CN113321717B - LOV protein mutant and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to LOV protein mutants and applications thereof. The invention discloses a preparation method of a gene and protein with good biocompatibility and automatic assembly with intracellular flavin to form a high-efficiency fluorescent probe, and a method and application for assembling the intracellular flavin to form a high-efficiency fluorescent probe. This series of fluorescent proteins can be used as fluorescent probes in the field of biological and medical molecular monitoring, and can also be used as a basis to develop more efficient fluorescent probes or photosensitizers for applications in biology, medicine and other fields.

Description

LOV protein mutants and their applications

technical field

The invention belongs to the technical field of biological materials, and particularly relates to LOV protein mutants and applications thereof.

Background technique

With the expansion and deepening of scientific research in the biological field, the application scope and application field of fluorescent probes are no longer limited to the biological field that was originally discovered, but also mainly used in the qualitative and quantitative analysis and detection of biological molecules, proteins, cells, etc. In experiment. Among them, researches such as expanding fluorescent probes in different wavelength bands and labeling organelles commonly used in biological research have also become hot spots. Commonly used fluorescent molecular probes for molecular detection and imaging of proteins typically consist of fluorescent chromophore-ligand conjugates, enabling them to target and thus label target proteins with high selectivity.

The most well-known fluorescent reporter protein is green fluorescent protein (GFP). GFP can be expressed across a broad host range of different organisms and used as real-time fluorescent probes to monitor protein expression, localization, folding, and interactions in living cells. However, GFP family proteins require molecular oxygen for fluorescence maturation, which hinders their application in strictly anaerobic microorganisms, resulting in limited applications as fluorescent reporter molecules.

The flavin mononucleotide-based photoreceptor protein (FbFP) was successfully adapted as an oxygen-independent fluorescent probe and also a photosensitizer in aerobic and anaerobic biological systems. The LOV domain belongs to FbFP. In recent years, modifying the LOV domain to play its role and expand its application has become a research direction that researchers are keen on. Their autofluorescence can not only be used for precise positioning and tracking of intracellular targets such as organelles, etc. , and may also become a gene-encoded photosensitizer valuable for clinical or life applications in various fields.

SUMMARY OF THE INVENTION

In view of the problems existing in the prior art, the present invention provides a LOV protein mutant and application thereof, aiming to solve some problems in the prior art or at least alleviate some of the problems in the prior art.

In order to overcome the deficiencies in the prior art that the application sensitivity and application range of fluorescent reporter proteins are limited by conditions such as themselves or application objects, the present invention evolves the rice LOV gene mutation into high-efficiency fluorescent proteins, namely LORO1.1-1.8. LORO1.1-1.8 can automatically assemble the intracellular pigment FMN in model organisms and express to form a highly efficient fluorescent protein with high fluorescence quantum yield, which further proves that LORO1.6 and LORO1.8 can act as fluorescent proteins in mammalian cells. probe.

By performing point saturation mutation on key sites in the LOV domain and combining its mutation effect, the mutants were analyzed by spectral analysis, fluorescence quantum yield and molar extinction coefficient and other parameters. Enhanced mutants with better activity are used to research and develop fluorescent protein bioprobes in the blue-violet light region, broaden the application range of FMN and LOV, and develop new intracellular pigment-type fluorescent proteins with high stability and strong brightness: LORO1.1-1.8. Because FMN is an endogenous pigment in cells, it is beneficial to bind to LORO1.1-1.8 as a fluorescent probe, and at the same time it binds to LORO1.1-1.8, which can be induced in vitro or in vivo by light at specific locations in cells or throughout the body Generate reactive oxygen species, which can act as both fluorescent probes and genetically encoded photosensitizers.

The present invention will provide a fluorescent protein with good biocompatibility and automatic assembly with intracellular pigment FMN with high pigment binding ability and high fluorescence quantum yield: LORO1.1-1.8. Among them, LORO1.6 and LORO1.8 can be used as fluorescent probes in mammalian cells.

The present invention is realized by the LOV protein mutant, the amino acid sequence of the mutant is any one of SEQ ID NO.2-SEQ ID NO.9.

The present invention also provides the application of the above-mentioned LOV protein mutants in the preparation of fluorescent probes.

Further, the LOV protein mutant is combined with FMN cytochrome as a fluorescent probe.

Further, the fluorescent probe is applied to mammalian cells.

Further, the mammalian cells include HEK293T cells.

The present invention also provides the application of the above-mentioned LOV protein mutants in the preparation of gene-encoded photosensitizers.

The present invention also provides a recombinant vector, transgenic cell or recombinant bacteria, comprising the nucleotide sequence expressing the above-mentioned LOV protein mutant.

A series of fluorescent proteins LORO1.1-1.8 provided by the present invention have the maximum absorption peak at 439-445 nm and the maximum emission peak at 493-502 nm in Tris-HCl buffer solution.

A series of fluorescent proteins LORO1.1-1.8 provided by the present invention have molar extinction coefficients of 29913-39883 M ^-1 cm ^-1 in Tris-HCl buffer solution.

The fluorescence quantum yield of a series of fluorescent proteins LORO1.1-1.8 provided by the present invention in Tris-HCl buffer solution is 0.36%-0.55%.

A series of fluorescent proteins LORO1.1-1.8 provided by the present invention are excited at 400 nm and start to emit at 420 nm, and the fluorescence intensity is 1.9-2.9 times that of the wild type.

The fluorescent proteins LORO1.6 and LORO1.8 provided by the present invention have better molecular brightness in mammalian cells (HEK293T).

To sum up, the advantages and positive effects of the present invention are:

The LOV domain photoreceptor protein is soluble and small in size, only about 100-140 amino acid residues, its light-absorbing cofactor FMN exists in almost all types of cells, and does not require oxygen to participate in the maturation of the chromophore . The invention mutates and functionally evolves a LOV sequence (LOC4349531 gene in the Oryza sativa Japonica Group) in the japonica rice genome, and successfully screen out a series of mutants that can efficiently express and form fluorescent proteins, which are named LORO1.1-1.8.

The high-efficiency fluorescent protein formed by combining FMN and LORO1.1-1.8 protein in the present invention can be applied to biological qualitative and quantitative analysis, and can also be used to label or detect the intramolecular interaction, metabolite concentration and the like of living cells. Or applied to the screening of anaerobic bacteria, fluorescence lifetime imaging, and the determination of the microenvironment and local oxygen environment in living cells. It is used in various fields such as biology, medicine, structure and so on.

Description of drawings

Fig. 1 is the amino acid sequence comparison diagram of LORO1.1-1.8 fluorescent protein;

Figure 2 is the UV absorption spectrum of LORO1.1-1.4 fluorescent protein;

Figure 3 is the UV absorption spectrum of LORO1.5-1.8 fluorescent protein;

Figure 4 is the ultraviolet fluorescence spectrum of LORO1.1-1.4 fluorescent protein;

Figure 5 is the ultraviolet fluorescence spectrum of LORO1.5-1.8 fluorescent protein;

Figure 6 is an image of HEK293T transfected mammalian cells with LORO1.6 fluorescent probe (Ex 395±40nm, spectroscope 425nm, Em510nm±40nm) in pcDNA3.1:LORO1.6;

Figure 7 is the image of HEK293T transfected mammalian cells with LORO1.8 fluorescent probe (Ex 395±40nm, spectroscope 425nm, Em510nm±40nm) in pcDNA3.1:LORO1.8.

Detailed ways

In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the examples. The equipment and reagents used in each example and test example can be obtained from commercial channels unless otherwise specified. The specific embodiments described herein are only used to explain the present invention, and are not intended to limit the present invention.

From the information contained in this application, various changes to the precise description of the present invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the processes, properties or components defined, as these embodiments and other descriptions are intended to be illustrative only of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention that are obvious to those skilled in the art or related fields are intended to be within the scope of the appended claims.

For a better understanding of the invention and not to limit the scope of the invention, all numbers expressing amounts, percentages, and other numerical values used in this application should in all cases be understood as modified by the word "about". Accordingly, unless expressly stated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At a minimum, each numerical parameter shall be deemed to have been obtained from the reported significant digits and by conventional rounding methods. In the present invention, "about" means within 10% of a given value or range, preferably within 5%.

In the following embodiments of the present invention, when the temperature is not particularly limited, the conditions are all at room temperature. Normal temperature refers to the natural room temperature in the four seasons without additional cooling or heating treatment. Generally, the normal temperature is controlled at 10-30°C, preferably 15-25°C. After the protein is purified, it needs to measure the spectral data or save it in an ice bath. Generally it is 4 ℃.

The genes, proteins or fragments thereof contemplated in the present invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, plants) using recombinant techniques.

The present invention discloses LOV protein mutants and their applications. The amino acid sequence of wild-type LOC4349531 involved in the present invention is shown in SEQ ID NO.1; the amino acid sequence of LORO1.1 is shown in SEQ ID NO.2; the amino acid sequence of LORO1.2 is shown in SEQ ID NO.3; The amino acid sequence of LORO1.3 is shown in SEQ ID NO.4; the amino acid sequence of LORO1.4 is shown in SEQ ID NO.5; the amino acid sequence of LORO1.5 is shown in SEQ ID NO.6; the amino acid sequence of LORO1.6 is shown in SEQ ID NO.6 The sequence is shown in SEQ ID NO.7; the amino acid sequence of LORO1.7 is shown in SEQ ID NO.8; the amino acid sequence of LORO1.8 is shown in SEQ ID NO.9. The amino acid sequence alignment is shown in Figure 1.

The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

Example 1

1. Construction of mutants

The gene sequences in this example were synthesized by Qingke Biotechnology. The LOC4349531 gene from Oryza sativa Japonica Group was inserted into pET28 by conventional genetic engineering method, using pET28a(+) as E. coli expression vector, and the resulting plasmid was called pET28-LOC4349531. This plasmid can express LOC4349531 in E. coli.

Through site-directed mutagenesis, it was found that the 39-position cysteine of LOC4349531 plays an important role in the ability of the LOV domain to bind the intracellular pigment FMN, and the 102-position glutamine affects the state of the chromophore FMN and the electron transfer reaction it participates in (Sequence Listing The first Met in is the promoter, starting from the second position is the first amino acid of the target protein). Therefore, point saturation mutations were carried out for these two sites respectively, and mutants with good pigment binding ability were obtained by screening, and then the two mutant effects were combined, expressed in E. coli, and the characteristic spectrum was measured. The rate-related proteins are called LORO1.1-1.8. The primer sequences involved in site-directed mutagenesis and point saturation mutagenesis are shown in the following table:

Table 1 Primers for site-directed mutagenesis

Table 2 LOC4349531 C39 and Q102 site mutation primers

To obtain the target fragment of site-directed mutagenesis by PCR, the plasmid carrying the LORO-X11 (ie LOC4349531) gene on the synthesized pET28 vector is used as the template, and the method of T4 polynucleotide kinase is used for site-directed mutagenesis. The entire plasmid fragment carrying the LORO-X11 gene mutation site was amplified by Taq polymerase PCR. Among them, the primer sequences involved in amplifying LORO 1.1 include LOC4349531-C39A-F, LOC4349531-C39-R, LOC4349531-Q102C-F and LOC4349531-Q102-R; the primer sequences involved in amplifying LORO 1.2 include LOC4349531-C39A-F, LOC4349531-C39-R, LOC4349531-Q102I-F and LOC4349531-Q102-R; primer sequences involved in amplifying LORO 1.3 include LOC4349531-C39A-F, LOC4349511-C39-R, LOC4349531-Q102L-F and LOC4349531-Q ; The primer sequences involved in amplifying LORO 1.4 include LOC4349531-C39A-F, LOC4349531-C39-R, LOC4349531-Q102V-F and LOC4349531-Q102-R; the primer sequences involved in amplifying LORO 1.5 include LOC4349531-C39P-F, LOC4349531 -C39-R, LOC4349531-Q102C-F and LOC4349531-Q102-R; primer sequences involved in amplifying LORO 1.6 include LOC4349531-C39P-F, LOC4349531-C39-R, LOC4349531-Q102I-F and LOC4349531-Q102-R; The primer sequences involved in amplifying LORO 1.7 include LOC4349531-C39P-F, LOC4349531-C39-R, LOC4349531-Q102L-F and LOC4349531-Q102-R; the primer sequences involved in amplifying LORO 1.8 include LOC4349531-C39P-F, LOC4349531- C39-R, LOC4349531-Q102V-F and LOC4349531-Q102-R.

PCR reaction conditions: pre-denaturation at 95°C for 5min; denaturation at 95°C for 20s; gradient annealing at 61°C for 10s; extension at 72°C for 3min30s (the extension reaction rate of Taq polymerase is about 2000bp/min); this is one cycle, with a total of 40 cycles , the final annealing temperature is 54°C; the extension is 10min at 72°C. The reaction system is shown in Table 3:

Table 3 PCR reaction system

The PCR products were separated by electrophoresis in a 1-1.2% agarose gel, the electrophoresis voltage was 12v/cm, and 1×TAE buffer. According to the DNA Marker molecular weight standard, it can be directly judged whether the PCR results are correct, and the target fragment was determined. Cut off the band glue of the target DNA fragment in the agarose gel on a UV gel cutter (remove the excess part as much as possible), put it into a clean centrifuge tube, and use the Kangwei DNA gel recovery kit for recovery.

The DNA eluate is the recovered product. The PCR recovery product lacks phosphate groups, so the recovered product needs to be phosphorylated, and the reaction system is shown in Table 4:

Table 4 Phosphorylation system

The phosphorylation reaction system was placed in a water bath at 37°C for 45 minutes, and then in a metal bath at 80°C for 5-8 minutes, and then the ligation reaction was carried out. The connection system is shown in Table 5:

Table 5 Connection system

After the ligation system was placed in a metal bath at 22°C for 40min, the ligation product was introduced into E. coli BL21 competent cells using the CaCl ₂ -MgCl ₂ chemical method, spread on LB plates containing Kan antibiotics, and inverted in a 37°C incubator. , cultured overnight, colonies can appear after 12-16h.

Pick a few single colonies from the plates that were cultured overnight, transfer them to 5mL LB liquid medium containing the corresponding antibiotics, and shake them in a shaker at 37°C and 250r/min for 5-6h. The bacterial solution was mixed with sterilized glycerol in a ratio of 1:1 and stored at -20°C.

Plasmids were extracted using the Kangwei Century Plasmid Mini-Extraction Kit.

The extracted plasmid was sent to Qingke Company for sequencing, and the obtained sequence was compared in DNAMAN to verify whether it was a correct site mutation.

2. Expanded culture and protein expression of mutants

Take an appropriate amount of the stored strains of the correct mutant type in LB liquid medium containing the corresponding Kan antibiotics at 250 r/min, incubate in a constant temperature shaker at 37°C for 5-6 hours, inoculate 1 mL of each bacterial solution in 300 mL of Kan antibiotics-containing medium. TB liquid medium in a conical flask, cultured at 250 r/min, 37 °C constant temperature shaker for about 4 hours, when the bacterial concentration (OD ₆₀₀ ) reaches about 0.6, take out the conical flask and place it in an ice-water bath for 30 min. Add 180 μL of IPTG to each bottle of TB expression medium on the ultra-clean workbench, and then place the medium on a 16°C constant temperature shaker for expression, 250 r/min, and express under dark conditions for 16-18 hours. After the expression was completed, centrifuge at 6500 r/min for 5 min in a large centrifuge cup, discard the supernatant, add an appropriate amount of single-distilled water, place it on a shaker to shake to disperse the cells, transfer it to a 50 mL centrifuge tube, and then centrifuge at 6500 r/min for 5 min, discard the supernatant, The collected cells can be stored at -20°C for later use.

3. Protein purification

(1) Resuspend the cells with 15 mL of Tris-HCl, and place them in an ice-water bath for sonication. The ultrasonic fragmentation power is 400W, and each cycle is: ultrasonic 1s interval 2s, 60 cycles/time, 6-9 times of treatment. The sonicated cell suspension was centrifuged at 12000 r/min, 4°C for 100 min, and the supernatant was taken and prepared to be applied to a pretreated nickel ion chromatography column.

(2) Pretreatment of ion chelate affinity chromatography column: first wash the column with water 3 times, add 3 times the column volume of ultrapure water filtered with 0.22 μm filter paper 3 times, and add 0.2 The mol/L NiCl ₂ solution is about 2 times the column volume, and then washed three times with water to remove the nickel ions that are not bound to the column, and then add 3 times the column volume of the loading buffer Tris-HCl to balance the nickel ion column.

(3) The protein supernatant after centrifugation is filtered with a filter head with a pore size of 0.22 μm, and the filtrate is added to the treated chromatographic column until the addition is complete (the chromatographic column can be properly blocked and incubated to improve the protein binding rate), and the filtrate After the flow is exhausted, add 3 column volumes of Tris-HCl to equilibrate the column and wash the impurity proteins non-specifically bound to the Ni column. Then add 5 times the column volume of Tris-HCl containing 50 mmol/L imidazole to wash the impurity protein, and finally use 1 column volume of Tris-HCl containing 500 mmol/L imidazole to elute the target protein.

(4) After collecting the target protein, treat the nickel ion chelation affinity chromatography column, rinse one column volume with 100mmol/L EDTA, and then wash the column 3 times with water. After washing once with L of NaOH, it was quickly washed with water to reduce the influence on the column material. After washing with water, it was sealed with 20% ethanol and stored at 4°C.

4. Determination of protein spectrum

Determination of absorption spectrum: Take 700 μL of protein sample and place it in a quartz cuvette with a light path of 10 mm, and measure its absorption spectrum in the range of 200-800 nm with an ultraviolet-visible spectrometer.

Determination of fluorescence spectrum: the emission spectrum was measured with a steady-state fluorescence spectrometer, the excitation wavelength was 400 nm, the emission wavelength was 420 nm, the scanning speed was 1200 nm/min, the slit width was 5 nm, and the gain was 2.

5. Calculation of fluorescence quantum yield and molar extinction coefficient

Calculation of fluorescence quantum yield:

The reference sample is SOPP3 in pET28a with a fluorescence quantum yield of Φ _F =0.40 in Tris-HCl buffer.

According to the formula Φ _F = F/A _absorption , by the Beer-Lambert law: I = -lg (A _transmission / A _incidence ) = εcb

When εcb<0.1, A _absorption = A _incidence · I

F = A _absorption · Φ _F = A _incidence · I · Φ _F

From this, it can be deduced that Φ _F1 = (F ₁ /F ₂ )·(A _{absorption 1} /A _{absorption 2} )·Φ _F2

where F is the fluorescence intensity of the emitted light, A _absorption is the intensity of the absorbed light, that is, the absorption photometric value of the incident light at the corresponding excitation wavelength, Φ _F1 and Φ _F2 are the fluorescence quantum yields of the test substance and the reference standard, respectively .

Calculation of pigmentation rate:

Pigmentation rate×100%=(A _{sample max} /ε _{sample max} )/(A ₂₈₀ /ε ₂₈₀ )

Among them, A _{sample max} and A ₂₈₀ are the absorption values at the large absorption peak and 280 nm of the sample to be tested, respectively.

ε _{sample max} and ε ₂₈₀ are the same.

Calculation of molar extinction coefficient:

The absorption spectrum of the purified protein was measured and calculated according to Beer's law A=εLC. Protein denaturation under 8mol/L urea and acidic conditions can dissociate non-covalently bound pigments, and covalently bound pigments are also in a free state. The absorption spectrum obtained at this time shows the characteristic absorption spectrum of free pigments. From the formula A=εLC, A ₁ /A ₂ =ε ₁ L ₁ C ₁ /ε ₂ L ₂ C ₂ can be obtained: ε ₂ =ε ₁ (C ₁ A ₂ /1.6C ₂ A ₁ ), where A ₁ and C ₁ is the absorbance and protein concentration before protein denaturation, A ₂ and C ₂ are the absorbance and protein concentration after protein denaturation; the volume expansion is 1.6 times due to the addition of urea, so the volume expansion coefficient is considered in the calculation, so divide by 1.6 ; L is the length of the cuvette, 10 mm; ε ₁ and ε ₂ are the molar extinction coefficients of the protein sample before and after denaturation, respectively.

The UV absorption spectrum and UV fluorescence spectrum of some mutants are shown in Figure 2-5, and the calculation of the biological property parameters of the fluorescent protein is shown in Table 6:

Table 6 Calculation of biological properties parameters of fluorescent proteins

Example 2

In this example, templates: pET28-LOC4349531 and its double mutants; pcDNA3.1 empty. Restriction site: HindIII and XBaI; construct a mammalian cell expression vector in the form of vector plasmid pcDNA3.1:LOV.

1. Construction of animal cell expression vector

The method for obtaining the target fragment is the same as that in Example 1. After the PCR product was recovered, double-enzyme digestion was carried out according to the system in Table 7, and then the enzyme-digested product was recovered in the same steps as the PCR product. The primer sequences required for constructing animal cell expression vectors are shown in Table 8.

Table 7 Double enzyme digestion reaction system

Table 8. Primer sequences required for constructing animal cell expression vectors

When constructing the vector plasmid pcDNA3.1:LOV, pET28-LOC4349531 and its double mutants were used as templates to amplify the target fragment. The pcDNA3.1 empty plasmid and the target fragment recovered by PCR were double digested with enzyme 1 (HindIII) and enzyme 2 (XBaI), respectively. After 4 hours of water bath in a constant temperature water bath at 37 °C, the enzyme cleavage product was placed in a constant temperature metal bath at 80 °C for 10 min to inactivate the restriction endonuclease activity, and the ligation reaction was carried out according to the connection system in Table 9.

Table 9 Connection system

The mammalian cell expression vector pcDNA3.1:LOV was constructed.

2. Animal cell culture and transfection

1) Cell recovery

First, the mammalian cell (HEK293T) laboratory was routinely sterilized, irradiated with ultraviolet light for more than 40 minutes, and the culture medium (DMEM) and serum were placed in a constant temperature water bath box at 37°C for preheating for 20 minutes for use. Take out the cryovial containing 1 mL of cryopreserved cells from the liquid nitrogen storage tank, immediately put it into a 37°C water bath, shake quickly for 1 min until the cryopreserved cells are completely thawed, and transfer the cell suspension into a medium containing 5 mL of culture medium (DMEM+10% serum) and record the recovery date. The culture flask was placed in a 5% CO ₂ , 37° C. constant temperature incubator for culture, and the culture medium (DMEM+10% serum) was changed every 18 h.

2) Cell passage

After the cells adhered to 80%-90%, take out the culture flask from the incubator and pour out the medium. Add 5 mL of PBS buffer to wash off residual serum, and discard the buffer. Slowly add 1 mL of trypsin solution along the wall (the side without cells), and place the culture flask in a 37°C incubator for about 3 minutes to fully digest the adherent cells. Add 5 mL of complete medium to the flask, and repeat for 2 min by pipetting to evenly disperse the cells. Aspirate all the cell suspension, inoculate 1 mL of the suspension into the original culture flask and another new culture flask, and inoculate the rest into a 12-well plate (1 mL for each well). Add 5 mL of complete medium to each of the two bottles and place them in a CO2 incubator for cultivation. (Diluted 1:5 or greater when cells are passaged).

3) Transfection of animal cells

The plasmid used for transfection needs to be detoxified, and the plasmid concentration is 1000ng/L. First, take out a sterile 1.5 mL EP tube, add 50 μL of the medium opti-MEM and 2 μL of P3000, and then add 1 μL of DNA to it, shake gently, and mix well. Take another sterile 1.5mL EP tube, add 2μL of Lip3000, and add 50μL of opti-MEM medium, shake gently, and mix well. Add the plasmid-containing mixture to the Lip3000-containing mixture, shake gently, and mix. The mixture was left at room temperature for 5-10 min. During this period, remove the 12-well plate from the constant temperature _CO2 incubator, and wash the cells in the 12-well plate once with PBS or serum-free medium. Add the transfection mixture to it, shake the culture plate gently, put it into a constant temperature CO ₂ incubator for 8 hours, and then change the culture medium to complete culture medium (DMEM+10% serum) and continue to culture for 24-30 hours. Observation under the conditions of Ex395±40nm, spectroscope 425nm, Em 510nm±40nm to obtain the cell image.

Compared with the control, only two mutants, LORO1.6 and LORO1.8, were bright when expressed in animal cells, as shown in Figures 6-7.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

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Met Glu Lys Asn Phe Val Ile Thr Asp Pro Arg Leu Pro Asp Asn Pro

1 5 10 15

Ile Ile Phe Ala Ser Asp Ser Phe Leu Gln Leu Thr Glu Tyr Asn Arg

20 25 30

Glu Glu Ile Leu Gly Arg Asn Pro Arg Phe Leu Gln Gly Pro Glu Thr

35 40 45

Asp Arg Ala Thr Val Arg Lys Ile Arg Asp Ala Ile Asp Asn Gln Ala

50 55 60

Glu Val Thr Val Gln Leu Ile Asn Tyr Thr Lys Ser Gly Lys Lys Phe

65 70 75 80

Trp Asn Leu Phe His Leu Gln Pro Met Arg Asp Gln Lys Gly Asp Val

85 90 95

Gln Tyr Phe Ile Gly Val Ile Leu Asp Gly

100 105

<210> 8

<211> 106

<212> PRT

<213> Japonica (Oryza sativa Japonica Group)

<400> 8

Met Glu Lys Asn Phe Val Ile Thr Asp Pro Arg Leu Pro Asp Asn Pro

1 5 10 15

Ile Ile Phe Ala Ser Asp Ser Phe Leu Gln Leu Thr Glu Tyr Asn Arg

20 25 30

Glu Glu Ile Leu Gly Arg Asn Pro Arg Phe Leu Gln Gly Pro Glu Thr

35 40 45

Asp Arg Ala Thr Val Arg Lys Ile Arg Asp Ala Ile Asp Asn Gln Ala

50 55 60

Glu Val Thr Val Gln Leu Ile Asn Tyr Thr Lys Ser Gly Lys Lys Phe

65 70 75 80

Trp Asn Leu Phe His Leu Gln Pro Met Arg Asp Gln Lys Gly Asp Val

85 90 95

Gln Tyr Phe Ile Gly Val Leu Leu Asp Gly

100 105

<210> 9

<211> 106

<212> PRT

<213> Japonica (Oryza sativa Japonica Group)

<400> 9

Met Glu Lys Asn Phe Val Ile Thr Asp Pro Arg Leu Pro Asp Asn Pro

1 5 10 15

Ile Ile Phe Ala Ser Asp Ser Phe Leu Gln Leu Thr Glu Tyr Asn Arg

20 25 30

Glu Glu Ile Leu Gly Arg Asn Pro Arg Phe Leu Gln Gly Pro Glu Thr

35 40 45

Asp Arg Ala Thr Val Arg Lys Ile Arg Asp Ala Ile Asp Asn Gln Ala

50 55 60

Glu Val Thr Val Gln Leu Ile Asn Tyr Thr Lys Ser Gly Lys Lys Phe

65 70 75 80

Trp Asn Leu Phe His Leu Gln Pro Met Arg Asp Gln Lys Gly Asp Val

85 90 95

Gln Tyr Phe Ile Gly Val Val Leu Asp Gly

100 105

Claims (9)

1. A LOV protein mutant, characterized in that: the amino acid sequence of the mutant is shown in SEQ ID NO.7. 2. The application of the LOV protein mutant as claimed in claim 1 in the preparation of fluorescent probes. 3 . The application according to claim 2 , wherein the LOV protein mutant is combined with FMN cytochrome to serve as a fluorescent probe. 4 . 4. The application according to claim 2, wherein the fluorescent probe is applied to mammalian cells. 5. The use according to claim 2, wherein the mammalian cells comprise HEK293T cells. 6. The use of the LOV protein mutant as claimed in claim 1 in the preparation of a gene-encoded photosensitizer. 7. A recombinant vector, characterized in that it comprises a nucleotide sequence expressing the LOV protein mutant of claim 1. 8. A transgenic cell, characterized in that it comprises a nucleotide sequence expressing the LOV protein mutant of claim 1. 9. A recombinant bacteria, characterized in that it comprises a nucleotide sequence expressing the LOV protein mutant as claimed in claim 1.

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