CN113480612B - C-terminal structural domain antiviral polypeptide of RIG-I, carrier and application thereof in preparation of antiviral drugs - Google Patents

Abstract

The invention provides an antiviral polypeptide, a carrier and application thereof in preparing an antiviral drug, wherein the polypeptide is a C-terminal domain (CTD) protein or polypeptide of RIG-I, and the amino acid sequence of the antiviral polypeptide is shown as SEQ ID NO. 3. The invention determines that a G4 structure exists on hepatitis C virus core RNA through a circular dichrograph and a nuclear magnetic resonance spectrometer, then utilizes a G4pull down experiment and a Western Blot to identify and obtain a binding protein RIG-I with the G4 structure, and proves that the RIG-I can be combined with the G4 structure through an immune confocal experiment and an EMSA experiment, and then finds that the RIG-I-CTD structural domain can stabilize the G4 structure and inhibit the replication of HCV through FRET and qRT-PCR experiments, thereby indicating that the CTD polypeptide can play an antiviral role as a potential drug.

Description

C-terminal structural domain antiviral polypeptide of RIG-I, carrier and application thereof in preparation of antiviral drugs

Technical Field

The invention relates to the technical field of biomedicine, in particular to a C-terminal domain (CTD) antiviral polypeptide of RIG-I, a carrier and application thereof in preparing antiviral drugs.

Background

Infection with Hepatitis C Virus (HCV) is prone to chronic Hepatitis, eventually leading to cirrhosis and hepatocellular carcinoma. Globally, it is estimated that more than 1.1 million people have serological evidence of current or past infection with HCV, while 8000 million people have chronic viral infections, estimated to cause 703800 deaths annually, accounting for one-third of the total mortality rate of HCC. HCV belongs to the flaviviridae family of single-stranded positive-strand RNA viruses, is about 9.6kb in length, and encodes polyproteins that can be cleaved by proteases into ten different proteins, including three structural proteins (nucleocapsid protein core, envelope proteins E1 and E2) and seven non-structural proteins (p 7, NS2, NS3, NS4A, NS4B, NS5A, NS 5B). The structural protein Core coded by the HCV Core gene has 191 amino acids in length, can form a nucleocapsid of the HCV virus, and prevents the genomic RNA of the virus from being damaged in the process of infecting a host.

G-quadruplexes (G4) are atypical secondary nucleic acid structures formed from guanine-rich DNA or RNA [7,8]The basic sequence formula is G _X N _1-7 G _X N _1-7 G _X N _1-7 G _X Wherein x is not less than 3, N representsThe loop region may consist of any base. The human genome has more than 375000G 4 motifs, is widely present in promoters, telomeres and oncogenes (such as c-Myc, c-Kit and the like), and has the functions of maintaining genome stability and regulating important life processes such as gene transcription, replication and the like. G-quadruplexes are also present in viral genes and can mediate the immune escape of the virus through interaction with G-quadruplex binding proteins. HCV RNA was found to be able to form the G4 structure, and core G4 was the most conserved and stable.

Due to the high variability of HCV, HCV control must rely on multiple pathways. The search for highly conserved structures on HCV genomic RNA and the screening of ligands that can bind these structures would also provide new avenues for combating HCV infection. Therefore, there is a need to develop a new antiviral drug.

Disclosure of Invention

The invention aims to provide a C-terminal structural domain (CTD) antiviral polypeptide of RIG-I, a carrier and application thereof in preparing antiviral drugs.

In a first aspect of the invention, a C-terminal domain (CTD) antiviral polypeptide of RIG-I is provided, wherein the antiviral polypeptide is a C-terminal domain (CTD) of retinoic acid inducible gene 1, and the amino acid sequence of the antiviral polypeptide is shown as SEQ ID NO. 3.

In a second aspect of the invention, there is provided a nucleic acid molecule encoding a gene encoding the antiviral polypeptide.

Further, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO:4, respectively.

In a third aspect of the invention, there is provided a vector comprising said encoding gene.

In a fourth aspect of the invention, there is provided a host bacterium comprising the vector.

Further, the host bacterium is one of DH5 α, top10, BL21, and Rosetta.

In a fifth aspect of the invention, there is provided a cell line comprising said vector.

In a sixth aspect of the invention, there is provided a use of said antiviral polypeptide, said nucleic acid molecule or said vector or said host bacterium or said cell line in the preparation of an antiviral medicament.

Further, the virus includes a virus containing a G4 structure.

In a seventh aspect of the present invention, there is provided an antiviral drug, wherein an active ingredient of the drug comprises the antiviral polypeptide.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

the invention provides an antiviral polypeptide, a carrier and application thereof in preparation of antiviral drugs, and finds that a C-terminal domain (CTD) of a retinoic acid inducible gene 1 can inhibit virus replication by stabilizing an HCV core RNA G4 structure, which indicates that the CTD polypeptide can play an antiviral role as a potential drug.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 shows the formation of RNA G4 structure of core gene of HCV type 1 a; FIG. 1A shows the result of G4 structure formation by a circular dichroism spectrometer, and FIG. 1B shows the result of verification by an 800-MHz NMR spectrometer;

FIG. 2 is a graph showing the binding of RIG-I to HCV 1a core G4 structure. G4 RIG-I is drawn in pull down experiment and verified by Western Blot experiment (figure 2A-B), and RIG-I and its structural domain eukaryotic expression plasmid and prokaryotic expression plasmid are constructed (figure 2C);

FIG. 3 is a graph of the effect of RIG-I and CTD domains on G4 structural stability; FRET experiments demonstrated the effect of RIG-I and CTD domains on the G4 structure (FIGS. 3A-B);

FIG. 4 is a graph of the detection of regulation of HCV RNA replication levels by RIG-I and CTD domains; wherein FIG. 4A and FIG. 4B are qRT-PCR validation results.

Detailed Description

The present invention will be specifically explained below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented thereby. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.

In order to solve the technical problems, the general idea of the embodiment of the application is as follows:

the invention determines that the HCV core RNA has a G4 structure by a circular dichroism chromatograph and a nuclear magnetic resonance spectrometer, obtains the binding protein RIG-I with the G4 structure by utilizing a G4pull down experiment and Western Blot identification, and proves that the RIG-I can be combined with the G4 structure by an immune confocal experiment and an EMSA experiment;

subsequently, the RIG-I-CTD domain can stabilize the G4 structure and inhibit the replication of HCV through FRET and qRT-PCR experiments.

Indicating that retinoic acid induces the C-terminal domain (CTD) of gene 1 to inhibit viral replication by stabilizing the viral RNA G4 structure. The mRNA nucleic acid sequence of RIG-I is shown as SEQ ID NO. 1. The amino acid sequence of RIG-I is shown in SEQ ID NO. 2. The amino acid sequence of the antiviral polypeptide (C-terminal structural domain of retinoic acid inducible gene 1) is shown as SEQ ID NO.3, and the nucleotide sequence is shown as SEQ ID NO. 4.

Therefore, retinoic acid induces the C-terminal domain (CTD) of gene 1, and is useful as a drug for inhibiting replication of a virus having a G4 structure such as HCV.

Therefore, the recombinant vector of the antiviral polypeptide can be constructed, introduced into host bacteria or target cells for expression, and the antiviral polypeptide, the nucleic acid molecule or the vector or the host bacteria or the cell line can be applied to the preparation of antiviral drugs.

The antiviral effect of the invention includes but is not limited to anti-HCV, and the like, specifically, the antiviral polypeptide can inhibit the replication of HCV, and the invention verifies the influence of RIG-I and CTD on the replication of HCV in human hepatoma cells Huh7.5.1 through qRT-PCR experiments.

In addition, the active component of the antiviral drug comprises the antiviral polypeptide. Pharmaceutical compositions comprising a pharmaceutically acceptable carrier, diluent or excipient are also within the scope of the present invention.

An antiviral polypeptide, a carrier and its use in preparing an antiviral drug of the present application will be described in detail with reference to the following examples and experimental data.

Example 1 formation of RNA G4 Structure of HCV type 1a core Gene

The embodiment of the invention verifies the formation of the HCV 1a core RNA G4 structure.

1. Synthesis of RNA sequence: HCV 1a core RNA G4 sequence (5 'biotin-GGGCUGCGGGUGGGCGGGA-3' shown in SEQ ID NO. 5) and its corresponding point mutation sequence (5 'biotin-GGACUGCGUGAGCGGGA-3' shown in SEQ ID NO. 6) were synthesized by Scyllaceae biotechnology Co.

2. The RNA sequence lyophilized powder was dissolved using a buffer containing 10mM Tris-HCl (pH 7.0), 100mM KCl, heated at 95 ℃ for 5min, and then slowly cooled to room temperature to form a G4 structure. Setting the running temperature of the circular dichroism instrument at 25 ℃, the scanning wavelength at 220 nm-320 nm, the optical path at 1.0mm and the scanning speed at 50nm/min, then sucking the mixed solution into a cuvette with 1.00mm, and measuring the CD spectra of different RNA sequences.

3. The HCV 1a core RNA G4 sample was dissolved in 10mM PBS (pH 7.0) containing 100mM KCl and 10% D2O to a final concentration of 0.5mM and ASO to a final concentration of 0.1mM. After heating at 95 ℃ for 5min, slowly cooling to room temperature to form a G4 structure. Signals were collected on a 800-MHz Bruker Avance III HD spectrometer instrument with a triple resonant 5mm HCN cryoprobe at 298K. Raw data were analyzed using MestReNova software.

As a result, as shown in FIG. 1, it was found that the RNA G4 sequence could form a G4 structure.

Example 2 verification of the binding of RIG-I to HCV 1a core G4 Structure

Firstly, using a G4pull down experiment to draw RIG-I, using a Western Blot experiment to verify, then using a fluorescence confocal microscope to observe the co-localization condition of the structures of RIG-I and G4, constructing RIG-I and a domain eukaryotic expression plasmid and a prokaryotic expression plasmid thereof, and further using an EMSA experiment to verify the combination of RIG-I and G4.

1. G4pull down experiment:

using the biotin-G4 sequence synthesized in example 1 and the corresponding point mutation sequence, 20. Mu.L of streptavidin-agarose was added to 1mL of PBS for resuspension, centrifuged at 2500rpm for 2min and then washed repeatedly 1 time, followed by addition of 600. Mu.L of PBS for resuspension, addition of 10. Mu.g of biotin-G4 and incubation in a 4 ℃ chromatographic cabinet for 2h in advance. The incubated G4-agarose was mixed well with the total protein from RIPA lysis of Huh7 cells and placed in a 4 ℃ chromatography cabinet and turned overnight. The protein-bound solution was centrifuged at 2500rpm for 3min, and the supernatant was carefully discarded. The solution was washed 1 time with 1mL PBS, centrifuged at 2500rpm for 3min, and unbound protein was washed off. To the bottom was added 50. Mu.L of 2 XSDS loading buffer and denatured by boiling at 100 ℃ for 5min.

2. Western Blot validation:

the biotin-G4-pulled proteins were subjected to a 10% SDS-PAGE gel separation experiment, 5% nonfat dry milk blocking for 2h after transfer to PVDF membrane, and incubated overnight at 4 ℃ with primary anti-RIG-I (1. The membrane was washed 4 times with TBST for 10min each time. A secondary antibody, HRP-goat anti-rabbit IgG (1 10000) was added and incubated at 37 ℃ for 1h. The membrane was washed 6 times with TBST for 5min each time. Mixing the solution A and the solution B according to the proportion of 1: and preparing a light-shielding color development liquid with the volume ratio of 1, adding the light-shielding color development liquid to the PVDF membrane, and carrying out ECL color development.

The results are shown in FIGS. 2A-2B, indicating that there is an interaction between RIG-I and G4 structures.

3. Constructing RIG-I and a structural domain eukaryotic expression plasmid and a prokaryotic expression plasmid thereof:

(1) Vector construction

According to a human RIG-I gene coding sequence (AF 038963.1) and a protein structure, RIG-I and a structural domain sequence thereof are amplified and cloned to a pFlag-CMV-2 eukaryotic expression vector and a pGEX-KG prokaryotic expression vector, and specifically:

the primer pairs and annealing temperatures required for the construction of the pFlag-CMV-2-RIG-I vector, the pGEX-KG-RIG-I vector, the pFlag-CMV-2-CTD vector and the pGEX-KG-2-CTD vector are specifically shown in the following Table 1; the sequence with homologous arm is constructed by homologous recombination, and the sequence with enzyme cutting site is constructed by enzyme connection.

TABLE 1

The results are shown in FIG. 2C, indicating successful vector construction.

(2) Protein expression

The expression of RIG-I and the structural domain protein thereof is induced by using an escherichia coli prokaryotic expression system, and then the protein is purified by using an affinity chromatography method.

Example 3 Effect of RIG-I and CTD domains on G4 structural stability

1. The effect of RIG-I and its domain on G4 structural stability was examined using FRET kinetic analysis: the dual-HCV core RNA G4 sequence (5 '-FAM-GGGCUGCGGGUGGGCGGGA-TAMRA-3' is shown in SEQ ID NO. 15) is synthesized. The dual-HCV core RNA G4 lyophilized powder was dissolved in buffer containing 10mM Tris-HCl (pH 7.0) and 100mM KCl, heated at 95 ℃ for 5min, and then slowly cooled to room temperature to form a structure.

ASO (5. The final concentrations of the double-labeled G4 sequence and ASO were 200nM and 2.0. Mu.M, respectively. GST-RIG-I and other proteins were added at a concentration of 1.0molar equiv, and GST-unloaded protein was added as a control. The protein was added and incubated for 30min at room temperature. Signals were collected from the start of ASO addition (t = 0). The reaction was carried out at 25 ℃ in a cuvette with a 1cm optical path, and the fluorescence signal was collected using a fluorescence spectrophotometer (F-4600). The excitation wavelength and emission wavelength were set to 494nm and 590nm, respectively.

2. The FRET principle is utilized to detect the unwinding action of RIG-I and the structural domain thereof on the G4 structure: the double-labeled dual-HCV core RNA G4-FB sequence (5 '-FAM-GGGCUGCGGGUGGGCGGGA-BHQ1-3' shown in SEQ ID NO. 17) was synthesized. The double-HCV core RNA G4-FB lyophilized powder was dissolved in buffer containing 10mM Tris-HCl (pH 7.0) and 100mM KCl, heated at 95 ℃ for 5min, and slowly cooled to room temperature to form a structure. 200nM of the dual-labeled HCV G4 was mixed with 1.0molar equiv protein and incubated on ice for 20min. The mixed liquid was aspirated into a cuvette having a 1cm optical path, and the fluorescence spectrum was scanned using a fluorescence spectrophotometer (F-4600). The excitation and emission wavelengths were 490nm and 522nm, respectively.

The results are shown in FIG. 3A, which indicates that the FRET experiment verifies that the RIG-I and CTD domains have unwinding effect on the G4 structure.

Example 4 detection of regulation of HCV RNA replication levels by RIG-I and its Domain

1. qRT-PCR: cells were harvested, 1mL Trizol added per well, incubated on ice for 3-5min, and transferred to RNase-free 1.5mL EP tubes. Continuously adding 200 mu L of chloroform into the EP tube, oscillating the tube body for 15s back and forth violently, and standing for 2-3min at room temperature. Centrifuging at 12000rpm at 4 deg.C for 15min, separating the tube into upper inorganic water phase, middle layer and lower organic phenol chloroform phase, and transferring the upper water phase into new RNase-free EP tube. Adding isopropanol with the same volume, turning upside down to mix thoroughly, standing at room temperature for 5min,12000rpm, and centrifuging at 4 deg.C for 15min. The white precipitate was washed by adding 1mL of pre-cooled 75% ethanol, centrifuged at 12000rpm for 5min, and repeated 1 time. The supernatant was aspirated off and the white precipitate was blown dry in a fume hood until the bottom precipitate became translucent. Adding 20 μ L of DEPC-ddH preheated 65 ℃ in advance ₂ Dissolving the precipitate with O, and measuring the RNA concentration with an ultraviolet spectrophotometer. RNA reverse transcription was performed according to the RNA reverse transcription kit (Toyobo Co., japan), and the expression level of HCV RNA was examined by qRT-PCR.

TABLE 2 primers used for RT-qPCR

The results are shown in FIG. 4, which shows that qRT-PCR verifies the effect of RIG-I and its C-terminal domain on HCV RNA replication. FIG. 4B shows that after qRT-PCR validated HCV G4 structural mutations, RIG-I and its C-terminal domain had an effect on HCV RNA replication. The C-terminal domain of RIG-I inhibits HCV replication by stabilizing the HCV core RNA G4 structure. The C-terminal domain polypeptide of RIG-I is suggested to play an antiviral role as a medicine.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

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<211> 132

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gln Glu Lys Pro Lys Pro Val Pro Asp Lys Glu Asn Lys Lys Leu Leu

1 5 10 15

Cys Arg Lys Cys Lys Ala Leu Ala Cys Tyr Thr Ala Asp Val Arg Val

20 25 30

Ile Glu Glu Cys His Tyr Thr Val Leu Gly Asp Ala Phe Lys Glu Cys

35 40 45

Phe Val Ser Arg Pro His Pro Lys Pro Lys Gln Phe Ser Ser Phe Glu

50 55 60

Lys Arg Ala Lys Ile Phe Cys Ala Arg Gln Asn Cys Ser His Asp Trp

65 70 75 80

Gly Ile His Val Lys Tyr Lys Thr Phe Glu Ile Pro Val Ile Lys Ile

85 90 95

Glu Ser Phe Val Val Glu Asp Ile Ala Thr Gly Val Gln Thr Leu Tyr

100 105 110

Ser Lys Trp Lys Asp Phe His Phe Glu Lys Ile Pro Phe Asp Pro Ala

115 120 125

Glu Met Ser Lys

130

<210> 4

<211> 396

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

caagaaaaac caaaacctgt ccctgataag gaaaataaaa aactgctctg cagaaagtgc 60

aaagccttgg catgttacac agctgacgta agagtgatag aggaatgcca ttacactgtg 120

cttggagatg cttttaagga atgctttgtg agtagaccac atcccaagcc aaagcagttt 180

tcaagttttg aaaaaagagc aaagatattc tgtgcccgac agaactgcag ccatgactgg 240

ggaatccatg tgaagtacaa gacatttgag attccagtta taaaaattga aagttttgtg 300

gtggaggata ttgcaactgg agttcagaca ctgtactcga agtggaagga ctttcatttt 360

gagaagatac catttgatcc agcagaaatg tccaaa 396

<210> 5

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gggcugcggg ugggcggga 19

<210> 6

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggacugcgug ugagcggga 19

<210> 7

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

acaaagacga tgacgacaag atgaccaccg agcagcgacg 40

<210> 8

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtcacaggga tgccacccgg tttggacatt tctgctggat c 41

<210> 9

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atactcgaga tgaccaccga gcagcgacg 29

<210> 10

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ataaagcttt ttggacattt ctgctggatc 30

<210> 11

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

acaaagacga tgacgacaag caagaaaaac caaaacctg 39

<210> 12

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gtcacaggga tgccacccgg tttggacatt tctgctggat c 41

<210> 13

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atactcgagc aagaaaaacc aaaacctgtc 30

<210> 14

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ataaagcttt ttggacattt ctgctggatc 30

<210> 15

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gggcugcggg ugggcggga 19

<210> 16

<211> 22

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

acccgcagcc cucccgccca cc 22

<210> 17

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gggcugcggg ugggcggga 19

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

raycactccc ctgtgaggaa c 21

<210> 19

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tgrtgcacgg tctacgagac ctc 23

<210> 20

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gaaggtgaag gtcggagtc 19

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gaagatggtg atgggatttc 20

Claims (3)

1. An application of antiviral polypeptide in preparing anti-HCV virus medicine, wherein the antiviral polypeptide is C-terminal structural domain (CTD) of retinoic acid inducible gene 1, and the amino acid sequence of the antiviral polypeptide is shown as SEQ ID NO. 3.

2. An application of nucleic acid molecule of antiviral polypeptide shown in SEQ ID NO.3 in preparing anti-HCV virus medicine.

3. Use of a vector or host bacterium or cell line comprising a nucleic acid molecule according to claim 2 in the preparation of a medicament against HCV virus.

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