CN111793721B

CN111793721B - Application of eEF1D protein in preparation of medicine for preventing or treating foot-and-mouth disease virus infection

Info

Publication number: CN111793721B
Application number: CN202010779829.1A
Authority: CN
Inventors: 郑海学; 刘会胜; 薛巧; 朱紫祥; 杨帆; 薛钊宁; 曹伟军; 刘湘涛
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2020-08-05
Filing date: 2020-08-05
Publication date: 2021-04-20
Anticipated expiration: 2040-08-05
Also published as: CN111793721A

Abstract

The invention belongs to the field of biotechnology, in particular to the application of an eEF1D protein in the preparation of a medicament for preventing or treating foot-and-mouth disease virus infection. In the present invention, it is found that the expression of eEF1D mRNA increases after infection with FMD virus, while the content of eEF1D protein decreases, that is, eEF1D mRNA and eEF1D protein can be used as detection markers after FMD virus infection to evaluate whether to be infected with FMD virus; secondly, eEF1D protein can significantly Reducing the structural protein VP1 of foot-and-mouth disease virus, the expression of foot-and-mouth disease virus mRNA and the titer of foot-and-mouth disease virus, significantly inhibited the replication of foot-and-mouth disease virus, and can be used to prevent or treat infection of foot-and-mouth disease virus; finally, knocking down the eEF1D protein promotes the structural protein of foot-and-mouth disease virus The expression of VP1 increases the mRNA expression and virus titer of foot-and-mouth disease virus. Therefore, the eEF1D protein can be knocked down in the production cell line by means of genetic engineering, and a vaccine with better performance than the existing production cell line for producing foot-and-mouth disease virus vaccine can be obtained. Cell line for the production of foot-and-mouth disease virus vaccine.

Description

Application of eEF1D protein in preparation of drugs for preventing or treating foot-and-mouth disease virus infection

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of eEF1D protein in preparation of a medicine for preventing or treating foot-and-mouth disease virus infection.

Background

The picornaviridae (RNA) family is a family consisting of the smallest group of RNA viruses, mainly including enterovirus, rhinovirus, cardiovirus, and aphthovirus, and has high homology in structural proteins of four genera of the picornaviridae. The foot-and-mouth disease belongs to the genus of foot-and-mouth disease virus (Aphthovius) of Picornaviridae (Picornaviridae), and is an important disease caused by the foot-and-mouth disease virus for infecting artiodactyl.

Foot-and-mouth disease is an acute, febrile and contagious infectious disease caused by infection of artiodactyl animals (pigs, cattle, sheep, camels, etc.) by Foot and Mouth Disease Virus (FMDV). The FMDV nucleic acid type is a single-stranded positive strand RNA and comprises seven serotypes, and cross protection reaction does not exist among all types. The foot-and-mouth disease has high incidence, rapid infection and serious harm, and is classified as a class A virulent animal infectious disease by the world animal health Organization (OIE), and is classified as a class A animal infectious disease in China. At present, vaccination is an effective means for specifically preventing Foot and Mouth Disease (FMD), and conventional vaccines such as FMD attenuated vaccine and inactivated vaccine have good immunogenicity and play an important role in the process of preventing and controlling FMD. However, foot-and-mouth disease has more serum and no cross protection, which brings huge challenges to the control measures mainly based on vaccine immunization, and the outbreak of FMD in some regions of the world seems to be related to the remaining live virus in the inactivated vaccine due to unsafe factors such as virus virulence reversion, incomplete virus inactivation, live virus escape processing factory and the like. Therefore, how to better inhibit the expression of viral proteins is an effective way to prevent or control viral infection.

Previous studies have shown that TANK binding kinase 1(TBK1) is the major kinase in phosphorylation IRF3/7 and is also a key kinase in TLR and RLR mediated IFN- β expression; TBK1 can be used as a novel E3 ubiquitin ligase, and can effectively degrade virus VP3 protein, further remarkably inhibit protein assembly of picornaviridae, particularly FMDV, and inhibit replication of picornaviridae virus, but TBK1 can only effectively degrade virus VP3 protein, and has weak effect on other structural proteins.

The translation of eukaryotic mRNA into protein on ribosome is a complex process, and requires multi-step reaction and participation of various factors, including three stages of translation initiation, extension and termination, which depend on the participation of translation initiation factor (eIF), extension factor (eEF) and release factor (eRF). Eukaryotic translation initiation factors mainly comprise eIF1, eIF1A, eIF2, eIF2B, eIF3, eIF4A, eIF4B, eIF4G, PABP and the like, elongation factors comprise eEF2, eEF1A, eEF1G, eEF1B2(eEF1B alpha), eEF1D (eEF1 delta or eEF1B beta), and release factors comprise eRF1 and eRF 3. Eukaryotic mRNA is m7G cap-dependent translation. Among them, the protein translation elongation factor eEF1D is a multifunctional protein that plays an important role in both protein translation elongation and cancer regulation. Research shows that Tat protein of human immunodeficiency virus interacts with eEF1D, so that translation of host protein is inhibited; whereas herpes simplex virus infection modifies eEF1D, increasing its molecular weight.

The invention firstly discovers that the expression content of eEF1D mRNA is obviously increased after the foot-and-mouth disease virus is infected, the content of eEF1D protein is reduced, and the content of the cracked eEF1D protein is increased, so that the eEF1D mRNA and the eEF1D protein can be used as detection markers after the foot-and-mouth disease virus is infected, and are used for evaluating whether the foot-and-mouth disease virus is infected; secondly, the eEF1D protein can obviously reduce the structural protein VP1 of the foot-and-mouth disease virus, the expression content of the mRNA of the foot-and-mouth disease virus and the titer of the foot-and-mouth disease virus, can obviously inhibit the replication of the foot-and-mouth disease virus, and can be used for preventing or treating the infection of the foot-and-mouth disease virus, so that the eEF1D protein or the eEF1D protein expression promoter can be used for preparing a composition for preventing or treating the infection of the foot-and-mouth disease virus; finally, the inventor also finds that the eEF1D protein is knocked down in a production cell line, the expression of a structural protein VP1 of the foot-and-mouth disease virus is obviously promoted, and the mRNA expression of the foot-and-mouth disease virus and the titer of the foot-and-mouth disease virus are improved, so that the eEF1D protein can be knocked down or the function of the eEF1D protein can be lost in the production cell line through a genetic engineering means, and the cell line with better performance than that of the existing production cell line for producing the foot-and-mouth disease virus vaccine is obtained and is used for efficiently producing the foot-and-mouth disease virus vaccine.

Disclosure of Invention

The invention firstly discovers that after the foot-and-mouth disease virus is infected, the expression content of eEF1D mRNA is obviously increased, the content of eEF1D protein is reduced, and the content of cracked eEF1D protein is increased. Although the invention is exemplified by foot-and-mouth disease virus, the foot-and-mouth disease virus belongs to picornaviridae, and structural proteins of viruses of four genera (enterovirus, rhinovirus, cardiovirus, aphthovirus) of picornaviridae have high homology, so the invention aims to provide an application of eEF1D mRNA as a marker for detecting infection of picornaviridae viruses.

Another objective of the invention is to provide an application of the eEF1D protein as a marker for detecting the infection of the picornaviridae virus.

Secondly, after the invention discovers that the over-expression of the eEF1D protein can obviously reduce the structural protein VP1 of the foot-and-mouth disease virus, reduce the expression content of the mRNA of the foot-and-mouth disease virus, reduce the titer of the foot-and-mouth disease virus, obviously inhibit the replication of the foot-and-mouth disease virus, and can be used for preventing or treating the infection of the foot-and-mouth disease virus. Although the invention is exemplified by foot-and-mouth disease virus, the foot-and-mouth disease virus belongs to the picornaviridae, and structural proteins of viruses of four genera (enterovirus, rhinovirus, cardiovirus and aphthovirus) of the picornaviridae have high homology, so the invention aims to provide the application of the eEF1D protein in preparing a medicament, a pharmaceutical composition or a vaccine composition for preventing or treating the picornaviridae virus infection.

The invention also aims to provide application of the eEF1D protein expression promoter in preparing a medicament, a pharmaceutical composition or a vaccine composition for preventing or treating the picornaviridae virus infection.

Another object of the present invention is to provide a method for over-expressing the eEF1D protein in a host cell in breeding animals resistant to picornaviridae.

Preferably, the host cell is derived from a bovine (Bovidae) animal, a porcine (Suidae) animal.

Preferably, the picornaviridae virus is a foot-and-mouth disease virus.

Preferably, the amino acid sequence of the eEF1D protein is shown as SEQ ID NO. 1.

Preferably, the nucleotide sequence encoding the eEF1D protein is shown in SEQ ID NO. 2.

Another objective of the invention is to provide a method for preparing animal breeding cells resisting infection of picornaviridae by overexpression of eEF1D protein.

Preferably, the method comprises

(1) Synthesizing a gene sequence of eEF 1D;

(2) carrying out enzyme digestion on a lentiviral vector plasmid by using endonuclease, then connecting the lentiviral plasmid with an eEF1D gene, transferring into competent cells, and extracting and constructing a successful lentiviral plasmid;

(3) when the host cells grow to about 80 percent, carrying out lentivirus packaging on the constructed lentivirus plasmid so as to obtain Lenti-eEF1D-puro lentivirus;

(4) Lenti-eEF1D-puro lentivirus infects cells, and puromycin is added for screening to obtain cells stably expressing eEF1D gene.

The invention finds that after the expression of eEF1D protein is reduced, the expression of a structural protein VP1 of the foot-and-mouth disease virus is obviously promoted, and the expression content of the foot-and-mouth disease virus mRNA and the titer of the foot-and-mouth disease virus are enhanced. Although the present invention is exemplified by foot-and-mouth disease virus, since foot-and-mouth disease virus belongs to the picornaviridae family and structural proteins of viruses of four genera (enterovirus, rhinovirus, cardiovirus, aphthovirus) of the picornaviridae family have high homology, it is another object of the present invention to provide a method for preparing a cell line that promotes replication of picornaviridae virus by loss of the function of eEF1D protein.

Preferably, the method for losing the function of the eEF1D protein is an RNA interference technology.

Preferably: when the host cell grows to 70% -80% confluence, the eEF1D siRNA is transfected with the liposomal reagent.

Preferably, the sequence of the eEF1D siRNA is: f: CCAUCCUGCUGGAUGUCAATT, R: UUGACAUCCAGCAGGAUGGTT are provided.

Preferably, the method for losing the function of the eEF1D protein is a CRISPR/Cas9 lentivirus knockout technology.

Preferably, the method comprises the steps of:

(1) synthesizing sgRNA aiming at an eEF1D gene sequence, and adding BbsI enzyme cutting sites at two ends of the sgRNA;

(2) the lentiviral vector plasmid and double-stranded DNA were digested with BbsI endonuclease, followed by ligation. Transferring into competent cells, and extracting and constructing successfully lentiviral plasmids;

(3) when the host cells grow to about 80%, carrying out lentivirus packaging on the constructed lentivirus plasmid so as to obtain Lenti-sgRNA-Cas9-puro lentivirus;

(4) the Lenti-sgRNA-Cas9-puro lentivirus infects cells, and puromycin is added for screening to obtain the polyclonal cell line with the eEF1D gene knocked down.

Preferably, the sequence of the sgRNA is: sgRNA 1: TCAAATACGATGATGCAGAG (SEQ ID No.3), or sgRNA 2: AAACTTCCTGGTGCACGAGA (SEQ ID No.4), or sgRNA 3: CTCGTGCACCAGGAAGTTTG (SEQ ID No.5) or sgRNA 4: TGAGCAGATGAACGGGCCTG (SEQ ID NO. 6).

Preferably, the picornaviridae virus is a foot-and-mouth disease virus.

Another object of the present invention is to provide a cell line for promoting replication of a Picornaviridae virus, prepared by the above method.

The invention has the beneficial effects that:

firstly, the invention discovers that the expression content of eEF1D mRNA is obviously increased after the foot-and-mouth disease virus is infected, the content of eEF1D protein is reduced, and the content of the cleaved eEF1D protein is increased, so that the eEF1D mRNA and the eEF1D protein can be used as detection markers after the foot-and-mouth disease virus is infected, and can be used for evaluating whether the foot-and-mouth disease virus is infected.

Secondly, the eEF1D protein can obviously reduce the structural protein VP1 of the foot-and-mouth disease virus, reduce the expression content of the mRNA of the foot-and-mouth disease virus, reduce the titer of the foot-and-mouth disease virus, can obviously inhibit the replication of the foot-and-mouth disease virus, and can be used for preventing or treating the infection of the foot-and-mouth disease virus; therefore, the eEF1D protein or the eEF1D protein expression promoter can be used for preparing a composition for preventing or treating foot-and-mouth disease virus infection and inhibiting the replication of the foot-and-mouth disease virus. Meanwhile, the eEF1D protein in the host cell is over-expressed by a genetic engineering means, so that the eEF1D protein can be used for constructing animal breeding cells for resisting the foot-and-mouth disease virus and breeding animals for resisting the foot-and-mouth disease virus infection.

Finally, after the eEF1D protein is knocked down, the expression of the structural protein VP1 of the foot-and-mouth disease virus is obviously promoted, and the expression of the mRNA of the foot-and-mouth disease virus and the titer of the foot-and-mouth disease virus are improved, so that the eEF1D protein can be knocked down in a production cell strain by a genetic engineering means, a cell line with better performance than that of the existing production cell line for producing the foot-and-mouth disease virus vaccine is obtained, the cell line is used for producing the foot-and-mouth disease virus vaccine, and the eEF1D protein inhibitor can be used for inhibiting the expression of the eEF1D protein to construct a cell line for promoting the replication of the foot-and-mouth disease virus and be used for producing the.

Because the foot-and-mouth disease virus belongs to the picornaviridae and the structural proteins of the viruses of four genera (enterovirus, rhinovirus, cardiovirus and aphthovirus) of the picornaviridae have high homology, the expression promoter of the eEF1D protein or the eEF1D protein can be used for preparing a composition for preventing or treating the infection of other picornaviridae viruses and inhibiting the replication of other picornaviridae viruses; meanwhile, the eEF1D protein in the host cell is over-expressed by a genetic engineering means, so that the eEF1D protein can be used for constructing animal breeding cells for resisting other picornaviridae viruses and animal breeding infected by other picornaviridae viruses; the eEF1D protein can be lost through a genetic engineering means or an eEF1D protein inhibitor is used for inhibiting the expression of eEF1D protein, so that a cell line for promoting the replication of other picornaviridae viruses is constructed and is used for producing vaccines of other picornaviridae viruses.

Drawings

FIG. 1 shows the qPCR detection results of eEF1D mRNA expression levels in cells at different time points of PK-15 cells infected with foot-and-mouth disease virus, and the results show that the foot-and-mouth disease virus infection can up-regulate the expression level of eEF1D mRNA in the cells;

FIG. 2 shows that the protein cleavage of eEF1D is increased and the protein expression level of eEF1D is reduced after foot-and-mouth disease virus infection, according to the detection result of Western blot degradation of the protein expression level of eEF1D in cells at different time points of PK-15 cells infected by the foot-and-mouth disease virus;

FIG. 3FLAG-eEF1D plasmid map;

FIG. 4 shows the influence of over-expressed eEF1D protein on the replication of foot-and-mouth disease virus, where a is the Western blot result, b is the qPCR result of foot-and-mouth disease virus mRNA expression, and c is the virus titer determination result of foot-and-mouth disease virus, and the results show that with the increase of FLAG-eEF1D expression level, the expression of structural protein of foot-and-mouth disease virus is reduced, the mRNA level and virus titer of foot-and-mouth disease virus are significantly reduced, and the replication of foot-and-mouth disease virus is significantly inhibited;

FIG. 5 shows the effect of down-regulated expression of eEF1D on the replication of foot-and-mouth disease virus, wherein a is the qPCR result of eEF1D mRNA expression, b is the Western blot result, c is the qPCR result of foot-and-mouth disease virus mRNA expression, and d is the virus titer determination result of foot-and-mouth disease virus, and the results show that the expression of structural proteins of foot-and-mouth disease virus is increased with the increase of the cleavage of eEF1D protein, the mRNA level and virus titer of foot-and-mouth disease virus are obviously increased, and the replication of foot-and-mouth disease virus is promoted.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments. The scope of the invention is not limited to the examples described below.

The term "overexpression" refers to the process of adding regulatory elements upstream of a target gene by artificial construction, so that the gene can realize mass transcription and translation under the condition of artificial control, thereby realizing the overexpression of a gene product. According to the invention, lentivirus plasmids are connected with eEF1D genes, a Lenti-eEF1D-puro lentivirus for over-expressing eEF1D is constructed, and cells are infected to obtain a polyclonal cell strain for over-expressing eEF1D genes. However, the present invention is not limited to the above-mentioned method, and overexpression of the eEF1D gene can be achieved by other technical means of gene editing.

The term "loss of function of a protein" refers to the loss of function of a protein encoded by a gene by knocking out, mutating or inserting a part of the gene in a gene segment encoding the protein, so that the protein encoded by the gene is subjected to frame shift mutation. The eEF1D gene in the host cell is knocked out in a targeted mode, so that the function of the eEF1D gene encoding protein is lost, and a cell line with the function of the eEF1D gene encoding protein lost is constructed and is used for producing the picornaviridae virus vaccine. However, the invention is not limited to the eEF1D gene knockout, and the function of the protein encoded by the eEF1D gene can be lost by other technical means and used for constructing a cell line with the function of the protein encoded by the eEF1D gene being lost.

The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials used in the following examples were all purchased from conventional biochemicals, unless otherwise specified.

The FLAG-eEF1D plasmid was constructed by Riboley Biotechnology, Inc., Lanzhou.

FMDV (O/BY/CHA/2010) was isolated BY this team and maintained in the foot-and-mouth disease and New disease epidemiology team and the national foot-and-mouth disease reference laboratory of Lanzhou veterinary institute, national academy of agricultural sciences.

Example 1 detection of changes in eEF1D mRNA and protein expression levels in cells following foot and mouth disease virus infection

1. Preparation of PK-15 cell samples infected with FMDV

Will be 6X 10⁵Individual PK-15 cells were plated in a single well of a 6-well plate for a total of 3 dishes. When the cells grow to 70-80% of fusion degree, respectively transfecting FLAG-

eEF1D plasmids

0, 1 and 2 mu g by using a liposome reagent, transfecting for 24h, inoculating 1MOI foot-and-mouth disease virus, and changing to a maintenance solution for culture after inoculating for 1 h.

qPCR detection of eEF1D mRNA expression levels

Cells infected with foot-and-mouth disease virus were harvested at 0, 4, 6, 8, 10 and 12h, respectively, and washed once with PBS. The RNA extraction process is as follows:

1mL Trizol reagent was added, the cells were lysed thoroughly by vigorous pipetting for 5-15min, and the liquid was transferred to a 1.5mL centrifuge tube without RNase.

250 μ L of chloroform was added, and the liquid was turned into pale red by vigorous shaking and allowed to stand at 4 ℃ for 10 min.

Centrifuging at 12000r/min at 4 deg.C for 15min, sucking 200 μ L of supernatant into a new RNase-free 1.5mL centrifuge tube, adding 200 μ L of isopropanol, slightly inverting for 8 times, standing at-20 deg.C for 30min, and centrifuging at 12000r/min at 4 deg.C for 15 min.

The supernatant was discarded, 1mL of 75% ethanol was added, the mixture was inverted 5 times, and centrifuged at 4 ℃ at 10000r/min for 5 min.

Discard the supernatant, suck the residual liquid, and dry the centrifuge tube. Then 25. mu.L of DEPC was added to dissolve the RNA.

The prepared total RNA (viral RNA and cellular RNA) is subjected to reverse transcription and then real-time quantitative PCR detection.

The experimental results are shown in fig. 1, and the expression level of eEF1D mRNA in PK-15 cells is increased along with the increase of the infection time of the foot-and-mouth disease virus, which indicates that the foot-and-mouth disease virus infection promotes the expression of eEF1D mRNA in host cells.

Western blot detection of eEF1D protein expression level

Preparation of protein samples: discarding cell culture supernatant after 12h infection of foot and mouth disease virus, washing a cell sample once with PBS, scraping cells by using a cell scraper, transferring the cells into a 1.5mL centrifuge tube, centrifuging at 2000rpm for 5min, discarding supernatant, and retaining cell precipitates to obtain a harvested cell sample (all on ice); adding appropriate amount of cell lysate according to the amount of collected cell precipitate, rapidly and repeatedly blowing and beating the resuspended cell sample, performing ice lysis for 5min, and performing ultrasonic instantaneous disruption (ice operation, ultrasonic for 2-3 times); centrifuging at 4 deg.C and 13000rpm for 10min, removing bottom precipitate, collecting supernatant, and preserving in another precooling centrifuge tube; adding 5 Xprotein sample buffer solution into the protein supernatant, boiling in boiling water for denaturation for 10min, centrifuging at 4 deg.C and 12000rpm for 5min, and performing protein electrophoresis on the supernatant.

SDS-PAGE gel electrophoresis and its electrophoretogram protein transfer: SDS-PAGE gels and running buffers were prepared according to the molecular cloning protocol. The loading of each protein sample was 40 μ g, while a separate well was selected and a pre-stained protein marker was added as an indicator. When the protein sample is concentrated with glue, 80V voltage is called; and after the protein sample enters the separation gel, regulating the voltage to 120V until the electrophoresis is finished. Before transfer, a suitable nitrocellulose membrane (NC membrane) is cut according to the size of SDS-PAGE gel, and is soaked in a transfer buffer for about 10min, and simultaneously 6 layers of filter paper are cut and soaked in the transfer buffer for several minutes. Placing the protein glue according to the sequence of 'positive glue and negative glue of the film': after the negative electrode, the sponge, the 3 layers of filter paper, the gel, the NC membrane, the 3 layers of filter paper, the sponge and the positive electrode are installed with the transfer sandwich, the sandwich is placed into a full-wet transfer printing tank, enough transfer printing buffer solution is added, a power supply is switched on (the constant current is 240mA or the constant voltage is 65V for transfer printing is 2-3h), and the temperature of the outside of the transfer printing tank is reduced by the aid of an ice bag. After the transfer is finished, the NC membrane is sealed by 5% TBST-skimmed milk, the reaction is carried out for 2-3h at room temperature, the sealing solution is discarded, TBST (pH7.6) buffer solution is used for rinsing for 3 times, residual skimmed milk sealing solution is washed away, and then antibody incubation is carried out. Antibody reaction: adding TBST to dilute the primary antibody, shaking gently at 4 ℃ overnight (or room temperature for 4-6h), and recycling the primary antibody. After the TBST is rinsed gently for 3 times, the mixture is washed with TBST for 2-3 times, and each time, rinsing is carried out for 10 min. After rinsing, adding HRP-labeled secondary antibody, reacting at room temperature for 2h, rinsing with TBST gently for 3 times after reaction, and rinsing with TBST for 3 times, each time for 10 min. After the completion of the rinsing, a color reaction was carried out. The color was developed in a dark room using an ECL color development kit. Mixing solution A and solution B in the kit in equal amount, slightly and uniformly wetting NC membrane, acting for 1-2min, placing the acting membrane in X-ray exposure clamp, and placing X-ray film at the top for exposure. And (3) firstly, placing the exposed film into a developing solution for developing, after the required protein band is displayed, slightly rinsing the film by using tap water, and then placing the film into a fixing solution for fixing for 2-3 min. And after the fixation is finished, the film is put into tap water for rinsing and further dried, a protein Marker is marked after the film is dried, and the film is scanned to store the result.

The experimental result is shown in fig. 2, with the increase of the foot-and-mouth disease virus infection time, the expression level of the eEF1D protein in the PK-15 cells is reduced, and the amount of the cleaved eEF1D protein is increased, which indicates that the foot-and-mouth disease virus infection promotes the self-cleavage of the eEF1D protein in the host cells. The experiments show that the eEF1D mRNA and the eEF1D protein can be used as detection markers for evaluating the infection degree of the foot-and-mouth disease virus.

Although the invention takes the foot-and-mouth disease virus as an example, the foot-and-mouth disease virus belongs to the picornaviridae, and structural proteins of viruses of four genera (enterovirus, rhinovirus, cardiovirus and aphthovirus) of the picornaviridae have high homology, so the eEF1D mRNA and the eEF1D protein can be used as detection markers for evaluating the infection degree of other picornaviridae viruses.

Example 2 evaluation of Effect of eEF1D protein on inhibiting replication of foot-and-mouth disease Virus

Construction of the FLAG-eEF1D plasmid

The FLAG-eEF1D plasmid is constructed by RiboLai Biotechnology GmbH, Lanzhou, the plasmid construction information is shown in figure 3, wherein the eEF1D gene is synthesized according to a swine eEF1D gene sequence, and the login number is as follows: XM _ 021090459.1.

2. Preparation of PK-15 cell samples infected with FMDV

Will be 6X 10⁵Individual PK-15 cells were plated in a single well of a 6-well plate in 3 dishes. When the cells grow to 70-80% of fusion degree, respectively transfecting FLAG-

eEF1D plasmids

3. Detection of viral protein content

The detection method was the same as that described in 3 of example 1.

The experimental result is shown in a in figure 4, along with the increase of the addition of the FLAG-eEF1D plasmid, the expression of the foot-and-mouth disease virus structural protein VP1 is obviously reduced, which indicates that compared with the normal PK-15 cell, the expression of the foot-and-mouth disease virus structural protein VP1 is obviously inhibited by adding the eEF1D protein exogenously. Similarly, the eEF1D protein expression promoter is used for promoting the expression of eEF1D protein in PK-15 cells, and can also inhibit the expression of the foot-and-mouth disease virus structural protein VP 1.

4. Foot and mouth disease virus mRNA detection

Cells were harvested after 0, 4, 6, 8, 10 and 12h of infection, RNA was extracted and reverse transcribed as follows. Then, SYBR Premix Ex Taq from Takara was used^TMThe kit carries out real-time quantitative PCR detection according to the following system and program, and compares the difference of virus mRNA levels among different groups.

Reverse transcription system:

reaction procedure: at 25 ℃ for 10 min; 60min at 37 ℃; 75 ℃ for 10 min.

Real-time quantitative PCR reaction system:

reaction procedure:

at 95 ℃ for 2 min; 95 ℃, 10s, 60 ℃, 34s, 40 cycles; melt cut; storing at 4 ℃.

The experimental result is shown in b in fig. 4, the content of the mRNA of the foot-and-mouth disease virus is significantly reduced with the increase of the addition of the FLAG-eEF1D plasmid, which indicates that the exogenous addition of the eEF1D protein significantly inhibits the expression of the mRNA of the foot-and-mouth disease virus and inhibits the replication of the foot-and-mouth disease virus relative to normal PK-15 cells. Similarly, the eEF1D protein expression promoter is used to promote the expression of eEF1D protein in PK-15 cells, and can also inhibit the expression of foot-and-mouth disease virus mRNA and inhibit the replication of foot-and-mouth disease virus.

5. Foot and mouth disease virus titer determination

Cell supernatants were collected after FMDV infection by TCID₅₀And (4) determining and performing virus titer analysis. Measurement of viral titer: BHK-21 cells were seeded into 96-well plates 16h in advance. After the cells formed a monolayer, BHK-21 cells were washed 3 times with PBS and inoculated with foot-and-mouth disease virus (10)^-1-10^-10) Two additional columns of negative control wells were provided. Infecting the wells, adding 100 μ L of virus filtrate or diluted virus diluent at multiple ratio into each well, adsorbing at 37 deg.C for 1h, and shaking gently once every 20min to ensure uniform virus adsorption. After 1h of adsorption, the supernatant was aspirated and the plate was washed gently with PBS 1 time. Adding virus maintaining liquid. Observing cytopathic condition every 12h after 48h, recording pathological change hole number after 72h, and calculating TCID₅₀Triplicate determinations were made and the mean was taken as the final virus titer.

The results of the experiments are shown in figure 4 c, with the increase of the amount of the FLAG-eEF1D plasmid, the TCID of the foot-and-mouth disease virus₅₀The obvious reduction shows that the over-expression of the eEF1D protein obviously inhibits the virus TCID of the foot-and-mouth disease virus₅₀。

The experiment shows that compared with normal PK-15 cells, the exogenously added eEF1D protein can obviously inhibit the replication of the foot-and-mouth disease virus, so that the exogenously added eEF1D protein or eEF1D protein expression promoter can obviously inhibit the replication of the foot-and-mouth disease virus and can be used for preventing or treating the infection of the foot-and-mouth disease virus.

Although the invention takes the foot-and-mouth disease virus as an example, the foot-and-mouth disease virus belongs to the picornaviridae, and structural proteins of four viruses (enterovirus, rhinovirus, cardiovirus and aphthovirus) in the picornaviridae have high homology, so that the exogenously added eEF1D protein or eEF1D protein expression promoter and the induced eEF1D protein overexpression can obviously inhibit the replication of other picornaviridae viruses, and can be used for preventing or treating the infection of other picornaviridae viruses.

The experiment proves that the eEF1D protein exogenous or eEF1D protein expression promoter can be used for preparing a medicament for preventing or treating the infection of the picornaviridae virus; and by over-expressing the eEF1D protein in the host cell, a cell line capable of inhibiting the replication of the picornaviridae virus can be constructed and used for breeding the anti-picornaviridae virus animals.

Example 3 construction of lentiviruses and cell lines overexpressing eEF1D

The gene sequence of eEF1D was synthesized by shanghai gimar pharmaceutical technology ltd.

The lentivirus vector plasmid is cut by restriction enzyme for 2h, and then the lentivirus plasmid and the eEF1D gene are subjected to ligation reaction. The ligation product is transferred into competent cells, and the successfully constructed lentiviral plasmid is extracted.

Enzyme digestion system (10 μ L):

ligation reaction (10 μ L):

and (3) when the host cells grow to about 80%, carrying out lentivirus packaging on the constructed lentivirus plasmid according to the instruction of a lentivirus packaging kit so as to obtain the Lenti-eEF1D-puro lentivirus. The lentivirus can promote the overexpression of eEF1D protein, and can be used for preparing vaccines or vaccine compositions for preventing or treating the infection of the picornaviridae virus.

The cell is infected by Lenti-eEF1D-puro lentivirus, puromycin (puromycin,2 mu g/ml) is added for screening, and then the polyclonal cell strain of the over-expressed eEF1D gene can be obtained. The cell strain can be stably passaged in the presence of puromycin, and can be used for breeding of anti-picornaviridae virus animals.

Example 4 evaluation of Effect of Down-regulated expression of eEF1D protein on promotion of foot-and-mouth disease Virus replication

Design of NC siRNA and eEF1D siRNA

The NC siRNA sequence and the eEF1D siRNA sequence are designed and synthesized by Shanghai Jima pharmaceutical technology GmbH. Wherein, NC siRNA: f: UUCUCCGAACGUGUCACGUTT, R: ACGUGACACGUUCGGAGA ATT, respectively; eEF1D siRNA: f: CCAUCCUGCUGGAUGUCAATT, R: UUGACAUCCAGCAGGAUGGTT are provided.

2. Preparation of PK-15 cell samples infected with FMDV

Will be 6X 10⁵Individual PK-15 cells were plated in a single well of a 6-well plate for a total of 2 dishes. When the cells grow to 70% -80% of fusion degree, respectively transfecting NC siRNA and eEF1D siRNA by using a liposome reagent for 36h, inoculating 1MOI foot and mouth disease virus, and changing to maintenance liquid for culture after 1h of inoculation.

qPCR detection of eEF1DmRNA expression levels

The procedure is as described in example 1, 2.

As shown in a of fig. 5, the NC siRNA does not inhibit the expression of eEF1D mRNA with the infection of foot and mouth disease virus, whereas the addition of eEF1D siRNA significantly reduces the expression level of eEF1D mRNA compared to NC siRNA, and the addition of eEF1D siRNA reduces the expression level of eEF1D mRNA more significantly compared to NC siRNA with the increase of infection time of foot and mouth disease virus, which indicates that eEF1D siRNA successfully inhibits the expression of eEF1D mRNA.

4. Detection of viral protein content

The detection method was the same as that described in 3 of example 1.

The experimental result is shown in b in fig. 5, with the infection of the foot-and-mouth disease virus, the NC siRNA enables the expression level of eEF1D protein to be significantly reduced, and the expression level of cleaved eEF1D protein is significantly increased, which indicates that with the infection of the foot-and-mouth disease virus, the NC siRNA group eEF1D protein is cleaved by the foot-and-mouth disease virus; compared with NC siRNA, the addition of eEF1D siRNA can obviously reduce the expression level of eEF1D protein, relatively reduce the expression level of cleaved eEF1D protein, and obviously increase the expression content of FMDV structural protein VP1, which indicates that the addition of eEF1D siRNA inhibits the expression level of eEF1D protein, promotes FMDV replication, and can be used for efficiently producing foot-and-mouth disease virus vaccines.

qPCR detection of FMDV mRNA expression levels

The procedure is as described in example 1, 2.

Experimental results as depicted in fig. 5 c, the addition of eEF1D siRNA significantly increased FMDV mRNA expression levels, and as the time to foot and mouth disease virus infection increased, FMDV mRNA expression levels were more significantly increased after addition of eEF1D siRNA compared to NC siRNA. Therefore, the expression of the eEF1D protein inhibits the increase of the replication of the foot-and-mouth disease virus; therefore, the protein eEF1D can be lost or an eEF1D protein inhibitor is used for inhibiting the expression of eEF1D protein, so that the replication of the foot-and-mouth disease virus is promoted, and the method can be used for efficiently producing the foot-and-mouth disease virus vaccine.

5. Foot and mouth disease virus titer determination

Cell supernatants were collected 12 hours after infection by TCID₅₀And (4) determining and performing virus titer analysis.

Experimental results as shown in d in fig. 5, the addition of eEF1D siRNA significantly increased the viral titer of foot and mouth disease, and as the infection time of foot and mouth disease virus increased, the viral titer of foot and mouth disease increased more significantly after adding eEF1D siRNA compared to NC siRNA. The expression of the eEF1D protein inhibits the increase of the virus titer of the foot-and-mouth disease virus; therefore, the replication of the foot-and-mouth disease virus can be promoted by losing the function of the eEF1D protein or inhibiting the expression of the eEF1D protein by using an eEF1D protein inhibitor.

The results show that the replication of the foot-and-mouth disease virus can be promoted after the expression level of the eEF1D protein is regulated, so that a cell line for promoting the replication of the foot-and-mouth disease virus can be successfully constructed by losing the function of the eEF1D protein or inhibiting the expression of the eEF1D protein by using an eEF1D protein inhibitor, and the cell line is used for producing a foot-and-mouth disease virus vaccine. Although the present invention is exemplified by foot-and-mouth disease virus, since foot-and-mouth disease virus belongs to the family picornaviridae and structural proteins of viruses of four genera (enterovirus, rhinovirus, cardiovirus, aphthovirus) of the family picornaviridae have high homology, cell lines that promote replication of viruses of other picornaviridae can be successfully constructed and used for production of vaccines against viruses of other picornaviridae by loss of the function of eEF1D protein or inhibition of eEF1D protein expression using eEF1D protein inhibitors, based on the examples described in the present invention.

Example 5 construction of a lentiviral knockout eEF1D cell line

Aiming at the eEF1D gene sequence of a pig, 4 pairs of sgRNA primer sequences are designed according to the design principle of sgRNA targets, and BbsI enzyme cutting sites are added at two ends of the sgRNA primer sequences. Wherein the sgRNA sequences are as follows:

sgRNA 1：TCAAATACGATGATGCAGAG；

sgRNA 2：AAACTTCCTGGTGCACGAGA；

sgRNA 3：CTCGTGCACCAGGAAGTTTG；

sgRNA 4：TGAGCAGATGAACGGGCCTG。

the lentiviral vector plasmid and the double-stranded DNA were digested with BbsI endonuclease for 2h, followed by overnight ligation. The ligation product is transferred into competent cells, and the successfully constructed lentiviral plasmid is extracted.

Enzyme digestion system (10 μ L):

ligation reaction (10 μ L):

and (3) when the host cells grow to about 80%, carrying out lentivirus packaging on the constructed lentivirus plasmid according to the instruction of a lentivirus packaging kit so as to obtain the Lenti-sgRNA-Cas9-puro lentivirus.

Lenti-sgRNA-Cas9-puro lentivirus infects cells, and puromycin (puromycin,2 mu g/ml) is added for screening to obtain the polyclonal cell strain with the eEF1D gene knocked down. The cell strain can be stably passaged in the presence of puromycin, and is used for high-efficiency production of the picornaviridae virus vaccine.

The above-mentioned embodiments only represent the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that other modifications can be made by those skilled in the art without departing from the spirit of the invention, and these are within the scope of the invention.

Sequence listing

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

Application of <120> eEF1D protein in preparation of drugs for preventing or treating foot-and-mouth disease virus infection

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 280

<212> PRT

<213> pig (Sus scrofa)

<400> 1

Met Ala Thr Asn Phe Leu Val His Glu Lys Val Trp Phe Asp Lys Phe

1 5 10 15

Lys Tyr Asp Asp Ala Glu Arg Lys Phe Tyr Glu Gln Met Asn Gly Pro

20 25 30

Val Ala Gly Ser Ser Arg Gln Glu Asn Gly Ala Ser Val Ile Leu Arg

35 40 45

Asp Ile Ala Arg Ala Arg Glu Asn Ile Gln Lys Ser Leu Ala Gly Ser

50 55 60

Ser Gly Pro Gly Ala Ser Ser Gly Pro Ser Gly Asp His Ser Glu Leu

65 70 75 80

Val Ile Arg Ile Ala Ser Leu Glu Val Glu Asn Gln Ser Leu Arg Gly

85 90 95

Val Val Gln Asp Leu Gln Gln Ala Val Ser Lys Leu Glu Ala Arg Leu

100 105 110

Ser Ala Leu Glu Lys Thr Ser Pro Ala His Arg Ala Thr Ala Pro Gln

115 120 125

Thr Gln His Val Ser Pro Met Arg Gln Val Glu Pro Pro Ser Arg Lys

130 135 140

Ala Ala Thr Ala Thr Glu Asp Asp Glu Asp Asp Asp Ile Asp Leu Phe

145 150 155 160

Gly Ser Asp Glu Glu Glu Asp Lys Glu Ala Ala Arg Leu Arg Glu Glu

165 170 175

Arg Leu Arg Gln Tyr Ala Glu Lys Lys Ala Lys Lys Pro Ala Leu Val

180 185 190

Ala Lys Ser Ser Ile Leu Leu Asp Val Lys Pro Trp Asp Asp Glu Thr

195 200 205

Asp Met Ala Gln Leu Glu Ala Cys Val Arg Ser Ile Gln Leu Asp Gly

210 215 220

Leu Thr Trp Gly Gly Ser Lys Leu Val Pro Val Gly Tyr Gly Ile Arg

225 230 235 240

Lys Leu Gln Ile Gln Cys Val Val Glu Asp Asp Lys Val Gly Thr Asp

245 250 255

Leu Leu Glu Glu Glu Ile Thr Lys Phe Glu Glu His Val Gln Ser Val

260 265 270

Asp Ile Ala Ala Phe Asn Lys Ile

275 280

<210> 2

<211> 843

<212> DNA

<213> pig (Sus scrofa)

<400> 2

atggccacaa acttcctggt gcacgagaag gtctggttcg acaagttcaa atacgatgat 60

gcagagagga aattctatga gcagatgaac gggcctgtgg ctggctcctc acgccaggag 120

aacggcgcca gcgtgatcct ccgtgacatc gcgagagcca gagaaaacat ccagaagtcc 180

ctggccggaa gctcaggccc tggggcctcc agcgggccca gtggagacca cagtgagctg 240

gtcatccgga tcgccagcct ggaagtggag aaccagagcc tgcgaggggt ggtacaggat 300

ctgcagcagg ccgtctccaa gctggaggcc cggctgagtg cgctcgagaa gacctcgccc 360

gcccaccgtg ccacagcccc acagacccag cacgtgtctc ccatgcgcca agtggagccc 420

cccagccgga aggcggccac ggccacagag gacgacgagg acgatgacat tgacctgttc 480

ggcagcgacg aagaggagga caaggaggct gcccggctgc gggaggagag gctgcggcaa 540

tacgccgaga aaaaggccaa gaagcccgcc ctggtggcca agtcctccat cctgctggat 600

gtcaagcctt gggacgacga gacggacatg gcccagctgg aggcctgtgt gcgctccatc 660

cagctggacg ggctgacctg gggcggctcc aagctggtgc ctgtgggcta cggcatccgc 720

aagctgcaga tccagtgcgt ggtggaggac gacaaggtgg gcaccgacct gctggaagag 780

gagatcacca agttcgagga gcacgtgcag agtgtggaca ttgccgcctt caacaagatc 840

tga 843

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tcaaatacga tgatgcagag 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aaacttcctg gtgcacgaga 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctcgtgcacc aggaagtttg 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tgagcagatg aacgggcctg 20

Claims

1. The application of a reagent for detecting eEF1D mRNA or eEF1D protein in preparing a foot-and-mouth disease virus infection diagnostic reagent is disclosed, wherein the amino acid sequence of the eEF1D protein is shown as SEQ ID NO. 1.

2. The use of claim 1, wherein the nucleotide sequence encoding the eEF1D protein is set forth in SEQ ID No. 2.

The application of eEF1D protein or eEF1D protein expression promoter in the preparation of drugs for preventing or treating foot-and-mouth disease virus infection, wherein the amino acid sequence of the eEF1D protein is shown as SEQ ID NO. 1.

4. The use of claim 3, wherein the nucleotide sequence encoding the eEF1D protein is as shown in SEQ ID No. 2.