CN117088988B - Fusion protein, plasmid and application thereof in HIV protease inhibitor screening and drug effect evaluation - Google Patents

Fusion protein, plasmid and application thereof in HIV protease inhibitor screening and drug effect evaluation Download PDF

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CN117088988B
CN117088988B CN202311361133.7A CN202311361133A CN117088988B CN 117088988 B CN117088988 B CN 117088988B CN 202311361133 A CN202311361133 A CN 202311361133A CN 117088988 B CN117088988 B CN 117088988B
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hiv protease
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protease
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CN117088988A (en
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简书令
方晨捷
倪思睿
孙慧敏
宋家升
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Zhejiang Difu Runsi Biotechnology Co ltd
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    • G01N2333/8142—Aspartate protease (E.C. 3.4.23) inhibitors, e.g. HIV protease inhibitors
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Abstract

The invention provides a fusion protein, a plasmid and application thereof in HIV protease inhibitor screening and drug effect evaluation. The HIV fluorescent reporter protein obtained after expression has fluorescence excitation activity same as or similar to that of the wild GFP fluorescent protein by inserting enzyme cleavage substrate sequence of the HIV protease into the gene sequence of the wild GFP fluorescent protein. The HIV protease is cloned and expressed on the plasmid of the HIV fluorescent reporter protein to obtain fusion protein, and the fluorescence can be used for detecting the activity of the HIV protein and the activity of the HIV protease inhibitor, so that a HIV protease inhibitor screening and efficacy evaluation platform can be formed.

Description

Fusion protein, plasmid and application thereof in HIV protease inhibitor screening and drug effect evaluation
Technical Field
The invention relates to the field of biotechnology, in particular to fusion protein, plasmid and application thereof in screening HIV protease inhibitors and evaluating drug effect.
Background
Acquired immunodeficiency syndrome (Acquired immunodeficiency syndrome, AIDS), also known as AIDS, is a global significant disease caused by the human immunodeficiency virus (Human immunodeficiency virus, HIV). United nations have indicated in 2018's aids report that HIV has caused 7000 tens of thousands of infections, about 3500 tens of thousands of deaths, during the last twenty years. The number of newly infected HIV in 2018 is 170 tens of thousands, and the number of patients dying from AIDS-related diseases worldwide is still up to hundreds of thousands. HIV has two subtypes, type 1 and type 2, of which type 1 is the major strain currently prevalent worldwide.
The genome of HIV consists of two identical positive strand RNAs, with a full length of about 9.7 kb. The genome is wrapped in a viral protein shell, and the periphery of the nucleocapsid is a phospholipid bilayer derived from a host cell membrane and a viral-encoded membrane protein. The HIV genome contains 3 structural genes gag, pol, and env, where the gag gene sequence encodes a nucleocapsid protein; the env gene sequence encodes two glycoproteins, envelope glycoproteins gp120 and gp41, necessary for the virus to infect cells; the pol gene sequence encodes enzymes necessary for viral replication, namely reverse transcriptase, binding enzyme and viral protease. HIV protease is an aspartic protease that is active only as a homodimer. It is capable of cleaving the gag gene and the multimeric protein expressed by the gag-pol gene of HIV into proteins required by the virus. The Gag region carries viral structural proteins, while the Pol region contains viral enzymes (protease, reverse transcriptase, RNase H and integrase).
The HIV protease plays a very critical role in the maturation and replication of the HIV virus, and inhibiting this enzyme results in the production of non-infectious progeny virus, thereby preventing further infection by the virus. In 1995, the first antiviral compound with a completely new mechanism of action, saquinavir, was FDA approved and was a peptidomimetic inhibitor of HIV protease. Other viral protease inhibitors and compounds have subsequently emerged for different steps of viral infection. More protease inhibitor drugs may provide more options for the collocation of therapeutic regimens. Therefore, it is important to develop a drug efficacy evaluation system for viral protease inhibitors.
Most cellular antiviral experiments of highly infectious viruses need to be performed in a laboratory of BSL-2+ or higher, however the resources of such a laboratory are rather scarce. The in-vitro large-scale screening and drug effect evaluation methods applied at present are mainly divided into two types according to detection principles: (1) competitive binding method: is a main in vitro screening method, which is to add the drug to be screened after the existing protease is immobilized and to competitively combine the immobilized protease with the existing effective drug. Wherein the medicine to be screened can be provided with detection marks such as biotin, fluorescent substances and the like for detection. It competes with existing protease inhibitors for binding sites, and therefore only congeners of known protease inhibitors can be screened. The screening and evaluation effects of the protease which cannot bind to the known protease inhibitor against different sequences are poor. (2) functional assay: the fluorescent quenching molecule containing the short peptide of the protease cleavage site is used as a substrate of the protease, and the fluorescent gene is separated from the quenching group to emit fluorescence after the protease is used for cutting the substrate, so that the activity of the screened medicine can be judged by detecting the fluorescence intensity, but the method is easy to be interfered by small molecular compounds to generate false positive. Meanwhile, many inhibitors with good in vitro activity have poor cell membrane permeability, and have no activity or weak activity in cells.
Therefore, it is necessary to develop a simple, safe, high-throughput and reproducible protease activity and related protease inhibitor screening and efficacy evaluation platform.
Disclosure of Invention
The invention aims to provide a fusion protein, a plasmid and application thereof in HIV protease inhibitor screening and drug effect evaluation.
In order to solve the problems, the invention provides the following technical scheme:
in a first aspect, the present invention provides a fusion protein comprising an HIV fluorescent reporter protein and an HIV protease PR linked by a self-cleaving 2A short peptide; the HIV fluorescent reporter protein is a recombinant GFP gene obtained by inserting an enzyme digestion sequence of HIV protease PR into a specific site of a wild GFP fluorescent protein sequence, and then the recombinant GFP gene is inserted into a eukaryotic expression vector and then expressed.
Further, the HIV fluorescent reporter protein has the same or similar fluorescence-stimulating activity as the wild-type GFP fluorescent protein.
It will be appreciated that the HIV fluorescent reporter protein of the present invention is engineered from wild-type GFP (Green fluorescent protein ) and the engineered protein does not affect the original fluorescent activity of the GFP fluorescent protein.
Further, the cleavage sequence of the protease PR is inserted into the wild-type GFP fluorescent protein at the position comprising site15, site20 and site24; wherein site15 is located between KQ and KN of the amino acid sequence shown as SEQ ID NO.4 (MADKQKN); site20 is located between EG and DT of the amino acid sequence shown as SEQ ID NO.5 (FEGDTL); site24 is located between QK and NG of the amino acid sequence shown as SEQ ID NO.6 (DKQKNGI).
In some technical schemes, the amino acid sequence of the wild GFP fluorescent protein is shown as SEQ ID NO. 1, site15 is positioned between amino acids 158 and 159 of the GFP amino acid sequence; site20 is located between amino acids 117 and 118 of the GFP amino acid sequence; site24 is located between amino acids 159 and 160 of GFP amino acids.
Further, the enzyme digestion amino acid sequence of the HIV protease PR is RVLAEA and/or TIMMQR.
In another aspect, the invention provides recombinant genes encoding the HIV fluorescent reporter proteins.
The invention also provides an expression vector which comprises a gene sequence for encoding the HIV fluorescent reporter protein and a gene sequence for HIV protease PR, and the expression vector co-expresses the HIV fluorescent reporter protein and the protease PR.
Further, the present invention provides a plasmid comprising the expression vector in which a plasmid expressing an HIV fluorescent reporter protein is linked to an HIV protease PR via a self-cleaving 2A short peptide.
The invention also provides application of the fusion protein, the recombinant gene, the expression vector or the plasmid in HIV protease inhibitor activity evaluation and screening.
In another aspect, the invention provides a method for determining HIV protease activity comprising the steps of:
the HIV protease PR is connected with the HIV fluorescent reporter protein through a 2A short peptide in a plasmid of an expression vector for expressing the HIV fluorescent reporter protein, so that the HIV protease PR and the HIV fluorescent reporter protein are co-expressed in cells, the activity of the HIV protease PR is characterized by measuring the fluorescence signal intensity of a co-expression system, and the lower the fluorescence signal intensity is, the higher the activity of the HIV protease PR is, and the two are in negative correlation.
In yet another aspect, the present invention provides a method for screening an inhibitor of HIV protease activity, comprising the steps of:
co-incubating an expression vector co-expressing an HIV fluorescent reporter protein and an HIV protease PR with a candidate protease inhibitor in cells as an experimental group while setting a negative control to which the candidate protease inhibitor is not added, and screening an HIV protease activity inhibitor according to a change in fluorescent signal intensity of the experimental group relative to the negative control;
the change in fluorescence signal intensity is an increase in fluorescence signal intensity.
The invention also provides a kit for screening inhibitors of HIV protease activity comprising an expression vector expressing an HIV fluorescent reporter protein and an HIV protease PR or a plasmid expressing an HIV fluorescent reporter protein.
The principle of the invention is as follows: after the HIV fluorescent reporter is expressed in the cell, it has the same or similar fluorescent activity as the wild-type GFP fluorescent protein. When the HIV fluorescent reporter is co-expressed with the HIV protease PR, the HIV protease PR undergoes self-cleavage to release itself to react with the cleavage substrate in the HIV fluorescent reporter, which results in the structure of the HIV fluorescent reporter being destroyed and the fluorescence being reduced or eliminated. Therefore, when the system does not contain HIV protease PR or inhibitor or the added candidate inhibitor has no inhibition effect, the HIV fluorescence reporter protein can normally emit fluorescence, and the normal fluorescence signal intensity is detected. After the system HIV fluorescent reporter protein and the HIV protease PR are co-expressed, an inhibitor or a candidate inhibitor is added, if the fluorescence signal intensity is improved, the fact that the inhibitor or the candidate inhibitor inhibits the activity of protease is indicated, so that the HIV protease PR cannot react with an enzyme-cleaved substrate, the complete structure of the HIV fluorescent reporter protein is reserved, the fluorescence effect can be exerted, the fact that the inhibitor or the candidate inhibitor has an inhibition effect on protease targets is indicated, and the fluorescence intensity is positively correlated with the inhibition effect.
Compared with the prior art, the invention has the following technical effects:
the fusion protein provided by the invention comprises an HIV fluorescent reporter protein and an HIV protease PR, wherein the HIV fluorescent reporter protein and the HIV protease PR are connected by a self-shearing 2A short peptide; the HIV fluorescent reporter protein is a recombinant GFP gene obtained by inserting an enzyme digestion sequence of HIV protease PR into a specific site of a wild GFP fluorescent protein sequence, and then the recombinant GFP gene is inserted into a eukaryotic expression vector and then expressed. The HIV fluorescent reporter protein obtained after expression has fluorescence excitation activity same as or similar to that of the wild GFP fluorescent protein by inserting enzyme cleavage substrate sequence of the HIV protease into the gene sequence of the wild GFP fluorescent protein. Further, by cloning and expressing HIV protease on a plasmid of HIV fluorescent reporter protein, the HIV fluorescent reporter protein and HIV protease PR are co-expressed to obtain fusion protein, and the fluorescence can be used for detecting the activity of HIV protease and the activity of HIV protease inhibitor, thus forming a platform for screening HIV protease inhibitor and evaluating the efficacy of the HIV protease inhibitor.
The method for determining the activity of the HIV protease and the method for screening the HIV protease activity inhibitor provided by the invention only need to use the expression vector for coexpression of the HIV fluorescent reporter protein and the HIV protease PR, and can obtain a qualitative result by observing fluorescent signals of cells, so that safety concern caused by using HIV live viruses can be avoided, the requirement on the biological safety level is low, the BSL-1 laboratory can meet the experimental requirement, and the method has the advantages of high efficiency, accuracy, stability, high flux and repeatability.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the comparison of the fluorescence effects of pGFP20-HIVcut1/cut2 and pGFP20-HIVcut1/cut 2-P2A-PR constructed in examples 1 and 2 of the present invention;
FIG. 2 is a graph showing the comparison of the fluorescence effects of pGFP15-HIVcut1/cut2 and pGFP15-HIVcut1/cut 2-P2A-PR constructed in examples 1 and 2 of the present invention;
FIG. 3 is a graph showing the comparison of the fluorescence effects of pGFP24-HIVcut1/cut2 and pGFP24-HIVcut1/cut 2-P2A-PR constructed in examples 1 and 2 of the present invention;
FIG. 4 is a graph showing the comparison of the fluorescence effects of the comparative example separately transfected with a plasmid expressing HIV protease PR and a plasmid expressing HIV reporter fluorescent protein and transfected with a plasmid co-expressing HIV protease PR and HIV reporter fluorescent protein.
Description of the embodiments
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments. It will be apparent that the embodiments described below are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The specific steps can be seen in: molecular cloning guidelines (Molecular Cloning: A Laboratory Manual) Sambrook, j., russell, davidw., molecular Cloning: A Laboratory Manual,3rd edition,2001,NY,Cold SpringHarbor).
The various biomaterials described in the examples were obtained by merely providing an experimental route for achieving the objectives of the specific disclosure and should not be construed as limiting the source of biomaterials of the present invention. In fact, the source of the biological material used is broad, and any biological material that is available without violating law and ethics may be used instead as suggested in the examples.
Example 1 HIV fluorescent reporter
The present invention first performed PCR amplification of GFP and pcDNA3.1 (+) vectors according to the Primer Star enzyme instructions. The GFP 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 pcDNA3.1 (+) -GFP plasmid as follow-up template and control.
Then, the cleavage substrates cut1 of HIV protease PR were inserted into the site15/20/24 positions of wild-type GFP (amino acid sequence shown in SEQ ID NO: 1) by PCR and homologous recombination technique (Uniclone One Step Seamless Cloning Kit kit), respectively: RVLAEA, cut2: TIMMQR constructs plasmid pGFP15/20/24-HIVcut1/cut 2. Table 1 shows the cleavage sites for HIV protease PR. Wherein, GFP15 is a modified HIV fluorescent reporter protein formed by inserting enzyme cleavage substrate sequences between amino acids 158 and 159 of GFP amino acid sequences; GFP20 is a modified HIV fluorescent reporter formed by inserting enzyme cleavage substrates between amino acids 117 and 118 of the GFP amino acid sequence; GFP24 is a engineered HIV fluorescent reporter formed by insertion of cleavage substrates between amino acids 159 and 160 of GFP.
TABLE 1 cleavage site of HIV protease PR
The constructed pGFP15/20/24-HIVcut1/cut2 plasmid was transfected. 293T cells have the advantages of fast growth and high protein expression levels and are therefore selected as the object of plasmid transfection. The transfection procedure was as follows:
and (3) performing experiments by using a six-hole cell plate, and performing plasmid transfection experiments when 293T cells grow to 70% -80%. Plasmid transfection was 2. Mu.g, and 10% FBS DMEM was used as the medium, and the specific experimental procedure was as follows: 1. adding a required culture medium into an EP tube, then adding lipo2000, gently blowing and mixing, and standing for 5 min; 2. adding plasmid into the above mixture, gently blowing, mixing, and standing for 25 min. 3. The cells were washed with PBS and then added to the medium, and the liquid in the EP tube was added uniformly to the wells and incubated in a carbon dioxide incubator at 37 ℃. 4. After culturing for 4-6 hours, washing with PBS, adding a culture medium, and culturing in a carbon dioxide incubator at 37 ℃. 5. Cell status was observed every 24 th h and the effect of fluorescence was photographed. The results are shown in FIGS. 1-3.
As can be seen from the graph, the multiple HIV fluorescence reporter proteins prepared in this example all showed fluorescence activity, wherein pGFP15-HIVcut1 and pGFP24-HIVcut2 showed the best fluorescence effect. This shows that the HIV fluorescent reporter protein constructed by the invention has the same or similar fluorescent activity as the original GFP protein at a specific site.
EXAMPLE 2 construction of HIV fluorescent reporter and HIV protease PR Co-expression plasmids
Subsequently, in order to verify the cleavage effect of HIV protease PR on cleavage sites in HIV fluorescent reporter protein, PCR amplification (Primer Star) and homologous recombination technology (Uniclone One Step Seamless Cloning Kit kit) were used to insert HIV protease PR after GFP of the plasmid of example 1, and P2A short peptide was used in the middle to construct plasmid pGFP15/20/24-HIVcut1/cut 2-P2A-PR. The amino acid sequence of the HIV protease PR used in this example is SEQ ID NO. 7 and the nucleotide sequence is SEQ ID NO. 8.
The constructed pGFP15/20/24-HIVcut1/cut2 and pGFP15/20/24-HIVcut1/cut 2-P2A-PR plasmid were transfected. Transfection procedure was as in example 1.
The experimental results are shown in FIGS. 1-3: as is evident from comparison of the fluorescence photographs of example 1, pGFP15-HIVcut1 and pGFP24-HIVcut2, which have the best initial fluorescence effects, have significantly reduced fluorescence effects after co-expression. This shows that after the HIV fluorescent reporter protein is co-expressed with the HIV protease PR, the HIV protease PR can self-shear to release the HIV protease PR to react with the enzyme-cleaved substrate in the HIV fluorescent reporter protein, so that the structure of the HIV fluorescent reporter protein is destroyed, and the fluorescence effect is weakened or vanished.
Comparative example
Cloning HIV Protease into pcDNA3.1 (+) plasmid multiple cloning site by PCR and homologous recombination to construct plasmid pPR for expressing HIV Protease PR, mixing pPR with plasmid pGFP15-HIVcut1 and plasmid pGFP24-HIVcut2 in equal proportion, and transfecting plated 293T cells. Transfection procedure was as in example 1.
As a result, as shown in FIG. 4, it was found that when HIV fluorescent reporter and HIV protease PR were co-transfected into two plasmids, there was little cleavage of HIV fluorescent reporter by HIV protease PR and no significant decrease in fluorescence intensity compared to when HIV fluorescent reporter was expressed by single transfection.
This shows that the difference in expression mode affects the function of the fusion protein, and the same plasmid is used for co-expressing the HIV fluorescence reporter protein and the HIV protease PR to obtain the fusion protein, so that the fluorescence effect of the fusion protein can be used for detecting the activity of HIV protease and the activity of HIV protease inhibitor, and a HIV protease inhibitor screening and efficacy evaluating platform can be formed.
In the foregoing embodiments, the descriptions of the embodiments are focused on, and for those portions of one embodiment that are not described in detail, reference may be made to the related descriptions of other embodiments.
While the invention has been described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes and substitutions of equivalents may be made and equivalents will be apparent to those skilled in the art without departing from the scope of the invention. Therefore, the protection scope of the invention is subject to the protection scope of the claims.

Claims (6)

1. A fusion protein comprising an HIV fluorescent reporter protein, an HIV protease, and a self-cleaving 2A short peptide linking the HIV fluorescent reporter protein and the HIV protease; the HIV fluorescent reporter protein is recombinant GFP protein obtained by inserting an enzyme digestion sequence of HIV protease into a specific site of a wild GFP fluorescent protein amino acid sequence;
the HIV fluorescent reporter protein has the same or similar fluorescence excitation activity as the wild GFP fluorescent protein;
the amino acid sequence of the wild GFP fluorescent protein is shown as SEQ ID NO. 1;
the enzyme cutting sequence of the HIV protease is inserted into a site15 or site24 of a wild GFP fluorescent protein; wherein site15 is located between amino acids 158 and 159 of the sequence of SEQ ID NO. 1; site24 is located between amino acids 159 and 160 of the sequence of SEQ ID NO. 1;
the HIV protease has an enzyme digestion sequence of RVLAEA or TIMMQR.
2. A recombinant gene encoding the fusion protein of claim 1.
3. An expression vector comprising the recombinant gene of claim 2, wherein the expression vector co-expresses the HIV fluorescent reporter protein and HIV protease.
4. Use of the fusion protein of claim 1, the recombinant gene of claim 2 or the expression vector of claim 3 in HIV protease inhibitor activity assessment and screening.
A method for determining hiv protease activity comprising the steps of:
culturing cells comprising the expression vector of claim 3, and characterizing HIV protease activity by measuring the fluorescent signal intensity of the co-expression system.
A method of screening for inhibitors of hiv protease activity comprising the steps of:
incubating cells comprising the expression vector of claim 3 with a candidate protease inhibitor as an experimental group while providing a negative control without the candidate protease inhibitor, and screening for HIV protease inhibitor based on the change in fluorescent signal intensity of the experimental group relative to the negative control;
if the fluorescence signal intensity of the experimental group is improved compared with that of the negative control, the candidate protease inhibitor has an inhibition effect on HIV protease, and the fluorescence intensity is positively correlated with the inhibition effect.
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Citations (5)

* Cited by examiner, † Cited by third party
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