CN118207181A - T4 phage recombinase UvsX mutant with wide temperature applicability and application thereof - Google Patents
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Abstract
The invention relates to a T4 phage recombinase UvsX mutant with wide temperature applicability and application thereof, belonging to site-directed mutagenesis of proteins by utilizing protein modification technology based on genetic engineering. The amino acid sequence of the mutant F203YV244A is shown as SEQ ID No.1, and the mutant F203YV244A replaces wild UvsX to be applied to conventional RPA in-vitro nucleic acid amplification reaction within a wider temperature range. The improved UvsX protein mutant F203YV244A can replace wild UvsX protein to perform RPA reaction, and the optimal reaction temperature range of the whole reaction is enlarged to 20-45 ℃ while the same amplification effect is achieved, so that the enzyme performance in a wide temperature range is enhanced, and the practical application scene and the use limit of the RPA reaction are widened.
Description
Technical Field
The invention belongs to a method for carrying out site-directed mutagenesis on protein by utilizing a protein transformation technology based on genetic engineering, and forms a T4 phage recombinase UvsX mutant with wide temperature adaptability and application thereof.
Background
The isothermal nucleic acid amplification technology is a novel technology introduced after the PCR technology, and provides simplified culture conditions for in vitro nucleic acid amplification. Among them, the Recombinase Polymerase Amplification (RPA) has the advantages of simple equipment requirement, fast reaction time, high sensitivity, good specificity and the like due to isothermal property, and is developed very rapidly. The reaction mechanism of the method depends on the homologous recombination process of natural cells, takes a T4 phage DNA replication mechanism system as a blue book, and suggests that the reaction temperature is between 37 ℃ and 42 ℃ and the reaction time is 15-30 min.
The UvsX protein is a DNA recombinase encoded by T4 phage and plays an important role in repair of DNA double strand breaks and restarting of replication forks, and is combined with upstream and downstream primers to form nucleoprotein filaments in the RPA reaction process, the obtained complex searches for homologous sequences in double-stranded DNA, once the homologous sequences are positioned, the complex invades the double-stranded DNA to form a D-ring structure, and the primers hybridize with template chains to initiate a strand exchange reaction.
The current techniques for isothermal amplification of recombinant polymerase based on the development of the company TwistDx Inc in the United kingdom in 2006 (Recombinase Polymerase Amplification, RPA) are described in detail in the United kingdom under the company TwistDx Inc "Recombinase Polymerase Amplification ASSAY DESIGN" (https:// www.twistdx.co.uk/en/support /). The optimum temperature of the reaction is about 37 ℃, and the amplification speed and the sensitivity are reduced at other temperatures. The basic response mechanism of RPA relies on the process of homologous recombination in the natural cellular DNA metabolism, requiring T4 phage DNA recombinase (UvsX), recombinase cofactor (UvsY), single chain binding protein (SSB) three critical proteins. UvsX is combined with a primer with the aid of UvsY to form a nucleoprotein filament, the obtained complex searches for a homologous sequence in double-stranded DNA, once the complex is found, the complex invades the double-stranded DNA to displace one DNA strand to form a D-ring structure, hybridization of the primer and a template strand starts a subsequent strand exchange reaction, the rest of the DNA strand in the displacement is stabilized by SSB, at the moment, uvsX completes its task, and is unloaded from the DNA, so that polymerase completes subsequent nucleic acid amplification. UvsX can be well combined with single-stranded DNA at an adaptive temperature, so that the stable operation of the whole system is ensured, and the in-vitro isothermal nucleic acid amplification is completed.
In the RPA reaction process, the formation of a recombinase-primer complex is a main speed limiting step, the reaction temperature is the most important external determining factor in the RPA reaction, the recommended temperature is limited to 37-42 ℃, and too low or too high temperature can cause the loss of enzyme activity in the preparation, so that the final presented result of a reaction system is affected. In the practical application process, the RPA reaction is influenced by the environmental temperature and the use limitation of instruments, so that false negative or false positive phenomena occur in the RPA reaction, and the wider reaction temperature is needed to ensure that the RPA reaction is normally carried out under various conditions.
Since the UvsX protein of the RPA starting from the T4 phage is combined with the primer to form the nucleoprotein filament to search for a homologous sequence in the double-stranded DNA, in order to ensure that the protein has better applicability, the applicable temperature of the UvsX needs to be widened, if the diagnosis of a sample can be efficiently completed under wider environmental conditions, the field application of the protein is greatly promoted, and the detection efficiency is improved.
Disclosure of Invention
The invention provides a T4 phage recombinant enzyme UvsX mutant with wide temperature applicability and application thereof, so as to improve detection stability and sensitivity of RPA reaction under the condition of wide temperature range.
The technical scheme adopted by the invention is as follows:
the amino acid sequence of the T4 phage recombinase UvsX mutant F203YV244A with wide temperature applicability is shown in SEQ ID No. 1.
The nucleotide sequence of the T4 phage recombinase UvsX mutant F203YV244A with wide temperature applicability is synthesized by using plasmid pET-28a in the escherichia coli BL21 (DE 3) as shown in SEQ ID No. 2.
The nucleotide sequence of the T4 phage recombinase UvsX mutant F203YV244A with wide temperature applicability is synthesized in the bacillus subtilis DB1342 by utilizing a plasmid pHT43 and is shown in SEQ ID No. 3.
The application of the T4 phage recombinase UvsX mutant F203YV244A with wide temperature applicability replaces the application of the wild type UvsX in the conventional RPA in-vitro nucleic acid amplification reaction within a wider temperature range.
The broader temperature range is 20-45 ℃.
The beneficial effects of the invention are as follows: the invention can obtain UvsX protein mutant F203YV244A with wide temperature adaptability by carrying out base site-specific mutation on protease UvsX, provides an original protein reagent for RPA reaction, can be combined on an upstream primer and a downstream primer under the conditions of lower temperature (constant temperature) and higher temperature (constant temperature), and can enable the primer to be replaced with a target section. The enzyme of the invention can be applied to the RPA reaction, and can also be applied to common PCR or other isothermal amplification technologies such as rolling circle nucleic acid amplification, loop-mediated isothermal amplification and the like, thus the invention enhances the performance of the enzyme in a wide temperature range, widens the application of the enzyme and overcomes the condition limitation of in vitro nucleic acid amplification.
The modified UvsX protein mutant F203YV244A designed and improved in the invention can replace wild UvsX protein to carry out RPA reaction, and can expand the optimal reaction temperature range of the whole reaction to 20-45 ℃ while achieving the same amplification effect, thereby enhancing the enzyme performance in a wide temperature range and widening the practical application scene and the use limit of the RPA reaction.
Drawings
FIG. 1 is an electrophoretogram of purified proteins expressed by wild-type and mutant UvsX;
FIG. 2 is a nucleic acid electrophoretogram of RPA amplification of Staphylococcus aureus at different temperatures;
FIG. 3 is a nucleic acid electrophoretogram of Salmonella RPA amplification at various temperatures.
Detailed Description
Expression and purification of T4 phage recombinase UvsX mutant F203YV244A with wide temperature applicability
(1) Recombinant expression of mutant genes
Extraction of plasmids: inoculating BL21 (DE 3) strain into 10ml LB liquid culture medium, adding kanamycin into the culture medium according to the ratio of 1000:1, shaking overnight at 37 ℃ for culture, and extracting pET-28a plasmid in the bacterial liquid by using a plasmid extraction kit;
Construction of expression plasmids: 50. the micro L enzyme cutting system comprises 10 mu L of plasmid, 5 mu L of buffer solution 10 XH buffer,1 mu L of restriction enzymes BamH1 and Xhol and sterile water; 37. performing enzyme digestion at the temperature of 30 min;
Integration of vector and UvsX mutant genes: 1. the enzyme-digested plasmid obtained in the previous step of the [ mu ] L, 7 [ mu ] L gene fragments, 1 [ mu ] L buffer solution 10×T4 digestion buffer,1 [ mu ] L T DNA ligase and incubation at 37 ℃ for 3 hours;
Transformation of recombinant plasmid: placing 10 mu L of the connection product in the last step in 100 mu L DH5 alpha escherichia coli competent cells, carrying out ice bath 30min and then carrying out water bath 45 s at 42 ℃, quickly placing back on ice, carrying out ice bath 2 minutes, adding 500 mu L of SOC liquid culture medium, culturing at 37 ℃ for 1 h, centrifuging at 5000 r/min at room temperature for 30 s, removing about 200 mu L of part of culture medium, coating on a flat plate in a resuspension manner, and placing in a constant-temperature incubator at 37 ℃ for overnight culture;
verifying the recombinant plasmid: selecting single bacterial colonies, uniformly mixing the single bacterial colonies in 10 mu L of sterile water, taking 1 mu L of the single bacterial colonies for PCR verification, inoculating the screened bacterial strains into a 10ml LB liquid culture medium, adding Kana into the culture medium according to a ratio of 1000:1, culturing and extracting plasmids overnight at 37 ℃, taking 2 mu L of plasmids, carrying out enzyme digestion verification on 1 mu L of 10 Xbuffer, 0.5 mu L of BamHI and XhoI and 6 mu L of sterile water, incubating for 30 min at 37 ℃, and carrying out agarose gel electrophoresis to verify whether the recombinant plasmids are successfully constructed;
(2) Protein expression purification
Protein expression: and (3) transforming 10 mu L of constructed recombinant plasmid into E.coli BL21 (DE 3) competent cells, coating the competent cells on an LB plate containing Kana, and culturing at 37 ℃ until the concentration OD 600 value of the bacterial liquid reaches 0.6-1.0. Taking 50 mu L of bacterial liquid as a control, and carrying out ultrasonic crushing on the rest part of the bacterial liquid in an ice bath under the following ultrasonic conditions: 300 W ultrasonic 3 s, intermittent 3 s, repeated 180 times, 2500G centrifugation for 30 minutes, and using Ni-NTA His Bind affinity column to purify the protein.
The invention is further illustrated by the following experimental examples.
Experimental example 1 preparation of wild-type and mutant UvsX
1) The wild UvsX Gene was synthesized based on the UvsX sequence recorded in GenBank (Gene ID: 1258704); in addition, the coding gene F203YV244A of the mutant F203YV244A is synthesized;
2) Connecting the sequences synthesized in 1) with an expression vector pET26b respectively, and obtaining expression vectors containing wild type UvsX and mutant UvsX respectively;
3) Respectively converting the connection products into escherichia coli BL21 (DE 3) to obtain recombinant strains respectively expressing wild-type UvsX and mutant UvsX;
4) Preparation of wild-type and mutant UvsX
Recombinant strains containing wild-type and mutant UvsX genes were inoculated into LB (100. Mu.g/mL Amp) medium at an inoculum size of 0.1%, respectively, and rapidly shaken at 37℃for 16 h. Then, the activated bacterial liquid is inoculated into fresh LB (containing 100 mug/mL-1 Amp) culture liquid with an inoculum size of 1%, after rapid shaking culture is carried out for about 2-3 h (OD 600 reaches 0.6-1.0), IPTG with a final concentration of 0.1 mM is added for induction, and shaking culture is continued at 20 ℃ for about 20 h.12000 Centrifuging at 5 min rpm to collect thalli; suspending the thalli with a proper amount of pH 7.0 Tris-HCl buffer solution, and then carrying out ultrasonic breaking on the thalli under a low-temperature water bath; after the above intracellular concentrated crude enzyme solution was centrifuged at 12,000 rpm for 10 min, the supernatant was aspirated and the target protein was eluted by affinity respectively with Nickel-NTA Agarose and imidazole of 0 to 500 mM. SDS-PAGE results show that the wild type UvsX gene and the mutant UvsX gene are expressed in the escherichia coli, and the products are single bands after purification as shown in figure 1.
Experimental example 2 in vitro nucleic acid amplification of recombinant proteins of the invention over a broad temperature range
(1) A conventional in vitro nucleic acid amplification reaction system (50. Mu.l) is composed of 60-90mM Tris buffer, 60-100 mM potassium acetate (optimally 60 mM), 1% -5% polyethylene glycol (molecular weight 30000-40000, optimally 3%), 5-20 mM ATP (optimally 10 mM), creatine phosphate 20-50. Mu.g/. Mu.l (optimally 30. Mu.g/. Mu.l), 200-500 mM dNTPs, 40-80 ng/. Mu.l UvsX protein (optimally 60 ng/. Mu.l), 500-1200 ng/. Mu.l gp32 (optimally 800 ng/. Mu.l), 20-80 ng/. Mu.l Bsu (optimally 50 ng/. Mu.l), 50-100 ng/. Mu.l exonuclease (optimally 85 ng/. Mu.l), 5-30 ng/. Mu.l RecQ protein (optimally 10 ng/. Mu.l), 5-30 mM dithiothreitol (optimally 10 mM);
the in vitro nucleic acid amplification reaction system at a wide temperature range (constant temperature) was divided by replacing the UvsX protein in the above system with mutant UvsX (optimally 500 ng/. Mu.l). Mixing, adding 2 μl Salmonella DNA,10-40 mM magnesium acetate (optimally 20-mM), and optimally reacting at 20-45deg.C.
(2) RPA amplification was performed at different temperatures using UvsX protein mutant F203YV244A instead of the unc signature gene of core protein UvsX on staphylococcus aureus as the amplification subject.
The fixed reaction time was 20min and the temperature of the UvsX protein mutant F203YV244A at RPA amplification of staphylococcus aureus was explored. As shown in FIG. 2, lane 1 is a Marker of 500 bp, in which No. 1 is an amplification temperature of 10 ℃, no. 2 is an amplification temperature of 15 ℃, no. 3 is an amplification temperature of 20 ℃, no. 4 is an amplification temperature of 25 ℃, no. 5 is an amplification temperature of 30 ℃, no. 6 is an amplification temperature of 35 ℃, no. 7 is an amplification temperature of 40 ℃, no. 8 is an amplification temperature of 45 ℃, no. 9 is an amplification temperature of 50 ℃, and No. 10 is an amplification temperature of 55 ℃. Lanes show clear bright bands in the range of numbers 3 to 8 as shown in fig. 2. It can be demonstrated that the RPA reaction amplifies double-stranded DNA at a temperature ranging from 20℃to 45℃and that the amplification temperature of the reaction can be adapted to a wide temperature range. When the temperature is lower than 20 ℃, the enzyme activity is lower, and the target fragment cannot be amplified.
(3) RPA amplification was performed at different temperatures using UvsX protein mutant F203YV244A instead of the core protein UvsX for the inv a signature gene of salmonella as the amplification subject.
The fixed reaction time was 20 min and the RPA optimum reaction temperature was explored. FIG. 3 shows that lane 1 is a 500 bp Marker, in which number 1 is an amplification temperature of 5 ℃, number 2 is an amplification temperature of 10 ℃, number 3 is an amplification temperature of 15 ℃, number 4 is an amplification temperature of 20 ℃, number 5 is an amplification temperature of 25 ℃, number 6 is an amplification temperature of 30 ℃, number 7 is an amplification temperature of 35 ℃, number 8 is an amplification temperature of 40 ℃, number 9 is an amplification temperature of 45 ℃, and number 10 is an amplification temperature of 50 ℃. Lanes show clear bright bands within the range of numbers 4-9 as shown in FIG. 3. It can be demonstrated that the RPA reaction amplifies double-stranded DNA at a temperature ranging from 20℃to 45℃and that the amplification temperature of the reaction can be adapted to a wide temperature range.
Claims (5)
1. The amino acid sequence of the T4 phage recombinase UvsX mutant F203YV244A with wide temperature applicability is shown in SEQ ID No. 1.
2. The nucleotide sequence of the broad temperature applicability T4 phage recombinase UvsX mutant F203YV244A as described in claim 1 is synthesized in the Escherichia coli BL21 (DE 3) by using plasmid pET-28a as described in SEQ ID No. 2.
3. The nucleotide sequence of the broad temperature applicability T4 phage recombinase UvsX mutant F203YV244A as described in claim 1 is synthesized in Bacillus subtilis DB1342 by using plasmid pHT43 as described in SEQ ID No. 3.
4. Use of the broad temperature applicability T4 phage recombinase UvsX mutant F203YV244A according to claim 1, characterized in that: it is applied to the conventional RPA in vitro nucleic acid amplification reaction in a wider temperature range instead of the wild-type UvsX.
5. The use according to claim 4, characterized in that: the broader temperature range is 20-45 ℃.
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