Foot-and-mouth disease virus VP1 protein modified by non-natural amino acids, preparation method and application

Abstract

The invention belongs to the field of biotechnology, and specifically relates to a method for introducing an early stop codon at a specific amino acid residue of a foot-and-mouth disease virus VP1 protein based on a genetic codon expansion technology, and using a biological orthogonal translation system to read through an amber stop codon UAG. Under the condition of the presence or absence of a non-natural amino acid, not only can the expression of the VP1 protein be conditionally controlled, and the non-natural amino acid can be introduced into the foot-and-mouth disease virus VP1 protein at a specific point, but also the foot-and-mouth disease virus VP1 protein can be specifically labeled with a non-natural amino acid containing a special group, and a stable covalent bond can be formed between proteins that interact with cells through light cross-linking, thereby providing a powerful means for studying the interaction between the foot-and-mouth disease virus and host proteins; and the method can also be used to prepare a replication-deficient recombinant foot-and-mouth disease virus and a vaccine with a mutation at a specific site of the VP1 protein.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a foot-and-mouth disease virus VP1 protein that is site-modified by using orthogonal unnatural amino acids (UAAs) based on the genetic code expansion (GCE) technology and a bio-orthogonal translation system to read through premature termination codons (PTC), as well as a preparation method and application thereof. Background Art

[0002] Foot and mouth disease virus (FMDV) is a single-stranded positive-strand RNA virus belonging to the Picornaviridae family and the genus Foot and Mouth Disease Virus. The virus particles are enveloped and icosahedral, and are composed of structural proteins VP0, VP3 and VP1, of which VP0 protein is composed of VP4 and VP2. The virus mainly infects even-toed ungulates such as pigs, cattle, and sheep. It has the characteristics of rapid replication, high contact infection, and strong pathogenicity, and can cause a large-scale epidemic in pigs. Foot and mouth disease outbreaks will restrict the trade of animals and their products, causing significant economic losses and social impacts. The World Organization for Animal Health (WOAH) has listed it as a legally notifiable infectious disease. VP1 protein is one of the main structural proteins of FMDV. It contains the RGD motif on a unique flexible loop structure (G-Hloop), which is a key motif that binds to integrin receptors on the cell surface to mediate virus adsorption to cells. VP1 protein is also involved in virus invasion and immune escape, and is a key protein that induces the host body to produce neutralizing antibodies. It is the preferred protein for the development of foot and mouth disease subunit vaccines. Therefore, in-depth research on the function of FMDV VP1 protein and its interacting proteins is of great significance for understanding the interaction mechanism between FMDV and the host.

[0003] The genetic codon expansion (GCE) technology developed in recent years is a technology that artificially expands the codon table of protein genetic coding and introduces new coding rules to modify or change proteins. This technology is achieved through engineered aminoacyl-tRNA synthetase (aa-tRNA synthetase, aaRS) / tRNA pairs. The modified aaRS can specifically aminoacylate the homologous tRNA with a specific UAA, and is dedicated to decoding the reallocated codon (usually the stop codon, and the matching aaRS / tRNA pair is called nonsense inhibitory aaRS / tRNA). The tRNA is then recognized by the cellular ribosomes. UAA, allowing UAA to correspond to designated codons during translation and to be site-specifically incorporated into synthetic peptides. All metazoan proteins are synthesized from 20 classical amino acids. Different combinations of these 20 amino acids give proteins a variety of structures and functions, but at the same time limit the development of protein properties. Although post-translational modification mechanisms have relieved this limitation to a certain extent, this relies on extremely robust and complex biological systems. The use of genetic codon expansion technology to directly incorporate UAAs into proteins has greatly expanded the properties of proteins, allowing UAAs to be specifically inserted into any site of interest on the protein peptide chain through an orthogonal translation system in living cells. An amber stop codon (TAG) is introduced into the genome of the target protein, and an exogenous UAAs bioorthogonal translation system is used to encode a variety of unnatural amino acids and insert them into the living organism. This technology has successfully site-specifically expressed hundreds of UAAs (including affinity-tagged and photoisomerizable amino acids, carbonyl amino acids, and glycosylated amino acids) in proteins in living cells, giving these proteins novel physical, chemical, and physiological properties. This technology can selectively introduce special chemical groups such as carbonyl, alkynyl and azide groups into proteins to achieve site-specific modification of proteins and improve the properties of proteins. At the same time, this technology can also be applied to site-specific labeling, site-specific modification, and replication control of living organisms (such as viruses and bacteria), and has been applied to many fields, such as the treatment of genetic diseases, protein interactions and biomedicine.

[0004] PTC technology is a technology for developing viral vaccines that introduces premature termination codons (PTC) into the viral genome to control viral replication and protein expression, making it dependent on exogenous non-natural amino acids. To truly apply GCE and PTC technologies to the development of viral vaccines, an urgent problem to be solved is how to select appropriate and efficient amino acid mutation sites. Achieving efficient incorporation of exogenous UAAs and high-level expression of target proteins will not only help study the biological functions of viral proteins, but will also greatly promote the application of PTC technology in viral vaccine research and development.

[0005] The present invention first selects 15 amino acid residues from the amino acid sequence of VP1 protein for codon point mutation based on protein 3D structure analysis and software prediction analysis, and screens amino acid sites with high UAA incorporation efficiency. This mutation method is a non-natural amino acid introduced at a specific site in the VP1 protein, and its unique azide group can specifically react with a fluorescent marker specific group to click, thereby marking the location of the VP1 protein in living cells. The method for preparing FMDV VP1 protein with non-natural amino acid site-specific modification has the following advantages: 1) the efficient insertion site of non-natural amino acids is screened in the coding region of the VP1 protein, and the conditional control of the expression of VP1 protein in the cell is realized; 2) the method of site-specific modification of proteins by non-natural amino acids is used to realize the intracellular fluorescent labeling of VP1 protein; 3) the site-specific modification of non-natural amino acids using photocrosslinking can be used to identify protein complexes that interact with VP1 protein, thereby determining key interacting proteins; 4) based on the above-mentioned efficient amino acid mutation sites, it can be used to prepare PTC recombinant FMDV, which is helpful for the development of viral vaccines and has broad application prospects. Summary of the invention

[0006] The present invention finds that by mutating the codon of the amino acid residue at at least one of the T43, H59, R67, K81, D85, K96, L98, Y107, P111, R135, R138, P142, R145, L148 and L151 positions of the FMDV VP1 protein of the amino acid sequence as shown in SEQ ID NO.1 to an amber codon TAG, and utilizing a bioorthogonal translation system, a non-natural amino acid can be site-specifically incorporated into the VP1 protein, so that conditional control of the expression of the VP1 protein and intracellular fluorescent labeling can be achieved, and an unknown protein interacting with the VP1 protein can be captured by a photocrosslinking reaction in living cells.

[0007] The specific content of the invention is:

[0008] In the first aspect, the present invention provides a foot-and-mouth disease virus VP1 protein modified with a non-natural amino acid, characterized in that at least one amino acid at positions T43, H59, R67, K81, D85, K96, L98, Y107, P111, R135, R138, P142, R145, L148 and L151 of the foot-and-mouth disease virus VP1 protein shown in SEQ ID NO.1 is mutated to a non-natural amino acid.

[0009] Preferably, the unnatural amino acids include: NAEK, DiZPK, DiZSeK, Bpa.

[0010] Preferably, the unnatural amino acids are NAEK and DiZPK.

[0011] In a second aspect, the present invention provides a nucleic acid molecule encoding the VP1 protein with the above-mentioned non-natural amino acid site-specific modification.

[0012] Preferably, the nucleic acid molecule is: the codon encoding at least one of the amino acids at positions T43, H59, R67, K81, D85, K96, L98, Y107, P111, R135, R138, P142, R145, L148 and L151 of SEQ ID NO.1 is mutated to the codon TAG.

[0013] In a third aspect, the present invention provides a method for preparing the foot-and-mouth disease virus VP1 protein modified with a non-natural amino acid as described in the first aspect, the method comprising the following steps:

[0014] (1) mutating the codon encoding at least one amino acid in positions T43, H59, R67, K81, D85, K96, L98, Y107, P111, R135, R138, P142, R145, L148 and L151 of the sequence shown in SEQ ID NO.1 to an amber codon TAG to obtain an FMDV VP1 protein gene coding sequence carrying an amber codon mutation;

[0015] (2) operably linking the gene sequence described in step (1) to a suitable vector to obtain a mutant sequence expression vector;

[0016] (3) The mutant sequence expression plasmid and the orthogonal translation system expression plasmid are co-transfected into host cells, and the non-natural amino acid-induced culture is used to screen and obtain the foot-and-mouth disease virus VP1 protein with site-specific modification of the non-natural amino acid.

[0017] Preferably, the unnatural amino acids include: NAEK, DiZPK, DiZSeK, Bpa.

[0018] Preferably, the unnatural amino acids are NAEK and DiZPK.

[0019] Preferably, the orthogonal translation system expression plasmid comprises:

[0020] Orthogonal tRNA / aminoacyl-tRNA synthetases;

[0021] The tRNA is derived from a prokaryotic organism;

[0022] The anticodon loop of the tRNA corresponds to the premature stop codon UAG;

[0023] The aminoacyl-tRNA synthetase is capable of binding an unnatural amino acid to the tRNA;

[0024] The tRNA and aminoacyl-tRNA synthetase are located on the same carrier.

[0025] In a fourth aspect, the present invention provides the use of the foot-and-mouth disease virus VP1 protein modified with a non-natural amino acid as described in the first aspect in screening for conditionally controlling the expression of the foot-and-mouth disease virus VP1 protein in vitro.

[0026] In a fifth aspect, the present invention provides the use of the foot-and-mouth disease virus VP1 protein modified with a non-natural amino acid site-specifically as described in the first aspect in intracellular fluorescence localization analysis of the foot-and-mouth disease virus VP1 protein labeled with a non-natural amino acid.

[0027] In a sixth aspect, the present invention provides the use of the foot-and-mouth disease virus VP1 protein site-specifically modified with non-natural amino acids as described in the first aspect in the preparation of a recombinant foot-and-mouth disease virus vaccine.

[0028] In a seventh aspect, the present invention provides the use of the foot-and-mouth disease virus VP1 protein modified with a non-natural amino acid as described in the first aspect in screening proteins that interact with the foot-and-mouth disease virus VP1 protein.

[0029] Preferably, the foot-and-mouth disease virus VP1 protein modified with non-natural amino acids is: at least one amino acid at positions T43, H59, R67, K81, D85, and K96 of the foot-and-mouth disease virus VP1 protein shown in SEQ ID NO.1 is mutated to a non-natural amino acid.

[0030] The beneficial effects of the present invention are:

[0031] (1) The present invention found that in the coding region of the VP1 protein whose amino acid sequence is shown in SEQ ID NO. 1, the efficient insertion sites of non-natural amino acids, T43, H59, R67, K81, D85, K96, L98, Y107, P111, R135, R138, P142, R145, L148 and L151, were screened, thereby achieving site-specific modification of FMDV VP1 protein with non-natural amino acids and conditional control of VP1 protein expression in cells;

[0032] (2) The method of site-specific modification of proteins with non-natural amino acids achieves intracellular fluorescent labeling of VP1 protein;

[0033] (3) The method for site-specific modification of proteins with non-natural amino acids can be used to identify protein complexes that interact with VP1 protein by using photo-crosslinking of non-natural amino acids for site-specific modification;

[0034] (4) The highly efficient amino acid mutation sites described above can be used to prepare PTC recombinant FMDV, which is helpful for the development of viral vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The 3D structure of the VP1 protein, where the red marks are amino acid residues T43, H59, R67, and K81;

[0036] Figure 2 Results of conservation analysis of the amino acid sequence of VP1 protein;

[0037] Figure 3 Results of screening for efficient insertion sites of some unnatural amino acids in VP1 protein;

[0038] Figure 4 Intracellular fluorescent labeling of FMDV VP1 protein modified with unnatural amino acids;

[0039] Figure 5 Photocrosslinking of FMDV VP1 protein modified with unnatural amino acids to identify interacting protein complexes;

[0040] Figure 6 Schematic diagram of the construction of recombinant PTC-FMDV mutants based on the VP1 mutation site using PTC technology. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical scheme and advantages of the present invention clearer, the embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. However, it will be appreciated by those skilled in the art that in the embodiments of the present invention, many technical details are provided to enable the reader to better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical scheme claimed in the present application can be implemented.

[0042] Description and explanation of relevant terms in the present invention:

[0043] The term "bioorthogonal translation system" refers to an engineered orthogonal aminoacyl-tRNA synthetase (aa-tRNAsynthetase, aaRS) / tRNA pair, in which the aaRS can specifically aminoacylate the homologous tRNA with a specific UAA, which is dedicated to decoding a specific codon (usually the stop codon UAG), and then the tRNA is recognized by the cellular ribosome. CUA , allowing site-specific incorporation of specific codons corresponding to UAA into synthetic peptides during translation.

[0044] The term "vector" refers to a nucleic acid carrier into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can transform, transduce or transfect the host cell so that the genetic material elements it carries can be expressed in the host cell. For example, vectors include: plasmids; bacteriophages; cosmids, etc.

[0045] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.

[0046] Biological materials: PK-15 cells and HEK-293 cells were purchased from ATCC, and the plasmid expressing tRNA synthetase / tRNA orthogonal pair (addgene: #91706) and pCMV-VP1-Flag expression plasmid were stored in our laboratory. Escherichia coli competent cells DH5α were purchased from Beijing Quanshijin Biotechnology Co., Ltd. Gene synthesis was completed by Beijing Liuhe BGI Gene Technology Co., Ltd. Primer synthesis and DNA sequencing were completed by Yangling Tianrun Aoke Biotechnology Co., Ltd.

[0047] Main reagents: Puromycin, neomycin (G418), and low melting point agarose were purchased from Ameresco; unnatural amino acid NAEK (CAS: 1167421-25-1) was purchased from Shanghai Zhezhi Biological Company; DiZPK (CAS No: 1337883-32-5) was purchased from MCE Company; DIBO-Alexa 488, Trizol reagent was purchased from Invitrogen; Viral RNA Kit extraction kit and plasmid mini-extraction kit were purchased from OMEGA; plasmid midi extraction kit was purchased from MN (Macherey-nagel) in Germany; agarose gel recovery kit was purchased from Promega; Q5 ultra-fidelity DNA polymerase, endonuclease, and HiFi DNA Assembly Master Mix was purchased from NEB; One step PrimeScript RT-PCR Kit (Perfect Real Time), DNA ladder, and T4 DNA ligase were purchased from TAKARA; DMEM, MEM cell culture medium, 0.25% trypsin, and fetal bovine serum (FBS) were purchased from Gbico; and JetPRIME cell transfection reagent was purchased from Polyplus-transfection, France.

[0048] The unnatural amino acid (UAA) described in the following examples is selected from NAEK or DiZPK.

[0049] Example 1 Selection of VP1 protein mutation site and construction of mutant expression plasmid

[0050] 1. Selection of VP1 protein mutation sites

[0051] According to the 3D structure analysis, amino acid conservation analysis and mutation site selection principle of VP1 protein, the amino acid residues close to the N-terminus of the amino acid sequence are preferentially selected. A suitable amino acid site is selected in the amino acid sequence encoded by VP1 protein, and the amino acid codon of the mutation site is mutated to a TAG codon to construct a VP1 protein mutant expression plasmid.

[0052] 2. Construction of mutant expression plasmid

[0053] According to standard molecular cloning techniques, TAG-VP1 mutant plasmids were constructed, and the codons of amino acids at positions T43, H59, R67, K81, D85, K96, L98, Y107, P111, E133, R135, R138, P142, R145, L148, and L151 in the VP1 coding gene sequence were point mutated to TAG. The TAG-VP1 mutants were named: T43TAG, H59TAG, R67TAG, G, K81TAG, D85TAG, K96TAG, L98TAG, Y107TAG, P111TAG, E133TAG, R135TAG, R138TAG, P142TAG, R145TAG, L148TAG and L151TAG; primers for constructing each mutant were designed respectively (as shown in Table 1), and the pCMV-VP1-Flag expression plasmid was used as a template to perform site-directed mutagenesis on the corresponding amino acid sites, and the codons of the selected amino acid sites were mutated to TAG. One-step point mutation PCR was performed to amplify the target plasmid, and TAG codons were introduced at the mutation site by PCR. The operation was performed according to the instructions of the kit. The PCR reaction system and reaction procedure are shown in Table 2-3, and the specific operation steps are as follows.

[0054] 1) Prepare the PCR reaction system according to the table below and perform PCR amplification of the target fragment.

[0055] Table 1 Primer sequence list for constructing TAGA-FMDV mutants

[0056]

[0057]

[0058] Table 2 PCR reaction system

[0059]

[0060] Table 3 PCR reaction program

[0061]

[0062] 2) After the PCR reaction was completed, 1 μL of Dpn I enzyme, 2 μL of 10× Buffer, 17 μL of H2O, and a total volume of 20 μL were added to each tube of sample and digested at 37°C for 30 min.

[0063] 3) Enzyme digestion of the vector plasmid (select appropriate endonuclease sites for double enzyme digestion based on the mutation site).

[0064] 4) 1% agarose gel electrophoresis, gel excision, recovery and purification of DNA fragments.

[0065] 5) Homologous recombination connection: according to HiFi DNA Assembly Master Mix Kit Instructions. Reaction system: 10ul purified DNA + 10ul HiFi DNA Assembly Master; reaction conditions: 50℃, 15-30min.

[0066] 6) The ligation product was transformed into E. coli competent cells Trans5α and cultured overnight in a 37°C incubator for 14 to 16 hours.

[0067] 7) Pick a single clone from the LB plate, shake the bacteria, and send the plasmid to the company for sequencing.

[0068] 8) After the correctly sequenced plasmid is transformed, the bacteria are inoculated into LB medium and the plasmid is extracted (endotoxin-free) after shaking, and then stored at -20°C.

[0069] In order to efficiently screen for efficient insertion sites of non-natural amino acids and design VP1 protein mutant expression plasmids, we analyzed the 3D structure and amino acid sequence conservation of VP1 protein. Combining the selection principle of mutation sites, we selected 16 amino acid sites and constructed mutant expression plasmids respectively. The 3D structure of VP1 protein is shown in Figure 1 , where the red marks are amino acid residues T43, H59, R67, and K81; the results of the amino acid sequence conservation analysis of VP1 protein are shown in Figure 2 .

[0070] Example 2 Selection of sites for site-specific modification of VP1 protein with non-natural amino acids

[0071] 1. Transfection of cells with VP1 mutant expression plasmid

[0072] 1) Prepare HEK293 cells at 5×10 5The cells were plated at a density of 1.54 cells / mL in 6-well cell culture plates.

[0073] 2) The cells were cultured at 37°C and 5% CO2. When the cell confluence reached 70-80%, the transfection complex was prepared.

[0074] 3) Follow the instructions of the JetPRIME transfection kit, which is briefly described as follows: add 200 μL of JetPRIME Buffer to a 1.5 mL EP tube, then add 2 μg of the TAG-VP1 mutant plasmid and the plasmid expressing the tRNA synthetase / tRNA orthogonal pair, mix well, add 8 μL of transfection reagent, and mix well again.

[0075] 4) Let stand at room temperature for 10 min and then add dropwise to the cell wells.

[0076] 5) 6 h after transfection, the medium was replaced with fresh DMEM complete medium containing 10% FBS and supplemented with 1 mM UAA (+UAA). The control group did not add UAA (–UAA).

[0077] 6) 24 h after transfection, cells were harvested and protein expression was detected by Western blotting.

[0078] 2. Western blotting:

[0079] 1) 24 hours after transfection, discard the cell culture medium, wash the cells once with PBS, centrifuge at 12000×g for 10 min, and discard the supernatant.

[0080] 2) Lyse cells: add protein lysis buffer, incubate on ice for 30 minutes, and collect the protein lysis buffer.

[0081] 3) Take the supernatant after centrifugation, add 5× Loading Buffer, mix well, and cook at 100℃ for 10 minutes.

[0082] 4) The protein samples were separated by SDS-PAGE electrophoresis, and then the proteins were electro-transferred to the NC membrane.

[0083] 5) Place the transferred NC membrane in blocking solution and block at room temperature for 1 to 2 hours.

[0084] 6) Dilute the primary antibody to an appropriate concentration with TBST, transfer the blocked NC membrane into the primary antibody solution, and incubate overnight at 4°C.

[0085] 7) Wash the membrane three times with TBST at room temperature, 10 min each time.

[0086] 8) Dilute the secondary antibody to an appropriate concentration with TBST, transfer the NC membrane into the secondary antibody solution, and incubate at room temperature for 1 h.

[0087] 9) Wash the membrane three times with TBST at room temperature, 10 min each time.

[0088] 10) Take out the NC membrane, add ECL luminescent solution, mix well, and stand at room temperature away from light for 1 to 2 minutes. Use the BIO-RAD protein imaging system to scan the protein bands and save them.

[0089] The results showed that the constructed 16 VP1 mutant expression plasmids were transfected into cells, and the unnatural amino acid group (+UAA) and the unnatural amino acid group (–UAA) were set up respectively. The Western blotting results showed that each VP1 mutant +UAA group could express normally, but could not express normally in –UAA group ( Figure 3 ), indicating that non-natural amino acids can be inserted into each site, and the insertion efficiency of each site is different. There are differences in the protein expression levels of the 16 selected mutation sites, especially at mutation sites such as H59, K81, D85, K96, L98, Y107, P111 and R135. The expression level of VP1 protein is equivalent to that of WT, while the protein expression level after mutation at E133 site is the lowest ( Figure 3 The results show that the addition of UAA can achieve conditional control of protein expression; on the other hand, it shows that the site described in the present application is an efficient insertion site for non-natural amino acids and does not affect the expression level of VP1 protein.

[0090] Example 3 Intracellular Fluorescence Labeling of VP1 Protein Expression by Non-natural Amino Acid Site-directed Modification

[0091] The VP1 mutant expression plasmid (taking P111TAG as an example) with the efficient insertion site selected in the results of Example 2 above was co-transfected into cells with the plasmid expressing the tRNA synthetase / tRNA orthogonal pair. 48 hours after cell transfection, the cells were washed twice with 1% FBSDPBS. A certain concentration of DIBO-Alexa fluor-488 was dissolved in 1% FBSDPBS and stained at room temperature in the dark for 1 hour. Washed 3 times with 1% FBSDPBS, then stained the cell nucleus with DIPA, and then washed 3 times with 1% FBSDPBS, observed with a fluorescence microscope and photographed.

[0092] In addition, the transfected cells were fixed with 4% paraformaldehyde at room temperature for 10-15 minutes, and then rinsed with PBS at room temperature for 3 times, 5 minutes each time. Permeabilization was performed with 0.5% Triton-X 100 / PBS at room temperature, and then rinsed with PBS for 3 times, 5 minutes each time. 10% BSA / PBS, blocked at 37°C for 30 minutes. VP1 antibody was diluted with primary antibody diluent, 100uL was added to a glass-bottomed dish, sealed in a wet box, and incubated at 37°C for 30 minutes. After rinsing with PBS 3 times, 100uL of the diluted Alexa Fluor 594-labeled secondary antibody was added, sealed in a wet box, and incubated at 37°C for 30 minutes. After rinsing with PBS and deionized water, the sealing agent was dropped into the wells of the glass-bottomed dish, and observed and photographed with a fluorescence microscope.

[0093] The results showed that after transfecting cells with the VP1 mutant expression plasmid P111TAG, adding DIBO-Alexa fluor-488 to living cells could specifically label the expression of VP1 protein in cells ( Figure 4 ). In addition, it can be observed that the VP1 protein fluorescently labeled with unnatural amino acids combined with DIBO-Alexa fluor-488 co-localizes with the VP1 protein labeled with indirect immunofluorescence, indicating that this technology can successfully achieve fluorescent labeling of proteins in living cells.

[0094] In this example, P111TAG is used as an example to perform intracellular fluorescent labeling of VP1 protein expression modified with non-natural amino acids. However, based on Example 2, other mutation sites described in this application, such as T43, H59, R67, K81, D85, K96, L98, Y107, R135, R138, P142, R145, L148 and L151, can achieve the same intracellular fluorescent labeling effect.

[0095] Example 4 Identification of protein complexes interacting with VP1 protein modified by non-natural amino acids

[0096] As in Example 2 above, the VP1 mutant expression plasmid and the plasmid expressing the tRNA synthetase / tRNA orthogonal pair were co-transfected into HEK293 cells. 24 hours after cell transfection, the culture medium was removed and the cells were rinsed once with PBS. For the cells in the illumination group, they were placed on ice and irradiated with UV365nm. After 10 minutes of 365nm ultraviolet light irradiation, the photo-crosslinking reaction of DiZPK was fully completed. The covalently cross-linked VP1-protein complex band was identified by Western blotting experiments, and the protein complex was further affinity purified, and LC-MS / MS analysis was performed to identify the unknown interacting proteins.

[0097] The results showed that after expressing DiZPK-tagged VP1 protein in cells for a period of time, ultraviolet UV365nm irradiation was used to induce the photo-crosslinking reaction of DiZPK, forming a covalent bond between the DiZPK-tagged VP1 protein and its interacting protein. The formed photo-crosslinked complex was detected by protein immunoblotting ( Figure 5 ). It was also found that the molecular weight of the photo-crosslinked complexes ranged from 60 kDa to 170 kDa, among which the photo-crosslinked complexes obtained by the VP1 proteins with mutation sites at H59, R67 and K81 were the most obvious, while the photo-crosslinked complexes captured by the VP1 proteins with mutation sites at T43, D85 and K96 were mainly around 60 kDa, indicating that there are differences in the protein complexes that can be obtained by inserting non-natural amino acids at different sites.

[0098] The above results show that the mutation sites T43, H59, R67, K81, D85, and K96 described in the present application can be used to capture protein complexes after introducing non-natural amino acids, and can be used to identify protein complexes that interact with VP1 protein. Among them, the schematic diagram of constructing recombinant PTC-FMDV mutants based on VP1 mutation sites using PTC technology is shown in the figure below. Figure 6 shown.

[0099] In summary, the present application found that by screening out efficient insertion sites of non-natural amino acids in the coding region of the VP1 protein whose amino acid sequence is shown in SEQ ID NO.1, the site-specific modification of FMDV VP1 protein with non-natural amino acids and conditional control of VP1 protein expression in cells were achieved, and it can also be used to prepare PTC recombinant FMDV, which is helpful for the development of viral vaccines; the method of site-specific modification of proteins with non-natural amino acids achieves intracellular fluorescent labeling of VP1 protein; and the site-specific modification of non-natural amino acids by photocrosslinking can be used to screen and identify protein complexes that interact with VP1 protein.

Claims

1. A foot-and-mouth disease virus VP1 protein modified with a non-natural amino acid, characterized in that: The amino acid at position K82 of the foot-and-mouth disease virus VP1 protein whose amino acid sequence is shown in SEQ ID NO.1 is mutated to the non-natural amino acid DiZPK.

2. The method for preparing the foot-and-mouth disease virus VP1 protein modified by non-natural amino acids as claimed in claim 1, characterized in that: The method comprises: (1) based on the nucleic acid molecule encoding the foot-and-mouth disease virus VP1 protein with an amino acid sequence as shown in SEQ ID NO.1, the codon encoding the K82 amino acid is mutated to the amber codon TAG to obtain a gene sequence encoding the foot-and-mouth disease virus VP1 protein with a site-specific modification of a non-natural amino acid; (2) operably linking the gene sequence described in step (1) to a vector to obtain a mutant sequence expression plasmid; (3) co-transfecting the mutant sequence expression plasmid and the orthogonal translation system expression plasmid into host cells, inducing the culture with unnatural amino acids, and screening to obtain the foot-and-mouth disease virus VP1 protein modified with unnatural amino acids; The orthogonal translation system expression plasmid comprises: Orthogonal tRNA / aminoacyl-tRNA synthetases; The tRNA is derived from a prokaryotic organism; The anticodon loop of the tRNA corresponds to the premature stop codon UAG; The aminoacyl-tRNA synthetase is capable of binding an unnatural amino acid to the tRNA; The tRNA and aminoacyl-tRNA synthetase are located on the same carrier.

3. Use of the foot-and-mouth disease virus VP1 protein modified with a non-natural amino acid as claimed in claim 1 for conditionally controlling the expression of the foot-and-mouth disease virus VP1 protein in vitro.

4. Use of the foot-and-mouth disease virus VP1 protein modified with a non-natural amino acid as claimed in claim 1 in screening proteins that interact with the foot-and-mouth disease virus VP1 protein.

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