Application of high-efficiency thermostable ribonuclease SiRe0902 in specific recognition of GG site

Abstract

The invention discloses an application of ribonuclease SiRe0902 with high efficiency and thermal stability in specific recognition of a GG site. The amino acid sequence of the gene is as shown in SEQ ID NO.2, and the nucleotide sequence of the gene is as shown in SEQ ID NO.1. The ribonuclease SiRe0902 has very efficient and specific RNA (Ribonucleic Acid) hydrolysis activity, and does not have non-specific cutting on DNA (Deoxyribose Nucleic Acid) at all. In addition, the enzyme is derived from thermophilic archaea, is detected to have good thermal stability, and has very good RNA cleavage activity under the conditions of 37 DEG C and 65 DEG C. Through detection, the enzyme can specifically recognize a GG sequence for cutting, and also has high cutting activity for the GG sequence in a secondary structural region. Therefore, the ribonuclease SiRe0902 is novel ribonuclease with thermal stability, has extremely high RNA hydrolytic activity under normal-temperature and high-temperature conditions, and meets the requirements in the fields of clinic, production, molecular cloning and the like.

Description

Technical Field

[0001] The invention belongs to the fields of biochemical engineering and biotechnology, and specifically relates to the application of highly efficient and heat-stable ribonuclease SiRe_0902 in specifically recognizing a GG site under high temperature conditions. Background Art

[0002] Ribonuclease (RNase) refers to an enzyme that can hydrolyze the phosphodiester bond of RNA. Different RNases have different specificities. Currently, commonly used ribonucleases include RNaseA, RNase H, and RNase T1. Ribonuclease is a protein with globulin properties, soluble in water and 50% acetone. The most suitable temperature for this enzyme is 60°C, and it loses its effect above 85°C. The most suitable pH value is 7.6. Among them, ribonuclease A (RNase A): derived from bovine pancreas, 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 final product of the reaction is a 3' pyrimidine nucleotide and an 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 placental RNase inhibitor (RNasin) or vanadyl-ribonucloside complex (VRC). Ribonuclease T1 (RNase T1): Derived from Aspergillus orjzae, it specifically acts on the 3' phosphate of guanine, and the cleavage site is the phosphodiester bond between the 3' phosphate of guanine and the 5' hydroxyl of the adjacent nucleotide. The final product of the reaction is 3' guanylate and an oligonucleotide fragment with 3' guanylate at the end. Ribonuclease H (RNase H): It was first discovered from calf thymus tissue, and its coding gene has been cloned into Escherichia coli. It can specifically degrade the RNA chain in the DNA:RNA hybrid double-stranded chain to produce oligonucleotides and mononucleotides with 3'-OH and 5'-phosphate ends. It cannot degrade single-stranded or double-stranded DNA or RNA.

[0003] At present, ribonucleases are widely used in various fields such as molecular cloning, clinical, food and medicine. In molecular cloning, ribonucleases can be used to ① remove RNA molecules from DNA: RNA hybrids or DNA preparations; ② determine the location of single-base mutations in RNA or DNA; ③ RNA detection is used in RNase protection assay. In addition, it is reported that ribonucleases can change host cell metabolism, inhibit virus synthesis, inhibit influenza virus proliferation in vitro, and inhibit vaccinia and herpes virus formation in chicken embryos. Clinical ribonucleases are beneficial for the treatment of epidemic encephalitis by intramuscular injection of 180 mg per day. However, the types of ribonucleases currently available on the market are limited, especially the commercialization of high-temperature resistant RNA endonucleases with sequence recognition sites is almost blank, and there is a great demand for ribonucleases with new functions. Summary of the invention

[0004] The present invention aims to provide an application of an efficient and thermostable ribonuclease SiRe_0902 in the specific recognition of GG sites. The ribonuclease SiRe_0902 is directly prepared from the thermophilic archaeon Sulfolobus islandicus REY15A, which is an efficient, heat-resistant and stable ribonuclease, and can specifically recognize the GG site in the RNA sequence for cutting, and can efficiently cut RNA with secondary structure at 37°C or 65°C. This has very good application prospects in the analysis of RNA with complex structures. In particular, the enzyme has a stable structure and still has very good activity after being placed at 4°C for 1 year, and has the characteristics of being developed into a commercial RNA endonuclease.

[0005] The first objective of the present invention is to provide the use of ribonuclease SiRe_0902 in specifically recognizing and cleaving the GG site, wherein the amino acid sequence of the ribonuclease SiRe_0902 is shown in SEQ ID NO.2.

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

[0007] The ribonuclease gene SiRe_0902 of the present invention comes from the thermophilic archaeon Sulfolobus icelandica REY15A. The present invention finds through molecular cloning, activity verification and other methods that the gene SiRe_0902 encodes an efficient thermostable ribonuclease. Under normal circumstances, the enzyme is modified and silenced by the nucleic acid-specific enzyme encoded by the downstream gene SiRe_0903. It has been experimentally verified that when the ribonuclease gene SiRe_0902 and the downstream SiRe_0903 are co-expressed and purified, it has no effect on the growth of the host, and there is no stable interaction between the two, so a large amount of nucleic acid-modified ribonuclease SiRe_0902 can be purified.

[0008] Preferably, the cleavage GG site is to cleave the phosphodiester bond in the middle of the 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 the specific recognition and cleavage of the GG site is 60-70° C., and the reaction time is 10-20 min.

[0012] Preferably, the reaction temperature for the specific recognition and cleavage of the GG site is 65° C. and the reaction time is 15 min.

[0013] The second object of the present invention is to provide the use of ribonuclease SiRe_0902 in hydrolyzing RNA in non-disease diagnosis and treatment, wherein 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° C., 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° C., and the hydrolysis time is 15 min.

[0016] Advantages of the present invention:

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

[0018] Ribonuclease SiRe_0902 can specifically recognize the GG site in the RNA sequence for cutting. At 65°C, when the concentration of RNA endonuclease SiRe_0902 is 5μg / mL and the reaction time is within 15min, its cutting of the substrate is completely sequence-dependent, and there is no random sequence hydrolysis activity. SiRe_0902 has the potential to be developed into a ribonuclease product. It can recognize and cut the GG sequence under high temperature conditions and has obvious advantages in opening the RNA structure.

[0019] The thermophilic archaeon 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. The applicant also holds it and guarantees that it will be released to the public within 20 years from the date of application. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] Figure 2 The effect of reaction time on the activity of ribonuclease SiRe_0902 (10 μg / mL) in cleaving RNA probes and total RNA samples.

[0022] Figure 3It is a test of the ability of ribonuclease SiRe_0902 (10μg / mL) to cut single-stranded DNA probes and double-stranded DNA probes.

[0023] Figure 4 This is the identification result of ribonuclease SiRe_0902 (5 μg / mL) recognizing the cleavage sequence. DETAILED DESCRIPTION

[0024] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[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 of the thermophilic archaeon Sulfolobus islandicus REY15A, which is often used for the expression and purification of target proteins in cells. The wild-type Sulfolobus islandicus REY15A genomic DNA was extracted using the Tiangen kit as a template for subsequent PCR cloning; primers for PCR amplification of the SiRe_0902-0903 operon (the nucleotide sequence of SiRe_0902 is shown in SEQ ID NO.1; the nucleotide sequence of SiRe_0903 is shown in SEQ ID NO.3) were designed, and the primer sequences are as follows: Upstream primer SiRe_0902-his-F (pSeSD): 5'GGAATTC CATATG CACCACCACCACCACCACAACTATAAAGAATGGATAGAG 3'; downstream primer SiRe_0903-R (pSeSD): 5'ACGC GTCGACTTATAGAAATTCCTTTATATCT 3'. Using SiRe_0902-his-F (pSeSD) and SiRe_0903-R (pSeSD) as primers and wild-type Icelandic Sulfolobus REY15A genomic DNA as template, Takara's PrimeSTAR enzyme was used to PCR amplify the coding region of the SiRe_0902-0903 operon. The PCR amplification system is shown in Table 1. The PCR amplification conditions are: first 95℃5min; then 95℃30s, 56℃30s, 72℃30s, a total of 30 cycles; finally 72℃10min. After the reaction, the PCR amplification product was subjected to 1.0% agarose gel electrophoresis, and the target gene fragment of about 700bp was recovered and purified. The recovered fragment was double-digested with NdeI and SalI endonucleases, and the digested product was subjected to 1.0% agarose gel electrophoresis, and the gel was recovered and purified. It was then ligated with the vector pSeSD that had also been treated with NdeI and SalI using T4 DNA ligase (TaKaRa), and the ligation product was transferred into Escherichia coli BW25113 competent cells. Positive transformants were screened, verified by PCR, and sent to a sequencing company for sequencing 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: High-efficiency expression of ribonuclease SiRe_0902

[0031] 2.1 Preparation of Sulfolobus icelandica REY15A competent cells

[0032] 1. One day before preparing competent cells, inoculate 2 mL of Icelandic Sulfolobus REY15A starter culture into 100 mL of SCVU (U 10 μg / mL) medium and culture at 78°C overnight;

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

[0034] 2. Determination of OD 600 Stop the culture at 0.2 (for ease of operation OD 600 Transfer the culture to 2×50mL centrifuge tubes at room temperature. Centrifuge at 6000rpm for 10min at room temperature to collect the cells. Discard the supernatant (absorb all the supernatant).

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

[0036] 4. Resuspend the cells with room temperature sucrose solution (20 mM) (do not vortex) to make the OD of competent cells 600 The value is 5-10. Store the competent cells at room temperature for one week or at -80°C.

[0037] 2.2 Electroporate the target recombinant plasmid into the expression strain REY15A

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

[0039] 2. Transfer 50 μL of competent cell-DNA suspension into the corresponding electroporation cup. The electroporation conditions are: 1.2KV, 600Ω, 25μF (Bio-Rad Gene pulserⅡ), time constant 12ms.

[0040] 3. Transfer the transformed cells to a 1.5 mL centrifuge tube containing 800 μL of incubation medium (see Table 2) preheated 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 No.: 71010-52-1) solution and 2×SCV, and mix them in a 1:1 volume ratio to obtain 1×SCV top gel. Add 6mL 1×SCV top gel to 50μL of electrotransformed cells, mix well, add to the preheated plate with SCV bottom gel, and fix at room temperature for 30 minutes. Incubate at 75℃ for 7 days and check for transformants;

[0042] Base gel: Prepare as shown in Table 2-4, 1.4% Gelriter solution is mixed with an equal volume of 2×SCV.

[0043] Top gel: 0.4% Gelrite and 2×SCV were mixed in equal volumes, with final concentrations of 0.2% and 1×SCV, respectively.

[0044] 2.3 Protein induction expression

[0045] The expression strain of Sulfolobus icelandica REY15A containing pSeSD-SiRe_0902-0903-Nhis was cultured in SCV medium until OD600 The induction agent D-arabinose was about 0.5, and the final concentration was 10mM. The cells were cultured at 75℃ for 48h. 500mL of bacterial solution was centrifuged at 4500rpm for 10min at room temperature to collect the cells, and the cells were resuspended in 30mL PBS buffer (50mM, pH 7.4), ultrasonicated at 400w for 5s, stopped for 5s, and broken for 10min. The cells were centrifuged at 10000rpm for 30min at 4℃ and the supernatant was collected.

[0046] 2.4 Protein purification and SDS-PAGE electrophoresis

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

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

[0049]

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

[0051] Table 3 SCV culture medium

[0052]

[0053] Table 4 Vitamin Mixture 100×

[0054]

[0055]

[0056] (Tock solution inmg / L distilledH 2 O; filter sterilize. )

[0057] Example 3: Preparation of active ribonuclease SiRe_0902

[0058] According to the preparation process of active ribonuclease SiRe_0902, the ribonuclease SiRe_0902 (SiRe_0902-modified) purified in step 2.4 of Example 2 was treated ( Figure 1 -A). First, 2.5 mL of desalted protein SiRe_0902-modified was mixed with 100 μL of phosphodiesterase PDE (2 U μL -1 ) and treated overnight at 4°C (12-16h). After treatment, the protein mixture was re-purified using a nickel ion affinity chromatography column. The purification steps refer to step 2.4 of Example 2. After purification, small nucleic acid molecules and phosphodiesterase PDE were filtered out, and finally the ribonuclease SiRe_0902 with nucleic acid modification removed was collected. The purified product was subjected to SDS-PAGE gel electrophoresis, and the purified protein size was about 15kD ( Figure 1 -B), as expected.

[0059] Example 4: Activity detection of ribonuclease SiRe_0902

[0060] In order to ensure the accuracy of the experimental results, the activity of the two states of ribonuclease SiRe_0902 was compared in this study. First, the protein concentration of the two states of SiRe_0902 (with nucleic acid modification and without nucleic acid modification) was determined using the BCA protein quantification kit (P0010, Biyuntian), and the mother liquor concentrations of both were adjusted to 0.3 mg / mL using Tris-Hcl (20mM, PH 8.0) buffer for later use. 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. The probe sequences are shown in Table 5.

[0061] Table 5 Probe sequence list

[0062]

[0063] The reaction conditions for the hydrolysis of the substrate by ribonuclease SiRe_0902 were as follows: ① The two SiRe_0902 proteins were diluted in a gradient manner to 50, 100, and 500 μg / mL, respectively, in a dilution buffer of 50 mM Tris-HCl, pH 7.5, 10 M MgCl 2 ② Add 1 μL of ribonuclease SiRe_0902 to the 10 μL reaction system, the concentration of the nucleic acid substrate is 50 nM, and the mixed system is reacted at 60°C for 15 min. ③ Detect the hydrolysis of the substrate: add 2 μL of nucleic acid loading to the 10 μL reaction system, 15% urea gel, run the gel at 110 V for 30 min, and detect the FAM signal exposure ( Figure 2-3 ).

[0064] Figure 2 and Figure 3 The results showed that SiRe_0902 with nucleic acid modification had no ribonuclease activity and had no effect on RNA or DNA probes. SiRe_0902 without nucleic acid modification had efficient ribonuclease activity and could specifically cleave RNA substrates at 65°C with high cleavage efficiency. Even at extremely low enzyme concentrations (10μg / mL), substrate hydrolysis could be completed within 15 minutes. In addition, we compared the activity of freshly prepared unmodified SiRe_0902 with that of SiRe_0902 that had been stored at 4°C for one year and found that it still had efficient RNA cleavage activity, indicating good stability ( Figure 2 -C).

[0065] Example 5: Specific detection of ribonuclease SiRe_0902 recognition sequence

[0066] According to the reaction conditions of Example 4 (reaction temperature changed to 65°C), SiRe_0902 has a certain sequence selectivity when cleaving RNA substrates. Using 50mM Tris-HCl, pH 7.5, 10mM MgCl 2 As a buffer, five probes with different 5' or 3' FAM labels were introduced ( Figure 4 -A) to detect the recognition sequence of SiRe_0902. The results of in vitro activity experiments showed that SiRe_0902 specifically recognized the GG sequence inside the RNA probe for cleavage. At 65°C, when the concentration of RNA endonuclease SiRe_0902 was 5μg / mL and the reaction time was within 15min, its cleavage of the substrate showed complete sequence dependence, and there was no random sequence hydrolysis activity ( Figure 4 -B). The above results show that the thermostable endonuclease SiRe_0902 has the potential to be developed 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 tested the activity of SiRe_0902 at 37°C (other reaction conditions were the same as in Example 4). The results showed that SiRe_0902 could not only hydrolyze single-stranded RNA without secondary structure at room temperature, but also cut RNA molecules with complex secondary structure, achieving full hydrolysis of GG sequence in RNA at room temperature ( Figure 4 -C). SEQ ID NO.1 (ribonuclease gene SiRe_0902 nucleotide sequence)

[0068] ATG AACTATAAAGAATGGATAGAGCAAGCTTTGGAAGATCTCGACACTGCAAAACTTCTTTTAACTAATGGGAAATATTACGCTTCTGCCTTCTATTCTCAGCAAGCAGTAGAGAAATCATTAAAGTCCCTTATAATTTATCTTGGAAAAGATCCTGGTAAGACGCATTCACTTACTGAACTTATTGAAATGGTAGAGAAGGAAGGAGTGACTATGCCAATAAATATTAAAGAGAATCTGATGGTTCTTTCACCCCATTTCATAATTTCAAGATATCCAGACGCTGCAAACGGAGTTCCCTTTAAGCAGTATAGTAAATCAATTTCTGAGGACCTTTATAATAGAGCAAAAGAGGTGATTGAATGGGTAAAGGAAAATCTGCAATAGSEQ ID NO.2 (Amino acid sequence of ribonuclease SiRe_0902)

[0069] MNYKEWIEQALEDLDTAKLLLTNGKYYASAFYSQQAVEKSLKSLIIYLGKDPGKTHSLTELIEMVEKEGVTMPINIKENLMVLSPHFIISRYPDAANGVPFKQYSKSISEDLYNRAKEVIEWVKENLQSEQ ID NO.3 (Nucleotide sequence of ribonuclease gene SiRe_0903)

[0070] ATGGGTAAAGGAAAATCTGCAATAGAGAGCCAAATGAAGTTAATAAATCTAGTAAAGGAAATAGTGGAAGAAATAGCTAAGGACTTCCAGCAGTTAGACGAAGTTTATATCTTTGGCTCTAGAGCTAAAGGAAATTATTTAGATACTAGTGATATAGATGTTATCTTCGTTTTCAAGGGCATAAAGGAAATGAACGTATTTGATAGGATGTATATGGTAAGTAAATACATAAAAGGAAATATAGATTATATAGTATTAAACGAGGACGAAAAGGATAGAATAAGAGAGAAGAAATTATTTTGGAAGAGGAATAAGGGATTTGT AGATATAAAGGAATTTCTATAA SEQ ID NO.4 (Amino acid sequence of ribonuclease SiRe_0903)

[0071] MGKGKSAIESQMKLINLVKEIVEEIAKDFQQLDEVYIFGSRAKGNYLDTSDIDVIFVFK GIKEMNVFDRMYMVSKYIKGNIDYIVLNEDEKDRIREKKLFWKRNKGFVDIKEFL

Claims

1. The use of ribonuclease SiRe_0902 in specific recognition and cleavage of GG sites, characterized in that: The amino acid sequence of the ribonuclease SiRe_0902 is shown in SEQ ID NO.

2.

2. The use 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 use according to claim 1, characterized in that: The cleavage GG site is to cleave the phosphodiester bond in the middle of the GG sequence.

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

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

6. The use according to claim 1, characterized in that: The reaction temperature for specifically recognizing and cutting the GG site is 37° C.-70° C., and the reaction time is 10-20 min.

7. The use according to claim 6, characterized in that: The reaction temperature for the specific recognition and cleavage of the GG site is 65° C., and the reaction time is 15 min; or the reaction temperature for the specific recognition and cleavage of the GG site is 37° C., and the reaction time is 9-12 min.

8. The use of ribonuclease SiRe_0902 in hydrolyzing 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 use according to claim 8, characterized in that: The concentration of the ribonuclease SiRe_0902 is 5-10 μg / mL, the hydrolysis temperature is 37° C.-65° C., and the hydrolysis time is 10-20 min.

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

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