CN113388626A - New application of novel coronavirus NSP13 gene - Google Patents

Abstract

The invention provides a new application of a novel coronavirus NSP13 gene, which is characterized in that the novel coronavirus NSP13 gene is prepared into a product allowing expression of SARS-CoV-2NSP13 protein, the SARS-CoV-2NSP13 protein expressed in eukaryotic cells can obviously inhibit the expression of eukaryotic expression plasmids, and the SARS-CoV-2NSP13 protein only inhibits the expression of the eukaryotic expression plasmids but does not inhibit the expression of genes in the genome of the eukaryotic cells, and further, the SARS-CoV-2NSP13 protein inhibits the expression of the eukaryotic expression plasmids at the mRNA level. The invention utilizes the discovery to introduce exogenous protein into eukaryotic cells for the first time to inhibit the expression of eukaryotic expression plasmids, fills up the prior technical gap of exogenous plasmid expression regulation and control, and provides a new thought for inhibiting the expression of free genes in the cells.

Description

New application of novel coronavirus NSP13 gene

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to a novel application of a novel coronavirus NSP13 gene.

Background

For most RNA viruses, viral RNA will typically form multiple cis-acting elements in some untranslated region or Open Reading Frame (ORF) sequence to play an important role in the viral replication cycle. SARS-CoV-2, a positive-strand RNA virus with an envelope, whose nonstructural protein, NSP13, contains 601 amino acids, is a protein with nucleoside triphosphate hydrolase (NTPase) activity and RNA helicase activity, and is also one of the two core components that make up the viral transcriptional replication machinery. The overall structure of SARS-CoV-2NSP13 is similar to that of NSP13 protein of Severe Acute Respiratory Syndrome (SARS) and middle east respiratory syndrome coronavirus (MERS), and is pyramid-shaped and composed of the following five structural domains: the zinc finger domain (ZBD) and the talk domain at the N-terminus, the 1B domain, and the helicase core part Rec a1 and Rec a2 domain at the C-terminus. During the viral life cycle, NSP13 exerts its helicase activity to unwind double-stranded RNA for the next round of RNA replication. In the course of the evolution of coronaviruses, the NSP13 protein shows high conservation, and at present, it is not clear for a moment what mechanism NSP13 specifically plays a biological function, so that the research on the structure and the function of the NSP13 protein is very important.

In 1952, the biologist in the united states j. reed burg introduced for the first time the term plasmid, i.e. used to describe genetic material outside of the chromosome. Plasmids are widely found in the kingdom biologies, from bacteria, fungi to plants and even mammals. The vast majority of plasmids are known as circular double-stranded DNA (covalently closed circular DNA, cccDNA), which is autonomously replicable, can maintain a constant copy number in daughter cells, and express the genetic information carried. In its simplest form, a plasmid comprises an Origin of Replication (ORI) responsible for the initiation of replication of the plasmid, a resistance gene for rapid selection and at least one cleavage site for insertion of a foreign gene, these elements ensuring the amplification of the plasmid in bacteria while allowing the isolation of the plasmid-carrying bacteria from plasmid-free bacteria, and a cleavage site for insertion of a foreign DNA fragment into the plasmid.

Because of these special properties of plasmids, they are often used as vectors for transporting foreign genes in genetic engineering research, and in recent years, plasmid vectors have attracted attention and have been developed for gene therapy, and compared with viral vectors, plasmids have the advantages of simplicity, low cost, rapidity, and safety, and can be used in combination with other synthetic vectors. Because some treatments do not require long-term introduction of a foreign gene or foreign nucleic acid contamination during the treatment, the prior art cannot flexibly eliminate the foreign gene. And in the biological world, some plasmids are pathogenic, e.g., bacterial toxins that cause insect disease or even death are plasmid-encoded; some plasmids endow the host with drug resistance inheritance, so that the host can show resistance to antibiotics, chemical drugs, heavy metals and other bactericides, can prevent the antibiotics from entering cells or modify the antibiotics to inactivate the antibiotics, even change target sites of the antibiotic action or generate target enzymes which can replace the antibiotic action in the host cells, so that the negative control action of the plasmid on a copy and transcription level is particularly important, and different experimental techniques need to be invented according to different experimental requirements.

Detection and degradation of foreign nucleic acids is an ancient host defense. However, the underlying mechanism is not fully understood. Usually, when a host cell faces DNA or RNA of a foreign virus or a transfected gene, natural immune signals of the host cell are triggered, and DNA receptors, namely some Toll-like receptors (TLRs) or RNA receptors RIG-like receptors, are activated, so that the up-regulation of some antiviral proteins is caused, such as some nucleases, topoisomerase and the like, so that foreign nucleic acids are degraded, the antiviral proteins are usually controlled by the host gene, and the fact that the expression of foreign plasmids can be inhibited by the foreign proteins under the condition of over-expression is not reported at present. Therefore, how to introduce exogenous protein to inhibit the expression of exogenous plasmid is a topic to be studied intensively.

Disclosure of Invention

The invention obtains the gene sequence information of SARS-CoV-2NSP13 protein by the gene group data of novel coronavirus SARS-CoV-2, then obtains the gene of SARS-CoV-2NSP13 protein by the gene synthesis method, and further obtains the eukaryotic expression plasmid pXJ40-HA-NSP13 which can express SARS-CoV-2NSP13 protein in eukaryotic cells by the molecular cloning method. The SARS-CoV-2NSP13 protein can obviously inhibit the expression of eukaryotic plasmid by co-transforming pXJ40-HA-NSP13 and other eukaryotic expression plasmids in cells. It is found through experiments that SARS-CoV-2NSP13 protein can inhibit the expression of eukaryotic expression plasmid, but not inhibit the expression of genome gene in eukaryotic cell. Finally, through further experiments, the SARS-CoV-2NSP13 protein is found to inhibit the expression of eukaryotic expression plasmid at mRNA level.

Based on the above findings, the present invention provides the following technical solutions:

in a first aspect, the invention provides a plasmid pXJ40-HA-NSP13 capable of expressing a novel coronavirus SARS-CoV-2NSP13 protein, which is used for preparing products for inhibiting the expression of eukaryotic expression plasmids. pXJ40-HA-NSP13 plasmid SARS-CoV-2NSP13 gene sequence is SEQ ID NO: 1.

further, the pXJ40-HA eukaryotic expression vector on the pXJ40-HA-NSP13 plasmid is suitable for replication and amplification in bacteria. And the expression vector can express SARS-CoV-2NSP13 protein in eukaryotic cells.

Further, the eukaryotic expression plasmid pXJ40-HA-NSP13 of SARS-CoV-2NSP13 can be amplified in large amounts in bacteria. And can express SARS-CoV-2NSP13 protein in eukaryotic cell.

In a second aspect, the present invention provides a method for constructing a eukaryotic expression plasmid of SARS-CoV-2NSP13, comprising the following steps: PCR amplifies new type coronavirus SARS-CoV-2NSP13 gene, connects the amplified segment to eukaryotic expression vector, transfers the connection product to the susceptible cell, carries on ampicillin resistance LB culture medium screening, colony PCR appraisal, plasmid extraction in turn, obtains the expression vector expressing SARS-CoV-2NSP13 protein.

In a third aspect, the invention provides a product capable of inhibiting the expression of foreign plasmids in eukaryotic cells, wherein the product comprises eukaryotic expression plasmids pXJ40-HA-NSP13 and pXJ40-HA-NSP13 of SARS-CoV-2NSP13 gene, and the product can express SARS-CoV-2NSP13 protein in eukaryotic cells. Furthermore, the SARS-CoV-2NSP13 protein can obviously inhibit the expression of pGL 3-IFN-beta-luc, pRL-TK, Flag-RIG-IN, Flag-MDA5, Flag-MAVS, Flag-TBK1, Flag-IKK epsilon and the like, thereby indicating that the product can inhibit eukaryotic exogenous plasmids.

In a fourth aspect, the invention provides a product that can inhibit the expression of foreign plasmids in eukaryotic cells, primarily at the mRNA level.

The invention has the following advantages and beneficial effects:

the invention discovers for the first time that SARS-CoV-2NSP13 protein, which is one of the most evolutionarily conserved non-structural proteins in coronavirus, can inhibit the expression of eukaryotic expression plasmid, and the inhibition effect occurs at mRNA level.

Drawings

FIG. 1 shows the construction strategy of recombinant plasmid of SARS-CoV-2NSP13 protein in example 1 of the present invention.

Fig. 2 shows the test results of example 2: the SARS-CoV-2NSP13 protein can inhibit the expression of pGL 3-IFN-beta-luc plasmid and pRL-TK plasmid. FIG. 2(A) shows the result of inhibition of expression of pGL 3-IFN-. beta. -luc plasmid by SARS-CoV-2NSP13 protein, and FIG. 2(B) shows the result of inhibition of expression of pRL-TK plasmid by SARS-CoV-2NSP13 protein.

Fig. 3 shows the test results of example 3: the SARS-CoV-2NSP13 protein can inhibit the expression of plasmids such as Flag-RIG-IN, Flag-MDA5, Flag-MAVS, Flag-TBK1, Flag-IKK epsilon and the like. Among them, FIG. 3(A) represents the result of inhibition of expression of Flag-RIG-IN plasmid by SARS-CoV-2NSP13 protein, FIG. 3(B) represents the result of inhibition of expression of Flag-MDA5 plasmid by SARS-CoV-2NSP13 protein, FIG. 3(C) represents the result of inhibition of expression of Flag-MAVS plasmid by SARS-CoV-2NSP13 protein, FIG. 3(D) represents the result of inhibition of expression of Flag-TBK1 plasmid by SARS-CoV-2NSP13 protein, and FIG. 3(E) represents the result of inhibition of expression of Flag-IKK epsilon plasmid by SARS-CoV-2NSP13 protein.

Fig. 4 shows the test results of example 4: the inhibition of plasmid expression by SARS-CoV-2NSP13 protein is independent of the promoter on the recombinant plasmid. FIG. 4 shows the inhibition of expression of pIFN-. beta. -Luc plasmid and expression of pEF 1. alpha. -Luc plasmid by SARS-CoV-2NSP13 protein.

Fig. 5 to 7 show the detection results of example 5: the SARS-CoV-2NSP13 protein can inhibit the expression of episome gene, but not inhibit the expression of endogenous gene. Wherein, FIG. 5 shows the inhibition result of SARS-CoV-2NSP13 protein on exogenous free Flag-RIG-I gene, FIG. 6 shows the effect of SARS-CoV-2NSP13 protein on the expression of cell endogenous RIG-I gene, and FIG. 7 shows the effect of SARS-CoV-2NSP13 protein on the expression of exogenous Firefoil luciferase gene integrated on cell genome.

Fig. 8 to 9 show the detection results of example 6: SARS-CoV-2NSP13 protein inhibits the expression of episome at the mRNA level. FIG. 8 shows that SARS-CoV-2NSP13 protein inhibits expression of pIFN-. beta. -Luc, pEF 1. alpha. -Luc plasmids at the mRNA level. FIG. 9 shows that SARS-CoV-2NSP13 protein inhibits the expression of Flag-RIG-I plasmid at the mRNA level.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the devices and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.

For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.

Unless otherwise stated, the following examples of the present invention are carried out at room temperature, which is natural room temperature in four seasons, without additional cooling or heating, and the temperature is generally controlled at 10-30 ℃, preferably 15-25 ℃.

The embodiment of the invention comprises the following parts:

a first part: constructing recombinant plasmid of SARS-CoV-2NSP13 protein. The eukaryotic expression plasmid pXJ40-HA-NSP13 of SARS-CoV-2NSP13 protein is obtained by a molecular cloning method.

A second part: the SARS-CoV-2NSP13 protein can inhibit the expression of pGL 3-IFN-beta-luc plasmid and pRL-TK plasmid. Firstly, firefly luciferase reporter plasmid (pGL 3-IFN-beta-luc) of IFN-beta promoter co-transferred in HEK293T cells, holothurian luciferase internal reference reporter plasmid (pRL-TK) and SARS-CoV-2NSP13 protein eukaryotic expression plasmid with dose gradient are subjected to luciferase activity detection, and SARS-CoV-2NSP13 protein is found to be capable of remarkably inhibiting the expression of the two plasmids in a dose-dependent manner.

And a third part: the SARS-CoV-2NSP13 protein can inhibit the expression of plasmids such as Flag-RIG-IN, Flag-MDA5, Flag-MAVS, Flag-TBK1, Flag-IKK epsilon and the like. The SARS-CoV-2NSP13 protein eukaryotic expression plasmid and a series of eukaryotic expression plasmids (Flag-RIG-IN, Flag-MDA5, Flag-MAVS, Flag-TBK1, Flag-IKKepsilon and other plasmids) are co-transformed IN HEK293T cells, and the SARS-CoV-2NSP13 protein can obviously inhibit the plasmid expression.

The fourth part: the inhibition of the expression of the plasmid by the SARS-CoV-2NSP13 protein is independent of the promoter on the recombinant plasmid. The SARS-CoV-2NSP13 protein eukaryotic expression plasmid and firefly luciferase reporter plasmid (pIFN-beta-Luc and pEF1 alpha-Luc) with different promoters are co-transformed in HEK293T cells, and the inhibition of plasmid expression by the SARS-CoV-2NSP13 protein is found to be independent of the promoter on the recombinant plasmid.

The fifth part is that: the SARS-CoV-2NSP13 protein can inhibit the expression of episome gene, but not inhibit the expression of endogenous gene. By detecting the influence of SARS-CoV-2NSP13 protein on the expression of free gene and endogenous gene integrated in eukaryotic cell genome, it is found that SARS-CoV-2NSP13 protein only inhibits the expression of free gene in cell, and has no influence on the expression of endogenous gene.

A sixth part: SARS-CoV-2NSP13 protein inhibits the expression of free genes at the mRNA level. The effect of SARS-CoV-2NSP13 protein on plasmid expression was detected at the mRNA level by qPCR and it was found that SARS-CoV-2NSP13 protein inhibits eukaryotic plasmid expression at the mRNA level.

The invention is illustrated in detail in the following examples.

Example 1: constructing recombinant plasmid of SARS-CoV-2NSP13 protein.

The gene sequence (SEQ ID NO.1) coding SARS-CoV-2NSP13 protein is cloned to pXJ40-HA vector by molecular cloning technology to obtain pXJ40-HA-NSP13 plasmid, and the construction strategy is schematically shown in figure 1.

(1) PCR amplification

Polymerase Chain Reaction (PCR) amplification was performed using a 50. mu.L reaction system, the amplification system used is shown in Table 1, and the reaction procedure is shown in Table 2.

TABLE 1 PCR amplification System

General System	50μL
		Form panel	2μL
Upstream primer	2μL
		Downstream primer	2μL
MIX	25μL
		Sterilization ddH2O	19μL

TABLE 2 PCR reaction procedure

And ∞ indicates that the storage time is uncertain.

(2) Double enzyme digestion

After a double enzyme digestion system is prepared, the PCR amplification product is subjected to enzyme digestion for 5-15 min at 37 ℃.

Table 3 shows the 50. mu.L double digestion system

General System	50μL
		Xho1	2μL
Not1	2μL
10 Xbuffer	5μL
		Vectors/plasmids	4μg(XμL)
Sterilization ddH2O	41-XμL

X represents the volume of the vector/plasmid.

(3) Glue recovery

(3.1) after the DNA electrophoresis is finished, the gel containing the target DNA fragment is cut off rapidly under an ultraviolet lamp, and it is recommended to suck up the liquid on the surface of the gel with a paper towel and cut up, and remove the excess gel as much as possible. The gel was weighed (the empty tube weight was removed) and 100mg of gel was equivalent to a volume of 100. mu.L as one gel volume.

(3.2) adding equal volume of buffer GDP, carrying out water bath at 50-55 ℃ for 7-10min, and properly adjusting time according to the size of the gel to ensure that the gel is completely dissolved. The sol solution is obtained by mixing the sol solution and the mixture in a reverse manner for 2 times during the water bath.

(3.3) collecting the droplets on the tube wall by brief centrifugation. The Fsatpure DNA Mini Columns-G adsorption column is placed in a 2mL collection tube, less than or equal to 700 mu L of sol solution is transferred to the Fsatpure DNA Mini Columns-G adsorption column, and the centrifugation is carried out for 30-60s at 12000 Xg. If the volume of the sol is larger than 700. mu.L, the adsorption column is placed back into the collection tube, the rest sol solution is transferred into the adsorption column, and the centrifugation is carried out for 30-60s at 12000 Xg.

(3.4) discarding the filtrate and placing the adsorption column in the collection tube. Add 300. mu.L of buffer GDP to the adsorption column. Standing for 1 min. 12000 Xg centrifugation for 30-60 s.

(3.5) discarding the filtrate and placing the adsorption column in the collection tube. Add 600. mu.L of buffer GW (to which absolute ethanol has been added) to the adsorption column. 12000 Xg centrifugation for 30-60 s.

(3.6) repeating the step (3.5).

(3.7) discarding the filtrate and putting the adsorption column back into the collection tube. Centrifuge at 12000 Xg for 2 min.

(3.8) placing the adsorption column in a 1.5mL sterilized centrifuge tube, adding 10-30 μ L elution buffer solution to the center of the adsorption column, and standing for 2 min. Centrifuging at 12000 Xg for 1min to obtain plasmid vector enzyme digestion product, discarding adsorption column, and storing the plasmid vector enzyme digestion product at-20 deg.C.

(4) Connection of

The cut product of the plasmid vector and the NSP13 fragment are placed in a connection system for connection for 1h at 22 ℃, and the connection system is shown in Table 4.

TABLE 4 connection System

(5) Transformation of

(5.1) thawing 100. mu.L of competent JM109 frozen at-80 ℃ on ice.

(5.2) adding the ligation product into the competent cells, flicking the tube to mix the competent cells and ligation product, and placing on ice for 30 min.

(5.3) then, the mixture was heat-shocked in a water bath at 42 ℃ for 90 seconds, cooled on ice for 5 minutes, added with 1mL of fresh LB medium, and cultured on a shaker at 37 ℃ for 1 hour with shaking at 220 rpm.

(5.4) the culture was centrifuged at 5000rpm for 5min, 900. mu.L of the supernatant was discarded by a pipette gun, and the remaining liquid was pipetted and spread evenly on an ampicillin-resistant LB solid plate using a glass rod. And (3) rightly placing the incubator at 37 ℃ for 15min, then inverting, observing the growth condition of bacteria the next day, and respectively picking a certain number of single colonies at different positions to culture in an ampicillin resistant LB culture medium.

(6) Colony PCR and agarose gel electrophoresis identification

4-5 colonies were picked, and colony PCR was performed to determine whether bands were present or absent by agarose gel electrophoresis, and the colony PCR system is shown in Table 5.

TABLE 5 colony PCR System

(7) Extraction of pXJ40-HA-NSP13 plasmid

(7.1) the bacterial liquid and LB culture medium which are successfully identified and transformed by agarose gel electrophoresis are enlarged and cultured for 12-16h at 37 ℃ according to a ratio of 1:100 (namely 100 mu L of bacterial liquid is added into 10mL of culture medium), and then pXJ40-HA-NSP13 plasmid is extracted according to a plasmid extraction kit. The method comprises the following steps:

(7.2) 5-15mL of overnight-cultured bacterial liquid was collected by centrifugation at 13000rpm for 1min, and the supernatant was discarded.

(7.3) to the centrifugal tube with the cell pellet, 500. mu.L of buffer solution P1 (ribonuclease (RNaseA) was added, and the bacterial solution was mixed well, suspended, and the bacterial pellet was lysed well.

(7.4) adding 500. mu.L of buffer solution P2 into the centrifuge tube, gently mixing for 8-10 times (without violent shaking) to fully dissolve the thalli, and standing at room temperature for 3-5min, wherein the solution becomes clear and viscous.

(7.5) adding 500. mu.L of buffer solution E3 into the centrifuge tube, immediately turning upside down and mixing uniformly for 8-10 times to avoid local precipitation, standing for 5min at room temperature, then centrifuging for 5min at 13000rpm, sucking supernatant, adding into a filter column, centrifuging for 1min at 13000rpm, and collecting filtrate in a new centrifuge tube.

(7.6) to the filtrate, 450. mu.L of isopropyl alcohol was added, and the mixture was thoroughly mixed by turning upside down.

(7.7) pipette 200. mu.L of buffer PS into the adsorption column, centrifuge at 13000rpm for 2min, and discard the waste.

(7.8) adding the mixed solution obtained in the step (7.6) into an adsorption column, centrifuging at 1300rpm for 1min, and discarding the waste liquid in the collection tube.

(7.9) Add 700. mu.L buffer PW (ethanol already added) to the adsorption column, centrifuge at 1300rpm for 1min, discard the waste liquid in the collection tube.

(7.10) repeating the step (7.8).

(7.11) spin the column at room temperature, centrifuge at 1300rpm for 1min to remove the remaining ethanol.

(7.12) the adsorption column was placed in a freshly sterilized 1.5mL centrifuge tube, 100. mu.L of elution buffer was added, the tube was left at room temperature for 2-5min, and centrifuged at 1300rpm for 2min to give pXJ40-HA-NSP13 plasmid, which was stored at-20 ℃.

Example 2: detecting whether pXJ40-HA-NSP13 expression plasmid can inhibit pGL 3-IFN-beta-luc and pRL-TK plasmid

HEK293T cells are inoculated to a 24-well plate and transfected with reporter plasmids pGL 3-IFN-beta-luc and pRL-TK respectively, and a dose gradient transfection pXJ40-HA or pXJ40-HA-NSP13 expression plasmid, IAV NS1 is used as a positive control for inhibiting pGL 3-IFN-beta-luc, after transfection for 24h, SEV treatment is added for 10h, three multiple wells are arranged in each group, then the cells are lysed, and luciferase activity is detected. As shown in FIG. 2, SARS-CoV-2NSP13 protein significantly inhibited the expression of pGL 3-IFN-. beta. -luc and pRL-TK plasmids dose-dependently. Example 3: detecting whether pXJ40-HA-NSP13 expression plasmid can inhibit the expression of Flag-RIG-IN, Flag-MDA5, Flag-MAVS, Flag-TBK1 and Flag-IKK epsilon

HEK293T cells were cultured IN 12-well plates and transfected with Flag-RIG-IN, Flag-MDA5, Flag-MAVS, Flag-TBK1, Flag-IKK epsilon and pXJ40-HA or pXJ40-HA-NSP13 plasmids, respectively, using PEI as the transfection reagent, 1g:3mL of plasmid to PEI, and opti-MEM as the transfection solvent. The cells were transfected for 6 hours and then replaced with fresh medium (DMEM), and the protein expression level was measured by Western blotting (Western Blot) 24 hours after transfection, as shown IN FIG. 3, the results showed that SARS-CoV-2NSP13 protein could significantly inhibit the expression of a series of plasmids such as Flag-RIG-IN, Flag-MDA5, Flag-MAVS, Flag-TBK1 and Flag-IKK epsilon.

Example 4: it was examined whether the suppression of plasmid expression by SARS-CoV-2NSP13 protein was dependent on the promoter on the recombinant plasmid.

When HEK293T cells are cultured in a 24-well plate and the cell density reaches 70% -80%, pXJ40-HA or pXJ40-HA-NSP13 is co-transfected with luciferase reporter genes pIFN-beta-Luc and pEF1 alpha-Luc plasmids containing different promoters, PEI is used as a transfection reagent, the dosage ratio of the plasmids to the PEI is 1g:3mL, and opti-MEM is used as a transfection solvent. After 24h of transfection, the IFN-beta promoter group is treated by SEV for 10h, and then cell sample extraction protein is collected; the EF1 alpha promoter group directly collects cell-like extracted protein, and detects the expression level of the luciferase protein (firefly luciferase) by a western blot method. FIG. 4(A) shows the result of inhibition of expression of pIFN-. beta. -Luc plasmid by SARS-CoV-2NSP13 protein, and FIG. 4(B) shows the result of inhibition of expression of pEF 1. alpha. -Luc plasmid by SARS-CoV-2NSP13 protein.

Example 5: detecting whether SARS-CoV-2NSP13 protein can inhibit the expression of endogenous gene:

(1) when HEK293T cells were cultured in a 24-well plate and the cell density reached 70% -80%, pXJ40-HA or pXJ40-HA-NSP13 was co-transfected with Flag-RIG-I plasmid using PEI as transfection reagent in a ratio of 1g to 3mL plasmid to PEI and opti-MEM as transfection solvent. And collecting cell samples after 24h to extract protein, and detecting the expression level of Flag-RIG-I protein by a western blot method.

(2) When HEK293T cells are cultured in a 24-well plate and the cell density reaches 70% -80%, pXJ40-HA or pXJ40-HA-NSP13 is transfected, PEI is used as a transfection reagent, the dosage ratio of the plasmid to the PEI is 1g:3mL, and opti-MEM is used as a transfection solvent. 24h after transfection, SEV treatment is added for 10h, and the expression level of endogenous RIG-I protein is detected by Western blotting.

(3) Constructing a stable transfer cell line HEK 293T-Luc: integrating a firefly luciferase gene into a 293T cell chromosome through a lentivirus system, culturing 293T-Luc cells in a 24-well plate, transfecting pXJ40-HA-NSP13 when the cell density is 70-80%, collecting cell samples after 24h to extract protein, and detecting the expression level of the luciferase protein through Western Blot.

As shown in FIGS. 5 to 7, SARS-CoV-2NSP13 protein can significantly inhibit the expression of eukaryotic plasmid, but has no effect on the expression of eukaryotic cell genome gene.

Example 6: detecting the step of expressing SARS-CoV-2NSP13 protein in the plasmid to inhibit the expression of eukaryotic plasmid:

(1) when HEK293T cells are cultured in a 24-well plate and the cell density reaches 70% -80%, the pXH40-NSP13 plasmid, pIFN-beta-Luc plasmid and pEF1 alpha-Luc plasmid are co-transfected, PEI is used as a transfection reagent, the dosage ratio of the plasmids to the PEI is 1 g/3 mL, and opti-MEM is used as a transfection solvent. After 24h of transfection, SEV treatment is added for 10h, then RNA samples are collected, RNA is extracted by a Trizol method, and the level of luciferase RNA of the firefly is detected by qPCR.

(2) When HEK293T cells were cultured in a 24-well plate and the cell density reached 70% -80%, pXJ40-HA or pXJ40-HA-NSP13 was co-transfected with Flag-RIG-I plasmid using PEI as transfection reagent in a ratio of 1g to 3mL plasmid to PEI and opti-MEM as transfection solvent. Collecting RNA samples after 24h, extracting RNA by a Trizol method, and detecting the level of RIG-I RNA by qPCR.

(3) When HEK293T cells are cultured in a 24-well plate and the cell density reaches 70% -80%, pXJ40-HA or pXJ40-HA-NSP13 is transfected, PEI is used as a transfection reagent, the dosage ratio of the plasmid to the PEI is 1g:3mL, and opti-MEM is used as a transfection solvent. After 24h of transfection, SEV treatment is added for 10h, RNA samples are collected, RNA is extracted by a Trizol method, and the level of endogenous RIG-I RNA is detected by qPCR. As shown in FIGS. 8 and 9, the SARS-CoV-2NSP13 protein inhibited the expression of eukaryotic plasmids at the mRNA level.

Sequence listing

<110> Wuhan university

<120> novel use of novel coronavirus NSP13 gene

<160> 1

<170> SIPOSequenceListing 1.0

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ctggttccgc aggaacacta tgtgcgtatc accggtctgt acccgaccct gaacattagc 780

gacgagttca gcagcaacgt tgcgaactat cagaaagtgg gtatgcaaaa atatagcacc 840

ctgcaaggtc cgccgggtac cggcaagagc cactttgcga tcggtctggc gctgtactat 900

ccgagcgcgc gtattgttta taccgcgtgc agccatgcgg cggtggatgc gctgtgcgaa 960

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aagctgaaag cgcacaagga caaaagcgcg cagtgcttca agatgtttta caaaggtgtg 1440

atcacccacg acgttagcag cgcgatcaac cgtccgcaaa ttggcgtggt tcgtgagttc 1500

ctgacccgta acccggcgtg gcgtaaggcg gtttttatca gcccgtataa cagccagaac 1560

gcggtggcga gcaaaattct gggtctgccg acccagaccg ttgatagcag ccaaggcagc 1620

gaatacgact atgtgatctt cacccaaacc accgagaccg cgcacagctg caacgtgaac 1680

cgttttaacg ttgcgattac ccgtgcgaag gttggtatcc tgtgcattat gagcgaccgt 1740

gatctgtacg ataaactgca gttcaccagc ctggaaattc cgcgtcgtaa cgttgcgacc 1800

ctgcag 1806

Claims (10)

1. Use of the NSP13 gene of a novel coronavirus, characterized in that: is used for preparing products for inhibiting the expression of eukaryotic expression plasmids, and the products allow the expression of SARS-CoV-2NSP13 protein.

2. Use of the novel coronavirus NSP13 gene according to claim 1, wherein: the product comprises an expression vector allowing expression of the SARS-CoV-2NSP13 protein.

3. Use of the novel coronavirus NSP13 gene according to claim 2, wherein: the expression vector is suitable for expressing SARS-CoV-2NSP13 protein in eukaryotic cells.

4. Use of the novel coronavirus NSP13 gene according to claim 2, wherein: the expression vector is suitable for replication and amplification in bacteria.

5. Use of the novel coronavirus NSP13 gene according to claim 1, wherein: the eukaryotic expression plasmid is one or more of pGL 3-IFN-beta-luc, pRL-TK, Flag-RIG-IN, Flag-MDA5, Flag-MAVS, Flag-TBK1 and Flag-IKK epsilon.

6. Use of the novel coronavirus NSP13 gene according to claim 1, wherein: the gene sequence of SARS-CoV-2NSP13 is SEQ ID NO: 1.

7. a product for use in inhibiting expression of a eukaryotic expression plasmid, said product comprising: comprises a polypeptide containing the sequence shown in SEQ ID NO: 1, and can normally express SARS-CoV-2NSP13 protein.

8. The product for inhibiting expression of a eukaryotic expression plasmid according to claim 7, wherein: the product comprises a recombinant plasmid which contains SARS-CoV-2NSP13 gene and can normally express SARS-CoV-2NSP13 protein.

9. The product for suppressing expression of a eukaryotic expression plasmid of claim 8, wherein: the recombinant plasmid takes pXJ40 as a skeleton vector.

10. A construction method of an expression vector capable of expressing SARS-CoV-2NSP13 protein is characterized by comprising the following steps: PCR amplifying SARS-CoV-2NSP13 gene, connecting the amplified segment to eukaryotic expression vector pXJ40, transforming the connection product to competent cell, and successively making ampicillin resistance LB culture medium screening, colony PCR identification and plasmid extraction to obtain expression vector for expressing SARS-CoV-2NSP13 protein.

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