CN109337887A

CN109337887A - A kind of encoding gene of Nucyep enzyme, recombinant expression carrier, recombination engineering and its preparation method and application

Info

Publication number: CN109337887A
Application number: CN201811245986.3A
Authority: CN
Inventors: 葛燕; 王辂; 李端华; 赵晨; 李进军; 刘涛
Original assignee: Sichuan Industrial Institute of Antibiotics
Current assignee: Chengdu University
Priority date: 2018-10-24
Filing date: 2018-10-24
Publication date: 2019-02-15
Anticipated expiration: 2038-10-24
Also published as: CN109337887B

Abstract

The invention belongs to technical field of bioengineering, and in particular to a kind of encoding gene of Nucyep enzyme, recombinant expression carrier, recombination engineering and its preparation method and application.The nucleotide sequence of the encoding gene of recombination Nucyep enzyme is as shown in Seq ID No.2, or the gene to have 90% or more homology with nucleotide sequence gene as shown in SEQ ID NO.2.The encoding gene of the Nucyep enzyme and the recombination of pET-24a (+) plasmid are constituted into recombinant expression carrier, and the recombinant expression carrier is transformed into Escherichia coli Rosseta (DE3) and constitutes recombination engineering.The advantages of recombination engineering of the present invention, is, optimizes the coding gene sequence of Nucyep enzyme, so that the recombination engineering, under lactose induction, it is only necessary to which several hours can produce a large amount of destination proteins.Expression product is inclusion body, avoids soluble nuclease to the toxic effect of host strain.And the present invention provides the renaturation and purification process of Nucyep enzyme inclusion body, high-efficient, process is simple, easily operated, is more applicable for the industrial production and popularization of Nucyep enzyme.

Description

A kind of encoding gene, recombinant expression carrier, recombination engineering and its system of Nucyep enzyme Preparation Method and application

Technical field

The invention belongs to field of biotechnology, and in particular to a kind of encoding gene of Nucyep enzyme, recombinant expression carrier, again Group engineering bacteria and its preparation method and application.

Background technique

Nuclease is the class of enzymes of catalytic phosphatase diester linkage hydrolysis using nucleic acid as substrate.General nuclease can be divided into DNA Enzyme, RNA enzyme and Non-specific nuclease three classes.Wherein the specificity of Non-specific nuclease is lower, can non-specifically degrade The nucleic acid of nearly all form, DNA and RNA including single-stranded, double-strand, threadiness, ring-type and supercoiled form, and to the sequence of nucleic acid Column do not require.Non-specific nuclease is widely present in virus, bacterium, in a variety of biologies such as fungi and zooblast.It is non-specific Nuclease plays an important role in many biochemical processes, duplication and reparation including DNA；Recombination, maturation and the volume of DNA/RNA Volume；Host resists foreign nucleic acid molecules etc..Non-specific nuclease is industrially mainly used for exogenous nucleic acid in biological products Removal, improve safety.Meanwhile non-specific nuclease also can apply to the development of new bio disinfectant, it can be cracked carefully The nucleic acid substances of bacterium and virus, to achieve the purpose that disinfection and sterilization.Compared to chemosterilant, non-specific nuclease is a kind of reason The environmentally protective disinfectant preparation thought, it is free from environmental pollution, it is without any side effects to body.Up to the present, it has been reported that a variety of Non-specific nuclease, such as ox pancreas islet DNase I, staphylococcus aureus (Staphylococcus aureus) nuclease and Serratia marcescens (Serratia marcescen) extracellular nuclease etc..So far, the non-specific nuclease of most study is spirit Bacillus nuclease.Bacterium prodigiosum nuclease has commercially produced product and releases at present, and trade name is Benzonase, it is as a kind of work Has the pollution of nucleic acid being used to remove in biological products in the industrial production.

+ 5 DEG C -+40 DEG C of temperature range of Benzonase application, however in the research and production process of biotech drug In, high-temperature process sample can be many times used, removal nucleic acid interference is wanted under the conditions of such, Benzonase is unable to satisfy Demand, there is an urgent need to develop go out a kind of novel to play active nuclease at a higher temperature.2013, Chinese Scientists Team has found one in yersinia enterocolitica (Yersinia enterocolitica subsp.palearctica) The Non-specific nuclease (Nucyep) of kind novel fire resistant.The enzyme is made of 283 amino acid, molecular size range 30.7KDa, Wide in range pH variation range (pH3.6~9.9) is adapted to, there is fabulous heat resistance, can be made in 0~100 degree Celsius of performance With.The genetic engineering bacterium of currently reported Nucyep is constructed using the gene order of source bacterial strain, is made using IPTG For inducer, induction time was up to for 20 several hours, and expression efficiency is low, and product yield is low, and it is raw to be not suitable for large-scale industrialization It produces.Therefore, it is badly in need of developing a kind of new method suitable for this kind of heat-resisting Non-specific nuclease is mass produced.

Summary of the invention

The heat-resisting non-specific nuclease in the source Yersinia enterocolitica subsp.palearctica (Nucyep) enzyme has the ability of nonspecific degradation nucleic acid, it may have good heat resistance.The purpose of the invention is to mention The production level of high Nucyep shortens induction time, increases efficiency up to standard and product yield.Present invention optimizes Nucyep enzymes Coding gene sequence constructs recombinant expression carrier and genetic recombination engineering bacteria, which can the production of high efficient expression purpose Object.The present invention also provides the preparation method of Nucyep enzyme, which includes the renaturation and purification process of Nucyep enzyme, should Method is efficient, simply, is more applicable for being mass produced.

The technical scheme adopted by the invention is as follows:

The present invention relates to a kind of from the heat-resisting non-of Yersinia enterocolitica subsp.palearctica Specific nucleic acid enzyme (Nucyep), specific involved amino acid sequence is as shown in SEQ ID NO.1.

The present invention relates to the coding gene sequence of one section of SEQ ID NO.1 peptide chain, the encoding gene sequences of the Nucyep enzyme Column are as shown in SEQ ID NO.2.

Due to the particularity of nucleotide sequence, the variant of polynucleotides shown in any SEQ ID NO.2, as long as itself and the sequence 90% or more homology is shown, the column of the scope of the present invention are belonged to.The variant of the nucleotide refers to a kind of with one Or the polynucleotide sequence that multiple nucleotide change.This variant may be substitution, missing or the insertion of one or more nucleotide, But not from the function of substantially changing the amino acid that it is encoded.

The present invention relates to a kind of Nucyep enzyme recombinant expression carrier, specific construction method includes: in sequence SEQ ID NdeI and XhoI restriction enzyme site is added at the both ends NO.2, as shown in SEQ ID NO.3.Artificial synthesized SEQ ID NO.3 gene order, SEQ ID NO.3 sequence is attached recombination through NdeI and XhoI restriction enzyme site and expression vector pET-24a (+).It is described heavy Group expression vector is named as pET-Nucyep.

The present invention also provides a kind of recombination engineering, the recombination engineering is by recombinant expression carrier pET- above-mentioned Nucyep is transformed into host strain and is prepared, and the host strain is Escherichia coli Rosetta2 (DE3).It is described heavy Group engineering bacteria is named as Rosetta-Nucyep.

The present invention also provides a kind of preparation methods of Nucyep enzyme, comprising the following steps:

(1) the above-mentioned recombination engineering of fermented and cultured, the lactose that 5g/L is added carry out inducing expression Nucyep as inducer Enzyme obtains the fermentation liquid containing a large amount of Nucyep enzymes by the induction of 4~6h；

(2) it is centrifuged the fermentation liquid, precipitating is taken, obtains the inclusion body of the enzyme containing Nucyep；

(3) renaturation is diluted to the inclusion body of the enzyme containing Nucyep, obtains the thick enzyme of active Nucyep.Specific renaturation mistake Journey includes: that inclusion body is resuspended with urea liquid, obtains inclusion body urea lysate, and inclusion body urea lysate instillation renaturation is delayed Fliud flushing, 4 DEG C stand overnight, and centrifuging and taking supernatant is to get the thick enzyme of Nucyep.The concentration of urea is 6~8mol/ in the urea liquid L.The ratio of the quality and urea liquid volume of inclusion body is 1g:50mL in the inclusion body urea lysate.It is described to forgive During body urea lysate instills renaturation buffer, the volume ratio of inclusion body urea lysate and renaturation buffer is 1:5 ~1:10.Renaturation buffer formula are as follows: 20mmol/L Tris-Cl, 20mmol/L MgCl₂, pH 7.2.

(4) the thick enzyme of Nucyep is crossed into anion-exchange column, affords purifying Nucyep enzyme.

The present invention also provides a kind of application of aforementioned prepared Nucyep enzyme in degradation nucleotide.The enzyme can efficiently and Non-specific degradative plasmid DNA, single-stranded salmon sperm dna and RNA.

The invention has the benefit that

Present invention optimizes the coding gene sequences of Nucyep enzyme, utilize Nucyep enzyme nucleotide sequence provided by the invention Construct genetic recombination engineered strain.The engineering bacteria using lactose as inducer, induction time be only 4~6h can be obtained it is rich Rich expression product, 1g thallus can obtain the inclusion body of 0.3~0.4g, greatly reduce cost of material and time cost.Induce table It is inclusion body up to product, avoids Nucyep enzyme to the toxic effect of host strain.Nucyep enzyme inclusion body egg provided by the invention White refolding method, can highly efficient renaturation inclusion body protein, finally obtain that activity is good, Nucyep enzyme that yield is high.Generally speaking, this is heavy Group engineering bacteria and Nucyep enzyme preparation method process are simple, and safety is good, easily operated, substantially reduce production cost, improve and produce Object yield is more applicable for the industrial production and popularization of Nucyep enzyme.

Detailed description of the invention

Fig. 1 is SDS- of the recombination engineering provided by the invention through lactose induction different time post-fermentation liquid total protein PAGE electrophoretogram；

Fig. 2 is the SDS-PAGE figure of the Nucyep enzyme of identification recombination engineering Rosseta-Nucyep expression of the invention；

Fig. 3 is the SDS-PAGE figure for the Nucyep enzyme that different denaturing conditions obtain；

Fig. 4 is the SDS-PAGE figure of the Nucyep enzyme purified using anion-exchange column Q-sepharose；

Fig. 5 is that Nucyep enzyme solution digests the agarose gel electrophoresis figure after different types of nucleotide chain；

Fig. 6 is the agarose gel electrophoresis figure of the hydrolysis result of the Nucyep enzyme solution of different volumes；

Fig. 7 is the agarose gel electrophoresis figure of the hydrolysis result of the Nucyep enzyme solution of same volume at different temperatures；

Fig. 8 is the calculated result figure of the hydrolysis result of the Nucyep enzyme solution of same volume at different temperatures.

Specific embodiment

With reference to the accompanying drawing and specific embodiment does further explaination to the present invention.

Embodiment 1

The purpose of the present embodiment is that providing a kind of Nucyep enzyme nucleotide sequence.

According to amino acid sequence (WP_050330881.1,283 ammonia of the NCBI Non-specific nuclease announced on the net Base acid), the amino acid sequence of Nucyep enzyme is optimized as follows: eliminating the signal peptide of 23 amino acid of former sequence N-terminal Sequence, then a methionine, the amino for the destination protein for finally needing to express are added in the N-terminal of remaining 260 amino acid Acid sequence is as shown in SEQ ID NO.1.

By advanced optimizing analysis, it converts the amino acid sequence of destination protein to the core for being best suited for host strain expression Nucleotide sequence, the nucleotide sequence after optimization is as shown in SEQ ID NO.2.

Specific restriction enzyme site, i.e. 5 ' ends of the sequence shown in SEQ ID NO.2 are added at the nucleic acid sequence both ends optimized Add CAT, 3 ' ends plus CTCGAG sequence, obtain both ends added with restriction enzyme site nucleotide sequence (as shown in SEQ ID NO.3, The base of the acceptance of the bid double underline of table 1 is the restriction enzyme site added).Both ends are sent to added with the nucleotide sequence of restriction enzyme site Biotech firm's synthesis, obtains the nucleotide sequence of Nucyep enzyme, and it is true to carry out gene sequencing verifying to obtained nucleotide sequence Recognize.

Table 1

Embodiment 2

The purpose of the present embodiment is that providing a kind of recombinant expression carrier of aforementioned Nucyep enzyme.

The both ends of Nucyep enzyme obtained in embodiment 1 are added with to nucleotide sequence (the SEQ ID of restriction enzyme site NO.3 it) carries out recombination with XhoI for NdeI by restriction enzyme with pET-24a (+) carrier to connect, using subsequent turn Change, clone, select positive clone molecule, extract plasmid and sequence verification, SEQ ID NO.3 is finally inserted into pET-24a (+) carrier In, the recombinant expression carrier of Nucyep enzyme is obtained, pET-Nucyep is named as.

Embodiment 3

The purpose of the present embodiment is that providing a kind of recombination engineering comprising aforementioned Nucyep enzyme.

2 μ L pET-Nucyep and 100 μ L Rosseta (DE3) competence are mixed, after placing 30min on ice, 42 DEG C Water-bath 90s, puts stand 5min on ice immediately.900 μ L SOC culture mediums are added, are put on 37 DEG C of shaking tables, 200rpm, are incubated for 1h, It takes and is coated in appropriate bacterium solution to LB (Kana+) plate, is inverted in 37 DEG C of incubators, overnight incubation selects positive clone molecule, surveys Sequence verifying, verifying are correctly the recombination engineering comprising pET-Nucyep, are named as recombination engineering Rosseta- Nucyep。

Embodiment 4

The purpose of the present embodiment is that induction recombination engineering Rosseta-Nucyep expresses Nucyep enzyme.

1, it induces

(1) it configures LB culture medium: successively weighing Tryptone 1g, Yeast extract 0.5g and NaCl 0.5g, shake Until solute dissolves, adjusting pH to 7.0 is added distilled water and is settled to 100mL, 120 DEG C of high-temperature sterilization 20min, uses visibly moved device Before, kanamycins is added in LB culture medium after sterilization, makes the final concentration of 50 μ g/mL of kanamycins；

(2) glycerol stock comprising recombination engineering Rosseta-Nucyep that will be frozen, 1:2000 is inoculated in by volume In LB liquid medium, 37 DEG C, 220rpm, in shaking table overnight (16~18h), level-one bacterium solution is obtained；

(3) it by the ratio of the 1:100 by volume of level-one bacterium solution obtained in step (2), accesses in fresh LB culture medium, 37 DEG C, 220rpm, bacterium is shaken to OD 600=1.3 or so；

(4) lactose solution that concentration is 300g/L is added into bacterium solution, so that the final concentration of 5g/L of lactose, 37 DEG C are shaken bacterium Start to induce, after induction, obtains fermentation liquid；

(5) it takes appropriate bacterium solution to add 5 × SDS loading buffer, after boiling water boiling 10min, is examined with SDS-PAGE electrophoresis Survey the destination protein expression in total protein.

Total protein in the fermentation liquid of different induction times is as shown in Figure 1,1 swimming lane is empty bacterium control；2 swimming lanes are engineering bacteria Control is not induced；3~8 swimming lanes are respectively the total protein after lactose induces 1,2,3,4,5,6 hour.As shown in Figure 1,4~6 are induced Hour can obtain a large amount of destination proteins.

2, destination protein is extracted and is detected

Sonicated cells leach protein: taking the fermentation liquid of the above-mentioned optimum efficiency of 4.5mL, and centrifugation is removed supernatant, precipitated, Cracking Buffer (25mM Tris-Cl, 1mM EDTA, 20mM MgCl is added₂, pH 8.0) and it is resuspended, precipitating and cracking Buffer Ratio be 1g:20mL, obtain full cell suspension, take a small amount of full cell suspension (sample 1) to retain spare, remaining full cell suspension is used Ultrasonication, centrifugation separate supernatant (sample 2) and precipitating (sample 3)；Add appropriate 5 × SDS loading into sample 1 and sample 2 Buffer, appropriate 1 × SDS loading buffer is added into sample 3, and boiling water heats 10min, is loaded to 12% SDS- 1~sample of sample 3 is added separately to swimming lane 1~3, conventionally carries out subsequent electrophoresis, dyeing and decoloration by PAGE glue. SDS-PAGE electrophoresis result is as shown in Figure 2.

1~3 respectively represents swimming lane 1~3 in Fig. 2.By in Fig. 2 it is found that the band of destination protein is most dense in swimming lane 3 (sample 3), Show that target protein is primarily present in insoluble sediment fraction, illustration purpose albumen is the i.e. sample 3 with existing for inclusion bodies In Nucyep enzyme content it is maximum.

Embodiment 5

The purpose of the present embodiment is that provide a kind of recombination engineering Rosseta-Nucyep renaturing inclusion bodies method and Method for purifying proteins.

1, inclusion body protein is extracted

The destination protein (sample 3) in fermentation liquid is extracted by the step 2 in embodiment 4, obtains inclusion body protein.It is lured through above-mentioned The fermentation liquid that guiding method obtains, 1g thallus can obtain the inclusion body protein of 0.3~0.4g.

2, the dissolution and renaturation of inclusion body protein

(1) above-mentioned inclusion body protein is washed twice with the cracking Buffer of the X-100 containing 1%Triton；

(2) to the inclusion body after washing in 1g:50mL ratio be added 8mol/L urea buffer (8 mol/L Urea, 0.1mol/L NaH₂PO4,0.01mol/L Tris-Cl, pH 8.0) dissolution is resuspended, centrifuging and taking supernatant is urinated to get to inclusion body Plain lysate.It is 2.0mg/mL or so that BCA method, which measures protein concentration,.

(3) make the destination protein renaturation in urea lysate with dilution method, can specifically there is different dilution ratio and sequence, Renaturation solution (20mmol/L Tris-Cl, 20mmol/L MgCl is such as added dropwise into inclusion body urea lysate₂, pH 7.2)； Or inclusion body urea lysate is added dropwise into renaturation solution.The ratio of inclusion body urea lysate and renaturation solution, can be 1:5, It is also possible to 1:10.It after being added dropwise to complete, is being stored at room temperature 2 hours, is being centrifuged, takes supernatant.

(4) sample that above-mentioned 5 kinds of Renaturations obtain is sequentially added into swimming lane 1~5, is detected with SDS-PAGE multiple Property after destination protein in solvable supernatant it is horizontal.

(5) by the sample of swimming lane 4, protein content, result 0.12mg/mL are measured with BCA method.

(6) anion-exchange column Q-sepharose is used, the enzyme solution (gained supernatant in step (3)) after renaturation is carried out pure Change, obtains albumen after purification.

SDS-PAGE result after different dilution ratio renaturation is shown in that Fig. 3, each Lane Sample are respectively as follows:

1, renaturation solution, 1:5, total protein, 10 μ L of loading are instilled in inclusion body urea liquid

2, renaturation solution is instilled in inclusion body urea liquid, 1:5 is centrifuged supernatant, 10 μ L of loading

3, inclusion body urea liquid is instilled in renaturation solution, 5:1 is centrifuged supernatant, 10 μ L of loading

4, inclusion body urea liquid is instilled in renaturation solution, 10:1 is centrifuged supernatant, 10 μ L of loading

5, inclusion body urea liquid is instilled in renaturation solution, 10:1 is centrifuged supernatant, 20 μ L of loading

Swimming lane 1 is control, includes solvable and insoluble all albumen samples in total protein, remaining swimming lane for renaturation and from Solvable supernatant protein sample after the heart.It is analyzed with optical density of the Quantity One software to swimming lane destination protein each in Fig. 3 It is found that the gray value of the destination protein of swimming lane 5 is higher, i.e., the soluble destination protein after renaturation is more, and foreign protein is few, i.e., renaturation is imitated Fruit is more preferable.It can be seen that instilling inclusion body urea liquid, the volume ratio of renaturation solution and inclusion body urea liquid into renaturation solution It is best to the renaturation effect of destination protein when for 10:1.

By sample carries out SDS-PAGE detection after sample and renaturation before albumen, renaturation after purification in step (5), as a result such as Fig. 4 institute Show, after being analyzed with optical density of the Quantity One software to each swimming lane destination protein, with the light of the destination protein of swimming lane 3 As a result density value is greater than 90%, i.e., the purification efficiency of albumen is greater than after purification divided by the OD value of the destination protein of swimming lane 1 90%.

Embodiment 6

The purpose of the present embodiment is to verify the activity of the Nucyep enzyme of recombination engineering Rosseta-Nucyep expression.

1, digestion validity check of the Nucyep enzyme to salmon sperm dna:

Salmon sperm dna, adjustment nucleic acid concentration to 1mg/mL are prepared with renaturation solution.Respectively by Nucyep enzyme solution 34,17,8.5, 4.25,2.125,1.0625,0.53125,0 μ L is added in 37 μ g salmon sperm dnas, adjusts total volume to 50 μ L with renaturation solution.37 DEG C, 30min, immediately plus 6 × nucleic acid loading buffer terminates reaction, obtains reaction solution.Above-mentioned 10 μ L of reaction solution is taken, according to Secondary loading carries out gel electrophoresis to each swimming lane of 1.0% Ago-Gel.As a result see Fig. 6.

1~8 respectively represents 34,17,8.5,4.25,2.125,1.0625,0.53125,0 μ L Nucyep enzyme solution in Fig. 6 Reaction solution after being added in 37 μ g salmon sperm dnas.

2, digestion validity check of the Nucyep enzyme to three kinds of various forms of nucleases

With Nucyep enzyme after purification, in 50 μ L systems, respectively with Plasmid DNA (double-stranded DNA), RNA, commodity salmon Smart DNA (single stranded DNA) is incubated for 30min at 37 DEG C altogether.After the completion of incubation, immediately plus 6 × nucleic acid loading buffer of 10 μ L Terminate reaction.10 μ L of loading, runs 1.0% agarose electrophoresis.As a result as shown in Figure 5.In Fig. 5 in 1,3 and 5 swimming lanes on sample Product are respectively untreated Plasmid DNA, RNA and commodity salmon sperm dna, and 2,4 and 6 swimming lanes are respectively after Nucyep digestion Plasmid DNA, RNA and commodity salmon sperm dna.As shown in Figure 5, relative to 1,3 and 5 swimming lanes, nucleic acid item is had no in 2,4 and 6 swimming lanes Band, show recombination engineering pET-Nucyep expression Nucyep enzyme can digestion double-stranded DNA, RNA and single stranded DNA, it is non-to meet its Specific nucleic acid enzyme viability.

Embodiment 7

The purpose of the present embodiment is to verify the heat resistance of the Nucyep enzyme of recombination engineering Rosseta-Nucyep expression.

1,2 μ L Nucyep enzymes (the Nucyep enzyme i.e. in embodiment 5 after optimum condition renaturation) are added to 37 μ g salmons In milt DNA, with renaturation solution (20mmol/L Tris-Cl, 20 mmol/L MgCl₂, pH 7.2) and total volume is adjusted to 50 μ L；

2, respectively at 37 DEG C, 60 DEG C, 75 DEG C and 90 DEG C progress endonuclease reactions, the digestion time is 30min, immediately plus 6 × Loading buffer is terminated, and is successively named as 1~No. 4 digested liquid, while loading is free of the salmon sperm dna conduct of Nucyep enzyme Control；

3, above-mentioned 10 μ L loading of digested liquid is taken, 1.5% agarose electrophoresis detects, as a result as shown in Figure 7.

1~5 respectively represents swimming lane 1~5 in Fig. 7, and the sample of swimming lane 5 is the control sample of non-digestion, swimming lane 1~4 in Fig. 7 In sample be 1~No. 4 digested liquid.Quantity One software analyzes the optical density of each swimming lane nucleic acid, each swimming lane Digesting efficiency may be calculated:

(X 1/2/3/4.).Sample in swimming lane 5 is control, and the enzyme activity of the Nucyep enzyme of this condition can be regarded as 100%, With the difference of the optical density of the band of the band and other swimming lanes of the nucleic acid of swimming lane 5 divided by the optical density of the band of swimming lane 5, obtain The ratio arrived, the enzyme activity of the enzyme under the reaction condition of the corresponding sample of as each swimming lane, i.e., Nucyep when 60 DEG C, 70 DEG C, 90 DEG C The enzyme activity of enzyme.Using 4 digesting efficiency of swimming lane as radix, the opposite digesting efficiency of each swimming lane is calculated.As a result as shown in Figure 8.Nucyep Opposite enzyme activity of the enzyme at 37 DEG C, 60 DEG C, 75 DEG C, 90 DEG C is 100%, 57%, 40%, 40% respectively.Offer of the present invention is provided Recombination engineering pET-Nucyep expression Nucyep enzyme at high temperature still have good digestion activity.

The present invention is not limited to above-mentioned optional embodiment, anyone can show that other are each under the inspiration of the present invention The product of kind form.Above-mentioned specific embodiment should not be understood the limitation of pairs of protection scope of the present invention, protection of the invention Range should be subject to be defined in claims, and specification can be used for interpreting the claims.

Sequence table

<110>Sichuan Inst. of Antibiotic Industry, China Medicine Group Corp.

<120>a kind of encoding gene of Nucyep enzyme, recombinant expression carrier, recombination engineering and its preparation method and application

<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

<210> 3

<211> 792

<212> DNA

<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 kind of encoding gene of Nucyep enzyme, it is characterised in that: the nucleotide sequence of the encoding gene such as SEQ ID No.2 It is shown, or the gene to have 90% or more homology with nucleotide sequence gene as shown in SEQ ID NO.2.

2. a kind of recombinant expression carrier, it is characterised in that: the recombinant expression carrier is by nucleotide sequence described in claim 1 It is inserted into carrier by NdeI and XhoI restriction enzyme site to recombinate, the carrier is pET-24a (+).

3. a kind of recombination engineering, it is characterised in that: the recombination engineering is turned by recombinant expression carrier as claimed in claim 2 It dissolves into host strain to be prepared, the host strain is Escherichia coli Rosetta2 (DE3).

4. a kind of preparation method of Nucyep enzyme, which comprises the following steps:

(1) recombination engineering described in fermented and cultured claim 3 uses lactose to carry out inducing expression Nucyep as inducer Enzyme obtains the zymocyte liquid containing Nucyep enzyme；

(2) it is centrifuged the fermentation liquid, takes precipitating, ultrasound is carried out after precipitating is resuspended using buffer or homogenized, centrifugation obtains The insoluble inclusion body of the enzyme containing Nucyep；

(3) dissolution and renaturation are carried out to above-mentioned inclusion body, obtains the thick enzyme of active Nucyep；

(4) the thick enzyme of Nucyep is crossed into anion-exchange column and obtains purifying Nucyep enzyme.

5. the preparation method according to claim 4, it is characterised in that: the renaturation process in the step (3) includes as follows Step is resuspended inclusion body with urea liquid, obtains inclusion body urea lysate, and inclusion body urea lysate is instilled renaturation buffering Liquid, 4 DEG C stand overnight, and centrifuging and taking supernatant is to get Nucyep enzyme；The concentration of urea is 6~8mol/L in the urea liquid.

6. preparation method according to claim 4 or 5, it is characterised in that: inclusion body in the inclusion body urea lysate Quality and urea liquid volume ratio be 1g:50mL.

7. preparation method according to claim 4 or 5, it is characterised in that: delay inclusion body urea lysate instillation renaturation During fliud flushing, the volume ratio of inclusion body urea lysate and renaturation buffer is 1:5~1:10；Renaturation buffer formula Are as follows: 20mmol/L Tris-Cl, 20mmol/L MgCl₂, pH 7.2.

8. a kind of the answering in degradation nucleic acid of Nucyep enzyme prepared by preparation method described in claim 4-7 any one With.

9. application as claimed in claim 8, which is characterized in that the nucleic acid is including but not limited to double-stranded DNA, single stranded DNA, or RNA。