CN115044571B - Extreme thermophilic archaea recombinant HhH-GPD protein and preparation method and application thereof - Google Patents

Extreme thermophilic archaea recombinant HhH-GPD protein and preparation method and application thereof Download PDF

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CN115044571B
CN115044571B CN202210709994.9A CN202210709994A CN115044571B CN 115044571 B CN115044571 B CN 115044571B CN 202210709994 A CN202210709994 A CN 202210709994A CN 115044571 B CN115044571 B CN 115044571B
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张立奎
殷有成
李铮
姜董豪
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Guangling College Of Yangzhou University
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Abstract

The invention discloses an extremely thermophilic archaeaSulfolobus islandicusREY15A recombinant HhH-GPD (Sis-HhH-GPD) protein, the amino acid sequence of which is shown as SEQ ID NO.2, and the nucleotide sequence of which is shown as SEQ ID NO. 1. The invention utilizes the genetic engineering technology to construct a genetically engineered bacterium for expressing the Sis-HhH-GPD protein, the genetically engineered bacterium can efficiently express the Sis-HhH-GPD protein, the subsequent purification steps are simple and feasible, and a large amount of recombinant enzyme proteins can be easily obtained. The recombinant Sis-HhH-GPD protein can specifically excise 1-meA in DNA, and the binding capacity of DNA containing 1-meA is significantly higher than that of DNA binding to normal DNA. The recombinant Sis-HhH-GPD protein provided by the invention is used as a DNA methylation detection reagent, and has a wide application prospect in the medical field and the molecular biology field.

Description

Extreme thermophilic archaea recombinant HhH-GPD protein and preparation method and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an extreme thermophilic archaea recombinant HhH-GPD protein, a preparation method and application thereof.
Background
It is known that base methylation is one of the common types of base damage in DNA, and methylation reagents present in cell endogenous and environmental causes methylation of bases in DNA, and the types of major base methylation include 7-methylguanine (N7-methylguanine, 7-meG), 3-methyladenine (3-methyladenine, 3-meA), 6-methylguanine (O6-methylguanine, O6-meG), 1-methyladenine (1-meA), 3-methylguanine (3-methylythosine, 3-meC), 4-methylthymine (O4-methylguanine, O4-meT) and Methyl Phosphotriesters (MPT). It was found that 7-meG, 3-meA and O6-meG are the major forms of DNA methylation, while 1-meA, 3-meC, O4-meT and MPT are the forms of relatively less DNA methylation. O6-meG and O4-meT have been found to have high levels of gene mutagenesis and gene toxicity, 3-meA, 1-meA, 3-meC and 3-meG are capable of preventing DNA replication and transcription, and have cytotoxicities and relatively low gene mutagenesis. Thus, different types of base methylation can lead to genetic mutations and interfere with the cellular DNA replication and repair process, necessitating detection of methylated bases in the DNA. The existing DNA methylation detection method mainly comprises ultra-high performance liquid chromatography-tandem mass spectrometry, photocrosslinking sequencing, single-cell real-time sequencing and the like. Although these methods can achieve the objective of detecting DNA methylation, they still have some limitations such as cumbersome steps or expensive equipment.
The DNA glycosidase is an enzyme for hydrolyzing N-C glycosidic bond in DNA, and then cutting off damaged base in DNA, thus having important functions in repairing damaged base of DNA, avoiding cell mutation and detecting DNA mutation. Currently, DNA glycosidases can be divided into monofunctional DNA glycosidases and bifunctional DNA glycosidases. The monofunctional DNA glycosidase cleaves only the damaged base in the DNA, thereby forming an abasic site. The bifunctional DNA glycosidase is capable of cleaving not only a specific base in DNA, but also further cleaving a phosphodiester bond at the resulting abasic site.
The HhH (Heix-hairpin-Heix) DNA glycosidase superfamily currently comprises six family members: nth, oggI, mutY/Mig, alkA, mpgII and OggII. HhH DNA glycosidase can cleave deaminated bases, oxidized bases, alkylated bases, and the like in DNA. HhH DNA glycosidases are widely dividedBacterial and eukaryotic sources of HhH DNA glycosidases have been more studied, whereas archaea HhH DNA glycosidases have been relatively less studied. Icelium roseum sulfuret ZuccSulfolobus islandicusREY15A is an important model organism for researching the replication and repair of archaea DNA, and the genome codes for an HhH-GPD (Sis-HhH-GPD) protein, belonging to the HhH DNA glycosidase superfamily.
Disclosure of Invention
Aiming at the technical problems to be solved, the invention provides an extreme thermophilic archaea recombinant HhH-GPD protein and a preparation method thereof, which overcome the defects of the prior art.
Another object of the present invention is to provide a method for detecting DNA methylation which is efficient, specific and sensitive, and which is practical without using expensive equipment and cumbersome steps.
The invention provides an extreme thermophilic archaea recombinant HhH-GPD protein, the amino acid sequence of which is shown as SEQ ID NO. 2.
The nucleotide sequence of the nucleic acid molecule for encoding the protein is shown as SEQ ID NO. 1.
The invention also provides a preparation method of the protein, the protein is produced by genetic engineering bacteria capable of expressing Sis-HhH-GPD (NCBI: ADX 84373), and the used bacterial strain isE. coliBL21 (DE 3) pLysS cells can express a target protein in a large amount in a short time.
The invention constructs a genetically engineered bacterium capable of over-expressing heat-resistant HhH-GPD protein by using a genetic engineering technology, and obtains electrophoresis-grade recombinant enzyme protein through the steps of induction, expression, nickel ion affinity purification and the like. The protein can specifically cut 1-meA in DNA under high temperature condition, and has higher affinity to DNA containing 1-meA, which is obviously higher than that of DNA combined with normal, thus providing a simple method for detecting methylated DNA. Therefore, the protein has application potential in the molecular biology field related to detection and repair of methylated bases in DNA.
The invention discloses a construction method of genetic engineering bacteria capable of expressing Sis-HhH-GPD, which comprises the following steps:
step (a)1. By using primers toS. islandicusPerforming PCR amplification by taking REY15A genome as a template, and detecting a PCR amplification result by agarose gel electrophoresis; the forward primer sequences used were: 5'-CGCGGATCCATGGTTCGTAAAATACTTGAC-3', the reverse primer sequence is: 5'-CCGCTCGAGTCACGAGGAATTTTCTCTATA-3';
step 2, carrying out double enzyme digestion on the PCR amplified product obtained in the step 1 and the pET-28a Plus vectorBamHI/XhoI) Reacting;
step 3, recovering the digested PCR product and the pET-28a Plus carrier fragment through agarose gel electrophoresis, and carrying out a connection reaction;
step 4, converting the connection product obtained in the step 3 into competent cellsE. coliCulturing overnight in DH5 alpha (i.e. coating on LB plate containing kanamycin, culturing overnight at 37 ℃), picking up clone, extracting plasmid for sequencing verification to obtain clone plasmid with correct gene sequence;
step 5, transforming the cloning plasmid with correct gene sequence into competent cellsE. coliObtaining genetic engineering bacteria capable of expressing Sis-HhH-GPD in BL21 (DE 3) plysS cells, and performing induced expression;
and step 6, purifying the enzyme through ultrasonic crushing and nickel ion affinity purification to obtain an electrophoresis grade pure protein sample Sis-HhH-GPD.
The invention further provides application of the protein in detecting DNA methylation.
The molecular weight of the protein Sis-HhH-GPD obtained by the above construction method is about 26 kDa, and 1-meA in DNA can be specifically excised and phosphodiester bonds can be further cleaved at the abasic site formed.
The protein Sis-HhH-GPD can be at 40 o C ～ 90 o Cutting 1-meA-containing DNA within the temperature range C, the optimum temperature for the cutting reaction being 70 o C。
The protein Sis-HhH-GPD is capable of cleaving 1-meA-containing DNA at a pH in the range of 8.0 to 9.5.
The cleavage reaction of the protein Sis-HhH-GPD does not require divalent metal ions.
The cleavage reaction of the protein Sis-HhH-GPD does not require the presence of a salt, which inhibits the activity of the enzyme.
The protein Sis-HhH-GPD is capable of binding to DNA containing 1-meA, and is incapable of binding to normal DNA.
The beneficial effects of the invention are as follows:
the invention utilizes the genetic engineering technology to construct a genetically engineered bacterium for expressing the Sis-HhH-GPD protein, the genetically engineered bacterium can efficiently express the Sis-HhH-GPD protein, the subsequent purification steps are simple and feasible, and a large amount of recombinant enzyme proteins can be easily obtained (about 2 mg recombinant proteins can be obtained from each liter of fermentation liquor); the recombinant Sis-HhH-GPD protein has high activity, and can specifically cut off 1-meA in DNA; the ability of the recombinant Sis-HhH-GPD protein to bind to DNA containing 1-meA is significantly higher than to bind to normal DNA.
Based on the research results, the recombinant Sis-HhH-GPD protein provided by the invention is used as a DNA methylation detection reagent, and has a wide application prospect in the medical field and the molecular biology field.
In a word, the extremely thermophilic archaea recombinant HhH-GPD protein can cut off and combine methylated bases in DNA, has the advantages of high expression quantity, easy purification, high activity and the like, has the possibility of market implementation in the aspect of detecting DNA methylation, and is expected to generate larger economic benefit.
Drawings
FIG. 1 is a schematic diagram showing the results of induction, expression and purification of Sis-HhH-GPD.
FIG. 2 is a schematic diagram of analysis of DNA cleavage by Sis-HhH-GPD.
FIG. 3 is a schematic diagram showing the effect of temperature on DNA cleavage by Sis-HhH-GPD.
FIG. 4 is a schematic representation of the effect of pH on DNA cleavage by Sis-HhH-GPD.
FIG. 5 is a schematic representation of the effect of divalent metal ions on DNA cleavage by Sis-HhH-GPD.
FIG. 6 is a schematic representation of the effect of salt concentration on DNA cleavage by Sis-HhH-GPD.
FIG. 7 is a schematic representation of the results of an analysis of SIS-HhH-GPD binding DNA.
Detailed Description
The following describes the technical scheme of the present invention in further detail by combining examples: the present embodiment is implemented on the premise of the technical scheme of the present invention, and a detailed implementation manner and a specific operation process are provided, but the protection rights of the present invention are not limited to the following embodiments.
The sources of reagents and materials involved in the examples are as follows:
OMEGA PCR kit is purchased from Tiangen Biochemical technology (Beijing) Co., ltd, pET-28a Plus vector is provided by the national academy of sciences high energy physics research, OMEGA gel recovery kit is purchased from Tiangen Biochemical technology (Beijing) Co., ltd,E. coliDH5 alpha competent cells were obtained from the whole gold organism company, OMEGA plasmid extraction kit was obtained from Tiangen Biochemical technology (Beijing) Limited, competent cellsE. coli BL21 (DE 3) plysS cells were purchased from full gold biosystems.
Except for the above-mentioned biological materials and reagents, the rest of the materials and reagents mentioned in the present invention are commercially available, and the public can purchase them from commercial sources both at home and abroad, and will not be described here. In the invention, "%" is volume percent.
EXAMPLE 1 Gene cloning of the Sis-HhH-GPD protein
(1) Designing primers
Downloading of sequencing-completed sulfolobus Iceliaceae from GenBankS. islandicusThe gene sequence of the Sis-HhH-GPD encoded by REY15A genome (NCBI: siRe_0278), a pair of primers containing two different restriction endonuclease cleavage sites was designed, and the forward primer sequence was: 5' -CGCGGATCCATGGTTCGTAAAATACTTGAC-3', the reverse primer sequence is: 5' -CCGCTCGAGTCACGAGGAATTTTCTCTATA-3', wherein the underlined bases are respectivelyBamHI andXhoi cleavage site.
(2) PCR amplification of the enzyme Gene
a) Using the above primer pair to download from GenBankS. islandicusPCR amplification was performed using REY15A genome as a template.
The PCR reaction system was 50. Mu.L:
10. mu M forward primer 2. Mu.L
10. Mu M reverse primer 2. Mu.L
S. islandicusREY15A genomic DNA (50 ng/. Mu.L) 1. Mu.L
ddH 2 O 20 μL
2 × Phanta Max Master Mix 25 μL
PCR reaction cycle parameters: 95 o C,3 min; cycling 34 times (95) o C，30 s；55 o C，30 s；72 o C，1 min）；72 o C extends for 5 min.
b) The result of PCR amplification of the enzyme gene was detected by agarose gel electrophoresis: after the reaction, 5. Mu.L of the PCR product was subjected to 1.0% agarose gel electrophoresis.
c) Purification of PCR products: the OMEGA PCR kit is adopted for recovery and purification, and specific steps are shown in the specification. The concentration of the PCR recovered product was determined using a Nanodrop 2000 ultra-micro spectrophotometer.
(3) Enzyme gene and plasmid vector
Double enzyme digestion is carried out on the PCR product and the pET-28a Plus vector respectivelyBamHI/XhoI) And (3) reacting.
The cleavage reaction system was 20. Mu.L:
16. Mu.L of PCR product or pET-28a Plus vector
10 X enzyme digestion buffer (Mg) 2+ plus) 2 μL
BamHI 1 μL
XhoI 1 μL
37 o C water bath 2 hr. After the completion of the cleavage, the cleavage product was subjected to 1.0% agarose gel electrophoresis. The OMEGA gel recovery kit is adopted for gel cutting recovery and purification, and specific steps are shown in the specification. The concentration was determined using a Nanodrop 2000 ultra-micro spectrophotometer.
(4) Ligase gene and vector: the digested PCR product and pET-30a (+) vector were recovered by agarose gel electrophoresis and subjected to ligation.
The ligation reaction was 10. Mu.L:
10 x Ligation Buffer 1 μL
2 mu L of pET-28a Plus vector after enzyme digestion
6 mu L of the digested PCR product
T4 DNA Ligase 1μL
22 o C reaction 2 hr.
(5) Transformation of recombinant plasmid: conversion of ligation products toE. coliDH5 alpha competent cells, and spread on LB plates containing kanamycin, 37 o C, overnight culture. Single colonies were picked and plasmids were extracted for sequencing verification.
mu.L of ligation product was pipetted into 50. Mu.LE. coliDH5 alpha competent cells were mixed well and placed in ice bath for 30 min.42 o C, standing in a water bath for 90 sec, and rapidly returning to ice for 2min. Adding 200 μLLB liquid culture medium at 37 o Culture 1 hr on a C shaker at 150 rpm. 100. Mu.L of the culture was pipetted onto LB medium plates containing kanamycin at a final concentration of 50. Mu.g/mL, 37 o Culturing 12 h-16 h to obtain 100-200 single colonies.
Subsequently, positive clones were validated: 4 individual colonies were selected and inoculated into 5 mL LB medium test tubes containing 50. Mu.g/mL kanamycin, 37 o C150 rpm overnight culture on shaker. Extracting plasmids by using an OMEGA plasmid extraction kit, and sequencing. And (3) comparing the sequencing result with NCBI annotated sequences, and verifying positive cloning to obtain a recombinant plasmid Sis-HhH-GPD-pET-28a Plus (+).
Example 2 Induction expression and purification of Sis-HhH-GPD protein
(1) The cloning plasmid with correct gene sequence is passed through 42 o C incubation for 90 sec conversion to competent cellsE. coli In BL21 (DE 3) plysS cells, genetically engineered bacteria capable of expressing HhH-GPD proteins were obtained, and IPTG (isopropyl-. Beta. -D-thiogalactoside) was used as an inducer to induce the expression of the enzyme.
Transformation of recombinant plasmid Sis-HhH-GPD-pET-28a Plus (+) intoE. coliBL21 (DE 3) pLysS expression strain, and then single colony of the expression strain was inoculated into LB liquid medium test tube containing 50. Mu.g/mL kanamycin and 34. Mu.g/mL chloramphenicol, 37 o C overnight culture, 1% inoculum size was transferred to 500 mL LB liquid medium containing 50. Mu.g/mL kanamycin and 34. Mu.g/mL chloramphenicol at 37 o C shaking culture to OD 600 About 0.6 mM IPTG was added and the incubation continued for 12 hours at room temperature to express the recombinant protein.
(2) The recombinant enzyme is purified by adopting the steps of ultrasonic cell disruption, heat treatment, nickel ion affinity purification and the like.
The cells were collected and suspended in buffer A (20 mM Tris-HCl pH 8.0, 500 mM NaCl and 10% glycerol). Ultrasonic disruption of cells, 12000 rpm, 4 o C centrifuging for 20 min to obtain supernatant, and subjecting the supernatant to 70 o C heat treatment for 20 min,12000 rpm, 4 o C, centrifuging for 20 min. The supernatant was passed through a Ni affinity column equilibrated with buffer A, and eluted with a gradient of buffer B (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol and 500 mM imidazole) to collect fractions. All fractions were subjected to SDS-PAGE gel electrophoresis, pooled and dialyzed overnight against storage buffer (20 mM Tris-HCl pH 8.0, 1 mM DTT, 50 mM NaCl and 50% glycerol), -80 o C, freezing and preserving. The protein concentration was determined by ultraviolet absorption. FIG. 1 is a diagram showing the result of SDS-PAGE gel electrophoresis of steps of induction expression of protein, cell disruption by ultrasonic waves, heat treatment, affinity purification by nickel ions, and the like.
The molecular weight of the Sis-HhH-GPD obtained by the construction method is about 26 kDa, the nucleotide sequence of the Sis-HhH-GPD is shown as SEQ ID NO.1, and the amino acid sequence of the Sis-HH-GPD is shown as SEQ ID NO. 2. The protein is capable of specifically cleaving 1-meA in DNA and is further capable of cleaving phosphodiester bonds at the abasic site formed.
Example 3 testing of Sis-HhH-GPD for DNA cleavage and binding
The activity of the Sis-HhH-GPD cleavage containing 1-meA ssDNA was analyzed and the effect of the optimal reaction temperature, optimal reaction pH and optimal divalent metal ion and salt concentration of the enzyme on the activity of the enzyme was studied. In addition, the present invention also detects the ability of Sis-HhH-GPD to bind DNA containing damaged bases.
Test 1: analysis of the DNA cleaved by Sis-HhH-GPD
Based on the fact that the biological company can only synthesize DNA containing 1-meA, the invention takes the DNA containing 1-meA as a representative of methylated DNA substrates, and tests the activity of the Sis-HhH-GPD for cutting DNA. The sequence of the oligonucleotide chain containing 1-meA, labeled with fluorescent Cy3, used in the present invention is as follows: 5'-Cy3CGA ACT GCC TGG AAT CCT GAC GAC XTG TAG CGA ACG ATC ACC TCA-3', wherein X is A, tg (thymol), 8oxoG, U (uracil), hx (hypoxanthine) or 1-meA. The complementary nucleotide sequence is 5' -TGA GGT GAT CGT TCG CTA CAY GTC GTC AGG ATT CCA GGC AGT TCG, where Y is A, G, C and T.
The reaction system was 10. Mu.L: 20 mM Tris-HCl pH 8.0, 100 nM ssDNA, 1000 nM Sis-HhH-GPD, 1 mM DTT and 8% glycerol, 70 o C, reacting for 30 min. After the completion of the reaction, 10. Mu.L of formamide containing 100 mmol/L EDTA was added to terminate the reaction, 95 o C treatment for 5 min to melt the double chains. The excised product was subjected to gel electrophoresis using 15% denaturing polyacrylamide gel (0.5×tbe) with urea concentration of 8M. As shown in FIG. 2, under the above reaction conditions, only 1000 nM Sis-HhH-GPD was used to cleave 77% of 1-meA from 100 nM DNA. However, sis-HhH-GPD cannot cleave DNA containing other damaged bases, nor does it cleave normal DNA.
Test 2: effect of reaction temperature on Sis-HhH-GPD Activity
The reaction system was 10. Mu.L: 20 mM Tris-HCl pH 8.0, 100 nM contains ssDNA of 1-meA, 1000 nM Sis-HhH-GPD, 1 mM DTT and 8% glycerol. The reaction temperatures were 30 respectively o C、40 o C、50 o C、60 o C、70 o C、80 o C or 90 o The reaction time of C was 30 min. After the completion of the reaction, 10. Mu.L of formamide containing 100 mmol/L EDTA was added to terminate the reaction, 95 o C, reacting for 5 min to enable the double chains to be melted. The excised product was subjected to gel electrophoresis using 15% denaturing polyacrylamide gel (0.5×tbe) with urea concentration of 8M. As shown in FIG. 3, the electrophoresis results show that Sis-HhH-GPD can be found at 30 o C ～90 o Excision of 1-meA from DNA at temperature C, which has an optimum reaction temperature of 70 o C。
Test 3: effect of reaction pH on Sis-HhH-GPD Activity
The reaction system was 10. Mu.L: 100 nM contains ssDNA of 1-meA, 1000 nM Sis-HhH-GPD, 1 mM DTT, 8% glycerol and buffers of different pH (20 mmol/L). The method for preparing buffers with different pH values is as follows: sodium phosphate buffer (pH 6.0,6.5); tris-HCl buffer (pH 7.0,7.5 and 8.0); glycine buffer (pH 8.5, 9.0); sodium bicarbonate buffer (pH 10.0). 70 o C, reacting for 30 min. After the completion of the reaction, 10. Mu.L of formamide containing 100 mmol/L EDTA was added to terminate the reaction, 95 o The double strand was melted by reaction at C for 5 min, and the excised product was subjected to gel electrophoresis using 15% denaturing polyacrylamide gel (0.5 XTBE) having a urea concentration of 8M. As shown in FIG. 4, the electrophoresis results show that Sis-HhH-GPD can cleave 1-meA from DNA within the pH range of 6.0 to 9.5, and the optimal reaction pH is 8.0 to 9.5.
Test 4: effect of divalent Metal ions on Sis-HhH-GPD Activity
The reaction system was 10. Mu.L: 20 mM Tris-HCl pH 8.0, 100 nM ssDNA containing 1-meA, 1000 nM Sis-HhH-GPD, 1 mM DTT, 8% glycerol and 5 mM divalent metal ion (divalent metal ion is Ca) 2+ 、Mg 2+ 、Zn 2+ 、Mn 2+ 、Ni 2+ 、Co 2+ Or Cu 2+ ）。70 o C for 30 min, after the reaction, 10. Mu.L of formamide containing 100 mmol/L EDTA was added to terminate the reaction, 95 o The double strand was melted by reaction at C for 5 min, and the excised product was subjected to gel electrophoresis using 15% denaturing polyacrylamide gel (0.5 XTBE) having a urea concentration of 8M. As a result, as shown in FIG. 5, the activity of 1-meA in the DNA excised by Sis-HhH-GPD was independent of divalent metal ions; zn (zinc) 2+ 、Ni 2+ 、Co 2+ And Cu 2+ To a different extent, the activity of the enzyme is inhibited, while Ca 2+ 、Mn 2+ And Mg (magnesium) 2+ Does not affect the activity of the enzyme.
Test 5: effect of salt concentration on Sis-HhH-GPD Activity
Reaction system10. Mu.L: 20 mM Tris-HCl pH 8.0, 100 nM containing ssDNA of 1-meA, 1000 nM Sis-HhH-GPD, 1 mM DTT, 8% glycerol and different concentrations of NaCl (50 mM, 100 mM, 200 mM or 400 mM); 70 o C, reacting for 30 min. After the completion of the reaction, 10. Mu.L of formamide containing 100 mmol/L EDTA was added to terminate the reaction, 95 o The double strand was melted by reaction at C for 5 min, and the excised product was subjected to gel electrophoresis using 15% denaturing polyacrylamide gel (0.5 XTBE) having a urea concentration of 8M. As shown in FIG. 6, the electrophoresis results show that the removal of 1-meA from DNA by Sis-HhH-GPD is independent of NaCl, and that the high concentration of NaCl inhibits the activity of the enzyme.
Test 6: analysis of Sis-HhH-GPD binding DNA
The gel blocking assay system was 10 μl:20 mM Tris-HCl pH 8.0, 1 mM DTT, 10% glycerol, 100 nM Cy3-labeled 1-meA ssDNA or normal ssDNA, and varying concentrations of Sis-HhH-GPD. After 10 min of room temperature binding, the blocking product was directly loaded and isolated by 4% non-denaturing polyacrylamide gel electrophoresis (0.1 XTBE). The DNA bands in the figures were then quantitatively analyzed by scanning with a molecular imager, and the binding rate of the enzyme to the substrate was calculated. As shown in FIG. 7A, sis-HhH-GPD was able to bind to 1-meA-containing DNA with high efficiency, but weakly to normal DNA (FIG. 7B).
The invention discovers that the Sis-HhH-GPD protein is a difunctional DNA glycosidase, can specifically cut 1-meA in DNA, and has significantly higher binding capacity to DNA containing 1-meA than to normal DNA, so the invention provides an important method for detecting DNA methylation. Compared with other DNA methylation detection methods, the method provided by the invention has the advantages of high sensitivity, low cost, easiness in operation and the like.
The foregoing is merely illustrative of the embodiments of the present invention, and the scope of the present invention is not limited thereto, and any person skilled in the art will appreciate that modifications and substitutions are within the scope of the present invention, and the scope of the present invention is defined by the appended claims.
Sequence listing
<110> university of Yangzhou Guangling university college
<120> extreme thermophilic archaea recombinant HhH-GPD protein, and preparation method and application thereof
<160> 2
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<211> 684
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<213> Artificial sequence (Artificial Sequence)
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atggttcgta aaatacttga cacattgctg gaaatatttg aaaacaataa aagcatattg 60
aaggaaaaag gttggatagt ttcgtccgaa acttcttatg aatggtggga cggactaaaa 120
agtgcagaag aaataatcat ttcagcaata ttggttcaaa tgtcaagatg ggaaattgta 180
aagggcaaag tagaggagat gaggagtaag ggtttgactg atttttataa attatacaat 240
actactgaac aagaattata tgatgtattg aaaggaatta acttctataa gactaaggtt 300
aagaggttaa ttaatttatc taaaatcata ataaatctag gtagtgttga gaaattttat 360
gacagaaatt tacttttaag cattgatggt ataggcgaag aaacagctga ctcaatcttg 420
cttttcgcag gtcacaaacc aaactttcca ccatcagagt acggtaagag agtattatct 480
agagtattag gaattagtat aaagaaaaag aatgaggtta aaagactagt agaggagaat 540
ttagagcgaa acgtctacga atacaaatta ctacacgctg gaatagtcac tgtaggtaga 600
gcattttgtt tcattgaaaa tcccaaatgt gaagactgta tcttgaagaa agtatgtaaa 660
tattatagag aaaattcctc gtga 684
<210> 2
<211> 227
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Met Val Arg Lys Ile Leu Asp Thr Leu Leu Glu Ile Phe Glu Asn Asn
1 5 10 15
Lys Ser Ile Leu Lys Glu Lys Gly Trp Ile Val Ser Ser Glu Thr Ser
20 25 30
Tyr Glu Trp Trp Asp Gly Leu Lys Ser Ala Glu Glu Ile Ile Ile Ser
35 40 45
Ala Ile Leu Val Gln Met Ser Arg Trp Glu Ile Val Lys Gly Lys Val
50 55 60
Glu Glu Met Arg Ser Lys Gly Leu Thr Asp Phe Tyr Lys Leu Tyr Asn
65 70 75 80
Thr Thr Glu Gln Glu Leu Tyr Asp Val Leu Lys Gly Ile Asn Phe Tyr
85 90 95
Lys Thr Lys Val Lys Arg Leu Ile Asn Leu Ser Lys Ile Ile Ile Asn
100 105 110
Leu Gly Ser Val Glu Lys Phe Tyr Asp Arg Asn Leu Leu Leu Ser Ile
115 120 125
Asp Gly Ile Gly Glu Glu Thr Ala Asp Ser Ile Leu Leu Phe Ala Gly
130 135 140
His Lys Pro Asn Phe Pro Pro Ser Glu Tyr Gly Lys Arg Val Leu Ser
145 150 155 160
Arg Val Leu Gly Ile Ser Ile Lys Lys Lys Asn Glu Val Lys Arg Leu
165 170 175
Val Glu Glu Asn Leu Glu Arg Asn Val Tyr Glu Tyr Lys Leu Leu His
180 185 190
Ala Gly Ile Val Thr Val Gly Arg Ala Phe Cys Phe Ile Glu Asn Pro
195 200 205
Lys Cys Glu Asp Cys Ile Leu Lys Lys Val Cys Lys Tyr Tyr Arg Glu
210 215 220
Asn Ser Ser
225

Claims (4)

1. Extreme thermophilic archaeaSulfolobusislandicus) The application of the recombinant HhH-GPD protein in detecting single-stranded DNA methylation is characterized in that the nucleotide sequence of the encoded protein is shown as SEQ ID NO.1, and the protein has the function of specifically cutting 1-meA in single-stranded DNA.
2. Use of an extreme thermophilic archaea recombinant HhH-GPD protein according to claim 1, for the detection of single-stranded DNA methylation, characterized in that: the environmental condition of 1-meA in the protein excised single-stranded DNA is 40 o C ～90 o C. pH 8.0-9.5, no salt and no divalent metal ion.
3. Use of an extreme thermophilic archaea recombinant HhH-GPD protein according to claim 1 or 2, for the detection of single-stranded DNA methylation, characterized in that: the protein is produced by genetic engineering bacteria capable of expressing the protein, and the competent cells areE. coli BL21 (DE3) pLysS。
4. The use of recombinant HhH-GPD protein from extreme thermophilic archaea according to claim 3 for the detection of single-stranded DNA methylation, characterized in that the construction method of said genetically engineered bacteria comprises the following steps:
step 1, using primers toS. islandicusPerforming PCR amplification by taking REY15A genome as a template, and detecting a PCR amplification result by agarose gel electrophoresis; the forward primer sequences used were: 5'-CGCGGATCCATGGTTCGTAAAATACTTGAC-3', reverse primer sequenceThe method comprises the following steps: 5'-CCGCTCGAGTCACGAGGAATTTTCTCTATA-3';
step 2, carrying out double enzyme digestion on the PCR amplified product obtained in the step 1 and the pET-28a Plus vectorBamHI/XhoI) Reacting;
step 3, recovering the digested PCR product and the pET-28a Plus carrier fragment through agarose gel electrophoresis, and carrying out a connection reaction;
step 4, converting the connection product obtained in the step 3 into competent cellsE. coliCulturing overnight in DH5 alpha, extracting plasmids, and carrying out sequencing verification to obtain cloned plasmids with correct gene sequences;
step 5, transforming the cloning plasmid with correct gene sequence into competent cells
E. coli In BL21 (DE 3) plysS cells, genetically engineered bacteria capable of expressing the extremely thermophilic archaea recombinant HhH-GPD protein are obtained.
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