Application of high-efficiency heat-stable ribonuclease SiRe_0902 in specific recognition of GG site

Abstract

The application discloses application of a high-efficiency and heat-stable ribozyme SiRe_0902 in specific recognition of a GG site. The amino acid sequence of the ribozyme SiRe_0902 is shown as SEQ ID NO. 2, and the nucleotide sequence is shown as SEQ ID NO. 1. The ribozyme SiRe_0902 has very high and specific hydrolysis activity on RNA, and has no non-specific cutting on DNA. In addition, the enzyme is derived from a thermophilic archaea, and is detected to have good heat stability and very good RNA cutting activity under conditions of 37 DEG C and 65 DEG C. The enzyme is detected to be capable of specifically recognizing and cutting a GG sequence, and also has high cutting activity on a GG sequence in a secondary structure region. Therefore, the ribozyme SiRe_0902 is a new heat-stable ribozyme, and has very high RNA hydrolysis activity under normal temperature and high temperature conditions, and meets the needs of clinical, production and molecular cloning fields.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of bio-chemical industry and biotechnology, and particularly relates to application of a high-efficiency and heat-stable ribonuclease SiRe_0902 in specific recognition of a GG site under high-temperature conditions. BACKGROUND

[0002] Ribonuclease (RNase) refers to an enzyme capable of hydrolyzing RNA phosphodiester bonds, and different RNases have different specificity. Currently, commonly used ribonucleases include RNase A, RNase H and RNase T1. Ribonuclease is a protein with globin properties, which is soluble in water and 50% acetone. The optimum temperature of the enzyme is 60℃, and it loses its function above 85℃. The optimum pH value is 7.6. Among them, ribonuclease A (RNase A) is derived from bovine pancreas and is an endoribonuclease that can specifically attack the 3' end of pyrimidine residues on RNA, cut the phosphodiester bond formed by cytosine or uracil and adjacent nucleotides, and the reaction end product is 3' pyrimidine nucleotide and oligonucleotide with a 3' pyrimidine nucleotide at the end. In the absence of auxiliary factors and divalent cations, the action of ribonuclease A can be inhibited by RNasin or vanadyl-ribonucloside complex (VRC). Ribonuclease T1 (RNase T1) is derived from Aspergillus orjzae, which specifically acts on the 3' end of guanine phosphate, and the cleavage site is the phosphodiester bond between the 3' phosphate of guanine and the 5' hydroxyl of adjacent nucleotides. The reaction end product is 3' guanosine and oligonucleotide fragments with a 3' guanosine at the end. Ribonuclease H (RNase H) was first discovered in calf thymus tissue, and its encoding gene has been cloned into E. coli. It can specifically degrade the RNA chain in DNA:RNA hybrid double-stranded DNA, producing oligonucleotides and mononucleotides with 3'-OH and 5'-phosphate ends, and it cannot degrade single-stranded or double-stranded DNA or RNA.

[0003] Ribozymes are widely used in molecular cloning, clinical, food and drug, and other fields. In molecular cloning, ribozymes can be used to remove RNA molecules from DNA: RNA hybrid or DNA preparation, determine the position of single base mutation in RNA or DNA, and detect RNA for RNase protection assay. In addition, ribozymes are reported to change host cell metabolism, inhibit virus synthesis, inhibit influenza virus proliferation in vitro, and inhibit the formation of vaccinia and herpes viruses in chicken embryos. Clinical use of ribozymes at a dose of 180 mg per day by intramuscular injection is beneficial for the treatment of epidemic encephalitis. However, there are limited types of commercially available ribozymes, especially high-temperature-resistant endoribonucleases with sequence recognition sites, and there is a great demand for ribozymes with new functions. SUMMARY

[0004] The present application aims to provide the application of the high-efficiency heat-stable ribozyme SiRe_0902 in specific recognition of GG sites. The ribozyme SiRe_0902 is directly prepared from the thermophilic archaea Sulfolobus islandicus REY15A, and has high efficiency, heat resistance and stability. It can specifically recognize and cut the GG site in the RNA sequence, and can efficiently cut the RNA with secondary structure at 37℃ or 65℃. This has very good application prospects in the analysis of RNA with complex structure. Especially important is that the enzyme structure is stable and still has very good activity after being placed at 4℃ for 1 year, which has the characteristics of developing into a commercial endoribonuclease.

[0005] The first object of the present application is to provide the application of the ribozyme SiRe_0902 in specific recognition and cutting of GG sites, wherein the amino acid sequence of the ribozyme SiRe_0902 is shown in SEQ ID NO. 2.

[0006] Preferably, the nucleotide sequence of the coding gene of the ribozyme SiRe_0902 is shown in SEQ ID NO. 1.

[0007] The ribonuclease gene SiRe_0902 of the present application is from the thermophilic archaea Sulfolobus islandicus REY15A. The present application discovers that the gene SiRe_0902 encodes a high-efficiency heat-stable ribonuclease through molecular cloning, activity verification and other methods. Under normal circumstances, the enzyme is modified and silenced by the nucleic acid-specific enzyme encoded by the downstream gene SiRe_0903. It is found through experiments that the ribonuclease gene SiRe_0902 and the downstream SiRe_0903 have no influence on the growth of the host when they are co-expressed and purified, and there is no stable interaction between them, so a large amount of nucleic acid-modified ribonuclease SiRe_0902 can be purified.

[0008] Preferably, the cleavage GG site is the phosphodiester bond in the middle of the cleavage GG sequence.

[0009] Preferably, the concentration of the ribonuclease SiRe_0902 is 5-10 μg / mL.

[0010] Preferably, the concentration of the ribonuclease SiRe_0902 is 5 μg / mL.

[0011] Preferably, the reaction temperature for specifically recognizing and cleaving the GG site is 60-70℃, and the reaction time is 10-20 min.

[0012] Preferably, the reaction temperature for specifically recognizing and cleaving the GG site is 65℃, and the reaction time is 15 min.

[0013] The second object of the present application is to provide the application of the ribonuclease SiRe_0902 in the hydrolysis of RNA in non-disease diagnosis and treatment, and the amino acid sequence of the ribonuclease SiRe_0902 is shown in SEQ ID NO. 2.

[0014] Preferably, the concentration of the ribonuclease SiRe_0902 is 5-10 μg / mL, the hydrolysis temperature is 60-70℃, and the hydrolysis time is 10-20 min.

[0015] Preferably, the concentration of the ribonuclease SiRe_0902 is 10 μg / mL, the hydrolysis temperature is 60℃, and the hydrolysis time is 15 min.

[0016] The advantages of the present application are:

[0017] The ribonuclease SiRe_0902 provided by the patent has very efficient and specific hydrolysis of RNA activity, and has no non-specific cutting of DNA. In addition, the enzyme is derived from a thermophilic archaea, and has been detected to have good thermal stability, and has very high RNA cutting activity at 37℃ and 65℃. Therefore, the ribonuclease SiRe_0902 is a new heat-stable ribonuclease, which has extremely high RNA hydrolysis activity and substrate sequence dependence under normal temperature and high temperature conditions, meeting the needs of clinical, production and molecular cloning fields.

[0018] The ribonuclease SiRe_0902 can specifically recognize the GG site in the RNA sequence for cutting. At 65℃, when the concentration of the RNA endonuclease SiRe_0902 is 5μg / mL, the cutting of the substrate by the ribonuclease SiRe_0902 within 15min shows complete sequence dependence, and there is no random sequence hydrolysis activity. The SiRe_0902 has the potential to be developed into a ribonuclease product, and can realize the recognition and cutting of the GG sequence under high temperature conditions, and has obvious advantages in opening the RNA structure.

[0019] The thermophilic archaea Sulfolobus islandicus REY15A is a model organism, which is disclosed in the literature: Genome analyses of Icelandic strains of Sulfolobus islandicus, model organisms for genetic and virus-host interaction studies, which is owned by the applicant and is guaranteed to be publicly released to the public for 20 years from the filing date. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 : A is the flowchart for preparing the unmodified active ribonuclease SiRe_0902. B is the SDS-PAGE electrophoresis diagram of the ribonuclease SiRe_0902. In the left electrophoresis diagram, lane 1 is the REY15A crude protein containing pSeSD-SiRe_0902-0903-NHis without L-Ara induction, lane 2 is the total protein of the cells after L-Ara induction, and lane 3 is the purified SiRe_0902 protein (with nucleic acid modification); the right electrophoresis diagram is the change of the molecular weight of the ribonuclease SiRe_0902 before and after PDEs treatment.

[0021] Figure 2 is the effect of reaction time on the activity of the ribonuclease SiRe_0902 (10μg / mL) in cutting the RNA probe and total RNA sample.

[0022] Figure 3The ability of ribonuclease SiRe_0902 (10 μg / mL) to cleave single-stranded DNA probes and double-stranded DNA probes was detected.

[0023] Figure 4 The identification result of the recognition and cleavage sequence of ribonuclease SiRe_0902 (5 μg / mL). DETAILED DESCRIPTION

[0024] The following examples are further illustrations of the present application and are not intended to limit the present application.

[0025] Example 1: Cloning and vector construction of ribonuclease SiRe_0902

[0026] The pSeSD plasmid (published in the literature "A synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus") is a high-copy expression vector for hyperthermophilic archaeon Sulfolobus islandicus REY15A, and is commonly used for the expression and purification of target proteins in cells. The wild-type Sulfolobus islandicus REY15A genomic DNA was extracted by a Tiangen kit as a template for subsequent PCR cloning; the primers for amplifying the SiRe_0902-0903 operon (the SiRe_0902 nucleotide sequence is shown in SEQ ID NO. 1; the SiRe_0903 nucleotide sequence is shown in SEQ ID NO. 3) were designed, and the primer sequences are as follows: the upstream primer SiRe_0902-his-F (pSeSD): 5' GGAATTC CATATG CACCACCACCACCACCACAACTATAAAGAATGGATAGAG 3'; the downstream primer SiRe_0903-R (pSeSD): 5' ACGC GTCGACTTATAGAAATTCCTTTATATCT 3'. The coding region of the SiRe_0902-0903 operon was amplified from the wild type S. islandicus REY15A genomic DNA using the primers SiRe_0902-his-F (pSeSD) and SiRe_0903-R (pSeSD) and Takara's PrimeSTAR enzyme according to the following PCR reaction system and conditions: first 95°C for 5 min; then 95°C for 30 s, 56°C for 30 s, 72°C for 30 s for 30 cycles; finally 72°C for 10 min. After the reaction, the PCR product was subjected to 1.0% agarose gel electrophoresis, and the about 700 bp target gene fragment was recovered and purified. The recovered fragment was treated with Ndel and Sail endonucleases, and the enzyme digestion product was subjected to 1.0% agarose gel electrophoresis and gel recovery and purification. Then it was ligated with the vector pSeSD treated with Ndel and Sail endonucleases using T4 DNA ligase (TaKaRa), and the ligation product was transformed into E. coli BW25113 competent cells, and positive transformants were screened, verified by PCR and sequenced by a sequencing company to obtain the recombinant plasmid pSeSD-SiRe_0902-0903-Nhis and the target strain containing the recombinant plasmid.

[0027] Table 1 PCR reaction system

[0028]

[0029]

[0030] Example 2: Efficient expression of ribonuclease SiRe_0902

[0031] 2.1 Preparation of S. islandicus REY15A competent cells

[0032] 1. One day before preparing the competent cells, 2 mL of S. islandicus REY15A starter culture was inoculated into 100 mL of SCVU (U 10 μg / mL) medium and cultured at 78°C overnight.

[0033] SCVU: 10 μL / mL Uracil (2 mg / mL stock) was added to 1 mL of SCV (Table 3).

[0034] 2. Measure OD 600 Stop the culture when OD 600 It can also be between 0.2 and 0.3), and the culture was transferred to 2 x 50 mL centrifuge tubes at room temperature. The cells were collected by centrifugation at 6000 rpm at room temperature for 10 min. The supernatant was discarded (all the supernatant was sucked out).

[0035] 3. Add 15 mL room temperature sucrose solution (20 mM) per tube, gently pipette to resuspend the cells. Spin at 6000 rpm for 10 min at room temperature, repeat twice.

[0036] 4. Resuspend the cells with room temperature sucrose solution (20 mM) (without vortexing) to make the OD value of the competent cells 5-10. Store the competent cells at room temperature for one week or at -80 °C. 600

[0037] 2.2 Electroporation of the recombinant plasmid of interest into the expression strain REY15A

[0038] 1. Take 0.8 pg (0.5-1 pg) of the recombinant plasmid pSeSD-SiRe_0902-0903-Nhis obtained in Example 1 and mix with 50 pL of competent cells of S. islandicus REY15A, incubate at room temperature for 30 min.

[0039] 2. Transfer 50 pL of the competent cell-DNA suspension into the corresponding electroporation cuvette. The electroporation conditions are: 1.2 KV, 600 W, 25 pF (Bio-Rad Gene pulser II), time constant 12 ms.

[0040] 3. Transfer the transformed cells into a 1.5 mL centrifuge tube containing 800 pL of incubation medium (see Table 2) pre-heated to 75 °C. After incubation at 75 °C for 1 hour (without shaking), remove the sample and place it at room temperature until plating.

[0041] 4. Heat the required 0.4% Gelrite (Sigma, CAS number: 71010-52-1) solution and 2x SCV, mix both in a 1 : 1 volume ratio to obtain a 1x SCV top agar. Add 6 mL of 1x SCV top agar to 50 pL of the electroporated cells, mix and add to the pre-heated plates with the bottom agar SCV, let it set at room temperature for 30 min. Incubate at 75 °C for 7 days and check the transformants;

[0042] Bottom agar: prepare as indicated in Tables 2-4, mix 1.4% Gelrite solution with an equal volume of 2x SCV.

[0043] Top agar: mix 0.4% Gelrite with an equal volume of 2x SCV, both at a final concentration of 0.2% and 1x SCV, respectively.

[0044] 2.3 Protein induced expression

[0045] S. islandicus REY15A expression strain containing pSeSD-SiRe_0902-0903-Nhis was grown in SCV medium to an OD​600 For 0.5, add inducer D-arabinose to a final concentration of 10 mM, and incubate at 75°C for 48 h. Centrifuge 500 mL of bacterial solution at 4500 rpm for 10 min at room temperature to collect the bacterial cells, resuspend the bacterial cells in 30 mL of PBS buffer (50 mM, pH 7.4), and break the cells by ultrasonic treatment at 400 W for 5 s, 10 min, 4°C, and 10000 rpm for 30 min to collect the supernatant.

[0046] 2.4 Protein purification and SDS-PAGE electrophoresis

[0047] The supernatant collected in step 2.3 was purified by a nickel ion affinity chromatography column, and the specific implementation is as follows: 5 column volumes were eluted with 10 mM imidazole, 20-30 column volumes were eluted with 20 mM imidazole, 1 column volume was eluted with 50 mM imidazole, and finally 2.5 mL of 300 mM imidazole was used for elution, and the last 2.5 mL of eluate was collected. Desalting was performed using a desalting column Sephadex G 25, and the specific operation method was performed according to the operation manual of GE Company. The purified expression product was subjected to SDS-PAGE gel electrophoresis, and the purified ribonuclease SiRe_0902 with nucleic acid modification (A) was obtained, with a protein size of about 15 kD, which was consistent with the theoretical expectation. Figure 1 -A), the purified protein size was about 15 kD, which was consistent with the theoretical expectation.

[0048] Table 2 Mineral Salt Solution (pH 3.5)

[0049]

[0050] (Add a solution of water and concentrated sulfuric acid diluted 1:1 to adjust the pH to 3.)

[0051] Table 3 SCV medium

[0052]

[0053] Table 4 Vitamin Mixture 100x

[0054]

[0055]

[0056] (Tock solution in mg / L distilled H2O; filter sterilized.)

[0057] Example 3: Preparation of active state ribonuclease SiRe_0902

[0058] The ribonuclease SiRe_0902 prepared according to the procedure of Example 2 Step 2.4 was treated (SiRe_0902-modification) Figure 1 -A) First, 2.5 mL of the desalted protein SiRe_0902-modification was mixed with 100 μL of phosphodiesterase PDE (2 U / μL -1 ) and treated at 4°C overnight (12-16 h). After treatment, the protein mixture was purified again using a nickel ion affinity column, and the purification steps are described in Example 2 Step 2.4. After purification, the nucleic acid small molecules and phosphodiesterase PDE were filtered out, and the ribonuclease SiRe_0902 without nucleic acid modification was finally collected. The purified product was subjected to SDS-PAGE gel electrophoresis, and the size of the purified protein was about 15 kD Figure 1 -B), which was consistent with the expectation.

[0059] Example 4: Activity detection of ribonuclease SiRe_0902

[0060] In order to ensure the accuracy of the experimental results, the activities of the ribonuclease SiRe_0902 in two states were compared in the study. First, the protein concentrations of the two states of SiRe_0902 (with and without nucleic acid modification) were determined using a BCA protein quantification kit (P0010, Biyun Tian), and the mother liquor concentrations of both were adjusted to 0.3 mg / mL using Tris-Hcl (20 mM, pH 8.0) buffer for standby. Four commonly used nucleic acid substrates were selected for detection in this study, including single-stranded RNA probes, total RNA, single-stranded DNA probes, and double-stranded DNA probes, and the probe sequences are shown in Table 5.

[0061] Table 5 Probe sequence table

[0062]

[0063] The reaction conditions for the ribonuclease SiRe_0902 to hydrolyze the substrate were as follows: ① The two SiRe_0902 proteins were gradient-diluted to 50, 100, and 500 μg / mL, respectively, and the dilution buffer was 50 mM Tris-HCl, pH 7.5, 10 M MgCl2. ② In a 10 μL reaction system, 1 μL of ribonuclease SiRe_0902 was added, and the concentration of the nucleic acid substrate was 50 nM. The mixed system was reacted at 60°C for 15 min. ③ Detection of substrate hydrolysis: 2 μL of nucleic acid loading was added to 10 μL of the reaction system, and 15% urea gel was run at 110 V for 30 min. FAM signal exposure detection Figures 2-3 ).

[0064] Figure 2and Figure 3 The results show that SiRe_0902 without nucleic acid modification has no ribonuclease activity and has no effect on RNA or DNA probes. SiRe_0902 without nucleic acid modification has high ribonuclease activity and can specifically cut RNA substrates at 65°C with high efficiency. Even at very low enzyme concentration (10 μg / mL), the hydrolysis of the substrate can be completed within 15 minutes. In addition, we compared the activity of freshly prepared SiRe_0902 without modification with SiRe_0902 stored at 4°C for one year, and found that it still has high RNA cutting activity, which indicates that it has good stability Figure 2 -C).

[0065] Example 5: Detection of the specificity of the ribonuclease SiRe_0902 recognition sequence

[0066] According to the reaction conditions of Example 4 (the reaction temperature is changed to 65°C), SiRe_0902 has certain sequence selectivity when cutting RNA substrates. Using 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2 as buffer, 5 5'-end or 3'-end FAM-labeled probes with different sequences are introduced to detect the recognition sequence of SiRe_0902. Figure 4 -A) The results of in vitro activity experiments show that SiRe_0902 specifically recognizes the GG sequence in the RNA probe for cutting. At 65°C, when the concentration of the RNA endonuclease SiRe_0902 is 5 μg / mL, the cutting of the substrate by SiRe_0902 within 15 minutes is completely sequence-dependent, and there is no random sequence hydrolysis activity Figure 4 -B). The above results show that the high-temperature resistant endonuclease SiRe_0902 has the potential to develop into a ribonuclease product, which can recognize and cut the GG sequence under high-temperature conditions, and has obvious advantages in opening the RNA structure.

[0067] Since the conventional enzyme reaction temperature is 37°C, we further detected the activity of SiRe_0902 at 37°C (other reaction conditions are the same as in Example 4). The results show that SiRe_0902 can not only hydrolyze single-stranded RNA without secondary structure at room temperature, but also cut RNA molecules with complex secondary structure, realizing the complete hydrolysis of GG sequences in RNA at room temperature Figure 4 -C) SEQ ID NO. 1 (nucleotide sequence of ribonuclease gene SiRe_0902)

[0068] ATG AACTATAAAGAATGGATAGAGCAAGCTTTGGAAGATCTCGACACTGCAAAACTTCTTTTAACTAATGGGAAATATTACGCTTCTGCCTTCTATTCTCAGCAAGCAGTAGAGAAATCATTAAAGTCCCTTATAATTTATCTTGGAAAAGATCCTGGTAAGACGCATTCACTTACTGAACTTATTGAAATGGTAGAGAAGGAAGGAGTGACTATGCCAATAAATATTAAAGAGAATCTGATGGTTCTTTCACCCCATTTCATAATTTCAAGATATCCAGACGCTGCAAACGGAGTTCCCTTTAAGCAGTATAGTAAATCAATTTCTGAGGACCTTTATAATAGAGCAAAAGAGGTGATTGAATGGGTAAAGGAAAATCTGCAATAG SEQ ID NO. 2 (Ribonuclease SiRe_0902 amino acid sequence)

[0069] MNYKEWIEQALEDLDTAKLLLTNGKYYASAFYSQQAVEKSLKSLIIYLGKDPGKTHSLTELIEMVEKEGVTMPINIKENLMVLSPHFIISRYPDAANGVPFKQYSKSISEDLYNRAKEVIEWVKENLQ SEQ ID NO. 3 (Ribonuclease gene SiRe_0903 nucleotide sequence)

[0070] ATGGGTAAAGGAAAATCTGCAATAGAGAGCCAAATGAAGTTAATAAATCTAGTAAAGGAAATAGTGGAAGAAATAGCTAAGGACTTCCAGCAGTTAGACGAAGTTTATATCTTTGGCTCTAGAGCTAAAGGAAATTATTTAGATACTAGTGATATAGATGTTATCTTCGTTTTCAAGGGCATAAAGGAAATGAACGTATTTGATAGGATGTATATGGTAAGTAAATACATAAAAGGAAATATAGATTATATAGTATTAAACGAGGACGAAAAGGATAGAATAAGAGAGAAGAAATTATTTTGGAAGAGGAATAAGGGATTTGT AGATATAAAGGAATTTCTATAA SEQ ID NO. 4 (Ribonuclease SiRe_0903 amino acid sequence)

[0071] MGKGKSAIESQMKLINLVKEIVEEIAKDFQQLDEVYIFGSRAKGNYLDTSDIDVIFVFK GIKEMNVFDRMYMVSKYIKGNIDYIVLNEDEKDRIREKKLFWKRNKGFVDIKEFL

Claims

1. The application of ribonuclease SiRe_0902 in the specific recognition and cleavage of GG sites in RNA in non-disease diagnosis and treatment, characterized in that, The amino acid sequence of the ribonuclease SiRe_0902 is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the ribonuclease SiRe_0902 is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The GG cleavage site refers to the cleavage of the phosphodiester bond in the middle of the GG sequence.

4. The application according to claim 1, characterized in that, The concentration of the ribonuclease SiRe_0902 is 5-10 μg / mL.

5. The application according to claim 4, characterized in that, The concentration of the ribonuclease SiRe_0902 is 5 μg / mL.

6. The application according to claim 1, characterized in that, The reaction temperature for specifically recognizing and cleaving GG sites is 37℃-70℃, and the reaction time is 9-20 min.

7. The application according to claim 6, characterized in that, The reaction temperature for specifically recognizing and cleaving GG sites is 65°C, and the reaction time is 15 min; or the reaction temperature for specifically recognizing and cleaving GG sites is 37°C, and the reaction time is 9-12 min.

8. The application of ribonuclease SiRe_0902 in the hydrolysis of RNA in non-disease diagnosis and treatment, characterized in that, The amino acid sequence of the ribonuclease SiRe_0902 is shown in SEQ ID NO.

2.

9. The application according to claim 8, characterized in that, The concentration of the ribonuclease SiRe_0902 is 5-10 μg / mL, the hydrolysis temperature is 37℃-65℃, and the hydrolysis time is 10-20 min.

10. The application according to claim 9, characterized in that, The concentration of the ribonuclease SiRe_0902 is 10 μg / mL, the hydrolysis temperature is 65℃, and the hydrolysis time is 15 min.

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