Recombinant human type VI collagen as well as production method and application thereof

Abstract

The invention discloses a recombinant human type VI collagen as well as a production method and application thereof. The recombinant human VI-type collagen is a truncated protein of natural human VI-type collagen, and the amino acid sequence of the recombinant human VI-type collagen is shown as SEQ ID No. 1. The recombinant human VI type collagen disclosed by the invention can be efficiently secreted and expressed in a commercial expression system, and the biological activities of hemostasis and cell adhesion promotion of the recombinant human VI type collagen are higher than those of natural human VI type collagen and other recombinant human VI type collagen.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and in particular relates to a novel genetically engineered recombinant collagen, a production method and use 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, and is one of the most critical raw materials in the biotechnology industry. It 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 VI collagen is commonly found in the media and adventitia of arterial smooth muscle and coexists with fibronectin. The two may form a final forked shortcut to reach the basement membrane. At present, the main problems in the production of type VI collagen are as follows: 1) Natural type VI collagen is difficult to obtain. Natural type VI collagen is isolated from human placenta and purified using a multi-step process. This collagen extraction method has certain limitations, such as low extraction efficiency and low purity. 2) Recombinant type VI collagen is difficult to express. The expression level of full-length type VI 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 VI collagen fragments is unclear. At present, it has only been reported that type VI collagen is related to the support of cell basement membrane and extracellular matrix, and there are almost no reports on the biological activity of type VI collagen fragments.

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

[0009] 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 VI collagen, which can be efficiently secreted and expressed in a commercial Escherichia coli expression system and has good biological activities of hemostasis and cell adhesion promotion.

[0010] The inventors conducted in-depth research to solve the above-mentioned technical problems, and found that a truncated protein (221 amino acid residues) from the α2 chain of natural human type VI collagen can be efficiently secreted and expressed in a commercial Escherichia coli expression system (the yield can be as high as about 15 g / L), and has good hemostatic and cell adhesion promoting biological activities, thereby completing the present invention.

[0011] That is, the technical solution of the present invention includes:

[0012] 1. A recombinant human type VI collagen, the amino acid sequence of which is shown in SEQ ID No.: 1.

[0013] SEQ ID No.: 1

[0014] GERGDQGGKGDPGRPGRRGPPGEIGAKGSKGYQGNSGAPGSPGVKGAKGGPGPRGPKGEPGRRGDPGTKGSPGSDGPKGEKGDPGPEGPRGLAGEVGNKGAKGDRGLPGP RGPQGALGEPGKQGSRGDPGDAGPRGDSGQPGPKGDPGRPGFSYPGPRGAPGEKGEPGPRGPEGGRGDFGLKGEPGRKGEKGEPADPGPPGEPGPRGPRGVPGPEGEPGPP

[0015] 2. A nucleic acid encoding the recombinant human type VI collagen according to claim 1.

[0016] 3. An expression vector comprising the nucleic acid of claim 2.

[0017] 4. A host cell into which the expression vector of claim 3 is introduced. The host cell may be a prokaryotic cell or a eukaryotic cell, including bacterial hosts such as Escherichia coli, Bacillus subtilis, 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 Escherichia coli.

[0018] 5. A method for producing the recombinant human type VI collagen according to claim 1, comprising the steps of culturing the host cell according to claim 4 to express the recombinant human type VI collagen according to claim 1, and collecting the culture containing the recombinant human type VI 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.

[0019] 6 . The production method according to claim 5 , further comprising the step of separating and purifying the culture to obtain purified recombinant human type VI collagen.

[0020] 7. The production method according to claim 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.

[0021] 8. Use of the recombinant human type VI collagen according to claim 1 in the preparation of skin care products, health care products, tissue engineering materials or medical devices.

[0022] 9. The use according to claim 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.

[0023] The beneficial effects of the present invention are: providing a recombinant type VI collagen, which can be efficiently secreted and expressed in a commercial Escherichia coli expression system and has good biological activities of hemostasis and cell adhesion promotion. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 2 The figure shows the electrophoresis result after recombinant collagen purification (2ul loading amount). DETAILED DESCRIPTION

[0026] Example 1 Preparation of recombinant collagen VI-1 in Escherichia coli expression system

[0027] 1. Experimental Methods

[0028] 1. Preparation of shuttle plasmid and E. coli expression strain

[0029] According to the amino acid sequence shown in SEQ ID No.1, the codon optimization of the Escherichia coli expression system was carried out to obtain the target gene sequence of recombinant collagen VI-1. The obtained target gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for gene synthesis. The synthesized gene was connected to the pet-28a plasmid to obtain the pET28a-VI-1 plasmid.

[0030] The pET28a-VI-α2 plasmid was transformed into competent E. coli, and the transformants were screened using kanamycin resistance as a screening marker to obtain the E. coli expression strain.

[0031] 2. Induced expression of target protein

[0032] (1) Pick a single colony of the constructed E. coli expression strain and add it to 5 ml of LB liquid medium (0.5% yeast extract, 1% peptone and 1% sodium chloride), and culture it at 37°C and 200 rpm for 18 h for activation;

[0033] (2) Transfer the inoculum to a 500 ml Erlenmeyer flask (containing 200 ml of LB culture medium) at 1% of the inoculum volume and culture at 37°C, 200 rpm for 12 h to serve as the seed for the tank.

[0034] (3) Prepare a high-density fermentation medium, the medium composition is as follows:

[0035] Yeast powder 20-60g / L, glycerol 20-40g / L, yeast peptone 5-10g / L, dipotassium hydrogen phosphate trihydrate 3.4-6.8g / L, potassium dihydrogen phosphate dihydrate 1.4-2.8g / L, calcium sulfate dihydrate 0.15-0.3g / L, ammonium sulfate 1.2-2.4g / L, sodium chloride 3-6g / L, citric acid 0.6-1.2g / L, magnesium sulfate heptahydrate 1.5-3g / L, triammonium citrate 2.5-5g / L, glycine 1.2-2.4g / L, leucine 0.5-1g / L, methionine 0.3-0.9g / L.

[0036] 3 L of the culture medium was added to a 5 L fermentation tank, sterilized at 121° C. for 20 min, and after cooling to 37° C., the pH was adjusted to 6.8, and the seeds prepared in (2) were added to the fermentation tank by flame inoculation for fermentation culture;

[0037] (4) Cultivate until OD 600 When the dissolved oxygen rebounds to 100%, 50% glycerol and 20% yeast powder are added, and 1mMol / L IPTG is added for induction;

[0038] (5) During the induction process, the dissolved oxygen was controlled to be not less than 30% and the pH was about 6.8. The induction was carried out for 12 hours, and the fermentation was terminated. The culture medium was centrifuged at 8000 rpm for 20 minutes, the supernatant was removed, the bacteria were retained and diluted a certain multiple, and the protein dilution was obtained by ultrasonic disruption. The protein yield and purity were detected by BCA method and SDS-PAGE method.

[0039] 3. Collagen purification

[0040] The collected protein dilution was passed through a 15KD ultrafiltration membrane, and then passed through a hollow fiber column and freeze-dried to obtain recombinant collagen VI-1.

[0041] 2. Experimental Results

[0042] The BCA test result of the recombinant collagen VI-1 fermentation tank prepared by the above method is as high as 30g / L. The test result of SDS-PAGE electrophoresis shows that the target protein accounts for more than 50%, the target protein yield in the fermentation broth can reach 15g / L, and the protein purity after ultrafiltration through the hollow fiber column can reach more than 80%. The SDS-PAGE electrophoresis of the protein before purification is shown in Figure 1 The purified protein SDS-PAGE electrophoresis is shown in Figure 2 .

[0043] Example 2 Control Preparation of Recombinant Type VI Collagen in Escherichia coli Expression System

[0044] The same design concept as that of recombinant collagen VI-1 was used to extract a protein sequence with a similar molecular weight from the α2 chain of natural type VI collagen and express it in the same way. The results of sequence design and expression are shown in the table below (Table 1). The SDS-PAGE electrophoresis of the protein before purification is shown in Figure 1 The purified protein SDS-PAGE electrophoresis is shown in Figure 2 .

[0045] Table 1 Different amino acid sequences and expression results

[0046]

[0047] Note: The amino acid sequence of VI-2 is shown in SEQ ID No.: 2; the amino acid sequence of VI-3 is shown in SEQ ID No.: 3.

[0048] As shown in Table 1, the yield of the recombinant collagen VI-1 is much higher than that of the recombinant collagens VI-2 and VI-3 of similar design, and the purity of the target protein is also higher. 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.

[0049] Example 3: Application of recombinant type VI collagen in hemostasis

[0050] Specific implementation method:

[0051] Establishment of animal model of liver bleeding and evaluation of hemostatic effect Fifty SD rats were randomly divided into groups according to their body weight using the random number table method, and the sample size of animals was determined by the unpaired experimental method. They were randomly divided into three groups. Among them, 30 rats in the experimental group used recombinant VI-1 collagen, VI-2 collagen and VI-3 collagen as filling materials for liver damage, 10 rats in the control group used hemostatic sponges as filling materials for liver damage, and 10 rats in the blank group had part of the liver removed without filling.

[0052] Construction of rat model of hepatic hemorrhage: animals were fasted for at least 2 hours before surgery, and SD rats were intraperitoneally injected with 1% sodium pentobarbital. After satisfactory anesthesia, the abdominal surgical area was prepared, disinfected with iodine, and the abdominal cavity was opened layer by layer to expose the liver. The abdominal cavity, abdominal wall and peritoneal fluid around the liver were wiped clean with disposable sterile cotton swabs, and sterile gauze was weighed and placed under the liver. Ophthalmic surgical scissors were used to cut 1 cm in front of the left lobe of the liver. 2The modeling was successful when the liver was successfully removed and bleeding was visible to the naked eye. The time was kept at the same time. Different groups used corresponding materials to fill the wound surface, and the blank group did not fill the material. The hemostasis time was recorded, and the gauze was removed and weighed after hemostasis. The abdominal cavity was sutured layer by layer, iodine disinfection, and sodium penicillin was injected intramuscularly for 3 consecutive days. The mental coat color and activity status of the rats and the healing of the epidermis were observed, and the abnormal reactions of the rats, the presence of exudate in the abdominal cavity and the infection were recorded in detail.

[0053] Evaluation of hemostasis effect: ① Hemostasis time: start timing when the liver is cut open, and stop timing when bleeding stops. The recorded time is the hemostasis time of each animal. ② Bleeding volume: weigh the sterile gauze and place it under the liver. After the bleeding stops, weigh the gauze under the liver again. The difference in gauze mass before and after is the bleeding volume.

[0054] The results are as follows:

[0055] Table 2 Statistics of liver bleeding time and bleeding weight of rats in each group

[0056] Group Bleeding time (S) Bleeding weight (g) Blank Group 120 3.42 VI-1 30 1.74 VI-2 100 3.01 VI-3 90 2.84 Control group 10 1.62

[0057] It can be seen from Table 2 that the recombinant collagen VI-1 can significantly shorten the bleeding time and reduce the amount of blood loss compared with other recombinant type VI collagens, and has a good hemostatic effect.

[0058] Example 4: Application of recombinant type VI collagen in the preparation of cell adhesion promoter

[0059] Specific implementation method:

[0060] Normally culture HSF (human skin fibroblast) cells, take the purified lyophilized product of recombinant VI collagen, dissolve it in ultrapure water, and dilute it with PBS solution to 0.25mg / L, 0.5mg / L, and 1mg / L, respectively, and act on HSF (human skin fibroblast) cells to promote cell adhesion experiments. The specific steps are as follows: human HSF cells are inoculated in a sterile 96-well plate, 200μL culture medium per well, and only PBS is added to the blank control group. After being placed at 37℃ for 2h, each group is washed 3 times with PBS to wash away the non-adherent cells, 100μL culture medium and 50μL MTT solution are added to each well, shaken, and incubated in an incubator for 4h, the mixed solution is aspirated, 150μL DMSO is added to dissolve the purple crystal formazan, shake for 10min, put it in an ELISA reader, and the detection wavelength is 570nm to measure the absorbance value of each well.

[0061] The data of promoting HSF (human skin fibroblast) cell adhesion by recombinant type VI collagen and natural type VI collagen (purchased from Coriong Matrigel, USA) are shown in Table 1, wherein, cell adhesion promotion rate = (experimental group OD-blank group OD) × 100% / blank group.

[0062] Table 3 Cell adhesion promoting effect of different concentrations of recombinant collagen on skin fibroblasts

[0063]

[0064] The results show that the recombinant collagen VI-1 can significantly promote HSF cell adhesion compared with other recombinant type VI collagens, and its adhesion-promoting effect is also significantly higher than that of natural human type VI collagen. It has good cell compatibility and exhibits a good cell adhesion-promoting effect.

[0065] Sequence information

[0066] SEQ ID No.: 1

[0067] GERGDQGGKGDPGRPGRRGPPGEIGAKGSKGYQGNSGAPGSPGVKGAKGGPGPRGPKGEPGRRGDPGT

[0068] KGSPGSDGPKGEKGDPGPEGPRGLAGEVGNKGAKGDRGLPGPRGPQGALGEPGKQGSRGDPGDAGPRG

[0069] DSGQPGPKGDPGRPGFSYPGPRGAPGEKGEPGPRGPEGGRDGDFGLKGEPGRKGEKGEPADPGPPGEPGPR

[0070] GPRGVPGPEGEPGPPSEQ ID No.:2

[0071] DIASTPHELYRNDYATMLPDSTEIDQDTINRIIKVMKHEAYGECYKVSCLEIPGPSGPKGYRGQKGAKGNM

[0072] GEPGEPGQKGRQGDPGIEGPIGFPGPKGVPGFKGEKGEFGADGRKGAPGLAGKNGTDGQKGKLGRIGPP

[0073] GCKGDPGNRGPDGYPGEAGSPGERGDQGGKGDPGRPGRRGPPGEIGAKGSKGYQGNSGAPGSPGVKGA

[0074] KGGPGPRGPKGEPGRRG

[0075] SEQ ID No.:3

[0076] PGDAGPRGDSGQPGPKGDPGRPGFSYPGPRGAPGEKGEPGPRGPEGGRGDFGLKGEPGRKGEKGEPADP

[0077] GPPGEPGPRGPRGVPGPEGEPGPPGDPGLTECDVMTYVRETCGCCDCEKRCGALDVVFVIDSSESIGYTNF

[0078] TLEKNFVINVVNRLGAIAKDPKSETGTRVGVVQYSHEGTFEAIQLDDERIDSLSSFKEAVKNLEWIAGGT

[0079] WTPSALKFAYDR。

Claims

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

1.

2. A nucleic acid encoding the recombinant human type VI 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, wherein the host cell is preferably Escherichia coli and Pichia pastoris, and more preferably Escherichia coli.

5. A method for producing the recombinant human type VI collagen according to claim 1, comprising the steps of culturing the host cell according to claim 4 to express the recombinant human type VI collagen according to claim 1, and collecting the culture containing the recombinant human type VI collagen; the expression may be constitutive expression, induced expression, or a combination of the two; wherein, The inducing agent for inducing expression can be IPTG, β-galactoside, methanol, ethanol and the like. 6 . The production method according to claim 5 , further comprising the step of separating and purifying the culture to obtain purified recombinant human type VI collagen.

7. The production method according to claim 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.

8. Use of the recombinant human type VI collagen according to claim 1 in the preparation of skin care products, health care products, tissue engineering materials 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 biomembranes, bone implants, vascular stents, hemostatic agents, procoagulants, interstitial scaffolds and collagen sponges.

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