CN114836401A

CN114836401A - Recombinant ssDNA (single-stranded deoxyribonucleic acid) nucleic acid cyclase, preparation method and application

Info

Publication number: CN114836401A
Application number: CN202210433225.0A
Authority: CN
Inventors: 王梁华; 孙铭娟; 高思懿; 陈涵; 杨尧; 杨佳平; 邓博文; 崔师海; 周世源; 蒋柏杨; 马振霞; 赵鲁明; 郭晗; 杨成芳; 邹彬
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2022-04-24
Filing date: 2022-04-24
Publication date: 2022-08-02

Abstract

The invention relates to the field of preparation of cyclized nucleic acid aptamers, and provides a recombinant ssDNA (single stranded deoxyribonucleic acid) nucleic acid cyclase, a preparation method and application thereof. The recombinant ssDNA (single stranded deoxyribonucleic acid) nucleic acid cyclase is a thermostable nucleic acid cyclase Circling strand separated from phage TS2126, a recombinant expression vector pET22 b-Circling strand is constructed by carrying out codon optimization and modification on the nucleic acid cyclase, and is transformed into an E.coil Escherichia coli expression system, IPTG is used as an inducer, and the expressed recombinant protein ssDNAligase has the amino acid sequence and the gene sequence shown as SEQ ID No.1 and SEQ ID No.2 respectively. Functional experiments prove that the recombinant protein has stable and efficient cyclization effects on ssDNA with different lengths, has catalytic efficiency similar to or even superior to that of commercialized similar products, and is suitable for cyclization modification and related function research of nucleic acid aptamers.

Description

Recombinant ssDNA (single-stranded deoxyribonucleic acid) nucleic acid cyclase, preparation method and application

Technical Field

The invention relates to the technical field of biology, relates to the field of preparation of cyclized nucleic acid aptamers, and particularly relates to a recombinant ssDNA (single-stranded deoxyribonucleic acid) nucleic acid cyclase, and a preparation method and application thereof.

Background

With the development of molecular biology techniques, particularly aptamer-related nucleic acid application techniques, more and more nucleic acid-based biological detection techniques and biosensor techniques have been widely studied and applied. Compared with the antibody with the chemical nature of protein, the aptamer with similar function has obvious advantages in yield and cost because of chemical synthesis, and the stability of the nucleic acid structure can be obviously improved by performing cyclization modification on the nucleic acid.

Currently common techniques for preparing circular nucleic acids include enzymatic synthesis using T4 DNA ligase or T4 RNA ligase, or chemical synthesis by completing the circularization through a carboxyl and amino amidation reaction, azide-alkyne click reaction, and chemical catalysts, among others. The enzyme synthesis method based on T4 ligase often needs some auxiliary nucleic acid chains to improve cyclization efficiency, is only suitable for cyclizing natural nucleic acid, and has low cyclization efficiency for short-chain oligonucleotides, but can hardly cyclize for modified oligonucleotides; the advantage of chemical synthesis is that it is not limited by the modified oligonucleotide, but it often requires modification of the Nucleic acid itself to achieve ligation, and the effect on Nucleic acid function, particularly on Nucleic acid aptamers, is difficult to control (Jiuxing Li, Mostafa modified-Elsabah, Freeman Paczkowski, and Yingfu Li. circular Nucleic Acids: Discovery, Functions and applications. ChemBiochem 2020,21, 1-21)).

Despite the research of various cyclases in the prior art, many suitable templates for natural single-stranded DNA, such as the single-stranded DNA independent ligation reaction mixture, method and kit disclosed in chinese patent document CN102317475A, do not include synthetic ssDNA, and do not involve cyclization of aptamers. The synthesis of ssDNA and circularization of aptamers has been an open problem in the field.

Disclosure of Invention

The present invention is based on the above-mentioned research, and the first objective of the present invention is to provide a recombinant ssDNA nucleic acid cyclase (ssDNAligase) capable of catalyzing ssDNA cyclization and ligation and having the function of preparing a cyclized aptamer, and the second objective of the present invention is to provide a preparation method and application of the recombinant ssDNA nucleic acid cyclase.

The basic principle of the invention is as follows: after codon optimization and modification are carried out on the nucleic acid cyclase Circligase (total length 1188bp, consisting of 395aa and molecular weight of 43.3KD) separated from the bacteriophage TS2126 through heat stability, a recombinant expression vector pET22b-Circligase is constructed through chemical synthesis, and is transferred into an E.coil Escherichia coli expression system, IPTG is used as an inducer, a recombinant ssDNAlid is expressed, and the function verification is carried out on the recombinant ssDNAlid.

In the first aspect of the invention, a recombinant ssDNA nucleic acid cyclase is provided, which is a recombinant protein ssDNAligase that is obtained by performing codon optimization on a thermostable nucleic acid cyclase (Circligase) isolated from phage strain TS2126 and transferring the codon optimized enzyme into an E.coil E.coli expression system for induced expression.

The amino acid sequence (Protein Length 415MW 46069.6PredictedpI 6.67) of the recombinant ssDNA nucleic acid cyclase is shown in SEQ ID No. 1:

MSSLAPWRTTSWSPLGSPPSLEDALRLARTTRAFAVRRDGEGRALVTYLYGTPELFSLPGARELRGIVYREEDGTVLSRPFHKFFNFGEPLAPGEEAFKAFRDSMVPLFVAEKVDGYLAQAYLDGGELRFASRHSLNPPLVGALLRKAVDEEAMARLGKLLAAEGGRWTALLEVVDPEAPVMVPYQEPGVYLLALRSIGEGHYLLPGVHFPLPEALRYVRWEPRMDFDPHRFRGEIRDLQGVEGYVVTDGAEFVKFKTGWAFRLARFLMDPEGVFLEAYAEDRLDDLVGALAGREDLLRAVARAQDYLAGLYGEAVGAGDALRRMGLPRKEAWARVQEEAGRWGGFAPAYARAAMAAYEGGEAREAFLVELRKRSARKALEALHLFPRVGGELRGPNSSSVDKLAAALEHHHHHH

the gene sequence of the coding recombinant ssDNA nucleic acid cyclase is shown as SEQ ID NO. 2:

ATGAGCAGCCTGGCGCCGTGGCGTACCACCAGCTGGAGCCCGCTGGGTAGCCCGCCGAGCCTGGAAGATGCGCTGCGTCTGGCGCGTACCACCCGTGCGTTTGCGGTTCGTCGTGATGGTGAAGGCCGTGCGCTGGTGACCTACCTGTATGGTACCCCGGAACTGTTTAGCCTGCCGGGTGCGCGTGAGCTGCGTGGTATCGTTTACCGTGAGGAAGACGGCACCGTGCTGAGCCGTCCGTTCCACAAGTTCTTTAACTTCGGCGAGCCGCTGGCGCCGGGTGAGGAAGCGTTCAAGGCGTTTCGTGATAGCATGGTGCCGCTGTTCGTTGCGGAGAAAGTGGACGGTTACCTGGCGCAGGCGTATCTGGATGGTGGCGAACTGCGTTTTGCGAGCCGTCACAGCCTGAACCCGCCGCTGGTTGGTGCGCTGCTGCGTAAGGCGGTGGACGAGGAAGCGATGGCGCGTCTGGGTAAACTGCTGGCGGCGGAAGGTGGCCGTTGGACCGCGCTGCTGGAAGTGGTTGATCCGGAGGCGCCGGTGATGGTTCCGTACCAGGAGCCGGGTGTTTATCTGCTGGCGCTGCGTAGCATCGGTGAAGGCCACTACCTGCTGCCGGGCGTTCACTTCCCGCTGCCGGAAGCGCTGCGTTATGTGCGTTGGGAGCCGCGTATGGACTTCGATCCGCACCGTTTTCGTGGTGAAATTCGTGACCTGCAAGGTGTTGAGGGCTACGTGGTTACCGATGGTGCGGAGTTCGTGAAGTTTAAAACCGGCTGGGCGTTCCGTCTGGCGCGTTTTCTGATGGACCCGGAAGGCGTGTTTCTGGAAGCGTATGCGGAGGATCGTCTGGATGATCTGGTTGGTGCGCTGGCGGGTCGTGAGGACCTGCTGCGTGCGGTGGCGCGTGCGCAGGATTACCTGGCGGGTCTGTATGGTGAAGCGGTGGGTGCGGGTGATGCGCTGCGTCGTATGGGTCTGCCGCGTAAAGAAGCGTGGGCGCGTGTGCAAGAGGAAGCGGGCCGTTGGGGTGGCTTCGCGCCGGCGTACGCGCGTGCGGCGATGGCGGCGTATGAGGGTGGCGAAGCGCGTGAGGCGTTCCTGGTTGAACTGCGTAAGCGTAGCGCGCGTAAAGCGCTGGAGGCGCTGCACCTGTTTCCGCGTGTGGGTGGCGAACTGCGTGGTCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGA。

in a second aspect of the present invention, there is provided a method for preparing the recombinant ssDNA nucleic acid cyclase, comprising the following steps:

A. construction of recombinant expression vector pET22b-Circligase

The recombinant ssDNA nucleic acid cyclase is synthesized by a whole gene, connected with pET22b through NdeI/EcoRI double enzyme cutting sites to construct a recombinant expression vector pET22b-Circligase, and transformed into an Escherichia coli competent cell E.coli DH5 alpha. The method comprises the following specific steps:

1) gene fragment and plasmid digestion

And (3) configuring an enzyme digestion reaction system to respectively carry out enzyme digestion on the ssDNA nucleic acid cyclase PCR product and the plasmid extraction product, and carrying out gel recovery through agarose gel electrophoresis.

Wherein the enzyme digestion reaction system of the ssDNA nuclease gene fragment comprises a gene fragment, an enzyme digestion buffer solution, a NdeI site enzyme digestion solution and a XhoI site enzyme digestion solution, and the volume ratio is 16:2:1: 1; the enzyme digestion reaction system of the pET22b plasmid extraction product comprises a plasmid, an enzyme digestion buffer solution, a Nde I site enzyme digestion solution and an Xho I site enzyme digestion solution, and the volume ratio is 16:2:1: 1; the enzyme digestion reaction conditions are as follows: incubation was carried out at 37 ℃ for 2.5 h.

2) The gene fragment is connected with plasmid

And incubating a reaction system consisting of T4 ligase, a connection buffer solution, a gene fragment enzyme digestion product and a plasmid enzyme digestion product according to the volume ratio of 1:2:9:8 at the constant temperature of 16 ℃ for 1.5h to complete the connection of the gene fragment and the plasmid.

3) Preparation of competent Escherichia coli E.coli DH5 alpha by calcium chloride method

Transferring the ligation product into competent escherichia coli with the volume 20 times that of the competent escherichia coli, adding an LB culture medium with the volume 100 times that of the ligation product, and performing shake cultivation at 37 ℃ and 180r/min for 45 min; taking a proper amount of bacterial liquid, coating the bacterial liquid on a plate containing ampicillin, culturing the bacterial liquid in a thermostat at 37 ℃ and 180r/min overnight, selecting bacteria on the plate, inoculating the bacteria on an LB culture medium containing ampicillin, culturing the bacteria in a shaker at 37 ℃ and 180r/min overnight to obtain an Escherichia coli E.coil DH5 alpha clone strain containing pET22 b-Circliase, and placing the Escherichia coli E.coil DH5 alpha clone strain containing 8% of glycerol at-80 ℃ for seed preservation.

Wherein, the LB culture medium formula is as follows: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride; ampicillin was added at a concentration of 100mg/ml to LB medium at a ratio of 1000: 1.

B. pET22b-Circligase coli E.coil BL21 expression strain construction

And B, performing plasmid extraction on the bacterial liquid obtained in the step A, verifying the purity of the bacterial liquid, transferring the bacterial liquid into escherichia coli E.coil BL21, adding an LB culture medium, performing shake cultivation, and screening by using ampicillin, wherein the specific steps are as follows:

1) taking a clone strain of Escherichia coli E.coil DH5 alpha containing pET22b-Circligase from a refrigerator at-80 ℃; according to the following steps of 1: 100 percent, inoculating into a test tube filled with 3ml of LB culture medium, shaking-culturing at 37 ℃ and 180r/min for 12-16h, and recovering bacteria.

2) And d, performing plasmid extraction on the bacterial liquid obtained in the step a by using a plasmid extraction kit (Axygen) and performing purity verification through agarose gel electrophoresis.

3) Transferring plasmid pET22b-Circligase into Escherichia coli E.coil BL21 by calcium chloride conversion method, adding 500 μ l LB culture medium, shake culturing at 37 deg.C and 180r/min for 45 min; taking 50 mul of coated plates, and culturing overnight in a thermostat at 37 ℃ and 180 r/min; the strain on the plate is inoculated into 4ml LB culture medium containing ampicillin and cultured for 12-16h at 37 ℃ and 180r/min overnight in a shaking table.

Wherein the LB culture medium comprises the following components in percentage by weight: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride; the concentration of the ampicillin is 100mg/ml, and the ampicillin and the LB culture medium are added according to the proportion of 1000: 1.

C. Inducible expression of recombinant proteins

Absorbing the expression bacterial liquid in the step B into LB culture medium with 100 times volume, carrying out shake culture at 37 ℃ and 180r/min until OD is reached ₆₀₀ And (4) approximately closing to 0.6, and adding an inducer IPTG 25 to induce the expression of the recombinant protein. The method comprises the following specific steps:

1) sucking 30 mul of expression bacterium liquid to a test tube filled with 3ml of LB culture medium, shaking-culturing for 3h at 37 ℃ and 180r/min until OD600 is approximately equal to 0.6, and adding 25 mul of IPTG (isopropyl-beta-thiogalactoside) as an inducer for continuous culture for 4 h; centrifuging at 5000r/min for 5min, discarding the supernatant, collecting thallus, and adding 1ml PBS for resuspension; the mixture was fully dissolved using an ultrasonicator, centrifuged at 12000r/min for 15min, and the supernatant and the precipitate were separately collected and examined by SDS-PAGE.

2) Optimizing expression conditions according to the result of the step a, enlarging expression scale: IPTG solution is 100mmol/L, and the final concentration is 1mmol/L after adding LB culture medium; the PBS solution was diluted 5-fold with 5XPBS solution, and the phosphate concentration was 10 mmol/L.

D. Recombinant protein purification

1) Taking supernatant obtained after induced expression and ultrasonic disruption as crude protein; taking Ni-NTA, and washing and balancing by using connecting buffer solution with 5 times of column volume; incubating the crude protein and the equilibrated Ni-NTA at 4 ℃ for 20 min; keeping the temperature low, and cleaning the equilibrium column by using a Binding buffer until the absorbance of the effluent liquid is not changed; eluting the equilibrium column with an elution buffer solution with 5 times of column volume to obtain a purified product;

2) ultrafiltering and concentrating the purified product twice at 5000g for 30 min; adding protein preservation buffer solution into the residual liquid, and ultrafiltering and concentrating for 30min twice to obtain purified sample with concentration of 1 mg/ml.

Wherein, in the above steps, the connection buffer solution comprises: 20mmol/L sodium phosphate, 500mmol/L sodium chloride and 20mmol/L imidazole; the elution buffer composition was: 20mmol/L sodium phosphate, 500mmol/L sodium chloride and 500mmol/L imidazole; the composition of the preservation buffer solution is as follows: 50mmol/LpH 7.5.5 Tris-HCl, 100mmol/LNaCl, 0.1mmol/L EDTA, 1mmol/LDTT, 0.1% Triton X100, 50% Glycerol.

In a third aspect of the present invention, there is provided the use of the recombinant ssDNA nucleic acid cyclase in cyclizing a nucleic acid aptamer, wherein the nucleic acid aptamer is a single-stranded DNA or RNA.

Preferably, the circularized ligation includes homo-di ligation, hetero-di ligation, homo-multimeric ligation, hetero-multimeric ligation: experiments prove that the recombinant ssDNA nucleic acid cyclase can catalyze the cyclization of ssDNA aptamers at different ends, and the catalytic activities of the ssDNA aptamers at different 5' ends with the same length are respectively G > A > T > C from high to low; to the same length of 3'

The catalytic activity of the ssDNA at the end is T > A > G > C from high to low.

(2) The single-stranded DNA or RNA is phosphorylated single-stranded DNA or RNA: the present invention is very catalytically active against phosphorylated ssDNA, whereas almost no formation of cyclization products is observed for non-phosphorylated ssDNA.

(3) The method is suitable for circularization of single-stranded DNA or RNA with different lengths: has good cyclization activity on ssDNA with different lengths (including but not limited to 16nt, 40nt and 60 nt).

(4) The recombinant ssDNA nucleic acid cyclase of the present invention requires Mn during the cyclization process ²⁺ And betaine presence: when Mn is present ²⁺ When the final concentration is 1.25mM-2.5mM, the enzyme has higher catalytic activity; when the final concentration of the betaine is 0.75-1mM, the enzyme has higher catalytic activity.

The invention has the following beneficial guarantee and effects:

the ssDNA nucleic acid cyclase synthesized by the invention is obtained by carrying out codon optimization and then recombinant expression on the thermostable nucleic acid cyclase Circligase of bacteriophage TS2126, can realize cyclized connection on ssDNA with different lengths, and can stably and efficiently prepare the circular nucleic acid. Compared with T4 ligase, the method has higher cyclization efficiency without auxiliary nucleic acid chains, and has good cyclization efficiency for short-chain oligonucleotides (> 15 nt). Compared with the commercialized cyclase of the same type, the cyclase has convenient protein purification, controllable cost, similar or even more excellent catalytic efficiency with the commercialized cyclase of the same type, and can be used for cyclization modification and related function research of the aptamer.

Drawings

FIG. 1 is a graph of the results of a small test SDS-PAGE of ssDNA nucleic acid cyclase fusion protein expression, in which M: protein Marker (Cat. No.: C600525); 1: total protein prior to induction; 2: supernatant at 20 ℃; 3: precipitating at 20 ℃; 4: supernatant at 37 ℃; 5: precipitating at 37 ℃.

FIG. 2 is a graph of the results of nickel agarose affinity chromatography purification of the ssDNA nucleic acid cyclase fusion protein, in which M: protein Marker (Cat. No.: C600525); 1: sampling; 2: flowing out; 3-4: 20mM Imidazole eluate fraction; 5-6: 50mM Imidazole eluate fraction; 7-8: 500mM Imidazole fraction.

FIG. 3 is a SDS-PAGE result of purified protein of final ssDNA nucleic acid cyclase, in which M: protein Marker (Cat. No.: C600525); 1: and fusing the target protein.

FIG. 4 is a Western Blot analysis of the final ssDNA nucleic acid cyclase purified protein, where M: protein Marker (Cat. No.: C600525); 1: and fusing the target protein.

FIG. 5 shows the results of the cyclization of ssDNA aptamers of different lengths catalyzed by ssDNA nucleocyclase;

FIG. 6 is a comparison of catalytic activities of ssDNA cyclase for ssDNA of different 5' termini of the same length;

FIG. 7 is a comparison of catalytic activity of ssDNA cyclase for ssDNA at different 3' termini of the same length

FIG. 8 is a graph of the effect of terminal phosphorylation on cyclization reactions;

FIG. 9 shows Mn ²⁺ The effect of concentration on the cyclization reaction;

FIG. 10 is a graph of the effect of betaine concentration on cyclization;

FIG. 11 shows the effect of enzyme concentration on cyclization reaction.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

The experimental procedures, in which specific conditions are not specified, in the following examples are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Percentages and parts are by volume 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 practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1 preparation of recombinant proteins of ssDNA nucleic acid cyclase

The cyclase used in the invention is a recombinant protein ssDNAligase obtained by performing codon optimization modification on a nucleic acid cyclase Circligase which is separated from phage TS2126 and is thermostable, and performing chemical synthesis and induced expression.

1. Construction of recombinant expression vector pET22b-Circligase

1) The Circligase codon is optimized and modified to be more beneficial to the expression of the Circligase codon in Escherichia coli, a polyhistidine tag is added, and the gene is obtained by chemical synthesis (entrusted Genscript Kinry). The recombinant is constructed by connecting NdeI/EcoRI double enzyme cutting sites with pET22b, transformed into E.coli DH5 alpha, amplified, sequenced and identified.

2) The enzyme digestion reaction systems configured according to the table 1 and the table 2 are respectively used for enzyme digestion (incubation at constant temperature of 37 ℃ for 2.5 h) of the gene PCR product and the plasmid product, and gel recovery is carried out through agarose gel electrophoresis.

TABLE 1 cleavage system for Circligase Gene fragments

Gene fragment	16μl
		Enzyme digestion buffer solution	2μl
NdeⅠ	1μl
		XhoⅠ	1μl
Total	20μl

TABLE 2 digestion system for pET22b plasmid extract

Plasmids	16μl
		Enzyme digestion buffer solution	2μl
NdeⅠ	1μl
		XhoⅠ	1μl
Total	20μl

3) A ligation reaction system was configured according to Table 3 and incubated at 16 ℃ for 1.5h to achieve ligation of the gene fragment and the plasmid.

TABLE 3 Gene fragment and plasmid ligation System

T4 ligase	0.5μl
		Ligation buffer	1μl
PCR cleavage product	4.5μl
		Plasmid cleavage product	4μl
Total	10μl

4) Preparing competent Escherichia coli E.coli DH5 alpha by calcium chloride method,

transferring 5 μ l of the ligation product into 100 μ l of competent Escherichia coli, adding 500 μ l of LB medium, shaking-culturing at 37 deg.C for 45min at 180 r/min; 50. mu.l of the bacterial suspension was spread on a plate containing ampicillin and cultured overnight in a 180r/min incubator at 37 ℃. The plate was inoculated to 4ml LB medium containing ampicillin and cultured overnight at 37 ℃ with a shaker at 180r/min to obtain E.coli E.coil DH 5. alpha. clonal strain containing pET22b-Circligase and was incubated at-80 ℃ with LB medium containing 8% glycerol.

Wherein, the LB culture medium formula is as follows: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride; ampicillin concentration is 100mg/ml, and the ampicillin and LB culture medium are added according to the proportion of 1000: 1;

2. construction of pET22b-Circligase coli E.coil BL21 expression Strain:

3. Inducible expression of recombinant proteins

1) Sucking 30 mul of expression bacterial liquid into a test tube filled with 3ml of LB culture medium, shaking-culturing at 37 ℃ and 180r/min for 3h until OD600 is approximately equal to 0.6, adding 25 mul of inducer IPTG, and continuing culturing for 4 h; centrifuging at 5000r/min for 5min, discarding the supernatant, collecting thallus, and adding 1ml PBS for resuspension; the protein was dissolved sufficiently by using an ultrasonicator, centrifuged at 12000r/min for 15min, and the supernatant and the precipitate were separately collected and examined by SDS-PAGE, and the target protein was found in the precipitate under the electrophoresis condition at 37 ℃ as shown in FIG. 1.

2) Optimizing expression conditions according to the result of the step 1) and enlarging expression scale.

IPTG solution is 100mmol/L, and the final concentration is 1mmol/L after adding LB culture medium. The PBS solution is obtained by diluting 5 times of 5xPBS solution, and the phosphate radical concentration of the PBS solution is 10 mmol/L.

4. Recombinant protein purification

1) Taking supernatant obtained after induced expression and ultrasonic disruption as crude protein for purification; taking 2ml of Ni-NTA, and washing and balancing by using a Binding buffer with 5 times of column volume; incubating the crude protein and the equilibrated Ni-NTA at 4 ℃ for 20 min; keeping the temperature low, and cleaning the equilibrium column by using a Binding buffer until the absorbance of the effluent liquid is not changed; the equilibrium column was eluted with 5 column volumes of Elution buffer to obtain 25ml of purified product. The result of the SDS-PAGE purification by nickel agarose affinity chromatography of the ssDNA nucleic acid cyclase fusion protein is shown in FIG. 2.

Wherein, the Binding buffer comprises the following components: 20mmol/L sodium phosphate, 500mmol/L sodium chloride and 20mmol/L imidazole; the Elution buffer consists of: 20mmol/L sodium phosphate, 500mmol/L sodium chloride and 500mmol/L imidazole; the composition of the preservation buffer solution is as follows: 50mmol/L of LTris-HCl (pH7.5), 100mmol/L of sodium chloride, 0.1mmol/L of EDTA, 1mmol/L of LDTT, 0.1% of Triton X100, 50% of Glycerol.

2) Ultrafiltering and concentrating the purified product twice at 5000g for 30 min; adding 10ml of protein preservation buffer solution into the residual liquid, and ultrafiltering and concentrating twice at 5000g for 30min to obtain 600 μ l of sample with the detected concentration of 1 mg/ml. The final SDS-PAGE result of the purified protein of ssDNA cyclase is shown in FIG. 3, and the molecular weight of the fusion target protein is about 45.0 kDa.

To further confirm that the purified protein was the target protein, the protein was developed using a TMB color kit according to the Western Blot procedure. As shown in FIG. 4, a distinct band appeared at the corresponding position, indicating that the protein is a recombinant target protein of ssDNA nucleic acid cyclase.

Example 2 Mass Spectrometry identification of recombinant proteins

The sequence coverage rate of the recombinant protein is determined to be 80% through biological medium spectrum identification. The mass spectrometry data identification results are as follows:

example 3 catalysis of cyclization of ssDNA aptamers of varying lengths

The ssDNA nucleic acid cyclase recombinant protein prepared by the method is used for cyclizing and enzyme cutting ssDNA aptamers with different lengths (16nt, 40nt and 60 nt). The cyclization and cleavage reaction systems are shown in tables 4 and 5, respectively:

TABLE 4 cyclization reaction System

Table 5 digestion system:

as shown in FIG. 5, the results of 20% native PAGE electrophoresis identification after circularization of ssDNA of different lengths show that the present invention has good circularization activity for ssDNA of different lengths (including but not limited to 16nt, 40nt, and 60 nt).

Example 4 catalysis of different terminal ssDNA aptamer cyclization

The cyclization and cleavage reaction was as in example 3, with a list of different terminal substrates as shown in Table 6:

TABLE 6 summary of sequences of different terminal substrates to be cyclized

Name (R)	Length of	End tip	Sequence of	Numbering
					P13SHBF-5D1(3D1)	16nt	5’g3’t	ggactcaggaggtggt	SEQ ID NO.3
P13SHBF-5D2	16nt	5’a3’t	agactcaggaggtggt	SEQ ID NO.4
					P13SHBF-5D3	16nt	5’t3’t	tgactcaggaggtggt	SEQ ID NO.5
P13SHBF-5D4	16nt	5’c3’t	cgactcaggaggtggt	SEQ ID NO.6
					P13SHBF-3D2	16nt	5’g3’a	ggactcaggaggtgga	SEQ ID NO.7
P13SHBF-3D3	16nt	5’g3’g	ggactcaggaggtggg	SEQ ID NO.8
					P13SHBF-3D4	16nt	5’g3’c	ggactcaggaggtggc	SEQ ID NO.9

The results of the electrophoretic identification of 20% urea denaturation PAGE after the enzyme digestion and cyclization of the gene fragment are shown in the figure 6 and the figure 7: from the electrophoresis results of 5D1-5D4, it can be found that the catalytic activities of the invention on ssDNA of different 5' ends with the same length are respectively G > A > T > C from high to low (FIG. 6); from the electrophoresis results of 3D1(5D1) -3D4, it can be found that the catalytic activity of the invention on ssDNA of different 3' ends with the same length is from high to low, which is T > A > G > C respectively (FIG. 7).

Example 5 Effect of terminal phosphorylation on cyclization reactions

The cyclization and cleavage reaction was as in example 3, with a list of different phosphorylated substrates as shown in Table 7:

TABLE 7 summary of the different phosphorylated substrates to be cyclized

Name (R)	Phosphorylation of	DNA/RNA	Sequence of	Numbering
					P13SHBF-5D1	Is that	DNA	ggactcaggaggtggt	SEQ ID NO.3
13SHBF	Whether or not	DNA	ggactcaggaggtggt	SEQ ID NO.3
					P13SHBF-5D1	Is that	RNA	ggacucaggagguggu	SEQ ID NO.10

The electrophoresis result of the 20% urea denaturation PAGE electrophoresis identification result obtained after the gene fragment is subjected to enzyme digestion and cyclization is shown in figure 8, and the electrophoresis result shows that the catalytic activity of the gene fragment is very strong on phosphorylated ssDNA, and the generation of a cyclization product can hardly be observed on non-phosphorylated ssDNA; furthermore, no significant cyclization products were observed for the effect of ssRNA cyclization affected by ssRNA degradation.

Example 6 Mn ²⁺ Effect of concentration on cyclization reaction

The cyclization reaction system is shown in Table 8, and the cleavage reaction system is the same as in example 3.

TABLE 8 Mn-containing ²⁺ Cyclization reaction system

Wherein the concentration varies with the amount x added (x ═ 0, 0.5, 1, 2).

The electrophoresis result is shown in fig. 9, wherein the 3/4 th lane x is 0; 5/6 lane x is 0.5; 7/8 lane x ═ 1; 9/10 lane x is 2. According to the electrophoresis result, the invention can be found in different Mn ²⁺ There is a clear difference in the catalytic activity of the substrate ssDNA under conditions where Mn is present ²⁺ The invention has higher catalytic activity when the final concentration is 1.25m M-2.5m M.

Example 7 Effect of betaine concentration on cyclization reaction

The cyclization reaction system is shown in Table 9, and the cleavage reaction system is the same as in example 3.

TABLE 9 betaine-containing cyclization reaction System

Wherein the Betaine (Betaine) concentration varies with the amount x added (x ═ 0, 1, 2, 3, 4, 5).

The electrophoresis result is shown in fig. 10, wherein the 3/4 th lane x is 0; 5/6 lane x ═ 1; 7/8 lane x ═ 2; 9/10 lane x ═ 3; 11/12 lane x is 4; 13/14 lane x is 5. According to electrophoresis results, the catalytic activities of the ssDNA of the substrate are obviously different under different betaine conditions, wherein the enzyme has higher catalytic activity when the final concentration of the betaine is 0.75M-1M.

Example 8 Effect of enzyme concentration on cyclization reaction

The cyclization reaction system is shown in Table 10, and the cleavage reaction system is the same as in example 3.

TABLE 10 cyclization reaction systems at different enzyme concentrations

Wherein the enzyme concentration varies with the amount x added (x ═ 0, 1, 1.5, 2).

The electrophoresis result is shown in fig. 11, wherein the 3/4 th lane x is 0; 5/6 lane x ═ 1; 7/8 lane x ═ 1.5; 9/10 lane x ═ 2; lane 11/12 shows a commercial enzyme. Electrophoresis results show that the catalytic activity of the ssDNA substrate is obviously different under different enzyme concentration conditions, wherein the catalytic activity of the method is higher when the final concentration of the cyclase is 0.0375 mu g/mu l.

Example 9 cyclization Effect of the invention in comparison with commercial kits

The cyclization reaction system and the cyclization reaction system of the commercial kit are shown in the table 11, and the enzyme digestion reaction system is the same as the example 3.

TABLE 11 cyclization reaction System of the present invention with commercial kits

In the above table, the enzymes correspond to the synthetase of the present invention and the commercial cyclase, respectively, and the concentrations of both are 0.025. mu.g/. mu.l. This experiment was performed on the same gel as in example 8, and referring to FIG. 11, the results of the present synthetase correspond to lane 5/6, and the results of the commercial cyclase correspond to lane 11/12. Electrophoresis results show that the low-concentration enzyme of the invention has similar cyclization effect with commercial kits.

The present invention is not limited to the embodiments described above, and those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are included in the scope defined by the claims of the present application.

Sequence listing

<110> China people liberation army navy military medical university

<120> recombinant ssDNA (single stranded deoxyribonucleic acid) nucleic acid cyclase, preparation method and application

<130> specification of claims

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Met Ser Ser Leu Ala Pro Trp Arg Thr Thr Ser Trp Ser Pro Leu Gly

1 5 10 15

Ser Pro Pro Ser Leu Glu Asp Ala Leu Arg Leu Ala Arg Thr Thr Arg

20 25 30

Ala Phe Ala Val Arg Arg Asp Gly Glu Gly Arg Ala Leu Val Thr Tyr

35 40 45

Leu Tyr Gly Thr Pro Glu Leu Phe Ser Leu Pro Gly Ala Arg Glu Leu

50 55 60

Arg Gly Ile Val Tyr Arg Glu Glu Asp Gly Thr Val Leu Ser Arg Pro

65 70 75 80

Phe His Lys Phe Phe Asn Phe Gly Glu Pro Leu Ala Pro Gly Glu Glu

85 90 95

Ala Phe Lys Ala Phe Arg Asp Ser Met Val Pro Leu Phe Val Ala Glu

100 105 110

Lys Val Asp Gly Tyr Leu Ala Gln Ala Tyr Leu Asp Gly Gly Glu Leu

115 120 125

Arg Phe Ala Ser Arg His Ser Leu Asn Pro Pro Leu Val Gly Ala Leu

130 135 140

Leu Arg Lys Ala Val Asp Glu Glu Ala Met Ala Arg Leu Gly Lys Leu

145 150 155 160

Leu Ala Ala Glu Gly Gly Arg Trp Thr Ala Leu Leu Glu Val Val Asp

165 170 175

Pro Glu Ala Pro Val Met Val Pro Tyr Gln Glu Pro Gly Val Tyr Leu

180 185 190

Leu Ala Leu Arg Ser Ile Gly Glu Gly His Tyr Leu Leu Pro Gly Val

195 200 205

His Phe Pro Leu Pro Glu Ala Leu Arg Tyr Val Arg Trp Glu Pro Arg

210 215 220

Met Asp Phe Asp Pro His Arg Phe Arg Gly Glu Ile Arg Asp Leu Gln

225 230 235 240

Gly Val Glu Gly Tyr Val Val Thr Asp Gly Ala Glu Phe Val Lys Phe

245 250 255

Lys Thr Gly Trp Ala Phe Arg Leu Ala Arg Phe Leu Met Asp Pro Glu

260 265 270

Gly Val Phe Leu Glu Ala Tyr Ala Glu Asp Arg Leu Asp Asp Leu Val

275 280 285

Gly Ala Leu Ala Gly Arg Glu Asp Leu Leu Arg Ala Val Ala Arg Ala

290 295 300

Gln Asp Tyr Leu Ala Gly Leu Tyr Gly Glu Ala Val Gly Ala Gly Asp

305 310 315 320

Ala Leu Arg Arg Met Gly Leu Pro Arg Lys Glu Ala Trp Ala Arg Val

325 330 335

Gln Glu Glu Ala Gly Arg Trp Gly Gly Phe Ala Pro Ala Tyr Ala Arg

340 345 350

Ala Ala Met Ala Ala Tyr Glu Gly Gly Glu Ala Arg Glu Ala Phe Leu

355 360 365

Val Glu Leu Arg Lys Arg Ser Ala Arg Lys Ala Leu Glu Ala Leu His

370 375 380

Leu Phe Pro Arg Val Gly Gly Glu Leu Arg Gly Pro Asn Ser Ser Ser

385 390 395 400

Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His

405 410 415

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atgagcagcc tggcgccgtg gcgtaccacc agctggagcc cgctgggtag cccgccgagc 60

ctggaagatg cgctgcgtct ggcgcgtacc acccgtgcgt ttgcggttcg tcgtgatggt 120

gaaggccgtg cgctggtgac ctacctgtat ggtaccccgg aactgtttag cctgccgggt 180

gcgcgtgagc tgcgtggtat cgtttaccgt gaggaagacg gcaccgtgct gagccgtccg 240

ttccacaagt tctttaactt cggcgagccg ctggcgccgg gtgaggaagc gttcaaggcg 300

tttcgtgata gcatggtgcc gctgttcgtt gcggagaaag tggacggtta cctggcgcag 360

gcgtatctgg atggtggcga actgcgtttt gcgagccgtc acagcctgaa cccgccgctg 420

gttggtgcgc tgctgcgtaa ggcggtggac gaggaagcga tggcgcgtct gggtaaactg 480

ctggcggcgg aaggtggccg ttggaccgcg ctgctggaag tggttgatcc ggaggcgccg 540

gtgatggttc cgtaccagga gccgggtgtt tatctgctgg cgctgcgtag catcggtgaa 600

ggccactacc tgctgccggg cgttcacttc ccgctgccgg aagcgctgcg ttatgtgcgt 660

tgggagccgc gtatggactt cgatccgcac cgttttcgtg gtgaaattcg tgacctgcaa 720

ggtgttgagg gctacgtggt taccgatggt gcggagttcg tgaagtttaa aaccggctgg 780

gcgttccgtc tggcgcgttt tctgatggac ccggaaggcg tgtttctgga agcgtatgcg 840

gaggatcgtc tggatgatct ggttggtgcg ctggcgggtc gtgaggacct gctgcgtgcg 900

gtggcgcgtg cgcaggatta cctggcgggt ctgtatggtg aagcggtggg tgcgggtgat 960

gcgctgcgtc gtatgggtct gccgcgtaaa gaagcgtggg cgcgtgtgca agaggaagcg 1020

ggccgttggg gtggcttcgc gccggcgtac gcgcgtgcgg cgatggcggc gtatgagggt 1080

ggcgaagcgc gtgaggcgtt cctggttgaa ctgcgtaagc gtagcgcgcg taaagcgctg 1140

gaggcgctgc acctgtttcc gcgtgtgggt ggcgaactgc gtggtccgaa ttcgagctcc 1200

gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactga 1248

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ggactcagga ggtggt 16

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agactcagga ggtggt 16

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tgactcagga ggtggt 16

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cgactcagga ggtggt 16

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ggactcagga ggtgga 16

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ggactcagga ggtggg 16

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ggactcagga ggtggc 16

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ggacucagga gguggu 16

Claims

1. A recombinant ssDNA nucleic acid cyclase characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. The gene encoding the recombinant ssDNA nucleic acid cyclase of claim 1, wherein the polynucleotide sequence of the gene is set forth in SEQ ID No. 2.

3. The method of preparing the recombinant ssDNA nucleic acid cyclase of claim 1, comprising the steps of:

A. construction of recombinant expression vector pET22b-Circligase

The recombinant ssDNA nucleic acid cyclase is synthesized by a whole gene, connected with pET22b through NdeI/EcoRI double enzyme cutting sites to construct a recombinant expression vector pET22b-Circligase, and transformed into an Escherichia coli competent cell E.coli DH5 alpha;

B. pET22b-Circligase coli E.coil BL21 expression strain construction

B, carrying out plasmid extraction on the bacterial liquid obtained in the step A, carrying out purity verification, transferring the bacterial liquid into escherichia coli E.coil BL21, adding an LB culture medium, carrying out shake cultivation, and screening by using ampicillin;

C. recombinant protein induced expression and purification

Absorbing the expression bacterial liquid in the step B into LB culture medium with 100 times volume, carrying out shake culture at 37 ℃ and 180r/min until OD is reached ₆₀₀ About 0.6, adding an inducer IPTG 25 to induce the expression of the recombinant protein;

and (3) purifying the supernatant obtained after induced expression and ultrasonic disruption as crude protein, and performing ultrafiltration and concentration on a purified product to obtain the recombinant ssDNA (single-stranded deoxyribonucleic acid) nucleic acid cyclase.

4. The method of claim 3, wherein the recombinant ssDNA nucleic acid cyclase is prepared by:

wherein, step A includes the following steps:

(1) gene fragment and plasmid digestion

Configuring an enzyme digestion reaction system to respectively carry out enzyme digestion on the ssDNA nucleic acid cyclase PCR product and the plasmid extraction product, carrying out gel recovery by agarose gel electrophoresis,

wherein the enzyme digestion reaction system of the ssDNA nuclease gene fragment comprises a gene fragment, an enzyme digestion buffer solution, a NdeI site enzyme digestion solution and a XhoI site enzyme digestion solution, and the volume ratio is 16:2:1: 1;

the digestion reaction system of the pET22b plasmid extraction product comprises a plasmid, digestion buffer solution, NdeI site digestion solution and XhoI site digestion solution, and the volume ratio is 16:2:1: 1;

the enzyme digestion reaction conditions are as follows: incubating at constant temperature for 2.5h at 37 ℃,

(2) the gene fragment is connected with plasmid

Incubating a reaction system consisting of T4 ligase, a connection buffer solution, a gene fragment enzyme digestion product and a plasmid enzyme digestion product according to the volume ratio of 1:2:9:8 at the constant temperature of 16 ℃ for 1.5h to complete the connection of the gene fragment and the plasmid,

(3) preparation of competent Escherichia coli E.coli DH5 alpha by calcium chloride method

Transferring the ligation product into competent Escherichia coli with the volume 20 times that of the competent Escherichia coli, adding LB culture medium with the volume 100 times that of the ligation product, and performing shake culture at 37 ℃ and 180r/min for 45 min; taking a proper amount of bacterial liquid, coating the bacterial liquid on a plate containing ampicillin, culturing the bacterial liquid in a thermostat at 37 ℃ and 180r/min overnight, selecting bacteria on the plate, inoculating the bacteria on an LB culture medium containing ampicillin, culturing the bacteria in a shaker at 37 ℃ and 180r/min overnight to obtain an Escherichia coli E.coil DH5 alpha clone strain containing pET22 b-Circliase, and placing the Escherichia coli E.coil DH5 alpha clone strain containing 8% of glycerol at-80 ℃ for seed preservation.

5. The method of claim 3, wherein the recombinant ssDNA nucleic acid cyclase is prepared by:

the specific operation steps of the step B are as follows:

b, carrying out plasmid extraction on the bacterial liquid obtained in the step A by using a plasmid extraction kit, and carrying out purity verification through agarose gel electrophoresis;

transferring the plasmid pET22b-Circligase into Escherichia coli E.coil BL21 by a calcium chloride conversion method, adding an LB culture medium, and carrying out shake cultivation at 37 ℃ and 180r/min for 45 min; taking a proper amount of bacterial liquid to coat a plate, and culturing overnight in a thermostat of 180r/min at 37 ℃; the strain on the plate is inoculated into LB culture medium containing ampicillin and cultured for 12-16h at 37 ℃ on a shaking table at 180r/min overnight.

6. The method of claim 4 or 5, wherein the recombinant ssDNA nucleic acid cyclase is prepared by:

wherein the LB culture medium comprises the following components in percentage by weight: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride;

the concentration of the ampicillin is 100mg/ml, and the ampicillin and the LB culture medium are added according to the proportion of 1000: 1.

7. The method of claim 3, wherein the recombinant ssDNA nucleic acid cyclase is prepared by:

in the step C, in the induction expression process, the concentration of an IPTG solution is 100mmol/L, and the final concentration is 1mmol/L after an LB culture medium is added;

the purification process comprises the following specific steps:

taking supernatant obtained after induced expression and ultrasonic disruption as crude protein; taking Ni-NTA, and washing and balancing by using connecting buffer solution with 5 times of column volume; incubating the crude protein and the balanced Ni-NTA at 4 ℃ for 20 min; keeping the temperature low, and washing the equilibrium column with a connecting buffer solution until the absorbance of the effluent liquid is not changed; eluting the equilibrium column with an elution buffer solution with 5 times of column volume to obtain a purified product;

ultrafiltering and concentrating the purified product twice at 5000g for 30 min; adding protein preservation buffer solution into the residual liquid, and performing ultrafiltration concentration twice at 5000g for 30min to obtain a purified sample with the concentration of 1 mg/ml.

8. The method of claim 3, wherein the recombinant ssDNA nucleic acid cyclase is prepared by:

wherein the ligation buffer consists of: 20mmol/L sodium phosphate, 500mmol/L sodium chloride and 20mmol/L imidazole;

the elution buffer composition was: 20mmol/L sodium phosphate, 500mmol/L sodium chloride and 500mmol/L imidazole;

the preservation buffer comprises the following components: 50mmol/L Tris-HCl pH7.5, 100mmol/L NaCl, 0.1mmol/L EDTA, 1mmol/L DTT, 0.1% TritonX100, 50% Glycerol.

9. The use of the recombinant ssDNA nucleic acid cyclase of claim 1 to cyclize an aptamer, wherein the aptamer is a single-stranded DNA or RNA.

10. Use according to claim 9, characterized in that:

wherein, the cyclization connection comprises same-second connection, different-second connection, same-poly connection and different-poly connection;

the single-stranded DNA or RNA is phosphorylated.