CN115747187B - Recombinase UvsX and expression gene and application thereof - Google Patents

Abstract

The invention belongs to the fields of enzyme engineering and microbial engineering, and particularly relates to a recombinase UvsX, an expression gene thereof and application thereof. The invention selects gene of Escherichia T6 virus coding recombinase UvsX as a template, obtains a recombinase UvsX strain with 12 times of improved expression quantity through codon preference optimization and expression vector construction optimization, and obtains the recombinant UvsX enzyme with the purity of more than 95 percent through optimizing expression conditions and purification technology. The recombinant enzyme UvsX plasmid obtained by the invention can be used for preparing recombinant strains, and the Escherichia coli is taken as a recombinant expression host, so that the efficient expression of the recombinant enzyme UvsX is realized.

Description

Recombinase UvsX and expression gene and application thereof

Technical Field

The invention belongs to the fields of enzyme engineering and microbial engineering, and particularly relates to a recombinase UvsX, an expression gene thereof and application thereof.

Background

In recent years, molecular biology has been rapidly developed and has been widely used in various fields such as IVD and pharmacy, wherein DNA amplification makes a very important cycle in molecular biology. The PCR technology invented in 1968 is the most widely used DNA amplification technology, which is a molecular biology technology for amplifying specific DNA fragments, and consists of three basic reaction steps of denaturation, annealing and extension, which can be regarded as specific DNA replication outside organisms and is widely used in molecular diagnosis, however, the technology requires an accurate temperature control system for accurately controlling the temperature in the three reaction steps, and the whole PCR reaction time is relatively long.

With the continuous development of molecular biology, a new isothermal nucleic acid amplification technology with short reaction time and without the help of a precise temperature circulation system is invented, namely a Recombinase Polymerase Amplification (RPA) technology.

The recombinase polymerase amplification technique is a technique for realizing exponential amplification of nucleic acids under isothermal conditions in the presence of a plurality of enzymes and proteins, and is called a nucleic acid detection technique that can replace PCR. It relies mainly on three enzymes: recombinant enzyme UvsX capable of binding single-stranded nucleic acids (oligonucleotide primers), single-stranded DNA binding protein GP32, and strand displacement DNA polymerase BsuL. The mixture of these three enzymes is also active at normal temperature, the optimal reaction temperature is around 37 ℃, and during amplification, the recombinase UvsX binds to and forms complexes with the primers, which are complementary to the sequence of the DNA template. The single-stranded DNA is combined with the untwisted single-stranded DNA to stabilize the same, and the strand displacement DNA polymerase Bsu is combined at the 3' -end of the nucleic acid protein complex to carry out strand extension to form a complementary strand, and the newly synthesized single strand is paired with the original complementary strand, thereby realizing the exponential growth of the DNA. Two advantages of RPA over PCR are: 1. the special equipment can be eliminated to provide temperature control, and the reaction speed is high, the reaction time is short, and the method is more suitable for POCT.

In the recombinant polymerase amplification technology, the recombinant enzyme UvsX plays an important role, the consumption of the recombinant enzyme UvsX is high in the reaction of RPA, but the expression level of the wild UvsX gene in escherichia coli is extremely low, and even if the expression level is low after codon optimization, the purity of subsequent purification is low, the purification difficulty is increased, and the purification steps are increased, so that the production cost of the induced growth is high, and the industrialized application is not satisfied.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention provides a recombinase UvsX and an expression gene and application thereof, wherein the gene of the Escherichia T6 virus encoding the recombinase UvsX is selected as a template through an OPTIMIZER website, and the recombinase UvsX with improved expression quantity is obtained through codon preference optimization and single-point mutation of TEV protease, and the nucleotide sequence of the recombinase UvsX is shown as SEQ ID NO. 1.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the first object of the invention is to provide an expression gene of recombinase UvsX, which comprises a nucleotide sequence shown in SEQ ID NO. 1.

ATGAGCATCGCGGATCTGAAAAGCCGTCTGATCAAAGCGAGCACCAGCAAAATGACCGCGGAACTGACCACCTCCAAATTCTTCAACGAAAAAGACGTTATCCGTACCAAAATCCCGATGCTGAACATCGCGATCAGCGGTGCGATCGATGGTGGCATGCAGTCTGGCCTGACCATCTTCGCGGGTCCGTCCAAACACTTCAAAAGCAACATGAGCCTGACCATGGTTGCGGCGTACCTGAACAAATACCCGGACGCGGTTTGCCTGTTCTACGATAGCGAATTCGGCATCACCCCGGCGTACCTGCGTAGCATGGGCGTGGATCCGGAACGTGTTATCCACACCCCGATCCAGAGCGTTGAACAGCTGAAAATCGACATGGTTAACCAGCTGGAAGCAATCGAACGTGGCGAAAAAGTTATCGTGTTCATCGACTCTATCGGTAACATGGCGTCTAAAAAAGAAACCGAAGATGCGCTGAACGAAAAAAGCGTTGCGGATATGACCCGTGCGAAAAGCCTGAAATCCCTGTTCCGTATCGTTACCCCGTACTTCAGCATCAAAAACATCCCGTGCGTTGCGGTGAACCACACCATCGAAACCATCGAAATGTTCAGCAAAACCGTGATGACCGGTGGCACCGGCGTTATGTACTCCGCGGACACCGTTTTCATCATCGGCAAACGTCAGATCAAAGATGGCAGCGATCTGCAGGGTTACCAGTTCGTTCTGAACGTTGAAAAATCCCGTACCGTTAAAGAAAAATCTAAATTCTTCATCGACGTTAAATTCGATGGCGGCATCGATCCGTACTCTGGTCTGCTGGATATGGCGCTGGAACTGGGCTTCGTGGTGAAACCGAAAAACGGTTGGTATGCGCGTGAATTCCTGGATGAAGAAACCGGTGAAATGATCCGTGAAGAAAAAAGCTGGCGTGCGAAAGATACCAACTGCACTACCTTCTGGGGTCCGCTGTTCAAACACCAGCCGTTCCGTGACGCGATCAAACGTGCTTACCAGCTGGGCGCGATCGATTCTAACGAAATCGTTGAAGCGGAAGTTGATGAACTGATCAACAGCAAAGTTGAAAAATTCAAAAGCCCGGAAAGCAAAAGCAAAAGCGCGGCGGATCTGGAAACCGATCTGGAACAGCTGAGCGATATGGAAGAATTCAACGAATAA（SEQIDNO.1）

The second object of the present invention is to provide a recombinant expression vector pET-32a-UvsX-TEV comprising a pET-32a vector and a target gene expression fragment inserted into the pET-32a vector; the pET-32a vector comprises a basic sequence, a multiple cloning site sequence and a promoter sequence of the pET-32a vector; the multiple cloning sites comprise Kpn I restriction sites and Xho I restriction sites; the target gene expression fragment comprises a nucleotide sequence shown as SEQ ID NO. 1; the 5 'end of the target gene expression fragment is provided with a Kpn I restriction enzyme site adhesive end, the 3' end of the target gene expression fragment is provided with an Xho I restriction enzyme site adhesive end, and the target gene expression fragment is inserted between the Kpn I restriction enzyme site and the Xho I restriction enzyme site of the multi-cloning site sequence in a forward direction; the restriction enzyme site Thrombin on the recombinant expression vector is mutated into a TEV Protease restriction enzyme site.

The third object of the present invention is to provide a method for increasing the expression level of recombinant enzyme UvsX, comprising the steps of:

s1, introducing a target gene fragment into a pET-32a vector to obtain a recombinant expression vector, wherein the target gene expression fragment contains a nucleotide sequence shown as SEQ ID NO.1, the 5 'end of the target gene expression fragment is provided with a Kpn I restriction site adhesive end, and the 3' end of the target gene expression fragment is provided with a Xho I restriction site adhesive end;

s2, transferring the recombinant expression vector into escherichia coli BL21 (DE 3) to obtain recombinant engineering bacteria;

s3, carrying out induction culture on the recombinant engineering bacteria to obtain the recombinant enzyme TrxA-UvsX;

s4, adding thrombin into the recombinase liquid TrxA-UvsX to cut the TrxA protein, thereby obtaining the recombinase UvsX.

Preferably, before the step of introducing the target gene expression fragment into the pET-32a vector to obtain the recombinant expression vector in the step S1, the method further comprises the following steps:

taking a nucleotide sequence shown in SEQ ID NO.1 as an amplification template, adding an upstream primer and a downstream primer, and performing PCR amplification to obtain a PCR amplification product, wherein the upstream primer comprises a Kpn I enzyme cutting site, and the downstream primer comprises an XhoI enzyme cutting site; inserting the PCR amplification product into a pET-32a vector, and converting the PCR amplification product into competent cells to obtain a cloning vector; and (3) carrying out double digestion on the cloning vector by using restriction enzymes Kpn I and Xho I to obtain a target gene expression fragment.

Preferably, the sequence information of the upstream primer is shown as SEQ ID NO.2, and the sequence information of the downstream primer is shown as SEQ ID NO.3.

ATCTGGGTACCATGAGCATCGCGGATCTGAA（SEQ ID NO.2）；

TGGTGCTCGAGTTATTCGTTGAATTCTTCCATATCGCTCAG（SEQ ID NO.3）。

Preferably, the step of performing induction culture on the recombinant engineering bacteria in the step S3 to obtain the recombinase UvsX includes: when the OD value of the colony of the recombinant engineering bacteria is 0.2-1.0, adding isopropyl thiogalactoside with the final concentration of 0.01-0.2 mM/L, inducing and expressing for 3-9 hours at 18-25 ℃, centrifugally separating and collecting thalli, ultrasonically crushing thalli, centrifugally separating and collecting supernatant to obtain a crude product, and purifying the crude product to obtain the recombinant enzyme TrxA-UvsX.

Preferably, in the step S4, thrombin is added into the recombinase liquid TrxA-UvsX to cut TrxA protein, the mass ratio of TEV enzyme to the recombinase liquid TrxA-UvsX is 1/300-1/100, the pH value of a cutting buffer solution is 6.5-9.0, the cutting temperature is 2-20 ℃, the cutting time is 8-20h, and separation and purification are carried out after cutting, so that the recombinase UvsX is obtained.

Preferably, the step of purifying the crude product to obtain the recombinase UvsX comprises: purifying the crude product by adopting a Ni affinity column to obtain the recombinase TrxA-UvsX; adding TEV enzyme for cutting, purifying the cut crude product by Ni column affinity flow through, and ultrafiltering and concentrating to obtain the recombinant enzyme UvsX.

It is a further object of the present invention to provide a recombinase UvsX, obtainable by a method as described in any of the above.

It is a further object of the present invention to provide the use of a recombinase UvsX as described above for the amplification of RPA by a recombinase polymerase.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses a recombinase UvsX, an expression gene and application thereof. The invention provides an expression gene of recombinase UvsX, and the nucleotide sequence is shown as SEQ ID NO. 1; the expression quantity of the recombinant enzyme UvsX is further improved by 12 times through reconstructing an expression vector, optimizing an induction expression condition and optimizing a purification process condition; compared with the commercially available recombinase UvsX, the activity of the recombinase UvsX obtained by the invention is also obviously improved. In addition, the recombinant enzyme UvsX plasmid obtained by the invention can be used for preparing recombinant vectors and recombinant strains, and the high-efficiency expression of the recombinant enzyme UvsX is realized by taking escherichia coli as a recombinant expression host.

Drawings

FIG. 1 is a vector pET-32a vector map and a polyclonal site map;

FIG. 2 is a diagram showing double enzyme digestion gel electrophoresis of the expression vector pET32a and the UvsX enzyme gene;

FIG. 3 is a PCR detection positive clone electrophoresis pattern of recombinant expression vector pET32 a-UvsX;

FIG. 4 is a map and polyclonal site map of the engineered expression vector pET32 a-UvsX-TEV;

FIG. 5 shows SDS-PAGE electrophoresis detection of the expression vector pET32a-UvsX-TEV fermentation broth;

FIG. 6 is a SDS-PAGE electrophoresis of the fermentation broth after purification with Ni;

FIG. 7 is a SDS-PAGE electrophoresis detection of the recombinant enzyme TrxA-UvsX purified by Ni column, after TEV cleavage and then purified by Ni column;

FIG. 8 is a nucleic acid electrophoretogram of the recombinases UvsX, trxA-UvsX with commercial UvsX under RPA reaction.

Detailed Description

The above-described aspects of the present invention will be described in further detail with reference to the following embodiments. It should not be construed that the scope of the above subject matter of the present invention is limited to the following examples.

Molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j. Sambrook, or according to the kit and product instructions; the reagents and biological materials, unless otherwise specified, are commercially available. Experimental materials and reagents involved in the invention:

1. strain and vector

Competent BL21 (DE 3), competent TOP10 were purchased from Shanghai Biotechnology and pET-32a plasmid from Bio-wind Corp.

2. Enzyme and kit

Restriction enzymes KpnI, xhoI, T4 DNAligenase, ex Taq enzyme were purchased from TAKARA, TEV enzyme was purchased from NEB, pfu was purchased from Promega, sanPrep nucleic acid purification kit, sanPrep column PCR product purification kit was purchased from Shanghai organisms.

3. Culture medium

LB medium formulation (L): 10g of tryptone, 5g of yeast extract and 10g of sodium chloride.

4. The recombinase UvsX activity assay reagent was purchased from twist dx company; the commercially available UvsX enzyme is available from TwitDx corporation.

EXAMPLE 1 acquisition and Synthesis of the Gene for recombinase UvsX

The invention utilizes an OPTIMIZER website to code a DNA sequence of recombinase UvsX of escherichia coli T6 phage (ATCC 11303-B6), the DNA sequence of the recombinase UvsX is shown as SEQ ID DN O.4, the gene sequence of the recombinase UvsX is obtained by optimizing according to codon preference of escherichia coli, the gene sequence of the recombinase UvsX is shown as SEQ ID NO.1, and the optimized gene of the recombinase UvsX (Kpn I cleavage site is designed at the 5 'end and Xho I cleavage site is designed at the 3' end) is connected to an expression vector PUC57 to obtain a cloning vector PUC57-UvsX, and the process is completed by Shanghai biological company.

ATGTCTATTGCAGATTTAAAATCCCGTTTGATTAAAGCTTCCACTTCTAAAATGACTGCTGAGCTGACTACATCTAAATTCTTTAATGAAAAGGATGTAATCCGTACAAAAATCCCAATGCTTAATATTGCTATTTCTGGTGCGATTGATGGCGGTATGCAGTCTGGTTTAACTATTTTCGCAGGGCCTTCTAAACACTTTAAATCAAATATGTCTTTGACTATGGTTGCAGCGTATTTGAACAAATATCCTGACGCGGTTTGTCTATTCTATGATAGCGAATTTGGTATTACTCCAGCTTATTTGCGATCCATGGGAGTTGACCCGGAACGTGTAATTCATACGCCAATCCAGTCAGTTGAACAGCTGAAAATTGATATGGTGAACCAGCTTGAAGCTATTGAGCGTGGTGAAAAAGTTATTGTATTCATCGACTCAATTGGTAATATGGCTTCCAAGAAAGAAACGGAAGATGCCTTGAATGAAAAATCTGTGGCAGATATGACTCGTGCTAAATCACTGAAGTCATTATTCCGTATTGTTACTCCTTATTTTAGCATTAAAAATATTCCATGTGTTGCGGTTAACCATACAATTGAAACAATTGAAATGTTTAGTAAAACCGTGATGACAGGTGGTACAGGCGTAATGTATTCGGCTGATACTGTATTCATTATCGGTAAGCGTCAGATTAAAGATGGTTCTGATCTTCAGGGGTATCAATTTGTTCTAAATGTAGAAAAATCTCGTACCGTTAAAGAAAAAAGTAAATTCTTTATTGATGTTAAATTTGACGGTGGTATCGATCCTTATTCTGGATTGTTAGATATGGCTCTAGAATTAGGATTTGTAGTAAAACCAAAAAATGGCTGGTATGCTCGTGAATTTCTTGACGAAGAAACTGGCGAGATGATTCGCGAAGAAAAATCTTGGCGTGCAAAAGATACTAACTGCACTACATTCTGGGGTCCTTTATTTAAGCATCAACCATTCCGAGATGCTATTAAACGTGCTTATCAGTTAGGTGCTATTGATAGTAATGAAATTGTTGAAGCTGAAGTTGATGAATTGATTAACTCAAAGGTTGAAAAATTTAAATCTCCAGAAAGTAAAAGTAAATCAGCTGCTGATTTAGAAACTGACCTAGAGCAGTTAAGTGATATGGAAGAATTTAATGAATAA（SEQ ID NO.4）；

Example 2 construction of recombinant enzyme UvsX expression vector and construction of recombinant bacterium

2.1 construction of the recombinant enzyme UvsX expression vector

The cloning vector PUC57-UvsX and pET-32a vector were double digested with restriction enzymes KpnI and XhoI, the digestion system was as shown in Table 1, and the conditions were: and (3) performing gel electrophoresis and recovery on the enzyme-digested product at 37 ℃ for 4 hours. The UvsX gene was ligated into pET-32a vector using T4 ligase, the ligation system is shown in Table 2 below, and the ligation conditions were: at 4℃overnight, the strain was inoculated with E.coli TOP10 strain (purchased from Shanghai Biotechnology) and the experimental procedure was as follows: all the connection products (10 mu L) are taken, added into 100 mu L of TOP10 competent cell solution, ice-bath is carried out for 30min, heat shock is carried out at 42 ℃ for 90s, then the mixture is kept in ice-bath for 15-20min, 800 mu L of LB culture medium is added, constant temperature culture is carried out for 1h at 37 ℃ and 200r/min, an EP tube is taken out, centrifugation is carried out for 5min at 4000r/min, 200 mu L of supernatant is reserved at the same time, after being uniformly mixed, bacterial precipitation is uniformly coated on an Amp resistance flat plate, single colony on the flat plate is picked up for the next day of constant temperature culture at 37 ℃ and cultured overnight in 10mL of LB culture medium, plasmid extraction is carried out on bacterial liquid by adopting SanPrep nucleic acid purification kit (plasmid extraction) to carry out double enzyme digestion identification and PCR identification respectively, and sequencing verification is carried out after the identification is correct, thus recombinant expression vector pET-32a-UvsX is successfully constructed.

TABLE 1 enzyme digestion system

Table 2 connection system

The double enzyme digestion identification comprises the following steps: the extracted plasmid is taken for double enzyme digestion, the system of double enzyme digestion is shown in table 1, and then the enzyme digestion plasmid is taken for 1% agarose gel nucleic acid electrophoresis detection, and the electrophoresis gel is observed under an ultraviolet lamp.

The PCR identification process comprises the following steps: the plasmids were taken and subjected to PCR reaction according to the reaction system of Table 3 and the amplification procedure of Table 4, wherein the upstream and downstream primers were SEQ ID NO.2 and SEQ ID NO.3, respectively. And then taking the PCR product to carry out 1% agarose gel electrophoresis detection, and observing the electrophoresis gel under an ultraviolet lamp.

TABLE 3 PCR reaction System

TABLE 4 PCR amplification procedure

The gene sequence information of the vector pET-32a can refer to the website: http:// www.biofeng.com/zaiti/dachang/pet32a.html.

Wherein, FIG. 1 is pET-32a vector map and polyclonal site map. FIG. 2 is a graph showing the results of double digestion of cloning vector pET32a-UvsX, from which it can be seen that vector pET32a-UvsX forms two bands after double digestion, respectively 5810bp pET-32a and 1182bp UvsX genes, and the results indicate that the double digestion verifies that vector pET32a-UvsX is constructed correctly. FIG. 3 is a diagram showing the results of PCR detection. The recombinant expression vector pET-32a-UvsX constructed can be amplified into 1182bp bands to obtain the recombinant expression vector, and the result shows that PCR verifies that the vector pET32a-UvsX is constructed correctly.

2.2 protease cleavage site mutation of recombinase UvsX expression vector

The Protease cleavage site Thrombin (Thrombin) of the recombinant expression plasmid pET32a-UvsX is mutated into a TEV Protease cleavage site, and the PCR product amplification process of the recombinant enzyme UvsX expression vector is as follows: (1) And carrying out PCR amplification by using an expression vector pET-32a-UvsX as a template and using a TEV protease cleavage site mutation primer, wherein an upstream primer of the TEV protease cleavage site mutation primer is shown as SEQ ID NO.5, and sequence information of a downstream primer is shown as SEQ ID NO. 6.

GCCCAGATCTGGGTACCGAGAATCTTTATTTTCAGGGGCCATGGCTGATATCGGATC（SEQ ID NO.5）；

GTGGTGGTGGTGGTGCTCGA（SEQ ID NO.6）。

The amplification system is shown in Table 5, and the amplification procedure is shown in Table 6; (2) Analyzing the obtained PCR amplified product through agarose electrophoresis, and purifying the target PCR product by using a SanPrep column type PCR product purification kit (purchased from Shanghai chemical industry), wherein the size of the PCR product is 1260bp; (3) Adding restriction enzyme Kpn I and Xho I into the purified PCR product for double digestion, and after enzymolysis for 2 hours, purifying and recovering double digestion products, and respectively carrying out gel recovery on a 1200bp fragment of the double digestion PCR product and a 5800bp large fragment of the double digestion pET-32a-UvsX plasmid; (4) And (3) adopting a PCR product and a pET-32a-UvsX vector fragment after T4 ligase is used for connecting enzyme digestion to obtain a recombinant expression vector pET-32a-UvsX-TEV. (5) The expression vector pET-32a-UvsX-TEV is thermally shocked and transformed into TOP10 competent cells, the cells are coated on a flat plate for overnight culture, single colonies are picked the next day, plasmids are extracted, double enzyme digestion identification is carried out, PCR identification is carried out, and sequencing is carried out after the identification is correct to verify the sequence correctness. In the experiment, transformation, plasmid extraction, double enzyme digestion verification, PCR identification and sequencing experiments were performed by the experimental method described in example 1.

The constructed pET-32a-UvsX-TEV vector map and the multiple cloning site are shown in FIG. 4.

TABLE 5 PCR product amplification System for recombinase UvsX expression vectors

TABLE 6 PCR amplification procedure

2.3 construction of recombinant enzyme UvsX expression engineering bacteria

The pET-32a-UvsX-TEV plasmid successfully constructed was transformed into E.coli BL21 (DE 3) strain (purchased from Shanghai Marine organism) under the transformation conditions of example 1, spread on LB plates, cultured overnight, and single colonies were picked up. Colony PCR verification is carried out by using the upstream primer and the downstream primer as SEQ ID NO.2 and SEQ ID NO.3 respectively, and the colony PCR verification is carried out in an experiment by adopting the experimental method described in the example 1. Determining positive transformants according to bacterial liquid PCR results, extracting plasmids of the positive transformants for sequencing identification, determining single bacterial colony with correct sequencing as UvsX enzyme recombinant engineering bacteria, and culturing until OD is 0.6-0.8, and preserving the seeds.

EXAMPLE 3 recombinant enzyme UvsX induced expression and affinity chromatography purification

3.1 expression of the recombinant enzyme UvsX induced

The recombinant expression plasmid pET32a-UvsX-TEV expresses the recombinant enzyme TrxA-UvsX in E.coli. The experimental procedure was as follows:

inoculating the UvsX enzyme recombinant engineering bacteria obtained by screening into 10mL of LB liquid medium, adding Kan antibiotics to a final concentration of 1mM, culturing at 37 ℃ at 250rpm for overnight, transferring the following day into 200mL of LB medium according to a ratio of 1:100, adding Kan antibiotics to a final concentration of 1mM, culturing at 37 ℃ at 250rpm until the OD value is 0.6-0.8, adding isopropyl thiogalactoside to a final concentration of 0.01mM/L-0.2mM/L, performing induced expression at 20 ℃ for 3-9h, centrifuging (4 ℃,800 rpm,5 min), separating and collecting thalli according to a mass ratio of thalli to Buffer A (Tirs-HCl 20mM, sodium chloride 500mM, imidazole, 10mM, and regulating pH to 7.0) of 1:10, mixing, ultrasonic crushing of escherichia coli, and crushing conditions: ultrasonic treatment in ice water bath for 3s, pause for 8s, power of 400W, and time of 20min. And (5) taking the bacterial cell disruption solution, and analyzing the expression quantity of the recombinant protein by SDS-PAGE electrophoresis.

SDS-PAGE electrophoresis results are shown in FIG. 5, whereinlane 1 shows the disruption of the inducing pre-cell;lane 2 is cell disruption induced for 3 h;lane 3 is cell disruption induced for 6h;lane 4 shows cell disruption induced for 9 h. The result shows that the target protein is expressed after induction for 6 hours, the expression quantity reaches the highest quantity, and the expression quantity is not increased any more after the induction time is increased.

3.2 purification of the recombinase UvsX

Purifying the crude enzyme solution by adopting a Ni affinity hanging column to obtain the recombinant enzyme TrxA-UvsX, wherein the experimental process is as follows:

(1) Centrifuging the above (ultrasonic disruption solution at 4deg.C and 12000rpm for 45min, collecting supernatant, filtering the supernatant with 0.22 μm filter membrane, and loading onto Ni chromatographic column;

(2) Eluting the TrxA-UvsX recombinase with Buffer B (Tirs-HCl 20mM, sodium chloride 500mM, imidazole 20mM, pH adjusted to 7.0), buffer C (Tirs-HCl 20mM, sodium chloride 500mM, imidazole 200mM, pH adjusted to 7.0);

(3) SDS-PAGE analysis of the purified samples;

(4) Adding TEV enzyme into the eluent TrxA-UvsX, wherein the mass ratio of the TEV enzyme to the recombinase TrxA-UvsX is 1/200, the pH value of a cutting buffer solution is 7.0, the cutting temperature is 4 ℃, and the cutting time is 16h; and (3) diluting with Buffer D (Tirs-HCl 20mM, sodium chloride 500mM, and pH is adjusted to 7.0) for 10 times after cutting, loading the mixture into a Ni chromatographic column for separation and purification, collecting flow-through liquid, and ultrafiltering to obtain the recombinase UvsX, and performing SDS-PAGE electrophoresis detection.

SDS-PAGE electrophoresis results are shown in FIGS. 6 to 7, wherein, as shown in FIG. 6,lane 1 is cell disruption;lane 2 is the supernatant after cell disruption;lane 3 is sediment after disruption;lane 4 is the 0.22 μm filter supernatant;lane 5 is fluid flow through;lane 6 is 50mM imidazole wash;lane 7 is the 200mM imidazole eluent group. The results show that the TrxA-UvsX protein is almost expressed as the supernatant, and the purified TrxA-UvsX recombinase has single band.

As shown in FIG. 7,lane 1 is TrxA-UvsX obtained by passing through a Ni column;lane 2 is the TEV-digested recombinase TrxA-UvsX;lane 3 is 200mM imidazole eluting collection column hanging protein;lane 4 is a Ni-column flow-through. As can be seen fromlane 2, most of TrxA-UvsX was cleaved by TEV into a UvsX enzyme with a molecular weight of 47KD and a TrxA protein with a molecular weight of 12KD, and a small part was not cleaved;lane 4 gives a single protein with a molecular weight of 47KD, which indicates that a UvsX enzyme band with the correct molecular size can be obtained through the purification step.

In conclusion, the above crude enzyme solution is purified by using Ni affinity column, and the recombinant enzyme TrxA-UvsX with purity of more than 90% can be obtained. Adding TEV enzyme into purified recombinase TrxA-UvsX for cutting, purifying the cut crude product by Ni column affinity flow-through, taking flow-through liquid, and concentrating by ultrafiltration to obtain recombinase UvsX with purity of more than 95%.

Example 4 enzyme Activity detection of recombinase UvsX

The RPA reaction determines the enzymatic activity of the recombinase UvsX by the following method: in RPA detection, a new coronal N protein gene plasmid is taken as a template, and a primer capable of amplifying 121bp DNA is designed, wherein primer information is shown as SEQ ID NO.8 and SEQ ID NO.9, and sequence information of the new coronal N protein gene plasmid is shown as SEQ ID NO. 7. Both template and primer are biosynthesized. The RPA reaction system is shown in tables 7-8 and the amplification procedure for the RPA reaction is shown in Table 9.

ACGTAGTCGCAACAGTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACTAAAGCATACAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGAACAAACCCAAGGAAATTTTGGGGA（SEQIDNO.7）；

AGGCAGCAGTAGGGGAACTTCTCCTGCTAGAAT（SEQIDNO.8）；

TTGGCCTTTACCAGACATTTTGCTCTCAAGCTG（SEQIDNO.9）。

TABLE 7 RPA reaction system (10X reactionbuffer)

Table 8 RPA reaction System formulation (50. Mu.L)

TABLE 9 RPA amplification procedure

After the RPA reaction, the reaction mixture was electrophoresed on a 1% lipo-glycogel and detected by electrophoresis in 0.5 XTBE buffer. As a result, FIG. 8 shows thatlanes 1 and 2 are commercial UvsX enzymes purchased,lanes 3 and 4 are recombinant UvsX enzymes purified after TEV cleavage, andlanes 5 and 6 are TrxA-UvsX enzymes purified by Ni column.Lanes 7, 8 are without UvsX enzyme. The result shows that the fusion expression TrxA-UvsX enzyme is inactive, the recombinant UvsX enzyme which cuts off the TrxA protein has activity, and the recombinant enzyme UvsX prepared by the method has the activity of the UvsX enzyme through the RPA reaction, so that compared with the commercial UvsX enzyme, the product obtained by the self-produced recombinant UvsX enzyme has brighter bands and fewer impurity bands.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims (8)

1. An expression gene of recombinase UvsX, which is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. A recombinant expression vector pET-32a-UvsX-TEV, which is characterized by comprising a pET-32a vector and a target gene expression fragment inserted into the pET-32a vector; the pET-32a vector comprises a basic sequence, a multiple cloning site sequence and a promoter sequence of the PET-32a vector; the multiple cloning sites comprise Kpn I restriction sites and Xho I restriction sites; the target gene expression fragment comprises a nucleotide sequence shown as SEQ ID NO. 1; the 5 'end of the target gene expression fragment is provided with a Kpn I restriction enzyme site adhesive end, the 3' end of the target gene expression fragment is provided with an Xho I restriction enzyme site adhesive end, and the target gene expression fragment is inserted between the Kpn I restriction enzyme site and the Xho I restriction enzyme site of the multi-cloning site sequence in a forward direction; the restriction enzyme site Thrombin on the recombinant expression vector is mutated into a TEV Protease restriction enzyme site.

3. A method for increasing the expression level of recombinase UvsX, comprising the steps of:

s1, introducing a target gene fragment into a pET-32a vector to obtain a recombinant expression vector, wherein the target gene expression fragment contains a nucleotide sequence shown as SEQ ID NO.1, the 5 'end of the target gene expression fragment is provided with a Kpn I restriction site adhesive end, and the 3' end of the target gene expression fragment is provided with a Xho I restriction site adhesive end;

s2, transferring the recombinant expression vector into escherichia coli BL21 (DE 3) to obtain recombinant engineering bacteria;

s3, carrying out induction culture on the recombinant engineering bacteria to obtain the recombinant enzyme TrxA-UvsX;

s4, adding TEV protease into the recombinase liquid TrxA-UvsX to cut the TrxA protein, thereby obtaining the recombinase UvsX.

4. The method for increasing the expression level of recombinant enzyme UvsX according to claim 3, wherein the step S1 of introducing the target gene expression fragment into the pET-32a vector to obtain the recombinant expression vector is preceded by the steps of:

taking a nucleotide sequence shown in SEQ ID NO.1 as an amplification template, adding an upstream primer and a downstream primer, and performing PCR amplification to obtain a PCR amplification product, wherein the upstream primer comprises a Kpn I enzyme cutting site, and the downstream primer comprises an XhoI enzyme cutting site; inserting the PCR amplification product into a pET-32a vector, and converting the PCR amplification product into competent cells to obtain a cloning vector; and (3) carrying out double digestion on the cloning vector by using restriction enzymes Kpn I and Xho I to obtain a target gene expression fragment.

5. The method for increasing the expression level of recombinase UvsX according to claim 4, wherein the upstream primer sequence is shown in SEQ ID NO.2 and the downstream primer sequence is shown in SEQ ID NO.3.

6. The method for increasing expression level of recombinant enzyme UvsX according to claim 3, wherein the step of inducing and culturing the recombinant engineering bacterium to obtain the recombinant enzyme UvsX in step S3 comprises: when the OD value of the colony of the recombinant engineering bacteria is 0.2-1.0, adding isopropyl thiogalactoside with the final concentration of 0.01-0.2 mM/L, inducing and expressing for 3-9 hours at 18-25 ℃, centrifugally separating and collecting thalli, ultrasonically crushing thalli, centrifugally separating and collecting supernatant to obtain a crude product, and purifying the crude product to obtain the recombinant enzyme TrxA-UvsX.

7. The method for increasing expression level of recombinant enzyme UvsX according to claim 3, wherein step S4 comprises adding TEV protease into the recombinant enzyme solution TrxA-UvsX to cleave TrxA protein, wherein the mass ratio of the added TEV enzyme to the recombinant enzyme solution TrxA-UvsX is 1/300-1/100, the pH value of cleavage buffer is 6.5-9.0, the cleavage temperature is 2-20 ℃, the cleavage time is 8-20h, and the separation and purification are performed after cleavage, thereby obtaining the recombinant enzyme UvsX.

8. The method for increasing the expression level of recombinase UvsX according to claim 6, wherein the step of purifying the crude product to obtain the recombinase UvsX comprises: purifying the crude product by adopting a Ni affinity column to obtain the recombinase TrxA-UvsX; adding TEV enzyme for cutting, purifying the cut crude product by Ni column affinity flow through, and ultrafiltering and concentrating to obtain the recombinant enzyme UvsX.

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