Novel recombinant human XV type collagen as well as production method and application thereof

Abstract

The invention discloses a novel recombinant human XV type collagen as well as a production method and application thereof. The amino acid sequence of the recombinant human XV type collagen is as shown in SEQ ID No. 1. The recombinant human XV type collagen is formed by splicing disconnected peptide fragments from a triple helix region of an alpha1 chain of the natural human XV type collagen, and the recombinant human XV type collagen can be secreted and expressed in a commercial pichia pastoris expression system at high yield and has good biological activity of resisting cancer and inhibiting proliferation and migration of vascular endothelial cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology and relates to a novel genetically engineered recombinant collagen, a production method and application thereof. Background Art

[0002] Collagen is a biological macromolecular protein, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. It is closely related to the formation and maturation of tissues, the transmission of information between cells, joint lubrication, wound healing, calcification, blood coagulation and aging. It is one of the most critical raw materials in the biotechnology industry and is widely used in medical materials, cosmetics, and food industries.

[0003] The collagen currently used in industry is mainly extracted from animal skin or bones through acid, alkali or enzymatic methods. Its main source is animal connective tissue. However, collagen extracted from animal tissues has risks such as animal-derived diseases. At the same time, large-scale preparation puts tremendous pressure on animal breeding on the supply side.

[0004] With the large-scale application of genetic engineering technology, genetic engineering recombinant expression of collagen has successfully solved the bottleneck of large-scale preparation of collagen by using modeled expression hosts to express exogenous proteins. Compared with traditional animal collagen extraction, genetic engineering recombinant preparation of collagen has the following advantages: 1. A wide variety of collagen is produced, which can be collagen from common sources such as cattle, pigs, and fish, and human collagen can also be produced, which has better safety and immune efficacy in the fields of medical devices. 2. It can effectively avoid animal-derived diseases. Recombinant collagen usually uses simple prokaryotic or eukaryotic cells as expression hosts. Its cell pathogens cannot be transmitted to each other due to the huge difference in cell structure with humans. 3. The production cycle is short, cost-saving, can be quickly amplified, and is suitable for industrial large-scale production. Compared with the long animal culture of 3-5 months, the microbial culture cycle only takes 2 or 3 days and the culture is simple. Large-scale culture can be carried out by providing simple C source and N source, which is easy to prepare at industrial level. Therefore, the preparation of recombinant collagen, especially recombinant human collagen, is currently a hot spot in collagen production research.

[0005] Recombinant human collagen is considered to be the collagen closest to the body's natural synthesis because its amino acid composition is similar or consistent with that of the human body. It has good biocompatibility and high safety while also having the efficacy and functions of animal collagen. There are a large number of literature reports on it, and commercial products have been successfully developed for use in the fields of beauty, cosmetics and medical devices.

[0006] Recombinant collagen can be obtained by constructing truncated proteins or truncated peptides of natural collagen. The uncertainty of the position and length of the truncated protein in natural collagen makes it possible to have tens of millions of recombinant collagen types in theory. However, there has been a lack of effective theoretical guidance on which of these recombinant collagens can have a higher secretion yield. The best results must be screened out through a large number of experimental verifications. This is the main reason why there are currently few types of recombinant collagen in mass production.

[0007] Type XV collagen combines with a variety of extracellular matrix proteins. This collagen not only has certain functions in maintaining the structure of skeletal muscle, heart, skin microvessels, and vitreous body of the eye. At present, the main problems of type XV collagen in production are as follows: 1) Natural type XV collagen is difficult to obtain and is difficult to separate. Currently, type XV collagen is widely and regionally distributed outside organs such as blood vessels and nerve tissues, and needs to be purified using a multi-step process. This collagen extraction method has certain limitations, such as low extraction efficiency and low purity. 2) Recombinant type XV collagen is difficult to express. The expression level of full-length type XV collagen in various existing expression systems is very low, which is difficult to meet the requirements of industrial production. 3) The biological activity of type XV collagen fragments is unclear. Currently, it is reported that the biological activity of type XV collagen includes playing a role in the adhesion of the basement membrane to the surrounding connective tissue and may be related to the tumorigenesis of keratinocytes and melanocytes.

[0008] Based on the above problems, how to obtain type XV collagen fragments so that they can be efficiently expressed in a microbial expression system (especially secretory expression in Escherichia coli or Pichia pastoris) and have clear and prominent biological activity is a technical problem that has always existed in this technical field.

[0009] The development of a method for preparing recombinant type XV collagen that has anti-cancer and endothelial cell proliferation and migration inhibitory effects has important theoretical and practical significance. Summary of the invention

[0010] In view of the technical problems existing in the above-mentioned prior art, the object of the present invention is to provide a new recombinant human type XV collagen, which can be efficiently secreted and expressed in a commercial Pichia pastoris expression system and has good biological activity of anti-cancer and inhibiting the proliferation and migration of vascular endothelial cells.

[0011] The inventors conducted in-depth research to solve the above technical problems, and found that the recombinant humanized collagen (758 amino acid residues) obtained by splicing peptide segments from the α1 chain of natural human type XV collagen can be efficiently secreted and expressed in a commercial Pichia pastoris expression system, and has good anti-cancer and biological activities of inhibiting endothelial cell proliferation and migration, thereby completing the present invention.

[0012] That is, the technical solution of the present invention includes:

[0013] 1. A recombinant human type XV collagen, the amino acid sequence of which is shown in SEQ ID No.: 1.

[0014] 2. A nucleic acid encoding the recombinant human type XV collagen described in item 1.

[0015] 3. An expression vector comprising the nucleic acid described in item 2.

[0016] 4. A host cell into which the expression vector described in item 3 is introduced. The host cell may be a prokaryotic cell or a eukaryotic cell, including bacterial hosts such as Escherichia coli, Bacillus subtilis, and Bacillus licheniformis, and eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, and plant cells, preferably Escherichia coli and Pichia pastoris, and more preferably Pichia pastoris.

[0017] 5. A method for producing the recombinant human type XV collagen described in item 1, comprising the steps of culturing the host cell described in item 4 to express the recombinant human type XV collagen described in item 1, and collecting the culture containing the recombinant human type XV collagen. The expression can be constitutive expression, induced expression, or a combination of the two. The inducer for inducing expression can be an inducer such as IPTG, β-galactoside, methanol, ethanol, etc.

[0018] 6. The production method according to item 5, further comprising the step of separating and purifying the culture to obtain purified recombinant human type XV collagen.

[0019] 7. The production method according to item 6, wherein the separation and purification adopts a combination of one or more of salting out, chromatography, affinity chromatography, acid-base precipitation, and membrane separation, preferably a combination of chromatography and membrane separation or a combination of ion exchange chromatography and membrane separation.

[0020] 8. Use of the recombinant human type XV collagen according to item 1 in the preparation of skin care products, health care products, tissue engineering materials, medicines or medical devices.

[0021] 9. The use according to item 8, wherein the tissue engineering material is selected from subcutaneous fillers, artificial bones, artificial skin, oral absorbable biomembranes, bone implants, vascular stents, hemostatic agents, procoagulants, interstitial scaffolds and collagen sponges.

[0022] 10. The use according to item 8, wherein the drug is used for preventing and fighting cancer or inhibiting the proliferation and migration of vascular endothelial cells.

[0023] The beneficial effects of the present invention are: providing a new recombinant human type XV collagen, which is spliced ​​from unconnected peptide segments from natural human type XV collagen, which can be efficiently secreted and expressed in a commercial Pichia pastoris expression system, and has good biological activity of anti-cancer and inhibiting the proliferation and migration of vascular endothelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The electrophoresis diagram shows the results of induced fermentation of recombinant collagen type XV-1 (2ul loading amount).

[0025] Figure 2 The electrophoresis diagram shows the results of purification of recombinant collagen type XV-1 (1-5ul loading amount).

[0026] Figure 3 The electrophoresis diagram shows the expression results of different recombinant collagen XV. DETAILED DESCRIPTION

[0027] Example 1 Preparation of recombinant collagen XV-1 in yeast expression system

[0028] 1. Experimental Methods

[0029] 1. Preparation of shuttle plasmid and yeast expression strain

[0030] According to the amino acid sequence shown in SEQ ID No.1, the codon optimization of the yeast expression system was carried out to obtain the target gene sequence of recombinant collagen XV-1. The obtained target gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for gene synthesis. The synthesized gene was connected to the pPicZαA plasmid to obtain the pPicZαA-I-α1 plasmid.

[0031] pPicZαA-I-α1 was linearized with Pme I and then transformed into Pichia pastoris X-33 competent cells. Bleomycin resistance was used as a selection marker to screen transformants to obtain a yeast expression strain.

[0032] 2. Induced expression of target protein

[0033] (1) Pick a single colony of the constructed yeast expression strain and add it to 5 ml of YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and culture it at 30°C and 200 rpm for 48 h for activation;

[0034] (2) Transfer the inoculum to a 500 ml Erlenmeyer flask (containing 200 ml of YPD culture medium) at a 1% inoculum volume and culture at 30°C and 200 rpm for 24 h to serve as the seed for the tank.

[0035] (3) Prepare 3 L of BSM medium and add it to a 5 L fermenter, sterilize at 121° C. for 20 min, and after cooling to 30° C., adjust the pH to 5.0, and add the seeds prepared in (2) to the fermenter by flame inoculation for fermentation;

[0036] (4) Cultivate until OD 600 When OD was 70, 50% glycerol was added until 600 When the temperature reaches about 120, stop adding, and wait until the dissolved oxygen rebounds to 100%, then start adding methanol for induction;

[0037] (5) During the induction process, the dissolved oxygen was controlled to be not less than 30% and the pH was about 5.0. The induction was continued for 40 h, and the fermentation was terminated. The culture medium was centrifuged at 12,000 rpm for 2 min, and the supernatant was taken for detection of protein yield and purity using the BCA method and SDS-PAGE method.

[0038] 2. Experimental Results

[0039] The BCA test result of the fermentation tank yield of recombinant collagen XV-1 prepared by the above method was as high as 25g / L, and the test result of SDS-PAGE electrophoresis showed that the target protein accounted for more than 50%, and the protein purity after ultrafiltration through a hollow fiber column could reach more than 90%.

[0040] Example 2 Preparation of recombinant collagen XV control protein in Pichia pastoris expression system

[0041] The same design concept as recombinant collagen XV-1 was used to extract peptide segments from the α1 chain of type XV collagen and splice them. The same method was used to express the peptides. The results of sequence design and expression are shown in the table below (Table 1). The protein electrophoresis is shown in Figure 3 .

[0042] Table 1 Expression results of different recombinant collagen XV

[0043]

[0044] Note: The amino acid sequence of recombinant collagen XV-2 is shown in SEQ ID No.: 2, and the amino acid sequence of recombinant collagen XV-3 is shown in SEQ ID No.: 3.

[0045] As can be seen from Table 1, the yield of the recombinant collagen XV-1 is much higher than that of the recombinant collagens XV-1-2 and XV-1-3 of similar design. Therefore, the recombinant collagen VI-1 provided by the present invention is easier to prepare on a large scale and has greater potential for mass production.

[0046] Example 3: Application of recombinant type XV collagen in anti-cancer

[0047] Specific implementation method: Human breast cancer cell inhibition tumor cell experiment

[0048] Each well was inoculated with 1.5 × 10 6 The 96-well culture plate of human breast cancer cells was placed in a CO2 incubator and cultured for 24h (saturated humidity, 37°C, 5% CO2). The blank group was normal cell culture fluid, and the experimental group was XV-1, XV-2, and XV-3 three recombinant XV collagens. Each recombinant XV collagen was set with 3 parallel wells, and 10μL of 25mg / mL recombinant XV collagen was added to each well. The culture time was 24h, 48h, and 72h. About 4 hours after the end of the culture, 20μL of MTT solution was added to each well and continued to culture. At the end of the culture, MTT was used to determine the cell inhibition rate. The results were statistically analyzed using SPSS16.0 statistical software, and the experimental data were expressed as mean ± standard deviation. The experimental results are shown in Table 2.

[0049] Inhibition rate (%) = (OD value of control group - OD value of experimental group) / OD value of control group

[0050] Table 2 Inhibitory effects of different recombinant collagens on human breast cancer cells

[0051] XV-1 XV-2 XV-3 24h 24.4±1.0 12.3±1.1 10.5±1.9 48h 40.3±0.3 15.4±1.2 30.4±1.4 72h 55.3±2.4 23.4±1.7 39.6±1.3

[0052] Example 4: Effect of recombinant human type XV collagen on inhibiting proliferation and migration of vascular endothelial cells

[0053] Specific implementation method:

[0054] 1. Experiment on inhibition of endothelial cell proliferation: MTT method was used to detect the effect of type XV collagen on endothelial cell proliferation.

[0055] Human umbilical vein endothelial cells were cultured at 1×10 4Density inoculated in 96-well plates for culture, discard the culture medium after 24 hours, add 100μl of Medium200 culture medium containing 4% LSGS to each well, add 100μL of each recombinant type XV collagen (concentration of 25mg / mL) to each well, and the negative control group is normally cultured endothelial cells. Each group has 6 replicate wells. After 48 hours of action, add 20μl of MTT solution (5μg / ml) to each well, culture at 37℃5% CO2 for 4 hours, and terminate the culture. Carefully aspirate and discard the supernatant in the well, add 100μl DMSO to each well, shake for 10 minutes, and measure the absorbance value (OD value) on the Bio-red 550 microplate reader after the precipitate is completely dissolved. The experiment is repeated three times.

[0056] Table 3 Effects of different recombinant collagens on endothelial cell proliferation

[0057] Group OD P Negative control 0.61±0.03 ＜0.001 XV-1 Collagen 0.84±0.02 ＜0.05 XV-2 Collagen 0.72±0.02 ＜0.05 XV-3 Collagen 0.74±0.02 ＜0.05

[0058] 2. Effect of recombinant human type XV collagen on endothelial cell migration

[0059] THP-1 cells were cultured at 1×10 5 The cells were inoculated into a 6-well culture plate at a concentration of 5×10 / ml. After 24 hours, 100 μL of each recombinant type XV collagen (concentration of 25 mg / mL) was added. Each group had 3 wells. After 18 hours, a microporous filter culture chamber (Transwell) was assembled into a 6-well culture plate. At the same time, the well-digested endothelial cells (ECV304) were inoculated into a concentration of 5×10 4 / ml, add 0.5mL to each chamber for monocyte-endothelial cell co-culture, remove the upper chamber after 6 hours, wipe off the endothelial cells that did not pass through the filter membrane with a cotton swab, fix, stain, count the number of cells in the upper, lower, left, right, and middle fields under an optical microscope, and take the average. Calculate the migration inhibition rate, migration inhibition rate = (the number of migrated cells in the control group - the number of migrated cells in the experimental group) / the number of migrated cells in the control group × 100%.

[0060] Table 4 Effects of different recombinant collagens on endothelial cell migration

[0061] Group Migration number Mobility Normal saline control group 75±6 / XV-1 Collagen 48±2 36％ XV-2 Collagen 60±3 20％ XV-3 Collagen 69±4 8％

[0062] It can be seen from Tables 3 and 4 that the XV-1 recombinant collagen has a better effect of inhibiting endothelial cell proliferation and migration than the XV-2 and XV-3 recombinant collagens.

[0063] Sequence information

[0064] SEQ ID No.: 1

[0065]

[0066]

[0067] SEQ ID No.:2

[0068]

[0069] SEQ ID No.:3

[0070]

Claims

1. A recombinant human type XV collagen, the amino acid sequence of which is shown in SEQ ID No.:

1.

2. A nucleic acid encoding the recombinant human type XV collagen according to claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. A host cell into which the expression vector according to claim 3 has been introduced; preferably Escherichia coli and Pichia pastoris, more preferably Pichia pastoris.

5. A method for producing the recombinant human type XV collagen according to claim 1, comprising the steps of culturing the host cell according to claim 4 to express the recombinant human type XV collagen according to claim 1, and collecting the culture containing the recombinant human type XV collagen. 6 . The production method according to claim 5 , further comprising the step of separating and purifying the culture to obtain purified recombinant human type XV collagen.

7. The production method according to claim 6, wherein: The separation and purification adopts one or more combinations of salting out, chromatography, affinity chromatography, acid-base precipitation and membrane separation.

8. Use of the recombinant human type XV collagen according to claim 1 in the preparation of skin care products, health care products, tissue engineering materials, medicines or medical devices.

9. The use according to claim 8, wherein The tissue engineering material is selected from subcutaneous fillers, artificial bones, artificial skins, oral absorbable biofilms, bone implants, vascular stents, hemostatic agents, procoagulants, interstitial scaffolds and collagen sponges.

10. The use according to claim 8, wherein The medicine is used for preventing and fighting cancer or inhibiting the proliferation and migration of vascular endothelial cells.

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