CN109593127B

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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

Translated fromChinese

本发明公开一种基因重组胶原样肽MJLGG‑34及其制备方法与应用，属于基因工程技术领域。通过基因重组得到的胶原样肽MJLGG‑34，与天然胶原蛋白相比，分子量小容易制备，生物活性稳定，具有更好的透皮率和更高的生物活性，在保证生物活性的同时皮肤更易吸收；其生物相容性和生物安全性均佳，具有良好的抗氧化、抗凋亡、抑制黑色素生成等生物活性，可显著抑制皮肤的氧化损伤、调节黑色素的生成及抵抗衰老，且无明显的毒副作用；可用于食品、保健品、化妆品等，应用范围广泛，具有良好的应用价值和产业化前景。本发明的制备方法安全、生产效率高，生产成本低，应用前景广阔。

The invention discloses a gene recombinant collagen-like peptide MJLGG-34, a preparation method and application thereof, and belongs to the technical field of genetic engineering. Compared with natural collagen, the collagen-like peptide MJLGG-34 obtained by genetic recombination has a small molecular weight and is easy to prepare, with stable biological activity, better skin penetration rate and higher biological activity, while ensuring biological activity. Absorption; its biocompatibility and biosafety are good, with good anti-oxidation, anti-apoptosis, inhibition of melanin production and other biological activities, can significantly inhibit the oxidative damage of the skin, regulate the production of melanin and resist aging, and no obvious It can be used in food, health products, cosmetics, etc., with a wide range of applications and good application value and industrialization prospects. The preparation method of the invention has the advantages of safety, high production efficiency, low production cost and broad application prospect.

Description

Translated fromChinese

基因重组胶原样肽MJLGG-34及其制备方法与应用Gene recombinant collagen-like peptide MJLGG-34 and its preparation method and application

技术领域technical field

本发明属于基因工程技术领域，特别涉及一种基因重组人胶原样肽MJLGG-34及其制备方法与应用。The invention belongs to the technical field of genetic engineering, and particularly relates to a gene recombinant human collagen-like peptide MJLGG-34 and a preparation method and application thereof.

背景技术Background technique

胶原蛋白是很多动物体内含量最丰富的蛋白质，有着丰富的多样性和组织分布的特异性。胶原蛋白的类型、质量以及分布的改变直接影响动物体正常的机能，且其具有良好的生物相容性、低免疫原性和可生物降解性等优良特性，在生物医药、组织工程、食品、化妆品等领域有着广泛的应用。目前已经有研究表明胶原蛋白是改善皮肤水分含量的功能因子，不仅能够补充合成胶原所需要的氨基酸，也能起到修补受损细胞作用，改善细胞的营养状况和微环境，从而令肌肤重新焕发青春。Collagen is the most abundant protein in many animals, with rich diversity and specificity of tissue distribution. Changes in the type, quality and distribution of collagen directly affect the normal function of the animal body, and it has excellent properties such as good biocompatibility, low immunogenicity and biodegradability, and is widely used in biomedicine, tissue engineering, food, Cosmetics and other fields have a wide range of applications. At present, studies have shown that collagen is a functional factor for improving skin moisture content. It can not only supplement the amino acids required for collagen synthesis, but also repair damaged cells, improve the nutritional status and microenvironment of cells, and rejuvenate the skin. youth.

动物体内的胶原蛋白以胶原原纤维或胶原纤维的形式存在。其基本结构单位是原胶原分子，长度约为300nm，直径约为1.5nm，分子量约为300KDa，由三股相互缠绕的多肽链组成。在电子显微镜下，胶原纤维呈现特有的横纹区带，区带间距为60～70nm，这取决于胶原的类型和生物来源。原胶原蛋白分子在胶原纤维中有规则地按四分之一错位，首尾相接，并排列组成纤维束。Collagen in animals exists in the form of collagen fibrils or collagen fibers. Its basic structural unit is a procollagen molecule, with a length of about 300 nm, a diameter of about 1.5 nm, and a molecular weight of about 300 KDa. It consists of three intertwined polypeptide chains. Under the electron microscope, collagen fibers showed characteristic striated zones with a spacing of 60-70 nm, depending on the type of collagen and biological origin. Procollagen molecules are regularly displaced by quarters in collagen fibers, end-to-end, and arranged to form fiber bundles.

由于胶原属于大分子化合物，需在体内代谢为小分子短肽而发挥作用，利用特异性胶原蛋白酶剪切胶原蛋白肽链可以得到保有胶原蛋白生物活性并有与人体皮肤结构生物相容性高的寡肽，可添加至化妆品作为有效成分。通过特定的氨基酸替代和改变产生各种肽类似物，能够精确调节潜在疗法的效力，溶解度，毒性和成本。对于大多数其他分子和生物化合物来说，这种可及性和控制不易实现。Since collagen is a macromolecular compound, it needs to be metabolized into small molecule short peptides to play a role in the body. Using specific collagenase to cut the collagen peptide chain can obtain collagen that retains the biological activity of collagen and has high biocompatibility with human skin structure. Oligopeptides that can be added to cosmetics as active ingredients. Generation of various peptide analogs through specific amino acid substitutions and alterations enables precise tuning of the potency, solubility, toxicity and cost of potential therapeutics. This accessibility and control is not easily achieved for most other molecules and biological compounds.

胶原蛋白生产主要有一下三大类：传统提取法、化学合成法及现代生物技术生产法。传统提取的胶原含有一定的免疫原性，增加了其应用的潜在风险，化学合成法在合成过程中外消旋作用及有毒试剂的使用导致明显的健康和环境问题，而重组蛋白具有可加工性、排异性低、无病毒隐患等特点。这些都预示着基因重组小分子胶原样肽有良好的应用价值和产业化前景。There are three main types of collagen production: traditional extraction method, chemical synthesis method and modern biotechnology production method. Traditionally extracted collagen contains a certain immunogenicity, which increases the potential risk of its application. The chemical synthesis method causes obvious health and environmental problems due to racemization and the use of toxic reagents in the synthesis process, while the recombinant protein has processability, It has the characteristics of low rejection and no hidden virus. All these indicate that the recombinant small molecule collagen-like peptide has good application value and industrialization prospect.

发明内容SUMMARY OF THE INVENTION

为了克服现有技术的缺点与不足，本发明的首要目的在于提供一种基因重组胶原样肽MJLGG-34。该胶原样肽MJLGG-34具有更好的透皮率和更高的生物活性。In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a gene recombinant collagen-like peptide MJLGG-34. The collagen-like peptide MJLGG-34 has better penetration rate and higher biological activity.

本发明的另一目的在于提供所述基因重组胶原样肽MJLGG-34的制备方法。该制备方法根据胶原样肽MJLGG-34的氨基酸序列及大肠杆菌密码子的偏好性，设计MJLGG-34在原核表达系统的基因序列，采用基因工程技术，结合蛋白内含肽(intein)的可诱导剪切功能实现目的多肽MJLGG-34的高效制备；该制备方法安全、生产效率高，生产成本低。Another object of the present invention is to provide a method for preparing the gene recombinant collagen-like peptide MJLGG-34. The preparation method designs the gene sequence of MJLGG-34 in a prokaryotic expression system according to the amino acid sequence of the collagen-like peptide MJLGG-34 and the codon preference of Escherichia coli, and adopts genetic engineering technology to combine the inducible intein. The shearing function realizes the efficient preparation of the target polypeptide MJLGG-34; the preparation method is safe, high in production efficiency and low in production cost.

本发明的再一目的在于提供所述基因重组胶原样肽MJLGG-34的应用。Another object of the present invention is to provide the application of the genetically recombined collagen-like peptide MJLGG-34.

本发明的目的通过下述技术方案实现：The object of the present invention is achieved through the following technical solutions:

一种基因重组胶原样肽MJLGG-34，其肽段设计参考III型胶原α1链为模板，综合分子量对透皮吸收率及抗氧化性的影响将大小定在3KD左右，在保留天然胶原蛋白胶原域Gly-X-Y序列的高度重复前提下，将除Pro以外的疏水氨基酸替换为亲水氨基酸以改善亲水性能。引入对Cu²⁺有高亲和作用的GHK序列，改变Cu²⁺的经皮传送，同时减少Cu²⁺结合络氨酸酶，抑制黑色素的生成。研究表明GHK-Cu比单独任何一种药物能更好地诱导胶原产生以及加快伤口的愈合。GHK-Cu通过上调MMP-2、TIMP-1和TIMP-2的mRNA及蛋白质的水平诱导胶原蛋白的重构，同时能增加皮肤硫酸钠和硫酸软骨素的合成，最终减少皮肤松弛和皱纹。同时尾端的Cys有利于维持胞内谷胱甘肽体系，发挥抗氧化的作用，同时也是最好的络氨酸酶制剂。其氨基酸序列如下所示：GEPGNPGHKGHKGQPGQPGPPGERGPPGPCCGGG。A genetically recombined collagen-like peptide MJLGG-34, its peptide segment design refers to the α1 chain of type III collagen as a template, and the effect of comprehensive molecular weight on transdermal absorption rate and antioxidant activity is set at about 3KD, while retaining the natural collagen collagen. Under the premise of the highly repetitive domain Gly-XY sequence, hydrophobic amino acids other than Pro were replaced with hydrophilic amino acids to improve the hydrophilic properties. The introduction of a GHK sequence with high affinity for Cu²⁺ changes the transdermal delivery of Cu²⁺ , while reducing the binding of Cu²⁺ to tyrosinase and inhibiting the production of melanin. 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 the mRNA and protein levels of MMP-2, TIMP-1 and TIMP-2, while increasing the synthesis of sodium sulfate and chondroitin sulfate in the skin, ultimately reducing skin laxity and wrinkles. At the same time, the Cys at the tail is beneficial to maintain the intracellular glutathione system and play an antioxidant role, and it is also the best tyrosinase preparation. Its amino acid sequence is as follows: GEPGNPGHKGHKGQPGQPPGPPGERGPPGPCCGGG.

优化后编码所述的基因重组胶原样肽MJLGG-34的核苷酸序列为：The optimized nucleotide sequence encoding the recombinant collagen-like peptide MJLGG-34 is:

GGTGAACCGGGCAACCCAGGTCACAAAGGCCACAAAGGCCAGCCGGGCCAGCCGGGTCCGCCGGGCGAACGTGGGCCGCCGGGCCCGTGCTGTGGTGGTGGC；GGTGAACCGGGCAACCCAGGTCACAAAGGCCACAAAGGCCAGCCGGGCCAGCCGGGTCCGCCGGGCGAACGTGGGCCGCCGGGCCCGTGCTGTGGTGGTGGC;

目的蛋白MJLGG-34与pET-32a载体构建后的融合蛋白Trx-6His-MJLGG-34的核苷酸序列如下：The nucleotide sequence of the fusion protein Trx-6His-MJLGG-34 constructed by the target protein MJLGG-34 and the pET-32a vector is as follows:

融合蛋白Trx-6His-MJLGG-34的氨基酸序列如下所示：The amino acid sequence of the fusion protein Trx-6His-MJLGG-34 is shown below:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFGEPGNPGHKGHKGQPGQPGPPGERGPPGPCCGGGMSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFGEPGNPGHKGHKGQPGQPGPPGERGPPGPCCGGG

所述的基因重组胶原样肽MJLGG-34的制备方法，包括以下步骤：The preparation method of the described gene recombinant collagen-like peptide MJLGG-34 comprises the following steps:

(1)设计并合成MJLGG-34基因：(1) Design and synthesis of MJLGG-34 gene:

采用两对互补引物退火合成MJLGG-34基因：The MJLGG-34 gene was synthesized by annealing two pairs of complementary primers:

引物T1+：Primer T1+:

5'-AATTCGGTGAACCGGGCAACCCAGGTCACAAAGGCCACAAAGGCCAGCCGGGCCAGC-3'；5'-AATTC GGTGAACCGGGCAACCCAGGTCACAAAGGCCACAAAGGCCAGCCGGGCCAGC-3';

引物T1-：Primer T1-:

5'-CCCGGCTGGCCCGGCTGGCCTTTGTGGCCTTTGTGACCTGGGTTGCCCGGTTCACCG-3'；5'-CCCGGCTGGCCCGGCTGGCCTTTGTGGCCTTTGTGACCTGGGTTGCCCGGTTCACCG -3';

引物T2+：Primer T2+:

5'-CGGGTCCGCCGGGCGAACGTGGGCCGCCGGGCCCGTGCTGTGGTGGTGGCtaaCTCGAGG-3'；5'-CGGGTCCGCCGGGCGAACGTGGGCCGCCGGGCCCGTGCTGTGGTGGTGGCtaaCTCGAGG -3';

引物T2-：Primer T2-:

5'-GATCCCTCGAGttaGCCACCACCACAGCACGGGCCCGGCGGCCCACGTTCGCCCGGCGGA-3'；5'-GATCCCTCGAG ttaGCCACCACCACAGCACGGGCCCCGGCGGCCCACGTTCGCCCGGCGGA-3';

GAATTC为EcoRI酶切位点，CTCGAG为XhoI酶切位点。GAATTC is the EcoRI restriction site,CTCGAG is the XhoI restriction site.

将引物T1+和引物T1-进行退火合成反应，同时将引物T2+和引物T2-进行退火合成反应，分别制得片段一和片段二；The primer T1+ and the primer T1- are subjected to an annealing synthesis reaction, while the primer T2+ and the primer T2- are subjected to an annealing synthesis reaction to obtain the fragment one and the fragment two respectively;

(2)构建重组载体pET-32a-MJLGG-34：(2) Construction of recombinant vector pET-32a-MJLGG-34:

用EcoRI酶和XhoI酶分别对质粒pET-32a和步骤(1)制得的MJLGG-34基因进行双酶切，再将双酶切后得到的MJLGG-34基因和双酶切后的质粒pET-32a连接，得到重组载体pET-32a-MJLGG-34；The plasmid pET-32a and the MJLGG-34 gene obtained in step (1) were subjected to double digestion with EcoRI enzyme and XhoI enzyme, respectively, and then the MJLGG-34 gene obtained after double digestion and the double digestion plasmid pET- 32a was connected to obtain the recombinant vector pET-32a-MJLGG-34;

(3)制备表达工程菌pET-32a-MJLGG-34/BL21(DE3)：(3) Preparation and expression of engineering strain pET-32a-MJLGG-34/BL21(DE3):

用重组载体pET-32a-MJLGG-34转化表达宿主大肠杆菌E.coli BL21(DE3)，得到表达工程菌pET-32a-MJLGG-34/BL21(DE3)；Transform the expression host E. coli BL21(DE3) with the recombinant vector pET-32a-MJLGG-34 to obtain the expression engineering strain pET-32a-MJLGG-34/BL21(DE3);

(4)表达和纯化：(4) Expression and purification:

①诱导表达工程菌pET-32a-MJLGG-34/BL21(DE3)表达由目的多肽、His标签蛋白和Trx标签蛋白等组成的融合蛋白；①Induce the expression of engineering bacteria pET-32a-MJLGG-34/BL21(DE3) to express a fusion protein composed of target polypeptide, His-tagged protein and Trx-tagged protein;

②得到的His标签融合蛋白用镍柱进行纯化：在蛋白上样后，带有组氨酸标签的蛋白特异性结合到镍柱，其他的杂蛋白流出；镍柱中的Ni²⁺也可以与咪唑结合，用咪唑梯度洗脱，咪唑竞争性结合到镍柱上，释放融合蛋白，然后收集洗脱液；②The obtained His-tagged fusion protein is purified with a nickel column: after the protein is loaded, the protein with histidine tag specifically binds to the nickel column, and other impurity proteins flow out; Ni²⁺ in the nickel column can also The imidazole is bound and eluted with an imidazole gradient, and the imidazole is competitively bound to the nickel column to release the fusion protein, and then the eluate is collected;

③目的蛋白的切割和纯化；③Cut and purify the target protein;

④利用高效液相色谱技术纯化目的蛋白，得到基因重组胶原样肽MJLGG-34。④The target protein was purified by high performance liquid chromatography to obtain the recombinant collagen-like peptide MJLGG-34.

步骤(1)中所述的退火合成反应的条件优选为：37℃，35min；98℃，3min；95℃，5min；The conditions of the annealing synthesis reaction described in step (1) are preferably: 37°C, 35min; 98°C, 3min; 95°C, 5min;

步骤(4)②纯化所用的平衡缓冲液、洗涤缓冲液的组成如下：The composition of the equilibration buffer and washing buffer used in step (4) ② purification is as follows:

Lysis平衡缓冲液(LE Buffer)：50mM Na₂HPO₄，0.3M NaCl，pH＝8.0；Lysis equilibration buffer (LE Buffer): 50 mM Na₂ HPO₄ , 0.3 M NaCl, pH=8.0;

洗涤缓冲液：50mM Na₂HPO₄，0.3M NaCl，10～50mM imidazole，pH＝8.0；Washing buffer: 50 mM Na₂ HPO₄ , 0.3 M NaCl, 10-50 mM imidazole, pH=8.0;

步骤(4)②中所述的洗脱镍柱的溶液的组成优选如下：50mM Na₂HPO₄，0.3M NaCl，250mM imidazole，pH＝8.0；The composition of the solution for eluting the nickel column described in step (4) ② is preferably as follows: 50mM Na₂ HPO₄ , 0.3M NaCl, 250mM imidazole, pH=8.0;

步骤(4)③中所述目的蛋白的切割和纯化步骤如下：The cleavage and purification steps of the target protein described in step (4) ③ are as follows:

1)将融合蛋白洗脱液透析到20mM Tris-HCl，pH 8.0或者1×PBS，pH 7.4中；1) Dialyze the fusion protein eluate to 20mM Tris-HCl, pH 8.0 or 1×PBS, pH 7.4;

2)小规模酶切优化：2) Small-scale enzyme digestion optimization:

a)在EK稀释/储存缓冲液(EK Dilution/Storage Buffer)中对肠激酶(EK，5IU/μL)进行4次连续稀释(0.001IU，0.01IU，0.1IU和1IU酶/μL)。a) 4 serial dilutions (0.001 IU, 0.01 IU, 0.1 IU and 1 IU enzyme/μL) of enterokinase (EK, 5 IU/μL) in EK Dilution/Storage Buffer.

b)设置5个反应，包括没有EK的反应用于对照：b) Set up 5 reactions, including the reaction without EK for control:

Fusion Protein(融合蛋白)Fusion Protein10μg10μg10×EK Cleavage/Capture Buffer10×EK Cleavage/Capture Buffer5μL5μLDiluted EK(add 2μL EK Dilution/Storage Buffer for the control)Diluted EK (add 2μL EK Dilution/Storage Buffer for the control)2μL2μLDeionized Water(去离子水)Deionized Water至50μLto 50 μLTotal volumeTotal volume50μL50μL

c)充分混合并在22℃下孵育1小时，3小时，5小时并过夜(～16小时)。c) Mix well and incubate at 22°C for 1 hour, 3 hours, 5 hours and overnight (~16 hours).

d)在SDS-PAGE凝胶上加载10μL以确定最佳切割结果；如果结果不合适，可以测试温度的优化。结果显示EK酶切优化条件为：1U肠激酶22℃条件下裂解100μg融合蛋白，酶切5h。d) Load 10 μL on an SDS-PAGE gel to determine optimal cleavage results; if results are not suitable, optimization of temperature can be tested. The results showed that the optimal conditions for EK digestion were as follows: 1U enterokinase was cleaved at 22℃ for 100μg fusion protein, and the digestion was performed for 5h.

3)根据最佳切割结果放大反应比例。3) Scale up the reaction ratio according to the best cleavage result.

4)放大反应酶切后，将酶切体系过事先平衡好的Ni-NTA树脂中，收集穿透液，用含250mmol/L咪唑的洗脱缓冲液洗脱柱子上的标签蛋白和酶切的碎蛋白，洗脱总体积为柱床的5倍，从而将目的蛋白与标签蛋白分离开来，得到纯化的目的蛋白。4) After the amplification reaction, pass the enzyme digestion system through the pre-equilibrated Ni-NTA resin, collect the permeate, and use the elution buffer containing 250mmol/L imidazole to elute the tagged protein and the enzyme-digested protein on the column. Crushed protein, the total elution volume is 5 times that of the column bed, so as to separate the target protein from the tag protein to obtain the purified target protein.

步骤(4)④中所述的利用高效液相色谱技术纯化目的蛋白MJLGG-34的步骤如下：The steps of utilizing high performance liquid chromatography to purify target protein MJLGG-34 described in step (4) ④ are as follows:

A、流动相A为在体积百分比100％乙腈中添加三氟乙酸(TFA)得到，TFA的终浓度为体积百分比0.1％；流动相B为100％水中添加三氟乙酸(TFA)，TFA的终浓度为体积百分比0.1％；流速1.0mL/min，25min线性梯度洗脱；A. Mobile phase A is obtained by adding trifluoroacetic acid (TFA) to 100% acetonitrile by volume, and the final concentration of TFA is 0.1% by volume; mobile phase B is adding trifluoroacetic acid (TFA) to 100% water, and the final concentration of TFA is The concentration is 0.1% by volume; the flow rate is 1.0 mL/min, and the 25min linear gradient elution;

B、线性梯度洗脱中流动相B由90～65％(v/v)，收集目的蛋白洗脱峰，光吸收检测波长为220nm；并进行质谱鉴定。B. In the linear gradient elution, the mobile phase B is 90-65% (v/v), and the target protein elution peak is collected, and the light absorption detection wavelength is 220 nm; and mass spectrometry identification is carried out.

所述的基因重组胶原样肽MJLGG-34应用于制备美容化妆品、保健品或食品原料，有如下作用：抗氧化、抑制人真皮成纤维细胞凋亡、抑制黑色素合成等功能。The genetically recombined collagen-like peptide MJLGG-34 is used in the preparation of cosmetic cosmetics, health care products or food raw materials, and has the following functions: anti-oxidation, inhibiting apoptosis of human dermal fibroblasts, inhibiting melanin synthesis and the like.

本发明相对于现有技术具有如下的优点及效果：Compared with the prior art, the present invention has the following advantages and effects:

(1)、本发明设计的胶原样肽基因MJLGG-34是全新的序列，长度远小于天然人胶原蛋白基因，分子水平操作更简易化，且其翻译的多肽分子量小，容易制备，生物活性稳定，在保证生物活性的同时皮肤更易吸收，可显著抑制皮肤的氧化损伤抵抗衰老；(1) The collagen-like peptide gene MJLGG-34 designed by the present invention is a brand-new sequence, the length is much smaller than that of the natural human collagen gene, the molecular level operation is simpler, and the molecular weight of the translated polypeptide is small, easy to prepare, and stable in biological activity , while ensuring biological activity, the skin is more easily absorbed, which can significantly inhibit the oxidative damage of the skin and resist aging;

(2)、本发明根据大肠杆菌密码子的偏好性，对胶原样肽的基因序列进行密码子优化，消除了胶原样肽表达中因密码子利用限制导致的低翻译效率，使其更适于在大肠杆菌中表达；(2) According to the codon preference of Escherichia coli, the present invention performs codon optimization on the gene sequence of collagen-like peptides, which eliminates the low translation efficiency caused by the restriction of codon utilization in the expression of collagen-like peptides, making it more suitable for Expressed in E. coli;

(3)、本发明方法所制备的基因重组人胶原样肽MJLGG-34，与动物来源的天然胶原蛋白相比较，其生物相容性和生物安全性均佳，具有良好的抗氧化、抗凋亡、抑制黑色素生成等生物活性，同时结果显示，制备的基因重组胶原样肽MJLGG-34通过减少中环腺苷酸依赖性蛋白激酶催化亚基(PRKACA)和小眼转录因子(MITF)的表达抑制黑素合成，无明显的毒副作用表现；可用于食品、保健品、化妆品等，应用范围广泛，具有良好的应用价值和产业化前景；(3) The recombinant human collagen-like peptide MJLGG-34 prepared by the method of the present invention has good biocompatibility and biosafety compared with natural collagen derived from animals, and has good anti-oxidation and anti-withering properties. At the same time, the results showed that the prepared gene recombinant collagen-like peptide MJLGG-34 inhibited the expression of middle cyclic adenylate-dependent protein kinase catalytic subunit (PRKACA) and microphthalmia transcription factor (MITF). Melanin synthesis, no obvious side effects; can be used in food, health products, cosmetics, etc., with a wide range of applications, with good application value and industrialization prospects;

(4)本发明所述重组人胶原样肽MJLGG-34的制备方法效率高、成本低，应用前景广阔。(4) The preparation method of the recombinant human collagen-like peptide MJLGG-34 of the present invention has high efficiency, low cost and broad application prospects.

附图说明Description of drawings

图1是质粒pET-32a及重组表达质粒pET-32a-MJLGG-34酶切示意图。Figure 1 is a schematic diagram of the digestion of plasmid pET-32a and recombinant expression plasmid pET-32a-MJLGG-34.

图2是SDS-PAGE检测His融合蛋白Trx-6His-MJLGG-34表达的鉴定图；其中，泳道1是IPTG诱导前全菌蛋白；泳道2是IPTG诱导前全菌上清；泳道3是IPTG诱导后的全菌蛋白；泳道4、5均是IPTG诱导后的全菌上清；泳道6是诱导后菌体破碎上清液；泳道7是诱导后菌体破碎沉淀。Figure 2 is the identification diagram of the expression of His fusion protein Trx-6His-MJLGG-34 detected by SDS-PAGE; wherein,lane 1 is the whole cell protein before IPTG induction;lane 2 is the supernatant of the whole cell before IPTG induction;lane 3 is IPTGinduction Swimming lanes 4 and 5 are the whole microbial supernatant after IPTG induction;Swimming lane 6 is the supernatant after the induction of the microbial cell disruption;Swimming lane 7 is the microbial cell crushing precipitate after the induction.

图3是SDS-PAGE检测His融合蛋白Trx-6His-MJLGG-34纯化的鉴定图；其中，泳道1是纯化前菌体破碎上清液；泳道2上柱样品流出液；泳道3是洗涤流出液；泳道4是250mmol/L咪唑纯化后的蛋白。Fig. 3 is the identification diagram of the purification of His fusion protein Trx-6His-MJLGG-34 detected by SDS-PAGE; wherein,lane 1 is the supernatant of the cell fragmentation before purification;lane 2 is the effluent of the column sample;lane 3 is the washing effluent ;Lane 4 is the protein purified with 250 mmol/L imidazole.

图4是制备的重组人胶原样肽MJLGG-34的飞行质谱鉴定图。Fig. 4 is the identification chart of flight mass spectrometry of the prepared recombinant human collagen-like peptide MJLGG-34.

图5为制备的重组人胶原样肽MJLGG-34的DPPH清除作用检测结果图；其中，A：样品一；B：样品二。Fig. 5 is a graph showing the detection result of the DPPH scavenging effect of the prepared recombinant human collagen-like peptide MJLGG-34; wherein, A: sample one; B: sample two.

图6为重组人胶原样肽MJLGG-34缓解H₂O₂导致的HaCAT细胞氧化应激损伤的检测结果图。Figure 6 is a graph showing the detection results of recombinant human collagen-like peptide MJLGG-34 alleviating H₂ O₂ -induced oxidative stress injury in HaCAT cells.

图7为重组人胶原样肽MJLGG-34抑制人黑色素瘤细胞的黑素生成检测结果图。FIG. 7 is a graph showing the detection results of recombinant human collagen-like peptide MJLGG-34 inhibiting the melanogenesis of human melanoma cells.

图8为重组人胶原样肽MJLGG-34减少人黑色素瘤细胞黑素合成信号通路中环腺苷酸依赖性蛋白激酶催化亚基和小眼转录因子表达的检测结果图。Figure 8 is a graph showing the detection results of recombinant human collagen-like peptide MJLGG-34 reducing the expression of cyclic adenylate-dependent protein kinase catalytic subunit and ommatidium transcription factor in the melanoma synthesis signal pathway of human melanoma cells.

具体实施方式Detailed ways

下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。The present invention will be described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

下列实施例中未注明具体实验条件的试验方法，通常按照常规实验条件或按照制造厂所建议的实验条件。所使用的材料、试剂等，如无特殊说明，为从商业途径得到的试剂和材料。The test methods that do not specify specific experimental conditions in the following examples are usually in accordance with conventional experimental conditions or in accordance with experimental conditions suggested by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, are the reagents and materials obtained from commercial sources.

实施例1Example 1

表达工程菌pET-32a-MJLGG-34/BL21(DE3)的构建和表达Construction and Expression of Expression Engineering Bacteria pET-32a-MJLGG-34/BL21(DE3)

具体步骤如下：Specific steps are as follows:

(1)设计并合成MJLGG-34基因：(1) Design and synthesis of MJLGG-34 gene:

按照大肠杆菌的密码偏好性设计编码MJLGG-34的cDNA，设计两对引物，采用两对互补引物退火合成MJLGG-34基因：The cDNA encoding MJLGG-34 was designed according to the code preference of E. coli, two pairs of primers were designed, and the MJLGG-34 gene was synthesized by annealing two pairs of complementary primers:

引物T1+：Primer T1+:

引物T1-：Primer T1-:

引物T2+：Primer T2+:

引物T2-：Primer T2-:

其中，GAATTC为EcoRI酶切位点，CTCGAG为XhoI酶切位点。Among them,GAATTC is the EcoRI restriction site, andCTCGAG is the XhoI restriction site.

退火各个成分的用量：(引物浓度为1OD溶于400μL ddH₂O，反应体系：20μL)The amount of each component for annealing: (primer concentration is 1OD dissolved in 400μL ddH₂ O, reaction system: 20μL)

片段一：引物T1+2μL、引物T1-5μL、T4PNK 1μL(10U)、ATP 20mM、ddH₂O补水至20μL；Fragment 1: primer T1+2μL, primer T1-5μL, T4PNK 1μL (10U), ATP 20mM, ddH₂ O to make up to 20μL;

片段二：引物T2+2μL、引物T2-2μL、T4PNK 1μL(10U)、ATP 20mM、ddH₂O补水至20μL；Fragment 2: Primer T2+2μL, Primer T2-2μL, T4PNK 1μL (10U), ATP 20mM, ddH₂ O water to 20μL;

退火反应程序：37℃，35min；98℃，3min；95℃，5min；Annealing reaction program: 37℃, 35min; 98℃, 3min; 95℃, 5min;

自然冷却至室温，退火产物备用。Naturally cooled to room temperature, and the annealed product was used for later use.

用EcoRI酶和XhoI酶分别对质粒pET-32a(购自上海生工生物工程有限公司)和步骤(1)制得的MJLGG-34基因进行双酶切，酶切反应体系：pET-32a 2μg、10×FD Buffer 5μL、EcoRI 1μL(10U/μL)、XhoI 1μL(10U/μL)、ddH₂O 42μL，以上体系放入37℃恒温水浴锅中反应2h；再将双酶切后得到的MJLGG-34基因和双酶切后的质粒pET-32a连接，连接体系20μL：片段一1μL、片段二1μL、酶切载体pET-32a 4μL、10×T4DNAligase Buffer 2μL、T4DNAligase 1μL(5U/μL)、ddH₂O补充至20μL，上述连接混合液放在16℃恒温1h即可，得到重组载体pET-32a-MJLGG-34，重组载体pET-32a-MJLGG-34酶切鉴定如图1所示；The plasmid pET-32a (purchased from Shanghai Sangon Bioengineering Co., Ltd.) and the MJLGG-34 gene obtained in step (1) were double-enzyme digested with EcoRI enzyme and XhoI enzyme, respectively. The enzyme digestion reaction system: pET-32a 2μg, 10×FD Buffer 5μL, EcoRI 1μL (10U/μL), XhoI 1μL (10U/μL), ddH₂ O 42μL, the above system was placed in a constant temperature water bath at 37°C for 2h reaction; MJLGG- The 34 gene was ligated with the double-enzyme-digested plasmid pET-32a, and the ligation system was 20 μL:fragment 1 1 μL,fragment 2 1 μL, digested vector pET-32a 4 μL, 10×T4DNAligase Buffer 2 μL,T4DNAligase 1 μL (5U/μL), ddH₂ O supplemented to 20 μL, and the above ligation mixture was kept at 16°C for 1 h to obtain the recombinant vector pET-32a-MJLGG-34. The restriction enzyme digestion identification of the recombinant vector pET-32a-MJLGG-34 is shown in Figure 1;

用重组载体pET-32a-MJLGG-34转化表达宿主大肠杆菌E.coli BL21(DE3)(购自上海生工生物工程有限公司)，得到表达工程菌pET-32a-MJLGG-34/BL21(DE3)；The recombinant vector pET-32a-MJLGG-34 was used to transform the expression host E. coli BL21(DE3) (purchased from Shanghai Sangon Bioengineering Co., Ltd.) to obtain the expression engineering strain pET-32a-MJLGG-34/BL21(DE3) ;

(4)表达和纯化：(4) Expression and purification:

从保种的EP管中取50μL表达工程菌菌液于5mL含有100μg/mL氨苄青霉素的LB培养基中，37℃、220rpm摇菌培养过夜，再扩大，以体积比为1：100的比例接于50mL含100μg/mL氨苄青霉素的LB培养基，37℃、220rpm摇菌至OD₆₀₀为0.8～1.0左右，加入异丙基-β-D硫代半乳糖苷(IPTG)至终浓度0.5mmol/L，37℃诱导表达6h。5000rpm离心30min收集菌体，将菌体重悬于10mL平衡缓冲液(50mM Na₂HPO₄，0.3M NaCl，pH＝8.0)中，然后超声破碎细胞，在冰上进行操作，破碎1秒，冷却3秒，总时间为30～45分钟。Take 50 μL of the expression engineered bacteria from the preserved EP tube and put it in 5 mL of LB medium containing 100 μg/mL ampicillin. In 50 mL of LB medium containing 100 μg/mL ampicillin, shake the bacteria at 37 °C and 220 rpm until the OD₆₀₀ is about 0.8 to 1.0, and add isopropyl-β-D thiogalactoside (IPTG) to a final concentration of 0.5 mmol/ L, 37 ℃ induced expression for 6h. The cells were collected by centrifugation at 5000 rpm for 30 min, and the cells were resuspended in 10 mL of equilibration buffer (50 mM Na₂ HPO₄ , 0.3 M NaCl, pH=8.0), and then the cells were disrupted by sonication, operated on ice, disrupted for 1 second, and cooled for 3 seconds, the total time is 30 to 45 minutes.

破碎产物在4℃，12000rpm离心15～20mim，收集上清，该上清称为破碎上清液。SDS-PAGE检测His融合蛋白Trx-6His-MJLGG-34的表达，鉴定结果如图2所示。表明His融合蛋白表达成功，且以上清表达为主。The crushed product was centrifuged at 4°C and 12000rpm for 15-20mim, and the supernatant was collected, which was called crushed supernatant. The expression of His fusion protein Trx-6His-MJLGG-34 was detected by SDS-PAGE, and the identification results are shown in Figure 2. It indicated that the expression of His fusion protein was successful, and the supernatant was mainly expressed.

融合蛋白Trx-6His-MJLGG-34的核苷酸序列如SEQ ID NO：3，其氨基酸序列为SEQID NO：4。The nucleotide sequence of the fusion protein Trx-6His-MJLGG-34 is as SEQ ID NO: 3, and the amino acid sequence thereof is SEQ ID NO: 4.

实施例2Example 2

重组人胶原样肽MJLGG-34的纯化、制备和鉴定Purification, preparation and identification of recombinant human collagen-like peptide MJLGG-34

轻轻颠倒翻转瓶子几次，使介质混合均匀，吸取2mL的介质加入到柱子(Ni-NTA柱，金斯瑞生物科技有限公司，10mL柱体积)中，让介质自由沉降，并放干储存液，加入4倍柱体积的平衡缓冲液平衡层析介质；用破碎上清液以1.0mL/min的流速上柱，收集流出液以待后续分析；用8倍柱体积的洗涤缓冲液(50mM Na₂HPO₄，0.3M NaCl，10～50mM imidazole，pH＝8.0)以1.0mL/min过柱，以洗去杂蛋白或未亲和结合的融合蛋白，收集流出液以待后续分析；用10倍柱体积的洗脱缓冲液以1.0mL/min过柱，从第二管开始收集洗脱液。Gently invert the bottle several times to mix the medium evenly, pipette 2mL of the medium into the column (Ni-NTA column, GenScript Biotechnology Co., Ltd., 10mL column volume), let the medium settle freely, and drain the storage solution , add 4 column volumes of equilibration buffer to equilibrate the chromatography medium; apply the crushed supernatant to the column at a flow rate of 1.0 mL/min, and collect the effluent for subsequent analysis;use 8 column volumes of washing buffer (50 mM Na₂ HPO₄ , 0.3M NaCl, 10-50mM imidazole, pH=8.0) through the column at 1.0mL/min to wash off impurity proteins or unaffinity-bound fusion proteins, and collect the effluent for subsequent analysis; use 10 times The column volume of elution buffer was passed through the column at 1.0 mL/min, and the eluate was collected from the second tube.

取样品流出液，洗涤流出液，洗脱流出液进行SDS-PAGE检测His融合蛋白的纯化效果(结果如图3所示)。表明在用250mM咪唑的洗脱液中得到了除去了大部分杂质的His融合蛋白。The sample effluent was taken, washed, and the eluted effluent was subjected to SDS-PAGE to detect the purification effect of the His fusion protein (the results are shown in Figure 3). It was shown that the His fusion protein with most impurities removed was obtained in the eluate with 250 mM imidazole.

对目的蛋白进行切割和纯化，步骤如下：To cut and purify the target protein, the steps are as follows:

2)小规模酶切优化：2) Small-scale digestion optimization:

d)在SDS-PAGE凝胶上加载10μL以确定最佳切割结果；结果显示EK酶切优化条件为：1U肠激酶22℃条件下裂解100μg融合蛋白，酶切5h。d) Load 10 μL on the SDS-PAGE gel to determine the best cleavage result; the results show that the optimal conditions for EK digestion are: 1 U enterokinase to cleave 100 μg of fusion protein at 22°C for 5 h.

再利用高效液相色谱(HPLC)纯化目的蛋白MJLGG-34，步骤如下：Then use high performance liquid chromatography (HPLC) to purify the target protein MJLGG-34, the steps are as follows:

B、线性梯度洗脱中流动相B由90～65％(v/v)，收集目的蛋白洗脱峰，光吸收检测波长为220nm。制备得到了纯度为质量百分比99.3％的目的多肽。B. In the linear gradient elution, the mobile phase B is 90-65% (v/v), and the elution peak of the target protein is collected, and the optical absorption detection wavelength is 220 nm. The target polypeptide with a purity of 99.3% by mass was prepared.

制备的目的多肽MJLGG-34进行质谱鉴定(结果如图4所示)，质谱检测分子量为3.215kDa.，其与理论值一致，表明经EK酶切并纯化后成功地将目的小肽与标签蛋白分离开来，得到正确的目的蛋白。The prepared target polypeptide MJLGG-34 was identified by mass spectrometry (the results are shown in Figure 4), and the molecular weight detected by mass spectrometry was 3.215kDa. separated to obtain the correct target protein.

实施例3Example 3

制备的重组人胶原样肽MJLGG-34的抗氧化能力Antioxidative ability of prepared recombinant human collagen-like peptide MJLGG-34

(1)DPPH的清除作用(1) The scavenging effect of DPPH

取多肽适量用PBS(pH7.5，0.02M)溶解成0.3mM的溶液，加入1.5％的胰蛋白酶水解4h后沸水浴5min灭活酶，终止水解制成样品一；另取多肽适量用PBS(pH7.5，0.02M)溶解成3mM溶液为样品二。Take an appropriate amount of polypeptide and dissolve it into a 0.3mM solution with PBS (pH 7.5, 0.02M), add 1.5% trypsin to hydrolyze it for 4 hours, and then inactivate the enzyme in a boiling water bath for 5 minutes, and stop the hydrolysis to preparesample 1; another appropriate amount of polypeptide is dissolved in PBS ( pH7.5, 0.02M) was dissolved into a 3mM solution as sample two.

分别取0～250μL样品一和样品二于1.5mL离心管，按浓度梯度分别加入250～500μL乙醇溶液，再加入1mL 0.1mM DPPH·乙醇溶液，混匀，暗处放置30min，不断震荡，测定517nm处的吸光值。Take 0-250 μL ofsample 1 andsample 2 respectively in a 1.5 mL centrifuge tube, add 250-500 μL of ethanol solution according to the concentration gradient, then add 1 mL of 0.1 mM DPPH·ethanol solution, mix well, place in the dark for 30 min, shake continuously, and measure 517 nm absorbance value at .

DPPH·清除能力计算公式为：DPPH·抑制率(％)＝(Ao—A1)/Ao×100The calculation formula of DPPH·scavenging ability is: DPPH·inhibition rate (%)=(Ao-A1)/Ao×100

其中Ao为未加样品的空白的吸光值，A1为加入样品后的吸光值。Wherein Ao is the absorbance value of the blank without sample added, and A1 is the absorbance value after adding the sample.

实验结果如图5所示，A图随着多肽水解液浓度的升高，清除DPPH的能力逐渐增强，在浓度为0.125mM时，清除率达到58.89％；B图完整的胶原样肽MJLGG-34的DPPH清除率同样显示出浓度依赖性，随着胶原样肽浓度的升高，清除DPPH的能力逐渐增强，在浓度为1.25mM时，清除率达到62.14％。The experimental results are shown in Figure 5. As the concentration of the polypeptide hydrolyzate increases, the ability to clear DPPH gradually increases in picture A, and when the concentration is 0.125 mM, the clearance rate reaches 58.89%; picture B, the intact collagen-like peptide MJLGG-34 The DPPH clearance rate also showed a concentration dependence, with the increase of collagen-like peptide concentration, the ability to clear DPPH gradually increased, and the clearance rate reached 62.14% when the concentration was 1.25mM.

实施例4Example 4

重组人胶原样肽MJLGG-34缓解H₂O₂导致的HaCAT细胞氧化应激损伤Recombinant human collagen-like peptide MJLGG-34 alleviates H₂ O₂ -induced oxidative stress injury in HaCAT cells

HaCAT细胞(购自中国科学院昆明细胞库)接种于6孔细胞培养板中，于37℃、5％CO₂培养箱中培养，待细胞生长至亚融合状态时，换入新鲜培养基。空白组(Blankcontrol)：生理盐水；模型组(Model)：生理盐水；胶原对照组(Collangen I)：200μmol·L^-1天然人I型胶原蛋白；重组胶原样肽组(Polypeptide)：200μmol·L^-1制备的MJLGG-34。除空白组外，其余三组在加药前，用300μmol·L^-1的H₂O₂处理12h。加药作用48h后，吸取一定量培养液于1mL离心管中留样待测。吸弃剩余培养液，用预冷的PBS缓冲液洗1～2次，刮下贴壁细胞，加入1×D-Hank’s缓冲液1mL，用移液枪轻轻混匀，反复冻融3次，3500r·min^-1、4℃离心10min，取上清液待测。按照试剂盒说明书操作，测定细胞裂解液中SOD的含量。HaCAT cells (purchased from Kunming Cell Bank, Chinese Academy of Sciences) were seeded in 6-well cell culture plates and cultured in a 37°C, 5%_CO2 incubator, and when the cells grew to a subconfluent state, they were replaced with fresh medium. Blank control: physiological saline; model group: physiological saline; collagen control group (Collangen I): 200 μmol·L^-1 natural human type I collagen; recombinant collagen-like peptide group (Polypeptide): 200 μmol·L^-1 MJLGG-34 prepared. Except the blank group, the other three groups were treated with 300 μmol·L^-1 of H₂ O₂ for 12 h before dosing. After 48 hours of dosing, a certain amount of culture medium was drawn into a 1 mL centrifuge tube to retain a sample for testing. Aspirate and discard the remaining culture medium, wash 1-2 times with pre-cooled PBS buffer, scrape off adherent cells, add 1 mL of 1×D-Hank's buffer, mix gently with a pipette, and freeze andthaw 3 times. Centrifuge at 3500r·min^-1 and 4°C for 10min, and take the supernatant for testing. According to the kit instructions, measure the SOD content in the cell lysate.

如图6所示，当用采用300μmol·L^-1H₂O₂处理细胞12h后，模型组SOD值非常低，约只有空白组SOD值(0.01449U/mg protein)的三分之一。当加入重组人胶原样肽MJLGG-34及天然I型胶原蛋白对细胞进行修复后，细胞内的SOD值有所增加，两者相对于模型组均表现出显著性差异，其中胶原样肽处理组增加了152.14％，I型胶原处理组增加了166.80％，这表明二者对H₂O₂造成的HaCAT氧化损伤有较好的修复作用。As shown in Figure 6, when cells were treated with 300 μmol·L^-1 H₂ O₂ for 12 h, the SOD value of the model group was very low, about one third of the SOD value of the blank group (0.01449U/mg protein). When the cells were repaired by adding recombinant human collagen-like peptide MJLGG-34 and natural type I collagen, the SOD value in the cells increased, and both showed significant differences compared with the model group, among which the collagen-like peptide treatment group It increased by 152.14%, and the collagen type I treatment group increased by 166.80%, which indicated that the two had better repairing effect on the oxidative damage of HaCAT caused by H₂ O₂ .

实施例5Example 5

制备的重组人胶原样肽MJLGG-34抑制人黑色素瘤细胞的黑素生成The prepared recombinant human collagen-like peptide MJLGG-34 inhibits the melanogenesis of human melanoma cells

调整人黑素瘤细胞A375(购自中国科学院昆明细胞库)浓度至1×10⁵个/mL，接种于6孔板，每孔2mL。贴壁后细胞分组分别用25～200μmol·L^-1的MJLGG-34处理(Polypeptide)，对照组(Collangen I)用200μmol·L^-1天然人I型胶原蛋白处理，空白组(control)用生理盐水处理，48h后用预冷的PBS洗涤3次刮下收集于无菌EP管中。黑色素含量的测定采用Tsuboi(1998)的方法，1500rpm离心5min弃上清，以0.5mL含10％DMSO的1mol·L^-1NaOH溶液裂解细胞，超声波破碎30min，90℃水浴2h，使细胞团块完全裂解后，3000rpm离心15min，取上清至96孔板于酶标仪450nm处测A值，计算细胞黑色素相对含量。The concentration of human melanoma cells A375 (purchased from Kunming Cell Bank, Chinese Academy of Sciences) was adjusted to 1×10⁵ cells/mL, and inoculated into 6-well plates with 2 mL per well. After adherence, the cells were grouped with 25-200 μmol·L^-1 MJLGG-34 (Polypeptide), the control group (Collangen I) was treated with 200 μmol·L^-1 natural human type I collagen, and the blank group (control) was treated with physiological After being treated with saline, washed 3 times with pre-cooled PBS after 48 h, scraped and collected in a sterile EP tube. The method of Tsuboi (1998) was used for the determination of melanin content. The supernatant was discarded by centrifugation at 1500 rpm for 5 min. The cells were lysed with 0.5 mL of 1 mol·L^-1 NaOH solution containing 10% DMSO. The cells were disrupted by ultrasonic for 30 min and water bathed at 90°C for 2 h to make the cells clump together. After complete lysis, centrifuge at 3000rpm for 15min, take the supernatant to a 96-well plate, measure the A value at 450nm of a microplate reader, and calculate the relative content of melanin in cells.

黑素合成相对含量(％)＝A1/A0×100Relative content of melanin synthesis (%)=A1/A0×100

式中Al是样品组的吸光度，A0是空白组的吸光值where Al is the absorbance of the sample group, A0 is the absorbance of the blank group

如图7所示，25～200μmol·L^-1范围内的重组人胶原样肽MJLGG-34孵育黑素瘤细胞48小时后，呈浓度依赖性抑制黑素细胞中的黑素合成(P＜0.05)，而200μmol·L^-1MJLGG-34对黑素合成抑制作用最强，与空白组相比，黑素含量减少约26.62％(73.38±0.017；P<0.01)。As shown in Figure 7, the recombinant human collagen-like peptide MJLGG-34 in the range of 25-200 μmol·L^-1 inhibited melanin synthesis in melanoma cells in a concentration-dependent manner after incubating melanoma cells for 48 hours (P<0.05 ), and 200μmol·L^-1 MJLGG-34 had the strongest inhibitory effect on melanin synthesis, compared with the blank group, the melanin content was reduced by about 26.62% (73.38±0.017; P<0.01).

实施例6Example 6

MJLGG-34减少人黑色素瘤细胞黑素合成信号通路中环腺苷酸依赖性蛋白激酶催化亚基(PRKACA)和小眼转录因子(MITF)的表达MJLGG-34 reduces the expression of cyclic adenylate-dependent protein kinase catalytic subunit (PRKACA) and microphthalmia transcription factor (MITF) in human melanoma cells in the melanin synthesis signaling pathway

调整人黑素瘤细胞A375浓度至1×10⁵个/mL，接种于6孔板，每孔2mL。贴壁后细胞分组，分别用200μmol·L^-1的MJLGG-34(Polypeptide)和200μmol·L^-1天然人I型胶原蛋白(Collangen I)处理，空白组(control)用等量的PBS处理，48h后弃上清，用预冷的PBS洗涤2次，将PBS弃去，加含PEMS的裂解液150μL/孔，摇匀冰上裂解30min；裂解完后，将细胞碎片和裂解液移至1.5mL离心管中；4℃，12 000r/min，离心15min，将离心后的上清液分装置200μLEP管中，-70℃保存备用。按BCA蛋白浓度测定试剂盒操作测定、配平蛋白浓度后进行SDS-PAGE电泳、转膜和Western-blotting反应，具体实验步骤按试剂盒操作进行。The concentration of human melanoma cells A375 was adjusted to 1×10⁵ cells/mL, and seeded in a 6-well plate with 2 mL per well. After adherence, the cells were grouped and treated with 200 μmol·L^-1 of MJLGG-34 (Polypeptide) and 200 μmol·L^-1 of natural human collagen I (Collangen I), respectively, and the blank group (control) was treated with the same amount of PBS, After 48 h, discard the supernatant, wash twice with pre-cooled PBS, discard the PBS, add 150 μL/well of lysis solution containing PEMS, shake well for lysis on ice for 30 min; after lysis, move the cell debris and lysis solution to 1.5 mL centrifuge tube; 4°C, 12 000 r/min, centrifugation for 15 min, the supernatant after centrifugation was divided into 200 μL EP tubes, and stored at -70°C for later use. According to the BCA protein concentration determination kit, SDS-PAGE electrophoresis, membrane transfer and Western-blotting reaction were performed after balancing the protein concentration. The specific experimental steps were carried out according to the kit operation.

如图8所示，由Western-blotting实验结果可知，制备的重组人胶原样肽MJLGG-34和天然I型胶原蛋白分别作用A375细胞48h后，与空白组相比，两种药物不同程度减少环腺苷酸依赖性蛋白激酶催化亚基(PRKACA)和小眼转录因子(MITF)的表达，且效果显著(P<0.05)；其中200μmol·L^-1MJLGG-34作用细胞48h后，与空白组相比，MITF含量减少约64.81％，PRKACA含量减少65.62％。As shown in Figure 8, the results of Western-blotting experiment showed that after the prepared recombinant human collagen-like peptide MJLGG-34 and natural type I collagen acted on A375 cells for 48 hours, compared with the blank group, the two drugs reduced the amount of cyclic activity to varying degrees. The expression of adenylate-dependent protein kinase catalytic subunit (PRKACA) and ommatidium transcription factor (MITF), and the effect was significant (P<0.05). Among them, 200μmol·L^-1 MJLGG-34 treated cells for 48 hours, which was different from the blank group. In contrast, the content of MITF was reduced by about 64.81%, and the content of PRKACA was reduced by 65.62%.

上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

序列表sequence listing

<110> 暨南大学<110> Jinan University

<120> 基因重组胶原样肽MJLGG-34及其制备方法与应用<120> Gene recombinant collagen-like peptide MJLGG-34 and its preparation method and application

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Gln Pro Gly Pro Pro Gly Glu Arg Gly Pro Pro Gly Pro Cys Cys GlyGln Pro Gly Pro Gly Glu Arg Gly Pro Pro Gly Pro Cys Cys Gly

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Gly GlyGly Gly

<210> 2<210> 2

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<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> 编码所述的基因重组胶原样肽MJLGG-34的核苷酸序列<223> Nucleotide sequence encoding said genetically recombinant collagen-like peptide MJLGG-34

<400> 2<400> 2

ggtgaaccgg gcaacccagg tcacaaaggc cacaaaggcc agccgggcca gccgggtccg 60ggtgaaccgg gcaacccagg tcacaaaggc cacaaaggcc agccgggcca gccgggtccg 60

ccgggcgaac gtgggccgcc gggcccgtgc tgtggtggtg gc 102ccgggcgaac gtgggccgcc gggcccgtgc tgtggtggtg gc 102

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<223> 融合蛋白Trx-6His-MJLGG-34的核苷酸序列<223> Nucleotide sequence of fusion protein Trx-6His-MJLGG-34

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atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60

gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120

ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180

atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240

ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300

aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat 360aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat 360

catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 420catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 420

ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg 480ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg 480

gctgatatcg gatccgaatt cggtgaaccg ggcaacccag gtcacaaagg ccacaaaggc 540gctgatatcg gatccgaatt cggtgaaccg ggcaacccag gtcacaaagg ccacaaaggc 540

cagccgggcc agccgggtcc gccgggcgaa cgtgggccgc cgggcccgtg ctgtggtggt 600cagccgggcc agccgggtcc gccgggcgaa cgtgggccgc cgggcccgtg ctgtggtggt 600

ggctaactcg ag 612ggctaactcg ag 612

<210> 4<210> 4

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<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> 融合蛋白Trx-6His-MJLGG-34的氨基酸序列<223> Amino acid sequence of fusion protein Trx-6His-MJLGG-34

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

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

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

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

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

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

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

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145 150 155 160145 150 155 160

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

165 170 175 165 170 175

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

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Pro Pro Gly Pro Cys Cys Gly Gly GlyPro Pro Gly Pro Cys Cys Gly Gly Gly

195 200 195 200

<210> 5<210> 5

<211> 57<211> 57

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> 引物T1+<223> Primer T1+

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aattcggtga accgggcaac ccaggtcaca aaggccacaa aggccagccg ggccagc 57aattcggtga accgggcaac ccaggtcaca aaggccacaa aggccagccg ggccagc 57

<210> 6<210> 6

<211> 57<211> 57

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> 引物T1-<223> Primer T1-

<400> 6<400> 6

cccggctggc ccggctggcc tttgtggcct ttgtgacctg ggttgcccgg ttcaccg 57cccggctggc ccggctggcc tttgtggcct ttgtgacctg ggttgcccgg ttcaccg 57

<210> 7<210> 7

<211> 60<211> 60

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> 引物T2+<223> Primer T2+

<400> 7<400> 7

cgggtccgcc gggcgaacgt gggccgccgg gcccgtgctg tggtggtggc taactcgagg 60cgggtccgcc gggcgaacgt gggccgccgg gcccgtgctg tggtggtggc taactcgagg 60

<210> 8<210> 8

<211> 60<211> 60

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> 引物T2-<223> Primer T2-

<400> 8<400> 8

gatccctcga gttagccacc accacagcac gggcccggcg gcccacgttc gcccggcgga 60gatccctcga gttagccacc accacagcac gggcccggcg gcccacgttc gcccggcgga 60

Claims

Translated fromChinese

1.一种基因重组胶原样肽MJLGG-34，其特征在于：其氨基酸序列如SEQ ID NO.1所示。1. A gene recombinant collagen-like peptide MJLGG-34, characterized in that: its amino acid sequence is as shown in SEQ ID NO.1.

2.编码权利要求1所述的基因重组胶原样肽MJLGG-34的核苷酸序列。2. The nucleotide sequence encoding the recombinant collagen-like peptide MJLGG-34 of claim 1.

3.根据权利要求2所述的核苷酸序列，其特征在于：编码所述的基因重组胶原样肽MJLGG-34的核苷酸序列如SEQ ID NO.2所示。3 . The nucleotide sequence according to claim 2 , wherein the nucleotide sequence encoding the gene recombinant collagen-like peptide MJLGG-34 is shown in SEQ ID NO.2. 4 .

4.一种基因重组胶原样肽MJLGG-34的制备方法，其特征在于：包括以下步骤：4. a preparation method of gene recombinant collagen-like peptide MJLGG-34, is characterized in that: comprise the following steps:

(1)设计并合成MJLGG-34基因：(1) Design and synthesis of MJLGG-34 gene:

GAATTC为EcoRI酶切位点，CTCGAG为XhoI酶切位点；GAATTC is the EcoRI restriction site,CTCGAG is the XhoI restriction site;

将引物T1+和引物T1-进行退火合成反应，同时将引物T2+和引物T2-进行退火合成反应，分别制得片段一和片段二；将片段一和片段二进行退火合成反应，获得MJLGG-34基因；The primer T1+ and primer T1- are subjected to annealing synthesis reaction, and primer T2+ and primer T2- are subjected to annealing synthesis reaction to obtain fragment one and fragment two respectively; fragment one and fragment two are subjected to annealing synthesis reaction to obtain MJLGG-34 gene ;

用重组载体pET-32a-MJLGG-34转化表达宿主大肠杆菌E.coliBL21(DE3)，得到表达工程菌pET-32a-MJLGG-34/BL21(DE3)；The recombinant vector pET-32a-MJLGG-34 was used to transform the expression host Escherichia coli E.coliBL21(DE3) to obtain the expression engineering strain pET-32a-MJLGG-34/BL21(DE3);

(4)表达和纯化：(4) Expression and purification:

①诱导表达工程菌pET-32a-MJLGG-34/BL21(DE3)表达由目的多肽、His标签蛋白和Trx标签蛋白组成的融合蛋白；①Induce the expression of engineering bacteria pET-32a-MJLGG-34/BL21(DE3) to express a fusion protein composed of the target polypeptide, His-tagged protein and Trx-tagged protein;

②得到的His标签融合蛋白用镍柱进行纯化：在蛋白上样后，带有组氨酸标签的蛋白特异性结合到镍柱，其他的杂蛋白流出；镍柱中的Ni²⁺也能与咪唑结合，用咪唑梯度洗脱，咪唑竞争性结合到镍柱上，释放融合蛋白，然后收集洗脱液；②The obtained His-tagged fusion protein is purified with a nickel column: after the protein is loaded, the protein with histidine tag specifically binds to the nickel column, and other impurity proteins flow out; Ni²⁺ in the nickel column can also interact with the nickel column. The imidazole is bound and eluted with an imidazole gradient, and the imidazole is competitively bound to the nickel column to release the fusion protein, and then the eluate is collected;

③目的蛋白的切割和纯化；③Cut and purify the target protein;

5.根据权利要求4所述的制备方法，其特征在于：5. preparation method according to claim 4, is characterized in that:

步骤(1)中所述的退火合成反应的条件为：37℃，35min；98℃，3min；95℃，5min。The conditions of the annealing synthesis reaction described in step (1) are: 37°C, 35min; 98°C, 3min; 95°C, 5min.

6.根据权利要求4所述的制备方法，其特征在于：6. preparation method according to claim 4, is characterized in that:

Lysis平衡缓冲液：50mM Na₂HPO₄和0.3M NaCl，pH＝8.0；Lysis equilibration buffer: 50 mM Na₂ HPO₄ and 0.3 M NaCl, pH=8.0;

洗涤缓冲液：50mM Na₂HPO₄，0.3M NaCl和10～50mM咪唑，pH＝8.0；Washing buffer: 50 mM Na₂ HPO₄ , 0.3 M NaCl and 10-50 mM imidazole, pH=8.0;

步骤(4)②中所述的洗脱镍柱的溶液的组成如下：50mM Na₂HPO₄，0.3M NaCl和250mM咪唑，pH＝8.0。The composition of the nickel column eluting solution described in step (4) ② is as follows: 50 mM Na₂ HPO₄ , 0.3 M NaCl and 250 mM imidazole, pH=8.0.

7.根据权利要求4所述的制备方法，其特征在于：7. preparation method according to claim 4, is characterized in that:

2)小规模酶切优化；2) Small-scale enzyme digestion optimization;

3)根据最佳切割结果放大反应比例；3) Amplify the reaction ratio according to the best cutting result;

8.根据权利要求7所述的制备方法，其特征在于：8. preparation method according to claim 7, is characterized in that:

步骤2)中所述的酶切优化后得到的酶切条件为1U肠激酶22℃条件下裂解100μg融合蛋白，酶切5h。The enzyme digestion conditions obtained after the enzyme digestion optimization described in step 2) were 1 U enterokinase to cleave 100 μg of the fusion protein at 22° C., and enzyme digestion for 5 hours.

9.根据权利要求4所述的制备方法，其特征在于：9. preparation method according to claim 4, is characterized in that:

步骤(4)④中所述的利用高效液相色谱技术纯化目的蛋白的步骤如下：The step of utilizing high performance liquid chromatography technology to purify the target protein described in step (4) is as follows:

A、流动相A为在体积百分比100％乙腈中添加TFA得到，TFA的终浓度为体积百分比0.1％；流动相B为100％水中添加TFA，TFA的终浓度为体积百分比0.1％；流速1.0mL/min，25min线性梯度洗脱；A. Mobile phase A is obtained by adding TFA to 100% acetonitrile by volume, and the final concentration of TFA is 0.1% by volume; mobile phase B is 100% water added with TFA, and the final concentration of TFA is 0.1% by volume; flow rate 1.0mL /min, 25min linear gradient elution;

B、线性梯度洗脱中流动相B由90～65％v/v，收集目的蛋白洗脱峰，光吸收检测波长为220nm；并进行质谱鉴定。B. In the linear gradient elution, the mobile phase B is 90-65% v/v, and the target protein elution peak is collected, and the light absorption detection wavelength is 220 nm; and mass spectrometry identification is carried out.

10.权利要求1所述的基因重组胶原样肽MJLGG-34的应用，其特征在于：10. the application of the recombinant collagen-like peptide MJLGG-34 of claim 1, is characterized in that:

所述的基因重组胶原样肽MJLGG-34用于制备具有抗氧化、降低黑素合成作用的化妆品、保健品或食品。The genetically recombined collagen-like peptide MJLGG-34 is used for preparing cosmetics, health care products or food with antioxidative and melanin synthesis reducing effects.