CN109234253B

CN109234253B - Mhp non-specific nuclease, coding gene and application thereof

Info

Publication number: CN109234253B
Application number: CN201811096592.6A
Authority: CN
Inventors: 吴文学; 李鹏; 张云科
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2018-09-19
Filing date: 2018-09-19
Publication date: 2021-02-19
Anticipated expiration: 2038-09-19
Also published as: CN109234253A

Abstract

The invention relates to an Mhp non-specific nuclease, an encoding gene and application thereof, wherein the amino acid sequence of the Mhp non-specific nuclease is shown as SEQ ID NO. 2. The Mhp non-specific nuclease provided by the invention can degrade various nucleic acid substrates, such as single-stranded DNA, double-stranded DNA, plasmids and RNA, has very high enzymatic activity and high temperature resistance, and can be widely applied to the fields of biological pharmacy, biological reagent research and development, industrialization and the like.

Description

Mhp non-specific nuclease, coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to Mhp non-specific nuclease, a coding gene and application thereof.

Background

Nucleases are enzymes that degrade nucleic acid substrates and catalyze the hydrolysis of phosphodiester bonds, and can be classified into dnases, rnases and nonspecific nucleases. Wherein the non-specific nuclease is a high-activity hydrolase, can non-specifically degrade almost all forms of nucleic acids, including single-stranded, double-stranded, linear, circular and supercoiled forms of DNA and RNA, and has no requirement on the sequence of the nucleic acids. The non-specific nuclease has a plurality of important functions, such as participation in DNA replication, recombination and repair, RNA splicing, maturation, degradation and interference, cell vegetative growth, apoptosis, inflammatory reaction, vascular thrombosis, host defense and other processes, thereby having important application value in the diagnosis and treatment of diseases such as cancer and the like; as a gene killer for controlling the self-destruction of the transformed microbial organisms; used for eliminating exogenous nucleic acid in recombinant protein drugs, reducing anaphylactic reaction and carcinogenic risk and improving the safety of biological products; the method is used for degrading the nucleic acid amplification products of PCR, LAMP and the like so as to avoid the pollution of the amplification products to a nucleic acid amplification laboratory or an operation table and reduce or eliminate non-specific amplification; the novel environment-friendly biological disinfectant can act on genetic materials of bacteria and viruses to crack DNA or RNA so as to achieve the aim of killing the bacteria and the viruses.

At present, nonspecific nuclease used at home and abroad is mainly from Benzonase endonuclease from Merck company, the Benzonase nuclease is endonuclease from Serratia marcocens and improved by genetic engineering, and can keep high activity under a wide range of conditions: maintaining high activity at a pH of 6-10 and a temperature of 0-42 deg.C; the activity was maintained in 1.0mM PMSF, 1.0mM EDTA and urea. However, the recombinant protein has a signal peptide, and the part exists in an inclusion body form, so that the renaturation is difficult, the price is high, and the large-scale application of the recombinant protein is limited. Therefore, it is necessary to develop a novel nonspecific nuclease and establish a compatible production process.

Disclosure of Invention

The invention aims to provide an Mhp non-specific nuclease, a coding gene and application thereof.

In order to achieve the object, the invention provides, in a first aspect, an Mhp non-specific nuclease derived from Mycoplasma Hyopneumoniae (Mhp), wherein the Mhp non-specific nuclease is:

(A) a protein consisting of an amino acid sequence shown as SEQ ID NO. 2; or

(B) 2, protein which is derived from (A) and has the same function by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 2.

Due to the specificity of the amino acid sequence, any fragment of the polypeptide containing the amino acid sequence shown in SEQ ID NO. 2 or its variants, such as conservative variants, bioactive fragments or derivatives thereof, is included in the scope of the present invention as long as the homology between the fragment of the polypeptide or the polypeptide variant and the amino acid sequence is above 95%. In particular, the alteration may comprise a deletion, insertion or substitution of an amino acid in the amino acid sequence; where conservative changes to a variant are made, the substituted amino acid has similar structural or chemical properties as the original amino acid, e.g., replacement of isoleucine with leucine, and the variant may also have non-conservative changes, e.g., replacement of glycine with tryptophan.

In a second aspect, the invention provides a gene encoding said Mhp non-specific nuclease. The nucleotide sequence is as follows:

i) 1, SEQ ID NO;

ii) a nucleotide sequence which is obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.1 and expresses the same functional protein;

iii) a nucleotide sequence which hybridizes with the sequence shown in SEQ ID NO.1 under stringent conditions in which hybridization is performed at 65 ℃ in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC containing 0.1% SDS solution and the membrane is washed with the solution and expresses the same functional protein; or

iv) a nucleotide sequence which has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses the same functional protein.

In a third aspect, the invention provides biological materials containing the genes encoding the Mhp non-specific nucleases, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, viral vectors or engineered bacteria.

Furthermore, the invention provides a recombinant expression vector containing the Mhp non-specific nuclease coding gene, and a starting vector of the recombinant expression vector is a vector capable of expressing a target protein in a eukaryotic expression system or a prokaryotic expression system.

Preferably, the starting vector is selected from pET series, pUC series or pBR322 vector, etc.

More preferably, the recombinant expression vector is pET28a-nuc obtained by inserting the coding gene of the Mhp non-specific nuclease between Nco I and Xho I cleavage sites of plasmid pET28 a.

Furthermore, the invention provides a recombinant genetic engineering bacterium, which is escherichia coli and carries an expression vector for expressing the Mhp non-specific nuclease.

In a specific embodiment of the invention, the recombinant genetically engineered bacterium is an engineered bacterium obtained by transforming Escherichia coli by using a recombinant expression vector pET28a-nuc, is named as Escherichia coli (Escherichia coli) BL21/Mhp-nuc, and is deposited in the China general microbiological culture Collection center, Anhui No.1 Hopkin 3 of the Ind region of Beijing, China academy of sciences, Microbiol research institute of China, postal code 100101, with the deposit number of CGMCC NO.16353, and the deposit date of 2018 months and 29 days.

In a fourth aspect, the invention provides a truncated protein of an Mhp non-specific nuclease, said truncated protein being:

(a) a protein consisting of the amino acid sequence shown in SEQ ID NO. 3; or

(b) 3, protein which is derived from (a) and has the same function by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 3.

In a fifth aspect, the invention provides any one of the following uses of the Mhp non-specific nuclease, a gene encoding the Mhp non-specific nuclease, a biological material containing the gene, or a truncated protein of the Mhp non-specific nuclease:

1) the application in degrading nucleic acid;

2) application in preparing nucleic acid degradation agent.

Wherein, the nucleic acid includes but is not limited to double-stranded DNA, single-stranded DNA, plasmid DNA, RNA or antisense RNA/DNA.

In a sixth aspect, the present invention provides a nucleic acid degradation agent, the effective component of which is the Mhp non-specific nuclease, truncated protein of the Mhp non-specific nuclease, and/or fusion protein obtained by linking a tag to the N-terminal and C-terminal of the truncated protein.

Further, the invention provides an application of the Mhp non-specific nuclease coding gene in preparation of the Mhp non-specific nuclease, wherein the application comprises the following steps: constructing a recombinant vector containing the Mhp non-specific nuclease gene, transforming the recombinant vector into escherichia coli, carrying out induction culture on the obtained recombinant genetic engineering bacteria (IPTG is used as an inducer), and separating a culture solution to obtain thalli cells containing the Mhp non-specific nuclease.

The invention relates to the acquisition of an Mhp non-specific nuclease nucleotide sequence and the construction of a high-yield strain, which specifically comprises the following steps:

(1) a primer is designed by utilizing a known mycoplasma hyopneumoniae non-specific nuclease gene, an Mhp genome is taken as a template, a gene fragment of the Mhp non-specific nuclease is amplified by a PCR technology, and the gene fragment is connected with a cloning vector for sequencing. Because the TGA codon expresses tryptophan in Mycoplasma hyopneumoniae and is a stop codon in an Escherichia coli expression system, the TGA codon inside the Mhp non-specific nuclease gene is mutated into TGG by an overlap extension PCR method by designing a mutation primer, and the nucleotide sequence of the Mhp non-specific nuclease is obtained as shown in SEQ ID NO: 1. The amino acid sequence corresponding to the mutated gene sequence is shown in SEQ ID NO. 2.

(2) Carrying out double enzyme digestion on the Mhp non-specific nuclease gene by using Nco I and Xho I, connecting with pET28a (+) subjected to double enzyme digestion, transforming to an Escherichia coli E.coli Transetta (DE3) expression vector, constructing a genetic engineering bacterium, and adding IPTG (isopropyl-beta-thiogalactoside) into a culture medium to induce and express recombinant protein, thereby obtaining a bacterial cell for producing the Mhp non-specific nuclease.

The research on the enzymological properties of the Mhp non-specific nuclease mainly comprises the following steps: the method uses Ni-NTA resin affinity chromatography to separate and purify Mhp non-specific nuclease, uses double-stranded DNA, single-stranded DNA, plasmid DNA and RNA as substrates, and researches the influence of the substrate specificity, degradation time, optimal temperature and different ion temperatures of the Mhp non-specific nuclease on the activity of the Mhp non-specific nuclease. The research result shows that: the Mhp non-specific nuclease has no substrate specificity, can degrade various nucleic acid substrates, has extremely quick degradation reaction, and can complete the degradation reaction after being uniformly mixed; the enzyme activity is extremely strong, 192ng Mhp nonspecific nuclease can completely reduce 1 mug nucleic acid substrate; has heat resistance, and can play a stronger role in nuclease activity within the temperature range of 17-57 ℃; the nuclease can exert the activity by low concentration of calcium ions or magnesium ions, and other ions such as copper ions, ferrous ions and ferric ions can hardly exert the degradation activity of the nuclease.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the invention provides a non-specific nuclease from mycoplasma hyopneumoniae and a coding gene thereof, and constructs a high-yield strain of Mhp non-specific nuclease. The Mhp non-specific nuclease not only has non-specificity, but also has wide applicability, such as rapid degradation reaction, high enzyme activity, high temperature resistance, low concentration of required ions and the like, and can be widely applied to the fields of biological pharmacy, biological reagent research and development, industrialization and the like.

The Mhp non-specific nuclease can effectively degrade a neutrophilic granulocyte trap, reduce trapping and killing of pathogenic microorganisms by the neutrophilic granulocyte, reduce expression of interferon, enhance infection and morbidity of the pathogenic microorganisms, be matched with pathogens to establish an animal infectious disease morbidity model, and be used as an antigen to prepare a vaccine for immunoprophylaxis of infectious diseases such as cow mastitis.

Drawings

FIG. 1 shows the SDS-PAGE detection result of the Mhp non-specific nuclease in example 2 of the present invention; wherein, M: a protein Marker; mhp 597: mhp non-specific nuclease.

FIG. 2 shows the result of the determination of the Mhp non-specific nuclease activity in example 3 of the present invention; wherein, A is degradation of dsDNA by Mhp non-specific nuclease in different time; degradation of ssDNA by Mhp non-specific nuclease in different time; degrading plasmid DNA by Mhp non-specific nuclease in different time; degrading RNA by Mhp non-specific nuclease in different time; degrading the nucleic acid substrate by Mhp non-specific nuclease with different concentrations; f, influence of different temperatures on the Mhp non-specific nuclease activity; g Ca of different concentrations²⁺Effect on Mhp non-specific nuclease activity; h Mg of different concentrations²⁺Effect on Mhp non-specific nuclease activity; i Cu of different concentrations²⁺Effect on Mhp non-specific nuclease activity; j Fe of different concentrations²⁺Effect on Mhp non-specific nuclease activity; k is Fe of different concentrations³⁺Effect on Mhp non-specific nuclease activity. The dsDNA is calf thymus DNA, the ssDNA is M13phage DNA, the plasmid DNA is puc19 plasmid DNA, and the RNA is extracted from PK15 cells. Control, no treatment of nucleic acid substrates.

FIG. 3 shows rMhp597 in example 3 of the present invention^δTMAnd rMhp597^δ315-377The result of the nuclease activity assay of the protein; wherein, A is 1 mu g rMhp597 in different time^δTMDegradation of 1. mu.g dsDNA; b is not simultaneousInternally 1. mu.g rMhMhp597^δ315-377Degradation of 1. mu.g dsDNA.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 acquisition of Mhp non-specific nuclease nucleotide sequences and Point mutations

Known Mycoplasma hyopneumoniae (Mhp) nonspecific nuclease gene (gene accession number AAZ53943.1) was used to design primers, Mhp597-F '(5'-ATGAAAATTAAAAAATTATTTCTTTTT-3') and Mhp 597-R' (5'-TTAATTCTGACTGTTTTGGCCT-3'), Mhp whole genome was used as a template to perform PCR amplification, and the obtained sequence was ligated to a cloning vector and sent to Meiji Biotech Ltd for sequencing. Translating the sequencing result into a protein sequence by using DNAStar software, finding a TGA stop codon inside a gene fragment of the Mhp non-specific nuclease, designing mutation primers, wherein the mutation primer list is shown in table 1, performing point mutation by adopting an overlap extension PCR method, mutating the TGA inside the gene into TGG, and obtaining the nucleotide sequence of the Mhp non-specific nuclease, wherein the result is shown as SEQ ID NO.1, and the corresponding amino acid sequence is shown as SEQ ID NO. 2.

The PCR amplification system and conditions were as follows:

the 25 μ L PCR reaction system was: DNA template 2.0. mu.L, 10. mu.M upstream primer 0.5. mu.L, 10. mu.M downstream primer 0.5. mu.L, 2 XTaq PCR StarMix12.5. mu.L, ddH₂O 9.5μL。

PCR cycling program: pre-denaturation at 94 ℃ for 5.0 min; denaturation at 94 ℃ for 30sec, annealing at 56 ℃ for 30sec, and extension at 72 ℃ for 1min for 35 cycles; extension at 72 ℃ for 5.0 min.

TABLE 1Mhp597 mutant primers

EXAMPLE 2 construction of engineered strains expressing Mhp non-specific nuclease

1. Amplification of Mhp non-specific nuclease Gene

Designing primers according to the sequence characteristics of the modified Mhp non-specific nuclease gene, wherein an upstream primer Mhp597-F contains an NcoI site Mhp597-F (5'-CATGCCATGGGCACCGGAC TCGGAGC TTATTT-3'), an original signal peptide is not amplified (1-18 amino acid excision is carried out, the total length is 54bp), a downstream primer Mhp597-R contains an XhoI site Mhp597-R (5'-CCGCTCGAGATTCTGACTGTTTTGG CCT-3'), and an Mhp non-specific nuclease gene (shown in SEQ ID NO: 1) is used as a template for PCR amplification. The PCR amplification system and conditions were the same as in example 1.

2. Cloning of Mhp non-specific nuclease Gene

And (2) recovering the PCR amplification product in the step (1) by adopting a common agarose gel DNA recovery kit of Beijing Tianzhu Biotechnology limited company, and connecting a pEASY-T1cloning vector of Beijing Quanji Biotechnology limited company, namely pEASY-T1-nuc. The method for connecting cloning vector and transforming product into competent Escherichia coli Trans-T1 comprises the following steps: accurately sucking 1 mu L of pEASY-T1Cloning Vector and 4 mu L of gene fragment glue recovery product, putting the products into a PCR reaction tube, gently mixing the products by using a small gun head, putting the reaction tube into a PCR instrument, reacting for 15min at 25 ℃, and putting the reaction tube on ice for later use; taking out 1 tube of Trans 1-T1 Escherichia coli competent cells (50 μ L) from-80 deg.C, placing on ice until they are melted for about 8min, adding all the above reaction products into the competent cells, flicking with finger, mixing, placing on ice for 30min, placing in 42 deg.C water bath, thermally shocking for 45s, rapidly placing on ice, and placing for 2 min; adding 500 μ L LB non-resistance culture medium, and culturing in a shaker at 37 deg.C and 200rpm for 1 h; 100 μ L of the bacterial culture was applied evenly on ampicillin (Amp +) resistant LB solid medium using a sterilized triangular rod and inverted at 37 ℃ for 16 hours. A single colony on the culture medium was picked up with a small gun head, cultured in a liquid LB medium for ampicillin resistance, and cultured on a constant temperature shaker at 37 ℃ and 200rpm for 10 hours.

3. Construction of Mhp non-specific nuclease expression vector

Double digestion of pEASY-T1-nuc and pET28a (+) with NcoI and XhoI, and ligation of the Mhp non-specific nuclease gene fragment obtained by double digestion and pET28a (+) vector with T4DNA ligase. The ligation product was transformed into competent Escherichia coli Transetta (DE3) expressing strain, the transformed strain was inoculated on LB agar plate containing kanamycin to a final concentration of 100. mu.g/ml, cultured overnight at 37 ℃, single colony was randomly picked up, cultured with shaking at 37 ℃ for 12 hours, plasmid was extracted using a plasmid miniprep kit from Beijing and Biochemical technologies, Inc., and sent to Meiji Biotechnology, Inc. for sequencing, indicating that the nucleotide sequence shown in SEQ ID NO:1 had been recombined into expression vector pET28a (+), to obtain Escherichia coli Transetta (DE3) containing the Mhp non-specific nuclease gene. The ligation product was transformed into competent E.coli Transetta (DE3) by: taking out a tube of 50 μ L Transetta (DE3) competent cells from-80 refrigerator, standing on ice for about 8min for thawing, sucking 5 μ L of the above ligation product to competent cells, flicking, mixing, and standing on ice for 30 min; the competent cells were heat-shocked in a 42 ℃ water bath for 45s and rapidly transferred to ice. Standing for 2 min; adding 500 μ L of non-resistant LB culture medium, culturing at 37 deg.C and 200rpm in shaker for 1 h; 100 mul of the bacterial liquid is sucked and placed on an LB solid culture medium with kanamycin resistance, the bacterial liquid is evenly smeared by a sterilized triangular rod, and the bacterial liquid is inversely cultured for 16h in an incubator at 37 ℃.6 single colonies with uniform growth were picked up with a sterilized pipette tip and inoculated into LB liquid medium containing kanamycin resistance, 5mL per tube, and cultured at 37 ℃ and 200rpm for 10 hours.

4. Inducible expression of Mhp non-specific nucleases

Inoculating the thallus with correct sequence identification (containing the nucleotide sequence shown in SEQ ID NO: 1) in the step 3 into LB liquid culture medium, and performing shaking culture at 37 ℃ until OD is reached₆₀₀When the concentration is about 4 hours, IPTG with the final concentration of 1.0mM is added, the shaking culture is continued for 12 hours at 37 ℃, the centrifugation is carried out (4 ℃, 8000rpm and 5 minutes), and the precipitate is collected to obtain somatic cells containing the Mhp non-specific nuclease coded by the nucleotide sequence shown in SEQ ID NO.1, and the somatic cells are named as Escherichia coli (Escherichia coli) BL 21/Mhp-nuc. The culture medium is sent to the China general microbiological culture Collection center for preservation, and the preservation number is CGMCC NO. 16353.

5. Separation and purification of Mhp non-specific nuclease

Resuspending the expression bacteria with 80mL PBS, and crushing for 40min by using an ultrasonic crusher; centrifuging at 12000rpm at 4 deg.C for 10min, collecting supernatant, adding saturated ammonium sulfate until final saturation is 40%, slightly blowing and sucking, mixing, and standing at room temperature for 20 min; centrifuging at 4 deg.C and 12000rpm for 10min, collecting supernatant, adding saturated ammonium sulfate to final saturation of 55%, and standing at room temperature for 20 min; centrifuging at 12000rpm at 4 deg.C for 10min, collecting precipitate, re-suspending the precipitate with 80mL PBS, dissolving completely, placing into dialysis bag, dialyzing and removing the dialysis bag in 3L PBS, changing solution once within 6h, and taking out protein solution after 24h for Ni column affinity chromatography.

Samples were taken and analyzed by 12% polyacrylamide gel electrophoresis to find that Ni column affinity chromatography could purify the nuclease protein to a very pure form (FIG. 1).

Example 3 investigation of the enzymatic Properties of Mhp non-specific nucleases

1. Degradation of different nucleic acid substrates by Mhp non-specific nuclease

Adding dsDNA (calf thymus DNA), ssDNA, plasmid DNA, and RNA as substrate into 1 μ g of Mhp597 protein, adding 10mM Ca²⁺、100mM Mg²⁺In the buffer (Table 2), the reaction was carried out for 0.25min, 1min, 2min, 3min, 4min and 5min, respectively, and then the reaction system was run on a 1% agarose gel for electrophoresis. The results show that 1. mu.g of Mhp non-specific nuclease can completely degrade 1. mu.g of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), plasmid DNA and RNA, and the degradation reaction time is very short, and the degradation reaction can be completed by uniformly mixing (FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D).

TABLE 2 degradation of different nucleic acid substrates by Mhp nonspecific nucleases

2. Effect of protein concentration on Mhp nonspecific nuclease Activity

In order to investigate the influence of different concentrations of Mhp non-specific nuclease on the degradation of substrate, the Mhp non-specific nuclease with final concentrations of 12ng/mL, 24ng/mL, 48ng/mL, 96ng/mL and 192ng/mL was used for nucleic acid degradation experiments, the reaction system is shown in Table 3, and then the reaction system was run through 1% agarose gel electrophoresis to detect the results. The results show that Mhp non-specific nuclease has strong nuclease activity, the protein concentration required for completely degrading 1 mu g dsDNA is 192ng/mL, namely the ng-grade protein amount can exert strong nucleic acid degradation capability, and the rMhp597 protein is a very efficient nuclease (FIG. 2E).

TABLE 3 Effect of protein concentration on Mhp nonspecific nuclease Activity

3. Effect of reaction temperature on Mhp nonspecific nuclease Activity

Temperature conditions have important influence on enzyme activity, in order to explore the influence of temperature on the degradation activity of Mhp non-specific nuclease nucleic acid, a mixed solution of a nucleic acid substrate and a reaction Buffer is prepared according to a reaction system, the mixed solution and the recombinant Mhp non-specific nuclease are respectively placed into a metal bath with the temperature of 17 ℃, the temperature of 27 ℃, the temperature of 37 ℃, the temperature of 47 ℃, the temperature of 57 ℃ and the temperature of 67 ℃ for incubation for 5min, then 1 mu g of Mhp non-specific nuclease is added into the mixed solution for reaction for 30min, see table 4, and then the reaction system is run on 1% agarose gel electrophoresis to detect results. The results show that Mhp non-specific nuclease is thermostable, and still has strong nuclease activity when the temperature reaches 57 ℃ (FIG. 2F).

TABLE 4 Effect of reaction temperature on Mhp nonspecific nuclease Activity

4. Effect of Metal ions on Mhp non-specific nuclease Activity

Many proteases require the assistance of metal ions for their activity, and in order to investigate whether the degradation of nucleic acid substrates by Mhp non-specific nucleases is metal ion dependent, recombinant Mhp non-specific nucleases, metal ions (Ca) at different concentrations (0.1M, 0.2M, 0.4M, 0.8M, 1.6M) were used²⁺、Mg²⁺、Cu²⁺、Fe²⁺、Fe³⁺) And nucleic acid substrates are uniformly mixed according to a system shown in the table 5, incubated at 37 ℃ for 30min, and then subjected to 1% agarose gel electrophoresis in a reaction system to detect results. The results show that in Ca²⁺Or Mg²⁺In the presence of Mhp non-specific nuclease, the nuclease activity is exerted, and in Cu²⁺、Fe²⁺And Fe3+, the Mhp non-specific nuclease cannot exert nuclease activity (FIG. 2G, FIG. 2H, FIG. 2I, FIG. 2J, FIG. 2K).

TABLE 5 Effect of Metal ions on Mhp nonspecific nuclease Activity

5. Functional region of Mhp non-specific nuclease

Structural analysis shows that the Mhp597N end has a 23aa transmembrane region and the C end has a 21aa strong basic polar region, and the proteins with the two special peptide segments removed are respectively truncated and expressed and the biological functions of the proteins are analyzed. The results showed that the N-terminal transmembrane region was removed (rMhp 597)^δTM) Still has extremely high nuclease activity, while the protein with the strongly basic polar region removed (rMhp 597)^δ315-377) No nuclease activity was observed (FIG. 3).

Truncated protein rMhp597 of Mhp non-specific nuclease^δTMThe amino acid sequence of (A) is shown in SEQ ID NO. 3.

6. Acid-base stability of Mhp non-specific nuclease

The Mhp non-specific nuclease is determined to have stable enzyme activity under the condition of pH6.5-8.5.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Ser Ser Leu Leu Glu Ile Ala Asn Phe Ala Ser Pro Ser Asn Pro Tyr

115 120 125

Leu Ile Gln Lys Gly Val Lys Leu Glu Trp Ala Arg Pro Leu Ala Ala

130 135 140

Val Lys Phe Asn Thr Lys Thr Glu Arg Lys Glu Asp Phe Thr Phe Val

145 150 155 160

Ile Gly His Phe Asp Ala Pro Gly Val Gln Lys Asn Arg Gly Glu Ile

165 170 175

Gln Asp Lys Glu Thr Ser Gln Gly Ser Gln Glu Ala Ala Glu Ala Arg

180 185 190

Asp Leu Ile Asn Leu Leu Val Glu Ile Asp Lys Lys Asp Gly Glu Asn

195 200 205

Lys Glu Ile Ile Phe Met Ala Asp Thr Asn Ile Arg Ala Glu Asn Ser

210 215 220

Glu Lys Leu Phe Lys Ser Thr Leu Asn Ser Tyr Arg Ser Leu Leu Asp

225 230 235 240

Lys Glu Glu Lys Thr Ser Leu Ser Asn Thr Phe Gly Lys Tyr Ser Asn

245 250 255

Ser Tyr Asp Lys Ile Phe Tyr Lys Gly Asn Leu Val Ala Asn Asn Gly

260 265 270

Gln Lys Phe Asp Ile Phe Ser Ile Phe Lys Lys Asn Ile Ala Asn Val

275 280 285

Glu Lys Tyr Asp Glu Leu Arg Lys Lys Asp Gly Arg Ser Met Asn Tyr

290 295 300

Lys Ile Asn Gln Leu Gly Glu Ile Asn Arg Ile Lys Gly Ile Ser Asp

305 310 315 320

His Thr Met Val Tyr Phe Asp Leu Glu Leu Gly Gln Asn Ser Gln Asn

325 330 335

Claims

1. The engineering bacterium is Escherichia coli (Escherichia coli) BL21/Mhp-nuc, and the preservation number is CGMCC NO. 16353.

A truncated protein of Mhp non-specific nuclease, characterized in that the amino acid sequence of said truncated protein is shown in SEQ ID NO. 3.

Use of an Mhp non-specific nuclease or a truncated protein according to claim 2, in any of the following applications:

1) the application in degrading nucleic acid;

2) the application in preparing nucleic acid degradation agent;

wherein the nucleic acid comprises double-stranded DNA, single-stranded DNA, plasmid DNA, or RNA;

the amino acid sequence of the Mhp non-specific nuclease is shown in SEQ ID NO. 2.

4. A nucleic acid degradation agent characterized in that the effective components are Mhp non-specific nuclease, truncated protein according to claim 2 and/or fusion protein obtained by connecting labels to the N-terminal or C-terminal of the truncated protein;

wherein, the amino acid sequence of the Mhp non-specific nuclease is shown as SEQ ID NO. 2.