CN111019962A - SOD-ELP fusion protein and preparation method thereof - Google Patents
SOD-ELP fusion protein and preparation method thereof Download PDFInfo
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- CN111019962A CN111019962A CN201911310455.2A CN201911310455A CN111019962A CN 111019962 A CN111019962 A CN 111019962A CN 201911310455 A CN201911310455 A CN 201911310455A CN 111019962 A CN111019962 A CN 111019962A
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- elp
- sod
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- fusion protein
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
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- C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Abstract
The invention discloses a SOD-ELP fusion protein and a preparation method thereof, wherein the SOD-ELP fusion protein is prepared from ELP20Or ELP40With SOD, purifying SOD-ELP by ITC three-cycle circulation40The fusion protein enables the purification process of the protein to be simple, economic and efficient, and can be used for preparing the recombinant human copper-zinc superoxide dismutase with high specific activity in large scale in industry. The fusion protein not only retains the activity of enzyme, but also has good heat resistance and biocompatibility, and greatly prolongs the simulated plasma half-life period of the enzyme. The invention further purifies the SOD-ELP40Is embedded in liposome to obtain high-entrapment efficiency liposome with transdermal effect. SOD-ELP prepared by the invention40Can be widely applied to the fields of cosmetics, foods, medicine and pharmacology and the like.
Description
Technical Field
The invention belongs to the field of protein engineering, relates to a SOD-ELP fusion protein and a preparation method thereof, and particularly relates to a method for purifying superoxide dismutase by a non-chromatographic method by utilizing the phase change characteristic of elastin-like protein.
Background
Superoxide Dismutase (SOD) is an active substance widely existing in life bodies, and can remove harmful substances generated in the metabolism process of animals, plants and microorganisms. In 1938 scientists discovered superoxide dismutase from bovine red blood cells and studied superoxide dismutase for over 80 years.
SOD is a kind of metalloenzyme widely existing in biology, has the functions of catalyzing superoxide anion free radical to generate disproportionation reaction and balancing oxygen free radical in organism, and is a key prevention line for organism to defend active oxygen poison. SOD has unique functions in the aspects of radiation resistance, aging resistance, tumor inhibition and the like due to the strong oxidation resistance, and has good application prospect in the fields of medicines, foods and cosmetics.
SOD is mainly classified into three types according to the difference of metal ions bound to its active center: Cu/Zn-SOD, Mn-SOD and Fe-SOD, wherein the Mn-SOD is more common in mitochondria of prokaryotic cells and eukaryotic cells, and the Fe-SOD is mainly distributed in a few plant cells and prokaryotic cells. The Cu/Zn-SOD protein contains two subunits, and each subunit contains one Cu2+And one Zn2+，Cu2+Mainly plays a role in catalyzing superoxideDisproportionation occurs from the group, and Zn2+Mainly plays a role in maintaining the structural stability of protein, and takes Cu/Zn-SOD as a main species in human cytoplasm.
Most of the SOD protein produced initially is extracted from animal and plant tissues, but the extraction process is relatively complex, the yield is low, the quality is unstable, the production cost is high, and the problems of immunogenicity and the like exist. Therefore, the production of human superoxide dismutase by genetic engineering techniques has become a new direction for research and development of SOD production in recent years, and the method is an effective way to reduce the cost and obtain non-antigenic human SOD. At present, the production of human superoxide dismutase by genetic engineering techniques is precedent, but still has many problems: mainly comprises the following steps: (1) the expression level of the target protein is not high (2) the purification process is complicated, and the target protein needs to be purified by a series of chromatographic methods such as ion exchange, molecular exclusion and the like.
Elastin-like polypeptides (ELP) are a synthetic, genetically engineered polypeptide polymer with many different forms, mainly (VPGXG) n pentapeptide repeats, and variations such as (KGGVG) n, (VGGVG) n, (GVGVP) n, (LGAGGAG) n, (LGAGGAGVL) n, etc. Currently, the most predominant form of ELP study consists of a series connection of Val-Pro-Gly-Xaa-Gly (vpgxg) n pentapeptide repeat units, where Xaa (x) refers to amino acids other than proline (Pro), typically valine (Val, V), alanine (Ala, a), glycine (Gly, G) leucine (Leu, L) isoleucine (Ile, I), lysine (Lys, K), phenylalanine (Phe, F), histidine (His, H), and several amino acid types, with repeat units n typically varying from 20 to 120. Xaa (X) may be composed of a single amino acid other than Pro in the repeating unit n of 20 to 120, or may be composed of amino acids such as Val, Ala, Gly, Ile, Leu, Lys, Phe, His, etc. in various proportions, but the first 3 and last 1 amino acids in VPGXG are always constant in the pentapeptide repeating unit and only the type or proportion of the 4 th amino acid is changed, and the repeating unit n is usually varied from 20 to 120. The ELP protein alone or together with foreign gene forms fusion protein expression at N-terminal or C-terminal, and has phase transition characteristic, that is, when reaching a certain temperature, the ELP protein or ELP fusion protein can be reversibly changed from solution to condensed state, and when the temperature is reduced to a certain temperature, the ELP or ELP fusion protein can be restored to solution state, when Xaa is hydrophobic group, the phase transition temperature can be reduced, otherwise, the ELP or ELP fusion protein with different phase transition temperature can be designed according to the polarity of Xaa, so that through the reversible change of the condensed state of the ELP, the recombinant protein can be separated by centrifugation, and the simple ITC (inversion transition cycle) non-chromatographic purification (Rodriguez-Cabelo JC, Arias FJ, Rodrio MA. et. Elastin-peptides in Drug Delivery. advanced Drug Delivery. modified recombinant protein) of the recombinant protein can be achieved. In addition, ELP has been shown to enhance the accumulation of different recombinant proteins in plants, which increases recombinant protein accumulation in the endoplasmic reticulum of plants, thereby solving the major limitation of low yield in plant-derived expression systems (Conley AJ, Joensuu JJ, Menassa r.et al.indicatono protein body formation in plant leaves by project-like polypeptidic usages.bmc biology.2009,7(48): 1-18). Fusion proteins of ELPs and SOD have not been reported, and it is unpredictable whether the ELPs can maintain the reversible phase transition characteristics of the ELPs after being fused with SOD, and can be used for expression and purification of SOD and whether the fusion proteins have enzyme activity.
Disclosure of Invention
The invention aims to provide an SOD-ELP fusion protein expressed by escherichia coli and a preparation method thereof, aiming at the defects in the prior art.
It is still another object of the present invention to provide a liposome comprising the SOD-ELP fusion protein.
The purpose of the invention can be realized by the following technical scheme:
a recombinant expression vector is characterized in that an elastin-like polypeptide coding gene is inserted between BamHI enzyme cutting sites and XhoI enzyme cutting sites of a prokaryotic cell expression vector pET-28a, and an SOD coding gene is inserted between NdeI enzyme cutting sites and BamHI enzyme cutting sites; the elastin-like polypeptide is in a pentapeptide repeating unit (VPGXG) n structure in series, the VPGXG is Val-Pro-Gly-Xaa-Gly, and the Xaa is any amino acid except Pro; the number n of the (VPGXG) pentapeptide repeating units in series is 20-120.
The Xaa is preferably one or more of Val, Ala, Gly, Leu, Ile, Lys, Phe and His.
The elastin-like polypeptide is preferably ELP20Or ELP40。
As a further preferred aspect of the present invention, the ELP is20Represents a total of 20 pentapeptide repeat units (VPGXG) in tandem, of which VPGVG has 10, VPGGG6 and VPGAG 4, abbreviated as ELP [ V10G6A4 ]]20 or ELP20The amino acid sequence is shown in SEQ ID NO. 1.
The ELP40Represents a total of 40 pentapeptide repeat units (VPGXG) in tandem, wherein the VPGHG has 33, the VPGVG has 7, and the abbreviation is ELP [ H33V7 ]]40 or ELP40The gene sequence is shown in SEQ ID NO. 2.
The accession number of the SOD coding gene sequence in GenBank is AY 049787. See SEQ ID NO.3
The recombinant expression vector is further preferably prepared by introducing ELP20Or ELP40Inserting the SOD coding gene between BamHI and XhoI enzyme cutting sites of pET-28a, and inserting the SOD coding gene into a plasmid containing ELP20Or ELP40The NdeI and BamHI enzyme cutting sites of the pET-28a expression vector are obtained; alternatively, the ELP was prepared by inserting the SOD encoding gene between the BamHI and XhoI sites of pET-28a40Inserted between NdeI and BamHI cleavage sites of pET-28a expression vector containing SOD.
A genetic engineering bacterium capable of expressing SOD-ELP fusion protein is an escherichia coli containing the recombinant expression vector.
A method for preparing SOD-ELP fusion protein comprises the following steps:
(1) transducing the recombinant expression vector of claim 1 into E.coli expression strain BL21(DE3), and performing IPTG induced expression on the identified positive transformant;
(2) collecting thalli, carrying out ultrasonic crushing, and centrifuging to remove insoluble substances to obtain a soluble cell lysate, namely a crude enzyme solution;
(3) and (4) circularly purifying the crude enzyme solution by three rounds of ITC to obtain the SOD-ELP fusion protein.
Among them, the induction conditions of the present invention are preferably: selecting positive monoclonal bacteria, adding 3mL of common LB culture solution containing 50 mu g/mL of kanamycin to a 15mL test tube, shaking the bacteria at 37 ℃ overnight, then adding 10mL of common LB culture solution containing 50 mu g/mL of kanamycin to a 50mL test tube, adding 0.5mL of bacterial solution, shaking the bacteria at 37 ℃ for 2h-3h, and adding 1mmol/L of IPTG (isopropyl thiogalactoside) to express the bacteria at 37 ℃ for 4h when the OD600 value reaches 0.4-0.6.
The preparation conditions of the crude enzyme solution are preferably as follows: centrifuging the cultured 1L fermentation liquid in a centrifuge at 3000rpm for 20min, collecting bacterial sludge, adding Escherichia coli lysate with three times volume for dissolving for a certain time, performing 1 time of ultrasonic-freeze thawing cycle, namely ultrasonic once (750W, 10min), freezing at-80 ℃, taking out and dissolving, centrifuging at 13000rpm for 20min, collecting supernatant, namely crude enzyme liquid, and placing in a refrigerator at 4 ℃ for later use.
The invention preferably obtains high-purity fusion protein through three rounds of ITC circulation:
a) taking a proper amount of the supernatant of the bacterial liquid, adding 1.5M NaCl, and carrying out ice bath at 4 ℃ for 2 h;
b) centrifuging at 13000rpm and 4 ℃ for 20min, and taking the supernatant;
c) adding 10mg/ml PEG-8000 into the supernatant, and performing water bath at 37 ℃ for 20 minutes;
d) centrifuging at 13000rpm and 37 ℃ for 20min, and discarding the supernatant;
e) the pellet was dissolved in an appropriate amount of PBS for one cycle, three times in total.
The SOD-ELP prepared by the method of the invention40The amino acid sequence of the fusion protein is shown in SEQ ID NO. 4.
The SOD-ELP of the invention40Liposomes of the fusion protein.
SOD-ELP prepared by thin film hydration method40The method of liposomes is as follows: lecithin in membrane material: the molar ratio of cholesterol is 1.5-3: 0.5-1.5: dissolving in chloroform at a certain volume, evaporating under reduced pressure to remove chloroform, forming lipid membrane in round-bottom flask, adding ELP at a certain volume40SOD fusion protein (0.25mg/ml, enzyme activity 8021U/mg, dissolved in PBS pH 6.8), after hydration and ice bath ultrasound, using filter membranes with pore size of 0.45 μm and 0.22 μm to sequentially filter, obtaining liposome with diameter of about 100-200 nm.
Wherein: lecithin: 0.48% -0.96%;
cholesterol: 0.16% -0.48%;
ELP40-SOD：0.025％。
the liposome is preferably prepared by a film method, the average particle size is 120-140 nm, the potential is-15 mV to-25 mV, the protein encapsulation rate is 80.8 +/-3.42 percent, and the activity encapsulation rate is 81.5 +/-2.17 percent. .
Has the advantages that:
the invention compares two SOD-ELP fusion proteins with different molecular weights, namely SOD-ELP20And SOD-ELP40The two fusion proteins have different numbers of pentapeptide repeating units (VPGXG) in series connection and different Xaa in the pentapeptide repeating units (VPGXG), and SOD-ELP is analyzed by comparing protein expression quantity40Has higher protein expression in engineering bacteria.
The invention compares the fusion protein with two different connection modes of carboxyl terminal or amino terminal of elastin-like protein polypeptide, namely SOD-ELP40(SEQ ID NO.4) and ELP40SOD (SEQ ID NO.5), as SOD-ELP by comparative analysis of the specific activity of the purified protein40The fusion protein in the connection mode has higher enzyme activity.
The invention provides an elastin-like protein ELP with a specific sequence40Has good biocompatibility, low immunogenicity, easy degradation, reversible heat phase transformation property and can convert the elastin like ELP40Construction of fusion protein SOD-ELP with superoxide dismutase40Expression, not only simply and rapidly purify the fusion protein through multiple rounds of ITC, but also retain the high enzyme activity and good heat resistance of the superoxide dismutase, and effectively prolong the half-life period of the superoxide dismutase. The recovery rate of the total enzyme activity of the superoxide dismutase is 28% -35%; the average purification multiple of the protein is 4.5, and the method can be used for preparing the recombinant humanized copper-zinc superoxide dismutase with high specific activity on a large scale in industry. The invention is not only suitable for the purification of SOD-ELP fusion protein, but also can be used for the separation and purification of other recombinant proteins.
Drawings
FIG. 1 shows that pET28 a-containing SOD-ELP40Total eggs of positive bacteriaThe electrophoretogram of the white SDS-PAGE,
1,2 are Bovine Serum Albumin (BSA) standard 1 mug, 2 mug, 3 is a sample without IPTG induction, 4,8,9 are SDS-PAGE electrophoretograms of whole bacteria, supernatant or precipitate expressed for 16h at 15 ℃ and 4h at 37 ℃ of 5,6,7,10,11, SOD-ELP40The highest expression level (indicated by the arrow) of (D) was determined to be 10 mg/L.
FIG. 2 shows pET28 a-containing SOD-ELP20SDS-PAGE electrophoresis picture of the total protein of the positive bacteria,
1,2 are Bovine Serum Albumin (BSA) standard 1 mug, 2 mug, 3 is a sample without IPTG induction, 4,8,9 are SDS-PAGE electrophoretograms of whole bacteria, supernatant or precipitate expressed for 16h at 15 ℃ and 4h at 37 ℃ of 5,6,7,10,11, SOD-ELP20The highest expression level (indicated by the arrow) of (D) was determined to be 6 mg/L.
FIG. 3 shows pET28 a-containing ELP40SDS-PAGE (sodium dodecyl sulfate-PAGE) electrophoretogram and ELP (Elephora-superoxide dismutase) of total protein of SOD positive bacteria40SOD protein purification results, ELP indicated by the arrow40SOD fusion proteins.
FIG. 4 three ITC cycle flow diagram
FIG. 5 shows SDS-PAGE of fusion proteins purified by three cycles of ITC cycles, wherein three cycles of ITC cycles of purified SOD-ELP are shown in lanes 1,2, 3, and 440A fusion protein.
FIG. 6SOD-ELP40Fusion protein pH stability Curve
FIG. 7SOD-ELP40Temperature stability curve of fusion protein
FIG. 8 with addition of Cu2+/Zn2+The mixed solution increases SOD-ELP40Time-dependent change curve of fusion protease activity
FIG. 9SOD-ELP40Fusion proteins mimic in vivo half-life
FIG. 10SOD-ELP40Toxicity of fusion proteins to HUVEC cells
FIG. 11SOD-ELP40Measurement of liposome particle size
FIG. 12SOD-ELP40Liposomes mimic half-life in vivo
FIG. 13SOD-ELP40Transdermal rate profile of liposomes
Detailed Description
The terms used in the present invention have the meanings and operation methods generally understood by those skilled in the art unless otherwise specified.
The present invention will be described in further detail with reference to the following examples of concrete embodiments and applications thereof and to the implementation data. It will be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way. (wherein, the SOD-ELP fusion protein in examples 5 to 12 is SOD-ELP40Fusion protein)
Example 1
1 entrusted Nanjing Kinsrui Biotechnology Ltd to synthesize ELP [ H33V7 in advance]The 40 gene (sequence information shown in SEQ ID NO.2) was inserted in advance between BamHI and XhoI of pET28a vector, wherein pET28a-ELP was formed40And (3) a carrier.
2. The Nanjing Kingsley Biotechnology Ltd was entrusted with the synthesis of SOD gene (sequence information shown in SEQ ID NO.3) (Cu, Zn Superoxide Dismutase, Cu, Zn-SOD, EC 1.15.1.1, GenBank: AY049787) and PCR primers (sequence information shown in SEQ ID NO.6 and SEQ ID NO.7), under the PCR conditions of pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 25s, annealing at 56 ℃ for 30s, elongation at 72 ℃ for 25s, cycling for 30 times, elongation at 72 ℃ for 10min, and heat preservation at 10 ℃.
3. The PCR product of SOD gene and pET28a-ELP were separately mixed40The vector was purified by NdeI and BamHI double digestion and kit, and the PCR digestion product and the product containing ELP [ H33V7 ] were ligated to SOD in accordance with the M2200S Quick digestion kit of NEB]40 pET28a-ELP40Connecting double enzyme digestion products of the vector according to the instruction of a kit, introducing DH5 α competence, culturing kanamycin-resistant plate bacterial colonies at 37 ℃, selecting a monoclonal bacterial colony, shaking the bacterial colony at 37 ℃ overnight in 3mL of common LB culture solution containing 50 mu g/mL kanamycin at the final concentration, after identifying the masculinity of the monoclonal bacterial colony by PCR, extracting an expression plasmid by using a plasmid extraction kit, and after identifying the masculinity by enzyme digestion, obtaining the plasmid pET28a-SOD-ELP40。
4. Extracting plasmid, transducing into Escherichia coli expression strain BL21 & DE3, selecting monoclonal bacteria, adding 3mL of common LB culture solution containing kanamycin with final concentration of 50 μ g/mL into 15mL test tube, and shaking at 37 deg.CAnd standing overnight, then adding 10mL of common LB culture solution containing kanamycin with the final concentration of 50 mu g/mL into a 50mL test tube, adding 0.5mL of bacterial solution, shaking the bacterial solution at 37 ℃ for 2h-3h, adding 1mmol/L of IPTG (isopropyl-beta-thiogalactoside) when the OD value reaches 0.4-0.6, shaking the bacterial solution at 37 ℃ for expression for 4h, or firstly cooling the bacterial solution to 15 ℃, and then adding 1mmol/L of IPTG at 15 ℃ for expression for 16 h. Collecting 1mL of expression bacteria, centrifuging at 8000rpm for 5min, adding 100 μ L of 1X electrophoresis buffer, ultrasonic crushing, decocting at 100 deg.C for 10min, centrifuging, and performing SDS-PAGE electrophoresis to identify SOD-ELP in the expression bacteria40The recombinant protein expression molecular weight and expression quantity of the exogenous gene.
BSA is taken as a measurement standard, a GS-900 imager (SH1WBA10698) is used for carrying out gel transmission image scanning, the expression level of a target gene in an SDS-PAGE electrophoresis picture is calculated, and the measurement result shows that the SOD-ELP expressed by pET28a vector is expressed for 4 hours at 37 ℃ in a common LB culture medium by using a common shaking table40The expression level is 10mg/L at most (FIG. 1)
Example 2
1 entrusted Nanjing Kinsrui Biotechnology Ltd to synthesize ELP [ V10G6A4 in advance]20 gene (sequence information shown in SEQ ID NO.1) was inserted between BamHI and XhoI of pET28a vector to form pET28a-ELP20And (3) a carrier.
2. The PCR primers (sequence information is shown in SEQ ID NO.6 and SEQ ID NO.7) of SOD (Cu, Zn Superoxide Dismutase, Cu, Zn-SOD, EC 1.15.1.1, GenBank: AY049787) synthesized by Nanjing Kingsley Biotechnology Limited are consigned, the PCR conditions are pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 25s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 25s, circulation for 30 times, extension at 72 ℃ for 10min and heat preservation at 10 ℃.
3. PCR products of SOD gene and ELP-containing [ V10G6A4 ] synthesized by request]pET28a-ELP of 2020The vector is purified by NdeI and BamHI double enzyme digestion and a kit, the enzyme digestion and the connection are respectively carried out according to the specification of M2200S Quick ligation kit of NEB company, the strain is cultured by introducing DH5 α competence and kanamycin resistance plate bacterial colony at 37 ℃, a monoclonal bacterium colony is selected to shake at 37 ℃ in 3mL of common LB culture solution containing 50 mu g/mL of kanamycin at the final concentration overnight, after the PCR identification of the monoclone bacterium, a plasmid extraction kit is used for extracting expression plasmid, and after the enzyme digestion identification is positive, the plasmid is pET28a-SOD-ELP20。
4. Extracting plasmids, transducing the plasmids into escherichia coli expression bacteria BL21 & DE3 competence, selecting monoclonal bacteria, adding 3mL of common LB culture solution containing 50 mu g/mL of kanamycin to a 15mL test tube, shaking the bacteria at 37 ℃ overnight, then adding 10mL of common LB culture solution containing 50 mu g/mL of kanamycin to a 50mL test tube, adding 0.5mL of bacterial liquid, shaking the bacteria at 37 ℃ for 2h to 3h, adding 1mmol/L of IPTG to express the bacteria at 37 ℃ for 4h when the OD value reaches 0.4 to 0.6, or firstly cooling the bacterial liquid to 15 ℃, and then adding 1mmol/L of IPTG to express the bacteria at 15 ℃ for 16 h. Collecting 1mL of expression bacteria, centrifuging at 8000rpm for 5min, adding 100 μ L of 1X electrophoresis buffer, ultrasonic crushing, decocting at 100 deg.C for 10min, centrifuging, and performing SDS-PAGE electrophoresis to identify SOD-ELP in the expression bacteria20The recombinant protein expression molecular weight and expression quantity of the exogenous gene.
Taking BSA as a measurement standard, performing gel transmission image scanning by a GS-900 imager (SH1WBA10698), measuring and calculating the expression level of a target gene in an SDS-PAGE electrophoretogram, and determining results show that the expression is performed for 4 hours at 37 ℃ in a common LB culture medium by using a common shaking table, and ELP expressed by a pET28a vector20The expression level of SOD fusion protein (FIG. 2) was up to 6 mg/L.
Thus, the elastin-like polypeptide ELP is preferred40Form fusion protein with SOD.
Example 3 fusion protein ELP40Construction and expression of SOD
For comparing two fusion proteins with different connection modes of SOD and elastin-like polypeptide, i.e. SOD-ELP40And ELP40The difference of SOD expression level, and the construction of the vector pET28a-ELP40-SOD。
1. The PCR primers (sequence information is shown in SEQ ID NO.6 and SEQ ID NO.7) of SOD (Cu, Zn Superoxide Dismutase, Cu, Zn-SOD, EC 1.15.1.1, GenBank: AY049787) synthesized by Nanjing Kingsley Biotechnology Limited are consigned, the PCR conditions are pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 25s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 25s, circulation for 30 times, extension at 72 ℃ for 10min and heat preservation at 10 ℃. The PCR product was inserted between BamHI and XhoI of pET28a vector to form pET28a-SOD vector.
2. Entrusted Nanjing Kinsrui Biotechnology LtdSynthesis of ELP [ H33V7 ] in advance]40 gene (sequence information is shown in SEQ ID NO. 2). Entrusted Nanjing King Shirui Biotechnology Ltd to synthesize ELP40The PCR primers (sequence information shown in SEQ ID NO.8 and SEQ ID NO.9) of (1) are subjected to pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 25s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 25s, circulation for 30 times, extension at 72 ℃ for 10min and heat preservation at 10 ℃.
3. Respectively subjecting ELP to40The PCR product of the gene and the synthesized pET28a-SOD vector are purified by NdeI and BamHI double enzyme digestion and kit, the enzyme digestion and connection are respectively carried out according to the M2200S Quick ligation kit of NEB company, the competence is led into DH5 α, the colony is cultured by a kanamycin-resistant plate at 37 ℃, the monoclonal colony is selected to shake bacteria at 37 ℃ in 3mL of common LB culture solution containing kanamycin with the final concentration of 50 mug/mL for overnight, after the PCR identification of the monoclone bacteria, the expression plasmid is extracted by using a plasmid extraction kit, and after the enzyme digestion identification is positive, the plasmid is pET28a-ELP40-SOD。
4. Extracting plasmids, transducing the plasmids into escherichia coli expression bacteria BL21 & DE3 competence, selecting monoclonal bacteria, adding 3mL of common LB culture solution containing 50 mu g/mL of kanamycin to a 15mL test tube, shaking the bacteria at 37 ℃ overnight, then adding 10mL of common LB culture solution containing 50 mu g/mL of kanamycin to a 50mL test tube, adding 0.5mL of bacterial liquid, shaking the bacteria at 37 ℃ for 2h to 3h, adding 1mmol/L of IPTG to express the bacteria at 37 ℃ for 4h when the OD value reaches 0.4 to 0.6, or firstly cooling the bacterial liquid to 15 ℃, and then adding 1mmol/L of IPTG to express the bacteria at 15 ℃ for 16 h. Collecting 1mL of expression bacteria, centrifuging at 8000rpm for 5min, adding 100 μ L of 1X electrophoresis buffer, ultrasonic crushing, decocting at 100 deg.C for 10min, centrifuging, and performing SDS-PAGE electrophoresis to identify ELP in the expression bacteria40-recombinant protein expression molecular weight and expression level of SOD foreign gene. (see FIG. 3)
Example 4 purification of fusion proteins
(I) bacterial liquid culture
200 mul of the gene carrying the target protein (plasmid pET28 a-SOD-ELP) is taken40) The Escherichia coli (E.coli) (frozen at-80 ℃) was inoculated into 500ml of LB medium, shake-cultured at 37 ℃ and 140rpm for 20 hours, inoculated into 1L of LB medium at an inoculum size of 2%, cultured at 37 ℃ until OD600 reached 0.6, and added with 1mmol/L of IPTG 3And (4) shaking the bacteria at 7 ℃ to express for 4h, and collecting the fermentation liquor. Centrifuging the cultured fermentation liquid in a centrifuge at 3000rpm for 20min, collecting bacterial sludge, adding Escherichia coli lysate with three times volume for dissolving for a certain time, performing ultrasonic-freeze thawing cycle for 1 time, namely performing ultrasonic once (750W, 10min), freezing at-80 deg.C, taking out for dissolving, centrifuging at 13000rpm for 20min, respectively collecting supernatant and precipitate, and storing in a refrigerator at-80 deg.C. The supernatant is the crude enzyme solution.
(II) ITC cycle purification of proteins
Optimization of ITC purification protocol: (see attached FIG. 4)
Final purification scheme:
1. taking a proper amount of the supernatant of the bacterial liquid, adding 1.5M NaCl, and carrying out ice bath at 4 ℃ for 1 h;
2. centrifuging at 13000rpm and 4 ℃ for 20min, and taking the supernatant;
3. adding 20mg/ml PEG-8000 into the supernatant, and water-bathing at 37 deg.C for 20 min;
4. centrifuging at 13000rpm and 37 ℃ for 20min, and discarding the supernatant;
the pellet was dissolved in an appropriate amount of PBS for one cycle, three times in total.
(III) enzyme activity determination: SOD enzyme activity unit definition measured by a modified pyrogallol autoxidation method (325nm method): under certain conditions, the enzyme quantity of the lml reaction solution for controlling the autoxidation rate of pyrogallol at the wavelength of 325nm to 50 percent per minute is determined as an activity unit, and the measured data calculates the enzyme activity according to the following formula:
a1: the auto-oxidation rate of pyrogallol, OD/min;
a2: adding SOD enzyme solution to obtain autoxidation rate OD/min;
a, the dosage of pyrogallol and ml;
b, the dosage of SOD enzyme solution and ml.
The protein concentration of the purified protein solution was measured by BCA kit method (THERMO) using BSA as a measurement standard. The enzyme specific activity (U/mg) was calculated.
SDS-PAGE was performed on the purified protein solution, and the band was subjected to the calculation of the purity of the protein electrophoresis band by Tanon3500 gel imaging system using Imagestudio software, and the activity of the fusion protein was measured by pyrogallol method, as shown in the following tables and attached FIGS. 3 and 5
As shown in the above table, the SOD-ELP is used40The fusion protein formed by the connection mode has higher enzyme specific activity, and SOD-ELP is used in the subsequent examples40Fusion protein formed by connecting mode.
Example 5SOD-ELP40pH stability of the fusion protein:
respectively adjusting the pH of 10mMPBS to 3.0, 5.0, 7.0, 7.4, 8.0, 9.0 and 11.0 by using dilute NaOH or HCl solution, ten-fold diluting the purified enzyme solution by using PBS with different pH values, setting 3 parallel pH gradients, measuring the enzyme activity of each group after incubating for 2h in a water bath kettle at 25 ℃, and taking the enzyme solution diluted by PBS with pH value of 7.4 as the initial enzyme activity to obtain a relation curve of the enzyme activity retention rate and the solution pH value, wherein the result is shown in figure 6.
As can be seen from FIG. 6, SOD-ELP40The enzyme activity can be kept above 90% in the range of pH 5-8, which shows that the pH in the range has little influence on the enzyme activity and the protein is stable. When the pH is higher>8 or pH<5, the enzyme activity is reduced rapidly.
Example 6SOD-ELP40Thermal stability of fusion proteins
Taking a proper amount of the purified enzyme solution, respectively carrying out water bath at 25 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ for 30min, cooling to room temperature, measuring the enzyme activity of each group, setting three temperature gradients in parallel, taking the enzyme solution in the water bath at 25 ℃ for 30min as the initial enzyme activity, and obtaining a relation curve of the enzyme activity retention rate and the temperature, wherein the result is shown in figure 7.
As can be seen from FIG. 7, when the temperature is lower than 60 ℃, the activity of ELP-SOD is more stable, and after the temperature exceeds 60 ℃, the enzyme activity is sharply reduced, and when the temperature is 80 ℃, the ELP-SOD fusion protein is almost completely inactivated.
Example 7Cu2+And Zn2+Addition of (2)Increase SOD-ELP40Enzyme activity
Purifying SOD-ELP40Protein and optimal final concentration of Cu2+/Zn2+(Cu2+Zn at a concentration of 0.9mM2+Concentration of 0.45mM), the final concentration of the protein is 0.04mg/ml, three groups are arranged in parallel, samples are taken when the reaction is carried out for 1h, 2h, 4h, 10h, 16h and 24h at 4 ℃, and the SOD enzyme activity is determined by a pyrogallol method, the result is shown in figure 9, the enzyme activity is rapidly increased within 1 hour, and the enzyme activity is not obviously increased in each hour, so that the reaction is carried out for 1 hour at 4 ℃, and the enzyme activity can reach 11444.6U/mg at the highest. Then, 5ml of the enzyme protein solution was put into a dialysis bag (8000-2+The enzyme activity after dialysis is 80-88% of the enzyme activity before dialysis and reaches above 9155.7U/mg.
Example 8SOD-ELP40Mimicking in vivo half-life of fusion proteins
Taking SPF-grade SD rat femoral artery blood (heparin sodium treatment test tube wall), standing at room temperature for 0.5h, centrifuging at 3000rpm for 10min to obtain rat plasma, and performing in vivo half-life simulation experiment with fresh rat plasma.
The experiment is divided into three groups, wherein the experiment group is SOD-ELP40(0.25mg/ml), the positive control group was purified SOD protein (obtained from Hangzhou Niulon Biotechnology Co., Ltd., prepared as 0.25mg/ml protein solution), and the blank control group was PBS solution. In the experiment, 50% of rat serum, 40% of PBS and 10% of corresponding protein (the volume of the negative control group is supplemented by PBS) in the volume of each group of sample liquid are respectively added, uniformly mixed and then placed in a constant-temperature incubator at 37 ℃ for culturing for 0, 2, 4, 6, 8, 10, 20, 32 and 48 hours, respectively sampling and determining enzyme activity, the experimental result is shown in figure 9, and the calculated half-life period is shown in table 1.
t1/2 half-life (time of continuous operation, h) for which the enzyme activity decreases to half of the initial enzyme activity);
e0: initial enzyme activity;
e: enzyme activity at t.
TABLE 1 simulated in vivo half-life calculation results
Note: p <0.05 compared to SOD
The results of the simulated in vivo half-life calculations for each group are shown in Table 1, SOD-ELP40The half-life period of the fusion protein is 59h, and the fusion protein is stable and long-acting antioxidant enzyme. As can be seen from FIG. 9, the SOD-ELP increased with the incubation time40The fusion protein and SOD pure product have good stability, for example, after incubation at 37 ℃ for 32h, the SOD enzyme activity is only 78.4% of the original enzyme activity, while the SOD-ELP40The fusion protein group is 84.1%, and therefore, the addition of the ELP in the fusion protein not only simplifies the protein purification process, but also effectively retains the enzyme activity and prolongs the simulated half-life period of the enzyme.
Example 9SOD-ELP40Fusion protein cytotoxicity assays
1) Human Umbilical Vein Endothelial Cells (HUVEC) were collected and prepared to 5X 10 using RPMI Medium Modified Medium containing 10% fetal bovine serum4Cell suspension per ml. The cell suspension was seeded in 96-well plates at 100. mu.L per well at 37 ℃ in 5% CO2Culturing for 24h under the condition;
2) the stock culture was aspirated, washed once with PBS, and 100. mu.L of ELP-SOD protease solution diluted with a medium (containing 2.5% fetal bovine serum) was added to each well. Four concentration gradients of 1.5%, 3%, 6%, 12% (corresponding to enzyme activities of 10.5, 21, 42, 84U/ml) were set, and nine-well replicates were set for each set at 37 deg.C with 5% CO2Culturing for 24h, 48h and 72h under the condition respectively. Adding 100 μ L of RPMI Medium Modified culture Medium (containing 2.5% fetal bovine serum) into each well of control group;
3) after the culture is finished, taking out the culture medium from the incubator, adding 10 mu L of prepared MTT solution (5mg/mL) into each hole, and putting the culture medium into a cell incubator to continue incubation for 4 hours;
4) removing culture solution in the holes by suction, adding 100 mu L DMSO into each hole, slightly beating the frame of the 96-hole plate to fully dissolve the culture solution, and measuring the absorbance at 490nm by using an enzyme-labeling instrument;
5) the growth inhibition rate (IC%) was calculated according to the following formula
The results of the experiment are shown in FIG. 10.
Example 10SOD-ELP40Preparation of fusion protein liposome
SOD-ELP prepared by thin film hydration method40Liposomes. Dissolving lecithin and cholesterol (mass ratio of 2:1) with total membrane concentration of 9.6mg/ml in chloroform at 30 deg.C, removing chloroform by rotary evaporation to form a uniform lipid membrane on the inner wall of eggplant-shaped bottle, and adding SOD-ELP at a certain volume40The fusion protein (0.25mg/ml, dissolved in PBS (pH6.8)) was hydrolyzed in a water bath at 37 ℃ for a certain period of time, the prepared liposome was sonicated with a sonicator under 260W for 10min, and then the liposome was sequentially filtered through a 0.45 μm filter and a 0.22 μm filter, and aseptically stored at 4 ℃ for further use. SOD-ELP measured by ZS90 (Malvern, UK) potentiometer40The mean particle size of the liposomes was 125nm, the potential was-19.7 mV, and PDI was 0.137, indicating that the liposomes had good homogeneity (see FIG. 11).
1. Calculation of enzyme Activity encapsulation efficiency
Taking a certain volume of SOD-ELP40The liposomes were centrifuged at 5000g for 20min in a 100KD ultrafiltration tube (Amicon, Ultra-4), the filtrate was collected and the volume of the filtrate was recorded, the filtrate was the free fusion protein solution, and the enzymatic activity of the filtrate was measured. SOD-ELP40Breaking membrane with 1% Triton x-100, diluting the membrane-broken liposome, and measuring SOD-ELP40Total activity of the enzyme(s).
A:SOD-ELP40Total activity of (1), U/ml;
a, SOD-ELP added during ultrafiltration40Liposome volume, ml;
b, the activity of the filtrate obtained by ultrafiltration is U/ml;
b, the volume of the filtrate obtained by ultrafiltration, ml.
2 determination of protein Encapsulated Rate
Taking a certain volume of SOD-ELP40The liposomes were centrifuged at 5000g for 20min in a 100KD ultrafiltration tube (Amicon, Ultra-4), the filtrate was collected and the volume of the filtrate was recorded, the filtrate was the free fusion protein, and the protein concentration of the filtrate was determined experimentally using BCA kit (THERMO).
a, SOD-ELP added during ultrafiltration40Liposome volume, ml;
C1：SOD-ELP40liposome Total protein concentration, mg/ml;
b, the volume of the filtrate obtained by ultrafiltration is ml;
c2: protein concentration of filtrate, ml.
SOD-ELP40The encapsulation efficiency of the liposome is high, wherein the protein encapsulation efficiency is 80.8 +/-3.42 percent, and the active encapsulation efficiency is 81.5 +/-2.17 percent.
Example 11SOD-ELP40Simulated in vivo half-life of liposomes prepared from fusion proteins
Taking SPF-grade SD rat femoral artery blood (heparin sodium treatment test tube wall), standing at room temperature for 0.5h, centrifuging at 3000rpm for 10min to obtain rat plasma, and performing in vivo half-life simulation experiment with the rat plasma.
The experiment is divided into three groups, wherein the experiment group is SOD-ELP40The liposome, positive control group was SOD liposome (protein from Hangzhou Niulon Biotechnology Co., Ltd.) prepared by the same method, and blank control was PBS solution. In the experiment, 50% of rat serum, 40% of PBS and 10% of corresponding liposome (the volume of the negative control group is supplemented by PBS) in the volume of each group of sample liquid are respectively added, uniformly mixed and then placed in a constant-temperature incubator at 37 ℃ for culturing for 0, 2, 4, 6, 8, 10, 20, 32 and 48 hours, respectively sampling and determining enzyme activity, the experimental result is shown in figure 12, and the calculated half-life period is shown in table 2. Half-life calculation formula:
t1/2 half-life (time of continuous operation, h) for which the enzyme activity decreases to half of the initial enzyme activity);
e0: initial enzyme activity;
e: enzyme activity at t.
TABLE 2 simulated in vivo half-life calculation results
Note: p <0.05 compared to SOD liposomes
The results of the simulated in vivo half-life calculations for each group are shown in Table 2, SOD-ELP40The fusion protein can effectively prolong the half-life period in blood plasma after being prepared into liposome. As can be seen from FIG. 11, although the incubation time increased, the SOD-ELP was added40The liposome prepared from fusion protein or SOD has good stability, but SOD-ELP40The half-life of the fusion protein liposome reaches 73.9h, which is much higher than that of SOD liposome, and the two groups have obvious difference (P)<0.05)。
Example 12 transdermal Effect of ELP-SOD liposomes
1 preparation of mouse Ex vivo skin
Dislocation of cervical vertebrae of 6 healthy spf male ICR mice, depilatory treatment of abdominal skin of the mice with depilatory, cleaning of the depilatory remained on the abdominal of the mice with physiological saline, peeling of the abdominal skin of the mice, scraping of tissues on the inner side of the in vitro skin with forceps, and soaking of the in vitro skin with 10mm of PBS for 10 min.
2 in vitro transdermal test
TP-6 transdermal diffusion instrument is adopted to carry out in-vitro release experiment, and the transdermal efficiency of carrying the ELP-SOD by the ELP-SOD liposome is inspected by using a Franz diffusion cell. The Franz diffusion cell is formed by joining an upper ground glass container and a lower ground glass container, the medicine release area is 1.13cm2, the volume of the receiving cell is 15ml, the skin is clamped between the two glass containers, the skin on the outer side of the abdomen faces upwards, and the skin is fixed by a stainless steel clamp.
The Franz receiving cell was filled with 15ml PBS, a magnetic rotor was added, the mouse skin was fixed between the receiving and feeding chambers (inner skin in contact with PBS,no air bubble remained between the isolated skin and PBS), adding 0.5ml SOD-ELP respectively40Liposomes or SOD-ELP 0.25mg/ml40Protein solution, the temperature of the water pool was adjusted to 37 ℃, the Franz diffusion cell was placed in the water pool, a magnetic stirrer was set at 350rpm, approximately 1ml of sample was taken every 2 hours (at the time of sampling, a dropper was required to extend from the sampling port into the interior of the diffusion cell and no air bubbles were generated) and then the volume was made up with an equal amount of PBS solution for 24 hours, with 6 replicates per group.
The SOD activity of the two groups of samples is measured, and the transmittance of the ELP-SOD liposome is calculated according to the following formula.
Transmittance of a × b/c × d
a: total Franz diffusion cell volume; ml of
b: sampling the measured SOD activity of the sample; u/ml
c: the supply chamber adds a sample volume; ml of
d: SOD activity of the sample before sample adding; u/ml
Results as shown in fig. 12, the prepared liposomes rapidly diffused transdermally into the sink, reaching up to 29.93% at 8 hours, compared to the fusion protein. Later, although there was a decrease, it was still nearly 20% higher than the fusion protein at a minimum of 24 h.
Sequence listing
<110> Nanjing university of science and technology
Point bucket Gene science and technology (Nanjing) Ltd
<120> SOD-ELP fusion protein and preparation method thereof
<160>9
<170>SIPOSequenceListing 1.0
<210>1
<211>100
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<213> Artificial Sequence (Artificial Sequence)
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Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val
1 5 10 15
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
20 25 30
Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly
35 40 45
Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala
50 55 60
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
65 70 75 80
Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val
85 90 95
Pro Gly Val Gly
100
<210>2
<211>606
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
gttccgggcc acggtgtccc aggtcacggc gtaccgggcc acggtgttcc tggtcacggc 60
gtgccgggcg tgggtgttcc gggccacggt gtcccaggtc acggcgtacc gggccacggt 120
gttcctggtc acggcgtgcc gggcgtgggt gttccgggcc acggtgtccc aggtcacggc 180
gtaccgggcc acggtgttcc tggtcacggc gtgccgggcg tgggtgttcc ggttcacggt 240
gtcccaggtc acggcgtacc gggccacggt gttcctggtc acggcgtgcc gggcgtgggt 300
gttccgggcc acggtgtccc aggtcacggc gtaccgggcc acggtgttcc tggtcacggc 360
gtgccgggcg tgggtgttcc gggccacggt gtcccaggtc acggcgtacc gggccacggt 420
gttcctggtc acggcgtgcc gggcgtgggt gttccgggcc acggtgtccc aggtcacggc 480
gtaccgggcc acggtgttcc tggtcacggc gtgccgggcg tgggtgttcc gggccacggt 540
gtcccaggtc acggcgtacc gggccacggt gttcctggtc acggcgtgcc gggctggccg 600
gtcgac 606
<210>3
<211>624
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<213> Artificial Sequence (Artificial Sequence)
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atggcgacga aggccgtggt tccgggccac ggtgtcccag gtcacggcgt accgggccac 60
ggtgttcctg gtcacggcgt gccgggcgtg ggtgttccgg gccacggtgt cccaggtcac 120
ggcgtaccgg gccacggtgt tcctggtcac ggcgtgccgg gcgtgggtgt tccgggccac 180
ggtgtcccag gtcacggcgt accgggccac ggtgttcctg gtcacggcgt gccgggcgtg 240
ggtgttccgg ttcacggtgt cccaggtcac ggcgtaccgg gccacggtgt tcctggtcac 300
ggcgtgccgg gcgtgggtgt tccgggccac ggtgtcccag gtcacggcgt accgggccac 360
ggtgttcctg gtcacggcgt gccgggcgtg ggtgttccgg gccacggtgt cccaggtcac 420
ggcgtaccgg gccacggtgt tcctggtcac ggcgtgccgg gcgtgggtgt tccgggccac 480
ggtgtcccag gtcacggcgt accgggccac ggtgttcctg gtcacggcgt gccgggcgtg 540
ggtgttccgg gccacggtgt cccaggtcac ggcgtaccgg gccacggtgt tcctggtcac 600
ggcgtgccgg gctggccggt cgac 624
<210>4
<211>360
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<213> Artificial Sequence (Artificial Sequence)
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His Met Met Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro
1 5 10 15
Val Gln Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val
20 25 30
Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe
35 40 45
His Val His Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly
50 55 60
Pro His Phe Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu
65 70 75 80
Glu Arg His Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly
85 90 95
Val Ala Asp Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp
100 105 110
His Cys Ile Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp
115 120 125
Leu Gly Lys Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly
130 135 140
Ser Arg Leu Ala Cys Gly Val Ile Gly Ile Ala Gln Gly Ser Val Pro
145 150 155 160
Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly
165 170 175
His Gly Val Pro Gly Val Gly Val Pro GlyHis Gly Val Pro Gly His
180 185 190
Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly Val Gly
195 200 205
Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val
210 215 220
Pro Gly His Gly Val Pro Gly Val Gly Val Pro Val His Gly Val Pro
225 230 235 240
Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly
245 250 255
Val Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His
260 265 270
Gly Val Pro Gly His Gly Val Pro Gly Val Gly Val Pro Gly His Gly
275 280 285
Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val
290 295 300
Pro Gly Val Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro
305 310 315 320
Gly His Gly Val Pro Gly His Gly Val Pro Gly Val Gly Val Pro Gly
325 330 335
His Gly Val Pro Gly His Gly Val Pro Gly His GlyVal Pro Gly His
340 345 350
Gly Val Pro Gly Trp Pro Val Asp
355 360
<210>5
<211>360
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
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His Met Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His
1 5 10 15
Gly Val Pro Gly His Gly Val Pro Gly Val Gly Val Pro Gly His Gly
20 25 30
Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val
35 40 45
Pro Gly Val Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro
50 55 60
Gly His Gly Val Pro Gly His Gly Val Pro Gly Val Gly Val Pro Val
65 70 75 80
His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His
85 90 95
Gly Val Pro Gly Val Gly Val Pro Gly His Gly Val Pro Gly His Gly
100 105 110
Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly Val Gly Val
115 120 125
Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro
130 135 140
Gly His Gly Val Pro Gly Val Gly Val Pro Gly His Gly Val Pro Gly
145 150 155 160
His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly Val
165 170 175
Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly
180 185 190
Val Pro Gly His Gly Val Pro Gly Trp Pro Val Asp Gly Ser Met Ala
195 200 205
Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly Ile
210 215 220
Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp Gly
225 230 235 240
Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu
245 250 255
Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe Asn
260 265 270
ProLeu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His Val
275 280 285
Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp Val
290 295 300
Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile Ile
305 310 315 320
Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys Gly
325 330 335
Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu Ala
340 345 350
Cys Gly Val Ile Gly Ile Ala Gln
355 360
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<213> Artificial Sequence (Artificial Sequence)
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atggcgacga aggccgtggt tc 22
<210>7
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<213> Artificial Sequence (Artificial Sequence)
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gtcgaccggc cagcccggca cg 22
<210>8
<211>22
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<213> Artificial Sequence (Artificial Sequence)
<400>8
gttccgggcc acggtgtccc ag 22
<210>9
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
gtcgaccggc cagcccggca cg 22
Claims (10)
1. A recombinant expression vector is characterized in that an elastin-like polypeptide coding gene is inserted between BamHI enzyme cutting sites and XhoI enzyme cutting sites of a prokaryotic cell expression vector pET-28a, and an SOD coding gene is inserted between NdeI enzyme cutting sites and BamHI enzyme cutting sites; the elastin-like polypeptide is in a pentapeptide repeating unit (VPGXG) n structure in series, the VPGXG is Val-Pro-Gly-Xaa-Gly, and the Xaa is any amino acid except Pro; the number n of the (VPGXG) pentapeptide repeating units in series is 20-120.
2. The recombinant expression vector according to claim 1, wherein Xaa is selected from one or more of Val, Ala, Gly, Leu, Ile, Lys, Phe, His.
3. The recombinant expression vector of claim 1, wherein the elastin-like polypeptide is selected from the group consisting of ELP20Or ELP40(ii) a The ELP20Represents a total of 20 pentapeptide repeat units (VPGXG) in tandem, of which VPGVG has 10, VPGGG6 and VPGAG 4, abbreviated as ELP [ V10G6A4 ]]20 or ELP20The amino acid sequence is shown as SEQ ID NO. 1; the ELP40Represents a total of 40 pentapeptide repeat units (VPGXG) in tandem, wherein the VPGHG has 33, the VPGVG has 7, and the abbreviation is ELP [ H33V7 ]]40 or ELP40The gene sequence is shown in SEQ ID NO. 2.
4. The recombinant expression vector according to claim 1, wherein the SOD encoding gene sequence has an accession number AY049787 in GenBank.
5. The recombinant expression vector according to any one of claims 1 to 4, characterized in that it is produced by subjecting ELP20Or ELP40Inserting the SOD coding gene between BamHI and XhoI enzyme cutting sites of pET-28a, and inserting the SOD coding gene into a plasmid containing ELP20Or ELP40The NdeI and BamHI enzyme cutting sites of the pET-28a expression vector are obtained; or by inserting SOD encoding gene between BamHI and XhoI cleavage sites of pET-28a and then ELP40Insert pET-28 containing SODa between NdeI and BamHI cleavage sites of the expression vector.
6. A genetically engineered bacterium capable of expressing SOD and elastin-like peptide to form fusion protein, characterized in that the genetically engineered bacterium is Escherichia coli containing the recombinant expression vector of any one of claims 1-5.
7. A method for preparing SOD-ELP fusion protein is characterized by comprising the following steps:
(1) transducing the recombinant expression vector of claim 1 into E.coli expression strain BL21(DE3), and performing IPTG induced expression on the identified positive transformant;
(2) collecting thalli, adding lysis solution, carrying out ultrasonic crushing and freeze thawing, and centrifuging to remove insoluble substances to obtain soluble cell lysis solution, namely crude enzyme solution;
(3) and (4) circularly purifying the crude enzyme solution by three rounds of ITC to obtain the SOD-ELP fusion protein.
8. The method of claim 7, wherein the three-cycle ITC purification process comprises:
a) taking a proper amount of the supernatant of the bacterial liquid, adding 1.5M NaCl, and carrying out ice bath at 4 ℃ for 2 h;
b) centrifuging at 13000rpm and 4 ℃ for 20min, and taking the supernatant;
c) adding 10mg/ml PEG-8000 into the supernatant, and performing water bath at 37 ℃ for 20 minutes;
d) centrifuging at 13000rpm and 37 ℃ for 20min, and discarding the supernatant;
e) the pellet was dissolved in an appropriate amount of PBS for one cycle, three times in total.
9. The SOD-ELP fusion protein prepared by the method of claim 7, wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO. 4.
10. A liposome comprising the SOD-ELP fusion protein of claim 9; preferably, the liposome is prepared by a film method, the particle size of the liposome is 120-140 nm, and the potential is-15 mV to-25 mV.
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