CN109593127B

CN109593127B - Gene recombinant collagen peptide MJ L GG-34 and preparation method and application thereof

Info

Publication number: CN109593127B
Application number: CN201811505180.3A
Authority: CN
Inventors: 马义
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2018-12-10
Filing date: 2018-12-10
Publication date: 2020-08-07
Anticipated expiration: 2038-12-10
Also published as: CN109593127A

Abstract

Compared with natural collagen, the collagen-like peptide MJ L GG-34 obtained through gene recombination has small molecular weight, easy preparation, stable bioactivity, better skin penetration rate, higher bioactivity, better biocompatibility and biological safety, good bioactivity of oxidation resistance, apoptosis resistance, melanin generation inhibition and the like, can obviously inhibit the oxidation damage of the skin, regulate the generation of melanin and resist aging, has no obvious toxic or side effect, can be used for food, health care products, cosmetics and the like, has wide application range, good application value and industrial prospect.

Description

Gene recombinant collagen peptide MJ L GG-34 and preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a gene recombinant human collagen-like peptide MJ L GG-34, and a preparation method and application thereof.

Background

Collagen is the most abundant protein in many animals, and has abundant diversity and specificity of tissue distribution. The change of the type, quality and distribution of the collagen directly influences the normal function of the animal body, has excellent characteristics of good biocompatibility, low immunogenicity, biodegradability and the like, and is widely applied to the fields of biomedicine, tissue engineering, food, cosmetics and the like. At present, researches show that the collagen is a functional factor for improving the moisture content of the skin, can supplement amino acid required for synthesizing the collagen, can also play a role in repairing damaged cells, and improves the nutritional status and microenvironment of the cells, so that the skin is revived.

Collagen in the animal body exists in the form of collagen fibrils or collagen fibers. The basic structural unit is a collagen molecule with the length of about 300nm, the diameter of about 1.5nm and the molecular weight of about 300KDa, and consists of three intertwined polypeptide chains. Under an electron microscope, the collagen fibers present specific striated zones, and the interval between the zones is 60-70 nm, which depends on the type and biological source of the collagen. The procollagen molecules are regularly staggered by one quarter in the collagen fibers, end to end, and grouped into bundles of fibers.

Because collagen belongs to macromolecular compounds and needs to be metabolized into small molecular short peptides in vivo to play a role, oligopeptide which has the bioactivity of collagen and high biocompatibility with a human skin structure can be obtained by shearing a collagen peptide chain by utilizing specific collagenase, and the oligopeptide can be added into cosmetics to serve as an effective component. The generation of various peptide analogs through specific amino acid substitutions and alterations allows for precise adjustment of potential therapeutic efficacy, solubility, toxicity and cost. This accessibility and control is not easily achieved for most other molecular and biological compounds.

Collagen production is mainly classified into three main groups: traditional extraction method, chemical synthesis method and modern biotechnology production method. The traditional extracted collagen has certain immunogenicity, potential risks of application are increased, obvious health and environmental problems are caused by racemization and toxic reagent in the synthetic process of the chemical synthesis method, and the recombinant protein has the characteristics of processability, low rejection, no virus hidden danger and the like. These indicate that the gene recombinant small molecule collagen-like peptide has good application value and industrialization prospect.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the invention mainly aims to provide a gene recombinant collagen-like peptide MJ L GG-34, wherein the collagen-like peptide MJ L GG-34 has better transdermal rate and higher bioactivity.

The invention also aims to provide a preparation method of the gene recombinant collagen-like peptide MJ L GG-34, the preparation method designs the gene sequence of MJ L GG-34 in a prokaryotic expression system according to the amino acid sequence of the collagen-like peptide MJ L GG-34 and the preference of an escherichia coli codon, and the efficient preparation of the target polypeptide MJ L GG-34 is realized by adopting a genetic engineering technology and combining the inducible shearing function of intein, and the preparation method is safe, high in production efficiency and low in production cost.

The invention further aims to provide application of the gene recombinant collagen-like peptide MJ L GG-34.

The purpose of the invention is realized by the following technical scheme:

a gene recombinant collagen-like peptide MJ L GG-34 has a peptide segment designed by referring to type III collagen α 1 chain as template, the influence of comprehensive molecular weight on transdermal absorption rate and oxidation resistance is determined to be about 3KD, and hydrophobic amino acids except Pro are replaced by hydrophilic amino acids to improve hydrophilic property on the premise of keeping the high repetition of natural collagen domain Gly-X-Y sequence²⁺GHK sequence with high affinity, alteration of Cu²⁺Transdermal delivery of while reducing Cu²⁺Combined with tyrosinase, can inhibit melanin generation. Studies have shown that GHK-Cu induces collagen production and accelerates wound healing better than either drug alone. GHK-Cu induces collagen remodeling by up-regulating mRNA and protein levels of MMP-2, TIMP-1 and TIMP-2, and simultaneously increases synthesis of sodium sulfate and chondroitin sulfate in the skin, and finally reduces skin laxity and wrinkles. Meanwhile, the Cys at the tail end is beneficial to maintaining an intracellular glutathione system and playing a role of antioxidation, and is also the best preparation of the tyrosine enzyme. The amino acid sequence is shown as follows: GEPGNPGHKGHKGQPGQPGPPGERGPPGPCCGGG are provided.

The optimized nucleotide sequence of the gene recombinant collagen peptide MJ L GG-34 is as follows:

GGTGAACCGGGCAACCCAGGTCACAAAGGCCACAAAGGCCAGCCGGGCCAGCCGGGTCCGCCGGGCGAACGTGGGCCGCCGGGCCCGTGCTGTGGTGGTGGC；

the nucleotide sequence of the fusion protein Trx-6His-MJ L GG-34 constructed by the target protein MJ L GG-34 and a pET-32a vector is as follows:

the amino acid sequence of the fusion protein Trx-6His-MJ L GG-34 is shown as follows:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFGEPGNPGHKGHKGQPGQPGPPGERGPPGPCCGGG

the preparation method of the gene recombinant collagen-like peptide MJ L GG-34 comprises the following steps:

(1) design and synthesis of MJ L GG-34 Gene:

and (3) annealing by adopting two pairs of complementary primers to synthesize MJ L GG-34 gene:

primer T1 +:

5'-AATTCGGTGAACCGGGCAACCCAGGTCACAAAGGCCACAAAGGCCAGCCGGGCCAGC-3'；

primer T1-:

5'-CCCGGCTGGCCCGGCTGGCCTTTGTGGCCTTTGTGACCTGGGTTGCCCGGTTCACCG-3'；

primer T2 +:

5'-CGGGTCCGCCGGGCGAACGTGGGCCGCCGGGCCCGTGCTGTGGTGGTGGCtaaCTCGAGG-3'；

primer T2-:

5'-GATCCCTCGAGttaGCCACCACCACAGCACGGGCCCGGCGGCCCACGTTCGCCCGGCGGA-3'；

GAATTCis an EcoRI enzyme cutting site,CTCGAGXhoI cleavage site.

Carrying out annealing synthesis reaction on the primer T1+ and the primer T1-, and simultaneously carrying out annealing synthesis reaction on the primer T2+ and the primer T2-, so as to respectively prepare a fragment I and a fragment II;

(2) construction of the recombinant vector pET-32a-MJ L GG-34:

carrying out double enzyme digestion on plasmid pET-32a and MJ L GG-34 gene prepared in the step (1) by using EcoRI enzyme and XhoI enzyme respectively, and then connecting MJ L GG-34 gene obtained after double enzyme digestion with plasmid pET-32a obtained after double enzyme digestion to obtain a recombinant vector pET-32a-MJ L GG-34;

(3) preparation of expression engineering bacterium pET-32a-MJ L GG-34/B L21 (DE 3):

transforming an expression host escherichia coli E.coli B L21 (DE3) by using a recombinant vector pET-32a-MJ L GG-34 to obtain an expression engineering bacterium pET-32a-MJ L GG-34/B L21 (DE 3);

(4) expression and purification:

① inducing expression engineering bacteria pET-32a-MJ L GG-34/B L21 (DE3) to express fusion protein composed of target polypeptide, His tag protein and Trx tag protein;

② the His-tagged fusion protein is purified by nickel column, wherein after loading the protein, the protein with histidine tag specifically binds to the nickel column, and other foreign proteins flow out, and Ni in the nickel column²⁺Or combined with imidazole, gradient elution is carried out by using imidazole, imidazole is competitively combined on a nickel column to release fusion protein, and then eluent is collected;

③ cutting and purifying the target protein;

④ purifying the target protein by high performance liquid chromatography to obtain gene recombinant collagen peptide MJ L GG-34.

The conditions of the annealing synthesis reaction described in step (1) are preferably: at 37 ℃ for 35 min; heating at 98 deg.C for 3 min; 95 ℃ for 5 min;

the composition of the equilibration buffer and the washing buffer used for the purification step (4) ② was as follows:

l ysis equilibration Buffer (L E Buffer) 50mM Na₂HPO₄，0.3M NaCl，pH＝8.0；

Washing buffer solution: 50mM Na₂HPO₄，0.3M NaCl，10～50mM imidazole，pH＝8.0；

The composition of the solution for eluting the nickel column described in the step (4) ② is preferably 50mM Na₂HPO₄，0.3M NaCl，250mM imidazole，pH＝8.0；

The cleavage and purification steps of the target protein in the step (4) ③ are as follows:

1) the fusion protein eluate was dialyzed into 20mM Tris-HCl, pH 8.0 or 1 × PBS, pH 7.4;

2) small-scale enzyme digestion optimization:

a) enterokinase (EK, 5 IU/. mu. L) was diluted 4 times in EK Dilution/Storage Buffer (EK Dilution/Storage Buffer) (0.001IU, 0.01IU, 0.1IU and 1IU enzyme/. mu. L).

b) 5 reactions were set, including reactions without EK for controls:

fusion Protein	10μg
		10×EK Cleavage/Capture Buffer	5μL
Diluted EK(add 2μL EK Dilution/Storage Buffer for the control)	2μL
		Deinized Water (Deionized Water)	To 50 μ L
Total volume	50μL

c) Mix well and incubate at 22 ℃ for 1 hour, 3 hours, 5 hours and overnight (-16 hours).

d) The optimal cleavage result was determined by loading 10. mu. L on SDS-PAGE gel, and if the result was not appropriate, optimization of temperature was tested, the result showed that EK digestion optimization was performed by 1U enterokinase at 22 ℃ to cleave 100. mu.g of fusion protein for 5 h.

3) The reaction ratio was enlarged according to the optimal cleavage results.

4) After the amplification reaction enzyme digestion, passing an enzyme digestion system through Ni-NTA resin which is balanced in advance, collecting penetration liquid, eluting the tag protein and the enzyme digested broken protein on a column by using an elution buffer solution containing 250 mmol/L imidazole, wherein the total volume of elution is 5 times of that of a column bed, so that the target protein and the tag protein are separated, and the purified target protein is obtained.

The step of purifying the target protein MJ L GG-34 by using a high performance liquid chromatography technology, which is described in the step (4) ④, is as follows:

A. mobile phase A was prepared by adding trifluoroacetic acid (TFA) to 100% acetonitrile by volume to give a final concentration of TFA of 0.1% by volume, and mobile phase B was prepared by adding trifluoroacetic acid (TFA) to 100% water to give a final concentration of TFA of 0.1% by volume, and eluting with a linear gradient of 1.0m L/min at a flow rate of 25 min;

B. collecting target protein elution peaks from 90-65% (v/v) of a mobile phase B in linear gradient elution, wherein the light absorption detection wavelength is 220 nm; and carrying out mass spectrum identification.

The gene recombinant collagen peptide MJ L GG-34 can be used for preparing cosmetic, health product or food material, and has the functions of resisting oxidation, inhibiting human dermal fibroblast apoptosis, inhibiting melanin synthesis, etc.

Compared with the prior art, the invention has the following advantages and effects:

(1) the designed collagen-like peptide gene MJ L GG-34 is a brand-new sequence, the length is far shorter than that of a natural human collagen gene, the molecular level operation is simpler and easier, the molecular weight of the translated polypeptide is small, the preparation is easy, the bioactivity is stable, the biological activity is ensured, the skin is easier to absorb, and the oxidative damage of the skin can be obviously inhibited to resist aging;

(2) according to the preference of the escherichia coli codon, the gene sequence of the collagen-like peptide is subjected to codon optimization, so that the low translation efficiency caused by codon utilization limitation in collagen-like peptide expression is eliminated, and the collagen-like peptide is more suitable for expression in the escherichia coli;

(3) compared with natural collagen from animals, the gene recombinant human collagen-like peptide MJ L GG-34 prepared by the method has good biocompatibility and biological safety, has good biological activities of oxidation resistance, apoptosis resistance, melanin generation inhibition and the like, and simultaneously has no obvious toxic or side effect expression by reducing the expression of cyclic adenosine monophosphate-dependent protein kinase catalytic subunit (PRKACA) and microphthalmia transcription factor (MITF), so that the gene recombinant collagen-like peptide MJ L GG-34 can be used for foods, health care products, cosmetics and the like, has wide application range, good application value and industrial prospect;

(4) the preparation method of the recombinant human collagen-like peptide MJ L GG-34 has high efficiency, low cost and wide application prospect.

Drawings

FIG. 1 is the restriction enzyme cutting schematic diagram of plasmid pET-32a and recombinant expression plasmid pET-32a-MJ L GG-34.

FIG. 2 is an identification chart of expression of His fusion protein Trx-6His-MJ L GG-34 detected by SDS-PAGE, wherein lane 1 is IPTG induced whole bacterial protein, lane 2 is IPTG induced whole bacterial supernatant, lane 3 is IPTG induced whole bacterial protein,

lanes

4 and 5 are IPTG induced whole bacterial supernatant, lane 6 is induced bacterial disruption supernatant, and lane 7 is induced bacterial disruption precipitate.

FIG. 3 is a graph showing the identification of the purification of His fusion protein Trx-6His-MJ L GG-34 by SDS-PAGE, wherein lane 1 is the supernatant from the disrupted cell before purification, lane 2 is the effluent from the column, lane 3 is the effluent from the washing, and lane 4 is the protein after purification with 250 mmol/L imidazole.

FIG. 4 is a graph of the identification of flight mass spectrum of the prepared recombinant human collagen-like peptide MJ L GG-34.

FIG. 5 is a graph showing the result of DPPH clearance assay of the prepared recombinant human collagen-like peptide MJ L GG-34, wherein A is sample I and B is sample II.

FIG. 6 shows that recombinant human collagen-like peptide MJ L GG-34 relieves H₂O₂The detection result of the oxidative stress injury of the HaCAT cells is shown.

FIG. 7 is a graph of the results of melanogenesis assay of recombinant human collagen-like peptide MJ L GG-34 inhibiting human melanoma cells.

FIG. 8 is a graph of the results of detecting that recombinant human collagen-like peptide MJ L GG-34 reduces the expression of cyclic adenosine monophosphate-dependent protein kinase catalytic subunit and microphthalmia transcription factor in the melanin synthesis signal pathway of human melanoma cells.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.

Example 1

Construction and expression of expression engineering bacterium pET-32a-MJ L GG-34/B L21 (DE3)

The method comprises the following specific steps:

(1) design and synthesis of MJ L GG-34 Gene:

designing cDNA encoding MJ L GG-34 according to the codon preference of Escherichia coli, designing two pairs of primers, and annealing by using two pairs of complementary primers to synthesize MJ L GG-34 gene:

primer T1 +:

5'-AATTCGGTGAACCGGGCAACCCAGGTCACAAAGGCCACAAAGGCCAGCCGGGCCAGC-3'；

primer T1-:

5'-CCCGGCTGGCCCGGCTGGCCTTTGTGGCCTTTGTGACCTGGGTTGCCCGGTTCACCG-3'；

primer T2 +:

5'-CGGGTCCGCCGGGCGAACGTGGGCCGCCGGGCCCGTGCTGTGGTGGTGGCtaaCTCGAGG-3'；

primer T2-:

5'-GATCCCTCGAGttaGCCACCACCACAGCACGGGCCCGGCGGCCCACGTTCGCCCGGCGGA-3'；

wherein,GAATTCis an EcoRI enzyme cutting site,CTCGAGXhoI cleavage site.

The amount of each annealing component (primer concentration 1OD dissolved in 400. mu. L ddH)₂O, reaction System 20. mu. L)

Fragment I, primer T1+ 2. mu. L, primer T1-5. mu. L, T4PNK 1. mu. L (10U), ATP 20mM, ddH₂O moisturizing to 20 mu L;

fragment two, primer T2+ 2. mu. L, primer T2-2. mu. L, T4PNK 1. mu. L (10U), ATP 20mM, ddH₂O moisturizing to 20 mu L;

and (3) annealing reaction procedures: at 37 ℃ for 35 min; heating at 98 deg.C for 3 min; 95 ℃ for 5 min;

naturally cooling to room temperature, and obtaining an annealing product for later use.

(2) Construction of the recombinant vector pET-32a-MJ L GG-34:

the plasmid pET-32a (purchased from Shanghai Bioengineering Co., Ltd.) and the MJ L GG-34 gene prepared in step (1) were subjected to double digestion with EcoRI enzyme and XhoI enzyme, respectively, in the digestion reaction systems pET-32a 2. mu.g, 10 × FD Buffer 5. mu. L, EcoRI 1. mu. L (10U/. mu. L), XhoI 1. mu. L (10U/. mu. L), ddH₂O42 mu L, putting the system into a37 ℃ constant temperature water bath kettle for reaction for 2h, connecting the MJ L GG-34 gene obtained after double enzyme digestion with the plasmid pET-32a obtained after double enzyme digestion, wherein the connection system is 20 mu L, and the connection system comprises a fragment I1 mu L, a fragment II 1 mu L, an enzyme digestion vector pET-32a 4 mu L, a 10 × T4DNaligase Buffer 2 mu L, a T4DNaligase 1 mu L (5U/mu L) and ddH₂Supplementing O to 20 mu L, and placing the ligation mixture at the constant temperature of 16 ℃ for 1h to obtain a recombinant vector pET-32a-MJ L GG-34, wherein the restriction enzyme of the recombinant vector pET-32a-MJ L GG-34 is shown in figure 1;

transforming an expression host escherichia coli E.coli B L21 (DE3) (purchased from Shanghai biological engineering Co., Ltd.) with a recombinant vector pET-32a-MJ L GG-34 to obtain an expression engineering bacterium pET-32a-MJ L GG-34/B L21 (DE 3);

(4) expression and purification:

taking 50 μ L bacteria of the expression engineering bacteria from the preserved EP tube, culturing in L B medium containing 100 μ g/m L ampicillin at 5m L at 37 deg.C and 220rpm overnight, expanding, inoculating to 50m L L B medium containing 100 μ g/m L ampicillin at a volume ratio of 1: 100, culturing at 37 deg.C and 220rpm to OD₆₀₀Adding isopropyl- β -D thiogalactoside (IPTG) to a final concentration of 0.5 mmol/L, performing induced expression at 37 deg.C for 6 h.5000rpm, centrifuging for 30min, collecting thallus, and resuspending in 10m L balance buffer (50mM Na₂HPO₄0.3M NaCl, pH 8.0), then sonicating the cells, performing the operation on ice, disruption for 1 second, cooling for 3 seconds, for a total time of 30-45 minutes.

And centrifuging the crushed product at 4 ℃ and 12000rpm for 15-20 mim, collecting a supernatant, wherein the supernatant is called a crushed supernatant, detecting the expression of the His fusion protein Trx-6His-MJ L GG-34 by SDS-PAGE, and identifying the result as shown in figure 2.

The nucleotide sequence of the fusion protein Trx-6His-MJ L GG-34 is shown as SEQ ID NO. 3, and the amino acid sequence thereof is SEQ ID NO. 4.

Example 2

Purification, preparation and identification of recombinant human collagen-like peptide MJ L GG-34

The bottle was turned over gently several times to mix the medium well, 2m L of the medium was pipetted into the column (Ni-NTA column, Kinsley Biotechnology Ltd., 10m L column volume), the medium was allowed to settle freely, the stock solution was drained, 4 column volumes of equilibration buffer were added to equilibrate the chromatography medium, the disruption supernatant was applied to the column at a flow rate of 1.0m L/min, the effluent was collected for subsequent analysis, 8 column volumes of wash buffer (50mM Na) were applied₂HPO₄0.3M NaCl, 10-50 mM imidazole, pH 8.0) was passed through the column at 1.0M L/min to wash away the contaminating proteins or fusion proteins not affinity bound, the effluent was collected for subsequent analysis, 10 column volumes of elution buffer were passed through the column at 1.0M L/min, and the eluate was collected from the second tube.

The effluent of the sample was sampled, washed, and the eluate was subjected to SDS-PAGE to examine the purification effect of the His fusion protein (the results are shown in FIG. 3). It was shown that His fusion protein with most of the impurities removed was obtained in the elution with 250mM imidazole.

The target protein is cut and purified, and the steps are as follows:

2) small-scale enzyme digestion optimization:

b) 5 reactions were set, including reactions without EK for controls:

d) The optimal cleavage result was determined by loading 10. mu. L on SDS-PAGE gel, and the results showed that the optimized EK cleavage conditions were cleavage of 100. mu.g of fusion protein with 1U enterokinase at 22 ℃ for 5 h.

3) The reaction ratio was enlarged according to the optimal cleavage results.

And purifying the target protein MJ L GG-34 by using high performance liquid chromatography (HP L C) according to the following steps:

B. and collecting target protein elution peaks from 90-65% (v/v) of a mobile phase B in linear gradient elution, wherein the light absorption detection wavelength is 220 nm. The target polypeptide with the purity of 99.3 percent is prepared.

The prepared target polypeptide MJ L GG-34 is subjected to mass spectrum identification (the result is shown in figure 4), the mass spectrum detection molecular weight is 3.215kDa, and the molecular weight is consistent with the theoretical value, which shows that the target small peptide and the tag protein are successfully separated after EK enzyme digestion and purification, and the correct target protein is obtained.

Example 3

Antioxidant capacity of prepared recombinant human collagen-like peptide MJ L GG-34

(1) DPPH scavenging action

Dissolving appropriate amount of polypeptide with PBS (pH7.5, 0.02M) to obtain 0.3mM solution, adding 1.5% trypsin for hydrolysis for 4 hr, inactivating enzyme in boiling water bath for 5min, and terminating hydrolysis to obtain sample I; another appropriate amount of polypeptide was dissolved in PBS (pH7.5, 0.02M) to obtain 3mM solution as sample two.

Respectively putting a first sample 0-250 mu L and a second sample in a 1.5m L centrifuge tube, respectively adding 250-500 mu L ethanol solution according to concentration gradient, then adding 1m L0.1.1 mM DPPH-ethanol solution, uniformly mixing, standing in the dark for 30min, continuously shaking, and measuring the light absorption value at 517 nm.

The formula for calculating DPPH.removing ability is DPPH.inhibiting rate (%) (Ao-A1)/Ao × 100

Where Ao is the absorbance of the blank without sample and A1 is the absorbance after sample addition.

The experimental result is shown in FIG. 5, the DPPH removing ability of the A picture is gradually enhanced along with the increase of the concentration of polypeptide hydrolysate, the DPPH removing rate reaches 58.89% at the concentration of 0.125mM, the DPPH removing rate of the B picture of the intact collagen-like peptide MJ L GG-34 also shows concentration dependence, the DPPH removing ability is gradually enhanced along with the increase of the concentration of collagen-like peptide, and the DPPH removing rate reaches 62.14% at the concentration of 1.25 mM.

Example 4

Recombinant human collagen-like peptide MJ L GG-34 relieves H₂O₂Resulting oxidative stress injury of HaCAT cells

HaCAT cells (purchased from Kunming cell Bank, Chinese academy of sciences) were seeded in 6-well cell culture plates at 37 ℃ with 5% CO₂Culturing in incubator, and when the cells grow to sub-confluent state, adding fresh culture medium, blank group (Blankcontrol): physiological saline, Model group (Model): physiological saline, collagen control group (Collagen I): 200. mu. mol. L^-1Natural human type I collagen, recombinant collagen-like peptide (Polypeptide) 200. mu. mol. L^-1Prepared MJ L GG-34, except blank group, the other three groups are treated with 300 mu mol L before adding medicine^-1H of (A) to (B)₂O₂Treating for 12h, adding medicine for 48h, sucking a certain amount of culture solution into a centrifugal tube of 1m L, reserving a sample to be tested, sucking and discarding the rest culture solution, washing for 1-2 times by using precooled PBS buffer solution, scraping adherent cells, adding 1 × D-Hank's buffer solution of 1m L, gently mixing by using a pipette gun, repeatedly freezing and thawing for 3 times, 3500 r.min^-1Centrifuging at 4 deg.C for 10min, and collecting supernatant. The SOD content in the cell lysate was determined according to the kit instructions.

As shown in FIG. 6, when the method is used, 300. mu. mol · L is used^-1H₂O₂After the cells are treated for 12 hours, the SOD value of the model group is very low and is about one third of the SOD value of a blank group (0.01449U/mg protein). when the cells are repaired by adding the recombinant human collagen-like peptide MJ L GG-34 and the natural type I collagen, the SOD value in the cells is increased, and the SOD value show significant difference relative to the model group, wherein the amount of the collagen-like peptide in the treatment group is increased by 152.14%, and the amount of the collagen in the treatment group is increased by 166.80%, which shows that the SOD value and the SOD value in the treatment group are H for the cells₂O₂The HaCAT oxidative damage caused by the method has better repairing effect.

Example 5

The prepared recombinant human collagen-like peptide MJ L GG-34 can inhibit melanogenesis of human melanoma cells

Adjusting the concentration of human melanoma cells A375 (purchased from Kunming cell Bank, Chinese academy of sciences) to 1 × 10⁵The cells per m L are inoculated on a 6-well plate, each well has 2m L, the cells after adherence are grouped by 25-200 mu mol L^-1MJ L GG-34 treatment (Polypeptide) of (9), control (Collagen I) with 200. mu. mol. L^-1The melanin content was determined by Tsuboi (1998) by centrifugation at 1500rpm for 5min and discarding the supernatant at 0.5m L with 10% DMSO 1mol L^-1And (3) cracking cells by using NaOH solution, carrying out ultrasonic disruption for 30min, carrying out water bath for 2h at 90 ℃ to ensure that cell aggregates are completely cracked, centrifuging for 15min at 3000rpm, taking supernatant to a 96-well plate, measuring an A value at 450nm of an enzyme labeling instrument, and calculating the relative content of melanin in the cells.

Relative content (%) of melanin synthesis equal to A1/A0 × 100

Wherein Al is the absorbance of the sample set, and A0 is the absorbance of the blank set

As shown in FIG. 7, 25 to 200. mu. mol · L^-1Recombinant human collagen-like peptide MJ L GG-34 in the range inhibits melanin synthesis in melanocytes concentration-dependently (P < 0.05) and 200. mu. mol. L after 48 hours of incubation of melanoma cells^-1MJ L GG-34 has the strongest inhibitory effect on melanin synthesis, and the melanin content is reduced by about 26.62% (73.38 +/-0.017; P)<0.01)。

Example 6

MJ L GG-34 can reduce the expression of cyclic adenosine dependent protein kinase catalytic subunit (PRKACA) and microphthalmia transcription factor (MITF) in the melanin synthesis signal pathway of human melanoma cells

Adjusting the concentration of human melanoma cells A375 to 1 × 10⁵Each m L, inoculated in 6-well plate, 2m L per well, grouped with 200. mu. mol L cells after adherence^-1MJ L GG-34 (polypide) and 200. mu. mol L^-1Treating natural human type I collagen (Collagen I), treating blank group (control) with PBS of equal amount, discarding supernatant after 48h, washing with precooled PBS for 2 times, discarding PBS, adding lysis solution containing PEMS 150 mu L/hole, shaking and evenly shaking for lysis on ice for 30min, transferring cell debris and lysis solution into a 1.5m L centrifuge tube after lysis is finished, centrifuging for 15min at 4 ℃ and 12000 r/min, separating centrifuged supernatant into a 200 mu L EP tube, storing at-70 ℃ for later use, carrying out SDS-charging after operation determination according to BCA protein concentration determination kit and balancing protein concentrationPAGE electrophoresis, membrane transfer and Western-blotting reaction, and the specific experimental steps are carried out according to the operation of a kit.

As shown in FIG. 8, the Western-blotting experiment results show that the prepared recombinant human collagen-like peptide MJ L GG-34 and the natural type I collagen act on A375 cells for 48h respectively, and compared with the blank group, the two drugs reduce the expression of the catalytic subunit of the cyclic adenosine monophosphate-dependent protein kinase (PRKACA) and the microphthalmia transcription factor (MITF) to different degrees, and the effect is obvious (P is P)<0.05) of which 200. mu. mol. L^-1After MJ L GG-34 acted on the cells for 48h, the MITF content was reduced by about 64.81% and the PRKACA content was reduced by 65.62% compared with the blank group.

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

Sequence listing

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of fusion protein Trx-6His-MJ L GG-34

<400>3

atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60

gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc120

ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180

atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240

ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300

aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat 360

catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 420

ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg 480

gctgatatcg gatccgaatt cggtgaaccg ggcaacccag gtcacaaagg ccacaaaggc 540

cagccgggcc agccgggtcc gccgggcgaa cgtgggccgc cgggcccgtg ctgtggtggt 600

ggctaactcg ag 612

<210>4

<211>201

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of fusion protein Trx-6His-MJ L GG-34

<400>4

Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp

1 5 10 15

Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp

20 25 30

Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp

35 40 45

Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn

50 55 60

Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu

65 70 75 80

Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

85 90 95

Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

100 105 110

Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro

115 120 125

Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln

130 135 140

His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met

145 150 155 160

Ala Asp Ile Gly Ser Glu Phe Gly Glu Pro Gly Asn Pro Gly His Lys

165 170 175

Gly His Lys Gly Gln Pro Gly Gln Pro Gly Pro Pro Gly Glu Arg Gly

180 185 190

Pro Pro Gly Pro Cys Cys Gly Gly Gly

195 200

<210>5

<211>57

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer T1+

<400>5

aattcggtga accgggcaac ccaggtcaca aaggccacaa aggccagccg ggccagc 57

<210>6

<211>57

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer T1-

<400>6

cccggctggc ccggctggcc tttgtggcct ttgtgacctg ggttgcccgg ttcaccg 57

<210>7

<211>60

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer T2+

<400>7

cgggtccgcc gggcgaacgt gggccgccgg gcccgtgctg tggtggtggc taactcgagg 60

<210>8

<211>60

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer T2-

<400>8

gatccctcga gttagccacc accacagcac gggcccggcg gcccacgttc gcccggcgga 60

Claims

1. A gene recombinant collagen peptide MJ L GG-34 is characterized in that the amino acid sequence is shown in SEQ ID NO. 1.

2. A nucleotide sequence encoding the genetically recombinant collagen-like peptide MJ L GG-34 as claimed in claim 1.

3. The nucleotide sequence of claim 2, wherein the nucleotide sequence encoding the genetically modified collagen-like peptide MJ L GG-34 is shown in SEQ ID NO. 2.

4. A preparation method of gene recombinant collagen-like peptide MJ L GG-34 is characterized by comprising the following steps:

(1) design and synthesis of MJ L GG-34 Gene:

primer T1 +:

5'-AATTCGGTGAACCGGGCAACCCAGGTCACAAAGGCCACAAAGGCCAGCCGGGCCAGC-3'；

primer T1-:

5'-CCCGGCTGGCCCGGCTGGCCTTTGTGGCCTTTGTGACCTGGGTTGCCCGGTTCACCG-3'；

primer T2 +:

5'-CGGGTCCGCCGGGCGAACGTGGGCCGCCGGGCCCGTGCTGTGGTGGTGGCtaaCTCGAGG-3'；

primer T2-:

5'-GATCCCTCGAGttaGCCACCACCACAGCACGGGCCCGGCGGCCCACGTTCGCCCGGCGGA-3'；

GAATTCis an EcoRI enzyme cutting site,CTCGAGis XhoI restriction site;

carrying out annealing synthesis reaction on the primer T1+ and the primer T1-, simultaneously carrying out annealing synthesis reaction on the primer T2+ and the primer T2-to respectively prepare a segment I and a segment II, and carrying out annealing synthesis reaction on the segment I and the segment II to obtain MJ L GG-34 gene;

(2) construction of the recombinant vector pET-32a-MJ L GG-34:

(4) expression and purification:

② the His-tagged fusion protein is purified by nickel column, wherein after loading the protein, the protein with histidine tag specifically binds to the nickel column, and other foreign proteins flow out, and Ni in the nickel column²⁺Can also be combined with imidazole, imidazole is used for gradient elution, imidazole is competitively combined on a nickel column to release fusion protein, and then eluent is collected;

③ cutting and purifying the target protein;

5. The method of claim 4, wherein:

the annealing synthesis reaction conditions in the step (1) are as follows: at 37 ℃ for 35 min; heating at 98 deg.C for 3 min; 95 ℃ for 5 min.

6. The method of claim 4, wherein:

l ysis equilibration buffer 50mM Na₂HPO₄And 0.3M NaCl, pH 8.0;

washing buffer solution: 50mM Na₂HPO₄0.3M NaCl and 10-50 mM imidazole, pH 8.0;

the composition of the solution for eluting the nickel column described in the step (4) ② was 50mM Na₂HPO₄0.3M NaCl and 250mM imidazole, pH 8.0.

7. The method of claim 4, wherein:

2) optimizing small-scale enzyme digestion;

3) amplifying the reaction ratio according to the optimal cutting result;

8. The method of claim 7, wherein:

the enzyme digestion condition obtained after the enzyme digestion optimization in the step 2) is that 100 mu g of fusion protein is cracked by 1U of enterokinase at the temperature of 22 ℃ and the enzyme digestion is carried out for 5 hours.

9. The method of claim 4, wherein:

the step of purifying the target protein by using the high performance liquid chromatography technique described in the step (4) ④ is as follows:

A. mobile phase A was obtained by adding TFA to 100% acetonitrile by volume at a final concentration of 0.1% by volume, and mobile phase B was obtained by adding TFA to 100% water at a final concentration of 0.1% by volume, eluting with a linear gradient of 25min at a flow rate of 1.0m L/min;

B. collecting target protein elution peaks from 90-65% v/v of a mobile phase B in linear gradient elution, wherein the light absorption detection wavelength is 220 nm; and carrying out mass spectrum identification.

10. The use of the genetically recombinant collagen-like peptide MJ L GG-34 as claimed in claim 1, wherein:

the gene recombinant collagen peptide MJ L GG-34 is used for preparing cosmetics, health products or foods with effects of resisting oxidation and reducing melanin synthesis.