Recombinant III-type human collagen as well as expression system and application thereof

Abstract

The invention provides recombinant III-type human collagen as well as an expression system and application thereof, and belongs to the technical field of biological medicines. The recombinant human III-type collagen is efficiently expressed and prepared on a large scale through a genetic engineering technology, specific fragments are screened for sequence optimization based on amino acid sequence characteristics of the human III-type collagen, and soluble expression efficiency of target protein in a host is improved in combination with escherichia coli codon preference modification; the metabolic activity and the product stability of the recombinant strain are enhanced by optimizing the accurate proportion of fermentation medium components and trace elements; and a high-efficiency purification process is established to ensure that a high-purity humanized collagen product is obtained. The method overcomes the immunogenicity risk of the traditional animal source collagen, and provides a safe and controllable collagen source for the fields of medical treatment, beauty and biological materials.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to recombinant type III human collagen, its expression system and applications. Background Art

[0002] Collagen is a natural biological protein with a very high content, accounting for about 30% of the total protein; it is an important functional protein widely distributed in connective tissues such as animal skin, bones, teeth, tendons, blood vessels, etc. Collagen plays an important supporting role in the connective tissues of the human body and other mammals. In addition to acting as a functional structural protein in the extracellular matrix to maintain the shape and structure of the skin and tissues and organs, the main functions of collagen in the body are to support and protect tissues (maintaining the shape and function of tissues), promote cell proliferation and differentiation (which is of great significance for tissue growth, repair and regeneration), transmit intercellular information (regulating the physiological functions and metabolic activities of cells), promote wound healing and tissue repair, etc.

[0003] Collagen has good biocompatibility, low antigenicity, high affinity for the skin, and controllable biodegradability, etc., and has broad application prospects in industries such as biomedicine, food, and daily chemicals. In the medical field, collagen is widely used in tissue engineering and regenerative medicine for repairing tissue defects and traumas; in the health care field, collagen is used for preventing and treating diseases such as arthritis and osteoporosis; in the beauty field, collagen is widely used in skin care, beauty, weight loss, etc.

[0004] Currently, most of the collagen and related products sold on the market are derived from mammalian tissues or organs, with those from pigs and cows accounting for the vast majority. Not only is the extraction efficiency limited, but especially the obtained collagen has a complex composition, and there are some inherent defects such as immune rejection, complex composition, large batch differences, and virus risks, etc., which limit its wide application. Summary of the Invention

[0005] The purpose of the present invention is to provide recombinant type III human collagen, its expression system and applications, achieving efficient recombinant expression.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a recombinant type III human collagen, and the recombinant type III human collagen is a truncated form of human type III collagen, and its amino acid sequence is as shown in SEQ ID NO.1.

[0008] The present invention also provides a gene encoding the above-mentioned recombinant type III human collagen, and the nucleotide sequence of the gene is as shown in SEQ ID NO.2.

[0009] The present invention also provides a recombinant expression vector, which is characterized in that the recombinant expression vector comprises an initial vector and the gene encoding recombinant type III human collagen.

[0010] Preferably, the initial vector is a pET series vector, a pGEX series vector or a pMAL series vector.

[0011] Preferably, the initial vector is a pET-28a vector.

[0012] The present invention also provides a recombinant strain expressing the above-mentioned recombinant type III human collagen, and the above-mentioned recombinant expression vector is transformed into the recombinant strain.

[0013] Preferably, the recombinant strain is induced to express by IPTG, and the concentration of IPTG is 0.5-2 mM.

[0014] The present invention also provides the application of the above-mentioned recombinant type III human collagen, recombinant expression vector or recombinant strain in the preparation of wound repair products, scaffold materials or cosmetics.

[0015] Advantages of the present invention:

[0016] The present invention efficiently expresses and scales up the preparation of recombinant human type III collagen by genetic engineering technology. Based on the amino acid sequence characteristics of human type III collagen, specific fragments are screened for sequence optimization, combined with the modification of Escherichia coli codon preference to improve the soluble expression efficiency of the target protein in the host; by optimizing the components of the fermentation medium and the precise ratio of trace elements, the metabolic activity and product stability of the recombinant strain are enhanced; an efficient purification process is established to ensure the acquisition of a highly pure humanized collagen product. This method overcomes the immunogenic risk of traditional animal-derived collagen and provides a safe and controllable source of collagen for the medical, cosmetic and biomaterial fields. Description of the Drawings

[0017] Figure 1 It is the plasmid map of pET28a(+);

[0018] Figure 2 It is the SDS-PAGE detection result diagram of the purification of human type III collagen. Among them: 1. Protein Marker; 2. BL21(DE3); 3. pET28a / BL21(DE3); 4. pET28a-IIICOLA / BL21(DE3); 5. Supernatant after ultrasonic disruption; 6. Precipitate after ultrasonic disruption; 7. Flow-through during loading; 8. Washing solution; 9. 500 mM elution solution;

[0019] Figure 3 It is the result diagram of the optimization of the fermentation medium;

[0020] Figure 4 It is a graph of the optimized result of trace element composition;

[0021] Figure 5 It is a graph of the experimental results of promoting human glial cell proliferation;

[0022] Figure 6 It is a graph of the restriction enzyme digestion identification result. Specific implementation manners

[0023] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0024] Embodiment 1

[0025] (1) Selection of human type III collagen fragment

[0026] Based on the amino acid sequence of human type III collagen (NCBI protein sequence number: NP_000081; protein sequence website: https: / / www.ncbi.nlm.nih.gov / protein / NP_000081.2#sequence_NP_000081.2), the amino acid sequence at positions 370 - 710 was selected for expression, with a total of 350 amino acids (Mw: 31093.74): SNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAA, as shown in SEQ ID NO.1.

[0027] (2) Codon optimization

[0028] The above amino acid sequence was optimized for codons in the Escherichia coli expression system, and the nucleic acid sequence is 1050bp as follows:

[0029] (3)Induced expression

[0030] 1) The optimized nucleic acid sequence was ligated to the pET28a(+) expression vector, and the insertion sites were NcoI and XholI. The constructed expression vector is shown as Figure 1 shown, and the restriction enzyme digestion identification was carried out. The results of restriction enzyme digestion identification are shown as Figure 6 shown.

[0031] 2) Transformation and screening: 0.5 μL of the expression vector plasmid was mixed with 100 μL of Escherichia coli BL21(DE3), and incubated on ice for 30 minutes; after taking out, it was placed in a water bath at 42 °C for 90 seconds; then transferred to an ice bath for 5 minutes; then added to 200 μL of LB medium and incubated at 37 °C for 1 hour; finally, 200 μL was taken out and spread on an LB plate containing 100 μg / L kanamycin, and inverted and placed in an incubator at 37 °C for overnight culture.

[0032] 3) Activation of single colonies: Single clone colonies were picked and cultured in 5 mL of LB medium at a culture temperature of 37 °C, a rotation speed of 200 rpm, and a culture time of 16 hours;

[0033] 4) Subculture: The bacterial liquid cultured for 16 hours was inoculated into 500 mL of LB medium at a ratio of 1:100, and the culture temperature was 37 °C and the rotation speed was 200 rpm;

[0034] 5) Induced expression: When the OD600 of the bacterial liquid reached 0.6 - 0.8, induced expression was carried out. The IPTG induction concentration was 1 mM, the culture temperature was 37 °C, the rotation speed was 200 rpm, and the culture time was 16 hours.

[0035] (4)Ultrasonic disruption

[0036] The above-mentioned bacterial cells were collected by centrifugation at 5000 rpm for 20 minutes, and resuspended with 50 mL of equilibration buffer (50 mM NaH2PO4; 300 mM NaCl; 10 mM imidazole); after ultrasonic treatment, centrifuged at 5000 rpm for 20 minutes and the precipitate was discarded, and the supernatant was centrifuged at 12000 rpm for 20 minutes, and the supernatant was collected for purification. [[ID=##]]

[0037] (5)Nickel column purification

[0038] 1) Column equilibration: The nickel column was equilibrated with equilibration buffer (50 mM NaH2PO4; 300 mM NaCl; 10 mM imidazole) at a flow rate of 1 ml / min until the baseline was stable.

[0039] 2) Loading: The supernatant of the ultrasonic disruption was loaded in a cycle at 1 ml / min for 2 hours.

[0040] 3) Washing: Rinse the nickel column with the washing solution (50 mM NaH2PO4; 300 mM NaCl; 20 mM imidazole) at a flow rate of 1 ml / min until the baseline is stable.

[0041] 4) Elution: Elute the target protein with the elution solution (50 mM NaH2PO4; 300 mM NaCl; 250 mM imidazole) at a flow rate of 1 ml / min and collect the elution peak.

[0042] 5) Rinse the nickel column: Rinse the nickel column with pure water at a flow rate of 3 ml / min until the baseline is stable.

[0043] 6) Column sealing: Rinse the nickel column with 20% ethanol at a flow rate of 3 ml / min until the baseline is stable.

[0044] (6) Electrophoresis detection

[0045] Detect the purity of the recombinant protein by SDS-PAGE electrophoresis. After software analysis, the purity is about 95%, as Figure 2 .

[0046] (7) Fermentation medium optimization

[0047] Select fermentation media with different compositions for optimization, and finally determine the fermentation medium formula. Compared with the LB medium, the cell concentration increases by 116% after 24-hour culture, as Figure 3 shown.

[0048] The composition of the optimized fermentation medium is shown in Table 1:

[0049] Table 1 Fermentation medium formula

[0050]

[0051] (8) Trace element formula optimization

[0052] Select trace elements with different compositions for optimization, and finally determine the trace element formula. Compared with the case without adding trace elements, the cell concentration increases by 3.7% after 24-hour culture; compared with the LB medium (with added trace elements), the cell concentration of this fermentation medium formula (with added trace elements) increases by 101% after 24-hour culture, as Figure 4 shown. The composition of the optimized trace elements is shown in Table 2:

[0053] Table 2 Trace element formula

[0054]

[0055] (9) Experiment on promoting the proliferation of human glial cells

[0056] Human keratinocytes (HaCaT) were digested with trypsin, and the cell concentration was adjusted to 5×10 4 cells / mL.

[0057] 100 μL of cell suspension (about 5000 cells / well) was added to each well and incubated in an incubator at 37 °C and 5% CO2 for 24 hours to allow the cells to adhere to the well.

[0058] The stock solution of type III collagen was diluted to the working concentration (10, 50, 100, 200 μg / mL) with serum-free DMEM.

[0059] The medium in each well was aspirated, and 100 μL / well of type III collagen with different concentrations was added. At the same time, 100 μL of complete medium was added to the blank control well.

[0060] The cells were further incubated in an incubator at 37 °C and 5% CO2 for 24 hours.

[0061] Detection by MTT method

[0062] After the incubation, 10 μL of MTT solution (5 mg / mL, final concentration 0.5 mg / mL) was added to each well.

[0063] Incubate at 37 °C and 5% CO2 for 4 hours, and observe the formation of purple formazan crystals during this period.

[0064] The medium was aspirated, and 100 μL of DMSO was added to each well. The 96-well plate was gently shaken for 10 minutes to completely dissolve the formazan crystals.

[0065] The absorbance (OD value) at 570 nm was measured using an enzyme-linked immunosorbent assay reader, and the reference wavelength was 630 nm.

[0066] The results of the experiment on promoting the proliferation of human glial cells are as Figure 5 , recombinant human type III collagen has the effect of promoting the proliferation of human glial cells, and there is a certain dose-dependent relationship.

[0067] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A recombinant type III human collagen, characterized in that, The recombinant type III human collagen is a truncated form of human type III collagen, and its amino acid sequence is as shown in SEQ ID NO.

1.

2. The gene encoding the recombinant type III human collagen as claimed in claim 1, characterized in that, The nucleotide sequence of the gene is as shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, The recombinant expression vector includes an initial vector and the gene encoding the recombinant type III human collagen described in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The initial vector is a pET series vector, a pGEX series vector or a pMAL series vector.

5. The recombinant expression vector according to claim 4, characterized in that, The initial vector is a pET-28a vector.

6. A recombinant strain expressing the recombinant type III human collagen as claimed in claim 1, characterized in that, The recombinant strain is transformed with the recombinant expression vector described in any one of claims 3 to 5.

7. The recombinant strain according to claim 6, wherein The recombinant strain is induced to express by IPTG, and the concentration of IPTG is 0.5 to 2 mM.

8. Use of the recombinant type III human collagen according to claim 1, the recombinant expression vector according to any one of claims 3 to 5, or the recombinant strain according to any one of claims 6 to 7 in the preparation of a wound repair product, a scaffold material or a cosmetic.

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