Recombinant humanized type I collagen and its preparation method and application

Abstract

The present invention belongs to the field of biomaterials, and specifically relates to a recombinant humanized type I collagen protein and a preparation method and application thereof. The present invention provides a recombinant humanized type I collagen protein, which is efficiently expressed through a eukaryotic expression system, and a recombinant humanized type I collagen protein with high purity is obtained after separation and purification, and has the effect of promoting the proliferation of skin fibroblasts and skin repair, and has broad application prospects in the fields of biomaterials and cosmetics.

Description

Technical Field

[0001] The invention belongs to the field of biomaterials, and in particular relates to a recombinant humanized type I collagen and a preparation method and application thereof. Background Art

[0002] Collagen is the most abundant protein in the human body, accounting for 25-30% of the total protein. As the most abundant protein in the extracellular matrix, collagen maintains the integrity of the cell structure and various physiological functions. More than 20 different types of collagen have been found, which can be roughly divided into the following categories: fibrous collagen, basement membrane collagen, microfibril collagen, anchoring fibril, transmembrane collagen, and incompletely characterized collagen. Type I collagen is a fibrous collagen that is widely found in skin, bones, and tendons. It can enhance tissue strength and elasticity, provide cells with sufficient nutrients, and maintain normal physiological functions.

[0003] At present, collagen is mainly derived from animal tissues. Naturally extracted collagen is a mixture of collagens of different molecular weights. It is insoluble in water and has poor biocompatibility. The traditional method of producing collagen is to use acid, alkali, and enzymatic hydrolysis to treat animal tissues to extract collagen derivatives. The collagen extracted by these methods has lost its original biological activity and cannot be used in the biomedical field to play its true function.

[0004] The best solution to the shortcomings and deficiencies in the current sources and extraction methods of collagen is to vigorously develop recombinant humanized collagen, especially recombinant humanized type I collagen. The present invention recombines the existing humanized type I collagen to construct a new recombinant type I humanized collagen, so that it can promote the proliferation of skin fibroblasts and the function of skin repair. Summary of the invention

[0005] The main purpose of the present invention is to provide a recombinant type I humanized collagen protein and a preparation method and application thereof. The recombinant type I humanized collagen protein obtained by large-scale production through a eukaryotic expression system has the effects of promoting the proliferation of skin fibroblasts and skin repair, and has broad application prospects in the fields of biomaterials and cosmetics.

[0006] One aspect of the present invention is to provide a recombinant humanized type I collagen, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] Among them, recombinant humanized type I collagen is based on human type I collagen, and is obtained by artificially inserting, deleting, replacing, and other changes at certain amino acid sites.

[0008] Optionally, an amino acid sequence having more than 90% sequence identity with the amino acid sequence shown in SEQ ID NO.1 and retaining the same biological function as the amino acid sequence shown in SEQ ID NO.1.

[0009] Optionally, an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO.1, while retaining the same biological function as the amino acid sequence shown in SEQ ID NO.1.

[0010] One aspect of the present invention is to provide a nucleic acid encoding recombinant humanized type I collagen, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0011] One aspect of the present invention is to provide a vector comprising a nucleic acid having a nucleotide sequence as shown in SEQ ID NO.2.

[0012] One aspect of the present invention is to provide a cell, wherein the cell comprises the above-mentioned vector. Preferably, the cell is Pichia pastoris.

[0013] One aspect of the present invention is to provide a method for preparing recombinant humanized type I collagen, specifically, the nucleic acid shown in SEQ ID NO.2 is introduced into a plasmid and then transformed into Pichia pastoris, the Pichia pastoris is cultured to suitable conditions for inducing expression, and finally the recombinant humanized type I collagen is obtained by separation and purification. Preferably, the purification is performed by cation exchange chromatography, and the protein corresponding to the elution peak is eluted and collected to obtain the recombinant humanized type I collagen.

[0014] Another aspect of the present invention provides an application of recombinant humanized type I collagen. Preferably, the application is used to prepare biomaterials and cosmetics that promote skin fibroblast proliferation and skin repair.

[0015] Beneficial technical effects of the present invention:

[0016] The present invention constructs a eukaryotic expression system to express recombinant type I humanized collagen, and finally obtains the recombinant type I humanized collagen by separation and purification. Circular dichroism analysis shows that the structure and properties of the recombinant type I humanized collagen are similar to those of natural type I collagen. Cell and skin tests show that the recombinant type I humanized collagen has the effects of promoting the proliferation of skin fibroblasts and promoting skin repair, and can be widely used in biomedical materials or cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 : Circular dichroism spectra of recombinant humanized type I collagen at different temperatures.

[0019] Figure 2 : Proliferation-promoting activity of recombinant humanized type I collagen in skin fibroblasts.

[0020] Figure 3 :WB quantitative results of TGM1 protein in mouse skin tissue. DETAILED DESCRIPTION

[0021] The following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1: Preparation of recombinant humanized type I collagen

[0023] The gene fragment was synthesized according to the nucleotide sequence shown in Seq ID NO.2, and the synthesized gene fragment was inserted between the restriction sites EcoRⅠ and NotⅠ on the pPIC9K plasmid to obtain a recombinant plasmid;

[0024] The recombinant plasmid was mixed with the GS115 competent cells, and after ice bath, the cells were electroporated, and then a sorbitol solution was added, mixed, and then transferred to a sterile EP tube, incubated at 30°C for 1h-2h, and then spread on an MD solid plate, and then inverted and cultured at 30°C for 2d-5d until a single colony grew;

[0025] Scrape off the His on the MD solid plate + After the transformant is diluted, it is spread on a YPD plate containing G418, and inverted and cultured at 30°C until a single colony appears, and then the single colony on the YPD plate is selected and transferred to a 96-well culture plate containing YPD medium, and cultured at 30°C to screen the recombinant yeast engineering bacteria;

[0026] The recombinant yeast engineering bacteria are selected and inoculated into BMGY induction medium, and cultured until the OD600nm absorbance reaches 2.0-6.0;

[0027] After centrifugation at 3000 rpm for 10 min, the cells were collected and re-inoculated into BMGY induction medium until the OD600nm absorbance reached 2.0, and culture was continued at 30°C and 220 rpm, with 0.5% methanol added every 24 h. After induction, the induction supernatant was collected by centrifugation;

[0028] The induced supernatant is purified by a cation exchange chromatography column, and the protein corresponding to the elution peak is eluted and collected, thereby obtaining recombinant type I humanized collagen with a purity of 98%.

[0029] Example 2: Circular dichroism of recombinant humanized type I collagen

[0030] The recombinant humanized type I collagen obtained in Example 1 was freeze-dried, a certain mass of freeze-dried powder was weighed, dissolved in a certain volume of deionized water, and prepared into a solution with a final concentration of 10 μM. The solution was placed in a micro-cuvette with a thickness of 1 mm to explore the effect of temperature on the conformation of recombinant humanized type I collagen. When the set temperature is reached and stabilized, incubate for 10-15 minutes, and perform circular dichroism on the recombinant humanized type I collagen, with a scanning wavelength range of 190-260 nm and an interval of 1 nm.

[0031] The conformation of recombinant humanized type I collagen was studied by circular dichroism analysis. The results showed that in a solution of a certain concentration, recombinant humanized type I collagen had a negative absorption peak between 190-200nm and a weak positive absorption peak at 220nm, with a typical characteristic circular dichroism peak of the collagen triple helix structure, which was similar to the circular dichroism peak of other collagens ( Figure 1 ).

[0032] Example 3: Experiment on promoting proliferation of skin fibroblasts by recombinant humanized type I collagen

[0033] The recombinant humanized type I collagen of Example 1 was used to promote cell proliferation. The cells used were mouse skin fibroblasts.

[0034] Mouse skin fibroblasts were cultured in complete medium at 37°C and 5% carbon dioxide, and the cell concentration was controlled to be 1.0×10 5 cells / ml-5.0×10 5 cells / ml, and used for biological activity determination 24h-36h after subculture; discard the culture medium in the culture flask, digest and collect the cells, and use complete cell culture medium to make 5.0×10 5 cells / ml-8.0×10 5Cell suspension of cells / ml was inoculated in a 96-well cell culture plate, 100 μL per well, and cultured at 37°C and 5% carbon dioxide; after 24 hours, PBS buffer was replaced and cultured at 37C and 5% carbon dioxide for 24 hours; the prepared cell culture plate discarded PBS buffer, added standard (commercially available animal-derived type I collagen) and recombinant type I humanized collagen of Example 1, and negative control added PBS, 100 μL per well, and cultured at 37°C and 5% carbon dioxide for 64h-72h; 20 μL of MTT solution was added to each well and cultured at 37°C and 5% carbon dioxide for 5h. The above operations were performed under sterile conditions. After discarding the liquid in the culture plate, 100 μL of DMSO was added to each well, and after mixing, the absorbance was measured at a wavelength of 570nm on an ELISA reader with 630nm as the reference wavelength, and the measurement results were recorded.

[0035] The results show that Figure 2 ), the recombinant humanized type I collagen of the present application has a significant effect of promoting the proliferation of skin fibroblasts compared with the standard product (Table 1).

[0036] Table 1: Experimental results of promoting the proliferation of mouse skin fibroblasts

[0037]

[0038] Example 4: Skin repair experiment with recombinant humanized type I collagen

[0039] The recombinant humanized type I collagen of Example 1 was subjected to a mouse skin repair test.

[0040] Several mice were divided into standard group, experimental group and control group, with 3 mice in each group. The mice underwent back hair removal, with an exposed area of ​​2×2 cm 2 Square area, each group used ultraviolet light to irradiate (200mJ / cm2) the back hair removal area, and redness and swelling appeared on the back of the mouse after irradiation, indicating that acute inflammation of the back skin of the mouse was successfully caused. Standard group, smeared commercially available animal-derived type I collagen. Experimental group, the recombinant type I humanized collagen of Example 1 was prepared into a gel and applied to the injured area on the back of the mouse. Control group, applied physiological saline. The frequency of the above-mentioned dressing change is 1 day / time, and the application is based on complete coverage of the damaged area. After 7 days, the skin repair is observed with the naked eye, specifically by wound healing rate = (damage area-remaining wound area) / total area (Table 2).

[0041] According to the recorded statistical results, both the experimental group and the standard group showed more significant skin repair effects than the control group, and the experimental group was slightly better than the standard group. The recombinant humanized type I collagen of the present application has a good skin repair effect (Table 2).

[0042] Table 2 (Wound healing rate = (injured area - wound remaining area) / total area) × 100%

[0043]

[0044] After 7 days, the back skin tissues of mice in each group were taken for protein extraction, and the TGM1 protein was quantified by WB experiment. The results showed that the TGM1 protein content in the experimental group and the standard group was significantly higher than that in the control group, and the TGM1 protein content in the experimental group was slightly higher than that in the standard group, which was consistent with the results of naked eye observation of skin repair.

[0045] It can be seen from this that the recombinant humanized type I collagen obtained in the present invention can effectively promote skin repair, and the repair effect is higher than that of conventional products sold on the market.

[0046] The above descriptions are only optional embodiments of the present invention, and are not intended to limit the patent scope of the present invention. All equivalent structural changes made using the contents of the present invention's specification and drawings, or directly / indirectly applied in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A recombinant humanized type I collagen, characterized in that: The amino acid sequence of the recombinant humanized type I collagen is shown in SEQ ID NO.

1. 2 . A nucleic acid encoding the recombinant humanized type I collagen according to claim 1 .

3. The nucleic acid according to claim 2, characterized in that The sequence of the nucleic acid is shown in SEQ ID NO.

2.

4. A carrier, characterized in that The vector comprises the nucleic acid according to claim 2 or 3.

5. A cell, characterized in that The cell comprises the nucleic acid of claim 2 or 3 or the vector of claim 4.

6. The cell according to claim 5, characterized in that The cell is Pichia pastoris.

7. A method for preparing recombinant humanized type I collagen according to claim 1, characterized in that: The nucleic acid shown in SEQ ID NO.2 is introduced into a plasmid and then transformed into Pichia pastoris. The Pichia pastoris is cultured to suitable conditions for inducing expression, and finally recombinant humanized type I collagen is obtained by separation and purification.

8. The preparation method according to claim 7, characterized in that: The method comprises inoculating the recombinant Pichia pastoris engineered bacteria into a BMGY induction medium, culturing until the OD600nm absorbance reaches 2.0-6.0; after centrifugation at a speed of 3000 rpm for 10 minutes, collecting the bacteria, re-inoculating into the BMGY induction medium, making the OD600nm absorbance reach 2.0, and continuing to culture at 30°C and 220 rpm, and adding 0.5% methanol every 24 hours. After induction, centrifugation is performed to collect the induced supernatant.

9. Use of the recombinant humanized type I collagen according to claim 1 in the preparation of biomaterials and cosmetics for promoting skin repair.

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