CN116496379A - Thermal stability Rnase inhiba mutant, preparation method and application - Google Patents
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of biology, in particular to a thermostable Rnaseinhibator mutant, a preparation method and application thereof, wherein the amino acid sequence of the Rnaseinhibator mutant is shown as SEQ ID NO. 1. The activity is equivalent to that of a wild murine RI, but the heat stability is obviously improved, and the heat resistance can reach 75 ℃; the temperature is kept for 12 hours at 55 ℃, the residual activity is more than 60 percent, and good conditions are provided for RNA high-temperature reaction.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a thermal stability Rnase inhibator mutant, a preparation method and application thereof.
Background
RI (RNase inhibitor) is a cytoplasmic protein that is widely found in a variety of mammalian tissues. It inhibits various ribonucleases (RNases) by binding tightly to intracellular and extracellular RNases. Some evidence suggests that RI is higher than RNase in tissues highly active in protein synthesis, while RI is significantly lower than RNase in catabolically active tissues where RNA accumulation is not required, and is widely used in various molecular biological applications to prevent RNase enzyme contamination due to its specific function in inhibiting RNase activity; specific fields of application include reverse transcription of mRNA, cell-free translation systems, preparation of RNase-free antibodies and in vitro viral replication. Ideally, RI for such applications would be able to inhibit a large number of rnases, such as eukaryotic RNase a, RNase B and RNase C, as well as prokaryotic rnases. In the prior art, naturally-derived RI or recombinant expressed porcine RI, human RI and murine RI are mostly used. When the temperature increases, the binding between RI and RNase becomes weak, and part of the RNase activity is released, so that a thermostable RNase inhibator is required.
Disclosure of Invention
In order to overcome the defects of the technology, the invention provides a thermostable RNase inhibitor mutant, a preparation method and application thereof, which are applied to a wild mouse RNase inhibitor by using a structure biological technology and a site-directed mutagenesis technology.
The thermostable Rnase inhibator mutant provided by the invention is a protein which is formed by inserting, substituting or deleting 1 or more amino acids in an amino acid sequence shown in SEQ ID NO.3, or adding or deleting 1 or more amino acids at one or two ends of the amino acid sequence shown in SEQ ID NO.3, has 80% identity with SEQ ID NO.3 and has enhanced MRI function and thermostability. Preferably 90% identical, more preferably 95% identical, and most preferably 99% identical.
In some embodiments, the Rnase inhibator mutants of the present invention have optimal activity at temperatures greater than 37 ℃, in some embodiments, the Rnase inhibator mutants have optimal activity at least 42 ℃, preferably at least 50 ℃, more preferably at least 60 ℃. In some specific embodiments, the Rnase inhiba mutant has an activity of at least 120% at 37 ℃ and preferably at least 130% and more preferably at least 150% at 50 to 65 ℃ of 37 ℃.
In some embodiments, the Rnase inhibator mutant of the present invention has a heat tolerance of no less than 50 ℃, and in some embodiments, the Rnase inhibator mutant of the present invention has a heat tolerance of no less than 60 ℃.
In some more specific implementations, the invention provides an Rnase inhiba mutant, the amino acid sequence of which is shown in SEQ ID No.1, or an amino acid sequence with 80% identity to the sequence shown in SEQ ID No.1, having MRI function and enhanced thermostability; preferably 85% identical, more preferably 90% identical, more preferably 95% identical, and most preferably 99% identical.
The invention also provides a nucleotide sequence for encoding the Rnase inhiba mutant of the invention, and it is well known to those skilled in the art that the nucleotide sequence for encoding the Rnase inhiba mutant is not limited to one type, and can be obtained by mutating one or more nucleotides from the nucleotide sequence of the Rnase inhiba mutant shown in SEQ ID NO.1 to form synonymous mutation, and can also encode the amino acid sequence of the mutant of the invention, or can be designed according to codon optimization.
In some specific embodiments, the invention also provides a nucleotide sequence encoding an Rnase inhiba mutant, which may be as shown in SEQ ID No.2, or a nucleotide sequence encoding an Rnase inhiba mutant amino acid sequence of the invention may be obtained by mutating one or more nucleotides of an Rnase inhiba mutant nucleotide sequence as shown in SEQ ID No.1 to form a synonymous mutation, or may be designed according to codon optimization. The sequence is a sequence obtained by codon optimization for an escherichia coli expression system, and can remarkably improve the expression efficiency of heterologous genes in host bacteria.
The invention also provides a recombinant vector comprising a nucleotide sequence encoding the Rnase inhibator mutant of the invention. The parent vector used to construct the expression vector of the present invention is not limited, and any conventional vector used for transformation of prokaryotes or eukaryotes may be used. Preferably a prokaryotic expression vector; more preferably pET series vectors; more preferably pET-28a.
The invention also encompasses a recombinant cell which can be constructed by inserting the recombinant vector into a random prokaryotic or eukaryotic cell, comprising a nucleotide sequence encoding the Rnase inhibator mutant of the invention, or a vector comprising a nucleotide sequence encoding the Rnase inhibator mutant. The engineering cell is preferably an escherichia coli cell; BL21 (DE 3) cells are more preferred. The recombinant engineering cell strain can express the recombinant expression vector rapidly and solubly.
On the other hand, the invention also provides a method for obtaining the Rnase inhibator mutant, which comprises the following steps:
mutant construction: the gene sequence of the MRI female parent is shown as SEQ ID NO.3, and is obtained by using gene synthesis and synthesized on a vector pET32a to generate pET32a-MRI plasmid, wherein the specific synthesis sequence comprises a purification tag 6 xHis sequence added at the 3' end and a TAA stop codon, the plasmid is transformed into competent cells of escherichia coli BL21 to carry out expression host construction, and the plasmid is induced to be expressed to generate MRI with the C-terminal fused 6 xHis purification tag. Construction of MRI mutant by taking plasmid pET32a-MRI as original template, and performing site-directed mutagenesis on MRI encoding gene according to PCR-mediated site-directed mutagenesis method. The QuikChange Primer Design website is utilized to design a mutation primer, and a reaction system (50 mu L) of site-directed mutagenesis PCR is carried out on the MRI encoding gene according to a PCR-mediated site-directed mutagenesis method. After mixing the components in Table 1-1 on ice, PCR amplification was performed as follows: (1) pre-denaturation at 98℃for 3min; (2) denaturation at 98℃for 20s, annealing at 60℃for 30s, and extension at 68℃for 6min; (3) step 2 is performed for 30 cycles; (4) extending at 68 ℃ for 10min; 5) Preserving heat at 4 ℃. After the reaction according to this procedure, a mixture of the amplified product and the template DNA is obtained. To 10. Mu.L of PCR amplification product, 1. Mu.L of DpnI and 9. Mu.L of the corresponding buffer were added according to the instructions of using DpnI digestive enzyme, and incubated overnight at 37℃to digest the template DNA molecule thoroughly. The overnight digested product was subjected to preliminary purification treatment using the Tiangen universal DNA purification recovery kit (DP 214), for specific steps as described in the specification. The purified PCR product is transformed into E.coli DH5 alpha competent cells, and the specific steps are as follows: (1) Taking 50 mu L of E.coli DH5 alpha competent cells out of a refrigerator at the temperature of minus 80 ℃ and placing the competent cells on ice, and standing for 5min to melt the competent cells; (2) Adding 10 mu L of purified PCR product into competent cells, gently mixing, and standing on ice for 60min; (3) Placing competent cells in a water bath at 42 ℃ for 45s, rapidly taking out, placing back on ice, and standing for 5min; (4) Adding 500 mu L of LB liquid medium without antibiotics, and incubating at 37 ℃ for 1-2h; (5) The liquid containing competent cells was centrifuged at 5000 Xg for 1min at 4℃to remove 400. Mu.L of supernatant; (6) Mixing 160 μl of liquid containing competent cells, uniformly coating on LB plate containing 100 μg/mL ampicillin, standing until the liquid is completely absorbed, and culturing at 37deg.C overnight;
screening of mutants: 6-10 individual clones per LB plate were inoculated overnight into LB medium containing 100. Mu.g/mL ampicillin, and plasmids were extracted using Plasmid DNAMidiprep Kits kit. The plasmid was sent to gene sequencing. Comparing the sequencing result by adopting SnapGene software, and converting the plasmid with correct sequencing into E.coli BL21 (DE 3) competent cells to obtain recombinant bacteria containing the target mutant genes;
fermentation and purification of mutants: inoculating the recombinant bacteria containing the target mutant genes into 1mL of LB liquid medium containing 50 mug/mL ampicillin, performing shake culture and activation at 37 ℃ and 180rpm until the OD value is 0.5-0.6, adding inducer IPTG with the final concentration of 0.05mM, continuously culturing at 28 ℃, and inducing enzyme expression; and (3) centrifugally collecting fermentation thalli after 10 hours, taking the thalli after fermentation, re-suspending the thalli by using a Ni affinity chromatography column combined buffer solution, crushing the thalli by using a cell high-pressure homogenizing crusher, centrifugally collecting crushed supernatant at a low temperature and high speed, and purifying and collecting the supernatant by using a Ni affinity chromatography purification column in an environment of 4 ℃ to obtain the RI mutant pure enzyme. The pure enzyme was stored using a storage buffer formulation of 15mM Tris-HCl, pH 7.5, 50mM KCl,0.1mM EDTA,10mM DTT,50% glycerol at-20 ℃.
The invention also provides application of the Rnase inhibator mutant, the recombinant vector or the recombinant cell in the field of biotechnology.
The invention also provides application of the Rnase inhibator mutant, the recombinant vector or the recombinant cell in the field of reverse transcription reaction.
The invention has the beneficial effects that: according to the invention, a new mutation site is found, and a molecular rationality design and functional screening are combined, so that a Rnase inhibator mutant with thermal stability is obtained after screening in a smaller range, the activity is equivalent to that of a wild murine RI, but the thermal stability is obviously improved, and the heat resistance can reach 75 ℃; the temperature is kept for 12 hours at 55 ℃, the residual activity is more than 60 percent, and good conditions are provided for RNA high-temperature reaction.
Drawings
FIG. 1 is a graph showing the activity of RNase inhibitors;
FIG. 2 is a thermal stability assay for RNase inhibitors.
Detailed Description
The present invention will be described in detail with reference to specific embodiments thereof, so that those skilled in the art can better understand the technical solutions of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. Percentages and parts are by weight unless otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.
EXAMPLE 1 mutant construction
The gene sequence of the MRI female parent is shown as SEQ ID NO.3, and is obtained by using gene synthesis and synthesized on a vector pET32a to generate pET32a-MRI plasmid, wherein the specific synthesis sequence comprises a purification tag 6 xHis sequence added at the 3' end and a TAA stop codon, the plasmid is transformed into competent cells of escherichia coli BL21 to carry out expression host construction, and the plasmid is induced to be expressed to generate MRI with the C-terminal fused 6 xHis purification tag. Construction of MRI mutant by taking plasmid pET32a-MRI as original template, and performing site-directed mutagenesis on MRI encoding gene according to PCR-mediated site-directed mutagenesis method. The QuikChange Primer Design website is utilized to design a mutation primer, and a reaction system (50 mu L) of site-directed mutagenesis PCR is carried out on the MRI encoding gene according to a PCR-mediated site-directed mutagenesis method. After mixing the components in Table 1-1 on ice, PCR amplification was performed as follows: (1) pre-denaturation at 98℃for 3min; (2) denaturation at 98℃for 20s, annealing at 60℃for 30s, and extension at 68℃for 6min; (3) step 2 is performed for 30 cycles; (4) extending at 68 ℃ for 10min; 5) Preserving heat at 4 ℃. After the reaction according to this procedure, a mixture of the amplified product and the template DNA is obtained. To 10. Mu.L of PCR amplification product, 1. Mu.L of DpnI and 9. Mu.L of the corresponding buffer were added according to the instructions of using DpnI digestive enzyme, and incubated overnight at 37℃to digest the template DNA molecule thoroughly. The overnight digested product was subjected to preliminary purification treatment using the Tiangen universal DNA purification recovery kit (DP 214), for specific steps as described in the specification. The purified PCR product is transformed into E.coli DH5 alpha competent cells, and the specific steps are as follows: (1) Taking 50 mu L of E.coli DH5 alpha competent cells out of a refrigerator at the temperature of minus 80 ℃ and placing the competent cells on ice, and standing for 5min to melt the competent cells; (2) Adding 10 mu L of purified PCR product into competent cells, gently mixing, and standing on ice for 60min; (3) Placing competent cells in a water bath at 42 ℃ for 45s, rapidly taking out, placing back on ice, and standing for 5min; (4) Adding 500 mu L of LB liquid medium without antibiotics, and incubating at 37 ℃ for 1-2h; (5) The liquid containing competent cells was centrifuged at 5000 Xg for 1min at 4℃to remove 400. Mu.L of supernatant; (6) 160. Mu.L of liquid containing competent cells was mixed and spread evenly on LB plate containing 100. Mu.g/mL ampicillin, and after all the liquid was absorbed, the plate was incubated at 37℃overnight with inversion.
EXAMPLE 2 screening of mutants
6-10 individual clones per LB plate were inoculated overnight into LB medium containing 100. Mu.g/mL ampicillin, and plasmids were extracted using Plasmid DNAMidiprep Kits kit. Carrying out gene transfer sequencing on plasmids (the amino acid sequence of an RI mutant (V3) is shown as SEQ ID NO.1, the amino acid sequence of an RI mutant (V7) is shown as SEQ ID NO. 4), comparing and processing the sequencing result by adopting SnapGene software, and converting plasmids with correct sequencing into E.coli BL21 (DE 3) competent cells to obtain recombinant bacteria containing target mutant genes;
EXAMPLE 3 fermentation and purification of mutants
Inoculating the recombinant bacteria containing the target mutant genes into 1mL of LB liquid medium containing 50 mug/mL ampicillin, performing shake culture and activation at 37 ℃ and 180rpm until the OD value is 0.5-0.6, adding inducer IPTG with the final concentration of 0.05mM, continuously culturing at 28 ℃, and inducing enzyme expression; and (3) centrifugally collecting fermentation thalli after 10 hours, taking the thalli after fermentation, re-suspending the thalli by using a Ni affinity chromatography column combined buffer solution, crushing the thalli by using a cell high-pressure homogenizing crusher, centrifugally collecting crushed supernatant at a low temperature and high speed, and purifying and collecting the supernatant by using a Ni affinity chromatography purification column in an environment of 4 ℃ to obtain the RI mutant pure enzyme. The pure enzyme was stored using a storage buffer formulation of 15mM Tris-HCl, pH 7.5, 50mM KCl,0.1mM EDTA,10mM DTT,50% glycerol at-20 ℃.
EXAMPLE 4 determination of RNase inhibitor Activity
The determination is described in the literature (J.biol. Chem.252:5904-5910 (1977)). The RNase inhibitor activity was measured by inhibiting 50% of 5ng of RNaseA activity, and the results are shown in FIG. 1. It was shown that after 25min incubation, the amount of enzyme required for RI mutant (V3) and RI mutant (V7) was not different from that of wild-type MRI, showing that the mutation sites did not cause a decrease in enzyme activity.
EXAMPLE 5 measurement of thermal stability of RNase inhibitor
RI mutant and corresponding parent MRI were taken, diluted with storage buffer to an enzyme activity concentration of 200U/μl, incubated at 35-65 ℃ for 30min, and incubated at 5 ℃ as an incremental unit for a total of 6 gradient increasing temperatures. The activity of RNaseA was measured at different temperatures, and the activity of RNase inhibitor after incubation at different temperatures was measured and compared with commercially available RNase inhibitor, and the results are shown in FIG. 2. The RI mutant (V3) is shown to show strong inhibition ability at 65 ℃ and can protect RNA from RNase degradation at high temperature, and the performance of the RI mutant is obviously superior to that of commercial products.
Finally, it should be noted that the above description is only a preferred embodiment of the present invention, and that many similar changes can be made by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (6)
1. A thermostable Rnase inhiba mutant characterized by: the amino acid sequence is shown as SEQ ID NO. 1.
2. A DNA molecule encoding the mutant of claim 1, wherein: the nucleotide sequence is shown as SEQ ID NO. 2.
3. A recombinant expression vector, characterized in that: comprising the Rnase inhiba mutant nucleotide sequence of claim 1.
4. A recombinant cell, characterized in that: is obtained by transforming the recombinant expression vector of claim 3 into an engineered cell.
5. A method of making the Rnase inhiba mutant of claim 1.
6. Use of an Rnase inhibator mutant according to claim 1, a recombinant vector according to claim 3 or a recombinant cell according to claim 4 in the field of reverse transcription reactions.
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| BOMBACI G，等: "NP_001165571", GENBANK * |
| LOMAX, J.E. 等: "3TSR_E", GENBANK * |
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