RNase inhibitor mutant as well as preparation method and application thereof

Abstract

The invention discloses an RNase inhibitor mutant as well as a preparation method and application of the RNase inhibitor mutant. Firstly, an RNase inhibitor mutant is disclosed, and point mutations K169N and K205R are introduced into a wild type RNase inhibitor. The invention further discloses a preparation method of the RNase inhibitor mutant and an application of the RNase inhibitor mutant. The RNase inhibitor mutant disclosed by the invention has the advantages of high stability of long-term storage at 37 DEG C and 2 hours of tolerance at 55 DEG C, and can be applied to the field of molecular in-vitro diagnosis such as extraction of various RNAs, in-vitro reverse transcription and translation, virus detection and the like.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and enzyme engineering, and more specifically, to an RNase inhibitor mutant and a preparation method and application thereof. Background Art

[0002] RNase inhibitor (RI) is a cytoplasmic protein mainly found in mammals, with a molecular weight of about 50 kDa. RI can bind to members of the pancreatic RNase superfamily in a 1:1 non-covalent manner to inhibit the activity of RNase, and its affinity can reach the fmol level. RI has a wide range of applications in molecular diagnosis, especially in reactions involving RNA. First, adding RI to various RNA extraction, in vitro reverse transcription and translation systems can inhibit the activity of RNase by binding to RI, thereby protecting RNA from degradation and ensuring the integrity of RNA. Secondly, RI does not inhibit the activity of other nucleases or polymerases. Therefore, adding RI to experiments such as RT-PCR, qRT-PCR and RT-LAMP can effectively protect RNA templates and improve detection sensitivity.

[0003] Therefore, the application of highly stable RNase inhibitors in various molecular diagnostic kits has received increasing attention. Summary of the invention

[0004] One object of the present invention is to provide a highly stable RNase inhibitor mutant.

[0005] Another object of the present invention is to provide a preparation method and application of the above-mentioned RNase inhibitor mutant.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention first provides an RNase inhibitor mutant, wherein the RNase inhibitor mutant introduces a point mutation into a wild-type RNase inhibitor, wherein the point mutation is represented by a triplet: letter-number-letter, wherein the number represents the position of the mutated amino acid, the letter before the number corresponds to the amino acid involved in the mutation, and the letter after the number represents the amino acid used to replace the amino acid before the number: K169N and K205R.

[0008] Furthermore, the wild-type RNase inhibitor is a wild-type human RNase inhibitor, and its amino acid sequence is shown in SEQ ID NO.3.

[0009] Furthermore, the amino acid sequence of the RNase inhibitor mutant is shown in SEQ ID NO.1.

[0010] The nucleotide sequence encoding the above-mentioned RNase inhibitor mutant is also within the protection scope of the present invention.

[0011] Furthermore, the nucleotide sequence encoding the above-mentioned RNase inhibitor mutant is shown in SEQ ID NO.2.

[0012] The recombinant expression plasmid or recombinant cell containing the above nucleotide sequence is also within the protection scope of the present invention.

[0013] In a specific embodiment of the present invention, the recombinant expression plasmid is pET28a-RI-Mut, that is, the nucleotide sequence encoding the RNase inhibitor mutant as shown in SEQ ID NO.2 is inserted between NdeI and BamHI of pET28a, and the other sequences of pET28a are kept unchanged.

[0014] In a specific embodiment of the present invention, the host cell of the recombinant cell is a modified BL21 Escherichia coli. In a specific embodiment of the present invention, the host cell is a BL21 (DE3) competent cell.

[0015] The present invention further discloses a method for preparing the above-mentioned RNase inhibitor mutant, and the preparation method comprises the following steps:

[0016] a1) constructing a recombinant expression plasmid comprising the nucleotide sequence shown in SEQ ID NO.2;

[0017] a2) transforming the recombinant expression plasmid into host cells, inducing expression, and obtaining bacterial cells;

[0018] a3) breaking the bacterial cells, obtaining the supernatant after centrifugation, and precipitating the protein to obtain the crude protein solution;

[0019] a4) Purifying the crude protein solution to obtain the RNase inhibitor mutant.

[0020] Furthermore, the protein precipitation is ammonium sulfate precipitation; and the purification includes Ni column and Q column purification.

[0021] The present invention further discloses the use of the above RNase inhibitor mutant, the above nucleotide sequence, or the above recombinant expression plasmid or recombinant cell in inhibiting RNase activity or preparing a product that inhibits RNase activity.

[0022] Furthermore, the application may be any of the following:

[0023] b1) Application in the field of molecular in vitro diagnostics;

[0024] b2) Application in RNA extraction;

[0025] b3) Application in in vitro reverse transcription;

[0026] b4) Application in in vitro translation;

[0027] b5) Application in virus detection.

[0028] Furthermore, the in vitro reverse transcription can be used in various RT-PCR, qRT-PCR and RT-LAMP experiments.

[0029] The beneficial effects of the present invention are as follows:

[0030] The RNase inhibitor mutant of the present invention introduces specific amino acid mutations K169N and K205R into the wild-type human RNase inhibitor, and the mutant can be overexpressed in a prokaryotic host and purified by affinity chromatography and ion exchange chromatography to obtain a large amount of protein. The RNase inhibitor mutant of the present invention has the advantages of high stability for long-term storage at 37°C and tolerance to 55°C for up to 2 hours, and can be applied to various molecular in vitro diagnostic fields such as RNA extraction, in vitro reverse transcription and translation, and virus detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The specific implementation modes of the present invention will be further described in detail below in conjunction with the accompanying drawings.

[0032] Figure 1 This is the SDS-PAGE gel image of the RNase inhibitor mutant of the present invention.

[0033] Figure 2 This is an electrophoresis diagram of the RNase inhibitory activity detected after the RNase inhibitor mutant of the present invention was pressurized.

[0034] Figure 3 This is the amplification map of the one-step qRT-PCR after pressurization of the RNase inhibitor mutant of the present invention. DETAILED DESCRIPTION

[0035] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0036] Example 1 Design of RNase inhibitor mutant

[0037] By comparing the sequences of 200 RNase inhibitors from different sources and analyzing the sequence logo, it was found that the amino acids at the K169 and K205 sites were mutated, so these non-conservative amino acids K169 and K205 were mutated to conservative amino acids N169 and R205 (i.e., K169N and K205R), respectively. In order to improve the stability of the wild-type RNase inhibitor, specific amino acid mutations K169N and K205R were introduced into the wild-type human RNase inhibitor (i.e., the amino acid sequence shown in SEQ ID NO.3) to enhance its long-term stability at 37°C and tolerance at 55°C. The amino acid sequence of the RNase inhibitor mutant is shown in SEQ ID NO.1, which is encoded by the nucleotide sequence shown in SEQ ID NO.2.

[0038] Structural stability prediction analysis of wild-type human RNase inhibitor and RNase inhibitor mutants revealed that the Gibbs free energy of the RNase inhibitor mutant was reduced, indicating that its structure was more stable.

[0039] Example 2 Preparation of RNase inhibitor mutants

[0040] 1. Construction of RNase inhibitor mutant recombinant expression plasmid

[0041] The whole gene synthesis RNase inhibitor mutant recombinant expression plasmid pET28a-RI-Mut was performed, that is, a nucleotide sequence encoding the RNase inhibitor mutant (abbreviated as RI-Mut) was inserted between NdeI and BamHI of pET28a (as shown in SEQ ID NO.2), and other sequences of pET28a were kept unchanged.

[0042] 2. Inducible Expression

[0043] The RNase inhibitor mutant recombinant expression plasmid pET28a-RI-Mut was transformed into the host competent cells and induced to express the target protein.

[0044] The specific steps are as follows:

[0045] 1. Thaw BL21(DE3) competent cells on ice.

[0046] 2. Take 1 μl of the recombinant expression plasmid pET28a-RI-Mut and add it to the melted BL21 (DE3) competent cells and place it on ice for 30 minutes.

[0047] 3. After ice bath, heat shock the competent cells in a 42°C water bath for 45 seconds, and then place them on ice for 2 minutes.

[0048] 4. Take 400 μl of LB liquid culture medium and add it to the cells obtained in step 3. Incubate at 37°C in a shaker at 220 rpm for 45 min.

[0049] 5. Take 200 μl of the cells incubated in step 4 and spread them on a kanamycin plate, and culture them in a 37°C constant temperature incubator overnight.

[0050] 6. Pick a single clone from the overnight cultured kanamycin plate and add it to 20 ml LB medium containing 50 μg / ml kanamycin. Place it in a 37°C shaker at 220 rpm and culture for 7 hours.

[0051] 7. Add 20 ml of the culture obtained in step 6 to 1 L LB medium containing 50 μg / ml kanamycin and culture in a 37°C shaker at 220 rpm for 2 hours. 600 =0.6, add 200 μl 0.5 M IPTG, adjust the shaking temperature and speed to 20°C, 200 rpm, and induce expression overnight for about 15 hr.

[0052] 8. Centrifuge the bacterial solution after induction expression in step 7 to collect the bacteria at 8000 rpm, 15 min, 4°C, and weigh the bacteria.

[0053] 3. Purification

[0054] After ammonium sulfate precipitation, purification methods such as Ni column and Q column were used to obtain RNaseinhibitor mutants with higher purity.

[0055] The specific steps are as follows:

[0056] 1. Sedimentation treatment

[0057] 1) Resuspend the cells in 30 ml PBS buffer and centrifuge at 12,000 rpm, 10 min, 4°C, and discard the supernatant to obtain the cells.

[0058] 2) Resuspend the cells in 30 ml of lysis buffer and add PMSF to a final concentration of 1 mM.

[0059] 3) Using a high-pressure homogenizer, the PMSF-added bacterial resuspension obtained in step 2) was crushed.

[0060] 4) The crushed liquid obtained in step 3) was centrifuged at 12000 rpm, 30 min, 4°C, and the precipitate was discarded to obtain the supernatant.

[0061] 5) Slowly add ammonium sulfate powder to the supernatant obtained in step 4) during stirring to a saturated concentration of 60%. After the ammonium sulfate is completely dissolved, place the mixture in a refrigerator at 4° C. for 1 hour to precipitate the protein.

[0062] 6) The precipitate sample obtained in step 5) was centrifuged at 12000 rpm, 30 min, 4°C, and the supernatant was discarded to obtain the protein precipitate.

[0063] 7) The protein precipitate obtained in step 6) was resuspended in 20 ml of Ni-binding buffer and filtered using a 0.45 μM filter to obtain a protein sample (crude protein solution).

[0064] 2. NI column purification

[0065] 1) Connect the 5 ml Ni pre-packed column to the AKTA system, and put the filtered and evacuated Ni-Binding buffer and Ni-Elution buffer into the A and B pumps respectively, and wash the AKTA system with the pumps.

[0066] 2) Equilibrate the Ni column with Ni-Binding buffer.

[0067] 3) Load the protein sample obtained in step 1 onto a Ni column.

[0068] 4) The target protein was eluted with Ni-Elution buffer (2 ml / min flow rate, 30 min elution time, 0-100% B pump gradient).

[0069] 5) Collect the target protein peak to obtain the protein sample after Ni column elution.

[0070] 3. Q column purification

[0071] 1) Connect the 5 ml Q pre-packed column to the AKTA system, and put the filtered and evacuated Q-Binding buffer and Q-Elution buffer into the A and B pumps respectively, and wash the AKTA system with the pumps.

[0072] 2) Equilibrate the Q column with Q-Binding buffer.

[0073] 3) Load the protein sample eluted from the Ni column obtained in step 2 onto the Q column.

[0074] 4) The target protein was eluted with Q-Elution buffer (2 ml / min flow rate, 30 min elution time, 0-100% B pump gradient).

[0075] 5) Collect the target protein peak to obtain a protein sample.

[0076] The protein sample was dialyzed into Storage buffer (50 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM DTT, 50% Glycerol), and the SDS-PAGE gel test results were as follows: Figure 1 As shown in the figure, the size of the protein sample obtained in this example is 52 kDa, which is consistent with the size of the expected RNase inhibitor mutant, indicating that the protein sample finally obtained in this example is an RNase inhibitor mutant. Further, sequencing verification is correct.

[0077] Example 3 Testing of RNase inhibitory activity of mutants after pressurization

[0078] Using the unpressurized 40U / μl RNase inhibitor mutant (abbreviated as RI-Mut) as a control, the 40U / μl RNase inhibitor mutant was placed at 37°C for one month and heated at 55°C for 2 hours to test the RNase inhibitory activity.

[0079] After preparing the system as shown in Table 1 and setting up duplicate wells, the system was incubated at 37°C for 15 minutes, and then 2 μl of 6xDNA Loading Buffer was added, mixed and directly subjected to agarose gel electrophoresis. The electrophoresis conditions were 200V, 10 minutes, and the gel imager was used to take pictures immediately after the electrophoresis.

[0080] Table 1 RNase inhibition activity test system

[0081] Components Volume (μl) RNase-free water 6 10×Tris-HCl (500mM) 1 HeLa RNA (500 ng / μl) 1 With or without RI-Mut (40U / μl) 1 With or without RNase A (10 ng / μl) 1

[0082] The results are as follows Figure 2 As shown, compared with the unpressurized RI-Mut, the RNase inhibitor mutant can still inhibit 10 ng RNase A after being placed at 37°C for one month and heated at 55°C for 2 hours, protecting RNA from degradation, indicating that the RNase inhibitor mutant has high stability.

[0083] Example 4: One-step qRT-PCR test after pressurization of RNase inhibitor mutant

[0084] Using the unpressurized 40U / μl RNase inhibitor mutant (abbreviated as RI-Mut) as a control, the one-step qRT-PCR test experiment was performed after heating the 40U / μl RNase inhibitor mutant at 55°C for 2 hours.

[0085] The qRT-PCR amplification system as shown in Table 2 was prepared and duplicate wells were set up for one-step qRT-PCR. The amplification procedure was shown in Table 3.

[0086] Table 2 qRT-PCR amplification system

[0087]

[0088]

[0089] Table 3 qRT-PCR amplification procedures

[0090]

[0091] Test results such as Figure 3 As shown, compared with the unpressurized RI-Mut, the RNase inhibitor mutant can still maintain no significant change in Ct value after heating at 55°C for 2 hours, indicating that the RNase inhibitor mutant has high stability.

[0092] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. An RNase inhibitor mutant, characterized in that: The RNase inhibitor mutant introduces a point mutation into the wild-type RNase inhibitor, and the point mutation is represented by a triplet: letter-number-letter, wherein the number represents the position of the mutated amino acid, the letter before the number corresponds to the amino acid involved in the mutation, and the letter after the number represents the amino acid used to replace the amino acid before the number: K169N and K205R.

2. The RNase inhibitor mutant according to claim 1, characterized in that The wild-type RNase inhibitor is a wild-type human RNase inhibitor, and its amino acid sequence is shown in SEQ ID NO.

3.

3. The RNase inhibitor mutant according to claim 1, characterized in that The amino acid sequence of the RNase inhibitor mutant is shown in SEQ ID NO.

1.

4. A nucleotide sequence encoding the RNase inhibitor mutant according to any one of claims 1 to 3.

5. The nucleotide sequence according to claim 4, characterized in that The nucleotide sequence of the RNase inhibitor mutant is shown in SEQ ID NO.

2.

6. A recombinant expression plasmid or recombinant cell comprising the nucleotide sequence of claim 4 or 5.

7. The method for preparing the RNase inhibitor mutant according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: a1) constructing a recombinant expression plasmid comprising the nucleotide sequence shown in SEQ ID NO.2; a2) transforming the recombinant expression plasmid into host cells, inducing expression, and obtaining bacterial cells; a3) breaking the bacterial cells, obtaining the supernatant after centrifugation, and precipitating the protein to obtain the crude protein solution; a4) Purifying the crude protein solution to obtain the RNase inhibitor mutant.

8. The preparation method according to claim 7, characterized in that: The protein precipitation is ammonium sulfate precipitation; preferably, the purification includes Ni column and Q column purification.

9. Use of the RNase inhibitor mutant according to any one of claims 1 to 3, the nucleotide sequence according to claim 4 or 5, and the recombinant expression plasmid or recombinant cell according to claim 6 in inhibiting RNase activity or preparing a product that inhibits RNase activity.

10. Use of the RNase inhibitor mutant according to any one of claims 1 to 3, the nucleotide sequence according to claim 4 or 5, the recombinant expression plasmid or recombinant cell according to claim 6 in any of the following: b1) Application in the field of molecular in vitro diagnostics; b2) Application in RNA extraction; b3) Application in in vitro reverse transcription; b4) Application in in vitro translation; b5) Application in virus detection.

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