WO2025081670A1 - Fluorescent reporter protein - Google Patents

Abstract

A fluorescent reporter protein. An exogenous amino acid sequence capable of being recognized and cleaved is inserted at a specific position inside a wild-type fluorescent protein, without affecting the light-emitting function of the fluorescent protein, so that a brand-new fluorescent reporter protein is formed. After the inserted exogenous amino acid sequence is recognized and cleaved, the structure of the fluorescent protein is directly damaged, resulting in fluorescence quenching. On the basis of this phenomenon, a variety of applications can be carried out, such as evaluating the activity of a protease, evaluating the effect of a protease inhibitor, and screening an antiviral drug.

Description

A fluorescent reporter protein Technical Field

The invention relates to the technical field of protein engineering, and in particular to a fluorescent reporter protein.

Background Art

Green fluorescent protein (GFP) is a protein composed of about 238 amino acids. It can be excited by light from blue to ultraviolet light and emit green fluorescence. Although many other marine organisms have similar green fluorescent proteins, traditionally, green fluorescent protein (GFP) refers to the protein first isolated from the Victoria jellyfish. This protein was first discovered in the Victoria jellyfish by Osamu Shimomura and others in 1962. The luminescence process also requires the help of the cold light protein aequorin, and this cold light protein can interact with calcium ions.

The wild-type green fluorescent protein found in the Victoria jellyfish has the maximum and second-largest excitation wavelengths of 395nm and 475nm, respectively. Its emission wavelength peaks at 509nm, which is in the blue-green position in the visible spectrum. The wild-type green fluorescent protein was originally a peptide chain of about 238 amino acids, about 25KDa. Then, according to certain rules, 11 β-folds formed a cylindrical fence around the outside; in the cylinder, the α-helix fixed the chromophore almost in the center. The chromophore is surrounded in the center, which can prevent dipolarized water molecules, paramagnetized oxygen molecules or cis-trans isomers from interacting with the chromophore, resulting in fluorescence quenching.

In cell biology and molecular biology, the green fluorescent protein (GFP) gene is often used as a reporter gene. The green fluorescent protein gene can also be cloned into vertebrates (such as rabbits) for expression and used to verify a certain experimental method. Through genetic engineering technology, the green fluorescent protein (GFP) gene can be transferred into the genome of different species and continuously expressed in offspring. Now, the green fluorescent protein (GFP) gene has been introduced and expressed in many species, including bacteria, yeast and other fungi, fish (such as zebrafish), plants, flies, and even mammalian cells such as humans.

In addition, GFP can emit light of different wavelengths through site-directed mutagenesis, such as blue for BFP (when Tyr-66 is replaced by His), cyan for CFP (when Tyr-66 is replaced by Trp), and yellow for YFP (when Thr203 is replaced by Tyr). GFP and its red, blue, yellow, and orange cousins have revolutionized biomedical research, making every major disease "light up" in the laboratory from a causal perspective for researchers to visualize, understand, and ultimately conquer them.

Based on the many advantages of GFP, such as broad spectrum, stability, easy vector construction and detection, and non-toxicity, GFP is widely used in the fields of cell screening, gene expression, cell labeling, genetic tracking, etc. As a living reporter protein, GFP also has a high fluorescence intensity, which is easy to observe in vivo and quantitatively detect with instruments. The tertiary structure of GFP is very important for the generation of fluorescence. Protein denaturation and separated chromophores cannot produce fluorescence. Usually, in order not to destroy the tertiary structure of GFP, multiple proteins are fused at its N-terminus and C-terminus. Although such modified fluorescent proteins do not affect the luminescence of GFP, the breakage of the added protein itself will not affect the luminescence of GFP.

In the process of virus replication and proliferation, the help of proteases is needed. For example, the protease of human immunodeficiency virus (HIV) can cleave the polyproteins expressed by the gag gene and gag-pol gene of HIV into the proteins required by the virus. HIV protease plays a very critical role in the maturation and replication of HIV virus. Inhibition of this enzyme will produce non-infectious progeny viruses, thereby preventing further infection of the virus. For another example, the polyprotein of the new coronavirus is cleaved by two viral proteases, papain-like protease (PLpro) and 3-chymotrypsin-like protease (3CLpro), which are responsible for protein cleavage at 3 sites and 11 sites respectively. 3CLpro is an enzyme necessary for coronavirus replication. Due to its strong cutting specificity, 3CLpro can avoid the possibility of off-target and can be used as an effective target for antiviral drugs.

Summary of the invention

The purpose of the present invention is to provide a modified fluorescent reporter protein, which forms a new fluorescent reporter protein by inserting an exogenous amino acid sequence that can be recognized and cleaved at a specific position inside a wild-type fluorescent protein without affecting the luminescent function of the fluorescent protein. When the inserted exogenous amino acid sequence is recognized and cleaved, the structure of the fluorescent protein is directly destroyed, resulting in fluorescence quenching. Based on this phenomenon, a variety of applications can be performed, such as evaluation of protease activity, evaluation of the effect of protease inhibitors, screening of antiviral drugs, etc.

The technical solution adopted by the present invention to solve its technical problem is:

A fluorescent reporter protein is obtained by modifying a fluorescent protein, specifically: inserting an exogenous amino acid sequence that can be recognized and cleaved by a protease into a specific position of a wild-type fluorescent protein, and the inserted exogenous amino acid sequence does not affect the luminescence function of the fluorescent protein; the specific position includes an insertion region and a separate insertion site outside the insertion region;

the insertion area is the first insertion area, the second insertion area or the third insertion area,

The first insertion region is amino acids 116 to 120 of the green fluorescent protein GFP amino acid sequence, or a corresponding amino acid region on a fluorescent protein amino acid sequence having a sequence identity of more than 95% compared with the green fluorescent protein GFP amino acid sequence;

The second insertion region is amino acids 154-160 of the green fluorescent protein GFP amino acid sequence, or a corresponding amino acid region on a fluorescent protein amino acid sequence having a sequence identity of more than 95% compared with the green fluorescent protein GFP amino acid sequence;

The third insertion region is amino acids 170-177 of the green fluorescent protein GFP amino acid sequence, or a corresponding amino acid region on a fluorescent protein amino acid sequence having a sequence identity of more than 95% compared with the green fluorescent protein GFP amino acid sequence;

The separate insertion sites outside the insertion region include independent site A and independent site B.

The independent site A is the 10th to 11th amino acids of the green fluorescent protein GFP amino acid sequence, or a corresponding amino acid region in a fluorescent protein amino acid sequence having a sequence identity of more than 95% compared with the green fluorescent protein GFP amino acid sequence;

Independent site B is amino acid 139-140 of the green fluorescent protein GFP amino acid sequence, or a corresponding amino acid region in a fluorescent protein amino acid sequence having a sequence identity of more than 95% compared with the green fluorescent protein GFP amino acid sequence;

The amino acid sequence of the green fluorescent protein GFP is shown in SEQ ID No.1.

In the present invention, the first insertion region is the 116th to 120th amino acid of the green fluorescent protein GFP amino acid sequence, or the corresponding amino acid region on the fluorescent protein amino acid sequence having more than 95% sequence identity with the green fluorescent protein GFP amino acid sequence; the corresponding amino acid region refers to the position corresponding to the 116th to 120th amino acid of the GFP amino acid sequence on the fluorescent protein amino acid sequence having more than 95% sequence identity with the green fluorescent protein GFP amino acid sequence. For other similar expressions, refer to the interpretation here.

In the present invention, the exogenous amino acid sequence that can be recognized and cleaved by the protease can be a combination of two or more different sequences.

The definition of the insertion region in the present invention can also be adopted in the following manner: the first insertion region is the amino acid sequence region shown in SEQ ID No. 2 (amino acids 116-120 of the GFP amino acid sequence), or an amino acid sequence region having a sequence identity of more than 85% compared with the amino acid sequence shown in SEQ ID No. 2;

The second insertion region is the amino acid sequence region shown in SEQ ID No.3 (amino acids 154-160 of the GFP amino acid sequence), or an amino acid sequence region having a sequence identity of more than 85% compared with the amino acid sequence shown in SEQ ID No.3;

The third insertion region is the amino acids 170-177 of the GFP amino acid sequence in the amino acid sequence region shown in SEQ ID No.4, or an amino acid sequence region having a sequence identity of more than 85% compared with the amino acid sequence shown in SEQ ID No.4.

After research, the present invention unexpectedly found that after inserting an amino acid sequence of a specific length in certain specific positions (3 regions and two separate insertion points) in the fluorescent protein, the fluorescent protein can still maintain the original luminescent function. Unlike the existing modification of the fluorescent protein at the outer ends, the fluorescent insertion site of the present invention is inside the fluorescent protein sequence. When the insertion sequence is damaged, the structure of the fluorescent protein is also damaged, resulting in fluorescence quenching. Such a discovery can bring new applications to fluorescent proteins, such as proteases related to viral maturation and replication. Such proteases can be used as targets for antiviral drugs. Then, the fluorescent reporter protein of the present invention can be used to screen new antiviral drugs. When the protease functions normally, the fluorescent protein is cut into two parts and loses its function, and the fluorescence disappears at this time; when the protease is inhibited, the fluorescent protein can emit fluorescence normally, and the inhibitory effect of the protease inhibitor can be positively reflected according to the fluorescence intensity of the fluorescent protein. However, the existing antiviral drug screening method can only be confirmed by repeated experiments and verifications due to microscopic invisibility, which is time-consuming and labor-intensive, and the results cannot be observed intuitively. The fluorescent reporter protein of the present invention can be overcome after application.

The amino acid sequence having a sequence identity of more than 95% compared with the amino acid sequence of green fluorescent protein GFP may be a variant of yellow fluorescent protein YFP, blue fluorescent protein BFP, cyan fluorescent protein CFP or green fluorescent protein GFP.

The length of the inserted exogenous amino acid sequence is less than or equal to 17 amino acids. If the inserted exogenous amino acid sequence is too long, the luminescence function of the fluorescent protein will be destroyed.

The foreign amino acid sequence includes a protease cleavage sequence that can be cleaved by a viral protease, and the protease includes a protease associated with viral replication.

Proteases associated with viral replication include proteases associated with new coronavirus replication, proteases associated with HIV replication, proteases associated with polio virus replication, proteases associated with dengue virus replication, proteases associated with West Nile virus replication, proteases associated with hepatitis C virus replication, proteases associated with herpes simplex virus replication, proteases associated with hand, foot and mouth disease virus replication, proteases associated with Japanese encephalitis virus replication, proteases associated with African swine fever virus replication, proteases associated with porcine reproductive and respiratory syndrome virus replication, proteases associated with foot-and-mouth disease virus replication, and proteases associated with feline coronavirus replication.

Proteases associated with the replication of the new coronavirus include 3CLpro.

The protease cleavage sequence that can be cleaved by viral proteases is selected from one or more combinations of the sequences shown in SEQ ID No.5-SEQ ID No.34.

The first insertion region comprises a plurality of insertion sites, and any two amino acids in the first insertion region constitute an insertion site.

The second insertion region comprises a plurality of insertion sites, and any two amino acids in the second insertion region constitute an insertion site.

The third insertion region comprises a plurality of insertion sites, and any two amino acids in the third insertion region constitute an insertion site.

Specifically, when the fluorescent protein is GFP:

The first insertion region includes four insertion sites, which are: amino acids 116-117 of the GFP amino acid sequence, amino acids 117-118 of the GFP amino acid sequence, amino acids 118-119 of the GFP amino acid sequence, and amino acids 119-120 of the GFP amino acid sequence.

The second insertion region includes 6 insertion sites, which are: amino acids 154-155 of the GFP amino acid sequence, amino acids 155-156 of the GFP amino acid sequence, amino acids 156-157 of the GFP amino acid sequence, amino acids 157-158 of the GFP amino acid sequence, amino acids 158-159 of the GFP amino acid sequence, and amino acids 159-160 of the GFP amino acid sequence.

The third insertion region includes 7 insertion sites, which are: amino acids 170-171 of the GFP amino acid sequence, amino acids 171-172 of the GFP amino acid sequence, amino acids 172-173 of the GFP amino acid sequence, amino acids 173-174 of the GFP amino acid sequence, amino acids 174-175 of the GFP amino acid sequence, amino acids 175-176 of the GFP amino acid sequence, and amino acids 176-177 of the GFP amino acid sequence.

The wild-type fluorescent protein is selected from one of green fluorescent protein GFP, yellow fluorescent protein YFP, blue fluorescent protein BFP and cyan fluorescent protein CFP.

A fluorescence detection kit comprises the fluorescent reporter protein.

A nucleotide encoding the fluorescent reporter protein.

The beneficial effect of the present invention is that the present invention forms a new fluorescent reporter protein by inserting an exogenous amino acid sequence that can be recognized and cleaved at a specific position inside the wild-type fluorescent protein without affecting the luminescence function of the fluorescent protein. When the inserted exogenous amino acid sequence is recognized and cleaved, the structure of the fluorescent protein is directly destroyed, resulting in fluorescence quenching. Based on this phenomenon, a variety of applications can be performed, such as the evaluation of protease activity, the evaluation of the effect of protease inhibitors, the screening of antiviral drugs, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a fluorescence image of GFP after the cleavage sequence of 3CLpro is inserted into different positions;

Figure 2 is a fluorescence image after the cleavage sequence of 3CLpro is inserted into the GFP insertion region 115-122aa;

Figure 3 is a fluorescence image after the cleavage sequence of 3CLpro is inserted into the GFP insertion region 153-161aa;

FIG4 is a fluorescence image after the cleavage sequence of 3CLpro is inserted into the GFP insertion region 169 to 178 aa;

Fig. 5 is a schematic diagram of the insertable region on GFP;

FIG6 is a fluorescence image of the protease cleavage sequence of HIV type 1 inserted into three insertion regions of GFP, GFP20-HIVcut1 represents the insertion of HIV protease cleavage sequence cut1:RVLAEA at site 20 of GFP, and so on;

FIG7 is a fluorescence image of the protease cleavage sequence of poliovirus inserted into three insertion regions of GFP, GFP20-PVcut1 represents the insertion of the PV protease cleavage sequence cut1:ALFQGP at the site 20 position of GFP, and so on;

FIG8 is a fluorescence image of the dengue virus protease cleavage sequence inserted into three insertion regions of GFP, GFP20-DVcut1 represents the insertion of the DV protease cleavage sequence cut1: AGRKSLTL at the site 20 position of GFP, and so on;

Figure 9 is a fluorescence image of the protease cleavage sequence of West Nile virus inserted into three insertion regions of GFP, GFP20-WNVcut1 represents the insertion of the WNV protease cleavage sequence cut1: SGKRSQIG at the site 20 position of GFP, and so on;

Figure 10 is a fluorescence image of the protease cleavage sequence of herpes simplex virus type 1 inserted into three insertion regions of GFP, GFP8-HSVcut1 represents the insertion of the HSV protease cleavage sequence cut1: LVNASSAA at the site 8 position of GFP, and so on;

FIG11 is a fluorescence image of the protease cleavage sequence of hand, foot and mouth disease virus after being inserted into three insertion regions of GFP, GFP20-EV71cut1 represents the insertion of the EV71 protease cleavage sequence cut1:AVTQGF at the site 20 position of GFP, and so on;

FIG12 is a fluorescence image of the protease cleavage sequence of Japanese encephalitis virus inserted into three insertion regions of GFP, GFP20-JEVcut1 represents the insertion of the JEV protease cleavage sequence cut1:AGKRSAIS at the site 20 position of GFP, and so on;

Figure 13 is a fluorescence image of the protease cleavage sequence of the feline coronavirus inserted into the three insertion regions of GFP, GFP20-FCoVcut1 represents the insertion of the FCoV protease cleavage sequence cut1:STLQSGLR at the site 20 position of GFP, and so on;

Figure 14 is a fluorescence image of the protease cleavage sequence of African swine fever virus inserted into three insertion regions of GFP, GFP20-ASFVcut1 represents the insertion of the ASFV protease cleavage sequence cut1: GYFNGGGDK at the site 20 position of GFP, and so on;

Figure 15 is a fluorescence image of the protease cleavage sequence of foot-and-mouth disease virus inserted into the three insertion regions of GFP, GFP20-FMDVcut1 It represents the insertion of FMDV protease cleavage sequence cut1:AEKQLKAR at site 20 of GFP, and so on;

Figure 16 is a fluorescence image of the protease cleavage sequence of porcine blue ear virus inserted into three insertion regions of GFP, GFP20-PRRSVcut1 represents the insertion of the PRRSV protease cleavage sequence cut1:SLLEGAFR at the site 20 position of GFP, and so on;

FIG17 is a fluorescence image of the hepatitis C virus protease cleavage sequence inserted into three insertion regions of GFP, GFP24-HCVcut1 represents the insertion of the HCV protease cleavage sequence cut1:DEMEECSQHL at the site 24 position of GFP;

FIG. 18 is a fluorescence image of different combinations of exogenous protease cleavage sequences after insertion into GFP

FIG. 19 shows the effect of the modified GFP when co-expressed with protease.

DETAILED DESCRIPTION

The technical solution of the present invention is further described in detail below through specific embodiments.

In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

pcDNA3.1(+), pCMV, and pCAGGS are the most commonly used mammalian expression vectors. The vectors have the advantages of high copy number and high expression level, and can be used for plasmid construction and exogenous gene expression in the present invention. The embodiment of the present invention takes the pcDNA3.1(+) expression vector as an example to illustrate the specific invention content. The specific plasmid construction process is as follows: 1. Use DNA polymerase (Primer Star) to perform PCR amplification to obtain the corresponding fragments; 2. Each fragment is recombined using homologous recombinase (Uniclone One Step Seamless Cloning Kit) and transformed into competent cells; 3. Pick a single colony and use universal vector primers and Taq enzyme to perform colony PCR, and send the PCR product with the correct band size for testing; 4. Extract plasmids from the colony clones with correct sequencing.

Amino acid sequence of GFP:

Embodiment 1:

This embodiment is implemented using the SARS-CoV-2 3C-like protease (3CLpro) and the enzyme's corresponding cleavage sequence AVLQSGFR (SEQ ID No. 5).

1. Preliminary search for the location of the protease cleavage sequence in GFP

According to the instructions of Primer Star enzyme (Takara), GFP and common expression vectors were PCR amplified. The present embodiment takes pcDNA3.1(+) vector (commercially available) as an example. The GFP gene sequence (SEQ ID NO.36) was cloned into the multiple cloning site (MCS) of pcDNA3.1(+) vector by homologous recombination (Uniclone One Step Seamless Cloning Kit). The pGFP plasmid was constructed and used as the template and control for subsequent experiments.

Subsequently, the 3CLpro cutting sequence SEQ ID NO.5: AVLQSGFR (coding nucleotide sequence is SEQ ID NO.35: gctgttttgcagagtggttttaga) was inserted into different positions of GFP through homologous recombination technology (Uniclone One Step Seamless Cloning Kit) to construct a series of plasmids expressing modified GFP, including pGFP1-COVID19, pGFP2-COVID19, pGFP3-COVID19,…, pGFP 45-COVID19. Among them, GFP1~45 represents the fluorescent protein containing the protease cleavage sequence formed after the protease cleavage sequence is inserted into the corresponding site1~45 in Table 1, for example: GFP8 represents the insertion of the protease cleavage sequence between the amino acids at positions 119~120aa (site8) of GFP; GFP8-COVID19 represents the insertion of the protease cleavage sequence AVLQSGFR between the amino acids at positions 119~120aa (site8) of GFP; Correspondingly, pGFP8-COVID19 represents the plasmid formed after the protease cleavage sequence AVLQSGFR is inserted between the amino acids at positions 119~120aa (site8) of GFP.

Table 1. Site 1 to 45 positions

The constructed pGFP1～45-COVID19 plasmid was transfected. 293T cells have the advantages of fast growth and high protein expression level, so the present invention selects 293T cells as the object of plasmid transfection. Prepare well-grown 293T cells, add 10% FBS DMEM culture medium to the 293T cells, and culture them in a 37°C, 5% CO2 incubator. When the 293T cells grow to 70% to 80% density, a transfection experiment is performed, and the amount of plasmid transfection is determined according to the size of the well plate. In this embodiment, the amount of plasmid transfection in a 6-well plate is 2μg. Observe the cell state every 24h after transfection, and take fluorescent photos after 48h.

As shown in Figure 1, among the 45 selected sites for inserting the 3CLpro protease cleavage sequence, only 19 sites, namely site8/11/15/19/20/23/24/25/28/31/34/35/36/37/38/39/40/43/45 (Note: "/" means "or", the same below) can be compatible with the exogenous protease cleavage sequence and have no effect or limited effect on the GFP fluorescence. After the 3CLpro protease cleavage sequence is inserted, the remaining sites cannot detect GFP fluorescence, such as site6/10/13/22/27. Among them, Site19 and Site25 are separate insertion sites that can still excite green fluorescence after the 3CLpro protease cleavage sequence is inserted.

When searching for the position where GFP can be inserted into the protease cleavage sequence, the inventors had the following ideas:

1. First, we randomly selected site 1 to 30 to construct the corresponding pGFP1 to 30-COVID19 plasmid for cell transfection. After transfection, we found that among the above 30 sites, site 8/20/23 sites were close to the position of GFP, site 15/24 sites were close to the position of GFP, and site 11/28 sites were close to the position of GFP. Therefore, it is speculated that there is a more flexible site region in GFP. After inserting the protease cleavage sequence in this region, the luminescence function of GFP is not affected and can still stimulate green fluorescence.

2. Subsequently, the upstream and downstream regions of the above sites were further explored to see whether there were other sites that were compatible with exogenous protease cleavage sequences. To this end, the inventors constructed pGFP31-45-COVID19 plasmids to transfect cells and observe fluorescence expression. After verification, it was found that there were indeed high flexibility regions in GFP, and three high flexibility regions were finally screened, as shown in Tables 2-4.

Correspondingly, as shown in Figure 2, when the insertion site is located at 116-120aa (site23/20/31/8), the modified GFP can still stimulate different degrees of green fluorescence. That is, there is an exogenous protease cleavage sequence in the GFP amino acid sequence that can be inserted into region 1: 116-120aa (see Table 2), and the amino acid sequence of this region is SEQ ID NO.2: EGDTL, and no fluorescence is detected in the neighboring sites Site22 and Site32/33.

Table 2. Location of region 1 and nearby sites

As shown in Figure 3, when the insertion site is located at 154-160aa (site34/35/36/15/24/43), the modified GFP can still stimulate different degrees of green fluorescence. That is, there is an exogenous protease cleavage sequence in the GFP amino acid sequence that can be inserted into region 2: 154-160aa (see Table 3), the amino acid sequence of which is SEQ ID NO.3: MADKQKN, and no fluorescence is detected at the neighboring sites Site42 and Site27.

Table 3. Location of region 2 and nearby sites

As shown in Figure 4, when the insertion site is located at 170-177aa (site37/38/39/11/39/28/40), the modified GFP can still stimulate different degrees of green fluorescence. There is an exogenous protease cleavage sequence in the GFP amino acid sequence that can be inserted into region 3: 170-177aa (see Table 4). The amino acid sequence of this region is SEQ ID NO.4: HNIEDGSV. No fluorescence is detected at the neighboring sites Site44 and Site41.

Table 4. Location of region 3 and nearby sites

The above results indicate that there are some regions on GFP where GFP can still emit green fluorescence after inserting several protease cleavage sequences. These regions include: region 1: 116-120aa, with the amino acid sequence shown in SEQ ID NO.2; region 2: 154-160aa, with the amino acid sequence shown in SEQ ID NO.3 and region 3: 170-177aa, with the amino acid sequence shown in SEQ ID NO.4 (Figure 5).

Example 2-29

In order to verify that the above three regions are compatible with exogenous viral protease cleavage sequences, the inventors verified the GFP fluorescence after other viral protease cleavage sequences were inserted into the above three regions of GFP, and the plasmid construction method was referred to Example 1. Other viral and protease cleavage sequences are shown in Table 5.

Table 5. Viruses and corresponding protease cleavage sequences

As shown in Figures 6 to 17, the results show that when different viruses and protease cleavage sequences of different sizes within the length defined by the present invention are inserted into the three insertion regions of GFP defined by the present invention, GFP can still emit green fluorescence.

In order to further verify that the above-mentioned sites/regions are compatible with the combination of different exogenous protease cleavage sequences and to explore the maximum insertion length, the present invention connected and inserted the protease cleavage sequences from two viral sources into GFP.

As shown in FIG18 , after the combination of different exogenous protease cleavage sequences was inserted into GFP, fluorescence expression was still detectable.

Verify the effect of modified GFP when co-expressed with protease

In order to verify the cleavage effect of protease on the modified GFP, the plasmids pGFP8-COVID19, pGFP20-WNVcut2 and pGFP15-EV71cut1 in Example 1 were selected, and the corresponding viral proteases were inserted after the GFP of these plasmids by PCR amplification (Primer Star) and homologous recombination technology (Uniclone One Step Seamless Cloning Kit). P2A was used to connect them in the middle to obtain the plasmids pGFP8-COVID19-P2A-3CLpro, pGFP20-WNVcut2-P2A-NS2B/NS3 and pGFP15-EV71cut1-P2A-3C that can co-express proteases.

The above plasmids were transfected into cells according to the method of Example 1, and the fluorescence intensity was observed.

The experimental results are as follows: As shown in Figure 20, after the GFP containing the protease cleavage sequence and the corresponding viral protease were co-expressed, the green fluorescence disappeared significantly, indicating that the protease can cleave the modified GFP, that is, the modified GFP can become a reporter protein indicating protease activity.

GFP gene sequence:

The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims (11)

A fluorescent reporter protein, characterized in that: it is formed by modifying a wild-type fluorescent protein, specifically: an exogenous amino acid sequence that can be recognized and cleaved by a protease is inserted into a specific position of the wild-type fluorescent protein, and the inserted exogenous amino acid sequence does not affect the luminescence function of the fluorescent protein; The specific position includes an insertion region and a separate insertion site outside the insertion region; the insertion area is the first insertion area, the second insertion area or the third insertion area, The first insertion region is amino acids 116 to 120 of the green fluorescent protein GFP amino acid sequence, or a corresponding amino acid region on a fluorescent protein amino acid sequence having a sequence identity of more than 95% compared with the green fluorescent protein GFP amino acid sequence; The second insertion region is amino acids 154-160 of the green fluorescent protein GFP amino acid sequence, or a corresponding amino acid region on a fluorescent protein amino acid sequence having a sequence identity of more than 95% compared with the green fluorescent protein GFP amino acid sequence; The third insertion region is amino acids 170-177 of the green fluorescent protein GFP amino acid sequence, or a corresponding amino acid region on a fluorescent protein amino acid sequence having a sequence identity of more than 95% compared with the green fluorescent protein GFP amino acid sequence; The separate insertion sites outside the insertion region include independent site A and independent site B. The independent site A is the 10th to 11th amino acids of the green fluorescent protein GFP amino acid sequence, or a corresponding amino acid region in a fluorescent protein amino acid sequence having a sequence identity of more than 95% compared with the green fluorescent protein GFP amino acid sequence; Independent site B is amino acid 139-140 of the green fluorescent protein GFP amino acid sequence, or a corresponding amino acid region in a fluorescent protein amino acid sequence having a sequence identity of more than 95% compared with the green fluorescent protein GFP amino acid sequence; The amino acid sequence of the green fluorescent protein GFP is shown in SEQ ID No.1. The fluorescent reporter protein according to claim 1, characterized in that the length of the inserted exogenous amino acid sequence is less than or equal to 17 amino acids. The fluorescent reporter protein according to claim 1 is characterized in that: the exogenous amino acid sequence includes a protease cleavage sequence that can be cleaved by a viral protease, and the protease includes a protease associated with viral replication. The fluorescent reporter protein according to claim 3 is characterized in that: the protease associated with virus replication includes a protease associated with the replication of the new coronavirus, a protease associated with the replication of HIV, a protease associated with the replication of polio virus, a protease associated with the replication of dengue virus, a protease associated with the replication of West Nile virus, a protease associated with the replication of hepatitis C virus, a protease associated with the replication of herpes simplex virus, a protease associated with the replication of hand, foot and mouth disease virus, a protease associated with the replication of Japanese encephalitis virus, a protease associated with the replication of African swine fever virus, a protease associated with the replication of porcine reproductive and respiratory syndrome virus, a protease associated with the replication of foot-and-mouth disease virus, and a protease associated with the replication of feline coronavirus. The fluorescent reporter protein according to claim 4 is characterized in that the protease associated with the replication of the new coronavirus includes 3CLpro. The fluorescent reporter protein according to claim 3 is characterized in that the protease cleavage sequence that can be cleaved by viral proteases is selected from one or more combinations of sequences shown in SEQ ID No.5-SEQ ID No.34. The fluorescent reporter protein according to claim 1 is characterized in that: the first insertion region comprises a plurality of insertion sites, and an insertion site is formed between any two amino acids in the first insertion region. The fluorescent reporter protein according to claim 1 is characterized in that: the second insertion region comprises a plurality of insertion sites, and an insertion site is formed between any two amino acids in the second insertion region. The fluorescent reporter protein according to claim 1 is characterized in that: the third insertion region comprises a plurality of insertion sites, and an insertion site is formed between any two amino acids in the third insertion region. A fluorescence detection kit, characterized by comprising the fluorescent reporter protein according to claim 1. A nucleotide, characterized in that it encodes the fluorescent reporter protein according to claim 1.