CN110452897B

CN110452897B - Antarctic yellow loofah CPD (cytochrome oxidase inhibitor) photorepair enzyme as well as preparation method and application thereof

Info

Publication number: CN110452897B
Application number: CN201910750009.7A
Authority: CN
Inventors: 张鹏英; 刘红伟; 刘胜浩; 陈靠山; 王会娟; 徐新慧
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2019-08-14
Filing date: 2019-08-14
Publication date: 2022-06-03
Anticipated expiration: 2039-08-14
Also published as: CN110452897A

Abstract

The invention relates to a Antarctic yellow loofah CPD (cytochrome oxydase D) photorepair enzyme as well as a preparation method and application thereof. The amino acid sequence of the Antarctic yellow loofah CPD photolyase is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO. 2. According to the invention, the CPD light repair enzyme gene is found in Antarctic yellow loofah moss for the first time, and is successfully cloned into an expression vector, so that a large amount of the CPD light repair enzyme of Antarctic yellow loofah moss is obtained, and the CPD light repair enzyme of Antarctic yellow loofah moss is expected to be applied to related fields such as cosmetics, biomedicine and the like, so that the disease caused by ultraviolet rays can be actively repaired, and the problem that skin cells cannot be repaired in time after being damaged due to the lack of DNA light repair enzyme related protein in a human body is solved.

Description

Antarctic yellow loofah CPD (cytochrome oxidase inhibitor) photorepair enzyme as well as preparation method and application thereof

Technical Field

The invention relates to a Antarctic yellow loofah CPD (cytochrome oxydase D) photorepair enzyme as well as a preparation method and application thereof, belonging to the technical field of biology.

Background

Ultraviolet rays can be classified into UVA (ultraviolet A, wavelength 320-400 nm, long wave), UVB (wavelength 280-320 nm, medium wave), and UVC (wavelength 100-280 nm, short wave). Sunlight is the most important source of natural ultraviolet radiation, and plants, animals and humans on earth cannot live if all the ultraviolet radiation of the solar radiation reaches the ground. Although the ozone layer absorbs UVC from solar radiation in large amounts, the remaining ultraviolet light (UVA and UVB) from intense sunlight can still damage cellular DNA. Four base pairs in an organism, UVB, have strong absorption, and therefore UVB can induce adjacent pyrimidine bases in the same strand of DNA to produce pyrimidine dimers, forming two photoproducts: the first is CPD photoproducts, which can cause temporary or short-term arrest in the cell cycle; the second is a 6-4 photoproduct, which can mutate or kill the cell. The light-induced damage changes the space structure of the DNA, and blocks the processes of DNA replication and transcription, thereby influencing the function of the protein. The light repair function is an important damage repair mode of DNA, and the light repair enzyme plays a key role in the DNA ultraviolet damage repair process, and can break sigma bonds between C5-C5 and C6-C6 of CPD to recover pyrimidine dimers into pyrimidine monomers, thereby playing a repair role. The specific repair process can be divided into the following two steps:

recognition of dimers by photorepair enzymes: DNA photorepair enzyme, a "structure-specific DNA binding protein", has specificity determined by the DNA backbone structure of the binding site. The structure of the dimer generated by adjacent thymine on the DNA chain is mainly cis-syn type, and the structure of the dimer is less trans-syn type due to ultraviolet radiation. The specificity of the combination of the DNA light repair enzyme and cis-syn type cyclobutane pyrimidine dipolymer is very strong. Not only does the pyrimidine dimer itself determine its binding to DNA photorepair enzymes, but the entire interaction of the bases attached to the same strand, and even the base pairs in opposite positions on the complementary strand, contributes.

The photolyase catalyzes the dimer cleavage: the process of cracking the dimer by using the photorepair enzyme involves two aspects of electron transfer and energy transfer: electron transfer, wherein the excited reduced flavin can spontaneously transfer an electron to the pyrimidine dimer; energy transfer, one prosthetic group (folic acid or deazaflavin) for light absorption and another prosthetic group (flavin) for photocatalysis. The light reactivation reaction is initiated by absorbing a blue photon, and the whole reaction can be divided into 5 steps: the light trapping prosthetic group MTHF or 8-HDF absorbs one photon. If light is directly absorbed by flavin, the quantum yield of the photorepair enzyme is generally 0.5-0.9, while 8-HDF absorbs photons, which is close to 1.② the energy of the MTHF or 8-HDF in the excited state is transferred to FADH-through dipole-dipole interaction. Energy transfer between prosthetic groups is not the rate-limiting step in the photorepair reaction. ③ the excited singlet state FADH-spontaneously transfers an electron to the substrate CPD to form the free radical FADH-. And fourthly, breaking two sigma bonds of C5-C5 and C6-C6 in the CPD which receives an electron to generate a pyrimidine monomer and a corresponding anion. Fifthly, electrons on pyrimidine anions return to FADH to regenerate active form FADH-, DNA recovers the double helix structure, and the light repair enzyme is dissociated from the repaired DNA to complete the light repair process.

DNA is a carrier of genetic material of organisms and is of great importance in the research of life sciences. Although basic research on DNA repair is focused on Escherichia coli and yeast, these results are of great significance to the study of human diseases. Through experiments, many researchers at home and abroad find that certain animal cells can form thymine dimers like bacteria after being irradiated by ultraviolet rays and then can be excised, so that DNA damage repair is realized. This would indicate that DNA repair would have immeasurable significance for the study of human diseases, especially cancer, and the study of DNA uv repair has become an important breakthrough in the treatment of skin cancer. The ultraviolet ray damage to skin is mainly reflected in the gene damage, and because of the lack of DNA (deoxyribonucleic acid) photorepair enzyme related protein in a human body, skin cells cannot be repaired in time after being damaged, so that various problems, skin inflammation, immunosuppression, aging acceleration, even canceration and the like occur in succession.

The DNA photorepair enzyme can specifically and effectively restore the connected dimer into a monomer structure and reduce the normal state of DNA. In recent years, with the improvement of the protection level of people, ultraviolet protection work is greatly emphasized, and meanwhile, sunscreen cream is updated and updated, and is gradually upgraded from simple physical protection to chemical repair. The traditional sunscreen cream isolates the skin from the outside by the basic physical principles of reflection, scattering and the like of ultraviolet rays, and the protection effect played by the mode is extremely limited. For those skin cells that have been damaged by uv light, improvement has also been achieved by chemical repair. Experiments have proved that the chemical sunscreen product can reduce the damage of DNA, and when the skin is irradiated by ultraviolet, the CPD level can be effectively reduced and the skin state can be improved after the skin is repaired by utilizing the photo-repair enzyme.

Antarctic, the continent surrounding the Antarctic, is located at the southern end of the Earth and is surrounded by three oceans. There are only 850 plants distributed, and the vast majority are lower, with only 3 flowering plants belonging to higher plants. Antarctic climate is extremely cold, dry, strong wind and strong ultraviolet radiation are the main factors that restrict the growth of terrestrial plants. The ozone layer has the function of shielding ultraviolet rays and can filter 99% of the ultraviolet rays. During the spring, the ozone reduction amount in the south pole area reaches 50% -60%, so that the ultraviolet radiation reaching the ground is obviously increased, and the brought strong ultraviolet radiation can cause fatal damage to organisms.

Recent studies have shown that plants on the Antarctic mainland may have originated independently from ancient terrestrial plants and have been isolated from plants other than the Antarctic mainland for as long as ten million years. Mosses belong to the lower group of higher plants and represent a transition from aquatic to terrestrial plant types. Its gametophyte is developed, sporophyte is degenerated and epiphytic, and its plant body only has the differentiation of pseudostem, pseudoleaf and pseudoroot, but has the functional characteristics of photosynthetic reaction system, phytohormone and cell division and differentiation similar to those of higher plant. The bryophyte is a green plant with the widest distribution and the largest quantity in the Antarctic continental land, and is a main producer in the Antarctic land ecosystem. In the long-term evolution process, moss plants gradually form various adaptation strategies from the molecular level to the cellular level in order to survive under extreme natural conditions, and the uniqueness, diversity and novelty in the aspects of species, metabolites, gene resources and the like are created.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a CPD (photoproduction enzyme) of Antarctic sphagnum luffa and a preparation method and application thereof. The invention screens the full-length coding gene of CPD (CPD photo-repairing enzyme) from the transcriptome of Antarctic yellow loofah moss, clones the coding gene, connects the cloned coding gene with a prokaryotic expression vector, induces and expresses protein, and applies the separated and purified product to the fields of cosmetics and biomedicine.

The technical scheme of the invention is as follows:

a photoproduction enzyme of Antarctic yellow loofah is disclosed, the amino acid sequence is shown in SEQ ID NO. 1.

The nucleotide sequence of the coding gene of the CPD (photoproduction enzyme) of the Antarctic yellow loofah moss is shown in SEQ ID No. 2.

In the invention, the total length of the coding gene of the Antarctic yellow loofah CPD photolyase is 1830bp, the coding gene consists of 609 amino acids, and the relative molecular mass of protein is 69.1 KDa.

A recombinant expression vector of Antarctic yellow loofah CPD (photo-repairing enzyme) comprises a coding gene of the Antarctic yellow loofah CPD.

Preferably, according to the present invention, the expression vector is pGEX-4T-1.

Preferably according to the invention, the recombinant expression vector further comprises a selectable marker gene, including but not limited to: a gene for an enzyme that produces a color change in the expressed strain, or a gene for a luminescent compound, or a gene with an antibiotic marker.

The application of the Antarctic yellow loofah CPD photolyase in preparing cosmetics or biomedicines for repairing ultraviolet injury.

Specifically, the technical scheme of the invention is that a gene with strong ultraviolet response is selected from a transcriptome of Antarctic yellow loofah moss, and the sequence comparison and function prediction of the gene are carried out on NCBI, so that the gene is preliminarily determined to be CPD (compact peripheral component interconnect) photorepair enzyme. Designing specific primer, cloning the cDNA full length of the target gene by PCR. And connecting the target fragment with a prokaryotic expression vector pGEX-4T-1 by a homologous arm recombination method, and screening the recombined vector for sequencing verification. Finally, the correct recombinant vector is introduced into Escherichia coli BL21(DE3) for protein expression. Separating and purifying target protein by affinity chromatography, and detecting enzyme activity in vitro. At the same time, the empty vector and the recombinant vector are respectively introduced into SY2 light repair deficient Escherichia coli for complementation experiment.

The preparation method of the Antarctic yellow loofah CPD photolyase comprises the following steps:

(1) extraction of total RNA of Antarctic yellow loofah moss

Extracting total RNA of Antarctic yellow loofah moss by using a Trizol method;

(2) obtaining of cDNA sequence of CPD light repair enzyme gene (PnPHR1) of Antarctic yellow loofah moss

Synthesizing cDNA (complementary deoxyribonucleic acid) from the extracted total RNA of Antarctic yellow loofah moss by using a reverse transcription kit, wherein the specific procedure of the reverse transcription is as follows: storing at 25 deg.C for 10min, 42 deg.C for 15min, 85 deg.C for 5min, and 4 deg.C; storing cDNA obtained by reverse transcription at-20 deg.C;

designing a homologous arm primer according to the characteristics of the expression vector and the sequence of the target fragment to amplify the target fragment in vitro, wherein the sequence of the PCR primer is as follows:

the upstream primer is:

5’-CCGCGTGGATCCCCGGAATTCCCGGGTCGAATGTACTTTGGGTCTACTACTTTCCCC-3’，

the downstream primer is:

5’-GCAGATCGTCAGTCAGTCACGATGCGGCCGTTAGTGGTGGTGGTGGTGGTGCACCTTCCGCCCTGCCGGCTT-3’，

wherein the introduced His tag sequence is underlined;

using cDNA obtained by reverse transcription as a template, and amplifying a target fragment in vitro by using the primers, wherein the PCR reaction program is as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 54 ℃, 2min at 72 ℃ and 35 cycles; storing at 72 deg.C for 7min and 4 deg.C; storing the PCR product at-20 deg.c;

(3) construction of CPD (compact plant-derived protein) photorepair enzyme recombinant expression vector of Antarctic yellow loofah moss

Firstly, linearizing pGEX-4T-1 plasmid by Ncol fast cutting enzyme, and then homologously connecting the linear pGEX-4T-1 plasmid and a PCR product by a homologously recombining kit to construct a recombining expression vector; transforming the connected recombinant expression vector into escherichia coli DH5 alpha, screening a positive recombinant by using a plate containing ampicillin resistance, selecting a positive strain, culturing and extracting a plasmid, introducing the plasmid into escherichia coli BL21(DE3) by using the same method, and sequencing and identifying;

(4) expression and purification of Antarctic yellow loofah CPD (cytochrome oxidase inhibitor) photorepair enzyme and protein identification

Inoculating recombinant Escherichia coli BL21(DE3) into LB culture medium containing ampicillin for culture, adding IPTG induced expression protein when thallus grows to logarithmic phase, centrifuging to collect thallus, breaking cell, and separating and extracting target protein by nickel column affinity chromatography; taking a proper amount of target protein to carry out SDS-PAGE constant pressure electrophoresis, staining the gel after electrophoresis by Coomassie brilliant blue, and observing the expression condition of the target protein after decoloration by a decoloration solution.

Has the advantages that:

according to the invention, the CPD light repair enzyme gene is found in Antarctic yellow loofah moss for the first time, and is successfully cloned into an expression vector, so that a large amount of the CPD light repair enzyme of Antarctic yellow loofah moss is obtained, and the CPD light repair enzyme of Antarctic yellow loofah moss is expected to be applied to related fields such as cosmetics, biomedicine and the like, so that the disease caused by ultraviolet rays can be actively repaired, and the problem that skin cells cannot be repaired in time after being damaged due to the lack of DNA light repair enzyme related protein in a human body is solved.

Drawings

FIG. 1 is an electrophoresis diagram of CPD photolyase SDS-PAEG of Antarctic yellow loofah; in the figure, M represents a standard molecular weight, 1 represents a supernatant sample obtained by cell disruption and centrifugation of blank control BL21(DE3)/pEGX-4T-1, 2 represents a supernatant sample obtained by cell disruption and centrifugation of recombinant Escherichia coli BL21(DE3)/pEGX-4T-1/PnPHR1, and 3 represents a purified protein sample obtained by separation and purification by affinity chromatography;

FIG. 2 shows oligo (dT)₁₆An absorbance change graph under the irradiation of an ultraviolet lamp; in the figure, the abscissa is time in min;

FIG. 3 is a graph showing the absorbance change of the catalytic reaction of the CPD photolyase of Antarctic yellow loofah and its substrate oligo (dT) 16; in the figure, the abscissa is time in min; the experimental group-1 shows that the reaction system contains the Antarctic yellow loofah CPD photolyase and is carried out under the illumination condition; an experimental group-2 shows that the reaction system contains the Antarctic yellow loofah CPD light repairing enzyme and is carried out under the dark condition; the control group-1 shows that the reaction system does not contain Antarctic yellow loofah CPD (cytochrome oxidase inhibitor) photorepair enzyme and is carried out under the illumination condition; the control group-2 shows that the reaction system does not contain Antarctic yellow loofah CPD (cytochrome oxidase inhibitor) photorepair enzyme and is carried out under the dark condition;

FIG. 4 is a complementary experimental diagram of a CPD photo-repairing enzyme SY2 of Antarctic yellow loofah moss; in the figure, A-1 is normal culture of the experimental group without ultraviolet irradiation, A-2 is visible light culture of the experimental group after ultraviolet irradiation, and A-3 is dark culture of the experimental group after ultraviolet irradiation; b-1 is normal culture without ultraviolet irradiation of a control group, B-2 is visible light culture after ultraviolet irradiation of the control group, and B-3 is dark culture after ultraviolet irradiation of the control group;

FIG. 5 is a histogram of the number of surviving colonies of experiments complemented by CPD photo-repairing enzyme SY2 of Antarctic yellow loofah; in the figure, A-1 is normal culture of the experimental group without ultraviolet irradiation, A-2 is visible light culture of the experimental group after ultraviolet irradiation, and A-3 is dark culture of the experimental group after ultraviolet irradiation; b-1 is normal culture without ultraviolet irradiation in the control group, B-2 is visible light culture after ultraviolet irradiation in the control group, and B-3 is dark culture after ultraviolet irradiation in the control group.

Detailed Description

The technical solution of the present invention is further described below with reference to the following examples and drawings, but the scope of the present invention is not limited thereto. The experimental methods mentioned in the examples are all routine experimental operations in the field if no special description is given; the reagents and drugs mentioned in the examples are all common commercial products unless otherwise specified.

Room temperature: having the meaning known to the person skilled in the art, generally 25. + -. 2 ℃.

Samples of Antarctic yellow Luffa cylindrica (Pohlia nutans) were from the 24 th Antarctic scientific investigation in China.

Example 1 obtaining of cDNA sequence of CPD photolyase from Antarctic yellow Luffa

Extraction of Antarctic yellow loofah moss total RNA

Culturing Antarctic yellow loofah moss (Pohlia nutans) according to conventional culture conditions, wherein the conventional culture conditions comprise the temperature of 16 +/-1 ℃, the relative humidity of 70 percent, the illumination intensity of 1000-; extracting total RNA of Antarctic yellow towel gourd moss (Pohlia nutans) by using a Trizol method, wherein the used reagents are Trizol, chloromethane and isopropanol. RNA is very easy to degrade, so that gloves are needed to be worn in the operation process to prevent RNA enzyme in sweat from degrading RNA, and all tools need to be strictly sterilized and subjected to RNA enzyme inactivation treatment in the extraction process.

The specific operation steps of extracting the total RNA of Antarctic yellow loofah moss by the Trizol method are as follows:

1. putting the tissues (leaves) into liquid nitrogen, grinding in a mortar, continuously putting the ground tissues into the liquid nitrogen, and putting the powder which is kept at low temperature and ground into a 1.5mL centrifuge tube;

2. adding 1mL Trizol into the centrifugal tube, mixing, shaking for 15s, and standing at room temperature for 5-10 min;

3. adding 200 μ L chloroform (chloroform), reversing up and down, shaking vigorously for 15s, and standing at room temperature for 15 min;

centrifuging at 12000rpm for 15min at 4.4 deg.C, sucking supernatant (400-;

5. adding 500 μ L isopropanol (precooled in advance), slightly inverting, and standing at room temperature for 10 min;

centrifuging at 12000rpm for 15min at 6.4 deg.C to obtain precipitate at the bottom or tube wall;

7. discarding the supernatant, adding 1mL of 75% ethanol, slightly inverting the upper part and the lower part, cleaning the precipitate, and fully and uniformly mixing;

centrifuging at 8.4 deg.C and 12000rpm for 5min, discarding supernatant, and air drying at room temperature (3-4 min);

9. dissolving the precipitate in 20-35 μ L DEPC-water;

10. and (3) determining the concentration of the RNA for reverse transcription or liquid nitrogen cryopreservation.

Second, obtaining cDNA sequence of Antarctic yellow loofah moss

The cDNA synthesis is completed by using 5 xAll-In-One RT MasterMix kit operation, and the specific reaction system for the synthesis is as follows:

RNA sample 2. mu.g, AccuRT Reaction Mix 4X 2. mu.L, RNase free H₂O is added to 8 mu L;

the reaction system is incubated at 42 ℃ for 2min, or placed at room temperature for 5min, and then the following reagents are added:

AccuRTReaction Stopper 5× 2μL

5×All-In-One RT MasterMix 4μL

RNase free H₂O 6μL

the reaction procedure is as follows: 10min at 25 ℃, 15min at 42 ℃, 5min at 85 ℃ and heat preservation at 4 ℃. The synthesized cDNA can be stored at-20 ℃.

Example 2 cloning of Antarctic yellow Luffa Lygodii CPD light repair enzyme gene (PnPHR1) and construction of recombinant expression vector

Cloning of CPD (cytochrome oxydase) photorepair enzyme gene of Antarctic yellow loofah moss

According to the CPD light repair enzyme gene sequence in Antarctic yellow loofah moss transcriptome data and the homologous recombination principle, PCR primers are designed as follows:

the upstream primer is as follows:

5’-CCGCGTGGATCCCCGGAATTCCCGGGTCGAATGTACTTTGGGTCTACTACTTTCCCC-3’，

the downstream primer is:

wherein the introduced His tag sequence is underlined;

the PCR reaction system is set by utilizing a PCR kit of Novozan company according to the instruction:

the reaction procedure is as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 54 ℃ and 2min at 72 ℃ for 35 cycles; storing at 72 deg.C for 7min and 4 deg.C. And (3) carrying out electrophoresis detection on the PCR product by using 0.8% agarose gel, cutting the gel and recovering a target fragment to obtain the Antarctic yellow loofah CPD photolyase gene.

Construction of recombinant expression vector

pGEX-4T-1 plasmid (Youbao organism) is firstly cut by NcoI fast cutting enzyme (Thermoscientific) in water bath at 37 ℃ for 2 hours to change the circular plasmid into a linear plasmid, the cut plasmid is recovered by 0.8 percent agarose gel, and then the CPD photolyase gene of the target fragment Antarctic yellow loofah is homologously connected with the pGEX-4T-1 linear plasmid by adopting a homologous arm recombination kit (Novozam).

The homologous ligation system is as follows:

after the system is prepared, the components are uniformly mixed by blowing and beating the components up and down for several times by using a pipette gun, so that bubbles are avoided (oscillation or vortex is avoided). After 30 minutes of reaction at 37 ℃, the reaction system is immediately placed in an ice water bath for 5 minutes to obtain the recombinant expression vector PnPHR1-pGEX-4T-1, and the recombinant expression vector can be directly used for transformation or stored at-20 ℃.

Transformation and screening of recombinant Escherichia coli

The recombinant expression vector PnPHR1-pGEX-4T-1 is used for transforming Escherichia coli DH5 alpha, and the steps are as follows:

(1) taking out competent cell Escherichia coli DH5 alpha from a refrigerator at-80 ℃, quickly inserting into ice, adding a recombinant expression vector PnPHR1-pGEX-4T-1 after a bacterium block is melted, slightly and uniformly mixing the bottom of an EP tube by poking with a hand, avoiding sucking with a gun, and standing in ice for 25 minutes;

(2) heat shock is carried out in 42 ℃ water bath for 45 seconds, and the mixture is quickly put on ice and stands for 2 minutes;

(3) adding 700 mu L of LB sterile medium without antibiotics into an EP tube, uniformly mixing, and recovering for 60 minutes at 37 ℃ and 200 rpm;

(4) centrifuging at 5000rpm for 1 min to collect thallus, collecting supernatant of about 100 μ L, lightly blowing and beating the resuspended thallus block, and spreading on LB solid plate containing ampicillin;

(5) the plates were placed upside down in a 37 ℃ incubator overnight.

And selecting a single colony, and carrying out colony PCR identification. Single colonies of PCR bands were picked and cultured overnight at 37 ℃ and 200rpm in 50mL of LB liquid medium containing benzyl group resistance. Extracting positive recombinant Escherichia coli DH5 alpha plasmid, transforming Escherichia coli BL21(DE3) by the method, sequencing and identifying to obtain recombinant Escherichia coli BL21(DE3)/pGEX-4T-1/PnPHR1, wherein the primer sequence used for sequencing and identification is as follows:

an upstream primer: 5'-CTGACTTCATGTTGTATGACGCTC-3' the flow of the air in the air conditioner,

a downstream primer: 5'-GTATCACGAGGCCCTTTCGT-3' are provided.

Sequencing results show that the total length of the CPD (PnPHR1) gene of the Antarctic yellow loofah is 1830bp, and the nucleotide sequence is shown as SEQ ID NO. 2. Performing function prediction on the target gene by using BLAST function of NCBI website (https:// www.ncbi.nlm.nih.gov /); reading frame prediction and amino acid translation are carried out on the cDNA sequence by using primer5.0 software, and https:// www.predictprotein.org/and http:// swissmodel.expasy.org/three-dimensional modeling of the secondary structure and the structural domain of the photorepair gene is analyzed on line; alignment of amino acid sequences was performed using DNAMAN software. The result shows that the Antarctic yellow loofah CPD (PnPHR1) has 609 amino acid compositions, the amino acid sequence is shown as SEQ ID NO.1, and the relative molecular mass of the protein is 69.1 KDa. Blast results show that the gene is a type II CPD light repair enzyme gene. Comparing the amino acid sequence of the CPD (PnPHR1) of Antarctic yellow loofah with the amino acid sequences of Dunaliella salina, Gliocladium sp, Proteus, Chlamydomonas and Paniculatum sp, finding that the CPD and PnPlr have the same conserved domain and the highest similarity with the Gliocladium sp, wherein the similarity is 44.35%.

Example 3 induced expression, separation and purification of Antarctic yellow Luffa CPD photolyase protein

Recombinant E.coli BL21(DE3)/pGEX-4T-1/PnPHR1 was inoculated into 50mL of LB liquid medium containing ampicillin and cultured overnight at 37 ℃ and 180 rpm. The next day, 5mL of activated bacterial liquid was taken and cultured in 500mL of medium until OD of the bacterial cells was reached₆₀₀When the concentration is 0.6-0.8, the bacterial solution is placed on ice for 30 minutes, then IPTG with the final concentration of 0.2mM is added, the culture condition is 16 ℃, the low-temperature induction is carried out at 100rpm for 20 hours, and the bacteria are centrifugally collected at 4 ℃ and 8000 rpm.

The thallus is resuspended in 40mL buffer solution, the thallus is broken for 40 minutes by 300W in ice water bath, and a proper amount of protease inhibitor PMSF is added before the breaking to ensure that the final concentration is 0.1-1 mM. The disrupted system was centrifuged at 12000rpm at 4 ℃ for 15min, and the supernatant was filtered through a 0.22 μm filter and applied to the column. The target protein is separated and purified by using the principle of affinity chromatography. Since the His tag was added in the primer design of the target fragment, the protein was separated and purified by Ni Sepharose column chromatography. The chromatographic column is first equilibrated with 3-5 column volumes of protein buffer solution, and the supernatant is then applied to the column and incubated at 4 deg.c for 30min to contact the target protein with the Ni column. The hetero-protein was eluted with 50mM imidazole and the protein of interest was eluted with 500mM imidazole. Ultrafiltering imidazole with ultrafilter tube, concentrating to obtain purified protein, measuring protein concentration, subpackaging, and freezing in liquid nitrogen.

The composition of the buffer was as follows:

the purified protein was verified by SDS-PAGE electrophoresis. 5% concentrated gel, 8% separation gel, protein sample and 5 x protein loading buffer mixed after boiling 10 minutes at 100 degrees C to denature protein. 20 μ L of the sample was applied, and after 30 minutes of 80V electrophoresis, the voltage was adjusted to 120V for 1 hour of electrophoresis. And (5) dyeing with Coomassie brilliant blue after electrophoresis is finished, and decoloring the destaining solution to observe the separation condition of the target protein.

The results are shown in FIG. 1, wherein M represents the standard molecular weight of protein, 1 represents the supernatant sample after cell disruption and centrifugation of blank control group BL21(DE3)/pEGX-4T-1, 2 represents the supernatant sample after cell disruption and centrifugation of recombinant Escherichia coli BL21(DE3)/pEGX-4T-1/PnPHR1, and 3 represents the purified protein sample after affinity chromatography separation and purification, the band of the purified protein is clear and single, and the molecular weight is consistent with the theoretical value, which indicates that the CPD photolyase of Antarctic yellow Luffa can be obtained by in vitro induction expression by the method.

Example 4 Antarctic yellow loofah CPD photolyase in vitro Activity assay

1. Preparation of pyrimidine dimers: artificial synthesis of 16 pyrimidine base single-stranded oligonucleotide oligo (dT)₁₆The dry powder is prepared into a solution with the concentration of 10 mu M at the concentration of 60 mu W/cm²Is irradiated at a short distance of 5cm lamp distance until oligo (dT)₁₆The absorbance of the solution at 260nm is no longerDecrease until, as shown in FIG. 2, oligo (dT)₁₆Under the irradiation of an ultraviolet lamp, the absorbance of the solution at 260nm is continuously reduced, the initial absorbance reduction range is large, the reduction range is gradually reduced from the third hour until the 6 th hour later, and the absorbance is not changed. The result shows that the pyrimidine base monomer forms pyrimidine dimer under the condition of ultraviolet irradiation, and the DNA damage product is successfully prepared.

2. In vitro photorepair reaction: oligo (dT) after UV irradiation₁₆The final concentration of the reaction substrate is 4 mu M, the final concentration of the antarctic yellow loofah CPD photolyase is 2mg/mL, the coenzyme FAD is 0.5mM, the catalysis function of the antarctic yellow loofah CPD photolyase is assisted, dithiothreitol DTT is 1mM, the BSA is 100mg/mL and is used as an electron donor of a reaction system, the reaction system is placed in a protein buffer solution and reacts for 4 hours under the irradiation of visible light at 30 ℃, and the change of absorbance of the reaction system at 260nm is detected at different time points respectively, so that the ultraviolet repair condition of the protease is observed.

The results are shown in FIG. 3: OD in contrast group without Antarctic yellow loofah CPD photolyase in reaction system₂₆₀Has no substantial increase in value of (A), OD under dark reaction conditions₂₆₀There is also a tendency to decline; OD (optical density) in experimental group containing Antarctic yellow loofah CPD (Complex planar light Detector) photorepair enzyme in reaction system under dark reaction condition₂₆₀Substantially no change in the overall, OD under light conditions₂₆₀The value is obviously increased, which indicates that after the CPD (cytochrome Diphyllophora and loofah) photorepair enzyme repair is carried out, the pyrimidine dimer covalent bond is broken and recovered into a pyrimidine monomer to cause OD (optical Density)₂₆₀And the increase indicates that the CPD (photoproduction repair) enzyme of Antarctic yellow loofah has the photoproduction activity.

SY2 complementation test detection: SY2 is E.coli.CPD light repair enzyme deficient strain (JM 107. DELTA. phr:: Cm)^rΔuvrA::Km ^rΔrecA::Tet^rSee document Visible Light-indelible PhotolyticeGene from the Goldfish Carassius auratus). SY2 competent cells are prepared, and a recombinant expression vector PnPHR1-pGEX-4T-1 and an empty vector pGEX-4T-1 are respectively transformed into SY2 competent cells to serve as an experimental strain and a control strain. Inoculating two strains of bacteria respectively to a culture medium containing ampicillinIn LB liquid Medium of (1), cultured at 37 ℃ and 180rpm to OD₆₀₀When the concentration is 0.6-0.8, IPTG is added to induce the cells to a final concentration of 0.2mM, after the cells are cultured for 20 hours, 1mL of each of the two cells is taken out and diluted with sterile water by about 10³Doubling and keeping the concentration of the two strains consistent, respectively taking 50 mu L of the bacterial liquid to coat an LB solid plate and standing for 30 minutes at room temperature, and respectively dividing the experimental strain and the control strain into three groups to carry out the following treatment, wherein each group of treatment is set to be 5 times. After the thalli absorb the water on the flat plate and adapt to a new environment, placing the first group of flat plates in a constant temperature incubator at 37 ℃ for inversion and normal culture; second set of plates at 17. mu.W/cm²Irradiating for 35 seconds under the ultraviolet intensity, and then placing the mixture in a constant temperature incubator at 37 ℃ irradiated by visible light for inversion for recovery culture; third set of plates at 17. mu.W/cm²After the irradiation for 35 seconds at the ultraviolet intensity, the cells were placed in a dark condition at 37 ℃ and then subjected to the recovery culture. And (4) counting the survival number of colonies on each plate by using a colony counter after the recovery culture for 30 hours, and comparing the survival rates of the thalli after the ultraviolet repair of the experimental strain and the control strain. The results are shown in FIG. 4: the survival rate of the colonies of the experimental group A-2 relative to the A-1 is 17.40%, the survival rate of the colonies of the experimental group A-3 relative to the A-1 is 3.54%, the survival rate of the colonies of the control group B-2 relative to the B-1 is 4.38%, the survival rate of the colonies of the control group B-3 relative to the B-1 is 3.85%, and the survival rate of the colonies of the experimental group under the visible light recovery culture condition is about 4 times that of the control group, so that the CPD photolyastra antarctica plays a repairing function in the photolysis defective escherichia coli, and the survival rate of the colonies is increased.

The SY2 competent cell is prepared as follows:

(1) SY2 is inoculated in LB liquid culture medium containing tetracycline to be activated overnight;

(2) transferring 500 μ L of the overnight activated SY2 bacterial solution in 50mLLB liquid, and culturing to obtain bacterial OD₆₀₀Stopping culturing when the culture temperature is 0.6-0.8%;

(3) placing the transfer solution in an ice bath for 10 minutes, and centrifuging at 4 ℃ and 4000rpm for 12 minutes;

(4) re-suspending and precipitating the pre-cooled TB solution, carrying out ice bath for 20-30 minutes, and then centrifuging at the temperature of 4000rpm for 12 minutes;

(5) repeating the step (4);

(6) resuspending the bacterial pellet with 4mL of pre-chilled TB solution, adding 280 mu LDMSO, and carrying out ice bath for 10 minutes;

(7) competent cells were split into 0.5ml LEP tubes (100. mu.L per tube) and snap frozen in liquid nitrogen and stored at-80 ℃.

Example 5 application of Antarctic yellow loofah CPD (cytochrome oxidase inhibitor) photorepair enzyme in cosmetic field

In this example, the CPD photolyase of Antarctic yellow loofah purified in example 3 was embedded with lecithin and cholesterol for relevant experimental detection.

The preparation method of the Antarctic yellow loofah CPD photolyase liposome comprises the following steps: according to the following steps: 2, weighing lecithin and cholesterol in a mass ratio, dissolving the lecithin and cholesterol in diethyl ether, adding the Antarctic yellow loofah CPD (Phyllostachys lutea) light repairing enzyme obtained by purification in the embodiment 3, performing ultrasonic emulsification to form water-in-oil type, distilling to remove a low-boiling organic phase to obtain Antarctic yellow loofah CPD light repairing enzyme liposome suspension, continuing to evaporate the suspension until white suspension is formed, centrifuging the white suspension, collecting precipitate, and performing freeze preservation to obtain the Antarctic yellow loofah CPD light repairing enzyme liposome.

Mouse skin section experiment: the mouse is Kunming mouse with body weight of 20-25g for 6-8 weeks. Shearing hairs in an area of about 3cm multiplied by 3cm from the back of the mouse, exposing the skin of the mouse, and performing ultraviolet ray damage and photorepair experiments, wherein the mouse in the experimental group is treated by the CPD photorepair enzyme liposome of Antarctic yellow loofah moss, and the control group is not treated by the CPD photorepair enzyme liposome of Antarctic yellow loofah. After the experiment operation is completed, the mice are immediately killed, the back skin of the mice is taken as a tissue section, hematoxylin-eosin staining is respectively carried out, and the epidermal tissues of the mice are observed. The experimental result shows that the epidermal tissue of the control group of mice is obviously thickened, and the epidermal tissue of the mice which are coated with the Antarctic yellow loofah CPD photo-repairing enzyme liposome for photo-repairing after ultraviolet irradiation has no obvious change, so that the cucurbita moschata CPD photo-repairing enzyme liposome prepared by the experiment can inhibit the skin hyperplasia effect caused by ultraviolet rays, and is expected to be applied to the field of cosmetics.

Example 6 application of Antarctic yellow loofah CPD (cytochrome oxidase inhibitor) photorepair enzyme in field of biological medicine

In the embodiment, the CCK-8 method is adopted to detect the protection effect of the CPD photolyase liposome of Antarctic sphagnum on HaCaT cells under ultraviolet irradiation. Wherein, the preparation method of the Antarctic yellow loofah CPD photolyase liposome is the same as that of the example 5.

Cultured HaCaT cells (Wuhan Punuo Seiki Life technologies, Ltd.) are spread on a 96-well plate, the density is controlled at 5000/well, after culturing for 4 hours by using a fresh medium, the cells are allowed to grow adherently, and then a CPD photolyase liposome of Antarctic yellow loofah moss is added, and a negative control (an equal amount of fresh medium is added) is set. Culturing in incubator for 2 hr, taking out, washing with fresh culture medium three times to remove residual liposome, and culturing without adding culture medium to 72 μ W/cm²The UV irradiation was carried out at an intensity for 95 seconds. Washing with fresh culture medium again to avoid influence of superoxide radical caused by ultraviolet, replacing with new culture medium, irradiating with visible light for 30min, and adding CO₂And (5) restoring the culture in the incubator for 2 h. Finally, 20 mu LCCK-8 is added, and the absorbance at 450nm is detected by a microplate reader after staining for 1 h. The result shows that the survival rate of the HaCaT cells in the experimental group is obviously higher than that of the negative control group, which indicates that the CPD (photopreparation enzyme) of Antarctic yellow loofah can repair the damage of ultraviolet rays to the cells, and the CPD can be expected to be applied to the field of biological medicines.

SEQUENCE LISTING

<110> Shandong university

<120> Antarctic yellow loofah CPD (cytochrome oxydase D) photorepair enzyme as well as preparation method and application thereof

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 609

<212> PRT

<213> Pohlia nutans

<400> 1

Met Tyr Phe Gly Ser Thr Thr Phe Pro Ile Arg Trp Asn Val Ala Val

1 5 10 15

Leu Arg Thr Ser Ser Tyr Val Glu Pro Cys Met Arg Ala Tyr Ser Leu

20 25 30

Cys Ile His Tyr Gln Val Asn Asn Ser Lys Val Gln Ser Arg Gly Phe

35 40 45

His Ser Val Gly Arg Val His Ser Phe Ala Ala Gly Lys Ser Ser Lys

50 55 60

Glu Thr Met Pro Pro Lys Lys Lys Leu Lys Gln Ser Phe Leu Val Lys

65 70 75 80

Glu Glu Asn Gln Gly Met Leu Lys Ala Ile Ala Glu Asp Glu Asp Gln

85 90 95

Pro Pro Arg Ala Arg Lys Leu Lys Pro Ser Val Asp Glu Glu Asp Glu

100 105 110

Ile Leu Ala Pro Lys Ala Glu Gly Arg Pro Pro Leu Asn Tyr Gly Val

115 120 125

His Pro Gly Arg Ile Gln Lys Leu Asn Pro Gly Gly Asn Gln Asn Gly

130 135 140

Pro Val Val Tyr Trp Met Ser Arg Asp His Arg Ser Arg Asp Asn Trp

145 150 155 160

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

165 170 175

Ala Val Ala Phe Asn Leu Val Glu Ser Phe Leu Glu Ala Arg Ala Arg

180 185 190

His Phe Gly Phe Met Leu Arg Gly Leu Arg Val Val Glu Arg Asn Leu

195 200 205

Gln Ala Val Asp Ile Pro Phe Phe Leu Phe Arg Gly Lys Ala Glu Glu

210 215 220

Thr Ile Pro Ala Phe Val Lys Lys Cys Asn Ala Ser Leu Leu Val Met

225 230 235 240

Asp Tyr Ser Ser Leu Arg Ile Gly Lys Gln Trp Arg Thr Ala Ile Cys

245 250 255

Gln Asn Val Pro Pro Ser Val Ala Val Ala Glu Val Asp Ala His Asn

260 265 270

Val Val Pro Ile Trp Cys Ala Ser Asp Lys Met Glu Tyr Gly Ala Arg

275 280 285

Thr Ile Arg Thr Lys Ile Thr Arg Gln Leu Pro Asp Phe Leu Asn Glu

290 295 300

Tyr Pro Val Leu Glu Asn Ser Gly Thr Pro Trp Glu Leu Asp Ala Pro

305 310 315 320

Asp Ala Ile Asp Trp Asp Ala Leu Ile Ala Asp Val Val Arg Val Gly

325 330 335

Ala Glu Val Pro Glu Val Thr Trp Leu Glu Ser Gly Glu Asp Ala Ala

340 345 350

Leu Glu Ala Leu Ala Gly Lys Ala Lys Gly Phe Val Asn Thr Arg Met

355 360 365

Lys Asn Tyr Glu Asn Arg Asn Asp Pro Ser Lys Pro Thr Gly Leu Ser

370 375 380

Gly Leu Ser Pro Tyr Leu His Tyr Gly Gln Ile Ser Ala Gln Arg Cys

385 390 395 400

Ala Leu Glu Ala Arg Lys Phe Arg Lys Ala His Thr Lys Ser Val Asp

405 410 415

Ala Phe Leu Glu Glu Leu Ile Val Arg Gly Gly Leu Ala Glu Asn Tyr

420 425 430

Cys His Tyr Gln Pro Asn Tyr Asp Asn Leu Lys Gly Ala Trp Ser Trp

435 440 445

Ala Gln Glu Ser Leu Arg Ile His Ala Ser Asp Lys Arg Glu Phe Thr

450 455 460

Tyr Thr Glu Ser Glu Leu Glu Ala Gly Lys Thr His Asp Lys Leu Trp

465 470 475 480

Asn Ala Ala Gln Leu Glu Met Val Tyr Tyr Gly Lys Met His Gly Phe

485 490 495

Met Arg Met Tyr Trp Ala Lys Lys Ile Leu Glu Trp Thr Glu Ser Pro

500 505 510

Glu Glu Ala Leu Arg Ile Ala Ile Tyr Leu Asn Asp Lys Tyr Glu Leu

515 520 525

Asp Gly Arg Asp Pro Asn Gly Tyr Val Gly Cys Met Trp Ser Ile Cys

530 535 540

Gly Ile His Asp Gln Gly Trp Lys Glu Arg Pro Val Phe Gly Lys Ile

545 550 555 560

Arg Tyr Met Asn Tyr Asn Gly Cys Lys Arg Lys Phe Asn Val Asp Gly

565 570 575

Tyr Ile Met Asn Val Ser Gln Met Val Ala Lys Thr Arg Lys Lys Leu

580 585 590

Gln Glu Gly Ile Ala Ser Thr Ser Pro Ser Asn Pro Ala Gly Arg Lys

595 600 605

Val

<210> 2

<211> 1830

<212> DNA

<213> Pohlia nutans

<400> 2

atgtactttg ggtctactac tttccccatc agatggaatg tagcagtcct ccgcacctcg 60

tcttacgtcg agccatgtat gcgcgcctat tcactatgta ttcactatca ggttaacaat 120

agcaaggtgc agtcccgcgg attccattct gtaggtcgcg tgcattcgtt tgcagctggc 180

aaatcatcga aagagacaat gcctcccaag aagaaactga agcaatcctt ccttgtcaag 240

gaggaaaatc aggggatgtt gaaggccata gctgaggatg aagatcagcc tcccagagcg 300

cggaaattga agccgtccgt tgatgaagaa gatgagatct tagcaccgaa agcagaaggc 360

cggccgcctc taaactatgg ggtgcatccc ggaaggattc agaaattgaa tcctggaggg 420

aaccagaacg ggccggtggt gtattggatg tcaagggatc atcggtctcg tgataactgg 480

gctctcctgc acgctgtgca tcaggctcgc gataagggtg ttgccgttgc agtggcgttc 540

aacttggtgg agagcttctt agaggcccgg gcacgccatt tcggtttcat gttgcgtgga 600

ctgcgcgttg tggaacgtaa cttgcaggct gtcgacattc ccttcttcct ctttcgaggt 660

aaggcagaag aaaccattcc agcatttgtg aagaagtgca atgcttcact tctggttatg 720

gactactcct ctctgcgcat tgggaagcag tggaggactg caatctgcca aaacgtaccg 780

ccctccgttg cagtagccga ggtggatgcc cacaatgtcg ttcctatttg gtgcgcttct 840

gataagatgg agtatggtgc ccgcaccatt cgaactaaga ttaccaggca actcccagat 900

ttcctgaatg aataccctgt gctggaaaac tcaggtacac catgggagtt ggatgcacct 960

gatgctattg attgggacgc tcttatagct gacgtcgtca gggtcggtgc tgaggtccct 1020

gaggtaactt ggctggaatc gggagaggat gccgctttgg aagcactcgc ggggaaggcg 1080

aaaggcttcg ttaacacccg gatgaagaat tacgaaaacc gtaacgatcc ctctaagcca 1140

accgggctgt caggtctttc accttatttg cactacggtc agatctcagc gcagcgatgt 1200

gctcttgagg caaggaagtt tcgaaaggct cacacaaagt cggtggatgc attcttggag 1260

gagttgattg tgcgcggtgg tttagccgaa aattactgtc attaccagcc caactatgac 1320

aatttaaaag gggcctggag ttgggctcaa gagtcgttga gaatccacgc aagtgacaaa 1380

cgagagttta cttatactga aagtgagctg gaagctggta agactcacga caagctttgg 1440

aatgctgctc agctcgagat ggtttactat gggaaaatgc atggtttcat gagaatgtac 1500

tgggcgaaga agattttgga gtggacggag tctcctgagg aagctcttcg tatagctatt 1560

taccttaatg acaagtacga attggacgga agagatccga atggctatgt tggctgcatg 1620

tggtcaatat gtggaattca tgatcagggc tggaaggagc gtcctgtgtt cggcaaaatt 1680

aggtatatga actacaatgg ttgcaaacga aagttcaatg ttgatggata cattatgaat 1740

gtgagtcaga tggtagccaa gactcggaaa aagctacaag agggcattgc cagtacttcg 1800

ccctccaatc cagcagggcg gaaggtgtaa 1830

Claims

1. A Antarctic yellow loofah CPD photolyase is characterized in that the amino acid sequence of the Antarctic yellow loofah CPD photolyase is shown in SEQ ID NO. 1.

2. The encoding gene of CPD (photoproduction enzyme) of Antarctic yellow towel gourd moss according to claim 1, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2.

3. A recombinant expression vector of Antarctic yellow loofah CPD photolyase, which is characterized in that the recombinant expression vector contains the coding gene of the Antarctic yellow loofah CPD photolyase of claim 2.

4. The recombinant expression vector of claim 3, wherein the expression vector is pGEX-4T-1.

5. The recombinant expression vector of claim 3, further comprising: a gene for an enzyme that produces a color change in the expressed strain, or a gene for a luminescent compound, or a gene with an antibiotic marker.

6. Use of the Antarctic yellow Luffa cylindrica CPD photolyase of claim 1 in the preparation of cosmetics or drugs for repairing UV damage.