CN110894224A - A kind of oyster ferritin with high thermal stability and preparation method thereof - Google Patents

Abstract

The invention discloses an oyster ferritin with high thermal stability and a preparation method. The amino acid sequence of the above oyster ferritin is shown in SEQ ID NO. , the nucleotide is mutated from gcc to gac. The method of the invention is as follows: using oyster as a raw material, recombinantly expressing oyster ferritin, mutating negatively charged aspartic acid at the triple axis of ferritin into uncharged glycine through point mutation, and sonicating cells, ammonium sulfate precipitation and Weak anion exchange column chromatography to obtain high-purity oyster ferritin with high thermal stability. The present invention improves the thermal stability of ferritin from 76°C to 87°C, while still having a certain physiological activity; the present invention has simple operation and strong controllability of conditions, and its greatest advantage is that oysters can be kept under relatively high temperature conditions. The physiological activity of ferritin still has unique self-assembly properties, and it has a good application prospect.

Description

Oyster ferritin with high thermal stability and preparation method thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to oyster ferritin with high thermal stability and a preparation method based on a genetic engineering means.

Background

Ferritin is purified and separated from spleen of vertebrate horse in 1937, and found to be widely present in spleen, liver, kidney, bone marrow, heart, pancreas, intestine, placenta and other metabolically active tissues and vertebrate blood, and has the characteristics of dilute acid resistance pH of 2, dilute alkali resistance pH of 12, high temperature resistance of 70-75 deg.C, etc. The ferritin is spherical in shape and consists of two parts, namely a protein shell and an iron core, wherein the protein shell is an inner cavity structure formed by highly symmetrical 24 subunits, and the iron core is a non-uniform iron core structure formed by thousands of ferric hydroxide molecules and hundreds of phosphate molecules and can be divided into an iron core surface layer with high phosphorus-iron ratio and an iron core inner layer structure with low phosphorus-iron ratio. Ferritin is widely found in animals, plants and microorganisms. When the content of ferrous iron ions in cells is high, ferritin is catalytically oxidized to generate nontoxic ferric iron through a ferrous iron redox center under the assistance of oxygen and is stored in the ferritin, and 4500 ferric irons can be stored in 1 molecule ferritin at most, which is characterized in that the ferritin is different from other enzymes at most. When cells need iron, ferritin reduces ferric iron into ferrous iron ions under the help of a reducing agent, and releases the ferrous iron ions from the interior of the cells for synthesis and utilization of other proteins, so ferritin has the double effects of removing the toxicity of the ferrous iron ions and regulating the balance of iron metabolism in the cells and has important biological activity. Durand et al have demonstrated that ion exchange chromatography and high performance liquid chromatography can be used to separate purified ferritin from oyster viscera. A20 kDa band was shown on SDS-PAGE. The results of the amino acid sequence alignment of the oyster ferritin cDNA show that the oyster ferritin cDNA have consistency with pinctada fucata martensii, lygodium japonicum and burgundy snail. Meanwhile, experiments prove that residues responsible for the center of the iron oxide enzyme exist in the oyster ferritin subunit.

At present, because the content of ferritin obtained by natural purification is limited and the ferritin is expensive, ferritin cannot be widely applied to experimental research and drug development systems. The heat denaturation temperature of the natural oyster ferritin is 76 ℃, although the natural oyster ferritin has relatively good heat resistance, the application of the ferritin is still limited along with the further increase of the temperature. Therefore, the prokaryotic expression of the gene can not only improve the expression efficiency, but also has relatively low price and is suitable for mass production.

Disclosure of Invention

The invention aims to provide oyster ferritin with high thermal stability and a preparation method thereof. The invention changes the amino acid sequence of wild oyster ferritin, thereby changing the environment near the triple axis of the oyster ferritin, improving the heat resistance of the oyster ferritin and enlarging the application range of the oyster ferritin. The invention carries out point mutation on the wild oyster ferritin for the first time, increases the heat resistance of the oyster ferritin and has higher practical significance and application prospect. Can be used for storing iron for human needs or as a carrier for transferring nutrient substances and the like.

The invention constructs a new ferritin by changing the amino acid sequence of wild oyster ferritin through point mutation. The wild oyster ferritin amino acid sequence is provided by NCBI website (Gene ID: LOC105327516), 120 th aspartic acid in the wild oyster ferritin amino acid sequence is mutated into glycine, and nucleotide is mutated from gcc into gac, thus obtaining the oyster ferritin with high thermal stability. Ferritin can be loaded with small molecular substances or other nutrients due to its unique structure, and thus can be used as a transport carrier to transport substances required by the body. Successful acquisition of this protein can expand the range of use of ferritin.

An oyster ferritin with high thermal stability has amino acid sequence shown in SEQ ID NO.2, and molecular weight of 480 kDa; or the amino acid sequence with the same function obtained by deleting, inserting or replacing the amino acid sequence shown as SEQ ID NO. 2.

The nucleotide sequence of the gene mGF1 for coding the oyster ferritin with high thermal stability is shown as SEQ ID No. 1.

The invention also provides a preparation method of the oyster ferritin with high thermal stability, which comprises the following steps:

s1, obtaining a nucleotide sequence for coding the oyster ferritin with high thermal stability, wherein the nucleotide sequence is shown as SEQ ID NO. 1;

s2, inserting the nucleotide sequence into a pET vector to obtain a recombinant plasmid;

s3, transforming the recombinant plasmid into escherichia coli BL21, and performing expression culture;

s4, extracting oyster ferritin with high thermal stability expressed by the escherichia coli BL 21;

preferably, the conditions for the expression culture in step S3 are: to the medium of E.coli BL21 containing the recombinant plasmid was added isopropyl thiogalactoside (IPTG) at a final concentration of 0.6mM, and the mixture was shake-cultured at 28 ℃ for 6 hours.

Preferably, the extraction process of the oyster ferritin with high thermal stability in step S4 is as follows: ultrasonically crushing Escherichia coli BL21 containing recombinant plasmids, centrifuging to obtain supernatant, and sequentially performing ammonium sulfate precipitation, dialysis desalting and weak anion exchange chromatography on the supernatant to obtain the oyster ferritin with high thermal stability.

Preferably, the weak anion exchange chromatography is a DEAE cellulose column chromatography.

Preferably, the rotation speed of the centrifugation is 10000 rpm; the ammonium sulfate is precipitated, and the saturation degree of the ammonium sulfate is 80 percent at 4 ℃.

The heat denaturation temperature of the oyster ferritin with high thermal stability prepared by the invention is measured, the absolute amount of the protein is 1mg, and the result of the measurement shows that the denaturation temperature of the oyster ferritin with high thermal stability is 87 ℃, which is 11 ℃ higher than that of the wild oyster ferritin. Meanwhile, the property analysis of the mutated ferritin after heat treatment shows that the oyster ferritin with high thermal stability still maintains the shell-shaped structure of the hollow protein under a certain temperature condition, and the phenomena of monomer depolymerization and macromolecular aggregation are not found.

The invention prepares acidified FeSO₄Slowly adding into oyster ferritin with high thermal stability at 25 deg.C, and measuring Fe catalyzed by ferritin²⁺Is oxidized into Fe³⁺The ability of the cell to perform. The result shows that the activity of the oyster ferritin with high thermal stability is 0.4370 ron/subbunit/s (the activity of the wild oyster ferritin is 0.6197 ron/subbunit/s), and the mutated ferritin still has strong oxidation activity.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention purifies the mutated oyster ferritin for the first time, increases the thermal denaturation temperature by 11 ℃, and ensures that the ferritin can still maintain the unique physiological activity at higher temperature.

2. The oyster ferritin with high thermal stability can be used as carrier for embedding active substances or medicines, and can maintain unique hollow cage structure and physiological activity after pasteurization due to the improvement of heat resistance, and can be applied to the development of functional foods.

3. The method for efficiently preparing the oyster ferritin based on the genetic engineering means is convenient and rapid, and a large amount of ferritin can be obtained by mass strain expression and purification, so that the raw material cost in the purification process is greatly reduced, and the manpower and resources are greatly saved. The invention has important significance for the research works such as developing ferritin carriers and regulating iron metabolism balance.

Drawings

FIG. 1 is a graph showing the thermal denaturation temperature (87 ℃) of oyster ferritin having high thermal stability obtained in example 3 of the present invention.

FIG. 2 is a graph showing the distribution of the particle size of oyster ferritin having high thermal stability without heat treatment according to example 3 of the present invention.

FIG. 3 is a graph showing the distribution of the particle size of oyster ferritin having high thermal stability obtained in example 3 of the present invention after heating at 60 ℃ for 10 min.

FIG. 4 is a graph showing the distribution of the particle size of the oyster ferritin having high thermal stability in example 3 of the present invention after heating at 70 ℃ for 10 min.

FIG. 5 is a graph showing the distribution of the particle size of oyster ferritin having high thermal stability obtained in example 3 of the present invention after heating at 80 ℃ for 10 min.

FIG. 6 is a graph showing the distribution of the particle size of oyster ferritin having high thermal stability obtained in example 3 of the present invention after heating at 90 ℃ for 10 min.

FIG. 7 is a graph showing the distribution of the particle size of oyster ferritin having high thermal stability obtained in example 3 of the present invention after heating at 100 ℃ for 10 min.

FIG. 8 is a transmission electron microscope negative staining image (TEM) of the oyster ferritin with high thermal stability obtained in example 3 of the present invention without heat treatment.

FIG. 9 is a transmission electron microscope negative staining image (TEM) of the oyster ferritin with high thermal stability obtained in example 3 of the present invention heated at 60 ℃ for 10 min.

FIG. 10 is a transmission electron microscope negative staining image (TEM) of the oyster ferritin with high thermal stability obtained in example 3 of the present invention heated at 70 ℃ for 10 min.

FIG. 11 is a transmission electron microscope negative staining image (TEM) of the oyster ferritin with high thermal stability obtained in example 3 of the present invention heated at 80 ℃ for 10 min.

FIG. 12 is a transmission electron microscope negative staining image (TEM) of the oyster ferritin with high thermal stability obtained in example 3 of the present invention heated at 90 ℃ for 10 min.

FIG. 13 is a transmission electron microscope negative staining image (TEM) of the oyster ferritin with high thermal stability obtained in example 3 of the present invention heated at 100 ℃ for 10 min.

FIG. 14 is a comparison of the oxidation activities of oyster ferritin having high thermal stability obtained in example 4 of the present invention and wild-type oyster ferritin.

Detailed Description

The present invention is further illustrated by the following specific examples, but the scope of the invention is not limited thereto. For process parameters not specifically noted, reference may be made to conventional techniques.

The invention constructs a novel ferritin by changing the amino acid sequence of the ferritin through point mutation. The molecular weight of the ferritin is 480kDa, and the amino acid sequence is shown in SEQ ID NO. 2. Wherein the 120 th aspartic acid is mutated into glycine, and the nucleotide is mutated from gcc into gac (the base sequence is shown as SEQ ID NO. 1).

The amino acid sequence of the wild oyster ferritin is provided by NCBI website (Gene ID: LOC 105327516). The construction of oyster ferritin having high thermal stability is as follows: using oyster ferritin cDNA as a template and SEQ ID NO.3 and SEQ ID NO.4 as primers to amplify a partial GF1DNA fragment 1 containing point mutation; using oyster ferritin cDNA as a template and SEQ ID NO.5 and SEQ ID NO.6 as primers to amplify a partial GF1DNA fragment 2 containing point mutation; amplifying the Pacific oyster point mutation ferritin gene mGF 1DNA by using a mixture (molar ratio is 1:1) of the GF1DNA fragment 1 and the GF1DNA fragment 2 as a template and using SEQ ID NO.3 and SEQ ID NO.6 as primers; constructing a prokaryotic expression system BL21-pET21a-mGF1 and carrying out fermentation culture in an LB culture medium; the oyster ferritin with high thermal stability is obtained by purifying through ultrasonic crushing, ammonium sulfate precipitation and DEAE weak anion exchange chromatography.

The invention measures the heat denaturation temperature of the oyster ferritin with high thermal stability, the absolute amount of the protein is 1mg, and the measurement result shows that the denaturation temperature of the oyster ferritin is improved by 11 ℃ due to mutation. Meanwhile, the property analysis of the ferritin after heat treatment shows that the ferritin still maintains the shell-like structure of the hollow protein under a certain temperature condition, and the phenomena of depolymerization of monomers and aggregation of macromolecules are not found.

The invention prepares acidified FeSO₄Slowly adding wild type Concha Ostreae ferritin and Concha Ostreae ferritin with high thermal stability at 25 deg.CMeasurement of ferritin catalyzed Fe²⁺Is oxidized into Fe³⁺The ability of the cell to perform. The results show that the oyster ferritin with high thermal stability still has certain oxidation activity although the activity is slightly reduced.

Example 1

A method for producing oyster ferritin having high thermal stability, comprising the steps of:

s1, construction of oyster ferritin prokaryotic expression BL21-pET21a-mGF1 engineering strain with high thermal stability, comprising the following steps:

s11, oyster RNA extraction: extracting mRNA from fresh Pacific oyster;

s12, construction and amplification of oyster ferritin mGF1 gene (SEQ ID No.1) with high thermal stability: and (5) carrying out reverse transcription by taking the mRNA in the step S11 as a template to obtain Pacific oyster cDNA: PrimeScript was obtained using the kit^TM1st StrandcDNA Synthesis Kit (6110A), Takara, Baozi, performed the reverse transcription process. Taking 2 μ L of Random 6mers, 1 μ L of dNTPmix, 2 μ g of template mRNA, ddH₂And (3) supplementing O to 10 mu L, uniformly mixing, incubating at 42 ℃ for 30min, heating to 65 ℃, preserving heat for 5min to inactivate enzyme activity, and then quickly cooling on ice to obtain the oyster cDNA.

Amplifying a part GF1DNA fragment 1(SEQ ID NO.7) containing point mutation by using the oyster cDNA as a template and SEQ ID NO.3 and SEQ ID NO.4 as primers; the amplification system is derived from a PCR kit except for the cDNA which is taken as a template: takara LA Baori medical Co

PCR kit (RR002A), using system: 10 μ L of Taq Buffer 2 μ L, 1 μ L of the above-obtained cDNA template, 1 μ L of dNTP2 μ L, 1 μ L of primer GF1-F (10nM) (SEQ ID NO.3), 1 μ L of primer GF1-T (10nM) (SEQ ID NO.4), 0.2 μ L of Tapase, and purification by ddH₂O was replenished to 20. mu.L. Placing into BIO-RAD PCR instrument, performing pre-denaturation at 95 deg.C for 10min, circulating, performing denaturation at 95 deg.C for 30s, annealing at 52 deg.C for 40s, and extending at 72 deg.C for 1min for 35 times, and finally extending at 72 deg.C for 10min, and amplifying to obtain part GF1DNA fragment 1 containing point mutation;

taking the oyster cDNA as a template, and taking SEQ ID NO.5 and SEQ ID NO.6 as primers,amplifying the partial GF1DNA fragment 2(SEQ ID NO.8) containing the point mutation; the method comprises the following steps: 10 XTaq Buffer 2. mu.L, cDNA template obtained above 1. mu.L, dNTP 2. mu.L, primer GF1-P (10nM) (SEQ ID NO.5) 1. mu.L, primer GF1-R (10nM) (SEQ ID NO.6) 1. mu.L, Tapase 0.2. mu.L, using ddH₂O was replenished to 20. mu.L. Placing into BIO-RAD PCR instrument, performing pre-denaturation at 95 deg.C for 10min, circulating, performing denaturation at 95 deg.C for 30s, annealing at 52 deg.C for 40s, and extending at 72 deg.C for 1min for 35 times, and finally extending at 72 deg.C for 10min, and amplifying to obtain partial GF1DNA fragment 2 containing point mutation;

amplifying oyster ferritin gene mGF 1DNA with high thermal stability by using a mixture (molar ratio is 1:1) of GF1DNA fragment 1 and GF1DNA fragment 2 as a template and SEQ ID NO.3 and SEQ ID NO.6 as primers; the method comprises the following steps: 10 XTaq Buffer 2. mu.L, partial GF1DNA fragment containing point mutations 120 ng, partial GF1DNA fragment containing point mutations 220 ng, dNTP 2. mu.L, primer GF1-F (10nM) (SEQ ID NO.3) 1. mu.L, primer GF1-R (10nM) (SEQ ID NO.6) 1. mu.L, Tap enzyme 0.2. mu.L, using ddH₂O was replenished to 20. mu.L. Placing in a BIO-RAD PCR instrument, performing pre-denaturation at 95 ℃ for 10min, then performing circulation, performing denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 40s, performing extension at 72 ℃ for min, performing circulation for 35 times, and finally performing extension at 72 ℃ for 10min, and amplifying to obtain the oyster ferritin mGF1 gene containing point mutation. Cloning a target gene mGF1, transferring the cloned gene into a clone strain Escherichia coli JM109, and carrying out PCR verification and sequencing on a bacterial liquid;

s13, construction and amplification of expression plasmid pET21a-mGF 1: amplifying and culturing the expression plasmid, and amplifying the target gene mGF 1; carrying out double digestion on plasmid pET21a and mGF 1DNA by using BamH I and Xho I respectively, and connecting double digested pET21a and GF1DNA fragments by using T4 DNAIgase to obtain recombinant plasmid pET21a-mGF 1; the recombinant plasmid pET21a-mGF1 was heat shock transformed into the clonal strain JM 109;

s14, construction of prokaryotic expression system BL21-pET21a-mGF 1: extracting the recombinant plasmid pET21a-mGF1, transforming the recombinant plasmid into an engineering strain BL21(DE3), preparing an engineering strain BL21-pET21a-mGF1, and storing the strain at-80 ℃;

s2, culturing engineering strains, inducing expression and separating and purifying: adding the engineering strain into LB liquid culture mediumCulturing at 37 deg.C and 180rpm for 3h, and measuring absorbance OD of fermentation liquid by ultraviolet spectrophotometer₆₀₀When OD is reached₆₀₀When the concentration is 0.6 ℃, adding isopropyl thiogalactoside (IPTG) with the final concentration of 0.6mM for induction expression, continuously culturing for 6h under the condition of 28 ℃ to obtain a zymophyte liquid, centrifuging for 15min under 10000rpm under the condition of 4 ℃, taking a precipitate, dissolving the precipitate in buffer 50mM Tris-HCl (pH 7) with the volume 5 times that of the crude protein extracting solution, controlling the power of 300W and the ultrasonic time to be 3s, stopping for 5s, and totally 15min (including the time of ultrasonic treatment and stopping), after ultrasonically crushing the obtained thalli after ultrasonically fermenting, centrifuging for 15min under 10000rpm for a thallus crushing liquid, adding ammonium sulfate with the saturation degree of 80% into a supernatant, stirring for 30min, standing for 6h under 4 ℃, centrifuging for 15min under 10000rpm, taking the precipitate, re-dissolving the precipitate in 50mM Tris-HCl (pH 7) with the mass 10 times that of the precipitate, dialyzing the solution by using a dialysis bag with the molecular weight of 3500Da, and taking the solution with the volume of 100 times, Dialyzing the solution with 0.05mol/L Tris-HCl (pH 7), changing the dialyzate every 6h, dialyzing for 6 times, and removing ammonium sulfate in the protein solution to obtain the crude extract of the oyster ferritin. Filtering the dialyzed liquid by using a 0.45-micron water-based filter membrane to obtain an oyster ferritin crude solution; purifying the crude oyster ferritin solution obtained in the step S5 by using a DEAE weak anion exchange column; eluting the positively charged ferritin in crude solution of oyster ferritin with 50mM Tris-HCl (pH 7) as mobile phase solution; the flow rate of the mobile phase is 2mL/min, and the protein elution peak is detected by ultraviolet at 280 nm; collecting protein elution peak, concentrating and desalting with 100kDa ultrafiltration centrifuge tube, and ultrafiltering the ferritin elution peak at 5000rpm for 10 min; after the ultrafiltration is carried out once, pure water is supplemented into the inner sleeve of the ultrafiltration centrifugal pipe to the volume before the centrifugation, and the centrifugation condition is repeated for three times; obtaining the oyster ferritin solution with high thermal stability which is purified after concentration and desalination.

Comparative example 1

S1, construction of a wild oyster ferritin prokaryotic expression BL21-pET21a-GF1 engineering strain, which comprises the following steps:

s11, extracting wild oyster ferritin RNA: extracting mRNA from fresh oyster;

s12, amplification of wild oyster ferritin GF1 gene: the mRNA described in step S11 isTemplate, inverse transcription to obtain oyster ferritin cDNA; PrimeScript was obtained using the kit^TM1st Strand cDNA Synthesis Kit (6110A), Takara, Baozi, performed a reverse transcription process. Taking 2 μ L of Random 6mers, 1 μ L of dNTP mix, 2 μ g of template mRNA, ddH₂And (3) supplementing O to 10 mu L, uniformly mixing, incubating at 42 ℃ for 30min, heating to 65 ℃, preserving heat for 5min to inactivate enzyme activity, and then quickly cooling on ice to obtain the oyster cDNA.

Amplifying GF1DNA (SEQ ID NO.9) by using the oyster cDNA as a template and SEQ ID NO.3 and SEQ ID NO.6 as primers; the method comprises the following steps: 10 μ L of Tap Buffer, 1 μ L of the above-obtained cDNA template, 2 μ L of dNTP, 1 μ L of primer GF1-F (10nM) (SEQ ID NO.3), 1 μ L of primer GF1-R (10nM) (SEQ ID NO.6), 0.2 μ L of Tap enzyme, and purification by ddH₂O was replenished to 20. mu.L. Placing into BIO-RAD PCR instrument, pre-denaturing at 95 deg.C for 10min, circulating, denaturing at 95 deg.C for 30s, annealing at 52 deg.C for 40s, and extending at 72 deg.C for 1min, for 35 times, and finally extending at 72 deg.C for 10 min. Amplifying to obtain the gene containing wild oyster ferritin GF 1. After cloning the target gene GF1, transferring the cloned gene into a clone strain GM109, and carrying out PCR verification and sequencing on bacterial liquid.

S13, construction and amplification of a wild oyster ferritin expression plasmid pET21a-GF 1: amplifying and culturing an expression plasmid, and amplifying a target gene GF 1; carrying out double digestion on plasmid pET21a and GF1DNA by using BamHI and XhoI respectively, and connecting double digested pET21a and GF1DNA fragments by using T4 DNA Ligase to obtain recombinant plasmid pET21a-GF 1; the recombinant plasmid pET21a-GF1 is transformed into a clonal strain JM109 by heat shock, and the conditions of the heat shock transformation are that the recombinant plasmid is heated at 42 ℃ for 45s and then placed on ice for 1 min;

s14, construction of prokaryotic expression system BL21-pET21a-GF 1: extracting the recombinant plasmid pET21a-GF1, transforming the recombinant plasmid into an engineering strain BL21(DE3) to prepare an engineering strain BL21-pET21a-GF1, and storing the strain at 80 ℃ below zero (the ratio of the bacteria liquid to 40% glycerol is 1: 1);

s2, culturing engineering strains, inducing expression and separating and purifying: adding the engineering strain into LB liquid culture medium, culturing at 37 deg.C and 180rpm for 3h, and measuring absorbance OD of the fermentation liquid by ultraviolet spectrophotometer₆₀₀When OD is reached₆₀₀When the concentration is 0.6mM, isopropyl thiogalactoside is added to the solution to a final concentration of 0.6mM(IPTG) and continuing to culture for 6h at the temperature of 28 ℃ to obtain the zymocyte liquid. Centrifuging at 10000rpm for 15min at 4 ℃, dissolving the precipitate in 50mM Tris-HCl (pH 7) buffer solution with the volume 5 times of the volume of the crude protein extract, performing ultrasonic treatment with the power of 300W for 3s, stopping the ultrasonic treatment for 5s, and performing total time of 15min (including ultrasonic treatment and stopping time), after performing ultrasonic disruption fermentation, centrifuging the obtained thallus at 10000rpm for 15min, adding ammonium sulfate with the saturation of 80% into the supernatant, stirring for 30min, standing at 4 ℃ for 6h, centrifuging at 10000rpm for 15min, re-dissolving the precipitate in 50 mM-HCl (pH 8) with the mass 10 times of the mass of the precipitate, dialyzing the solution by using a dialysis bag with the molecular weight of 3500Da, dialyzing the solution by using 0.05mol/L Tris-HCl (pH 7) with the volume of 100 times and replacing the dialysate every 6h, dialyzing for 6 times, removing ammonium sulfate in the protein solution, obtaining the crude extract of the oyster ferritin. Filtering the dialyzed liquid by using a 0.45-micron water-based filter membrane to obtain an oyster ferritin crude solution; purifying the crude oyster ferritin solution obtained in the step S5 by using a DEAE weak anion exchange column; eluting the positively charged protein in the crude oyster ferritin solution by using 50mM Tris-HCl (pH 7) as a mobile phase solution; eluting the negatively charged protein adsorbed on the DEAE column with 50mM Tris-HCl (pH 7) containing 1M NaCl at a flow rate of 2mL/min, and detecting the protein elution peak at 280nm by UV; collecting protein elution peak, concentrating and desalting with 100kDa ultrafiltration centrifuge tube, and ultrafiltering the ferritin elution peak at 5000rpm for 10 min; after the ultrafiltration is carried out once, pure water is supplemented into the inner sleeve of the ultrafiltration centrifugal pipe to the volume before the centrifugation, and the centrifugation condition is repeated for three times; obtaining the wild oyster ferritin solution which is purified after concentration and desalination.

Example 3

The oyster ferritin solution having high thermal stability obtained in example 1 was sampled and measured for thermal stability using a differential scanning calorimeter. The thermostable oyster ferritin solution was diluted to 5mg/mL with 50mM Tris-HCl (pH 7), and 200 μ L of the sample was accurately pipetted, placed in an aluminum pan and immediately sealed. The deionized water with the same mass is used as a reference, the scanning starting temperature is 20 ℃, the termination temperature is 100 ℃, and the heating rate is 1 ℃/min. The results of the experiment are shown in FIG. 1.

The oyster ferritin solution with high thermal stability obtained in example 1 was diluted to 0.5mg/mL with 50mM Tris-HCl (pH 7), heated at 60, 70, 80, 90 and 100 ℃ for 10min while untreated oyster ferritin solution with high thermal stability was used as a control, centrifuged at 10000rpm for 10min, and the supernatant was taken out after the precipitation to prepare a sample a to be tested. Analysis of the oyster ferritin aggregation phenomenon with high thermal stability using dynamic light scattering: 200 mu L of the sample A to be measured is placed in a micro measuring tube, the measuring angle is 90 degrees, the measuring time is 30s, and each experiment is repeated three times to obtain an average value. The results of the curve fitting using the software are shown in FIGS. 2-7.

The oyster ferritin solution with high thermal stability obtained in example 1 was diluted to 0.2mg/mL with 50mM Tris-HCl (pH 7), heated at 60, 70, 80, 90 and 100 ℃ for 10min while untreated oyster ferritin solution with high thermal stability was used as a control, centrifuged at 10000rpm for 10min, and the supernatant was removed after precipitation and used as sample B. Analysis of the appearance of oyster ferritin with high thermal stability using transmission electron microscopy: dripping 10 mu L of the sample B to be detected on a copper net attached with a carbon film, and sucking the sample B to be detected by filter paper after 5 minutes; 10 mu L of uranyl acetate (2%) is dropped on a copper net and kept for 5 minutes, and then is sucked dry by filter paper; the protein morphology was observed on a transmission electron microscope using a voltage of 80 kV. The same samples were observed in different areas to avoid experimental errors, and the results are shown in FIGS. 8-13.

Example 4

The oyster ferritin solution having high thermal stability obtained in example 1 and the wild-type oyster ferritin solution shown in comparative example 1 were sampled, and the samples were diluted to 0.5mg/mL with 50mM Mops (pH 7) according to the ratio of ferritin: FeSO₄(10mM, prepared using pH 2.0 ultrapure water) acidified FeSO was added at a ratio of 5:1 (volume ratio)₄Slowly dropping into ferritin solution, detecting ultraviolet absorption value at 300nm with ultraviolet spectrophotometer, stopping when the light absorption value rises until it is gentleThe data is scanned and saved. The data were derived to determine the rate of oxidative precipitation of ferritin. The results of the experiment are shown in FIG. 14.

FIG. 1 shows that the heat denaturation temperature of oyster ferritin having high thermal stability is 87 ℃. FIGS. 2 to 7 show that the particle size of oyster ferritin with high thermal stability is about 12nm when untreated, and the particle size of ferritin slightly increases with increasing temperature, but no significant aggregation of macromolecules is observed. FIGS. 8 to 13 show that the oyster ferritin with high thermal stability shows a clear white protein shell-like structure after negative dyeing when untreated; with the temperature rise, the ferritin still maintains a distinct and uniformly distributed structure, distinct aggregation occurs at 100 ℃, but the morphology of the protein is still visible. FIG. 14 shows that the rate of iron oxidation precipitation of oyster ferritin with high thermal stability is 0.4370 ron/subBunit/s, which is slightly lower than that of wild type oyster ferritin (0.6197 ron/subBunit/s), but still has strong oxidation activity.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.

Sequence listing

<110> university of Dalian Industrial university

<120> oyster ferritin with high thermal stability and preparation method thereof

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caaatcaaca tggaactgta tgcctgctat acctaccagt ccatggccta ctacttcgat 120

agagatgatg tggcccttcc gggattcagc aagttcttca agaattcatc agatgaagaa 180

cgggaacatg ctgaaaaact gatgaagtac caaaataaga gaggagggcg tgtcgtgctc 240

caagacatca agaaacccga ccgtgatgag tggggcaccg gcttggacgc catgcaggtg 300

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agtcaccagg atgcacagat gtgcgatttc cttgagaccc actacttgga ggagcaagtg 420

aacgccatca aggagatatc ggaccacatc actcagttga agagagtggg cagtgggctg 480

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Met Ala Glu Ser Gln Cys Arg Gln Asn Tyr His Gln Glu Ser Glu Ala

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Gly Ile Asn Arg Gln Ile Asn Met Glu Leu Tyr Ala Cys Tyr Thr Tyr

20 25 30

Gln Ser Met Ala Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu Pro Gly

35 40 45

Phe Ser Lys Phe Phe Lys Asn Ser Ser Asp Glu Glu Arg Glu His Ala

50 55 60

Glu Lys Leu Met Lys Tyr Gln Asn Lys Arg Gly Gly Arg Val Val Leu

65 70 75 80

Gln Asp Ile Lys Lys Pro Asp Arg Asp Glu Trp Gly Thr Gly Leu Asp

85 90 95

Ala Met Gln Val Ala Leu Gln Leu Glu Lys Thr Val Asn Gln Ser Leu

100 105 110

Leu Asp Leu His Lys Val Ala Gly Ser His Gln Asp Ala Gln Met Cys

115 120 125

Asp Phe Leu Glu Thr His Tyr Leu Glu Glu Gln Val Asn Ala Ile Lys

130 135 140

Glu Ile Ser Asp His Ile Thr Gln Leu Lys Arg Val Gly Ser Gly Leu

145 150 155160

Gly Glu Tyr Glu Tyr Asp Arg Arg Leu Asp Ser

165 170

<210>3

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

atggccgaat cccaatgtcg 20

<210>4

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

atcctggtga ctgccagcga ccttgtgaag 30

<210>5

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

cttcacaagg tcgctggcag tcaccaggat 30

<210>6

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

ttacgagtcg aggcggcgat 20

<210>7

<211>372

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

atggccgaat cccaatgtcg ccaaaattac caccaggaga gcgaagctgg catcaaccgc 60

caaatcaaca tggaactgta tgcctgctat acctaccagt ccatggccta ctacttcgat 120

agagatgatg tggcccttcc gggattcagc aagttcttca agaattcatc agatgaagaa 180

cgggaacatg ctgaaaaact gatgaagtac caaaataaga gaggagggcg tgtcgtgctc 240

caagacatca agaaacccga ccgtgatgag tggggcaccg gcttggacgc catgcaggtg 300

gcgctacagc tggagaagac tgtgaaccag tctctgcttg accttcacaa ggtcgctggc 360

agtcaccagg at 372

<210>8

<211>174

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

cttcacaagg tcgctggcag tcaccaggat gcacagatgt gcgatttcct tgagacccac 60

tacttggagg agcaagtgaa cgccatcaag gagatatcgg accacatcac tcagttgaag 120

agagtgggca gtgggctggg agagtatgaa tacgatcgcc gcctcgactc ctaa 174

<210>9

<211>516

<212>DNA

<213> Pacific oyster (Crassostra gigas)

<400>9

atggccgaat cccaatgtcg ccaaaattac caccaggaga gcgaagctgg catcaaccgc 60

caaatcaaca tggaactgta tgcctgctat acctaccagt ccatggccta ctacttcgat 120

agagatgatg tggcccttcc gggattcagc aagttcttca agaattcatc agatgaagaa 180

cgggaacatg ctgaaaaact gatgaagtac caaaataaga gaggagggcg tgtcgtgctc 240

caagacatca agaaacccga ccgtgatgag tggggcaccg gcttggacgc catgcaggtg 300

gcgctacagc tggagaagac tgtgaaccag tctctgcttg accttcacaa ggtcgctgac 360

agtcaccagg atgcacagat gtgcgatttc cttgagaccc actacttgga ggagcaagtg 420

aacgccatca aggagatatc ggaccacatc actcagttga agagagtggg cagtgggctg 480

ggagagtatg aatacgatcg ccgcctcgac tcctaa 516

Claims (10)

1. An oyster ferritin with high thermal stability is characterized in that the amino acid sequence is shown in SEQ ID NO. 2.

2. The oyster ferritin with high thermal stability according to claim 1, characterised in that the denaturation temperature is 87 ℃ when the absolute amount of oyster ferritin with high thermal stability is 1 mg.

3. The oyster ferritin with high thermal stability according to claim 1, characterised in that its activity is 0.4370 ron/subbunit/s.

4. The gene mGF1 encoding the oyster ferritin having high thermostability according to claim 1, whose nucleotide sequence is shown in SEQ ID No. 1.

5. A method for preparing oyster ferritin with high thermal stability is characterized by comprising the following steps:

s1, obtaining a nucleotide sequence for coding the oyster ferritin with high thermal stability according to claim 1, wherein the nucleotide sequence is shown as SEQ ID NO. 1;

s2, inserting the nucleotide sequence into a pET vector to obtain a recombinant plasmid;

s3, transforming the recombinant plasmid into escherichia coli BL21, and carrying out expression culture;

s4, extracting oyster ferritin with high thermal stability expressed by the Escherichia coli BL 21.

6. The method for preparing oyster ferritin with high thermal stability according to claim 5, wherein the conditions of the expression culture in step S3 are: to the culture medium of E.coli BL21 containing the recombinant plasmid was added isopropyl thiogalactoside to a final concentration of 0.6mM, and the mixture was shake-cultured at 28 ℃ for 6 hours.

7. The method for preparing oyster ferritin with high thermal stability in claim 5, wherein the extraction process of oyster ferritin with high thermal stability in step S4 is as follows: ultrasonically crushing Escherichia coli BL21 containing recombinant plasmids, centrifuging to obtain supernatant, and sequentially performing ammonium sulfate precipitation, dialysis desalting and weak anion exchange chromatography on the supernatant to obtain the oyster ferritin with high thermal stability.

8. The method for preparing oyster ferritin with high thermal stability according to claim 7, wherein the weak anion exchange chromatography is DEAE cellulose column chromatography.

9. The method for preparing oyster ferritin with high thermal stability in claim 7 wherein the rotation speed of the centrifugation is 10000 rpm.

10. The method for preparing oyster ferritin with high thermal stability as claimed in claim 7 wherein the ammonium sulfate is precipitated at 4 ℃ and 80% saturation with ammonium sulfate.

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