CN120923600A - An ultra-stable red fluorescent protein - Google Patents

Abstract

The invention discloses a super-stable red fluorescent protein. The fluorescent protein disclosed by the invention is a protein with an amino acid sequence SEQ ID No. 4. Experiments prove that the fluorescent protein has excellent electron microscope sample preparation characteristics, can retain more fluorescent signals after osmium acid treatment and can resist the embedding of Epon resin, meanwhile, the fluorescent protein has excellent thermal stability, can retain more fluorescent signals at 90 ℃, and in addition, the fluorescent protein has higher chemical stability and light stability. Based on the relevant properties of the fluorescent protein, the fluorescent protein can be used in the fields of protein labeling, various types of fluorescent imaging and super-resolution photoelectric associated imaging, can be used for tracking the structure and morphological changes of samples such as proteins, subcellular organelles, local areas of cells, cells and the like, can be used for packaging relevant viruses or transgenic animals, labeling specific proteins, specific organelles or specific cells, and can realize rapid transparent tissue imaging and expansion super-resolution imaging.

Description

Super-stable red fluorescent protein

Technical Field

The invention relates to a super-stable red fluorescent protein in the field of biological imaging.

Background

The fluorescent protein can visualize, track and quantify molecules and events in living cells with high spatial resolution and high time resolution, thereby thoroughly widening the application range of microscopic imaging technology. Among all properties, the stability of fluorescent proteins plays a crucial role in microscopy imaging techniques. Recently, with the development of green fluorescent protein-StayGold and its monomer variants, which have the best photostability, long-term living cell imaging technology has changed over the course of the day, but red fluorescent protein is superior to green fluorescent protein in autofluorescence, light scattering, phototoxicity, etc. Therefore, a stable red fluorescent protein has been desired.

The super-resolution photoelectric correlation microscopic imaging technology not only can accurately position the target protein, but also can provide an ultra-micro environment where the target protein is located. The inventor firstly realizes super-resolution photoelectric associated imaging after embedding the high-temperature polymerized hydrophobic resin-Epon in the conventional electron microscope-resistant sample fluorescent protein mEosEM developed in 2020. Compared with other super-resolution photoelectric associated imaging technologies, the super-resolution photoelectric associated imaging technology after the Epon resin is embedded can better retain the ultrastructure of a sample, and is more beneficial to continuous ultrathin section and three-dimensional electron microscope reconstruction. After mEosEM, scientists have found that some of the existing fluorescent proteins, such as mKate2, mWasabi, coGFPv0, mCherry2, mEosEM-E, mScarlet series, hfYFP, etc., can still retain fluorescent signals during conventional electron microscopy sample preparation. However, after fixation with 1% osmium acid, the residual fluorescence signal ratio was only about 10%. If Epon embedded, the fluorescence signal will remain less. Therefore, more fluorescent proteins which can withstand conventional electron microscope sampling are required to widen the biological application range of the super-resolution photoelectric correlation imaging technology.

Tissue transparency technology is one of the key elements of current three-dimensional fluorescence imaging technology for organisms, because the technology can enable biological tissues to become transparent, thereby realizing visualization of deep tissues and complex tissue structures. These methods include removing lipids and other light scattering components from the tissue, allowing the light to penetrate deeper into the tissue and reducing light scattering. Due to the limitation of the thermal stability of the current fluorescent proteins, the tissue transparentization technology is carried out at room temperature or below 37 ℃ so as to avoid the loss of fluorescent signals caused by the severe change of the temperature of the fluorescent proteins as far as possible. Thus, the tissue transparentization process is extremely time consuming, often requiring a week to several weeks to achieve optimal transparency. Increasing the temperature may accelerate the tissue transparentization process, but may affect the stability of the fluorescent protein. Green thermostable fluorescent proteins have been successfully developed heretofore. Despite these advances, there remains a great need for red thermostable fluorescent proteins in order to speed up the tissue transparentization process without affecting the imaging quality.

Structured light illumination imaging (SIM) is widely regarded as the preferred super-resolution imaging method for live cell imaging due to its high imaging efficiency and low phototoxicity. Compared with two-dimensional structured light illumination imaging, three-dimensional structured light illumination imaging puts more stringent requirements on the light stability of fluorescent probes due to its unique imaging process. First, three-dimensional structured light illumination imaging captures and merges 15 images (5 phases×3 angles) per frame, the number of images being significantly greater than two-dimensional structured light illumination imaging. Secondly, in the three-dimensional imaging process, the three-dimensional structured light irradiates the fluorescent probe above and below the focal plane at the same time when imaging. This omnidirectional excitation further exacerbates the risk of photobleaching of the fluorescent probe. Although StayGold and its variants as reported so far can achieve long duration three-dimensional structured light illumination imaging of a single channel, there is a lack of a red light stable fluorescent protein co-labeled with Staygold to achieve long duration three-dimensional structured light illumination imaging.

In stimulated emission depletion ultra-high resolution imaging (STED), a tightly focused excitation beam is raster scanned over the sample, followed by a doughnut-shaped depletion beam, which effectively reduces the excitation spot. However, extremely high demands are made on the photostability of fluorescent probes. Although organic dyes are commonly used for STED due to their photostability, fluorescent probes have significant advantages in specificity and gene targeting. Thus, there is a significant need for red light stable fluorescent proteins to expand the spectral range of fluorescent probes used in STED microscopes.

Disclosure of Invention

The core technical problem to be solved by the invention is how to improve the photo-thermal stability, osmium acidity resistance and chemical stability of the red fluorescent protein.

In order to solve the technical problems, the invention firstly provides a protein, which is A1), A2) or A3):

A1 A protein having an amino acid sequence of SEQ ID No. 4;

a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID No.4 in the sequence table, keeps the 163 th amino acid residue unchanged and has the same function;

a3 A fusion protein obtained by ligating a tag to the N-terminal or/and the C-terminal of A1) or A2).

The protein in A2) has 75% or more identity with the amino acid sequence of the protein shown in SEQ ID No.4 and has the same function. The identity of 75% or more is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

The present invention also provides the protein-related biomaterial, which is any one of the following B1) to B9):

B1 A nucleic acid molecule encoding said protein;

B2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

B4 A viral vector comprising B1) said nucleic acid molecule, or a viral vector comprising B2) said expression cassette, or a viral vector comprising B3) said recombinant vector;

B5 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

B6 A transgenic cell line comprising the nucleic acid molecule of B1) or a transgenic cell line comprising the expression cassette of B2);

b7 A transgenic tissue comprising the nucleic acid molecule of B1) or a transgenic tissue comprising the expression cassette of B2);

b8 A transgenic organ comprising B1) said nucleic acid molecule, or a transgenic organ comprising B2) said expression cassette;

b9 A transgenic animal comprising the nucleic acid molecule of B1) or a transgenic animal comprising the expression cassette of B2).

In the above biological material, the nucleic acid molecule of B1) may be B11) or B12) or B13) as follows:

b11 A cDNA molecule or a DNA molecule of SEQ ID No.3 in the sequence table;

b12 A cDNA molecule or a DNA molecule shown in SEQ ID No.3 of the sequence Listing;

b13 A cDNA molecule or a DNA molecule having 75% or more identity to the nucleotide sequence defined in b 11) or b 12) and encoding said protein.

Wherein the nucleic acid molecule may be DNA such as cDNA, genomic DNA or recombinant DNA, or RNA such as mRNA or hnRNA.

The nucleotide sequence encoding the protein of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the protein shown in SEQ ID No.4 isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein shown in SEQ ID No.4 while maintaining the amino acid residue at position 163 of SEQ ID No.4 unchanged and having the same protein function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein consisting of the amino acid sequence shown in SEQ ID No.4 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

B2 The expression cassette containing the nucleic acid molecule encoding the protein refers to a DNA capable of expressing the protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the gene encoding the protein but also a terminator for terminating transcription of the gene encoding the protein. In addition, the expression cassette may further comprise an enhancer sequence.

Recombinant vectors containing the protein-encoding gene expression cassettes can be constructed using existing expression vectors.

The vector may be a plasmid, cosmid, phage or viral vector. The plasmid may be specifically a pET-28a (+) vector.

B3 Specifically, the recombinant vector can be pET-28a (+) -mBaoHong, wherein pET-28a (+) -mBaoHong is obtained by replacing a DNA fragment between BamHI and NotI recognition sequences of the pET-28a (+) vector with mBaoHong genes.

The microorganism may be yeast, bacteria, algae or fungi. The bacterium may be E.coli.

The cells may be plant cells or animal cells. In one embodiment of the present invention, hela cells are taken as a reference example.

The transgenic cell line, transgenic tissue, transgenic organ, and transgenic animal may each include no propagation material, or may include propagation material.

The use of said proteins as fluorescent proteins is also within the scope of the present invention.

The invention also provides a method for locating a target protein, which comprises the steps of connecting a coding gene of the protein and a coding gene of the target protein, introducing the obtained product into an isolated target cell, target tissue, target organ or target individual, enabling the target cell, the target tissue, the target organ or the target individual to express a fusion protein formed by the protein and the target protein, and detecting fluorescent signals of the protein in the target cell, the target tissue, the target organ or the target individual to realize the locating of the target protein.

In the above method, the gene encoding a protein and the gene encoding a target protein may be introduced into the target cell, the target tissue, the target organ or the target individual by an expression vector containing the gene encoding a protein and the gene encoding a target protein.

In the above method, the method may use Epon resin embedding method to embed the target cell, the target tissue, the target organ or the target individual.

The method may employ an osmium acid fixation method to fix the cells of interest, the tissue of interest, the organ of interest, or the individual of interest.

In the above method, the target cell, the target tissue, the target organ, or the target individual may be in an environment of 90 ℃ or less than 90 ℃.

The color-developing agent containing the protein or the biological material also falls within the scope of the present invention.

The invention also provides any one of the following applications of the protein or the biological material:

X1) protein labelling;

X2) fluorescence imaging;

x3) photo-electric correlated microscopy imaging;

x4) tracking the structure and/or morphology of proteins, subcellular organelles, local areas of cells, or small animal embryos;

X5) resolving the structure and/or localization of the target protein;

X6) preparing a protein-tagged product;

X7) preparing a fluorescent imaging product;

x8) preparing a photo-electric correlation microscopic imaging product;

X9) preparing a structural and/or morphological product that tracks proteins, subcellular organelles, local areas of cells, cells or small animal embryos;

X10) preparing a structure and/or localization product for resolving the target protein;

X11) preparing related viruses or transgenic animals, and marking specific proteins, specific cells or specific organelles to realize rapid transparent tissue imaging and expansion super-resolution imaging.

The above-mentioned fluorescence imaging includes conventional fluorescence imaging and ultra-high resolution fluorescence imaging.

The conventional fluorescence imaging described above includes, but is not limited to, wide-field fluorescence imaging, confocal microscopy imaging, total internal reflection fluorescence imaging, living cell fluorescence imaging, and the like.

The ultra-high resolution fluorescent imaging includes, but is not limited to, optical fluctuation ultra-high resolution imaging (SOFI), structured light illumination imaging (SIM), nonlinear structured light illumination imaging (PA NL-SIM), stimulated emission depletion ultra-high resolution imaging (STED), reversible saturable optical fluorescence transition super-resolution imaging (RESOLFT), and the like.

The above-mentioned photo-electric correlated microscopy imaging may be super-resolution photo-electric correlated microscopy imaging.

The fluorescence imaging in the photoelectric correlation microscopy imaging includes, but is not limited to, wide-field fluorescence imaging, confocal microscopy imaging, total internal reflection fluorescence imaging, living cell fluorescence imaging, optical fluctuation ultra-high resolution imaging (SOFI), structured light illumination imaging (SIM), nonlinear structured light illumination imaging (PA NL-SIM), stimulated emission depletion ultra-high resolution imaging (STED), reversible saturated optical fluorescence transition super-resolution imaging (RESOLFT), and the like.

The electron microscope imaging in the photoelectric correlation microscopic imaging includes but is not limited to scanning electron microscope, transmission electron microscope and refrigeration electron microscope. The electron microscope sample embedding method comprises, but is not limited to, hydrophobic resin, hydrophilic resin and low-temperature resin embedding method.

The fluorescent protein provided by the invention can be applied to photoelectric correlation microscopic imaging, including but not limited to two-dimensional mirror-electron microscope combined imaging, three-dimensional continuous slice mirror-electron microscope combined imaging and the like.

Experiments prove that the protein shown in SEQ ID No.4 can be used as fluorescent protein, and the fluorescent protein has good photo-thermal stability, osmium acidity resistance and chemical stability. Based on the characteristics, the fluorescent protein can be independently used in the fields of protein marking, fluorescent imaging, living cell dynamic imaging and super-resolution photoelectric correlation microscopic imaging, can be used for tracking the structural and morphological changes of samples such as proteins, subcellular organelles, cell local areas, cells, small animal embryos and the like, can be used for packaging related viruses or transgenic animals, marking specific proteins, specific cells or specific organelles, realizing rapid transparent tissue imaging and swelling super-resolution imaging, and has high fluorescence intensity after conventional chemical electron microscope sample preparation. Therefore, the fluorescent protein has wide application prospect.

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

Drawings

FIG. 1 is a photostability test of fluorescent proteins. * Indicating that significance analysis reached p <0.00001.

FIG. 2 shows the thermal stability of fluorescent proteins. The temperature is kept for 1h at intervals of 5 ℃ in the interval of 60 ℃ to 90 ℃.

FIG. 3 shows an anti-osmium acid test of fluorescent proteins, with an anti-osmium acidity test time of 10min. * Indicating that significance analysis reached p <0.00001.

FIG. 4 shows the percent fluorescence retention under a fluorescence microscope after transfection of Hela cells with fluorescent proteins. mScarlet-3 is mScarlet. * Indicating that significance analysis reached p <0.00001.

FIG. 5 is a chemical stability test of fluorescent proteins. Wherein, the pH represents the change condition of the pH value of the system after adding guanidine hydrochloride, guanidine thiocyanate or urea with corresponding concentration.

Detailed Description

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were each set up for at least three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

The preparation of the mito-mCherry2 vector comprises the steps of replacing a DNA fragment between NheI and AgeI recognition sequences of a pEGFP-N1 vector with a mitochondrial localization sequence to obtain a recombinant vector mito-EGFP, and replacing a DNA fragment containing an EGFP gene between AgeI and NotI recognition sequences of the mito-EGFP with an mCherry2 gene to obtain the recombinant vector mito-mCherry2.

The mitochondrial localization sequences were as follows:

ATGTCCGTCCTGACGCCGCTGCTGCTGCGGGGCTTGACAGGCTCGGCCCGGCGGCTCCCAGTGCCGCGCGCCAAG ATCCATTCGTTGGGGGATCTGTCCGTCCTGACGCCGCTGCTGCTGCGGGGCTTGACAGGCTCGGCCCGGCGGCTCCCAG TGCCGCGCGCCAAGATCCATTCGTTGGGGGAT.

The mCherry2 gene sequence is as follows:

ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGC

TCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGA

AGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGT

GAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTC

GAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCG

GCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCC

CGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAG

ACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACA

ACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAG。

examples 1, mBaoHong are fluorescent proteins with good photo-thermal stability and strong fluorescent signals

1. Mutant production and construction of recombinant vector

According to the protein sequence information of the disclosed fluorescent protein mScarlet3, the gene sequence of mScarlet3 is artificially synthesized and constructed on a carrier targeting mitochondria to obtain mito-mScarlet3. Mutation of methionine residue 163 of mScarlet to histidine residue gave mBaoHong, and the recombinant vector containing mBaoHong gene was designated mito-mBaoHong.

Wherein, the amino acid sequences of mScarlet and mBaoHong are respectively shown as SEQ ID No.2 and SEQ ID No.4 in the sequence table, and the DNA sequences of mScarlet gene and mBaoHong gene are respectively shown as SEQ ID No.1 and SEQ ID No.3 in the sequence table.

Construction of recombinant vector:

Mito-mScarlet A DNA fragment between AgeI and NotI recognition sequences of the Mito-mCherry2 vector is replaced by mScarlet gene, and the obtained recombinant vector is Mito-mScarlet3, and the recombinant vector can express mScarlet protein.

Mito-mBaoHong the DNA fragment between the AgeI and NotI recognition sequences of the Mito-mCherry2 vector is replaced with mBaoHong gene, and the recombinant vector obtained is Mito-mBaoHong, and the recombinant vector can express mBaoHong protein.

Mito-mScarlet-H the DNA fragment between AgeI and NotI recognition sequences of the Mito-mCherry2 vector is replaced by mScarlet-H gene, and the obtained recombinant vector is Mito-mScarlet-H, and the recombinant vector can express mScarlet-H protein.

MScarlet-H Gene:

ATGGTGAGCAAGGGAGAGGCCGTGATCAAGGAGTTCATGAGATTCAAGGTGCACATGGAGGGAAGCATGAACGGACACGAGTTCGAGATCGAGGGCGAGGGCGAGGGAAGGCCATACGAGGGGACCCAGACAGCAAAGCTGAAGGTGACAAAGGGCGGACCCCTGCCTTTTAGCTGGGACATCCTGAGCCCACAATTTATGTATGGCAGCAGAGCCTTTattAAGCACCCCGCCGACATCCCCGACTACTACAAACAATCCTTTCCCGAGGGCTTTAAATGGGAACGGGTGATGAACTTCGAAGACGGCGGCGCCGTGACCGTGACCCAGGATACCAGCCTGGAAGACGGAACCCTGATCTACAAAGTGAAGCTCAGAGGCACCAACTTCCCCCCCGACGGCCCCGTTATGCAGAAGAAGACAATGGGTTGGGAGGCCAGCACCGAGAGACTCTACCCCGAGGACGGCGTGCTCAAGGGCGACATTAAGCATGCACTGCGCCTGAAGGACGGCGGAAGATACCTGGCCGACTTCAAGACCACCTACAAGGCCAAAAAGCCCGTGCAGATGCCCGGCGCATACAACGTGGACAGAAAGCTGGACATAACCAGCCACAACGAAGACTACACCGTGGTGGAGCAATACGAGAGAAGCGAAGGAAGGCACAGCACAGGAGGCATGGATGAGCTGTATAAA.

mScarlet-H protein:

MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIK HPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPE DGVLKGDIKHALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYK.

The mito-mScarlet-I3 is that the DNA fragment between AgeI and NotI recognition sequences of the mito-mCherry2 vector is replaced by mScarlet-I3 gene, and the obtained recombinant vector is mito-mScarlet-I3, and the recombinant vector can express mScarlet-I3 protein.

MScarlet-I3 Gene:

ATGGATAGCACCGAGGCAGTGATCAAGGAGTTCATGCGGTTCAAGGTGCACATGGAGGGCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCTCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAGGGCCTTCATCAAGCACCCCGCCGACATCCCCGACTACTGGAAGCAGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGATCTTCGAGGACGGCGGCACCGTGTCCGTGACCCAGGACACCTCCCTGGAGGACGGCACCCTGATCTACAAGGTGAAGCTCCGCGGCGGCAACTTCCCTCCTGACGGCCCCGTAATGCAGAAGCGGACAATGGGCTGGGAAGCATCCACCGAGCGGTTGTACCCCGAGGACGTCGTGCTGAAGGGCGACATTAAGATGGCCCTGCGCCTGAAGGACGGCGGCCGGTACCTGGCGGACTTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGATGCCCGGCGCCTTCAACATCGACCGCAAGTTGGACATCACATCCCACAACGAGGACTACACCGTGGTGGAACAGTACGAACGCTCCGTGGCCCGCCACTCCACCGGCGGCTCCGGTGGCTCCTAA.

mScarlet-I3 protein:

MDSTEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIKH PADIPDYWKQSFPEGFKWERVMIFEDGGTVSVTQDTSLEDGTLIYKVKLRGGNFPPDGPVMQKRTMGWEASTERLYPED VVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHSTGGSGGS.

Mito-mScarlet the DNA fragment between the AgeI and NotI recognition sequences of the Mito-mCherry2 vector is replaced with mScarlet gene, and the recombinant vector obtained is Mito-mScarlet, and the recombinant vector can express mScarlet protein.

MScarlet Gene:

ATGGTGAGCAAGGGAGAGGCCGTGATCAAGGAGTTCATGAGATTCAAGGTGCACATGGAGGGAAGCATGAACGGACACGAGTTCGAGATCGAGGGCGAGGGCGAGGGAAGGCCATACGAGGGGACCCAGACAGCAAAGCTGAAGGTGACAAAGGGCGGACCCCTGCCTTTTAGCTGGGACATCCTGAGCCCACAATTTATGTATGGCAGCAGAGCCTTTACCAAGCACCCCGCCGACATCCCCGACTACTACAAACAATCCTTTCCCGAGGGCTTTAAATGGGAACGGGTGATGAACTTCGAAGACGGCGGCGCCGTGACCGTGACCCAGGATACCAGCCTGGAAGACGGAACCCTGATCTACAAAGTGAAGCTCAGAGGCACCAACTTCCCCCCCGACGGCCCCGTTATGCAGAAGAAGACAATGGGTTGGGAGGCCAGCACCGAGAGACTCTACCCCGAGGACGGCGTGCTCAAGGGCGACATTAAGATGGCACTGCGCCTGAAGGACGGCGGAAGATACCTGGCCGACTTCAAGACCACCTACAAGGCCAAAAAGCCCGTGCAGATGCCCGGCGCATACAACGTGGACAGAAAGCTGGACATAACCAGCCACAACGAAGACTACACCGTGGTGGAGCAATACGAGAGAAGCGAAGGAAGGCACAGCACAGGAGGCATGGATGAGCTGTATAAA.

mScarlet protein:

MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTK HPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPE DGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYK.

Mito-mScarlet-I the DNA fragment between AgeI and NotI recognition sequences of the Mito-mCherry2 vector is replaced with mScarlet-I gene, and the obtained recombinant vector is Mito-mScarlet-I, and the recombinant vector can express mScarlet-I protein.

MScarlet-I Gene:

ATGGTGAGCAAGGGAGAGGCCGTGATCAAGGAGTTCATGAGATTCAAGGTGCACATGGAGGGAAGCATGAACGGACACGAGTTCGAGATCGAGGGCGAGGGCGAGGGAAGGCCATACGAGGGGACCCAGACAGCAAAGCTGAAGGTGACAAAGGGCGGACCCCTGCCTTTTAGCTGGGACATCCTGAGCCCACAATTTATGTATGGCAGCAGAGCCTTTattAAGCACCCCGCCGACATCCCCGACTACTACAAACAATCCTTTCCCGAGGGCTTTAAATGGGAACGGGTGATGAACTTCGAAGACGGCGGCGCCGTGACCGTGACCCAGGATACCAGCCTGGAAGACGGAACCCTGATCTACAAAGTGAAGCTCAGAGGCACCAACTTCCCCCCCGACGGCCCCGTTATGCAGAAGAAGACAATGGGTTGGGAGGCCAGCACCGAGAGACTCTACCCCGAGGACGGCGTGCTCAAGGGCGACATTAAGATGGCACTGCGCCTGAAGGACGGCGGAAGATACCTGGCCGACTTCAAGACCACCTACAAGGCCAAAAAGCCCGTGCAGATGCCCGGCGCATACAACGTGGACAGAAAGCTGGACATAACCAGCCACAACGAAGACTACACCGTGGTGGAGCAATACGAGAGAAGCGAAGGAAGGCACAGCACAGGAGGCATGGATGAGCTGTATAAA.

mScarlet-I protein:

MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIK HPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPE DGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYK.

Mito-oScarlet the DNA fragment between the AgeI and NotI recognition sequences of the Mito-mCherry2 vector is replaced with oScarlet gene, and the recombinant vector obtained is Mito-oScarlet, and the recombinant vector can express oScarlet protein.

OScarlet Gene:

ATGGTGAGCAAGGGAGAGGCCGTGATCAAGGAGTTCATGAGATTCAAGGTGCACATGGAGGGAAGCATGAACGGACACGAGTTCGAGATCGAGGGCGAGGGCGAGGGAAGGCCATACGAGGGGACCCAGACAGCAAAGCTGAAGGTGACAAAGGGCGGACCCCTGCCTTTTAGCTGGGACATCCTGAGCCCACAATTTATGTATGGCAGCAGAGCCTTTACCAAGCACCCCGCCGACATCCCCGACTACTACAAACAATCCTTTCCCGAGGGCTTTAAATGGGATCGGGTGATGAACTTCGAAGACGGCGGCGCCGTGACCGTGACCCAGGATACCAGCCTGGAAGACGGAACCCTGATCTACAAAGTGAAGCTCAGAGGCACCAACTTCCCCCCCGACGGCCCCGTTATGCAGAAGAAGACAATGGGTTGGGAGGCCAGCACCGAGAGACTCTACCCCGAGGACGGCGTGCTCAAGGGCGACATTAAGATGGCACTGCGCCTGAAGGACGGCGGAAGATACCTGGCCGACTTCAAGACCACCTACAAGGCCAAAAAGCCCGTGCAGATGCCCGGCGCATACAACGTGGACAGAAAGCTGGACATAACCAGCCACAACGAAGACTACACCGTGGTGGAGCAATACGAGAGAAGCGAAGGAAGGCACAGCACAGGAGGCATGGATGAGCTGTATAAA.

oScarlet protein:

MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTK HPADIPDYYKQSFPEGFKWDRVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPE DGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYK.

2. Determination of fluorescent protein Properties

1. Construction of recombinant vectors

The mScarlet, mScarlet-I, oScarlet, mScarlet-H, mScarlet-I3, mScarlet3 and mBaoHong genes were constructed on pET-28a (+) vector as follows:

The DNA fragment between BamHI and NotI recognition sequences of the pET-28a (+) vector is replaced by mScarlet genes, the obtained recombinant vector is marked as pET-28a (+) -mScarlet, the recombinant vector contains mScarlet genes shown as SEQ ID No.1 and can express fusion protein formed by fusing mScarlet proteins shown as SEQ ID No.2 with polypeptides containing 6 XHis tags (the sequences of the fusion protein are SEQ ID No. 5) at the C end;

the DNA fragment between BamHI and NotI recognition sequences of the pET-28a (+) vector is replaced by mBaoHong genes, the obtained recombinant vector is marked as pET-28a (+) -mBaoHong, and the recombinant vector contains mBaoHong genes shown as SEQ ID No.3 and can express fusion protein formed by fusing mBaoHong proteins shown as SEQ ID No.4 with the C end of polypeptide containing a 6 XHis tag (the sequence of the fusion protein is SEQ ID No.5 in a sequence table);

The DNA fragment between BamHI and NotI recognition sequences of the pET-28a (+) vector is replaced by mScarlet-H gene, the obtained recombinant vector is marked as pET-28a (+) -mScarlet-H, the recombinant vector contains mScarlet-H gene and can express fusion protein formed by fusing mScarlet-H protein with the C end of polypeptide containing 6 XHis tag (the sequence of which is SEQ ID No.5 in a sequence table);

The DNA fragment between BamHI and NotI recognition sequences of pET-28a (+) vector is replaced by mScarlet-I3 gene, the obtained recombinant vector is marked as pET-28a (+) -mScarlet-I3, the recombinant vector contains mScarlet-I3 gene and can express fusion protein formed by C-terminal fusion mScarlet-I3 protein of polypeptide containing 6 XHis tag (the sequence of which is SEQ ID No.5 in a sequence table);

The DNA fragment between BamHI and NotI recognition sequences of the pET-28a (+) vector is replaced by mScarlet genes, the obtained recombinant vector is marked as pET-28a (+) -mScarlet, and the recombinant vector contains mScarlet genes and can express fusion protein formed by C-terminal fusion mScarlet protein of polypeptide containing a 6 XHis tag (the sequence of the fusion protein is SEQ ID No.5 in a sequence table);

The DNA fragment between BamHI and NotI recognition sequences of pET-28a (+) vector is replaced by mScarlet-I gene, the obtained recombinant vector is marked as pET-28a (+) -mScarlet-I, the recombinant vector contains mScarlet-I gene and can express fusion protein formed by fusing mScarlet-I protein with C end of polypeptide containing 6 XHis tag (the sequence of which is SEQ ID No.5 in a sequence table);

The DNA fragment between BamHI and NotI recognition sequences of pET-28a (+) vector is replaced by oScarlet gene, the obtained recombinant vector is marked as pET-28a (+) -oScarlet, the recombinant vector contains oScarlet gene and can express fusion protein formed by fusing oScarlet protein with 6 XHis tag-containing polypeptide (the sequence of which is SEQ ID No. 5) at the C end.

2. Expression and purification of fluorescent proteins

Expression of fluorescent protein:

And (3) respectively introducing the recombinant vectors in the step (1) into BL21 escherichia coli to obtain corresponding recombinant bacteria. The recombinant bacteria are inoculated into 1mL LB culture medium containing kana antibiotics according to the standard of 1ng/100 mu L, and after the bacteria liquid is turbid and expanded into 100mL LB culture medium containing kana antibiotics, the shake culture is continued at 37 ℃ until the OD value of the bacteria liquid is about 0.6-0.8, and the shake culture is completed, and the continuous culture is carried out for 16-20h at 16 ℃ with the addition of IPTG with the final concentration of 1mM, so as to induce the expression of a large amount of proteins.

Purification of fluorescent protein:

After completion of protein-induced expression, the resulting bacterial solution was centrifuged at 7000rpm at 4℃for 5min, and the supernatant was discarded to collect the cells, and after the completion of the collection, the cells were washed with Binding buffer (20 mM Tris-HCl pH=7.4, 150mM NaCl) and resuspended and centrifuged again. The cells were resuspended in 10mL Binding buffer again and subjected to ultrasound disruption in an ice-water bath (2 s excess, 4s rest, total ultrasound duration 30min; power: 60%). After completion of the sonication, the protein supernatant was collected by centrifugation at 10000rpm for 30min at 4 ℃.

The nickel column was treated by eluting ethanol from the column, washing 25 column volumes with ddH ₂ O, adding 0.2MNiSO ₄·6H₂ O5 column volumes, washing 25 column volumes with ddH ₂ O, and washing 5 column volumes with Binding buffer. The protein supernatant was transferred to a pretreated nickel column and incubated in a silent mixer for 2-3h at 4 ℃. After the incubation, the hybrid proteins were gradient eluted, first with a pre-chilled low concentration imidazole solution (20 mM imidazole, 20mM Tris-HClpH=7.4, 150mM NaCl), then with a 35mM imidazole solution (35 mM imidazole, 20mM Tris-HClpH=7.4, 150mM NaCl) followed by further washing, finally with a 40mM imidazole solution (40 mM imidazole, 20mM Tris-HClpH=7.4, 150mM NaCl). After the completion of washing, the target protein was eluted with 300mM high concentration imidazole solution (300 mM imidazole, 20mM Tris-HCl pH=7.4, 150mM NaCl) and collected, and the protein was concentrated to about 1mL by centrifugation at 4000rpm for 10min at 4℃with a retention amount of 10KD of the ultrafilter, followed by addition of an imidazole-free Binding buffer under the above conditions three times. After concentration, protein concentration was determined using Nanodrop and split-filled into 100 μl of protein each to centrifuge tubes for storage at-80 ℃.

The protein purification system used AKTA pure, first prepared 1L ddH ₂ O, binding buffer (20 mM Tris-HClpH=7.4, 150mM NaCl), 0.5M NaOH aqueous solution, required 0.22 μm pore size filter membrane for suction filtration, and 500mL ultrasonic 20% ethanol aqueous solution. The A pump used for AKTA pure was cleaned with ddH ₂ O, the flow rate was set at 1mL/min, and the gel column Superdex-75Increate 10/300GL was connected to the system during flow. The column was washed with ddH ₂ O in an amount of 1 column volume, and then 1 column volume was washed with Binding buffer at a flow rate of 0.8mL/min and a pressure limit of 5.0MPa. The sample loading head is cleaned, the sample loading volume is 2mL, the operation is carried out according to a set program, the purified protein is collected, marked clearly and concentrated to 5mg/mL, and the protein is stored at-80 ℃. After the end of the procedure, 1 column volume was washed with ddH ₂ O, 0.5M aqueous NaOH, ddH ₂ O, and 20% aqueous ethanol at a flow rate of 0.8mL/min, and the gel column was stored in 20% aqueous ethanol and removed. Finally, pump A is used for pumping wash by using 20% ethanol, and after the whole system is stored in 20% ethanol water solution, data are stored.

The resulting purified fusion proteins were mScarlet fusion protein, mScarlet-I fusion protein, oScarlet fusion protein, mScarlet-H fusion protein, mScarlet-I3 fusion protein, mScarlet3 fusion protein, and mBaoHong fusion protein, respectively.

3. Light stability test

The recombinant vector mito-mBaoHong is transfected into Hela cells, and the light stability of the recombinant vector is compared with that of fluorescent protein mScarlet-H with the best light stability and the best anti-electron microscope sample preparation effect at present under the same parameter setting condition. As a result, as shown in FIG. 1, under the same parameter setting conditions, the light stability of mBaoHong was much higher than mScarlet-H (a in FIG. 1), and the time for fluorescence to drop to half value was longer than mScarlet-H (b in FIG. 1).

The parameters were set by selecting 561nm excitation channel and setting the intensity to 5.006mW.

4. Thermal stability and osmium acid resistance test

And (3) thermal stability test, namely diluting the purified fluorescent protein obtained in the step (2) to 0.02mg/mL by using a Binding buffer (pH=7.4), detecting a fluorescent signal after heat preservation for 1h from 60 ℃ to 90 ℃ by using an enzyme-labeling instrument, setting an excitation wavelength to 561nm, setting an emission wavelength to 590nm, and eliminating background influence by using the Binding buffer (pH=7.4) as a blank control. As shown in FIG. 2, mBaoHong retains fluorescence after 90℃treatment far higher than mScarlet-H and mScarlet.

The osmium acid resistance test was performed at room temperature, the purified fusion protein obtained in the step 2 was diluted to 0.02mg/mL with Binding buffer (pH=7.4), then OsO ₄ having a final concentration of 1g/100mL was added and a control (OsO ₄ having a final concentration of 1g/100mL was added to the Binding buffer (pH=7.4)), fluorescence was recorded after 10 minutes using a bottom reader of an enzyme-labeled instrument, excitation wavelength was set to 561nm, emission wavelength was set to 590nm, and background influence was eliminated using the Binding buffer (pH=7.4) as a blank control. As shown in FIG. 3, mBaoHong has a much higher percent fluorescence retention after treatment with 1% osmium acid than mScarlet-H, mScarlet, mScarlet-I, oScarlet, mScarlet3, mScarlet-I3.

5. Anti-electron microscope sample preparation detection

And (3) respectively transfecting the recombinant vector (mito-mScarlet,mito-mScarlet-I,mito-mScarlet3,mito-mScarlet-I3,mito-oScarlet,mito-mScarlet-H,mito-mBaoHong) in the step one into Hela cells, performing digestion and fixation for electron microscopy sample preparation after 48 hours, and performing fluorescence detection and comparison on ultrathin sections. The method comprises the following specific steps:

when the density of the Hela cells in the growth log phase reaches about 80%, the recombinant vector is transfected into the Hela cells, the cells are fixed after 48 hours of transfection for fully folding and fully maturing fluorescent proteins, and the electron microscope sample preparation is started, wherein the sample preparation steps are as follows in sequence:

a. Cells transfected with fluorescent protein for 48h were trypsinized and centrifuged at 1000 Xg for 10min, after which the medium was aspirated and fixative (final concentration 4% PFA+0.25% GA+0.01M PBS) was added and incubated overnight at 4 ℃.

B. The fixative was aspirated and washed three times with 0.01M PBS for 10min each.

C. 1g/100mL osmium acid (solvent is Binding buffer (pH=7.4)) was added and fixed at 4℃for 1h.

D. the fixative was removed and washed three times with ddH ₂ O for 10min each.

E. Adding 2% alcohol uranium dye solution (obtained by dissolving uranium with ethanol, wherein the concentration of uranium is 2g/100 ml), and carrying out 4 ℃ light-proof uranium dyeing for 1h.

F. The 2% alcohol uranium dye solution was removed and washed three times with ddH ₂ O for 10min each.

G. Gradient dehydration, using 30%, 50%, 70%, 80%, 90% ethanol aqueous solution for 10min each, 100% ethanol for two times for 10min each.

H. the anhydrous acetone is dehydrated for 3 times and 10 minutes each time.

I. and (3) respectively treating the permeate solutions with acetone and resin proportions of 3:1, 1:1 and 1:3 for 1h, 2h and 3h, wherein the resin formula is Epon 812 11.05g,DDSA 6.1879g,NMA 6.5426g,DMP-30.3914 g.

J. The resin was used 3 times, 12h each.

K. The resin coated samples were placed in a 60 ℃ oven for polymerization for 14-16h.

After the polymerization was completed, the resin block was trimmed using an ultra-thin microtome, and the sample was cut into an ultra-thin slice having a thickness of 100nm, and a fluorescent signal after fluorescent protein electron microscopy was examined using a fluorescent microscope.

As shown in FIG. 4, the 561nm excitation channel is selected under the same parameter setting condition, the light intensity is set to be 10mW, and the fluorescence intensity after the mito-mBaoHong sample preparation is the strongest and is far higher than that of the template mScarlet3 and other fluorescent proteins, mScarlet, mScarlet-I, oScarlet, mScarlet-3 and mScarlet-I3.

6. Chemical stability test

And (3) chemical stability test, namely diluting the purified fluorescent protein obtained in the step (2) to 0.02mg/mL by using Binding buffer (pH=7.4), adding guanidine hydrochloride, guanidine thiocyanate or urea into the fluorescent protein, detecting fluorescent signals of the purified fluorescent protein after being treated for 16 hours by using a microplate reader through 1M-8M guanidine hydrochloride, 1M-4M guanidine thiocyanate and 1-10M urea respectively, setting an excitation wavelength to 561nm, setting an emission wavelength to 590nm, and using the Binding buffer (pH=7.4) as a blank control to eliminate background influence. As shown in FIG. 5, mBaoHong showed higher fluorescence retention after 90℃treatment than mScarlet-H and mScarlet3.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims (10)

1. Protein, A1), A2) or A3) as follows:

A1 A protein having an amino acid sequence of SEQ ID No. 4;

a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID No.4 in the sequence table, keeps the 163 th amino acid residue unchanged and has the same function;

a3 A fusion protein obtained by ligating a tag to the N-terminal or/and the C-terminal of A1) or A2).

2. A biological material related to the protein of claim 1, which is any one of the following B1) to B8):

b1 A nucleic acid molecule encoding the protein of claim 1;

B2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

B4 A viral vector comprising B1) said nucleic acid molecule, or a viral vector comprising B2) said expression cassette, or a viral vector comprising B3) said recombinant vector;

B5 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

B6 A transgenic cell line comprising the nucleic acid molecule of B1) or a transgenic cell line comprising the expression cassette of B2);

b7 A transgenic tissue comprising the nucleic acid molecule of B1) or a transgenic tissue comprising the expression cassette of B2);

b8 A transgenic organ comprising the nucleic acid molecule of B1) or a transgenic organ comprising the expression cassette of B2).

3. The biological material according to claim 2, wherein the nucleic acid molecule B1) is B11) or B12) or B13) as follows:

b11 A cDNA molecule or a DNA molecule of SEQ ID No.3 in the sequence table;

b12 A cDNA molecule or a DNA molecule shown in SEQ ID No.3 of the sequence Listing;

b13 A cDNA molecule or a DNA molecule which has 75% or more identity to the nucleotide sequence defined in b 11) or b 12) and which encodes a protein according to claim 1.

4. Use of the protein of claim 1 as a fluorescent protein.

5. A method for localizing a target protein, comprising ligating the gene encoding the protein according to claim 1 with the gene encoding the target protein, introducing the ligated gene into a target cell, target tissue, target organ or target individual, allowing the target cell, target tissue, target organ or target individual to express a fusion protein formed by the protein according to claim 1 and the target protein, and detecting a fluorescent signal of the protein according to claim 1 in the target cell, target tissue, target organ or target individual to localize the target protein.

6. The method according to claim 5, wherein the gene encoding the protein of claim 1 and the gene encoding the target protein are introduced into the target cell, the target tissue, the target organ or the target individual via an expression vector containing the gene encoding the protein and the gene encoding the target protein.

7. The method according to claim 5 or 6, wherein the method comprises embedding the target cell, the target tissue, the target organ or the target individual by using an Epon resin embedding method;

and/or, the method employs an osmium acid fixation method to fix the cells of interest, the tissue of interest, the organ of interest, or the individual of interest.

8. The method according to claim 5 to 7, wherein the target cell, the target tissue, the target organ or the target individual is in an environment of 90℃or less than 90 ℃.

9. A color developer comprising the protein of claim 1 or the biomaterial of claim 2 or 3.

10. Use of a protein according to claim 1 or any of the following biological materials according to claim 2 or 3:

X1) protein labelling;

X2) fluorescence imaging;

x3) photo-electric correlated microscopy imaging;

x4) tracking the structure and/or morphology of proteins, subcellular organelles, local areas of cells, or small animal embryos;

X5) resolving the structure and/or localization of the target protein;

X6) preparing a protein-tagged product;

X7) preparing a fluorescent imaging product;

x8) preparing a photo-electric correlation microscopic imaging product;

X9) preparing a structural and/or morphological product that tracks proteins, subcellular organelles, local areas of cells, cells or small animal embryos;

X10) preparing a structure and/or localization product for resolving the target protein;

X11) preparing related viruses or transgenic animals, and marking specific proteins, specific cells or specific organelles to realize rapid transparent tissue imaging and expansion super-resolution imaging.