CN109337887B

CN109337887B - Nucyep coding gene, recombinant expression vector, recombinant engineering bacterium, and preparation method and application thereof

Info

Publication number: CN109337887B
Application number: CN201811245986.3A
Authority: CN
Inventors: 葛燕; 王辂; 李端华; 赵晨; 李进军; 刘涛
Original assignee: Chengdu University
Current assignee: Chengdu University
Priority date: 2018-10-24
Filing date: 2018-10-24
Publication date: 2021-04-27
Anticipated expiration: 2038-10-24
Also published as: CN109337887A

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a coding gene, a recombinant expression vector, a recombinant engineering bacterium of Nucyep, and a preparation method and application thereof. The nucleotide sequence of the coding gene of the recombinant Nucyep is shown as Seq ID No.2, or the gene with more than 90 percent of homology with the gene with the nucleotide sequence shown as SEQ ID No. 2. The coding gene of Nucyep and pET-24a (+) plasmid are recombined to form a recombinant expression vector, and the recombinant expression vector is transformed into Escherichia coli Rosseta (DE3) to form recombinant engineering bacteria. The recombinant engineering bacterium has the advantages that the coding gene sequence of Nucyep is optimized, so that the recombinant engineering bacterium can generate a large amount of target protein only in a few hours under the induction of lactose. The expression product is an inclusion body, so that the toxic effect of the soluble nuclease on host bacteria is avoided. The invention provides a renaturation and purification method of the Nucyep inclusion body, which has the advantages of high efficiency, simple process and easy operation and is more suitable for the industrial production and popularization of the Nucyep.

Description

Nucyep coding gene, recombinant expression vector, recombinant engineering bacterium, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a coding gene of Nucyep, a recombinant expression vector, a recombinant engineering bacterium, a preparation method and an application thereof.

Background

The nuclease is an enzyme which takes nucleic acid as a substrate and catalyzes hydrolysis of phosphodiester bonds. Nucleases can be classified into three general categories, dnases, rnases and nonspecific nucleases. Wherein the nonspecific nuclease has low specificity, can nonspecifically 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. Non-specific nucleases are widely found in many organisms such as viruses, bacteria, fungi and animal cells. Non-specific nucleases play an important role in many biochemical processes, including DNA replication and repair; recombination, maturation and editing of DNA/RNA; host resistance to foreign nucleic acid molecules, and the like. The nonspecific nuclease is mainly used for removing exogenous nucleic acid in biological products in industry, and improves the safety. Meanwhile, the non-specific nuclease can be applied to the development of a novel biological disinfectant, and can crack nucleic acid substances of bacteria and viruses, so that the aims of disinfection and sterilization are fulfilled. Compared with chemical disinfectants, the non-specific nuclease is an ideal green environment-friendly disinfectant, does not pollute the environment and has no toxic or side effect on organisms. To date, various nonspecific nucleases have been reported, such as bovine islet DNase I, Staphylococcus aureus (Staphylococcus aureus) nuclease, and Serratia marcescens (Serratia marcocen) extracellular nuclease. To date, the most studied non-specific nuclease is the bacillus prodigiosus nuclease. Currently, there is a commercial product of the bacillus prodigiosus nuclease, which is called Benzonase, and is used as a tool in industrial production for removing nucleic acid pollution in biological products.

The applied temperature range of Benzonase is +5 ℃ to +40 ℃, however, in the research and production process of biotechnology drugs, samples are often treated at high temperature, under the condition that nucleic acid interference is required to be removed, Benzonase cannot meet the requirement, and a novel nuclease which can exert activity at higher temperature is urgently needed to be developed. In 2013, a novel thermostable non-specific nuclease (nucieep) was found by the group of chinese scientists in Yersinia enterocolitica subsp. The enzyme consists of 283 amino acids, has the molecular weight of 30.7KDa, can adapt to a wide pH change range (pH 3.6-9.9), has excellent heat resistance, and can play a role at 0-100 ℃. The currently reported genetic engineering bacteria of Nucyep adopt a gene sequence of a source strain to construct, IPTG is used as an inducer, the induction time is as long as 20 hours, the expression efficiency is low, the product yield is low, and the Nucyep genetic engineering bacteria are not suitable for large-scale industrial production. Therefore, there is a need to develop a new method suitable for large-scale production of such thermostable nonspecific nucleases.

Disclosure of Invention

A heat-resistant non-specific nuclease (nucysep) enzyme derived from Yersinia enterocolitica subsp. The invention aims to improve the production level of Nucyep, shorten the induction time and increase the standard reaching efficiency and the product yield. The invention optimizes the coding gene sequence of Nucyep, constructs a recombinant expression vector and a gene recombinant engineering bacterium, and the recombinant engineering bacterium can efficiently express a target product. The invention also provides a preparation method of the Nucyep, which comprises a renaturation and purification method of the Nucyep, and the method is efficient, simple and more suitable for large-scale production.

The technical scheme adopted by the invention is as follows:

the invention relates to heat-resistant non-specific nuclease (Nucyep) derived from Yersinia enterocolitica subsp.pallactca, and the specific related amino acid sequence is shown as SEQ ID NO. 1.

The invention relates to a coding gene sequence of a peptide chain of SEQ ID NO.1, wherein the coding gene sequence of Nucyep is shown as SEQ ID NO. 2.

Due to the specificity of the nucleotide sequence, any variant of the polynucleotide shown in SEQ ID NO.2 is within the scope of the present invention as long as it has more than 90% homology with the sequence. A variant of a nucleotide refers to a polynucleotide sequence with one or more nucleotide changes. Such variants may be substitutions, deletions or insertions of one or more nucleotides which do not substantially alter the function of the amino acid encoded thereby.

The invention relates to a Nucyep recombinant expression vector, and a specific construction method comprises the following steps: NdeI and XhoI enzyme cutting sites are added at two ends of the sequence SEQ ID NO.2, and are shown in SEQ ID NO. 3. Artificially synthesizing a gene sequence of SEQ ID NO.3, and connecting and recombining the gene sequence of SEQ ID NO.3 with an expression vector pET-24a (+) through NdeI and XhoI enzyme cutting sites. The recombinant expression vector is named pET-Nucyep.

The invention also provides a recombinant engineering bacterium, which is prepared by transforming the recombinant expression vector pET-Nucyep into a host bacterium, wherein the host bacterium is Escherichia coli Rosetta2(DE 3). The recombinant engineering bacterium is named as Rosetta-Nucyep.

The invention also provides a preparation method of Nucyep, which comprises the following steps:

(1) fermenting and culturing the recombinant engineering bacteria, adding 5g/L lactose as an inducer to perform induced expression of Nucyep, and performing induction for 4-6 h to obtain fermentation liquor containing a large amount of Nucyep;

(2) centrifuging the fermentation liquor, and taking the precipitate to obtain an inclusion body containing Nucyep;

(3) and (3) diluting and renaturing the inclusion body containing the Nucyep to obtain the active Nucyep crude enzyme. The specific renaturation process comprises the following steps: and (3) resuspending the inclusion body by using a urea solution to obtain an inclusion body urea solution, dripping the inclusion body urea solution into a renaturation buffer solution, standing overnight at 4 ℃, centrifuging and taking supernatant to obtain the Nucyep crude enzyme. The concentration of urea in the urea solution is 6-8 mol/L. The ratio of the mass of the inclusion bodies in the inclusion body urea dissolving solution to the volume of the urea solution is 1g:50 mL. In the process of dropping the inclusion body urea solution into the renaturation buffer solution, the volume ratio of the inclusion body urea solution to the renaturation buffer solution is 1: 5-1: 10. The prescription of the renaturation buffer solution is as follows: 20mmol/L Tris-Cl, 20mmol/L MgCl₂The pH was 7.2.

(4) And (3) passing the crude Nucyep enzyme through an anion exchange column, and eluting to obtain purified Nucyep.

The invention also provides an application of the prepared Nucyep in nucleotide degradation. The enzyme can degrade plasmid DNA, single-stranded salmon sperm DNA and RNA with high efficiency and non-specificity.

The invention has the beneficial effects that:

the invention optimizes the coding gene sequence of Nucyep, and constructs a gene recombination engineering strain by utilizing the Nucyep nucleotide sequence provided by the invention. The engineering bacteria adopt lactose as an inducer, rich expression products can be obtained only within 4-6 h of induction time, 0.3-0.4 g of inclusion bodies can be obtained from 1g of bacteria, and the raw material cost and the time cost are greatly reduced. The induction expression product is an inclusion body, so that the toxic effect of Nucyep on host bacteria is avoided. The Nucyep inclusion body protein renaturation method provided by the invention can efficiently renature the inclusion body protein, and finally obtains the Nucyep with good activity and high yield. On the whole, the recombinant engineering bacterium and the preparation method of the Nucyep are simple in process, good in safety, easy to operate, capable of greatly reducing the production cost and improving the product yield, and more suitable for the Nucyep industrial production and popularization.

Drawings

FIG. 1 is SDS-PAGE electrophoresis of total protein in the fermented liquid after lactose induction of recombinant engineering bacteria for different time;

FIG. 2 is an SDS-PAGE picture identifying Nucyep expressed by Rosseta-Nucyep of the recombinant engineered bacterium of the present invention;

FIG. 3 is an SDS-PAGE image of Nucyep obtained under different renaturation conditions;

FIG. 4 is an SDS-PAGE picture of Nucyep purified using an anion exchange column Q-sepharose;

FIG. 5 is a photograph of agarose gel electrophoresis of Nucyep solution after enzymatic digestion of different types of nucleotide chains;

FIG. 6 is an agarose gel electrophoresis image of the enzymatic hydrolysis effect of Nucyep solution with different volumes;

FIG. 7 is an agarose gel electrophoresis image of the enzymolysis effect of Nucyep liquid with the same volume at different temperatures;

figure 8 is a graph of the results of the calculation of the enzymolysis effect of the same volume of nuciep solution at different temperatures.

Detailed Description

The invention is further explained below with reference to the drawings and the specific embodiments.

Example 1

The purpose of this example is to provide a Nucyep nucleotide sequence.

The amino acid sequence of Nucyep was optimized as follows based on the amino acid sequence of the nonspecific nuclease published on NCBI web (WP _050330881.1, 283 amino acids): the signal peptide sequence of 23 amino acids at the N end of the original sequence is removed, a methionine is added at the N end of the remaining 260 amino acids, and the amino acid sequence of the target protein to be expressed is shown as SEQ ID NO. 1.

Through further optimization analysis, the amino acid sequence of the target protein is converted into a nucleotide sequence most suitable for host bacteria expression, and the optimized nucleotide sequence is shown as SEQ ID NO. 2.

Adding specific enzyme cutting sites at two ends of the optimized nucleic acid sequence, namely adding CAT at the 5 'end of the sequence shown in SEQ ID NO.2 and adding CTCGAG sequence at the 3' end to obtain a nucleotide sequence with the enzyme cutting sites at two ends (shown in SEQ ID NO.3, the bases marked by double underlines in Table 1 are the added enzyme cutting sites). And (3) sending the nucleotide sequence with the enzyme cutting sites added at the two ends to a biological company for synthesis to obtain a nucleotide sequence of Nucyep, and carrying out gene sequencing verification and confirmation on the obtained nucleotide sequence.

TABLE 1

Example 2

The purpose of this example is to provide a recombinant expression vector for the aforementioned Nucyep.

The nucleotide sequence (SEQ ID NO.3) with enzyme cutting sites added at two ends of the Nucyep obtained in the embodiment 1 and a pET-24a (+) vector are recombined and connected through NdeI and XhoI serving as restriction enzymes, and finally the SEQ ID NO.3 is inserted into the pET-24a (+) vector through subsequent transformation, cloning, selection of positive clones, plasmid extraction and sequencing verification to obtain a recombinant expression vector of the Nucyep, which is named as pET-Nucyep.

Example 3

The present embodiment aims to provide a recombinant engineered bacterium comprising the Nucyep.

mu.L of pET-Nucyep was mixed with 100. mu.L Rosseta (DE3) to be competent, and after 30min on ice, it was put on water bath at 42 ℃ for 90s and immediately kept on ice for 5 min. Adding 900 mu L of SOC culture medium, placing on a shaker at 37 ℃, incubating for 1h at 200rpm, taking a proper amount of bacterial liquid to an LB (Kana +) plate for coating, inversely placing in an incubator at 37 ℃, culturing overnight, selecting positive clones, sequencing and verifying, wherein the correct recombinant engineering bacteria containing pET-Nucyep are named as recombinant engineering bacteria Rosseta-Nucyep.

Example 4

The purpose of this example is to induce the recombinant engineered bacterium Rosseta-Nucyep to express Nucyep.

1. Induction of

(1) Preparing an LB culture medium: weighing 1g of Tryptone, 0.5g of Yeast extract and 0.5g of NaCl in sequence, shaking the container until solute is dissolved, adjusting pH to 7.0, adding double distilled water to fix the volume to 100mL, sterilizing at 120 ℃ for 20min, and before use, adding kanamycin into the sterilized LB culture medium to ensure that the final concentration of kanamycin is 50 mug/mL;

(2) inoculating the frozen glycerol strain containing the recombinant engineering bacteria Rosseta-Nucyep into an LB liquid culture medium according to the volume ratio of 1:2000, keeping the temperature at 37 ℃ and the rpm at 220rpm, and standing overnight (16-18 h) in a shaking table to obtain a first-level bacterial liquid;

(3) inoculating the primary bacterial liquid obtained in the step (2) into a fresh LB culture medium according to the volume ratio of 1:100, and shaking the bacteria until the OD 600 is about 1.3 at 37 ℃ and 220 rpm;

(4) adding a lactose solution with the concentration of 300g/L into the bacterial liquid to ensure that the final concentration of lactose is 5g/L, shaking the bacteria at 37 ℃ to start induction, and obtaining fermentation liquid after the induction is finished;

(5) adding 5 xSDS loading buffer into proper amount of bacterial liquid, boiling for 10min, and detecting the expression of target protein in total protein by SDS-PAGE electrophoresis.

Total protein in fermentation broths at different induction times is shown in FIG. 1, lane 1 is empty bacteria control; lane 2 is the control of no induction of the engineering bacteria; lanes 3-8 show total protein after 1, 2, 3, 4, 5, and 6 hours of lactose induction. As shown in FIG. 1, a large amount of the target protein can be obtained after 4-6 hours of induction.

2. Extraction and detection of target protein

Carrying out ultrasonic cell disruption and protein extraction: 4.5mL of the above-mentioned fermentation broth with the optimum effect was centrifuged, the supernatant was removed to obtain a precipitate, and lysis Buffer (25mM Tris-Cl,1mM EDTA, 20mM MgCl) was added₂pH 8.0), the ratio of precipitate to lysis Buffer was 1g:20mL to obtain a whole cell suspension, taking a small amount of the whole cell suspension (sample 1) for storage, crushing the rest whole cell suspension by using ultrasonic waves, centrifuging, and separating a supernatant (sample 2) and a precipitate (sample 3); adding a proper amount of 5 xSDS loading buffer into the sample 1 and the sample 2, adding a proper amount of 1 xSDS loading buffer into the sample 3, heating the mixture in boiling water for 10min, loading the mixture to 12% SDS-PAGE gel, adding the samples 1 to 3 into the lanes 1 to 3 respectively, and performing subsequent electrophoresis, dyeing and decoloring according to a conventional method. The results of SDS-PAGE are shown in FIG. 2.

In FIG. 2, 1 to 3 represent lanes 1 to 3, respectively. As can be seen from FIG. 2, the band of the target protein in lane 3 (sample 3) is the most concentrated, indicating that the target protein is mainly present in the insoluble precipitated fraction, indicating that the target protein is present in the form of inclusion bodies, i.e., the Nucyep content in sample 3 is the greatest.

Example 5

The purpose of this example is to provide an inclusion body renaturation method and a protein purification method of recombinant engineered bacterium Rosseta-Nucyep.

1. Extraction of Inclusion body proteins

The target protein (sample 3) in the fermentation broth was extracted according to step 2 of example 4 to obtain inclusion body protein. 1g of thalli of the fermentation liquor obtained by the induction method can obtain 0.3-0.4 g of inclusion body protein.

2. Solubilization and renaturation of Inclusion body proteins

(1) Washing the inclusion body protein twice by using a lysis Buffer containing 1% Triton X-100;

(2) washed inclusion bodies were mixed at a ratio of 1g:50mL of Urea buffer (8mol/L Urea, 0.1mol/L NaH) was added₂PO4, 0.01mol/L Tris-Cl, pH 8.0), and centrifuging to obtain supernatant, thus obtaining the inclusion body urea solution. The protein concentration measured by BCA method is about 2.0 mg/mL.

(3) The dilution method can be used for renaturing target protein in urea solution, and specifically can have different dilution ratios and orders, such as adding renaturation solution (20mmol/L Tris-Cl, 20mmol/L MgCl) dropwise into inclusion body urea solution₂pH 7.2); or dripping the inclusion body urea solution into the renaturation solution. The ratio of the inclusion body urea solution to the renaturation solution can beAt a ratio of 1:5, or 1: 10. After completion of the dropwise addition, the mixture was allowed to stand at room temperature for 2 hours, centrifuged, and the supernatant was taken.

(4) The samples obtained by the 5 renaturation modes are respectively and sequentially added into lanes 1-5, and the level of the target protein in the soluble supernatant after renaturation is detected by SDS-PAGE.

(5) The protein content of the sample in lane 4 was measured by BCA method and found to be 0.12 mg/mL.

(6) And (4) purifying the renatured enzyme solution (the supernatant obtained in the step (3)) by using an anion exchange column Q-sepharose to obtain the purified protein.

The results of SDS-PAGE after renaturation at different dilution ratios are shown in FIG. 3, and the lane samples are:

1. adding renaturation liquid 1:5 and total protein into inclusion body urea solution, loading 10 μ L

2. Dripping renaturation solution into the inclusion body urea solution at a ratio of 1:5, centrifuging the supernatant, and loading 10 mu L

3. Dripping inclusion body urea solution into the renaturation solution at a ratio of 5:1, centrifuging the supernatant, and loading 10 mu L of the supernatant

4. Dripping inclusion body urea solution into the renaturation solution at a ratio of 10:1, centrifuging the supernatant, and loading 10 mu L of the supernatant

5. Dripping inclusion body urea solution into the renaturation solution at a ratio of 10:1, centrifuging the supernatant, and loading 20 mu L of the supernatant

Lane 1 is a control, and all protein samples, both soluble and insoluble, are included in the total protein, and the remaining lanes are soluble supernatant protein samples after renaturation and centrifugation. When the optical density of the target protein in each lane in fig. 3 is analyzed by the Quantity One software, the gray level of the target protein in lane 5 is higher, that is, the amount of soluble target protein after renaturation is more, the amount of impure protein is less, and the renaturation effect is better. It can be seen that when the inclusion body urea solution is dropped into the renaturation solution, the volume ratio of the renaturation solution to the inclusion body urea solution is 10:1, the renaturation effect on the target protein is optimal.

And (3) performing SDS-PAGE detection on the purified protein, the renaturation pre-sample and the renaturation post-sample in the step (5), wherein the result is shown in FIG. 4, after the optical density of the target protein in each lane is analyzed by using Quantity One software, the optical density value of the target protein in the lane 3 is divided by the optical density value of the target protein in the lane 1, and the result is more than 90%, namely the purification efficiency of the purified protein is more than 90%.

Example 6

The purpose of this example is to verify the activity of the recombinant engineered bacterium Rosseta-Nucyep expressing Nucyep.

1. And (3) checking the enzyme digestion effect of Nucyep on salmon sperm DNA:

preparing salmon sperm DNA by using the renaturation solution, and adjusting the nucleic acid concentration to 1 mg/mL. Nucyep solution 34, 17, 8.5, 4.25, 2.125, 1.0625, 0.53125 and 0. mu.L are added to 37. mu.g of salmon sperm DNA, respectively, and the total volume is adjusted to 50. mu.L with a renaturation solution. The reaction was terminated by immediately adding 6 Xnucleic acid loading buffer at 37 ℃ for 30min to obtain a reaction solution. mu.L of the reaction solution was sequentially applied to lanes of 1.0% agarose gel and subjected to gel electrophoresis. The results are shown in FIG. 6.

FIGS. 6, 1 to 8, represent reaction solutions obtained by adding 34. mu.L, 17. mu.L, 8.5. mu.L, 4.25. mu.L, 2.125. mu.L, 1.0625. mu.L, 0.53125. mu.L and 0. mu.L of Nucyep solution to 37. mu.g of salmon sperm DNA.

2. Nucyep test for enzyme digestion effect of three different types of nuclease

The purified Nucyep was incubated with plasmid DNA (double-stranded DNA), RNA, and commercial salmon sperm DNA (single-stranded DNA) in a 50. mu.L system at 37 ℃ for 30 min. After completion of the incubation, the reaction was terminated by adding 10. mu.L of 6 × nucleic acid loading buffer. Load 10. mu.L, run 1.0% agarose electrophoresis. The results are shown in FIG. 5. The samples in

lanes

1, 3 and 5 of FIG. 5 are untreated plasmid DNA, RNA and commercial salmon sperm DNA, respectively, and

lanes

2, 4 and 6 are Nucyep-cut plasmid DNA, RNA and commercial salmon sperm DNA, respectively. As can be seen from FIG. 5, compared with

lanes

1, 3 and 5, no nucleic acid band is found in

lanes

2, 4 and 6, which indicates that the Nucyep expressed by the recombinant engineered bacterium pET-Nucyep can cleave double-stranded DNA, RNA and single-stranded DNA, and conforms to the property of non-specific nuclease.

Example 7

The purpose of this example is to verify the heat resistance of the Nucyep expressed by the recombinant engineered bacterium Rosseta-Nucyep.

1. mu.L of Nucyep (i.e., Nucyep renatured under the optimum conditions in example 5) was added to 37. mu.gIn salmon sperm DNA, renaturation solution (20mmol/L Tris-Cl, 20mmol/L MgCl)₂pH 7.2) to a total volume of 50. mu.L;

2. carrying out enzyme digestion reaction at 37 ℃, 60 ℃, 75 ℃ and 90 ℃ respectively, wherein the enzyme digestion time is 30min, immediately adding 6 × loading buffer to terminate, sequentially naming the solution as No. 1-4 enzyme digestion solution, and simultaneously loading salmon sperm DNA without Nucyep as a reference;

3. 10. mu.L of the above digestion solution was applied and detected by 1.5% agarose electrophoresis, and the results are shown in FIG. 7.

In FIG. 7, 1 to 5 represent lanes 1 to 5, respectively, in FIG. 7, the sample in lane 5 is a control sample without enzyme digestion, and the samples in lanes 1 to 4 are enzyme digestion solutions 1 to 4. The Quantity One software analyzes the optical density of nucleic acid in each lane, and the enzyme digestion efficiency of each lane can be calculated as follows:

(X is 1/2/3/4.). The sample in the lane 5 is a control, the enzyme activity of the Nucyep under the condition is calculated as 100%, and the ratio obtained by dividing the difference value of the optical density of the nucleic acid band in the lane 5 and the optical density of the bands in other lanes by the optical density of the band in the lane 5 is the enzyme activity of the enzyme under the reaction condition of the sample corresponding to each lane, namely the enzyme activity of the Nucyep at 60 ℃, 70 ℃ and 90 ℃. The relative cleavage efficiencies of the lanes were calculated based on the cleavage efficiencies of lane 4. The results are shown in FIG. 8. The relative enzyme activities of Nucyep at 37 ℃, 60 ℃, 75 ℃ and 90 ℃ are respectively 100%, 57%, 40% and 40%. The Nucyep expressed by the recombinant engineering bacteria pET-Nucyep provided by the invention still has good enzyme digestion activity at high temperature.

The present invention is not limited to the above-described alternative embodiments, and various other forms of products can be obtained by anyone in light of the present invention. The above detailed description should not be taken as limiting the scope of the invention, which is defined in the claims, and which the description is intended to be interpreted accordingly.

Sequence listing

<110> Sichuan institute of antibiotic industry, general corporation of Chinese pharmaceutical industries

<120> encoding gene of Nucyep enzyme, recombinant expression vector, recombinant engineering bacterium, preparation method and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 261

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Ser Ala Pro Lys Thr Ile Leu Ser Ala Pro Thr Val Thr Glu Gln

1 5 10 15

Asn Leu Pro Ala Ala Ala Ile Asp Asn Cys Leu Val Gly Cys Pro Thr

20 25 30

Gly Gly Ser Asp Gln Thr Val Ile Arg Asp Val Tyr Thr Leu Asn Asn

35 40 45

Asn Ser His Thr Lys Phe Ala Asn Trp Val Ala Tyr Lys Val Thr Lys

50 55 60

Ser Ser Gln Ala Ser Asn His Pro Arg Lys Trp Ala Gln Asp Pro Asp

65 70 75 80

Leu Pro Asp Ser Asp Thr Leu Ala Pro Ala Asp Tyr Thr Gly Ala Asn

85 90 95

Gln Lys Leu Ala Val Asp Arg Gly His Gln Ala Pro Leu Ser Leu Leu

100 105 110

Ala Gly Asn Glu Asp Ser Gln Ala Leu Asn Tyr Leu Ser Asn Ile Thr

115 120 125

Pro Gln Lys Ala Ala Leu Asn Gln Gly Ala Trp Val Arg Leu Glu Asp

130 135 140

Gln Glu Arg Asn Leu Ala Asn Arg Pro Asp Val Thr Ala Val Tyr Ser

145 150 155 160

Val Thr Gly Pro Leu Phe Glu Arg His Ile Ala Thr Leu Pro Ala Lys

165 170 175

Pro Thr Val Glu Ile Pro Ser Gly Tyr Trp Lys Ile Ile Phe Ile Gly

180 185 190

Thr Ser Pro Asp Lys Gly Gln Tyr Ala Ala Phe Leu Met Asp Gln Asn

195 200 205

Thr Ala Lys Ser Ala Asn Phe Cys Asp Tyr Gln Val Asn Val Asp Thr

210 215 220

Ile Glu Ala Lys Thr Asn Pro Gln Leu Thr Ile Trp Ser Asn Leu Pro

225 230 235 240

Ala Asp Val Ala Gln Ile Ile Lys Ser Gln Lys Gly Thr Leu Ala Gln

245 250 255

Thr Ile Gly Cys Asp

260

<210> 2

<211> 783

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgagtgcac cgaaaaccat tctgagcgcc ccgaccgtga ccgaacagaa tctgccggca 60

gcagccattg ataattgcct ggtgggctgt ccgaccggcg gtagtgatca gaccgtgatt 120

cgtgatgttt ataccctgaa taataacagt cataccaaat ttgccaactg ggtggcatat 180

aaagttacca aaagtagcca ggccagcaat catccgcgta aatgggccca ggatccggat 240

ctgccggata gcgataccct ggcaccggcc gattataccg gcgccaatca gaaactggcc 300

gttgatcgtg gccatcaggc cccgctgagt ctgctggcag gtaatgaaga tagtcaggcc 360

ctgaattatc tgagtaatat taccccgcag aaagcagccc tgaatcaggg tgcctgggtt 420

cgtctggaag atcaggaacg caatctggca aatcgtccgg atgttaccgc agtttatagt 480

gttaccggcc cgctgtttga acgtcatatt gccaccctgc cggcaaaacc gaccgtggaa 540

attccgagtg gttattggaa aattattttc attggcacca gtccggataa aggtcagtat 600

gccgcatttc tgatggatca gaataccgcc aaaagtgcca atttttgtga ttatcaggtt 660

aatgttgaca ccattgaagc aaaaaccaat ccgcagctga ccatttggag taatctgccg 720

gcggatgttg cacagattat taagagtcag aaaggtaccc tggcacagac cattggctgc 780

gat 783

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<211> 792

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<213> Artificial Sequence (Artificial Sequence)

<400> 3

catatgagtg caccgaaaac cattctgagc gccccgaccg tgaccgaaca gaatctgccg 60

gcagcagcca ttgataattg cctggtgggc tgtccgaccg gcggtagtga tcagaccgtg 120

attcgtgatg tttataccct gaataataac agtcatacca aatttgccaa ctgggtggca 180

tataaagtta ccaaaagtag ccaggccagc aatcatccgc gtaaatgggc ccaggatccg 240

gatctgccgg atagcgatac cctggcaccg gccgattata ccggcgccaa tcagaaactg 300

gccgttgatc gtggccatca ggccccgctg agtctgctgg caggtaatga agatagtcag 360

gccctgaatt atctgagtaa tattaccccg cagaaagcag ccctgaatca gggtgcctgg 420

gttcgtctgg aagatcagga acgcaatctg gcaaatcgtc cggatgttac cgcagtttat 480

agtgttaccg gcccgctgtt tgaacgtcat attgccaccc tgccggcaaa accgaccgtg 540

gaaattccga gtggttattg gaaaattatt ttcattggca ccagtccgga taaaggtcag 600

tatgccgcat ttctgatgga tcagaatacc gccaaaagtg ccaatttttg tgattatcag 660

gttaatgttg acaccattga agcaaaaacc aatccgcagc tgaccatttg gagtaatctg 720

ccggcggatg ttgcacagat tattaagagt cagaaaggta ccctggcaca gaccattggc 780

tgcgatctcg ag 792

Claims

1. A gene encoding nuciep, comprising: the nucleotide sequence of the coding gene is shown as SEQ ID No. 2.

2. A recombinant expression vector characterized by: the recombinant expression vector is formed by inserting the nucleotide sequence of claim 1 into pET-24a (+) vector through NdeI and XhoI enzyme cutting sites.

3. A recombinant engineering bacterium, which is characterized in that: the recombinant engineering bacteria is prepared by transforming the recombinant expression vector of claim 2 into host bacteriaEscherichia coliRosetta2(DE3)。

4. A preparation method of Nucyep is characterized by comprising the following steps:

(1) fermenting and culturing the recombinant engineering bacteria of claim 3, and performing induced expression on Nucyep by using lactose as an inducer to obtain a zymocyte liquid containing Nucyep;

(2) centrifuging the fermentation liquor, taking the precipitate, carrying out ultrasonic or homogenate treatment after using buffer solution to resuspend the precipitate, and centrifuging to obtain an insoluble inclusion body containing Nucyep;

(3) dissolving and renaturing the inclusion body to obtain crude enzyme with active Nucyep;

(4) the crude enzyme of Nucyep was passed through an anion exchange column to obtain purified Nucyep.

5. The method of claim 4, wherein: the renaturation process in the step (3) comprises the following steps of resuspending the inclusion bodies by using a urea solution to obtain an inclusion body urea solution, dripping the inclusion body urea solution into a renaturation buffer solution, standing overnight at 4 ℃, centrifuging and taking supernatant to obtain Nucyep; the concentration of urea in the urea solution is 6-8 mol/L.

6. The method of claim 5, wherein: the ratio of the mass of the inclusion bodies in the inclusion body urea dissolving solution to the volume of the urea solution is 1g:50 mL.

7. The method of claim 5, wherein: dripping the inclusion body urea solution into a renaturation buffer solution, wherein the volume ratio of the inclusion body urea solution to the renaturation buffer solution is 1: 5-1: 10; the prescription of the renaturation buffer solution is as follows: 20mmol/L Tris-Cl, 20mmol/L MgCl₂The pH was 7.2.

8. Use of Nucyep prepared by the preparation method of any one of claims 4 to 7 for degrading nucleic acids for non-therapeutic purposes.

9. The use of claim 8, wherein the nucleic acid is selected from the group consisting of double-stranded DNA, single-stranded DNA, and RNA.