CN107955806B - Preparation method and application of superoxide dismutase Cu, Zn SOD (superoxide dismutase) from deep-sea cucumber - Google Patents

Abstract

The invention discloses a preparation method and application of superoxide dismutase Cu, Zn SOD derived from abyss sea cucumber. In the method, the total RNA of Paelopatides sp. is extracted, reverse transcribed into cDNA by RT-PCR, and amplified by PCR. The gene sequence PaSOD encoding superoxide dismutase was obtained, and a prokaryotic expression recombinant plasmid was constructed in the pCold II vector, which was introduced into Escherichia coli Chaperone Competent Cell pG‑KJE8/BL21 for soluble expression of the recombinant protein. The recombinant protein prepared by this method overcame The source of raw materials is difficult to obtain, and the recovery process of protein denaturation and renaturation is complicated and cumbersome. The purified protein has high purity, good activity, wide temperature adaptation range, and can resist high-concentration digestive enzyme digestion. This protein is widely used in biology and food. , medicine, beauty and other fields to lay the foundation.

Description

Preparation method and application of superoxide dismutase Cu, Zn SOD (superoxide dismutase) from deep-sea cucumber

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a preparation method and application of superoxide dismutase (Cu, Zn) SOD derived from deep-sea cucumber.

Background

Superoxide dismutase (SOD), referred to as SOD, is a biological enzyme widely existing in the natural world and can be classified into copper-zinc SOD, manganese SOD, iron SOD, nickel SOD and the like according to the types of metals contained in the SOD. Most of the SOD sold in the market at present are extracted from blood, and belong to copper-zinc SOD (Cu, Zn-SOD). The Cu, Zn-SOD molecule consists of two subunits, each subunit contains a copper ion and a zinc ion, and the molecular weight is about 32000 Da. SOD is a biological enzyme, the chemical nature of which is protein, and the toxicity of which is widely studied at home and abroad. Experiments show that it has no toxic side effect on human body and is a pure natural bioactive substance.

Cu, Zn-SOD is a special biological enzyme, so the Cu, Zn-SOD has a plurality of special functions, the living environment of the earth is increasingly worsened, and a large amount of free radicals are induced to generate, thereby having influence on the health of human bodies. Studies have shown that, by supplementing SOD in appropriate amounts, the human body may enter some special digestive absorption mechanisms to make certain amounts of SOD enter the body to function, thereby effectively protecting human cells from excessive oxygen free radicals, slowing down the aging process of the cells, and prolonging the lifespan (Cicek E, Yildiz M, Delibas N, et al. the effects of 201Tl muscular perfusion on oxidative damagein tissues [ J ]. West Indian Medical Journal,2009,58(1): 50.). In addition, domestic and foreign SOD applications are also focused on several aspects as medicinal enzyme raw materials, cosmetic additives, functional food enhancers, etc. The main bottleneck of the prior SOD application technology is that the traditional SOD extracted from animals and plants has poor stability, difficult activity maintenance and the like. Therefore, it is imperative to develop high-efficiency and high-quality SOD genes and to utilize genetically engineered bacteria to ferment and produce and extract SOD. The deep-brillouin organism living environment is severe, and because of the limitation of sampling technology and sample preservation, the development of the functional genes of the deep-brillouin organism has the phenomenon of deficient research at home and abroad at present. The reports on SOD are relatively less, especially on Cu, Zn-SOD. In addition, the methods for producing proteins generally used at present are often performed in the form of inclusion bodies, and the expressed proteins are often inactive and cannot be recovered or are recovered with renaturation. Has the defects of low success rate, complex operation, long time consumption, large enzyme activity loss and the like.

Disclosure of Invention

In view of the above, the invention provides superoxide dismutase Cu, Zn-SOD from sea cucumber with deep-sea cucumber and a preparation method thereof, aiming at the problems that the existing SOD has poor stability, the activity is difficult to maintain, high-quality SOD genes are difficult to obtain, the process of recovering renaturation of protein is complex and fussy and the like.

In order to solve the technical problems, the invention discloses a coding protein of superoxide dismutase from the deep-sea cucumber Paelopidides sp, which has an amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence which is formed by replacing, deleting or adding one or more amino acids in the sequence and has similar or equivalent functions.

The invention also discloses a gene for coding the protein.

Further, the nucleotide sequence has a nucleotide sequence shown in SEQ ID NO. 1.

The invention also discloses a pCold II vector containing the gene.

The invention also discloses a Chaperone component Cell pG-KJE8/BL21 expression host Cell containing the vector.

The invention also discloses a preparation method of the superoxide dismutase Cu, Zn-SOD from the deep-sea cucumber, which is implemented according to the following steps:

step 1, obtaining Paelopides sp total RNA and cDNA;

step 2, obtaining and sequence analyzing a Paelopides sp superoxide dismutase PaSOD gene;

and 3, soluble expression and purification of the Paelopdes sp.

Further, the PCR reaction system used in the acquisition of Paelopdes sp. superoxide dismutase PaSOD gene in said step 2 was 50. mu.L, including 10. mu.L PrimeSTAR GXL Buffer, 4. mu.L dNTP, 1. mu.L each of F and R primers, 1. mu.L template cDNA, 2. mu.L template cDNA

GXL DNA Polymerase (TaKARa Co., Ltd.) using H₂O made up the total volume to 50. mu.L.

Further, the primer pair in the PCR reaction system is a primer F and a primer R, wherein,

and (3) primer F: CG (CG)GGATCCATGTCTGTCCACGCCGTTTGTGTATT, the nucleotide sequence of which is shown in SEQ ID NO. 3;

and (3) primer R: AACTGCAGTATCTTTTTGATCCCAATTACA, the nucleotide sequence is shown in SEQ ID NO.4Shown in the specification;

underlined GGATCC represents the BamH I site and CTGCAG represents the Pst I site.

Further, the PCR reaction conditions used in the obtaining of the paelopdis sp. Storing at 98 deg.C for 10s, 55 deg.C for 15s, 68 deg.C for 10s, 30cycles, 4 deg.C.

Further, the purification in step 3 is performed by using a Ni-NTA column.

Compared with the prior art, the invention can obtain the following technical effects:

the recombinant protein prepared by the method overcomes the defects that the raw material source is difficult to obtain, the protein renaturation recovery process is complex and fussy and the like, the purified protein has high purity, good activity, wide temperature application range and strong digestive enzyme tolerance, and lays a foundation for the wide application of the protein in the fields of biology, food, medicine, cosmetology, agriculture and the like.

Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is an SDS-PAGE electrophoresis of recombinant expression of the Paelopdes sp. Wherein M is a protein Marker; 1: the non-induced expression of the recombinant vector pCold II in the Chaperone component Cell pG-KJE8/BL 21; 2: the recombinant vector pCold II is induced to express in Chaperone component Cell pG-KJE8/BL 21; 3: after the recombinant plasmid pCold II is induced and expressed in the Chaperone component Cell pG-KJE8/BL21, carrying out ultrasonic disruption on the recombinant plasmid pCold II to obtain an inclusion body; 4: after the recombinant plasmid pCold II is induced and expressed in the Chaperone component Cell pG-KJE8/BL21, the supernatant is subjected to ultrasonic disruption; 5: purifying the obtained target protein PaSOD by a nickel column medium;

fig. 2 is a graph showing the thermal stability of the present Paelopatides sp. Wherein the abscissa represents different temperatures and the ordinate represents the relative activity of PaSOD;

fig. 3 is a graph showing the acid-base stability of the present invention, PaSOD, the superoxide dismutase, wherein the abscissa indicates different pH and the ordinate indicates the relative activity of PaSOD.

Detailed Description

The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.

The experimental procedures for the following examples, in which the specific conditions are not specified, were generally carried out according to the experimental conditions described in the molecular cloning laboratory manual (15, SammBruker, Lassel, Huangpetang (eds.), molecular cloning instructions, scientific Press, 2002, third edition) or according to the instructions recommended by the reagent or instrument manufacturers.

In order to achieve the purpose, the invention adopts the following specific measures, and the specific steps are as follows:

example 1 obtaining of paelopathides sp total RNA and cDNA of deep-crouin sea cucumber:

deep-Yuan sea cucumber Paelopidides sp, obtained from 6500m deep-Yuan Hai (latitude: 10 ℃ 57.1693 'N, longitude: 141 ℃ 56.1719' E), was ground by in situ addition of RNAlater and immediately stored in liquid nitrogen. After returning to the laboratory, 50-100mg of sample tissue was taken, the residual RNAlater was blotted using RNase FREE test paper and the tissue was placed in 2ml centrifuge tubes. 1000ul of QIAzol was added and stirred for 20-40 seconds using a Ruptor to break the sample sufficiently. The homogenate was allowed to stand at room temperature for more than 10 min. The treated homogenate was subjected to total RNA extraction according to QIAGEN (QIAGEN) Total RNA extraction kit. Detecting no degradation of RNA by electrophoresis, and performing reverse transcription to synthesize cDNA. Total RNA to cDNA reverse transcription was performed according to TAKARA reverse transcription Kit (PrimeScriptTM 1st Strand cDNA Synthesis Kit) Kit protocol. The resulting cDNA was used for PCR amplification in example 2.

Among them, the sea cucumber sample was sufficiently ground in QIAzol of the early stage and extracted with QIAGEN (QIAGEN) kit of the later stage with good effect.

Example 2 acquisition and sequence analysis of the paelopdes sp.

The PasSOD gene was amplified using the cDNA sequence obtained in example 1 as a template and primers F and R, wherein,

and (3) primer F: CG (CG)GGATCCATGTCTGTCCACGCCGTTTGTGTATT, the nucleotide sequence of which is shown in SEQ ID NO. 3;

and (3) primer R: AACTGCAGTATCTTTTTGATCCCAATTACA, the nucleotide sequence of which is shown in SEQ ID NO. 4;

underlined GGATCC represents the BamH I site and CTGCAG represents the Pst I site.

The PCR reaction system was 50. mu.L, including 10. mu.L PrimeSTAR GXL Buffer, 4. mu.L dNTP, 1. mu.L each of F and R primers, 1. mu.L of template cDNA, 2. mu.L

GXL DNA Polymerase (TaKARa Co., Ltd.) using H₂O made up the total volume to 50. mu.L. The PCR reaction conditions are as follows: storing at 98 deg.C for 10s, 55 deg.C for 15s, 68 deg.C for 10s, 30cycles, 4 deg.C. The PCR product is purified by tapping and then mixed with pMD^TM18-T Vector is connected and transformed into escherichia coli DH5 α competent cells, a single colony is selected for PCR, then a positive clone with DNA fragments is selected for sequencing, the property of the positive clone is double-stranded linear DNA, the length of the positive clone is 459bp, the nucleotide sequence is shown as SEQID NO.1 and is marked as PaSOD, namely the nucleotide sequence of superoxide dismutase Cu and Zn-SOD from the deep-pool sea cucumber Paelopides sp, and the amino acid sequence is shown as SEQ ID NO. 2.

The PCR reaction adopts PrimeSTAR GXL high fidelity enzyme, the amplification efficiency is high, and the fidelity is good.

Example 3 soluble expression and protein purification of the paelopdis sp.

Amplifying and culturing DH5 α with correct sequencing, extracting plasmids, carrying out Bam H I and Pst I double enzyme digestion reaction on the plasmids and pCold II vector plasmids by PaSOD, recycling PaSOD target genes and linear pCold II vectors by glue, connecting the PaSOD target genes and the linear pCold II vectors by TAKARA T4 ligase (TAKARA company) overnight, transforming the connecting products into escherichia coli DH5 α competent cells, selecting single colonies, carrying out PCR, and selecting positive clone sequencing of target DNA fragments.

The correctly sequenced DH5 α was subjected to scale-up culture, plasmids were extracted, E.coli Chaperone Comentcell pG-KJE8/BL21 was transformed, and after plating, single colony PCR was selected to verify the correctness of the transformation, and the correctly transformed colonies were inoculated into LB medium containing 20. mu.g/ml chloramphenicol, 100. mu.g/ml ampicillin, 0.5mg/ml L-Arabidopsis, and 2ng/ml Tetracyclin, and when cultured with shaking at 37 ℃ until OD600 became 0.4-0.6, they were left at 15 ℃ for 30 minutes, and then 0.1mM IPTG was added, and cultured with shaking at 15 ℃ for 24 hours.

After centrifugation at 8000g for 5min to collect cells and washing the cells once with 1 XPBS, the bacterial cells were resuspended in 20 mM binding Buffer (50mM sodium phosphate Buffer, 300mM sodium chloride, pH 7.4), sonicated on ice until the cell suspension became translucent, centrifuged at 14000rpm for 20min, and the pellet was discarded. The resulting supernatant was filtered through a 0.45 μm disposable filter and then purified by using a fresh Ni-NTA column, and the operation and the reagents used were carried out according to the instructions. The purified fusion protein was dialyzed against 1 × TBS as a dialysis solution, and the dialysis solution was replaced at 4, 8, and 14 hours. The dialyzed protein was concentrated by centrifugation at 7000g using a 3kDa ultrafiltration tube (Millipore Co.) for 20 min. The final recombinant fusion protein PaSOD has a molecular weight of about 19kDa and a purity of more than 95% as shown in FIG. 1, which is analyzed by 12% SDS-PAGE electrophoresis.

In the method, a pCold II cold shock expression vector is selected to be combined with a Chaperone component Cell pG-KJE8/BL21 expression host to carry out high-efficiency soluble expression of the target protein.

The construction of a high-efficiency expression system of the protein is a very important technology for the later protein function research. The pCold II cold shock expression vector selected by the invention contains a cold shock gene cspA promoter, when the culture temperature is switched to low temperature, the growth of escherichia coli is temporarily stopped, the expression of most of escherichia coli proteins is reduced, and the target gene can be efficiently expressed. The advantage of selecting Chaperone component Cell pG-KJE8/BL21 as the expression host bacterium is that: conventional methods for protein expression are often performed as inclusion bodies, and the expressed protein is often inactive, unrecoverable, or amenable to renaturation. The host bacterium product contains chaperone plasmid and can express a group of molecular chaperones participating in protein folding, and the target protein and the molecular chaperones are co-expressed, so that the recovery rate of soluble protein can be increased. Thereby simplifying the whole operation process, reducing the inactivation speed of enzyme preparations and improving the protein yield.

EXAMPLE 4 enzymatic Properties of recombinant superoxide dismutase PaSOD

a) The determination principle and method are as follows:

the enzymological property research of the recombinant superoxide dismutase PaSOD is carried out by adopting a total superoxide dismutase (product number: A001-1 hydroxylamine method) detection kit of Nanjing construction company. The principle is that a reaction system of xanthine and xanthine oxidase generates superoxide anion free radicals which oxidize hydroxylamine to form nitrite, the nitrite is purple red under the action of a color developing agent, and the absorbance of the nitrite is measured by using a visible spectrophotometer at OD550 nm. When the tested sample contains SOD, it has specific inhibiting action to superoxide anion free radical, so that the produced nitrite is reduced, and the absorbance value of the test tube is lower than that of the control tube in the colorimetric process, and the SOD activity in the tested sample can be obtained by formula calculation. SOD enzyme activity is expressed in U/mg. The corresponding PaSOD is defined as an enzyme activity unit when the inhibition rate of the superoxide anion free radical in the reaction reaches 50% per milligram of protein. The enzyme activity calculation formula is as follows:

SOD activity (U/mg)

b) Thermal stability of recombinant superoxide dismutase PaSOD

mu.L of control (1 XTBS) and 30. mu.L of protein sample were taken, treated at 0 ℃,5 ℃, 10 ℃,20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃ for 20min, respectively, pH was 7.4, immediately placed in ice, and the residual activity of PaSOD was detected. Each spot was replicated three times and the average of each control and assay was taken for enzyme activity calculation. The result shows that the temperature adaptation range of the enzyme is wide, more than 80% of enzyme activity can be maintained after the enzyme is treated for 20min within the range of 0-60 ℃, and the optimal temperature is around 40 ℃, as shown in figure 2.

c) Acid-base stability of recombinant superoxide dismutase PaSOD

The residual SOD activity was determined by incubating 30. mu.L of protein with equal amounts of buffer at different pH values for 1h at 25 ℃. Three replicates were set at each pH, and the enzyme activity was calculated from the average of each set of controls and measurements. The pH buffers used were 0.2M citric acid-sodium citrate buffer (pH4 and 5), 0.2M phosphate buffer (pH6, pH7, pH7.4 and 8), 0.2M Tris-HCl buffer (pH8.5 and 9) and 0.2M glycine/NaOH (pH10 and 12). The results show that the enzyme had an optimum pH of 8.5 and maintained an activity of 40% or more after 1 hour of treatment at a pH of 5-9, as shown in FIG. 3.

d) Digestive enzyme tolerance

The purified protein was incubated with a digestive enzyme (trypsin: chymotrypsin: 2400: 400; raw) at 37 ℃ and ph7.4 at a ratio of 1:77(w/w) for 1h, 2h, and 3h, respectively, and then the residual enzyme activity was measured. As shown in Table 1, the results indicate that PaSOD has a strong ability to resist the action of digestive enzymes.

TABLE 1

e) Computational analysis

The temperature and pH value action points are respectively provided with three repeated average values to calculate the enzyme activity, the point with the highest SOD enzyme activity is set as 100 percent of the enzyme activity, and the rest is obtained by the percentage of the enzyme activity occupying the highest enzyme activity. In a digestive enzyme experiment, the enzyme activity for digesting for 0h is set as 100%, and the rest is obtained by taking the percentage of the enzyme activity accounting for 0 h.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.

Sequence listing

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Claims (3)

1. A gene of superoxide dismutase Cu, Zn SOD derived from deep-sea cucumber is characterized in that the gene is marked as PaSOD and has a nucleotide sequence shown in SEQ ID NO. 1.

2. A pCold II vector containing the gene of claim 1.

3. A Chaperone comparative Cell pG-KJE8/BL21 expression host Cell comprising the vector of claim 2.

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