work has shown that deer antler peptide can inhibit the migration of osteosarcoma cells, and the signaling pathway involved in the process is TGF-beta. Protein interaction analysis predicts that its target is tβRI, so a biological probe that can detect deer antler peptide is needed. Summary of the invention

[0003] To solve the above problems, the present invention provides a biological probe for detecting antler polypeptides based on cpFP technology and a recombinant plasmid thereof, which is designed and prepared based on circularly permuted fluorescent proteins (cpFP) technology and conventional biological subcloning technology. The biological probe protein can be expressed in living cells by itself, and based on the specific interaction relationship between proteins, the presence and intensity of the fluorescent signal can quantitatively reflect the interaction between the antler polypeptide and the receptor protein. The probe protein can also be expressed by prokaryotic cells, and then the antler polypeptide detection can be achieved by scanning the fluorescence emission wavelength intensity, and applied to the detection of antler polypeptides in liquids. It has the advantages of high time resolution, low cost, and accurate non-destructive detection at the single cell level.

[0004] The present invention realizes the visualization of the mechanism of action of antler polypeptide by constructing biological probes based on the specific interaction between proteins. The cpFP probe tool for visualization of the mechanism of action of antler polypeptide comprises three parts: a cyclized rearranged fluorescent protein and the interacting proteins TβRI-R and TβRI-F connected at both ends thereof, which are the target protein antler polypeptides. The DNA sequences of these two parts are sheared and connected and reconstructed by using subcloning technology, i.e., polymerase chain reaction technology (PCR), nucleic acid specific enzyme cutting and connection experimental technology, and a recombinant plasmid is formed with a pcDNA3.1 (+) or pRSET-B vector. The TβRI-R and TβRI-F sequences intercepted by the probe design are only the necessary parts for binding to antler polypeptides, which can specifically bind to antler polypeptides, which can not only eliminate the interference of TβRI protein function changes on the probe work, but also reduce the influence of increased exogenous TβRI protein levels on cells. The detection principle is that when the detection domain specifically binds to the antler polypeptide, the fluorescent protein forms a closed ring structure and emits fluorescence, thereby achieving the purpose of detecting antler polypeptides.

[0005] The technical solution of the present invention is:

[0006] A biological probe for identifying antler polypeptides, including a cyclic rearranged fluorescent protein cpFP and interacting proteins TβRI-R and TβRI-F connected at both ends thereof for identifying target protein antler polypeptides. The cyclic rearranged fluorescent protein cpFP is formed by connecting the original N and C ends of the fluorescent protein body with a linker and reopening an N and C end near its chromophore.

[0007] The amino acid sequence of the TβRI-R binding region is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4.

[0008] The amino acid sequence of the TβRI-F binding region is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6.

[0009] The amino acid sequence of the linker is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.8.

[0010] Amino acid sequence of TβRI-R (SEQ ID NO.3):

[0011] EIDLIPRDRPFVCAPSSKTGSVTTT

[0012] Corresponding DNA sequence (SEQ ID NO.4):

[0013] GAAATTGACTTAATTCCTCGAGATAGGCCGTTTGTATGTGCACCCTCTCA

[0014] AAAACTGGGTCTGTGACTACAACA

[0015] TβRI-F amino acid sequence (SEQ ID NO.5):

[0016] FTCVTDGLCFVSVTETTDKVIH

[0017] Corresponding DNA sequence (SEQ ID NO.6):

[0018] TTTACTTGTGTGACAGATGGGCTCTGCTTTGTCTCTGTCACAGAGACCACA

[0019] GACAAAGTTATACAC

[0020] Linker amino acid sequence is (SEQ ID NO.7):

[0021] GGTGGS

[0022] The corresponding DNA sequence is (SEQ ID NO.8):

[0023] GGCGGCACCGGCGGCAGC

[0024] The cyclic permuted fluorescent protein cpFP is selected from circularly permuted blue fluorescent protein (cpBFP), circularly permuted green fluorescent protein (cpGFP), circularly permuted red fluorescent protein (cpRFP), circularly permuted cyan fluorescent protein (cpCFP), and circularly permuted yellow fluorescent protein (cpYFP).

[0025] In a preferred embodiment, the cyclized permuted fluorescent protein is a red mCherry cyclized fluorescent protein.

[0026] In a preferred embodiment, the amino acid sequence of the biological probe for recognizing velvet antler polypeptide is shown as SEQ ID NO.1, and the nucleotide sequence thereof is shown as SEQ ID NO.2.

[0027] The complete amino acid sequence of the biological probe for recognizing velvet antler polypeptide is (SEQ ID NO.1): MEIDLIPRDRPFVCAPSSKTGSVTTTGYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGGSGGMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGLMCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNGFTCVTDGLCFVSVTETTDKVIH

[0028] The complete nucleotide sequence of the biological probe for identifying pilose antler polypeptide is (SEQ ID NO.2): AAGCTTATGGAAATTGACTTAATTCCTCGAGATAGGCCGTTTGTATGTGCACCCTCTTCAAAAACTGGGTCTGTGACTACAACATTGGCGCGCCAAATCGATGGCTATAACAGCCATAACGTGTATATTATGGCGGATAAACAGAAAAACGGCATTAAAGTGAACTTTAAAATTCGCCATAACATTGAAGATGGCAGCGTGCAGCTGGCGGATCATTATCAGCAGAACACCCCGATTGGCGATGGCCCGGTGCTGCTGCCGGATAACCATTATCTGAGCTATCAGAGCGCGCTGAGCAAAGATCCGAACGAAAAACGCGATCATATGGTGCTGCTGGAATTTGTGACCGCGGCGGGCATTACCCATGGCATGGATGAACTGTATAAAGGCGGCAGCGGCGGCATGGTGAGCAAAGGCGAAGAACTGTTTACCGGCGTGGTGCCGATTCTGGTGGAACTGGATGGCGATGTGAACGGCCATAAATTTAGCGTGAGCGGCGAAGGCGAAGGCGATGCGACCTATGGCAAACTGACCCTGAAATTTATTTGCACCACCGGCAAACTGCCGGTGCCGTGGCCGACCCTGGTGACCACCTTTACCTATGGCCTGATGTGCTTTGCGCGCTATCCGGATCATATGAAACGCCATGATTTTTTTAAAAGCGCGATGCCGGAAGGCTATGTGCAGGAACGCACCATTTTTTTTAAAGATGATGGCAACTATAAAACCCGCGCGGAAGTGAAATTTGAAGGCGATACCCTGGTGAACCGCATTGAACTGAAAGGCATTGATTTTAAAGAAGATGGCAACATTCTGGGCCATAAACTGGAATATAACGGCATCGATTTTACTTGTGTGACAGATGGGCTCTGCTTTGTCTCTGTCACAGAGACCACAGACAAAGTTATACACTAGGAATTC

[0029] On the other hand, the present invention provides a recombinant plasmid comprising the complete nucleotide sequence of the biological probe for recognizing velvet antler polypeptide.

[0030] The recombinant plasmid vector is a eukaryotic expression vector or a prokaryotic expression vector. The eukaryotic expression vector is selected from pcDNA3.1(+) vector, pcDNA TM 3.3 vector, pCMVp-NEO-BAN vector, CMV4 vector; the prokaryotic expression vector is selected from pET-32a(+) vector, pET-30a vector, pRSET-B vector, PGEX vector.

[0031] In a preferred embodiment, the recombinant plasmid vector is a pcDNA3.1(+) vector or a pRSET-B vector.

[0032] Beneficial effects of the present invention:

[0033] The present invention provides a biological probe for visual detection of antler polypeptides based on cpFP technology, which reflects the changes of antler polypeptides through the presence and intensity of fluorescent signals, thereby realizing visual detection of antler polypeptides. The constructed probe tool is transfected into living cells, and the fluorescent protein reconstructed fusion probe structure is expressed by itself. A fluorescence microscope or a fluorescence spectrophotometer is used to dynamically detect and analyze the changes of the fluorescent signal, thereby detecting the mechanism of action of the antler polypeptides. At the same time, the expression of the probe fusion protein can be realized through a prokaryotic expression system, and the antler polypeptide can be detected by scanning the intensity of the fluorescence emission wavelength, which can be applied to the detection of antler polypeptides in liquids. The probe realizes the dynamic detection of antler polypeptides in living cells and outside cells, has the characteristics of simple operation, low cost, no damage to cells, simple verification results, etc., and provides a visual monitoring tool for the research and detection of antler polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1( a ) is a structural diagram of a biological probe for detecting velvet antler polypeptides based on cpFP technology.

[0035] FIG1( b ) is a diagram showing the working principle of the biological probe for detecting antler peptides based on the cpFP technology.

[0036] Figure 2(a) to Figure 2(c) It is a fluorescence image based on cpFP technology to detect the transfer of velvet antler polypeptide biological probe into different cells.

[0037] FIG3(a) is the fluorescence spectrum scanning result of the velvet antler polypeptide biological probe based on cpFP technology under different proteins.

[0038] FIG3( b ) is the fluorescence spectrum scanning result of the velvet antler polypeptide biological probe based on cpFP technology at different pH values.

[0039] FIG3(c) is the fluorescence spectrum scanning result of the velvet antler polypeptide biological probe based on cpFP technology at different temperatures. DETAILED DESCRIPTION

[0040] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and technical solutions.

[0041] A biological probe for identifying antler polypeptide, as shown in FIG1(a), includes a cyclic rearranged fluorescent protein cpFP and interacting proteins TβRI-R and TβRI-F for identifying antler polypeptide connected at both ends thereof. The cyclic rearranged fluorescent protein cpFP is formed by connecting the original N and C ends of the fluorescent protein body with a linker and reopening an N and C end near its chromophore. Its working principle is shown in FIG1(b).

[0042] Example

[0043] The present invention mainly obtains a plasmid by synthesizing a probe DNA sequence in a certain company, and then transforms the recombinant plasmid into DH5α for screening and amplification experiments to obtain the target probe.

[0044] Test Example 1:

[0045] After the probe prepared in this example is transfected into living cells using the liposome transfection method, the cells can express the reconstructed fusion fluorescent protein. The probe has stability characteristics and can work in a variety of living cells. It can be normally expressed and produce fluorescence in human neuroblastoma cells SH-SY5Y, human osteosarcoma cells U2OS, and Hela cells. Figure 2(a) to Figure 2(c) shown.

[0046] Test Example 2:

[0047] The probe was expressed using a prokaryotic expression system, namely, the BL21 competent cell, and then the probe fusion protein was purified. In an in vitro specificity experiment using different proteins to detect the probe, the results showed that the probe could specifically detect antler polypeptides as shown in Figure 3(a). In an experiment to detect the stability of antler polypeptides at different temperatures and pH values, the probe was also found to have good stability as shown in Figures 3(b) and 3(c).

[0048] In summary, after the probe is transfected into cells, the probe protein can be stably expressed in living cells. The transfected cells are given excitation light of 580nm wavelength, and the emission fluorescence image of 610nm wavelength is collected simultaneously by using a fluorescence microscope. The antler polypeptide is detected by the presence or absence of the emitted fluorescence signal. When the red fluorescence is enhanced, it indicates that the antler polypeptide has entered the cell, otherwise, it indicates that the antler polypeptide has not entered the cell. At the same time, after the probe is expressed and purified by using a prokaryotic expression system, a fluorescence spectrophotometer is used to scan the fluorescence intensity value of the emission wavelength of the fluorescent protein under different excitation wavelengths to detect whether the liquid contains antler polypeptide.

Claims

1. A biological probe for identifying velvet antler polypeptides, characterized in that: The biological probe for identifying velvet antler polypeptides comprises a cyclic rearranged fluorescent protein cpFP and interactive protein sequences TβRI-R and TβRI-F for identifying the target protein velvet antler polypeptides connected to both ends thereof; wherein the cyclic rearranged fluorescent protein cpFP is formed by connecting the original N and C ends of the fluorescent protein body with a linker and reopening an N and C end near its chromophore; wherein, The amino acid sequence of the TβRI-R binding region is shown in SEQ ID NO. 3, and the nucleotide sequence is shown in SEQ ID NO.

4. The amino acid sequence of the TβRI-F binding region is shown in SEQ ID NO. 5, and the nucleotide sequence is shown in SEQ ID NO.

6.

2. The biological probe for identifying velvet antler polypeptide according to claim 1, characterized in that: The cyclized rearranged fluorescent protein is selected from cyclized green fluorescent protein, cyclized red fluorescent protein, cyclized cyan fluorescent protein, cyclized blue fluorescent protein or cyclized yellow fluorescent protein.

3. The biological probe for identifying velvet antler polypeptide according to claim 1, characterized in that: The cyclized rearranged fluorescent protein is an mCherry cyclized fluorescent protein that emits red fluorescence.

4. The biological probe for identifying velvet antler polypeptide according to claim 1, 2 or 3, characterized in that: The amino acid sequence of the linker is shown in SEQ ID NO.

7.

5. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the nucleotide sequence of the biological probe for recognizing velvet antler polypeptide as described in any one of claims 1 to 4, and the vector of the recombinant plasmid is selected from pcDNA3.1 (+) vector, pcDNA™ 3.3 vector, pCMVp-NEO-BAN vector, CMV4 expression vector, pET-32a (+) vector, pET-30a vector, pRSET-B vector or PGEX vector.

6. The recombinant plasmid according to claim 5, characterized in that The vectors of the recombinant plasmid are pcDNA3.1 (+) vector and pRSET-B vector.

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