CN117327164A - Red fluorescent reporter protein, plasmid and application thereof - Google Patents

Abstract

The invention discloses a red fluorescent reporter protein, a plasmid and application thereof, wherein an exogenous amino acid sequence which can be identified and cracked by protease is inserted into a specific position inside a wild red fluorescent protein without affecting the luminous function of the red fluorescent protein, so that a brand new red fluorescent reporter protein is formed, and when the inserted exogenous amino acid sequence is identified and cracked, the structure of the red fluorescent protein is directly damaged, thereby causing fluorescence quenching. Based on this phenomenon, various applications such as evaluation of protease activity, evaluation of protease inhibitor effect, screening of antiviral drugs, and the like can be performed.

Description

Red fluorescent reporter protein, plasmid and application thereof

Technical Field

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

Background

In 1999, matz et al isolated from coral worm a protein that fluoresced red under ultraviolet radiation-Red Fluorescent Protein (RFP). The protein encoded by the RFP gene consists of more than 200 amino acids, and like GFP, the RFP also forms a beta barrel-shaped structure on a secondary structure, and the structural basis of the RFP has a great influence on luminescence. The RFP emission wavelength is 560-610 nm. It has higher fluorescence intensity, lower imaging background, and can excite and emit longer wavelengths than GFP. Because the conjugated structure is critical for the color development of RFP fluorescent proteins, the insertion of an amino acid sequence within the RFP amino acid sequence is likely to cause the destruction of the RFP excitation fluorescence.

Many viruses require the aid of proteases in the replication and proliferation process. Some proteases of viruses have self-cleaving function to cleave their polyprotein precursor into functionally active proteins, and inhibition of these protease activities can prevent replication and proliferation of the virus. For example: human immunodeficiency virus (HIV, human immunodeficiency virus) proteases are capable of cleaving the gag gene of HIV and the multimeric protein expressed by the gag-pol gene into proteins required by the virus. HIV protease plays a very critical role in the maturation and replication process of HIV virus, and inhibiting the enzyme can generate non-infectious progeny virus, thereby preventing the virus from further infection; as another example, the multimeric proteins of the novel coronaviruses are cleaved by two viral proteases, papain-like protease (PLpro) and 3C-like protein (3 CLpro,3-chymotrypsin like protease), which are responsible for protein cleavage at 3 and 11 sites, respectively. Among them, 3CLpro is widely considered as an excellent drug target because of its strong cleavage specificity, which can avoid the possibility of off-target.

However, at present, antiviral drugs with protease as targets are developed, and early candidate drug screening mainly expresses protease, and candidate drugs are screened and evaluated at a non-cellular level. The probability of screening leakage and false screening is very high, because the antiviral drugs mainly need to enter living cells to inhibit target proteins in the animal body, and the candidate drugs enter the living cells in the mode is deleted, so that the actual activity level of the drugs cannot be evaluated or screened, and therefore, the development of a simple, safe, high-flux and repeatable protease activity and related protease inhibitor activity screening platform is necessary.

Disclosure of Invention

The invention aims to provide a red fluorescent reporter protein, a plasmid and application thereof, wherein an exogenous amino acid sequence which can be identified and cleaved by protease is inserted into a specific position inside a wild red fluorescent protein without affecting the luminous function of the red fluorescent protein, so that a brand-new red fluorescent reporter protein is formed, when the inserted exogenous amino acid sequence is identified and cleaved, the structure of the red fluorescent protein is directly destroyed, and fluorescence quenching is caused, and based on the phenomenon, various applications such as evaluation of protease activity, evaluation of protease inhibitor effect, screening of antiviral drugs and the like can be performed.

The technical scheme adopted for solving the technical problems is as follows:

the red fluorescent reporter protein is prepared by modifying red fluorescent protein, and specifically comprises the following components: inserting an exogenous amino acid sequence which can be recognized and cleaved by protease at a specific position of the red fluorescent protein, wherein the inserted exogenous amino acid sequence does not influence the light-emitting function of the red fluorescent protein;

the specific location includes an insertion region;

the insertion region is selected from one of a first insertion region, a second insertion region, a third insertion region, and a fourth insertion region,

the amino acid sequence of the first insertion region is PAGGL,

the amino acid sequence of the second insertion region was KKPAKNLKMPGV,

the amino acid sequence of the third insertion region is RIKEAD,

the amino acid sequence of the fourth insertion region was VARYCDLPS.

The amino acid sequence of the red fluorescent protein is the amino acid sequence shown as SEQ ID No.1 or the amino acid sequence with more than 95 percent of sequence identity compared with the amino acid sequence shown as SEQ ID No. 1.

The red fluorescent reporter protein is prepared by modifying red fluorescent protein, and specifically comprises the following components: inserting an exogenous amino acid sequence which can be recognized and cleaved by protease at a specific position of the red fluorescent protein, wherein the inserted exogenous amino acid sequence does not influence the light-emitting function of the red fluorescent protein;

the specific location includes a separate insertion site;

the individual insertion sites include independent site a, independent site B,

independent site A is amino acid 145-146 of RFP amino acid sequence, or corresponding amino acid region on amino acid sequence with sequence identity over 95% compared with RFP amino acid sequence;

independent site B is amino acid 208-209 of RFP amino acid sequence, or corresponding amino acid region on amino acid sequence with sequence identity over 95% compared with RFP amino acid sequence;

the RFP amino acid sequence is shown in SEQ ID No. 1.

According to the invention, after research, it is unexpectedly found that after certain specific positions (4 regions and two independent insertion points) in the red fluorescent protein are inserted into amino acid sequences with specific lengths, the red fluorescent protein can still maintain the original light-emitting function, and when the insertion sequences are damaged, the structure of the red fluorescent protein is simultaneously damaged, so that fluorescence quenching is caused. The discovery can bring brand-new application to the red fluorescent protein, such as protease related to virus maturation and replication, the protease can be used as a target point of antiviral drugs, so that the red fluorescent reporter protein can serve for screening brand-new antiviral drugs, and when the protease normally functions, the red fluorescent protein is cut into two parts to lose functions, and fluorescence disappears; the red fluorescent protein can normally emit fluorescence when protease is inhibited, and the inhibition effect of the protease inhibitor can be positively reflected according to the fluorescence intensity of the red fluorescent protein. The series of operations can be carried out at the cellular level, the whole process of the candidate medicine playing a role in the cell can be completely simulated, and the existing screening method of the antiviral medicine is carried out at the non-cellular level, so that the process of entering the cell by the candidate medicine is deleted, the screening leakage and the screening error of the candidate medicine are easily caused, protease is required to be expressed in vitro, the time and the labor are consumed, the defects of the existing method can be completely overcome after the red fluorescent reporter protein is applied, and the screening of the protease inhibitor antiviral candidate medicine can be carried out simply and conveniently at high flux.

The length of the inserted exogenous amino acid sequence is less than or equal to 16 amino acids. Preferably, the inserted exogenous amino acid sequence is 10 amino acids or less in length.

The exogenous amino acid sequence includes a protease cleavage sequence that is cleavable by a viral protease.

The viral protease is derived from one of all positive-strand RNA viruses, partial negative-strand RNA viruses and partial DNA viruses, and specifically comprises one of picornaviridae viruses, calicividae viruses, togaviridae viruses, flaviviridae viruses, enteroviridae, retrovirus viruses, coronaviridae viruses, herpesviridae viruses and poxviridae viruses. Preferably, the viral Protease is derived from one of a novel coronavirus Protease (e.g., 3 CLpro), an aids virus Protease (e.g., protease), a hand-foot-and-mouth disease virus Protease (e.g., 3C), a dengue virus Protease (e.g., NS2B-NS 3), a herpes simplex virus Protease (e.g., UL 26), a hepatitis C virus Protease (e.g., NS 3/4A).

The protease cleavage sequence that is cleavable by a viral protease is selected from the group consisting of one or more of the sequences set forth in SEQ ID No.2-SEQ ID No. 12.

An insertion site is formed between any two amino acids in the first insertion region, the second insertion region, the third insertion region, and the fourth insertion region.

A nucleotide encoding said red fluorescent reporter protein.

A plasmid comprising the nucleotide sequence encoding the red fluorescent reporter protein.

Further, the plasmid also comprises a coding nucleotide sequence of the viral protease, and the plasmid coexpresses the red fluorescent reporter protein and the viral protease.

An antiviral drug screening method taking protease as a target spot comprises the following steps:

(1) Constructing a co-expression plasmid for co-expressing the red fluorescent reporter protein and the viral protease;

(2) Screening: spreading cells to be transfected in a porous plate, then adding a co-expression plasmid for transfection, culturing the transfected cells, simultaneously adding a drug to be screened into the porous plate, taking a group without the drug to be screened as a contrast, observing the state of the cells, performing fluorescence photographing, and detecting the fluorescence intensity; if the fluorescence intensity is increased compared with the control, the drug to be screened has an inhibition effect on viral protease, and if the fluorescence intensity is increased, the inhibition effect on viral protease by the drug to be screened is stronger.

A method for evaluating the forward visual inhibition effect of a protease inhibitor comprises the following steps:

(1) Constructing a co-expression plasmid for co-expressing the red fluorescent reporter protein and the viral protease;

(2) Evaluation: spreading the cells to be transfected in a cell culture dish, then adding a co-expression plasmid for transfection, culturing the transfected cells, simultaneously adding an inhibitor into the cell culture dish, taking the cell without the inhibitor as a control, observing the state of the cells, taking a fluorescent photograph, and detecting the fluorescence intensity; the forward direction of the fluorescence intensity reflects the inhibition effect of the inhibitor, and the higher the fluorescence intensity is, the stronger the inhibition effect of the inhibitor on viral protease is.

The beneficial effects of the invention are as follows:

1. according to the invention, the exogenous amino acid sequence which can be identified and cleaved by protease is inserted into a specific position inside the wild red fluorescent protein, so that the luminous function of the red fluorescent protein is not influenced, a brand new red fluorescent report protein is formed, and when the inserted exogenous amino acid sequence is identified and cleaved, the structure of the red fluorescent protein is directly damaged, so that fluorescence quenching is caused.

2. The invention can screen antiviral drugs based on modified red fluorescence by taking protease as a target point, the inhibition effect of the antiviral drugs on the protease is positively correlated with the fluorescence intensity, the inhibition effect can be judged very conveniently and intuitively qualitatively, and the inhibition effect can be analyzed quantitatively by measuring the fluorescence value.

3. According to the screening method of the antiviral drugs and the evaluation method of the inhibition effect of the protease inhibitor, a qualitative result can be obtained by observing fluorescent signals of cells, safety concern caused by using live viruses can be avoided, the requirement on biological safety level is low, the BSL-1 laboratory can meet the experimental requirement, and the screening method has the advantages of high efficiency, accuracy, stability, high flux and repeatability.

Drawings

FIG. 1 is a fluorescent image after insertion of the cleavage sequence of 3CLpro in different positions of RFP;

fig. 2 is RFP insertion region one: fluorescence map after insertion of a cleavage sequence for 3CLpro in the position of the amino acid sequence PADGGL;

fig. 3 is RFP insertion region two: fluorescence map after insertion of cleavage sequence for 3CLpro in amino acid sequence KKPAKNLKMPGV position;

fig. 4 is RFP insertion region three: fluorescence map after insertion of a cleavage sequence for 3CLpro in the amino acid sequence RIKEAD position;

fig. 5 is RFP insertion region four: fluorescence map after insertion of cleavage sequence for 3CLpro in amino acid sequence VARYCDLPS position;

FIG. 6 is a fluorescent image of the insertion of different viral protease cleavage sequences into RFP site 15;

FIG. 7 is a fluorescent image of different viral protease cleavage sequences inserted into RFP site 30;

FIG. 8 is a fluorescent image of the insertion of different viral protease cleavage sequences into RFP site 34;

FIG. 9 is a fluorescent image of the insertion of different viral protease cleavage sequences into RFP site53 sites;

FIG. 10 is a fluorescence plot of engineered RFPs upon co-expression of 3 CLpro;

FIG. 11 is a fluorescent image of a plasmid following disruption of expression of novel coronavirus 3 CLpro;

FIG. 12 shows the fluorescence results at various times after the addition of the 3CLpro inhibitor Enstralvir 1. Mu.M-100. Mu.M after transfection of cells with the plasmid pRFP15-P2A-3CLpro of the invention;

FIG. 13 shows the fluorescence results at various times after the addition of the 3CLpro inhibitor Enstralvir 1. Mu.M-100. Mu.M after transfection of cells with the plasmid pRFP30-P2A-3CLpro of the invention;

FIG. 14 is a fluorescent image after insertion of two viral protease cleavage sequences linked at the RFP site30 site.

Detailed Description

The technical scheme of the invention is further specifically described by the following specific examples.

In the present invention, the materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.

The sources of the materials, the reagents and the instruments used in the embodiment of the invention are respectively as follows: 293T cells (purchased from the department of Chinese sciences cell bank). Cell culture plates were purchased from corning. Uniclone One Step Seamless Cloning Kit kit was purchased from Jinsha organisms. PrimerStar enzyme was purchased from Takara. The endotoxin-free plasmid miniprep kit was purchased from Tiangen Biochemical technologies Co. Primers were synthesized by the same company as the Optimus of the family Prinsepia. Protease inhibitor GC376 was purchased from ala Ding Shenghua technologies limited. Ensitrelivir was purchased from MCE company. Carbon dioxide cell incubators were purchased from Thermo Fisher company. Fluorescence microscopy was purchased from OLYMPUS corporation under model CKX53.

Example 1

Search for the position of the insertable protease cleavage sequence in RFP:

RFP and a usual expression vector were PCR amplified according to the Primer Star enzyme protocol, and pcDNA3.1 (+) vector was used as an example of the present invention. The RFP gene sequence is cloned between Multiple Cloning Sites (MCS) of pcDNA3.1 (+) vector by homologous recombination mode (Uniclone One Step Seamless Cloning Kit kit) to construct pRFP plasmid as follow-up template and control.

The cleavage sequence of the novel coronavirus 3CLpro is then inserted in a different position of the RFP by homologous recombination technique (Uniclone One Step Seamless Cloning Kit kit): AVLQSFFR (SEQ ID NO. 2), the coding nucleotide sequence of AVLQSFFR is: gctgttttgcagagtggttttaga (SEQ ID NO. 13), a series of plasmids pRFP1, pRFP2, pRFP3, …, pRFP 64 expressing engineered RFPs was constructed. Wherein RFPs 1 to 45 represent fluorescent proteins containing protease cleavage sequences formed after insertion of the 3CLpro cleavage sequences into the corresponding sites 1 to 64 in Table 1, for example: RFP8 represents the cleavage sequence SEQ ID NO.2 with 3CLpro inserted between amino acids at positions 115-116 aa (site 8) of RFP.

TABLE 1 site1 to 64 site positions

The constructed pRFP 1-64 plasmid was transfected. Well-grown 293T cells were prepared, 2% FBSDMEM medium was added to the 293T cells, and the mixture was placed at 37℃with 5% CO ₂ Culturing in an incubator. Transfection experiments were performed when 293T cells grew to a density of 70% -80%, and plasmid transfection was determined according to the well plate size, in this example, the 6 well plate plasmid transfection was 2. Mu.g. Cell status was observed every 24h after transfection, and fluorescent photographing was performed 48h later.

As can be seen from FIG. 1, of the 64 selected sites inserted into the 3CLpro protease cleavage sequence, 31 sites, i.e., sites 10, 13, 14, 15, 20, 22, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 39, 40, 41, 47, 48, 52, 53, 54, 55, 58, 59, 60, 61, 62, 63 are compatible with the exogenous protease cleavage sequence and have no or limited effect on RFP fluorescence. The remaining sites, after insertion of the 3CLpro protease cleavage sequence, were not able to detect fluorescence of RFP, e.g.site 3/49/64, etc.

In finding the position of the RFP insertable protease cleavage sequence, the inventors thought to be as follows:

1. first, site 1-22 was randomly selected to construct the corresponding pRFP 1-22 plasmid for cell transfection. After transfection, it was found that of the 22 sites above, sites 10, 13, 14, 15, 20, 22 were compatible with the exogenous protease cleavage sequence, i.e., RFP10/13/14/15/20/22 was detectable to excite red fluorescence.

2. Subsequently, the inventors further explored whether additional sites were present upstream and downstream of site10, 13, 14, 15, 20, 22RFP positive sites for compatible cleavage by exogenous proteases. For this, the inventors constructed pRFP 23-64 plasmid transfected cells and observed the fluorescent expression. It was verified that sites 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 39, 40, 41, 47, 48, 52, 53, 54, 55, 58, 59, 60, 61, 62, 63 are compatible with the exogenous protease cleavage sequence in addition to the above sites, and that sites 20, 28, 29, 30, 31 constitute region one (amino acids PADGGL, P150-L155) (fig. 2), sites 13, 32, 33, 34, 35, 36, 47, 48, 58, 59, 60 constitute region two (amino acids KKPAKNLKMPGV, K182-V193) (fig. 3), sites 14, 38, 39, 40, 41 constitute region three (amino acids riad, R202-D207) (fig. 4), and sites 15, 52, 53, 54, 55, 61, 62, 63 constitute region four (amino acids VARYCDLPS, V219-S227) (fig. 5). Therefore, we speculate that the presence of a region of high flexibility in RFP is compatible with the shorter exogenous amino acid sequence, which in the present invention is the exogenous protease cleavage sequence.

RFP amino acid sequence:

MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILA

TSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIR

GVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSDMALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARYCDLPSKLGHK*(SEQ ID NO.1)。

nucleotide sequence encoding RFP:

ATGGTGAGCGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACCGT

GAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACC

CAGACCATGAGAATCAAGGTGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCT

GGCTACCAGCTTCATGTACGGCAGCAGAACCTTCATCAACCACACCCAGGGCATCCCCG

ACTTCTTTAAGCAGTCCTTCCCTGAGGGCTTCACATGGGAGAGAGTCACCACATACGAA

GACGGGGGCGTGCTGACCGCTACCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCT

ACAACGTCAAGATCAGAGGGGTGAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAA

AACACTCGGCTGGGAGGCCAACACCGAGATGCTGTACCCCGCTGACGGCGGCCTGGAA

GGCAGAAGCGACATGGCCCTGAAGCTCGTGGGCGGGGGCCACCTGATCTGCAACTTCA

AGACCACATACAGATCCAAGAAACCCGCTAAGAACCTCAAGATGCCCGGCGTCTACTAT

GTGGACCACAGACTGGAAAGAATCAAGGAGGCCGACAAAGAGACCTACGTCGAGCAG

CACGAGGTGGCTGTGGCCAGATACTGCGACCTCCCTAGCAAACTGGGGCACAAGTGA(SEQ ID NO.14)。

referring to FIG. 2, it can be seen that the modified RFP can still excite red fluorescence to different degrees when the insertion site is located at 150-155 aa. Namely, the amino acid sequence of RFP has an insertion region I of an exogenous protease cleavage sequence, P150-L155, and the amino acid sequence of the region is shown as SEQ ID NO.15: as shown by PAGGL, the RFP27 and RFP44 formed by inserting the exogenous protease cleavage sequence into the adjacent site of the region can not detect fluorescence.

Referring to FIG. 3, it can be seen that the modified RFP can still excite red fluorescence to different degrees when the insertion site is located at 182-193 aa. Namely, the amino acid sequence of RFP has an insertion region II of an exogenous protease cleavage sequence, wherein the amino acid sequence of the region II is shown as SEQ ID NO.16: KKPAKNLKMPGV, both RFP64 and RFP37 formed by insertion of the cleavage sequence of the foreign protease into the adjacent site of the region were not detected in fluorescence.

Referring to FIG. 4, it can be seen that the modified RFP can still excite red fluorescence to different degrees when the insertion site is located at 202-207 aa. Namely, an exogenous protease cleavage sequence insertionable region III of the RFP amino acid sequence is R202-D207, and the amino acid sequence of the region is shown as SEQ ID NO.17: as shown in RIKEAD, RFP22 formed by inserting the exogenous protease cleavage sequence into the adjacent site of the region can detect fluorescence, and other sites can not detect fluorescence.

Referring to FIG. 5, it can be seen that the modified RFP can still excite red fluorescence to different degrees when the insertion site is located at 219-227 aa. Namely, the RFP amino acid sequence has an insertion region IV of an exogenous protease cleavage sequence V219-S227, and the amino acid sequence of the region is shown as SEQ ID NO.18: VARYCDLPS, no fluorescence was detected by the engineered RFPs formed by insertion of the exogenous protease cleavage sequence into the region adjacent to the site.

Example 2

To further verify that the above regions of high flexibility are compatible with shorter exogenous amino acid sequences, we have found other viruses with self-cleaving proteases and corresponding protease cleavage sequences (Table 2), which were inserted into the sites of the high flexibility region above RFP.

The inventors randomly selected four sites, site15, site30, site34, site53, and inserted the protease cleavage sequences of the viruses shown in Table 2 into the sites, respectively, to form an engineered RFP containing the protease cleavage sequences of the specific viruses, named RFP-virus-cleavage sequences, such as RFP15-DV-cut1, representing insertion of protease NS2B-NS3 cleavage sequence cut1 of dengue virus into site15, and cloned it into pcDNA3.1+, transfected cells and observed for fluorescent expression.

Since most protease cleavage sequences are generally within 10 amino acids in length, to verify that RFP can be compatible with the maximum length of exogenous amino acid sequences, we insert the protease cleavage sequence of the novel coronavirus (SEQ ID NO. 2) into site30 site in RFP after ligation with the protease cleavage sequences corresponding to other viruses, named RFP30-COVID19+ virus-cleavage sequence, clone it into pcDNA3.1+, transfect cells and observe fluorescent expression.

TABLE 2

The results were as follows:

as shown in FIGS. 6-9, the modified RFPs formed after the protease cleavage sequences of the above viruses were inserted into the above-mentioned region sites of RFPs, and the modified RFPs formed by the combination of the remaining sites and the protease cleavage sequences except for RFP15-HCV-cut1, RFP15-HCV-cut2 and RFP30-HCV-cut2 were able to detect red-excited fluorescence to different extents.

As shown in FIG. 14, when the length of the inserted exogenous amino acid sequence reaches 14, 14 and 16 amino acids after the two linked viral protease cleavage sequences are inserted into the RFP site30 site, the excitation of red fluorescence is still detected, but the fluorescence intensity is reduced.

Example 3

Fluorescence Effect of the engineered RFP when coexpressed with 3CLpro

One of the important factors in the screening method of antiviral drugs of the present invention is that the fluorescence emitted by RFP can still be detected after insertion of the protease cleavage sequence. Another factor is that the antiviral drug screening method of the present invention also requires RFPs containing protease cleavage sequences that can be efficiently cleaved by the corresponding protease. In the first aspect, because the cleavage sequence of the foreign protease is inserted into the RFP, it is likely to break the secondary structure of the RFP, thus affecting the excitation fluorescence generation of the RFP. The cleavage efficiency of the protease also affects the signal-to-noise ratio of the whole antiviral drug screening system if the first aspect is satisfied.

To verify the cleavage effect of 3CLpro on the modified RFP, plasmid pRFP15/20/30/34 (remark: "/" means "or", the same applies hereinafter) of example 1 was randomly selected, 3CLpro was inserted after RFP of these plasmids using homologous recombination technique (Uniclone One Step Seamless Cloning Kit kit), P2A (2A peptide) was selected for ligation in the present example, and plasmid pRFP15/20/30/34-P2A-3CLpro capable of expressing 3CLpro was obtained.

Cell transfection was performed on the constructed pRFP15/20/30/34-P2A-3CLpro plasmid by the method of reference example 1, and fluorescence intensity was observed.

The results were as follows:

referring to FIG. 10, red fluorescence was not detected after co-expression of both RFP15/20/30/34 and 3CLpro proteases, i.e., plasmids pRFP15-P2A-3CLpro, pRFP15-P2A-3CLpro, pRFP30-P2A-3CLpro, pRFP34-P2A-3CLpro, and the expressed 3CLpro protease could cleave the engineered RFP after transfection of cells, resulting in quenching of fluorescence.

Example 4

To further verify that protease cleavage inhibited the fluorescent expression of RFP in the above system, the inventors added 5 consecutive stop codons TGA after P2A on the basis of pRFP15/20/30/34-P2A-3CLpro plasmid to disrupt the expression of 3CL protein.

The constructed pRFP15/20/30/34-P2A-3CLpro (5 xStop) disruption plasmid was transfected. Plasmid transfection was performed as in example 1, and the cell status was observed every 24 hours after transfection, and fluorescence photographing was performed for 48 hours.

The experimental results are as follows:

referring to FIG. 11, pRFP15/20/30/34-P2A-3CLpro (5 xStop) was added 5 consecutive stop codons TGA after P2A, and the red fluorescence of RFP destroyed plasmid was restored to the original state. It follows that the above system is indeed that the protease cleaves the modified RFP resulting in no more excitation light being produced by the RFP.

Example 5

Evaluation of inhibitory Effect of inhibitor

The 293T cells were plated and plasmid transfection was performed when the 293T cells grew to a density of 70% -80%, and pRFP15-P2A-3CLpro plasmid and pRFP30-P2A-3CLpro plasmid were selected for transfection with the results of combination examples 3 and 4 showing superior performance. Preparing culture medium containing different concentrations of inhibitor 4 hr after transfection, washing cells with PBS, adding culture medium containing different concentrations of novel coronavirus 3CLpro protease inhibitor into cells, standing at 37deg.C, 5% CO ₂ Culturing in an incubator. Cell status was observed every 24h and fluorescent photographed. Addition of inhibitor EnstrelvirConcentration reference IC50 values of 13nM were added and the experimental groups were set to add Enstrelvir at concentrations of 1nM, 5nM, 10nM, 100nM, 1. Mu.M, 10. Mu.M, 50. Mu.M, respectively. Control groups were added with 10 μl DMSO.

The results were as follows:

as shown in FIG. 12, cells transfected with pRFP15-P2A-3CLpro plasmid had higher fluorescence intensity with increasing inhibitor concentration at 24h and 48h post-transfection at the same excitation light intensity when inhibitor Enstrelvir was added. At 10. Mu.M, 293T cells cultured for 24h and 293T cells cultured for 48h showed very weak red fluorescence, and when Enstrelvir was added at 50. Mu.M or more, stronger red fluorescence was observed.

As shown in FIG. 13, cells transfected with pRFP30-P2A-3CLpro plasmid had higher fluorescence intensity with increasing inhibitor concentration at 24h and 48h post-transfection at the same excitation light intensity when inhibitor Enstrelvir was added. At 10. Mu.M, 293T cells cultured for 48h showed very weak red fluorescence, and when Enstrelvir was added at 50. Mu.M or more, stronger red fluorescence was observed.

Therefore, the fluorescent protein containing the protease cleavage sequence and the plasmid coexpression of the fluorescent protein and the corresponding protease are enough to be used for screening and activity detection of low-flux and high-flux protease inhibitor antiviral drugs.

The above-described embodiment is only a preferred embodiment of the present invention, and is not limited in any way, and other variations and modifications may be made without departing from the technical aspects set forth in the claims.

Claims (15)

1. The red fluorescent reporter protein is characterized by being prepared by modifying red fluorescent protein, and specifically comprises the following components: inserting an exogenous amino acid sequence which can be recognized and cleaved by protease at a specific position of the red fluorescent protein, wherein the inserted exogenous amino acid sequence does not influence the light-emitting function of the red fluorescent protein;

the specific location includes an insertion region;

the insertion region is selected from one of a first insertion region, a second insertion region, a third insertion region, and a fourth insertion region,

the amino acid sequence of the first insertion region is PAGGL,

the amino acid sequence of the second insertion region was KKPAKNLKMPGV,

the amino acid sequence of the third insertion region is RIKEAD,

the amino acid sequence of the fourth insertion region was VARYCDLPS.

2. The red fluorescent reporter protein is characterized by being prepared by modifying red fluorescent protein, and specifically comprises the following components: inserting an exogenous amino acid sequence which can be recognized and cleaved by protease at a specific position of the red fluorescent protein, wherein the inserted exogenous amino acid sequence does not influence the light-emitting function of the red fluorescent protein;

the specific location includes a separate insertion site;

the individual insertion sites include independent site a, independent site B,

independent site A is amino acid 145-146 of RFP amino acid sequence, or corresponding amino acid region on amino acid sequence with sequence identity over 95% compared with RFP amino acid sequence;

independent site B is amino acid 208-209 of RFP amino acid sequence, or corresponding amino acid region on amino acid sequence with sequence identity over 95% compared with RFP amino acid sequence;

the RFP amino acid sequence is shown in SEQ ID No. 1.

3. The red fluorescent reporter protein of claim 1 or 2, wherein the inserted exogenous amino acid sequence is 16 amino acids or less in length.

4. The red fluorescent reporter protein of claim 1 or 2, wherein the exogenous amino acid sequence comprises a protease cleavage sequence that is cleavable by a viral protease.

5. The red fluorescent reporter protein of claim 4, wherein the viral protease is from one of a coronaviridae virus, a picornaviridae virus, a calicividae virus, a togaviridae virus, a flaviviridae virus, an enteroviridae, a retrovirus, a herpesviridae, a poxviridae virus.

6. The red fluorescent reporter protein of claim 4, wherein the protease cleavage sequence cleavable by a viral protease is selected from the group consisting of one or more of the sequences set forth in SEQ ID No.2-SEQ ID No. 12.

7. The red fluorescent reporter protein of claim 1, wherein an insertion site is defined between any two amino acids within the first insertion region, the second insertion region, the third insertion region, and the fourth insertion region.

8. A nucleotide, characterized in that: encoding the red fluorescent reporter protein of any one of claims 1-7.

9. A plasmid comprising a nucleotide sequence as claimed in claim 8.

10. The plasmid of claim 9, further comprising a nucleotide sequence encoding a viral protease, wherein the plasmid co-expresses a red fluorescent reporter protein and a viral protease.

11. The plasmid of claim 10, wherein the viral protease is from one of the group consisting of coronaviridae, picornaviridae, caliciviridae, togaviridae, flaviviridae, enteroviridae, retrovirus, herpesviridae, poxviridae.

12. The screening method of the antiviral drug taking protease as a target spot is characterized by comprising the following steps of:

(1) Constructing a co-expression plasmid for co-expressing the red fluorescent reporter protein of claim 1 or 2 and the viral protease;

(2) Screening: spreading cells to be transfected in a porous plate, then adding a co-expression plasmid for transfection, culturing the transfected cells, simultaneously adding a drug to be screened into the porous plate, taking a group without the drug to be screened as a contrast, observing the state of the cells, performing fluorescence photographing, and detecting the fluorescence intensity; if the fluorescence intensity is increased compared with the control, the drug to be screened has an inhibition effect on viral protease, and if the fluorescence intensity is increased, the inhibition effect on viral protease by the drug to be screened is stronger.

13. The screening method of claim 12, wherein the viral protease is derived from one of a coronaviridae virus, a picornaviridae virus, a caliciviridae virus, a togaviridae virus, a flaviviridae virus, an enteroviridae virus, a retrovirus family virus, a herpesviridae virus, a poxviridae virus.

14. A method for evaluating the effect of a protease inhibitor on forward visual inhibition, comprising the steps of:

(1) Constructing a co-expression plasmid for co-expressing the red fluorescent reporter protein of claim 1 or 2 and the viral protease;

(2) Evaluation: spreading the cells to be transfected in a cell culture dish, then adding a co-expression plasmid for transfection, culturing the transfected cells, simultaneously adding an inhibitor into the cell culture dish, taking the cell without the inhibitor as a control, observing the state of the cells, taking a fluorescent photograph, and detecting the fluorescence intensity; the forward direction of the fluorescence intensity reflects the inhibition effect of the inhibitor, and the higher the fluorescence intensity is, the stronger the inhibition effect of the inhibitor on viral protease is.

15. The method for evaluating the effect of forward visual inhibition according to claim 14, wherein the viral protease is derived from one of coronaviridae, picornaviridae, caliciviridae, togaviridae, flaviviridae, enteroviridae, retrovirus, herpesviridae, poxviridae.