CN114891124A

CN114891124A - Transdermal peptide oligopeptide-5 fusion polypeptide and preparation method thereof

Info

Publication number: CN114891124A
Application number: CN202210651356.6A
Authority: CN
Inventors: 刘月月; 张志乾; 吴奕瑞; 宋丹丹; 姚宛霏; 江翱; 王海梅; 邱�益
Original assignee: Guangzhou Qianxiang Biotechnology Co Ltd
Current assignee: Guangzhou Qianxiang Biotechnology Co Ltd
Priority date: 2022-06-10
Filing date: 2022-06-10
Publication date: 2022-08-12

Abstract

The invention relates to the field of protein, in particular to a transdermal peptide oligopeptide-5 fusion polypeptide and a preparation method thereof. The transdermal drug delivery system has the advantages of easy operation, small damage and the like, and prevents the digestion and degradation of gastrointestinal tracts and livers. The establishment of the transdermal peptide-oligopeptide-5 fusion expression system not only exerts the transdermal function of cTAT, but also promotes the fusion polypeptide to penetrate the skin and enter cells so as to exert the effects of anti-wrinkle and whitening. It has the advantages of no irritation, no skin allergy and injury, good compatibility between medicine and material, stable physicochemical properties, rapid action, and long action time. Although there are many kinds of transdermal peptides, TAT is most widely used due to its low toxicity and high cell penetration, and backbone cyclization contributes to improving translocation ability, and has no influence on cell viability after long-term action on cells. The fusion polypeptide has low toxicity, high cell penetrability and the like, and has greater development and application values and market prospects.

Description

Transdermal peptide oligopeptide-5 fusion polypeptide and preparation method thereof

Technical Field

The invention relates to the field of protein, in particular to a transdermal peptide oligopeptide-5 fusion polypeptide and a preparation method thereof.

Background

Transdermal Drug Delivery Systems or Transdermal absorption Systems (TDDS/TTS for short) refer to a method for applying drugs by Transdermal application or application, and the drugs are absorbed from the skin into the systemic blood circulation to achieve effective blood concentration and achieve disease treatment or prevention. Transdermal administration has the advantages of easy operation, small damage and the like, and digestion and degradation of gastrointestinal tracts and livers are avoided.

The skin is the largest organ, and whether transdermal administration is used for treating skin diseases or for the purpose of beautifying and caring skin, how to promote transdermal absorption of active ingredients is the focus of research of researchers.

The traditional penetration enhancer has the defects of easy skin allergy and inflammation generation, high concentration, toxicity and ineffectiveness to macromolecules, and needs an instrument for assistance, and the physical penetration enhancing method has high cost and is inconvenient to carry, and the stratum corneum is easy to thin after long-term use, and the skin infection is easy to cause. The biological penetration promoting technology utilizes polypeptide molecules with skin penetration capacity, and the polypeptide molecules have about 10 to 30 amino acids, so that the transdermal penetration of macromolecules can be effectively promoted. The transdermal peptide has the advantages of no irritation, no skin allergy or injury, good compatibility between the medicine and the material, stable physicochemical property, quick action and long action time.

Transdermal peptides are polypeptides that have enhanced transdermal functionality, enhance and/or facilitate the permeation of active molecules through the skin, allow for increased doses of active analytes entering the systemic circulation or active molecules reaching target organs, tissues and cells, and can be used for transdermal administration of large molecular weight drugs such as polypeptides, proteins and nucleic acid molecules. Transdermal peptides are of many types, and TAT is most widely used for its low toxicity and high cell penetration.

The amino acid sequence of the cyclic cTAT (cyclization of Tat peptide) is CYGRKKRRQRRRC, the cysteine sulfydryl of the main chain forms a disulfide bond to generate cyclization, the cyclic cTAT has excellent cell penetrating capability, the transdermal capability of the cyclic cTAT is far higher than that of the linear TAT, and the cTAT does not have obvious influence on the cell vitality after being acted on the cell for a long time. Oligopeptide-5 is a cosmetic peptide with effects of removing wrinkle and whitening skin, is prepared by prokaryotic expression system (E.coli) recombinant expression, is a non-glycosylated polypeptide consisting of 169 amino acid residues, and has a molecular weight of 19.2 kDa.

Oligopeptide-5 (Oligopeptide-5, peptide-5 for short) is prepared by prokaryotic expression system recombinant expression, and the non-glycosylated polypeptide consisting of 169 amino acid residues has a molecular weight of 19.2 kDa. Oligopeptide-5 is a cosmetic peptide with anti-wrinkle and skin whitening effects.

Many chemical permeation enhancers are used to try to open the skin barrier, but most of the effects are not ideal. Without the aid of physical methods, it is difficult for chemical dermal penetration enhancers to effectively bring hydrophilic drugs (especially drugs with molecular weights greater than 500 Da) into the blood circulation through intact skin and to achieve the blood levels required for treatment; the existing chemical transdermal enhancer easily causes skin injury, allergy, inflammation, allergy, systemic toxicity and the like, has the defects of ineffectiveness to macromolecules and need of instrument assistance, and is greatly limited in use; the common physical methods include iontophoresis, microneedles and the like to prove that the transdermal enhancer has the effect of enhancing transdermal penetration of various drug molecules, but the physical methods have many defects, including the use of special instruments, high cost, poor flexibility of dosage forms and the like, the physical transdermal enhancement method has high cost and inconvenience for carrying, the cuticle is easy to thin and cause skin infection after long-term use, and the physical transdermal enhancer has pain feeling in different degrees and is not suitable for household use and the like.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a transdermal peptide oligopeptide-5 fusion polypeptide and a preparation method thereof.

The invention provides a fusion polypeptide comprising a cTAT and peptide 5.

The present invention does not limit the order of linkage of cTAT and peptide5 in the provided fusion polypeptide. In some embodiments, the N-terminal to C-terminal connection order is cTAT, peptide 5. In the invention, linker does not exist in the middle of fusion expression of cTAT and peptide 5. Wherein the cTAT is cyclized, and the cTAT-oligopeptide-5 fusion polypeptide has the characteristics of low toxicity, high cell penetrability and the like after the fusion polypeptide is formed.

Specifically, the fusion polypeptide has an amino acid sequence shown as SEQ ID NO. 1: CYGRKKRRQRRRCHKYILTYILPTLLYRSCFHIICLVGYISLACNDMTPEQMATNVNCSSPERHTRSYDYMEGGDIRVRRLFCRT are provided.

In the fusion polypeptide, the amino acid sequence of cTAT is CYGRKKRRQRRRC (SEQ ID NO. 3). The amino acid sequence of peptide5 is:

HKYILTYILPTLLYRSCFHIICLVGYISLACNDMTPEQMATNVNCSSPERHTRSYDYMEGGDIRVRRLFCRT(SEQ ID NO.4)。

the invention also provides: a nucleic acid encoding a fusion polypeptide according to the invention.

In the present invention, the sequence of the nucleic acid encoding the fusion polypeptide is: tgttacggccgtaagaaacgccgacagcgtcgccggtgccacaaatacattctgacctacattctgccgaccctgctgtatcgcagctgcttccacataatttgcctagttggttacatctccctggcttgtaatgatatgacacccgaacaaatggccacgaacgtcaattgctcatcgccagagcgccatactcgaagttatgactacatggaaggcggagatatacgggtaagacggttattctgtcgtacc (SEQ ID NO. 2).

The invention also provides an expression vector comprising a backbone vector and a nucleic acid of the invention.

The expression vector is a plasmid vector, and the expression vector comprises but is not limited to pET series vectors. In some embodiments, the backbone vector of the expression vector comprises the pET28a plasmid. The pET28a plasmid is used in the amplification, storage and protein expression of nucleic acid segment. Wherein the insertion site of the nucleic acid fragment is between the multiple cloning sites Not I and BamH I.

In the present invention, other vectors are used in addition to the pET28a expression vector, but the expression effect is not satisfactory, and for example, the present invention attempts to use competence such as rosetta and BL21(DE3) plys as the expression vector, but the expression effect is not satisfactory.

The invention also provides a host transformed or transfected with the expression vector.

The host of the present invention includes a storage host for a plasmid vector, or an expression host for the fusion polypeptide. In embodiments of the invention, the host is a bacterial, fungal or animal cell. In some embodiments, the host is a bacterium. In some embodiments, the strain comprises escherichia coli. In particular, the invention takes Escherichia coli BL21 DE3 as a host for expressing the fusion polypeptide.

The construction method of the host comprises the steps of transforming the plasmid vector into escherichia coli competence and constructing the expression host of the fusion polypeptide through recovery.

The invention also provides a preparation method of the fusion polypeptide, which comprises culturing the host to obtain a culture containing the fusion polypeptide.

The culture medium is LB liquid culture medium (added with antibiotic kana50mg/ml), and the culture temperature is 26-37 ℃.

After the culture, the method also comprises a step of inducing protein expression. The inducer for induction is IPTG. In some embodiments, the concentration of IPTG induced expression of the fusion polypeptide is between 0.1mM and 1.5 mM.

The protein tag expressed by the polypeptide fusion comprises His tag.

The preparation method of the fusion polypeptide further comprises the step of purifying the culture to obtain the fused protein. The fusion polypeptide prepared by the method has a purification tag which is His tag; the purification mode of the fusion polypeptide comprises nickel column purification, and the column packing of the purification method is Ni ⁺ Column, imidazole gradient elution.

The invention also provides the fusion polypeptide, the nucleic acid, the expression vector, the host, the fusion polypeptide prepared by the preparation method and/or application of the culture containing the fusion polypeptide prepared by the preparation method in preparation of anti-wrinkle and/or whitening products.

When the fusion polypeptide is used for treating skin wrinkles, the wrinkled skin can be smooth and glossy, and the wrinkles slowly fade and disappear; can also prolong the period of the new skin which is smooth, fine and glossy. When the whitening effect is exerted, the skin which is suntanned or becomes black and yellow by pigmentation can be gradually whitened, and the white, clean, smooth and glossy state of the skin is maintained, so that the skin is not easy to become black and yellow.

The invention provides an anti-wrinkle and/or whitening product, which comprises the fusion polypeptide.

The cyclization of TAT gives it superior cell penetration, a much higher transdermal capacity than linear TAT. TAT is most widely used for its low toxicity and high cell penetration. And the cyclization of the main chain is beneficial to improving the translocation capability, and experiments prove that the long-time action of cTAT on cells has no influence on the cell viability. Thus, cTAT was screened as the target transdermal peptide small molecule of the present invention. The invention fuses the three components as fusion polypeptide, but not simply mixes the three components because of doing so, improves the protein expression quantity and solubility, has better biocompatibility, does not cause skin allergy and damage due to the characteristics of low toxicity, no irritation, high cell penetrability and the like, has good compatibility with materials, has stable physicochemical property, takes effect quickly and has long acting time.

The cyclization principle of the cTAT is that cysteine forms a disulfide bond, and the disulfide bond can form the ring under alkaline and non-reducing conditions, so that the cyclization can be realized in one step.

The product of the invention comprises cosmetics, medicines, health products and foods. The dosage forms comprise injection and oral preparation;

the cosmetic comprises a cosmetic matrix and a fusion polypeptide; the medicine comprises the fusion polypeptide and pharmaceutically acceptable auxiliary materials; the health product comprises the fusion polypeptide and auxiliary materials acceptable in the health product; the food comprises the fusion polypeptide and acceptable auxiliary materials in the food.

The invention also provides an anti-wrinkle whitening method, and the product is administered. The administration mode includes application, smearing, acupuncture, massage, etc.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts according to the drawings:

a in FIG. 1 shows PCR amplification of a gene fragment of interest; b in FIG. 1 shows a pET28a vector fragment;

FIG. 2 shows transformation of DH5 alpha competent Kana resistance screening positive clone after seamless ligation of the target fragment with the vector;

FIG. 3 shows pET28a-peptide5-cTAT recombinant plasmid transformation BL21(DE3) competence and kana resistance selection;

FIG. 4 shows the expression of IPTG 37 ℃ induced fusion polypeptide in BL21(DE3), the sample is whole cell lysate, and the molecular weight of the fusion polypeptide is 27.4 KDa;

FIG. 5 shows protein nickel column purification and SDS-PAGE detection;

FIG. 6 shows a summary of fluorescence imaging test results;

FIG. 7 shows a mean fluorescence intensity quantitative statistical chart;

FIG. 8 shows a mean osmolarity histogram;

FIG. 9 shows a statistical graph of mean permeation flux.

Detailed Description

The invention provides a transdermal peptide-oligopeptide-5 fusion polypeptide and a preparation method thereof, and a person skilled in the art can realize the transdermal peptide-oligopeptide-5 fusion polypeptide by appropriately modifying process parameters according to the contents in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.

Example 1

1. Construction of transdermal peptide-oligopeptide 5 fusion expression vector: pET28a is used as a vector to construct a cTAT-peptide-5 fusion expression vector, and a target gene fragment is seamlessly cloned and connected with a pET28a vector obtained by PCR amplification. And transforming the recombinant product into DH5 alpha competence and plating, selecting a positive clone colony, and carrying out PCR identification and amplification on a fragment containing the target gene by using universal primers T7 and T7 ter. And 4-10 positive clone colonies which are identified by PCR and accord with the theoretical molecular weight band are selected and sequenced, and the comparison consistency of the sequencing result and the target fragment sequence reaches 100%. The plasmid of pET28a-peptide5-cTAT vector is transformed into BL21 DE3 competence, and kana resistance is screened to successfully transform into a positive clone colony of the recombinant plasmid.

2. Transdermal peptide-oligopeptide 5 induced expression: selecting and streaking the monoclonal colony, inoculating the colony in 3-5 ml LB liquid culture medium (added with antibiotic kana50mg/ml), shaking to culture at 37 deg.c for 3-12 hr to detect OD value of 0.2-1.0, diluting with fresh culture medium in the ratio of 1:10 to 1:100 (added with antibiotic kana50mg/ml), and expanding culture at 37 deg.c to OD ₆₀₀ When the concentration is about 0.2-1.0, IPTG is added, the induction concentration of IPTG is 0.1-1.5 mM, and the culture is carried out respectively at the temperature of 26-37 ℃.

3. Protein purification: by using Ni ⁺ Column purification, gradient elution with 20 mM-200 mM imidazole.

4. SDS-PAGE detection: collecting bacterial liquid, centrifuging at 12000rpm for 1-8 min and adding lysine buffer in 50-200 μ l, boiling in water at 80-120 deg.c for 5min, cooling on ice and centrifuging instantaneously. SDS-PAGE was used to determine whether the protein was expressed. 8 to 16 percent of prefabricated glue, the sample loading amount is 10 to 30 mul, 120 to 200V and 30 to 60 min.

Example 2 transdermal peptide Permeability efficacy evaluation based on skin model

The test is based on a 3D epidermis model

And (3) uniformly coating the sample on the surface of the model by adopting a surface administration mode, and collecting the model tissue and the culture solution after incubation for different times. The permeability of the sample to be detected is visually observed by using a fluorescence microscope observation method, and meanwhile, the permeability of the sample to be detected in the culture solution is detected by using a fluorescence microplate reader, so that the permeability of the sample is comprehensively judged. The test system comprises: the model used in the test is a 3D skin model

Batch number: ES210405 (cantonese buxi). The main reagents are as follows: PBS (Boshide), EpiRecovery broth (Guangdong Boxi). The main equipment is as follows: CO 2 ₂ Incubator (Thermo, 150I), clean bench (Suzhou Antai, SW-CJ-1F), fluorescence microscope (Olympus), multifunctional microplate reader (Perkin Elmer).

Sample information is shown in table 1:

table 1: information of sample to be measured

Sample name	Sample class	Sample State	Solubility in water	Storage conditions	Period of validity
						Oligopeptide-5, transdermal peptide-oligopeptide-5	Raw materials	Powder of	Dissolving in water	Low temperature preservation	/

Table 2: permeability and efficacy testing

Preparation of working solution

Sample preparation:

p5: weighing 3.75mg of sample P, and adding the sample P into 0.3mL of PBS for full dissolution;

T-P5: a4.53 mgT-P (equimolar) sample was weighed into 0.3ml PBS and the molar concentrations of the two sample solutions were the same.

The experimental steps are as follows:

1) according to the experimental design of table 2, the model was transferred to a 6-well plate (3.7mL of the corresponding grouping of episrecovery culture solution was added in advance) and the test group number was noted on the 6-well plate.

2) Adding 25 μ L of prepared sample working solution to the surface of the model, clamping with forceps to obtain nylon membrane, covering each model surface with the nylon membrane, uniformly distributing the sample on the model surface, and placing in CO ₂ Incubator (37 ℃, 5% CO) ₂ ) And (4) respectively incubating for 1h, 2h and 8 h.

3) Permeation detection

After the administration incubation is finished, collecting the culture solution in an EP tube, storing a sample for detecting the permeation quantity in a refrigerator at 4 ℃ after the collection, and detecting by a fluorescent microplate reader the next day; meanwhile, the 3D skin model was collected in EP tubes, 1mL paraformaldehyde was added to each tube, fixed at room temperature, frozen sectioned and photographed with a fluorescence microscope.

Results of the experiment

After the incubation culture is finished, the fluorescence detection of the frozen section and the measurement of the permeation quantity are carried out, and the results are summarized in table 3 and fig. 8.

Table 3: summary of fluorescence intensity values

Sample name	Mean fluorescence intensity	SD
			BC	10906.00	78.08
P5-1h	55916.67	2719.29
			T-P5-1h	164141.33	11333.47
P5-2h	93120.00	2388.39
			T-P5-2h	295334.33	22701.83
P5-8h	237328.67	5239.99
			T-P5-8h	781895.67	25982.63

Remarking: when statistically analyzed by the t-test method, the significance of the sample group was shown as a value of P-value < 0.05 and a value of P-value < 0.01, compared to the BC group.

Table 4: summary of osmolarity results

Remarking: the fluorescence intensity of each group was measured, and the absolute osmolarity of each group was calculated using the sample dilution as a standard. Absolute osmolarity-the mean value of the concentration corresponding to the sample group at fixed time-the mean value of the concentration corresponding to the time point BC group.

Table 5: penetration results summary sheet

Sample name	Average penetration amount (μ g)	SD
			BC	0	0
P5-1h	0.36	0.02
			T-P5-1h	1.30	0.10
P5-2h	0.69	0.02
			T-P5-2h	2.41	0.20
P5-8h	1.91	0.06
			T-P5-8h	6.54	0.22

Remarking: absolute permeation rate is the average permeation concentration × total volume of liquid (3.7 mL).

After the sample acts on the 3D epidermis model for 1h, the penetration amount of P5 is 0.36 mu g, and the penetration amount of T-P5 is 1.30 mu g; after 2 hours of action, the penetration amount of the P5 group is 0.69 mu g, and the penetration amount of the T-P5 group is 2.41 mu g; after 8 hours of action, the penetration amount of P5 was 1.91. mu.g, and the penetration amount of T-P5 was 6.54. mu.g.

Conclusion

Based on 3D epidermis model

The penetration test result shows that the penetration amount of the sample P5 is 0.36 mu g, 0.69 mu g and 1.91 mu g after 1h, 2h and 8 h; the penetration of sample T-P5 was 1.30. mu.g, 2.41. mu.g and 6.54. mu.g after 1h, 2h and 8 h. The two groups of samples acted on the 3D epidermal model for the same time (1h, 2h and 8h), and the permeation amount of the TP-5 fusion polypeptide was obviously higher than that of the P5 group.

In conclusion, the samples P5 and T-P5 have certain permeation behaviors, the sample permeation amount is increased continuously with the increase of time, and the fusion expression of T-P5 obviously improves the permeability of P5.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

<110> Dry-phase Biotechnology Ltd of Guangzhou City

<120> transdermal peptide oligopeptide-5 fusion polypeptide and preparation method thereof

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Cys Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Cys His Lys Tyr

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Ile Leu Thr Tyr Ile Leu Pro Thr Leu Leu Tyr Arg Ser Cys Phe His

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attctgccga ccctgctgta tcgcagctgc ttccacataa tttgcctagt tggttacatc 120

tccctggctt gtaatgatat gacacccgaa caaatggcca cgaacgtcaa ttgctcatcg 180

ccagagcgcc atactcgaag ttatgactac atggaaggcg gagatatacg ggtaagacgg 240

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Claims

1. A fusion polypeptide comprising a cTAT and peptide 5; wherein:

the cTAT has an amino acid sequence shown as SEQ ID NO. 3;

peptide5 has an amino acid sequence shown in SEQ ID NO. 4.

2. The fusion polypeptide of claim 1, wherein the fusion polypeptide has an amino acid sequence as set forth in SEQ ID No. 1.

3. A nucleic acid encoding the fusion polypeptide of claim 1.

4. The nucleic acid of claim 3, wherein the nucleic acid has the nucleotide sequence set forth in SEQ ID No. 2.

5. An expression vector comprising a backbone vector and the nucleic acid of claim 3 or 4.

6. The expression vector of claim 5, wherein the backbone vector comprises the pET28a plasmid.

7. A host transformed or transfected with the expression vector of claim 5 or 6.

8. The method for producing the fusion polypeptide according to claim 1 or 2, wherein the host according to claim 7 is cultured to obtain a culture containing the fusion polypeptide.

9. Use of the fusion polypeptide of claim 1 or 2, the nucleic acid of claim 3 or 4, the expression vector of claim 5 or 6, the host of claim 7, the fusion polypeptide prepared by the preparation method of claim 8, and/or the culture containing the fusion polypeptide prepared by the preparation method of claim 8 in the preparation of anti-wrinkle and/or whitening products.

10. An anti-wrinkle and/or whitening product comprising the fusion polypeptide of claim 1 or 2.