Application of inhibitor for resisting Tancheng virus 1 nucleoprotein

Abstract

The invention belongs to the technical field of biological medicine, provides an application of an inhibitor for resisting Tancheng virus 1 nucleoprotein, and provides an application of a compound candesartan in the inhibitor for resisting Tancheng virus 1 nucleoprotein, and the compound candesartan is obtained by the following steps: expressing and purifying Tancheng virus 1 nucleoprotein by using an escherichia coli system, and purifying the Tancheng virus 1 nucleoprotein to obtain the Tancheng virus 1 nucleoprotein. A molecular thermal drift experiment TSA technology is used for screening natural drugs and FDA drug libraries, an effective screening system is established, a compound candesa capable of interacting with nucleoprotein is found, affinity determination is further carried out through experiments such as a biofilm interference technology BL I, the affinity of the compound candesa and the nucleoprotein is analyzed, and the research result shows that the compound candesa can interact with the nucleoprotein. Meanwhile, functional characteristics of nucleoprotein are utilized for verification and analysis, and it is verified that the compound candesa can effectively inhibit combination of the nucleoprotein and nucleic acid and can inhibit the nuclease hydrolysis capacity of the nucleoprotein.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically is an application of an anti-Tacheng virus 1 nucleoprotein inhibitor. Background Art

[0002] The Nairoviridae family contains a variety of viruses, some of which are distributed worldwide. For example, Crimean-Congo hemorrhagic fever virus (CCHFV) is a medically important virus in this family. It is widely endemic in Asia, Africa, the Middle East, and Southeast Europe. In addition, Tamdy virus (TAMV) is a tick-borne virus of the genus Ortho-Nairovirus in the Nairoviridae family. It was detected in the Xinjiang Uygur Autonomous Region of my country. It is mainly transmitted by Hyalomma asiaticum and Dermacentor nuttalli as the main tick vectors. Tacheng tick virus 1 is one of the viruses of the genus Tamdy virus that can cause disease in humans discovered in the Xinjiang Uygur Autonomous Region of my country in 2019. It belongs to the genus Ortho-Nairovirus in the Nairoviridae family. The clinical symptoms are mainly fever and muscle aches. In severe cases, neurological disorders such as meningitis may occur.

[0003] However, there is currently no effective specific treatment for this type of virus, and the development of effective antiviral drugs is of great significance. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an application of an anti-Tacheng virus 1 nucleoprotein inhibitor to solve the problems raised in the background technology.

[0005] The invention provides the use of a compound Candesartan as an inhibitor of nucleoprotein against Tacheng virus 1.

[0006] Preferably, the compound Candesartan is used as an anti-Tacheng virus 1 nucleoprotein inhibitor to prepare a pharmaceutical composition for treating or preventing diseases caused by Tacheng virus 1 infection.

[0007] Preferably, the pharmaceutical composition comprises candesartan as an active ingredient.

[0008] Preferably, the compound candesartan is obtained by the following steps:

[0009] S1. Express and purify the Tacheng virus 1 nucleoprotein using the E. coli system;

[0010] S2. A thermal drift experiment based on protein stability was performed using a fluorescent quantitative PCR instrument, and through screening of natural medicines and FDA drug libraries, a compound called candesartan that significantly changed the Tm value of Tacheng virus 1 nucleoprotein was selected as a candidate inhibitor;

[0011] S3, determination of the affinity of compound candesartan to nucleoprotein by biolayer interferometry;

[0012] S4. The inhibitory effect of compound candesartan on nucleoprotein was verified by gel retardation experiment.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1. The present invention uses TSA technology to find that the candidate compound CC can change the Tm value of nucleoprotein, and the change value is greater than 3 degrees Celsius, indicating that the compound candesartan interacts with the nucleoprotein, and the affinity Kd of the compound candesartan ester to the nucleoprotein is 7.8 μM, which has a high affinity.

[0015] 2. The present invention uses gel migration imaging to find that the compound candesartan can effectively inhibit the binding ability of nucleoproteins to nucleic acids, and can also effectively inhibit the enzymatic hydrolysis of nucleic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a gel filtration chromatography purification diagram of Tacheng virus 1 nucleoprotein of the present invention;

[0017] Figure 2 The results of compound screening based on protein stability using TSA technology in the present invention;

[0018] Figure 3 The TSA results of candesartan ester and nucleoprotein of the present invention are as follows;

[0019] Figure 4 The affinity constant of nucleoprotein and candesartan is determined based on BLI in the present invention;

[0020] Figure 5 The present invention utilizes EMSA to analyze the inhibitory effect of candesartan cilexetil on the nuclear protein nucleic acid binding ability and the nucleolytic ability. DETAILED DESCRIPTION

[0021] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0022] The invention provides the use of a compound Candesartan as an inhibitor of nucleoprotein against Tacheng virus 1.

[0023] The compound Candesartan is used as an anti-Tacheng virus 1 nucleoprotein inhibitor for preparing a pharmaceutical composition for treating or preventing diseases caused by Tacheng virus 1 infection.

[0024] The pharmaceutical composition comprises candesartan as an active ingredient.

[0025] The compound candesartan is obtained by the following steps:

[0026] S1. Express and purify the nucleoprotein of Tacheng virus 1 using the E. coli system;

[0027] S2. A thermal drift experiment based on protein stability was performed using a fluorescent quantitative PCR instrument, and through screening of natural medicines and FDA drug libraries, a compound called candesartan that significantly changed the Tm value of Tacheng virus 1 nucleoprotein was selected as a candidate inhibitor;

[0028] S3, determination of the affinity of compound candesartan to nucleoprotein by biolayer interferometry;

[0029] S4. The inhibitory effect of compound candesartan on nucleoprotein was verified by gel retardation experiment.

[0030] Example 1: Expression and purification of Tacheng virus 1 nucleoprotein using E. coli system

[0031] The expression, extraction and crude purification of nuclear proteins are used for protein preparation. The specific steps are as follows:

[0032] The target gene sequence of TcTV-1 NP protein was obtained from the NCBI website, and the sequence was optimized for the codons preferred by E. coli. The optimized sequence was commissioned to be synthesized by a company. The nucleotide sequence of the optimized nucleoprotein is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The target gene was then constructed into the vector pET-28a with polyhistidine and SUMO fusion tags constructed in this experiment by seamless cloning. The plasmid extraction process is used to construct the expression vector. The specific operation steps refer to the plasmid extraction kit operating procedures.

[0033] SEQ ID NO.1 is as follows:

[0034]

[0035]

[0036] SEQ ID NO.2 is as follows:

[0037]

[0038]

[0039] The fusion plasmid pET-21a-10×His-SUMO-TcTV-1NP plasmid was transferred into competent cells of E. coli expression strain for transformation, ice bathed for 30 minutes, heat shocked at 42°C for 90 seconds, ice bathed for 5 minutes, and then 600 μl LB liquid was added for recovery for 1 hour;

[0040] Spread the revived bacterial solution on an agar plate containing 50 μg / ml kanamycin and culture at 37°C overnight. Pick the transformed positive clone colonies and add them to 50 ml of fresh LB culture medium (add kanamycin to inhibit the growth of bacteria) and culture them at 37°C overnight;

[0041] A single clone of each bacterial strain was selected and placed in LB medium, and kanamycin (final concentration was 50 μg / ml) was added. The culture was cultured in a shaker at 220 rpm and 37°C for 3.5 hours. When the OD value of the bacterial solution reached about 0.8, the inducer IPTG was added for induction culture.

[0042] Protein extraction: After the culture, the bacteria were collected by centrifugation, resuspended in lysis buffer (5 mM imidazole, 20 mM Tris-Hcl pH = 7.5, 300 mM NaCl), and PMSF was added with a final concentration of 2 mM. The cells were crushed by a combination of low-temperature and high-pressure crushing and ultrasonic crushing. The cells were centrifuged at 25,000 g for 35 minutes at 4°C, the supernatant was collected, and the precipitate was discarded.

[0043] The supernatant was initially purified by Ni-NTA affinity chromatography: the centrifuged supernatant was incubated with the balanced Ni-NTA filler at 4°C for 1 hour. The incubated mixture was passed through a gravity chromatography column, and then eluted with buffers of different imidazole concentrations (5mM, 20mM, 40mM, 250mM, 500mM), and the corresponding eluates were collected. The elution solutions of each concentration were retained, mixed with the protein loading buffer, denatured at 100°C for 10 minutes, and the purification effect of the target protein was detected by SDS-PAGE.

[0044] In order to obtain high-purity target protein, AKTA purification system was used for secondary purification - ion exchange chromatography. The specific steps of ion exchange chromatography are as follows: install the ion exchange column according to the operating standards of AKTA purification system and clean it with ultrapure water; use high salt buffer and low salt buffer to clean and balance the ion preloaded column to remove the impurities in the preloaded column; select a suitable concentrator according to the sample volume and the molecular weight of the target protein to perform protein buffer replacement balance; when the balance is stable to each baseline, load the protein sample onto the ion preloaded exchange column, and then use high salt buffer for gradient elution, collect the protein elution peak and perform SDS-PAGE gel electrophoresis for identification.

[0045] like Figure 1As shown, in order to further ensure the purity of the protein, gel filtration chromatography is performed; the specific operating steps of gel filtration chromatography are as follows: the target protein peak after ion exchange chromatography purification is collected, concentrated and the protein buffer is replaced with gel filtration chromatography buffer, and the final concentration volume depends on the chromatography column; the gel filtration chromatography buffer is used to balance the gel filtration column; after the baseline is balanced, the concentrated protein is loaded into the chromatography column, the target protein is eluted at a flow rate of 0.5 ml / min, and the protein elution peak is collected.

[0046] Embodiment 2: Figure 2-3 As shown, nuclear protein-based thermal shift assay for drug screening

[0047] A system was established using a fluorescent quantitative PCR instrument to test the interaction between different compounds and nuclear proteins, using SYRO-Orange as the fluorescent dye. 96-well or 384-well tests can be performed each time to increase the number of test samples.

[0048] The interaction between the compound and the nucleoprotein was characterized by the change in the Tm value of the nucleoprotein, and the compound was selected based on the Tm change value greater than 3°C. Among them, compound No. 2 was candesartan, which caused the nucleoprotein Tm change value to be 3.8°C.

[0049] Example 3: Determination of affinity between nucleoprotein and compound by BLI technology

[0050] Sample preparation: Prepare nuclear protein samples, ensure that the purity of the samples is greater than >95% and the concentration is >5 mg / mL; prepare the compounds to be measured at a concentration of 20 mM / L and perform gradient dilution according to experimental requirements.

[0051] Equilibration probe: The Ni-NTA biosensor was placed in blank buffer for 60 seconds for baseline equilibrium to eliminate nonspecific adsorption and background signals on the sensor surface;

[0052] Nucleoprotein-probe coupling: Add the sample solution containing nucleoprotein to the biosensor to allow the biomolecule to bind to the probe on the sensor surface. Binding time and temperature and other conditions need to be optimized according to the experimental purpose and sample characteristics, and the sample binding time is finally determined to be 300 seconds;

[0053] Washing uncoupled nuclear proteins: Rinse the biosensor with buffer to remove unbound nuclear proteins to reduce interference from nonspecific binding. The washing time is 120 seconds.

[0054] Compounds bind to nucleoproteins: Add the sample solution containing the compound to be tested into a black 96-well deep-well plate, place the biosensor in it, and observe the binding process between the two biomolecules. The binding time is 500 seconds and the temperature is 25°C;

[0055] Mutual dissociation of compound and nucleoprotein: The NTA sensor was rinsed with blank buffer to remove the bound biomolecules and observe the dissociation process between the two biomolecules. The dissociation time was 500 seconds and the temperature was 25°C.

[0056] like Figure 4 As shown, the BLI instrument is used to collect data on biomolecular interactions in real time, including parameters such as binding curves, binding rates, dissociation rates, and affinity. Professional data analysis software can be used to analyze the collected data. During the analysis process, attention should be paid to the quality and reliability of the data, and interference from nonspecific binding and noise should be excluded. The results show that the affinity Kd of the compound candesartan ester with nucleoprotein is 7.8μM.

[0057] Example 4: Analysis of the inhibitory effect of the compound Candesartan on the function of nucleoprotein

[0058] Through EMSA (gel retardation assay), it is carried out at 4°C, the buffer used is 0.5x TBE solution, and the gel type is 2.5% agar gel. When a specific protein binds to the labeled nucleic acid probe, the molecular weight of the formed protein-nucleic acid complex increases, and the migration speed in the gel slows down, thereby distinguishing it from the nucleic acid probe that is not bound to the protein. Figure 5 As shown, the inhibitory analysis of candesartan cilexetil on the nuclear protein nucleic acid binding ability and nucleolytic ability was analyzed by EMSA.

[0059] Importantly, it should be noted that the construction and arrangement of the present application shown in a plurality of different exemplary embodiments are only exemplary. Although only a few embodiments are described in detail in this disclosure, it should be readily understood by those who refer to this disclosure that many modifications are possible without substantially departing from the novel teachings and advantages of the subject matter described in this application. Without departing from the scope of the present invention, other replacements, improvements, changes and omissions may be made in the design, operating conditions and arrangement of the exemplary embodiments. Therefore, the present invention is not limited to specific embodiments, but extends to a variety of modifications still falling within the scope of the appended claims.

[0060] Additionally, in order to provide a concise description of exemplary embodiments, all features of an actual embodiment (ie, those features that are not relevant to the best mode presently contemplated for carrying out the invention or those that are not relevant to implementing the invention) may not be described.

[0061] It should be understood that in the development of any actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but for those of ordinary skill having the benefit of this disclosure, without undue experimentation, the development effort will be a routine task of design, fabrication, and production.

[0062] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. An application of an anti-Tacheng virus 1 nucleoprotein inhibitor, characterized in that: The use of the compound Candesartan as an inhibitor of the nucleoprotein of Tacheng virus 1.

2. The use of an anti-Tacheng virus 1 nucleoprotein inhibitor as claimed in claim 1, characterized in that: The compound Candesartan is used as an anti-Tacheng virus 1 nucleoprotein inhibitor for preparing a pharmaceutical composition for treating or preventing diseases caused by Tacheng virus 1 infection.

3. The use of an anti-Tacheng virus 1 nucleoprotein inhibitor as claimed in claim 2, characterized in that: The pharmaceutical composition comprises candesartan as an active ingredient.

4. The use of an anti-Tacheng virus 1 nucleoprotein inhibitor as claimed in claim 1, characterized in that: The compound Candesartan is obtained by the following steps: S1. Express and purify the nucleoprotein of Tacheng virus 1 using the E. coli system; S2. A thermal drift experiment based on protein stability was performed using a fluorescent quantitative PCR instrument, and through screening of natural medicines and FDA drug libraries, a compound called candesartan that significantly changed the Tm value of Tacheng virus 1 nucleoprotein was selected as a candidate inhibitor; S3, determination of the affinity of compound candesartan to nucleoprotein by biolayer interferometry; S4. The inhibitory effect of compound candesartan on nucleoprotein was verified by gel retardation experiment.

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