A naturally occurring fusion protein in the form of TA system SO_1440 gene and its application in the control of eukaryotic cell virus infection

Abstract

The present invention discloses a naturally occurring fusion protein TA system SO_1440 gene and its application in the control of eukaryotic cell virus infection. The fusion protein SO_1440 is derived from marine Shewanella Shewanella oneidensis . The present invention found that: under normal circumstances, the toxin domain of the fusion protein SO_1440 is in a silent state. However, when the antitoxin domain is removed, the toxin domain is activated and exerts its toxicity by cutting intracellular RNA. The antitoxin domain can be further truncated, and when the N-terminal 79 amino acids are truncated, it can still silence the toxin by interfering with the dimerization of the toxin domain. At the same time, the linker region of the antitoxin and the toxin can be replaced with the recognition sequences of different proteases, which still does not affect the silencing of the toxin by the antitoxin region, suggesting that the fusion protein in its natural state has certain application prospects in the control of eukaryotic cell virus infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and biotechnology, and specifically relates to a toxin-antitoxin coding gene in the form of a naturally occurring fusion protein and its application in controlling eukaryotic cell infection. Background Art

[0002] In the past few years, efforts have been focused on the discovery and development of antiviral drugs targeting key steps in the viral replication cycle, including viral entry, RNA translation and post-transcriptional processing, reverse transcription, genome integration, viral assembly and release. A key step in the replication cycle of many viruses is the processing of polypeptide precursors by virally encoded proteases to achieve viral infection, such as yellow fever virus (YFV), retroviruses (such as HIV-1), hepatitis C virus (HCV), flavivirus (DFV), West Nile virus (WNV) and dengue virus (DFV). Based on this characteristic, it is of great significance to develop effective methods to eliminate virus-infected cells and achieve control of viral infection by targeting virus-specific proteases.

[0003] The toxin-antitoxin system is a binary system widely distributed in the genomes of bacteria and archaea. It consists of adjacent toxin genes and antitoxin genes, one of which encodes a stable toxin protein (Toxin) and the other encodes an unstable antitoxin molecule (Antitoxin). The main physiological function of the toxin is to function as an RNA endonuclease, which cuts intracellular RNA in an activated state to inhibit cell growth or even cause cell death. It has been confirmed that toxin proteins can also function by cutting RNA molecules in eukaryotic cells. This result raises an interesting possibility that mRNA interferase toxins from bacteria can be used as gene therapy tools to fight cancer, AIDS or other human diseases. Recently, a novel method has been reported, which artificially constructs a fusion protein of the toxin-antitoxin system MazE-MazF (Jung-Ho Park, Yoshihiro Yamaguchi, et al. Intramolecular regulation of thesequence-specific mRNA interferase activity of MazF fused to a MazE fragmentwith a linker cleavable by specific proteases[J]. Appl Environ Microbiol.2012 Jun;78(11):3794-9. ), adds a recognition site for a specific viral protease at the junction of the protein, and activates the toxin to eliminate the infected cells when the virus infects the cells, which may treat and prevent HIV-1 and hepatitis C virus (HCV) infection. The study also demonstrated that the use of MazF-based enzyme toxins to treat mixed cultures of healthy cells and HCV-infected cells has a "curative effect", leading to specific eradication of infected cell populations. However, the current method of artificially constructing toxin-antitoxin fusion proteins has certain risks. Whether the toxin can remain silent before viral infection and whether the toxic release of the toxin can be achieved after the toxin infection requires careful design and verification. Therefore, very few families of TA systems can clear virally infected tissues and cells by constructing fusion proteins. The present invention discovered a type of naturally occurring TA system in the form of a fusion protein from marine Shewanella, that is, the toxin and antitoxin are encoded in the same protein and connected by a shorter linker. It has been confirmed that the naturally occurring fusion protein has stable structure and physicochemical properties, and the linker region between the toxin and the antitoxin can be replaced with the recognition sequence of a variety of viral proteases. This provides more possibilities for TA in the form of fusion proteins to control viral infections in eukaryotic cells. Summary of the invention

[0004] The object of the present invention is to provide a toxin-antitoxin encoding gene in the form of a naturally occurring fusion protein SO_1440 The nucleotide sequence is shown in SEQ ID NO. 1. The protein SO_1440 encoded by the gene has an amino acid sequence shown in SEQ ID NO. 2.

[0005] The present invention is derived from marine Shewanella Shewanella oneidensis A fusion toxin-antitoxin gene was developed SO_1440 , a fusion protein SO_1440 Recombinant gene expression vectors and genetically engineered bacteria.

[0006] Therefore, the present invention also provides biological materials containing the coding gene of the above-mentioned toxin-antitoxin fusion protein SO_1440.

[0007] Preferably, the biological material is an expression cassette, a recombinant plasmid vector, a host cell or a genetically engineered bacterium.

[0008] Preferably, the host bacteria of the genetically engineered bacteria is Escherichia coli.

[0009] The present invention also provides a strategy for constructing a truncated mutant of the antitoxin region of the fusion protein SO_1440, which is more effective in releasing the toxin during virus infection.

[0010] The present invention also provides the addition of linker regions recognized by different viral proteases and the detection of the effects of toxin toxicity under normal conditions to ensure its safety and effectiveness.

[0011] On the other hand, the present invention also provides the use of the above-mentioned toxin-antitoxin fusion protein SO_1440 or its encoding gene, or a biological material containing its encoding gene in the preparation of drugs for anti-viral infection.

[0012] On the other hand, the present invention also provides a drug for antiviral infection, which contains the above-mentioned toxin-antitoxin fusion protein SO_1440.

[0013] The fusion protein SO_1440 of the present invention is derived from marine Shewanella Shewanella oneidensis. The present invention discovered through molecular cloning, activity verification and other methods that the toxin domain of the fusion protein SO_1440 is in a silent state under normal circumstances. However, when the antitoxin domain is removed, the toxin domain is activated, and exerts its toxicity by cutting intracellular RNA. Experimental verification has found that the antitoxin domain can be further truncated, and when the N-terminal 79 amino acids are truncated, the toxin can still be silenced by interfering with the dimerization of the toxin domain. At the same time, the linker region of the antitoxin and the toxin can be replaced with the recognition sequences of different proteases, which still does not affect the silencing of the toxin by the antitoxin region, suggesting that the fusion protein in its natural state has certain application prospects in the control of eukaryotic cell virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the domain composition of the fusion protein SO_1440 and the toxicity identification diagram of each domain.

[0015] Figure 2 This is a diagram showing the in vitro RNA cleavage activity of the fusion protein SO_1440 and the toxin domain (HEPN).

[0016] Figure 3 This is a diagram showing the silencing effect of the truncated antitoxin domain (MNT) on the toxin.

[0017] Figure 4 This is a graph showing the results of truncated antitoxin being able to silence the toxin.

[0018] Figure 5 This is a stability (toxicity) test chart after the linker region of the fusion protein SO_1440 was replaced with different viral protease recognition sequences. DETAILED DESCRIPTION

[0019] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0020] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the reagents and materials used are all commercially available unless otherwise specified.

[0021] Example 1: Cloning and vector construction of fusion protein SO_1440

[0022] 1. Construction of pHGE recombinant plasmid carrying antitoxin domain, toxin domain and full-length fusion protein

[0023] The fusion protein form of MNT-HEPN is a unique evolutionary form of the TA system. First, the cytotoxicity of each domain of the fusion protein SO_1440 (amino acid sequence as shown in SEQ ID NO.2) was tested ( Figure 1pHGE plasmid (source document "Shi, M., Wu, L., Xia, Y., Chen, H., Luo, Q., Sun, L., and Gao, H. (2013) Exoprotein production correlates with morphotype changes of non-motile Shewanella oneidensis mutants. J Bacteriol 195: 1463-1474. ") is a commonly used plasmid for identifying the toxicity of toxins and antitoxins. The wild-type Shewanella oneidensis MR-1 genomic DNA was extracted using the Tiangen kit as a template for subsequent PCR cloning; PCR amplification was designed separately. SO_1440 The primers for the gene (whose nucleotide sequence is shown in SEQ ID NO.1) and the antitoxin domain (MNT) and the toxin domain (HEPN) are as follows:

[0024] SO_1440-MNT-F: CCG GAATTC TTGTGGCTGTATGGTTCGCAGGC; (SEQ ID NO.3, the underline indicates the restriction site);

[0025] SO_1440-MNT-R: CGC GGATCC TTAAATAATAAAGTGGTTTTGGGC; (SEQ ID NO. 4, the underline indicates the restriction site);

[0026] SO_1440-HEPN-F: CCG GAATTC TTGGATAATGCCTATATCACGTC; (SEQ ID NO.5, the underline indicates the restriction site);

[0027] SO_1440-HEPN-R: CGC GGATCC TTAGTCACTATTCAGTGATAAGC; (SEQ ID NO.6, the underline indicates the restriction site);

[0028] Using SO_1440-MNT-F and SO_1440-MNT-R as primers and MR-1 genomic DNA as template, PCR was used to amplify the MNT domain of the fusion protein SO_1440; using SO_1440-HEPN-F and SO_1440-HEPN-R as primers, PCR was used to amplify the HEPN domain of the fusion protein SO_1440; using SO_1440-MNT-F and SO_1440-HEPN-R as primers, PCR was used to amplify the full length of the fusion protein SO_1440; PCR amplification conditions were: 95℃ for 5 min; then 95℃ for 30 s, 56℃ for 30 s, 72℃ for 30 s, for a total of 30 cycles; and finally 72℃ for 10 min. After the reaction was completed, the PCR amplification products were subjected to 1.0% agarose gel electrophoresis to recover and purify the target gene fragment. EcoRI and B H The vector pHGE was digested with endonuclease, and the double-digested vector was purified by 1.0% agarose gel electrophoresis and gel recovery. The recombinant product of the fragment and the vector was transferred into Escherichia coli BW25113 competent cells by homologous recombination, and positive transformants were screened, PCR verified, and sent to a sequencing company for sequencing to obtain the recombinant plasmid.

[0029] >SO_1440 (SEQ ID NO.1)

[0030] ATGACTATCGAGCAGAGCCAGCATTTGCACCAACTAAACGCTTTAGCTGCAAGCCCTCCTGAGGTGGCGGTCTTGTGGCTGTATGGTTCGCAGGCGAAAGGAAATGCTAGCGAGCATAGCGATTGGGATTTAGCCGTCGCATTCGACCCGGTAAAGGCGGACAGTGTGCTTGATACTCGTATTAGAGCTGAACTGCTTGCTATGGATTGGCAAAGGGCATTGGGGCTAGCCGAAGGAAAGCTGTCTGTTGTCGATATTAATCTAGCACCTATCCCTTTAGCCTTCGGCATCATTAATGCCAACAAACTAATCTTTAGTCGAGATGAAGGTCGAAGAATGCAAGAAGAGTCACGCATTATGTCGCAAATGGAACTCGATTATTCAGCCCAAAACCACTTTATTATTTTGGATAATGCCTATATCACGTCATTGCGCTTAAGCCTTAGCCGGTATCAAGCCGAGCTTGGGGAATTGCGTCAATTACTCTGTCAGCGTCCACTATCGAATCTAGAAGAACGCGCCGCAGAGAGGACGTTACAGGTCTCTATTGAAGCTTGTATTGGCATTGCCAAGCATTGGGCTAAAGCACTGGCTGGACATAGCCCTCAGGATGCCTACCAAGCATTTGAGATCCTGTGTCAACGAGGAGCACAACCAGCTGATGAATTAAATGGCTGGCGTAAAGTGATAGGCCTGCGTAATGCACTGGTACATGATTATCTGAATATTGACCCTGAGATCATCCGCAGTGTGATCACACAAGGTTACAGCGATAGATTATTTATCTTTGCAGAGCAGGGTTTAGAGTGCTTATCACTGAATAGTGACTAA。

[0031] >SO_1440(SEQ ID NO.2)

[0032] MTIEQSQHLHQLNALAASPPEVAVLWLYGSQAKGNASEHSDWDLAVAFDPVKADSVLDTRIRAELLAMDWQRALGLAEGKLSVVDINLAPIPLAFGIINANKLIFSRDEGRRMQEESRIMSQMELDYSAQNHFIILDN AYITSLRLSLSRYQAELGELRQLLCQRPLSNLEERAAERTLQVSIEACIGIAKHWAKALAGHSPQDAYQAFEILCQRGAQPADELNGWRKVIGLRNALVHDYLNIDPEIIRSVITQGYSDRLFIFAEQGLECLSLNSD.

[0033] 2. Construction of pET28b recombinant plasmid carrying toxin domain and full-length fusion protein

[0034] pET28b plasmid is often used for the expression and purification of target proteins in cells. This plasmid was used to construct SO_1440 and a separate toxin domain by restriction enzyme ligation. NcoI and HindIII The recovered fragments were double digested with endonucleases, and the digested products were subjected to 1.0% agarose gel electrophoresis and gel recovery and purification. NcoI and HindIII The enzyme-digested vector pET28b was ligated with T4 DNA ligase (TaKaRa), and the ligation product was transformed into Escherichia coli BL21 (DE3) competent cells. Positive transformants were screened, PCR verified, and sent to a sequencing company for sequencing to obtain recombinant plasmids pET28b-SO_1440-Chis and pET28b-HEPN-Chis and the target strain containing the recombinant plasmid.

[0035] Example 2: Activity verification of fusion protein SO_1440 and toxin domain

[0036] 1. Cytotoxicity detection of each domain of fusion protein

[0037] First, the cytotoxicity of the pHGE recombinant plasmid carrying the MNT antitoxin domain, the HEPN toxin domain, and the full-length fusion protein SO_1440 was determined. The recombinant strain cultured overnight was transferred, the initial OD was adjusted to 0.1, 0.5 mM IPTG was added as an inducer, and the cells were collected after 2 hours of induction for gradient dilution and plated. The results showed that the expression of the MNT antitoxin domain and the full-length fusion protein SO_1440 alone was non-toxic to the cells, while the expression of the HEPN toxin domain alone had obvious cytotoxicity ( Figure 1The above results suggest that under certain activation conditions, the fusion protein can release the toxin domain to exert cytotoxic effects.

[0038] 2. Expression and purification of fusion protein and toxin domain

[0039] The recombinant plasmids pET28b-SO_1440-Chis and pET28b-HEPN-Chis constructed by the above method were used to purify the fusion protein and the toxin domain HEPN in the BL21 (DE3) host. 400 mL of the recombinant strain was cultured until OD 600 The concentration of the inducing agent IPTG was about 0.8, and the final concentration was 0.5 mM. The inducing agent IPTG was added and cultured at 37℃ for 2-3 h. The cells were collected by centrifugation at 8000 rpm and 4℃ for 10 min, and the cells were resuspended with 30 mL (50 mM, pH 7.4) PBS buffer. Ultrasonication was performed at 400 w for 5 s, stopped for 5 s, and broken for 10 min. The supernatant was centrifuged and the supernatant was collected. The collected supernatant was purified by nickel ion affinity chromatography. The specific implementation plan is as follows: 10 mM imidazole was used for elution for 5 column volumes, 20 mM imidazole was used for elution for 20-30 column volumes, 50 mM imidazole was used for elution for 1 column volume, and finally 2.5 mL 300 mM imidazole was used for elution. The last 2.5 mL eluate was collected. Desalting was performed with a desalting column SephadexG25. The specific operation method was carried out according to the operation manual of GE. The purified expression products were subjected to SDS-PAGE gel electrophoresis to obtain purified SO_1440 protein and HEPN domain protein ( Figure 1 (c) The protein sizes are approximately 30 kDa and 15 kDa, respectively, which is consistent with theoretical expectations.

[0040] 3. In vitro activity detection of fusion proteins and toxin domains

[0041] HEPN family toxins mainly exert cytotoxicity through RNA cleavage. In order to ensure the accuracy of the experimental results, the RNase activity of the full-length fusion protein and the single toxin domain was compared in this study. First, the concentrations of the two proteins were measured using the Bio-Tech BSA protein quantification kit, and the mother liquor concentrations of both were adjusted to 0.3 mg / mL for later use. ompA The first 300 bp of the gene (Gene ID: 945571) was used as a template and transcribed using an in vitro transcription kit to obtain RNA substrate.

[0042] hydrolysis ompAThe reaction conditions of the mRNA substrate are as follows: ① The two proteins were diluted in a gradient to 1, 5, 20, 75, 300 μg / mL, respectively, and the dilution buffer was 50 mM Tris-HCl, pH 7.5, 10 M MgCl2. ② 1 μL of ribonuclease SiRe_0917 was added to the 10 μL reaction system, the concentration of the nucleic acid substrate was 50 nM, and the mixed system was reacted at 60℃ for 15 minutes. ③ Detect the hydrolysis of the substrate: 2 μL of nucleic acid loading was added to the 10 μL reaction system, 15% urea gel, 110V voltage was used for 60 minutes, and the gel was illuminated after EB staining.

[0043] Figure 2 The results showed that the full-length fusion protein had no RNA cleavage activity, while the HEPN protein expressed alone had significant RNA cleavage activity. Similar to the toxin MazF, the HEPN domain exerts cytotoxicity by cleaving mRNA, indicating that HEPN can also function when activated in eukaryotic cells.

[0044] Example 3: Function and stability testing of fusion protein truncations

[0045] In order to ensure that the toxin domain can fully release the toxicity after being separated from the antitoxin domain, it is first necessary to ensure that the antitoxin domain cannot antagonize the toxicity of the toxin after being separated from the toxin domain. We constructed a dual-plasmid system to split the antitoxin domain (pHGE-MNT) and the toxin domain (pBAD-HEPN). Through cytotoxicity detection, it was found that the antitoxin could not neutralize the toxicity of the toxin after separation, and could release the toxicity of the toxin very well. Furthermore, we gradually truncated the N-terminal MNT antitoxin domain of the fusion protein SO_1440, hoping to reduce the unnecessary effects of the antitoxin on the cell after being removed. Using the pHGE recombinant plasmid construction method mentioned above, the MNT domain was truncated and the silencing of the toxin in the body at different stages was tested. The results showed that the 1-83 amino acids of the truncated MNT domain can still antagonize the antitoxin toxicity ( Figure 3 Using density gradient centrifugation, it was found that the truncated MNT C-terminal domain silences the toxin mainly by interfering with the dimerization of the toxin ( Figure 4 ).

[0046] Example 4: Stability test after fusion protein linker replacement

[0047] In the natural fusion protein SO_1440, the MNT domain and the HEPN domain are connected by a shorter linker. In order to verify whether this fusion protein can be used to prevent and treat viral infections in eukaryotic cells, we first replaced the linker region with the protease recognition sequences of several common viruses. This ensures that the linker region is cut when the virus infects the cell, and the toxin and antitoxin domains are separated, thereby achieving directional activation of the toxin during viral infection. In order to test whether the toxin is silent under normal conditions after replacing the protein linker region, we used the pHGE vector to construct SO_1440 with different protein linkers replaced. Cytotoxicity tests found that SO_1440 can still remain in an inactive state under normal conditions after introducing linkers recognized by different eukaryotic virus proteases. It can be seen that its safety and stability have natural advantages over artificial TA fusion proteins ( Figure 5 ).

[0048] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering and technical personnel in the field should fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of toxin-antitoxin fusion protein SO_1440 or biological materials containing its encoding gene in the preparation of drugs for controlling eukaryotic cell virus infection; the amino acid sequence of the toxin-antitoxin fusion protein SO_1440 is the amino acids shown at positions 84 to 276 of the sequence shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The biological material is an expression box, a recombinant plasmid vector, a host cell or a genetically engineered bacterium.

3. The use according to claim 2, characterized in that: The host bacteria of the genetically engineered bacteria is Escherichia coli.

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