CN113321717B - LOV protein mutant and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to an LOV protein mutant and application thereof. The invention discloses a preparation method of a gene and a protein which have good biocompatibility and can be automatically assembled with intracellular flavin to form a high-efficiency fluorescent probe, and a method and application of the gene and the protein which can be used for assembling the intracellular flavin to form the high-efficiency fluorescent probe. The series of fluorescent proteins can be applied to fluorescent probes in the field of biological and medical molecular monitoring, and fluorescent probes or photosensitizers with higher performance can be developed on the basis of the fluorescent probes or photosensitizers, so that the fluorescent proteins can be applied to the fields of biology, medicine and the like.

Description

LOV protein mutant and application thereof

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to an LOV protein mutant and application thereof.

Background

With the development and deepening of scientific research in the biological field, the application range and the application field of the fluorescent probe are not limited to the originally discovered biological field any more, but are mainly applied to biological qualitative and quantitative analysis and detection experiments of biomolecules, proteins, cells and the like. Among them, the development of fluorescent probes with different wave band ranges and the research of labeling organelles and the like which are commonly used in biological research also become hot spots. Commonly used fluorescent molecular probes for molecular detection and imaging of proteins are usually composed of fluorescent chromophore-ligand conjugates, enabling them to target and thus label target proteins with high selectivity.

The best known fluorescent reporter protein at present is Green Fluorescent Protein (GFP). GFP can be expressed in a wide host range of different organisms and is used as a real-time fluorescent probe to monitor protein expression, localization, folding and interaction in living cells. However, GFP family proteins require molecular oxygen for fluorescent maturation, which hinders their use in strictly anaerobic microorganisms, resulting in limited use as fluorescent reporters.

The flavin mononucleotide based photoreceptor protein (FbFP) was successfully adapted as an oxygen independent fluorescent probe in aerobic and anaerobic biological systems and is also a photosensitizer. The LOV domain belongs to FbFP, and the modification of the LOV domain to play the role and the expansion of the application of the LOV domain in recent years become the enthusiastic research direction of researchers, so that the autofluorescence can be utilized for accurate positioning and tracing of target objects in cells such as organelles and the like, and the LOV domain can become a gene coding photosensitizer which is valuable to clinical or life application in various fields.

Disclosure of Invention

The invention provides an LOV protein mutant and application thereof aiming at solving part of problems in the prior art or at least alleviating part of problems in the prior art.

In order to overcome the defects that the application sensitivity, application range and the like of the fluorescent reporter protein in the prior art are limited by conditions such as self or application objects, the invention mutates and evolves the rice LOV gene into a high-efficiency fluorescent protein, namely LORO 1.1-1.8. LORO1.1-1.8 can be expressed to form high-efficiency fluorescent protein with high fluorescence quantum yield by the automatic assembly of cytochrome FMN in a model organism, and further proves that LORO1.6 and LORO1.8 can be used as fluorescent probes in mammalian cells.

By carrying out point saturation mutation on key sites in an LOV structural domain and combining the mutation effect, further carrying out analysis on parameters such as spectral analysis, fluorescence quantum yield, molar extinction coefficient and the like on the mutant, screening the mutant with enhanced fluorescence and better activity expressed in escherichia coli, researching and developing a fluorescent protein biological probe in a blue-violet light region, widening the application range of FMN and LOV, and developing novel intracellular pigment type fluorescent protein with high stability and stronger brightness: LORO 1.1-1.8. Since FMN is an endogenous pigment in cells, it is advantageous to bind to LORO1.1-1.8 as a fluorescent probe, and at the same time it binds to LORO1.1-1.8, it can produce active oxygen in cells or at specific locations throughout the body by light induction in vitro or in vivo, and thus can be used as both a fluorescent probe and a gene-encoded photosensitizer.

The invention provides a fluorescent protein which has good biocompatibility and high pigment binding capacity and high fluorescence quantum yield and is formed by automatically assembling the fluorescent protein with intracellular pigment FMN: LORO 1.1-1.8. Wherein LORO1.6 and LORO1.8 can be used as fluorescent probes for mammalian cells.

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

The invention also provides application of the LOV protein mutant in preparation of a fluorescent probe.

Further, the mutant LOV protein binds to FMN cytochrome as a fluorescent probe.

Further, the fluorescent probe is applied to mammalian cells.

Further, the mammalian cells include HEK293T cells.

The invention also provides application of the LOV protein mutant in preparing gene-coded photosensitizer.

The invention also provides a recombinant vector, a transgenic cell or a recombinant bacterium, which comprises a nucleotide sequence for expressing the LOV protein mutant.

According to the series of fluorescent proteins LORO1.1-1.8, in a Tris-HCl buffer solution, the maximum absorption peak is 439-445 nm, and the maximum emission peak is 493-502 nm.

The molar extinction coefficient of a series of fluorescent proteins LORO1.1-1.8 provided by the invention in a Tris-HCl buffer solution is 29913-39883M^-1cm^-1。

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

The series of fluorescent proteins LORO1.1-1.8 provided by the invention start to emit at 420nm under the excitation of 400nm, and the fluorescence intensity is 1.9-2.9 times of that of a wild type.

The fluorescent proteins LORO1.6 and LORO1.8 provided by the invention have better molecular brightness in mammalian cells (HEK 293T).

In summary, the advantages and positive effects of the invention are:

the LOV domain light receptor protein has solubility and small protein mass, only about 100-140 amino acid residues, and the light absorption cofactor FMN of the LOV domain light receptor protein exists in almost all types of cells and does not need oxygen to participate in the maturation of chromophores. The invention carries out mutation and functional evolution on an LOV sequence (LOC 4349531 gene in Oryza sativa Japonica Group) in a Japonica rice genome, successfully screens a series of mutants capable of efficiently expressing and forming fluorescent protein, and is named LORO 1.1-1.8.

The invention combines FMN and LORO1.1-1.8 protein to form high-efficiency fluorescent protein, which can be applied to biological qualitative and quantitative analysis, and can also be used for marking or detecting interaction, metabolite concentration and the like in living cell molecules. Or the method is applied to screening of anaerobic bacteria, fluorescence lifetime imaging, determination of microenvironment, local oxygen environment and the like in living cells, has great application value in all aspects, and can be used for developing fluorescent probes with more efficient performance on the basis, and can be applied to various fields of biology, medicine, structure and the like.

Drawings

FIG. 1 is a comparison of the amino acid sequences of the fluorescent proteins LORO 1.1-1.8;

FIG. 2 is a UV absorption spectrum of a fluorescent protein LORO 1.1-1.4;

FIG. 3 is a UV absorption spectrum of a fluorescent protein LORO 1.5-1.8;

FIG. 4 is a UV fluorescence spectrum of a fluorescent protein LORO 1.1-1.4;

FIG. 5 is a UV fluorescence spectrum of a fluorescent protein LORO 1.5-1.8;

FIG. 6 is an image of HEK293T transfected mammalian cells with LORO1.6 fluorescent probe (Ex 395 + -40 nm, spectroscope 425nm, Em 510nm + -40 nm) in pcDNA3.1: LORO 1.6;

FIG. 7 is an image of HEK293T transfected mammalian cells with LORO1.8 fluorescent probe (Ex 395 + -40 nm, spectroscope 425nm, Em 510nm + -40 nm) in pcDNA3.1: LORO 1.8.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein 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 procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.

For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated 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 the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.

In the following examples of the present invention, the temperature is not particularly limited, and all of the conditions are normal temperature conditions. The normal temperature refers to the natural room temperature condition in four seasons, no additional cooling or heating treatment is carried out, and the normal temperature is generally controlled to be 10-30 ℃, preferably 15-25 ℃. After the protein is purified, the spectral data needs to be measured or stored under the ice bath condition. Typically 4 deg.c.

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

The invention discloses an LOV protein mutant and application thereof. The amino acid sequence of the wild type LOC4349531 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. 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 FIG. 1.

The technical solution 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 sequence in this example was synthesized by Oncorhynchus bio. A conventional genetic engineering method is adopted, pET28a (+) is used as an escherichia coli expression vector, a LOC4349531 gene in Oryza sativa Japonica Group is inserted into pET28, and the obtained plasmid is named as pET28-LOC 4349531. The plasmid can express LOC4349531 in Escherichia coli.

The cysteine at position 39 of LOC4349531 was found to play an important role in the ability of the LOV domain to bind intracellular pigment FMN by site-directed mutagenesis, and glutamine at position 102 affected the state of chromophore FMN and the electron transfer reaction in which it participates (Met at the first position in the sequence listing is the promoter, and amino acid 1 from the second position to the protein of interest). Then point saturation mutation is carried out on the two sites respectively, mutants with good pigment binding capacity are obtained by screening, the two mutation effects are combined, the two mutations are expressed in escherichia coli, a characteristic spectrum is measured, and the related protein with high fluorescence quantum yield is called LORO 1.1-1.8. The primer sequences involved in site-directed mutagenesis and point saturation mutagenesis are shown in the following table, respectively:

TABLE 1 site-directed mutagenesis primers

TABLE 2 LOC 4349531C 39 locus and Q102 locus mutation primers

The target fragment of site-directed mutagenesis obtained by PCR was site-directed mutagenesis using T4 polynucleotide kinase, using a plasmid carrying LORO-X11 (i.e., LOC4349531) gene on a synthesized pET28 vector as a template. The entire plasmid fragment carrying the mutation site of LORO-X11 gene was amplified by Taq polymerase PCR. Wherein, the primer sequence related to the amplification LORO1.1 comprises LOC4349531-C39A-F, LOC4349531-C39-R, LOC4349531-Q102C-F and LOC 4349531-Q102-R; the primer sequence related to the amplification of LORO1.2 comprises LOC4349531-C39A-F, LOC4349531-C39-R, LOC4349531-Q102I-F and LOC 4349531-Q102-R; the primer sequence related to the amplification of LORO1.3 comprises LOC4349531-C39A-F, LOC4349531-C39-R, LOC4349531-Q102L-F and LOC 4349531-Q102-R; the primer sequence related to the amplification of LORO1.4 comprises LOC4349531-C39A-F, LOC4349531-C39-R, LOC4349531-Q102V-F and LOC 4349531-Q102-R; the primer sequence related to the amplification of LORO1.5 comprises LOC4349531-C39P-F, LOC4349531-C39-R, LOC4349531-Q102C-F and LOC 4349531-Q102-R; the primer sequence related to the amplification of LORO1.6 comprises LOC4349531-C39P-F, LOC4349531-C39-R, LOC4349531-Q102I-F and LOC 4349531-Q102-R; the primer sequence related to the amplification of LORO1.7 comprises LOC4349531-C39P-F, LOC4349531-C39-R, LOC4349531-Q102L-F and LOC 4349531-Q102-R; the primer sequence related to the amplification of LORO1.8 comprises LOC4349531-C39P-F, LOC4349531-C39-R, LOC4349531-Q102V-F and LOC 4349531-Q102-R.

And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20 s; gradient annealing at 61 ℃ for 10 s; extending for 3min30s at 72 deg.C (the reaction speed of Taq polymerase extension is about 2000 bp/min); thus, the cycle is completed for 40 times, and the final annealing temperature is 54 ℃; extension at 72 ℃ for 10 min. The reaction system is shown in Table 3:

TABLE 3 PCR reaction System

And (3) carrying out electrophoretic separation on the PCR product in 1-1.2% agarose gel, wherein the electrophoretic voltage is 12v/cm, 1 × TAE buffer solution can directly judge whether the PCR result is correct according to the DNA Marker molecular weight standard, determining a target fragment, cutting off the band gel of the target DNA fragment in the agarose gel (cutting off redundant parts as much as possible) in an ultraviolet gel cutter, putting the band gel into a clean centrifugal tube, and recovering by using a Kangwei DNA gel recovery kit.

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

TABLE 4 phosphorylation System

And (3) carrying out a phosphorylation reaction system in a water bath at 37 ℃ for 45min, then carrying out a metal bath at 80 ℃ for 5-8min, and then carrying out a connection reaction. The ligation system is shown in Table 5:

TABLE 5 connection System

Placing the connection system in a metal bath at 22 deg.C for 40min, and then using CaCl₂-MgCl₂Introducing the ligation product into E.coli BL21 competent cells by a chemical method, coating the competent cells on an LB plate containing Kan antibiotics, inverting the competent cells in a constant temperature incubator at 37 ℃, and culturing overnight for 12-16h to obtain colonies.

Selecting a plurality of single colonies on the overnight-cultured plate, transferring the single colonies into 5mL LB liquid culture medium containing corresponding antibiotics, carrying out shaking culture in a shaking table at 37 ℃ and 250r/min for 5-6h, and after the bacterial liquid is concentrated, taking 200 mu L of bacterial liquid and sterilized glycerol, and mixing the bacterial liquid and the sterilized glycerol in a ratio of 1: mixing at a ratio of 1, and storing at-20 deg.C.

Plasmids were extracted using a kang century plasmid miniprep extraction kit.

The extracted plasmid is sent to a promoter company for sequencing, and the obtained sequence is compared in DNAMAN to verify whether the site mutation is correct.

2. Amplification of mutants and protein expression

Taking a proper amount of the correct mutant type stored strains, putting the proper amount of the mutant type stored strains into LB liquid culture medium containing corresponding Kan antibiotics for 250r/min, culturing for 5-6h in a constant temperature shaking table at 37 ℃, inoculating 1mL of each strain into 300mL of TB liquid culture medium conical flask containing the Kan antibiotics, culturing for about 4h in a constant temperature shaking table at 37 ℃ at 250r/min until the concentration (OD) of the strain is reached₆₀₀) When the temperature reaches about 0.6, taking out the conical flask, placing the conical flask in an ice-water bath for 30min, adding IPTG 180 mu L into each bottle of TB expression culture medium on an ultra-clean workbench, then placing the culture medium on a constant temperature shaking table at 16 ℃ for expression, and expressing the culture medium for 16-18h at 250r/min under the dark condition. After the expression is finished, centrifuging for 5min by a big centrifuge cup 6500r/min, discarding the supernatant, adding a proper amount of single distilled water, placing on a shaking table, shaking to disperse the cells, transferring into a 50mL centrifuge tube, centrifuging for 5min by 6500r/min, discarding the supernatant, and storing the collected cells at-20 ℃ for later use.

3. Purification of proteins

(1) The cells were resuspended in 15mL Tris-HCl and sonicated in an ice water bath. The ultrasonication power was 400W, and each cycle was: the ultrasound is 1s at intervals of 2s, 60 cycles/time, and the treatment is 6-9 times. The cell suspension after the ultrasonic disruption is centrifuged for 100min at 12000r/min and 4 ℃, and the supernatant is taken and prepared to be applied to a pretreated nickel ion chromatographic column.

(2) Pretreatment of the ion chelating affinity chromatography column: washing the column with water for 3 times, adding 3 times of ultrapure water (3 times of column volume) filtered by 0.22 μm filter paper for 3 times, adding 0.2mol/L NiCl after water flow out₂The solution is about 2 times of the column volume, and is washed for 3 times to remove nickel ions which are not bound on the column, and then a sample loading buffer Tris-HCl with 3 times of the column volume is added to balance the nickel ion column.

(3) Filtering the centrifuged protein supernatant with a filter head with the pore diameter of 0.22 mu m, adding the filtrate into a treated chromatographic column until the filtrate is completely added (the chromatographic column can be properly blocked for incubation, so as to improve the protein binding rate), adding a Tris-HCl equilibrium chromatographic column with the column volume of 3 times after the filtrate is completely drained, and washing the hybrid protein non-specifically bound with the Ni column. And adding 5 times of column volume of Tris-HCl containing 50mmol/L imidazole, washing the hybrid protein, and finally eluting the target protein by using 1 time of column volume of Tris-HCl containing 500mmol/L imidazole.

(4) And (3) after the target protein is collected, processing the nickel ion chelating affinity chromatographic column, washing the column by using 100mmol/L EDTA for one column volume, washing the column for 3 times, and if the pigment is combined on the column, washing the column by using 100mmol/L NaOH which is 1 time of the column volume for 1 time, and then quickly washing the column to reduce the influence on column materials. Washing with water, sealing with 20% ethanol, and storing at 4 deg.C.

4. Determination of protein spectra

Measurement of absorption spectrum: 700 mul of protein sample is placed in a quartz cuvette with a 10mm light path, and the absorption spectrum of the protein sample in the range of 200 and 800nm is measured by an ultraviolet visible spectrometer.

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

5. Calculation of fluorescence quantum yield and molar extinction coefficient

Calculation of fluorescence quantum yield:

the reference sample is SOPP3 in pET28a and the fluorescence quantum yield in Tris-HCl buffer solution is phi_F＝0.40。

According to the formula phi_F＝F/A_{Absorption of}From Beer-Lambertlaw, I ═ lg (A)_{Through the use of}/A_{Incident light})＝εcb

When ε cb is<At 0.1, A_{Absorption of}＝A_{Incident light}·I

F＝A_{Absorption of}·Φ_F＝A_{Incident light}·I·Φ_F

From which phi can be derived_F1＝(F₁/F₂)·(A_{Absorption 1}/A_{Absorption 2})·Φ_F2

Wherein F is the fluorescence intensity of the emitted light, A_{Absorption of}For the intensity of the absorbed light, i.e. the absorbance value, Φ, of the incident light at the respective excitation wavelength_F1And phi_F2The fluorescence quantum yields of the test substance and the reference standard are respectively.

Calculation of the pigmentation ratio:

pigmentation rate × 100% (A)_{Sample max}/ε_{Sample max})/(A₂₈₀/ε₂₈₀)

Wherein A is_{Sample max}And A₂₈₀Respectively the absorption values at the large absorption peak and 280nm of the sample to be detected,

ε_{sample max}And ε₂₈₀The same is true.

Calculation of molar extinction coefficient:

the absorption spectrum of the purified protein was determined and calculated according to Beer's law, a ═ epsilon LC. The protein was denatured under acidic conditions with 8mol/L urea to dissociate the non-covalently bound dye, and the covalently bound dye was also in a free state, and the absorption spectrum obtained at this time was a characteristic absorption spectrum of the free dye. Is represented by formula A ═ ε LC, A₁/A₂＝ε₁L₁C₁/ε₂L₂C₂It is possible to obtain: epsilon₂＝ε₁(C₁A₂/1.6C₂A₁) Wherein A is₁And C₁Is the absorbance and protein concentration before protein denaturation, A₂And C₂Is the absorbance and protein concentration after protein denaturation; since urea is added, the volume expansion is 1.6 times, and therefore the volume expansion coefficient is considered in calculation, and is divided by 1.6; l is the length of the cuvette 10 mm; epsilon₁And ε₂Respectively are the molar extinction coefficients before and after the denaturation of the protein sample to be detected.

The ultraviolet absorption spectrum and the ultraviolet fluorescence spectrum of the partial mutant are shown in FIGS. 2-5, and the calculation of the biological property parameters of the fluorescent protein is shown in Table 6:

TABLE 6 calculation of biological Property parameters of fluorescent proteins

Example 2

In this embodiment, the template: pET28-LOC4349531 and double mutants thereof; pcDNA3.1 is unloaded. Enzyme cutting site: HindIII and XBaI; construction of a mammalian cell expression vector forming the vector plasmid pcDNA3.1: LOV form.

1. Construction of animal cell expression vectors

The target fragment was obtained in the same manner as in example 1. After the PCR product is recovered, double enzyme digestion is carried out according to a system shown in a table 7, and then the steps of enzyme digestion products and the PCR products are recovered. The sequences of primers required for the construction of animal cell expression vectors are shown in Table 8.

TABLE 7 double restriction reaction System

TABLE 8 primer sequences required for the construction of animal cell expression vectors

When constructing the vector plasmid pcDNA3.1: LOV, pET28-LOC4349531 and double mutant thereof are used as templates to amplify the target fragment. The pcDNA3.1 empty plasmid and the target fragment recovered by PCR were digested simultaneously with enzyme 1(HindIII) and enzyme 2(XBaI), respectively. After being put in a water bath at 37 ℃ for 4 hours, the enzyme digestion product is put in a metal bath at 80 ℃ for 10 minutes, the activity of the restriction enzyme is inactivated, and the ligation reaction is carried out according to the ligation system shown in the table 9.

TABLE 9 connection System

Construct the mammalian cell expression vector pcDNA3.1: LOV.

2. Culture and transfection of animal cells

1) Cell resuscitation

The method comprises sterilizing mammal cell (HEK293T) laboratory, irradiating with ultraviolet for more than 40min, and preheating culture solution (DMEM) and serum in constant temperature water bath at 37 deg.C for 20 min. Taking out the cryopreservation tube containing 1mL of cryopreserved cells from the liquid nitrogen preservation tank, immediately putting the tube into a 37 ℃ water bath kettleThe frozen cells were shaken rapidly for 1min until they were completely thawed, and the cell suspension was transferred into a cell culture flask containing 5mL of culture medium (DMEM + 10% serum) and the recovery date was recorded. And the flask was placed in 5% CO₂The culture was carried out in a 37 ℃ incubator, and the medium (DMEM + 10% serum) was changed every 18 hours.

2) Cell passage

And after the adherence of the cells reaches 80% -90%, taking out the culture bottle from the culture box, and pouring out the culture medium. 5mL of PBS buffer was added and the serum was washed off by pipetting and the buffer was decanted off. 1mL of a trypsin solution was slowly added along the wall (cell-free side), and the flask was placed in a 37 ℃ incubator for about 3min to sufficiently digest adherent cells. 5mL of complete culture medium is added into the bottle, and the mixture is repeatedly blown and beaten uniformly for 2min to uniformly disperse the cells. The cell suspension was aspirated completely, and 1mL of the suspension was inoculated into the original flask and another new flask, respectively, and the remainder was inoculated into 12-well plates (1 mL per well). 5mL of complete medium was added to each flask and incubated in a CO2 incubator. (cells were diluted 1: 5 or more at passage).

3) Transfection of animal cells

The plasmid used for transfection needs to be endotoxin-free, and the plasmid concentration is 1000 ng/L. First, a sterile 1.5mL EP tube was taken out, 50. mu.L of the culture medium opti-MEM and 2. mu. L P3000 were added thereto, and 1. mu.L of DNA was added thereto and gently shaken and mixed. Another 1 sterile 1.5mL EP tube was added with 2. mu.L of Lip3000 and 50. mu. L L of culture medium opti-MEM, gently shaken, and mixed well. And adding the mixed solution containing the plasmid into the mixed solution containing the Lip3000, slightly shaking and uniformly mixing. The mixture was left at room temperature for 5-10 min. During this period, the temperature of CO will be constant₂The 12-well plates in the incubator were removed and the cells in the 12-well plates were washed once with PBS or serum-free medium. Adding transfection mixture, shaking the culture plate gently, and adding constant temperature CO₂After culturing for 8h in the incubator, the culture solution is changed into complete culture solution (DMEM + 10% serum) to continue culturing for 24-30h, and an inverted fluorescence microscope is used for observing under the conditions of Ex 395 +/-40 nm, spectroscope 425nm and Em 510nm +/-40 nm to obtain a cell imaging graph.

Compared to the control, only two mutants, lor 1.6 and lor 1.8, showed brightness when expressed in animal cells, as shown in fig. 6-7.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> LOV protein mutant and application thereof

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100 105

<210> 6

<211> 106

<212> PRT

<213> Japonica rice (Oryza sativa Japonica Group)

<400> 6

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 Cys Leu Asp Gly

100 105

<210> 7

<211> 106

<212> PRT

<213> Japonica rice (Oryza sativa Japonica Group)

<400> 7

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 rice (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 rice (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. An LOV protein mutant characterized by: the amino acid sequence of the mutant is shown in SEQ ID NO. 7.
2. 2. Use of the mutant LOV protein of claim 1 in the preparation of a fluorescent probe.
3. 3. Use according to claim 2, characterized in that: the mutant LOV protein binds FMN cytochrome and serves as a fluorescent probe.
4. 4. Use according to claim 2, characterized in that: the fluorescent probe is applied to mammalian cells.
5. 5. Use according to claim 2, characterized in that: the mammalian cells include HEK293T cells.
6. 6. Use of a mutant LOV protein according to claim 1 for the preparation of a gene-encoded photosensitizer.
7. 7. A recombinant vector characterized by: comprising a nucleotide sequence that expresses a mutant LOV protein of claim 1.
8. 8. A transgenic cell, characterized by: comprising a nucleotide sequence that expresses a mutant LOV protein of claim 1.
9. 9. A recombinant bacterium, which is characterized in that: comprising a nucleotide sequence that expresses a mutant LOV protein of claim 1.

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