CN112195165A

CN112195165A - Anti-aging secretory Klotho protein, and coding gene, recombinant expression vector and application thereof

Info

Publication number: CN112195165A
Application number: CN202011104235.7A
Authority: CN
Inventors: 陈宏远; 芮雯
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2020-10-15
Filing date: 2020-10-15
Publication date: 2021-01-08

Abstract

The invention discloses an anti-aging secretory Klotho protein, and a coding gene, a recombinant expression vector and application thereof. The invention adopts gene engineering technology and strategy, optimizes the codon sequence of a target gene by adopting high-fidelity Taq polymerase, adds a histidine tag, and screens to obtain the recombinant anti-aging protein expressed by a prokaryotic expression vector, wherein the amino acid sequence of the recombinant anti-aging protein is shown in SEQ ID NO. 1. The invention also provides a gene for coding the anti-aging secretory Klotho protein, and the sequence of the gene is shown in SEQ ID NO. 2. The Klotho protein obtained in the invention has good anti-aging function, and can be used for anti-aging, regulation of generation of nitric oxide, oxidation stress resistance, regulation of calcium and phosphorus metabolism, anti-inflammation, cancer inhibition and the like.

Description

Anti-aging secretory Klotho protein, and coding gene, recombinant expression vector and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-aging secretory Klotho protein, and a coding gene, a recombinant expression vector and application thereof.

Background

Klotho is the name of the goddess of the line of spinning life in ancient Greek myth, and the gene name is to use the length of the spinning thread to indicate the length of the life. This gene was discovered and identified in 1997 in a mouse model of human aging and was the earliest anti-aging gene found in mammals. The product encoded by this gene was later demonstrated to be an important anti-senescence protein. The gene-deficient mice show aging phenomena in 4 weeks after birth, such as shortened lifespan, growth retardation, vascular calcification, arteriosclerosis, skin atrophy, osteopenia, emphysema, hearing impairment, motor neuron degeneration, infertility, tumorigenesis, and death due to aging at 2 months of age.

The human Klotho gene is located at human 13q12, 50kb in length, and the coding region contains 5 exons and 4 introns, transcribing 3036 base mRNA. In addition, there is an alternative splice site in exon 3 region, and thus, transmembrane and secretory Klotho proteins can be encoded. The former can be used as co-receptor of Fibroblast growth factor receptor (as Fibroblast growth factor-23, FGF-23) to mediate FGF-23 to exert biological activity so as to regulate phosphorus metabolism steady state, and the latter is formed from 549 amino acids, and only contains N-terminal signal sequence and extracellular region KL1, and has no extracellular region KL2, transmembrane region and intracellular region, and said protein is produced by that the extracellular region of membrane type Klotho protein is anchored on the cell membrane and the metalloproteases of ADAM10 and ADAM17, etc. are hydrolyzed and fallen off.

The Klotho gene expression is regulated by various factors, such as active peptide calcitonin gene related peptide, FGF-2 and the like, which can up-regulate the Klotho expression, and oxidative stress, inflammatory reaction renin-angiotensin and ureoxin which can down-regulate the Klotho expression. Improving the expression of endogenous klotho genes and providing exogenous klotho gene support, and can effectively delay cell aging.

Mizuno et al studied the effect of thyroid hormone on Klotho mRNA expression and found that thyroid hormone significantly increased the expression level of transmembrane-type Klotho mRNA in 3T3-L1 adipocytes in a dose-and time-dependent manner, while the expression level of secretory Klotho mRNA tended to increase; xiao L P et al found: klotho mRNA and protein expression levels were significantly increased in the FGF-2 transgenic mouse kidney, suggesting that FGF-2 overexpression may increase Klotho gene and protein expression.

The Klotho protein can up-regulate the expression of SOD2, and enhance the capability of eliminating harmful free radicals, thereby protecting cells from being damaged by oxidative stress. Yamamoto et al have demonstrated through experimentation that Klotho protein can significantly reduce the production of ROS, thereby slowing oxidative stress in cells or the body. Oxidative stress can induce apoptosis, and the oxidative stress state of the organism can be reflected by detecting the expression of apoptosis genes. Sun W L et al found that Klotho protein inhibited the expression of the Tac-induced apoptosis-inhibiting gene Bcl-2. After Tac treatment, the expression levels of the pro-apoptotic markers Bax, Caspase-9 and Caspase-3 were significantly increased, and they were significantly decreased in Klotho protein-treated cells. Therefore, the Klotho protein can regulate the process of apoptosis by regulating the expression of an apoptosis inhibiting gene Bcl-2 and apoptosis promoting markers Bax, Caspase-9 and Caspase-3, thereby reflecting the state of oxidative stress. The Klotho protein has the effects on oxidative stress and possible molecular mechanisms for regulating the body anti-oxidative stress, and the action mechanisms of the Klotho protein relate to FoxO, cAMP signal pathways and Keap1-Nrf2/ARE signal pathways.

The results of the beta-galactose staining method related to aging show that: klotho has obvious inhibition effect on the aging of HUVEC (human umbilical vein endothelial cells), and the expression degree of Klotho on p53 and p21 is obviously reduced. Wangjian et al indicate that the enhancement of Akt activity will shorten the life of human endothelial cells; but the existence of the Klotho gene leads to the reduction of Akt activity, thereby controlling cell senescence; at the same time, Akt activity is reduced, and the expression degree of P53 and P21 is reduced. The Klotho protein, as a circulating factor or hormone, inhibits the amplification of the intracellular insulin/insulin-like growth factor-1 (insulin/IGF-1) signaling cascade, an effect which is related to its anti-aging properties. In addition, the secretory klotho also has the effects of regulating the activity of an ion channel, inhibiting oxidative stress, inhibiting Wnt signal transduction, inhibiting cell inflammation and apoptosis, increasing nitric oxide synthesis, promoting angiogenesis, enhancing the strength of skeletal muscle and the like.

Since the earliest cloning of human Klotho gene by Matsumura Y, high fidelity cloning and prokaryotic expression of the gene have been reported. The reason is as follows: although prokaryotic protein expression systems are the most common and cost-effective protein expression systems. The method has the advantages of clear genetic background, low cost, high expression amount, relatively simple separation and purification of expressed products and the like, and has the defects of lack of processing mechanisms after protein translation, such as disulfide bond formation, protein glycosylation and correct folding, and small probability of obtaining the protein with biological activity. Purification tags selected by prokaryotic expression, such as GST (26kd) and MBP (44kd) fusion protein expression tags, have the potential of influencing the structural function of the target protein due to the large molecular weight. The influence factors of protein expression are more: including the problems of difficult protein renaturation of inclusion body purification, preference of host bacteria codon and soluble protein expression but expression quantity, etc.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide the anti-aging secretory Klotho protein.

Another object of the present invention is to provide a gene encoding the anti-aging secretory Klotho protein.

The invention also aims to provide a recombinant expression vector containing the gene for coding the anti-aging secretory Klotho protein and a recombinant bacterium.

The invention further aims to provide the gene and the recombinant expression vector of the anti-aging secretory Klotho protein and application of recombinant bacteria expressing the Klotho protein.

The purpose of the invention is realized by the following technical scheme: an anti-aging secretory Klotho protein, the amino acid sequence of which is shown as follows (SEQ ID NO. 1):

MPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRAMPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRAEPGDGAQTWARFSRPPAPEAAGLFQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSYNNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHFRDYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRLAPGIRGSPRLGYLVAHNLLLAHAKVWHLYNTSFRPTQGGQVSIALSSHWINPRRMTDHSIKECQKSLDFVLGWFAKPVFIDGDYPESMKNNLSSILPDFTESEKKFIKGTADFFALCFGPTLSFQLLDPHMKFRQLESPNLRQLLSWIDLEFNHPQIFIVENGWFVSGTTKRDDAKYMYYLKKFIMETLKAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQDKMLLPKSSALFYQKLIEKNGFPPLPENQPLEGTFPCDFAWGVVDNYIQVSQLTKPISSLTKPYHHHHHHH。

the gene for coding the anti-aging secretory Klotho protein.

The nucleotide sequence of the gene for coding the anti-aging secretory Klotho protein is preferably as shown in the specification (SEQ ID NO. 2):

ATGCCCGCCTCCGCCCCCCCCCGGCGGCCCCGGCCCCCCCCCCCCTCCCTGTCCCTGCTGCTGGTCCTGCTGGGGCTGGGGGGGCGGCGGCTGCGGGCCATGCCCGCCTCCGCCCCCCCCCGGCGGCCCCGGCCCCCCCCCCCCTCCCTGTCCCTGCTGCTGGTCCTGCTGGGGCTGGGGGGGCGGCGGCTGCGGGCCGAGCCCGGGGACGGGGCCCAGACGTGGGCCCGGTTTTCCCGGCCCCCCGCCCCCGAGGCCGCCGGGCTGTTTCAGGGGACGTTTCCCGACGGGTTTCTGTGGGCCGTCGGGTCCGCCGCCTACCAGACGGAGGGGGGGTGGCAGCAGCATGGGAAGGGGGCCTCCATATGGGACACGTTTACGCATCATCCCCTGGCCCCCCCCGGGGACTCCCGGAATGCCTCCCTGCCCCTGGGGGCCCCCTCCCCCCTGCAGCCCGCCACGGGGGACGTCGCCTCCGACTCCTACAATAATGTCTTTCGGGACACGGAGGCCCTGCGGGAGCTGGGGGTCACGCATTACCGGTTTTCCATATCCTGGGCCCGGGTCCTGCCCAATGGGTCCGCCGGGGTCCCCAATCGGGAGGGGCTGCGGTACTACCGGCGGCTGCTGGAGCGGCTGCGGGAGCTGGGGGTCCAGCCCGTCGTCACGCTGTACCATTGGGACCTGCCCCAGCGGCTGCAGGACGCCTACGGGGGGTGGGCCAATCGGGCCCTGGCCGACCATTTTCGGGACTACGCCGAGCTGTGCTTTCGGCATTTTGGGGGGCAGGTCAAGTACTGGATAACGATAGACAATCCCTACGTCGTCGCCTGGCATGGGTACGCCACGGGGCGGCTGGCCCCCGGGATACGGGGGTCCCCCCGGCTGGGGTACCTGGTCGCCCATAATCTGCTGCTGGCCCATGCCAAGGTCTGGCATCTGTACAATACGTCCTTTCGGCCCACGCAGGGGGGGCAGGTCTCCATAGCCCTGTCCTCCCATTGGATAAATCCCCGGCGGATGACGGACCATTCCATAAAGGAGTGCCAGAAGTCCCTGGACTTTGTCCTGGGGTGGTTTGCCAAGCCCGTCTTTATAGACGGGGACTACCCCGAGTCCATGAAGAATAATCTGTCCTCCATACTGCCCGACTTTACGGAGTCCGAGAAGAAGTTTATAAAGGGGACGGCCGACTTTTTTGCCCTGTGCTTTGGGCCCACGCTGTCCTTTCAGCTGCTGGACCCCCATATGAAGTTTCGGCAGCTGGAGTCCCCCAATCTGCGGCAGCTGCTGTCCTGGATAGACCTGGAGTTTAATCATCCCCAGATATTTATAGTCGAGAATGGGTGGTTTGTCTCCGGGACGACGAAGCGGGACGACGCCAAGTACATGTACTACCTGAAGAAGTTTATAATGGAGACGCTGAAGGCCATAAAGCTGGACGGGGTCGACGTCATAGGGTACACGGCCTGGTCCCTGATGGACGGGTTTGAGTGGCATCGGGGGTACTCCATACGGCGGGGGCTGTTTTACGTCGACTTTCTGTCCCAGGACAAGATGCTGCTGCCCAAGTCCTCCGCCCTGTTTTACCAGAAGCTGATAGAGAAGAATGGGTTTCCCCCCCTGCCCGAGAATCAGCCCCTGGAGGGGACGTTTCCCTGCGACTTTGCCTGGGGGGTCGTCGACAATTACATACAGGTCTCCCAGCTGACGAAGCCCATATCCTCCCTGACGAAGCCCTACCATCATCATCATCATCATCAT。

a recombinant expression vector contains the gene for coding the anti-aging secretory Klotho protein.

The construction method of the recombinant expression vector comprises the following steps:

(1) using hKlotho plasmid as template, and F₁And R₁Performing PCR amplification as a primer to obtain a PCR amplification product A₁(ii) a Then the amplification product A₁Performing electrophoresis, and recovering the target fragment B₁(ii) a Wherein, F₁And R₁The nucleotide sequence of (A) is shown in SEQ ID NO.3 and SEQ ID NO. 4;

(2) subjecting the target fragment B obtained in step (1)₁Performing double enzyme digestion by Xho I and Bam H I, performing electrophoresis after enzyme digestion, and recovering a target fragment C₁(ii) a At the same time, pcDNA3.1 plasmid is double digested by Xho I and Bam H I, and then target fragment C is digested by T4 DNA ligase₁Connecting with the pcDNA3.1 plasmid after double enzyme digestion to obtain pcDNA3.1-Klotho plasmid;

(3) using the pcDNA3.1-Klotho plasmid obtained in the step (2) as a template and F₂And R₂Performing PCR amplification as a primer to obtain a PCR amplification product A₂(ii) a Then the amplification product A₂Performing electrophoresis, and recovering the target fragment B₂(ii) a Wherein, F₂And R₂The nucleotide sequences of (A) are shown as SEQ ID NO.5 and SEQ ID NO. 6;

(4) subjecting the target fragment B obtained in step (3)₂Using EcoRI and XhoI to perform double enzyme digestion, performing electrophoresis after enzyme digestion, and recovering a target fragment C₂(ii) a Meanwhile, the pET-28a (+) plasmid is subjected to double digestion by EcoRI and XhoI, and then the target fragment C is subjected to T4 DNA ligase₂And the plasmid is connected with pET-28a (+) after double enzyme digestion to obtain a recombinant expression vector pET-28 a-Klotho.

The construction method of the recombinant expression vector also comprises the step of recovering the target fragment B in the step (1)₁And (2) recovering the target fragment C₁And (3) recovering the target fragment B₂And the target fragment C recovered in the step (4)₂And (5) further purifying.

The purification is preferably carried out by using a conventional plasmid purification kit.

The construction method of the recombinant expression vector also comprises the step of verifying the pcDNA3.1-Klotho plasmid obtained in the step (2); the method specifically comprises the following steps: transforming the pcDNA3.1-Klotho plasmid into DH5 alpha competent cells, screening positive clones with penicillin, carrying out plasmid amplification, selecting positive colonies for enzyme digestion identification and sequencing, wherein the enzyme digestion identification and sequencing result is correct and is the required pcDNA3.1-Klotho plasmid.

The system for PCR amplification in the step (3) is as follows: PCR amplification System: 25mM dNTP0.5. mu.L, primer F ₂2 μ L, primer R ₂2 μ L, template 5 μ L, ddH2O 20 μ L, 5 u/. mu.L Taq DNA polymerase 0.5 uL.

The PCR reaction conditions in step (3) are as follows: 94 ℃ for 4 min; 30s at 94 ℃; 30s at 60 ℃; 72 ℃ for 80 s; 30 cycles; 72 ℃ for 7 min.

A recombinant bacterium for expressing Klotho protein is a recombinant bacterium obtained by transforming Escherichia coli with the recombinant expression vector.

The Escherichia coli is preferably Escherichia coli DH5 alpha or BL21(DE 3).

The gene for coding the anti-aging secretory Klotho protein, the recombinant expression vector and the application of at least one of recombinant bacteria for expressing the Klotho protein in preparing the Klotho protein.

A preparation method of Klotho protein comprises the following steps: transforming the recombinant bacteria for expressing the Klotho protein into escherichia coli competent cells, culturing, adding IPTG (isopropyl-beta-thiogalactoside) for induced expression, centrifugally collecting bacteria, crushing, separating and purifying to obtain the Klotho protein; wherein the conditions for inducing expression are: the induction temperature is 20-37 ℃, the induction time is 6-12 h, and the final concentration of IPTG is 0.1-2.0 mmol/L.

The condition of inducing expression is preferably that the inducing temperature is 30 ℃, the inducing induction is carried out for 8h, and the final concentration of IPTG is 0.8 mmol/L.

The anti-aging secretory Klotho protein, the gene for coding the anti-aging secretory Klotho protein, the recombinant expression vector and at least one of recombinant bacteria for expressing the Klotho protein are applied to the preparation of anti-aging, nitric oxide generation regulation, anti-oxidative stress, calcium and phosphorus metabolism regulation, anti-inflammation and/or anti-tumor drugs.

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

1. the invention adopts gene engineering technology and strategy, optimizes the codon sequence of a target gene and adds histidine tags (six histidines are added to the C terminal of a recombinant gene to ensure that chimera is easy to separate and purify) by adopting high-fidelity Taq polymerase, optimizes and screens a recombinant anti-aging protein expressed by a prokaryotic expression vector, establishes an anti-aging model by adopting a proper cell strain to evaluate the anti-aging biological activity of the recombinant anti-aging protein, and specifically comprises the following steps: a target gene is cloned to a prokaryotic expression vector pET-28a (+), a recombinant expression plasmid pET-28a-Klotho is constructed and is induced and expressed in escherichia coli BL21(DE3), an expression product is purified by a Ni ion affinity chromatography method, a prokaryotic vector expression system is constructed, and the anti-aging activity of the prokaryotic vector expression system is investigated after the purification.

2. The invention constructs a prokaryotic vector expression system, the protein expression quantity of the prokaryotic vector expression system is high, and further detection through an aging model established by 2 different cell lines shows that: the purified protein has good anti-aging function.

3. The protein with stable expression and high purity obtained in the invention is beneficial to the application of the protein in the aspects of industrial production, anti-aging, regulation of generation of nitric oxide, anti-oxidative stress, regulation of calcium and phosphorus metabolism, anti-inflammation, cancer inhibition and the like.

4. The Klotho recombinant of the invention has the following characteristics: (1) can realize high-efficiency expression in prokaryotic expression vectors, the yield is 120mg/L, and the mass production and expression of target genes can be realized; (2) the separation and purification process after expression is simple, and the target protein with high purity can be obtained; (3) the method for producing the target protein by adopting the genetic engineering is feasible, has low production cost and is easy for large-scale industrial production; (4) the recombinant Klotho protein shows better anti-aging activity in vitro cell activity detection.

Drawings

FIG. 1 is a graph showing the results of PCR using the Klotho gene (the size of the Klotho gene is about 1700 bp).

FIG. 2 is a graph showing the Western Blot identification of recombinant Klotho protein.

FIG. 3 is a graph of the anti-aging effect of recombinant Klotho protein on HSF cells after irradiation; wherein A is the anti-aging effect of the recombinant Klotho protein with different concentrations on the HSF cells after radiation; b is the anti-aging effect of the positive control Vc with different concentrations on the irradiated HSF cells.

FIG. 4 is a graph of the anti-photoaging repair effect of recombinant Klotho protein on HaCaT cells after irradiation; wherein A is the anti-photoaging repair effect of the recombinant Klotho protein with different concentrations on the HaCaT cells after radiation; b is the anti-photoaging repair effect of positive control Vc with different concentrations on HaCaT cells after radiation.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. 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. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.

The construction process of the pcDNA3.1-Klotho eukaryotic expression vector related by the invention is as follows:

construction of fusion expression plasmid pcDNA3.1-Klotho of Gene and pcDNA3.1 to contain secreted Klotho GeneThe hKlotho Plasmid (purchased from Addgene: www.addgene.org, Plasmid #17713) was used as template (Primer design was performed according to the general principle of Primer design, using Primer 5.0, and the forward Primer F was used₁5'-TGGACCCACCTTGAGTTTTC-3' (SEQ ID NO.3), downstream primer R₁: 5'-GGAGGGAAGCCATTTTTCTC-3' (SEQ ID NO.4)), and performing a PCR reaction under the following reaction conditions: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 30s, 30 cycles, and finally 5min at 72 ℃. And the results were analyzed by electrophoresis on a 1% agarose gel. The PCR product was purified by a plasmid purification kit and digested simultaneously with both Xho I and BamH I, as well as with plasmid pcDNA3.1(pcDNA3.1 plasmid was purchased from Oligoengine). And (3) recovering a target fragment (a band between 1500 and 2000 bp) by 1.5 percent agarose gel electrophoresis, purifying by using a PCR purification kit, and connecting the target fragment with the pcDNA3.1 vector which is subjected to double enzyme digestion and purification by using T4 DNA ligase. The ligation product was transformed into DH 5. alpha. competent cells, penicillin-screened positive clones and plasmid-amplified. And selecting positive colonies for enzyme digestion identification and sequencing. The clone with correct enzyme cutting identification and sequencing result is the clone containing the target gene Klotho.

Example 1 construction and expression of recombinant prokaryotic expression vectors

(1) Construction of recombinant expression vectors

Primers were designed based on the known Klotho sequence number (Gen bank. nc — 000013) and optimized and BL21(DE3) vector cloning site:

carrying out PCR amplification by using the constructed pcDNA3.1-Klotho as a template and using the following sequences (upstream and downstream primers) as primers to obtain a PCR amplification product; then, the PCR amplification product was double-digested with EcoR I and XhoI enzymes, and pET-28a (+) plasmid was also simultaneously double-digested, and after the target fragment (the size of Klotho gene is 1764bp, FIG. 1) recovered by electrophoresis was purified by PCR purification kit, it was ligated with double-digested and purified pET-28a (+) vector (purchased from EMD Biosciences (Novagen)) by T4 DNA ligase to obtain pET-28 a-Klotho. Wherein, specific primers of the CDS sequence of the target gene are as follows:

upstream primer sequence F₂：5’-TATGAATTCATGCCCGCCAGCGCCCCCCCCAG-3’(SEQ ID NO.5)；

Downstream primer sequence R₂：5’-TATCTCGAGTCAGTGGTAGGGCTTGGTCAGG-3’(SEQ ID NO.6)。

PCR amplification System: dNTP (25mM) 0.5. mu.L, forward primer 2. mu.L, reverse primer 2. mu.L, template 5. mu.L, ddH2O 20. mu.L, Taq DNA polymerase (5 u/. mu.L, available from Eurogentec) 0.5. mu.L.

And (3) PCR reaction conditions: 94 ℃ for 4 min; 30s at 94 ℃; 30s at 60 ℃; 72 ℃ for 80 s; 30 cycles; 72 ℃ for 7 min.

And (3) carrying out analysis and identification by 1.2% agarose gel electrophoresis, cutting and recovering the gel, purifying a PCR product, and detecting the purity and the concentration of the PCR product. The recombinant prokaryotic expression vector pET-28a-Klotho of the gene is constructed. Double enzyme digestion and one-way sequence sequencing identification are adopted, and a recombinant prokaryotic expression vector pET-28a-Klotho with correct sequencing is selected for carrying out the next experiment.

(2) Transformation and inducible expression of expression vectors

The constructed gene-containing pET-28a-Klotho plasmid was transformed into BL21(DE3) competent cells from 100. mu.L of ice-thawed BL21(DE3) competent cells, and allowed to stand on ice for 25 min. Then, the mixture was spread evenly on LB plates (containing 50. mu.g/mL kanamycin sulfate), and then placed upside down in a 37 ℃ incubator overnight. And (3) selecting a single colony, inoculating the single colony in an LB culture medium, and performing shake culture at 180rpm and 37 ℃ for 10-12 h. IPTG induced expression is carried out after the amplification culture, and the selected temperatures are respectively as follows: the final concentration of IPTG is as follows at 20-37 ℃: 0.1 to 1.0 mmol/L. Inducing with IPTG (0.1, 0.2, 1mM) with different concentrations at 20 deg.C for 6,8, 12 h; inducing with IPTG (0.1, 0.2, 1mM) with different concentrations at 30 deg.C for 4,6,8,10,12 hours; induction was carried out at 37 ℃ for 4,6,8,10 and 12 hours with IPTG (0.1, 0.2 and 1mM) concentrations. The cells were collected by centrifugation at 9000rpm for 10min at 4 ℃. The wet weight of the cells was weighed, 1 XPBS buffer was added at a ratio of 1:10(w/w), and the cells were resuspended using a shaker. Carrying out low-temperature ultrasonication for 30min, centrifuging at the temperature of 4 ℃ for 17000g for 30min, and respectively taking a precipitate and a supernatant to carry out SDS-PAGE detection. SDS-PAGE, stained with Coomassie Brilliant blue and analyzed.

Example 2 recombinant prokaryotic vector protein expression, purification and identification

(1) SDS-PAGE analysis of expressed proteins

Crushing the thallus collected in the example 1, centrifuging to collect precipitate, washing the precipitate with PBS buffer solution containing 2M urea, centrifuging at 4 ℃ for 17000g for 30min, and collecting the precipitate; then washing with PBS buffer solution containing 0.4% (w/v) sodium deoxycholate, centrifuging at 4 deg.C for 17000g for 30min, and collecting precipitate; washing with PBS buffer solution for 2-3 times, centrifuging at 4 deg.C and 17000g for 30min, and collecting precipitate; finally, the pellet was dissolved in PBS buffer, and DTT (dithiothreitol) was added to the solution at a final concentration of 1mM, and the mixture was stirred overnight at 4 ℃. The supernatant was collected by centrifugation at 9000rpm for 20min at 4 ℃. NI-NTA packing is filled in a column, and after washing 2-3 column volumes with pure water, the column is equilibrated with PBS buffer solution with pH 8.0. And stirring the balanced filler and a sample to be detected in ice bath for 3h, precipitating for 2h, loading the precipitate on a column, washing the column to a baseline by using PBS (phosphate buffer solution), eluting the foreign protein by using PBS (phosphate buffer solution) containing 30mM imidazole, and eluting the target protein by using PBS (phosphate buffer solution) containing 400mM imidazole. After the imidazole removal, the protein concentration was determined by BCA method, and the elution peak from each step was sampled and purified for detection by 12% SDS-PAGE electrophoresis. The optimal conditions selected according to the experimental conditions are as follows: IPTG 0.8mM, temperature 30 ℃ for 8 hours, protein yield 120 mg/L.

(2) Western-blotting identification

After SDS-PAGE separation, membrane transfer, sealing, washing 3 times with 1 XTSST buffer solution for 8min each time, incubating the primary antibody overnight at 4 ℃, washing 3 times with 1 XTSST buffer solution for 8min each time, incubating the secondary antibody goat anti-mouse IgG (1:8000, v/v) for 1h at room temperature, washing 3 times with 1 XTSST buffer solution for 10min each time, taking the membrane, and ECL chemiluminescence developing. The results are shown in FIG. 2.

Example 3 anti-aging Activity assay for recombinant Klotho protein

(1) Establishment of cell aging model

The aging model of human immortalized skin keratinocyte (HaCaT) and Human Skin Fibroblast (HSF) and cell damage is irradiated by medium wave ultraviolet (the cells are respectively purchased from Shanghai Aimo and Shanghai Qiaoxin bamboo biotechnology limited company, and the aging model is established by a UVB irradiation method): the cell culture is carried out at 37 ℃ and 5% (v/v) CO₂The cell culture box is used for conventional culture and is respectively arrangedAt different energies 700-1000 mJ/cm²Irradiating under a UVB lamp, wherein the distance between a lamp source and a culture bottle is 15cm, after irradiating an experimental group, adding a PBS buffer solution for washing, then removing the PBS buffer solution, adding 100 mu L of a culture solution (a DMEM medium and 10% (v/v) newborn calf serum), and normally culturing; after 24h, the MTT method detects that the proliferation inhibition rate of two groups of cells is (1-irradiation group OD value/blank control group OD value) × 100%, and the radiation energy when the proliferation inhibition rate is 50% is selected as the experimental energy (80 mJ/cm) of the aging model²)。

(2) The aging repair capacity of the recombinant Klotho protein on HSF and HaCaT cells is respectively detected, 3 groups are respectively set for each cell experiment, namely a positive control group (adding DMEM complete culture solution containing Vc (vitamin C) with different concentrations, wherein the final concentration of Vc is 0.15, 0.3, 0.6, 1.2, 2.4 and 4.8mM), an experimental group (adding DMEM complete culture solution containing recombinant Klotho protein samples with different concentrations, the final concentration of the recombinant Klotho protein samples is 12.5, 25, 50, 100, 200 and 400pM), a blank irradiation control group, and 3 parallel multiple holes are set for each group. The results are shown in FIG. 3.

Taking HSF and HaCaT cells in logarithmic growth phase, digesting with 0.25% (w/v) trypsin, adjusting cell concentration to 1 × 10⁴And (2) inoculating the cells/mL into a 96-well plate, culturing the cells 100 mu L per well conventionally until the cells are attached to the wall, performing energy irradiation treatment (the method is the same as the step (1)) after the cells are cultured conventionally until the cells are attached to the wall, adding medicine protection (500pM recombinant Klotho protein, prepared in example 2) for further culturing for 24 hours, adding 20 mu L MTT and 180 mu L basal medium (DMEM medium + 10% (v/v) newborn bovine serum) per well, culturing for further 4 hours, collecting cell upper layer culture solution, measuring the contents of SOD (superoxide dismutase), GSH-Px (glutathione peroxidase) and MDA (malondialdehyde) according to a kit method, adding 150 mu L DMSO (dimethyl sulfoxide) per well into the culture plate continuously, shaking for 10 minutes, detecting the OD value at the wavelength of 490nm by using a microplate reader, and calculating the repairing effect of the samples on cell aging according to the following formula: sample repair effect-irradiation plus sample OD value/irradiation without sample OD value x 100%. When ratio of>100 percent, which indicates that the medicine has the repairing effect on cell damage. The results are shown in FIG. 4 and Table 1.

TABLE 1 Effect of treatment with 500pM recombinant Klotho protein on MDA, SOD and GSH-Px in HSF and HaCaT cells

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

<110> university of Guangdong department of pharmacy

<120> anti-aging secretory Klotho protein, and coding gene, recombinant expression vector and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 588

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Klotho protein

<400> 1

Met Pro Ala Ser Ala Pro Pro Arg Arg Pro Arg Pro Pro Pro Pro Ser

1 5 10 15

Leu Ser Leu Leu Leu Val Leu Leu Gly Leu Gly Gly Arg Arg Leu Arg

20 25 30

Ala Met Pro Ala Ser Ala Pro Pro Arg Arg Pro Arg Pro Pro Pro Pro

35 40 45

Ser Leu Ser Leu Leu Leu Val Leu Leu Gly Leu Gly Gly Arg Arg Leu

50 55 60

Arg Ala Glu Pro Gly Asp Gly Ala Gln Thr Trp Ala Arg Phe Ser Arg

65 70 75 80

Pro Pro Ala Pro Glu Ala Ala Gly Leu Phe Gln Gly Thr Phe Pro Asp

85 90 95

Gly Phe Leu Trp Ala Val Gly Ser Ala Ala Tyr Gln Thr Glu Gly Gly

100 105 110

Trp Gln Gln His Gly Lys Gly Ala Ser Ile Trp Asp Thr Phe Thr His

115 120 125

His Pro Leu Ala Pro Pro Gly Asp Ser Arg Asn Ala Ser Leu Pro Leu

130 135 140

Gly Ala Pro Ser Pro Leu Gln Pro Ala Thr Gly Asp Val Ala Ser Asp

145 150 155 160

Ser Tyr Asn Asn Val Phe Arg Asp Thr Glu Ala Leu Arg Glu Leu Gly

165 170 175

Val Thr His Tyr Arg Phe Ser Ile Ser Trp Ala Arg Val Leu Pro Asn

180 185 190

Gly Ser Ala Gly Val Pro Asn Arg Glu Gly Leu Arg Tyr Tyr Arg Arg

195 200 205

Leu Leu Glu Arg Leu Arg Glu Leu Gly Val Gln Pro Val Val Thr Leu

210 215 220

Tyr His Trp Asp Leu Pro Gln Arg Leu Gln Asp Ala Tyr Gly Gly Trp

225 230 235 240

Ala Asn Arg Ala Leu Ala Asp His Phe Arg Asp Tyr Ala Glu Leu Cys

245 250 255

Phe Arg His Phe Gly Gly Gln Val Lys Tyr Trp Ile Thr Ile Asp Asn

260 265 270

Pro Tyr Val Val Ala Trp His Gly Tyr Ala Thr Gly Arg Leu Ala Pro

275 280 285

Gly Ile Arg Gly Ser Pro Arg Leu Gly Tyr Leu Val Ala His Asn Leu

290 295 300

Leu Leu Ala His Ala Lys Val Trp His Leu Tyr Asn Thr Ser Phe Arg

305 310 315 320

Pro Thr Gln Gly Gly Gln Val Ser Ile Ala Leu Ser Ser His Trp Ile

325 330 335

Asn Pro Arg Arg Met Thr Asp His Ser Ile Lys Glu Cys Gln Lys Ser

340 345 350

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

355 360 365

Asp Tyr Pro Glu Ser Met Lys Asn Asn Leu Ser Ser Ile Leu Pro Asp

370 375 380

Phe Thr Glu Ser Glu Lys Lys Phe Ile Lys Gly Thr Ala Asp Phe Phe

385 390 395 400

Ala Leu Cys Phe Gly Pro Thr Leu Ser Phe Gln Leu Leu Asp Pro His

405 410 415

Met Lys Phe Arg Gln Leu Glu Ser Pro Asn Leu Arg Gln Leu Leu Ser

420 425 430

Trp Ile Asp Leu Glu Phe Asn His Pro Gln Ile Phe Ile Val Glu Asn

435 440 445

Gly Trp Phe Val Ser Gly Thr Thr Lys Arg Asp Asp Ala Lys Tyr Met

450 455 460

Tyr Tyr Leu Lys Lys Phe Ile Met Glu Thr Leu Lys Ala Ile Lys Leu

465 470 475 480

Asp Gly Val Asp Val Ile Gly Tyr Thr Ala Trp Ser Leu Met Asp Gly

485 490 495

Phe Glu Trp His Arg Gly Tyr Ser Ile Arg Arg Gly Leu Phe Tyr Val

500 505 510

Asp Phe Leu Ser Gln Asp Lys Met Leu Leu Pro Lys Ser Ser Ala Leu

515 520 525

Phe Tyr Gln Lys Leu Ile Glu Lys Asn Gly Phe Pro Pro Leu Pro Glu

530 535 540

Asn Gln Pro Leu Glu Gly Thr Phe Pro Cys Asp Phe Ala Trp Gly Val

545 550 555 560

Val Asp Asn Tyr Ile Gln Val Ser Gln Leu Thr Lys Pro Ile Ser Ser

565 570 575

Leu Thr Lys Pro Tyr His His His His His His His

580 585

<210> 2

<211> 1764

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Klotho Gene

<400> 2

atgcccgcct ccgccccccc ccggcggccc cggccccccc ccccctccct gtccctgctg 60

ctggtcctgc tggggctggg ggggcggcgg ctgcgggcca tgcccgcctc cgcccccccc 120

cggcggcccc ggcccccccc cccctccctg tccctgctgc tggtcctgct ggggctgggg 180

gggcggcggc tgcgggccga gcccggggac ggggcccaga cgtgggcccg gttttcccgg 240

ccccccgccc ccgaggccgc cgggctgttt caggggacgt ttcccgacgg gtttctgtgg 300

gccgtcgggt ccgccgccta ccagacggag ggggggtggc agcagcatgg gaagggggcc 360

tccatatggg acacgtttac gcatcatccc ctggcccccc ccggggactc ccggaatgcc 420

tccctgcccc tgggggcccc ctcccccctg cagcccgcca cgggggacgt cgcctccgac 480

tcctacaata atgtctttcg ggacacggag gccctgcggg agctgggggt cacgcattac 540

cggttttcca tatcctgggc ccgggtcctg cccaatgggt ccgccggggt ccccaatcgg 600

gaggggctgc ggtactaccg gcggctgctg gagcggctgc gggagctggg ggtccagccc 660

gtcgtcacgc tgtaccattg ggacctgccc cagcggctgc aggacgccta cggggggtgg 720

gccaatcggg ccctggccga ccattttcgg gactacgccg agctgtgctt tcggcatttt 780

ggggggcagg tcaagtactg gataacgata gacaatccct acgtcgtcgc ctggcatggg 840

tacgccacgg ggcggctggc ccccgggata cgggggtccc cccggctggg gtacctggtc 900

gcccataatc tgctgctggc ccatgccaag gtctggcatc tgtacaatac gtcctttcgg 960

cccacgcagg gggggcaggt ctccatagcc ctgtcctccc attggataaa tccccggcgg 1020

atgacggacc attccataaa ggagtgccag aagtccctgg actttgtcct ggggtggttt 1080

gccaagcccg tctttataga cggggactac cccgagtcca tgaagaataa tctgtcctcc 1140

atactgcccg actttacgga gtccgagaag aagtttataa aggggacggc cgactttttt 1200

gccctgtgct ttgggcccac gctgtccttt cagctgctgg acccccatat gaagtttcgg 1260

cagctggagt cccccaatct gcggcagctg ctgtcctgga tagacctgga gtttaatcat 1320

ccccagatat ttatagtcga gaatgggtgg tttgtctccg ggacgacgaa gcgggacgac 1380

gccaagtaca tgtactacct gaagaagttt ataatggaga cgctgaaggc cataaagctg 1440

gacggggtcg acgtcatagg gtacacggcc tggtccctga tggacgggtt tgagtggcat 1500

cgggggtact ccatacggcg ggggctgttt tacgtcgact ttctgtccca ggacaagatg 1560

ctgctgccca agtcctccgc cctgttttac cagaagctga tagagaagaa tgggtttccc 1620

cccctgcccg agaatcagcc cctggagggg acgtttccct gcgactttgc ctggggggtc 1680

gtcgacaatt acatacaggt ctcccagctg acgaagccca tatcctccct gacgaagccc 1740

taccatcatc atcatcatca tcat 1764

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer F1

<400> 3

tggacccacc ttgagttttc 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer R1

<400> 4

ggagggaagc catttttctc 20

<210> 5

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer sequence F2

<400> 5

tatgaattca tgcccgccag cgcccccccc ag 32

<210> 6

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer sequence R2

<400> 6

tatctcgagt cagtggtagg gcttggtcag g 31

Claims

1. An anti-aging secreted Klotho protein characterized by: the amino acid sequence is shown in SEQ ID NO. 1.

2. A gene encoding the anti-aging secretory Klotho protein of claim 1.

3. The gene encoding the anti-aging secretory Klotho protein of claim 2, characterized in that: the nucleotide sequence is shown as SEQ ID NO. 2.

4. A recombinant expression vector characterized by: contains the gene encoding the anti-aging secretory Klotho protein of claim 2 or 3.

5. The method of constructing the recombinant expression vector of claim 4, comprising the steps of:

(4) subjecting the target fragment B obtained in step (3)₂Using EcoRI and XhoI to perform double enzyme digestion, performing electrophoresis after enzyme digestion, and recovering a target fragment C₂(ii) a Meanwhile, the pET-28a (+) plasmid is subjected to double digestion by EcoRI and XhoI, and then the target fragment C is subjected to T4 DNA ligase₂Connecting with pET-28a (+) plasmid after double enzyme digestion to obtainTo the recombinant expression vector pET-28 a-Klotho.

6. A recombinant bacterium which expresses Klotho protein, characterized in that: is obtained by transforming Escherichia coli with the recombinant expression vector of claim 4.

7. The recombinant bacterium that expresses Klotho protein according to claim 6, wherein: the Escherichia coli is Escherichia coli DH5 alpha or BL21(DE 3).

8. Use of at least one of the gene encoding an anti-aging secretory Klotho protein of claim 2 or 3, the recombinant expression vector of claim 4, and the recombinant bacterium expressing a Klotho protein of claim 6 or 7 for producing a Klotho protein.

9. A preparation method of Klotho protein is characterized by comprising the following steps: transforming the recombinant bacteria for expressing the Klotho protein of claim 6 or 7 into competent cells of Escherichia coli, culturing, adding IPTG for induced expression, centrifuging to collect bacteria, crushing, separating and purifying to obtain the Klotho protein; wherein the conditions for inducing expression are as follows: the induction temperature is 20-37 ℃, the induction time is 6-12 h, and the final concentration of IPTG is 0.1-2.0 mmol/L.

10. Use of at least one of the anti-aging secretory Klotho protein of claim 1, the gene encoding the anti-aging secretory Klotho protein of claim 2 or 3, the recombinant expression vector of claim 4, and the recombinant bacterium expressing the Klotho protein of claim 6 or 7 for the preparation of anti-aging, nitric oxide production, antioxidant stress, calcium and phosphorus metabolism, anti-inflammatory, and/or anti-tumor medicaments.