RNaseIII mutant with high stability, gene and preparation method of RNaseIII mutant

Abstract

The invention discloses an RNase III mutant with high stability, a gene and a preparation method of the RNase III mutant. Wherein the RNase III mutant is obtained by mutating the 46th amino acid of a wild type RNase III amino acid sequence from serine to lysine, and the amino acid sequence of the RNase III mutant is as shown in SEQ ID NO: 1. The enzymatic property of the RNase III mutant is the same as that of wild type RNase III, and the pH stability and the temperature stability of the RNase III mutant are obviously higher than those of the wild type RNase III; according to the preparation method of the RNase III mutant, a gene engineering means is adopted, RNase III mutant genes are cloned to an expression vector, host cells are transformed for prokaryotic expression, a large number of high-stability RNase III mutants can be obtained, a basis is provided for preparation of siRNA and development of mRNA vaccines, and the production cost is reduced.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and specifically relates to an RNase III mutant with high stability, a gene and a preparation method thereof. Background Art

[0002] Ribonuclease (RNase) is a key factor in bacterial post-transcriptional regulation, which can control the degradation and steady-state levels of various types of RNA to maintain various life activities. The catalytic characteristics and functions of RNase have been explored in many bacteria. Among them, ribonuclease III (RNase III) is a type of double-stranded specific endoribonuclease, which is widely present in prokaryotes and eukaryotes. Bacterial RNase III can regulate the synthesis of its own mRNA and the degradation and maturation of specific RNA. In Escherichia coli growing in the logarithmic phase, more than 10% of the mRNA steady-state levels are regulated by RNase III. RNase III can cut longer double-stranded RNA into a group of small interfering RNA (siRNA) fragments consisting of 18-25bp in a reaction buffer containing manganese ions, which is more suitable for RNA interference experiments in mammalian cells. RNase III can also degrade residual dsRNA in the development of mRNA vaccines, and plays an important role in the development of mRNA vaccines.

[0003] Wild-type RNaseⅢ has poor stability and low enzyme activity in acidic or alkaline solutions, which is not conducive to reactions in acidic or alkaline substrate solutions. In addition, wild-type RNaseⅢ has good enzyme activity only at 37°C, and performs poorly at room temperature (20°C), which is not conducive to reaction experiments and increases energy consumption and heating equipment requirements. Therefore, a method for mass production of RNaseⅢ suitable for room temperature and with high stability is needed. Summary of the invention

[0004] The technical problem solved by the present invention is to provide a highly stable RNase III mutant, a gene and a preparation method thereof. The enzymatic properties of the RNase III mutant are the same as those of the wild-type RNase III, and its pH stability is significantly higher than that of the wild-type RNase III, and it can be used for room temperature reactions. The preparation method of the RNase III mutant adopts genetic engineering means to clone the RNase III mutant gene into an expression vector, transform the host cell for prokaryotic expression, and obtain a large number of highly stable RNase III mutants, which provide basic raw materials for the preparation of small interfering RNA (siRNA) and the development of mRNA vaccines, thereby reducing production costs.

[0005] In order to solve the above problems, one aspect of the present invention provides a highly stable RNase III mutant, wherein the RNase III mutant is obtained by mutating the 46th amino acid of the wild-type RNase III amino acid sequence from serine (S) to lysine (K), and the amino acid sequence of the RNase III mutant is shown in SEQ ID NO: 1. The present invention mutates the 46th amino acid of the wild-type RNase III whose amino acid sequence is shown in SEQ ID NO: 2, and mutates the 46th amino acid of the wild-type RNase III from serine to lysine. Compared with the wild-type RNase III, the obtained RNase III mutant has similar enzymatic properties, and its pH stability is significantly higher than that of the wild-type RNase III, and has good enzyme activity at room temperature.

[0006] The RNase III mutant has high pH stability and temperature stability, with a pH stability range of pH 4-11 and a temperature stability range of 15-45°C.

[0007] The sequence structure of SEQ ID NO: 1 is as follows:

[0008] MNPIVINRLQRKLGYTFNHQELLQQALTHRSASSKHNERLEFLGDKILSYVIANALYHRFPRVDEGDMSRMRATLVRGNTLAELAREFELGECLLRLGPGELKSGGFRRESILA DTVEALIGGVFLDSDIQTVEKLILNWYQTRLDEISPGDKQKDPKTRLQEYLQGRHLPLPTYLVVQVRGEAHDQEFTIHCQVSGLSEPVVGTGSSRRKAEQAAAEQALKKLELE.

[0009] The sequence structure of SEQ ID NO:2 is as follows:

[0010] MNPIVINRLQRKLGYTFNHQELLQQALTHRSASSKHNERLEFLGDSILSYVIANALYHRFPRVDEGDMSRMRATLVRGNTLAELAREFELGECLLRLGPGELKSGGFRRESILA DTVEALIGGVFLDSDIQTVEKLILNWYQTRLDEISPGDKQKDPKTRLQEYLQGRHLPLPTYLVVQVRGEAHDQEFTIHCQVSGLSEPVVGTGSSRRKAEQAAAEQALKKLELE.

[0011] Another aspect of the present invention provides an RNase III mutant gene with high stability, wherein the RNase III mutant gene encodes the above-mentioned RNase III mutant with high stability.

[0012] The coding gene of the above amino acid sequence is optimized according to the codon preference of Escherichia coli to obtain a DNA sequence suitable for expression in Escherichia coli. Preferably, the nucleotide sequence of the RNase III mutant gene is shown in SEQ ID NO:3.

[0013] The sequence structure of SEQ ID NO:3 is as follows:

[0014] .

[0015] Another aspect of the present invention provides a recombinant vector, the recombinant vector contains the above-mentioned RNase III mutant gene. The recombinant vector can be a plasmid, a derivative of lambda phage or an animal or plant virus, preferably, the recombinant vector is a plasmid. The plasmid is preferably pET28a.

[0016] Another aspect of the present invention provides a host cell, wherein the host cell contains the above-mentioned RNase III mutant gene. The host cell may be one of Escherichia coli, Bacillus subtilis, Agrobacterium tumefaciens, yeast, and animal or plant cells. Preferably, the host cell is Escherichia coli.

[0017] Another aspect of the present invention provides a method for preparing a highly stable RNase III mutant, comprising the following steps:

[0018] S1. constructing a highly stable RNaseⅢ mutant genetically engineered strain, wherein the highly stable RNaseⅢ mutant genetically engineered strain contains the above-mentioned RNaseⅢ mutant gene;

[0019] S2. inoculating the highly stable RNase III mutant genetically engineered strain into a seed culture medium for seed culture;

[0020] S3. Inoculate the highly stable RNase III mutant genetically engineered strain after seed culture into a fermentation medium for fermentation culture to obtain the highly stable RNase III mutant.

[0021] The present invention utilizes a genetic engineering method to first construct a genetic engineering strain having an RNase III mutant gene, and then culture and ferment the genetic engineering strain to achieve prokaryotic expression of the RNase III mutant. A large number of RNase III mutants can be obtained through separation and purification. The enzymatic properties of the RNase III mutant are the same as those of the wild-type RNase III, and the pH stability of the RNase III mutant is significantly higher than that of the wild-type RNase III. The pH application range is wide, and the method can be used for mass production, thereby providing a large amount of raw materials for the development of clinical diagnostic reagents and reducing production costs.

[0022] Preferably, step S1 comprises the following steps:

[0023] S101. synthesizing the RNase III mutant gene;

[0024] S102. Cloning the RNase III mutant gene into an expression vector to obtain a recombinant vector, wherein the expression vector is pET28a;

[0025] S103. Transform the recombinant vector into competent cells, screen positive transformants, and obtain the highly stable RNase III mutant genetic engineering strain, wherein the competent cells are Escherichia coli BL21 (DE3) competent cells.

[0026] Further preferably, step S1 comprises the following steps:

[0027] S101. Synthesize an RNase III mutant gene according to the sequence of SEQ ID NO:3, and amplify the RNase III mutant gene using the RNase III mutant gene as a template through an upstream primer and a downstream primer, wherein the upstream primer has a sequence as described in SEQ ID NO:4, and the downstream primer has a sequence as described in SEQ ID NO:5,

[0028] The sequence structure of SEQ ID NO:4 is as follows:

[0029] 5'-GGGAATTCCATATGAACCCTATCGTCATT-3'.

[0030] The sequence structure of SEQ ID NO:5 is as follows:

[0031] 5'-CCGCTCGAGTTATTCCAGCTCCCAGTTT-3'.

[0032] S102. After double digestion of the amplified product and the expression vector, the amplified product and the expression vector are connected with a ligase to clone the RNase III mutant gene into the expression vector;

[0033] S103. The expression vector is transferred into competent cells, and positive transformants are screened to obtain the highly stable RNase III mutant genetic engineering strain, wherein the competent cells are Escherichia coli BL21 (DE3) competent cells.

[0034] Preferably, step S2 specifically includes:

[0035] The highly stable RNase III mutant genetic engineering strain is inoculated into LB medium and cultured at 27-42° C. and 110-210 rpm for 9-18 hours.

[0036] Further preferably, step S2 is specifically:

[0037] The highly stable RNase III mutant genetic engineering strain was inoculated into LB medium and cultured at 37° C. and 140 rpm for 10 hours.

[0038] Preferably, step S3 comprises the following steps:

[0039] S301. The bacterial solution obtained from the seed culture in step S2 is transferred into a TB liquid culture medium at a volume fraction of 1%-10%, and cultured at 27-42°C and 110-210 rpm stirring conditions until the OD600 is 0.7-0.8;

[0040] S302. The bacterial solution obtained in step S301 is cultured at 8-18°C and 100-200 rpm for 18-20 hours to obtain a fermentation liquid;

[0041] S303. The fermentation broth is centrifuged, the supernatant is removed, potassium phosphate buffer is added to suspend the cells, and the cells are broken;

[0042] S304. The disrupted cells are centrifuged, and the supernatant is purified by affinity chromatography to obtain the highly stable RNase III mutant.

[0043] Further preferably, step S3 comprises the following steps:

[0044] S301. The bacterial solution obtained from the seed culture in step S2 is transferred into 1 L of TB liquid culture medium (50 μg / mL kanamycin) at a volume fraction of 5%, and cultured at 37° C. and 140 rpm stirring conditions until the OD600 is 0.7-0.8;

[0045] S302. The bacterial solution obtained in step S301 is cultured at 8-18°C and 140 rpm for 19-21 hours to obtain a fermentation liquid;

[0046] S303. Centrifuge the fermentation broth at 8000 rpm for 15 min, remove the supernatant, add 0.1 M potassium phosphate to suspend the cells, and crush the cells using an ultrasonic cell crusher in an ice bath;

[0047] S304. The disrupted cells were centrifuged at 4°C and 10,000 rpm for 30 min, the supernatant was collected and purified by affinity chromatography to obtain the highly stable RNase III mutant.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] 1. The present invention mutates the 46th amino acid of the wild-type RNase III from serine to lysine. The obtained RNase III mutant has similar enzymatic properties to the wild-type RNase III and the existing imported RNase III, and its pH stability is significantly higher than that of the wild-type RNase III. It has a wide temperature application range, a pH stability range of pH 4-11, and a temperature stability range of 15-45°C.

[0050] 2. The present invention utilizes genetic engineering methods to first construct a genetically engineered strain having an RNase III mutant gene, and then culture and ferment the genetically engineered strain to achieve prokaryotic expression of the RNase III mutant. A large number of RNase III mutants can be obtained through separation and purification. The enzymatic properties of the RNase III mutant are the same as those of the wild-type RNase III, and its pH stability is significantly higher than that of the wild-type RNase III. It has a wide temperature application range and can be mass-produced by this method, providing a large amount of raw materials for the development of clinical diagnostic reagents and reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the electrophoresis diagram of PCR identification of the mutant genetic engineering strain in Example 2 of the present invention, wherein lane 1 is the standard DNA molecular weight, and lane 2 is the RNase III gene in the mutant genetic engineering strain.

[0052] Figure 2 This is the SDS-PAGE electrophoresis diagram of the purified highly stable RNase III mutant in Example 2 of the present invention. Lane 1 is the standard protein molecular weight; Lane 2 is the lysate before passing through the nickel column; Lane 3 is the effluent after passing through the nickel column; Lane 4 is the purified highly stable RNase III mutant.

[0053] Figure 3 This is an electrophoresis diagram of the degradation effect of mutant and wild-type RNase III on dsRNA in Example 3 of the present invention. A is the electrophoresis result of dsRNA degradation by wild-type RNase III, B is the electrophoresis result of dsRNA degradation by mutant RNase III, lane 1 is the standard protein molecular weight; lane 2 is dsRNA; lane 3 is degraded by RNase III.

[0054] Figure 4 This is the pH stability curve of the highly stable RNase III mutant in Example 3 of the present invention.

[0055] Figure 5 This is the temperature stability curve of the highly stable RNase III mutant in Example 3 of the present invention. DETAILED DESCRIPTION

[0056] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] Example 1 Design of RNase III mutant gene with high stability

[0058] Based on the amino acid sequence of the RNase III mutant with high stability of the present invention, the sequence is optimized according to the codon preference of Escherichia coli to obtain a RNase III mutant gene with high stability. The amino acid sequence of the RNase III mutant with high stability is shown in SEQ ID NO: 1, and the nucleotide sequence of the optimized RNase III mutant gene is shown in SEQ ID NO: 3.

[0059] The sequence structure of SEQ ID NO: 1 is as follows:

[0060] MNPIVINRLQRKLGYTFNHQELLQQALTHRSASSKHNERLEFLGDKILSYVIANALYHRFPRVDEGDMSRMRATLVRGNTLAELAREFELGECLLRLGPGELKSGGFRRESILA DTVEALIGGVFLDSDIQTVEKLILNWYQTRLDEISPGDKQKDPKTRLQEYLQGRHLPLPTYLVVQVRGEAHDQEFTIHCQVSGLSEPVVGTGSSRRKAEQAAAEQALKKLELE.

[0061] The sequence structure of SEQ ID NO:3 is as follows:

[0062] .

[0063] Example 2 Preparation of RNase III mutants with high stability

[0064] The method for preparing the highly stable RNase III mutant described in this embodiment comprises the following steps:

[0065] 1. Construction of highly stable RNaseⅢ mutant genetic engineering strains:

[0066] 1) performing full gene synthesis on the RNase III mutant gene according to the sequence of SEQ ID NO:3, and amplifying the RNase III mutant gene using the RNase III mutant gene as a template using an upstream primer and a downstream primer, wherein the upstream primer has a sequence as described in SEQ ID NO:4, and the downstream primer has a sequence as described in SEQ ID NO:5,

[0067] The sequence structure of SEQ ID NO:4 is as follows:

[0068] 5'-GGGAATTCCATATGAACCCTATCGTCATT-3'.

[0069] The sequence structure of SEQ ID NO:5 is as follows:

[0070] 5'-CCGCTCGAGTTATTCCAGCTCCCAGTTT-3'.

[0071] 2) The amplified product and the expression vector (pET28a) were double-digested and then ligated with ligase TaKaRa, and the RNaseⅢ mutant gene was cloned into the expression vector pET28a to obtain a recombinant plasmid, which was recorded as pET28a-RNaseⅢ.

[0072] 3) The recombinant plasmid pET28a-RNaseⅢ was transformed into Escherichia coli BL21 (DE3) competent cells (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and positive transformants were screened on kanamycin resistance plates to obtain highly stable RNaseⅢ mutant genetic engineering strains. The results of PCR identification and electrophoresis of mutant genetic engineering strains are shown in Figure 1 shown.

[0073] 2. Seed culture of highly stable RNaseⅢ mutant genetically engineered strains:

[0074] The monoclonal RNaseⅢ mutant genetic engineering strain obtained by screening was selected and inoculated into LB medium and cultured at 37°C and 120 rpm for 12 h.

[0075] 3. Fermentation culture of highly stable RNaseⅢ mutant genetically engineered strains:

[0076] 1) The bacterial solution obtained from the seed culture was transferred into 1 L of TB liquid medium (50 μg / mL kanamycin) at a volume fraction of 5%, and cultured at 37°C and 120 rpm until the OD600 was 0.7-0.8;

[0077] 2) transferring the bacterial solution obtained in step 1) to 16° C. and 120 rpm and culturing for 18-20 hours to obtain a fermentation liquid;

[0078] 3) The fermentation broth was centrifuged at 6000 rpm for 20 min, the supernatant was removed, 0.05 M Tris-HCl (pH 8.0) was added to buffer the suspended cells, and the recombinant E. coli was broken using a homogenizer under ice bath conditions;

[0079] 4) The broken suspension was centrifuged for 30 minutes at 4°C and 10,000 rpm, and the supernatant was taken for centrifugation. The RNase III mutant in the supernatant was purified by affinity chromatography to obtain a highly stable RNase III mutant. The affinity chromatography method was as follows: first, the equilibrated nickel column was rinsed with 0.05M Tris-HCl (pH8.0) buffer, and the sample was loaded at a flow rate of 5 mL / min; after loading, the impurities were washed away with 0.05M Tris-HCl (pH8.0, containing 15mM imidazole) buffer; and the RNase III mutant was eluted with 0.05M Tris-HCl (pH8.0, 500mM imidazole) buffer. The eluted RNase III mutant was desalted by a G25 gel column to obtain a pure highly stable RNase III mutant, which was stored at -20°C. The SDS-PAGE analysis results of the highly stable RNase III mutant are shown in the figure. Figure 2 As shown by Figure 2 It can be seen that the purification effect has been achieved.

[0080] Example 3 Determination of the enzymatic properties of highly stable RNase III mutants

[0081] RNaseⅢ mutant activity detection method:

[0082] Step 1: Prepare the reaction mixture: 2 μL of 1 μg / μL dsRNA, 2 μL of 0.5M Tris-HCl (pH 8.0), 2 μL of 0.5M MnCl2, 2 μL of diluted RNase III, and 12 μL of sterile water. The reaction mixture should be prepared and placed on ice.

[0083] Step 2: Incubate at 37°C for 20 min, add 2 μL of 500 mM EDTA in the reaction stop solution to inactivate RNase III. Figure 3 As shown), after 20 min, the reaction of wild-type RNase III substrate was not complete (as shown Figure 3 A), there are still incompletely reacted bands, while the mutant RNase III bands reacted completely (such as Figure 3 B), are degraded into small fragments of 18-25 bp.

[0084] The enzyme activity of RNase III mutant was defined as the amount of enzyme required to degrade 1 μg dsRNA into siRNA within 20 minutes at 37°C.

[0085] Calculation formula for RNaseⅢ mutant activity:

[0086] Enzyme activity (U / L) = [luminescence value of siRNA / (luminescence value of siRNA+luminescence value of dsRNA)] × 2 (unit IU).

[0087] pH stability: The enzyme solution was diluted with 0.5 M buffer of different pH values, placed at 37°C for 1 hour, and the enzyme activity was determined according to the standard enzyme activity determination method. Plot the pH value as the horizontal axis and the enzyme activity as the vertical axis (e.g. Figure 4 The results showed that the wild-type RNaseⅢ maintained a high stability at pH 7-9, and the pH dropped rapidly in buffers with a pH lower than 7 and higher than 9. The mutant RNaseⅢ maintained a high stability at pH 4-11. This indicates that the RNaseⅢ mutant has a wide range of pH stability and its enzyme activity is very stable under both weakly acidic and weakly alkaline conditions.

[0088] Temperature stability: The reaction system was placed at different temperatures for 20 min, and the enzyme activity was measured according to the standard activity measurement method. The temperature was used as the horizontal axis and the enzyme activity was used as the vertical axis (e.g. Figure 5 The results showed that the wild-type RNaseⅢ maintained high stability between 30-40℃, and its activity decreased rapidly below 30℃ and above 40℃. The RNaseⅢ mutant could maintain good activity between 15-45℃. The RNaseⅢ mutant had high temperature stability and could react at room temperature (20℃).

[0089] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A highly stable RNase III mutant, characterized in that: The RNase III mutant is obtained by mutating the 46th amino acid in the wild-type RNase III amino acid sequence from serine to lysine. The amino acid sequence of the RNase III mutant is shown in SEQ ID NO:

1.

2. The RNase III mutant with high stability according to claim 1, characterized in that: The RNase III mutant has high pH stability and temperature stability, with a pH stability range of pH 4-11 and a temperature stability range of 15-45°C.

3. A highly stable RNase III mutant gene, characterized in that: The RNase III mutant gene encodes the RNase III mutant with high stability as claimed in claim 1.

4. The RNase III mutant gene with high stability according to claim 3, characterized in that: The nucleotide sequence of the RNaseⅢ mutant gene is shown in SEQ ID NO:

3.

5. A recombinant vector, characterized in that: The recombinant vector contains the RNase III mutant gene as described in claim 3 or 4.

6. A host cell, characterized in that The host cell contains the RNase III mutant gene as described in claim 3 or 4.

7. A method for preparing a highly stable RNase III mutant, characterized in that: The following steps are involved: S1. constructing a genetically engineered strain containing the RNase III mutant gene as described in claim 3 or 4; S2. inoculating the genetically engineered strain into a seed culture medium to obtain a bacterial solution; S3. Inoculate the bacterial liquid into a fermentation medium for fermentation culture to obtain the highly stable RNaseⅢ mutant.

8. The method for preparing a highly stable RNase III mutant according to claim 7, characterized in that: Step S1 specifically comprises: cloning the RNase III mutant gene as described in claim 3 or 4 into pET28a to obtain a recombinant vector, transforming the recombinant vector into Escherichia coli BL21 (DE3) competent cells, screening positive transformants, and obtaining a genetically engineered strain.

9. The method for preparing a highly stable RNase III mutant according to claim 7, characterized in that: Step S2 specifically includes: inoculating the genetically engineered strain into LB culture medium, culturing for 9-18 hours at 27-42° C. and 110-210 rpm to obtain a bacterial solution.

10. The method for preparing a highly stable RNase III mutant according to claim 7, characterized in that: Step S3 is specifically as follows: 1) Inoculate the bacterial solution into TB liquid culture medium at a volume fraction of 1-10%, and culture at 27-42°C and 110-210 rpm until the OD600 is 0.7-0.8; 2) culturing the bacterial solution obtained in step 1) at 8-18° C. and 110-210 rpm for 16-18 hours to obtain a fermentation solution; 3) The fermentation broth is centrifuged, the cells are broken, and centrifuged again. The supernatant is purified by affinity chromatography to obtain the highly stable RNase III mutant.

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