RNase inhibitor mutant, method for preparing the same and use thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and enzyme engineering. More particularly, it relates to a RNase inhibitor mutant, its preparation method and application. BACKGROUND

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

[0003] Therefore, the application of RNase inhibitor with high stability in various molecular diagnostic kits is increasingly valued. SUMMARY

[0004] One object of the present application is to provide a RNase inhibitor mutant with high stability.

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

[0006] To achieve the above objects, the present application adopts the following technical solutions:

[0007] The present application first provides a RNase inhibitor mutant, which introduces a point mutation in 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.

[0008] Further, 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] Further, the amino acid sequence of the RNase inhibitor mutant is shown as SEQ ID NO. 1.

[0010] The nucleotide sequence encoding the RNase inhibitor mutant is also within the protection scope of the present application.

[0011] Further, the nucleotide sequence encoding the RNase inhibitor mutant is shown as SEQ ID NO. 2.

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

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

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

[0015] The present application further discloses a preparation method of the RNase inhibitor mutant, which comprises the following steps:

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

[0017] a2) transforming the recombinant expression plasmid into a host cell, inducing expression to obtain a bacterial body;

[0018] a3) crushing the bacterial body, centrifuging to obtain supernatant, precipitating protein to obtain a crude protein solution;

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

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

[0021] The present application further discloses the application of the RNase inhibitor mutant, the nucleotide sequence, or the recombinant expression plasmid or the recombinant cell in inhibiting RNase activity or preparing a product for inhibiting RNase activity.

[0022] Further, the application can be an application in any of the following:

[0023] b1) an application in the field of molecular in vitro diagnosis;

[0024] b2) an application in RNA extraction;

[0025] b3) an application in in vitro reverse transcription;

[0026] b4) an application in in vitro translation;

[0027] b5) an application in virus detection.

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

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

[0030] The RNase inhibitor mutant of the present application introduces specific amino acid mutations K169N and K205R in 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 application has high stability for long-term storage at 37℃ and the advantage of tolerance to 55℃ for up to 2 hours, and can be applied to various fields of molecular in vitro diagnosis such as RNA extraction, in vitro reverse transcription and translation, and virus detection. BRIEF DESCRIPTION OF DRAWINGS

[0031] The specific embodiments of the present application will be further described in detail below with reference to the accompanying drawings.

[0032] Figure 1 Figure 4 is an SDS-PAGE gel map of the RNase inhibitor mutant of the present application.

[0033] Figure 2 Figure 6 is an electrophoresis map of the RNase inhibitor mutant of the present application after pressure for detection of RNase inhibition activity.

[0034] Figure 3 Figure 8 is an amplification map of one-step qRT-PCR of the RNase inhibitor mutant of the present application after pressure. DETAILED DESCRIPTION

[0035] In order to more clearly illustrate the present application, the present application will be further described below with reference to the preferred embodiments and the accompanying drawings. Like components are denoted by the same reference numerals in the drawings. Those skilled in the art should understand that the specific descriptions below are illustrative and not limiting, and should not limit the scope of protection of the present application.

[0036] Design of RNase inhibitor mutant

[0037] By sequence alignment and sequence logo analysis of two hundred different sources of RNase Inhibitor, it was found that the amino acids at K169 and K205 sites were variable, so these non-conserved amino acids K169 and K205 were mutated to conservative amino acids N169 and R205 (i.e. K169N and K205R) respectively. To improve the stability of 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] The structure stability prediction analysis of wild-type human RNase inhibitor and RNase inhibitor mutant found that the Gibbs free energy of RNase inhibitor mutant was reduced, indicating that its structure was more stable.

[0039] Preparation of RNase inhibitor mutant

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

[0041] The RNase inhibitor mutant recombinant expression plasmid pET28a-RI-Mut was constructed by inserting the nucleotide sequence encoding RNase inhibitor mutant (referred to as RI-Mut) between Ndel and BamHI of pET28a (as shown in SEQ ID NO. 2), and keeping other sequences of pET28a unchanged.

[0042] II. Induction of expression

[0043] The RNase inhibitor mutant recombinant expression plasmid pET28a-RI-Mut was transformed into 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 recombinant expression plasmid pET28a-RI-Mut and add to thawed BL21(DE3) competent cells, ice bath for 30 min.

[0047] 3. After ice bath, put the competent cells into 42°C water bath for 45 s, then put on ice for 2 min.

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

[0049] 5. Take 200 μl cells after incubation in step 4 and spread on kanamycin plate, incubate in 37°C constant temperature incubator overnight.

[0050] 6. Pick one single colony on the kanamycin plate after overnight incubation and add to 20 ml LB medium containing 50 μg / ml kanamycin, put into 37°C shaker at 220 rpm for 7 hr.

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

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

[0053] III. Purification

[0054] After ammonium sulfate precipitation, purify by Ni column and Q column, etc. to obtain RNase inhibitor mutant with high purity.

[0055] The specific steps are as follows:

[0056] 1. Precipitation treatment

[0057] 1) Resuspend the bacterial cells with 30 ml PBS buffer, centrifuge at 12000 rpm for 10 min at 4°C, discard the supernatant to obtain the bacterial cells.

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

[0059] 3) Crush the bacterial cell suspension with PMSF obtained in step 2) by high pressure homogenizer.

[0060] 4) Centrifuge the broken solution from step 3) at 12000 rpm for 30 min at 4°C, discard the precipitate and keep the supernatant.

[0061] 5) Slowly add the ammonium sulfate powder to the supernatant from step 4) under stirring, the saturation concentration is 60%, after the ammonium sulfate is completely dissolved, put it in the refrigerator at 4°C for 1 hr to precipitate the protein.

[0062] 6) Centrifuge the precipitate from step 5) at 12000 rpm for 30 min at 4°C, discard the supernatant and keep the protein precipitate.

[0063] 7) Resuspend the protein precipitate from step 6) with 20 ml Ni-binding buffer, and filter it with a 0.45 μM filter head to obtain the protein sample (crude protein solution).

[0064] 2, NI column purification

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

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

[0067] 3) Load the protein sample from step 1) onto the Ni column.

[0068] 4) Elute the target protein 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 a 5 ml Q pre-packed column to the AKTA system, put the A and B pumps into the filtered and degassed Q-Binding buffer and Q-Elution buffer respectively, and pump the AKTA system.

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

[0073] 3) Load the protein sample after Ni column elution from step 2) onto the Q column.

[0074] 4) Elute the target protein 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 the 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 result was detected by SDS-PAGE gel as shown in Figure 1 From the figure, it can be seen that the size of the protein sample obtained in this embodiment is 52 kDa, which is consistent with the size of the expected RNase inhibitor mutant, indicating that the protein sample obtained in this embodiment is the RNase inhibitor mutant. Further, sequencing verification is correct.

[0077] Example 3 Test of RNase inhibitor mutant after pressure on RNase inhibition activity

[0078] Take 40 U / μl RNase inhibitor mutant (referred to as RI-Mut) without pressure treatment as a control, and test the RNase inhibition activity of 40 U / μl RNase inhibitor mutant after being placed at 37°C for one month and heated at 55°C for 2 hours.

[0079] Prepare the system as in Table 1 and set up duplicate wells, then incubate the system at 37°C for 15 min after preparation, then add 6x DNA Loading Buffer 2 μl, mix well and directly perform agarose gel electrophoresis. The electrophoresis condition is 200 V, 10 min, and immediately take a photo with a gel imager after electrophoresis.

[0080] Table 1 RNase inhibition activity test system

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

[0082] The results are shown in Figure 2 Compared with RI-Mut without pressure, the RNase inhibitor mutant after being placed at 37°C for one month and heated at 55°C for 2 hours can still inhibit 10 ng RNase A and protect RNA from degradation, indicating that the RNase inhibitor mutant has high stability.

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

[0084] The 40U / ul RNase inhibitor mutant (referred to as RI-Mut) without pressure treatment was used as a control, and the 40U / ul RNase inhibitor mutant was heated at 55℃ for 2 hours before one-step qRT-PCR test.

[0085] The qRT-PCR amplification system as shown in Table 2 was prepared, and after the multi-well was set, one-step qRT-PCR was performed, and the amplification procedure is shown in Table 3.

[0086] Table 2 qRT-PCR amplification system

[0087]

[0088]

[0089] Table 3 qRT-PCR amplification procedure

[0090]

[0091] The test results are shown in Table 4. Figure 3 Compared with the RI-Mut without pressure treatment, the RNase inhibitor mutant after heating at 55℃ for 2 hours can still keep the Ct value without obvious change, which indicates that the RNase inhibitor mutant has high stability.

[0092] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. For those skilled in the art, on the basis of the above description, other different forms of changes or variations can also be made, and it is impossible to enumerate all the embodiments here. Any changes or variations derived from the technical solutions of the present application still fall within the protection scope of the present application.

Claims

1. An RNase inhibitor mutant, characterized in that, The RNase inhibitor mutant introduces point mutations into the wild-type RNase inhibitor. The point mutations are represented by a triplet: letter-number-letter, where the number indicates 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 indicates the amino acid used to replace the amino acid before the number: K169N and K205R. The amino acid sequence of the RNase inhibitor mutant is shown in SEQ ID NO.

1.

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. A polynucleotide encoding the RNase inhibitor mutant of claim 1 or 2.

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

2.

5. A recombinant expression plasmid or recombinant cell comprising the polynucleotide of claim 3 or 4.

6. The method for preparing the RNase inhibitor mutant according to claim 1 or 2, characterized in that, The preparation method includes the following steps: a1) Construct a recombinant expression plasmid containing the polynucleotide sequence shown in SEQ ID NO.2; a2) Transform the recombinant expression plasmid into host cells, induce expression, and obtain bacterial cells; a3) Disrupt the bacterial cells, centrifuge to obtain the supernatant, precipitate the protein, and obtain the crude protein solution; a4) The crude protein solution was purified to obtain the RNase inhibitor mutant.

7. The preparation method according to claim 6, characterized in that, The protein precipitate was an ammonium sulfate precipitate.

8. The preparation method according to claim 6, characterized in that, The purification process includes Ni column and Q column purification.

9. The use of the RNase inhibitor mutant of claim 1 or 2, the polynucleotide of claim 3 or 4, the recombinant expression plasmid or recombinant cell of claim 5 in inhibiting RNase activity or in preparing products that inhibit RNase activity.

10. The use of the RNase inhibitor mutant of claim 1 or 2, the polynucleotide of claim 3 or 4, the recombinant expression plasmid or recombinant cell of claim 5, in any of the following: b1) Application in RNA extraction; b2) Application in in vitro reverse transcription.

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